A multi-epitope subunit vaccine based on CU/ZN-SOD, OMP31 and BP26 against Brucella melitensis infection in BALB/C mice.

Brucellosis, a zoonosis caused by Brucella, is highly detrimental to both humans and animals. Most existing vaccines are live attenuated vaccines with safety flaws for people and animals. Therefore, it is advantageous to design a multi-epitope subunit vaccine (MEV) to prevent Brucella infection. To this end, we applied a reverse vaccinology approach. Six cytotoxic T cell (CTL) epitopes, seven T helper cell (HTL) epitopes, and four linear B cell epitopes from CU/ZN-SOD, Omp31, and BP26 were obtained. We linked the CTL, HTL, B-cell epitopes, the appropriate CTB molecular adjuvant, and the universal T helper lymphocyte epitope, PADRE, with linkers AAY, GPPGG, and KK, respectively. This yielded a 412-amino acid MEV construct, which we named MEVcob. The immunogenicity, stability, safety, and feasibility of the construct were evaluated by bioinformatics tools (including the AlphaFold2 prediction tool, the AlphaFold2 tool, NetMHC-I pan 4.0 server, IEDB MHC-I server, ABCpred service, and C-ImmSim server); the physicochemical properties, secondary and tertiary structures, and binding ability of MEVocb to toll-like receptor 4 (TLR4) was analyzed. Then, codon adaptation and computer cloning studies were performed. MEVocb is highly immunogenic in immunostimulation experiments, The proteins translated by these sequences were relatively stable, exhibiting a high antigenic index. Furthermore, mouse experiments confirmed that the MEVocb construct could raise IFN-γ, IgG, IgG2a, IgG1, IL-2, TNF-α levels in mice, indicating that induced a specific humoral and cellular immune response in BALB/c mice. This vaccine induced a statistically significant level of protection in BALB/c mice when challenged with Brucella melitensis 043 in Xinjiang. Briefly, we utilized immunoinformatic tools to design a novel multi-epitope subunit candidate vaccine against Brucella. This vaccine aims to induce host immune responses and confer specific protective effects. The study results offer a theoretical foundation for the development of a novel Brucella subunit vaccine.

A health promotion perspective for the control and prevention of Brucellosis (Brucella melitensis); Israel as a case study.

BACKGROUND: Brucellosis (Brucella melitensis) is endemic in many countries around the world, therefore, identifying what is required to control and prevent the disease is essential. The health promotion concept and five areas of action, presented in the Ottawa Charter (1986) may help understand how to go forward in the prevention of the disease. Israel serves as a case study. AIM: To identify barriers to the control and prevention of brucellosis (Brucella melitensis) in Israel by analyzing trends in incidence in conjunction with interventions implemented over the last seven decades, applying the health promotion areas of action. METHODS: 1. A document review approach was adopted to develop a list of interventions implemented in Israel to prevent and control brucellosis and identify barriers to implementation. These were analysed using the health promotion areas of action. 2. Data from the mandatory reporting of infectious diseases in Israel regarding brucellosis in humans between 1951 and 2021 are presented and analyzed in conjunction with the interventions implemented. RESULTS: A large range of interventions were implemented following outbreaks of the disease. These interventions followed the health promotion areas of action, including mainly: policy, education and environment and brought about a decrease in the disease among both animals and humans. However, major interventions were discontinued after a few years. In addition, we identified some areas of action that could be much improved on. The interventions, in many cases were not simultaneously implemented or coordinated, decreasing the chances of them having the expected long term impact. CONCLUSIONS: Control and prevention of the disease in Israel is partial. Areas of action that could be improved include enforcement of regulations, strengthening community action and improving personal skills. Simultaneous and continuous implementation of the interventions may achieve the goals of sustained prevention and control. There seems to be a lack of a long-term strategy and an integrated holistic intervention approach that may contribute to the control and prevention of the disease.

Protection of palmitic acid treatment in RAW264.7 cells and BALB/c mice during Brucella abortus 544 infection.

