Hiv-1 Membrane Antigen Transformation And Immunogenicity Evaluation

HIV Env located on the surface of the viron is the main target of the neutralizingantibodies, but under the immune pressure the wild type Env usually eludes the control of theimmune response, which may be related to the complex conformation of Env and theglycoside chains on its surface. We expected to expose epitopes of the four knownneutralizing antibodies and the potential neutralizing antibodies in the CD4-induced region,by deglycosylation or removal of complex amino acid residues through site-directedmutagenesis, and by deletion of variable loops. And we wanted to link the humoral andcellular immune responses together in order to find a new immunogen which could be able toinduce effective and balanced humoral and cellular immune responses. Besides, we clonedthe wild type and mutated Envs into another expression vector to analyze the effects of theEnv mutations on the assembly of functional pseudovirus.Based on the four known neutralizing antibody epitopes in the three-dimensionalstructure of HIV-1 Env and others'work, we designed four groups of mutations. And throughdifferent combination we designed five mutant clones including: M2, M5-1, M5-2, M4 andM7. And then we deleted the V1V2 variable loops of the wild type and the mutants to designanother six new mutants including: dWt, dM2, dM5-1, dM5-2, dM4 and dM7. Throughsite-directed mutagenesis and DpnI selection, we got all the 11 mutants. We primarilyidentified the mutants by restriction endonuclease and finally determined them by sequencing.We extracted the plasmids of Wt and mutants from E. coli, and then transfected 293T cellswith them and investigated whether the plasmids could express in vitro by using WesternBlot (WB). We extracted Wt and mutational plasmids and immunized rabbits. The antibodylevels were detected by using ELISA assay. Neutralizing antibody levels of rabbit sera wereassesed by single-cycle infection neutralizing assay. Correlationship between the ELISAresults and the neutralizing assay results was analyzed. Mice were immunizedintramuscularly followed by electroporation.Sera and spleen white cells were collected forneutralizing assay and ELISPOT assay. Peptide pool SHIVchnl9 and peptide Env34 wereemployed in the ELISPOT assay. We cloned all the envs into pcDNATM3.1 D/V5-His-TOPO(?) expression vector, and cotransfected 293T with pSG3^△Env. We collectedsupernatant and infected TZM-bl cells and investigated the influence of the Env mutations onassembly of functional pseudovirus.The WB results show that all the mutants and those envs cloned into pcDNA^TM3.1D/V5-His-TOPO(?) vector can express in vitro and there is no difference in the expressionquantity between them and the Wt. And the results present that most of the mutants movefaster than the Wt in electrophoresis, which further prove that we have succeeded inintroducing the mutations into the Env. The ELISA results show that compared to the DNAimmunized rabbits without electroporation, higher antibody titers could be gotten even withsmaller injection dose ofplasmids and shorter vaccination period from those immunized withplasmids followed by electroporation. Although we got different results from differentELISA kits, the results of different kits showed similar trend. High levels of antibody weredetected in the rabbit sera of Wt, N201Q, G2, M2, M5-1 and dM5-1, but there were notapparent advantages over the Wt. Different antibody levels were detected between tworabbits immunized with the same plasmid. And the neutralizing antibody levels of the tworabbits of the same group were different too. There were no significant correlationshipbetween the ELISA results and the neutralizing assay results. As far as the two rabbits of thesame group were concerned, the one with a higher level of neutralizing antibody wasdetected a higher level of antibody by ELISA assay. The mice ELISPOT results showed thatcompared to the wild type group, the cellular immune response of most of the mutant groupsdecreased, including M5-1, dM5-1, M5-2, dM5-2, M7, dM7, dWt, M4 and dM4. Amongthese groups, only the decrease of groups dM5-1, M5-2, M7, dM7 and dM4 were statisticallysignificant (p＜0.05). The cellular immune response of groups M2, dM2 and G2 werestronger than that of Wt group. Only the enhancement of M2 group was statisticallysignificant compared with the Wt group. The cellular immune responses for the four peptidepools were different. The sequence from strong to weak was peptidel＞peptide3＞peptide2＞peptide4. The response for the peptide 1 was the strongest, which took more than 75% of thesum of the four. The correlation of the cellular response for each peptide pool with that of thetotal present a sequence: peptidel＞peptide3＞peptide2＞peptide4. The correlation coefficientof the cellular response for Env34 and the total of the four peptide pools was 0.942(p＜0.01). In both of the twoneutralization assays, the neutralizing antibody levels of groups dWt, M2, M5-2, M5-1, dM7and N201 were higher than that of the Wt group. But only the enhancement of groups dWtand M5-2 were statistically significant. Besides, the enhancement of group M5-1 in the Wtneutralizing assay and groups M2 and N201Q in the 74-2 neutralizing assay were statisticallysignificant. In the two neutralizing assays the neutralizing antibody levels of groups dM2,dM5-2, M4, dM4 and M7 decreased compared to the group Wt. Amongst these groups, onlythe decrease of groups M4 and dM4 were statistically significant. The results of single-cycleinfection assay showed that except the G2 mutant, other mutations weakened the functionalpseudovirus assembly ability of Env.From the animal experiment results, we can conclude that different mutations postdifferent effects on the immunogenicity of the Env. The M2 mutation elevates the ability ofEnv to induce cellular and humoral immune response. The dWt mutant can induce strongerhumoral response and similar cellular response, compared with the Wt. Although thosemutants including: N201Q, M5-1, M5-2 and dM7 induce weaker cellular response than theWt, they can induce stronger humoral response.dM2 and G2 mutants can not induce humoralimmune response as strong as Wt, but they can induce similar cellular immune response.Those mutations including dM5-2, M4, dM4 and M7 weaken the immunogenicity of the Envto induce humoral and cellular immune response. We can conclude from the single-cycleinfection assay that the G2(N620Q, N632Q) mutation has no effect on the assembly offunctional pseudovirus, but the two single sites mutation F174A and N201Q respectivelymake the Env lose the ability of functional pseudovirus assembly.