Brucella Vaccine Research Articles (Page 1)

Brucella-infected macrophage-derived Exosomes enhance the anti-Brucella acquired immune response.

Brucellosis, caused by the intracellular pathogen Brucella, remains a significant health challenge, alongside substantial economic impacts on livestock industries. Despite antibiotic treatments, the absence of licensed human vaccines necessitates innovative preventive strategies. In this study, we employed reverse vaccinology to design a novel multi-epitope vaccine (MEV) targeting Brucella melitensis. Three immunogenic proteins-outer membrane protein OMP31, LPS assembly protein LptE, and the type IV secretion system protein VirB2-were selected as vaccine candidates. Comprehensive bioinformatics analysis identified six cytotoxic T lymphocyte (CTL) epitopes, nine helper T lymphocyte (HTL) epitopes, seven linear B-cell epitopes, and five conformational B-cell epitopes. The incorporation of molecular adjuvants (cholera toxin B subunit and PADRE) served to further enhance the immunogenicity of the vaccine. Given that Brucella is an intracellular parasite, TAT cell-penetrating peptides were added to further enhance the intracellular delivery of MEV. The constructed MEV has been shown to have excellent antigenicity (VaxiJen score >0.8), stability (instability index <40), solubility (Protein-Sol score: 0.87) and hydrophilicity (GRAVY index: -0.319), and is non-allergenic. Structural optimization, including disulfide bond engineering (11 pairs of residues), improved molecular stability, with molecular docking and dynamics simulations confirming robust interactions with immune cell receptors (docking score: -311.85). Using SnapGene 7.1.2, we performed in silico cloning simulation of the codon-optimized multi-epitope vaccine (MEV) sequence into the pMV261 shuttle vector, generating a recombinant BCG (rBCG) construct. Immunoinformatics simulations (C-ImmSim) demonstrated potent immune activation, with significant increases in cytotoxic T cells (1050 cells/mm³), memory helper T cells (1150 cells/mm³), and IFN-γ production (2 × 10^6 ng/ml), alongside sustained IgG/IgM titers over 350 days(1 × 10^5 cells/mm3) . Furthermore, the recombinant BCG multi-epitope Brucella vaccine, developed through bioinformatics approaches, demonstrates promising characteristics and immunogenicity. Nevertheless, its immunological efficacy requires to further experimental validation.

Brucellosis, a global zoonosis caused by Brucella species, currently lacks safe vaccines for human use, while existing veterinary live-attenuated vaccines pose infection risks. Although ferritin nanoparticle materials have shown significant advantages in delivery, there is not much research on loading complex polysaccharide antigens. Here, we engineered a bioconjugate nanovaccine (Fn-OPS) through a bacterial glycosylation-driven synthetic strategy, which couples Yersinia enterocolitica O:9 (YeO9) O-specific polysaccharide (OPS)─a structural analogue of Brucella antigens─to a self-assembled ferritin nanoparticle (Fn). This nanocarrier platform combines the symmetrical architecture and antigen-presenting advantages of Fn with the precision of enzymatic glycosylation, addressing the limitations of complex chemical synthesis methods. After confirming the presence of monodisperse nanoparticles with stability at room temperature and great in vivo safety, we performed murine immunization studies and demonstrated a robust activation of T follicular helper (Tfh) cells and germinal center B cells, leading to the production of high-titer IgG antibodies that are cross-reactive with Brucella lipopolysaccharide. This immune response provides strong protection against Brucella infection. This work establishes a ferritin-based nanoconjugate platform for targeted delivery of complex polysaccharide antigens. It advances a scalable, biosafe strategy for Brucella vaccines, expanding the toolkit for targeted antigen delivery in conjugate vaccine design and broadening applications in infectious disease prophylaxis.

