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206 Design and in Vitro Characterization of Clade AG and B DNA Vaccines for AIDS
J. Smith*1, D. Ellenberger2, B. Li2, W. Heneine2, S. Butera1, T. Folks2, and H. L. Robinson1
1Emory Vaccine Ctr., Emory Univ., Atlanta, GA, USA and 2CDC, Atlanta, GA, USA
Background: We have designed candidate clade AG and B HIV-1 DNA vaccines to be tested in conjunction with matched recombinant MVA. Recently, priming with DNA and boosting with MVA has been shown to protect animals from disease against a highly pathogenic 89.6P SHIV challenge. To take this approach forward into humans, we will express several viral proteins from 1 multigenic construct to maximize the number of potential epitopes. In addition we have also codon optimized the env gene in the context of the multigenic construct to increase Env expression.
Methods: All vaccine inserts were first subcloned in 5 (gag/pol) and 3 (tat/rev/vpu/env) halves and then assembled into the pGA expression vector. Both codon optimized and native sequences were inserted for gag/pol and env. Expression levels were assayed from transient transfections in 293T cells by ELISA and Western blot. Safety mutations were introduced into the zinc finger of gag and also in RT by site directed mutagenesis. The efficiency of the mutations was assayed by an RT-PCR packaging assay and AMP-RT assay, respectively. Production of VLPs was assayed by electron microscopy and centrifugation through sucrose gradients.
Results: High levels of Gag, Pol, and Env were expressed from a single plasmid in cell culture. The zinc finger mutations reduced packaging of viral RNA 10,000-fold from that of wt HIV-1. The RT mutations completely abrogated RT activity. VLP production was markedly increased with addition of a protease inhibitor to the cultures or with a genetic mutation in the catalytic site of protease. Codon optimization of env resulted in an increase in Env production with little loss of Gag production. A mixture of gag aggregates and VLP are produced from constructs with active protease, and inactivation of protease either genetically or with specific inhibitors shifts the ratio toward VLP production.
Conclusions: Our multigenic approach to HIV-1 DNA vaccines expresses high levels of Gag and Env and the safety mutations are effective. Codon optimization in a multigenic construct is possible and results in an increase in expression of the optimized env. VLPs are produced by the constructs in greater numbers in the absence of an active protease.
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