New Insights On Antiviral Probiotics

Imad Al Kassaa
New Insights
on Antiviral
Probiotics
From Research to Applications
New Insights on Antiviral Probiotics
Telegram: @Microbiology_Channel
Imad Al Kassaa
New Insights on Antiviral

Probiotics
From Research to Applications
Imad AL KASSAA
Laboratoire de Microbiologie Environnement et Santé (LMSE)
Doctoral School of Sciences and Technology/Faculty of Public Health
Lebanese University
Tripoli, Lebanon
ISBN 978-3-319-49687-0 ISBN 978-3-319-49688-7 (eBook)

DOI 10.1007/978-3-319-49688-7
Library of Congress Control Number: 2016959215
© Springer International Publishing AG 2017

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I would like to thank all the authors who
took part in the success of this book, mainly
Dr. Mohammad-Bachar ISMAIL and the
promising M. Mazen ZAYLAA who works in
Pasteur Institute in Lille on the probiotics
effects on Crohn’s disease. I would like also
to thank my leader Pr. Monzer HAMZEH
for the great support he presented and for
his patience throughout my scientific career.
Great thanks go as well to my sincere friend
Pr. Fouad DABBOUSSI. Finally, I would
like to thank my wife Hawa DHAYBI for her
patience and for the calm ambiance she
created to help me writing this book. Tala,
Omar and Ali, my dear children, thank you
for tolerating my long absence.
I would like also to thank Dr. Khaled EL

OMARI and Ms Yemen SAYOUR for their
support.
Foreword
In 1908, Elie Metchnikoff, a Russian researcher who was a Nobel laureate and pro-
fessor at the Pasteur Institute in Paris, discovered that the lifespan of Bulgarian
people was related to the consumption of fermented milk containing lactic acid
bacteria.
In 1900, Henry Tissier, a researcher at the Pasteur Institute, isolated a bacterial
strain from a breastfed child belonging to the Bifidobacterium genus, which he
called Bacillus bifidus communis. The researcher declared that this strain reduced
the infectious incidence of pathogenic bacteria, in particular Clostridium difficile,
which causes acute and inflammatory diarrhea. Moreover, Tissier recommended the
administration of such strains to children with this symptom.
After many years of research, these beneficial strains were considered an alterna-
tive treatment and were named “probiotics”, meaning “for life”. This term was
introduced in 1965 by Lilly and Stillwell. Nevertheless, probiotics and antibiotics
were defined as microbes, of molecule derived from microbes, which inhibit the
growth of other microorganisms.
Probiotics belong to several genera, such as Lactobacillus, Bifidobacterium,
Propionibacterium, and Enterococcus as Gram-positive bacteria and Escherichia
coli as Gram-negative bacteria (E. coli Nissle) and yeast like Saccharomyces bou-
lardii, which may or may not be present in the resident intestinal microflora of
humans and animals.
Probiotics were considered to be vectors that can transport active molecules to
the gut or vaginal ecosystem. Moreover, they can enhance immunity and exclude
undesirable bacteria by direct or indirect mechanisms. Indeed, probiotics inhibit
pathogenic bacteria, neutralize toxins, improve food digestibility, and enhance the
immune system. In addition, probiotics can be considered a source of vitamins
(mainly B group vitamins) and minerals.
In general, probiotics have a variety of mechanisms which can lead to beneficial
effects. However, this depends on the bacterial strain in question.
vii
viii Foreword
The emergence of multidrug-resistant bacteria and the limited number of antivi-

ral agents are major threats to public health. In addition, the increase of cancers
related to viral infections requires scientists to find new solutions. Indeed, research-
ers are focusing on the place of probiotics and some of these metabolites in control-
ling these complicated problems. Hence, the authors of New Insights on Antiviral
Probiotics have promoted the importance of the role and place of probiotics in the
treatment of respiratory and enteric viral infections, as well as the different mecha-
nisms of action. One chapter is devoted to the use of probiotics as vaccine vectors
in preventing some viral infections (influenza, HIV, HPV, rotavirus). Chronic dis-
eases related to viral infection are the subject of another chapter. The majority of
viruses involved in this type of pathology are discussed (HPV, EBV, herpes, HIV,
HTLV, etc.), as well as the impact of probiotics on reducing cancer development
during infection. The authors have also drawn attention to the importance of probi-
otic metabolites in inhibiting viral infections. Finally, the authors have included a
chapter on the methods used to evaluate probiotic strains with antiviral effects. This
chapter is a valuable tool and an excellent reference for researchers.
I am convinced that this is a book with great scientific value, which will be highly
useful to the scientific community and will bring much new information on the role
of probiotics in the treatment and prevention of viral infections.
Monzer HAMZE
Head of Laboratoire de Microbiologie Sante et Environnement (LMSE)
Lebanese University
Tripoli, Lebanon
Preface
It has been more than 20 years since viruses were first considered a threat to public
health. The rate of viral infections is increasing dramatically worldwide, and defini-
tive solutions seem to be far from reality. Moreover, numerous factors – pollution,
immunosuppressive drugs, and non-equilibrate diets – have impaired immunity and
thus amplified the risk of infection, while also causing the appearance of new patho-
gens. In addition, antiviral agents are rare because of the genetic variation of many
viruses. Furthermore, vaccines are considered a last resource for microbiologists
attempting to prevent complicated viral infections. However, it is not possible to
defeat some viruses due to their genetic variation.
More than a century ago, scientists began using, by chance, lactic acid bacteria
naturally present in fermented products to fight viral infections. Researchers have
focused during the last 20 years on the importance of probiotics in bacterial infec-
tions and chronic diseases, including cancers. In fact, antiviral probiotics appeared
first in 1990, when they acted as agents to help protect the intestinal epithelium from
viral infection and to help to decrease diarrhea. Noting this effectiveness, some
researchers conducted further studies to determine the mechanisms causing this
antiviral effect.
This book highlights probiotics with antiviral effects, which can be named
“antiviral probiotics” due to their direct and indirect effects on viral particles.
New Insights on Antiviral Probiotics contains five chapters that discuss the different
applications of this kind of probiotics in infectious and chronic viral diseases. The
third chapter focuses on the use of probiotic strains as vaccine vectors. The two last
chapters prove the importance of the antiviral metabolites of certain probiotics and
the methods used to characterize bacterial strains as antiviral probiotics.
Imad AL KASSAA
Laboratoire de Microbiologie Environnement et Santé (LMSE)
Lebanese University
Tripoli, Lebanon
ix
Contents
1 Antiviral Probiotics: A New Concept in Medical Sciences . . . . . . . . . . 1

Imad AL KASSAA
1.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Part I-A: Probiotics and Respiratory Infections . . . . . . . . . . . . . . . . . 4
1.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.2 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3 Part I-B Probiotics and Viral Gastroenteritis . . . . . . . . . . . . . . . . . . . 14
1.3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.2 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . 35
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2 The Use of Probiotics as Vaccine Vectors to Prevent
Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Bachar ISMAIL
2.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.1.1 Mucosal Immunity and Vaccines . . . . . . . . . . . . . . . . . . . . . . 49
2.1.2 Probiotic Bacteria as Vaccine Delivery Vehicles:
A Promising Strategy for Mucosal Vaccination . . . . . . . . . . . 49
2.1.3 Parameters that Modulate the Immune Responses
Induced by Recombinant Probiotic Vaccines. . . . . . . . . . . . . 50
2.1.4 Probiotics as Vaccine Vectors to Prevent Viral Infections . . . 51
2.2 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3 Probiotics: Role in the Prevention of Chronic Viral Diseases . . . . . . . . 61
Imad AL KASSAA and Mazen ZAYLAA
3.1 General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2 Cancer Related to Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3 The Impact of Probiotics in Cancers Related to Human
Papillomavirus (HPV) Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
xi
xii Contents
3.4 The Impact of Probiotics in Cancers Related to Human T-Cell

Lymphotropic/Leukemia Virus (HTLV) Infection . . . . . . . . . . . . . . . 69
3.5 Probiotics as a Novel Prevention Strategy Against
Type 1 Diabetes Related to Viral Infection . . . . . . . . . . . . . . . . . . . . 71
3.6 Probiotics as a Treatment and Prevention Strategy for Liver
Complications Caused by Hepatitis B and C Virus . . . . . . . . . . . . . . 72
3.7 Treatment and Prevention Strategy of Herpes Simplex
Viruses 1 and 2 Using Probiotic Strains . . . . . . . . . . . . . . . . . . . . . . 74
3.8 Probiotics and Human Immune Deficiency Virus (HIV) . . . . . . . . . . 76
3.9 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4 The Antiviral Activity of Probiotic Metabolites . . . . . . . . . . . . . . . . . . . 83
Imad AL KASSAA
4.1 Antiviral Activity of Probiotic Metabolites . . . . . . . . . . . . . . . . . . . . 84
4.1.1 Non-organic Substances. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.1.2 Organic Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.2 Probiotics and Their Proteinaceous Metabolites . . . . . . . . . . . . . . . . 91
4.3 Unspecified Antiviral Metabolites by Assessment
of Probiotic/LAB Native Supernatants . . . . . . . . . . . . . . . . . . . . . . . 92
4.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
5 Methods and Techniques to Evaluate the Antiviral Activity
of a New Probiotic Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Imad AL KASSAA
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.2 Evaluation of a Potential Probiotic Strain . . . . . . . . . . . . . . . . . . . . . 101
5.2.1 Isolation and Characterization of Probiotic Strains . . . . . . . . 101
5.2.2 Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.2.3 Screening Tests to Confirm Potential Probiotic
Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
5.2.4 Safety of Selected Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . 103
5.2.5 Antibiotic Resistance Profile . . . . . . . . . . . . . . . . . . . . . . . . . 103
5.2.6 In Vivo Studies in Animal Models and Human Trials . . . . . . 104
5.3 Evaluation of Antiviral Probiotics (AvPrs) . . . . . . . . . . . . . . . . . . . . 104
5.3.1 In Vitro Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.3.2 Antiviral Assays for Bacterial Cells . . . . . . . . . . . . . . . . . . . . 106
5.3.3 In Vivo Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.3.4 Clinical Trials (CTs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Chapter 1
Antiviral Probiotics: A New Concept
in Medical Sciences
Imad AL KASSAA
Contents
1.1 Overview .......................................................................................................................... 3
1.2 Part I-A: Probiotics and Respiratory Infections ............................................................... 4
1.2.1 Introduction ....................................................................................................... 4
1.2.2 Conclusion and Perspectives ............................................................................. 13
1.3 Part I-B Probiotics and Viral Gastroenteritis ................................................................... 14
1.3.1 Introduction ....................................................................................................... 14
1.3.2 Conclusion and Perspectives ............................................................................. 35
References ................................................................................................................................. 35
Abstract In recent decades, probiotics have shown beneficial effects on animal and
human health. Probiotics can protect the host against several health threats, includ-
ing infectious diseases. Before 1995, researchers believed that the effect of probiot-
ics was only on gut microbiota which can restore the gut flora and thus prevent
pathogenic bacteria from triggering gastroenteritis. Recent studies have shown that
the immunomodulatory activity is the most important mechanism of action of pro-
biotics. From this information, researchers started to evaluate the effect of some
immunobiotics, not only on pathogenic bacteria but also on viruses, including
enteric and respiratory viruses. Several studies have confirmed the potential antivi-
ral activity of some probiotics due to the immunomodulatory effect. These studies
were conducted on humans (clinical trials) and in animal models. In this chapter,
probiotics with antiviral effect against respiratory and enteric viruses will be pre-
sented and discussed, as well as their mechanisms of action.
Keywords Antiviral probiotics • Respiratory viruses • Immunomodulation • Gut

microbiota • Immunobiotics • Enteric viruses • Antiviral probiotics • Viral trapping
• Norovirus • Rotavirus • Immunomodulation • Gut microbiota
Abbreviations
AAstV Avastrovirus
AdVs Enteric adenoviruses
AEnP Anti-EnV probiotics
© Springer International Publishing AG 2017 1

I. Al Kassaa, New Insights on Antiviral Probiotics,
DOI 10.1007/978-3-319-49688-7_1
2 1 Antiviral Probiotics: A New Concept in Medical Sciences
AMPs Antimicrobial peptides

AMs Animal models
AVs Arboviruses
BALF Bronchoalveolar lavage fluid
BCS Bacterial cell suspension
BLISs Bacteriocin-like inhibitory substances
CA16 Coxsackievirus type A strain 16
CFU Colony-forming unit
CRFK Crandell-Reese feline kidney
CTs Clinical trials
CXCL1 Neutrophil chemokine
DLP Double-layered particle
EnVs Enteric viruses
EU European Union
EV71 Enterovirus 71
EVs Enteroviruses
GIT Human gastrointestinal tract
GRAS Generally recognized as safe
HBGAs Histo-blood group antigens
HFMD Hand, foot, and mouth disease
HRoV Human rotavirus vaccine
ID Infectious diseases
IFN-α Interferon-α
IgA Immunoglobulin A
IL-10 Interleukin 10
IL-2 Interleukin 2
IL-6 Interleukin 6
IL-8 Interleukin 8
IVA-H1N1 Influenza virus type A
LAB Lactic acid bacteria
LPS Lipopolysaccharide
MAstV Mamastrovirus
MMTV Mouse mammary tumor virus
MuNoVs Murine noroviruses
NK cells Natural killer cells
NRPS Non-ribosomal peptide synthetase
NSP Nonstructural protein
NVs Noroviruses
PVR Poliovirus receptor
RB Rice bran
ROS Reactive oxygen species
RoVs Rotaviruses
RSBCT Randomized single-blind controlled trial
RSV Respiratory syncytial virus
1.1 Overview 3
RTIs Respiratory tract infections

RVs Respiratory viruses
TGEV Transmissible gastroenteritis virus
TGFβ Transforming growth factor beta
TLR Toll like receptors
TNF-α Tumor necrosis factor alpha
VLPs Viruslike particles
VP Viral protein
VP1 Viral protein 1
1.1 Overview
Respiratory infections and gastroenteritis constitute the major causes of mortality

and morbidity worldwide, both in developing and developed countries [1]. Despite
the widespread adoption of vaccines strategies, some pathogens remain a threat to
public health worldwide. The US National Institutes of Health (NIH; Bethesda,
MD, USA) declared the emergence of 16 new infectious diseases, six of which have
been considered reemerging infections [2]. In the United States of America (USA),
mortality caused by infectious diseases (IDs) was amounted to 170,000 deaths in
2000 [3]. An increase in immunocompromised patients plays a crucial role in the
emergence and/or reemergence of IDs; therefore, post-infection complications can
lead to death. Public health is faced with two major obstacles to eradicating IDs: (1)
Antibiotic therapies, which have been saving infected patient for several years.
Unfortunately, the rapid emergence of resistant bacteria is occurring worldwide,
endangering the efficacy of antibiotics, which have transformed medicine and saved
millions of lives [4]. (2) The lack of antiviral agents against infectious viruses,
which leads to a high treatment level between populations even in the presence of
some vaccines covering a few virus types [5]. Several strategies have been devel-
oped to overcome this crisis, e.g., (i) the use of bacteriophages as antibacterial
agents [4], (ii) the extraction and purification of antimicrobial peptides [6], and (iii)
the prevention of IDs by using vaccines and/or recombinant vaccine strategies [7].
Preventing infectious diseases occurring seems to be the perfect method of avoiding
ID complications, since all of the abovementioned strategies have inconveniences
such as side effects and stability in the host.
Immune system boosting is the essential key factor in ID prevention. Dietary
balance in meals, administration of supplements such as fiber, and probiotics are
three methods to enhance and stimulate the immune system, thus protecting the
mucosa against the entry of pathogens. Probiotics have demonstrated their capacity
to stimulate and modulate the immune system [8]. In addition to the antibacterial
activity of probiotics, some strains showed an effective antiviral activity which can
be a solution to the lack of antiviral agents [9].
In this chapter, we focus on probiotics which have been shown to be effective as
antiviral agents against respiratory and enteric viruses. In addition, we give details
of some clinical trials and both in vitro and in vivo experiments which have con-
firmed this efficacy.
Immunobiotic Cancer prevention

Restoration of the normal
Action Cholesterol reduction
intestinal microbiota after
Local and distant Prevention of Crohn
Antibiotherapy
effects disease
Reduction of
Positive effect on
lactose intolerance LAB – probiotics
the intestinal
Increase of lactose
microbiota
digestion
Prevention of microbial Production of inhibitory

Reduction of pH
and viral infections in metabolites (bacteriocins)
intestines and urogenital Production of vitamins
tracts, vagina. . . B and C
Fig. 1.1 Potential functions attributed to LAB probiotics [5]
1.2 Part I-A: Probiotics and Respiratory Infections
1.2.1 Introduction
Lactic acid bacteria (LAB) can be found in many ecosystems, including human and
animal flora. LAB, as well as their metabolites such as bacteriocins, are generally
recognized as safe (GRAS) [10]. The antibacterial activity of probiotics has been
confirmed in a large number of research studies. This activity may occur through
several mechanisms: (1) Pathogens exclusion: probiotic strains have a high affinity
for adhesion to epithelial cells. Thus, probiotics will saturate the receptors and exert
a barrier effect against the pathogens involved in infections. (2) Nutrient competi-
tion: probiotic strains can ingest many essential molecules, and consequently patho-
gens cannot grow in this ecosystem. (3) Production of antimicrobial compounds,
such as lactic acid, hydrogen peroxide, bacteriocin-like inhibitory substances
(BLISs), NRPS, and bacteriocins [11–13] (Fig. 1.1).
In addition to food applications, the use of LAB is growing, in particular as probiot-
ics for controlling, for example, gastroenteritis, inflammatory pathologies of the diges-
tive tract [14, 15] and to stimulate the local and systemic immune response [16, 17].
In recent decades, some probiotics have shown an antiviral activity and several
mechanisms have been demonstrated. In respiratory tract infections (RTIs), the
majority of probiotics can inhibit the most important respiratory viruses (RVs) by
immunomodulatory mechanisms [18] (Fig. 1.2). This antiviral mechanism might be
explained due to the entry routes of RVs. RVs infect the mucosal cells of the RT, and
for this reason, probiotic strains and their antimicrobial compounds cannot directly
1.2 Part I-A: Probiotics and Respiratory Infections 5
Fig. 1.2 Suggested mechanisms of antiviral probiotics against respiratory viruses. This figure
shows the antiviral mechanisms of some probiotic strains used against viral respiratory infec-
tions. Although there is a difference between the probiotic colonization ecosystem and the target
RV ecosystem, several studies have showed that there is a relationship between gut microbiota
and other tissues. Probiotics can inhibit viruses and/or help the immune system defend itself
against RVs. First, the RVs interact with the respiratory epithelium, which generates an innate
immune response by activating the IFN signaling and other proinflammatory cytokines. Once
cytokines have been secreted, macrophages and NK cells will be recruited to phagocytize and kill
both viruses and viral-infected cells. To trigger a specific immune response, the immune system
needs proinflammatory cytokines, energy, and some cofactor elements. Hence, probiotics can
provide some elements to boost the immune response: A. Probiotics interact with the gut epithe-
lium and are recognized by intestinal DCs (IDCs); this interaction results in the production of
IL-12 and IFNγ by IDCs, which can modulate both the respiratory and gut immune response. B.
Secretion of IFNγ and IL-12 by intestinal DCs; these two proinflammatory cytokines have dual
functions: IFNγ and IL-12 can circulate in the bloodstream to reach the respiratory epithelium
and therefore help alveolar macrophages and NK cells eliminate RVs. C. The proinflammatory
cytokines (IFNγ and IL-12) secreted in the gut ecosystem after colonization of some probiotic
strains help the immune system to generate a specific Th1/Th17 immune response; the number of
CD4+ and CD8+ increases and becomes more efficient. In addition, CD4+ will secrete IL-17,
which enhances the innate immune response. D. Some probiotic strains, via induction of IFNγ
and IL-17 production, can stimulate the overexpression of innate immunity-related genes such as
the overexpression of TLR7, even in the lung. This overexpression of TLR7 amplifies the innate
immune responses. E. Probiotics can help B lymphocytes differentiate and become plasma cells,
which can secrete specific sIgA. In our case, some studies showed the impact of some probiotics
in increasing sIgA in lung tissues. However, until now there is no explanation of the real mecha-
nisms of how intestinal probiotics can help secretion of sIgA which are specific to elimination of
RVs. This effect can be explained by the capacity of some probiotics to enhance cytokine produc-
tion, which can improve the rapid differentiation of B lymphocytes to plasma cells in lung
tissues.
interact with viruses by physical contact. Probiotic strains can finally reduce or erad-
icate virus infectivity by immunomodulatory activity, which has led scientists to call
them “immunobiotics” [19]. In this chapter, the majority of anti-RV probiotics will
be mentioned with other information, such as the source of probiotic strain, target
virus, experimental model, and mode of antiviral activity (Table 1.1).
1.2.1.1 Lactobacillus Probiotic Strains in Viral Respiratory Infections
Lactobacillus is the most studied genus of anti-RVs probiotics, followed by the

Bifidobacterium genus. It was reported that Lactobacillus plantarum L-137 (L.
plantarum L-137) isolated from fermented foods showed proinflammatory activity
which can decrease the titer of influenza virus type A (IVA-H1N1) in mouse lungs
[20]. Another strain, L. plantarum YU, isolated from Japanese fermented foods,
showed anti-H1N1 activity by activating the Th1 immune response [22].
Recently, several studies have reported that L. plantarum species reduce the
signs of influenza-like symptoms and even increase body weight and survival rate
in a mouse model [21, 23, 24]. In addition, mice infected by a lethal pneumovirus
survived when they were protected using a combination of two lactobacilli strains,
L. plantarum NCIMB 8826 isolated from human saliva and L. reuteri F275 iso-
lated from the human gastrointestinal tract (GIT) [25]. It has not been reported that
L. plantarum probiotic strains were assessed in a human experimental model,
which may be due to the undesirable acid or metabolites secreted by this species.
L. rhamnosus strains are the most important probiotics for human applications.
Furthermore, the majority of L. rhamnosus strains are immunobiotics, due to their
ability to stimulate and enhance the host immune system. In animal experiments, L.
rhamnosus GG (LGG), a famous probiotic strain, was evaluated and showed an
anti-influenza virus activity on intranasal and oral administration [26, 27].
In mice infected with respiratory syncytial virus (RSV), heat-killed L. rhamno-
sus CRL1505 and CRL1506 showed a good inhibitory effect and increased the body
weight of mice [34]. These two strains were considered good immunobiotics in
RSV infection due to their IFN-α stimulation, which decreases the viral load in
mouse lungs [34]. The majority of clinical trials in children were performed using
LGG [28, 29]. LGG reduces the number of upper and lower viral RTIs in children,
reduces the days of absence from daycare and decreases antibiotic use [28]. The
administration route of LGG in clinical trials in children was often by drinking
probiotic-inoculated milk [28, 30–32].
The antiviral activity of LGG was also assessed in adults and the elderly. Several
clinical trials were conducted in order to improve the beneficial effect of this strain
in the treatment and prevention of viral infections. Kekkonen et al. studied 141
marathon runners (22–69 years old) who drank two bottles of milk daily. The
probiotic group drank a milk inoculated with LGG (4.1010 CFUs). The results
showed that the ingestion of LGG did not decrease RTI episodes or the severity of
symptoms [33]. However, the combination of LGG with Bifidobacterium animalis
ssp. Lactis Bb12 (B. animalis ssp. Lactis Bb12) decreased the duration and symp-
tom severity of URTIs significantly [62].
Table 1.1 Probiotics assessed against viral respiratory infections
1.2
Probiotic strains Origin Infection Mechanisms Experiment References

Lactobacillus plantarum L-137 Fermented food IVA-H1N1 Proinflammatory activity Mouse lungs Murosaki et al. [20]
Th1 immune response 7-week-old Maeda et al. [21]
C57BL/6 mice
L. plantarum YU Japanese H1N1 Suppressing viral proliferation in Normal mouse Kawashima et al. [22]
fermented food infection lung and increasing specific IgA experiment
secretion
L. plantarum CNRZ 1997 – H1N1 strain Proinflammatory response HT-29 cell culture; Kechaou et al. [23]
A PBMC and in vivo
using BALB/c mice
L. plantarum DK 119 Fermented Influenza Increase of IFNγ and IL-2 In vivo normal mice Park et al. [24]
Korean cabbage virus A
L. plantarum NCIMB 8826 Human saliva Pneumovirus Increasing infected mice survival BALB/c and Gabryszewski et al. [25]
L. reuteri F275 Human GIT via TLR-dependent inflammatory C57BL/6 mice,
response MyD88−/− mice on
Part I-A: Probiotics and Respiratory Infections
a C57BL/6
LGG Human feces H1N1 Increase of mRNA of IL-1β and BALB/c mice Harata et al. [26]
activation of lung NK cells Kawase et al. [27]
URTIs and Reducing absence days RDBPC Hatakka et al. [28];
LRTIs Reducing antibiotics intake Luoto et al. [29];
Decrease in severity of symptoms Hojsak et al. [30, 31];
Kumpu et al. [32];
Kekkonen et al. [33]
L. rhamnosus CRL 1505 – RSV Innate immunity stimulation and BALB/c mice Tomosada et al. [34]
L. rhamnosus CRL 1506 induction of IFN-α production via
TLR3/RIG-I-triggered antiviral
respiratory immune response
LcS Human and H1N1 Innate immunity stimulation and BALB/c mice Hori et al. [35]
mouth flora induction of IFN-α production
7
(continued)
8
Table 1.1 (continued)

L. casei DN-114,001 Fermented food RTIs Decrease in illness period by 1 day RDBPC: Cobo Sanz et al. [36]
251 participants Merenstein et al. [37]
(children from 3 to
12 years of age, 142
receiving probiotics
and 109 in the
placebo group)
Decrease in RTI duration and RDBPC: Adults and Guillemard et al. [38];
1
CIDs elderly Tiollier et al. [39];

Turchet et al. [40]
Lpp 06Tca19 Camel milk H1N1 Increase in IL-12, IFNγ, and NK BALB/c mice Takeda et al. [41]
Lpp06Tca22 cell concentration in BALF
Decrease in TNF-α
L. fermentum CJL-112 Korean fermented H1N1 Increase in IgA BALB/c mice Yeo et al. [42];
food Enhancement of Th1 immune HD-11 cell culture
response
Increase in NO production
L. fermentum −1 – H1N1 Increase in IgA and IL-12 BALB/c mice Youn et al. [43]
production
L. fermentum CECT5716 with Human breast URTIs Decrease in the incidence of RDBPC: Maldonado et al. [44];
galactooligosaccharides as milk and LRTIs URTIs and LRTIs 215 children
prebiotic (6 months old)
Potentiation if the immunological 50 volunteers Olivares et al. [45]
response of an anti-influenza
vaccine and increase in Th1
immune response
Antiviral Probiotics: A New Concept in Medical Sciences
1.2
L. fermentum VRI003 (PCC) PCC®; RTIs In the duration of RTIs RDBPC of 20 Cox et al. [46]
Probiomics Ltd. Insignificance on the incidence of endurance athletes
et al. RTIs
URTIs Reduction of episodes and RDBPC of 99 West et al. [47]
duration of URTIs cyclists (35 to
59 years old)
L. acidophilus L-92 Japanese healthy H1N1 Activation of NK cells in lungs BALB/c mice Goto et al. [48]
volunteer Increase in IFN-α secretion
L. salivarius – UTIs Insignificant results 66 endurance Gleeson et al. [49]
athletes
L. brevis KB-290 Japanese pickle IgA and IFN-α increase in mice BALB/c mice Waki et al. [50]
“Suguki” lungs
L. gasseri TMC0356 Intestine of H1N1 Decrease in the severity of BALB/c mice Kawase et al. [27]
healthy adults symptoms and viral titer:
Stimulation of IL-12, IL-6, IFN-c,
and IgA production
Part I-A: Probiotics and Respiratory Infections
L. pentosus S-PT84 Kyoto pickles H1N1 Activation of NK cells in lungs BALB/c mice Izumo et al. [51]
Increase in IFN-α and IgA
secretion
L. pentosus b240 Fermented tea H1N1 Increase in mRNA expression of BALB/c mice Kiso et al. [52]
leaves antiviral gene:
Egr1 (critical regulator of host
inflammatory chemokines) and
Rsad2 (an interferon-stimulated
gene (ISG))
B. longum BB536 Japanese healthy H1N1 Increase in IFNγ and IL-6 BALB/c mice Iwabuchi et al. [53]
infant Decrease in symptoms and body
weight loss
B. longum (Bifico®) – H1N1 Increase in gene expression such BALB/c mice Wu et al. [54]
as TLR7
9
10