Brucella spp., the causative agent of zoonotic brucellosis, poses a significant threat to both animal husbandry and human health. Given the limitations of current antibiotic treatments for human brucellosis, there is an urgent need to develop effective human vaccines. Using reverse vaccinology and bioinformatics approaches, we designed a multi-epitope vaccine (MEV) specifically targeting the CcmA, ccmC, and BepC membrane proteins, with the aim of providing robust protection against human infection.We screened 11 CTL, 9 HTL, 2 conformational B cell epitopes, and 9 linear B cell epitopes, linked them with GGGS,GPGPG and KK. and incorporated HMGN1 (high mobility group nucleosome binding protein 1) as an adjuvant and PADRE (a universal HTL epitope to enhance cross-population immunogenicity) to boost immunogenicity. Analyses showed the 638-amino-acid MEV had high antigenicity (1.2542, exceeded the threshold of 0.4), was non-toxic(ToxinPred2) and non-allergenic(AllergenFP), had low human homology(BLASTP e-value < 0.005), Instability index 26.80 (< 40),and good solubility(0.896, > 0.5).Molecular docking and dynamics confirmed its stability. Codon optimization for E. coli enhanced expression potential. Immunoinformatics simulations showed the MEV effectively increased antibody level,T cell counts, and proinflammatory cytokines.In conclusion, the MEV shows promise for brucellosis vaccine development. To fully assess its efficacy and safety, a series of in vivo experiments will be conducted using animal models, which will serve as a critical step towards validating its potential as a viable vaccine candidate.

Brucellosis is a zoonosis that occurs worldwide, and vaccination is the main strategy for controlling it. In China, the Brucella abortus A19-ΔVirB12 strain is utilized in main vaccines. However, a high-sensitivity nucleic acid detection method to effectively differentiate Brucella infections from immunization with the A19-ΔVirB12 strain is lacking. Therefore, in this study, a duplex droplet digital PCR (ddPCR) assay was established using primers and probes targeting the VirB8 gene and the deleted VirB12 gene in the A19-ΔVirB12 strain. The specificity of the method was tested using genomic DNA of Mycobacterium bovis, Escherichia coli (O:157), Salmonella spp., Streptococcus spp., and A19-ΔVirB12 Brucella. Only A19-ΔVirB12 amplified VirB8 gene. The detection limits of the method for VirB8 and VirB12 were 2.13 × 100 and 2.26 × 100 copies/μL, respectively. In the detection of DNA in epidemic-related samples, the positive rate of ddPCR was much higher than that in the samples analyzed using the commercial fluorescence quantitative reagent kits. Meanwhile, the ddPCR of the A19-ΔVirB12 Brucella vaccine strain was identified in the clinical samples. In summary, the ddPCR method with high sensitivity and specificity was established, which will support the future identification of A19-ΔVirB12 Brucella vaccine strains in immunized and wild-type Brucella.

ABSTRACTThe Brucella abortus A19 attenuated live vaccine poses potential infection risks during practical applications and interferes with serological diagnostics, thereby affecting quarantine measures and the establishment of disease‐free zones. Consequently, this study aimed to reduce its potential virulence, enhance its protective efficacy and differentiate it from wild‐type strains by knocking out the immunosuppressive virulence gene btpB in the A19 strain. Using homologous recombination, we successfully obtained the A19ΔBtpB deletion strain. In a mouse model, the A19ΔBtpB strain demonstrated improved safety and significantly increased TLR2 and TLR4 expression levels in splenic lymphocytes, suggesting attenuated immune suppression. The A19ΔBtpB strain induced Brucella‐specific IgG antibodies comparable to those of the A19 strain but exhibited superior performance in promoting cellular immunity. It effectively induced Th1‐type cytokine (IL‐6 and TNF‐α) production, reduced Th2‐type cytokine (IL‐4 and IL‐10) expression and promoted IFN‐γ expression in T lymphocytes. Notably, the A19ΔBtpB deletion strain provided better protection against the virulent M28 strain in mice than did the A19 strain. In addition, an indirect ELISA diagnostic method based on BtpB protein has been developed, effectively distinguishing vaccine and wild‐type strains in the infection of Brucella. The A19ΔBtpB strain thus represents a promising candidate for a Brucella gene‐deletion vaccine, offering both theoretical and empirical support for future vaccine development.

