Study Of Virological And Immunological Characteristics In HIV-1-Infected Long-term Nonprogressors And Isolation Of Neutralizing Antibodies To HIV-1

Objective:Understanding the mechanisms contributing to natural viral control in LTNPs might be informative for inducing antiretroviral therapy(ART)-free HIV-1 remission or making effective HIV-1 prevention and treatment.To date,no clear pattern has emerged explaining this favored clinical status.We aimed to clarify factors associated with viremia control by investigating the virological and immunological characteristics in LTNPs and identify neutralizing antibodies from LTNPs,in order to provide new insight into the strategies for HIV-1 prophylaxis and therapy.Methods:LTNPs were enrolled and classified as VC-LTNPs(viremic control)and non-VC-LTNPs based on viral load dynamics.The longitudinal evolution of cellular HIV-1 DNA and immune cell profiles in VC-LTNPs and non-VC-LTNPs was monitored.Plasma HIV-1-specific antibody titers and avidity,antibodies engaging in broadly neutralization and the inhibition of HIV-1 cell-to-cell transmission were evaluated in LTNPs and typical progressors(TPs).The plasma levels of twelve biomarkers were compared among the study groups.In LTNPs,dynamic changes of these immunological parameters were investigated and the interplay between viral load and immunological parameters was analyzed.For isolation of neutralizing antibodies,antigen-specific memory B cells were sorted from PBMC of a HIV-1-infected LTNPs by flow cytometry,and were seeded into 96-well plates at 1 cell per well.Ig genes were amplified by RT-PCR,cloned into expression vectors,re-expressed in 293T cells or CHO-S cells,and purified with a rProtein-A column.The biological activity of neutralizing antibodies was identified by ELISA and neutralization assay.Results:(1)Compared with non-VC-LTNPs,VC-LTNPs had limited and stable HIV-1 reservoirs(p=0.018).(2)Although the immune cell profiles of VC-LTNPs and non-VC-LTNPs were similar at baseline,VC-LTNPs had lower levels of CD8+T cell count and percentage of activated CD8+T cells,and contained higher CD4/CD8 ratio and percentage of CD8+CD28+T cells than non-VC-LTNPs during follow-up.The CD4/CD8 ratio was negatively associated with HIV-1 RNA viral load,and the proportion of activated CD8+T cells were positively associated with HIV-1 RNA viral load.(3)The titers of HIV-1-specific and neutralizing antibodies were higher in TPs than in LTNPs(p<0.05)and were positively correlated with HIV-1 RNA viral load.(4)A proliferation-inducing ligand(APRIL),B cell-activating factor(BAFF)and interferon gamma-induced protein-10(IP-10)levels differed significantly among the groups at baseline(p<0.05).APRIL levels were significantly elevated in VC-LTNPs,and were inversely associated with HIV-1 RNA viral load(r=-0.60 78,p<0.0001),the proportion of CD8+CD38+T cells(r=-0.3276,p=0.0106),and inflammatory biomarkers BAFF(r=-0.3072,p=0.0160),IP-10(r=-0.4784,p=0.0016)and MCP-1(r=-0.4317,p=0.0048),and were positively correlated with CD4+T cell count and CD4/CD8 ratio(r=0.2918,p=0.0225;r=0.321 1,p=0.0116).(5)The neutralizing potency and breadth of plasma were associated with duration of HIV-1 infection(r=0.2999,p=0.0234;r=0.3333,p=0.0106).Six samples with 50%inhibition more than 60 and breadth above 50%were identified from 59 plasms samples.(6)181 pairs of Ig genes were amplified and cloned into expression vectors.VH genes derived from six germlines,including IGHV1-2、IGHV1-69、IGHV3-11、IGHV3-15、IGHV4-38 and IGHV4-59,with somatic hypermutation(SHM)from 6.5%to 32%;VL genes originated from two germlines,IGKV1-5*03 和 IGKV1-33*01,with 4%and 21%SHM respectively.(7)Eight antibodies(7D5,53,8A4,8E5,7F,8F10,8H4,and 8H5)were prepared and identified,and 7D5 had the highest binding activity and neutralizing activity,which might relate with its high SHM.(8)Two antibody candidates,8G2 and 8H9 were screened,and they carried high SHM and long HCDR3.Conclusions:The size of HIV-1 reservoirs was associated with plasma HIV-1 RNA viral load and disease progression.The proportion of activated T cells was positively correlated with HIV-1 reservoirs size,and the levels of APRIL were significantly and negatively correlated with HIV-1 reservoirs level,suggesting that low immune activation and high APRIL levels are associated with viral control.APRIL is a discriminant biomarker in LTNPs,and further study of this biomarker could provide new insight into the mechanisms of natural viral control.The development of neutralization potency and breadth in bNAbs might need long-term co-evolution of the HIV-1 and antibodies,and LTNPs provide adequate conditions for the generation and maturation of bNAbs.The characteristics of antibodies identified imply that SHM might impact the binding and neutralizing activities of bNAbs.Therefore,screening antibodies with high SHM tends to obtain great potency and breadth,and might further afford more potential strategies for HIV-1 prevention and treatment.