Vaccine 36 (2018) 2928–2936

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Review

Brucellosis vaccines based on the open reading frames from genomic

island 3 of Brucella abortus
Leonardo Gómez, Francisco Alvarez, Daniel Betancur, Angel Oñate ⇑
Laboratory of Molecular Immunology, Faculty of Biological Sciences, Department of Microbiology, University of Concepción, Concepción, Chile

a r t i c l e i n f o a b s t r a c t

Article history: Brucella abortus is the etiological agent of brucellosis, a zoonotic disease affecting cattle and humans. This
Received 4 December 2017 disease has been partially controlled in cattle by immunization with live attenuated B. abortus S19 and
Received in revised form 2 February 2018 RB51 strains. However, use of these vaccine strains has been associated with safety issues in animals
Accepted 4 April 2018
and humans. New vaccines have since emerged in the prevention of brucellosis, particularly DNA vacci-
nes, which have shown effectiveness and a good safety profile. Their protection efficacy in mice is asso-
ciated with the induction of Th1 type and cytotoxic T cell mediated immune response against structural
Keywords:
antigens and virulence factors expressed during B. abortus infection. Some antigenic candidate for vaccine
Brucellosis
Brucella abortus
design against brucellosis (mainly DNA vaccines) have been obtained from genomic island 3 (GI-3) of B.
Vaccines abortus, which encodes several open reading frames (ORFs) involved in the intracellular survival and vir-
Genomic island 3 ulence of this pathogen. The immunogenicity and protection conferred by these DNA vaccines in a mur-
Intracellular survival ine model is reviewed in this article, suggesting that some of them could be safe and effective vaccine
Protective response candidates against to prevent B. abortus infection.
Th1 immunity Ó 2018 Elsevier Ltd. All rights reserved.
Interferon gamma

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2928
2. Bovine brucellosis vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2929
3. Vaccines against Brucella based on genomic island 3 (GI-3) open reading frames . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2931
3.1. Subunit of flagellar protein FlgJ vaccine encoded by BAB1_0260 ORF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2932
3.2. Hypothetical protein encoded by BAB1_0263 ORF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2932
3.3. Hypothetical protein with an Src homology 3-like domain encoded by BAB1_0267 ORF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2933
3.4. ImmA/IrrE metallo-endopeptidase family encoded by BAB1_0270 ORF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2933
3.5. Hypothetical protein GcrA encoded by BAB1_0278 ORF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2933
3.6. Multi-epitope and multivalent DNA vaccines based on GI-3 ORFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2934
4. Conclusions and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2934
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2935
Conflict of interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2935
Authors contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2935
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2935

1. Introduction humans in several regions of the world. It is a Gram-negative bac-

terium characterized as a small, microaerophilic, non-spore-
Brucella abortus is a facultative intracellular pathogen that forming, slow growing, coccobacillus [1]. This pathogen is one of
causes brucellosis, an endemic zoonosis affecting bovines and the most virulent species of Brucella genus infecting humans [2];
infection usually occurs through ingestion of contaminated food
or through direct contact with infected animals [3]. After initial
⇑ Corresponding author. contact with the host, Brucella adheres to and penetrates the
E-mail address: aonate@udec.cl (A. Oñate).