Construction of dual-signal electrochemical sensors based on the coordination chemical reaction of Fe2+ and K3[Fe(CN)6] for detecting Brucella vaccine and wild strains

Pathogenic bacteria of the genus Brucella cause a severe threat for public health and agricultural economics. The World Health Organization considers brucellosis to be one of the most serious and also neglected zoonotic diseases. The use of traditional whole-cell brucellosis vaccines complicates the differentiation between infected and vaccinated animals (DIVA). Moreover, diagnostics based on lipopolysaccharide of Brucella are susceptible to false positive results. Structural features of Brucella O-antigens make synthetic oligosaccharides promising agents for the development of diagnostic tools and vaccines against brucellosis. Here we report the synthesis of spacer-armed di-, tri-, tetra- and penta-4,6-dideoxy-4-formamido-α-(1→2)-d-mannopyranosides which are related to the A-epitope of Brucella O-antigen. The key α-(1→2)-linked disaccharide thioglycoside donor was synthesized by employing the strategy of orthogonal glycosylation of thioglycoside acceptor with trichloroacetimidate donor. Sequential block-wise assembly yielded a series of desired compounds, which were subsequently deprotected and converted into target molecules and then into their fluorescein-labeled conjugates. The obtained conjugates were employed as tracers in a fluorescence polarization assay (FPA) to detect anti-Brucella immunoglobulins. Among the studied compounds, the trisaccharide conjugate showed the greatest difference in median FP signals between Brucella-positive and Brucella-negative sera samples making it a promising candidate for developing FP diagnostic assays. The decreased FP signal in the cases of tetra- and pentasaccharide tracers can be associated with the known “propeller-effect” due to the rotational mobility of the part bearing the fluorescent label and of the fluorescein itself and/or the enlarging of the distance between the fluorescein part and the antibody-oligosaccharide complex. This observation demonstrates the advantages of using synthetic relatively small synthetic tracers with well-defined structure in comparison with heterogeneous fluorescein-labelled O-polysaccharides which are in use today in spite of the fact that they contain poorly characterized amounts of label attached along the polysaccharide chains.

IntroductionBrucella abortus and Mycobacterium bovis, the causative agents of bovine brucellosis and tuberculosis respectively, are zoonotic bacterial pathogens that both contribute to major economic losses in the cattle industry and pose a human health risk worldwide. Co-infections of cattle with B. abortus and M. bovis have been identified in various developing countries, necessitating the development of an efficacious strategy for controlling both important zoonotic diseases even in the event of co-infection. Brucella abortus strain RB51, a live attenuated vaccine for bovine brucellosis that is currently used in the US, is highly effective at preventing reproductive failure due to brucellosis in cattle. Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine strain of M. bovis that provides protection against bovine tuberculosis in cattle but is not currently licensed for use in the US.MethodsThe study presented here compares functional Th1 responses of RB51 + BCG vaccinated cattle to responses of RB51-only and BCG-only vaccinated cattle to evaluate the feasibility of a combined vaccination strategy for controlling both bovine brucellosis and tuberculosis.ResultsThis work identified that peripheral blood mononuclear cells (PBMC) from RB51 vaccinates proliferate not only in response to stimulation with killed RB51 but also in response to mycobacterial antigen PPDb. Combination vaccinates show significantly more CD4+ T cell proliferation than single BCG vaccinates when stimulated with PPDb, while no differences were observed between RB51 and combination vaccinates stimulated with RB51.Discussion/conclusionSignificantly enhanced BCG-specific Th1 responses in combination vaccinates compared to BCG-only vaccinates suggest that combining vaccinations for B. abortus and M. bovis may alter the host CD4+ T cell response.

UGPase: A novel molecule that regulates LPS synthesis, virulence, and immunogenicity of Brucella melitensis.

Brucella bacteria are adept at evading the human immune system, leading to a chronic infectious disease known as brucellosis, which poses significant global health challenges. This study addresses a notable gap in bibliometric analyses concerning the immune response to brucellosis. We conducted a comprehensive literature screening from the Web of Science Core Collection, covering publications from 1980 to 2024. Relevant publications were analyzed using R software, VOSviewer, and CiteSpace for bibliometric analysis. A total of 733 publications were included in this study, revealing an average annual growth rate of 5.91% in publications. The United States led in publication volume and citations, followed by China and France. Prominent research institutions included INSERM (France) and CONICET (Argentina). Infection and Immunity was identified as a leading journal in the field, publishing 97 papers with 5,184 citations. Keyword co-occurrence analysis delineated three main research clusters: Brucella vaccine development and immune protection, Brucella molecular mechanisms and intracellular survival strategies, and host innate immunity and Brucella interaction mechanisms. Burst analysis highlighted increasing attention on keywords such as "protection," "type IV secretion system," "pathogenesis," and "prediction" since 2018. This bibliometric analysis sheds light on the global research landscape regarding the immune response in human brucellosis, pinpointing trends and gaps, with a focus on immune escape mechanisms and the future development of safe, effective vaccines.

