Restriction Factors: From Intrinsic Viral Restriction to Shaping Cellular Immunity Against HIV-1

Antiviral restriction factors are host cellular proteins that constitute a first line of defense blocking viral replication and propagation. In addition to interfering at critical steps of the viral replication cycle, some restriction factors also act as innate sensors triggering innate responses against infections. Accumulating evidence suggests an additional role for restriction factors in promoting antiviral cellular immunity to combat viruses. Here, we review the recent progress in our understanding on how restriction factors, particularly APOBEC3G, SAMHD1, Tetherin, and TRIM5α have the cell-autonomous potential to induce cellular resistance against HIV-1 while promoting antiviral innate and adaptive immune responses. Also, we provide an overview of how these restriction factors may connect with protein degradation pathways to modulate anti-HIV-1 cellular immune responses, and we summarize the potential of restriction factors-based therapeutics. This review brings a global perspective on the influence of restrictions factors in intrinsic, innate, and also adaptive antiviral immunity opening up novel research avenues for therapeutic strategies in the fields of drug discovery, gene therapy, and vaccines to control viral infections

HIV-1 Gag mutations alone are sufficient to reduce darunavir susceptibility during virological failure to boosted PI therapy

Virological failure (VF) to boosted PIs with a high genetic barrier is not usually linked to the development of resistance-associated mutations in the protease gene. From a cohort of 520 HIV-infected subjects treated with lopinavir/ritonavir or darunavir/ritonavir monotherapy, we retrospectively identified nine patients with VF. We sequenced the HIV-1 Gag-protease region and generated clonal virus from plasma samples. We characterized phenotypically clonal variants in terms of replicative capacity and susceptibility to PIs. Also, we used VESPA to identify signature mutations and 3D molecular modelling information to detect conformational changes in the Gag region. All subjects analysed harboured Gag-associated polymorphisms in the absence of resistance mutations in the protease gene. Most Gag changes occurred outside Gag cleavage sites. VESPA analyses identified K95R and R286K (P < 0.01) as signature mutations in Gag present at VF. In one out of four patients with clonal analysis available, we identified clonal variants with high replicative capacity and 8- to 13-fold reduction in darunavir susceptibility. These clonal variants harboured K95R, R286K and additional mutations in Gag. Low susceptibility to darunavir was dependent on the Gag sequence context. All other clonal variants analysed preserved drug susceptibility and virus replicative capacity. Gag mutations may reduce darunavir susceptibility in the absence of protease mutations while preserving viral fitness. This effect is Gag-sequence context dependent and may occur during boosted PI failure

Control of HIV-1 Pathogenesis in Viremic Nonprogressors Is Independent of Gag-Specific Cytotoxic T Lymphocyte Responses

Viremic nonprogressors (VNPs) constitute a very scarce group of untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain stable CD4+ T cell counts despite high levels of HIV-1 replication. The specific factors associated with this atypical control of the HIV infection have been poorly described. Since specific T cell responses seem to be one of the main causes of HIV-1 control in elite controllers, we studied whether HIV-1 Gag-specific cytotoxic T lymphocyte (CTL) responses could also modulate disease control in VNPs. We characterized the immune responses from four VNPs compared to those of five standard progressors (SPs) during the first years of HIV-1 infection. We observed no differences in the breadth and frequency of Gag-specific cellular responses. Furthermore, we obtained 217 HIV-1Gag clonal sequences in which the viral variability of Gag increased over 3 years of infection for synonymous and nonsynonymous mutations in both VNPs and SPs. VNPs evolution rates in gag were comparable to SPs. This observation is in line with a similar accumulation of CTL putative escape mutations in Gag epitopes targeted by CTL responses. Altogether, the absence of viral pathogenesis in VNP individuals seems to be independent of HIV-Gag-specific CTL responses. This novel information guides to the study of alternative mechanism of HIV-1 pathogenesis control.IMPORTANCE Control of HIV infection has been widely studied in elite controllers or long-term nonprogressor models. However, there is a less-known group of individuals, termed viremic nonprogressors (VNPs), who maintain stable CD4+ T cell counts despite high plasma viremia. The mechanisms involved in this remarkable control of HIV-1 pathogenesis clearly have implications for the development of new drugs and vaccines. We show here for the first time that VNPs have immune responses and HIV-gag evolution similar to those of standard progressors. Remarkably, we demonstrate that the mechanism of pathogenesis control in these individuals differs from some elite controllers that are reported to have improved immune control. This is noteworthy since it opens the door to new, as-yet-unknown mechanisms for HIV control. Our novel results advance the understanding of mechanisms involved in viremic nonprogression and suggest that there are alternative mechanisms to the adaptive immune responses for an effective control of viral pathogenesis.We thank the founders for support of this project. We also thank all the centers and investigators involved in CoRIS. M.S. was supported by a Sara Borrell grant (CD11/00286). The RIS cohort (CoRIS) is supported by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en Sida (RD06/006 RD12/0017/0018, RD16/0025/0041) as part of the Plan Nacional R+D+I and cofinanced by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). This study was supported by the National Health Institute Carlos III (PI14/01058) and the Gilead Fellowship Program GLD 15/00298. J.G.P. holds a Miguel Servet II contract (CPII15/00014) funded by ISCIII. E.J.-M. is supported by Redes Temáticas de Investigación en SIDA (ISCIII RETIC RD16/0025/0041); Acción Estratégica en Salud; Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011; and Instituto de Salud Carlos III, Fondos FEDER. M.S., A.G.-M., and E.J.-M. performed the experiments. J.D., P.V., and B.A. selected and designed the cohorts. M.S., B.C., J.G.P., and J.M.-P. designed the experiments and drafted the paper. P.V. and B.A. are members of CoRISpe and the HIV HGM BioBank Study Group.S