https://doi.org/10.1016/j.vaccine.2018.04.014
0264-410X/Ó 2018 Elsevier Ltd. All rights reserved.
 L. Gómez et al. / Vaccine 36 (2018) 2928–2936
mucous membrane and travels through the lymphatic system and
bloodstream and finally reaching the bone marrow, lymph nodes,
liver and spleen. In some case, the bacterium can be found in
bones, joints, male reproductive organs, placenta and the fetuses
of pregnant females [4]. In these tissues, Brucella survives and
replicates within various cell types, including but not limited to
dendritic and epithelial cells, placental trophoblasts, and macro-
phages [5]. The ability of this pathogen to adapt to intracellular
environments is essential for establishment of infection. The infec-
tion in human exhibit an acute phase, associated with undulant
fever, fatigue and headaches, followed by a chronic phase, which
can manifest into arthritis, orchitis, hepatitis, endocarditis or neu-
robrucellosis [6]. Due to these complications, brucellosis is consid-
ered a crippling disease, annually affecting more than half a million
people in the world [3]. In bovines, brucellosis causes abortion in
pregnant females, stillborn or weak calves, retention of placentas
and reduced milk yield. Besides, B. abortus can be found in the male
reproductive organs causing infertility [7]. All these complications
produce major economic losses and public health problems neces-
sitating the containment and treatment of the disease in humans
and bovines [8].
Prevention of brucellosis primarily depends on immunization;
which plays a key role in containing the disease in animals before
it spreads to humans. In order to develop safe and effective vacci-
nes against bovine and human brucellosis, significant efforts must
be made to identify the genes that encode B. abortus virulence fac-
tors. Using these antigenic factors as vaccine candidates, the host
immune responses can be directed towards these virulence factors.
To this end of identifying antigenic targets for the development of
vaccines against brucellosis, this study will discuss the immuno-
genicity and protection conferred in murine model by vaccines that
were developed based on the open reading frames (ORFs) from
genomic island 3 (GI-3) of B. abortus. The results suggest that some
these constructs discussed in this reports could be used as safe and
effective vaccines against B. abortus.
2. Bovine brucellosis vaccines
Prevention of bovine brucellosis primarily done by using
licensed live attenuated B. abortus S19 and RB51 vaccine strains.
B. abortus S19 is a smooth attenuated strain, effective against bru-
cellosis in cattle [9]; however, it can induce abortions in immu-
nized females and is infective in humans. Additionally, the
presence of lipopolysaccharide (LPS) in this strain makes it difficult
to differentiate between immunized versus naturally infected ani-
mals because of the anti-LPS antibody cross-reactivity [10].
Table 1
Live attenuated vaccines based on genes deleted from Brucella abortus.
Target Mice model Route Vaccine doses (CFU)  mice
Dpgk BALB/c i.p. 1  105
C57BL/6
129/Sv
Dpgm BALB/c i.p. 1  107
DznuA BALB/c i.p. 1  108
2 (1  108)
DznuA BALB/c i.p. 2 (1  108)
DpurE
DwbkA BALB/c i.p. 1  106
S19 DvjbR::Kan BALB/c i.p. Encapsulated (1  10 ) 5
Non-encapsulated (1  105)
Significant values (*); Intraperitoneal (i.p.) route; weeks (wks).
a
Protection levels are measured by units of protection, represented by the difference between the mean of log10 CFU/spleen values of the negative control group and the
mean of log10 CFU/spleen values of the immunized group.
2929
Although S19 strain is used in several countries because of its pro-
tective efficacy in cattle, the aforementioned diagnostic and safety
issues allowed for the introduction of B. abortus RB51 rough strain
[11]. This vaccine strain has similar protective efficacy but is less
abortive in cattle than the B. abortus S19 strain [11]. Furthermore,
since B. abortus RB51 is a mutant lacking the O-antigen of LPS, it
does not interfere with the diagnosis between immunized versus
infected bovines. However, RB51 strain is infectious to humans
and resistant to rifampicin; one of the major antibiotics used to
treat human brucellosis [9,12]. These vaccines have been shown
to induce protective immunity in mice, associated with strong
CD4+ Th1 cell-mediated immune response with production of
IFN-c (but not IL-4), and specific CD8+ cytotoxic T cells [13]. In cat-
tle, immunization with B. abortus RB51 and S19 vaccines induces
an immune response characterized by proliferation of CD4+ and
CD8+ T-cells; IFN-c and IL-17A production by CD4+ T-cells and;
CD4+ and CD8+ memory cells [14].
Safety limitations of B. abortus S19 and RB51 strains-based vac-
cines warranted the search for safer and more effective experimen-
tal vaccines. Some of these newer vaccine strains are based on
attenuated strains, protein subunits or DNA fragments. Their pro-
tections levels are defined by the difference between the mean of
log10 CFU/spleen values of the negative control group (non immu-
nized group) and the mean of log10 CFU/spleen values of the immu-
nized group (1, 2 and 3). These vaccines have shown a high
variability in the protection levels depending on the type of vac-
cine, route of administration, doses or the challenge time used in
each experiment [15]. Among them, main attenuated B. abortus
2308 strains described are those mutated for the gene encoding
for phosphoglycerate kinase (Dpgk), phosphoglucomutase (Dpgm),
zinc uptake system (DznuA) and glycosyltransferase wbkA (DwbkA)
[16–19]. Besides these vaccines, promising results have been
achieved using live attenuated S19 DvjbR::Kan in encapsulated or
non-encapsulated alginate microspheres. This vaccine based on
vjbR, a regulatory system of quorum sensing (QS) controlling the
expression of Brucella virulence factors genes, is safer than S19 vac-
cine and it induces protection level of immunity in mice (Table 1)
[20]. Although attenuated strains are usually more immunogenic
vaccines, there is a risk that mutations can be reversed, thereby
promoting infection and miscarriage in immunized animals [21].
These safety issues were absent in those immunized with subunit
vaccines such as those containing periplasmic binding protein P39,
periplasmic peptidyl cis–trans isomerase SurA, as well as diverse
outer membrane proteins (Omps). These vaccines have shown to
provide protection in mice (Table 2) [22–30]. The search for safe
and effectiveness vaccine candidates has been extended to DNA
vaccines as well; a strategy that continues to raise considerable
Strain challenge/Doses (CFU)  mice Challenge time Protection levelsa Ref.
B. abortus 2308/1 x 106 2 wks 0.96* [16]
1.36*
3.28*
B. abortus 2308/5  105 2 wks 2.25* [17]
4 wks 1.93*
B. abortus 2308/5  10 4
4 wks 1.78* [18]
1.67*
B. abortus 2308/5  104 4 wks 0.79* [18]
B. abortus 2308/1  106 2 wks 2.22* [19]
4 wks 1.76*
B. abortus 2308/1  10 5
1 wk 3.86* [20]
3.06*
2930 L. Gómez et al. / Vaccine 36 (2018) 2928–2936

Table 2
Subunit vaccines based on the Brucella abortus proteins.

Target Mice model Route Vaccine doses/adjuvant  mice Strain challenge/Doses (CFU)  mice Challenge time Protection levelsa Ref.
BLS BALB/c i.p. rBLS (10 lg)/PBS B. abortus 544/4  10 4
30 d 0.37 NS
[22]
rBLS (10 lg)/IFA 1.26*
rBLS (10 lg)/MPA 1.31*
rBLS (10 lg)/Al 1.40*
MBP-L7/L12 BALB/c i.p. MBP-L7/L12 (100 lg)/IAS B. abortus/1  106 1 wk 0.84* [23]
2 wks 1.13*
4 wks 1.21*
P39 BALB/c i.m. rP39 (20 lg)/CpG (20 lg) B. abortus 544/5  104 4 wks 2.48* [24]
8 wks 1.21*
DnaK BALB/c i.p. rDnaK (30 lg)/CFA-IFA B. abortus 2308/1  10 4
30 d 0.9*/0.95* [25]
SurA BALB/c i.p. rDnaK (30 lg)/CFA-IFA B. abortus 2308/1  104 30 d 0.97*/1.45* [25]
Omp16 BALB/c i.p. L-Omp16 (10 lg)/ IFA B. abortus 544/4  104 30 d 1.44* [26]
L-Omp16 (30 lg)/IFA 1.78*
Omp19 BALB/c i.p. L-Omp19 (10 lg)/IFA B. abortus 544/4  104 30 d 1.38* [26]
L-Omp19 (30 lg)/IFA 1.72*
Omp25 BALB/c i.d. rOmp25 (30 lg)/CFA-IFA B. abortus 544/1  109 1 wks 2.4* [27]
Omp28 BALB/c i.p. rOmp28 (100 µg/mL)/IFA B. abortus 544/1  104 10 d 2.6* [28]
Omp28 Swiss Albino i.m. rOmp28 B. abortus 544/1  105 2 wks 0.437NS [29]
rOmp28/CpG 0.966*
rOmp28 3 wks 0.439NS
rOmp28/CpG 0.898*
BAB2_0122(FliN) BALB/c i.p. rFliN (30 lg)/CFA-IFA B. abortus 544/2  105 30 d 1.32* [30]

