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Cyclophilin A as genetic adjuvant, induced broader and long-lasting Gag-specific cellular immune response based on CyPA-Gag specific interaction

Investigations into the correlation between cyclophilin A and HIV gag protein have been around for decades. Previous studies demonstrated that the human peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA) interacts with the N-terminal domain of HIV-1 Gag CA. It is reported that, when dendritic cells (DC) succumb to HIV-1 infection under co-transfected with SIV Vpx protein, HIV-1 induces DC maturation and a series of innate responses, including the induction of an antiviral type I interferon response and activation of T cells. These processes are dependent on the interaction of newly synthesized HIV-1 capsid with cellular cyclophilin A (CyPA). This consequence of infection might not be benefit for AIDS progress, but we could take advantage of this specific interaction to promote vaccine immunogenicity. Therefore, we investigated the suitability of CyPA as a genetic adjuvant for an HIV-1 Gag DNA vaccine. 
We designed DNA vaccine, single expression cassette that including Gag gene and CyPA gene respectively. As, pDRVI4.0-Gag, pDRVI4.0-Env and pDRVI4.0-hCyPA. We also constructed dual expression cassette that expressed both Gag and CyPA gene in one plasmid and under each CMV promoter control. The vaccination scheme show out in the below. Antigen-specific cellular immune response was detected by IFN-γ ELISPOT; humoral response and antibody affinity were detected by ELISA. The interaction between CyPA and Gag was achieved by Co-IP analysis in vitro.
1.In CyPA specific adjuvanticity analysis, we found CyPA could not enhance Env or irrelevant antigen (EGFP) immunity but promote HIV Gag-specific cellular immune responses.
2.In the dual expression cassette regimen, the cellular immune responses showed that, Gag/CyPA could stimulate high level Gag-specific cellular immune responses, compared with Gag alone.
3.Moreover, based on CyPA mutations Co-IP analysis, we assume the adjuvant effect of CyPA is based on Gag-CyPA specific interaction.
4.In assessing the potential effect of CyPA on the breadth of T-cell responses in mice, CyPA broadened the T-cells’ response spectrum of Gag peptides, with 8 pools showing significant enhancement through CyPA use. However, the dominate broader-spectrum was not focus on the Gag-CyPA binding loop sites.
5.In the longitudinal analysis of immunogenicity, Gag-specific cellular immune responses induced by Gag/CyPA and Gag/CyPA-3mut were maintained at high level from 150 days after the final inoculation, compared with Gag alone.

There is the first report that Cyclophilin A could augment HIV-1 Gag specific cellular immune response as genetic adjuvant in multiplex DNA immunization strategies. The adjuvanticity of CyPA is specific, broad and long-lasting. And the breadth and longitude of T cell responses elicited by DNA HIV vaccines is thought to be important for pathogen control, perhaps by preventing the selection of escape mutants.