B. animalis ssp. Lactis BB12 Chr. Hansen, RTIs Reduction in the number of viral RDBPC: 109 Taipale et al. [55]
Horsholm, RTIs healthy newborns
Denmark, No effect on acute otitis
B. animalis ssp. lactis B1–04 Bl-04; Danisco URTIs Reduction of URTI episodes DBPCT: 460 West et al. [56]
USA, Madison, physically active
WI adults
Combination
LGG with Bifidobacterium – URTIs Decrease in the severity and RDBPC: 231 adult Smith et al. [57]
1
animalis ssp. Lactis Bb12 Viral RTIs duration of symptoms students Rautava et al. [58]
Reduction in antibiotic RDVPC:
consumption and decrease in the
incidence of acute otitis
Bifidobacterium animalis ssp. Peruvian mother’s Viral RTIs Reduction in viral RTI symptoms RDBPC: 201 Weizman et al. [59]
Lactis Bb12 with milk and antibiotic consumption healthy infants
L. reuteri ATCC DSM 1793
B. bifidum with L. acidophilus Infloran, Berna, Viral RTIs Reduction in symptoms and RDBPC: 80 children Rerksuppaphol and
Switzerland decrease in school absence Rerksuppaphol [60]
frequency
LGG, L. rhamnosus Lc705, B. – Human Reduction in symptoms of HBoV RDBPC: 269 Lehtoranta et al. [61]
breve 99 and P. freudenreichii bocavirus infection but not picornavirus otitis-prone children
JS infection
Lactobacillus casei (L. casei) is a beneficial bacterium found naturally in both

the mouth and intestines of humans. L. casei may be found in “raw or fermented
dairy and fresh or fermented plant products” [63].
L. casei Shirota (LcS) is the major probiotic strain among this species. This
strain has been isolated from mouth flora [64]. The intranasal administration of LcS
in H1N1-infected mice showed a decrease in the viral titer in a nasal wash. Moreover,
LcS increases the secretion of antiviral cytokines such as interferon alpha (IFN-α).
Furthermore, LcS stimulates the innate immune response [35]. LcS has shown an
immunomodulatory activity against RVs. However, in clinical trials, in particular in
the elderly group, LcS has shown insignificant results in comparison with the pla-
cebo group [65, 66].
Another probiotic strain, L. casei DN-114,001, showed good antiviral activity in
clinical trials. L. casei DN-114,001 was evaluated in children, adults, and the elderly
in separate studies. In children clinical trials, L. casei DN-114,001 decreased the
symptoms and duration of RTIs significantly [36, 37]. In the adult and elderly
groups, the administration of L. casei DN-114,001 decreased the duration of RTIs
and common infectious diseases (CIDs) [38–40, 67].
L. paracasei, in particular L. paracasei ssp. Paracasei (Lpp), was evaluated for
its antimicrobial activity in animal models [24, 41]. After oral administration in
mice, the Lpp 06Tca19 and Lpp 06Tca22 strains, isolated from fermented camel
milk, showed a significant decrease in TNF-α in bronchoalveolar lavage fluid
(BALF). This effect led to an increase in the mice’s survival and a decrease in the
macrophage and neutrophil concentrations in BALF [41].
L. fermentum is a species which can be found in human and animal flora [68]. This
species is usually used as a probiotic in humans. In RTIs, L. fermentum was evaluated
in both human clinical trials, in particular in children and adults [45, 46], and in
animal models in order to investigate the mechanism of viral inhibition [42, 43].
L. fermentum-1 and L. fermentum CJL-112 were assessed in H1N1-infected mice.
The results have shown an important reduction in the viral load, with high stimulation
of IgA and Il-12 secretion which allows an increase in the mice’s survival [42, 43].
L. fermentum CECT5716 was evaluated only in human clinical trials [44, 45].
Two hundred and fifteen healthy infants (6 months old) took 2.108 CFUs/daily with
galactooligosaccharides as prebiotics. This trial showed a significant decrease in the
incidence of URTIs and LRTIs in infants [44]. L. fermentum VRI003 and L. fermen-
tum PCC are two probiotic strains which showed a significant decrease in the dura-
tion of RTI symptoms in healthy, physically active adults. However, these two
strains did not reduce the incidence of RTIs [46, 47].
L. acidophilus is a famous probiotic strain used in pharmaceutical supplements
[69]. A few studies evaluated the antiviral activity of L. acidophilus, because this spe-
cies is usually used for gastrointestinal problems [47, 60]. All antiviral clinical trials
used in humans were conducted using a combination formula with other probiotic
strains, while one animal experiment was conducted using L. acidophilus L-92, iso-
lated from a healthy Japanese volunteer, which showed an anti-IFV A (H1N1) activity
by increasing active NK cells in lungs. Moreover, L. acidophilus L-92 showed an
increase in IFN-α secretion [48].
L. salivarius resides in the mouth and small intestine. It mainly plays a role in pro-
tection against several kinds of pathogens [70]. Sixty-six endurance athletes partici-
pated in a clinical trial; 33 of them have taken 2.1010 CFUs of L. salivarius probiotic
strains daily for 16 weeks, while the control group (n =33) took a placebo. The results
showed a nonsignificant change in comparison with the placebo group [49]. These
results can lead to the conclusion that the influence of probiotics in RTIs could be
directly related to the species or to a new characteristic presented in a specific strain.
L. brevis KB-290 (isolated from a traditional Japanese pickle called “suguki”), L.
gasseri TMC0356 (isolated from the intestine of healthy adults), L. pentosus S-PT84
(isolated from Kyoto pickles), and L. pentosus b240 (isolated from fermented tea
leaves) are probiotic strains evaluated in mouse model experiments [27, 50–52].
These strains showed a strong anti-IFV activity, in particular against the H1N1
strain. The anti-H1N1 activity of L. brevis KB-290 was reported to increase IFN-α
and IgA secretion in mouse lungs after oral administration of this strain [50]. The
oral administration of L. gasseri TMC0356 in intranasally H1N1-infected mice
showed a positive effect on influenza symptoms. L. gasseri TMC0356 can decrease
the viral titers by interacting with the intestinal immunity system, in particular in
Peyer’s patch, resulting in high production of IL-12, IL-6, IFN-c, and IgA [27]. Kiso
et al. reported that the oral administration of L. pentosus b240 increased protection
in mice against a lethal dose of H1N1. The primary mechanism of this effect was by
upregulation of antiviral genes such as Egr1 (a critical regulator of host inflamma-
tory chemokines) and Rsad2 (an interferon-stimulated gene (ISG)) [52]. Izumo
et al. reported a new antiviral activity mechanism in a mouse experimental model
infected by the H1N1 strain. They showed that the antiviral activity was created by
activation of lung NK cells after intranasal administration of L. pentosus S-PT84 in
BALB/c mice. Moreover, this strain can increase the production of IFN-α and IgA
and decrease the allergic reaction by modulating the Th1/Th2 balance [51].
1.2.1.2 Bifidobacteria Probiotic Strains in Viral Respiratory Infections
Bifidobacterium is a very important bacterium in animal and human flora. This

genus has beneficial effects for the host, and it was used for the first time as a com-
mercial probiotic [71]. Bifidobacteria promote good digestion, boost the immune
system, and inhibit almost all intestinal pathogens [72].
In viral RTIs, the bifidobacterial strains were used in combination in several
human clinical trials to assess antiviral activity and investigate the mechanism of
such activity [55, 60]. The combination was conducted using lactobacilli probiotic
strains [28, 58, 60]. In a few studies, the antiviral mechanisms of bifidobacteria were
investigated in animal models, in particular mouse experiments.
Bifidobacterium longum (B. longum) BB536, isolated from Japanese healthy
infants, showed an anti-IFV A/PR/8/34 (H1N1) activity after oral administration for
2 weeks before infection. This antiviral activity was created by decreasing
proinflammatory cytokines, such as IFNγ and IL-6, in BALB/c mice. Furthermore,
this strain showed the capability to significantly decrease the symptom score and
body weight loss [53]. In another BALB/c mouse experiment, Wu et al. reported
that the administration of a Bifico® strains mixture, in particular B. longum, led to

upregulation of the expression of several genes involved in antiviral responses, such
as TLR7, MyD88, IRAK4, TRAF6, and NF-KB [54].
In a double-blind, placebo-controlled study, 109 healthy newborns aged 1 month
participated; 55 candidates were subjected to 109 CFUs/day of B. animalis ssp. Lactis
BB12 and the control group (n =54) received control tablets as a placebo. Taipale
et al. reported that the B. animalis ssp. Lactis BB12 strain reduced the number of
viral RTI episodes, while there was no effect on the occurrence of acute otitis [55].
Several studies investigated the combination of B. animalis ssp. Lactis BB12 with
other probiotic strains to determine the possibility of the strongest antiviral activity.
The combination of L. reuteri ATCC DSM 1793 (isolated from Peruvian mother’s
milk) with B. animalis ssp. Lactis BB12 was evaluated in a double-blind, placebo-
controlled, randomized trial of 201 healthy infants aged between 4 and 10 months. This
combination reduced the viral RTI symptoms, fever, and antibiotic consumption [59].
Rautava et al. showed that the combination of B. animalis ssp. Lactis BB12 and
the LGG strain reduced antibiotic consumptions. Moreover, this combination
reduced the incidence of acute otitis media in the first 7 months of life [58].
In another double-blind randomized controlled trial, a combination of B. bifidum
with L. acidophilus probiotic strains (Infloran, Bern, Switzerland) was administered
to 80 healthy children aged between 8 and 13 years old. Reduced viral RTI symp-
toms and a decrease in the school absence rate were the main outcomes of this
combination [60].
B. animalis ssp. Lactis B1–04 reduced viral URTI episodes in a clinical trial of
460 physically active adults (18–60 years old) [56]. Lehtoranta et al. reported the
efficacy of a combination of LGG, L. rhamnosus Lc705, B. breve 99, and
Propionibacterium freudenreichii JS in nasopharynx bocavirus infection, in particu-
lar in otitis-prone patients [61]. This study was a randomized, double-blind, placebo-
controlled trial on 269 otitis-prone children (aged 9 months to 5.6 years) with
6 months of probiotic intervention. The authors showed the specific antiviral activ-
ity of this combination against bocavirus but not picornavirus. Moreover, this com-
bination seems to be effective in children, but not in the elderly [61].
Each of the abovementioned probiotic strains seems to have one or many specific
antiviral mechanisms. Furthermore, the viral specificity is directly related to the
strain used or the combination of several probiotic strains in the same or different
genus types. Moreover, the antiviral effect of probiotics by immunomodulatory
mechanisms depends on the immune system status, which can be explained in the
study conducted by Lehtoranta et al., who showed that the combination of four
probiotic strains worked very well in children but not in the elderly [61].
1.2.2 Conclusion and Perspectives
Lactobacillus and Bifidobacterium genera have the strongest antiviral activity

against respiratory viruses, in particular against influenza virus type A. This antivi-
ral activity depends on the strain’s specificity and the situation of the host immune
system. Clinical trials (CTs) are the most used evaluation method in these studies.
The alleviation of symptoms was measured in these CTs in order to investigate the
impact of suggested probiotic strains against RVs. The main mechanism of such
probiotics is immunomodulation. The use of probiotics in respiratory infections
leads to the activation of many signals for innate immunity and the production of
IgA antibodies in respiratory tissue. Anti-inflammatory probiotics in respiratory
viral infections are not welcome, since they block the immune responses against the
virus. However, in respiratory inflammation, probiotics that stimulate the produc-
tion of anti-inflammatory (IL-10, TGFβ) cytokines play a crucial role in suppressing
inflammation.
We recommend avoiding antibiotic treatment for respiratory infections except in
the case of a confirmed bacterial infection. Although the antibiotic treatment may
prevent respiratory bacterial superinfection, this antibiotic therapy eradicates the
microbiota, in particular Gram-positive probiotic strains. Anti-RoV probiotics
should be evaluated in depth in germ-free mice and/or antibiotic-treated mice in
order to determine the complete mechanism of such probiotics. Furthermore, the
molecules responsible for this immunomodulatory activity should be investigated
and purified for in vivo experiments.
1.3 Part I-B Probiotics and Viral Gastroenteritis
1.3.1 Introduction
In 1907, Elie Metchnikoff observed the healthy effect of fermented dairy products
in his population. Recently, researchers have confirmed the beneficial effects of
fermented foods, in particular the role of lactic acid bacteria (LAB), such as
Lactobacillus, Bifidobacterium and other LAB, which have probiotic properties
[73]. Gram-positive Lactobacillus and Bifidobacterium bacteria are not occasional
contaminants, but they are two genera that colonize the primary microbiota of
humans.
The colonization, development, and maturation of the newborn’s gastrointestinal
tract that begins immediately at birth and continues for 2 years is modulated by
numerous factors, including mode of delivery, feeding regime, maternal diet/weight,
probiotic and prebiotic use, and antibiotic exposure pre-, peri-, and postnatally [74].
This microbiota plays a major role in the host’s defense against pathogens [75]. This
microbiota can maintain the health of the gut ecosystem and preserve defensive
readiness against enteric pathogens and sometimes prevent enteric chronic diseases
[76]. Microbial concentrations are distributed along the digestive tract, with 103
bacterial cells/mm3 in duodenum and stomach, 102 to 103 bacterial cells/mm3 in the
fasting ileus and distal ileum, and 1010 to 1012 bacterial cells/mm3 in the colon [77].
Unfortunately, due to an incorrect feeding regime, humans are losing the primary
microbiota which is related to an increase in diseases, including infectious diseases.
To restore this primary microbiota, several health organizations suggested using
probiotics as a dietary supplement.
1.3 Part I-B Probiotics and Viral Gastroenteritis 15
1.3.1.1 The Importance of Microbiota
Recent studies have demonstrated the symbiotic relationship between intestinal

microorganisms that benefits their human host. Intestinal microorganisms, referred
to as intestinal microbiota, have several mechanisms of maintaining gut health. The
intestinal microbiota degrades indigestible dietary substances, such as fiber, and
converts it into an energy source for gut cells and the immune system [78, 79].
The other role of intestinal microbiota is to develop the gut immune system. Hooper
et al. showed that germ-free infected mice have severely compromised immune responses
and a reduction in the level of secretory immunoglobulin A (IgA) and the number of
intestinal T cells compared with wild-type mice [80, 81]. In addition, intestinal micro-
biota can protect the host against pathogens by inducing intestinal epithelial cells to
secrete antimicrobial proteins, such as angionin and C-type lectin RegIIIγ [82, 83].
1.3.1.2 Histo-Blood Group Antigens (HBGAs) and Gram-Negative

Bacteria in Gut Microbiota
The ABO groups or “blood group antigens” in human red cells were discovered by
Karl Landsteiner in 1900 [84]. Subsequently, this kind of antigens, called histo-
blood group antigens (HBGAs), has been found in other tissues and biological flu-
ids, such as gut and saliva [85]. Some hosts lack the function of the fut1 gene and
are called “nonsecretory hosts” [86]. The biological role of HBGAs has not yet been
completely defined. In infectious diseases, the presence of A and B HBGAs can
inhibit the in vitro motility of carcinoma cells, and their absence is associated with
an unfavorable prognosis [86]. Returning to infectious diseases, HBGAs play a cru-
cial role in bacterial and viral pathogenesis. Specific strains of pathogens bind to
carbohydrates of the HBG family. Several studies have shown that a large number
of pathogens bind HBG as the first step of pathogenesis, such as uropathogenic
strain of E. coli R45, S. pneumoniae, S. aureus, Salmonella typhimirium, and
Campylobacter jejuni [87–90].
The HBGAs may not only provide an attachment receptor to pathogens, since
they may be present on the pathogens themselves. Gram-negative bacteria can pres-
ent this type of antigen (in some strains may can be on the LPS molecules) [91];
humoral immune responses can thus be generated. For example, E. coli 086 pres-
ents B HBGA; thus, an anti-B response will be evoked in A and O HBGA individu-
als. Thus, B group individuals were more susceptible to infection caused by E. coli
086 [92]. In this chapter, the role of HBGA in viral infection will be discussed.
1.3.1.3 Viral Gastroenteritis and the Role of Gram-Negative Bacteria

of the Gut Microbiota
The gut microbiota contains a large number of microbes, forming the biggest
ecosystem in humans and animals [73]. Gastrointestinal infections have a great
impact on public health both in developing and developed countries [93]. Viruses
are the most frequent causative agent involved in gastroenteritis, in particular in

infants and children [94]. The sources of enteric viruses (EnVs) are usually con-
taminated food and water through ingestion by the orofecal route [95].
After the occurrence of infection, treatment of the symptoms is the only way to
prevent infection complications. In addition to antipyretic drugs, rehydration ther-
apy is the most common treatment for viral gastroenteritis. The absence of specific
antiviral agents against EnVs requires scientists to find an alternative which can
prevent or help in the treatment of such infections [5].
The definition of EnVs is viruses that are able to replicate in the intestinal epithe-
lium, even if only several types are the causing of gastroenteritis [96]. Noroviruses
(NoVs), rotaviruses (RoVs), arboviruses (AVs), enteric adenoviruses (AdVs), and
enteroviruses (EVs) are the viruses most frequently responsible for gastrointestinal
infections worldwide [97]. Reoviruses are one of the many EnVs that replicate in
the intestinal tract, but they are generally asymptomatic. Another type of enteric
virus, such as poliovirus, can cause severe disease after dissemination to peripheral
tissues [97]. Certain retroviruses, including mouse mammary tumor virus (MMTV),
can be transmitted orally from an infected mother through her milk, after which they
infect the gastrointestinal tract [98].
Does “Gut Microbiota” Enhance or Inhibit Viral Gastroenteritis?
Upon EnVs ingestion, viruses will “communicate” with gut microbiota resident in
the intestinal lumen, which vary from one host to another. The result of this interac-
tion seems to be dependent on the composition of the microbiota. In some hosts,
good microbiota (a high number of commensal bacteria) is an inconvenience; while
in other hosts, a complex microbiota (containing a large variety of microbial genera
and species) is beneficial in defending against EnVs and preventing viral gastroen-
teritis. This hypothesis is supported by several studies that have demonstrated that
the intestinal microbiota is important and plays various roles in reducing infection
by enteric viruses [97].
The role of commensal bacteria in the persistence of enteric viral infections has
previously been shown in a series of recent studies published in 2011, using poliovi-
rus, reovirus and mouse mammary tumor virus (MMTV) as EnV models [99–101].
The replication of poliovirus in the intestine was reduced in antibiotic-treated
mice, while the reconstitution of the intestinal microbiota restored poliovirus infec-
tion [100]. Moreover, Kuss et al. showed that intraperitoneal infection with poliovi-
rus was independent of the presence or absence of intestinal microbiota, which
highlights the important role of the microbiota in reducing poliovirus infection
[100]. In a recent study [101], showed that the use of broad-spectrum antibiotics in
a mouse model infected with rotavirus decreased the infectivity of the latter. They
reported that viral antigens were reduced in feces, and there was delayed shedding
of viruses compared with the control mice group [101].
In another study, murine Norovirus (MuNoV) was used to investigate the impor-
tance of intestinal microbiota in viral infectivity [102–104]. Jones et al. showed that
depletion of intestinal microbiota reduced the replication of MuNoV in the distal

ileum, mesenteric lymph nodes, and colon compared with the control group [103].
The MuNoV was reduced in feces shedding in antibiotic-treated mice compared
with colonized mice as shown by Kernbauer et al. [104]. A recent study conducted
by Baldridge et al. showed that the use of a broad-spectrum antibiotic in mice did
not help MuNoV to establish persistent infection, while the transplantation of intes-
tinal microbiota from another healthy mouse rescued the infectivity of this virus.
Moreover, they reported that systemic infection with the MuNoV was independent
of the presence or absence of gut microbiota as shown by poliovirus infection [102].
Direct and Indirect Mechanisms of Intestinal Microbiota, Which Enhance EnV

Infection
What Is the Direct Mechanism of the Gut Microbiota in Viral Infectivity?

Virion stabilization and promotion of virus attachment are the two direct mecha-
nisms by which the intestinal microbiota enhance EnV infections [100, 105]. These
findings were investigated using an in vivo poliovirus model (in mice) and an
in vitro model (cell culture testing). Kuss et al. studied the stability of the poliovirus
virion in the presence or absence of intestinal microbiota. They found that the isola-
tion of poliovirus (before progeny virion production) from colonized mice was
more viable and resistant to high temperatures and became bleach resistant. These
results were also seen when the poliovirus was incubated with dead Gram-negative
bacteria [100].
Robinson et al. conducted an in-depth study of the mechanisms of intestinal
microbiota. They found that surface compounds of Gram-negative bacteria played
a crucial role in poliovirus stability. The authors showed that the bacterial LPS
bound the viral protein 1 (VP1) at threonine 99. The LPS-bound virus increases the
thermostability and chlorine resistance as well as decreasing the viral genome
release [105]. Moreover, the pretreatment of poliovirus particles with Gram-
negative bacteria/or LPS molecules promotes poliovirus attachment to the host
cells [105]. The poliovirus receptor (PVR) seems to be an important element in the
abovementioned host cell attachment. The authors showed that poliovirus cannot
attach to the permissive cells which were pretreated with anti-PVR antibodies. This
result was independent of the presence of LPS binding to poliovirus. Moreover,
non-PVR-expressing cells also showed the same results [105]. Using another viral
model and after a long history of in vitro culture difficulties, B cells seem to be
permissive cells for MuNoV [103].
Tan and Jiang showed that human and MuNoV required commensal bacteria to
infect human B cells. These findings were supported by the reduction of norovirus
(NoV) infectivity when infected stool was filtered with a 0.22 μm filter. The infec-
tivity was rescued when live/dead commensal bacteria were added to the stool
[106]. The authors showed that LPS molecules were not the bacterial compound
that played the cofactor role in norovirus infection. Norovirus and B cells were
incubated with LPS, and the results showed that the LPS did not initiate the norovirus
infection. Moreover, the authors showed that the histo-blood group antigen (HBGA)
glycan was a cofactor of the norovirus infection [106]. A recent study showed that
a variety of commensal bacteria express these glycans and can bind norovirus in a
virus-strain-specific manner [107]. The majority of Gram-negative bacteria express
this kind of glycan, as shown in several recent studies [103, 108].
Indirect Mechanisms
As shown in Fig. 1.3, the gut microbiota can inhibit and/or reduce the antiviral
immune response via an indirect mechanism. The gut microbiota can sometimes
create a tolerogenic microenvironment that helps the virus infect cells and suppress
antiviral antibody production, and sometimes it can block virus-induced IFN signal-
ing [97].
(i) Tolerogenic microenvironment
The gut microbiota, in particular Gram-negative bacteria, induces a tolerogenic
microenvironment that allows persistent enteric virus infection [109, 110]. Briefly,
the capacity of enteric viruses to bind Gram-negative bacteria by LPS-VP (viral
protein) binding can skew the immune response. The story begins when the enteric
virus binds the microbiota LPS. The LPS will be recognized by the TLR4, which
induces the production of IL-6. The B cells have IL-6R; when the IL-6 binds to the
IL-6R of the B cells, the B cells produce IL-10, which is an anti-inflammatory cyto-
kine. This action blocks the antiviral immunity response and leads to viral persis-
tence. This information is supported by several studies using an MMTV and
norovirus model in solenocyte and B-cell culturing, respectively [99, 111, 112]. In
another study, the norovirus infection occurred in germ-free mice which were also
deficient in production of IL-10. This study supports and confirms that the produc-
tion of IL-10 by the presence of gut microbiota, in particular Gram-negative
bacteria, was the essential key to viral persistence through the creation of a tolero-
genic microenvironment [113, 114].
(ii) Viral antibody production
[101] in the case of rotavirus infection, showed that the fecal and serum IgA titer
was higher in germ-free mice compared with the control mice group. This data sug-
gests that gut microbiota suppress the antiviral humoral response. In contrast to the
rotavirus case, the MuNoV infection of antibiotic-treated mice reduced the serum
IgG titer after 35 days of infection compared with the colonized mice group [101].
These findings will be investigated in depth to identify exactly which bacterial com-
pound is responsible for this mechanism and to determine if this interaction occurred
in a virus-strain-specific manner.
(iii) Blocking of the IFN signaling
Several studies have reported that the IFNλ, which is considered to be in type
III of IFNs, activates the same intracellular signaling pathway and many of the
same biological activities as other IFN types, including antiviral activity, in a
Fig. 1.3 Suggested mechanisms of antiviral probiotics against enteric viruses. Since probiotics
and EnVs have the same route of entry, probiotics can interact with viral particles in several ways.
The advantage here is the capacity of probiotics to colonize the gut ecosystem, which is the target
of EnVs: A. Some probiotic strains can colonize the gut ecosystem and then form a carbohydrate
biofilm which probably saturates host IEC receptors as well as viral receptors. B. Probiotics protect
the host IECs against damage and lesions. Several studies have confirmed that some probiotic
strains play a crucial role in tissue restoration, especially by inducing mucin secretion by IECs and
strengthening cell tight junctions. C. The immunomodulatory effect is the principal mechanism of
antiviral probiotics (AvPr). These probiotics can stimulate the secretion of proinflammatory cyto-
kines, especially from DCs such as IL-6, Il-12, and IFNγ. In addition, AvPr can boost innate
immune cells, such as macrophages and NK cells. The latter also produce IFN-α, which is an
antiviral cytokine. D. AvPr help the immune system to react with more rapid specific responses.
The Th2 response is essential for B lymphocytes to be able to differentiate into plasma cells with
specific sIgA secretion. E. AvPr can inhibit or decrease viral infectivity and spreading by superpro-
duction of mucin and by changing the morphology of villi, which can skew viral attachment. F.
TLR3 is the PPR of viral MAMPs, especially for RNA viruses. Hence, the overexpression of TLR3
induced by some AvPr can amplify the innate immune response by catching a large number of viral
particles. G. Some AvPr interact physically with viral particles. Indeed, several studies have
showed that some probiotic strains can bind or trap viral particles on their cell wall. Moreover,
these trapped viruses lose some pathogenic characteristics and consequently lose cell infectivity.
H. Finally, AvPr can play an indirect role in preventing and/or decreasing viral infection, especially
against enteric viruses, by excluding the colonization of Gram-negative bacteria (See Fig. 1.3).
wide variety of target cells [115]. Baldridge et al. reported the importance of
IFN type I, II, and III responses in reducing MuNoV infection as well as viral
persistence. TLR4 is required for bacterial regulation of viral persistence.
Therefore, the presence of LPS/Gram-negative bacteria were dispensable in this
regulation [102].
Moreover, a recent study showed that the type III IFN response was essential in
reducing MuNoV infectivity in the colon [116]. Briefly, EnVs are recognized by a
variety of TLRs, such as TLR3, TLR9, etc. These TLRs stimulate the IFN produc-
tion by the B cells or other secretory cells. The IFNs, in particular type III IFNγ,
bind to the IFN receptor present on the enterocytes, which can reduce the viral
persistence. Several studies have reported that commensal bacteria, in particular
Gram-negative bacteria recognized by TLR4, bind to the EnVs and then the immune
system will be skewed. Thus, the TLR4s inhibit the production of IFNγ, allowing
viral persistence. This data was confirmed using MuNoVs as an EnV model [102,
116–118]. Pott et al. reported that IFNγ also controls rotavirus infection in mice;
thus, it will be interesting to determine whether this response is similarly regulated
by the interactions between the enteric virus and commensal bacteria [119].
1.3.1.4 Role of Probiotics in Gut Microbiota
In addition to the immunomodulatory effect of probiotics, these beneficial bacteria

have several mechanisms to defend gut pathogens and infections.
In the previous part, the studies have shown that the composition of the intestinal
microbiota can help EnVs to persist and sometimes amplifies their infectivity.
Remarkably, the presence of Gram-negative bacteria in the microbiota is essential to
save the infectivity of EnVs. Therefore, changing the intestinal microbiota composi-
tion seems to be effective in preventing or inhibiting enteric viral infection. Otherwise,
a high percentage of Gram-positive bacteria may be a solution in viral gastroenteritis
treatment and/or prevention. From this hypothesis, the importance of Gram-positive
bacteria, in particular lactic acid bacteria (LABs) – which is considered to be GRAS –
in preventing and even treating this type of infection will be discussed in this part.
The implantation of probiotics in the digestive tube is clearly beneficial, since they
have the ability to form a biofilm on the enterocytes and prevent the adhesion and
proliferation of other bacteria such as Gram-negatives [120]. Moreover, as shown in
Fig. 1.3, probiotics can exclude commensal and pathogenic bacteria by several mecha-
nisms such as the immunomodulatory effect (immunobiotic action), reduction of pH,
production of antimicrobial compounds (hydrogen peroxide, lactic acid, NRPS, bacte-
riocins, etc.), trophic competition, and biofilm formation (receptor competing) [121].
Anti-enteric Viruses Probiotics
Anti-EnV probiotics (AEnPs) are divided into two categories according to the direct
or indirect antiviral mechanisms. In this section, direct mechanisms will be dis-
cussed in detail.
The probiotics with antiviral effects, called further antiviral/anti-EnV probiotics,
can inhibit viral infections with several direct mechanisms. The antiviral compound
secreted by these probiotics will be discussed in Chap. 4. In this section, the immu-
nomodulation and physical interaction will be presented and discussed (Table 1.2).
Table 1.2 Probiotics assessed against viral gastroenteritis infections
Rotavirus infection
LGG Human feces RoVs Reduction in symptoms and RDBCT: 64 children Grandy et al. [122]
Porcine RoVs duration of gastroenteritis
Reduction in healthcare- RDBPC: 1092 children Szajewska et al. [123]
associated diarrhea including
rotaviral gastroenteritis
Reduction in diarrhea RDBPC: 287 children Guandalini et al. [124]
duration
Reduction in diarrhea RDBPC: 71 well-nourished Isolauri et al. [125]
duration children
Increase in sIgA in sera RDBPC: 49 children Majamaa et al. [126]
Reduction in diarrhea RDBPC: 204 children Oberhelman et al. [127]
episodes
Reduction in symptoms and PrTBPC: 39children Pant et al. [128]

duration of gastroenteritis
Reduction in symptoms and DBCT: 123 children Rautanen et al. [129]
duration of gastroenteritis and
acidosis correction
Reduction in symptoms and PrTBPC: 40 children Raza et al. [130]
duration of gastroenteritis
Enhancement of short-term RDBPC: 64 children Ritchie et al. [131]
recovery following acute
diarrheal illness
Insignificant results, due to RDBPC: 179 children Salazar-Lindo et al.
malabsorption of lactose [132]
(suggestion of authors)
Reduction in RoV-induced IPEC-J2 cell lines Liu et al. [133]
IL-6 response
21
(continued)
22