Significant values (*); Not-significant value (NS); Intramuscular (i.m.) route; Intraperitoneal (i.p.) route; Intradermal (i.d.) route; Phosphate Buffer Saline (PBS); Complete
Freund’s Adjuvants (CFA); Incomplete Freund’s Adjuvants (IFA); Monophosphoryl lipid A (MPA); Aluminum hydroxide gel (Al); Maltose binding protein (MBP); Immune-plus
adjuvant system (IAS); days (d); weeks (wks).
a
Protection levels are measured by units of protection, represented by the difference between the mean of log10 CFU/spleen values of the negative control group and the
mean of log10 CFU/spleen values of the immunized group.

Table 3
DNA vaccines based in the genes of Brucella abortus.

Target Mice model Route Vaccine doses x mice Strain challenge/Doses Challenge time Protection levelsa Ref.
(CFU)  mice
BLS BALB/c i.m. pcDNA-BLS (100 µg) B. abortus 544/1.12  105 30 d 1.25*/1.65* [32]
sodC BALB/c i.m. pcDNA-SOD (100 µg) B. abortus 2308/1 x 104 2 wks 2.16* [33]
L7/L12 BALB/c i.m. pcDNA3.1-L7/L12 (100 µg) B. abortus 544/5  105 4 wks 1.59* [34]
L7/L12-omp16 BALB/c i.m. Divalent pcDNA3.1-L7/L12- B. abortus 544/5  105 4 wks 2.05* [35]
Omp16 (100 µg)
Omp16 BALB/c i.m. pcDNA3.1-Omp16 (100 µg) B. abortus 544/5 x 105 4 wks 1.25* [35]
P39 BALB/c i.m. pCIP39 (100 µg) B. abortus 544/5  104 4 wks 0.15NS [36]
8 wks 0.73*
BCSP31, SOD, and L7/L12 C57BL/6 i.m. Combined DNA (150 µg) B. abortus 2308/5  106 4 wks 3.58* [37]
Ribosomal L9protein BALB/c i.m. pVaxL9 (100 µg) B. abortus 544/2  105 30 d 1.96* [38]
i.m./s.c. pVaxL9 + rL9 (25 µg) # 2.40*
i.m. pVaxL9-EP (100 µg) ## 2.55*

Significant values (*); Not-significant value (NS); Prime boost using DNA immunization and recombinant protein L9 (#); DNA electroporated (##); Intramuscular (i.m.) route;
Subcutaneous (s.c.) route; days (d); weeks (wks).
a
Protection levels are measured by units of protection represented by the difference between the mean of log10 CFU/spleen values of the negative control group and the
mean of log10 CFU/spleen values of the immunized group.

Fig. 1. Genetic organization of Brucella abortus 2308 strain open reading frames in GI-3. This genomic island is inserted in tRNA-phe, flanked by direct repeat sequences (DR1
and DR2) and it contains thirty ORFs named from the BAB1_0250 encoding of a P4-like integrase (int) to the BAB1_0279 encoding of Btp1 protein (orange). The position of
ORFs used to develop the vaccines discussed in this work are indicated in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
 L. Gómez et al. / Vaccine 36 (2018) 2928–2936 2931

Table 4
Open reading frames encoded in genomic island 3 from Brucella abortus 2308.

ORFs Protein Potential function (s) UniProt CDD/ Pfam

entry SPARCLE entry
entry
BAB1_0250 Bacteriophage P4 integrase These proteins cleave DNA substrates by a series of staggered cuts, during which the Q2YP86 cd00801 PF13356
protein becomes covalently linked to the DNA through a catalytic tyrosine residue at PF00589
the carboxy end of the alignment
BAB1_0251 Hypothetical protein No putative conserved domains are detected Q2YP85 NA NA
BAB1_0252 AlpA transcriptional Helix-Turn-Helix DNA-binding domain of transcription regulators from the MerR Q2YP84 COG3311 NA
regulator superfamily, mediate responses to stress including exposure to heavy metals, drugs,
or oxygen radicals
BAB1_0253 Hypothetical protein No putative conserved domains are detected Q2YP83 NA NA
BAB1_0254 Hypothetical protein No putative conserved domains are detected Q2YP82 NA NA
BAB1_0255 TraI and Toprim_3 domain- TraI is a bifunctional protein that catalyzes the unwinding of duplex DNA as well as Q2YP81 cl26921 PF13362
containing protein acts as a sequence-specific DNA trans-esterase, providing the site- and strand- cd01029
specific nick required to initiate DNA transfer
Toprim domain is found in a wide variety of enzymes involved in nucleic acid
manipulation
BAB1_0256 Pseudogene
BAB1_0257 Pseudogene
BAB1_0258 Hypothetical protein with Most proteins with this domain function as sequence-specific DNA binding domains, Q2YPB3 cl21459 NA
winged helix-turn-helix such as in transcription regulators
domain
BAB1_0259 Hypothetical protein No putative conserved domains are detected Q2YPB2
BAB1_0260 Flagellar protein FlgJ Flagellum-specific peptidoglycan hydrolase and flagellar rod assembly protein FlgJ Q2YPB1 COG1705 PF01832
BAB1_0261 Pseudogene
BAB1_0262 Pseudogene
BAB1_0263 Hypothetical protein No putative conserved domains are detected Q2YPB0 NA NA
BAB1_0264 Hypothetical protein No putative conserved domains are detected. Q2YPA9 NA NA
BAB1_0265 Hypothetical protein with SANT (SWI3, ADA2, N-CoR and TFIIIB) DNA-binding domain is found in regulatory Q2YPA8 cd00167
SANT domain transcriptional repressor complexes where it binds DNA
BAB1_0266 Hypothetical protein No putative conserved domains are detected Q2YPA7 NA NA
BAB1_0267* Hypothetical protein No putative conserved domains are detected* Q2YPA6 NA NA
BAB1_0268 Hypothetical protein No putative conserved domains are detected Q2YPA5 NA NA
BAB1_0269 Hypothetical protein No putative conserved domains are detected Q2YPA4 NA NA
BAB1_0270 Zinc-dependent ImmA/IrrE family metallo-endopeptidase Q2YPA3 COG2856 PF06114
metallopeptidase
BAB1_0271 Abortive infection protein This family of proteins confers resistance to bacteriophage Q2YPA2 cl16749 PF14253
AbiH
BAB1_0272 Abortive infection protein This family of proteins confers resistance to bacteriophage Q2YPA1 cl16749 PF14253
AbiH
BAB1_0273 Transcriptional regulator XRE (Xenobiotic Response Element) family transcriptional regulator controls the Q2YPA0 cd00093 PF13560
expression of genes involved in stress response
BAB1_0274 Hypothetical protein No putative conserved domains are detected Q2YP99 NA NA
BAB1_0275 Hypothetical protein No putative conserved domains are detected. Q2YP98 NA NA
BAB1_0276 Hypothetical protein No putative conserved domains are detected Q2YPC7 NA NA
BAB1_0277 Hypothetical protein No putative conserved domains are detected Q2YPC6 NA NA
BAB1_0278 Hypothetical protein GcrA is a master cell cycle regulator involved in controlling cell cycle progression Q2YPC5 COG5352 PF07750
encoding of GcrA protein. and asymmetric polar morphogenesis. GcrA acts as an activator of components of
the replisome and the segregation machinery
BAB1_0279 Toll/interleukin-1 receptor Tir domain is responsible for signal transduction in all toll-like receptor (TLR); Q2YPC4 PTZ00121 PF13676
(Tir) domain-containing similar to human Mal (MyD88-adapter-like) which is required for TLR4 signal PLN03194
protein. transduction

