The Construction Of A HIV-1 Thai-B Infectious Clone And Its Initial Application In The Neutralizing Antibody Detection And Vaccine Development

The construction of a HIV-1 Thai-B infectious clone and its initial application in the neutralizing antibody detection and vaccine developmentMolecular epidemiological studies reported the most circulating HIV-1 clades in China included Thai-B, CRF07_BC, CRF08_BC and CRF01_AE. In particular, CRF07_BC and CRF08_BC were unique subtypes which were recombined by Thai-B and C clades and only caused pandemic in China, subtype Thai-B emerged as the predominant subtype in China as a shift toward the Thai-B genotype from the former predominant prototype subtype B occurred.No doubt, HIV infectious clone was a very powerful tool to study the mechanism of virus recombinations, surveil and analyze the new emerging circulating isolates. Above all, to date, no HIV-1 thai-B infectious clone was constructed successfully. On the basis of construction of a thai-B infectious clone, we established a neutralizing system with pseudoviruses carrying exchangeable gp120 regions; we also designed a panel of HIV-1 DNA vaccines against circulating HIV-1 isolates in China and made initial research on their immunogenicities and protective activities.Part I. Construction and characterization of a HIV-1 Thai-B infectious molecular cloneAfter amplifying the full-length genome of CNHN24 in two half-molecules, We successfully constructed Thai-B infectious molecular clone using low copy plasmid pLG338, structural proteins of HIV were detected from the lysate of transfected 293T cells by Western blotting, infectious clone derived virions were also determined using p24 detection kit, cloned virus could infect and replicated in PBMCs and MT4 cells, its ability of causing syncytium in infected MT2 cells indicated it was a SI (syncytium inducing) strain. Cloned virus was certified as a R4 tropism by coreceptor usage experiments. No rearrangments, insertions, and large fragments deletions were found in the sequencing results of this infectious clone. In comparison with the primary isolate, truncation of 13 amino acids in the V1 region caused three N-linked glycosylation deletions, it was also indicated the length and glycosylation level of V1 correlated with the immune escape from the neutralizing antibodies. Successful construction of this infectious clone will provide a powerful tool to study the HIV pathogenesis.PartII. Establishment of a neutralizing system with Chimeric pseudoviruses carrying exchangeable gp120 regions.HIV pseudovirus was an important tool to evaluate the antibody protection and relative functions of Envelope region, pseudovirus system contained a backbone plasmid and a envelope vector, pseudovirus particles was produced by contransfecting these two plasmids into 293T cells, derived virions were able to infect the cells but unreplicable due to the lack of envelope region in their genomes. Two unique enzyme sites NheI and SphI were introduced into the leader sequence and cleavage site separately based on the SF162 envelope clone, in particular, Nhe and SphI were very rare in the HIV envelope genes. The expressions of gp160 glycoproteins from modified SF162 envelope clones were detected by Western Blotting. Several chimeric pseudoviruses containing different gp120 isolates were constructed and their activities against neutralizing antibodies were evaluated on the Tzm-bl cells. It was also found that the infectivity of resulting pseudovirus correlated with the virion-associated Env gp160 cleavage efficiency. These chimeric pseudoviruses constructs provided useful research tools to test the protective functions of neutralizing antibodies and perform further research on the pathogenicity of env.Part III. Establishing an initial research on the HIV vaccines against circulating HIV-1 isolates in China focused on the Env glycoproteinThe most circulating HIV-1 isolates in China were composed by clades Thai-B, BC and AE. In order to construct the DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying gp120 regions from isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed respectively, gp120 glycoproteins expressions of these DNA vaccines were detected by western blotting in transfected 293T cells. Immunizations of White New Zealand rabbits were performed as the strategy of"DNA prime plus Protein boost", the levels of antibodies responses and neutralizing antibodies against homologous gp120 were relative lower after 4 DNA primes, how ever, and the antibodies titers went up quickly with 2 protein boosts. Obviously, effect of this combination approach was much better than sole DNA and protein using. Protective activities of immunized rabbits sera against several pseudoviruses and laboratory strains were also evaluated on the Tzm-bl cells, the neutralizing titers and spectrums of sera from the rabbits which received DNA prime plus protein boost were broader than those received DNA and protein immunization separately. So it was indicated that polyvalent DNA prime plus protein boost was an effective immunization strategy to broad the protective spectrum, and relative further research about it should be performed on the basis of the pilot study.