Immunization with recombinant Brucella spp. outer membrane

Frontiers in cellular and infection microbiology, 2017

In this study, we explore the regulatory roles of pro-inflammatory cytokine tumor necrosis factor alpha (TNF) in the innate immunity of macrophages against B. abortus infection. We show that infection of macrophage with B. abortus induces marked expression and secretion of TNF which subsequently binds to TNF receptor 1 (TNFR-1) and activates a downstream signaling cascade of the innate immunity. Blocking of TNF signaling resulted in a notable increase of B. abortus survival which was associated with an increase of anti-inflammatory cytokine interleukin 10 (IL-10), a beneficial effector of Brucella survival, as well as remarkable decrease of reactive oxygen species (ROS) and nitric oxide (NO), antibrucella molecules. However, surprisingly, the interference of TNF did not show any influence on phagolysosome and cell death events. Furthermore, the transcriptional factor NF-kB was found to be a main mediator of TNF signaling when blocking of NF-kB pathway drastically suppressed the TNF-...

Frontiers in Immunology, 2017

Most pathogens infect through mucosal surfaces, and parenteral immunization typically fails to induce effective immune responses at these sites. Development of oral-administered vaccines capable of inducing mucosal as well as systemic immunity while bypassing the issues of antigen degradation and immune tolerance could be crucial for the control of enteropathogens. This study demonstrates that U-Omp19, a bacterial protease inhibitor with immunostimulatory features, coadministered with Salmonella antigens by the oral route, enhances mucosal and systemic immune responses in mice. U-Omp19 was able to increase antigen-specific production of IFN-γ and IL-17 and mucosal (IgA) antibody response. Finally, oral vaccination with U-Omp19 plus Salmonella antigens conferred protection against virulent challenge with Salmonella Typhimurium, with a significant reduction in bacterial loads. These findings prove the efficacy of this novel adjuvant in the Salmonella infection model and support the potential of U-Omp19 as a suitable adjuvant in oral vaccine formulations against mucosal pathogens requiring T helper (Th)1-Th17 protective immune responses.

Clinical and Developmental Immunology, 2012

The outer membrane vesicles (OMVs) from smoothB. melitensis16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs fromB. melitensis16 M; some of them are well-knownBrucellaimmunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strainB. melitensis16 M just as well as the group immunized with live strainB. melitensisRev1 (P<0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Journal of immunology research, 2018

is the protozoan parasite that causes Chagas disease, which is considered by the World Health Organization to be a neglected tropical disease. Two drugs exist for the treatment of Chagas disease, nifurtimox and benznidazole; they are only effective in the acute phase, and a vaccine is currently not available. In this study, we used the recombinant enolase from H8 strain (MHOM/MX/1992/H8 Yucatán) (rTcENO) and its encoding DNA (pBKTcENO) to immunize mice and evaluate their protective effects in an experimental murine model of acute phase infection. Our results showed that mice vaccinated with rTcENO or its encoding DNA were able to generate typical specific antibodies (IgG1, IgG2a, and IgG2b), suggesting that a mixed Th1/Th2 immune response was induced. The parasite burden in the blood was reduced to 69.8% and 71% in mice vaccinated with rTcENO and pBKTcENO, respectively. The group vaccinated with rTcENO achieved 75% survival, in contrast to the group vaccinated with pBKTcENO that sho...

BioMed Research International

In this paper, we first used recombinant influenza viral vector (rIVV) subtype H5N1 expressing from the open reading frame of NS1 80 and NS1 124 amino acids of Brucella outer membrane proteins (Omp) 16 and 19, ribosomal L7/L12, and Cu-Zn superoxide dismutase (SOD) proteins to develop a human brucellosis vaccine. We made 18 combinations of IVVs in mono-, bi-, and tetravalent vaccine formulations and tested them on mice to select the safest and most effective vaccine samples. Then, the most effective vaccine candidates were further tested on guinea pigs. Safety of the rIVV-based vaccine candidate was evaluated by a mouse weight-gain test. Mice and guinea pigs were challenged with the virulent strain B. melitensis 16M. The protective effect of the rIVV-based vaccine candidate was assessed by quantitation of Brucella colonization in tissues and organs of challenged animals. All vaccine formulations were safe in mice. Tested vaccine formulations, as well as the commercial B. melitensis R...

Clinical and Developmental Immunology, 2013

CD8+ T cells have been reported to play an important role in defense againstB. abortusinfection in mouse models. In the present report, we use CD8 knockout mice to further elucidate the role of these cells in protection fromB. melitensisinfection. Mice were immunized orally by administration ofB. melitensisWR201, a purine auxotrophic attenuated vaccine strain, then challenged intranasally withB. melitensis16M. In some experiments, persistence of WR201 in the spleens of CD8 knockout mice was slightly longer than that in the spleens of normal mice. However, development of anti-LPS serum antibody, antigen-induced production ofγ-interferon (IFN-γ) by immune splenic lymphocytes, protection against intranasal challenge, and recovery of nonimmunized animals from intranasal challenge were similar between normal and knockout animals. Further, primaryBrucellainfection was not exacerbated in perforin knockout and Fas-deficient mice and these animals’ anti-Brucellaimmune responses were indistin...

