Genome-wide analysis of Brucella melitensis growth in spleen of infected mice allows rational selection of new vaccine candidates.

Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates.

Design a novel of Brucellosis preventive vaccine based on IgV_CTLA-4 and multiple epitopes via immunoinformatics approach.

Brucellosis is a zoonotic disease caused by Brucella, which is difficult to eliminate by conventional drugs. Therefore, a novel multi-epitope vaccine (MEV) was designed to prevent human Brucella infection. Based on the method of "reverse vaccinology", cytotoxic T lymphocyte epitopes (CTLEs), helper T lymphocyte epitopes (HTLEs), linear B-cell epitopes (LBEs) and conformational B-cell epitopes (CBEs) of four Brucella proteins (VirB9, VirB10, Omp 19 and Omp 25) were obtained. In order to keep the correct protein folding, the multiple epitopes was constructed by connecting epitopes through linkers. In view of the significant connection between human leukocyte antigen CTLA-4 and B7 molecules found on antigen presenting cells (APCs), a new vaccine (V_C4MEV) for preventing brucellosis was created by combining CTLA-4 immunoglobulin variable region (IgV_CTLA-4) with MEV protein. Immunoinformatics analysis showed that V_C4MEV has a good secondary and tertiary structure. Additionally, molecular docking and molecular dynamics simulation (MD) revealed a robust binding affinity between IgV_ CTLA-4 and the B7 molecule. Notably, the vaccine V_C4MEV was demonstrated favorable immunogenicity and antigenicity in both in vitro and in vivo experiments. V_C4MEV had the potential to activate defensive cells and immune responses, offering a hopeful approach for developing vaccines against Brucella in the upcoming years.

Construction of recombinant Omp25 or EipB protein loaded PLGA nanovaccines for Brucellosis protection.

Safe and effective vaccine candidates are needed to address the limitations of existing vaccines against Brucellosis, a disease responsible for substantial economic losses in livestock. The present study aimed to encapsulate recombinant Omp25 and EipB proteins, knowledged antigen properties, into PLGA nanoparticles, characterize synthesized nanoparticles with different methods, and assessed theirin vitro/in vivoimmunostimulatory activities to develop new vaccine candidates. The recombinant Omp25 and EipB proteins produced with recombinant DNA technology were encapsulated into PLGA nanoparticles by double emulsion solvent evaporation technique. The nanoparticles were characterized using FE-SEM, Zeta-sizer, and FT-IR instruments to determine size, morphology, zeta potentials, and polydispersity index values, as well as to analyze functional groups chemically. Additionally, the release profiles and encapsulation efficiencies were assessed using UV-Vis spectroscopy. After loading with recombinant proteins, O-NPs reached sizes of 221.2 ± 5.21 nm, while E-NPs reached sizes of 274.4 ± 9.51 nm. The cumulative release rates of the antigens, monitored until the end of day 14, were determined to be 90.39% for O-NPs and 56.1% for E-NPs. Following the assessment of thein vitrocytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells,in vivoimmunization experiments were conducted using concentrations of 16µg ml-1for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing producedBrucella-specific IgG antibody levels for 3 times in contrast to control. Furthermore, it was also demonstrated that vaccine candidates stimulated Th1-mediated cellular immunity as well since they significantly raised IFN-gamma and IL-12 cytokine levels in murine splenocytes rather than IL-4 following to immunization. Additionally, the vaccine candidates conferred higher than 90% protection from the infection according to challenge results. Our findings reveal that PLGA nanoparticles constructed with the encapsulation of recombinant Omp25 or EipB proteins possess great potential to triggerBrucella-specific humoral and cellular immune response.

Detection of Brucella S19 Vaccine Strain DNA in Domestic and Wild Ungulates from Brazilian Pantanal.

