Study On The Development Of Broadly Neutralizing Antibody Responses And Transmissions Of Recombinant Strains In HIV-1 Superinfected MSM In China

Objective:Preventative HIV vaccine is one of the main challenges in current public health.Although highly effective antiretroviral therapy can control the replication of HIV(Human immunodeficiency virus,HIV)and delay disease progression,it cannot be cured and requires lifelong treatment.bNAbs can neutralize the diverse global viruses,which is the focus of the design of effective and preventative HIV vaccines.Animal studies have shown that bNAbs are able to effectively prevent HIV infection,but in human clinical bNAbs trials,due to the short half-life of bNAbs,viral rebound and resistance to bNAbs after a short period of control of virus replication,which makes the treatment more difficult in the future.Although more than 30 candidate vaccines have been tested in clinical trials,none can induce protective neutralizing antibodies.Therefore,exploring how to select HIV env immunogen and how to induce bNAbs will become the major focus of vaccine design in the future.Up to now,hundreds of bNAbs have been isolated from HIV-infected individuals,which mainly bind to conserved regions on HIV envelope proteins:V1V2,V3,CD4bs(CD4 binding site,CD4bs),MPER(Membrane proximal external region,MPER),gp120-gp41 interface and silent face.It has been found that broadly cross-neutralizing antibodies in HIV-1 patients may be composed by broadly neutralizing monoclonal antibodies or polyclonal antibodies that recognize different epitopes on HIV env.The kinetic analysis of the plasma breadth has shown that only about 15-30%HIV-1-infected individuals can produce bNAbs,which could neutralize a variety of heterologous viruses after 2-4 years of infection,while only about 1-2%HIV-1 infected individuals can produce bNAbs,which could neutralize more than 90%of global viruses.These people are called elite neutralizers”who may provide a new strategy to isolate and identify HIV-1 monoclonal antibodies and provide the best immunogen for the HIV vaccine design and induction of bNAbs.Many studies have shown that host factors(such as HLA,ethnicity,etc.),immune environment(such as immune cells,immune factors,effector functions,etc.)and antigen-specificity characteristics(such as virus subtype,length of infection,viral load and env diversity,etc.)are associated with the development of bNAbs.These factors may jointly promote the maturation of bNAbs,or they may be independent factors.The HIV-1 env diversity may be associated with the development of the bNAbs.HIV escapes from the selective pressure of immune system while generating a new antigen determinant to induce a new lineage of neutralizing antibodies.The co-evolution process of bNAbs and viruses in HIV-1 single infected individuals,provided the precise mechanisms,namely,through the creation or exposure of bNAb epitopes,or through the generation of multiple immunotypes(or epitope variants).The multiple transmitted/founder viruses(T/F viruses)and superinfection also expose the immune system to diverse and persistent immunogenic stimulation.Recent studies have shown that superinfection may induce specific neutralizing antibodies through different env immunogens and increase the neutralization breadth,but it does not guarantee the development of bNAbs.The broadly neutralizing activity in superinfected individuals is mainly attributable to antibody lineages that target the epitopes of superinfecting viruses.However,some studies have found that superinfection is not associated with neutralization width.Therefore,before designing sequential immunization strategies,it is necessary to fully consider whether bNAbs can be induced and what the role of neutralizing antibodies induced by each antigen play in increasing the neutralization width,which will help to improve the effectiveness of vaccine immunotherapy.Different HIV antigenic determinants can induce different immune responses.Superinfection,which occurs during natural HIV infection,provides the best sequential immune model for the design of HIV vaccine in the future.On the other hand,superinfection makes the viral outcomes more diverse and complicated.Superinfection is the prerequisite and basis for the generation of recombination.The increase of CRF and URFs in the HIV-1 infected population also indirectly indicates the occurrence of HIV superinfection,but there is no direct evidence to support the relationship between multiple infections and URF.In recent years,the number of new HIV-1 infections in the MSM population has increased,and MSM has become an important way of HIV transmission in China.This population has high-risk behaviors such as multiple sexual partners,unprotected anal sex,and substance abuse,which increases the opportunity of superinfection.As previously reported by our team,the frequency of superinfection in the MSM population in Liaoning was 15.6%during the 2-year follow-up.In order to further explore the role of superinfection on the development of bNAbs,this study expanded screening of superinfection from an HIV-1 seronegative MSM cohort,evaluated the level of neutralizing antibody response in MSM population with the global panel,and analyzed the relationship between superinfection and bNAb.Then we focus on an elite neutralizer with superinfection to analyze the co-evolution of bNAbs and viruses before and after superinfection,which may provide a