Generation and Characterization of Novel Chimeric Recombinant Proteins of Staphylococcus

aureus and Clostridium perfringens for Diagnostic and Protective Application

Staphylococcus aureus and Clostridium perfringens are two important food borne
pathogens containing toxin molecules having potential as biological warfare agents and
responsible for many serious community and nosocomially acquired infections. Both the
organisms infect the gastrointestinal tracts and cause enterotoxaemia. Staphylococcal
entertoxins (SEA – SEH), clostrdial enterotoxin and epsilon toxin have thermal and
environmental stability, substantial accessibility (from soil and food) and most importantly
high potency making these two organisms suitable for creating biological emergencies in
the form of outbreaks. In addition, novel and accessible technologies give rise to
proliferation of such weapons that have implications for regional and global security. The
easy access to a wide range of disease-producing biological agents, their non-detection by routine
security systems, and their easy transportation from one location to another are other attractive
features (Atlas, 1998). Their properties of invisibility and virtual weightlessness render detection
and verification procedures ineffectual and make non-proliferation of such weapons impossibility.
Genetic engineering and information are increasingly open to misuse in the development and
improvement of infective agents as bioweapons. Such misuse could be envisaged in the
development of antibiotic-resistant micro-organisms, and in the enhanced invasiveness and
pathogenicity of commensals
Foodborne pathogens are estimated to be responsible for some 6.5 to 33 million cases on human
illnesses and up to 9000 deaths in the USA per annum (Buzby et al, 1996). The costs of human
illnesses attributed to foodborne causes are between US$2.9 and 6.7 billion, and are attributed to
six bacterial pathogens-Salmonella typhosa, Campylobacter jejuni, Escherichia coli 0157H:H7,
Listeria monocytogenes, Staphylococcus aureus and Clostridium perfringens found in animal
products. Consequently, there is the dangerous risk that such organisms could be used in biological
warfare and bioterrorism given that Salmonella, Campylobacter and Listeria have been
encountered in outbreaks of foodborne infections, and that cases of food poisoning have been
caused by Clostridium, Escherichia and Staphylococcus.( Biological warfare,
bioterrorism, biodefence and the biological and toxin weapons convention

Edgar J. DaSilvaVol.2 No3, Issue of December 15, 1999. )

The genus Clostridium consists of a diverse group of gram-positive bacteria which are
spore forming, and anaerobic. Many of these anaerobes are pathogenic for both humans
and other animals causing some of the dangerous diseases such as tetanus and botulism.
Clostridium perfringens is sulfite-reducing ubiquitous clostridium commonly inhabiting
the gastrointestinal tract of humans and other animals, as well as soil, food and sewage. It
has been shown to be a cause of human diseases such as gas gangrene (clostridial
myonecrosis), food poisoning, necrotizing enterocolitis of infants, enteritis necroticans
(pigbel), lamb dysentery, ovine enterotoxemia (struck), pulpy kidney disease of sheep,
and other enterotoxemic diseases of lambs and calves (Julian and Stewart. 1991). It is
also one of the most common causes of foodborne illnesses. Drug resistant C.
perfringens strains are becoming a major health concern. A study by Teuber indicated
that copious use of antibiotics in agriculture is promoting a large antibiotic resistance
problem in foodborne pathogens, including C. perfringens (Teuber, M. 1999).
A surveillance summary made by Centre for Disease Control and Prevention (CDC) in
the U. S. revealed that approximately 32% (89.4 million) of the U. S. population was
colonized with S. aureus by the year 2007. There was an estimated 292,000
hospitalizations with the diagnosis of S. aureus infection annually, in the U. S. alone. C.
perfringens poisoning is another most commonly reported food borne illness; at least 10
to 20 outbreaks have been reported annually in the U. S. alone since past 2 decades.
Antibiotic-associated diarrhoea (AAD) cases are serious complications
of treatment in which up to 80% in some series, the organism
responsible is undiagnosed. Although toxin-producing strains of
Clostridium difficile are the proposed aetiological agents, it remains
unclear which other species are the most significant causes of disease
(L. Joshy et al.). Clostridium perfringens and Staphylococcus aureus are
the most frequently cited alternative causes of AAD (Wilcox MH, 2000).
Though AAD or food poisoning cases are reported from many parts of India, there is no
designated laboratory for investigation and at the same time, no proper diagnostic
tools/systems available to delineate the cause of these infections/ intoxications. Most
probably, many of the food borne outbreaks go unreported because the implicated foods
or patient faeces are not tested routinely for these pathogens or their toxins.

The genus Clostridium consists of relatively large, Gram-positive, rod-shaped bacteria in the Phylum Firmicutes
(Clostridia is actually a Class in the Phylum). All species form endospores and have a strictly fermentative type
of metabolism. Most clostridia will not grow under aerobic conditions and vegetative cells are killed by exposure
to O2, but their spores are able to survive long periods of exposure to air. The clostridia are ancient organisms
that live in virtually all of the anaerobic habitats of nature where organic compounds are present, including soils,
aquatic sediments and the intestinal tracts of animals. Most of the clostridia are saprophytes, but a few are
pathogenic for humans, primarily Clostridium perfringens, C. difficile, C. tetani and C. tetani. Those that are
pathogens have primarily a saprophytic existence in nature and, in a sense, are opportunistic pathogens.
Clostridium perfringens, which produces a huge array of invasins and exotoxins, causes wound and surgical
infections that lead to gas gangrene, in addition to severe uterine infections. Clostridial hemolysins and
extracellular enzymes such as proteases, lipases, collagenase and hyaluronidase, contribute to the invasive
process. Clostridium perfringens also produces an enterotoxin and is an important cause of food poisoning.
Usually the organism is encountered in improperly sterilized (canned) foods in which endospores have
germinated.
Clostridium perfringens, previously known as Clostridium welchii, is the most common
cause of clostridial gas gangrene (80-90% of cases). Other clostridia species responsible
for the condition include Clostridium novyi (40%), Clostridium septicum (20%),
Clostridium histolyticum (10%), Clostridium bifermentans (10%), and Clostridium fallax
(5%). Clostridial gas gangrene is a highly lethal necrotizing soft tissue infection of
skeletal muscle caused by toxin- and gas-producing Clostridium species. The synonym
clostridial myonecrosis better describes both the causative agent and the target tissue.
Prior to the advent of antibiotics and mobile army surgical hospitals, as many as 5% of
battlefield injuries were complicated by this condition. However, the incidence rate
dropped to less than 0.01% during the Vietnam War. Presently, 90% of contaminated
wounds demonstrate clostridial organisms, but fewer than 2% develop clostridial
myonecrosis. This underscores the importance of host and local wound factors in the
development of this process, rather than the mere presence of the organisms in the
wound.

Clostridium perfringens is a common intestinal

inhabitant of man. However, C. perfringens type A
(CPA) isolates produce lethal necrotizing antigens and
the heat resistant forms of the organism are associated
with pathogenic outcome in humans. It has been
reported that about 2 to 5 per cent of all CPA produce
enterotoxin1-3. It may be responsible for 5 to 20 per
cent of all cases of antibiotic associated diarrhoea
(AAD) and sporadic non-food borne diarrhoea4 though
C. difficile is implicated in the most severe cases of
53
AAD and specific treatment is usually successful5.
C. difficile is much less often found in diarrhoea or
colitis where pseudomembranes are absent and
interestingly, some C. difficile negative cases of AAD
present with bloody diarrhoea6.

TOXINS

Strains of C. perfringens are classified as 5 biotypes A – E depending on the differential

production of four major (i.e. lethal) exotoxins (alpha, beta, epsilon and iota). In addition,
strains of C. perfringens may also produce a number of other toxins, including:
neuraminidase and enterotoxin (Cpe).

Infections due to C. perfringens show evidence of tissue necrosis, bacteremia,

emphysematous cholecystitis, and gas gangrene, which is also known as clostridial
myonecrosis.

Clostridum perfringens types A,B,C,D, and E produce at least 12 different antigens,

referred to as toxins, that may be involved in pathogenesis. These antigens have been
given the names alpha, beta, epsilon, and iota toxin (‘major’ toxins), and delta, theta,
kappa (collagenase), lambda (protease), mu (hyaluronidase), nu (deoxyribonuclease),
gamma and eta toxin (‘minor’ toxins). An enterotoxin and neuraminidase are also
produced by certain strains.( Pharmacology & Therapeutics
Volume 10, Issue 3, 1980, Pages 617-655Clostridium perfringens toxins (type A, B, C,
D, E) James L. McDonela)

http://www.clostridia.net/Cperfringens.htm
Clostridium perfringens type A & antibiotic associated
diarrhoea
Chetana Vaishnavi, Sukhminderjit Kaur & Kartar Singh
Indian J Med Res 122, July 2005, pp 52-56
Staphylococci (staph) are Gram-positive spherical bacteria that occur in microscopic clusters resembling grapes.
Bacteriological culture of the nose and skin of normal humans invariably yields staphylococci. In 1884, Rosenbach
described the two pigmented colony types of staphylococci and proposed the appropriate nomenclature:
Staphylococcus aureus (yellow) and Staphylococcus albus (white). The latter species is now named
Staphylococcus epidermidis. Although more than 20 species of Staphylococcus are described in Bergey's Manual
(2001), only Staphylococcus aureus and Staphylococcus epidermidis are significant in their interactions with
humans. S. aureus colonizes mainly the nasal passages, but it may be found regularly in most other anatomical
locales, including the skin, oral cavity and gastrointestinal tract. S epidermidis is an inhabitant of the skin.

Skin infections (see above) are the most common type of disease produced by
Staphylococcus. Staph infections of the skin can progress to impetigo (a crusting of the
skin) or cellulitis (inflammation of the connective tissue under the skin, leading to
swelling and redness of the area). In rare situations, a serious complication known as
scalded skin syndrome (see below) can develop. In breastfeeding women, Staph can
result in mastitis (inflammation of the breast) or in abscess of the breast. Staphylococcal
breast abscesses can release bacteria into the mother's milk.

When the bacteria enter the bloodstream and spread to other organs, a number of serious
infections can occur. Spread of the organisms to the bloodstream is known as bacteremia
or sepsis. Staphylococcal pneumonia predominantly affects people with underlying lung
disease and can lead to abscess formation within the lungs. Infection of the heart valves
(endocarditis) can lead to heart failure. Spread of Staphylococci to the bones can result in
severe inflammation of the bones known as osteomyelitis. When Staph bacteria are
present in the blood, a condition known as staphylococcal sepsis (widespread infection of
the bloodstream) or staphylococcal bacteremia exists. Staphylococcal sepsis is a leading
cause of shock and circulatory collapse, leading to death, in people with severe burns
over large areas of the body. When untreated, Staph aureus sepsis carries a mortality
(death) rate of over 80%. Although not common, Staph aureus has been reported as a
cause of chorioamnionitis and neonatal sepsis in pregnancy, but group B streptococci are
the most common bacterial cause of this life-threatening condition for the fetus.

Staphylococcal infections are contagious and can be transmitted from person to person.
Since pus from infected wounds may contain the bacteria, proper hygiene and
handwashing is required when caring for Staph-infected wounds.

Staphylococcal food poisoning is an illness of the bowels that causes nausea, vomiting,
diarrhea, and dehydration. It is caused by eating foods contaminated with toxins produced
by Staphylococcus aureus. Symptoms usually develop within one to six hours after eating
contaminated food. The illness usually lasts for one to three days and resolves on its own.
Patients with this illness are not contagious, since toxins are not transmitted from one
person to another.