L. reuteri SD 2222 Human origin RoVs Reduction in symptoms, RDBPC: 66 children Shornikova et al. [134]
especially watery diarrhea
S. thermophilus Fermented food RoVs Reduction in symptoms and RDBPC Saavedra et al. [135]
duration of hospitalization
L. reuteri DSM 12246 Human origin RoVs Reduction in symptoms and RDBPC: 40 children Shornikova et al. [136]
duration of hospitalization
L. acidophilus La5 Dairy products RoVs Reduction in symptoms, RDBPC Sugita and Togawa [137]
especially watery diarrhea
E. faecium PCK38 Sausages RoVs and High attachment Cell culture: cell line: Maragkoudakis et al.
1
TGEV Increase in NO production H4 CLAB [138]

and improvement of ROS TLT PSI
release PoM2 GIE
L. fermentum Kasseri cheese RoVs and High attachment Cell culture: cell line: Maragkoudakis et al.
ACA-DC179 TGEV Increase in NO production H4 CLAB [138]
release PoM2 GIE
L. pentosus PCA227 Unknown RoVs and High attachment Cell culture: cell line: Maragkoudakis et al.
release PoM2 GIE
L. plantarum PCA236 Kasseri cheese RoVs and High attachment Cell culture: cell line: Maragkoudakis et al.
release PoM2 GIE
L. plantarum PCS22 Slovenian cheese RoVs and High attachment Cell culture: cell line: Maragkoudakis et al.
release PoM2 GIE
S. boulardii Lychees RoVs Decrease in fever and RDBPC: 64 children Grandy et al. [122]
reduction of diarrhea duration
L. acidophilus – RoVs Decrease in fever and RDBPC: 64 children Grandy et al. [122]
B. bifidum – RoVs Decrease in fever and RDBPC: 64 children Grandy et al. [122]
L. reuteri (Probio-16) Porcine feces Porcine RoVs Reduction of viral titer in cell Vero TF-104 cell line Seo et al. [139]
culture
L. reuteri DSM 17938 – RoVs Decrease in the number of RDBPC: 74 children Francavilla et al. [140]
patients with acute diarrhea
L. acidophilus LB Lactéol Fort RoVs Reduction in diarrhea RDBPC: 73 children Simakachorn et al. [141]
sachets duration
LGG Human feces Murine RoVs Decrease in barrier BALB/c mice Hagbom et al. [142]
permeability Pant et al. [143];
Decrease in vacuolation in the Zhang et al. [144]
jejunum
Stimulation of IgA secretion
Reduction in diarrhea
duration
L. casei DN-114,001 Fermented food Murine RoVs Change in the morphology of Germ-free suckled rats Guérin-Danan et al.
intestinal villi and decrease in [145]
cell damage
(continued)
23
24

L. reuteri DSM 17938 – Murine RoVs Reduction in duration of BALB/c mice Preidis et al. [146]
diarrhea and decrease in cell
damage
LGG Human feces Porcine RoVs Improvement in specific Weaned piglets Mao et al. [147]
immune response
Increased production of IgA
Inhibition of NSP4 function
Increased production of
mucin 1 and 2
1
Restoration of cell
morphology and tight-
junction function
E. coli Nissle Human feces Porcine RoVs Decrease in IgA secretion Neonatal gnotobiotic piglets Kandasamy et al. [148]
Production of IL-10 and IL-6 PBMC experiment
L. ruminis SPM0211 Young Korean Human RoVs Enhancement of Type I IFN Neonatal normal mice Kang et al. [149]
Wa response
Rice Ban (prebiotic) – Human RoVs Increase in IFNγ production Vaccinated gnotobiotic Yang et al. [150]
by CD4+ and CD8+ piglets
LGG Human feces RoVs Increase in TLR3, IFN-α, Lgr5+ (intestinal organoid Aoki-Yoshida et al. [151]
CXCL1 gene expression from stem cells)
B. longum SPM1205 Young Korean Human RoVs Enhancement of Type I IFN Neonatal mouse model Kang et al. [152]
and SPM1206 Wa response and inhibition of Caco-2 cells
rotavirus replication
B. longum subsp. Infant feces Murine RoVs Immunomodulatory effect MA-104 and HT-29 cell lines Muñoz et al. [153]
Infantis CECT 7210 and inhibition of viral McN mouse model
replication in both in vitro
and in vivo experiments
Norovirus infection
LcS Human feces HuNoVs Decrease in fever caused by OCC: 77 elderly patients Nagata et al. [154]
NoV infection
Increase in lactic acid in feces
Lactobacilli and
bifidobacteria become
dominant in gut flora
Not specified Increased IL-12 production PCCO: 10 elderly patients Takeda et al. [155]
by macrophages
Activation of NK cells
L. lactis ssp. Lactis – FCV Physical interaction between Cell culture: CRFK cell line Aboubakr et al. [156]
LM0230 bacterial cells and FCV
particles
E. coli Nissle 1917 Human feces NoVs: GI.1and Physical interaction: P-particles (same Rubio-del-Campo et al.
L. lactis MG1363, Cheese GII.4 NoV p-particles attached to conformation as VLP) [157]
L. acidophilus LA-5, Dairy products probiotic strains Cell line: HT-29

L. bulgaricus Dairy products
ATCC11842T
L. plantarum 299v Probiotics
L. plantarum 299v Adh- Derived from L.
plantarum 299v
L. casei 431 Infant feces
ATCC55544
L. casei BL23 Dairy products
CECT5275
L. casei VSL#3 Dairy products
LGG ATCC53103 Human feces
L. rhamnosus HN001 Dairy products
(continued)
25
26

L. paracasei ATCC Emmenthal cheese MuNoV Viral mRNA hydrolysis Cell culture Hoang et al. [158]
334 Decrease in VP1 expression Cell line: RAW264.7
Strain exhibit an
antiviral intracellular
protein (3D8 scFv)
B. adolescentis Adult intestine VLPs (MuNoV) Physical interaction Cell culture Li et al. [159]
LMG10502 Cell lines: Caco-2 and HT-29
E. faecium NCIMB – TGEV Physical interaction Cell culture Chai et al. [160]
10415 Immunomodulatory effect by Porcine testicular cells
1
enhancement of NO
production and secretion of
IL-6 and IL-8
L. reuteri Protectis Human feces CA16 Physical interaction Cell culture Ang et al. [161]
(ATCC 55730) EV71 Decrease in viral load Human rhabdomyosarcoma
Not LcS Not CB2 (RD) cells
Caco-2
Combination
L. acidophilus, – RoVs Reduction in duration of RSBPC: 75 children Teran et al. [162]
L. rhamnosus, diarrhea
B. longum, S. boulardii
Indirect Mechanism of Anti-EnV Probiotics
The microbiota diversity and composition are directly related to the incidence of
gastroenteritis, including viral infection [163]. For example, the presence of bifido-
bacteria genera in the first months in the gut microbiota of infants prevents the
majority of intestinal infections [164]. Therefore, almost all intestinal probiotics can
play a crucial role in preventing or treating viral gastroenteritis by indirect mecha-
nisms. These probiotics reduce the “viral infection cofactor,” which is the LPS and
HBGA molecules present in Gram-negative bacteria and some commensal bacte-
ria, respectively [108]. Otherwise, orally administered probiotics can change the
composition of the gut microbiota by increasing the number of probiotic cells and
decreasing commensal and Gram-negative bacteria.
Direct Mechanism of Anti-EnV Probiotics
The meaning of direct mechanism is when the EnVs interact directly with probiotic
cells and/or their metabolic compounds. As shown in Fig. 1.4, probiotics can inter-
act and inhibit EnVs by several mechanisms. Indeed, it is depending to the specific-
ity probiotic strain and viral type. Before talking about the direct mechanism or
direct interaction of these probiotics, the viral infection steps should be presented.
In general, EnVs can infect target cells by five steps called the viral replication
cycle. The viral replication cycle starts by viral attachment to host cells (1), followed
by penetration and uncoating (2), viroplasm formation (3), and finishing with virus
particle maturation (4) and release (5) [165]. Each EnV has its own specificity in
infection mechanisms and/or the replication cycle. For this reason, the following
information will discuss the direct mechanism of probiotics regarding the type of EnV.
1.3.1.5 Probiotic Strains Against Rotavirus (RoV) Infections
RoVs are the major cause of diarrhea and acute gastroenteritis in infants and young
children [165]. RoVs are naked viruses containing dsRNA. The RoV virion or particle
consists of three protein layers called a triple-layered particle (TLP) [166]. The viral
protein (VP) and nonstructural protein (NSP) are the two main viral proteins found in
RoVs. For TLP, the main protein forming the external layer is VP7, with VP4 which
forms the viral spike. VP6 forms the second layer of the RoV particle. Thus, the VP6
layer constitutes the double-layered particle (DLP) of the RoV. Kam et al. (2014)
showed that, in actively transcribing DLP, the middle VP6 layer order decreased,
while the number of cores increased. Thus, the transcribed mRNAs released from
these cores translated later to the viral protein (VP and NSP) in host cells [167].
The RoV replication cycle starts with the attachment to the host cells mediated
by VP4 and VP7 molecules which play a role in the penetration and uncoating of
RoV. The third step consists of the synthesis of ssRNA (mRNA), which is mediated
by VP1, VP3, and VP2 molecules. Viroplasm formation (viral protein (NSP2,
Fig. 1.4 Exclusion of commensal bacteria by probiotics in the gut ecosystem. AvPr can play an
indirect role in preventing and/or decreasing viral infection, especially against enteric viruses, by
excluding the colonization of Gram-negative bacteria (see Fig. 1.3) in the gut ecosystem, which
were considered a cofactor in some enteric virus infections. Thus, proinflammatory probiotics
(which induce a proinflammatory response) are welcome in viral gastroenteritis because they can
trigger proinflammatory immunity to eliminate EnVs. Probiotic strains capable of binding host
cells very well and then creating a microenvironment which prevents many kinds of commensal
and pathogenic bacteria from proliferating, including Gram-negative bacteria. Probiotics have a
stronger capacity to adhere to host cells than Gram-negative bacteria (probably because of the high
hydrophobicity of their cell walls), which can decrease the number of Gram-negative bacteria.
Probiotics can act in different ways: A. Biofilm formation: This biofilm can protect host cells
against other commensal bacteria, because this biofilm covers the majority of host cell receptors.
B. By the immunomodulatory effect, probiotics can stimulate the innate immune response, espe-
cially of phagocytes. C. At the same time, probiotics induce the secretion of antimicrobial peptides
(AMPs) such as β-defensins and cathelicidins which target commensal bacteria. However, there is
no explanation of the resistance of some probiotic strains against these AMPs. D. Overproduction
of mucin can also prevent commensal bacteria adhesion. E. The co-aggregation capacity of probi-
otic strains leads to the trapping of other microbes, as well as commensal or Gram-negative bacte-
ria. F. Probiotics can secrete several enzymes to compete with other commensal bacteria for
nutrients present in the gut ecosystem. In addition, the majority of probiotic strains possess argi-
nine dehydrogenase, which is important in this mechanism. G. Probiotic strains can secrete a
variety of antimicrobial substances, such as hydrogen peroxide, lactic acid, non-ribosomal peptide
synthetase (NRPS), bacteriocins, and bacteriocin-like inhibitory substances (BLIS).
NSP5) and viral RNA interact with each other to form cytoplasmic inclusion bod-
ies), RNA packaging, minus ssRNA synthesis (RNA replication), and DLP forma-
tion constitutes the fourth step. Finally, RoV will be released from host cells after
maturation of virus particles (from DLPs to TLPs) [165, 168].
Several studies have shown the effectiveness of probiotics in the treatment and
prevention of acute diarrhea including RoV infections. Human, murine, and porcine
rotaviruses were used in these studies. The majority of investigations were based on
the symptoms, such as duration of diarrhea, duration of hospitalization, virus shed-
ding in feces, and sometimes immunomodulation. A few studies conducted an in-
depth investigation of the mechanism of action of some probiotics, in particular the
interaction between virus-probiotic-host cells.
Clinical Trials (CTs)
Lactobacillus and Bifidobacterium strains were the most studied genera in rotavirus
infections. Lactobacillus rhamnosus GG (LGG) is the best studied probiotic which
showed a significant reduction of diarrhea duration and rotavirus infectivity [122,
123]. Effects of various probiotic strains on rotaviruses have been conducted using
double-blind placebo-controlled randomized trials since 1991 [124, 125, 134].
Guandalini et al. showed that LGG administration reduced the diarrhea duration in
neonatal patients with rotavirus infection [124].
In 49 children, the administration of 1010–1011 CFUs/ml of LGG twice daily for
5 days reduced the duration of acute diarrhea from 2.7 to 1.8 days, accompanied by
an increase of IgA-specific responses [126]. In other RCTs, LGG reduced the dura-
tion of diarrhea caused by rotavirus gastroenteritis and improved the health recovery
of infected children [127–132].
The L. reuteri SD 2222 strain was administered in patients aged 6–36 months
with watery diarrhea caused by rotavirus. This strain showed a strongly reduction of
diarrhea duration up to 5 days [134]. Saavedra et al., Shornikova et al., and Sugita
and Togawa showed the anti-rotaviral activity in clinical trials of the following pro-
biotic strains: Streptococcus thermophilus (S. thermophiles), L. reuteri DSM 12246,
and L. acidophilus La5 [135–137]. Another study showed that LGG strains and L.
casei Shirota (LcS) have an antiviral activity against rotaviruses and transmissible
gastroenteritis virus (TGEV). The LGG strain showed the strongest activity, because
of their strongest attachment capability to different cell lines. In addition to the
attachment effect, the induction of reactive oxygen species (ROS) release seems to
play a role in such activity [138]. Teran et al. conducted a randomized single-blind
controlled trial (RSBCT) in 75 Bolivian children aged from 28 days to 24 months.
A 1-gram mix of probiotic strains was administered to the probiotic group (n =25)
for 5 days. The mix contained the following strains: L. acidophilus, L. rhamnosus,
B. longum, and Saccharomyces boulardii (S. boulardii). The second group (n =25)
was given nitazoxanide (an antiparasitic agent) at the dose of 15 mg/kg. The third
group (n =25) was subjected to the normal protocol of rehydration. The results
showed that the duration of diarrhea was reduced to 48 h compared with 54 h and
79 h for the nitazoxanide and rehydration groups, respectively [162]. Moreover, a
study conducted by Grandy et al. in RDBPC trial showed the effectiveness of pro-
biotic strains against rotavirus infections using S. boulardii alone and S. boulardii
with a mixture of probiotic strains. The results showed that the two probiotic prepa-
rations reduced the infection symptoms (p = 0.0042) [122]. L. reuteri, called

Probio-16, showed antiviral activity against porcine rotavirus; this activity was
poorly demonstrated [139].
L. reuteri DSM 17938 was evaluated in RCTs on 74 children with rotavirus
infection, the results showed a decrease in the number of patients with acute diar-
rhea [140]. Simakachorn et al. conducted an RCT on 73 children with rotavirus
infection. The children were administered six sachets containing 109 of heat-treated
L. acidophilus LB cells and 160 mg of twofold concentrated neutralized CFCS. The
duration of the diarrhea decreased from 74 to 42.9 h [141].
Recent studies have started in-depth investigations of the interaction between
rotaviral particles and probiotic strains. The cell culture was used to demonstrate
what is happening between the rotaviral particles and probiotic strains. Various cell
lines were evaluated; pig and human epithelial cells were used in some studies.
Maragkoudakis et al. showed that LGG and LcS presence decreased the ROS release
which can reduce cell damage [138]. Liu et al. studied the mechanism of antiviral
activity of LGG in a new cell line called the porcine small intestinal epithelial cell
line (IPEC-J2) as a model to study the impact of LGG on innate immunity during a
rotavirus infection. They demonstrated that LGG presence reduced the rotavirus-
induced IL-6 response [133].
Animal Models (AMs)
The animal model was established for several reasons. First, the animal model
allows us to conduct an in-depth investigation of the mechanism of action of probi-
otic strains before and after viral infection. Moreover, the animal model (in vivo
model) facilitates monitoring of the probiotic’s effect during the animal’s life cycle.
The probiotics with antiviral activity were evaluated in vivo using a mouse model in
most studies. Hagbom et al. confirmed that the neonatal mice and rats provide a
reliable animal model for studying the rotavirus infection and also immune responses
during this infection [142]. In a murine infected model, LGG has decreased both the
barrier permeability in murine intestine and epithelium vacuolation in the jejunum.
Furthermore, LGG was able to reduce the duration of acute diarrhea, and finally
LGG was able to stimulate the secretion of IgA [143, 144]. L. casei DN-114,001
was administered in germ-free suckling rats infected further by rotavirus. The
results showed that L. casei DN-114,001 changed the morphology of the intestinal
villi and decreased intestinal cell lesions [145]. L. reuteri DSM 17938 was also
evaluated in normal mice infected by rotavirus. The results showed that L. reuteri
DSM 17938 has decreased the intestinal cell lesions and consequently reduced the
duration of acute diarrhea [146].
Recently, Mao et al. studied the effect of LGG on the intestinal physiology, mor-
phology and primary immune-specific responses of weaned piglets infected by the
porcine rotavirus. This study showed that LGG administration in the weaned piglets
group enhances specific immune responses by increasing rotavirus-specific IgA
secretion. In addition, LGG decreased the NSP4 (rotavirus enterotoxin) – consid-
ered an intracellular receptor essential for DLP particles to interact with viroplasms
and modulate intracellular Ca2+ and RNA replication [165] – in the jejunal mucosa
induced by rotavirus infection [147]. The production of mucin 1 and mucin 2 and
morphological improvement of the jejunal mucosa were evaluated in the presence
of LGG. The results showed that LGG enhanced the production of mucin and recu-
perated the integrity of both the villus and the tight junction by stimulation of occlu-
sion and other gene expression assisting the morphological jejunal defense against
rotavirus [147].
E. coli Nissle (EcN) – Gram-negative probiotic strain – was evaluated alone or in
combination with LGG in neonatal gnotobiotic piglets. The viral shedding titer was
lower using EcN in comparison with LGG, LGG+EcN, and without probiotic
strains. This result was correlated with the reduction of the specific IgA responses in
the small intestine in EcN colonized piglets. The in vitro investigation using mono-
nuclear cell culture, EcN, showed stimulation effects on the production of anti-
inflammatory cytokines such as IL-6 and IL-10 [148]. These findings support the
hypothesis conducted by Stephanie Karst in 2016 which showed that Gram-negative
bacteria improve viral infection by various direct and indirect mechanisms [97].
Yang et al. evaluated the impact of dietary rice bran (RB) on the human rotavirus
vaccine (HRoV) in vaccinated gnotobiotic pigs. They found that the RB-supplemented
diet enhanced the vaccination responses in gnotobiotic pigs. In addition, the levels
of IFNγ production from CD4+ and CD8+ were increased in intestinal and systemic
lymphoid tissues [169]. In 2015, the authors showed that RB plays a role as a pre-
biotic for some probiotic strains. The LGG+EcN colonized gnotobiotic pigs were
supplemented with RB daily, followed by human rotavirus (HuRoV) orally chal-
lenged. The RB showed a prebiotic effect promoting the growth of LGG and EcN in
the gut. Moreover, RB-fed pigs had a lower mitotic index and villus width. The RB
and/or probiotic strains increased immunomodulation by enhancing the secretion of
IFNγ and HuRoV-Ab [150].
L. ruminis species have shown antiviral activity for the first time against the
human rotavirus Wa strain. L. ruminis SPM0211 showed an anti-HuRoV activity
which was explained by an immunomodulatory effect enhancing the Type I IFNs
immune response [149].
To finish the last investigation of the antiviral mechanism of LGG, a new experi-
mental model was developed in order to understand the beneficial interaction
between pathogens and probiotics. An ex vivo experiment called intestinal organ-
oid (derived from Lgr5+ stem cells) was conducted by Aoki-Yoshida et al. [151].
The LGG strains showed an increase in TLR3 gene expression – TLR3 is the essen-
tial key in innate immune responses following the recognition of rotavirus – in
murine intestine both in in vivo and ex vivo experiments, without alteration of other
TLR gene expressions. Moreover, LGG increased the mRNA levels of interferon-α
(IFN-α) and a neutrophil chemokine (CXCL1). Furthermore, other probiotic
strains, B. bifidum and L. paracasei, failed to increase the TLR3 mRNA levels
ex vivo [151]. These findings confirm the hypothesis about the specificity of probi-
otic strains against viruses. Thus the antiviral activity occurred in a “virus-strain-
specific manner.”
Bifidobacterial probiotic strains were also evaluated against RoVs using in vitro
and in vivo experiments. B. longum SPM1205 and SPM1206 showed antiviral activ-
ity against the HuRoV Wa strain in an infected neonatal mouse model and Caco-2
cells. The two bifidobacterial strains showed an immunomodulatory effect on the
type I IFNs immune responses [152]. A complete genome sequence of B. longum
subsp. Infantis CECT 7210 was conducted in 2015 by [170]. This strain had previ-
ously showed, in a study conducted by Muñoz et al. [153], a direct effect on rotavi-
rus strains in both in vitro (MA-104 and HT-29 cell culture) and in vivo (McN mouse
model) experiments. The immunomodulatory mechanism was the main effect of this
strain [153]. After complete sequencing of the B. longum subsp. Infantis CECT
7210 strain, they reported that there were 360 more elements (genes) in this strain
compared with the complete genome sequence of B. longum 157F [170]. Thus, more
in-depth research must be conducted on this strain to identify the detailed mecha-
nism of antiviral activity, and more specifically the anti-HRoV activity.
1.3.1.6 Probiotic Strains Against Norovirus Infections
Noroviruses (NoVs) are naked RNA viruses belonging to the calicivirus family.
NoVs are transmitted via the fecal–oral route and cause gastrointestinal disease with
vomiting and acute diarrhea lasting 24–48 h [57]. NoVs cause 267 million infections
each year and over 200,000 deaths, mostly in infants and the elderly [171, 172].
NoVs need host receptors to start the infection cycle. Debbink et al. reported that
HBGA (See sec. I-B2) is a diverse family of carbohydrates expressed in mucosal
surfaces, which are the main receptors of NoVs, in particular for the GII.4 genotype
considered to cause the majority of human NoV infection because they can bind to
A, B, and O secretors which are the majority (80 %) of the population [57]. The
expression of HBGAs depend on the fut2 gene which codes for an enzyme called
fucosyltransferase. The GI.1 genotype (Norwalk virus) cannot infect patients with a
nonfunctional fut2 gene (called a “nonsecretory host”). However, some NoV strains
are capable of binding other receptors such as Lewis carbohydrates [173, 174].
The immune responses are very important to blockade NoVs infection and viral
spreading. The IgA genogroup-specific secretion is the main humoral immune
response against NoVs [175]. The CD4+Th1 response is essential in the cellular
immune response against NoVs which increases IFNγ and IL-2 production [176].
The development of antiviral treatments and vaccines to fight NoV infection has
been hindered because of their extreme genetic diversity. Recently, the uncultivable
nature of NoVs has been resolved by using a B-cell model. Thereby, the pathogen-
esis and replication cycle have been understood deeply in cell cultures and animal
models [177]. The prevention strategies seem to be most effective mainly in infants
and the elderly. To prevent and treat HuNoVs, several researchers have worked on
the role of probiotics in such infection. The probiotic effectiveness in NoV infec-
tions was evaluated using both in vitro and in vivo experiments and clinical trials.
LcS introduced in fermented milk alleviated fever in NoV-infected elderly
patients. The probiotic group (n =39) showed fast recuperation compared with the
control group. Moreover, the acetic acid concentration in feces has increased, and
thereby Bifidobacterium and Lactobacillus genera became dominant [154]. Takeda
et al. reported that the administration of LcS improves the natural killer (NK) cell
activity by producing the IL-12 by macrophages in response to LcS [155].
Lactococcus lactis ssp. Lactis LM0230 (L. lactis ssp. Lactis LM0230) – probi-
otic strains – were evaluated for antiviral activity against feline calicivirus (FCV), a
HuNoV surrogate. This strain, “bacterial cell suspension (BCS)” and its metabolites
“bacterial growth medium cell-free filtrate (BGMF)” were added to Crandell-Reese
feline kidney (CRFK) cells line. The results showed that CRFK pretreated by BCS
and BGMF caused nonsignificant decreases in the FCV titer. The pretreatment of
FCV by BCS resulted in a decreased FCV titer after 24 h. The co-incubation of FCV
and BCS in CRFK cells showed 100 % virus titer reduction (7.5 log TCID50/0.1 ml)
[156]. The effect of BGMF will be discussed in Chap. 4.
In order to investigate the physical interaction between probiotic cells and NoV
particles, Rubio-del-Campo et al. used a p-particles model designed from the
C-terminal protruding P-domain of the NoV VP1 capsid protein. The p-particles
exhibit the same surface conformation of viruslike particles (VLPs), and therefore
these p-particles can bind to the HBGAs. In this study, 11 probiotic strains were
tested: E. coli Nissle 1917 L. lactis MG1363, L. acidophilus LA-5, L. bulgaricus
ATCC11842T, L. plantarum 299v, L. plantarum 299v Adh- (an isogenic derivative
of 299v strain with decreased adhesion capacities), L. casei 431 ATCC55544, L.
casei BL23 CECT5275, L. casei VSL#3,
LGG ATCC53103, and L. rhamnosus HN001. The Norwalk virus (GI.1) and
GII.4 (HuNoV) were used in these experiments. The results showed that the probi-
otic strains possessed the capacity to bind to both GI.1 and GII.4 p-particles.
Furthermore, L. rhamnosus, L. casei BL23 CECT5275, L. casei VSL#3 showed the
highest binding effect of both p-particles. As unexpected results, the E. coli Nissle
1917 – Gram-negative probiotic – showed the poorest binding capacity to GI.1 and
GII.4, although other studies showed that Gram-negative bacteria can bind entero-
viruses via LPS molecules or HBGAs [97, 107]. In contrast, in HT-29 culture cells,
E. coli Nissle 1917 was more efficient in NoV p-particles blocking, resulting in low
host cell binding, while the other probiotic strains showed a low inhibition effect.
The low adhesion capacity of probiotic strains to host cells did not affect p-particles
binding; this suggestion was confirmed by the L. plantarum 299v adh- (probiotic
strain with low attachment capacity) which showed high GI.1 p-particles binding
compared with L. plantarum 99v (normal attachment capacity). In order to investi-
gate the interaction between probiotic strains and NoV p-particles in more depth,
an exclusion assay (HT-29 cells incubated with bacteria followed by P-particles
challenge) and displacement test (HT-29 cells incubated with p-particles followed
by bacterial challenge) were performed. The results showed that the probiotic
strains enhanced the NoV p-particle attachment of monolayer surfaces. These
results are not clear, since they disagree with other studies. The probable hypothe-
sis is that the attached probiotic strains can bind to the NoV p-particles on their
peptidoglycans (teichoic acid), which can lead to higher p-particle retention on the
HT-29 surfaces [157].
A recent study has evaluated an engineered probiotic strain of L. paracasei which

can produce the 3D8 scFv protein (an antiviral protein that can penetrate into host
cells and hydrolyze nucleic acid molecules) against MuNoV. The results showed
that L. paracasei 3D8 scFv retained its cell-penetrating effect, and therefore the
intracellular nucleic acids have been hydrolyzed. The pretreatment of RAW264.7
cells with this engineered probiotic strain prevented the cell apoptosis caused by
MuNoV infection. Moreover, L. paracasei 3D8 scFv has decreased mRNA expres-
sion of the viral capsid protein (VP1) [158].
Recently, B. adolescentis showed an antiviral activity against MuNoV as a
HuNoV surrogate. The results showed that the inhibition did not occur in the viral
binding step. Using VLPs as model, B. adolescentis decreased the attachment of
HuNoV GI.1 VLPs to both Caco-2 and HT-29 cells, while no effect was shown in
the presence of GII.4 VLPs [159].
1.3.1.7 Probiotics and Other Enteric Viruses
Astroviruses are nonenveloped viruses with positive-sense ssRNA. The Astroviridae

