Novel strategies to develop better brucellosis vaccines using Brucella abortus RB51 and B. neotomae

Neha Dabral, Purdue University

Abstract

The first part of the study was undertaken to investigate whether overexpression of two glycosyltransferases, WbkA and WbkE, in strain Brucella abortus RB51 would result in the expression of O-polysaccharide and restore the smooth phenotype. No O-polysaccharide expression was detected on overexpression of wbkA or wbkE in RB51. However, wbkA overexpression (strain RB51WbkA) leads to the development of extremely mucoid bacterial colonies that form clumps/strings in liquid culture. This mucoid phenotype is attributed to the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine. The clumping RB51WbkA strain exhibited increased adherence to polystyrene matrices; however, it was similar to strain RB51 in its attenuation characteristic and conferred a similar level of protection against virulent B. abortus 2308 as strain RB51. The second part of the study was carried out to determine whether increasing the amount of bactoprenol primed molecules in strain RB51WboA would lead to the expression of higher levels of O-polysaccharide and confer it a smooth phenotype. We generated strain RB51WboAKF by overexpressing wbkF gene, which encodes undecaprenyl-glycosyltransferase involved in bactoprenol priming for subsequent O-polysaccharide polymerization, in strain RB51WboA. Strain RB51WboAKF expressed high levels of O-polysaccharide and its Western blot reactivity profile with the O-polysaccharide-specific monoclonal antibody was similar to that of the smooth Brucella strains. Immunoelectron microscopy revealed that the O-polysaccharide was present mostly on the bacterial cell surface. However, RB51WboAKF strain exhibited rough phenotypic characteristic in acriflavine agglutination test. Although there was no difference between strains RB51 and RB51WboAKF in their ability to persist in spleens of BALB/c mice, strain RB51WboAKF was more resistant to the bactericidal effect of polymyxin B. Mice immunized with strain RB51WboAKF developed increased levels of smooth LPS-specific serum antibodies, primarily of IgG2a and IgG3 type, when compared with those immunized with strain RB51WboA. Levels of serum IL-12p70, IFN-γ and IL-10 were higher in mice immunized with strain RB51WboAKF when compared to the mice immunized with strain RB51. Splenocytes from the RB51WboAKF vaccinated group of mice secreted higher levels of antigen-specific IFN-γ, IL-10 and TNF-&agr; when compared to those of the RB51 or RB51WboA vaccinated groups. Also, increased numbers of antigen-specific IFN-γ secreting CD4+ and CD8+ T lymphocytes were detected in RB51WboAKF immunized mice. Importantly, immunization of mice with strain RB51WboAKF conferred greater protection against virulent B. abortus 2308 and B. melitensis 16M than immunization with RB51 or RB51WboA. The final part of the study was conducted to test the ability of orally inoculated gamma-irradiated strains B. neotomae and B. abortus RB51 in a prime-boost immunization approach to induce mucosal as well as systemic protection against virulent B. abortus 2308. Heterologous prime-boost vaccination regimens and homologous prime-boost vaccination of mice with B. neotomae led to the production of serum and mucosal antibodies specific to the smooth LPS. All oral vaccines induced antigen-specific CD4+ and CD8 + T cells capable of secreting IFN-γ and TNF-&agr;. Upon intra-peritoneal challenge, mice vaccinated with B. neotomae showed the highest level of resistance against virulent B. abortus 2308 colonization in spleen and liver. Experiments with vaccination with different doses of B. neotomae showed that all tested doses of 109, 10 10 and 1011 CFU-equivalent conferred significant protection against the intra-peritoneal challenge. However, only the highest tested dose of 1011 CFU-equivalent of B. neotomae afforded protection against intranasal challenge. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Vemulapalli, Purdue University.

Subject Area

Molecular biology|Immunology

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