Epidemiological Characteristics Of HIV-1 And Virus Neutralizing Sensitivity To M36.4 Antibody In The China-Myanmar Border Area

BackgroundAcquired immunodeficiency syndrome(AIDS)is a major global infectious disease caused by the human immunodeficiency virus(HIV)that seriously threatens human health.Affected by the proliferation of drugs in the "Golden Triangle",Myanmar is one of the countries with the most severe HIV-1 epidemic and the most complicated genotype in the world.Yunnan,China,is located in the China-Myanmar border area.It is the earliest epidemic area of HIV-1 in China.It is particulary severe and important for AIDS prevention and control in this area.The number of newly reported foreign HIV-infected persons in this area each year has exceeded the number of HIV infected Chinese.In 2017,the proportion of Myanmar nationals among the newly reported infections in the border city of Dehong Prefecture reached 71%.HIV-1 infected Burmese have played an important role in the transmission of HIV-1 in the China-Myanmar border area.The changes in the cross-border transmission of HIV-1 in the entire China-Myanmar border area are worthy of attention.However,it is not clear about the cross-border transmission of HIV-1 in this area and the degree of mutual influence.At the same time,the pre-treatment drug resistance of some cities in the region already exceeds the moderate epidemic level,and a comprehensive and systematic description of drug resistance during the epidemic period is still needed.Broad-spectrum neutralizing antibodies(bNAbs)have brought new options for the treatment of HIV-1,especially for the treatment of drug-resistant viruses.Due to its good neutralization effect,excellent safety,and participation in the host's immune response,bNAbs are constantly innovating and developing.The authors of the subject combination synthesized the M36.4 antibody against the co-receptor binding region,which showed broad-spectrum neutralizing activity against a variety of HIV pseudoviruses.In our previous trials,the multivalent bispecific antibody composed of M36.4 has a good neutralizing effect on a variety of clinical isolates in China.However,the form of recombinant strains of the imported infections in Myanmar is complex and closely related to the Yunnan strains of China,and the neutralizing effect of antibodies against these strains is unknown.At the same time,M36.4 has not yet clarified the binding site and the mechanism of virus resistance antibody,and further exploration is needed.ObjectiveAnalyze the sequence and epidemiological characteristics of HIV-1 infector at the China-Myanmar border area,reveal the cross-border transmission situation and the transmission pattern of the mainly epidemic strains in this area.Furthermore,by constructing pseudovirus from Myanmar samplies,detecting the neutralization sensitivity and revealing the escape site to M36.4 antibody.Provide scientific reference for precise intervention in the cross-border transmission of HIV-1.Methods1.The construction of the HIV-1 molecular network and transmission mode analysis in the China and Myanmar border area:Download the published sequences of the pol districts of people infected in Myanmar and Yunnan saved in the search laboratory in the HIV database.Further,from March 2019 to April 2020,continuous sampling was carried out in Dehong prefecture on the China-Myanmar border,and the newly reported plasma samples of HIV-1 infected persons from Myanmar were collected for sequencing and molecular traceability.By constructing a molecular communication network from 1994 to 2020,the potential communication relationship between sequences is inferred,and factors related to cross-border communication are explored.Furthermore,based on the Bayesian random search variable selection method,the temporal and spatial dynamics of the strains are analyzed,and the asymmetric migration model(Markov jump)is used to estimate the number of migration events expected between all regions to quantify the spread of the main epidemic strains in China.Calculating the Bayesian factor(BF)of all inter-regional transmission events gives statistical support for this transmission relationship.2.Analysis of the epidemiological characteristics of PWLH youths(16-25y)in the China-Myanmar border area from 2009 to 2017:Screen the sequence of untreated HIV-1 infected persons in the youths(16-25 years old)in Dehong Prefecture,a border city between China and Myanmar,to represent the local pre-treatment prevalence of transmission drug resistance(TDR),and analyze the correlation of drug resistance with clinical and virological characteristics factor.By constructing molecular network,to analyze the factors related to cross-border transmission of youths,and