The Pantanal region, the largest floodplain in the world, has a huge biodiversity and is an important livestock center. Bovine brucellosis has been reported in the region over the last three decades, posing implications for cattle industry as well as for the maintenance of biodiversity. We aimed to investigate the presence of B. abortus S19 vaccine strain DNA in unvaccinated domestic and wild ungulates from the Brazilian Pantanal. Fifty-two heifers, 63 ovine, 24 domestic pigs, 28 feral pigs, and three Pampas deer were sampled. Brucella spp. was detected through bcsp31 PCR of blood samples in 45.3% (77/170) of the sampled animals, of which 36.4% (28/77) showed positivity in ery PCR corresponding to B. abortus S19 strain. Feral pigs presented the highest occurrence of positive samples in bcsp31 PCR (75%), followed by ovine (47.6%), domestic pigs (41.7%), and unvaccinated heifers (30.8%). We did not observe positivity in Pampas deer. Our results strongly suggest that vaccination against bovine brucellosis may promote spill-over of B. abortus S19 strain in the Pantanal region. Moreover, our data indicate that wild strains of Brucella circulates in the Pantanal Biome.

Immunogenicity of Brucella Trivalent Immunogen-Containing Polyethyleneimine Nanostructure Targeted with LPS in a Mouse Model.

Brucella is a facultative intracellular gram-negative coccobacillus. It is nonsporulating and reproduced in macrophage phagosomes. The use of nanostructures as drug and vaccine carriers has recently received attention due to their ability to control the release profile and protect the drug molecules. This study presents a suitable nano-polyethyleneimine formulation to be used as an immunoadjuvant and LPS along with trivalent candidate antigens of TF, BP26, and omp31 to selectively stimulate the immune response. After designing and evaluating the immunogenic structure by databases and bioinformatics software, recombinant protein cloning and gene expression were performed in Escherichia coli BL21 bacteria. This protein was extracted from the cultured cells, purified by Ni-NTA column. After placing the antigen inside the polyethyleneimine nanostructure, various properties of the nanoparticles, including their size, zeta potential, and retention rate for injection and inhalation of mice, diffusion efficacy, and antigen binding evaluation were evaluated. Mice were treated with different groups of antigens and nanoparticles on days 0, 10, 24, and 38. Two weeks after the last injection, the level of cytokines were investigated in spleen cells, including IFN-γ, IL-4, and IL-12. The serum concentration of IgG2a and IgG1 antibodies were also assessed. The response was consistent with significant production of IgG1, IgG2a, IFN-γ21, IL-12, and IL-4 compared to the controls (P < 0.05). Compared to the positive and negative control groups, recombinant protein and nanoparticles showed a good response in subsequent injections with live bacterial strains. The present study also revealed the potential of the developed recombinant protein as a candidate in the design and manufacture of subunit vaccines against Brucella species. This protein stimulates cellular and humoral immune responses compared to the positive control groups. These findings can be useful in the prevention and control of brucellosis and pave the way for further research by researchers around the world.

A subunit vaccine based on Brucella rBP26 induces Th1 immune responses and M1 macrophage activation.

Brucellosis is a global zoonotic infection caused by Brucella bacteria, which poses a significant burden on society. While transmission prevention is currently the most effective method, the absence of a licenced vaccine for humans necessitates the urgent development of a safe and effective vaccine. Recombinant protein-based subunit vaccines are considered promising options, and in this study, the Brucella BP26 protein is expressed using prokaryotic expression systems. The immune responses are evaluated using the well-established adjuvant CpG-ODN. The results demonstrate that rBP26 supplemented with a CpG adjuvant induces M1 macrophage polarization and stimulates cellular immune responses mediated by Th1 cells and CD8 + T cells. Additionally, it generates high levels of rBP26-specific antibodies in immunized mice. Furthermore, rBP26 immunization activates, proliferates, and produces cytokines in T lymphocytes while also maintaining immune memory for an extended period of time. These findings shed light on the potential biological function of rBP26, which is crucial for understanding brucellosis pathogenesis. Moreover, rBP26 holds promise as an effective subunit vaccine candidate for use in endemic areas.