Toxic shock syndrome is an illness caused by toxins secreted by Staph aureus bacteria
growing under conditions in which there is little or no oxygen. Toxic shock syndrome is
characterized by the sudden onset of high fever, vomiting, diarrhea, and muscle aches,
followed by low blood pressure (hypotension), which can lead to shock and death. There
may be a rash resembling sunburn, with peeling of skin. Toxic shock syndrome was
originally described and still occurs especially in menstruating women using tampons.

Since the 1970s, S. aureus strains have emerged resistant to the penicillinase-stable
penicillins (cloxacillin, dicloxacillin, methicillin, nafcillin, and oxacillin). The resistance
is the result of a supplemental penicillin binding protein (PBP 2a) encoded by the
chromosomal mecA gene. These strains historically are termed methicillin resistant S.
aureus (MRSA) and are resistant to all beta-lactam agents. Laboratory confirmation of
these strains can be problematic. The resistant strains are often heteroresistant. That is,
two populations coexist, on susceptible and the other resistant. Each cell has the genetic
information for resistance, but only a very small number express this resistance in vitro (1
in 104 to 1 in 108). Successful detection of MRSA largely depends on promoting the
growth of the resistant population. This is done by lowering the incubation temperature
to 35oC, using a 0.5 McFarland suspension directly from the colonies, supplementing
with 2% sodium chloride, and incubating for a full 24 hours in ambient air. Even with
these refinements, the heterogeneous expression of some isolates may be interpreted as
susceptible. The oxacillin-salt screening plate which supplemented with 4% sodium
chloride and 6 µg/ml of oxacillin can be used to improve detection of these strains. The
growth of more than one colony indicates resistance.

Most isolates of S. aureus are susceptible to vancomycin. The MIC is typically

between 0.5 and 2 micrograms/mL (μg/mL). In contrast, S. aureus isolates for which
vancomycin MICs are 8-16 μg/mL are classified as vancomycin intermediate (VISA),
and isolates for which vancomycin MICs are ≥32 μg/mL are classified as vancomycin-
resistant (VRSA). The glyopeptide class of antibiotics includes both vancomycin and
teicoplanin. And, some of the original VISA strains were also intermediate for
teicoplanin, hence the name, GISA. However, not all strains of VISA show intermediate
susceptibility to teicoplanin.

CLSI (formerly NCCLS) lists only susceptible disk diffusion interpretive

criteria (in mm) for vancomycin and Staphylococcus spp. Insufficient numbers of non-
susceptible isolates from patients have been isolated to develop resistant and intermediate
breakpoints. Organisms for which the vancomycin zone diameters are ≥15mm are
considered susceptible, although this breakpoint is unreliable for detecting VISA strains.

Invasion of Host Tissues

The invasion of host tissues by staphylococci involves the production of a large

number of extracellular proteins.

The α, β, δ -Toxins

The α-toxin is expressed as a monomer that binds to the eukaryotic cell

membranes, the subunits oligomerize to form heptameric rings with a central pore
through which cellular contents leak. This toxin also induces the death of innate and
acquired immunity cells, interferes with the metabolism of arachidonic acid, exocytosis
and induces dysfunctions in contractility, leading to bacterial spread and alterations of the
hemostasis (28a).

The β-toxin or sphingomyelinase C is a haemolysin, which targets lipid-rich

membranes. It causes lysis of erythrocytes and mononuclear cells, and also induces a
strong inflammatory response. The majority of human isolates of S. aureus do not
express ß-toxin, but it is frequently found among strains responsible for bovine mastitis
(250a).

The δ -toxin consisting of a peptide 26aa, but its role in disease is unknown.

Phospholipase C

S. aureus secretes a phospholipase C, which specifically hydrolysis membrane

lipid and protein-containing glycosyl phosphatidylinositol (92b).

Metalloproteases

The aureolysine, member of the thermolysines family, is an extracellular and

zinc dependent metalloprotease. This enzyme destroys host defences molecules (14a).

Hyaluronidase and hyaluronate lyase

These enzymes digest hyaluronic acid, polymer present in the vitreous humour,
skin, bones and synovial fluid, promoting the infection process by dispersal and tissue
degradation (123a).
Exfolative Toxins

There are two major biologically and serologically distinct S. aureus exfolative
toxin isoforms, exfolative toxin A and exfolative toxin B, that are primarily responsible
for the skin manifestations of staphylococcal scalded skin syndrome and bullous
impetigo. Five percent of clinical S. aureus isolates produce either exfoliative toxin A,
exfoliative toxin B or both toxins. They cleave the stratum granulosum from the stratum
spinosum by targeting desmosomes (4a).

Epidermal Cell Differentiation Inhibitor

The epidermal cell differentiation inhibitors which are mono-ADP

ribosyltransferases belonging to the Rho family, are found in 8% of disease strains and in
3.7% of nasal carrier strains under 3 isoforms: epidermal cell differentiation inhibitors-A,
B and C. Although epidermal cell differentiation inhibitor-A inhibits the differentiation of
keratinocytes in vitro, the role of epidermal cell differentiation inhibitors in human
diseases is not established (453a).

Avoidance of Host Defenses

The S. aureus wall consists of two major components, i.e. peptidoglycan and
lipoteichoic acid, both analogous to the lipopolysaccharide in gram-negative bacteria.
They are able to induce the release of cytokines by macrophages, the activation of the
complement system and the platelets aggregation, thus triggering a disseminated
intravascular coagulation (236a).

Capsular Polysaccharides

Bacterial biofilms provide a niche for bacterial adherence and persistance on

implantable medical devices. S. aureus synthesizes exopolysaccharides, or glycocalix,
components of this biofilm. These capsular polysaccharides are found in 90% of clinical
strains. Eleven serotypes have been described, type 5 and 8 are most common among
human isolates (80%). Their role in virulence, however, is controversial, because these
capsular polysaccharides are also a target for protective antibodies.

Enterotoxins
 S. aureus can produces a large diversity of exoproteins belonging to the family
of superantigens, stimulating polyclonal T-cell proliferation through co-ligation between
major histocompatibility complex (MHC) class II molecules on antigen-presenting cells
and the variable portion of the T-cell antigen receptor β chain or α chain with no need for
prior antigen-presenting cells processing. T-cell/ antigen-presenting cells activation by
these toxins leads to the release of large amounts of various cytokines/lymphokines
which are deleterious for the host (99). Twenty different enterotoxins have been
described, among them, staphylococcal toxic shock syndrome toxin (TSST-1),
staphylococcal enterotoxin A, B, C or enterotoxins coded by egc cluster (179a). Beside
their superantigenic properties, they are also pyrogenic and enteropathogenic for the
majority, thus explaining their implication in both staphylococcal toxic shock symdrome
(TSS) and food poisoning. The enterotoxins have also been implicated in a number of
autoimmune disorders (rheumatic arthritis, etc.) and other abnormal immunologic states
such as psoriasis, atopic dermatitis and Kawasaki syndrome (255a).

Protein A

Protein A is a multifunctional virulence factor produced by almost all clinical

isolates: it inhibits opsonophagocytosis via binding of immunoglobulin Fc fragment, it is
also a B cell “superantigen” promoting B cell activation. It is a MSCRAMM, allowing
the attachment of vonWillebrand factor which is present on the injured endothelium.
Finally, protein A plays a pro-inflammatory role, via the activation of the tumor necrosis
factor receptor (TNFR1) present on the respiratory epithelium.

The Fatty Acid Modifying Enzyme and Lipases

More than 80% of strains of S. aureus express fatty acid modifying enzyme
which modify antibacterials lipids and thus may contribute to bacterial survival, including
in abscesses.

V8 Protease

The V8 protease is an extracellular serine protease witch possesses many

structural similarities with exfoliative toxin. It cleaves the peptide bonds, inactivating in
vitro and in vivo the action of antibodies and may protect against antimicrobial peptides
such as neutrophil defensin proteins and bactericidal platelet proteins thus contributing to
tissue proteins destruction during the invasion (310a).

Leukocidins

These toxins consist of two separately secreted and non-associated proteins

(class S and class F components) acting synergistically and promoting eukaryotic cell
lysis. They are: γ-haemolysin, Panton Valentine leukocidin (PVL) and leukocidins
LukD/E and LukM/F. The γ haemolysin is produced by almost all (≥ 99%) S. aureus
strains.

The Panton-Valentine leukocidin is a pore forming toxin which damages

human neutrophils, recovered from less than 2% of S. aureus strains It has been
epidemiologically linked to primary skin and soft tissue infections and to deep seated
infections such as necrotizing pneumonia and severe recurrent osteomyelitis occurring in
young immunocompetent patients. The genes coding for leukotoxins LukE-LukD were
detected in a high prevalence (82%) among blood isolates and 61% among nasal isolates
(422a).

Lina G, Vandenesch F, Etienne J. A brief history of Staphylococcus aureus Panton

Valentine leucocidin

John JF, Lindsay JA. Clones and Drones: Do Variants of Panton-Valentine

Leukocidin Extend the Reach of Community-Associated Methicillin-Resistant
Staphylococcus aureus? J Infect Dis 2008;197:175-178.

Staphylokinase

The staphylokinase, 136AA protein encoded by a bacteriophage, has two

important properties. Acting on the immunity, staphylokinase inhibits neutrophils
bactericides peptides by linking to the α-defensin. On the haemostasis, staphylokinase
acts as a plasminogen activator which causes dissolution of fibrin clots, and lyse fibrin
(34a).
 Throughout in vitro growth and during an infection, Staphylococcus aureus
responds to environmental signals by selectively generating specific virulence proteins. A
complex regulatory network, based on the accessory gene regulator (agr), a quorum-
sensing system, settles precise and flexible coordination of protein expression in response
to the microenvironment. Thus during infection, the early expression of the MSCRAMM
proteins facilitates initial colonization of tissue sites, whereas the later elaboration of
toxins facilitates spread and promotes acquisition of additional nutrients of S. aureus.

Literature Survey:
In 2005, Clostridium Perfringens Type A Toxoid from Novartis Animal
Health is the first commercial product for cattle to receive a
conditional license by the USDA to aid in the control of disease
syndromes caused by the alpha toxin of C. perfringens.

Vaccine. 2009 Nov 5;27 Suppl 4:D44-7.

Clostridium perfringens vaccines.

Titball RW.

Source

School of Biosciences, Geoffrey Pope Building, University of Exeter, Devon EX4 4QD,
United Kingdom. R.W.Titball@exeter.ac.uk

Abstract

Both Clostridium perfringens spores and toxins have reportedly been considered as a
biological warfare agents. The spores may be incorporated into weapons which cause
traumatic injury, and the resulting delivery of spores deep into tissues would result in the
development of gas gangrene. Of the C. perfringens toxins, the epsilon-toxin is of
particular concern and now appears on the list of CDC select agents. Currently there are
no licensed vaccines suitable for use in humans which protect against either gas gangrene
or epsilon-toxin. However, vaccines being developed for use in animals have the
potential to be developed for use in humans.
Immunization against Clostridium
perfringens cells elicits protection
against Clostridium tetani in mouse
model: identification of cross-reactive
proteins using proteomic methodologies
Syed Imteyaz Alam , Sunita Bansod and Lokendra Singh

BMC Microbiology 2008, 8:194

Several striking findings have emerged from the complete

genome sequencing data of these clostridial pathogens
[7,8]. Many homologous ORFs have been identified in the
genomes of C. tetani and C. perfringens by comparative
genomic analysis of the two genomes. Of the total 2372
ORFs observed in C. tetani E88, 1705 ORFs had a close
homologue in C. perfringens genome showing significant
sequence similarity [8]. This suggested a likelihood of
extensive sharing of common epitopes between homologous
proteins of these two medically important pathogens.
To examine this hypothesis, we probed the total
cellular proteins of C. tetani with antisera raised against
whole cells of C. perfringens ATCC13124. Cross-reactive
proteins have been identified and protection against challenge
with C. tetani to animals actively immunized with C.
perfringens whole cell has been reported.

