Immunoescape of HIV-1 in Env-EL9 CD8 + T cell response restricted by HLA-B*14:02 in a Non progressor who lost twenty-seven years of HIV-1 control

Background: Long-Term Non-Progressors (LTNPs) are untreated Human Immunodeficiency virus type 1 (HIV-1) infected individuals able to control disease progression for prolonged periods. However, the LTNPs status is temporary, as viral load increases followed by decreases in CD4 + T-cell counts. Control of HIV-1 infection in LTNPs viremic controllers, have been associated with effective immunodominant HIV-1 Gag-CD8 + T-cell responses restricted by protective HLA-B alleles. Individuals carrying HLA-B*14:02 control HIV-1 infection is related to an immunodominant Env-CD8 + T-cell response. Limited data are available on the contribution of HLA-B*14:02 CD8 + T -cells in LTNPs. Results: In this study, we performed a virological and immunological detailed analysis of an HLA-B*14:02 LNTP individual that lost viral control (LVC) 27 years after HIV-1 diagnosis. We analysed viral evolution and immune escape in HLA-B*14:02 restricted CD8 + T -cell epitopes and identified viral evolution at the Env-EL9 epitope selecting the L592R mutation. By IFN-γ ELISpot and immune phenotype, we characterized HLA- B*14:02 HIV-1 CD8 + T cell responses targeting, Gag-DA9 and Env-EL9 epitopes before and after LVC. We observed an immunodominant response against the Env-EL9 epitope and a decreased of the CD8 T + cell response over time with LVC. Loss of Env-EL9 responses was concomitant with selecting K588R + L592R mutations at Env-EL9. Finally, we evaluated the impact of Env-EL9 escape mutations on HIV-1 infectivity and Env protein structure. The K588R + L592R escape variant was directly related to HIV-1 increase replicative capacity and stability of Env at the LVC. Conclusions: These findings support the contribution of immunodominant Env-EL9 CD8 + T-cell responses and the imposition of immune escape variants with higher replicative capacity associated with LVC in this LNTP. These data highlight the importance of Env-EL9 specific-CD8 + T-cell responses restricted by the HLA-B*14:02 and brings new insights into understanding long-term HIV-1 control mediated by Env mediated CD8 + T-cell responses.Molecular Virology Laboratory was supported by grants SAF (2016-77894-R) from Ministerio de Economía y Competitividad (MINECO), ISCIII through the projects PI 13/02269, PI17/00164, PI16/0684, PI19/01127 (Co-funded by European Regional Development Fund/European Social Fund "Investing in your future"). The RIS-RETIC grants RD12/0017/0028, RD16/0025/0020 and RD16CIII/0002/0005. LTD was supported by the Instituto de Salud Carlos III (ISCIII) under grant agreement “CD20/00025” through the Sara Borrell Program. O.B.L was funded by an AGAUR-FI_B 00582 Ph.D. fellowship from the Catalan Government and the European Social Fund. M.A. was funded by grants RYC-2015-18241 and PID2019-107931GA-I00 from the Spanish Government and, ED431F 2018/08 from the “Xunta de Galicia”. ERM was supported by the Spanish National Research Council (CSIC). JGP laboratory was supported by National Health Institute Carlos III grant PI17/00164 and Redes Temáticas de Investigación en SIDA (ISCIII RETIC RD16/0025/0041). The funders had no role in study design, data collection and analysis, the decision to publish or drafting of the manuscript.S

Identification of a Cluster of HIV-1 Controllers Infected with Low Replicating Viruses

<div><p>Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in <i>env</i> gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70’s-early 80’s). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.</p></div

Correlation between sampling time and the genetic distance with reference and viral cluster strains.

<p>MRCA-to-tip distances were extracted from MrBayes phylogenetic tree using TreeStat v.1.2. •, values obtained for the nucleotide sequences collected at the beginning of HIV-1 epidemic (years 1981–1995) were plotted against sampling time and a linear regression analysis was performed. Red Δ, values obtained from the cluster nucleotide sequences. The red dashed line and the arrow permit the extrapolation of the year from cluster nucleotide sequences.</p

C2-V5 nucleotide sequences from patient LTNP_1.

<p>LTNP_1 samples were obtained from PBMC DNA (2005 sample), cell-free supernatant of PBMC co-culture from a 2004 sample and plasma RNA corresponding to a 2010 sample. Shaded boxes indicated amino acid mutations characteristics of the cluster viruses in this C2-V5 region. V3, V4 and V5 regions are indicated by boxes.</p

Biological characterization of the recombinant viruses.

<p>A) Infectivity of <i>env</i> recombinant viruses in TZM-bl cells. Cells were infected with 15 units of HIV-1 p24 antigen (75 pg) of virus-supernatants. Luciferase activity was measured 48 hours post-infection and the results were normalized to the value obtained with the WT virus (89ES061). Results represented the median and SE of two independent assays with thee replicates. B) Replication kinetics of <i>env</i> recombinant viruses in U87-CD4/CCR5 cells. Cells were infected with 100 units of HIV- 1 p24 antigen (500 pg) of virus supernatants. Cultures were followed during 14 days, and HIV-1 production was quantified by the RT-activity in the supernatant with in-house Syber green I based real-time PCR enhanced RT assay (SGPERT). Cluster’s recombinant viruses (red) were compared with recombinant viruses from chronic progressor patients (green), with a recombinant virus obtained from laboratory strain SF-162 (black), and with the laboratory infectious clone 89ES061 (black) where the nucleotide sequences were cloned.</p

Identification of amino acid positions associated with the cluster viruses.

<p>Identification of amino acid positions associated with the cluster viruses.</p

Phylogenetic tree from the Bayesian MCMC (MrBayes) analysis.

<p>The 50% majority rule consensus was constructed and posterior probabilities are indicated by asterisks in nodes (black <b>*</b> upper 0.85 and red <b>*</b> upper 0.95). MRCA including the vast of majority sequences analyzed (black ♦) and MRCA from the cluster viruses (red ♦) are marked. Branch lengths represent the mean value observed for that branch among the post-burning sampled trees. The branch colors identified nucleotide sequences origin: black correspond to ancient sequences from North-America (before 1991) and from Europe (before 1995), blue are Spanish sequences (from 1989 and 2005) and red Spanish cluster sequences (from 2004–2005, except As7 which was from 1989). Green sequences are from elite suppressors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077663#pone.0077663-Cormier1" target="_blank">[54]</a> and yellow sequences from elite controller patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077663#pone.0077663-Cormier1" target="_blank">[54]</a>. Gray lines are D subtype sequences used as outgroup in the bottom of the tree.</p

Host genetic factors of the cluster patients.

<p>Host genetic factors of the cluster patients.</p

Epidemiological, clinical and virological characteristics of the cluster patients.

<p>Epidemiological, clinical and virological characteristics of the cluster patients.</p

Comparison of the <i>env</i> gene amino acid sequences derived from cluster viruses with subtype B consensus sequence.

<p>35 common mutated positions detected in at least 5 of the cluster viruses are shown in color amino acids. Boxes marked the unusual amino acid whose presence in the cluster is statistically significant when compared with the reference amino acid sequence sets used in the study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077663#pone-0077663-t003" target="_blank">Table 3</a>).</p