Effects of vaccination with Brucella melitensis, strains Rev 1 ΔeryCD and Rev 1, on the reproductive system of young male goats.

The present study evaluates the effects of vaccination with Brucella melitensis strains Rev 1 ΔeryCD and Rev 1 on the reproductive system of male goats. Three groups, each of them consisting of 15 six-month-old brucellosis-free male goats, were studied. The first group was vaccinated with the Rev 1 ΔeryCD strain, the second group received Rev 1 and the third group was inoculated with sterile physiological saline solution. The dose of both strains was of 1×109CFU/ml. Over the course of the five months of this study, three males from each group were euthanized every month. Their reproductive tracts, spleens, and lymph nodes were collected to analyze serology, bacteriology PCR, histology, and immunohistochemistry. Results show that vaccination with B. melitensis strains Rev 1 ΔeryCD and Rev 1 does not harm the reproductive system of male goats. Strain B. melitensis Rev 1 ΔeryCD displayed a lower capacity to colonize the reproductive tract than strain Rev 1, which was attributed to its limited catabolic action toward erythritol.

Targeting resident memory T cell immunity culminates in pulmonary and systemic protection against Brucella infection.

Brucellosis remains the most common zoonotic disease globally. Currently no vaccines for humans exist, and conventional brucellosis vaccines for livestock fail to confer complete protection; hence, an improved vaccine is needed. Although Brucella infections primarily occur following a mucosal exposure, vaccines are administered parenterally. Few studies have considered mucosal vaccinations, or even targeting of tissue-resident memory T (TRM) cells. TRM cells protect against viral infections, but less is known of their role in bacterial infections, and even less for brucellosis. Oral prime, nasal boost with a newly developed Brucella abortus double mutant (znBAZ) confers nearly complete protection against pulmonary challenge with wild-type (wt) B. abortus 2308, and its protective efficacy is >2800-fold better than the RB51 vaccine. Vaccination with znBAZ potently stimulated CD8+ T cells, whereas mucosal vaccination with RB51 induced mostly CD4+ T cells. Subsequent analysis revealed these pulmonary CD44+ CD69+ CD8+ T cells to be either CD103+ or CD103- TRM cells, and these sequestered to the lung parenchyma as CXCR3lo and to the airways as CXCR3hi. Both CD8+ TRM subsets contained single-positive IFN-γ and TNF-α, as well as, polyfunctional cells. IL-17-producing CD8+ TRM cells were also induced by znBAZ vaccination, but in vivo IL-17 neutralization had no impact upon protection. In vivo depletion of CD4+ T cells had no impact upon protection in znBAZ-vaccinated mice. In contrast, CD4+ T cell depletion reduced RB51's protective efficacy in spleens and lungs by two- and three-logs, respectively. Although anti-CD8 mAb-treated znBAZ-vaccinated mice showed a significantly reduced pulmonary efficacy, this treatment failed to completely deplete the lung CD8+ T cells, leaving the CD103+ and CD103- CD8+ TRM cell ratios intact. Only znBAZ-vaccinated CD8-/- mice were fully sensitive to pulmonary challenge with virulent wt B. abortus 2308 since CD8+ TRM cells could not be induced. Collectively, these data demonstrate the key role of mucosal vaccination for the generation of CD8+ TRM cells in protecting against pulmonary challenge with virulent B. abortus.

Lactococcus-based vaccine against brucellosis: IgG immune response in mice with rOmp16-IL2 fusion protein.