Control of Clostridium perfringens

Vaccines using
an Indirect Competitive ELISA for the
Epsilon
Toxin Component
Examination of the Assay by a Collaborative Study
U. Rosskopf-Streicher, P. Volkers, E. Werner
Pharmeuropa Bio 2003-2

Clostridium perfringens vaccine for veterinary use

(0363). In: Ph. Eur. 4th ed. Strasbourg: Council of
Europe; 2002:2250-51.
Investigations on the replacement of the mouse neutralisation test for proving
vaccine batches of
Clostridium (C.) perfringens toxoid vaccines were performed since several years. The
European Pharmacopoeia
(Ph. Eur.) monograph Clostridium perfringens vaccines for veterinary use (0363) is
prescribing a potency test by
immunisation of rabbits and checking the induction of specific antibodies against the
toxins in a mouse neutralisation
test. Since the monograph was revised, immunochemical methods are favoured to
detect directly specific antibodies
in the rabbit sera.
An indirect competitive ELISA using a monoclonal antibody was established at the
Paul-Ehrlich-Institut for the
detection of antibodies against the epsilon toxin component of C. perfringens. It was
revised using the Clostridia rabbit
antiserum Ph. Eur. Biological Reference Preparation (BRP) Batch 1 as reference
serum. With a defined content of
11 International Units (IU) of C. perfringens epsilon antitoxin this reference serum
enables the calculation of the
potency of rabbit sera under test.
For the collaborative study vaccine products of different composition licensed for the
German and European markets
were used. Seven international laboratories were included. Aim was to make a
prediction on the transferability and
precision of the test method.
The results showing a satisfactory intermediate precision and transferability of the
test confirmed the applicability of the
ELISA method for the batch control of C. perfringens vaccines. Therefore a
replacement of the mouse neutralisation test
is available.

Vaccine
Volume 11, Issue 12, 1993, Pages 1253-1258

A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens

protects mice against experimental gas gangrene

E.D. Williamsona and R.W. Titball

Fragments of the alpha-toxin of Clostridium perfringens have been produced using

genetic manipulation techniques. Antibody which cross-reacted with the alpha-toxin was
induced after immunization with fragments representing the N- (Cpa1–249) and C-terminal
(Cpa247–370) domains of the toxin. Smaller fragments of the alpha-toxin did not induce
cross-reacting antibody. Anti-Cpa1–249 serum neutralized phospholipase C activity but not
haemolytic activity of the toxin. Anti-Cpa247–370 serum neutralized both the phospholipase
C and haemolytic activities. Only immunization with Cpa247–370 induced protection against
the lethal effects of the toxin. Immunization with Cpa247–370 also provided protection in a
mouse model against at least 10 LD100 doses of C. perfringens type A. This result
confirms the essential role of this toxin in the pathogenesis of gas gangrene.

Staphylococcus aureus is an important pathogen in the hospital and in the community,

and it is increasingly resistant to multiple antibiotics. A nonantimicrobial approach to
controlling S aureus is needed. The most extensively tested vaccine against S aureus,
which is a capsular polysaccharide-based vaccine known as StaphVAX, showed promise
in an initial phase 3 trial, but was found to be ineffective in a confirmatory trial, leading
to its development being halted. Likewise, a human IgG preparation known as INH-A21
(Veronate) with elevated levels of antibodies to the staphylococcal surface adhesins ClfA
and SdrG made it into phase 3 testing, where it failed to show a clinical benefit. Several
novel antigens are being tested for potential inclusion in a staphylococcal vaccine,
including cell wall-anchored adhesin proteins and exotoxins. Given the multiple and
sometimes redundant virulence factors of S aureus that enable it to be such a crafty
pathogen, if a vaccine is to prove effective, it will have to be multicomponent,
incorporating several surface proteins, toxoids, and surface polysaccharides.

Infectious Disease Clinics of North America

Volume 23, Issue 1 , Pages 153-171, March 2009

Staphylococcal Vaccines and

Immunotherapies
• Adam C. Schaffer, MD

Affiliations

o Department of Medicine, Brigham and Women's Hospital and Harvard

Medical School, PBB-B-422, 75 Francis street, Boston, MA 02115, USA
 ,

• Jean C. Lee

The Lancet Infectious Diseases, Volume 2, Issue 4, Page 201, April 2002
<Previous Article|Next Article>
doi:10.1016/S1473-3099(02)00254-2 Cite or Link Using DOI

Staphylococcal vaccine one step closer to

reality
Martina Habeck
American scientists report in the New England Journal of Medicine that a new vaccine,
StaphVax, provided immunity to Staphylococcus aureus infection in most dialysis
patients who participated in a phase III study; the patients were protected for up to 10
months.
USE OF A
STAPHYLOCOCCUS AUREUS
CONJUGATE VACCINE IN PATIENTS
RECEIVING HEMODIALYSIS
Henry Shinefield, M.D., Steven Black, M.D., Ali Fattom, Ph.D., Gary Horwith, M.D.,
Scott Rasgon, M.D., Juan Ordonez, M.D., Hock Yeoh, M.D., David Law, M.D., John B.
Robbins, M.D., Rachel Schneerson, M.D., Larry Muenz, Ph.D., Steve Fuller, Ph.D.,
Joanie Johnson, R.N., Bruce Fireman, M.A., Harry Alcorn, Pharm.D., and Robert Naso,
Ph.D
N Engl J Med, Vol. 346, No. 7

n patients with decreased resistance to infection, Staphylococcus aureus is a major cause

of bacteremia and its complications. The capsular polysaccharides are essential for the
pathogenesis of and immunity to S. aureus infection and are targets for vaccines. S.
aureus capsular polysaccharides alone are poor immunogens. Their immunogenicity is
enhanced by conjugation with carrier proteins.15-17 Monovalent vaccines containing S.
aureus type 5 or type 8 capsular polysaccharide bound to recombinant exoprotein A, a
nontoxic variant of Pseudomonas aeruginosa exotoxin A expressed in Escherichia coli,
are immunogenic and well tolerated in healthy adults and in patients with end-stage renal
disease.18 In a prior study of patients with end-stage renal disease who were receiving
peritoneal dialysis, a low dose of a conjugate antistaphylococcal vaccine was not
efficacious.

VIENNA, Aug 28 (Reuters) – Austrian biotech firm Intercell <ICEL.VI> said on

Thursday its partner Merck & Co <MRK.N> has started a Phase II clinical trial to
evaluate a vaccine candidate against Staphylococcus aureus infections.

Bacteria have evolved an elaborate small molecule-based communication system called

quorum sensing (QS). QS plays an important role in mediating bacterial virulence, and it
constitutes a newly emerging target for development of antibacterial agents. Park and
colleagues report an immunopharmacotherapeutic approach for the attenuation of (QS) in
the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer
monoclonal antibody was elicited against a rationally designed hapten and was shown to
efficiently inhibit QS in vitro. Importantly, the anti-autoinducer antibody suppressed S.
aureus pathogenicity in an abscess formation mouse model and provided mice with a
complete protection against a lethal S. aureus challenge. These findings build a strong
foundation for further investigation of using immunopharmacotherapy for the treatment
of bacterial infections in which the expression of virulence factors is under QS control.

Infection Control by Antibody Disruption of Bacterial Quorum

Sensing Signaling

Junguk Park1, 2, 3, 5, Reshma Jagasia1, 2, 3, 5, Gunnar F. Kaufmann1, 2, 3, John C.

Mathison2, Diana I. Ruiz1, Jason A. Moss1, 2, 3, Michael M. Meijler1, 2, 3, Richard J.
Ulevitch2 and Kim D. Janda1, 2, 3, 4, ,

Chemistry & Biology, Volume 14, Issue 10, 1119-1127, 26 October 2007

Summary
• Quorum sensing (QS) is the process through which bacteria communicate
utilizing small diffusible molecules termed autoinducers. It has been demonstrated
that QS controls a plethora of microbial processes including the expression of
virulence factors. Here we report an immunopharmacotherapeutic approach for
the attenuation of QS in the Gram-positive human pathogen Staphylococcus
aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited
against a rationally designed hapten, and efficiently inhibited QS in vitro through
the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus
RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an
abscess formation mouse model in vivo and provided complete protection against
a lethal S. aureus challenge. These findings provide a strong foundation for
further investigations of immunopharmacotherapy for the treatment of bacterial
infections in which QS controls the expression of virulence factors.

Antibodies to Block Staph Virulence

Michael Otto1, ,

1
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases,
The National Institutes of Health, 903 South 4th Street, Hamilton, MT 59840, USA

Corresponding author
Summary
• Antibodies rationally designed against staphylococcal virulence signals inhibit the
development of experimental disease by passive immunization [1], indicating
great potential for therapeutic approaches against staphylococcal and other
bacterial infections.

Abstract

Vaccination has proved relatively unsuccessful against the common mammalian

commensal bacteria Staphylococcus, despite almost a century of experimentation. Recent
progress in clinical trials, animal models and molecular laboratories offers hope that these
organisms might be partially or wholly controlled by augmenting host responses.

Abstract

Staphylococcal vaccination has proved relatively unsuccessful despite almost a century of

work. Recent progress however offers hope that these organisms might be partially or
wholly controlled by augmenting the host responses.

Staphylococcal vaccinesColin A. MichieTrends in Immunology

Volume 23, Issue 9, 1 September 2002, Pages 461-463
·

SEB Vaccines:

Infect Immun. 1991 September; 59(9): 2978-2986

Biodegradable and biocompatible poly(DL-lactide-co-glycolide)

microspheres as an adjuvant for staphylococcal enterotoxin
B toxoid which enhances the level of toxin-neutralizing
antibodies.
J H Eldridge, J K Staas, J A Meulbroek, T R Tice and R M Gilley

Department of Medicine, The University of Alabama, Birmingham 35294.

