Development Of An Indirect Enzyme-linked Immunosorbent Assay To Distinguish HIV-1 Recent And Long-term Infection

BackgroundHuman immunodeficiency virus(HIV)is the pathogen of acquired immunodeficiency syndrome(AIDS).In general,the incubation period of HIV infection is about 8-10 years.The traditional immunoassay for detecting anti-HIV antibody can only determine HIV infection,but cannot distinguish recent or long-term HIV infection.It cannot accurately monitor HIV epidemic trend and evaluate the effect of prevention.Several laboratory detection methods have been developed in recent years to estimate the incidence of HIV infection based on cross-sectional samples.However,no assay fulfills the "gold standard".Our previous study found that a 57-mer peptide of HIV-1 transmembrane glycoprotein gp41(gp41-p57)could distinguish recent HIV-1 infection from long-term HIV-1 infection.Further studies are needed to determine the immunodominant epitopes(IDEs)and investigate the influence of the differences of HIV-1 gp41-p57 sequences among different genotypes of HIV on HIV incidence assay performance.ObjectivesWe aimed to identify the major IDEs and the impact of HIV genotype specific sequences within gp41-p57 peptide in differentiating recent and long-term HIV-1 infection,and to establish a prototype laboratory immunoassay based on the gp41 peptides derived from the predominent HIV-1 genotypes in China to distinguish recent and long-term HIV-1 infection.The results could provide new tools to monitor the rate of HIV incidence infection and to evaluate the effect of interventions.Methods1.We synthesized 10 truncated peptides based on gp41-p57 peptide to characterize anti-HIV antibody titers of plasma samples from individuals of recent and long-term HIV-linfection to identify the IDEs of HIV-1 gp41 protein.2.We synthesized three peptides of gp41-p57 derived from CRF01_AE,CRF07_BC and CRF08 BC as well as subtype B,which were predominant HIV genotypes in China.These peptides were used to detect individuals infected with different HIV-1 genotypes to characterize the genotype specific difference when distinguishing HIV recent and long-term infection.3.We constructed a prokaryotic expression vector to express the recombinant protein MP4,which contains HIV-1 gp41 IDEs of HIV-1 genotype CRF01_AE,CRF07_BC/CRF08_BC,subtype D and subtype B.The immunological activity of recombinant protein MP4 was determined by western blot and indirect ELISA.4.An indirect ELISA based on the recombinant protein MP4 was established to distinguish recent vs.long-term HIV-1 infection.The sensitivity and specificity as well as positive predictive value and negative predictive value of the immunoassay were evaluated by comparing with the commercial LAg-avidity EIA kit.Results1.Motifs QKFLG and GK?C were identified as the two major IDEs of gp41-p57 for differentiating recent vs.long-term HIV-1 infection.The surrounding amino acids EAQQHLLQLT and WNSSWSN of p57could affect the recognition or binding of gp41 against anti-HIV antibodies.Compared with the LAg-Avidity EIA,the p57-based immunoassay showed the highest coefficient value(?=0.923)among the 11 peptides analyzed.The yoden index and the area under the receiver operator characteristic curve(AUC)of p57-based immunoassay was 92.32%and 0.987,respectively,making the gp41-p57 suitable for distinguishing recent and long-term HIV-1 infection.2.The amino acid sequences of gp41-p57 vary among different HIV-1 genotypes,resulting in discrepant sensitivity of the immunoassays.Using a mixture of peptides gp41-p57 or the recombinant protein MP4 derived from the dominant HIV-1 genotypes as antigen could improve the sensitivity of the immunoassay.3.Compared with the LAg-Avidity EIA kit,the MP4-based immunoassay showed a high coefficient value of agreement in detecting both the 37 follow-up samples and the 159 cross-sectional samples(?=f0.890 and ?=0.730,respectively),with a sensitivity of 90.91%and 96.85%,a specificity of 100.00%and 71.88%,a predictive value of recent infection of 88.24%and 85.19%,and a predictive value of long-term infection of 100.00%and 85.19%,respectively.The MP4-based immunoassay was repeated three times in detecting 159 cross-sectional samples.Only 7 samples(4.40%)were not completely consistent,indicating a high reproducibility.ConclusionThe IDEs and HIV-1 genotype differences of the gp41-p57 peptide for differentiating HIV-1 recent and long-term infection were identified.An indirect ELISA based on the recombinant protein MP4 was established and evaluated to distinguish HIV-1 recent and long-term infection.