The Correlation Between The Variation Of Virus Envelope And The Neutralizing Antibodies Response Of Plasma During Early Stages Of Primary HIV-1 CRF01_AE Subtype Infection

Objective: The virus replicates rapidly and the plasma viraemia increases exponentially to reach a peak after HIV-1 infection.Following the viral load decreases over 12-20 weeks to reach a more stable level.Now we think that in the acute phase plasma virus drops rapidly is the result of the interaction first between host immune and virus,which the outcome strongly influences the pathogenesis and clinical course of the infection.So for acute phase continuous observation of the HIV-1 infection virus in the natural course of evolution,to reveal the HIV-1 infection initially host immune response to control virus and provide key clues for vaccine research acquired immune response of the protective measures as definitive the value.Of HIV-1 infection cases in recent years studies have found that in the acute phase of infection the virus evolution features can reflect the virus escaping the acute phase of the host immune response mechanism,so as to provide some reference for vaccine research of replying to virus mutation and selecting effective neutralization sites.But the studies of different subtypes virus of acute infection reported inconsistent results are obvious,and failed to clarify acute viral escape immune selection mechanism,this may also be a hindrance to vaccine research at present one of reasons.The envelope of HIV-1 is the key structure to the virus infected target cell,and identified target areas by cellular and humoral immune response.During early stages of infection,research for the mutation of envelope gene driven by immune response can obtain the key evolutionary characteristics of domains and time of immune escape.But existing research results suggest that in acute phase of infection,virus env protein interact with immune selection may be different of virus subtype and individual infected,also prompt for the study of this pandemic strain evolution characteristics in the acute phase of infection,and clear the evolutionary trajectory of strain in our country.Through sexual transmission has become our country the main route of transmission of HIV-1 infection in recent years,which of infection CRF01_AE subtype is majority one of virus strains,the same as among men who sex with men(MSM),the new infection rate of which are rising rapidly up.Our research team previous found that the CRF01_AE subtype strains among MSM in china are divided into two different clusters,one ofwhich is the spread everywhere.Our team has selected one of the popular cluster 1 case of primary infection,which strains as the 12 th five year major projects mucosal vaccine strains.But the research is absence for envelope variation and host immune response during early HIV-1 CRF01_AE subtype infection,therefore tracking the evolution of envelope of mucosal vaccine strain and immune response in acute infection so as to deal with the virus mutation escape for vaccine and select effective sites of immune response for research of mucosal vaccine provided the scientific reference basis.Studied in this paper based on the selected for CRF01_AE mucosal vaccine strain,env gene of which were be tracking the evolution during 30 days to 2 years of infection,and combined with the development process of host plasma immune response,to analyze the relationship between env escape mutation and immune response,and make use of chimeric pseudotyped virus confirmed env gene change leading to escape neutralization as well as the first and the main mechanism in early infection,providing scientific reference basis for study of the mucosal vaccine strains.Methods: 1?Object Among MSM high-risk population,we found a primary infection of HIV-1 CRF01_AE subtype by regular voluntary test,strains of which selected as mucosal strains for vaccine research.Patient was male,39 years old.The first HIV-1 antibody screening result is negative,western blot confirmation trial(WB)result is uncer-tainty(gp160,p24,p17 strip positive),and the virus nucleic acid test is positive.According Fiebig stage,the disease process of patient was ? stage,about 30 days post-infection.HIV antibody was seroconversion after 54 days infection,and WB confirmation test result is positive(all bands).Continuous follow-up to infection after 2 years,we collected 7 plasma samples,which were test HIV-1 antibody,CD4 count,viral load and WB.VL average was 4.52 log10(copies/ml)during 2 years p.i.and about 133 days after infection VL reach a stable level named as viral load set point(VLS).The disease process is a typical development.The genotype of HLA is A * 0201;A * 2402;B * 4011;B * 5101;Cw * 0801;Cw * 0801;Bw4Bw6.The patient does not receive antiviral treatment during study.2? Experimental methods a)Construct env expression plasmid and analyze sequences of envelope i.Extract HIV-1 RNA from 7 plasma samples,and amplify env gene by Platinum?Taq High Fidelity Enzyme.And purify above PCR product by QIAquick PCR Purification/Gel Extraction Kit.ii.Purified PCR products were digested by Bam H ?and Not ?,and cloned into the pc DNA3.1 vector by T4 DNA ligase.iii.Pick a single medium-sized colony into a 5 ml LB liquid medium containing ampicillin 1%,37? 