ABSTRACT

Microspheres composed of biocompatible, biodegradable poly(DL-lactide-co-glycolide)

(DL-PLG) and staphylococcal enterotoxin B (SEB) toxoid were evaluated as a vaccine
delivery system when subcutaneously injected into mice. As measured by circulating
immunoglobulin G (IgG) antitoxin titers, the delivery of SEB toxoid via DL-PLG
microspheres, 1 to 10 microns in diameter, induced an immune response which was
approximately 500 times that seen with nonencapsulated toxoid. The kinetics, magnitude,
and duration of the antitoxin response induced with microencapsulated toxoid were
similar to those obtained when an equal toxoid dose was administered as an emulsion
with complete Freund adjuvant. However, the microspheres did not induce the
inflammation and granulomata formation seen with complete Freund adjuvant. The
adjuvant activity of the microspheres was not dependent on the superantigenicity of SEB
toxin and was equally effective at potentiating circulating IgG antitrinitrophenyl levels in
response to microencapsulated trinitrophenyl-keyhole limpet hemocyanin. Empty DL-
PLG microspheres were not mitogenic, and SEB toxoid injected as a mixture with empty
DL-PLG microspheres was no more effective as an immunogen than toxoid alone.
Antigen-containing microspheres 1 to 10 microns in diameter exhibited stronger adjuvant
activity than those greater than 10 microns, which correlated with the delivery of the 1- to
10-microns, but not the greater than 10-microns, microspheres into the draining lymph
nodes within macrophages. The antibody response induced through immunization with
microencapsulated SEB toxoid was protective against the weight loss and splenic V beta
8+ T-cell expansion induced by intravenous toxin administration. These results show that
DL-PLG microsphere vaccine delivery systems, which are composed of pharmaceutically
acceptable components, possess a strong adjuvant activity for their encapsulated antigens.

Infection and Immunity, April 2001, p. 2031-2036, Vol. 69, No. 4

0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.4.2031-2036.2001

Mucosal Vaccination with Recombinantly Attenuated

Staphylococcal Enterotoxin B and Protection in a Murine
Model
Bradley G. Stiles,* Anthony R. Garza, Robert G. Ulrich, and James W. Boles

Toxinology and Aerobiology Division, United States Army Medical Research Institute of
Infectious Diseases, Fort Detrick, Maryland 21702-5011

Received 25 September 2000/Returned for modification 14 December 2000/Accepted 3

January 2001

Previous work in our laboratory revealed that mice parenterally vaccinated with
recombinantly attenuated staphylococcal enterotoxin (SE) or toxic shock syndrome toxin
1 develop protective antibodies against a lethal intraperitoneal (i.p.) toxin challenge. This
study investigated the efficacy of nasal and oral immunizations with an SEB vaccine
(SEBv) toward an i.p. or mucosal (via an aerosol) toxin challenge. Both vaccination
routes, with the immunoadjuvant cholera toxin (CT), elicited comparable SEB-specific
immunoglobulin A (IgA) and IgG levels in saliva. Nasal or oral inoculations also
generated SEB-specific IgA, IgG, and IgM in the serum, but the nasal route yielded
higher specific IgG titers. SEBv alone, when given nasally or orally, did not induce any
detectable SEB-specific antibody. Mice vaccinated mucosally were protected against a
50% lethal dose of wild-type SEB given i.p. or mucosally, thus demonstrating that nasal
or oral administration of this SEBv, with CT, elicits systemic and mucosal antibodies to
SEB that protect against SEB-induced lethal shock.

Infect. Immun., 11 1996, 4686-4693, Vol 64, No. 11

Copyright © 1996, American Society for Microbiology

Immunogenicity and efficacy against lethal aerosol

staphylococcal enterotoxin B challenge in monkeys by
intramuscular and respiratory delivery of proteosome-
toxoid vaccines

GH Lowell, C Colleton, D Frost, RW Kaminski, M Hughes, J Hatch, C Hooper, J

Estep, L Pitt, M Topper, RE Hunt, W Baker and WB Baze
Division of Pathology, Walter Reed Army Institute of Research, Washington, D.C. 20307, USA.

Staphylococcal enterotoxin B (SEB), a primary cause of food poisoning, is also a

superantigen that can cause toxic shock after traumatic or surgical staphylococcal wound
[correction of would] infections or viral influenza-associated staphylococcal
superinfections or when aerosolized for use as a potential biologic warfare threat agent.
Intranasal or intramuscular (i.m.) immunization with formalinized SEB toxoid formulated
with meningococcal outer membrane protein proteosomes has previously been shown to
be immunogenic and protective against lethal respiratory or parenteral SEB challenge in
murine models of SEB intoxication. Here, it is demonstrated that immunization of
nonhuman primates with the proteosome-SEB toxoid vaccine is safe, immunogenic, and
protective against lethal aerosol challenge with 15 50% lethal doses of SEB. Monkeys (10
per group) were primed i.m. and given booster injections by either the i.m. or intratracheal
route without adverse side effects. Anamnestic anti-SEB serum immunoglobulin G (IgG)
responses were elicited in all monkeys, but strong IgA responses in sera and bronchial
secretions were elicited both pre- and post-SEB challenge only in monkeys given booster
injections intratracheally. The proteosome-SEB toxoid vaccine was efficacious by both
routes in protecting 100% of monkeys against severe symptomatology and death from
aerosolized-SEB intoxication. These data confirm the safety, immunogenicity, and
efficacy in monkeys of parenteral and respiratory vaccination with the proteosome-SEB
toxoid, thereby supporting clinical trials of this vaccine in humans. The safety and
enhancement of both bronchial and systemic IgA and IgG responses by the proteosome
vaccine delivered by a respiratory route are also encouraging for the development of
mucosally delivered proteosome vaccines to protect against SEB and other toxic or
infectious respiratory pathogens.

Infection and Immunity, May 2002, p. 2278-2281, Vol. 70, No. 5

0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.5.2278-2281.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Protection against Bacterial Superantigen Staphylococcal

Enterotoxin B by Passive Vaccination
Ross D. LeClaire,1 Robert E. Hunt,2 and Sina Bavari1*
U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland
21702-5011,1 Medical Research and Evaluation Facility, Battelle, Columbus, Ohio
43201-26932

Received 31 August 2001/ Returned for modification 19 November 2001/ Accepted 25

January 2002

We investigated the ability of two overlapping fragments of staphylococcal enterotoxin B

(SEB), which encompass the whole toxin, to induce protection and also examined if
passive transfer of chicken anti-SEB antibodies raised against the holotoxin could protect
rhesus monkeys against aerosolized SEB. Although both fragments of SEB were highly
immunogenic, the fragments failed to protect mice whether they were injected separately
or injected together. Passive transfer of antibody generated in chickens (immunoglobulin
Y [IgY]) against the whole toxin suppressed cytokine responses and was protective in
mice. All rhesus monkeys treated with the IgY specific for SEB up to 4 h after challenge
survived lethal SEB aerosol exposure. These findings suggest that large fragments of SEB
may not be ideal for productive vaccination, but passive transfer of SEB-specific
antibodies protects nonhuman primates against lethal aerosol challenge. Thus, antibodies
raised in chickens against the holotoxin may have potential therapeutic value within a
therapeutic window of opportunity after SEB encounter.

Superantigen Vaccines: A Comparative

Study of Genetically Attenuated
Receptor-Binding Mutants of
Staphylococcal Enterotoxin A
1. Sina Bavari,
2. Beverly Dyas and
3. Robert G. Ulrich
4. J Infect Dis. (1996) 174 (2): 338-345.

+ Author Affiliations

1. Department of Cell Biology and Biochemistry and of Molecular Biology and

Immunology, Army Medical Research Institute of Infectious Diseases, Frederick,
Maryland
1. Reprints or correspondence: Drs. Sina Bavari or Robert G. Ulrich, Depts. of Cell
Biology and Biochemistry or Molecular Biology and Immunology, respectively,
AMRIID, Bldg. 1425, Frederick, MD 21702-5011.
Abstract

Superantigens exert their pathologic effects by direct binding to major histocompatibility

complex (MHC) class II molecules and T cell antigen receptors (TCR), thus
circumventing the normal, antigen-specific immune response. A direct link between
disease and toxin suggests an excellent opportunity for vaccine intervention. Site-directed
mutants of staphylococcal enterotoxin A (SEA) that have attenuated binding to either the
TCR or the MHC class II molecule were developed. Both kinds of SEA mutants induced
high levels of antibody against SEA when used as vaccines, and the immunized animals
were fully protected when challenged with wild type toxin. However, a residual lethality
was associated with the attenuated TCR-binding mutant. These results, combined with an
understanding of the molecular nature of superantigen and receptor interactions, indicate
that targeting MHC class II binding by site-directed mutagenesis will produce the most
effective vaccine.

Infect. Immun., 05 1996, 1706-1713, Vol 64, No. 5

Copyright © 1996, American Society for Microbiology

Intranasal and intramuscular proteosome-staphylococcal

enterotoxin B (SEB) toxoid vaccines: immunogenicity and
efficacy against lethal SEB intoxication in mice

GH Lowell, RW Kaminski, S Grate, RE Hunt, C Charney, S Zimmer and C

Colleton
Division of Pathology, Walter Reed Army Institute of Research, Washington, DC 20307, USA.

Intranasal or intramuscular (i.m.) immunization of mice and i.m. immunization of rabbits

with formalinized staphylococcal enterotoxin B (SEB) toxoid in saline elicited higher
anti-SEB serum immunoglobulin G (IgG) titers when the toxoid was formulated with
proteosomes. In addition, intranasal immunization of mice with this proteosome-toxoid
vaccine elicited high levels of anti-SEB IgA in lung and intestinal secretions, whereas the
toxoid without proteosomes did not. Two i.m. immunizations with proteosome-toxoid
plus alum also induced higher murine serum responses than alum-adjuvanted toxoid
without proteosomes. Furthermore, proteosome-toxoid delivered intranasally in saline or
i.m. with either saline or alum afforded significant protection against lethal SEB
challenge in two D-galactosamine-sensitized murine models of SEB intoxication, i.e., the
previously described i.m. challenge model and a new respiratory challenge model of
mucosal SEB exposure. Efficacy correlated with the induction of high serum levels of
anti-SEB IgG. In contrast, intranasal or i.m. immunization with toxoid in saline without
proteosomes was not significantly protective in either challenge model. Proteosome-
toxoid plus alum given i.m. also elicited more significant protection against respiratory
challenge than the alum-adjuvanted toxoid alone. The capacity of proteosomes to enhance
both i.m. and intranasal immunogenicity and efficacy of SEB toxoid indicates that testing
such proteosome-SEB toxoid vaccines in the nonhuman primate aerosol challenge model
of SEB intoxication prior to immunogenicity trials in humans is warranted. These data
expand the applicability of the proteosome mucosal vaccine delivery system to protein
toxoids and suggest that respiratory delivery of proteosome vaccines may be practical for
enhancement of both mucosal and systemic immunity against toxic or infectious diseases.

Immune Protection against

Staphylococcal Enterotoxin-Induced
Toxic Shock by Vaccination with a
Venezuelan Equine Encephalitis Virus
Replicon
1. John S. Lee1,
2. Beverly K. Dyas2,
3. Steve S. Nystrom2,
4. Cathleen M. Lind1,
5. Jonathan F. Smith1,a and
6. Robert G. Ulrich2
7. J Infect Dis. (2002) 185 (8): 1192-1196.

+ Author Affiliations
1
1. Virology Division, United States Army Medical Research Institute of Infectious
Diseases, Fort Detrick, Frederick, Maryland
2. 2Toxinology Division, United States Army Medical Research Institute of
Infectious Diseases, Fort Detrick, Frederick, Maryland

+ Author Notes

• ↵a Present affiliation: AlphaVax, Durham, North Carolina.

1. Reprints or correspondence: Dr. John S. Lee, Virology Division, US Army

Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD
21702 (John.Lee@det.amedd.army.mil).