BACKGROUND: We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. OBJECTIVES: We aimed to investigate the efficacy of PA against B. abortus infection using a murine cell line and a murine model. METHODS: Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O2⁻) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. RESULTS: PA did not affect the growth of B. abortus. PA treatment in macrophages did not change B. abortus uptake but it did attenuate the intracellular survivability of B. abortus. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression. Compared to control cells, reduced nitrite accumulation, augmented O2⁻, and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus-infected cells. Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. CONCLUSIONS: Our results suggest that PA participates in the control of B. abortus within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus infection by fatty acids.

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.

Interplay of oxidative stress and antioxidant bio markers in oil adjuvant Brucella melitensis vaccinated and challenged mice.

The intracellular nature of Brucella leads to rise in oxidative stress due to bacterial invasion, particularly at the site of predilection spleen and lymph nodes. The present study aimed to evaluate the erythrocytic and tissue specific oxidative stress responses induced during oil adjuvant killed Brucella melitensis vaccination. The results of the study clearly implicated a significant increase in level of catalase, and superoxide dismutase (SOD) activity and lipid peroxidation (LPO), and total protein content in erythrocytes after vaccination. The activity of glutathione-S-transferase (GST) was unaltered during the period of experiment. The catalase activity and GSH content was significantly increased in lung and spleen tissues. The tissues GST levels increased significantly in all tissues, while tissue SOD level increased significantly only in lung tissues. Thus, it can be inferred that oil adjuvant based Brucella vaccine induces negligible signs of inflammatory pathophysiology and supports the development of significant level of protection against virulent Brucella challenge.

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.

Improved influenza viral vector based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensis infection in pregnant sheep and goats.

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse consequences of B. melitensis infection.

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.

Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells.

Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

Use of S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol as an adjuvant improved protective immunity associated with a DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus in mice.

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Humoral responses were characterized by the stimulation of IgG2a and IgG1 and by the presence of SOD-specific secretory IgA in nasal and bronchoalveolar lavage fluids. Furthermore, T-cell proliferative responses and increased production of gamma interferon were also observed upon splenocyte restimulation with recombinant SOD. Cytotoxic responses were also stimulated, as demonstrated by the lysis of RB51-SOD-infected J774.A1 macrophages by cells recovered from immunized mice. The pcDNA-SOD/BPPcysMPEG formulation induced improved protection against challenge with the virulent strain B. abortus 2308 in BALB/c mice over that provided by pcDNA-SOD, suggesting the potential of this vaccination strategy against Brucella infection.

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.

Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection.

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.

Co-immunization with interlukin-18 enhances the protective efficacy of liposomes encapsulated recombinant Cu-Zn superoxide dismutase protein against Brucella abortus.

Brucellosis is a worldwide zoonotic disease caused by Brucella abortus and a number of closely related species. Brucellosis has severe impact on the health and economic prosperity of the developing countries due to the persistent nature of infection and unavailability of effective control measures. The Cu-Zn superoxide dismuatse (SOD) protein of Brucella have been extensively studied as a major antigen involved in bacterial evading mechanism of host defence. Being a critical pro-inflammatory cytokine interleukin-18 (IL-18) plays key role in induction of immune mediated protection against intracellular pathogens. In the present study, we aimed to investigate the immunogenic potential of fusogenic liposomes (escheriosomes) encapsulated recombinant Cu-Zn SOD (rSOD) protein alone or in combination with recombinant IL-18 (rIL-18). Escheriosomes encapsulated rSOD mediated immune responses were further increased upon co-immunization with rIL-18. Furthermore, immunization with escheriosomes encapsulated rSOD alone or in combination with rIL-18, increased resistance in mice against challenge with B. abortus 544.

Efficacy of moxifloxacin or gatifloxacin as prophylaxis against experimental murine Brucella melitensis infection.

The prophylactic potential of moxifloxacin and gatifloxacin was assessed in comparison with doxycycline, an established therapeutic antibiotic, to limit or control infection by Brucella melitensis in an experimental mouse model, determined by reduced bacterial burden in the spleen. Although moxifloxacin was found to have a small protective effect when administered 6 h following infection, neither moxifloxacin nor gatifloxacin showed significant efficacy in vivo. In comparison, doxycycline provided significant protection when prophylaxis was started at 6 h, 7 days or 14 days following infection. Overall, these results confirm the utility of doxycycline in the prophylaxis of brucellosis and suggest that neither moxifloxacin nor gatifloxacin are likely to be valuable for post-exposure prophylaxis of Brucella infection.