Viral and Cellular factors leading to the Loss of CD4 Homeostasis in HIV-1 Viremic Nonprogressors

Human immunodeficiency virus type 1 (HIV-1) viremic nonprogressors (VNPs) represent a very rare HIV-1 extreme phenotype. VNPs are characterized by persistent high plasma viremia and maintenance of CD41 T-cell counts in the absence of treatment. However, the causes of nonpathogenic HIV-1 infection in VNPs remain elusive. Here, we identified for the first time two VNPs who experienced the loss of CD41 homeostasis (LoH) after more than 13 years. We characterized in deep detail viral and host factors associated with the LoH and compared with standard VNPs and healthy controls. The viral factors determined included HIV-1 coreceptor usage and replicative capacity. Changes in CD41 and CD81 T-cell activation, maturational phenotype, and expression of CCR5 and CXCR6 in CD41 T-cells were also evaluated as host-related factors. Consistently, we determined a switch in HIV-1 coreceptor use to CXCR4 concomitant with an increase in replicative capacity at the LoH for the two VNPs. Moreover, we delineated an increase in the frequency of HLA-DR1CD381 CD41 and CD81 T cells and traced the augment of naive T-cells upon polyclonal activation with LoH. Remarkably, very low and stable levels of CCR5 and CXCR6 expression in CD41 T-cells were measured over time. Overall, our results demonstrated HIV-1 evolution toward highly pathogenic CXCR4 strains in the context of very limited and stable expression of CCR5 and CXCR6 in CD41 T cells as potential drivers of LoH in VNPs. These data bring novel insights into the correlates of nonpathogenic HIV1 infection. Importance: The mechanism behind nonpathogenic human immunodeficiency virus type 1 (HIV-1) infection remains poorly understood, mainly because of the very low frequency of viremic nonprogressors (VNPs). Here, we report two cases of VNPs who experienced the loss of CD41 T-cell homeostasis (LoH) after more than 13 years of HIV-1 infection. The deep characterization of viral and host factors supports the contribution of viral and host factors to the LoH in VNPs. Thus, HIV-1 evolution toward highly replicative CXCR4 strains together with changes in T-cell activation and maturational phenotypes were found. Moreover, we measured very low and stable levels of CCR5 and CXCR6 in CD41 T-cells over time. These findings support viral evolution toward X4 strains limited by coreceptor expression to control HIV-1 pathogenesis and demonstrate the potential of host-dependent factors, yet to be fully elucidated in VNPs, to control HIV-1 pathogenesis.This research was supported by a Gilead Fellowship (grant GLD15/0298) and La Caixa Foundation (grant LCF/PR/PR16/11110026). M.C.-L. is a Beatriu de Pinós postdoctoral fellow (grant BP 00075) supported by the Government of Catalonia’s Secretariat for Universities and Research of the Ministry of Economy and Knowledge. J.G.P. was supported by the ISCIII (grant CP15/00014). E.J.-M. was funded by Redes Temáticas de Investigación en SIDA (ISCIII RETIC RD16/0025/0041); Acción Estratégica en Salud; Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008–2011; and Instituto de Salud Carlos III. E.J.-M. was cofunded by European Regional Development Fund/European Social Fund (FEDER) “Investing in your future.” J.M.-P. is supported by the Spanish Ministry of Science and Innovation (grant PID2019-109870RB-I00). J.G.P. and M.C.-L. designed the study, supervised experiments and data. J.G.P., M.C.-L., and A.K. contributed to data interpretation. M.C.-L., R.P., E.J.-M., M.P., and C.C. performed experiments, analyzed, and interpreted the data. J.D. carried out the clinical follow-up and patient identification. M.C.-L., D.O., M.P., and C.C. performed data analysis. M.C.-L., A.K., M.P., C.L.-G., B.C., J.M.-P., and J.G.P. performed manuscript writing, critical revision, and discussion. We declare no conflict of interest.S

Gag-protease coevolution analyses define novel structural surfaces in the HIV-1 matrix and capsid involved in resistance to Protease Inhibitors

