Impact Of HIV-1Env Quasispecies Variations On Antiretroviral Therapy And Fitness

Background and objectiveThe extensive heterogeneity observed in an HIV-infected individual because of the viral characteristics of the rapid viral turnover and a high rate of mutation. The high diversity of HIV results in adaptation to most environmental changes.Thus, HIV-1quasispecies are evolutionary and clinically relevant since this genetic variability can in a sense respond to selective pressure, which have the ability to maintain replication and pathogenicity. The emergence of HIV variants and drug-resistant strains is the main reason of treatment failure, which induces the viral escape and changed fitness. Currently, it is still unclearly that the impact of HIV-1quasispecies variations on antiretroviral therapy and fitness. For the presence of HIV-1drug resistance of the3TC regimens, the chronically HIV-1infected patients who received long-term antiretroviral therapy were studied to determine whether HIV-1quasispecies variation influences the treatment outcome and viral fitness. This study will provide important information for monitoring HIV prevalence, implementation of effective antiretroviral therapy and development of novel anti-HIV drugs and vaccines.Methods1. Retrospective inclusion of the chronically HIV-1infected patients who received long-term antiretroviral therapy from a established the National HIV Drug Resistance cohort in Anhui and Henan province were studied. Totally,46patients were included, which can be divided into virologic suppression group (VS,26patients) and treatment failure group (TF,20patients) according to the treatment outcome.2. HIV-1RNA extraction from plasma and single genome amplication were performed to obtain the HIV-1env sequences of each patient. We analyzed the characteristics of HIV-1env quasispecies from the horizontal and longitudinal level, in order to determine the correlation between HIV-1quasispecies variation and treatment outcome as well as the dynamic changes during antiretroviral therapy.3. The treatment failure related positive selection sites were screened using bioinformatics methods. Then these positive selection sites were associated with treatment outcome to acquire treatment-related potential adaptive mutation sites.4. The recombinant HIV-1virus were constructed to contain the above potential adaptive mutation sites. Then the infection ability assay and a multiple-cycle, tecombinant-virus, growth competition assay were applied to determine the impact of quasispecies variation on fitness and treatment outcome.Results1. The impact of HIV-1env quasispecies variation at baseline on treatment outcome.A total of638single genomes were obtained for HIV-1gp160at baseline. Among these, sequences that derived from plasmas of virologic suppression group (352) and TF (286) contributed to the cross-sectional analysis. Specific env sequence characteristics such as overall diversity, the accumulation rates of synonymous substitutions per potential synonymous site (dS), nonsynonymous substitution per potential nonsynonymous site (dN), the number of putative N-linked glycosylation sites (PNGS), and the length of amino acid and cell tropism were compared between VS group and TF group. Firstly, it is showed that there is higher diversity, dN and dS in gp160, gp120and gp41regions of TF group compared to VS in nucleotide level. The higher diversity were exist in TF group before ART; Then, increased amino acid length and more PNGS were observed in the C3region in TF group compared to VS group, which can be explained that length polymorphisms in the constant and variable envelope regions and more PNGS may also contribute to structural diversity. Finally,9featured sites in amino acid alignments of TF sequences those are distinctly relative to VS group, which mainly located in V1/V2and gp41region.To evaluate baseline variables relative to treatment outcome, logistic regression analysis were applied. Variables included were HIV-1CD4+, VL, gp160diversity, have related drug resistant mutation and have co-receptor CXCR4. Gp160diversity was the only variable to remain in the final model, and higher gp160diversity significantly increased the risk for treatment failure. Gp160diversity was the best single variable to predict TF for75.6%of individuals in this cohort, and an area under the curve (AUC) of0.794(CI95%,0.657-0.931) was obtained in the ROC curve, and selecting a0.50cut-off yielded a sensitivity and positive predictive value of75.0%, and a specificity and negative predictive value of80.0%.2. Dynamics of HIV-1env quasispecies characteristics from treatment failure patients during Antiviral Treatment.From the first section we can know that the higher diversity were observedin treatment failure patients. To investigate the dynamics of these HIV-1quasispecies during ART,12patients who received59monthes ART were included in this section. Similarly, the specific env sequence characteristics that mentioned in the first section were also applied on this part. A total of552single genomes were obtained for HIV-1gp160from2treatment point per patient during ART. First of all, in the longitudinal dataset, HIV-1quasispecies from TF patients showed no differences in diversity, dN and dS in gp160, gp120and gp41regions during ART. The viral load and viral quasispecies diversity were not reduced by treatment and host immune response. Subsequently, the amino acid length and PNGS of gp120were increased with time. HIV-1quasispecies may change amino acid length and PNGS to adapt the new host environment. And the cell tropism of HIV-1in TF patients keeps stable during ART. Eventually,22featured sites in amino acid from TF patients were detected during ART compared to baseline, which mainly located in V1/V2gp41region that included two insert mutations.3. The impact of treatment outcome related HIV-1env gene mutation on viral fitness.According to the results from the above two sections, a selection pressure based analysis was performed.to screen treatment failure related adaptive mutation and reveal the relationship between HIV-1env variation and viral fitness. Totally,36and10potential adaptive mutations related to treatment failure from gp120and gp41region were obtained respectively. Then4fitness mutaions (G145N, E150S,1371V and N392S) of gp120and5fitness mutaions (A96N, E151K, N166K, E223G and V321L) of gp41region were further predicted using Bayesian network. Those high frequency mutations that still existed during ART including G145N,137IV, A607N, E662K and N677K were selected for the recombinant-virus construction. Then the TCID50assay and the growth competition assay were applied to determine the impact of quasispecies variation on fitness. An increased fitness in both A607N and N677K mutant virus were observed upon the growth competition assay, though their TCID50decreased.ConclusionsWe found the HIV-1env quasispecies diversity influence the treatment outcome and fitness. Higher gp160diversity before ART will significantly increased the risk for treatment failure, which displayed as HIV-1env diversity cannot be restricted by ART and still evolved during ART. Some mutations that increased viral fitness already existed before ART, which contribute to the adaptation of the changed host environment after ART. And higher diversity provides a necessary condition for selecton of drug resistant mutations by drug pressure. Therefore, HIV-1quasispecies diversity should also be considered as an important factor for treatment outcome.