| Previous research showed that HIV-1 infected individuals can develop neutralizing antibodies. HIV-1 envelope protein is the major target of neutralizing antibodies, and the neutralizing antibody epitopes are mainly located in gp120 and the outer domain of gp41. So far, the knowledge of epitopes for neutralizing antibodies is mainly from the HIV-1 B subtype that is circulated in Europe and North America. A few broadly neutralizing monoclonal antibodies(bnmAb) are basically isolated from the individuals of B subtype infection, only recently several ones are isolated from subtype C infection. The major subtypes of HIV-1 epidemic in China are B'subtype, B'C and AE CRFs. The antibody response, strength and specificities in HIV-1 infected individuals in China have not been systematicaly studied. This study was to analyze the cross-reactive antibodies reponse and investigate the magnitude and strength in HIV-1 infected individuals in China using the immunogen from HIV-1 circulating isolates in China and HIV-1-pseudotype virus system. It is useful to find the neutralizing antibody epitopes in serum from Chinese HIV-1 infected individuals.The main results are as follows:1. constructed eukaryotic expression plasmids that express recombinant gp120 protein from HIV-1 B'subtype and B'C subtype isolates in China, the secreted gpl20 recombinant proteins with His tag were obtained by transient transfection of CHO-K1 cell. Western Blot results showed that the gp120 protein from HIV-1 B'subtype can be recognized by HIVIg, suggesting that it has the correct conformation; the gp120 protein from HIV-1 B'C subtype can be recognized by anti-His tag mAb, but not by HIVIg, which needs further identification. Using His tag affinity purification method, we obtained gp120 protein with a certain purity, which is useful for serological analysis.2. Using three V3 peptides from different HIV-1 isolates as the immunogen of ELISA, we identified 15 cross-reactive sera in 80 sera from HIV-1 infected individuals.Serum 15,28,29,38,44,73 can cross-react with all three peptides; serum 4,19,22,34 can cross-react with two peptides that were from the North American subtype B consensus sequence and the North American subtype B isolate sequence; serum 16,21,42,49,80 can cross-react with two peptides that were from North American subtype B consensus sequence and the Chinese B' subtype isolate sequence. There was no serum can cross-react with peptides that were from the North American subtype B isolate sequence and the Chinese B' subtype isolate sequence. The average serum reactivities of three peptides were ranked in the following order:North American subtype B consensus sequence> Chinese B'subtype isolate sequence>North American subtype B isolate sequence. The difference is statistically significant, the result showed that there could be more conserved epitopes in North American B subtype consensus sequence. The V3 cross-reactive sera also can recognize gp120 from three different HIV-1 isolates.3. Constructed and optimized the HIV-1 pseudotype virus sytem. The system consisted of ENV clones from three major prevalent HIV-1 subtypes in China, including three clones of B'subtype, three clones of AE subtype, five clones of B'C subtype, and two clones of B subtype in North America. The results of ENV sequence analysis showed that the position of cysteines is conserved in all ENV clones, but the length of the variable region is changeable. Overall, the length of V1/V2 region is longer in the clones of B'C subtype; the length of V3 region is conserved in all clones; the length of V4 and V5 region is moderate variable, but some clones contain longer sequence. We predicted the number of the potential N-linked glycosylation sites(PNLG) in ENV clones. The clones of B'C subtype contains more glycosylation sites in gp120 than other subtype clones. There was no obvious difference in the number of PNLG in gp41 among the subtypes. Optimized the ENV/Backbone plasmids ratio, incubation time of transfection complex, and DEAE Dextran concentration, respectively, and established the best experimental contition.Using different anti-HIV reagents, we testified the effectiveness of the pseudovirus system.The results showed that the system is opt for characterizing the neutralizing antibodies, evaluating the effect of HIV-1 vaccine candidate, and testing anti-HIV-1 drug.4. Using HIV-1 pseudovirus system, we investigated the cross-clade neutralizing activity of a panel of 80 sera from HIV-1 infected individuals in China.There were 42 sera with non-ENV-specific neutralization for MLV/HIV chimeric pseudovirus, suggesting that these sera may contain anti-HIV-1 drugs. The remaining 38 sera were not detected the neutralization for the MLV/HIV chimeric pseudovirus and the neutralization susceptibility of five HIV-1 isolates from different subtypes to these sera was ranked as following order:JR-FL>HXB2>CNE50>CNE6> CNE55. Identified several cross-clade neutralizing sera with potent neutralizaiton activities to one or several HIV-1 pseudovirus isolates, including:serum 29 exhibited potent cross-clade neutralizing activities to all five HIV-1 isolates; serum 15 exhibited potent neutralizing activities to CNE50 and JR-FL; serum 1,2 and 13 exhibited potent neutralizing activity to JR-FL; serum 7 exhibited potent neutralizing activity to CNE55; serum 44 exhibited potent neutralizing activity to HXB2.
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