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185 Attenuated HIV-1 nef DNA Vaccine Construct Induces Memory T-Cell Response
B. Majumdar1, B. Gray1, S. Mahalingam2, S. McBurney1, T. Schaefer1, T. Dentchev3, T. Reinhart1, and V. Ayyavoo*1
1Univ. of Pittsburgh Graduate Sch. of Publ. Hlth., PA, USA; 2Ctr. for DNA Fingerprinting and Diagnostics, Hydrabad, India; and 3Univ. of Pennsylvania, Philadelphia, USA
Background: Development of an HIV vaccine should include an important focus on broadly cross-reactive CTL responses as 1 major component. Nef is 1 of the early gene products produced in abundance, and a majority of the HIV-1-positive patients generate Nef-specific CTL responses, indicating that Nef is a potent immunogen. Nef is also known to down-regulate the expression of CD4 and MHC-I molecules. We propose development of an attenuated nonfunctional, yet immunodominant Nef as part of an HIV-1 multigene vaccine.
Methods: We have cloned and expressed multiple nef variants and analyzed their ability to down-regulate CD4 and MHC-I molecules. Mice were immunized with these nef constructs and assessed for their ability to induce antigen-specific primary and memory T-cell responses by IFN-gamma ELISPOT. Furthermore, we have mapped the immunodominant epitopes present in Nef using overlapping peptides.
Results: Functional analysis indicated that patients’ nef isolates did not down-regulate expression of either CD4 or MHC-I molecules, whereas nef from NL43 and 89.6 down-regulated both CD4 and MHC-I expression. Evaluation of T-cell immune responses in mice showed that nonfunctional nef (Nef-R3) is capable of inducing significant T-cell immune response measured by IFN-gamma secreting cells by ELISPOT. CD8-specific epitope mapping studies indicate that Nef-R3 has multiple CTL epitopes spanning throughout the gene. Further analysis of memory T-cell responses indicated that Nef-R3 and Nef-89.6 established memory T-cell response.
Conclusions: Results from this study indicate that a nonfunctional nef construct is capable of inducing significant CTL responses against multiple Nef variants, suggesting that Nef could be a potential immunogen in an HIV-1 vaccine. This attenuated vaccine strategy could be used for development of immunogens for pathogenic genes.
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