Research On Pharmacodynamics Of Recombinant Adenovirus Expressing HIV-1gag Gene

AIDS is one of the most serious diseases threatening human health nowadays.Since HIV was identified as the pathogen of AIDS, scientists have been focused ondeveloping effective strategies for prevention and treatment of AIDS. However, dueto the features of high variability and drug resistance of HIV effective drugs thatcould cure AIDS is still no available. Towards a long-term strategy for preventionand control of AIDS, the discovery of effective HIV vaccines is the best hope toconquer AIDS.We developed sequential and repeated immunization with multi-vector based HIVvaccines, the vaccinated animals could maintain high level of cellular immuneresponses for long time. The main objective of this study is to evaluate thepre-clinical immunogenicity of recombinant adenovirus vaccine expressing gag gene（Ad5-HIVgag）, and provide evidence for the clinical application of this vaccine.Adenoviral vector is widely used in gene therapy and vaccines research.Adenoviral vector has many advantages. It is safe, it can infect a wide variety of celltypes and tissues including both dividing and non-dividing cells for gene transfer orexpression. And it can be produced to high titers and purified easily. The viruschromosome remains episomal in the transduced cell, thus avoiding the possibilityof insertional mutagenesis. Besides, the adenovirus double-stranded DNA is easy forgenetic recombination. The core protein （Gag） is highly conservative, rich in CTLepitopes and is the major target of cellular immune responses. Ad5-HIVgag isnon-replicate recombinant virus which lacks of E1and E3region, and is insertedwith HIV-1subtype B gag consensus sequence.Firstly the methodological verification of PCR, restriction enzyme digestions,Western blot and ELISpot assay was performed and the products inspectioninstruction was prepared. Then three lots of purified rAd5-HIVgag vaccines wereverified by these assays.The effects of cellular immune responses in mice and monkeys were evaluated.The mice were randomly divided into six groups of10. Different amount of thepurified rAd5-HIVgag (2×10⁶VP,2×10⁷VP,2×10⁸VP,2×10⁹VP,2×10¹⁰VP) orPBS were administered two or three times. Gag-specific cellular immune responseswere detected by ELISpot assay at one week post last immunization. The results showed that significant Gag-specific cellular immune responses were detected inimmunized mice. And the mice immunized with2×10⁷VP of rAd5-HIVgag elicitedhighest level of cellular immune responses. To observe the specific cellular immuneresponses in Macaca fascicular is immunized with different dosage of Ad5-HIVgagby repeated in tramuscular injection, the Macaca fascicularis were randomly dividedinto four groups of6. Different amount of the purified Ad5-HIVgag （0.99×1011VP,4.94×1011VP,24.68×1011VP） or PBS were administered in3weeks interval andfive times. Gag-specific cellular immune responses and the level of anti-adenovirusneutralizing antibodies at different time points were detected by ELISpot assay andneutralization assay respectively. The results showed that significant Gag-specificcellular immune responses were detected in all Ad5-HIVgag immunized groups at5weeks post first immunization and then increased at8weeks. The Gag-specificcellular immune responses declined at12weeks and then increased with time. Theseindicated that Gag-specific cellular immune responses could be induced in Macacafascicularis immunized with Ad5-HIVgag in a certain range of dosages. Thepresence of anti-adenovirus neutralizing antibodies induced by vaccination withadenovirus vectors in Ad5-na ve animals did not further reduce Gag-specific cellularimmune responses.In summary, Ad5-HIVgag vaccine can induce Gag-specific cellular immuneresponses. It may be an effective vaccine for the treatment of AIDS. According toChinese registration administration of biological product and technical requirements,we organized the data about the quality control and pharmacodynamic research ofAd5-HIVgag and submitted the application of clinical research to Beijing DrugAdministration.