Abstract

A candidate vaccine against staphylococcal enterotoxin B (SEB) was developed using a

Venezuelan equine encephalitis (VEE) virus vector. This vaccine is composed of a self-
replicating RNA, termed “replicon,” containing the VEE nonstructural genes and cis-
acting elements and a gene encoding mutagenized SEB (mSEB). Cotransfection of baby
hamster kidney cells with the mSEB replicon and 2 helper RNA molecules resulted in the
release of propagation-deficient mSEB-VEE replicon particles (mSEB-VRPs). Mice
inoculated subcutaneously with mSEBVRPs were protected (15 of 20 mice) from a
challenge with 5 median lethal dose units of wildtype (wt) SEB. T cells from mice
vaccinated with mSEB-VRP responded normally both in vitro to wt SEB and in recall
response to the inactivated mSEB polypeptide. The profile of cytokines measured after
challenge with wt SEB suggested that the mode of protection was predominantly Th1
dependent. Our results suggest that the VEE replicon is a practical and convenient model
system for evaluating efficacy of vaccines for the control of bacterial diseases.

Humanlike immune response of human

leukocyte antigen-DR3 transgenic mice
to staphylococcal enterotoxins: A novel
model for superantigen vaccines
1. Luis DaSilva1,
2. Brent C. Welcher1,
3. Robert G. Ulrich1,
4. M. Javad Aman1,
5. Chella S. David2 and
6. Sina Bavari1

J Infect Dis. (2002) 185 (12): 1754-1760.

+ Author Affiliations
1
1. United States Army Medical Research Institute of Infectious DiseasesFrederick,
Maryland
2. 2Department of Immunology, Mayo Medical SchoolRochester, Minnesota
1. Reprints or correspondence: Dr. Sina Bavari, United States Army Medical
Research Institute of Infectious Diseases, Dept. of Cell Biology and
Biochemistry, 1425 Porter St., Frederick, MD 21702-5011 (bavaris@ncifcrf.gov).

Abstract

This study examined the biologic responses of transgenic mice expressing human
leukocyte antigen (HLA)-DR3 and human CD4 molecules, in the absence of murine
major histocompatibility complex (MHC) class II molecules (Ab0), to staphylococcal
enterotoxins (SEs) and evaluated protective immunity of a nonsuperantigen form of SEB
against wild-type holotoxin. HLA-DR3 transgenic mice responded to several log lower
concentrations of SEs and secreted higher levels of proinflammatory cytokines than did
wild-type mice. Vaccination of transgenic mice with a nonsuperantigenic form of SEB
induced high levels of neutralizing anti-SEB antibodies, which protected the mice from a
surge in proinflammatory cytokine secretion after SEB challenge. The humanlike
responses of the transgenic mice to SEs support the hypothesis that these mice represent
an appropriate model to examine vaccines and therapeutics against SEs. This is thought
to be the first report of examination of a vaccine against SEB in the context of human
MHC class II receptors.

Vaccine
Volume 15, Issue 2, February 1997, Pages 133-139

doi:10.1016/S0264-410X(96)00166-1 | How to Cite or Link Using Cited By in Scopus

DOI
Copyright © 1997 Published by Elsevier Science Ltd. (22)
Permissions & Reprints

Paper
Staphylococcal enterotoxin B mutants (N23K and F44S): biological effects and vaccine
potential in a mouse model

Mary Alice Woody, Teresa Krakauer and Bradley G. Stiles

Department of Immunology and Molecular Biology, United States Army Medical

Research Institute of Infectious, Diseases, Fort Detrick, Frederick, MD 21702-5011, USA

Received 12 February 1996;

revised 5 July 1996;
accepted 5 July 1996. ;
Available online 9 December 1997.

Abstract

Superantigens produced by Staphylococcus aureus can cause food poisoning and toxic
shock syndrome. The biological activities and vaccine potential of mutant staphylococcal
enterotoxin B (SEB) proteins, N23K and F44S, were studied in a lipopolysaccharide-
potentiated mouse model. Although 10 μg of SEB per mouse is equivalent to 30 LD50, the
same intraperitoneal dose of either mutant protein was nonlethal and did not elevate
serum levels of tumor necrosis factors (TNF). N23K, F44S, and SEB were serologically
identical in an enzyme-linked immunosorbent assay with polyclonal anti-SEB.
Immunization with alum containing N23K, F44S, or SEB elicited an anti-SEB response
that protected 80–87% of the mice against a 10 μg SEB challenge. Controls lacking an
anti-SEB titer did not survive. Pooled sera from immunized mice effectively blocked
SEB-induced T-cell proliferation in vitro. Naive mice survived a lethal SEB challenge
when given pooled antisera 1, 2, or 4 h later, whereas the antisera failed to protect
animals when administered 6 or 8 h after the toxin. Lethality at the later times was
consistent with increased serum levels of TNF observed 6 h after SEB injection. These
studies suggest that the N23K and F44S mutant proteins of SEB are less biologically
active than the wild-type toxin, yet retain epitopes useful for eliciting a protective
antibody response.

Cooperativity of Staphylococcal aureus

Enterotoxin B Superantigen, Major
Histocompatibility Complex Class II,
and CD80 for Immunotherapy of
Advanced Spontaneous Metastases in a
Clinically Relevant Postoperative
Mouse Breast Cancer Model 1
1. Beth A. Pulaski,
2. David S. Terman,
3. Saleem Khan,
4. Eric Muller, and
5. Suzanne Ostrand-Rosenberg2

+ Author Affiliations

1. Department of Biological Sciences, University of Maryland Baltimore County,

Baltimore, Maryland 21250 [B. A. P., E. M., S. O-R.]; Enerjen, Carmel,
California 93921 [D. S. T.]; and Department of Molecular Genetics and
Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15261 [S. K.]

Cancer Res May 5, 2000 60; 2710

Abstract

One of the leading causes of death for women is metastatic breast cancer. Because most
animal tumors do not accurately model clinical metastatic disease, the development of
effective therapies has progressed slowly. In this study, we establish the poorly
immunogenic mouse 4T1 mammary carcinoma as a postsurgical animal model. 4T1
growth characteristics parallel highly invasive human metastatic mammary carcinoma
and, at the time of surgery, the extent of disease is comparable with human stage IV
breast cancer. Progress in understanding the immune response has led to innovative
immune-based anticancer therapies. Here, we test in this postsurgical model, a novel cell-
based vaccine, combining MHC class II, CD80 (B7.1), and SEB superantigen. Effective
treatment of tumor-bearing mice with this immunotherapy requires expression of all three
molecules. Mean survival time is extended from 5–7.5 weeks for control-treated mice to
6–10.5 weeks for therapy-treated mice. Increased survival is accompanied by a maximum
of 100-fold decrease in clonogenic lung metastases. These therapeutic effects are
particularly noteworthy because: (a) the postoperative model demonstrates that early
metastases responsible for morbidity are established by 2 weeks after tumor inoculation
with 7 × 103 parental 4T1 cells into the mammary gland; (b) the immunotherapy is started
4 weeks after tumor inoculation when the mice contain extensive, pre-established,
disseminated metastases; and (c) CD4+ and CD8+ T cells are required for the effect.

Footnotes
Infect. Immun., 08 1995, 2880-2885, Vol 63, No. 8
Copyright © 1995, American Society for Microbiology

Humoral immunity to aerosolized staphylococcal enterotoxin B

(SEB), a superantigen, in monkeys vaccinated with SEB
toxoid-containing microspheres

J Tseng, JL Komisar, RN Trout, RE Hunt, JY Chen, AJ Johnson, L Pitt and DL

Ruble
Department of Experimental Pathology, Walter Reed Army Institute of Research, Washington, D.C. 20307-
5100, USA.

Staphylococcal enterotoxin B (SEB) toxoid-containing microspheres were tested for

efficacy in rhesus monkeys as a vaccine candidate for respiratory SEB toxicosis and toxic
shock. Forty monkeys were randomly separated into 10 groups of four monkeys each: 9
groups were vaccinated with the microspheres via combinations of mucosal and
nonmucosal routes, and 1 group served as nonvaccinated controls. Both vaccinated and
nonvaccinated monkeys were then challenged with a high lethal dose of SEB aerosol.
Monkeys primed with an intramuscular dose of the microspheres followed by an
intratracheal booster all survived the SEB challenge. Overall, monkeys with an
intratracheal booster generally had the highest antibody levels, which is consistent with
their high survival rate and lower rate of illness. Protective immunity was correlated with
antibody levels in both the circulation and the respiratory tract. The protection was not
due to the depletion or anergy of SEB-reactive T cells, since SEB-induced proliferation in
cultures of circulating lymphocytes was not significantly reduced after the microsphere
vaccination. It is evident that the nonsurvivors did not die of systemic anaphylaxis or
hypersensitivity because the monkeys did not die immediately after SEB challenge and
there were no significant differences in histamine levels between the vaccinated and
control monkeys before and after SEB challenge. The antibodies seemed to neutralize the
SEB that got into the airway and the circulation.

Advanced Drug Delivery Reviews

Volume 57, Issue 9, 17 June 2005, Pages 1424-1439
New Frontiers in Vaccines for Emerging Pathogens

doi:10.1016/j.addr.2005.01.017 | How to Cite or Link Using DOI Cited By in Scopus (19)

Copyright © 2005 Elsevier B.V. All rights reserved.
Permissions & Reprints

Vaccines against the category B toxins: Staphylococcal enterotoxin B, epsilon toxin and
ricin
,
Nicholas J. Mantis

Division of Infectious Disease, Wadsworth Center, New York State Department of

Health, Albany, NY 12208, United States

Received 29 June 2004;

accepted 25 January 2005.
Available online 28 April 2005.

Abstract

The threat of bioterrorism worldwide has accelerated the demand for the development of
therapies and vaccines against the Category B toxins: staphylococcal enterotoxin B
(SEB), epsilon toxin (ETX) produced by Clostridium perfringens types B and D, and
ricin, a natural product of the castor bean. The diverse and unique nature of these toxins
poses a challenge to vaccinologists. While formalin-inactivated toxins can successfully
induce antibody-mediated protection in animals, their usefulness in humans is limited
because of potential safety concerns. For this reason, research is now aimed at developing
recombinant, attenuated vaccines based on a detailed understanding of the molecular
mechanisms by which these toxins function. Vaccine development is further complicated
by the fact that as bioterrorism agents, SEB, ETX and ricin would most likely be
disseminated as aerosols or in food/water supplies. Our understanding of the mechanisms
by which these toxins cross mucosal surfaces, and importance of mucosal immunity in
preventing toxin uptake is only rudimentary.

Keywords: Vaccine; Biodefense; Toxins; Immun

Clinical Immunology
Volume 108, Issue 1, July 2003, Pages 51-59

doi:10.1016/S1521-6616(03)00066-4 | How to Cite or Link Using Cited By in Scopus

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Copyright © 2003 Elsevier Science (USA). All rights reserved. (28)
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Regular article
Generation of protective immunity by inactivated recombinant staphylococcal
enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity

James W. Bolesa, M. Louise M. Pitta, Ross D. LeClairea, Paul H. Gibbsa, Edna

Torresa, Beverly Dyasa, Robert G. Ulricha and Sina Bavari , , a
a
United States Army Medical Research Institute of Infectious Diseases, Frederick, MD
21702, USA

Received 19 April 2002;

accepted 13 March 2003. ;
Available online 8 May 2003.