GI-3 ORFs were analyzed using Kyoto Encyclopedia of genes and genomes (KEGGs), NCBI data bases and UniProt.
Not Available (NA).
*
BAB1_0267 ORF encode a Src Homology 3 (SH3)-type domain-containing protein with 100% identity to B. abortus (GenBank accession number: WP_002968149.1;
pfam08239) and 99.1% of identity with B. suis bv. 5 str. 513 (UniProt Entry: D0PAU5), B. neotomae 5 K33 (UniProt Entry: C9V8E3) and B. abortus NCTC 8038 (UniProt Entry:
A0A0M1WE22) strains.

interest due to their ease of production, administration, genetic
stability, absence of requirement for refrigeration and for their
capacity to induce both humoral and cellular immunity [31]. Sev-
eral Brucella DNA vaccines, such as BLS, Cu-Zn superoxide dismu-
tase (sodC), L7/L12, Omp16 or combined DNA based on BCSP31-
SOD-L7/L12 have proven to be protective in BALB/c mice (Table 3)
[32–38]. These vaccines allow for the expression of individual or
multivalent B. abortus antigens thus inducing a strong Th1-type
immune response thereby effective in eliminating the infecting
pathogen [39]. Therefore, considering the advantages of DNA vac-
cines, identification of genes relevant to B. abortus virulence is cru-
cial for the development of successful DNA vaccines for brucellosis
prevention.
3. Vaccines against Brucella based on genomic island 3 (GI-3)
open reading frames
GI-3 is a discrete DNA segment present in the chromosome I of
B. abortus, B. melitensis and B. ovis. GI is acquired by these species
through horizontal transfer events, but the GI es absent in other
Brucella species that may be due to spontaneous elimination from
bacterial chromosomes, together to speciation events occurring
within the genus [40]. The GI-3 has a size of 20.9 kb with a 52.3%
guanine-cytosine (GC) content, and it contains several ORFs, which
in the case of B. abortus have been annotated from BAB1_0250 to
BAB1_0279 [40,41]. The genomic structure of this GI is shown in
Fig. 1, and the ORFs potential functions are described in Table 4.
2932 L. Gómez et al. / Vaccine 36 (2018) 2928–2936

Table 5
Functions of GI-3 ORFs experimentally studied in Brucella abortus.

GI-3 ORFs Protein Biological functions Ref.

BAB1_0260 Flagellar protein FlgJ
BAB1_0263 Hypothetical protein
BAB1_0267 Hypothetical protein
BAB1_0270 Zinc-dependent metallopeptidase
BAB1_0273 Transcriptional regulator
BAB1_0278 Hypothetical protein encoding of
GcrA protein.
BAB1_0278a Hypothetical protein, homologous to
an ABC-type transporter
BAB1_0279 Btp1 (Toll/interleukin-1 receptor
(Tir) domain-containing protein).
Function has been not studied in B. abortus 2308. FlgJ protein is a protective antigen used as a [30]
subunit protein vaccine in the BALB/c mice
Deletion of BAB1 0278 not affect the ability of B. abortus 2308 to growth within non- [42]
professional and professional phagocytes. Its deletion not reduce the virulence of B. abortus
2308 in BALB/c mice
BAB1_0267 is involved in the intracellular survival of B. abortus 2308 in macrophages and [43]
epithelial cells. Deletion of this ORF reduce the virulence of B. abortus 2308 in BALB/c mice
BAB1_0270 is involved in the intracellular survival of B. abortus 2308 in macrophages and [43]
epithelial cells. Deletion of this ORF reduce the virulence of B. abortus 2308 in BALB/c mice
Function has been not studied in B. abortus 2308. BAB1_0273 ORF was used to design a multi- [66,67]
epitope DNA vaccine and multivalent DNA vaccines
Deletion of BAB1 0278 affect the ability of B. abortus 2308 to growth within non-professional [42]
and professional phagocytes. Deletion of this ORF reduce the virulence of B. abortus 2308 in
BALB/c mice
Function has been not studied in B. abortus 2308. BAB1_0278a ORF product is a protective [65]
antigen used as a DNA vaccine in BALB/c mice
Btp1 inhibits Toll-like receptor (TLR) signaling and Nuclear Factor-jB (NF-jB) activation. It [44,46,47,48]
reduces host inflammatory responses and dendritic cells maturation. Btp1 protein, also is a
effector secreted by T4SS virB