This study was designed to introduce the recombinant Lactococcus lactis MG1363 as a cell factory candidate for production of recombinant Brucella melitensis Omp16-Human IL2 (r-Omp16-IL2) and to suggest it as a promising safe, non-pathogenic mucosal live vaccine against brucellosis. Three groups of BALB/c mice (10 mice per group) were intragastrically administrated with phosphate-buffered saline (PBS), L. lactis harboring the empty pAMJ2008 plasmid and with L. lactis expressing rOmp-IL2. The first two groups were classified as control groups and the third one is indicated as treatment group. Another group was injected by the intraperitoneal (i.p.) route with purified rOmp16-IL2 protein. The total serum IgG of each group was assessed with indirect ELISAs at two days before immunization and also two weeks after the last immunization. Results showed that BALB/c mice intragastrically administrated with L. lactis expressing rOmp-IL2 had dominant IgG response compared to the control (PBS administrated) group (P < 0.05). The level of IgG was significantly increased by intraperitoneally injection of recombinant Omp-IL2 in adjuvant compared to the intragastrically administration of PBS and L. lactis/pAMJ2008 as control groups, and also compared to L. lactis/pAMJ2008-rOmp-IL2 (P < 0.05). Our findings provide the use of L. lactis rOmp16-IL2 as a new promising alternative safe strategy than presently live attenuated vaccines toward developing an oral vaccine or subunit-based vaccine against brucellosis.

Design of a new multi-epitope vaccine against Brucella based on T and B cell epitopes using bioinformatics methods.

Brucellosis is one of the most serious and widespread zoonotic diseases, which seriously threatens human health and the national economy. This study was based on the T/B dominant epitopes of Brucella outer membrane protein 22 (Omp22), outer membrane protein 19 (Omp19) and outer membrane protein 28 (Omp28), with bioinformatics methods to design a safe and effective multi-epitope vaccine. The amino acid sequences of the proteins were found in the National Center for Biotechnology Information (NCBI) database, and the signal peptides were predicted by the SignaIP-5.0 server. The surface accessibility and hydrophilic regions of proteins were analysed with the ProtScale software and the tertiary structure model of the proteins predicted by I-TASSER software and labelled with the UCSF Chimera software. The software COBEpro, SVMTriP and BepiPred were used to predict B cell epitopes of the proteins. SYFPEITHI, RANKpep and IEDB were employed to predict T cell epitopes of the proteins. The T/B dominant epitopes of three proteins were combined with HEYGAALEREAG and GGGS linkers, and carriers sequences linked to the N- and C-terminus of the vaccine construct with the help of EAAAK linkers. Finally, the tertiary structure and physical and chemical properties of the multi-epitope vaccine construct were analysed. The allergenicity, antigenicity and solubility of the multi-epitope vaccine construct were 7.37-11.30, 0.788 and 0.866, respectively. The Ramachandran diagram of the mock vaccine construct showed 96.0% residues within the favoured and allowed range. Collectively, our results showed that this multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for future laboratory experiments.

Immunization effect of lipopolysaccharide antigen in conjugation with PLGA nanoparticles as a nanovaccine against Brucella melitensis infection.

Brucella is an infectious disease with difficult treatment faced with drug resistance and recurrence of infection. Despite advances in the development of effective vaccines against brucellosis infections, there is still a need for more effective vaccine against brucellosis. In this study, we developed a nanovaccine for delivery of lipopolysaccharide Brucella melitensis antigen to the immune system using PLGA nanoparticles to prevent Brucella infection, which is associated with the stimulation of both humoral and cellular immune systems. In particular, we determined the rate of produced immunoglobulines and their functional effectiveness on the immune system by carring out opsonophagocytosis and challenge tests. According to the results, it was determined that PLGA improve the delivery of LPS antigen to the immune system to enhance the production of immunoglobulins levels and their efficiency to remove Brucella bacteria.

Evaluation of real-time PCR as an alternative for potency testing of Brucella abortus vaccines.

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

Comparative genomic analysis between newly sequenced Brucella abortus vaccine strain A19 and another Brucella abortus vaccine S19.