Brucellosis, primarily caused by B. melitensis, is a widespread zoonotic disease with significant economic and public health burdens on a global scale. Given the limitations of existing vaccines and the potential advantages associated with subunit vaccines, prioritizing the development of efficacious Brucellosis vaccines is crucial for effective disease control. This study examined the immunogenicity and protective efficacy of the Lpp-OmpA-BtaE fusion protein, a construct combining the lipoprotein outer membrane protein A (Lpp-OmpA) with Brucella trimeric autotransporter E (BtaE), co-administered with Freund’s adjuvant (FA), in protecting against B.melitensis infection in a murine model.Vaccination with Lpp-OmpA-BtaE + FA conferred superior protection compared to the Lpp-OmpA + BtaE construct alone. Notably, Lpp-OmpA-BtaE + FA immunization elicited robust B-cell responses characterized by the generation of specific antibodies with a Th1-biased profile, as evidenced by elevated IgG2a/IgG1 ratios. Moreover, Lpp-OmpA-BtaE + FAinjection significantly increased interferon-γ (IFN-γ)/interleukin-4 (IL-4)levels and CD8+/CD4+ T-cell ratio, further supporting the induction of cell-mediated immune responses. Furthermore, it effectively reduced bacterial burdens in the spleens of infected mice. These findings suggest that the Lpp-OmpA platform fused with BtaE represents a promising candidate for the development of an efficacious Brucellosis vaccine, potentially leading to a reduction in morbidity and mortality associated with the disease.

Brucella inactivated vaccine elicits immunity against B. melitensis infection in mice and guinea pigs.

Strong immune response and protection against Brucella abortus by Omp25 and BP26 mRNA vaccine candidates.

Background: Brucellosis poses a significant public health challenge, necessitating effective vaccine development. Current vaccines have limitations such as safety concerns and inadequate mucosal immunity. This study aims to develop an FcRn-targeted mucosal Brucella vaccine by fusing the human Fc domain with Brucella’s multi-epitope protein (MEV), proposing a novel approach for human brucellosis prevention. Methods: The study developed a recombinant antigen (h-tFc-MEV) through computational analyses to validate antigenicity, structural stability, solubility, and allergenic potential. Molecular simulations confirmed FcRn binding. The vaccine was delivered orally via chitosan nanoparticles in murine models. Immunization was compared to MEV-only immunization. Post-challenge assessments were conducted to evaluate protection against Brucella colonization. Mechanistic studies investigated dendritic cell activation and antigen presentation. Results: Computational analyses showed that the antigen had favorable properties without allergenic potential. Molecular simulations demonstrated robust FcRn binding. In murine models, oral delivery elicited enhanced systemic immunity with elevated serum IgG titers and amplified CD4+/CD8+ T-cell ratios compared to MEV-only immunization. Mucosal immunity was evidenced by significant IgA upregulation across multiple tracts. Long-term immune memory persisted for six months. Post-challenge assessments revealed markedly reduced Brucella colonization in visceral organs. Mechanistic studies identified FcRn-mediated dendritic cell activation through enhanced MHC-II expression and antigen presentation efficiency. Conclusions: The FcRn-targeted strategy establishes concurrent mucosal and systemic protective immunity against Brucella infection. This novel vaccine candidate shows potential for effective human brucellosis prevention, offering a promising approach to address the limitations of current vaccines.

