| Objective:In recent years,new recombinant strains of HIV-1 have been increasing both domestically and internationally,new recombinant strains can affect the replication adaptability,immune escape and virulence of the virus,which increases the difficulty of AIDS prevention and control and the difficulty of vaccine development.Eliminating virus-infected cells by inducing/enhancing HIV-specific T cell responses is an important means of vaccine development and immunotherapy.There are differences in genetic background and epidemic subtype virus characteristics among different populations.It is of great significance to clarify the differences in T cell epitopes among different subtypes of HIV-infected patients for the development of T cell-based immune response vaccines and immunotherapy.Due to the high variability,high replication rate and high recombination rate of HIV-1 gene,when different subtypes are co-circulated in the same area,it is very easy to recombine between subtypes to form new recombinant strains.There are 132 circulating recombinant forms(CRFs)and a large number of unique recombinant forms(URFs)reported worldwide.The HIV-1 genotypes prevalent in China are very complex,the most common are CRF01＿AE,CRF07＿BC,CRF08＿BC and B subtype strains,the fourth national HIV epidemiological survey showed that CRF55＿01B has been found in 25provinces in China,and the prevalence rate of CRF55＿01B ranged from 1.5%to 12.5%.CRF55＿01B has the highest proportion and wide distribution in new CRFs,and the proportion of MSM population in some southern provinces and cities was more than 10%.The CRF55＿01B recombinant strain was identified by our team from men who have sex with men(MSM)in Shenzhen,Guangdong Province in 2013.CRF01＿AE was used as the backbone to insert B subtype in the pol region,which was traced back to about 2000year.From 2005 to 2009,it showed an exponential growth.After 2010,it rapidly increased and has now become the fifth epidemic strain in China.It has been found that CRF55＿01B has become the third dominant strain after CRF01＿AE and CRF07＿BC in Shenzhen,and has been recombined with CRF01＿AE,CRF07＿BC and B subtype strains.It was reported that the viral load of newly diagnosed CRF55＿01B infected patients was significantly higher than that of CRF07＿BC at the baseline of diagnosis and treatment,and the CD4~+T cells were significantly lower than that of CRF07＿BC,while the viral load was significantly higher than that of CRF01＿AE at the baseline of treatment.The number of CD4~+T cells at baseline was 200-350/μl,and the decrease rate of CD4~+T cells was slower than that of CRF01＿AE.It can be seen that CRF55＿01B infection is’double high’,but the mechanism is not clear.The high level of viral replication in CRF55＿01B infected patients and the slow decline in the number of CD4~+T cells lead to a longer asymptomatic period,which may be related to the rapid spread of CRF55＿01B virus in China in recent years.Human leukocyte antigen I(HLA-I)-restricted T cell response plays an important role in controlling the disease progression of HIV-1 infected patients.It has been clearly reported that the disease progression of HLA-B infected patients is slow,such as HLA-B*27,B*57 and B*5801 in Caucasian population,B*13 in African population and HLA-B*51 in Asian population.Due to the high variability of HIV gene,the virus can constantly mutate under the pressure of immune selection in patients to evade the immune recognition of the host.Some mutations can reduce the replication ability of the virus,and the virus can partially restore the reduced replication ability through some compensation mutations.The virus gradually undergoes adaptive evolution,the immune escape ability is enhanced,and the viral load may be higher.The’double high’clinical phenotype of CRF55＿01B strain may be related to the increased adaptability,replication ability and immune escape ability of the recombinant virus.Exploring the variation of T cell epitopes in CRF55＿01B and other subtypes in China,especially the internationally reported HLA-restricted T cell epitopes such as HLA-B*27,B*57,B*5801,B*13 and B*51,not only helps to analyze the mechanism of the’double high’phenotype of CRF55＿01B strain,but also has important implications for understanding the virulence and adaptive evolution of new HIV recombinant strains and other viruses.In this study,we compared the variation of T cell epitopes of Gag,Pol and Nef protein between CRF55＿01B and CRF01＿AE,B,CRF07＿BC subtype viruses by cross-sectional analysis,especially the variation of HLA-restricted T cell epitopes known to have a clear protective effect,analyzed the effect of amino acid variation on viral replication ability,clarified the relationship between the second generation recombination breakpoint of CRF55＿01B subtype virus and amino acid polymorphism and T cell response,analyzed