calculate the degree of clustering of regions and transmission routes in the networks.3.Analysis of the neutralization sensitivity of M36.4 antibody to Myanmar strainsSelect representative strains from the samples of newly entered Myanmar infected persons from 2019 to 2020,and construct pseudo virus strains containing the env area.The neutralization sensitivity of M36.4 antibody to pseudovirus was tested on the TZM-bl cell line.As a control,a mD1.22 antibody against the CD4 binding region(CD4bs)of Env gp120,and three entry inhibitors:T20,C34,and polyethylene glycol(PEG)-C34.The HIV-1 SF33 strain was stably passaged on the MT2 cell line,and the SF33 strain was given in vitro antibodies(M36.4 and mD1.22)selective pressure for 12 weeks or more.The full-length sequence of the env of the virus was sequenced every 10 days until the antibody escape site was screened.A pseudovirus with escape site mutations is constructed by point mutation.The changes in antibody neutralization sensitivity before and after mutation were detected on the TZM-bl cell line.Results1.HIV-1 Molecular Transmission Network in Yunnan and Myanmar,from 1994 to 2020A total of 5380 sequences were included in the study of molecular communication networks,with 3892 Chinese nationals and 1488 Myanmar nationals.HIV-1 genotypes include CRF01_AE 1324(24.6%),CRF07_BC 533(9.9%),CRF08_BC 1268(23.6%),URF 1191(22.1%),B genotype 249(4.6%),C genotype 592(11.0%),others 222(4.1%).The proportions of genotype B,CRF01_AE and CRF08 BC of Myanmar nationality have decreased year by year,while the proportions of URF and C have increased significantly,while the proportions of CRF01_AE,genotype B,URF and CRF07_BC of Chinese nationality have decreased year by year,and the proportions of genotype C.RF08_BC and C have increased significantly.The overall drug resistance rate was 6.5%,and NNRTI-resistant drugs were the main ones;there were significant differences in the distribution of drug resistance sites in Myanmar and Yunnan.A total of 488 molecular networks were constructed(the optimal gene threshold is 1%),including 1915(36%)subjects,forming 5257 edges.After 2014,the network access rate has decreased significantly,and men,MSM,and C genotypes are more likely to access the networks.13.7%of the networks contain both Chinese and Myanmar nationals who are infected.Among them,Myanmar nationality,MSM,C genotype and BC reorganization are at high risk of entering the China-Myanmar mixed network.Among all infected persons,4.04%of Chinese nationals are linked to Myanmar nationality,and 7.99%of Myanmar nationals are linked to Chinese nationality.The risk factors related to cross-border nodes are Myanmar nationality,MSM and BC reorganization.The cumulative cross-border node ratio(23.4%?5.13%)and cross-border border ratio(20.18%?9.07%)show a downward trend year by year.From 2019 to 2020,18%of the newly reported Myanmar HIV-1 infected persons entered the molecular network.The molecular traceability results showed that 85%of the virus strains may originate from China,and the B genotypes,CRF07_BC,and CRF08_BC are more likely to originate from China.2.HIV-1 transmission patterns in the China-Myanmar border areaThe tMRCA of HIV-1 in this region is estimated by Bayesian phylogeographic analysis:(1)Genotype B entered Dehong from Thailand(1985.4,95%CI 1982.0-1988.8).(2)Genotype C entered Myanmar from India(1980.76,95%CI 1976.12-1985.4).(3)CRF01_AE 2 clusters entered the Honghe area from Vietnam(1997.7,95%CI1997.1-1998.3);CRF01_AE 4 clusters came from Kunming(1999.9,95%Cl 1996.7-2003.1);the unclassified CRF01_AE entered from Thailand Myanmar(1992.5,95%CI 1992.0-1993.0).(4)Unclustered CRF07_BC originated from Kunming area(1994.3,95%CI 1993.4-1995.3),and appeared in Myanmar in 1996(95%CI 1995.3-1996.7);CRF07 BC MSM cluster entered from Kunming area(2003.4,95%CI 2001.2-2005.7),appeared in Myanmar in 2003.8(95%CI 2001.1-2006.5).(5)CRF08_BC originated from Yunnan(1992.8,95%CI 1991.1-1994.5),and appeared in Myanmar in 1996.2(95%CI 1994.3-1998.1).Unclustered CRF01_AE,B and C genotypes have stable transmission clusters in Myanmar,while the remaining genotypes have not yet formed stable transmission clusters in Myanmar.Calculate the strain flow by BSSVS method:CRF01_AE other cluster and the C genotype into Yunnan in the entire epidemic history accounted for 97.7%and 63.6%of the total transmission events of this genotype,respectively;CRF01_AE 2 clusters,CRF01_AE 4 clusters,CRF07_BC other,CRF07_BC MSM cluster,CRF08_BC mainly spread within Yunnan.In the entire epidemic history,transmission events from Yunnan into Myanmar accounted for 28.6%.5.0%9.5%,7.3%,16.4%of the total transmission events of this genotype,respectively.In the B genotype,the transmission incidents