Efficacy of ciprofloxacin versus doxycycline as prophylaxis against experimental murine Brucella melitensis infection.

The prophylactic potential of ciprofloxacin was assessed in comparison with doxycycline, an established therapeutic antibiotic, to limit or control infection by Brucella melitensis in an experimental mouse model. Ciprofloxacin treatment reduced bacterial loads in the spleens of challenged mice when administered prior to or at the same time as the bacterial challenge. In comparison, doxycycline provided much greater reductions in bacterial counts, even when treatment was initiated after infection. Doxycycline was able to protect against B. melitensis when administered for 5 days from 24 h after infection and for at least 28 days after cessation of the antibiotic. Overall, these results confirm that ciprofloxacin is less effective than doxycycline but suggest that it may have some utility in providing protection against low-level infections. Combination studies are indicated.

Serological responses of rams to a Brucella ovis-vitamin E adjuvant vaccine.

Rams which were vaccinated at 6-8 months of age with a water-in-oil Brucella ovis-vitamin E adjuvant vaccine had significantly higher serum antibody levels than rams vaccinated with a commercial B. ovis bacterin or B. melitensis Rev 1. The adjuvant vaccine did not cause abscesses at the site of injection as some water-in-oil emulsions do. Two years after vaccination, the vitamin E adjuvant-vaccinated rams had higher antibody level than the other groups. This was most likely due to a secondary response to naturally occurring infection with B. ovis.

Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice.

The BALB/c mouse was selected as a model for infection with Brucella abortus on the basis of protracted nonclinical infection produced by strain 2308, virulent for cattle, and relatively rapid clearance of strain 19, an attenuated strain used to vaccinate cattle. Protection in mice vaccinated with strain 19 was compared with that obtained with nonliving vaccines at early (1 week) and later (4 weeks) intervals after challenge with strain 2308 and assessed by enumeration of B. abortus organisms in the spleen. Mice challenged 4 weeks after vaccination with strain 19 exhibited significant protection at 1 and 4 weeks postinfection (p.i.), with an increased magnitude of protection at the later time. When challenged 6 weeks after vaccination with strain 19, the level of protection diminished between 1 and 4 weeks p.i. and at the later time was not always significantly different from controls. Mice immunized 4 weeks earlier with nonliving vaccines in mineral oil with t trehalose dimycolate (TDM) and muramyl dipeptide (MDP) demonstrated patterns of protection similar to those obtained following the 6 week vaccination-challenge interval with strain 19. Vaccination with cell envelopes derived from strain 2308 produced equivalent protection at 1 week p.i. whether administered in phosphate-buffered saline, incomplete Freund adjuvant, or the TDM and MDP adjuvant. Equivalent protection also followed vaccination with strain 2308 killed whole cells, cell envelopes, or outer membrane proteins in phosphate-buffered saline or in the TDM and MDP adjuvant. The TDM and MDP adjuvant alone induced nonspecific resistance, which peaked at 1 day p.i. and was still present at 1 week p.i., although by this time its magnitude was significantly less than the protection induced by antigen combined with the adjuvant. These data, together with the results of antibody assays and passive and adoptive transfer studies, suggested that protection at 1 week p.i. could be accounted for largely by an effect of O antibodies, with T cell-mediated immune responses having a subsidiary role.

Protection of rams against epididymitis by a Brucella ovis-vitamin E adjuvant vaccine.

Rams vaccinated at 7 and 8 months of age with a B. ovis-vitamin E adjuvant vaccine had increased antibody titers compared with Freund's incomplete adjuvant or commercial bacterin vaccinated rams. The percent overall infectivity in an experimental infection of B. ovis-vitamin E adjuvant vaccinated rams was 22% compared to 44% for B. ovis-Freund's incomplete adjuvant or bacterin vaccinated rams and 67% for control.