Akoolo, Lavoisier (2015-08). Role of Brucella Toll/Interleukin-1 Receptor (TIR) Domain Containing Protein (?TcpB) Deletion Mutant in Protective Immunity against Brucellosis. Doctoral Dissertation.
Thesis
Brucellosis is an important zoonotic disease affecting about 500,000 people annually. The development of safer and more efficacious Brucella Live attenuated vaccines addresses safety concerns that include the identification of reproducible and reliable surrogates of protection and mechanisms to bolster longevity. Brucella encodes a toll interleukin receptor domain containing protein (TcpB/Btp-1). These proteins subvert host innate immunity by abrogating NF-?B mediated cytokine production, by binding to and/or causing the degradation of signaling molecules TIRAP (MAL) and MyD88. TcpB has also been shown to directly reduce the CTL killing activity of infected host cells. In the current study, we investigated the effect of deleting tcpB from Brucella on invitro and invivo immune responses. We also evaluated an in vitro murine Brucella growth inhibition co-culture assay to determine the capacity of immune splenocytes from mice exposed to the tcpB mutant or wild type to control the growth of Brucella melitensis in murine bone marrow derived macrophages. A tcpB knockout constructed by gene replacement in the Brucella abortus S19 genetic background was used to vaccinate C57BL/6 mice assessed for development of CD4+ memory T cells. Mice vaccinated with the mutant displayed an elevated Th1 response, compared to the parental S19 and non- vaccinated controls, as manifested by multiple factors. These include; elevated IFN-? early post vaccination, and a significant elevation of memory CD4+CD44+CD62L+ within CD4+T cell population in splenocytes derived from mice vaccinated with the mutant. S19 and S19?tcpB strains induced a significant increase in the IgG2a levels post vaccination. Consistent with a shift to a Th1 response, S19?tcpB induced a higher response later in vaccination. Splenocytes obtained from mice vaccinated with the S19?tcpB mutant exhibited significantly higher levels of killing activity compared to cells derived from S19 vaccinated mice and PBS controls. Consistent with enhanced immune protection, fewer bacteria were recovered from the spleens of mice vaccinated with the S19?tcpB mutant, which had reduced inflammatory lesions consistent with reduced bacterial burden. These results provide strong evidence that tcpB deletion improves immunogenicity, longevity and protective efficacy of S19 and that ex vivo co- cultivation may be employed to predict potential vaccine efficacy.