Abstract

At this time there are no vaccines or therapeutics to protect against staphylococcal

enterotoxin B (SEB) exposure. Here, we report vaccine efficacy of an attenuated SEB in
a nonhuman primate model following lethal aerosol challenge and identify several
biomarkers of protective immunity. Initial in vitro results indicated that the mutation of
key amino acid residues in the major histocompatibility complex (MHC) class II binding
sites of SEB produced a nontoxic form of SEB, which had little to no detectable binding
to MHC class II molecules, and lacked T-cell stimulatory activities. When examined in a
mouse model, we found that the attenuated SEB retained antigenic structures and elicited
protective immune responses against wild-type SEB challenge. Subsequently, a vaccine
regimen against SEB in a nonhuman primate model was partially optimized, and
investigations of immune biomarkers as indicators of protection were performed. SEB-
naïve rhesus monkeys were vaccinated two or three times with 5 or 20 μg of the
attenuated SEB and challenged by aerosol with wild-type SEB toxin. Unlike exposure to
the native toxin, the vaccine did not trigger the release of inflammatory cytokines (TNFα,
IL6, or IFNγ). All rhesus monkeys that developed anti-SEB serum titers ≥104 and elicited
high levels of neutralizing antibody survived the aerosol challenge. These findings
suggest that the attenuated SEB is fully protective against aerosolized toxin when
administered to unprimed subjects. Moreover, experiments presented in this study
identified various biomarkers that showed substantial promise as correlates of immunity
and surrogate endpoints for assessing in vivo biological responses in primates, and
possibly in humans, to vaccines against SEs.
Protein Expression and Purification
Volume 24, Issue 2, March 2002, Pages 302-312

doi:10.1006/prep.2001.1556 | How to Cite or Link Using DOI Cited By in Scopus (18)

Copyright © 2002 Elsevier Science (USA). All rights reserved.
Permissions & Reprints

Regular Article
Production and Purification of a Recombinant Staphylococcal Enterotoxin B Vaccine
Candidate Expressed in Escherichia coli

J. Daniel Coffmana, Jianwei Zhua, John M. Roacha, Sina Bavarib, Robert G. Ulrichb
and Steven L. Giardinaa, 1
a
Biopharmaceutical Development Program, SAIC Frederick, National Cancer Institute at
Frederick, Frederick, Maryland, 21702-1201
b
United States Army Medical Research Institute of Infectious Diseases, Ft. Detrick,
Maryland, 21702-5011

Received 2 May 2001;

revised 20 September 2001.
Available online 1 March 2002.

Abstract

An attenuated, recombinant form of Staphylococcus enterotoxin B (rSEB) was

overexpressed in Escherichia coli under transcriptional control of the T7 promoter. The
28-kDa rSEB was partially purified from soluble, intracellular protein by tangential flow
filtration and differential ammonium sulfate precipitation. The intermediate product was
then further purified using low-pressure liquid chromatography including hydrophobic
interaction, cation exchange, and size-exclusion matrices. The final vialed product was
>95% pure as determined by Coomassie blue-stained sodium dodecyl sulfate-
polyacrylamide gel electrophoresis, high-pressure size-exclusion chromatography, and
capillary zonal electrophoresis. The endotoxin level was <0.6 EU/mg. Final estimated
yield of purified rSEB was 147 mg/L of starting culture. Purified rSEB was stable,
elicited an immune response in mice, and protected mice against a lethal challenge with
the native toxin.
Vaccine
Volume 16, Issue 19, November 1998, Pages 1857-1864
Proceedings of the International Society for Vaccines Symposium on Vaccinology

doi:10.1016/S0264-410X(98)00176-5 | How to Cite or Link Using Cited By in Scopus

DOI
Copyright © 1998 Published by Elsevier Science Ltd. (54)
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Paper
Development of engineered vaccines effective against structurally related bacterial
superantigens

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$ 31.50

,
Robert G. Ulrich , Mark A. Olson and Sina Bavari

Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick,
MD 21702, USA

Available online 2 December 1998.

Abstract

Staphylococcal and streptococcal superantigens are exotoxins that may be linked to many
human pathologies involving impaired immune functions. Despite considerable sequence
divergence, bacterial superantigens share extensive secondary and tertiary structure and
use similar structural strategies to bind major histocompatibility complex class II
receptors. We produced by site-directed mutagenesis of the conserved receptor-binding
surfaces of the superantigens staphylococcal enterotoxins A and B. These vaccines
protected immunized mice and rhesus monkeys from lethal toxic shock. In addition,
antibodies produced against each superantigen recognized and neutralized distantly
related superantigens. This antibody cross-reactivity was additive in that mixtures of
superantigens used in immunization were more effective than single-component vaccines
in protecting mice from challenges with individual or mixed superantigens. We conclude
that an optimal combination of these genetically attenuated superantigen vaccines may
protect against all structurally related superantigens.
Infection and Immunity, March 1999, p. 1521-1525, Vol. 67, No. 3
0019-9567/99

Correlation of Temperature and Toxicity in Murine Studies of

Staphylococcal Enterotoxins and Toxic Shock Syndrome
Toxin 1
Bradley G. Stiles,1,* Yvette G. Campbell,1 Robert M. Castle,1 and Sara A. Grove2

Department of Immunology and Molecular Biology, U.S. Army Medical Research

Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011,1 and Shippensburg
University, Shippensburg, Pennsylvania 172572

Received 1 September 1998/Returned for modification 17 November 1998/Accepted 22

December 1998

This study describes a quick (<12 h) assay for detecting temperature decreases in
BALB/c and C57BL/6 mice injected intraperitoneally (i.p.) with staphylococcal
enterotoxin A (SEA), SEB, or SEC3 or toxic shock syndrome toxin 1 and a potentiating
dose of lipopolysaccharide (LPS). Toxin-specific antisera effectively neutralized the
temperature fluctuations in this model. Orally administered SEA or SEB (50 µg/animal),
with or without LPS, did not have an effect on temperature or lethality. Versus wild-type
mice, transgenic knockout mice lacking the p55 receptor for tumor necrosis factor (TNF)
or gamma interferon were protected against an i.p. challenge of SEA plus LPS. The p75
receptor for TNF and intercellular adhesion molecule 1 have a negligible role in this toxic
shock model.

Infection and Immunity, June 2010, p. 2801-2811, Vol. 78, No. 6

0019-9567/10/$12.00+0 doi:10.1128/IAI.01121-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Potent Neutralization of Staphylococcal Enterotoxin B by

Synergistic Action of Chimeric Antibodies
Mulualem E. Tilahun,1,2 Govindarajan Rajagopalan,3 Nalini Shah-Mahoney,1
Rebecca G. Lawlor,2 Ashenafi Y. Tilahun,3 Chen Xie,1 Kannan Natarajan,4 David H.
Margulies,4 David I. Ratner,1 Barbara A. Osborne,2 and Richard A. Goldsby1,2*

Department of Biology, Amherst College, Amherst, Massachusetts 01002,1 Department

of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst,
Massachusetts 01003,2 Department of Immunology, Mayo Clinic College of Medicine,
Mayo Clinic, Rochester, Minnesota 55905,3 Molecular Biology Section, Laboratory of
Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, Maryland 208924

Received 5 October 2009/ Returned for modification 28 October 2009/ Accepted 8

March 2010
Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by
Staphylococcus aureus, is an important cause of food poisoning and is a class B
bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the
T-cell population by cross-linking T-cell receptors (TCRs) with class II major
histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in
the induction of antigen independent proliferation and cytokine secretion by a significant
fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell
activation by blocking the toxin's interaction with the TCR or MHC-II and provide
protection against the debilitating effects of this superantigen. We derived and searched a
set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies
that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-
SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies
inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive
manner. In order to engineer antibodies more suitable than mouse MAbs for use in
humans, the genes encoding the VL and VH gene segments of a synergistically acting
pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions
of human Ig and human IgG1, transfected into mammalian cells, and used to generate
chimeric versions of these antibodies that had affinity and neutralization profiles
essentially identical to their mouse counterparts. When tested in cultures of human
peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic
mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-
cell activation and cytokine production.

Inhibition of Toxic Shock by Human

Monoclonal Antibodies against
Staphylococcal Enterotoxin B
Eileen A. Larkin1,2, Bradley G. Stiles1,3*, Robert G. Ulrich1,2

Background

Staphylococcus aureus is implicated in many opportunistic bacterial infections around the

world. Rising antibiotic resistance and few alternative methods of treatment are just two
looming problems associated with clinical management of S. aureus. Among numerous
virulence factors produced by S. aureus, staphylococcal enterotoxin (SE) B is a secreted
protein that binds T-cell receptor and major histocompatibility complex class II,
potentially causing toxic shock mediated by pathological activation of T cells.
Recombinant monoclonal antibodies that target SEB and block receptor interactions can
be of therapeutic value.

Methodology/Principal Findings
The inhibitory and biophysical properties of ten human monoclonal antibodies, isolated
from a recombinant library by panning against SEB vaccine (STEBVax), were examined
as bivalent Fabs and native full-length IgG (Mab). The best performing Fabs had binding
affinities equal to polyclonal IgG, low nanomolar IC50s against SEB in cell culture assays,
and protected mice from SEB-induced toxic shock. The orthologous staphylococcal
proteins, SEC1 and SEC2, as well as streptococcal pyrogenic exotoxin C were recognized
by several Fabs. Four Fabs against SEB, with the lowest IC50s, were converted into native
full-length Mabs. Although SEB-binding kinetics were identical between each Fab and
respective Mab, a 250-fold greater inhibition of SEB-induced T-cell activation was
observed with two Mabs.

Conclusions/Significance

Results suggest that these human monoclonal antibodies possess high affinity, target
specificity, and toxin neutralization qualities essential for any therapeutic agent.

Larkin EA, Stiles BG, Ulrich RG (2010) Inhibition of Toxic Shock by Human
Monoclonal Antibodies against Staphylococcal Enterotoxin B. PLoS ONE 5(10): e13253.
doi:10.1371/journal.pone.0013253

Epsilon toxin:

Veterinary Record 1998;142:722-725 doi:10.1136/vr.142.26.722

• Papers and Articles

Protection of goats against experimental

enterotoxaemia by vaccination with
Clostridium perfringens type D epsilon
toxoid
1. F. A. Uzal, MedVet, MSc, PhD1 and
2. W. R. Kelly, MSc, PhD1

+ Author Affiliations
 1
1. Department of Veterinary Pathology, The University of Queensland, Brisbane,
Qld 4072, Australia

Abstract

Enterotoxaemia in goats is mainly characterised by enterocolitis, and it has been

suggested that the poor efficacy of commercial vaccines in preventing the disease is due
to the local action of Clostridium perfringens toxin/s within the intestine, where
circulating antibodies might not exert their action. Five goat kids were vaccinated with an
incomplete Freund's adjuvant C perfringens type D epsilon toxoid vaccine on three
occasions at three-week intervals, four similar kids were vaccinated with a commercial
enterotoxaemia vaccine at the same times, and five other unvaccinated kids were used as
controls. All the animals were challenged intraduodenally, one week after the last
vaccination, with C perfringens type D filtered culture supernatant. At the time of
challenge, the level of epsilon toxin antibodies in the serum of the Freund's adjuvant-
vaccinated kids ranged between 2.45 and 230 iu/ml, while the kids that received the
commercial vaccine had levels between 0.22 and 1.52 iu/ml, and the unvaccinated kids
had levels below 0.03 iu/ml. No clinical or postmortem changes were observed in the
kids that received the Freund's adjuvant-vaccine. Three of the four kids that received the
commercial vaccine developed mild, pasty diarrhoea, with a slight reddening of the
colonic mucosa being observed postmortem. All the unvaccinated kids developed severe
diarrhoea, respiratory distress and central nervous system signs, and were killed
humanely between six and 24 hours after challenge. The postmortem changes consisted
of pseudomembranous colitis, lung oedema and perivascular oedema of the brain.
Moderate to high serum levels of anti-epsilon antibody appeared to protect the goats
against both the systemic and the intestinal effects of C perfringens type D toxins.

