Characterization Of Humoral Immune Responses Against Capsid Protein P24 And Transmembrane Glycoprotein Gp41 Of Human Immunodeficiency Virus Type 1

Background:The human immunodeficiency virus type one(HIV-1)is the causal pathogen of the acquired immunodeficiency syndrome(AIDS).The gp41 glycoprotein,the transmembrane portion of HIV-1 envelope protein gp160,is the most conservative antigen that has been widely used as coating antigen in the HIV-1 immunoassay.Neutralizing human monoclonal antibodies(mAbs)have also been identified to direct to HIV-1 gp41,making it the primary target for vaccine development.Furthermore,the capsid protein p24 of HIV-1 is the most abundant viral protein and highly conservative among different subtypes of HIV-1 and the earliest immunological biomarker detected after HIV-1 infection.In the previous report,the switch of humoral immune response patterns from polyclone-like reaction to monoclone-like reaction or reaction against conformational epitopes was also reported,suggesting that the specific peptides carrying the major IDEs of HIV-1 p24 and gp41 may be useful biomarkers for improving HIV-1 detection and distinguishing different HIV-1 infection.Objective:We have now designed and synthesized peptides representing major B cell linear IDEs of HIV-1 p24 and gp41,respectively guided by the previous report and the information from the HIV-1 database.We tend to explore the pattern as well as the characterization of the humoral immune responses,and further analyze the relation between the different pattern and any useful biomarkers.These results are of major importance for development of an improved HIV-1 p24 detection assay and in developing immunoassay to distinguish different HIV-1 infection.Method:The p24 gene was amplified and constructed into pGEX-4T-1 vector.Recombinant p24 protein was successfully expressed in E.coli by induction in the soluble form and the overall purity was conducted by one-step affinity chromatography purification.The molecular size and activity were determined by SDS-PAGE and indirect ELISA,respectively.Plasma samples were collected from different HIV-1 infected individuals and were divided into recent or long-term infection.According to the previous report and the information from the HIV-1 database,three peptides representing major B cell linear IDEs of HIV-1 p24 and gp41,respectively were synthesized.We tend to explore the pattern as well as the characterization of the humoral immune responses among different individuals in different infection period.We screened out a peptide that can be a useful biomarker in distinguish the infection period according to the immune patterns of the samples from individuals from different phases of HIV-1 infection.A simple and quicker method for distinguishing recent and long-term HIV-1 infection was initially established in laboratory through optimizing the condition.Sensibility and specificity were also evaluated comparing with two other commercial ELISA-kits.Result:The recombinant plasmid was successfully constructed and transformed into E.coli competent cells.The molecular size of the fusion protein was agreed with expectation and the result of indirect ELISA indicated the activity of the usion protein.The majority(88.3%,233/264)of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs,but did react with the recombinant full-length p24.In contrast,87.7%(236/269)of the samples reacted with at least one of the linear gp41 peptide.With the development of the infection,the titer of special antibodies against gp41-pl increased steadily.The correlation coefficient between postinfection day and optical density(OD)value was 0.78.While defining both gp41 and gp41-pl tested positive for antibodies as recent infection,gp41 tested negative and gp41-pl tested positive for antibodies as long-term infection,a newly gp41-based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection was initially setted up.Conclusion:The patterns of humoral immune responses against capsid protein p24 and transmembrane glycoprotein gp41 of HIV-1 were different,based on which a new immunoassay that can be used to distinguish recent and long-term HIV-1 infection was initially setted up.