BACKGROUND: Brucellosis is a bacterial disease caused by Brucella infection. Brucella abortus strain A19 is a spontaneously attenuated vaccine strain that has been used in vaccination of cattle against brucellosis. Until now, the physiological and molecular mechanisms of A19 are still unknown. RESULTS: In this paper, the whole-genome sequence of B. abortus A19 was performed using Illumina Hiseq 4000 and PacBio sequencing technology and comparative genomics analysis were carried out with the whole genome sequences of B. abortus strains S19. This analysis indicated that the two vaccine strains have a high degree of similarity in genomic structure. We further analysis of the difference in genomic structure between A19 and S19. And found some differential genes such as eryC, eryD and eryF. Of the other different proteins between A19 and S19, such as outer membrane protein, 2-isopropylmalate synthase, citramalate synthase, GntR family transcriptional regulator and ABC transporters, no clear effects related to bacterial virulence were found, pending further investigation. CONCLUSION: The data presented here provide a reasonable basis for designing Brucella vaccines that can be used in other strains.

Potential druggable proteins and chimeric vaccine construct prioritization against Brucella melitensis from species core genome data.

The Brucella melitensis chronic infection and drug resistance emerged as a severe health problem in humans and domestic cattle. The pathogens fast genome sequences availability fetched the possibility to address novel therapeutics targets in a rationale way. We acquired the core genes set from 56 B. melitensis publically available complete genome sequences. A stringent bioinformatics layout of comparative genomics and reverse vaccinology was followed to identify potential druggable proteins and multi-epitope vaccine constructs from core genes. The 23 proteins were shortlisted as novel druggable targets based on their role in pathogen-specific metabolic pathways, non-homologous to human and human gut microbiome proteins and their druggability potential. Furthermore, potential chimeric vaccine constructs were generated from lead T and B-cell overlapped epitopes in combination with immune enhancer adjuvants and linkers sequences. The molecular docking and MD simulation analyses ensured stable molecular interaction of a finally prioritized vaccine construct with human immune cells receptors.

B Cells Inhibit CD4+ T Cell-Mediated Immunity to Brucella Infection in a Major Histocompatibility Complex Class II-Dependent Manner.

Brucella spp. are facultative intracellular bacteria notorious for their ability to induce a chronic, and often lifelong, infection known as brucellosis. To date, no licensed vaccine exists for prevention of human disease, and mechanisms underlying chronic illness and immune evasion remain elusive. We and others have observed that B cell-deficient mice challenged with Brucella display reduced bacterial burden following infection, but the underlying mechanism has not been clearly defined. Here, we show that at 1 month postinfection, B cell deficiency alone enhanced resistance to splenic infection ∼100-fold; however, combined B and T cell deficiency did not impact bacterial burden, indicating that B cells only enhance susceptibility to infection when T cells are present. Therefore, we investigated whether B cells inhibit T cell-mediated protection against Brucella Using B and T cell-deficient Rag1-/- animals as recipients, we demonstrate that adoptive transfer of CD4+ T cells alone confers marked protection against Brucella melitensis that is abrogated by cotransfer of B cells. Interestingly, depletion of CD4+ T cells from B cell-deficient, but not wild-type, mice enhanced susceptibility to infection, further confirming that CD4+ T cell-mediated immunity against Brucella is inhibited by B cells. In addition, we found that the ability of B cells to suppress CD4+ T cell-mediated immunity and modulate CD4+ T cell effector responses during infection was major histocompatibility complex class II (MHCII)-dependent. Collectively, these findings indicate that B cells modulate CD4+ T cell function through an MHCII-dependent mechanism which enhances susceptibility to Brucella infection.

Elicitation of Th1/Th2 related responses in mice by chitosan nanoparticles loaded with Brucella abortus malate dehydrogenase, outer membrane proteins 10 and 19.