Journal of Animal Science, Vol 75, Issue 9 2328-2334, Copyright © 1997 by American Society of Animal
Science

JOURNAL ARTICLE

Vaccination schedules to raise antibody concentrations against

epsilon-toxin of Clostridium perfringens in ewes and their
triplet lambs

C. de la Rosa, D. E. Hogue and M. L. Thonney

Department of Animal Science, Cornell University, Ithaca, NY 14853-4801, USA.

The objective of this experiment was to compare vaccination schedules for ewes and their
lambs to raise antibody concentrations to epsilon-toxin of Clostridium perfringens, the
causative agent of enterotoxemia. Half of 200 Finnsheep x Dorset ewes were vaccinated
with C. perfringens type D toxoid vaccine 3 wk before lambing. Serum samples were
obtained from 20 ewes that were to be vaccinated and 20 ewes that would remain
unvaccinated before treatment and at wk 2, 1, and 0 before the start of lambing. Antibody
concentrations in sera of unvaccinated ewes remained at 2 IU/mL, but they peaked in
vaccinated ewes at 15 IU/mL by wk 1 before lambing. Lambs from each of the first 13
and the first 14 sets of triplets from vaccinated and unvaccinated ewes, respectively,
received one of three vaccination treatments: no vaccine (control), vaccination on d 1 and
21 of age, or vaccination on d 21 and 42 of age. Antibody concentrations declined in sera
of vaccinated ewes from 8.5 IU/mL immediately after lambing to 3 IU/mL 12 wk later.
Vaccination of lambs did not increase sera antibody concentration. However, prepartum
vaccination of ewes significantly increased lamb antibody concentrations (19 IU/mL)
compared with lambs reared by unvaccinated ewes (2 IU/mL). Vaccination of ewes
resulted in lambs with higher antibody concentrations until wk 10 postpartum.
Concentrations declined to .6 IU/mL in all lambs at 12 wk. Because concentrations of .2
IU/mL may be protective, these results indicate that vaccination of ewes before lambing
imparts passive protection in lambs to 12 wk of age, whereas vaccination of young lambs
provides no added protection.

Journal of Comparative Pathology

Volume 116, Issue 1, January 1997, Pages 63-71

doi:10.1016/S0021-9975(97)80044-8 | How to Cite or Link Using Cited By in Scopus

DOI
Copyright © 1997 Published by Elsevier Ltd. (26)
Permissions & Reprints

Effects of the intravenous administration of Clostridium perfringens type D epsilon toxin

on young goats and lambs

F.A. Uzala and W.R. Kellya

a
Department of Veterinary Pathology, The University of Queensland, Brisbane, Qld
4072, Australia

Received 7 May 1996;

accepted 30 September 1996.
Available online 6 June 2006.

Summary

Young goats (n=18) and lambs (n=10) were compared in respect of the effects of
Clostridium perfringens type D epsilon toxin. Toxin produced neurological signs within
0·5–3 h of intravenous injection in (1) all of six kids given doses of 250, 185 or 120
mouse lethal doses 50% (MLD50)/kg body weight, (2) two of the three kids given 60
MLD50/kg, and (3) all of five lambs given 250 or 120 MLD50/kg. Six kids and three lambs
given 45, 30 or 15 MLD50/kg, one lamb given 60 MLD50/kg, and three kids and one lamb
given saline (controls) all remained clinically normal. Gross post-mortem changes were
observed only in the kids and lambs that showed clinical signs. In the kids these changes
consisted of severe acute interstitial and alveolar oedema of the lungs. However, only two
out of five lambs that presented clinical signs showed pulmonary oedema. No
histological changes were observed in the brain of any of the kids inoculated with epsilon
toxin. In the brain of four out of the five lambs given doses of 120 or 250 MLD50/kg,
there were histological lesions consisting of perivascular proteinaceous oedema and
haemorrhages. These results show that kids and lambs are equally susceptible to the
intravenous injection of epsilon toxin, but that they differ in the histological response of
the central nervous system to the toxin.

Infection and Immunity, November 2005, p. 7413-7421, Vol. 73, No. 11

0019-9567/05/$08.00+0 doi:10.1128/IAI.73.11.7413-7421.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Epsilon-Toxin Is Required for Most Clostridium perfringens

Type D Vegetative Culture Supernatants To Cause Lethality
in the Mouse Intravenous Injection Model
Sameera Sayeed,1 M. E. Fernandez-Miyakawa,2 Derek J. Fisher,1,3 Vicki Adams,4
Rachael Poon,4 Julian I. Rood,4 Francisco A. Uzal,2 and Bruce A. McClane1,3*

Department of Molecular Genetics and Biochemistry,1 Molecular Virology and

Microbiology Graduate Program, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261,3 California Animal Health and Food Safety Laboratory
System, San Bernardino Branch, School of Veterinary Medicine, University of
California, Davis, San Bernardino, California 92408,2 ARC Centre for Structural and
Functional Microbial Genomics, Department of Microbiology, Monash University,
Victoria, Australia4

Received 3 June 2005/ Returned for modification 27 June 2005/ Accepted 27 July 2005

Clostridium perfringens type D enterotoxemias have significant economic impact by

causing rapid death of several domestic animal species. Consequently, domestic animals
are commonly vaccinated, at varying efficacy, with inactivated type D vegetative
supernatants. Improved type D vaccines might become possible if the lethal toxins
produced by type D isolates were characterized and the contributions of those toxins to
supernatant-induced lethality were established. Therefore, the current study evaluated the
presence of lethal toxins in supernatants prepared from late-log-phase vegetative cultures
of a large collection of genotype D isolates. Under this growth condition, most genotype
D isolates produced variable levels of at least three different lethal toxins, including
epsilon-toxin (ETX). To model the rapid lethality of type D enterotoxemias, studies were
conducted involving intravenous (i.v.) injection of genotype D vegetative supernatants
into mice, which were then observed for neurotoxic distress. Those experiments
demonstrated a correlation between ETX (but not alpha-toxin or perfringolysin O) levels
in late-log-phase genotype D supernatants and lethality. Consistent with the known
proteolytic activation requirement for ETX toxicity, trypsin pretreatment was required for,
or substantially increased, the lethality of nearly all of the tested genotype D vegetative
supernatants. Finally, the lethality of these trypsin-pretreated genotype D supernatants
could be completely neutralized by an ETX-specific monoclonal antibody but not by an
alpha-toxin-specific monoclonal antibody. Collectively, these results indicate that, under
the experimental conditions used in the present study, ETX is necessary for the lethal
properties of most genotype D vegetative supernatants in the mouse i.v. injection model.

Veterinary Record 1998;143:472-474 doi:10.1136/vr.143.17.472

• Papers and Articles

Variability of serum antibody responses

of goat kids to a commercial
Clostridium perfringens epsilon toxoid
vaccine
1. F. A. Uzal, PhD1,
2. D. A. V. Bodero, BSc, DipIp2,
3. W. R. Kelly, PhD1 and
4. K. Nielsen, PhD3

+ Author Affiliations
1
1. Division of Veterinary Pathobiology
2
2. Division of Fann Animal Studies, School of Veterinary Sciences, Brisbane,
Queensland 4072, Australia
3. 3Animal Diseases Research Institute, Canadian Food Inspection Agency, Box
11300, Station H, Nepean, Ontario, Canada K2H 8P9

Abstract

Twenty-nine Angora goats were used in a trial of a commercial enterotoxaemia (pulpy

kidney disease) vaccine. The animals were allocated to four groups, of which three
received an initial dose of vaccine, two also received a booster of the same vaccine either
28 or 42 days after the first vaccination, and the fourth remained as an unvaccinated
control group. An indirect ELISA technique was used to measure the titres of
Clostridium perfringens type D epsilon antitoxin in serum samples taken before
vaccination and 17, 28, 42, 59, 70, 86, 98 and 128 days after vaccination. There was a
wide range of antibody titres after vaccination, and the great majority of the vaccinated
animals had titres below the protective level, arbitrarily set at 0.25 iu/ml, by day 98.

Biologicals
Volume 18, Issue 4, October 1990, Pages 263-270
doi:10.1016/1045-1056(90)90028-X | How to Cite or Link Using Cited By in Scopus (11)
DOI
Copyright © 1990 Published by Elsevier Science Ltd.
Permissions & Reprints

Original paper
In vitro tests for the measurement of clostridial toxins, toxoids and antisera : II. Titration
of clostridium perfringens toxins and antitoxins in cell culture

P. A. Knight , J. Queminet , J. H. Blanchard and J. H. Tilleray

Wellcome Biotechnology Ltd, Langley Court, Beckenham, Kent, BR3 3BS, U.K.

Received 15 November 1989;

accepted 2 August 1990.
Available online 13 April 2004.

Abstract

The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon
toxins has been investigated.

Neutralization experiments with monoclonal antibodies have shown that the entities
responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin
preparations are identical. However, the cytopathic effects of the same preparations are
caused by other entities. Therefore, titrations based upon lethal and dermonecrotic
indicators of beta toxin are equally valid but those based on cytopathic effects are not.

Similar experiments with Cl perfringens epsilon preparations have shown that their
lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It
follows that all three activities can be valid indicators for toxin neutralization tests.

Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the
levels of test prescribed by the European Pharmacopoeia have produced consistent results
which agree closely with the dermonecrotic test. This test has, in turn, been shown to
reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at
lower levels of test have also produced results in close agreement with the in vivo test.

It is concluded that cell culture titration offers a valid in vitro alternative to the use of
mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in
the course of potency tests and field trials of Cl perfringens epsilon vaccines.

ALTEX. 2004;21 Suppl 3:65-9.

Quality assurance of C. perfringens
epsilon toxoid vaccines--ELISA versus
mouse neutralisation test.
Rosskopf-Streicher U, Volkers P, Noeske K, Werner E.

Source

Paul-Ehrlich-Institut, D-Langen, Germany. rosut@pei.de

Abstract

Clostridium (C.) perfringens is a Gram-positive anaerobic spore-forming bacterium.

