Nanoparticle-Based Vaccines for Brucellosis: Calcium Phosphate Nanoparticles-Adsorbed Antigens Induce Cross Protective Response in Mice.

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.

A comparison between adjuvant and delivering functions of calcium phosphate, aluminum hydroxide and chitosan nanoparticles, using a model protein of Brucella melitensis Omp31.

Vaccination is the most efficient and economic approach used to hinder infection and intense consequences caused by viruses, bacteria, or other pathogenic organisms. Since the intrinsic immunogenicity of recombinant antigens is usually low, safe and potent vaccine adjuvants are needed to ensure the success of those recombinant vaccines. Nanoparticles (NPs) have attracted much interest as adjuvants and delivery systems. Previous studies have shown that calcium phosphate (CP), aluminum hydroxide (AH) and chitosan (CS) NPs are promising delivery systems for immunization. In addition, it has been determined that Omp31 is a good candidate for inducing protection against Brucella (B) melitensis and B. ovis. Our aim in the present study was to compare the functions of CP, AH and CS NPs for stimulation of the immune response and protection against B. melitensis by using omp31 as a model protein. Based on the cytokine profile and subclasses of the antibody, vaccination with Omp31 load CP (CP/Omp31) and Omp31 load AH (AH/Omp31) NPs induced T helper type 1 (Th1)-T helper type 2 (Th2) immune response, whereas immunization by Omp31 load CS (CS/Omp31) NPs induced Th1 immune response. CP/Omp31 NPs elicited protection toward B. melitensis challenge equivalent to the vaccine strain B. melitensis Rev.1. Compared to CS/Omp31 NPs, CP/Omp31 NPs elicited a low increase in protection level against B. melitensis 16 M. In conclusion, the obtained results indicated that CP NPs were potent antigen delivery systems to immunize brucellosis.

Development and evaluation of in murine model, of an improved live-vaccine candidate against brucellosis from to Brucella melitensis vjbR deletion mutant.

Brucellosis is an infectious disease that brings enormous economic burdens for developing countries. The Brucella melitensis (B. melitensis) M5-90 vaccine strain (M5-90) has been used on a large scale in China, but may cause abortions if given to pregnant goats or sheep subcutaneously during the late stages of gestation. Moreover, the vaccine M5-90 cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent M5-90 vaccine is required. In this study, a vjbR mutant of M5-90 (M5-90ΔvjbR) was constructed and overcame these drawbacks. M5-90ΔvjbR strain showed reduced survival capability in murine macrophages (RAW 264.7) and BALB/c mice and induced high protective immunity in mice. In addition, M5-90ΔvjbR induced an anti-Brucella-specific immunoglobulin G (IgG) response and stimulated the expression of gamma interferon (INF-γ) and interleukin-4 (IL-4) in vaccinated mice. Furthermore, M5-90ΔvjbR induced IgG response and stimulated the secretion of IFN-γ and IL-4 in immunized sheep. Moreover, the VjbR antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90ΔvjbR is an ideal live attenuated and efficacious live vaccine candidate against B. melitensis 16 M infection.

[Different aluminum adjuvants significantly enhances the effect of immunization on Brucella Omp31].

Objective To investigate the effect of aluminum phosphate (AP) and aluminum hydroxide (AH) as adjuvants on Brucella outer membrane protein 31 (Omp31) in inducing humoral and cellular immune responses and immune protection. Methods AP and AH adjuvants were prepared and separately mixed with Brucella Omp31 protein to measure the adsorption rates. The AP- and AH-absorbed Omp31 protein were intraperitoneally injected into BLAB/c mice at 0, 2, and 4 weeks, and meanwhile, unabsorbed Omp31 protein and PBS were used as controls. The levels of serum IgG, IgG1, IgG2a and genital tract secretion sIgA were determined by ELISA at 0, 2, 4 and 6 weeks. Spleen cells were collected for culture at 6 weeks, and the cells were stimulated by Omp31 for 48 hours followed by the analysis of IFN-γ and IL-10 levels in the supernatants by ELISA, and the determination of lymphocyte proliferation by CCK-8 assay. The mice were challenged with Brucella at 6 weeks, and bacterial content in spleen tissue was determined 1 and 2 weeks later. Results AP and AH could absorb over 70% and 85% of the Omp31 protein, respectively, for solutions at all the tested concentrations. ELISA suggested that serum IgG, IgG1, IgG2a and genital tract sIgA levels peaked 2 weeks after the last immunization for both AP and AH groups, and antibody level was higher in the AP and AH groups than the control groups, and higher in the AH group than in the AP group. CCK-8 assay showed that the proliferating rate of lymphocytes induced by the AH group was significantly higher than that by the AP group, and the AH group also showed significantly higher IFN-γ level in the supernatant than the AP group, but no significant difference in IL-10 level. The AH group had remarkably lower bacterial load in the spleen than the AP group 2 weeks after challenged by Brucella 16M strain. Conclusion Both AP and AH adjuvants effectively enhanced immunogenicity and immune protection of the Brucella Omp31 protein, and AH was superior to AP in this respect.

Identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against Brucella melitensis through reverse vaccinology and immunoinformatics approach.