Brucella spp. is the causative agent of brucellosis, one of the worldwide diseases. The pathogen infects humans and animals mainly through the digestive or respiratory tract. Therefore, induction of mucosal immunity is required as the first line of defense. In this study, three Brucella abortus recombinant proteins, malate dehydrogenase (rMdh), outer membrane proteins (rOmp) 10 and 19 were loaded in mucoadhesive chitosan nanoparticles (CNs) and induction of mucosal and systemic immunity were investigated after intranasal immunization of BALB/c mice. These antigens were also coimmunized as cocktail (rCocktail) to evaluate multiple antigen specific vaccine candidates. At 6-weeks post-immunization (wpi), antigen specific total IgG was increased in all of the immunized groups, predominantly IgG1. In addition, spleenocyte from rMdh-, rOmp19-, and rCocktail-immunized groups significantly produced IFN-γ and IL-4 suggesting the induction of a mixed Th1-Th2 response. For mucosal immunity, anti-Mdh IgA from nasal washes and fecal excretions, and anti-Omps IgA from sera, nasal washes, genital secretions and fecal excretions were significantly increased in single antigen immunized groups. In the rCocktail-immunized group, anti-Mdh IgA were significantly increased while anti-Omps IgA was not. Collectively, this study indicates that comprise of B. abortus antigen-loaded CNs elicited the antigen-specific IgA with a Th2-polarized immune responses and combination of the highly immunogenic antigens elicited IgG specific to each type of antigen.

Development of attenuated live vaccine candidates against swine brucellosis in a non-zoonotic B. suis biovar 2 background.

Brucella is a genus of gram-negative bacteria that cause brucellosis. B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1-3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2ΔwbkF) or only producing internal O-chain precursors (Bs2Δwzm) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2ΔwadB and Bs2ΔwadD). We also investigated mutants in the pyruvate phosphate dikinase (Bs2ΔppdK) and phosphoenolpyruvate carboxykinase (Bs2ΔpckA) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (108 and 105 CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2ΔwbkF, Bs2Δwzm, Bs2ΔwadB and the Rev1 control (105 CFU). As described before for B. abortus, B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2ΔppdKΔpckA had the same metabolic phenotype as Bs2ΔppdK and ppdK mutation was enough to generate attenuation. At 105 CFU, Bs2ΔppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2ΔwbkF, the latter seems a better R vaccine candidate than Bs2Δwzm. However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2ΔwbkF is advantageous over Bs2ΔwadB or Bs2ΔppdK requires experiments in the natural host.

Live vaccine consisting of attenuated Salmonella secreting and delivering Brucella ribosomal protein L7/L12 induces humoral and cellular immune responses and protects mice against virulent Brucella abortus 544 challenge.

The present study employs the Brucella abortus L7/L12 antigen in a Salmonella secretion platform and investigates its ability to induce protective immune responses against wild type challenge in BALB/c mice. The highly conserved L7/L12 open reading frame was PCR amplified from B. abortus and cloned into a prokaryotic expression vector, pJHL65, directly under the beta-lactamase secretory signal. The plasmid constructs pJHL65::L7/L12 was then transformed into an attenuated Salmonella Typhimurium strain, JOL1800 (∆lon, ∆cpxR, ∆asd, and ∆rfaL), and protein secretion was verified by Western blot. Three mice groups were inoculated with either phosphate-buffered saline (PBS), vector-only control, or the vaccine strain secreting L7/L12 antigen. Assessment of humoral and cell-mediated immune responses revealed successful elicitation of Brucella antigen-specific Th1 and Th2 immune responses that were significantly higher than PBS and vector control groups. The immune responses were confirmed by splenocyte proliferation assay, flow cytometry analysis for CD4+ and CD8+ markers, and RT-PCR based cytokine profiling upon restimulation with L7/L12 purified antigen. Results indicate that immunization with Salmonella secreting L7/L12 antigen demonstrated significant enhancement of cell-mediated immune (CMI) responses in immunized mice. The overall effectiveness of the immunization was evaluated by challenging with virulent B. abortus that revealed significant reduction in Brucella colonization in spleen and liver tissues in Salmonella L7/L12 immunized mice. Delivery of Brucella protective antigen L7/L12 using the Salmonella secretion system can effectively accomplish immunogenic advantages of both Salmonella and L7/L12 to derive robust CMI responses and induce humoral immunity to protect against Brucella infection in the mouse model.

The candidate vaccine strain Brucella ovis ∆abcBA is protective against Brucella melitensis infection in mice.

Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log10 of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis.

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.