Although several studies demonstrated the roles of these ORFs in
the intracellular survival and virulence for B. abortus [42–44], the
specific contribution of this GI to the physiology of B. abortus is still
unknown. It is known that GIs contribute to bacterial adaptation,
genome plasticity and evolution, antibiotic resistance or virulence
genes dissemination [45]. It has been shown that GI-3 encodes the
Btp1 protein (also named BtpA or Tcp1), an important virulence
factor that subverts the host immune response [44]. This protein
possesses a domain with homology to the TIR (Toll/Interleukin-1
Receptor) region of Toll-like receptors (TLR), a crucial protein in
the innate immunity signaling [46]. Brucella Btp1 (TIR domain-
containing protein) inhibits Toll-like receptor (TLR) signaling and
Nuclear Factor-jB (NF-jB) activation, reducing host inflammatory
responses and dendritic cells maturation [44,46,47]. Btp1 protein is
an effector secreted by type IV secretion system (T4SS) encoded in
virB operon, functionally used for molecular mimicry [48].
Basic Local Alignment Search Tool (BLAST) analyses of amino
acid sequences encoded by GI-3 ORFs and experimental evidence
has allowed to find their potential functions in B. abortus fitness;
some of them involved in intracellular survival and virulence of
this species (see below and Tables 4 and 5). Considering that some
GI-3 ORFs studies contribute to infective process of this pathogen,
several experimental vaccines (mainly DNA vaccines), have been
developed against them. Some of these vaccines, described below,
have shown a promising immunogenicity and protection levels in
mice, measured by statistical significance (P value), against B. abor-
tus infection (Table 6).
3.1. Subunit of flagellar protein FlgJ vaccine encoded by BAB1_0260
ORF
Although Brucella species have been described as non-motile
bacteria, its genome contains all the necessary genes to express a
functional flagellum, which is detected only under very specific
conditions during infection or in the early log phase of a growth
curve [49,50]. An important component of this flagellum is
encoded in the BAB1_0260 ORF of B. abortus GI-3. ORF
BAB1_0260 encodes FlgJ, a mannosyl-glycoprotein endo-beta-N-
acetylglucosamidase, a flagellum-specific protein that plays a key
role as peptidoglycan hydrolase allowing to hydrolyze the peptido-
glycan layer to assemble the rod structure in the periplasmic space
[51,52]. Interestingly, this FlgJ protein is not codified in the flagel-
lar gene clusters (located in chromosome II) but in the GI-3, where
some ORFs involved in bacterial intracellular survival are located
[42,43]. Li et al., demonstrated the immunogenicity of this protein
[30] by developing a subunit vaccine expressing the FlgJ recombi-
nant protein (rFlgJ) in Escherichia coli ER2566 as a N-terminally
His-tagged fusion protein. Later, rFlgJ protein was purified and for-
mulated with complete or incomplete Freund’s adjuvant (CFA or
IFA) and its immunogenicity and protection levels conferred in
BALB/c mice after an intramuscularly immunization was evalu-
ated. Their results showed that this vaccine significantly increased
the levels of anti-B. abortus rFlgJ IgG antibodies and IFN-c pro-
duced by splenocytes in immunized mice. This immunization con-
ferred significant levels of protection [equivalent to 1.63 log10
reduction of pathogenic challenge strain] (Table 6), a protection
lower than B. abortus S19 vaccine strain. Therefore, since FlgJ flag-
ellar protein is a protective antigen in the murine model and, con-
sidering the safety and efficacy of subunit vaccines [30], this
antigen could be considered as candidate for use in future studies
of vaccines against bovine or human brucellosis.
3.2. Hypothetical protein encoded by BAB1_0263 ORF
This ORF encodes a hypothetical protein whose function in B.
abortus is unknown. However, the amino acid sequence it codifies
shares 42% homology with Lsr2 protein of Saccharopolyspora ery-
thraea strain NRRL 23,338 [53]. Lsr2 is a regulatory protein
involved in multiple cellular processes, including cell wall biosyn-
thesis, resistance to antibiotics and DNA-binding [54–56]. Based on
this ORF, Sislema-Egas et al. [53] developed a DNA vaccine by clon-
ing the complete BAB1_0263 ORF into pVAX1 vector (a plasmid
with human cytomegalovirus (CMV) promoter and high copy num-
ber origin of replication gene generally used for DNA immunization
purposes) to evaluate its immunogenicity in intramuscularly
immunized BALB/c mice. Their results showed that this DNA vac-
cine induced a significant production of serum IgG antibodies
against B. abortus recombinant protein encoded by BAB1_0263;
antibody response was dominated by IgG2a isotype, as well as an
increased lymphoproliferative response of IFN-c-producing
splenocytes. However, this specific humoral and cellular immunity
induced by this DNA vaccine against the recombinant antigen
encoded by BAB1_0263 was unable to confer statistically signifi-
cant levels of protection in BALB/c mice challenged with the patho-
genic B. abortus 2308 strain (Table 6). Therefore, although this
vaccine induced a Th1-type phenotype generally associated with
resistance to, it did not provide significant protection; supposedly
because the product encoded by this ORF is not involved in the
infection processes or in establishing this pathogen. This assump-
tion is based on the fact that the BAB1_0263 ORF deletion of B.
 L. Gómez et al. / Vaccine 36 (2018) 2928–2936 2933

Table 6
Experimental vaccines based on the GI-3 ORFs of Brucella abortus.