family consists of two genera, Mamastrovirus (MAstV) and Avastrovirus (AAstV),
based on mammalian and avian species, respectively [178]. Astroviruses can infect
a wide variety of mammalian species, such as cats [179], dogs [180], mice [181],
sheep [182], and cattle [183]. These mammals are always in direct contact with
humans. HAstVs are one of the most important causes of acute gastroenteritis in
newborn and infant patients [184]. Cross-species transmission is frequent, in par-
ticular in poultry as avian species [185] and between pigs, cats, and humans as
mammalian species [186]. Thus, the zoonotic potential of these viruses is high, and
future nonhuman-to-human transmissions are likely to occur [178]. Some authors
have speculated that probiotics, which may interfere with the biological cycle of
enteric viruses at many different stages, may be useful as a measure to prevent and/
or treat intestinal viral infections [187, 188].
E. faecium NCIMB 10415 is the first probiotic strain authorized by the European
Union (EU) as a probiotic feed additive for animals, including piglets. E. faecium
NCIMB 10415 has shown an immunomodulatory effect in several studies [189].
Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus,
causes 100 % mortality in newborn piglets after severe gastroenteritis. TGEV can
also infect respiratory tissues in some cases [190]. Chai et al. showed the antiviral
activity of E. faecium NCIMB 10415 against TGEV using in vitro swine testicle
(ST) cell lines. They showed that this strain has a double antiviral mechanism. First,
the strain can trap virus particles on its cell wall and consequently prevent infection.
The second mechanism is the stimulation of eukaryotic cells that produce NO, IL-6,
and IL-8 [160].
In addition to gastrointestinal infections, enteroviruses can cause extraintestinal
infections. Via the orofecal route, Coxsackievirus type A strain 16 (CA16) and
enterovirus 71 (EV71) cause hand, foot, and mouth disease (HFMD) [191]. This
viral infection results in morbidity and mortality in several regions, including Asia
References 35
Pacific and Europe [192]. HFMD can lead to neurological complications and car-
diopulmonary dysfunction resulting from acute EV71 infection [193].
Liu et al. evaluated a bivalent vaccine against EV71, which has completed the
phase III clinical trials [161, 194]. Since CA16 and EV71 act by the orofecal route,
Ang Yin et al. evaluated the impact of colonization of the probiotic strain on HFMD
using in vitro human skeletal muscle and colon cell lines. The authors showed that
the use of L. reuteri Protectis (ATCC 55730) [195], decreased the viral load.
Moreover, this antiviral activity is dose-dependent. The authors suggested that L.
reuteri Protectis interacted physically with CA6, CA16, and EV71 and impaired
viral entry to eukaryotic cells. This antiviral activity seems to be virus probiotic
strain specific, since no antiviral effect was shown using Coxsackievirus B strain 2
(target virus) in the presence of another probiotic strain LcS [161].
1.3.2 Conclusion and Perspectives
Probiotics exhibit direct and indirect mechanisms in eradicating enteric viruses. The
effectiveness of probiotics in the gut ecosystem is more relevant, since they interact
with viral infections by several mechanisms, including immunomodulation, which
is almost the only mechanism available for probiotics in respiratory infections.
The impact of enteric viruses can be decreased by changing the microbiota com-
position. Otherwise, HBGA and LPS are molecules that can be presented by Gram-
negative bacteria and are considered a secondary receptor for enteric viruses such as
NoVs and RoVs. For this reason, using probiotics can change the microbiota to
Gram-positive dominant flora, which blocks the Gram-negative cofactor of viral
infection.
Furthermore, the physical interaction of probiotics has been confirmed in several
studies which confirm the capacity of some probiotic strains to trap viruses.
The use of antibiotics in viral gastroenteritis is a double-edged sword. Broad-
spectrum antibiotic therapy kills probiotic strains or inhibits their multiplication. In
contrast, using anti-Gram-negative antibiotics such as polymyxin B or other non-
broad-spectrum antibiotics can be a crucial factor in blocking the viral cycle.
Moreover, using probiotic strains with antibiotic resistance should be taken into
consideration when treating viral gastroenteritis to keep probiotics live and eradi-
cate Gram-negative resident flora. The antibiotic resistance of commercial probiotic
strains can be found in a review conducted by Sharma et al. [196].
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Chapter 2
The Use of Probiotics as Vaccine Vectors
to Prevent Viral Infections
Bachar ISMAIL
Contents
2.1 Overview .......................................................................................................................... 48
2.1.1 Mucosal Immunity and Vaccines ....................................................................... 49
A Promising Strategy for Mucosal Vaccination ................................................ 49
Induced by Recombinant Probiotic Vaccines .................................................... 50
2.1.4 Probiotics as Vaccine Vectors to Prevent Viral Infections ................................. 51
2.2 Conclusion ....................................................................................................................... 56
References ................................................................................................................................. 57
Abstract Vaccine is one of the most important strategies to struggle infectious dis-
eases. In last decades, several types of vaccine have been used in clinical pathology to
prevent complicated infections especially viral infections which cannot be treated with
specific molecules such as human papillomavirus, rotavirus, and human immunodefi-
ciency virus. The initial vaccination approaches used attenuated or inactivated patho-
gens. While inactivated vaccines are killed pathogens, attenuated vaccines consist of
live microbes that lose their pathogenicity but preserve their antigenicity. Several fac-
tors hinder the development of efficient mucosal vaccines. Therefore, scientists try to
overcome this problem by using probiotic bacteria as delivery systems of heterologous
antigens which may help in designing such vaccines. In this chapter, we review the use
of live probiotic strains as mucosal vaccine vectors to prevent viral infections.
Keywords Mucosal vaccine • vaccine vectors • recombinant probiotic-based

vaccines • lactic acid bacteria • viral infections • influenza viruses • rotaviruses
• papillomaviruses
Abbreviations
CV-N Cyanovirin-N
DCpep DC-targeting peptide
DCs Dendritic cells
GRAS Generally regarded as safe
HA Hemagglutinin
DOI 10.1007/978-3-319-49688-7_2
48 2 The Use of Probiotics as Vaccine Vectors to Prevent Viral Infections
HIV Human immunodeficiency virus

HPV Human papillomavirus
IL-12 Interleukin 2
IL-1β Interleukin 1 beta
IL-2 Interleukin 2
IL-6 Interleukin 2
LTB Heat-labile toxin B
MPER Membrane proximal external region
sIgA Secretory IgA
SlpA Surface layer protein
TLR Toll-like receptors
2.1 Overview
Vaccination is a cornerstone strategy in reducing the burden of infectious diseases

and their associated devastating epidemics worldwide [1]. The benefits of effective
vaccines are not confined to human beings, since vaccination can also prevent infec-
tions and death in animals (especially domestic animals), thus avoiding important
economic losses [2].
Vaccines actively stimulate the host immune system to elicit protective humoral and
sometimes, to a lesser degree, cell-mediated immune responses [3]. Induction of
humoral immunity results in the production of specific neutralizing antibodies that block
infection. Cell-mediated immunity relies essentially on the activation of cytotoxic
T-lymphocytes that control infection once replication has been initiated intracellularly.
The initial vaccination approaches used attenuated or inactivated pathogens.
While inactivated vaccines are killed pathogens, attenuated vaccines consist of live
microbes that lose their pathogenicity but preserve their antigenicity. Importantly,
recent advances in the field of biological sciences, essentially immunology, micro-
biology, and molecular biology, have offered the potential to develop additional
vaccination strategies. Among them is the use of live attenuated viral and bacterial
pathogens as vectors, or delivery vehicles, to deliver heterologous antigens. In such
approaches, heterologous antigen-encoding genes are inserted and subsequently
expressed by the live recombinant vector [4].
Most currently available vaccines are delivered parenterally, while a few others
are administered through needle-free vaccine delivery methods (e.g., via mucosal
surfaces through oral administration). Indeed, several factors hinder the develop-
ment of efficient mucosal vaccines. However, the use of probiotic bacteria as deliv-
ery systems of heterologous antigens may help in designing such vaccines.
In this chapter, we review the use of live probiotic strains as mucosal vaccine
vectors to prevent viral infections. We first briefly describe mucosal immunity and
the advantages of mucosal vaccines. Then we discuss the potential use of recombi-
nant probiotics as mucosal vaccine delivery vehicles and present the factors that
modulate their induced immune responses in the host. Finally, we investigate the
2.1 Overview 49
protective effects of recombinant probiotic-based vaccines during viral infections,

focusing on the human immune deficiency virus (HIV), influenza viruses, rotavi-
ruses, and papillomaviruses.
2.1.1 Mucosal Immunity and Vaccines
Mucosal (gastrointestinal, respiratory, genital) surfaces are an essential entry route

through which most infectious agents invade the body. However, these surfaces are pro-
tected by an extensive mucosal immune system that plays a central role in defending the
mucosae. Complex and multiple immune effector mechanisms are elicited at mucosal
surfaces to confer mucosal immune protection. Local production of secretory IgA
(sIgA) antibodies by mucosal plasma cells has pivotal importance in this process [5].
Since the majority of infections initiate infectious processes at the mucosal sur-
faces, mucosal immunization may be a promising way to combat a variety of infec-
tious agents. Importantly, compared to parenteral vaccines, those vaccines
administered via mucosal routes show several advantages: they are simpler to
administer (orally, nasally), practical for mass vaccination (no requirement for
trained staff), and noninvasive and carry a lower risk of transmission of blood-
transmissible infections (through contaminated needles) [6, 7]. Moreover, while
systemic immunization is generally ineffective in stimulating mucosal immune
responses, mucosal immunization was shown to elicit both mucosal (at local and
distant sites) and systemic immunity [5]. However, in spite of these advantages, few
mucosal vaccines are currently commercially approved (against poliovirus, rotavi-
rus, influenza, Salmonella typhi, and Vibrio cholerae), while most immunization
procedures are performed via parenteral administration routes [8, 9].
The scarcity of mucosal vaccines may be due to a variety of limitations, including
possible enzymatic-induced degradation in harsh host environments (e.g., the GI tract),
low adherence and poor transport of antigens across mucosal surfaces, weak immuno-
genicity, and induction of antigen-specific tolerance rather than protective immune
responses [5, 6, 10]. In line with this, it was shown that soluble nonreplicating antigens
do not stimulate strong immune responses, are generally required to be administered in
multiple high doses, and may result in systemic nonresponsiveness [11].

A Promising Strategy for Mucosal Vaccination
To overcome the limitations that hinder the efficacy of mucosal vaccines, specific
approaches have been proposed that can promote antigen delivery, uptake, and presen-
tation at mucosal sites in order to stimulate appropriate immune responses. These
include the use of vaccine delivery systems [6, 10], which can be live microorganisms
or synthetic (nonliving) vehicles such as polymer-based delivery systems (biodegrad-
able micro- and nanoparticles) or lipid-based delivery systems (liposomes). So far, live
bacterial vectors (including attenuated pathogenic or commensal nonpathogenic bacte-

ria) are considered the most characterized vehicles for mucosal vaccine delivery [12].
Live attenuated bacteria can be used as carriers or delivery vehicles for heterolo-
gous antigens via the introduction of antigen-encoding genes, thus creating live
recombinant vectors. Previous procedures employed serial passages or chemical
mutagenesis to generate attenuated bacterial strains [13, 14]. However, advances in
biotechnology allowed bacterial attenuation by the use of genetic deletions to induce
well-characterized mutations in genes coding for essential virulence factors such as
Salmonella typhimurium [15, 16].
Live attenuated bacterial vaccine vectors are no longer pathogenic, but remain
immunogenic. Their use is a promising approach, since they are (i) able to stimulate
potent immune responses as in natural infections and (ii) possess potent intrinsic
adjuvant properties [4]. At the practical level, they also present several advantages,
including inexpensive and easy production, simplicity of administration, possible
accommodation of multiple heterologous genes, induction of immune responses
against both homologous and heterologous antigens, and sensitivity to antibiotics
with the possibility of treatment if adverse reactions occur [12, 17]. However, the
selection and construction of live attenuated bacterial vectors are tedious and time-
consuming steps [18]. Moreover, such vectors may present two important risks. The
first is the possible instability of the attenuated strain, with potential reversal of
attenuation to the full virulent phenotype in the host after administration. The sec-
ond is possible virulence in susceptible hosts, such as in partially immunocompetent
individuals (young infants, elderly) or immunocompromised subjects due to the
retention of a residual virulence level by attenuated strains [18, 19].
In order to overcome these problems, scientists exploited the use of safe non-
pathogenic bacteria as antigen delivery vehicles [20]. In this context, live nonpatho-
genic probiotics, especially lactic acid bacteria (LAB), have gained increased
interest during the past two decades. Indeed, these microorganisms show important
health-promoting properties, and, due to their long-standing and wide use in the
fermentation and preservation of food products, many of them have a generally-
regarded-as-safe (GRAS) status. These characteristics make them attractive alterna-
tive vector candidates in the field of vaccine development [21]. In this way, a variety
of probiotics are being engineered to successfully express bacterial, parasitic, and
viral antigens to function as novel vaccine delivery vehicles [21, 22].

Induced by Recombinant Probiotic Vaccines
Protective immune responses elicited by recombinant probiotics depend on several

parameters including (i) the bacterial strain, (ii) the administration route, (iii) the
amount of the synthetized antigen, and (iv) the mode of antigen production and
presentation [23–26]. The latter can occur in three different types: (i) intracellularly,
which protects antigen protection against harsh host environmental conditions but
requires cellular lysis to allow its release; (ii) extracellularly, in which the antigen is
2.1 Overview 51
released into the external medium and contacts the host mucosal surfaces directly
but can be highly exposed to harsh conditions; and (iii) cell wall anchored, where
the antigen can interact with host tissues and is generally protected against proteo-
lytic degradation [23, 27]. Although some data suggests that cell wall-anchored
antigens elicit stronger immune responses [27], other data underlines that it is dif-
ficult to conclude which location of an expressed antigen is the best in providing
optimal mucosal immunization [25].
Immune responses induced by recombinant probiotic vaccines can be promoted
by co-expression of the desired antigen with immunostimulatory cytokines, chemo-
kines, bacterial antigens, or molecules that bind specifically to dendritic cells (DCs)
[25, 28]. Indeed, it was shown that mice immunized with probiotic strains express-
ing heterologous antigens along with proinflammatory cytokines such as interleukin
(IL)-2 and IL-6 [29], IL-12 [30, 31], or IL-1β [32] show more effective immune
responses than mice immunized with strains that express the antigen only. Similarly,
CCL3 (MIP-1α), CXCL9 (MIG), and CXCL10 (IP-10) chemokines were success-
fully used as vaccine adjuvants and showed immunostimulatory properties in vitro
and/or in vivo [33, 34]. Bacterial toxins, such as cholera toxin, E. coli heat-labile
toxin B (LTB), and bacterial flagellar antigens [35–37], also promote immune
responses in mice vaccinated with probiotic strains and co-expressing such bacterial
products concomitantly with the antigen of interest. The use of probiotic strains for
specific delivery of the desired antigen fused to a DC-targeting peptide (DCpep) is
also an alternative promising approach allowing faster and more efficient transport
of the immunogenic material into mucosal DCs [38]. These cells, in turn, play a
pivotal role in the activation of potent protective adaptive immune responses.
2.1.4 Probiotics as Vaccine Vectors to Prevent Viral Infections
Several recent publications report the potential of probiotics as mucosal vaccine

vectors for host protection against a variety of viral infections with high efficacy in
animal models [39]. Below, we discuss these protective effects in the case of HIV,
influenza virus, rotavirus, and papillomavirus infections.
2.1.4.1 HIV
Development of an efficient vaccine against HIV remains a global health priority.

Since HIV is transmitted mostly by the mucosal route, a vaccine vector that colo-
nizes and replicates in the mucosa would be ideal against this virus [40].
Several studies examined the use of probiotics, especially LAB, as delivery vehi-
cles for a mucosal vaccine against HIV. In 2003, Xin et al. investigated whether
Lactococcus lactis (L. lactis) engineered to produce a surface-anchored HIV-1 enve-
lope protein might serve as an anti-HIV vaccine. In this regard, they modified this
LAB strain to express the V2–V4 loop of HIV Env. They showed that oral adminis-
tration of this recombinant vector can induce an effective HIV-specific mucosal and
systemic immunity and confers host protection against an HIV Env-expressing vac-
cinia virus after intraperitoneal challenge in mice [41]. However, the cholera toxin,
which is not acceptable for use in humans, was co-administered as a mucosal adju-
vant in this study. L. lactis was also used as a vaccine after genetic modification to
encode the gp120 antigen of HIV-1, and the in vivo immune responses induced by
this strain were compared to those elicited by Escherichia coli (E. coli) expressing
the same antigen [42]. Following intramuscular immunization, mice receiving L.
lactis vectors developed similar humoral immunity as revealed by gp120 antibody
titers, but a weaker cellular immune response than mice receiving E. coli vectors.
Kajikawa et al. constructed a genetically modified Lactobacillus acidophilus
(L. acidophilus) strain that expressed, on its cell surface, Gag antigen from HIV-1
fused with Salmonella enterica serovar typhimurium flagellin (FliC) as an adjuvant.
In vitro, heterologous antigens expressed by this strain were able to induce matura-
tion of human DCs. In vivo, intragastric immunization of mice with this recombinant
LAB led to a strong activation of TLR5, the specific flagellin receptor, and produced
an enhanced Gag-specific IgA response by activated B lymphocytes [43]. More
recently, the same group employed L. acidophilus as an oral mucosal vaccine plat-
form in which they inserted a linear epitope from the membrane proximal external
region (MPER) of HIV-1 into the highly expressed bacterial surface layer protein
(SlpA) [44]. Mice immunization via the intragastric route with such recombinant
lactobacilli induced MPER-specific antibodies, but did not stimulate a T-cell response.
This T-cell-independent antibody response may support the use of this strain as a vac-
cine platform, since a growing body of evidence underlines that vector-directed
responses that activate T-cells may promote HIV infection [45]. However, both anti-
body and T-cell responses against SlpA were also evoked in immunized mice.
Rather than coding for HIV antigens, some probiotic strains were also engi-
neered to express molecules that can inhibit HIV entry, as well as proteins display-
ing HIV virucidal activity. Examples include recombinant Lactobacillus jensenii (L.
jensenii) coding two-domain CD4 (2D CD4) proteins that inhibit the entry of HIV-1
into target cells [46] or coding for the potent HIV inhibitor cyanovirin-N (CV-N)
with the ability to colonize the vagina and produce full-length CV-N when adminis-
tered intravaginally to mice during the estrus phase [47].
2.1.4.2 Influenza Viruses
Influenza viruses are a great health problem in both human and animals. Considering
their high ability to spread and mutate inside their hosts, vaccination may be a very
efficient technique to combat these viruses. Several probiotic strains were used as
delivery systems for influenza virus antigens, especially hemagglutinin (HA) that
has shown itself to be an effective candidate vaccine antigen.
Significant mucosal and systemic immune responses leading to complete protec-
tion of immunized mice from a lethal dose of the highly pathogenic avian influenza
H5N1 were obtained using recombinant L. lactis expressing HA antigen [48]. In this
study, L. lactis bacilli were engineered to either intracellularly express or secrete the
antigen, and these vectors were loaded on mucoadhesive polymers and packaged in
2.1 Overview 53
enteric-coated mini-capsules. After oral administration, high levels of HA-specific

serum IgG and fecal IgA were detected in immunized mice, and the best results were
obtained with capsules containing L. lactis strains that secrete the HA antigens.
Protective immune responses against H5N1 were also efficiently induced using
two recombinant LAB strains, L. acidophilus ATCC 4356 (LA4356-pH) and L. del-
brueckii subsp. lactis D17 (DLD17-pH) [49]. Both strains were manipulated to
express the hemagglutinin 1 (HA1) and then orally administered to BALB/c mice.
Immunized mice showed mucosal and systemic immune responses with a significant
increase in the levels of specific anti-HA1 IgA and IgG antibodies in the mucosa and
serum, respectively. However, the authors suggested that DLD17-pH could be more
promising as an oral vaccine candidate against H5N1, since it induced more effective
protective responses compared with LA4356-pH. More recently, Szatraj et al. express
avian influenza hemagglutinin (H5) and chicken IL-2 in L. lactis, and animal trials
conducted in mice demonstrated that immunization with this recombinant strain by
intragastric gavage induces H5-specific serum IgG and IgA [50].
A recombinant Lactobacillus plantarum (L. plantarum) NC8 strain modified to
express the HA gene of H9N2 was also used as a vaccine vector against this virus.
Mice orally immunized with this recombinant strain were completely protected
against a lethal challenge with mouse-adapted H9N2. Moreover, macroscopic exam-
ination of their lungs showed that they have reduced pulmonary pathology after
challenge. Vaccinated mice produced efficient mucosal, humoral, and cellular
immunity with anti-HA-specific serum IgG and mucosal IgA antibodies, as well as
CD8+ T-cell responses [51]. More recently, similar promising results against H9N2
were obtained in mouse and chicken models immunized orally with L. plantarum
NC8 expressing HA fused to the specific DCpep that effectively target antigens to
DCs [52]. The authors of this study suggest that this strategy may reduce the number
of vaccinations and the quantity of L. plantarum required for oral administration.
Protection against influenza viruses was conferred after oral and nasal inocula-
tions of a vaccine consisting of recombinant Lactobacillus casei (L. casei) display-
ing the highly conserved matrix protein 2 with the cholera toxin subunit A1 (CTA1)
on its surface. Effective systemic and mucosal immune responses with high levels
of M2-specific serum IgG and mucosal IgA were detected in vaccinated mice.
Vaccination elicited significant levels of protection against lethal challenges of
divergent influenza subtypes, including H1N1, H5N1, H5N2, H9N2, and H7N3
[53]. However, the authors underline that the efficacy of the intranasal administra-
tion of this vaccine was superior to the oral vaccination route.
2.1.4.3 Rotaviruses
Rotaviruses are considered the main cause of infectious severe diarrhea in human
infants and animals worldwide. Two live attenuated rotavirus vaccines, Rotarix and
RotaTeq, are available worldwide [54, 55]. Recently, there have been several
attempts to develop new potential vaccines against rotaviruses using recombinant
antigens, especially capsid viral proteins, and employing probiotic bacteria as
delivery vehicles.
In 2005, Perez et al. investigated the immunogenicity of L. lactis expressing the

cytoplasmic, secreted, or cell wall-anchored forms of the rotavirus VP7 outer capsid
antigen in animals [56]. They showed that intragastric immunization with this
recombinant strain can induce the production of neutralizing antibodies and demon-
strated that the secreted form of VP7 was more immunogenic than other forms. In
the same way, an additional study showed that L. lactis expressing the VP4 capsid
antigen of the porcine rotavirus on the outer side of the cell wall induced specific
anti-VP4 local and systemic mucosal IgA and serum IgG antibodies in orally immu-
nized mice [57].
Porcine rotavirus VP4 was also expressed in another LAB strain, L. casei, with
or without LTB from E. coli [35]. The efficacy of these strains in acting as antigen
delivery systems for vaccination was then assessed in immunized mice. After oral
administration, both strains were able to induce specific anti-rotavirus mucosal and
systemic immune responses with high levels of antiVP4 serum IgG and mucosal
IgA. However, the protective IgA titers were greater in animals immunized with
strains expressing the VP4–LTB fusion protein than those expressing the VP4 anti-
gen only, thus highlighting the efficacy of LTB as a mucosal adjuvant.
Using L. lactis, Marelli et al. design different expression vectors that were used
to express the rotavirus spike-protein subunit VP8* in the cytoplasm, secreted, or as
a cell wall-anchored form [58]. The authors then assessed the efficacy of each of
these recombinant strains in orally immunized mice. They found that the strain
expressing the cytoplasmic form of VP8 elicited significant levels of intestinal IgA
antibodies, while the strains producing the surface-anchored form of this antigen
induced both mucosal and systemic anti-VP8 antibodies. In addition, the authors
demonstrated that mucosal IgA elicited by the cytoplasmic VP8-expressing strains
and serum IgG elicited by the cell wall-anchored VP8-expressing strains induce a
respective blockade of 50 and 100 % of rotavirus infection in fetal monkey kidney
cells in vitro.
Although the studies cited above demonstrate that recombinant probiotic strains
expressing heterologous rotavirus antigens provide protective responses, they do
not report any in vivo challenge experiments.
The nonpathogenic Bacillus subtilis (B. subtilis) probiotic strain engineered to
express either the bovine or murine rotavirus VP6 inner capsid antigen was exam-
ined as a potential recombinant vaccine against rotavirus [37]. The ability to confer
protection was tested in a mouse challenge model after intranasal inoculation with
vegetative cells or spores of the recombinant strains. The results showed that B.
subtilis spore-based rotavirus vaccines, but not vegetative cell-based vaccines, gen-
erate protective immune responses in immunized mice challenged orally with the
virus. Indeed, although the systemic responses induced by the spore and vegetative
forms were comparable, the mucosal responses were not. This was reflected in the
fecal IgG and IgA levels that show a significant increase in spore-vaccinated but not
in vegetative cell-vaccinated animals. Moreover, effective protection was obtained
when whole cholera toxin or a heat-labile toxin from a mutant form of E. coli was
added as mucosal adjuvants in the context of this expression system.
2.1 Overview 55
2.1.4.4 Human Papillomavirus
Human papillomavirus (HPV) is the most common sexually transmitted infection.

This group comprises more than 150 related viruses. Among them, HPV type 16
(HPV-16) is the major etiological agent of cervical cancer, the second most common
cause of cancer death in women [59, 60]. Three prophylactic vaccines (Gardasil,
Gardasil 9, and Cervarix) are currently available and have been shown to prevent
genital infection by some HPV types, including HPV-16, and to reduce their related
cancers [61]. However, these vaccines do not confer protection in patients who are
already infected prior to vaccination, thus highlighting the importance of the devel-
opment of therapeutic HPV vaccines [61, 62].
HPV-16 encodes the E7 oncoprotein, which is essential to the carcinogenesis
process. This immunogenic antigen is constitutively produced in cervical carcino-
mas, thereby forming an attractive candidate for the development of a therapeutic
vaccine for HPV-related cancers.
The production of the HPV-16 E7 antigen was previously reported in both safe
nonfood-grade Streptococcus gordonii (S. gordonii) [63] and food-grade L. lactis
[64] probiotic strains. Mice and monkeys vaccinated with E7-expressing S. gordo-
nii showed protective immune responses [65, 66]. In L. lactis, the intracellular
fabrication of E7 results in its rapid degradation, while secreted and surface-
anchored forms were stable and highly produced [64, 67]. In mice, intranasal
administration of L. lactis expressing different forms of E7 leads to the develop-
ment of both humoral and cell-mediated immune responses, as respectively
reflected in the production of anti-E7 antibodies and secretion of cytokines such
as IL-2 and IFN-γ [67, 68]. However, significantly higher responses were observed
in animals immunized with strains expressing E7 as a surface-anchored protein
[68]. Protection mediated by mucosally co-administered recombinant Lactococcus
lactis strains expressing cell wall-anchored E7 and a secreted form of IL-12 was
tested in mice challenged with lethal levels of the tumor cell line TC-1 expressing
the E7 antigen. The results demonstrated that half of the immunized mice remained
free of TC-1-induced tumors, and this antitumor activity appeared to be long last-
ing. In addition to these prophylactic effects, the Lactococcus strains constructed
in this study showed therapeutic properties, as almost one-third of the mice
showed tumor regression when immunized 7 days after TC-1 injection. The
authors found that these vaccination-related antitumor effects occurred through
cell-mediated CD4+- and CD8+-dependent immune responses [30]. Similar antitu-
mor effects were more recently reported in mice intranasally immunized with
recombinant L. lactis strains that simultaneously carry the HPV-16 E7 protein and
the IL-12 gene [31]. Another study showed that immune responses and antitumor
effects induced by recombinant lactococci displaying the E7 antigen at their
surface and secreting biologically active IL-12 were higher after intranasal immu-
nization than after intragastric administration [24]. This study also demonstrated
that intranasal administration of L. plantarum-expressing surface-anchored E7
can induce specific immune responses and causes significant regression of
HPV-induced tumors in intranasally vaccinated mice. These responses were sig-

nificantly higher than those induced by L. lactis anchoring this antigen and admin-
istered by the same route [24].
L. casei was also used as a delivery system of HPV-16 E7 [69–71]. C57BL/6
mice orally immunized with this recombinant strain displayed E7-specific serum
IgG and mucosal IgA antibodies and showed systemic and local cellular immu-
nities that were significantly increased after boosting [71]. Immunized mice also
show mucosal cytotoxic lymphocytes against HPV-16 E7-positive cells [69],
and vaccinated animals with TC-1-induced tumors showed reduced tumor for-
mation and an increased survival rate [71]. Mice protection against HPV-16-
induced tumors was also obtained using nongenetically modified lactic acid
bacteria displaying a stable E7 antigen at its surface using an L. casei-derived
cell wall anchor [72]. More recently, a phase I/IIa study involving patients with
HPV-16-positive cervical intraepithelial neoplasia grade 3 showed that oral vac-
cination with attenuated L. casei expressing a modified full-length HPV-16 E7
protein showed an E7-specific mucosal immune responses in uterine cervical
lesions [70].
The L1 and L2, major and minor HPV-16 capsid proteins, respectively, were also
expressed in L. casei. In the cytoplasm of these bacteria, HPV-16 L1 was able to
self-assemble into viruslike particles (VLPs) which display conformational epit-
opes. Anti-L1 IgG antibodies were detected in the sera of mice subcutaneously
immunized with this recombinant strain [73]. A partial HPV-16 L2 protein was also
expressed on the surface of L. casei, and oral immunization of BALB/c mice with
this strain elicited L2-specific serum IgG and vaginal IgG and IgA antibodies. In
addition, vaccination-generated antibodies display cross-neutralizing activities, and
the vaccinated mice were protected against genital infection by HPV-16, HPV-18,
HPV-45, and HPV-58 pseudovirions [74].
2.2 Conclusion
The data reviewed above shows promising effects for the use of probiotic strains,
especially LAB, as efficient vehicles for the delivery of viral antigens toward muco-
sal sites. These attractive vaccine vectors seem to be able to induce both mucosal
and systemic immunity and elicit antiviral protective responses in animal models.
However, such vaccines are not available commercially, and only a few of them are
currently in the subject of clinical trials, especially those consisting of L. casei
expressing the HPV-16 E7 antigen (clinical trials NCT02195089 and
UMIN000001686). Nevertheless, continuous optimization of these carriers by
searching for and identifying the most effective strains, the most appropriate immu-
nization modalities, and the most potent and safe adjuvants will increase their effi-
cacy in the future. These represent important steps toward the development of new,
safe, practical, and effective mucosal vaccines to prevent viral infections and reduce
their devastating mortality and morbidity.
References 57
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Chapter 3
Probiotics: Role in the Prevention of Chronic
Viral Diseases
Imad AL KASSAA and Mazen ZAYLAA
Contents
3.1 General Introduction ...................................................................................................... 63
3.2 Cancer Related to Viral Infections ................................................................................. 64
Papillomavirus (HPV) Infection .................................................................................... 67
T-Cell Lymphotropic/Leukemia Virus (HTLV) Infection .............................................. 69
3.5 Probiotics as a Novel Prevention Strategy Against
Type 1 Diabetes Related to Viral Infection .................................................................... 71
3.6 Probiotics as a Treatment and Prevention Strategy for
Liver Complications Caused by Hepatitis B and C Virus .............................................. 72
Viruses 1 and 2 Using Probiotic Strains ........................................................................ 74
3.8 Probiotics and Human Immune Deficiency Virus (HIV) ............................................... 76
3.9 Conclusion and Perspectives .......................................................................................... 77
References ............................................................................................................................... 78
Abstract Viral infections are the most critical among infectious diseases, especially
those that can lead to chronic diseases. The control and the prevention of chronic
diseases represent a challenge for public health organizations. These chronic
diseases are the major cause of death worldwide. To achieve the greatest impact,
public health campaigns should focus on creating novel treatment and prevention
strategies against chronic viral diseases. Probiotics are defined as live microorgan-
isms with beneficial effects for humans. Probiotic strains have shown antiviral
activity against a variety of infectious viruses such as respiratory and enteric viruses.
In this chapter, we discuss the possible role of probiotic strains in chronic viral
infections and their benefits in therapy strategies against such diseases. Data from
numerous studies has shown that the use of probiotic as therapeutic agents is safe
and inexpensive and can avoid the need for invasive treatment for several chronic
viral infections caused by HIV, HCV, HTLV, HPV, CVB4, etc. The principal mecha-
nisms of the antiviral activity of the probiotic strains studied until now were the
production of antiviral compounds, the immunomodulatory effect, and virus trap-
ping by the probiotic cell wall.
Keywords chronic viral diseases • antiviral probiotics • Cancer • Lactabocillus