the biological characteristics of CRF55＿01B subtype virus,and laid a foundation for disease progression evaluation and immunotherapy.Study methods:1.Subjects:In this study,199 newly diagnosed HIV-infected patients with CRF01＿AE,CRF07＿BC,CRF55＿01B and B subtype viruses by genotype drug resistance test were selected from Shenzhen city from 2015 to 2020.HIV infection was confirmed by Western Blot.At the time of inclusion,no antiretroviral drugs were taken,and recent infection(within 6 months)was determined by affinity test.Plasma samples of infected patients were collected and frozen at-80°C.All participants recruited in this project received written informed consent.2.Plasma HIV-RNA extraction and gag,pol,nef nested PCR amplification:HIV-RNA was extracted from plasma according to the instructions of QIAamp Viral RNA Mini Kit.The gag,pol and nef gene fragments were amplified by nested PCR.3.PCR product identification and sequencing:TAE buffer was used to configure 1.0%agarose gel.According to the gene fragment,2000 bp and 10000 bp DNA ladders were selected as quantitative markers.Each well was added with 5μl of the second round PCR product,and electrophoresis was performed at 120 V for 30 min.Observe whether there is a target band in the Ultra-Violet Product gel analysis system in UV mode.The second round of positive amplification products were directly sequenced by BGI(China).4.Constructing phylogenetic tree to determine subtypes:The virus sequence was assembled using Contig Express software,and then manually edited and corrected.HIV-1reference sequence was downloaded from Los Alamos HIV sequence database(http://www.hiv.lanl.gov)).HIV-1 Align tool(HIValign(lanl.gov))was used to compare HIV-1 gene sequence and reference sequence.The phylogenetic trees of gag,pol and nef sequences were constructed by IQ-TREE software to determine the subtype of HIV virus.5.Determination of epitope polymorphism and escape mutation:All HIV-1 sequences were translated from nucleotide sequences into protein sequences and aligned using the HIV-1Align tool(HIValign(lanl.gov)).Download the best epitopes of HLA-B*57,B*5801,B*27,B*51 and B*13 restricted HIV Gag,Pol and Nef proteins from HIV Database(https://www.hiv.lanl.gov/content/immunology/tables/optimate-ctl-summary.html)for analysis.Variation is defined as at least one amino acid is inconsistent with the epitope in the database.Variation rates were used to evaluate the polymorphism of epitopes.The escape mutation is defined as the presence of a previously reported variation in the epitope that can reduce the T cell response.6.CRF55＿01B subtype virus second-generation recombinant subtype determination and polymorphism analysis:The recombination subtype and recombination breakpoint of CRF55＿01B were determined by Simplot software,and the amino acid polymorphism of CRF55＿01B recombination breakpoint region and non recombination region was compared by Entropy entropy value(HIV Sequence Database:Entropy-Two Submission Form(lanl.gov)).The number of T cell response peptides that have been studied was downloaded from HIV Database(Interactive Epitope Maps(lanl.gov))to compare the effect of the second-generation recombinant strain breakpoint region of CRF55＿01B on T cell immune response.7.Statistical analysis:Nonparametric Mann-Whitney U test was used to compare the mutation rates of different subtypes,and P<0.05 was considered statistically significant.Results:1.Virus phylogenetic analysis and subtype judgmentIn this study,a cross-sectional analysis of 199 newly diagnosed HIV-infected patients with CRF01＿AE,CRF07＿BC,CRF55＿01B and B subtype viruses identified by genotype drug resistance test in Shenzhen from 2015 to 2020 was conducted.176 gag sequences,116 pol sequences and 187 nef sequences were obtained by PCR amplification.By constructing a phylogenetic tree,it was determined that the gene regions were mainly CRF55＿01B,CRF01＿AE,B,CRF07＿BC subtype viruses,and CRF01＿AE lineage 4 was the main epidemic cluster of CRF01＿AE subtype viruses.Recombination was found in 6.82%(12/176)of the gag region,23.28%(27/116)of the pol region,and 8.02%(15/187)of the nef region.2.CRF55＿01B and CRF01＿AE,B,CRF07＿BC subtype viruses have different variation rates in some known protective epitopesBy comparing the variation rates of 32 HLA-B*27,B*57,B*5801,B*13 and B*51restricted epitopes in each gene segment of different subtypes,it was found that compared with CRF01＿AE lineage 4,B and CRF07＿BC subtype viruses,CRF55＿01B subtype viruses had 21.88%(7/32),40.62%(13/32)and 46.88%(15/32)epitopes respectively,which were significantly different from the above subtypes.The total variation rate of CRF55＿01B subtype virus was significantly higher than that of B subtype virus.There was no difference with CRF07＿BC and CRF01＿AE lineage 4 subtype viruses.Among the epitopes with different mutation