GI-3 ORFs Type of Mice Route Vaccine doses x Strain Challenge/Doses Challenge Protection Ref.
vaccine Model mice (CFU)  Mice time levelsa
BAB1_0260 Subunit BALB/c i.p. rFlgJ (30 lg)/CFA- B. abortus 544/2  105 30 d 1.63* [30]
protein IFA
BAB1_0263 DNA BALB/c i.m. pVF263 (100 lg) B. abortus 2308/1  104
15 d 0.93 NS
[53]
BAB1_0267 DNA BALB/c i.m. pV267 (100 lg) B. abortus 2308/1  104 15 d 0.63NS [61]
BAB1_0270 DNA BALB/c i.m. pV270 (100 lg) B. abortus 2308/1  104 15 d 0.91* [61]
BAB1_0278 DNA BALB/c i.m. pVF278 (100 lg) B. abortus 2308/1  104 2 wk 1.28* [53]
BAB1_0278a DNA BALB/c i.m. pV278A (100 lg) B. abortus 2308/1  104 2 wk 1.33* [65]
BAB1_0260, BAB1_0270, BAB1_0273, DNA Multi- BALB/c i.m. pV-MEB (100 lg) B. abortus 2308/1  104 2 wk 1.14* [66]
BAB1_0278sodC epitope
BAB1_0273sodC DNA BALB/c i.m. pV273-sod (100 B. abortus 2308/1  104 15 d 0.40NS [67]
multivalent lg)
BAB1_0278sodC DNA BALB/c i.m. pV278-sod (100 B. abortus 2308/1  104 15 d 0.32NS [67]
multivalent lg)
BAB1_0273BAB1_278sodC DNA BALB/c i.m. pV273-278-sod B. abortus 2308/1  104 15 d 0.28NS [67]
multivalent (100 lg)

Significant values (*); Non-significant values (NS); Intraperitoneal (i.p.) route; Intramuscular (i.m.) route; Complete Freund’s Adjuvants (CFA); Incomplete Freund’s Adjuvants
(IFA); days (d); weeks (wk).
a
Protection levels are measured by units of protection, represented by the difference between the mean of log10 CFU/spleen values of the negative control group and the
mean of log10 CFU/spleen values of the immunized group.