basis for the design of sequential immunization strategies for heterologous immunogens to induce the generation of bNAbs.In order to further explore the impact of superinfection on the MSM population,we tracked the sexual partners of superinfected individuals,collected detailed epidemiological and sexual behaviors information,and applied phylogenetic analysis and recombination analysis to reveal the relationship between superinfection and URFs.It is emphasized that superinfected individuals with active sexual behavior should be taken as the major monitoring and treatment population in the future,and provide a theoretical basis for controlling and reducing the development and transmission of recombinant strains among the population.Methods1.Study subjectsPart 1:Subjects were recruited from 2008 to 2013 from a large-scale prospective HIV-negative MSM high-risk population at the First Affiliated Hospital of China Medical University in Liaoning Province and established a follow-up cohort of 200HIV-1 patients with acute HIV infection by serological testing and nucleic acid screening.In this study,52 HIV-infected individuals were selected from this cohort,and the inclusion criteria were 1)the estimated time of HIV-1 infection was less than 3 months;2)Follow-up time was>2 years,and there were longitudinal sampling points during the follow-up period.Plasma samples,CD4~+T cell count,viral load and related epidemiological data were collected.The patient does not receive antiviral treatment during the study.Part 2:Three elite neutralizers(one single infected individual,one co-infected individual,and one superinfected individual)were selected according to the results in Part 1,and the results suggested that superinfection may be related to the development of bNAbs.So,we selected one superinfected individual 300446 to explore the new mechanism of development of bNAbs.Patient 300446 was a superinfected individual who was initially infected with CRF07?BC strain and superinfected with CRF01?AE strain about 1 year later.We collected 21 plasma samples and CD4~+T cell counts and viral load during 7.66-years ART native period.According to the time of superinfection and dynamic process of neutralizing breadth,a series of sampling points were selected for the neutralization experiment and SGA.The patient does not receive antiviral treatment during the study.Part 3:The CRF01?AE/CRF07?BC recombinant strain detected in superinfected individual 320819 and shared breakpoints in 1.06kb pol gene with strains of five patients from the MSM population in 2008-2017.Therefore,the five patients 320639 320392,328575,301538,and 328576 were also included in this study.Longitudinal plasma samples were collected from superinfected patient 320819 during a 4-year follow-up period,and one plasma sample was collected from the other five patients at baseline or seroconversion.CD4~+T cell count,viral load,and detailed social behaviors,and epidemiological information were collected at each sampling time point.The patient does not receive antiviral treatment during the study.All subjects provided informed consent for this study.This study was approved by the Medical Research Ethics Committee of the First Affiliated Hospital of China Medical University.2.Experimental methods2.1 RNA extractionHIV-1 RNA was extracted from plasma with QIAamp Viral RNA Mini Kit(Qiagen,German)according to the manufacturer's recommendation and obtained 60ul RNA.2.2 Reverse transcription and nested PCR amplification of HIV env C2-V4 region and pol-RT regioncDNA was synthesized with Transcriptor First Strand cDNA Synthesis Kit using reverse primer 07Rev12 and rev2-1.Env and pol amplicons were amplified by nested PCCR from cDNA with KOD-plus-Neo per 25ul reaction.2.3 PCR products purification and Library QuantificationThe PCR products were purified according to the instructions of Beckman purification kit developed by Beckman Coulter Company.Qubit fluorescence quantitative analyzer and Agilent 2100 Bioanalyzer Instrument were used to quantify the PCR products accurately.KAPA Library Quantification Kits were performed for accurate q PCR-based library quantification.2.4 NGSBased on the above quantitative results of PCR products,the Input DNA for each sample is 150ng and 60ul volume.The library preparation according to the Tru Seq Nano DNA Library Prep Protocol instructions.The main operation steps were as follows:repair ends and size selection,adenylate 3'ends with Poly"A",ligate indexed paired-end adapters,PCR amplification,validate library,normalize and pool libraries and sequencing on Illumina Miseq.2.5 Bioinformatics AnalysisFirst,use fast QC to view the quality of raw data,and then download the Virtual Box to build the working environment of the analysis software Qiime2,and then,import the separated samples to be analyzed in Qiime2.DADA2 plugin was used to denoise,duplicate,trim and assemble paired-end sequences to obtain the sequences and proportions of HIV quasispecies of each sample.2.6 Identification of multiple infectionWe discarded the sequences with the frequency<1%and length<350bp.The filtered sequences were compared with the sequences in our laboratory and the reference sequences downloaded from the Los Alamos HIV database.The maximum likelihood tree,genetic distance and Highlighter analysis were used to identify HIV-1 multiple infection.2.7 Construct env expression plasmid and analyze sequences of envelope Amplification of HIV env full