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203 Engineered HIV-1 Subtype C Envelope Genes for Vaccine Applications
Y. Lian*1, Y. Sun1, L. Leung1, E. Kan1, A. Fong1, S. Engelbrecht2, F. Treurnicht2, J. zur Megede1, E. Janse van Rensburg2, I. Srivastava1, and S. W. Barnett1
1Chiron Corp., Emeryville, CA, USA and 2Univ. of Stellenbosch, South Africa
Objective: HIV-1 subtype C infections are on the rise in Africa and Asia. Our goal is to develop a subtype C envelope-based vaccine that can elicit immune responses to protect against HIV infection and disease.
Methods: Envelope gene expression cassettes with both the native and codon-optimized HIV-1 subtype C sequences were designed, constructed, and evaluated in vitro. Eleven full-length viral env sequences were cloned from 8 various South African isolates. Seventeen native env genes were prepared as gp120, gp140, and gp160 by PCR using proviral DNAs. The gp160 gene derived from TV1 isolate, clone 8.2, was selected to be codon-optimized. These gene cassettes were designed as gp120 and gp140 variants with V2 or V1/V2 region deletions. All the env genes, both native and optimized, were cloned into the Chiron pCMVlink vector. Transient transfection, Western blot, and Env capture ELISA were performed using 293T cells to evaluate the integrity and relative expression levels of the various constructs, and the gene products were tested in an in-house CD4 binding assay.
Results: The Env proteins expressed from constructs derived from the native TV1-clone 8.2 were found to migrate at the expected molecular weights as determined by Western blot. These proteins were further shown to bind soluble CD4. This would indicate that the expressed protein is maintained in a functional conformation. This result was also confirmed by a cell-fusion assay using the TV1, c8.2, gp160 plasmid (Himathongkham). Furthermore, all of the codon-optimized env plasmids showed 5- to 10-fold increased levels of expression relative to the native forms.
Conclusions: The various subtype C env gene cassettes expressed high levels of proteins that were of the predicted sizes and were shown to bind CD4. These materials were found suitable for DNA vaccines and Env protein production. Accordingly, the constructs encoding codon-optimized subtype C envelopes and mixtures of subtype B and C Env vaccines were used to immunize rabbits using a DNA prime-protein boost regimen. Both the levels of antibody and the relative abilities of these antibodies to neutralize homologous and heterologous virus isolates will be assessed.
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