Despite the major role of Gag in establishing resistance of HIV-1 to protease inhibitors (PIs), very limited data are available on the total contribution of Gag residues to resistance to PIs. To identify in detail Gag residues and structural interfaces associated with the development of HIV-1 resistance to PIs, we traced viral evolution under the pressure of PIs using Gag-protease single genome sequencing and coevolution analysis of protein sequences in 4 patients treated with PIs over a 9-year period. We identified a total of 38 Gag residues correlated with the protease, 32 of which were outside Gag cleavage sites. These residues were distributed in 23 Gag-protease groups of coevolution, with the viral matrix and the capsid represented in 87% and 52% of the groups. In addition, we uncovered the distribution of Gag correlated residues in specific protein surfaces of the inner face of the viral matrix and at the Cyclophilin A binding loop of the capsid. In summary, our findings suggest a tight interdependency between Gag structural proteins and the protease during the development of resistance of HIV-1 to PIs

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.Instituto de Salud Carlos III | Ref. PI13/02269Instituto de Salud Carlos III | Ref. PI17/00164Instituto de Salud Carlos III | Ref. PI16/0684Instituto de Salud Carlos III | Ref. PI19/01127Ministerio de Economía y Competitividad | Ref. RD12/0017/0028Ministerio de Economía y Competitividad | Ref. RD16/0025/0020Generalitat de Catalunya | Ref. AGAUR-FI_B 00582Agencia Estatal de Investigación | Ref. RYC-2015-18241Agencia Estatal de Investigación | Ref. PID2019-107931GA-I00Xunta de Galicia | Ref. ED431F 2018/08Ministerio de Economía y Competitividad | Ref. RD16/0025/004

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

Antigen production after latency reversal and expression of inhibitory receptors in CD8+ T cells limit the killing of HIV-1 reactivated cells

The so-called shock and kill therapies aim to combine HIV-1 reactivation by latency-reversing agents (LRA) with immune clearance to purge the HIV-1 reservoir. The clinical use of LRA has demonstrated detectable perturbations in the HIV-1 reservoir without measurable reductions to date. Consequently, fundamental questions concerning the limitations of the recognition and killing of LRA-reactivated cells by effector cells such as CD8+ T cells remain to be answered. Here, we developed a novel experimental framework where we combine the use of cytotoxic CD8+ T-cell lines and ex vivo CD8+ T cells from HIV-1-infected individuals with functional assays of LRA-inducible reactivation to delineate immune barriers to clear the reservoir. Our results demonstrate the potential for early recognition and killing of reactivated cells by CD8+ T cells. However, the potency of LRAs when crossing the barrier for antigen presentation in target cells, together with the lack of expression of inhibitory receptors in CD8+ T cells, are critical events to maximize the speed of recognition and the magnitude of the killing of LRA-inducible provirus. Taken together, our findings highlight direct limitations in LRA potency and CD8+ T cell functional status to succeed in the cure of HIV-1 infection

Enhancement of Antiviral CD8 + T-Cell Responses and Complete Remission of Metastatic Melanoma in an HIV-1-Infected Subject Treated with Pembrolizumab

Background: Pembrolizumab is an immune checkpoint inhibitor against programmed cell death protein-1 (PD-1) approved for therapy in metastatic melanoma. PD-1 expression is associated with a diminished functionality in HIV-1 specific-CD8 + T cells. It is thought that PD-1 blockade could contribute to reinvigorate antiviral immunity and reduce the HIV-1 reservoir. Methods: Upon metastatic melanoma diagnosis, an HIV-1-infected individual on stable suppressive antiretroviral regimen was treated with pembrolizumab. A PET-CT was performed before and one year after pembrolizumab initiation. We monitored changes in the immunophenotype and HIV-1 specific-CD8 + T-cell responses during 36 weeks of treatment. Furthermore, we assessed changes in the viral reservoir by total HIV-1 DNA, cell-associated HIV-1 RNA, and ultrasensitive plasma viral load. Results: Complete metabolic response was achieved after pembrolizumab treatment of metastatic melanoma. Activated CD8 + T-cells expressing HLA-DR + /CD38 + transiently increased over the first nine weeks of treatment. Concomitantly, there was an augmented response of HIV-1 specific-CD8 + T cells with TNF production and poly-functionality, transitioning from TNF to an IL-2 profile. Furthermore, a transient reduction of 24% and 32% in total HIV-1 DNA was observed at weeks 3 and 27, respectively, without changes in other markers of viral persistence. Conclusions: These data demonstrate that pembrolizumab may enhance the HIV-1 specific-CD8 + T-cell response, marginally affecting the HIV-1 reservoir. A transient increase of CD8 + T-cell activation, TNF production, and poly-functionality resulted from PD-1 blockade. However, the lack of sustained changes in the viral reservoir suggests that viral reactivation is needed concomitantly with HIV-1-specific immune enhancement