lengthcDNA was synthesized with Superscript?Reverse T-ranscriptase Kit using reverse primer 1.R3.B3R.Env region were amplified from cDNA with Platinum(?)Taq High Fidelity per 25ul reaction.And purify above PCR product by QIAquick PCR Purification/Gel Extraction Kit.LigationThe purified PCR products were ligated expression vectors which were digested with Bam H?and Not?restriction enzymes using the In-Fusion(?)HD Cloning Kit.The ligation products were then transformed into E.coli DH5?competent cells and uniformly coated in LB agarose culture dishes containing 1%ampicillinPlasmid identification and isolationA medium-sized colony was selected from the plate and inoculated in 5ml LB liquid medium containing ampicillin 1%for enrichment culture.After shaking at 37?250r/min for about 16 hours,1ul bacterial liquid samples were taken for PCR identification,and the qualified samples were selected to extract plasmids according to the instructions of QIAprep Spin Miniprep Kit.Sequencing and analysisEventually plasmids were sequencing for acquired env sequences by four primers.Analyze sequences by software:Sequence 4.10,Bio Edit 7.1,Fast Tree v2.1.9,Highlighter,RIP(Recombinant Identification Program)and jp HMM(jumping profile Hidden Markov Model).Online software Sequence Harmony was used to quantify amino acid diversity and predict neutralization epitopes.Site-directed mutagenesisUsing representative clone strains 1.29Y-3 and 1.88Y-4 as templates,amplifying env region that contains D461G/Del by Overlap-PCR with Platinum(?)Taq High Fidelity Enzyme.Then by purifying and sequencing PCR product to confirm.2.8 Construct env pseudotyped virus and neutralization testThe env pseudoviruses were produced by co-transfection of env expression plasmid and backbone vector p NL43-Luc-E-R-luciferase in 293T cells.After 48 hours of transfection at 37?,the supernatants were harvested to obtain the pseudoviruses suspension express the HIV-1 env protein.For TCID50 measurements,ghost cells were added to serial dilutions of pseudovirus-containing culture supernatants,after 48-hour incubation,Bright-Glotm reagent and Infinite M200 instrument were used to detect fluorescence intensity and calculate TCID50.The serial dilution plasma samples from 0.25 years to 7.66 years infection and pseudotyped virus were incubated at 37?for 1 hour and then added to prepared ghost cells.The cultures were incubated for 48 hours at 37?,after which the luciferase activity was determined to calculate the 50%inhibitory dilution(ID50)of neutralizing antibodies.2.9 Statistical analysisThe ID50 value was converted into a score,and the differences in neutralizing antibody responses were compared by Mann-Whitney U test between good neutralizer and poor neutralizer,superinfected individuals and non-superinfected individuals.Logistic regression model was used to analyze the factors related to the development of bNAbs.All the statistical analyses and graphical presentations were carried out in SPSS20.0 software and Graph Pad Prism 8.0,A p-value lower than 0.05(<0.05)is statistically significant2.10 Amplification of HIV 5'/3'half-genomecDNA was synthesized with Superscript?Reverse Transcriptase Kit using reverse primer 07Rev8 and 1.R3.B3R.5'/3'half-genome sequences were amplified from cDNA with Platinum(?)Taq High Fidelity per 25ul reaction.According to the Poisson distribution,the possibility of PCR amplification using a single cDNA template can be higher than 80%when the PCR product is less than 30%.The virus cDNA was diluted in multiple proportions,and 96 replicas were set under the dilution gradient conforming to Poisson distribution.Nested PCR amplification was performed according to the above operating procedure.2.11 Phylogenetic and recombination analysis of HIV-1 near full length or half genomeEach 5'/3'half-genome was sequenced using 16 sequencing primers,and assembled by Sequencher 4.10 software.Near full length genome and half genome sequences were manually corrected using the Bio Edit software and blasted with reference sequences downloaded from the Los Alamos HIV database.The maximum likelihood trees were constructed by Fast Tree and recombinant strains were identified by HIV Database online software Highlighter,RIP and jp HMM,and finally confirmed by Sim Plot.2.12 Estimated of the most recent common ancestorThe most recent common ancestor was estimated by BEAST and using IQ tree to select the optimal nucleic acid replacement model,opening the fasta files by BEAUTi,selecting operation parameters.The Bayesian Markov Chain Monte(MCMC)analysis was computed at least 2×10~8 states and saved it as.xml file.BEAST runs the.xml files and saved it as.log and.trees files.Opening the.log files with Tracer,the effective sample size of all parameters>200 is considered significant.The maximum clade credibility trees were viewed and edited using Fig Tree.Results:1.Impact of HIV-1 superinfection on the development of bNAbs in Chinese MSM populationa)The median infection time of 52 HIV-1 infected patients was 3.82 years(2.24-8.01years),CRF01?AE and CRF07?BC subtype were the major epidemic strains(80.8%,42/52).Among them,11 cases were HIV-1 dual infection with an incidence of 21.2%.In terms of subtypes,6 patients were identified as intrasubtype multiple infection and 5patients were identified as intersubtype multiple infection,the proportion of them was similar;In terms of infection type,2 patients were identified as coinfection and 9 patients