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Design and Construction of Novel Envelope Genes from the HIV-1SF162 Isolate for Use in DNA Vaccines and Production of Protein Vaccines Y. LIAN*, L. LEUNG, Y. SUN, J. ZUR MEGEDE, S. HILT, E. KAN, A. FONG, I. SRIVASTAVA, L. STAMATATOS, and S. W. BARNETT. Chiron Corp., Emeryville, CA A major goal for envelope–based vaccine strategies for protection against HIV-1 is the induction of broad and potent neutralizing antibody responses against diverse primary isolates. For this purpose, we must determine which Env structures will most efficiently present important neutralizing epitopes, and we must achieve high level expression of the env genes for gene delivery vaccine applications (e.g. DNA) and for Env protein production. For these studies, both intact and variable (V) region-deleted gene cassettes were constructed based on sequences encoding the full-length (gp160), the secreted oligomeric (gp140), and monomeric (gp120) proteins. The amino acid sequences used in these constructs were based upon those of the full-length infectious molecular clone of the macrophage-tropic, CCR5 co-receptor-dependent, subtype B HIV-1SF162 isolate. However, in order to achieve high level expression and to overcome the Rev-dependence of env gene expression, the DNA sequences were modified to conform with the codon usage of highly expressed human genes. Transient expression assays performed in 293T cells showed increased expression levels from the modified env plasmids of up to 13-fold as compared to the native constructs. Rabbits were immunized with eight different plasmids encoding the SF162 gp120, gp140, and gp160, with or without deletion of the V2-loop. Induction of high Env-specific antibody titers were observed after only two immunizations with the gp120 and gp140 constructs with slight boosting after the third; modest antibody titers were induced with the gp160. These animals will be subsequently boosted with oligomeric V2-deleted SF162 Env (o-gp140) protein purified from stable CHO cell lines. Pilot studies performed in parallel in two rhesus macaques that were immunized with V2-deleted SF162 gp140 plasmid DNA showed substantial induction of neutralizing activity against several R5 primary HIV-1 strains after a single booster immunization with the recombinant V2-deleted o-gp140 protein. These results indicate that a DNA prime/protein boost vaccine regimen employing these Env immunogens holds promise for the induction of broadly neutralizing antibodies. Key Words: antibodies, DNA vaccines, envelope |
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© 7th
Conference on Retroviruses and Opportunistic Infections, |