Brucella melitensis is an intracellular pathogen resides in the professional and non-professional phagocytes of the host, causing zoonotic disease brucellosis. The stealthy nature of the Brucella makes it's highly pathogenic, and it is hard to eliminate the bacteria completely from the infected host. Hitherto, no licensed vaccines are available for human brucellosis. In this study, we identified potential antigens for vaccine development from non-classically secreted proteins through reverse vaccinology approach. Based on the systemic screening of non-classically secreted proteins of B. melitensis 16M, we identified nine proteins as potential vaccine candidates. Among these, Omp31 and Omp22 are known immunogens, and its role in the virulence of Brucella is known. Roles of other proteins in the pathogenesis are yet to be studied. From the nine proteins, we identified six novel antigenic epitopes that can elicit both B-cell and T-cell immune responses. Among the nine proteins, the epitopes were predicted from Omp31 immunogenic protein precursor, Omp22 protein precursor, extracellular serine protease, hypothetical membrane-associated protein, iron-regulated outer membrane protein FrpB. Further, we designed a multitope vaccine using Omp31 immunogenic protein precursor, Omp22 protein precursor, extra cellular serine protease, iron-regulated outer membrane protein FrpB, hypothetical membrane-associated protein, and LPS-assembly protein LptD and polysaccharide export protein identified in the previous study. Epitopes were joined using amino acid linkers such as EAAAK and GPGPG. Cholera toxin subunit B, the nontoxic part of cholera toxin, was used as an adjuvant and it was linked to the N-terminal of the multitope vaccine candidate. The designed vaccine candidate was modeled, validated and the physicochemical properties were analyzed. Results revealed that the vaccine candidate is soluble, stable, non-allergenic, antigenic and 87% of residues of the designed vaccine candidate is located in the favored region. In conclusion, the computational analysis showed that the newly designed multitope protein could be used to develop a promising vaccine for human brucellosis.

Evaluation of the immunogenicity and the protective efficacy in mice of a DNA vaccine encoding SP41 from Brucella melitensis.

INTRODUCTION: Brucella melitensis is a facultative intracellular Gram-negative bacterial pathogen that may enter the host via ingestion or inhalation, or through conjunctiva or skin abrasions. Some Brucella spp surface proteins (SPs) play an important role in bacterial adhesion and invasion and thus represent targets for the host immune system. Brucella spp surface protein with apparent molecular mass of 41 kDa interacts selectively with HeLa cells. METHODOLOGY: To evaluate the role of SP41 (41 kDa) as a DNA vaccine against Brucella spp., pCISP41, a plasmid construct for protein expression in mammalian cells, was established. Exogenous SP41 was detected in pCISP41-transfected Vero cell line by immune blotting using specific polyclonal antibody. The protective role of pCISP41 against B. melitensis 16M in mice was evaluated by measuring B and T cell responses in comparison to those achieved with attenuated B. melitensis Rev. 1 vaccine. RESULTS: BALB/c mice injected with pCISP41 were able to develop SP41-specific serum immunoglobulin G (IgG) antibodies. In addition, splenocytes from DNA-SP41-vaccinated mice elicited a T-cell-proliferative response and also induced gamma interferon (IFN-γ) production, but not interleukin-5 (IL-5), suggesting the induction of a T-helper-1-dominated immune response. Vaccination with attenuated B. melitensis Rev.1 strain induced better protection levels than DNA vaccination with SP41 against B. melitensis 16M in mice. CONCLUSIONS: Such responses play an important role against intracellular infecting agents such as Brucella spp. Altogether, our data suggest that SP41 may represent a promising candidate for DNA vaccination against brucellosis, but more investigation to increase its protective efficacy should be done.

[Experimental evaluation of the brucellosis therapeutic vaccine efficacy].

Use of antibiotics can't completely solve the problem of brucellosis treatment, especially its chronic forms, because antibacterial preparations do not eliminate main pathogenetic factor of the disease--sensibilization of the macroorganism. It makes actual the question about complex immuno- and antibacterial therapy. Long-term clinical experience proved high effectiveness of a therapeutic brucellosis vaccine. Earlier this preparation was manufactured in Research Institute of Vaccines and Sera in Tbilisi (Georgia). To date new composition of components of the vaccine has been developed, and manufacturing and control methods have been improved. Marked desensitizing effect of the vaccine and its stimulatory action on cellular and humoral immunity has been observed. In 2002 technological normative documentation for manufacturing and use of the vaccine was developed in the Research Institute of Microbiology (Kirov) and production of the vaccine began.

Enhanced cellular response in mice treated with a Brucella antigen-liposome mixture.

The capacity of liposomes constituted by dycetyl-phosphate (0.009 mM), cholesterol (0.017 mM), lecithin (0.003 mM), and myristic (0.1 mM), stearic (0.1 mM), or oleic acid (0.1 mM) to modify the lymphocyte response to Brucella melitensis antigens in mice was studied. Mice treated with antigens mixed with liposomes containing myristic, stearic or oleic acid had higher antibody titres than mice given antigen suspended in a saline solution. Liposomes alone, without Brucella antigens, resulted in increased 3H-thymidine incorporation by lymphocytes both in vivo and in vitro. The addition of polyclonal activators (LPS and ConA) caused a further increase of 3H-thymidine uptake. Moreover, spleen lymphocytes from mice inoculated with Brucella antigens mixed with the liposomes had a significantly lower population of B lymphocytes (10%), and a notable increase in the Tc lymphocytes (20%). Autoradiography of sections of popliteal ganglia of treated mice showed that the radioactivity was concentrated mainly in the membrane structures of the cell.