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59 Detailed Analysis of CD4+ T-Helper Responses to Env and Gag Proteins of Simian Immunodeficiency Virus Reveals a Relative Lack of Broadly Reactive CD4+ T-Helper Epitopes in the SU Domain of Env
S. Sarkar*, V. Kalia, M. Murphey-Corb, and R. C. Montelaro
Univ. of Pittsburgh, PA, USA
Background: Immunization with live-attenuated simian immunodeficiency viruses (SIV) is 1 of the most effective strategies for eliciting enduring broadly protective immunity in an SIV/macaque model and provides an important tool to study immune mediators of protection. Antigen-specific CD4+ T-helper (Th) cells are gaining progressive significance in effective control of primate lentiviral infections. Functional characterization, fine specificity analysis, and quantification of virus-specific Th-cell responses will provide important information on the role of these cells in anti-virus immune mechanisms. To better understand the fine specificity of viral antigen-specific CD4+ Th cells associated with protective immunity, we analyzed peptide epitope-specific Th responses in macaques inoculated with reference attenuated SIV.
Methods: Proliferative responses of Th cells from 8 inoculated animals were assessed using an exhaustive panel of 20-mer overlapping peptides representing the entire sequence of SIV Env and Gag proteins. Ki67 and CFSE were used as proliferation markers in 4-color FACS-based assay. Broadly reactive Th peptides were selected using Student’s paired t test (p < 0.05). The Th peptides were further analyzed for amphipathicity, conservation, and glycosylation.
Results: A unique set of responder peptides was identified for each macaque, which was then used to define a group of 26 broadly reactive peptide epitopes across all 8 inoculated animals. A majority of these broadly reactive peptide epitopes (20 out of 26) concentrated to Gag and the transmembrane (TM) domain of Env and mapped to highly conserved regions of these proteins possessing high amphipathicity. Interestingly, SU contained only 6 of the 17 broadly reactive CD4+ Th epitope peptides in Env. These Th epitopes were confined to 2 nonglycosylated regions in the carboxyl terminus of SU, which are fairly conserved. This was in contrast to the uniform localization of Th epitope peptides in 5 distinct regions of the TM and 7 distinct regions of Gag.
Conclusions: Our study revealed a relative lack of broadly reactive CD4+ T-helper epitopes in SU, which is the largest exposed protein on the surface of the virus. Moreover, these Th epitopes of SU were excluded from the heavily glycosylated regions. Similar observations were also made for TM. This is suggestive of another potential immune escape mechanism developed by lentiviruses and provides important implications for vaccine design.
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