Recombinant vaccines for brucellosis
Abstract
Brucellosis is a worldwide zoonotic disease caused by members of the genus Brucella, which are Gram-negative, facultative intracellular bacteria. Humans generally acquire Brucella infection through consumption of contaminated milk or milk products, and less commonly through inhalation or direct mucosal contact with the infected material via conjunctiva or breached skin. Brucellosis has severe economic and public health consequences, particularly in many developing countries. Of the nine species of Brucella, B. abortus, B. melitensis, and B. suis are highly virulent to humans and animals. These three Brucella species are considered potential bioterrorism agents and they belong to the NIAID Category B priority pathogens list. Attenuated, live Brucella strains such as B. abortus RB51 and 19, and B. melitensis Rev.1 are being used as vaccines to control brucellosis in domestic animals. However, these animal vaccines are not fully effective and do not provide significant cross-protection against multiple Brucella spp. At present, there is no vaccine for human brucellosis and the current animal vaccines are too virulent to be used in humans. Although seemingly attractive, DNA or subunit vaccines do not yet provide a viable alternative for the live attenuated vaccine due to the absence of well-defined protective antigens for Brucella species. The long-term goal of this study is to develop a safe and broadly protective vaccine against brucellosis. Our strategy to achieve this goal was through enhancing the vaccine efficacy of strain RB51, by overexpressing specific protective antigens and increasing the safety of the generated recombinant vaccine by gamma-irradiation. In the first part of the present study, we engineered three recombinant RB51 strains, RB51/Omp28, RB51/RBS31, and RB51/L7L12, that overexpressed three previously described protective antigens Omp28, Omp31, and cytosolic L7/L12 protein, respectively. When used as live vaccines, all three recombinant RB51 vaccines induced significant levels of antigen-specific antibody and T-cell responses. Similarly, gamma-irradiated recombinant vaccines induced comparable levels of immune responses to those of live vaccines. Mice vaccinated with live and gamma-irradiated recombinant vaccines showed significant resistance against virulent B. abotus 2308, and the B. melitensis 16M challenge. However, the levels of protection conferred by these recombinant vaccines were not significantly improved when compared to the control RB51 vaccine. In the second part of this study, we desired to identify other novel protective antigen(s) of Brucella species which might provide a broad level of protection against multiple virulent Brucella species. Trimeric autotransporter adhesins (TAAs) are recently identified virulence factors found in some Gram-negative bacteria. Screening of a TAA domain annotation database indicated the presence of putative TAA proteins of variable sizes in all Brucella species sequenced so far. The largest TAA protein, consisting of 1333 amino acids with a predicted molecular mass of 147 kDa, was found in B. abortus strain 2308. Vaccination of mice with live recombinant RB51/BrAd vaccine, overexpressing a putative Brucella TAA protein, conferred significantly enhanced level of protection as compared to the control RB51 vaccine against virulent B. abortus 2308, B. melitensis 16M, and B. suis 1330 challenge. Gamma-irradiated RB51/BrAd vaccine also provided significantly enhanced protection against challenge with B. abortus and B. suis. In the third part of the study, we analyzed the feasibility of using the recombinant strain RB51/BrAd as an oral vaccine. When inoculated via the oral route, the recombinant RB51/BrAd vaccine induced strong mucosal as well as systemic immune responses and conferred systemic protection against intraperitoneally inoculated virulent B. abortus2308 in mice. In spite of the robust mucosal and systemic immune response, oral RB51/BrAd vaccines failed to provide significant protection against local lung infection after intranasal challenge with virulent B. abortus 2308. In conclusion, studies carried out in this dissertation generated a highly efficacious recombinant RB51/BrAd vaccine which conferred significant protection against three highly virulent Brucella species as compared to control RB51 vaccine. Our study also demonstrated the feasibility of using the recombinant strain RB51/BrAd as an oral vaccine for brucellosis.
Degree
Ph.D.
Advisors
Vemulapalli, Purdue University.
Subject Area
Biology|Veterinary services|Immunology
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