250r/min overnight,after shaking cu-lture take 500 ul to identify before extracting plasmids from liquid by QIAprep Spin Miniprep Kit.Eventually plasmids were sequencing for acquired env sequences by primers CMV-profor,BGH,UIF(5'-GTCTAAAGCCATGTGTAAAG-3'),UIR(5'-CCTATTAACCCTCCTACT-3').iv.Analyze sequences by software: Sequence 4.10,Bio Edit 7.1,MEGA 5 and internet tools(SNAP,N-Glycosite,et al)b)Construct env pseudotyped virus and neutralization test i.Clones with correctly oriented inserts were screened for infectivity by co-transfection with an env-deficient HIV-1(p NL43)backbone vector in 293 T cell by PEI.ii.Infectivity was determined by titration in ghost cells,and calculate 50% tissue culture infective dose(TCID50).env clones conferring the highest infectivity were selected for further characterization.The tropism of env clones were test by infecting ghost of expression co-receptor CCR5 or CXCR4.iii.Plasma samples from 54 days to 2 years infection and pseudotyped virus were incubated at 37? for 1 h and then added to prepared ghost cells.Cells were harvested at 48 h after infection and fluorescence intensity was test to calculate the 50% inhibitory dilution(ID50)of neutralizing anti-bodies.c)Construct chimeric pseudotyped virus and neutralization test i.Amplify V4 region of variation by Platinum? Taq High Fidelity Enzyme.And amplify backbone gene.ii.Purify above PCR products and clone into pc DNA3.1 vector by in-fusion ligase.Clones with correctly V4 region inserts were used to product chimeric pseudotyped virus.Then to test the sensitivity of plasma neutraliza-tion of chimeric pseudotyped virus.d)construct gp120 glycoprotein and the affinity assay.Amplify gp120 and clone into pc DNA3.1 vector,and were used to product gp120 glycoprotein.After verify by WB,the affinity assay was used to evaluate the affinity of antibody and virus.Results: 1? The evolution of envelope in early infection a)This study CRF01_AE subtype mucosal vaccine strain found that homol-ogy of envelope gene is high in 30 day infection,may be resulted from a single strain.Before 97 days after infection,envelope evolution was uni-directional,amino acid mutations random distribution in addition to the individual for fixed point mutation.From infected 97 days,the evolution of envelope gradually inclined to one direction,and to 2 years showed obviously one direction and selection pressure of host immune,under which the structure area appeared different degree of amino acid change fixed point mutation,deletion and glycosylation.b)V4 area,which variation,is the earliest and most significant structural of envelope.V4 region appeared an amino acid fixed point mutation at 54 days after infection,then continued in the deletion of amino acids,in addition to new point mutation and glycosylation during 223 days to 2 years.c)From 223 days infection,V1V2 region also showed by immune selection pressures,and continuously amino acids fixed point mutation and glyco-sylation.2? The process of emerge and development of plasma neutralization response a)This study found that there were detectable neutralization(ID50 = 469)of 54 days plasma to30 days virus,and up to peak in 223 days infection.But before 557 days infection,neutralization of plasma is not effective to virus of the same period.b)The plasma neutralizing activity to early virus were detected but weaker than to 30 days virus and ineffective to contemporaneous or later virus before 557 days infection and low effective to contemporaneous virus af-ter 557 days,and not detected cross neutralization to other subtypes virus during 2 years.c)V4 region was the first and most significant variation of structure,con-sistent with the development of host immune response,suggesting that V4 region variation could be the first and main escape mechanism to immune response in early infection.3? The correlation between variation of V4 region and virus escape neutralization a)The neutralization sensitivity of the V4 chimeric pseudotyped virus were significantly less than 30 days virus to plasma before 557 days infection.b)The sensitivity of plasma neutralization was consistent between V4 chi-meric virus and contemporaneous virus before 133 days infection,and during 133 days to 557 days,the sensitivity of V4 chimeric virus was higher than contemporaneous virus,implied that variation of V4 is the main mechanism of escape neutralization before 133 days,in addition to V4 region there may be other domains involved escape neutralization.c)Before 97 days of infection,the affinity of gp120 and plasma,and since 97 days of infection,plasma could not absorb the gp120 glycoprotein.Conclusion: 1? The envelope of CRF01_AE mucosal vaccine strain,V4 region was detectable of amino acids fixed mutations at 54 days p.i,and subsequently amino acid deletions,mutations and the change of N-linked glycosylation amount oc-curred persistently in V4 region,which was the most significant domain of virus envelope variation.V1V2 region was another significant domain of en-velope variation,which developed amino acid fixed mutations and changed N-linked glycosylation amount from 223 days p.i.2? The stronger effective autologous neutralization against 30 days virus were detected at 54 days p.i and developed to peak at 223 days p.i,subsequently to maintain strong activity.But autologous neutralization against contemporane-ous virus was not observed before 557 days p.i.During 557 days to 2 years,the plasma neutralizing antibodies activity against contemporaneous virus was observed much lower compared with virus 30 days,and the titers of neutral-izing antibodies were not detected against other subtypes viruses.3? The variation of V4 region of the mucosal vaccine strain can cause the virus to escape autologous plasma neutralizing antibodies response during early infection,the coevolution of virus and antibody did not promote the antibody to mature broadly neutralizing antibody,therefore,the V4 region might not a good target for HIV-1 vaccine.