imported from Dehong into Myanmar accounted for 58.5%of the total transmission incidents of this genotype,and the transmission incidents imported into Dehong from Myanmar accounted for 24.7%of the total transmission incidents of this genotype.3.2009-2017 Epidemiological characteristics of PWLH in Dehong,YunnanThe most common route of infection for the 573 youths(16-25y)in Dehong Prefecture,from 2009 to 2017,was heterosexual transmission(70.51%),followed by PWID(19.20%)and MSM(8.90%).HIV-1 genotype is mainly unique recombinant type(URF)(33.7%),CRF 35.0%,C genotype 27.4%,and B genotype 3.8%.The average transmissible resistance(TDR)rate from 2009 to 2017 was 6.3%,and the transmissible resistance rate increased significantly(3.48%-9.48%;p<0.05).The resistance rates of NNRTIs,NRTIs and PIs were 3.49%,2.62%and 0.52%,respectively.Among those infected with TDRM,most(94.40%,n=34)only developed resistance to a single drug category,and the most common resistance mutations were Y181I/C and K103N.The average CD4 counts of youths with TDRMs were low(373/mm3 and 496/mm3,p=0.013).83 molecular networks have been constructed through HIV-TRACE(44%network access rate),49%are China-Myanmar mixed networks(41/83),MSM,Burmese nationality and drug-resistant strains have a high risk of cross-border transmission.Chinese male PWID network homogeneity(clustering degree)is 0.30,Myanmar male PWID is 0.20,MSM is 0.14.4.Neutralizing Sensitivity of M36.4 to virus from MyanmarThe full-length sequence of the env of the sample from 2019 to 2020 was sequenced.Four pseudoviruses were constructed to represent the current status of virus strains in this population,namely CRF07_BC.CRF08_BC,URF₀1C.URF_BC.The average IC50 of M36.4 against the 4 pseudoviruses is 0.11?M;the average IC50 of the control antibody mD1.22 is 0.37?M;the average IC50 of the control drug entry inhibitor PEG40K-C34 is 0.99?M,the average IC50 of C34 is 0.005?M,and the average IC50 of T20 is 0.28?M.5.Screening of M36.4 escape siteSF33 was cultured with M3 6.4 for 140 consecutive days,21 generations of MT2 cells,the concentration of M36.4 antibody in the DMEM reached 59 folds(177nM)of its IC50 against the SF33 strain,and virus-mediated cell fusion can be still observed.Five mutations in the env region were obtained by multi-time sequencing.A pseudovirus containing mutation sites was constructed through point mutation of the SF33env plasmid,and an effective antibody escape site was finally screened.The amino acid at position 327 of SF33env was mutated from R to K,located at the 31st position in its V3-loop.The neutralization sensitivity of the M36.4 and mD1.22 antibodies to the SF33env pseudovirus was reduced by 11.5 folds and 60 folds,and there was no significant effect on the three entry inhibitors.Conclusions1.1994 to 2020,the HIV-1 drug resistance rate in the China-Myanmar border area has reached a moderate level of drug resistance,and there is a drug resistance transmission network;the subtypes are complex and diverse,and the proportion of unknown recombination is high.The proportion of cross-border communication nodes decreased year by year from 1994 to 2010,and stabilized at 5%after 2010.The cross-border HIV-1 transmssion bridge population in the China-Myanmar border area is PWID,MSM,Burmese nationality,mainly carrying C and BC recombination type,and the bridge area is Dehong.2.In the China-Myanmar border,China's main CRF strains have unbalanced two-way transmission between China and Myanmar.CRF01_AE 2 clusters,CRF01_AE 4 clusters,CRF07_BC other clusters,CRF07_BC MSM clusters,CRF08_BC mainly spread in Yunnan;CRF01_AE other clusters and C subtypes are mainly imported from Myanmar;B subtypes have a higher proportion of two-way transmission.The quantified flow of the virus spreading between the areas on the China-Myanmar border can provide a reference for the individualized interventions of the corresponding subtypes.3.From 2009 to 2017,the rate of transmissible drug resistance among young HIV-1 infected persons has reached the epidemic level of moderate drug resistance,and the CD4 counts of infected persons carrying drug-resistant sites are low,suggesting the development of antiretroviral in HIV recombination hotspots.Drug resistance needs to be considered before treatment.There are multiple ways of mixed transmission between MSM and Myanmar male PWID population,and the proportion of heterosexual transmission between Myanmar females and Chinese males is relatively high.4.M36.4 antibody has a good inhibitory effect on 4 kinds of Myanmar immigration pseudoviruses(01C recombinant type,BC recombinant type,CRF07_BC,CRF08_BC).The V3 loop R3 1K mutation can cause a significant increase in the resistance of the virus to M36.4 and mD1.22 antibodies.