• Oncovirus

DOI 10.1007/978-3-319-49688-7_3
62 3 Probiotics: Role in the Prevention of Chronic Viral Diseases
Abbreviations
AFB1 Aflatoxin B1
AIDS Acquired immune deficiency syndrome
ALT Alanine aminotransferase
AST Aspartate aminotransferase
ATCC American Type Culture Collection
ATL Adult T-cell leukemia
CLD Chronic liver disease
CPE Cytopathic effect
CSF Cerebrospinal fluid
CVB3 Coxsackievirus B3
EBV Epstein–Barr virus
GGT Gamma glutamyl transferase
H2O2 Hydrogen peroxide
HAM/TSP Myelopathy/tropical spastic paraparesis
HBV Hepatitis B virus
HCC Hepatocellular carcinoma
HCs HTLV-1 carriers
HCV Hepatitis C virus
HHV4 Human herpesvirus 4
HHV8 Human herpesvirus 8
HPV Human papillomavirus
HR High risk
HSV-1 Herpes simplex viruses 1
HTLV-1 Human T-cell lymphotropic virus type 1
IARC International Agency for Research on Cancer
KHSV Kaposi’s sarcoma-related herpesvirus
LDH Lactate dehydrogenase
LR Low risk
MHC Major histocompatibility complex
NK cells Natural killer cells
PBMCs Peripheral blood mononuclear cells
PRA Plaque reduction assay
pRb Retinoblastoma protein
T1D Type 1 diabetes
TGF-α Transforming growth factor alpha
TNF alpha Tumor necrosis factor alpha
3.1 General Introduction 63
3.1 General Introduction
Chronic diseases are the major cause of death worldwide. Infectious diseases are caused
by infectious agents including viruses, bacteria, and parasites. Physicians and research-
ers have hypothesized that infection may play a major role in certain chronic disorders.
Preventing or treating infection or strengthening the immune response to infection offers
an opportunity to upset the continuum and thus prevent or minimize chronic diseases.
The causal associations between infection and chronic diseases are characterized by a
diverse spectrum of agents, pathways, outcomes, and cofactors. Nevertheless, imple-
menting and maintaining infection control measures is changing disease patterns, so that
today chronic diseases represent the major health burden of established economies (>90
million people in the United States) and are a rapidly growing burden in developing
economies [1]. Moreover, it has recently been found that no less than 13 of 39 infectious
agents induce chronic diseases. For example, hepatitis B infection (HBV) has come to
explain a proportion of chronic liver disease (CLD) and hepatocellular carcinoma
(HCC) in zones with endemic disease. Creating novel treatment and prevention strate-
gies against infectious viruses using probiotics can influence health across populations,
creating opportunities to decrease the effects of chronic disease.
According to the FAO/WHO definition, probiotics are “Live microorganisms
which, when administered in adequate amounts, confer a health benefit on the host”
[2]. Toward the start of the twentieth century, Elie Metchnikoff presented a novel
theory about the health impacts of probiotics. He theorized that the consumption of
fermented milk products led to improved health and a longer lifespan among
Bulgarian laborers. In addition, he expressed that the microorganisms present in
yoghurt could protect the digestive tract from the damaging impact of other patho-
genic microorganisms [3].
Probiotic strains have certain properties, such as resistance to bile, hydrochloric
acid, and pancreatic juice, in addition the capacity to endure stomach and duode-
num conditions, activation of the immune system, subsequent enhancement of
intestinal capacity through adhesion, and colonization of the intestinal epithelium.
In addition, probiotic strains rival pathogens and balance permeability, produce lac-
tic acid, and display anticarcinogenic and antipathogenic activity [4]. Lactobacillus,
Bifidobacterium, Escherichia, Enterococcus, Bacillus, Streptococcus, and some
Saccharomyces species have been known to act as probiotics [5]. Powders, fluids,
gels, glues, granules, sachets, and a few varieties of food are available commercial
items containing probiotics [6]. Several studies and clinical trials have been con-
ducted to survey the impact of different strains of probiotics in the treatment and
prevention of diseases including specific types of diarrhea, inflammatory bowel dis-
ease, tumors, vaginosis, hepatic infection hypersensitivity, modulation of the
immune system, and several other pathologies [3].
Microbiota plays a major role in shaping innate and adaptive immunity and
maintaining immune homeostasis. An increasing number of studies have examined
the therapeutic potential of commensal bacteria in modulating the mucosal immune
responses [7]. Several studies published a few decades ago showed that microbiota
have the potential to modulate the outcome of certain viral infections [7]. Human
clinical studies examining the therapeutic potential of probiotics in the prevention

and treatment of viral infections are still in the early stages [7]. Below, we will
examine the evidence for the role of probiotics in the prevention and treatment of
chronic viral diseases (Table 3.1).
3.2 Cancer Related to Viral Infections
After dietary elements and tobacco smoke, infectious disease is the third leading
cause of tumors worldwide. The proportion of cancers related to infectious diseases
was assessed to be 10 % in the US population in 1981 [25] and 29.4 % (31.7 % in
men and 25.3 % in women) in the Chinese population in 2005 [26]. In the world-
wide population, it was assessed to be 15.6 % in 1990 and 16.1 % in 2008.
Specifically, this creates the impression that chronic infections with Helicobacter
pylori, human papillomaviruses (HPV), and both hepatitis B (HBV) and C (HCV)
infections are each accountable for 5 % of all human cancers and represented 15.6 %
of human tumors worldwide in 2002 and 14.7 % in 2008 [27, 28].
Twelve HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 have been
assigned by the International Agency for Research on Cancer (IARC) to Group 1, as
their human carcinogenicity has been sufficiently demonstrated. The general portion
of malignancy related to HPV infection was assessed to be 5.2 % and 4.8 % in 2002
and 2008, respectively (Fig. 3.1). Continuous infection of the uterine cervix is
responsible for 100 % of cervical tumors, although different factors could interact
with HPV in the etiology of cervical tumors, which is the third most common malig-
nancy in the female population worldwide in terms of mortality. Moreover, HPV can
target different locales in the anogenital area of women and/or men (vulva, vagina,
and penis), the upper aerodigestive tract (mouth and oropharynx), and the skin [29].
Herpesviridae may cause two vital tumors related to infections, both of which
are assigned to IARC Group 1. Both have been associated with around 1 % of every
human malignancy. One is the Epstein–Barr virus infection (EBV) or human her-
pesvirus 4 (HHV4), which causes infectious mononucleosis throughout adolescence
and young adulthood, while, in certain geographical zones, it is associated with
several forms of lymphoma. The most essential EBV-related malignancy is Burkitt’s
lymphoma. In addition to lymphomas, EBV is associated with nasopharyngeal car-
cinoma. The other cancer-related infection in this family is Kaposi’s sarcoma-related
herpesvirus (KHSV), or human herpesvirus 8 (HHV8), which has been found in
patients affected by acquired immune deficiency syndrome (AIDS) [29].
HTLV-1 (human T-cell lymphotropic infection type 1) has been associated with
adult T-cell leukemia/lymphoma and is assigned to IARC Group 1, while HTLV-2
is in Group 3. HIV-1 (human immune deficiency virus type 1) is the etiological
agent of AIDS which, because of immune deficiency, has been associated with
several types of human tumors and particularly with KHSV-related Kaposi’s sar-
coma and non-Hodgkin’s lymphoma. HIV-2 is also possibly carcinogenic [29].
The hepatitis B and C viruses (HBV and HCV) are hepatotropic viruses, an
infection with which might develop into chronic viral hepatitis. They are very
Table 3.1 Probiotic strains used in chronic viral diseases
3.2
Probiotic strains Origin Diseases Mechanisms Experiment References

B. adolescentis SPM1005-A Human feces HPV-associated cervical cancer Suppressed of E6 and E7 In vitro ( SiHa cells) [8]
oncogene expression
Probiotic drink (L. casei Human feces HPV-associated cervical cancer Clearance of HPV-related 54 women with an HPV [9]
Shirota) cytological abnormalities + low-grade squamous
intraepithelial
lesion- Belgium
Vaginal lactobacilli (L. The vagina of HPV-associated cervical cancer Independent of pH and In vitro: human normal [10]
crispatus strain SJ-3C-US a healthy lactate cytotoxic effects on fibroblast-like cervical
and L. gasseri ATCC 33323) woman cervical tumor cells (normal cervical) and
HeLa (cervical tumor)
Cancer Related to Viral Infections
Kefir – HTLV-1-positive malignant Downregulation of TGF-α In vitro (HuT-102 cells) [11]

T-lymphocytes expression
L. casei strain Shirota (LcS) Human feces Human T-cell lymphotropic Increase the NK cell activity, Ten patients with HAM/ [12]
virus type 1 (HTLV-1)- improvements in spasticity TSP
associated myelopathy/tropical (modified Ashworth scale
spastic paraparesis (HAM/TSP) scores) and urinary symptoms
B. adolescentis SPM1605 Human feces Coxsackievirus B3 infection Decrease the amplified viral In vitro (HeLa cells) [13]
sequences
VSL#3 probiotic mix – HCV-related chronic hepatitis Improve the level of aspartate 20 patients with [14]
aminotransferase and alanine HCV-related chronic
aminotransferase hepatitis
VSL#3 probiotic mix – HCV-related cirrhosis Improve the level of aspartate 16 patients with [14]
aminotransferase, alanine HCV-related cirrhosis
aminotransferase, and
gamma glutamyl transferase
L. rhamnosus LC705 and – Liver cancer Reduces the biologically 90 healthy young men [15]
Propionibacterium effective dose of aflatoxin from China
freudenreichii subsp. exposure
Shermanii
65
(continued)
66
Probiotic strains Origin Diseases Mechanisms Experiment References
L. rhamnosus GG and L. Human feces Liver cancer Enhanced protective against In vivo (200 Wistar [16]
casei strain Shirota aflatoxin B1 rats)
B. adolescentis SPM0212 Human feces HBV infection Decreased the extracellular In vitro (HepG2.2.15 [17]
HBsAg level cells)
L. gasseri CMUL57, L. The vagina of HSV-2 infection Anti-HSV-2 activity due to In vitro ( Vero cell [18]
acidophilus CMUL67, and a healthy physical contact between monolayers)
L. plantarum CMUL140 woman lactobacilli cell wall and viral
3
envelope
B. adolescentis SPM 0214 Human feces HSV-1 infection Anti-HSV-1 activity In vitro ( Vero cell [19]
monolayers)
L. crispatus ATCC33820 and The vagina of HSV-1 and HSV-2 infection HSV-2 replication was In vitro and in vivo [20]
L. gasseri ATCC33323 a healthy inhibited and inhibited (BALB/c mouse model)
woman HSV-1 infection in vivo
L. brevis CD2, L. salivarius The vagina of HSV-2 infection HSV-2 replication was In vitro ( Vero cells ) [21]
FV2, and L. plantarum FV9 a healthy inhibited and inhibited
woman HSV-2 infection
B. bifidum with – HIV infection Decrease in the diarrhea with 77 HIV-infected [22]
Streptococcus thermophilus increasing in the mean CD4+ children (2–12 years)
T-cell count
Lactobacillus rhamnosus The distal HIV infection Increasing in the mean CD4 24 women in Nigeria [23]
GR-1 urethra of a cell count and resolve of with HIV/AIDS
healthy woman Diarrhea, flatulence, and
L. reuteri RC-14 The vagina of a nausea
healthy woman
Lactobacillus casei 393 Dairy products HIV infection Blocking HIV-1 transmission In vitro ( TZM-bl cells) [24]
and increasing the CD4 counts
Probiotics: Role in the Prevention of Chronic Viral Diseases
3.3 The Impact of Probiotics in Cancers Related to Human Papillomavirus (HPV) Infection 67
6.00
5.2
5.00 4.8 4.9
4.7
Attributable cancer (%)
4.00
3.00
2.00
1 0.9
1.00
0.03 0.02
0.00
HPV HBV+HCV EBV HTLV-1
Fig. 3.1 Proportion of cancers attributable to infectious agents assigned to IARC Group 1, in rela-
tion to the total number of cancer cases in the worldwide population in 2002 [28] (blue columns)
and 2008 [27] (red columns)
distinctive viruses, HBV being a DNA virus belonging to the family Hepadnaviridae,
while HCV is an RNA virus belonging to the family Flaviviridae. Both pathogens
are assigned to IARC Group 1, and, on the whole, they were evaluated as respon-
sible for 4.9 % of malignancies in the worldwide population in 2002 [28] and 4.7 %
in 2008 [27]. They have been associated with 85.5 % of hepatocellular carcinomas
(HCCs), 54.4 % of which are attributable to HBV and 31.1 % to HCV [28].
The essential action to prevent infection-related tumors is to prevent the infec-
tious disease. Vaccines assume a principal role in the strategy available to prevent
certain tumor-related diseases. For certain viruses, for example, HCV, HIV, HPV,
and HTLV, vaccination is still in progress and has encountered technical problems.
On the other hand, different vaccines are broadly utilized worldwide and hold great
promise in tumor prevention. The problem remains one of vaccine availability and
costs in developing and underdeveloped countries.

Papillomavirus (HPV) Infection
Cervical cancer is the second most common disease of the female reproductive
organs, with an annual frequency of up to 570,000 cases in 2008, with a mortality
rate of roughly 25 % [30]. Most cervical malignancies affect the anogenital area or
mucosal cell infection with human papillomavirus (HPV) [31]. More than 200 dif-
ferent HPV types have been distinguished; 30 HPV types infect the anogenital skin
and oral mucosa and can be further named as low risk (LR) or high risk (HR) based
on the clinical prognosis of their associated lesions [32]. Around 99.7 % of cervical
tumors contain viral DNA of the HR type, with type 16 being the most common,
followed by types 18, 31, 33, and 45. The expression of two viral genes, E6 and E7,
which bind to p53 and retinoblastoma protein (pRb) and neutralize their capacity,
separately clarify the malignant phenotype of the HR types [33]. Binding to the
tumor suppressor p53, which leads to it degradation through an ubiquitin proteolytic
pathway, is the most significant role of the protein E6. Degradation of p53 bypasses
the normal development stop signals at the G1/S and G2/M checkpoints and is the
significant reason for chromosomal risk, with mutational results for HPV-positive
cells [34]. The protein E7 connects with pRb and discharges transcription factor
E2F, which induces the expression of genes involved in cell differentiation and mul-
tiplication [35]. Consequently, research into inhibitors of the oncogenic proteins E6
and E7 of HPV type 16 is always in progress.
Cha et al. (2012) surveyed the inhibitory impacts on the HPV oncogenic mRNA
and consequently on the production of oncogenic proteins. As mentioned above, the
carcinogenesis of cervical cancer is connected to the overexpression of the viral onco-
genic proteins E6 and E7 that deactivate the tumor suppressor, p53 and pRb, apoptosis
blocking, and reduction of immune recognition. There have been a few attempts to
decrease the expression of these two typical genes of HR-HPV-16 and HR-HPV18
[8]. Cha et al. demonstrated that the downregulation of expression of both genes at the
mRNA and protein levels in SiHa cells can be caused by Bifidobacterium adolescentis
SPM1005-A (B. adolescentis SPM1005-A). Specifically, the expression of both genes
was diminished fundamentally by B. adolescentis SPM1005-A treatment for 48 h.
The qRT-PCR results demonstrated that the E6 and E7 mRNA levels decreased at the
same time. Moreover, Western blot test showed that the E6 protein expression
decreased after 24 and 48 h. Furthermore, the decrease in the HPV-16 E6 and E7 gene
expressions and protein levels were not connected to cell morphology or to significant
cytotoxic impacts of B. adolescentis SPM1005-A in SiHa cells. However, it was not
explained how B. adolescentis SPM1005-A controls the expression of E6 and E7 or
its mechanism of action [8]. This study demonstrated that the probiotic B. adolescen-
tis SPM1005-A had an antiviral activity by suppressing E6 and E7 oncogene expres-
sion. The outcomes recommend that B. adolescentis SPM1005-A could potentially be
used for HPV-associated cervical tumor prevention.
Another study by Verhoeven et al. (2013) was conducted to investigate the poten-
tial impact of probiotics on human papillomavirus (HPV)-related precancerous
types in cervical cytology. They conducted a controlled pilot study, in which 54
women diagnosed with HPV + low-grade squamous intraepithelial lesion in their
Pap smear were monitored for 6 months. During the study period, the intervention
group took the probiotic Lactobacillus casei Shirota (LcS). The outcome measures
were the control Pap smear and HPV status after 6 months. The probiotic group had
twice as high a probability of clearance of cytological abnormalities (60 vs. 31 %, P
0.05). HPV was cleared in 19 % of the control patients versus 29 % in the probiotic
group (P 0.41). This exploratory pilot study proposes that the probiotic led to
improvements in the clearance of HPV-related cytological abnormalities [9].
Motevaseli et al. (2013) evaluated the antiproliferative impact of the supernatants,
cytoplasmic extracts, cell-wall extracts, and live lactobacilli on normal and tumor
cervical cell lines. Inhibition of tumor cell development by culture supernatants was
higher than that of pH- and lactate-adjusted controls. Nonetheless, the impact of the
supernatants on normal cells was identical to those of lactate-adjusted controls,
3.4 The Impact of Probiotics in Cancers Related to Human T-Cell 69
showing that normal vaginal lactobacilli (L. crispatus strain SJ-3C-US and L. gasseri
ATCC 33323) have cytotoxic impacts on cervical tumor cells, but not on typical
cells, and that this cytotoxicity is independent of pH and lactate [10]. However, some
probiotic activity results from lactate creation and the pH of the culture [36].
Considering that vaginal lactobacilli colonize the cervix of healthy adults,
Motevaseli et al. (2013) evaluated the impact of these lactobacilli (L. crispatus
strain SJ-3C-US and L. gasseri ATCC 33323) in normal and tumor cervical cell
lines. Interestingly, live lactobacilli co-culture with a normal cervical cell line did
not show any cytotoxic impact after 24 h; however, strong development inhibitors
of cervical tumor cells (HeLa) were present. The authors showed that live lactobacilli
strains (LS) have an anti-apoptotic impact on HeLa cells by decreasing the expres-
sion and action of caspase-3. The decrease of LDH discharged by skimming dead
cells shows a lower proportion of apoptotic cells among LS-treated cells. It should
be noted that this anti-apoptotic impact was clearly lactate dependent. Apoptosis
was inhibited by supernatants, which was reliable in higher β human chorionic
gonadotropin expression since hCG inhibits apoptosis [10].

T-Cell Lymphotropic/Leukemia Virus (HTLV) Infection
The most commonly detected retrovirus in people is the human T-cell lymphotropic/
leukemia virus (HTLV), which was discovered in 1980 in the blood of a patient with
cutaneous T-cell lymphoma [37]. Five to ten percent of infected people go on to
develop adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical
spastic paraparesis as another type of myeloma, although the rest remain asymp-
tomatic carriers [38]. The latency time of this retrovirus is long and can extend up
to 50 years. The average age of onset of ATL is 55 years, and the male to female rate
is 1.4:1. There are four subtypes of ATL: acute, chronic, smoldering, and lymphoma
relating to an arrangement of the quantity of irregular T-cells in peripheral blood,
serum lactic acid dehydrogenase levels, tumor lesions in different organs, and clini-
cal course. The clinical signs of ATL include malaise, fever, hacking cough, somno-
lence, lymphoadenopathy, hepatosplenomegaly, hypercalcemia, and jaundice.
Lymphocytes have a soluble type of the interleukin-2 receptor α [11]. The infection
ordinarily changes CD4+ lymphocytes, with a small incidence in CD8+ lympho-
cytes. The treatment of ATL remains disappointing.
Kefir is delivered by including kefir grains (a mass of proteins, polysaccharides,
bacteria, and yeast) to pasteurized milk; it appears to control a few cell types of
tumors. Some of the bacteria identified in kefir include L. brevis, L. helveticus, L. kefir,
L. kefiranofaciens, L. kefirgranum, L. parakefir, L. acidophilus, L. lactis subsp. lactis,
L. lactis subsp. cremoris, Streptococcus thermophilus, Enterococcus durans,
Leuconostoc mesenteroides, Bacillus subtilis, Micrococcus spp., and Escherichia coli.
Rizk et al. (2009) showed that the cell-free fraction of kefir had an antiproliferative
impact on malignant T-lymphocytes infected with HTLV-1. The cell-free fraction of
the kefir treatment decreases the proliferation of carcinogenic cells at the different
concentrations used (20, 40, 60, and 80 μg/μL). The cytotoxicity of the compound was
analyzed by deducting the percentage of remaining viable cells 6, 24, and 48 h follow-
ing incubation with several supernatant concentrations. Its impact on the cytotoxicity
and multiplication of normal human lymphocytes was additionally examined. The
cell viability remained above 90 % at 80 μg/μL, which is the highest concentration of
the kefir cell-free fraction used. Thus, no toxic impact on normal cells was seen.
Furthermore, the proliferation of normal lymphocytes was decreased by only around
8 %, which is not a significant different. Investigation of the cell cycle dispersion at
which the inhibition occurred showed that cell cycle inhibition occurred at the G0/G1
stage, which shows up in the pre-G1 increase. This G0/G1 capture may show that the
cell-free fraction of kefir could initiate apoptosis in HTLV-1-infected and malignant
cell lines. The transcriptional level of TGF-α was investigated in order to further study
the effect of the cell-free fraction of kefir on the proliferation of HuT-102 cell line. The
downregulation of TGF-α expression was caused by the kefir cell-free fraction, at all
noncytotoxic concentrations used. This study showed the positive impacts of a kefir
cell-free fraction in one malignant cell line tainted with the HTLV-1 infection. Kefir is
powerful in inhibiting the multiplication and prompting the apoptosis of HTLV-1-
positive cancer T-lymphocytes. Hence, in vivo trials are strongly encouraged [11].
Another type of cancer related to HTLV-1 is myelopathy/tropical spastic parapa-
resis (HAM/TSP) which is a chronic dynamic myelopathy characterized by spastic
paraparesis, sphincter dysfunction, and mild sensory disturbances in the lower
extremities [39]. Although the precise mechanism causing HAM/TSP is still obscure,
virus–host immunological interactions are considered the most critical reason for
this disease, since in HAM/TSP patients, the middle HTLV-1 provirus load is more
than ten times higher than in healthy HTLV-1 carriers (HCs) and is additionally asso-
ciated with an increased risk of progression to disease. The anti-HTLV-1 antibody
titer frequently reaches a high level in HAM/TSP patients; large populations of acti-
vated T-cells, both in peripheral blood mononuclear cells (PBMCs) and cerebrospi-
nal fluid (CSF), and spontaneous proliferation of PBMCs in vitro have been reported.
HTLV-1-specific CD8+ cytotoxic T-lymphocytes (CTLs) are abundant and activated
in PBMCs in HAM/TSP patients, and these CTLs are especially aggregated in CSF
cells. It has been shown that HTLV-1 Tax11–19-specific CD8+ T-cells have the poten-
tial to produce proinflammatory cytokines [12]. To manage such immunological pro-
cedures, some therapeutic trials using new treatments, such as prednisolone, plasma
exchange, and interferon (IFN)-α, have been conducted successfully.
Matsuzaki et al. (2005) showed that clinical improvement was seen in all HAM/
TSP patients following 4 weeks of daily oral administration of Lactobacillus casei
strain Shirota (LcS). Ten patients with HAM/TSP were treated in an uncontrolled
preparatory trial by oral administration of live LcS containing milk. The HTLV-1
provirus load, motor function, neurological discoveries, and immunological param-
eters were assessed after 4 weeks. Despite the fact that LcS did not change the fre-
quencies or total quantities of all examined cell surface phenotypes of peripheral
blood mononuclear cells, the NK cell activity was fundamentally increased follow-
ing 4 weeks of oral administration of an LcS regimen. Improvements in spasticity
(changed Ashworth scale scores) and urinary side effects were additionally seen
after LcS treatment. No side effects were seen in any of the ten patients taking LcS
3.5 Probiotics as a Novel Prevention Strategy Against Type 1 Diabetes Related 71
throughout the study period. This outcome showed that LcS might be a safe and
helpful addition to the treatment of HAM/TSP [12].
3.5 Probiotics as a Novel Prevention Strategy Against Type 1

Diabetes Related to Viral Infection
Type 1 diabetes (T1D) is an autoimmune illness characterized by advanced β-cell

destruction in the pancreas and decreased insulin generation by these cells. Genetic
susceptibility and environmental elements are factors that facilitate the disease pro-
cess [40]. β-cell damage is interceded by immunological mechanisms. The procedure
generally advances gradually and can start before clinical type 1 diabetes is analyzed.
The start of the subclinical stage is characterized by the presence of autoantibodies
against insulin and different autoantigens of pancreatic cells in peripheral blood.
These antibodies likewise predict the advancement of clinical disease [41]. This dis-
ease affects around 15 million people worldwide. It is characterized by a life expec-
tancy reduced by an average of 10 years and the induction of associated diseases
known as “diabetic complications” because the majority of patients develop the dis-
ease in early childhood or later in childhood and it causes prolonged impacts [42].
Occasionally, the presentation of type 1 diabetes involves a role of infectious
agents – specifically viruses – in the later phases of the disease pathway. Adams
affirmed in 1926 that the relationship between acute respiratory complaints and
diabetes was “self-evident” [43]. A few years later, Gamble and Taylor reported the
occasional presentation of diabetes in children from 1955 to 1968 [43]. The report
of an increased incidence of type 1 diabetes after an epidemic of Coxsackievirus B5
firmly established enteroviruses as the likely infectious agents. Enteroviruses have
been found most often, with connections between the rate of type 1 diabetes and the
rate of infection with Coxsackievirus B4 [44]. In human in vitro systems, the model
CVB3 (Nancy strain) has the capacity to replicate in β- and non-β-cells and to deci-
mate human islets 72 h after disease. On the other hand, the E2 strain of CVB4 has
diabetogenic characteristics. The model strains of CVB3, CVB4 and CVB5 could
infect human β-cells and cause cell death [44].
There are various major mechanistic hypotheses for the etiological role of viruses
in type 1 diabetes:
1. Direct infection of β-cells, stimulation of autoantigen induction in the context of an
enhanced immune response and inflammation, upregulation of major histocompat-
ibility complex (MHC) class I particles in β-cells, and initiation of autoimmunity
2. Viral infection, where the virus can infect tissue and cause tissue lesions, and then the
sequestered antigens are induced, resulting in activation of autoreactive T-cells [45]
3. Molecular mimicry, where viral proteins mimic the amino acid sequence of auto-
antigens – for instance, the homology between the Coxsackievirus protein P2-C
and the islet antigen glutamic acid decarboxylase 65 [46], leading to the activa-
tion of T-cells to cross-react with and damage host tissue
4. Viral persistence
Coxsackievirus B3 (CVB3) is a human enterovirus in the Picornaviridae family.