Disease caused by C. perfringens infection is called enterotoxaemia. C. perfringens
strains are classified on the basis of the lethal exotoxins formed by the bacteria. Epsilon
toxin is one of the major lethal toxins and is formed by C. perfringens types B and D. C.
perfringens is an ubiquitous bacterium. Infection occurs via food, water, animal litter or
soil. Affected animals include mainly sheep, pigs and cattle. C. perfringens infection
manifests as pulpy kidney disease and diarrhoea in suckling lambs. Enterotoxaemia
development is peracute in most cases. Animals die suddenly while grazing on the
pasture, without any prior signs of disease. Therefore, treatment is possible only in very
rare cases. Suitable immunoprophylactic measures are the treatment of choice to combat
the disease: Vaccines and immunosera have therefore been used extensively for a long
time. The requirements for quality, efficacy and safety testing of the inactivated vaccines
are laid down in the Ph. Eur. in the monograph: Clostridium perfringens vaccines for
veterinary use. After a marketing authorisation is attained, the product batches must be
tested in laboratory animal models for their potency against all vaccine components
(Pharmeuropa, 1997). For potency testing (batch control) of C. perfringens types B and
D, the induction of specific antibodies against epsilon toxin in rabbits must be verified.
For this purpose, 10 rabbits are immunised twice with the product to be tested. Their
blood is taken 14 days after the last immunisation and the serum is pooled. The pooled
serum is then tested for its protective effect. This is done by means of the toxin
neutralisation test in mice (optionally also in guinea pigs) in comparison with an
international reference serum. The evaluation criterion is the death rate of the mice in the
test and reference groups after administration of lethal doses of epsilon toxin. The exact
efficacy of the test serum is given in International Units (IU). The tested serum must
show a minimum content of 5 IU. This in vivo method requires a very high number of
experimental animals. Approximately 400 mice (or 50 guinea pigs) are used per vaccine
batch. The monograph for C. perfringens vaccines, which has recently been revised,
expressly indicates that a validated serological method may be used for batch testing. In
addition, a reference serum known as clostridium multicomponent serum has been
available since 2000. The objective is to test vaccine batches against this reference and by
means of a competitive ELISA developed in the precursor project, using a monoclonal
antibody for direct determination of specific antitoxins in rabbit sera. This ELISA method
was subjected to an international validation to verify whether the protocol and the
precision can be transferred within and between the participating laboratories.

Journal of Biological Standardization

Volume 17, Issue 2, 1989, Pages 117-124

doi:10.1016/0092-1157(89)90002-4 | How to Cite or Link Using DOI Cited By in Scopus (8)

Copyright © 1989 Published by Elsevier Science Ltd.
Permissions & Reprints

The detection of Clostridium perfringens epsilon antitoxin in rabbit serum by monoclonal

antibody based competition ELISA

Purchase
$ 31.50

Marcjanna G. Sojka†, Victor J. White†, Christopher J. Thorns† and Peter L. Roeder†

†
Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw,
Weybridge, Surrey KT15 3NB, UK

Received 4 August 1988.

Available online 9 February 2004.

Abstract

A competitive enzyme-linked immunosorbent assay (CELISA) has been developed,

standardized and compared with the toxin neutralization (TN) test performed in mice for
the measurement of antibody responses in rabbits vaccinated with clostridial vaccines. In
CELISA, sera were tested at a single dilution for their ability to compete with the reaction
between a monoclonal antibody, which neutralizes epsilon toxin, and epsilon toxoid
coated on to a solid phase. The results of the two tests correlated well. CELISA was
specific, rapid, reproducible and simple to perform and offered an alternative to the TN
test that reduced the requirement for experimental animals in the potency testing of
clostridial vaccines.

Article Outline
Production of a non-toxic site-directed mutant of Clostridium
perfringens -toxin which induces protective immunity in
mice
Petra C. F. Oyston, Dean W. Payne, Helen L. Havard, E. Diane Williamson and Richard
W. Titball

Microbiology 144 (1998), 333-341

Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 0JQ, UK

ABSTRACT

A panel of ten site-directed mutants of Clostridium perfringens -toxin was generated. All
of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by
a neutralizing mAb raised against wild-type native -toxin. The cytotoxicity of the site-
directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting
in loss of activity in the assay was identified. This non-toxic protein was derived by
substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice
with the non-toxic mutated -toxin resulted in the induction of a specific antibody
response and immunized mice were protected against 1000 LD50 doses of wild-type
recombinant -toxin.

The control of necrotic enteritis in

sucking piglets by means of a
Clostridium perfringens toxoid vaccine
1. S Springer,
2. H.-J Selbitz

FEMS Immunology & Medical Microbiology

Volume 24, Issue 3, pages 333–336, July 1999

Necrotic enteritis in sucking piglets constitutes a serious problem in piglet rearing units
because of the high morbidity and mortality associated with the disease. The primary
causal agent is Clostridium perfringens type C. The β-toxin plays a decisive role in the
pathogenesis of this disease. A toxoid vaccine for use in sows has been developed and
studied in field trials. The European Pharmacopoeia Monograph on vaccines for use in
animals lays down a method of the efficacy testing based on the immunization of rabbits,
the collection of pooled sera and the subsequent assay of anti-toxin antibodies in mice
using an appropriate test toxin. The vaccine is regarded as effective if it induces a
minimum of 10 IU of β-anti-toxin per ml of rabbit serum. We have established a range of
17.14–98.23 IU β-anti-toxin per ml rabbit serum induced by a sample of C. perfringens
toxoid vaccine. The vaccine has been used under field conditions in different rearing
units at the same time, mostly in the form of emergency vaccinations following the
outbreak of disease. The outcome of vaccination was evaluated by recording the total
numbers of piglets born alive and the piglet losses. Use of the vaccine, coupled with other
measures, resulted in an approximately 30% reduction in the number of losses.

Biologicals
Volume 19, Issue 4, October 1991, Pages 281-286

doi:10.1016/S1045-1056(05)80016-8 | How to Cite or Link Using DOI Cited By in Scopus (8)

Copyright © 1991 Published by Elsevier Ltd.
Permissions & Reprints

Original Paper
An alternative to the toxin neutralization assay in mice for the potency testing of the
Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium
perfringens Type D epsilon components of multivalent sheep vaccines

K.R. Wood

Hoechst Animal Health, Walton Manor, Walton, Milton Keynes, Bucks, MK7 7AJ, U.K.

Received 19 January 1991;

accepted 31 May 1991.
Available online 21 November 2005.

Abstract

Potency testing of veterinary vaccines containing clostridial antigens currently requires

the vaccination of laboratory rabbits followed by the determination of specific antitoxin
concentration in the rabbit sera by toxin neutralization test in mice. ELISAs are described
as an alternative method to toxin neutralization for the determination of Clostridium
tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens
Type D epsilon antitoxins. The assays were found to be rapid, specific and economical
and showed good correlation with the toxin neutralization test.

Infect Immun. 1990 August; 58(8): 2487-2492

Anti-idiotypic antibody-induced protection against Clostridium

perfringens type D.
D A Percival, A D Shuttleworth, E D Williamson and D C Kelly
Defence Microbiology Division, Chemical Defence Establishment, Porton Down,
Salisbury, Wiltshire, United Kingdom.

ABSTRACT

A monoclonal antibody (BALB/c mouse) with specificity for a neutralizing epitope on the
epsilon-toxin produced by Clostridium perfringens type D was used to raise anti-idiotypic
antibodies (anti-Id) in different strains of mice and rabbits. These were purified and used
in cross-immunization studies to induce anti-(anti-idiotype). All strains of mice and
rabbits immunized with BALB/c-derived anti-Id showed a high-titer antibody response
directed towards the active site of the toxin. This protected the animals against toxin
challenge and against an oral dose of the vegetative organisms. Animals immunized with
other anti-Id preparations showed no specific antibody response and were not protected.
Guinea pig peritoneal macrophages have a cell surface receptor for the toxin, and
incubation of these cells with BALB/c anti-Id allowed them to survive toxin challenge,
indicating that occupation of the receptors by the anti-Id prevented binding by the toxin.
In conclusion, we have shown that an internal-image anti-Id preparation will induce
protective immunity in syngeneic and xenogeneic animals and furthermore that immunity
to a single epitope on the exotoxin is sufficient to protect against the toxin and clinical
sequelae evoked by the disease-causing organism itself.

Infection and Immunity, April 2007, p. 1785-1793, Vol. 75, No. 4

0019-9567/07/$08.00+0 doi:10.1128/IAI.01643-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Neutralizing Antibodies against

Clostridium perfringens Epsilon-Toxin
Mark S. McClain1* and Timothy L. Cover1,2,3

Departments of Medicine,1 Microbiology and Immunology, Vanderbilt University School

of Medicine, Nashville, Tennessee 37232,2 Veterans Affairs Tennessee Valley Healthcare
System, Nashville, Tennessee3

Received 11 October 2006/ Returned for modification 13 November 2006/ Accepted 15

January 2007

The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness

(enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this
study, we examined the activities of two neutralizing monoclonal antibodies against the
C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards
cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma
membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-
aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent
assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope
recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145.
The antibody competition ELISA and analysis of mutant toxins suggest that the second
neutralizing monoclonal antibody also recognizes an epitope in close proximity to this
region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop
corresponding to the putative membrane insertion domain of the toxin. Identifying the
epitopes recognized by these neutralizing antibodies constitutes an important first step in
the development of therapeutic agents that could be used to counter the effects of the
epsilon-toxin.

Journal of Biological Standardization

Volume 16, Issue 3, 1988, Pages 207-216

doi:10.1016/0092-1157(88)90008-X | How to Cite or Link Using DOI Cited By in Scopus (5)

Copyright © 1988 Published by Elsevier Science Ltd.
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The production and evaluation of monoclonal antibodies to Clostridium perfringens type

D epsilon toxin

C. D. H. Boarer†, M. G. Sojka†, V. J. White† and P. L. Roeder†

†
Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw,
Weybridge, Surrey KT15 3NB, UK

Received 8 January 1988.

Available online 27 April 2004.

Abstract

The production of five monoclonal antibodies to the epsilon prototoxin of Clostridium

perfringens is described. All five monoclonal antibodies located three proteins in a
trypsinized preparation of epsilon prototoxin. These proteins were located at 37·6 kDal,
35·6 kDal and 33·7 kDal by Western blots. Two of the monoclonal antibodies, M26/2 and
M27/12, neutralized epsilon toxin in the mouse lethality assay. Four of the five
monoclonal antibodies are considered suitable as reagents to detect epsilon toxin in assay
procedures.

Veterinary Research Communications

Volume 23, Number 3, 143-150, DOI: 10.1023/A:1006206216220

Antibody Response in Goats Vaccinated

with Liposome-adjuvanted Clostridium
perfringens Type D Epsilon Toxoid
F.A. Uzal, J.P. Wong, W.R. Kelly and J. Priest

A trial was performed using 20 goats to evaluate the antibody responses to a liposome-
adjuvanted Clostridium perfringens epsilon toxoid vaccine (LIPV). The antibody
response was compared with those produced by epsilon toxoid vaccines prepared using
aluminium hydroxide (ALV) and incomplete Freud's adjuvant (FAV). The animals were
allocated to four groups at the beginning of the trial. The animals in group 1 were
vaccinated with ALV, while the animals in group 2 received FAV and those in groups 3
and 4 were vaccinated with LIPV. The animals in groups 1 to 3 received three doses of
the corresponding vaccine at intervals of three weeks, while those in group 4 received
only 1 dose of vaccine at the beginning of the trial. A blood sample was obtained from all
the goats at the beginning of the trial and then weekly for 8 weeks. The samples were
analysed for epsilon toxoid antibodies by an indirect ELISA technique. No major clinical
abnormalities were observed in the animals after vaccination, with the exception of those
that received the FAV, which experienced transient lameness. The highest antibody
response was observed in the animals vaccinated with FAV, but they presented moderate
to severe inflammatory tissue reactions at the injection site. Moderately high antibody
responses were obtained with the ALV, with which only minor local reactions were
observed. No significant antibody responses were obtained with the LIPV, nor were local
reactions observed.

Objectives:
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