Abstract Bacteriophages are considered ideal vaccine platforms owing to their safety, intrinsic adjuvant properties, stability, and low‐cost production. One of the best strategies to prevent brucellosis in humans and animals is vaccination. For several years, researchers have dedicated their efforts to enhance the effectiveness and safety of the Brucella vaccine. This study was designed to evaluate the immunogenicity of a phage vaccine displaying multiepitopes from six different Brucella protective proteins in a mouse model. This study used immunoinformatics to predict T‐ and B‐cell epitopes. Subsequently, a multiepitope protein was synthesized and recombinant phages displaying the multiepitope protein were prepared. The multiepitope protein display on the phage was confirmed by Western blot analysis. Six groups of BALB/c mice (6 mice per group) received multiepitope phage (as a vaccine), helper phage, and PBS as controls subcutaneously or orally. An ELISA assay was used to analyze the humoral response in mouse serum, while an interferon‐gamma ELISpot assay was performed on mouse splenocytes to evaluate the cell‐mediated immune response. Mice immunized with multiepitope phage showed significant serum levels of specific IgG and significant numbers of specific IFN‐producing T cells in splenic lymphocytes (p‐value &lt;0.05). The oral administration route provided a much stronger cellular response than a subcutaneous injection (about 10‐fold), which is important for combating brucellosis infection. These findings provide the first evidence that a multiepitope‐displayed phage vaccine may be a promising avenue for developing a safe and efficient vaccine against Brucella species.

Brucellosis is one of the most common zoonotic diseases caused by Brucella spp. However, there is currently no Brucella vaccine available for humans. Although some attenuated live vaccines have been approved for animals, their protective efficacy is suboptimal. In previous studies, we utilized an epitope- and structure-based vaccinology platform to identify the immunodominant epitopes of Brucella antigens OMP19, OMP16, OMP25, and L7/L12, and constructed the multi-epitope vaccine MEV-Fc against Brucella. In this study, OMP19, OMP16, OMP25, and L7/L12, and MEV-Fc was expressed and purified via an Escherichia coli expression system, which validated that MEV-Fc possesses high immunological efficacy and exerts a significant protective effect in BALB/c mice within the Brucella infection model. MEV-Fc enhanced Th1 and Th2 immune responses and strongly induced the production of the pro-inflammatory cytokine IFN-γ. Furthermore, MEV-Fc protected mice against Brucella infection compared to control group (PBS). In conclusion, our results provide new insights and data support for the development of human Brucella vaccines.

Introduction. Brucellosis remains one of the most common highly dangerous zoonotic infections. Resistance to the pathogenic microorganisms of the genus Brucella depends on the appropriate cell-mediated immunity, which includes the activation of the bactericidal mechanisms of phagocytes. Despite the repeatedly proven role of neutrophils in the fight against many bacterial pathogens, the functions of these immunocompetent cells in the setting of brucellosis have long remained unstudied.Objective. The study aimed to examine the functional and metabolic activity of neutrophils in young cattle sensitized with a non-agglutinogenic strain of Brucella.Materials and methods. The functional and metabolic state of neutrophils in young cattle immunized against brucellosis with a vaccine produced from the non-agglutinogenic RB-51 strain of Brucella abortus was assessed on days 7, 14, 21, 28, 35 after immunization using nitroblue tetrazolium (NBT) test, as well as based on the level of the enzymatic activity of myeloperoxidase and the content of non-enzymatic cationic proteins. The measurements were made photometrically in the spontaneous and stimulated variants of the test, with subsequent calculation of stimulation coefficients. Disintegrated and corpuscular antigens prepared from Brucella vaccine strains with different antigen structures were used as reaction stimulants.Results. It was found that the functional and metabolic status of neutrophils in young cattle immunized with the non-agglutinogenic strain of Brucella is characterized by increased neutrophil activity in the NBT test on days 7 and 35 of the experiment, by the absence of significant changes in the enzymatic activity of myeloperoxidase and a decrease in the content of non-enzymatic cationic proteins on days 7–14 after vaccination.Conclusion. The most pronounced increase in stimulation coefficients was observed when using disintegrated Brucella antigens as a reaction stimulant. The highest stimulation coefficients were registered on day 28 after vaccination during the assessment of the oxygen-dependent metabolism of neutrophils with the NBT test and on day 14 during the assessment of the oxygen-independent metabolism.