CVB3 has a single-stranded positive-sense RNA genome enclosed in a non-enveloped
icosahedral capsid composed of four capsid proteins (VP1–VP4) [47]. The range of
clinical diseases caused by CVB3 varies from mild to significant cases including
acute and chronic inflammations. CVB3 might spread from the intestinal tract to the
internal organs and induce severe cardiac damage and aseptic meningitis [48]. The
antiviral drug pleconaril is a potential medication for treatment of enteroviral dis-
eases in newborn and young children, but experience in its use remains limited [49].
Ribavirin, a nucleoside antimetabolite drug that interferes with the duplication of
viral material, is effective against various DNA and RNA viral infections, particularly
in influenza, flaviviruses, and numerous pathogens that cause different viral hemor-
rhagic fevers [13]. In addition, one study reported that ribavirin suppresses the cyto-
pathic effect (CPE) of CVB3 [50]. CVB3 infection in murine myocarditis has been
treated with ribavirin which suppresses viral replication, bringing about a reduction in
myocardial damage. However, this antiviral agent is not a particular therapeutic choice
in clinical practice to treat CVB3 infections, and there is no vaccine for CVB3. Thus,
novel methodologies for control of CVB3 deserve to be investigated [13].
Kim et al. (2014) investigated the antiviral activity of probiotics isolated from
young Koreans against Coxsackievirus B3 (CVB3). The plaque reduction assay
(PRA) was used to measure the impact of probiotics against CVB3, whereas the
cellular toxicity of such strain was evaluated using the 3-(4,5-dimethylthiazole-
2-yl)- 2,5-diphenyltetrazolium bromide (MTT) test on HeLa cells. Among 13 pro-
biotic strains, three B. adolescentis, two B. longum, and one B. pseudocatenulatum
had an antiviral impact against CVB3, while the others showed no such activity. B.
adolescentis SPM1605 showed the best inhibitory properties against CVB3. When
the limit cycle (CT) values for the treated B. adolescentis SPM1605 tests were con-
trasted with the outcomes for the non-treated specimens, it was shown that the
amplified viral sequences of CVB3 had their duplicate number reduced by B. ado-
lescentis SPM1605. In addition, the gene expression in infected HeLa cells was
likewise halved. Infectious diseases caused by Coxsackieviruses could be treated
with B. adolescentis SPM1605 by suppressing CVB3 [13].
In a recent study, Al Kassaa et al. (2015) showed that anti-HSV-2 L. gasseri
CMUL57 had no effect on CVB4. The authors suggest that the antiviral activity is
strain dependent [18].
3.6 Probiotics as a Treatment and Prevention Strategy

for Liver Complications Caused by Hepatitis B and C
Virus
Long-term hepatocellular lesions, cirrhosis, and hepatocellular carcinoma (HCC)

are the most common virus-associated chronic liver diseases. There are caused by
certain viruses like the hepatitis B and C virus (HBV and HCV). The Iranian popu-
lation shows a high prevalence of these viruses [51]. Patients infected with HBV
and HCV have an elevated level of plasma endotoxins [51]. Furthermore, these
3.6 Probiotics as a Treatment and Prevention Strategy for Liver Complications Caused 73
patients present high levels of the proinflammatory cytokines that cause liver dam-
age in the longer term [52]. On the other hand, cirrhosis, a vascular disease, is char-
acterized by features such as portal hypertension and hyperdynamic syndrome [3].
Similar to most liver diseases, a lack of equilibrium in normal gut flora and impair-
ment of the intestinal barrier cause endotoxemia, a high level of proinflammatory
cytokines, and induction of NO synthesis [53]. HCC is the third most common
cause of cancer mortality worldwide and is the most common liver cancer occurring
after cirrhosis with a high incidence [54]. The presence of viral antigens in chronic
hepatitis B or C virus infections, carcinogenic mycotoxins, and compounds that
produce reactive oxygen species are the major risk factors.
For instance, the conversion of G nucleotides to T in the P53 gene after attach-
ment of an aflatoxin, a strong mycotoxin, causes a reduction in P53 transcription. In
addition, the inhibitory effects of aflatoxins on c-myc and bcl2 result in cell prolif-
eration and tumor progression [3, 15]. These risk factors lead to and accelerate the
formation and progression of cirrhosis, which occurs in 80–90 % of HCC patients.
The 5-year cumulative risk for the development of HCC in patients with cirrhosis
ranges between 5 and 30 % [55].
In order to investigate the efficacy of treatment with probiotic supplements for
patients with various types of chronic liver disease due to viral infections, Loguercio
et al. (2005) treated 20 patients with HCV-related chronic hepatitis and 16 with
HCV-related cirrhosis with VSL#3 (Streptococcus thermophilus, B. breve, B.
longum, B. infantis, L. acidophilus, L. plantarum, L. casei, and L. bulgaricus) for 4
months. The results of routine liver tests, including aspartate aminotransferase
(AST) and alanine aminotransferase (ALT) levels, improved in the two groups, but
gamma glutamyl transferase (GGT) improvement was observed only in the HCV-
related chronic hepatitis group. No effects were observed on the plasma levels of
tumor necrosis factor alpha (TNF alpha), interleukin IL-6, and IL-10 in HCV
patients. However, the routine liver damage test results were improved at the end of
the treatment. This type of liver damage requires more study to properly assess the
benefits of probiotic therapy [14].
Few studies have been conducted on the impact of probiotics on the toxicity of
aflatoxin in liver diseases and hepatocellular carcinoma. In order to investigate the
impact of probiotic administration on the intestinal absorption of aflatoxin B(1), the
authors measured a biomarker, aflatoxin B(1)-N(7)-guanine, excreted in urine. El
Nezami et al. (2006) studied 90 healthy young men from China, who were randomly
assigned to two groups; one group received a mixture of L. rhamnosus LC705 and
Propionibacterium freudenreichii subsp. shermanii strains for 5 weeks, and the
other group received a placebo preparation. The probiotic group had a higher per-
centage of samples with negative AFB(1)-N(7)-guanine values than the placebo
group, and a statistically significant decrease in the concentration of urinary AFB(1)-
N(7)-guanine was observed in the urine samples of the probiotic group compared to
the placebo. The reduction was 36 % at week 3 and 55 % at week 5 [15]. A probiotic
supplement reduces the biologically effective dose of aflatoxin exposure and may
thus constitute an effective dietary approach to decreasing the risk of liver disease.
Kumar et al. (2011) investigated the effect of probiotics (L. rhamnosus GG
(LGG) and LcS) on chemoprevention of aflatoxin B1 (AFB1)-induced hepatocellu-
lar carcinoma. They measured the genotoxicity (DNA damage) in hepatic cells.
There was a decrease of approximately one-third in the tumor incidence compared
to the AFB1 control group. In addition, there was a decrease in the expression of the
c-myc, bcl-2, cyclin D1, and rasp-21 levels in the treatment group compared to the
AFB1 control group [16]. The results suggest the enhanced protective potential of
probiotic fermented milk against AFB1-induced molecular alterations in hepatic
cells during carcinogenesis.
Several studies have shown that certain probiotic strains can eliminate aflatoxins
by several mechanisms including aflatoxin adsorption [56].
Lee et al. (2013) explored the antiviral activity of B. adolescentis SPM0212 iso-
lated from healthy Koreans against HBV and its mechanism of action. The cell
extract of B. adolescentis SPM0212 dose dependently decreased the extracellular
HBsAg level by up to 50 %. The HBV gene expression in HepG2.2.15 cells was
also inhibited by 40 %. This extract significantly increased the expression level of
myxovirus resistance A, which is an IFN-inducible antiviral effector. Thus, the cell
extract of B. adolescentis SPM0212 inhibits HBV, and its antiviral mechanism is
associated with the Mx GTPase pathway [17].

Viruses 1 and 2 Using Probiotic Strains
Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are important human pathogens
belonging to the α-Herpesviridae subfamily. HSV-1 and HSV-2 contain a large
double-stranded DNA molecule [57]. Skin and mucosal membranes are the most
common sites of HSV primary infections. Orofacial infections and encephalitis are
regularly associated with HSV-1 infection, and it is characterized by lesions of the
buccal and gingival mucosa and high fever. However, genital tract infections are
usually caused by HSV-2 infection and are characterized by macules and papules,
followed by vesicles and pustules, and are the major cause of genital ulcers world-
wide. HSV-1 encephalitis is the most common cause of fatal sporadic encephalitis.
More than 70 % of untreated patients die and only 2–5 % of surviving patients
return to normal neurological function [57].
HSV-2 is one of the most common sexually transmitted infections [58]. Several
studies suggest that more than 45 million people in the United States are infected
with HSV-2, and the estimated incidence of new infections is 1 million annually.
More than 90 % of the general population has antibodies against HSV-1. Around
30 % of patients who have been exposed to HSV-1 develop recurrent infections, and
this level is continuously increasing [59].
Significant neurological morbidity is caused by HSV infection. Latent infections
in the neurons of the peripheral nervous system are characterized by both viruses
HSV-1 and HSV-2. After reactivation of the HSV in a nerve cell, lesions of the skin
and mucous membranes are caused by the migration of the virus down the axon to
infect peripheral tissue. This latency increases the pathogenicity of HSV and enables
these viruses to be used as therapeutic tools [59, 60].
3.7 Treatment and Prevention Strategy of Herpes Simplex Viruses 1 and 2 Using 75
The nucleoside analogs acyclovir (ACV) and penciclovir (PCV) are the most com-
monly used agents for the management of the HSV virus, but mutations in viral thymi-
dine kinase have caused increased resistance of HSV to ACV [61, 62]. Resistant HSV
infections are managed with the pyrophosphate analog foscarnet (FOS) that is a more
toxic and less bioavailable agent. However, FOS resistance is increasing rapidly [63].
There is a need for new antiviral compounds with different mechanisms of action.
Al Kassaa et al. (2015) demonstrated that L. gasseri CMUL57 (L. gasseri
CMUL57), L. acidophilus CMUL67, and L. plantarum CMUL140 isolated from
Lebanese female vaginal flora showed antiviral activity against HSV-2 virus. These
bacteria were noncytotoxic to Vero cells and Hela cells. The anti-HSV-2 activity
was examined by co-incubating the lactobacilli with the virus prior to inoculating
the mixture in Vero cell monolayers. The antiviral activity in this study is not due to
the lactic acid, bacteriocins, and hydrogen peroxide which are considered as antivi-
ral substances. This study showed that the anti-HSV-2 activity is due to physical
contact between the lactobacilli cell walls and the HSV-2 viral envelope [18].
An et al. (2012) tested the anti-HSV-1 activity of Bifidobacterium spp. isolated
from fecal samples of healthy Koreans. The (PRA) method and yield reduction
assay in Vero cells was used to evaluate the antiviral activity of these bacteria against
the HSV-1 virus. B. adolescentis SPM 0214 was not toxic against Vero cells. The
inhibition of the plaque and yield formation after treatment with a high concentra-
tion of B. adolescentis SPM 0214 demonstrated the antiviral activity of B. adoles-
centis SPM 0214 against the HSV-1 virus [19].
Zabihollahi et al. (2012) evaluated the antiviral activity in vitro of both vaginal
and nonvaginal lactobacilli against the HSV-2 virus using the PRA method and the
BALB/c mouse model to study the anti-HSV-1 activity by monitoring skin lesions
and the development of the immune response. The results of this study show that
HSV-2 replication was inhibited by 50 % after treatment with the supernatant of L.
crispatus. In addition, the inhibition of HSV-2 infection before the entry of the virus
into the cells was observed after treatment with culture supernatants of L. gasseri
and L. crispatus. The inhibitory activity was not related to the presence of lactobacilli
cells, because the inhibition of viral replication was seen in the presence of neutral
pH supernatants. High potential for the inhibition of HSV-1 infection in vivo was
shown by the presence of living L. gasseri [20].
Conti et al. (2009) showed the protective activity of vaginal Lactobacillus
strains (L. brevis CD2, L. salivarius FV2, L. plantarum FV9) against the HSV-2
virus. The mode of action of the vaginal Lactobacillus strains affects different viral
multiplication phases. The results of this study demonstrate that the adhesion
capacity of Lactobacillus strains plays an important role in the inhibition of the
early phases of viral infection. L. brevis CD2 increases the inhibition of HSV-2,
binding strongly adhesive bacteria with it. However, L. salivarius FV2 shows low
inhibition activity due to weak adhesive activity. In addition, Conti et al. (2009)
demonstrated that the presence of Lactobacillus cells was not related to the inhibi-
tion of HSV-2 viral replication, because the decrease in HSV-2 replication was
observed when HSV-2 was cultured in cells with neutral pH culture supernatants of
lactobacilli. The high antiviral activity strain was observed with L. brevis CD2,
which does not produce hydrogen peroxide, and lactic acid was neutralized. They
reported that the anti-HSV-2 activity was attributed to molecules other than H2O2
and lactic acid [21].
3.8 Probiotics and Human Immune Deficiency Virus (HIV)
The human immune deficiency virus (HIV) is a member of the family Retroviridae
that can cause acquired immune deficiency syndrome (AIDS) in humans. AIDS is
the most advanced stage of HIV infection characterized by increased risk of oppor-
tunistic infections and HIV-related cancers [64]. HIV contains a linear single-
stranded RNA as its genetic material. Two distinct major strains of HIV have been
characterized: HIV-1 and HIV-2. HIV-1 is more common worldwide, while HIV-2
is found in West Africa. At the end of 2010, approximately 34 million people were
living with HIV globally, 35 % of whom are pregnant women, and around 1.8 mil-
lion people died from AIDS-related causes [65].
An interaction between HIV infection and different chronic diseases has been
shown in several studies. For example, people with HIV infection have an increased
risk of other chronic infectious diseases such as tuberculosis and other chronic dis-
eases such as cancer, diabetes, and cardiovascular diseases [66]. Advanced immu-
nodeficiency is associated with an increase in diarrhea [5]. There is no vaccine for
HIV. For this reason, prevention of HIV diseases is only possible by avoiding expo-
sure to the virus. Highly active antiretroviral therapy is the major treatment for HIV
infection. This therapy seems highly beneficial to many HIV-infected patients, but
has been found to be very expensive and causes many negative health effects, such
as diarrhea, nausea, flatulence, and discomfort associated with the physical and
mental status of the person [67]. In addition, the antiretroviral treatments of HIV
infection increase the risk of hyperlipidemia and diabetes. The interest of using
probiotics in the treatment of human immunodeficiency virus (HIV)-associated dis-
eases and infection has recently increased. The CD4 receptor is the primary receptor
for the entry of T-tropic HIV into its target cells in vivo. It has been shown that
T-tropic isolates often appear in association with a decline in CD4+ T-lymphocytes
during disease progression. The most important marker of the disease progression
and the treatment efficacy is the CD4 count [24].
The use of probiotics has not been shown to have negative health effects and is
considered to be safe for HIV patients [68, 69]. Many clinical studies have demon-
strated that probiotics have a beneficial effect on HIV-induced diarrhea. Salminen
et al. (2004) examined the efficacy and safety of ameliorating gastrointestinal symp-
toms in HIV-infected patients on antiretroviral therapy using the LGG strain.
However, no significant differences were detected in gastrointestinal symptoms and
diarrhea between the LGG group and a small placebo-controlled group [67].
Trois et al. (2008) studied the effect of supplementing B. bifidum with S. ther-
mophilus to assess the benefits in terms of reduction of diarrhea and the immune
response determined by CD4+ cells in 77 HIV-infected children in a randomized,
double-controlled trial. A decrease in diarrhea with an increase in the mean CD4+
T cell count was seen in the probiotic group compared with the control group [22].
3.9 Conclusion and Perspectives 77
Anukam et al. (2008) showed the benefits of probiotic yoghurt containing probi-
otic L. rhamnosus GR-1 and L. reuteri RC-14 on the quality of life of 24 women in
Nigeria with HIV/AIDS having clinical signs of moderate diarrhea. They reported an
increase in the mean CD4+ cell count in 11/12 (92 %) probiotic-treated women com-
pared to 3/12 (25 %) of the women receiving the control yoghurt. Furthermore, all
probiotic-treated women showed a decrease in diarrhea, flatulence, and nausea [23].
The HIV-1 CD4+ receptor was detected on the lactobacilli cell surface. Furthermore,
viral binding to lactobacilli appeared to employ the CD4+ receptor, and Lactobacillus
casei 393 inhibited the infection of cells with HIV-1 pseudovirus in vitro [24].
Su et al. (2013) examined the role of extracellular proteins of L. casei 393 in
blocking HIV-1 transmission and increasing the CD4+ counts. The monoclonal
antibody of the CD4+ receptor was able to partially inhibit HIV-1 binding to L.
casei 393. In addition, L. casei 393 decreased HIV-1 pseudovirus infection of
TZM-bl cells in vitro by 60–70 %. They suggest that this probiotic strain can use
this receptor to bind HIV and block HIV infection. This may in turn increase the
CD4+ T-lymphocyte count in patients with HIV. This data provides direct evidence
that L. casei 393 expresses the CD4+ receptor and utilizes it to block HIV transmis-
sion. HIV is transmitted through the mucosal surfaces and causes severe damage to
the gut, which has led some scientists to believe that the use of probiotics may help
counter its devastating effects and infection [24].
3.9 Conclusion and Perspectives
Several studies conducted on different infectious diseases have confirmed the positive
impact of probiotic strains on chronic viral diseases. Compared to antiviral therapy and
surgery, the administration of probiotics is safer, less expensive, and considered a non-
invasive strategy. Despite, these aforementioned probiotics colonized the gut or vaginal
ecosystem; their antiviral effect can be appearing against viruses which cause systemic
chronic diseases. Probiotics have potential applications in HPV-related cervical cancer
and HSV-1, HSV-2, and Coxsackievirus B3 infection prevention and treatment. For
HPV and HSV-2 infection, the probiotic can interact directly with the virus and/or
genital tract epithelium including innate immunity. However, probiotics which have
showed an effect on HIV and HCV, for example, have colonized also the gut ecosys-
tem. On the other hand, probiotics could help to improve the quality of life of HIV/
AIDS patients, in particular by resolving diarrhea, flatulence, nausea, and increase in
the mean CD4 cell count. In addition, they can prevent HCV-related chronic hepatitis,
HCV-related cirrhosis, and HCV-related liver cancer. Indeed, this interaction is not
fully clarified. Numerous mechanisms may be involved in the inhibitory and preventive
effect of probiotics against chronic viral diseases: secretion of antiviral compounds
with the ability to block intracellular viral replication, by immunomodulatory effect,
virus trapping, and other unknown mechanisms. Antiviral probiotics are a new concept
for the natural treatment of chronic viral infections and should be used as prevention
agent to healthy patient and co-treatment for infected patient.
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Chapter 4
The Antiviral Activity of Probiotic Metabolites
Imad AL KASSAA
Contents
4.1 Antiviral Activity of Probiotic Metabolites ..................................................................... 84
4.1.1 Non-organic Substances ...................................................................................... 84
4.1.2 Organic Substances.............................................................................................. 85
4.2 Probiotics and Their Proteinaceous Metabolites.............................................................. 91
of Probiotic/LAB Native Supernatants ............................................................................ 92
4.4 Conclusion ....................................................................................................................... 93
References ................................................................................................................................. 94
Abstract One of the most important characteristics of lactic acid bacteria (LAB) is
the production of a large variety of active substances, such as acids, active ribo-
somal proteins, non-ribosomal peptide synthetase (NRPS), hydrogen peroxide, and
other metabolites. In recent decades, several studies have evaluated the importance
of these active substances in both the medical and food sectors. LAB have been used
for several years in food fermentation to give good taste and protect the food against
spoilage and pathogenic microorganisms. In this chapter, we focus on the antiviral
activity of LAB metabolites.
Keywords Antiviral metabolites • antiviral probiotics • Antiviral peptide •

antiviral bacteriocins • metabolites evaluation
Abbreviations
CA16 Coxsackievirus A 16
CFS Cell-free supernatant
CRFK Crandell–Reese feline kidney
EMCV Murine encephalomyocarditis virus
FCV Feline calicivirus
FDA Food and Drug Administration
GRAS Generally recognized as safe
H2O2 Hydrogen peroxide
DOI 10.1007/978-3-319-49688-7_4
84 4 The Antiviral Activity of Probiotic Metabolites
kDa Kilodalton
LabyA1 Labyrinthopeptin A1
lcFOS Long-chain fructooligosaccharides
NRPs Non-ribosomal peptides
PEDV Porcine epidemic diarrhea virus
scGOS Short-chain galactooligosaccharides
SFV Semliki Forest virus
SHV-1 Suid herpesvirus
SIV Simian immunodeficiency virus
VSV Vesicular stomatitis virus
4.1 Antiviral Activity of Probiotic Metabolites
4.1.1 Non-organic Substances
Hydrogen peroxide (H2O2) is an antimicrobial substance produced by several bacte-

rial species, such as Streptococcus pyogenes (S. pyogenes) and S. pneumoniae,
which are opportunistic or pathogenic bacteria [1, 2]. Some probiotic strains also
produce H2O2 as a defense mechanism to support a microenvironment containing
other non-catalase bacteria. Moreover, H2O2 allows some bifidobacterial species to
survive in microaerophilic conditions [3]. In humans and animals, H2O2 plays an
important role, especially in the vaginal ecosystem. Some lactobacilli produce H2O2
as a natural microbicide within the vaginal ecosystem, and it is toxic to a number of
microorganisms, including HIV-1 and HSV-2 [4, 5].
The Food and Drug Administration (FDA) has classified organic acids as “gener-
ally recognized as safe” (GRAS) for humans. The majority of microorganisms are
sensitive to organic acids that kill microorganisms via a mechanism in which undis-
sociated molecules flow through their cell membranes and are ionized inside.
Therefore, cells will react immediately by releasing hydrogen ions, which leads to
a pH decrease and thus to cell damage [6]. Production of lactic acid is an important
defense mechanism that prevents the growth of acid-sensitive microorganisms, such
as those associated with infectious diseases [7]. Physiological concentrations of
lactic acid, but not acetic acid, significantly reduce the viability of microorganisms,
as reported by Conti et al. [5]. This data hypothesizes that the antimicrobial activity
is not directly related to the pH value but rather to the nature of organic acid.
Lactic acid, a final product of carbohydrate metabolism, is produced by all LAB
species. In the human ecosystem, lactic acid is responsible for the physiologically
acidic vaginal pH value (≤ 4.5). The acidic pH inactivates HIV [8] and HSV-2 [9].
Moreover, HSV-2 is irreversibly inactivated by lactic acid concentrations with pH
values corresponding to those observed in the healthy human vagina [5]. It appears
that lactobacilli could produce compounds that could help the host cells prevent
viral replication [10]. In relation to this, a nonprotein cell wall component extracted
4.1 Antiviral Activity of Probiotic Metabolites 85
from a vaginal strain of L. brevis strongly reduced HSV-2 replication in a cell culture
[10], while acid Lactobacillus metabolic products decreased the activation of
T-lymphocytes, which may result in a decrease in lymphocyte susceptibility to
HIV-1 infection [11]. Straube et al. (2011) reported that lactic acid had an antiviral
effect against naked viruses, such as FCV and the ECHO virus, and noted that the
inactivation of enteroviruses depends on the virus type, since the ECHO virus
showed more stability than FCV in the presence of D/L lactic acid [12].
4.1.2 Organic Substances
The proteinaceous substances secreted by LAB and probiotic strains are the mole-
cules most characterized for their antimicrobial activity and thus for their antiviral
effect. However, the direct effects of different non-proteinaceous compounds, such
as polyphenols [13] or theaflavins [14], against rotaviruses have been reported previ-
ously. In contrast, proteinaceous substances of non-microbial origin have been little
described in terms of their antiviral activity. In the literature, the most frequently
reported protein is lactoferrin, which confers at least part of the antiviral properties
of breast milk [15] and prevents the adsorption of rotaviruses into the target cells due
to its capacity to bind virus particles [16]. K-Casein showed an antiviral activity
against HuRoVs. K-Casein can bind rotavirus particles via glycan residues [17].
(i) Bacteriocins
Bacteriocins are antimicrobial peptides synthesized by the ribosome route.
According to Tagg et al. (1976), bacteriocins are active against bacteria related to
the producing strain [18]. However, recent studies have shown that some bacterio-
cins produced by lactic acid bacteria belonging to the genera Lactobacillus (bacte-
riocin OR7) (bacteriocin XDSM) and Enterococcus (enterocins E50–52 and
enterocin E760) have a much wider activity spectrum, including Gram-positive and
Gram-negative bacteria such as Campylobacter jejuni, Yersinia spp., Salmonella
spp., Escherichia coli O157: H7, Shigella dysenteriae, Morganella morganii,
Staphylococcus aureus, and Listeria spp. [19–23].
Klaenhammer (1993) [24] proposed the classification of bacteriocins into four
classes based on their primary amino acid sequences, molecular weight (kDa), struc-
ture, and stability with both heat and pH variations. This classification has undergone
several changes due to the abundance of scientific results. A subsequent classification
into three classes was proposed by Cotter et al. [25]. According to Cotter et al. (Table
4.1), class I is “lantibiotics,” which are small hydrophobic peptides (<5KDa) that
contain unusual amino acids: lanthionine and beta-methyl-lanthionine. This class was
further divided into six subclasses according to Rea et al. [26] (Table 4.2). Class II are
non-lantibiotic peptides; this class was divided into four subclasses IIa, IIb, IIc, and
IId. The general characteristics of class II are determined by thermostability and
molecular weight <10 KDa. Table 4.2 shows the different characteristics of the four
subclasses of class II. Class III contain thermolabile bacteriocins, which are also con-
sidered to be proteins and not peptides, due to their high molecular weight (>30KDa).
Table 4.1 Bacteriocin classification by Cotter et al. [25]

Classes Characteristics
I Lantibiotics: small hydrophobic peptides (<5KDa) containing unusual amino acids
lanthionine and beta-methyl-lanthionine
II Small non-lantibiotic peptides (<10 kDa), thermostable, divided in four subgroups
Class IIa pediocin-like
Class IIb two-peptide bacteriocins
Class IIc circular bacteriocins
Class IId non-pediocin-like one-peptide linear bacteriocin
III Thermolabile proteins with molecular weight greater than 30 kDa
Table 4.2 Lantibiotic subclasses Rea et al. [26]

Bacteriocins Subclasses Examples
Lantibiotics I Nisin A, nisin F, nisin Q, nisin Z, epidermin, streptin,
planosporicin, Pep5
II Lacticin 481, mersacidin, cinnamycin, lactocin S,
cytolysin A1 et A2, lacticin 3147 A1 et A2
III SapB, AmfS et SapT
IV –
Labyrinthopeptins – Labyrinthopeptin A1, labyrinthopeptin A2
Sactibiotics – Subtilosin A, thuricin CD
Bacteriocins produced by lactic acid bacteria have several advantages. Indeed,

they allow bacteriocinogenic lactic acid bacteria to have a competitive effect on access
to nutrients in natural ecosystems containing a diversity of microorganisms [27]. They
are of interest to humans, including in food preservation, but can also be used as
alternatives to antibiotics or in combination with natural antibiotics [25, 28, 29].
While the antibacterial activity of bacteriocins has been partially explained, their
antiviral activity remains to be understood. The repertoire of bacteriocins endowed
with antiviral activities includes several reported or/and speculated as well as
unknown models (Table 4.3). Bacteriocins, including staphylococcin 188, enterocin
AAR-71, enterocin AAR-74, and erwiniocin NA4, have been evaluated against the
coliphage HSA virus isolated from a raw wastewater sample (collected from a local
sewage treatment plant).
Staphylococcin 188 and enterocin AAR-74 were shown to reduce viral progeny by
a factor of 10, while enterocin AAR-71 and erwiniocin NA4 eliminated viral progeny
completely [33]. In addition, staphylococcin 188 was active against the influenza
virus and Newcastle disease virus when studied using in vitro and in vivo models [48].
Enterocin ST5Ha (ST5Ha) exhibited antiviral activity against HSV-1 [34]. Specifically,
enterocin ST5Ha at 8645 mg/ml reduced the viability of a confluent cell (CC50) by
50 %. This concentration was higher than the antiviral concentrations of the bacterio-
cins ST4V and CRL35, which were 1600 mg/ml and 2500 mg/ml, respectively [35,
49]. The antiviral activity of enterocin CRL35 and ST4V has been observed against
thymidine kinase-positive and deficient strains of herpes simplex types 1 and 2 in Vero
and BHK-21 cells, affecting intracellular viral multiplication and inhibiting the late
Table 4.3 Antiviral metabolites of probiotic strains
LAB/probiotic metabolites Producing strain/metabolites Target virus Experiments Reference
H2O2 and lactic acid L. brevis CD2, L. salivarius FV2, L. HSV-2 Vero cell line [5]
plantarum FV9
H2O2 L. acidophilus (human vagina) HIV-1 CEM cell line (T [4 ]
lymphoblast)
Lactic acid LAB from breast milk HIV-1 TZM-bl cells [8]
Lactic acid Lactic acid solution (ACIDOFORM) HSV-2 CaSki cells [9]
Infected mice
D/L lactic acid Lactic acid solution MuNoV, H1N1 MDCK, CRFK cell lines [30]
Heat-resistant nonprotein cell L. brevis CD2 HSV-2 Vero cells [31]
surface bacterial component
Lactic acid D/L lactic acid solution 0.1–0.4 % FCV and ECHO virus CRFK cell line [12]
w/w, pH 6.0–3.2
Surfactin Bacillus subtilis/NRPS SFV, HSV-1 and 2, BHK21, Vero, ML, [32]
SHV-1, VSV, SIV, BHK21, MT-4,
HTLV-1, FCV, and transformed T-cell, CRFK,
EMCV and Hep-2 cell lines,
respectively, to viruses
order
CVB4 Vero cell lines Al Kassaa,
unpublished data
Staphylococcin 188 Staphylococcus aureus AB188/ BLIS Newcastle virus – [33]
Influenza virus
Coliphage HSA
Enterocin AAR-71 Enterococcus faecalis/BLIS Coliphage HSA E. coli ATCC 0241 [33]
Class IIa Culture
(continued)
87
88
LAB/probiotic metabolites Producing strain/metabolites Target virus Experiments Reference