Vaccines, 2021

Vaccination of cattle and buffaloes with Brucella abortus strain 19 has been the mainstay for control of bovine brucellosis. However, vaccination with S19 suffers major drawbacks in terms of its safety and interference with serodiagnosis of clinical infection. Brucella abortus S19∆per, a perosamine synthetase wbkB gene deletion mutant, overcomes the drawbacks of the S19 vaccine strain. The present study aimed to evaluate the potential of Brucella abortus S19Δper vaccine candidate in the natural host, buffaloes. Safety of S19∆per, for animals use, was assessed in guinea pigs. Protective efficacy of vaccine was assessed in buffaloes by immunizing with normal dose (4 × 1010 colony forming units (CFU)/animal) and reduced dose (2 × 109 CFU/animal) of S19Δper and challenged with virulent strain of B. abortus S544 on 300 days post immunization. Bacterial persistency of S19∆per was assessed in buffalo calves after 42 days of inoculation. Different serological, biochemical and pathological s...

PLoS ONE, 2011

Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5610 4 CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-c in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of protective immunity.

Pathogens, 2020

Currently used Brucella vaccines, Brucella abortus strain 19 and RB51, comprises of live attenuated Brucella strains and prevent infection in animals. However, these vaccines pose potential risks to recipient animals such as attenuation reversal and virulence in susceptible hosts on administration. In this context, recombinant subunit vaccines emerge as a safe and competent alternative in combating the disease. In this study, we formulated a divalent recombinant vaccine consisting of Omp25 and L7/L12 of B. abortus and evaluated vaccine potential individually as well as in combination. Sera obtained from divalent vaccine (Omp25+L7/L12) immunized mice group exhibited enhanced IgG titers against both components and indicated specificity upon immunoblotting reiterating its authenticity. Further, the IgG1/IgG2a ratio obtained against each antigen predicted a predominant Th2 immune response in the Omp25+L7/L12 immunized mice group. Upon infection with virulent B. abortus 544, Omp25+L7/L12...

Frontiers in Immunology

Brucellosis is an important zoonotic disease that causes great economic losses. Vaccine immunisation is the main strategy for the prevention and control of brucellosis. Although live attenuated vaccines play important roles in the prevention of this disease, they also have several limitations, such as residual virulence and difficulty in the differentiation of immunisation and infection. We developed and evaluated a new bacterial ghost vaccine of Brucella abortus A19 by a new double inactivation method. The results showed that the bacterial ghost vaccine of Brucella represents a more safe and efficient vaccine for brucellosis. We further characterised the antigenic components and signatures of the vaccine candidate A19BG. Here, we utilised a mass spectrometry-based label-free relative quantitative proteomics approach to investigate the global proteomics changes in A19BGs compared to its parental A19. The proteomic analysis identified 2014 proteins, 1116 of which were differentially ...

Alexandria Journal of Medicine, 2019

Brucellosis is an infectious disease caused by different Brucella species. The outer membrane proteins 25 and 31 play a significant role in stimulation of immunity against Brucella. Herein, the humoral and cellular immune responses of selected recombinant proteins emulsified in chitosan nanoparticles as individual (univalent) and simultaneous (divalent) injections were assessed. The humoral and cellular immune responses were measured by enzyme-linked immunosorbent assay in 12 groups (individual and simultaneous injections of rOMP25 and rOMP31 in different protein concentrations) and lymphocyte proliferation was measured using 3-4,5-dimethylthiazol-2-yl2,5-diphenyltetrazolium bromide (MTT) assay based on average Optical density (OD) of stimulated cells/average OD of unstimulated cells. Finally, data were analyzed using one-way analysis of variance. rOMP25 + rOMP31 group stimulated higher titer of INF-γ than other groups, whilst there were no statistically significant differences between all uni/divalent immunized groups. Tumor necrosis factor alpha titer showed no significant difference between divalent immunized groups (except rOMP31 + rOMP25(1) and rOMP31 + rOMP25(3)) and positive control group. Interleukin-4 analysis results demonstrated that there were no significant differences between positive control and uni/divalent vaccinated groups. In addition, analysis of antibody responses revealed rOMP25 + rOMP31, rOMP25 + rOMP31(2), and rOMP25 + rOMP31(3) groups induced higher level of total antibody compared to other immunized groups, although both univalent and divalent immunized mice induced higher IgG2a titer than IgG1 with the mean of IgG2a/IgG1 ratio~1.01 indicating strong bent of Th1 immune response. The cell proliferation assay demonstrated the vaccination with rOMP25 + rOMP3, rOMP25 + rOMP31(2), and rOMP25 + rOMP31(3) elicited vigorous antigen-specific cell proliferative. rOMP25 + rOMP3, rOMP25 + rOMP31(2), and rOMP25 + rOMP31(3) treatments could be used as potential candidates for developing new subunit vaccines.