were identified as superinfection,which accounted for a higher proportion.Among superinfected patients,3 cases acquired the second strain within one year and 6 cases acquired the second strain between one to two years.b)Among the 52 HIV-1 infected patients,good neutralizers(20 cases)had a highly broad and potent neutralizing antibody response than poor neutralizers(32 cases)(P<0.01),and 9 cases of good neutralizers were HIV-1 multiple infection(2 cases were coinfection and 7 cases were superinfection).Logistic analysis further suggested that multiple infection and superinfection occurring within 1 to 2 years were associated with the development of bNAbs.c)According to the virus subtype of initial infection,infection time,and viral setpoint,each superinfected individual had matched three single infected individuals,evaluate whether there was a difference in plasma neutralization activity between superinfection and single infection at an average of 4.69 years post initial infection.It was found that the neutralization breadth and potency of superinfection were significantly higher than those of single infection(p<0 01).It is suggested that the cross-neutralizing antibodies may be more likely to occur in superinfected individuals.2.Study on the mechanism of development of bNAbs in an HIV-1 intersubtype superinfected individual in Liaoning,Chinaa)Elite neutralizer 300446 initially infected a CRF01?AE strain at 0.25 years and superinfected a CRF07?BC strain between 1.07 to 1.29 years.He first developed potent broadly cross-neutralizing activity at 2.54 years and peaking at 6.62 years,neutralizing90%global panel viruses.b)CRF07?BC lineage did not show an obvious escape from neutralization,while CRF01?AE lineage showed a typical and persistent escape from neutralization,and the neutralization titer of autologous neutralizing antibody against the superinfecting virus was significantly higher than that of primary infecting virus,suggesting that the development of neutralizing breadth may be related to antibodies that were induced by the superinfecting virus.c)The primary infecting virus and superinfecting virus evolved rapidly by mutation,insertion,deletion,and recombination during the period of 7.66 years,and the neutralization escape pathways were different between the two lineages,but the escape mutations in early HIV Infection were mainly concentrated in the V5 region.d)The amino acid 461 in HIV-1envelope V5 highly variable region is the key epitope that mediated neutralization escape of CRF01?AE lineage virus,suggesting that there may develop neutralizing antibodies targeting this epitope in the superinfected individual.3.Study on the generation and transmission of recombinant strains of an HIV-1intersubtype superinfection in Liaoning,Chinaa)Before diagnosis of HIV infection,one superinfected individual and five CRF01?AE/CRF07?BC infected patients all had high-risk behaviors,such as seeking male sexual partners through the Internet and social circle,unprotected anal sex,positive syphilis and substance abuse,which could increase the risk of generation or transmission of HIV strains in the population.b)Six near-full length genomes were composed of CRF01?AE and CRF07?BC strains,and had common recombinant breakpoints and homologous parental strains.3'half-genome sequences were obtained from the longitudinal samples from superinfected donor 320819 and baseline/seroconversion samples from five recipients.Recombination analysis showed that quasispecies were complex and diverse in superinfected donor and these recombinants shared some breakpoints in tat,env,nef.Recombinant forms of strains obtained from early sampling points of the donor were identical or similar like those in five recipients.c)The topological structure of the donors and five recipients in MCC trees presented paraphyletic-monophyletic or paraphyletic-polyphyletic relationships,which suggested that there was a direct or indirect transmission relationship between the donor and five recipients.The BEAST analysis confirmed that the estimated time to the most recent common ancestor of CRF01?AE and CRF07?BC strains of the donor were earlier than the emergence of recombinants from five recipients.Conclusion:1.The development of bNAbs may be related to superinfection in HIV-1 infected individuals among Chinese MSM population.Compared with the single infected individuals,the superinfected individuals may have more complex and diverse quasispecies and may be more likely to develop broadly neutralizing activity.2.The broadly neutralizing activity in HIV-1 superinfected patients is mainly induced by the superinfecting virus.The amino acid 461 in the HIV-1 env V5 region is the key epitope that mediated neutralization escape,suggesting that there may develop neutralizing antibodies targeting this epitope in the superinfected individual.3.HIV-1 superinfection can generate a series of recombinant viruses with similar breakpoints,which is the prerequisite and basis for the generation and transmission of recombinant strains.The URFs with similar structures were detected in different acutely-infected individuals,suggesting that these URFs may derive from superinfection.This finding emphasizes that surveillance,management,and early initiation of antiretroviral therapy should be strengthened for superinfected patients,as well as partner testing and tracking to prevent the generation and transmission of HIV recombinant strains among people.