InvestigatingHIV-1DNAvaccineadjuvant And Gene Expression Profiling Of Two Viral Vectors

Despite decades of global research efforts, an efficacious HIV vaccine has remained elusive thus far.Plasmid DNA vaccines are a promising modality for immunization against a variety of human pathogens.However, poor delivery efficiency has impaired their practical use. Viral-based vaccine is also an excellent vector had been applied in clinial trials. So far, we still faced the plight that poor immunogenicity of DNA vaccine and unclear mechanism of viral vector stimulation immune system.To enhance the immunogenicity of DNA vaccine, we investigated the immunological characteristics and potential mechanism of candidate adjuvants, to seek suitable DNA vaccine adjuvant. In this study, we set up mouse bone marrow-derived dendritic cell in vitro, combined with TLR signaling pathway activation and DC maturation and cytokines secretion, evaluated oligodexynucleotide CpG, Polysaccharide nucleic acid fraction of bacillus calmette guerin (BCG-PSN), flagellin, Pseudomonas aeruginosa mannose sensitive hamemagglutination (PA-MSHA) and Cholera toxin B subunit (CTB), these five different species adjuvants. Based on the in vitro results, we chose PA-MSHA and CTB to analyze the effect of immunostimulation co-immunized with HIV DNA vaccine in mouse model. Both in vitro and in vivo data showed that, PA-MSHA and CTB could stimulate TLR signaling pathway, and promoted BMDC maturation and secreted several species cytokines. PA-MSHA showed a dose-dependent effect when co-administrated with HIV Env DNA vaccine, low dosage PA-MSHA enhanced the specific cellular and antibody responses that induced by HIV DNA vaccine, however, high dose PA-MSHA exhibited immune suppression that impaired HIV DNA vaccine immunogenicity. It's firstly report that CTB induced high levels of antigen-specific cellular and humoral immune responses co-inoculated with DAN vaccine intramuscularly in vivo; however, CTB was investigated as a classical mucosal adjuvant usually. And the strength of immune responses might correlate with CTB induced inflammatory responses.It is reported that, when dendritic cells succumb to HIV-1 infection co-transfected with SIV Vpx protein, HIV-1 induces DC maturation, stimulate 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). In this study, we investigated the suitability of CyPA as a genetic adjuvant for an HIV-1 Gag DNA vaccine.In CyPA specific adjuvanticity analysis, we found CyPA could not enhance Env or irrelevant antigen immunity but augment HIV Gag-specific cellular immune responses. 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. Moreover, based on CyPA mutations Co-IP analysis, we assume adjuvant effect of CyPA is based on Gag-CyPA specific interaction.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. In the longitudinal analysis of immunogenicity, Gag-specific cellular immune responses induced by CyPA were maintained at high level from 210 days after the final inoculation, compared with Gag alone. There is firstly report that Cyclophilin A could augment HIV-1 Gag specific cellular immune response as genetic adjuvant in multiplex DNA immunization strategies. And this adjuvanticity is specific, broad, long-lasting and based on Gag-CyPA interaction.To assess HIV vaccine candidate viral vectors, we investigated the characterizes of gene expression profiling of two licensed vaccine, yellow fever attenuated live vaccine YFV-17D and vaccinia post immunization in two clinical trials. We analyzed gene ontology and pathway in each time point, and explained the dynamic process by time-course analysis and dynamic transcription factors prediction.YFV stimulated immune system activation and hundreds of genes up-/down-regulated at early stage, but most of them involved cellular macromolecule metabolic process and regulation of cellular metabolic process. More genes enriched in RNA metabolic process, transcription, DNA-dependent, gene expression processing at dayl to day3, it might indicate immune system is preparing for consequence processes. Most important events showed up during day 5 to day 7 after vaccine immunization, including defense response, response to virus, innate immune response and several cytokine-mediated signaling pathways. In addition to some cytokine receptor activity and inflammatory responses, most of the different genes change back to cell cycle, mitosis and response to wounding at day 28. Vaccinia did not induce such robustness immune response, almost nothing changed before 3 days post-immunization. The expression peak presented at day 9 to day 14, most of genes involved lymphocyte differentiation, proliferation, T cell activation, cytokines production and lymphocyte homeostasis, and till day 28, there also had defense responses, dendritic cell migration and chemotaxis, TLR signaling pathway activation and T/B cell regulation. The time-course analysis showed the gene expression patterns of YFV stimulated were more complicated than vaccinia did. And based on dynamic regulation prediction, YFV activated amount of transcription factors, like STAT1, IRF9, IRF7, FOS and JUNB up-/down-regulation at various time points, however, vaccinia could not argument transcription factors significantly.In this study, we provide a new approach of improvement of HIV DNA vaccine immunogenicity and optimization of adjuvant screening, moreover, to develop candidate viral vector using system vaccinology.