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205 Improved Human Immunodeficiency Virus 1 Gag and Pol Expression Cassettes
J. zur Megede*, G. R. Otten, L. Leung, H. Liu, B. Doe, and S. W. Barnett
Chiron Corp., Emeryville, CA, USA
Background: The World Health Organization (WHO) estimated the number of people worldwide that are infected with HIV-1 to exceed 36.1 million. The development of a safe and effective HIV vaccine is therefore essential at this time. Recent studies have demonstrated the importance of CTL in controlling the HIV-1 replication in infected patients. Furthermore, CTL reactivity with multiple HIV antigens will be necessary for the effective control of virus replication. Therefore the inclusion of HIV-1 Gag and Pol, beside Env for the induction of neutralizing antibodies, into the vaccine is very important.
Methods: Several expression cassettes encoding HIV-1SF2 Gag and Pol were designed. These included gagprotease and gagpolDELTAintegrase with frameshift (+FS) and gagpolDELTAintegrase in frame ((FS). Versions of gagpolDELTAintegrase((FS) were also designed with attenuated or nonfunctional protease. The nucleic acid sequences were codon modified for highly expressing human genes. Mice were immunized with titrated DNA doses and humoral and cellular immune responses were evaluated by ELISA and intracellular cytokine staining.
Results: In vitro expression data showed improved expression of p55Gag using gag alone, gagprotease(+FS), and gagpolDELTAintegrase(+FS) over gagpolDELTAintegrase((FS). Western blot revealed improved expression of p55Gag and p66RT using gagpolDELTAintegrase((FS) with nonfunctional protease over both other versions with fully functional or attenuated protease gene. In mice, the plasmid gagpolDELTAintegrase((FS) was less potent for the induction of humoral and cellular immune responses against Gag compared with the other cassettes containing gag alone or gagprotease(+FS) and gagpolDELTAintegrase(+FS). No difference in immunogenicity was found using either gag alone, gagprotease(+FS) or gagpolDELTAintegrase(+FS).
Conclusions: Our in vitro and in vivo experiments suggest, that the expression and immunogenicity of Gag using gagpolDELTAintegrase((FS) is less efficient compared with gag alone, gagprotease(+FS) or gagpolDELTAintegrase(+FS). The immune responses in mice correlated with relative levels of expression in vitro. Vaccine studies in rhesus monkeys are planned to further address these questions in depth.
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