rates,71.43%(5/7)of the epitopes of CRF55＿01B subtype virus were significantly lower than those of CRF01＿AE lineage 4 subtype virus,100%(13/13)were significantly higher than those of B subtype virus,and 73.33%(11/15)were significantly higher than those of CRF07＿BC subtype virus.Overall,the variation level of CRF55＿01B subtype virus was higher than that of B and CRF07＿BC subtype viruses,and lower than that of CRF01＿AE lineage 4 subtype virus.3.CRF55＿01B was different from CRF01＿AE,B,CRF07＿BC subtype virus in the escape mutation rate of some known protective epitopesThe ability of amino acid mutations in the epitope to escape T cell response is different.Some mutations have no effect on T cell response,and the ability of some mutations to induce T cell response is significantly reduced.For this reason,we further analyzed the mutation rates of different subtypes that have been verified as epitope escape mutations in the above 32 best epitopes.It was found that compared with CRF01＿AE lineage 4,B and CRF07＿BC subtype viruses,CRF55＿01B subtype virus has 18.75%(6/32),37.50%(12/32)and 46.88%(15/32)of the epitopes in the above subtypes were significantly different,but there was no significant difference in the overall escape mutation rate.Among the epitopes with different escape mutation rates,66.67%(4/6)epitopes of CRF55＿01B subtype virus were significantly lower than those of CRF01＿AE lineage 4 subtype virus,75%(9/12)epitopes were significantly higher than those of B subtype virus,66.67%(10/15)epitopes were significantly higher than those of CRF07＿BC subtype virus.In summary,the immune escape ability of CRF55＿01B subtype virus may be stronger than that of CRF07＿BC and B subtype viruses,and lower than that of CRF01＿AE lineage 4subtype virus.4.Compared with CRF01＿AE lineage 4,B,CRF07＿BC subtype viruses,the types and frequencies of amino acid mutations in CRF55＿01B subtype virus were differentWe further analyzed the differences in the frequency and types of epitope amino acid mutations with different epitope mutation rates and escape mutation rates.Compared with CRF01＿AE lineage 4,B and CRF07＿BC subtype viruses,CRF55＿01B subtype virus has8,15 and 17 different epitopes.The variation characteristics are subtype-specific,that is,the mutation sites,amino acid mutation types and the proportion of intra-epitope mutations are different in different subtypes.5.The adaptive evolution of CRF55＿01B subtype virus occurringCompared with CRF01＿AE lineage 4,B,CRF07＿BC subtype virus,CRF55＿01B subtype virus carried significantly more mutations in the gag gene than B and CRF07＿BC subtype virus.The mutations that reduce replication capacity and compensation mutations coexist,the virus has adaptive evolution.The variation of CRF55＿01B subtype virus in pol and nef genes affecting viral replication was not different from other subtypes.6.The recombinant breakpoint of CRF55＿01B subtype virus recombinant strain related to amino acid polymorphism.A total of 11 recombinant strains of CRF55＿01B subtype virus were found in this study,including one case of CRF55＿01B and CRF07＿BC subtype virus recombination,and 5cases of CRF55＿01B and CRF01＿AE subtype virus recombination in the gag region;4cases of CRF55＿01B and CRF01＿AE subtype virus recombination,7 cases of CRF55＿01B and CRF07＿BC subtype virus recombination were foundin in pol region;six cases of recombination between CRF55＿01B and CRF07＿BC subtype virus were found in the nef region.It was found that the amino acid polymorphism in the recombination breakpoint region of gag gene was significantly higher than that in the non-recombination region,recombination was prone to occur at high amino acid variability.The amino acid polymorphism in the recombination breakpoint region of nef gene was significantly lower than that in the non-recombination region,while the recombination breakpoint in pol region was not related to amino acid polymorphism.It was found that the 116-156 of pol region and the 65-79 and 121-150 of nef region were the hot spots of T cell response,suggesting that the recombinant breakpoint may be related to immune escape.Conclusions:1.The ability of CRF55＿01B subtype virus to escape T cell response may be higher than B and CRF07＿BC subtype virus,lower than CRF01＿AE lineage 4 subtype virus;the type and frequency of amino acid mutations have obvious subtype differences.2.The mutation of the ability of CRF55＿01B subtype virus to reduce viral replication and the corresponding compensation mutation coexist in the population,it is suggested that adaptive evolution of viruses may be related to high viral load.3.Recombination occurs between CRF55＿01B and CRF01＿AE,CRF07＿BC subtype virus,some recombinant breakpoint regions are hot spots for T cell response,and viruses may escape T cell response through recombination. |
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