abortus 2308 strain does not affect its ability to survive inside modification, protein turnover and accompanying functions. It
human epithelial cells (HeLa), murine macrophages (J774) or has been described that ImmA and IrrE are metalloproteases that
BALB/c mice [42]. Results indicate that the immune response cleave repressor proteins, leading to the transcriptional induction
induced by this vaccine would not be triggered during the infec- of various genes required to regulate horizontal gene transfer or
tion, making this ORF an ineffective target for the design of a Bru- survival to environmental stress, activating the bacterial SOS
cella vaccine. response [62,63]. Ortiz-Román et al. [43] demonstrated that dele-
tion of this ORF reduced the ability of B. abortus 2308 to survive
and replicate within human epithelial cells (HeLa) and murine
3.3. Hypothetical protein with an Src homology 3-like domain encoded
macrophages (J774), in addition to a reduced survival of this strain
by BAB1_0267 ORF
in BALB/c mice. In addition, mutant strains for this ORF are unable
to escape the phagolysosomal degradation, preventing its subse-
This ORF encodes a conserved hypothetical protein with high
quent establishment in vacuoles that present markers for macro-
identity (99.1%) to Src Homology 3 (SH3)-type domain-
phage endoplasmic reticulum (data not published). B. abortus
containing protein of B. suis bv. 5 str. 513, B. neotomae 5K33 and
ORF BAB1_0270 was also targeted for the development of a DNA
B. abortus NCTC 8038 strains (Table 4). The SH3-like domains are
vaccine [61]. Results showed that immunized BALB/c mice signifi-
present in eukaryotic cell adapter proteins where they act on intra-
cantly increased titers of IgG and IgG2a antibodies anti- B. abortus
cellular signaling pathways and cytoskeleton regulation [57]. In
recombinant BAB1_0270 product, the production of IFN-c and
bacteria, proteins with SH3-like domains (SH3b) appear to be
TNF-a, and lymphoproliferative splenocyte responses. Polarization
involved in the modulation of host immune responses and/or cell
towards the Th1 phenotype response in this experimental model
invasion [58–60]. The importance of this ORF in the intracellular
conferred statistically significant levels of protection (0.91 log10),
survival and virulence of B. abortus 2308 in BALB/c mice was
against the pathogenic strain B. abortus 2308. Therefore, the zinc-
demonstrated by Ortiz-Román et al. [43]. Besides, deletion of the
dependent metalloproteinase encoded by ORF BAB1_0270 has pro-
BAB1_0267 ORF significantly reduced the ability of B. abortus
ven to be necessary for intracellular survival, and represents a
2308 to develop its replication niche in vacuoles that present endo-
viable candidate for a vaccine against brucellosis. These results
plasmic reticulum markers (data not published). Evidence has
showed the importance of this zinc-dependent metalloprotease
demonstrated the importance of this hypothetical protein in intra-
for Brucella virulence, raising the questions regarding oxidative
cellular pathogen establishment. Development of a DNA vaccine
and/or acid stress from phagosome/lysosome fusion triggering its
based on the BAB1_0267 ORF, cloned into pVAX1 plasmid, and
expression. Some of the other questions left unanswered are: Is
the immunization of BALB/c mice with this DNA vaccine induced
this protein involved in the transcriptional regulation of the
a significant titer of specific IgG and IgG1 antibodies against B.
remaining ORFs of GI-3? Does this protein have a function similar
abortus recombinant BAB1_0267, and an elevated lymphoprolifer-
to IrrE protein? In the eventuality that it cleaves a repressor, which
ative response was associated to low levels of IFN-c. This response
repressor protein is cleaved?
conferred non-significant levels of protection (0.63 log10) in ani-
mals tested with the pathogenic B. abortus 2308 strain (Table 6).
These results demonstrated that vaccination with DNA vectors
3.5. Hypothetical protein GcrA encoded by BAB1_0278 ORF
encoding this ORF triggered a poor immune response, ratifying
the importance of IFN-c for inducing a Th1-dependent immunity,
The GcrA protein is a master cell cycle regulator involved in the
required for B. abortus clearance in BALB/c mice [61].
control of cell cycle progression and asymmetric polar Brucella
morphogenesis. This protein acts as an activator of replisome com-
3.4. ImmA/IrrE metallo-endopeptidase family encoded by BAB1_0270 ponents and bacterial cell segregation machinery [64]. Céspedes
ORF et al. [42] demonstrated that the deletion of B. abortus 2308 ORF
BAB1_0278 significantly reduced bacterial duplication and growth
This ORF encodes zinc-dependent metalloprotease, which is a capacity within professional and non-professional phagocytes. Due
member of the ImmA/IrrE metalloendopeptidases family. The to the role of this ORF for intracellular survival of this bacterium,
ImmA/IrrE family of proteins are involved in posttranslational Sislema-Egas et al. [53] developed the pVF278 DNA vaccine, using
2934 L. Gómez et al. / Vaccine 36 (2018) 2928–2936
pVAX1 plasmid encoding the complete BAB1_0278 ORF. Their
results demonstrated that immunization with this vaccine
increased IgG antibody titer against the product of B. abortus
recombinant BAB1_0278; IgG2a being the dominant isotype, and
a significant increase of lymphoproliferative responses. This
immune response was capable to confer significant levels of pro-
tection (1.28 log10) to BALB/c mice when challenged with patho-
genic B. abortus 2308 strain. Although the authors did not report
IFN-c production (a critical cytokine in resistance against this
pathogen), they concluded that the protective response was inde-
pendent of IFN-c, and more associated with TNF-a production.
Results demonstrated that the protein encoded by BAB1_0278
ORF is immunogenic and provides protection, making it a target
for the design of vaccines against brucellosis. In parallel,
Riquelme-Neira et al. [65] developed a DNA vaccine that included
a genomic segment located after BAB1_0277 ORF and part of
BAB1_0278 ORF, which they named BAB1_0278a. This segment
could be a new ORF encoding a hypothetical protein, homologous
to an ABC-type transporter [65]. Their results showed that mice
immunized with this vaccine significantly increased the produc-
tion of serum IgG and IgG2a levels of anti-B. abortus recombinant
BAB1_0278a product, increased T-cell proliferation, increased
IFN-c production and protection (1.33 log10) against the B. abortus
pathogenic 2308 strain (Table 6). Unlike Sislema-Egas et al. [53],
these authors reported the production of IFN-c, indicating that
protective responses are dependent on this cytokine, which is crit-
ical in host resistance to brucellosis. Therefore, the immunogenic-
ity induced by the vaccines reported by Sislema-Egas et al. [53] and
Riquelme-Neira et al. [65] demonstrated that these DNA vaccines
are effective inducers of a Th1-type response, which is generally
protective against brucellosis. It must be emphasized that these
vaccines provided the highest levels of protection reported for
DNA vaccines to date, based on B. abortus ORFs located at GI-3.
3.6. Multi-epitope and multivalent DNA vaccines based on GI-3 ORFs
Multi-epitopes and multivalent DNA vaccines based on GI-3
ORFs have been used previously in vaccine design against brucel-
losis. Escalona et al. [66] used bio-informatic tools to develop a
chimerical DNA vaccine containing twenty-one B. abortus 2308
immunodominant epitopes for class I and class II Major Histocom-
patibility Complex proteins (MHC I and MHC II). These immun-
odominant epitopes were present in the products of BAB1_0260,
BAB1_0270, BAB1_0273, BAB1_0278 GI-3 ORFs and SOD protein.
The sequence containing the immunodominant epitopes was
cloned into pVAX1 plasmid and BALB/c mice were immunized with
this recombinant vector vaccine. Their results showed that intra-
muscular immunization with this vaccine significantly increased
the production of serum IgG2a anti-B. abortus multi-epitopes, lym-
phoproliferative responses and IFN-c levels (Table 6). This
immunogenicity conferred significant levels of protection (1.14
log10) in BALB/c mice that were challenged with B. abortus 2308
strain. According to these authors, these could be the first results
for a multi-epitope chimeric DNA vaccine designed using an in sil-
ico technique, with potential to prevent brucellosis. On the other
hand, Gómez et al. [67] evaluated the efficacy of three multivalent
DNA vaccines in BALB/c mice. These vaccines were designed by