International Journal of Nanomedicine, 2020

Introduction: Vaccine formulation with appropriate adjuvants is an attractive approach to develop protective immunity against pathogens. Calcium phosphate nanoparticles (CaPNs) are considered as ideal adjuvants and delivery systems because of their great potential for enhancing immune responses. In the current study, we have designed nanoparticle-based vaccine candidates to induce immune responses and protection against B. melitensis and B. abortus. Materials and Methods: For this purpose, we used three Brucella antigens (FliC, 7α-HSDH, BhuA) and two multi-epitopes (poly B and poly T) absorbed by CaPNs. The efficacy of each formulation was evaluated by measuring humoral, cellular and protective responses in immunized mice. Results: The CaPNs showed an average size of about 90 nm with spherical shape and smooth surface. The CaPNs-adsorbed proteins displayed significant increase in cellular and humoral immune responses compared to the control groups. In addition, our results showed increased ratio of specific IgG2a (associated with Th1) to specific IgG1 (associated with Th2). Also, immunized mice with different vaccine candidate formulations were protected against B. melitensis 16M and B. abortus 544, and showed same levels of protection as commercial vaccines (B. melitensis Rev.1 and B. abortus RB51) except for BhuA-CaPNs. Discussion: Our data support the hypothesis that these antigens absorbed with CaPNs could be effective vaccine candidates against B. melitensis and B. abortus.

Comparative Clinical Pathology, 2017

Brucellosis in humans is one of the most important zoonotic diseases worldwide. This disease causes weakness, fever, pain, sweats, and complications in the liver, spleen, and lymphatic system; Brucellosis requires long-term antibiotic therapy. This study attempts to identify Brucella abortus via an immunofluorescence (IF) test considering antibody against an outer membrane protein with 16-kDa molecular weight (OMP16). The B. abortus OMP16 protein was synthesized through cloning and expressing of the related gene and finally was purified to immunize rabbit in order to produce a polyclonal antibody. Fluorescein isothiocyanate (FITC) was attached to the antibody and the prepared complex was applied to screening of Brucella spp., as an IF test. Polyclonal antibody obtained from the sera of immunized rabbit with the purified OMP16 showed a strong reactivity in ELISA and properly identified the B. abortus strain in IF test. So, this high titer of antibody could be helpful for tracing of B. abortus via laboratory diagnostic tests.

Frontiers in Biology, 2012

Brucella spp. are zoonotic, facultative intracellular pathogens, which cause animal and human disease. Animal disease results in abortion of fetuses; in humans, it manifests flu-like symptoms with an undulant fever, with osteoarthritis as a common complication of infection. Antibiotic regimens for human brucellosis patients may last several months and are not always completely effective. While there are no vaccines for humans, several licensed live Brucella vaccines are available for use in livestock. The performance of these animal vaccines is dependent upon the host species, dose, and route of immunization. Newly engineered live vaccines, lacking well-defined virulence factors, retain low residual virulence, are highly protective, and may someday replace currently used animal vaccines. These also have possible human applications. Moreover, due to their enhanced safety and efficacy in animal models, subunit vaccines for brucellosis show great promise for their application in livestock and humans. This review summarizes the progress of brucellosis vaccine development and presents an overview of candidate vaccines.

Immunology & Cell Biology, 2016

Brucellosis remains a significant zoonotic threat worldwide. Humans and animals acquire infection via their oropharynx and upper respiratory tract following oral or aerosol exposure. After mucosal infection, brucellosis develops into a systemic disease. Mucosal vaccination could offer a viable alternative to conventional injection practices to deter disease. Using a nasal vaccination approach, the ΔznuA B. melitensis was found to confer potent protection against pulmonary Brucella challenge, and reduce colonization of spleens and lungs by more than 2500‐fold, with >50% of vaccinated mice showing no detectable brucellae. Furthermore, 10‐fold more brucellae‐specific, interferon‐γ (IFN‐γ)‐producing CD8+ T cells than CD4+ T cells were induced in the spleen and respiratory lymph nodes. Evaluation of pulmonary and splenic CD8+ T cells from mice vaccinated with ΔznuA B. melitensis revealed that these expressed an activated effector memory (CD44hiCD62LloCCR7lo) T cells producing elevated...