Enterocin AAR-74 Enterococcus faecalis/BLIS Coliphage HSA E. coli ATCC 0241 [33]
Class IIa Culture
Erwiniocin NA4 Erwinia carotovora NA4/BLIS Coliphage HSA E. coli ATCC 0241 [33]
Culture
Enterocin ST5Ha Enterococcus faecium ST5Ha/ HSV-1 – [34]
bacteriocin
Enterocin ST4V Enterococcus mundtii ST4V/ HSV-1 and HSV-2 – [35]
bacteriocin
Enterocin CRL35 Enterococcus mundtii CRL35/ HSV-1 and HSV-2 – [36]
Class IIa bacteriocin
Enterocin NKR-5-3C Enterococcus faecium HSV-1 – Al Kassaa et al.
NKR-5-3/bacteriocin (Unpublished data)
4
Subtilosin Bacillus amyloliquefaciens HSV-1 – [37]

KATMIRA 1933 KATMIRA 1933/NRPS
Bacteriocin B1 Lactobacillus delbrueckii/bacteriocin Influenza virus – [38]
Labyrinthopeptins A1 Actinomadura namibiensis DSM HIV, HSV-1, and HSV-2 MT-4 cells and PBMCs; Férir et al. [39]
6313/lantibiotic HEL cell lines
respectively to viruses
order
11mer digestion peptide Bifidobacterium longum subsp. RoV HT-29 and MA-104 cell [40]
infantis CECT 7210/protease lines
Proteinaceous substances L. casei, B. adolescentis RoV MA104 cell line [41]
Surface VHH expression LGG RoV Mouse pup [42]
Boiled yoghurt supernatant LAB supernatant IFV Infected mice [43]
The Antiviral Activity of Probiotic Metabolites
MRS CFSs B. breve DSM 20091, B. longum Q VSV IPEC-J2 and alveolar [44]
46, L. paracasei A14, L. paracasei macrophages 3D4/2
F19, L. rhamnosus Q 85, L.
plantarum M1.1, and L. reuteri DSM
12246
MRS and yoghurt supernatants L. acidophilus, L. rhamnosus, L. Influenza virus A/ Vero and MDCK cell lines [45]
(molecules with MW< plantarum, S. thermophilus, and B. PR/8/34, influenza virus
3000 Da) bifidum A/WS/33, influenza virus
B/Lee/40, Coxsackievirus
A 16 (CA16), CB3, CB4,
and porcine epidemic
diarrhea virus (PEDV) CV
777
CFS/lactic acid Lactococcus lactis subsp. lactis FCV CRFK cell line [46]
LM0230
CFS L. curvatus 1 MuNoV RAW 264.7 [30]

Unknown molecules
MW= 2 kDa
CFSs B. subtilis 168 and E. faecalis 19,433 MuNoV 1 RAW 264.7 and LLCMK2 [47]
Tulane virus cell lines
89
stages of replication [35, 36, 49]. Remarkably, the amino acid sequence of CRL35 is
expected to play a role in anti-HSV-1 and anti-HSV-2 activities. Derivatives of CRL35
without at least two cysteine residues were assayed and shown to have no antibacterial
activity; and the authors hypothesized that these derivatives will also have no anti-
herpes activity [50].
Bacteriocin ST5Ha at 50 mg/ml reduced the viral production of HSV-1 in a cell
culture by 50 % (EC50), with a selectivity index (CC50/CE50) of 173 [34]. The
pediocin-like enterocin NKR-5-3 C was shown to display strong anti-Listeria [34]
activity. Anti-HSV-1 of NKR-5-3 C was assessed and its CC50 was < 1200 μg/ml,
while the CE50 value was 30 mg/ml (Al Kassaa et al. unpublished data).
Labyrinthopeptin A1 (LabyA1) is a prototype peptide of a novel class of carbacy-
clic lantibiotics [39]. LabyA1 exhibited a consistent and broad anti-HIV activity
(EC50: 0.70–3.3 mM) and anti-HSV activity (EC50: 0.29–2.8 mM) in cell cultures
[39]. LabyA1 inhibited viral cell-to-cell transmission between persistently HIV-
infected T-cells and uninfected CD4+ T-cells (EC50: 2.5 mM) and inhibited the
transmission of HIV captured by DC-SIGN+−cells to uninfected CD4+ T-cells
(EC50: 4.1 mM) [39]. A synergistic effect in anti-HIV-1 and anti-HSV-2 activity
was demonstrated using LabyA1 in dual combination with tenofovir, acyclovir,
saquinavir, raltegravir, and enfuvirtide [39].
In contrast to bacteria, the mode of action of bacteriocins against viruses remains
to be determined. According to Waschman et al. [36], bacteriocins could lead to
aggregation of viral particles by blocking the receptor sites on host cells, or they
may inhibit key reactions in the multiplication cycle. Recently, a noncytotoxic class
IV bacteriocin produced by L. delbrueckii subsp. bulgaricus 1043 was isolated and
shown to be virucidal on the influenza virus [38].
The antiviral activity is related to the nature of bacteriocins. This hypothesis was
supported by [30], which have reported that sakacin A nisin did not reduce the viral
infectivity of MuNoV, H1N1, feline herpesvirus (FHV), and Newcastle disease
virus (NDV).
(ii) Non-ribosomal peptides (NRPs)
NRPs are biologically active and natural compounds. NRPs have a broad spectrum
of clinical applications. Some NRPs are used as antibiotics (daptomycin), antitumor
drugs (bleomycin), antifungal drugs, or immunosuppressants (cyclosporin) [51]. This
diverse bioactivity can be explained by how nature synthesizes these molecules.
NRPs are produced by the secondary metabolism of bacteria and fungi by the con-
secutive condensation of amino acids, which is achieved by large multimodular
enzymes, non-ribosomal peptide synthetases (NRPSs) [51, 52]. Several studies have
reported that some NRPSs can be used as antiviral agents against enveloped viruses.
Vollenbroich et al. (1997) [32] showed that the biosurfactant “surfactin” (an antifun-
gal NRPS produced by Bacillus subtilis) can inhibit Semliki Forest virus (SFV),
herpes simplex virus (HSV-1, HSV-2), Suid herpesvirus (SHV-1), vesicular stomati-
tis virus (VSV), simian immunodeficiency virus (SIV), feline calicivirus (FCV), and
murine encephalomyocarditis virus (EMCV). Furthermore, surfactin can also inhibit
Coxsackievirus B4 at low concentrations (Al Kassaa; unpublished data).
4.2 Probiotics and Their Proteinaceous Metabolites 91
4.2 Probiotics and Their Proteinaceous Metabolites
Recent clinical trials have demonstrated that some probiotic strains are able to
improve acute rotaviral diarrhea [53, 54]. It is assumed that the gut microbiota bal-
ance, the enhancement of the mucosal barrier, and the modulation of the immune
response afford protection against rotaviral diarrhea [55]. However, few studies
have investigated the mechanisms underlying the protective effect of probiotics
against viruses (see Chap. 1).
Lee et al. [54] found that B. longum (IBG) and L. acidophilus (LA) strongly
inhibited rotavirus infection in the Vero cell line, but they did not identify the mech-
anism further.
Bifidobacterium longum subsp. infantis CECT 7210 showed the ability to inhibit
rotavirus replication both in vitro and in vivo. Chenoll et al. [40] studied the antivi-
ral mechanism of the abovementioned strain. For this reason, the supernatant was
collected and tested against RoV in both HT-29 and MA-104 cell lines. The results
showed that the supernatant possessed antiviral activity and the protease digestions
revealed both the proteinaceous nature of the active substance and the fact that the
molecule responsible for inhibiting rotavirus replication was released in the super-
natant. The peptide was further purified and characterized. The authors showed that
this antiviral peptide contains 11 amino acids with the sequence MHQPHQPLPPT,
with a molecular weight of 1.282 KDa. After several experiments, they showed that
Bifidobacterium longum subsp. infantis CECT 7210 secretes a protease that digests
the casein already present in MRS broth. The antiviral peptide was one of the results
of the casein digestion [40].
In addition to the blocking effect of L. casei and B. adolescentis anti-rotavirus,
Olaya Galán et al. (2016) demonstrated another mechanism of these two strains.
They showed that the two strains secreted proteinaceous substances that interfere
with the final amount of intracellular NSP4 – described previously as rotaviral
enterotoxin [56] – and therefore the cell lysis as well as the diarrhea duration
decreased. Moreover, these metabolites can also regulate Ca2+ and prevent its releas-
ing from the endoplasmic reticulum of enterocytes, resulting in a decrease in cell
damage and electrolyte losses [41].
VHHs are nanobodies that have previously been shown to be useful in the treat-
ment of rotavirus-associated diarrhea [57, 58]. These nanobodies are fragments of
antibodies derived from immunoglobulins without light chains that can be found in
camelids [59]. LGGs, which have previously shown antiviral activity by several
mechanisms [60–64], were selected to express antiviral VHH in a study conducted
by [42]. Several LGG strains (GG (CMC), GG (ATCC 53103), GG (NCC 3003),
and GG (UT)) were evaluated for their capability to possess nanobodies on their
cell wall. Only the GG (UT) strain was able to display ARP1 (anti-rotaviral VHH)
on the bacterial surface, because this strain has the inactivated welE and welF EPS
genes. The lack of EPS expression allows an efficient display of ARP1 on its sur-
face. In an in vivo experiment using a mouse pup model, the GG (UT) strains
seemed to confer a level of protection against rotavirus-induced diarrhea. The
absence of EPS did not affect the antiviral activity, but more investigation should
be conducted into this strain, especially as regards its adhesion capacity to entero-
cytes [42].

of Probiotic/LAB Native Supernatants
Several studies have shown antiviral activity on the probiotic and/or LAB superna-
tants, without in-depth investigations or without specifying the antiviral molecules.
Therefore, the search for antiviral substances with high efficacy, low toxicity,
and minor side effects should be continued, and there is an urgent need for antiviral
molecules against viral pathogens in the human and animal fields. In 1986, a screen-
ing of the antiviral activity (anti-influenza virus) of boiled yoghurt, among different
milk preparations, was conducted by [43]. The results showed the survival rate of
infected mice treated with boiled yoghurt was longer than the control infected mice.
Moreover, the hemagglutinin titer in treated mice was lower than in the control
infected mice [43]. In another study, the cell-free supernatant (CFS) of seven probi-
otic strains B. breve DSM 20091, B. longum Q 46, L. paracasei A14, L. paracasei
F19, L. rhamnosus Q 85, L. plantarum M1.1, and L. reuteri DSM 12246, cultured
in MRS broth, was evaluated for its antiviral activity against vesicular stomatitis
virus (VSV), an enveloped virus belonging to the Rhabdoviridae family. The authors
showed that all probiotic strains exhibit antiviral activity in their supernatants, and
the viral infectivity decreased by about 68 %. The authors suggested that this activ-
ity was related to three possible mechanisms: trapping of viral particles by direct
interaction of probiotic strains, cross talk between bacterial and host cells, and,
finally, the neutralization of viral particles by probiotic metabolites such as organic
acids or proteinaceous molecules [44].
Choi et al. investigated both the yoghurt and MRS culture CFSs of several probi-
otic strains: L. acidophilus, L. rhamnosus, L. plantarum, S. thermophilus, and B.
bifidum. The CFSs were tested against a large variety of RNA viruses: influenza
virus A/PR/8/34; influenza virus A/WS/33; influenza virus B/Lee/40; Coxsackievirus
A 16 (CA16), CB3, and CB4; and porcine epidemic diarrhea virus (PEDV) CV 777.
In addition to the non-cytotoxicity on Vero and MDCK cell lines, these CFSs
showed antiviral activity by inhibiting viral multiplication. Moreover, the CFSs
from yoghurt showed the strongest activity [45]. Furthermore, the authors showed
that the CFSs from yoghurt filtered by Amicon (Millipore Co., Billerica, USA) with
MWCO < 3000 Da exhibit the same antiviral activity. The authors suggested that
the MW of the antiviral compound(s) was less than 3000 Da.
Another study showed the antiviral activity of CFS of Lactococcus lactis subsp.
lactis LM0230 – probiotic strain – cultured in MRS broth. This CFS was evaluated
in Crandell–Reese feline kidney (CRFK) against feline calicivirus (FCV) as a NoV
surrogate. The results showed that the native CFS reduced the viral load. However,
the neutralized CFS (pH=7.0) had no effect on the viral load [46]. The authors
4.4 Conclusion 93
suggested that the lactic acid was essential for denaturation of the viral capsid as
reported by Rodger et al. [65].
After sakacin A and nisin evaluation, Lange-Starke et al. [30] evaluated the anti-
viral activity of both D/L lactic acid and a large variety of sausage LAB CFSs
against MuNoV, H1N1, FHV, and NDV. The D/L lactic acid was evaluated against
MuNoV and H1N1. Lactic acid reduced the viral load by 3.25 log units and 2.5 log
units of MuNoV and H1N1, respectively. The active concentration of lactic acid was
not cytotoxic. In addition, the LAB CFSs were tested against MuNoV. The results
showed that only the L. curvatus 1 culture supernatant was active on
MuNoV. Furthermore, the heat treatment, catalase treatment, freezing, and neutral-
ization of L. curvatus 1 CFS indicated that the antiviral compound was a protein.
The separation of this CFS by the chromatographic method supposed that the MW
of this antiviral compound was 2 kDa. This MW is very close to a bacteriocin, but
the heat lability of this compound eliminates this hypothesis, since the thermolabile
bacteriocins have a MW > 30 kDa [24].
Another study conducted by Shearer et al. [47] reported the antiviral activity of
B. subtilis 168 and E. faecalis 19,433 CFSs against MuNoV 1 and Tulane virus. The
CFSs showed no direct effect on viral particles. However, the CFSs showed a slight
inhibition of viral propagation on RAW 264.7 and LLCMK2 cell lines [47].
One study investigated the antiviral effect of prebiotics. The authors did not
demonstrate the impact of prebiotics on rotaviral infection in G14 pregnant
Lewis rats in the presence of B. breve M-16 V as a probiotic strain. The diffi-
culty was faced on the stool consistency induced by the prebiotic short-chain
galactooligosaccharides (scGOS) and long-chain fructooligosaccharides
(lcFOS) [66].
4.4 Conclusion
Some LAB and probiotic metabolites can be active against several types of viruses,
such as enveloped and naked viruses found in both the medical and food sectors.
Organic acids, hydrogen peroxide, and proteinaceous molecules were the most
important antiviral compounds of probiotic metabolites. The D/L lactic acid and
bacteriocins are the most studied because of their safety in use. The antiviral activity
seems to have molecule specificity, since LAB CFSs containing lactic acid and
some bacteriocins showed no antiviral activity. Furthermore, the activity depends on
the virus type and/or subtypes. The lab experiments in the evaluation of these
metabolites are complicated. The cytotoxicity of some metabolites can skew the
view of researchers to see direct effects of these metabolites on the target virus. As
indicated by Liévin-Le Moal and Servin (2014) , some CFSs of lactobacilli had a
cytotoxic effect on Caco-2 cell lines, which can cover the initial antiviral effect of
these CFSs, for example, in the rotavirus replication cycle [67]. Fractionated CFS is
very important in identifying the real antiviral compound and conducting an in-
depth investigation of the mechanism of action of such molecules.
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Chapter 5
Methods and Techniques to Evaluate
the Antiviral Activity of a New Probiotic Strain
Imad AL KASSAA
Contents
5.1 Introduction .................................................................................................................... 100
5.2 Evaluation of a Potential Probiotic Strain ...................................................................... 101
5.2.1 Isolation and Characterization of Probiotic Strains ......................................... 101
5.2.2 Identification .................................................................................................... 102
5.2.3 Screening Tests to Confirm Potential Probiotic Characteristics ...................... 103
5.2.4 Safety of Selected Probiotics ........................................................................... 103
5.2.5 Antibiotic Resistance Profile ........................................................................... 103
5.2.6 In Vivo Studies in Animal Models and Human Trials ..................................... 104
5.3 Evaluation of Antiviral Probiotics (AvPrs) .................................................................... 104
5.3.1 In Vitro Evaluation .......................................................................................... 105
5.3.2 Antiviral Assays for Bacterial Cells ................................................................ 106
5.3.3 In Vivo Evaluation ........................................................................................... 108
5.3.4 Clinical Trials (CTs) ........................................................................................ 110
5.4 Conclusion ..................................................................................................................... 111
References ............................................................................................................................... 111
Abstract The “antiviral probiotic” term is not yet used in science nor approved by
FDA and WHO. Indeed, the evaluation of antiviral activity of probiotic strains
needs to be standardized and approved. Until now, “antiviral probiotics” are not
used either in the medical or food sectors. Furthermore, this type of probiotic is not
widely recognized by health organizations such as the WHO and FDA. However,
antiviral probiotics (AvPrs) have shown an efficient antiviral effect in the prevention
and treatment of several viral infections. In last decade, many studies have been
conducted to evaluate the antiviral activity of some probiotic strains. Few studies
have showed the mechanisms behind such activity. The needs and the importance of
antiviral probiotics have encouraged researchers to deeply investigate the antiviral
mechanism. A probiotic strain needs to be tested and evaluated by many experiment
to be recognized and approved as antiviral probiotics. Besides cytotoxicity, probi-
otic characteristics and immunomodulation effect of probiotic strain, choosing cell
line, indicator virus and addition time of bacterial cells are the essential criteria for
this selection.
Keywords Probiotic strain • antiviral probiotics • in vitro evaluation • in vivo

evaluation antiviral methods

DOI 10.1007/978-3-319-49688-7_5
100 5 Methods and Techniques to Evaluate the Antiviral Activity of a New Probiotic Strain
Abbreviations
AvPrs Antiviral probiotics

BLIS Bacteriocin-like inhibitory substances
CFS Cell-free supernatant
CMUL Collection Microbiologie Universite Libanaise
CPE Cytopathic effect
CT Clinical trial
DBPC Double-blind placebo controlled
DCs Dendritic cells
FDA Food and Drug Administration
HAM/TSP (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP)
HCV Hepatitis C virus
HSV Herpes simplex virus
HTLV-1 human T-cell lymphotropic virus type 1
IgA Immunoglobulin A
IL Interleukin
LcS L. casei Shirota
LGG L. rhamnosus GG
MOI Multiplicity of infection
MuNoV Murine norovirus
NK Natural killer
NoV Norovirus
PBMC Peripheral blood mononuclear cells
PBS Phosphate buffer salt
PCCO Placebo-controlled crossover
PFGE Pulsed-field gel electrophoresis
PRA Plaque reduction assay
PrTBPC Prospective triple-blind placebo controlled
RDBPC Randomized double-blind placebo controlled
RoV Rotavirus
RSBC Randomized single-blind controlled
WBC White blood cells
WHO World Health Organization
5.1 Introduction
Public health is in great need of alternatives to chemical molecules used as drugs and
food preservatives. Probiotics are used in human and animal health to prevent diseases,
including infectious diseases. These probiotic strains have been evaluated and studied
to confirm their ability to modulate immune responses and inhibit or kill pathogenic
5.2 Evaluation of a Potential Probiotic Strain 101
bacteria. To date, antiviral probiotics are not used either in the medical or food sectors.
Furthermore, this type of probiotic is not widely recognized by health organizations
such as the WHO and FDA. However, antiviral probiotics (AvPrs) have shown an
efficient antiviral effect in the prevention and treatment of several viral infections (see
Chap. 1). To promote the recognition and thus the use of AvPrs, scientists should stan-
dardize the methods used (1) in the selection and characterization of probiotic strains,
(2) the detection and characterization of antiviral metabolites, (3) the immunomodula-
tory effect in vitro, (4) cytotoxicity, (5) antiviral experiments both in vivo and in vitro
and in clinical trials, and finally (6) the definition of antiviral mechanisms.
5.2 Evaluation of a Potential Probiotic Strain
5.2.1 Isolation and Characterization of Probiotic Strains
Species of Lactobacillus and Bifidobacterium are most commonly used as probiotics,

but yeasts such as Saccharomyces species and some E. coli and Bacillus species are also
used as probiotics. LAB have been used for the preservation of food by fermentation for
thousands of years. Furthermore, they can have a dual function by acting as agents for
food fermentation and by maintaining host health. Probiotics can provide taste and low
pH and can thus prevent the multiplication of food and spoilage microorganisms.
The important question here is: Are all LAB or yeast strains considered to be
probiotics? The answer: There are criteria and specific characteristics that must be
met in order to consider a strain to be a probiotic.
The target host and domain of application first determine the genus and species of
the desired probiotic. For example, in the animal sector, species from other bacterial
genera, such as Streptococcus, Bacillus, and Enterococcus, have been used, but not for
human applications, except for a few studies, because there are concerns regarding the
safety of such probiotic genera [1]. However, Enterococcus LAB SF 68 is the only
strain used in fermented food (Bioflorin, Cerbios-Pharma) for human consumption [2].
The isolation and selection of potential probiotic strains starts with choosing the
sampling methods and origin. Otherwise, when the desired probiotic strains are
used in human applications (maintenance of the gut ecosystem), the sampling
should be from healthy human feces. Thus, the origin of probiotic strains and the
target application should be chosen consistently. After isolation, the strain should be
identified by Gram stain and catalase test. The Gram-positive and catalase-negative
bacteria should be characterized in depth. The use of biochemical identification may
help in the preliminary identification; however, molecular analysis, such as the com-
plete sequence of the 16S rDNA gene, is more reliable.
This safety evaluation should be performed when the evaluated strain belongs to a
genus with health concerns, such as the Enterococcus genus. The most important
approach that should be used is the detection of intrinsic properties, such as virulence
genes. Virulence genes are very variable from one genus to another. For Enterococcus,
the most important virulence genes are cylA, which is responsible for the transportation
and activation of cytolysin, cylB and cylM involved in posttranslational modification,
the esp gene responsible for a cell wall protein involved in immune evasion, agg
encodes for an aggregation protein responsible for adherence to eukaryotic cells, gelE
is responsible for toxin production that hydrolyzes gelatin and other compounds, and,
finally, the cpd, ccf, and cad genes code for sex pheromones, which are responsible for
facilitating conjugation [3]. In fact, the guidelines, recommended criteria, and methods
for the selection and use of probiotics in food were already created in 2002 by a work-
ing group convened by the FAO/WHO [1].
The guidelines for the “Evaluation of Probiotics in Food” draft contain the steps
to be followed in order to consider a strain to be a probiotic (Fig. 5.1).
5.2.2 Identification
The first step is the identification of the potential probiotic strain at all levels: genus,
species, and strain. The identity of such a strain is very important to link it to another
strain which shows probiotic characteristics, for example, L. rhamnosus, which is the
Strain identification by phenotypic and genotypic methods

• Genus,species,strain
• Deposit strain in international culture collection
Functional characterization Safety assessment

• In vitro tests • In vitro and/or animal
• Animal studies • Phase 1 human study
Double blind, randomized, placebo-controlled (DBPC) phase 2 human trial or

other appropriate design with sample size and primary outcome appropriate
to determine if strain/product is efficacious
Preferably second Phase 3, effectiveness trial is

independent DBPC appropriate to compare
Probiotic food
study to confirm probiotics with standard
results treatment of a specific condition
Labeling
• Contents-genus, species, strain designation

• Minimum numbers of viable bacteria at end of shelf-life
• Proper storage conditions
• Corporate contact details for consumer information
Fig. 5.1 Guidelines for the evaluation of probiotic methods (WHO/FDA)

5.2 Evaluation of a Potential Probiotic Strain 103
same species as LGG. The identification should be performed both by phenotypic and
genotypic methods. Furthermore, DNA/DNA hybridization is the gold-standard method
used to identify and compare strains. However, this method is complicated, especially
in small laboratories; complete sequencing of the 16S rRNA gene can replace DNA/
DNA hybridization. Moreover, strain typing is recommended by PFGE. The identified
strains should be registered in an international collection before or after publication.
5.2.3 Screening Tests to Confirm Potential Probiotic