fusion of the sequences of BAB1_0273 or/and BAB1_0278 ORFs to
sodC gene by rigid linkers based on the prolines and alanines
(PAPAP), which were chemically synthetized and cloned into the
pVAX1 plasmid. Their results showed these multivalent DNA vac-
cines induced a Th1 immunity in immunized mice, a response
associated with increased IgG2a levels against B. abortus recombi-
nant multivalent proteins, IFN-c and lymphoproliferative
response. However, this immunogenicity was unable to confer sta-
tistically significant levels of protection (0.40, 0.32, and 0.28 log10);
hence these are vaccines with lower protection levels (Table 6).
Results contrary to those obtained using individual GI-3 ORFs vac-
cines, showing that some multivalent vaccines can induce less pro-
tection levels than monovalent DNA vaccines encoding for sodC
gene or BAB1_0278 ORF [33,53]. A minor protective response,
probably because the combination of these antigens could have
triggered a phenomenon of immunological interference associated
with the competition between the antigens expressed by these
vaccines [68]. Therefore, these multivalent DNA vaccines demon-
strate that the immunogenicity induced by them is essential to
induce an immune response, but it is not a parameter sufficient
to achieve an effective protective immunity [69] against B. abortus
[67].
Interestingly, these vaccines were designed using the
BAB1_0273, ORF encoding the XRE (Xenobiotic Response Element)
family transcriptional regulator that controls the expression of
genes involved in stress response (Table 4). This protein has a
helix-turn-helix (HTH) domain that could be involved in the
repression of the GI-3 ORFs, which under certain conditions of
environmental stress could be cleaved by an ImmA/IrrE zinc-
dependent metalloprotease (ORF BAB1_0270). If, in fact, the pro-
duct of BAB1_0270 cleaves the repressor, it might allow the
expression of several genes associated with Brucella survival in a
hostile environment.
4. Conclusions and future perspectives
Alternative vaccination strategies have been developed for bru-
cellosis prevention, including live attenuated strains, protein sub-
units and DNA vaccines. These vaccine candidates are
summarized in Tables 1–3, which have demonstrated their ability
to elicit variable levels of protection in mice against intracellular or
cell envelope targets of B. abortus. Some of these vaccines are con-
sidered excellent vaccine candidates due to their ability to confer
higher than 2 log10 levels of protection [17,19,24,26,28,33,35,39]
or even values higher than 3 log10 of protection [16,20,37]. Besides
these vaccines candidates, in this review we have showed the
importance of the GI-3, a genomic region of B. abortus used as
source to develop promising vaccines, mainly DNA vaccines, in
the prevention of B. abortus infection.
This GI contains some ORFs encoding proteins involved in intra-
cellular survival and virulence of B. abortus (Table 5), supporting
the idea that GI-3 could, in fact, be an island of pathogenicity. Thus,
some GI-3 ORFs based vaccines induced immunogenicity capable
of conferring protection associated with Th1-dependent immunity
in BALB/c mice model (Table 6). These vaccines have shown vari-
able levels of protection; being the rFlgJ subunit protein vaccine
encoded by the BAB1_0260 ORF was the one that provided the best
protection levels in BALB/c mice [30], followed by DNA vaccines
based on the ORFs BAB1_0278 and BAB1_0278a, and the multi-
epitope DNA vaccine [53,65,66]. These differences in protective
efficacy may be associated with the nature of the antigen, the type
of vaccine (DNA or protein) as well as the use of Freund’s adjuvant,
a component used to increase the immunogenicity of rFlgJ subunit
vaccine [30,70]. These interesting results of immunogenicity have
shown that some targets encoded in these ORFs of the GI-3 are pro-
tective antigens against B. abortus. However, we must also consider
that these vaccines only reduced the bacterial burden in mice
spleen as a small percentage of bacteria still survived in this organ
suggesting a possible source for recrudescence of infection. This
remnant surviving Brucella must be cautiously considered in order
to develop better vaccines that will completely clear the infection.
In addition, of all the GI-3 ORFs used to develop vaccines, it is inter-
esting to determine the immune response induced by a vaccine
based on the Btp1 protein, a virulence factor involved in the host’s
 L. Gómez et al. / Vaccine 36 (2018) 2928–2936
immune suppression [43,45,46]. Therefore, from the analysis
reported in this review, many questions arise regarding the regula-
tion and importance of the GI-3 in the intracellular adaptation of B.
abortus, and its contribution to the development of vaccines to pre-
vent brucellosis.
As shown in Tables 1–3 and 6, several experimental vaccines
can confer significant protection against B. abortus infection.
However, a meta-analysis by Carvalho et al. [15] showed high
variability in protection achieved by Brucella vaccines, concluding
that so far there is no clear trend in improving protective immune
response by these vaccines. This variability in the protection
levels measured by reduction of B. abortus burden in mice spleens
has shown dissimilar results depending on the type of vaccine,
route of administration, doses and challenge duration used in
each experiment. Besides, experimental protocols used to test
the efficacy of these vaccines are not standardized for BALB/c
mice, generating results that are often not fully reproducible
[71]. In this context, vaccines developed from GI-3 ORFs are no
exception, showing variable levels of protection (Table 6). In addi-
tion to these drawbacks, we have to question whether the
immunogenicity and the levels of protection conferred by these
experimental vaccines in BALB/c mice will replicate in the natural
host and in humans. We postulate that all these differences in
efficacy may be due to significant discrepancies in the innate
and adaptive immune responses between mice, humans and
ruminants, however, it is clear that mice are a well-established
model for brucellosis studies, although the advantages and limita-
tions of the model they must be properly understood within the
appropriate experimental context [72]. Undoubtedly, the informa-
tion described in this review shows that new technologies must
be implemented for the development of an effective and safe vac-
cine to prevent bovine and human brucellosis. However, the
development of these vaccines is associated with intrinsic diffi-
culties presented by B. abortus, a pathogen that is highly adapted
to intracellular life, and that has developed a strategy to modulate
the host immune response [2–4]. For this reason, we must iden-
tify the virulence factors involved during the early stages of infec-
tion in order to develop vaccines against these targets. Moreover,
we must identify the variables influencing protection in BALB/c
mice to generate more reproducible data and to develop a
method, which will allow us to predict the performance of these
experimental vaccines in cattle and humans. These improvements
will help us to finally design approach strategies to develop effec-
tive and safe vaccines against Brucellosis.
Finally, incidence of human brucellosis has spurred us to
develop several experimental vaccines based on B. abortus 2308
GI-3 ORFs that provide effective protection in BALB/c mice. Based
on the literature, several challenges remain, including the correla-
tion between the protective response observed in BALB/c mice to
that of in cattle and humans. Also, unraveling the functions of
several GI-3 ORFs that have not yet been studied would identify
potential targets for therapy against this disease which by itself
is another frontier to conquer. This search, within and without
GI-3 will allow for the development of successful multivalent
and multi-epitope vaccines, prime boost strategies, and vaccines
complemented with adjuvants based on the nano- and micro-
encapsulation technologies that are currently being implemented.
Thus, significant advancements are necessary to develop a safe
and effective vaccine to prevent bovine and human brucellosis.
Acknowledgements
This work was supported by Grant 1130093 from the Fondo
Nacional de Desarrollo Científico y Tecnológico (FONDECYT),
Santiago, Chile.
2935
Conflict of interest
The authors declare no conflict of interest regarding the publi-
cation of this paper.
Authors contributions
All authors contributed equally to this work.
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