ABSTRACT
Objective To establish a pseudovirus-based neutralization assay. Methods The functional gp160 genes were amplified from plasmids containing HIV-1 gene. The products were ligased into pcDNA3.1 plasmid and positive clones were screened by digestion with restriction enzymes. The pseudoviruses were harvested by co-transfection of the positive clone and pSG3△env plasmid. The neutralizing activities of monoclonal antibodies and HIV-1 antibody positive plasma were measured by these pseudoviruses. Results The four strains of psedoviruses (CHB01, CHB02, CHBC03 and CHAE04) had been successfully obtained. Monoclonal antibody 4E10 could neutralize all of 4 pseudoviruses while 2G12 could not neutralize any pesudoviruses. Monoclonal antibody 2F5 could neutralize pseudovirus CHB01, CHB02 and CHAE04 but not CHBC03, while IgG1b12 could neutralize pseudovirus CHB01, CHB02 and CHBC03 but not CHAE04. The neutralizing activities of 43 of HIV-1 antibody positive plasma against different subtypes of pseudovirus were significant differences and the cross-neutralization effects for some samples exist. Conclusion The harvested pseudoviruses could be used in the neutralization assay. However, the neutralizing characteristics of different pseudoviruses may be different.
ABSTRACT
Objective To study the influence of site-directed deglycosylation of the HIV-1 envelope (Env) on its immunogenicity and assembly of functional pseudovirus. Methods Site-directed deglycosylation were performed using cycling mutagenesis and selection of mutants with DpnⅠ. Single-cycle infection assay was employed to analyze the effect of the mutations on the ability of functional pseudovirus assembly. The influence of deglycosylations on the immunodeficiency of Env was evaluated using pseudovirusbased neutralization assay and ELISPOT assay. Results Mutant N197Q induced higher neutralization activities for both pseudoviruses, but lower Env-specific T-cell response. And N197Q rendered the Env to lose the ability of functional pseudovirus assembly. Mutant G2 induced higher neutralization activities for pseudovirus 74-2 but lower for pseudovirus Wt, and had almost no influence on Env-specific T-cell response and functional pseudovirus forming. Conclusion The site-directed deglycosylation of the HIV-1 Env affects the pseudovirus forming and its immunogenicity.
ABSTRACT
Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around Nab epitopes and deletions of variable regions in env.The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γELISPOT.Overall,five mutants(dWt,M2,M5-2,M5-1 and dM7)induced highed neutralization activities for both pseudoviruses than plasmid Wt,while only two of the mutants(dWt and M5-2)showed significant differences(P<0.05).Two mutants(M2 and dM2)induced more Env-specific T cells than plasmid Wt.Statistically however,significance was only reached for mutant M2.Thus,properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.