False-positive serologic results (FPSRs) of brucellosis occur from time to time in various livestock with all the consequences (quarantine, compulsory slaughter, etc.) that follow true-positive laboratory results. A method based on the Polyacrylamide Gel Electrophoresis/Western Blot of a protein panel for resolving the FPSRs in the diagnosis of brucellosis was developed. Within the context of limited positive serum sample availability in Europe, the method successfully discriminates Brucella-positive sera from samples containing antibodies raised against infections caused by other Gram-negative bacteria causing FPSRs. An average CV% of 1.36 was determined for both repeatability and reproducibility for the whole separation mw range, and the test achieved 1.00 Diagnostic Sensitivity and 1.00 Diagnostic Specificity. The method with pre-prepared WB panels provides a rapid (less than 3 h), easily standardizable, and validatable alternative to existing confirmation methods. The whole WB process of the Brucella proteins and the subsequent densitometry can be accomplished with commercially available equipment, ready-to-use reagents, and open-source software, providing cost-effectiveness. The results of this study could attract broader attention, since molecular species in the 35.0-75.0 kDa range can serve as antigens in Brucella serology and the same fraction can be considered in the development of synthetic Brucella vaccines.

Brucellosis is still the most common zoonosis worldwide despite advanced technology and animal husbandry. Since there is still no effective Brucella vaccine for humans, it is crucial to control the disease in ruminants through eradication and vaccination. Although some countries around the world have achieved this circumstance, every country aims to become free of Brucellosis through vaccination, animal movements, and various eradication measures. For this purpose, the Brucella abortus S19 strain has been used safely for about 100 years. However, due to the O-polysaccharide (OPS) antigen in its structure, the antibody response created by the vaccine causes confusion in serological tests. For this purpose, researchers have provided both mucosal immunity and short-term antibody response by using the B. abortus S19 vaccine in conjunctival form instead of subcutaneous form. This study aimed to determine how long the post-vaccination titer levels persisted in animals vaccinated with vaccines from 3 different companies and different routes. In this study, a total of 115 calves aged 3 to 4.5 months were created in five groups, with 23 animals in each group: group 1 (vaccine brand A), group 2 (vaccine brand B), and group 3 (vaccine brand C) received the two-dose conjunctival vaccine, group 4 received the single-dose subcutaneous vaccine (vaccine brand C), and group 5 received the subcutaneous vaccine (vaccine brand C) plus the booster dose conjunctival vaccine (vaccine brand B). Brucellosis antibody titers were monitored each 21 days until the cattle were 26-28 months old. The collected sera were analyzed using the Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT), and Complement Fixation Test (CFT), which are the preferred serological methods for Brucellosis eradication plans worldwide. In the conjunctival vaccination groups, only 3 (13%) of the animals in group 1 developed antibody titers one month after vaccination, and there was no antibody response detected against Brucellosis in group 2 and group 3. In animals that were stimulated conjunctivally, the threshold value of 30 International CFT Units (ICFTUs) (for distinguishing between infective titers and vaccination titers) was observed in one animal each in group 1 and group 2 and 0 animal in group 3. It was found that antibody titers turn to Brucellosis negative in all conjunctival vaccine groups at 7 months after vaccination. In groups 4 and 5, the first-month serological screening detected over 30 ICFTUs in 17 (89.47%) animals and 16 (69.5%) animals, respectively. In group 4, CFT titers were found to fall below 30 on the 17th month and 9.3 on the 22nd month. On the 14th month, the CFT titers of group 5 were found to be below 30, and all animals in this group turned negative after the 19th month. It was found that the single dose B. abortus S19 subcutaneous vaccination in calves caused persistent antibodies in 5% of the population. It is believed that persistent and high antibody titers created by subcutaneous vaccines will cause false positivity and create confusion in Brucellosis eradication programs. Therefore, although there is no clear distinction between vaccinated and infected animals, it has been observed that conjunctival Brucellosis vaccines create more stable antibody titers and decrease rapidly compared to subcutaneous vaccines. Based on the results of this study and the advantages of conjunctival vaccines, more effective eradication programs and antibody monitoring can be carried out in vaccinated herds where Brucellosis outbreaks are observed.