Characteristics
Probiotics for human use should require proof of efficacy in human trials. For this
reason, several in vitro tests give primary answers on the functionality of this strain
in the human body:
(a) Resistance to gastric acidity
(b) Bile acid resistance
(c) Adherence to mucus and epithelial cells (cell cultures)
(d) Antimicrobial activity against pathogenic microorganisms
(e) Co-aggregation with pathogens
(f) Bile salt hydrolase activity to resist digestive tract complications
5.2.4 Safety of Selected Probiotics
Lactobacillus and Bifidobacterium genera are the most used probiotic types in both
clinical and food applications. These two genera are known as safe; otherwise, they
have no side effects. In general, there are four types of side effects of probiotics in
immunocompromised persons: (1) systemic infection, (2) damage to metabolic
activities, (3) excessive immunomodulation, and (4) gene or plasmid transfer [4]. To
evaluate the safety of potential probiotic strains, three approaches can be used: (1)
intrinsic properties; (2) survival, activity in the intestine, dose–response relation-
ships, and fecal and mucosal recovery; and (3) interaction with the host [5]. The
following characteristics are recommended by the working group of the FDA/WHO
concerning the safety of characterized strains:
5.2.5 Antibiotic Resistance Profile
The antimicrobial profile of probiotics is one of the most important debates in this
field. Benign antibiotic resistance can help probiotics resist some conditions, espe-
cially when patients take antibiotics during infections. However, the increased anti-
biotic resistance of probiotic strains has led to the need for further evaluation of
probiotics so that their possible negative consequences do not outweigh their bene-
fits [6]. The excessive use of probiotics, especially in food, could help to increase
the antibiotic resistance transmitted by such a route. For this reason, it is imperative
that probiotics are well researched and documented for their antibiotic resistance
profile. The ability to transfer antibiotic-resistant determinants must be considered
an important parameter for the selection of probiotic strains [6]
(a) Assessment of certain metabolic activities (e.g., D-lactate production, bile salt
deconjugation)
(b) Evaluation of side effects in human trials
(c) Epidemiological surveillance of adverse reactions in consumers (post-
marketing) non-toxicogenic strains
(d) Nonhemolytic strains
(e) No adverse reactions in immunocompromised animals
5.2.6 In Vivo Studies in Animal Models and Human Trials
Animal models have many advantages for evaluating novel probiotic strains.
This type of experiment is fast and easily monitored, provides substantiation of
in vitro effects, and confirms the mechanism of probiotics already seen in in vitro
evaluations. On the other hand, human clinical trials require many experimental
phases:
Phase 1, “safety studies,” focuses on the side effects of strains. Patients should
be followed and monitored for any adverse reactions. Phase 2, “efficacy studies,”
generally in the form of randomized, double-blind, placebo-controlled (DBPC)
designs, which measure efficacy compared with a placebo. In addition, phase 2
studies measure adverse effects. In phase 3, “effectiveness studies,” it is recom-
mended to conduct two independent clinical trials to confirm the results and effec-
tiveness. In phase 4, “surveillance studies and publications,” the researchers are
encouraged to publish their studies in peer-reviewed journals, even if they have
negative results (http://www.who.int/foodsafety/fs_management/en/probiotic_
guidelines.pdf).
5.3 Evaluation of Antiviral Probiotics (AvPrs)
The majority of studies start from step 2. They evaluate already confirmed probi-
otic strains, such as LGG, LcS, E. coli Nissle, etc. For novel probiotic strains,
researchers should initiate their research by following the first step, which is
related to the selection and evaluation of probiotic characteristics. What is the
second step?
The second step is the evaluation of the antiviral activity of probiotic strains that
should be conducted in in vitro and in vivo experiments and can be confirmed by
human clinical trials.
5.3 Evaluation of Antiviral Probiotics (AvPrs) 105
5.3.1 In Vitro Evaluation
5.3.1.1 Choose the Target Virus and Cell Culture Lines
First, the desired application of the potential antiviral probiotic should be considered. For
example, L. gasseri CMUL57, isolated from the vagina of healthy Lebanese women,
was evaluated for potential antiviral activity by Al [7]. The authors chose HSV-2 as the
target or indicator virus. They considered that HSV-2 has many complicated health con-
cerns for women worldwide. Furthermore, HSV-2 causes very complicated genital
infections. In addition, the origin of this selected probiotic strain is similar to the target
site of HSV-2. All probiotic strains evaluated against enteric viruses were isolated form
human feces or from dairy products [8–10]. When the selected strains were isolated
from animal feces, the application should be for animal sectors. Furthermore, infectious
animal viruses should be used, such as porcine rotavirus, murine rotavirus, feline calici-
virus, etc. [11, 12]. However, human strains can be used for animals, as reported by
[13–18]. The virus should be titered in the desired cell culture, and it should be decided
which multiplicity of infection (MOI) is needed in the experiment.
After the determination of the target virus, the cell lines should be defined. The
selection of the cell lines is related to the target virus, since each virus can be cul-
tured in specific cell line(s). In addition, cell line selection should be as closely
related as possible to the nature of the host tissue targeted by the indicator virus. For
example, the evaluation of probiotic strains isolated from healthy human feces
against human rotavirus should be performed on intestinal epithelial cells such as
Caco-2, HT-29, etc. [9, 14, 16, 19–22]. However, sometimes the virus type obligates
researchers to use a cell line which is not coherent with the target tissue. For exam-
ple, HSV-2 is highly infective in Vero cell lines but not in HeLa cell lines [7].
Furthermore, the use of PBMC is very useful for the immunomodulatory effect of
such probiotics [23, 24]. In addition, NoV can be cultured only in Lymphocyte B
cells as reported by Karst et al. [25].
5.3.1.2 Determination of Antiviral Activity Mechanisms
AvPrs can inhibit viruses by several mechanisms. The physical mechanisms corre-
spond to the direct interaction between the AvPr cell wall and viral particles, result-
ing in viral trapping or viral neutralization [7, 26, 27]. The indirect physical
mechanism corresponds to the capacity of AvPr to saturate host cell receptors. This
mechanism reflects the interference with virus attachment or entry into the cells,
perhaps by steric hindrance [26].
The other mechanisms correspond to the indirect influence of AvPrs on viral parti-
cles. For example, AvPrs can cross talk with host cells or innate immune cells such as
macrophages and DCs, resulting in modulation of antiviral immunity [14, 15, 23, 28–
30]. Another indirect mechanism corresponds to the secretion of antiviral compounds,
such as lactic acid, hydrogen peroxide, bacteriocins, BLIS, and NRPS [31–35].
5.3.1.3 Determination of Bacterial Cells and CFS Cytotoxicity
The cytotoxicity of bacterial cells and/or their metabolites should be determined

before antiviral evaluation. The cytotoxicity can be measured by both quantitative
and qualitative methods. The qualitative method corresponds to the observation of
the cytopathic effect (CPE) induced by bacterial strains and/or their metabolites. It
is a very easy and inexpensive method. However, CPE observation is a subjective
method and can differ from one person to another. Another qualitative method was
used to evaluate cell viability using viable staining such as alamarBlue reagent
(Invitrogen, United States) or trypan blue (Sigma-Aldrich, Germany).
The quantitative method should be used to standardize the methods and out-
comes. MTT is the method most often used to quantify the viable cells after expo-
sure to any kind of xenobiotic or cells, including bacterial cells [36, 37]. The MTT
assay is based on the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-z-yl)-
2,5-diphenyltetrazolium bromide. Al Kassaa et al. used the “UptiBlue” kit (Uptima,
Interchim, France) to evaluate the cytotoxicity of L. gasseri CMUL57 on the Vero
cell line [7]. The authors used this type of kit to measure the kinetics of cell viability
over time, because the UptiBlue kit is not cytotoxic like the MTT kit.
5.3.2 Antiviral Assays for Bacterial Cells
The antiviral assays can be performed in different ways. The bacterial cells can be
added to the cell culture at different times.
5.3.2.1 Co-incubation (Co-treatment)
First, the bacterial cells should be incubated with viral particles in the cell culture
medium used (specific to the cell line type). However, it is preferable that the medium
contains the minimum glucose concentration to avoid excessive lactic acid secretion
by the multiplication of bacterial cells, unless heat-killed bacteria are used. After the
co-incubation of the bacterial cells and viral particles for a minimum of 2 h, the
mixture should be poured into the cell line with 80 % confluent and then incubated
for 24 h or more depending on the virus type [7, 26, 27, 38, 39]. The co-incubated
mixture should be filtered using a 0.45/0.22 μm filter to eliminate bacterial presence
in cultured cells [19]. The virus titers of the co-incubated virus and control virus
(without interaction with bacteria) can be determined by four methods:
1. Spearman–Karber TCID50 (50 % tissue culture infectious dose) titration
method [40]
2. Plaque reduction assay (PRA), as described by [40]
3. MTT or UptiBlue cell viability quantification [7]
4. Immunostaining assay [19, 39]
The reduction of infectious viral particles indicates the activity of bacterial cells
by direct physical mechanisms such as virus trapping or viral lysis [7, 11, 19, 20, 22,
26, 38, 39].
5.3.2.2 Pretreatment of Eukaryotic Cells with Probiotic Strains
The probiotic strain should be poured on cultured cells with a known concentration.
First, it should be defined how many bacterial cells are needed to attach one cultured
cell. For example, in some cases, ten bacterial cells are needed to attach one cul-
tured cell, which is expressed as 10:1 bacteria/cell. The incubation time depends on
the probiotic strains. It is preferable to conduct an adhesion assay beforehand to
exactly measure the ability of these strains to attach to the cultured cell line and how
much time they need. After the incubation time, the cultured cells should be washed
two or three times with equilibrated PBS followed by viral challenge with the
defined MOI. The results can be extracted by the methods discussed in Sect. 3.2.1.
The pretreatment experiment can provide information about the mechanism of
probiotic strains [7, 26, 27, 38, 39]. If the probiotic strains can inhibit or reduce viral
infectivity in this experiment, this means that bacterial cells could interfere with viral
binding to eukaryotic cells. Another hypothesis can be suggested, namely, that bacte-
rial cells inhibit the spread of virions (new virus particles) between cultured cells.
5.3.2.3 Posttreatment
In this experiment, the cultured cells should be challenged by the target virus followed
by incubation for a minimum of 1 h (depending on the virus type). The incubation
time gives the virus the advantage of infecting the cells before adding probiotic strains.
After viral infection, the cells should be washed two or three times with equilibrated
PBS followed by addition of the probiotic strains. Here, the probiotic strains can be
kept in the infected cell well or the latter should be washed two or three times with
PBS to eliminate nonattached bacteria. In general, when heat-killed bacteria were
used, the bacterial cells were kept in infected cells without washing [7, 26, 27, 38, 39].
In this experiment, the results can provide information about the possible inter-
vention of probiotic strains in the viral cycle and/or the spread of virions between
cultured cells.
5.3.2.4 Inactivated/Killed Strains Vs. Viable Strains
The most important issue in this type of experiment is the choice of the bacterial strains
condition. In fact, this is a matter of debate among researchers. Several studies have
used viable probiotic strains. However, they have used cell culture media containing
bacteriostatic antibiotics such as gentamicin or streptomycin [19]. It should be noted
that the use of antibiotics in the cell culture media before antiviral assays cannot affect
bacterial cells, since the cultured cells will be washed twice with PBS, which elimi-
nates antibiotic presence in the wells. On the other hand, some studies have used viable
bacterial strains using antibiotic-free cell culture media which allow the proliferation of
probiotic strains [7, 14, 16, 21, 23, 26, 39, 41, 42]. Some studies have shown a compari-
son of the antiviral effect of probiotic strains in killed and inactivated conditions [19,
39]. However, there was no indication of the bacterial strain condition in some studies
[11, 38]. Furthermore, several studies have evaluated bifidobacterial species in antiviral
assays. However, in the use of viable bifidobacterial strains in cell cultures, the 5 %
CO2 atmosphere of the cell line culture condition should be considered an inhibitor of
bifidobacterial strains. However, bifidobacterial strains have kept all structures, includ-
ing cell wall molecules which are essential to the immunomodulatory effect. In such
studies, the authors considered these bifidobacterial strains to be viable strains [16, 20].
On the other hand, in this kind of experiment, the viral challenge in the cell culture
resulted in the production of a large number of virions after a complete viral cycle.
Logically, it is very important to let the bacterial cells proliferate to defend viral inva-
sion, even in any kind of antiviral mechanism as mentioned above. This kind of probi-
otic enhancement could not be achieved using inactivated probiotic strains.
The viable probiotic strains can be used to evaluate the impact of bacterial multipli-
cation on antiviral activity. Moreover, the production of antiviral metabolites (see Chap.
4) can be detected in such a condition. In addition, the live probiotic strains can keep all
cell wall components intact. These cell wall molecules can play a role in antiviral activ-
ity, especially when the immunomodulatory effect is the antiviral mechanism of such
strains [9, 14, 23, 41]. However, live bacteria can lead to cytotoxicity of cell lines used
in antiviral assays. This cytotoxicity can be induced after excessive proliferation of
probiotic strains and production of high amounts of organic acid and/or hydrogen per-
oxidase, which can affect the cell line and thus affect the interpretation of the results.
In contrast, the use of killed or inactivated bacteria has many different conse-
quences. The cytotoxicity of undesired metabolites secreted by bacterial strains can
be prevented when using inactivated bacteria, whereas the inactivation method may
influence the relevance of results. For example, heat killing is one famous method
of inactivating bacteria [39]. This method can lead to a partial or total degradation
of some cell wall components, which may be very important in such an antiviral
effect. Thus, another type of inactivation should be used to keep the bacterial cell
wall intact. Ang et al. used formaldehyde solution as an inactivation method to keep
all probiotic structures intact [19]. In addition, bacteriostatic antibiotics have also
been used in antiviral assays to prevent undesired microbial contamination and
sometimes to inhibit bacterial proliferation.
5.3.3 In Vivo Evaluation
Why Animal Models?

The animal model is the in vivo experiment which can confirm the efficacy of
any drug, including probiotics. In this model, the choice of animal type or animal
condition should be taken into consideration.
5.3.3.1 Mouse Model
BALB/c mice are useful in research into both cancer and immunology. Furthermore,
they are non-genetically modified and also very useful in the production of mono-
clonal antibodies [43]. For antiviral probiotic evaluation and after in vitro testing,
BALB/c mice were the most commonly used mouse model in antiviral assays for all
viral types, such as respiratory viruses [24, 44, 45], enteric viruses [13], and other
viruses such as HSV-2 and HIV [27]. Another type of mouse model corresponds to
the C57BL/6 substrain. This type of mouse can be genetically modified due to its
black color, which facilitates transgenic modification. In addition, C57BL/6 is very
useful in drug evaluation, it is very sensitive to pain and cold, and it can drink alco-
hol voluntarily [46]. To our knowledge, two studies have used C57BL/6 in antiviral
evaluation of probiotic strains [44, 45]. It should be noted that Gabryszewski et al.
used a genetically modified C57BL/6, which has a lack of MyD88 (C57BL/6-
MyD88-) [44]. Some studies have used germ-free mice to demonstrate the effect of
gut microbiota on the antiviral effect of probiotic strains [47], since gut microbiota
was considered essential to immunological response by inducing cross talk with
immunological actors [48]. In contrast, the use of germ-free mice is useful to evalu-
ate the impact of gut microbiota in enhancing viral infections [49].
5.3.3.2 Other Animal Types
In antiviral probiotic evaluations, piglets were used to investigate the efficacy of

probiotics on porcine RoVs. Weaned and neonatal gnotobiotic piglets are most
often used [15, 23]. For the evaluation of RoV vaccine enhancement by probiotic
strains, Yang et al. used vaccinated gnotobiotic piglets [50].
5.3.3.3 Animal Model Outcomes
The animal outcomes should be extracted after administration of probiotic strains,

usually compared with different animal groups: a control group that receives only
probiotic strains and an infected group that does not receive probiotic strains. The
outcomes can be divided as follows:
• Symptoms (diarrhea, fever, weight, etc.) [18, 28, 51]
• Blood indicators (WBC, inflammation markers, cytokines, antibodies, etc.)
• BALF (sIgA, cytokines, macrophages, etc.) [9, 29]
• Feces (microbiota composition, probiotic enumeration, viral shedding, etc.) [16]
• Tissue histology (inflammation score, tumor score, immunohistological assays, etc.)
[15, 47, 52].
It should be noted that the viral type should be compatible with the animal model,
for example, for piglets as animal models, a porcine virus such as porcine NoV,
RoV, and swine flu virus should be used, while in a mouse model, the researchers
use MuRoV as an indicator virus.
5.3.4 Clinical Trials (CTs)
5.3.4.1 CT Types
A clinical trial is prospectively planned experiment for the purpose of evaluating poten-
tially beneficial therapies or treatments. In general, these studies are conducted under
as many controlled conditions as possible, so that they provide definitive answers to
predetermined and well-defined questions. Clinical trials are very important to confirm
the antiviral activity of probiotics, as shown in both in vitro and in vivo experiments.
The majority of studies have used randomized double-blind placebo-controlled
(RDBPC) trials to evaluate antiviral probiotics in infected patients and control
groups [8, 53–57]. RDBPC trials are very useful in such evaluations and are consid-
ered the gold standard in clinical trials in epidemiological studies.
RDBPC studies remain the most convincing research design in which random
assignment of the intervention can eliminate the influence of unknown or immea-
surable confounding variables that may otherwise lead to biased and incorrect esti-
mation of the treatment effect [58]. In addition, blinding can also eliminate
confounding by co-interventions. Furthermore, the administration of a placebo to a
control group results in a very accurate comparison of outcomes. Therefore, this
type of trial is able to demonstrate causality.
RSBC trials were also used in antiviral probiotic evaluation [59]. This type of
clinical trial is very similar to RDBCs (without a placebo control group). The differ-
ence here is only that the researchers are aware of the treatment, while the patients
are unaware of it.
The prospective triple-blind placebo-controlled trial (PrTBPC) is a different type
of clinical trial. This type of trial is useful when patients take something outside of
their routine, such as a rehydration protocol [60]. The triple blinding means that the
subject, the person administering the treatment, and the person evaluating the
response to the treatment are all unaware of which subjects are receiving a particular
treatment or lack of treatment. Two PrTBPCs were conducted to evaluate the anti-
viral activity of LGG and L. casei DN-114,001 against rotavirus infection in a popu-
lation of children [60, 61].
In some circumstances, blinding does not required either double or single blind-
ing. For example, the comparison of the efficacy of a medication to intensive physi-
cal therapy sessions requires a non-blind trial. In this case, an open-case trial or
open-label controlled trial is more convincing. The effect of a continuous intake of
LcS in fermented milk by elderly patients in a noroviral gastroenteritis outbreak was
evaluated using an open-case controlled trial [62].
The last trial used for antiviral probiotics is the placebo-controlled crossover
(PCCO) trial. This is a longitudinal study. The crossover patients serve as their own
control. In addition, the number of patients in this trial can be lower than in other
types of clinical trials. Takeda et al. conducted a PCCO trial on ten elderly people
who received LcS fermented milk and a placebo randomly in two different periods.
This trial allowed the authors to evaluate the activation of NK and IL-12 production
in subjects who took a placebo and/or LcS fermented milk [63].
References 111
5.3.4.2 Population
Researchers should select the population that will give relevant results. For
example, to assess probiotics against rotavirus, infected children aged from more
than 3 months old should be studied [8, 54, 61, 64, 65]. For norovirus infection,
elderly persons should be the selected population [62, 63]. In addition, the virus
identification should be confirmed by the standard methods. In some cases, anti-
viral probiotics were assessed in chronically infected people. For example, HPV-
positive women were subjected to B. adolescentis SPM1005-A [66], human
T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spas-
tic paraparesis (HAM/TSP) patients were subjected to LcS [67], and cirrhosis or
hepatitis related to chronic HCV infection [68] and liver cancer patients were
subjected to a combination of lactobacilli and propionibacteria strains [69].
5.4 Conclusion
Antiviral probiotics can prove themselves in medical treatment or prevention. In the

near future, AvPrs will have a great opportunity in the medical and food sector.
Researchers should follow standardized instructions to confirm the potential antivi-
ral activity of probiotics or novel probiotic strains. In summary, to characterize a
novel antiviral probiotic, the potential probiotic strain should be identified by the
molecular method and compared to other strains. The probiotic strains should
exhibit many characteristics, such as high adhesion capacity to host cells, resistance
in the digestive tract, an immunomodulatory effect, secretion of antimicrobial com-
pounds, and non-cytotoxicity. On the other hand, the antiviral activity of probiotic
strains should pass through three major phases: in vitro, in vivo, and, finally, human
clinical trials. The in vitro phase corresponds to the different cell culture experi-
ments, like cell line pretreatment and posttreatment by viable or inactivated probi-
otic strains, followed by viral infection. In addition, the co-incubation of probiotic
cells with target viruses represents the last in vitro experiment. The second phase
corresponds to the in vivo experiment. In this phase, the animal model type is very
important, and thus the selection of the desired outcomes to be analyzed. Finally,
human clinical trials represent the third phase and can be conducted using several
CT types, such as RDBPCs, PCCOs, OCCs, PrRDBCs, and RSBPCs.
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Index
A in vitro evaluation, 105–106

AAstV. See Avastrovirus (AAstV) in vivo evaluation, 108–109
Acquired immune deficiency syndrome and viral gastroenteritis
(AIDS), 64. See also Human avastrovirus (AAstV), 34
immune deficiency virus (HIV) gram-negative bacteria, gut microbiota,
Antibiotics, efficacy of, 3 15–20
Anti-enteric viruses probiotics (AEnPs), gut microbiota, 20–27
20–26 histo-blood group antigens (HBGAs),
direct mechanism of, 27 gut microbiota, 15
indirect mechanism of, 27 mamastrovirus (MAstV), 34
Antimicrobial compounds, production of, 4 microbiota, 15
Anti-RVs probiotics, 6 against norovirus infections, 32–33
Antiviral activity against rotavirus (RoV) infections,
antiviral probiotics (AvPrs) 27–32
bacterial cells, 106–108 and viral respiratory infections
in vitro evaluation, 105–106 bifidobacteria probiotic strains in,
in vivo evaluation, 108–109 12–13
clinical trials (CTs), 110–111 Lactobacillus probiotic strains in, 6–12
potential probiotic strain Avastrovirus (AAstV), 34
antibiotic resistance profile, 103–104 AvPrs. See Antiviral probiotics (AvPrs)
identification, 102–103
in vivo studies, in animal models and
human trials, 104 B
isolation and characterization, 101–102 Bacterial cells, antiviral assays for
safety, 103 co-incubation (co-treatment), 106–107
screening tests, 103 eukaryotic cells with probiotic strains, 107
of probiotic metabolites inactivated/killed strains vs. viable strains,
non-organic substances, 84–85 107–108
organic substances, 85–90 posttreatment, 107
probiotic/LAB native supernatants, Bacterial cell suspension (BCS), 33
92–93 Bacterial growth medium cell-free filtrate
and probiotics, 90–92 (BGMF), 33
probiotic strains, 87–88 Bacterial vaccine vectors, 50
Antiviral probiotics (AvPrs) Bacteriocin-like inhibitory substances
bacterial cells, 106–108 (BLISs), 4
evaluation Bacteriocins, 4, 85, 90

DOI 10.1007/978-3-319-49688-7
118 Index
BALF. See Bronchoalveolar lavage fluid Hepatitis B and C virus, 72–74

(BALF) Hepatitis B infection (HBV), 63
Bifidobacterium animalis, 13 Hepatocellular carcinoma (HCC), 63
Bifidobacterium longum, 12 Herpes simplex viruses 1 and 2 (HSV-1 and
Bronchoalveolar lavage fluid (BALF), 11 HSV-2), 74–76, 90
Histo-blood group antigen (HBGA), 18
H1N1, 90
C Human-cell lymphotropic/leukemia virus
Cancer, 64–67 (HTLV) infection, 69–71
Chronic liver disease (CLD), 63 Human herpesvirus 8 (HHV8), 64
Coxsackievirus type A strain 16 (CA16), 34 Human immune deficiency virus (HIV),
Crandell-Reese feline kidney (CRFK) cells 51–52, 76–77
line, 33 Human papillomavirus (HPV) infection,
Cytopathic effect (CPE), 106 55–56, 67–69
Hydrogen peroxide (H2O2), 4, 84
D
Dendritic cells (DCs), 51 I
IDs. See Infectious diseases (IDs)
Immunobiotics, 6
E Immunomodulation, 14, 20, 29–31, 35, 103
Enteric viruses (EnVs) Infectious diseases (IDs), 3, 14, 15, 48, 63, 64,
anti-enteric viruses probiotics, 20–26 67, 77, 84
biological cycle of, 34 Influenza viruses, 52–53
capacity of, 18 Interferon (IFN) signaling, blocking
and gut microbiota, 16 of, 18–20
isolation, 105 Intestinal microbiota, direct and indirect
mechanisms of antiviral probiotics, 19 mechanisms, 17–20
and probiotics, 34–35 In vitro evaluation
sources of, 16 antiviral activity mechanisms, 105
Enterovirus 71 (EV71), 34 bacterial cells, 106
and CFS cytotoxicity, 106
target virus and cell culture lines, 105
F In vivo evaluation, animal models, 109
Feline calicivirus (FCV), 33, 90
Feline herpesvirus (FHV), 90
Food and Drug Administration (FDA), 84 K
Kaposi’s sarcoma-related herpesvirus
(KHSV), 64
G
Generally recognized as safe (GRAS), 4, 50, 84
Gram-negative bacteria, 18 L
Gut microbiota Lactic acid, 4
anti-enteric viruses probiotics (AEnPs), Lactic acid bacteria (LAB) probiotics, 4
20–26 Lactobacillus casei, 11
direct mechanism of, 27 Lactobacillus paracasei, 11
indirect mechanism of, 27 Lactobacillus plantarum, 6
gram-negative bacteria, 15–20 Lactobacillus rhamnosus GG (LGG), antiviral
histo-blood group antigens (HBGAs), 15 activity of, 6
viral gastroenteritis, 15–20
M
H Mamastrovirus (MAstV), 34
Hand, foot, and mouth disease (HFMD), 34 Mouse mammary tumor virus (MMTV), 16
Hemagglutinin (HA), 52, 53 Mucosal immunity, 49
Index 119
Murine encephalomyocarditis virus T

(EMCV), 90 Tolerogenic microenvironment, 18
Murine norovirus (MuNoV), 90 Transmissible gastroenteritis virus
(TGEV), 34
Type 1 diabetes (T1D), 71–72
N
National Institutes of Health (NIH), 3
Needle-free vaccine delivery methods, 48 V
Newcastle disease virus (NDV), 90 Vaccine delivery vehicles, 49–50
Non-Hodgkin’s lymphoma, 64 Vaccine vectors
Non-ribosomal peptides (NRPs), 90 and mucosal immunity, 49
Non-ribosomal peptide synthetase and mucosal vaccines, 49–50
(NRPS), 4, 20 to prevent viral infections
Noroviruses (NoVs), 16, 17 HIV, 51–52
Nutrient competition, 4 human papillomavirus, 55–56
influenza viruses, 52–53
rotaviruses, 53–54
P recombinant probiotic
Pathogens exclusion, 4 vaccines, 50–51
Poliovirus, 16 Vesicular stomatitis virus
Poliovirus receptor (PVR), 17 (VSV), 90, 92
Probiotics, 76–77. See also Antiviral Viral antibody production, 18
probiotics (AvPrs) Viral capsid protein (VP1), 17, 27, 34
definition, 3 Viral gastroenteritis
Prospective triple-blind placebo-controlled avastrovirus (AAstV), 34
trial (PrTBPC), 110 gut microbiota
Protective immune responses, 50 gram-negative bacteria, 15–20
histo-blood group antigens
(HBGAs), 15
R probiotics role, 20–27
Randomized double-blind placebo-controlled mamastrovirus (MAstV), 34
(RDBPC) trials, 110 microbiota, 15
Recombinant probiotic vaccines, 50–51 against norovirus infections, 32–33
Reovirus, 16 against rotavirus (RoV)
Respiratory syncytial virus (RSV), 6 infections, 27–32
Respiratory tract infections (RTIs), 4 Viral infections
Respiratory viruses (RVs), 4, 5, 13 cancer, 64–67
Rotavirus (RoVs), 16, 18, 20, 27–32, 53–54, general introduction, 63–64
91, 105 hepatitis B and C virus, 72–74
animal models (AMs), 30–32 herpes simplex viruses 1 and 2 (HSV-1 and
clinical trials (CTs), 29–30 HSV-2), 74–76
double-layered particle (DLP), 27 human-cell lymphotropic/leukemia virus
nonstructural protein (NSP), 27 (HTLV) infection, 69–71
triple-layered particle (TLP), 27 human immune deficiency virus (HIV),
RSV. See Respiratory syncytial virus (RSV) 76–77
RTIs. See Respiratory tract infections (RTIs) human papillomavirus (HPV) infection,
67–69
probiotics, 76–77
S type 1 diabetes (T1D), 71–72
Secretory IgA (sIgA) antibodies, 49 Viral trapping, 105
Semliki Forest virus (SFV), 90 VP1. See Viral capsid protein (VP1)
Simian immunodeficiency virus (SIV), 90 VSV. See Vesicular stomatitis
Suid herpesvirus (SHV-1), 90 virus (VSV)

New Insights On Antiviral Probiotics

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New Insights On Antiviral Probiotics

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Imad Al Kassaa

New Insights on Antiviral

ISBN 978-3-319-49687-0 ISBN 978-3-319-49688-7 (eBook)

Library of Congress Control Number: 2016959215

© Springer International Publishing AG 2017

Printed on acid-free paper

This Springer imprint is published by Springer Nature

I would like also to thank Dr. Khaled EL

The emergence of multidrug-resistant bacteria and the limited number of antivi-

1 Antiviral Probiotics: A New Concept in Medical Sciences . . . . . . . . . . 1

3.4 The Impact of Probiotics in Cancers Related to Human T-Cell

Keywords Antiviral probiotics • Respiratory viruses • Immunomodulation • Gut

© Springer International Publishing AG 2017 1

AMPs Antimicrobial peptides

RTIs Respiratory tract infections

Respiratory infections and gastroenteritis constitute the major causes of mortality

Immunobiotic Cancer prevention

Prevention of microbial Production of inhibitory

Fig. 1.1 Potential functions attributed to LAB probiotics [5]

1.2 Part I-A: Probiotics and Respiratory Infections

1.2.1.1 Lactobacillus Probiotic Strains in Viral Respiratory Infections

Lactobacillus is the most studied genus of anti-RVs probiotics, followed by the

Probiotic strains Origin Infection Mechanisms Experiment References

Table 1.1 (continued)

CIDs elderly Tiollier et al. [39];

Table 1.1 (continued)

Lactobacillus casei (L. casei) is a beneficial bacterium found naturally in both

1.2.1.2 Bifidobacteria Probiotic Strains in Viral Respiratory Infections

Bifidobacterium is a very important bacterium in animal and human flora. This

that the administration of a Bifico® strains mixture, in particular B. longum, led to

1.2.2 Conclusion and Perspectives

Lactobacillus and Bifidobacterium genera have the strongest antiviral activity

1.3 Part I-B Probiotics and Viral Gastroenteritis

1.3.1.1 The Importance of Microbiota

Recent studies have demonstrated the symbiotic relationship between intestinal

1.3.1.2 Histo-Blood Group Antigens (HBGAs) and Gram-Negative

1.3.1.3 Viral Gastroenteritis and the Role of Gram-Negative Bacteria

are the most frequent causative agent involved in gastroenteritis, in particular in

Does “Gut Microbiota” Enhance or Inhibit Viral Gastroenteritis?

depletion of intestinal microbiota reduced the replication of MuNoV in the distal

Direct and Indirect Mechanisms of Intestinal Microbiota, Which Enhance EnV

What Is the Direct Mechanism of the Gut Microbiota in Viral Infectivity?

1.3.1.4 Role of Probiotics in Gut Microbiota

In addition to the immunomodulatory effect of probiotics, these beneficial bacteria

Anti-enteric Viruses Probiotics

Reduction in symptoms and PrTBPC: 39children Pant et al. [128]

Probiotic strains Origin Infection Mechanisms Experiment References

TGEV Increase in NO production H4 CLAB [138]

Probiotic strains Origin Infection Mechanisms Experiment References

L. acidophilus LA-5, Dairy products probiotic strains Cell line: HT-29

Probiotic strains Origin Infection Mechanisms Experiment References

Indirect Mechanism of Anti-EnV Probiotics

Direct Mechanism of Anti-EnV Probiotics

1.3.1.5 Probiotic Strains Against Rotavirus (RoV) Infections

Clinical Trials (CTs)

rations reduced the infection symptoms (p = 0.0042) [122]. L. reuteri, called

Animal Models (AMs)

1.3.1.6 Probiotic Strains Against Norovirus Infections

A recent study has evaluated an engineered probiotic strain of L. paracasei which

1.3.1.7 Probiotics and Other Enteric Viruses

Astroviruses are nonenveloped viruses with positive-sense ssRNA. The Astroviridae

1.3.2 Conclusion and Perspectives

1. Fonkwo PN. Pricing infectious disease. EMBO Rep. 2008;9:S13–7. doi:10.1038/embor.2008.110.