Plasma proteomic profiling identifies CD33 as a marker of HIV control in natural infection and after therapeutic vaccination

Altres ajuts: National Institutes of Health (NIH), P01-AI131568Biomarkers predicting the outcome of HIV-1 virus control in natural infection and after therapeutic interventions in HIV-1 cure trials remain poorly defined. The BCN02 trial (NCT02616874), combined a T-cell vaccine with romidepsin (RMD), a cancer-drug that was used to promote HIV-1 latency reversal and which has also been shown to have beneficial effects on neurofunction. We conducted longitudinal plasma proteomics analyses in trial participants to define biomarkers associated with virus control during monitored antiretroviral pause (MAP) and to identify novel therapeutic targets that can improve future cure strategies. BCN02 was a phase I, open-label, single-arm clinical trial in early-treated, HIV infected individuals. Longitudinal plasma proteomes were analyzed in 11 BCN02 participants, including 8 participants that showed a rapid HIV-1 plasma rebound during a monitored antiretroviral pause (MAP-NC, 'non-controllers') and 3 that remained off ART with sustained plasma viremia <2000 copies/ml (MAP-C, 'controllers'). Inflammatory and neurological proteomes in plasma were evaluated and integration data analysis (viral and neurocognitive parameters) was performed. Validation studies were conducted in a cohort of untreated HIV-1+ individuals (n = 96) and in vitro viral replication assays using an anti-CD33 antibody were used for functional validation

IRF7 expression correlates with HIV latency reversal upon specific blockade of immune activation

The persistence of latent HIV reservoirs allows for viral rebound upon antiretroviral therapy interruption, hindering effective HIV-1 cure. Emerging evidence suggests that modulation of innate immune stimulation could impact viral latency and contribute to the clearing of HIV reservoir. Here, the latency reactivation capacity of a subclass of selective JAK2 inhibitors was characterized as a potential novel therapeutic strategy for HIV-1 cure. Notably, JAK2 inhibitors reversed HIV-1 latency in non-clonal lymphoid and myeloid in vitro models of HIV-1 latency and also ex vivo in CD4+ T cells from ART+ PWH, albeit its function was not dependent on JAK2 expression. Immunophenotypic characterization and whole transcriptomic profiling supported reactivation data, showing common gene expression signatures between latency reactivating agents (LRA; JAK2i fedratinib and PMA) in contrast to other JAK inhibitors, but with significantly fewer affected gene sets in the pathway analysis. In depth evaluation of differentially expressed genes, identified a significant upregulation of IRF7 expression despite the blockade of the JAK-STAT pathway and downregulation of proinflammatory cytokines and chemokines. Moreover, IRF7 expression levels positively correlated with HIV latency reactivation capacity of JAK2 inhibitors and also other common LRAs. Collectively, these results represent a promising step towards HIV eradication by demonstrating the potential of innate immune modulation for reducing the viral reservoir through a novel pathway driven by IRF7

Methylation regulation of Antiviral host factors, Interferon Stimulated Genes (ISGs) and T-cell responses associated with natural HIV control

GWAS, immune analyses and biomarker screenings have identified host factors associated within vivoHIV-1 control. However, there is a gap in the knowledge about the mechanisms that regulate the expression of such host factors. Here, we aimed to assess DNA methylation impact on host genome in natural HIV-1 control. To this end, whole DNA methylome in 70 untreated HIV-1 infected individuals with either high (>50,000 HIV-1-RNA copies/ml, n = 29) or low (<10,000 HIV-1-RNA copies/ml, n = 41) plasma viral load (pVL) levels were compared and identified 2,649 differentially methylated positions (DMPs). Of these, a classification random forest model selected 55 DMPs that correlated with virologic (pVL and proviral levels) and HIV-1 specific adaptive immunity parameters (IFNg-T cell responses and neutralizing antibodies capacity). Then, cluster and functional analyses identified two DMP clusters: cluster 1 contained hypo-methylated genes involved in antiviral and interferon response (e.g.PARP9,MX1, andUSP18) in individuals with high viral loads while in cluster 2, genes related to T follicular helper cell (Tfh) commitment (e.g.CXCR5andTCF7) were hyper-methylated in the same group of individuals with uncontrolled infection. For selected genes, mRNA levels negatively correlated with DNA methylation, confirming an epigenetic regulation of gene expression. Further, these gene expression signatures were also confirmed in early and chronic stages of infection, including untreated, cART treated and elite controllers HIV-1 infected individuals (n = 37). These data provide the first evidence that host genes critically involved in immune control of the virus are under methylation regulation in HIV-1 infection. These insights may offer new opportunities to identify novel mechanisms ofin vivovirus control and may prove crucial for the development of future therapeutic interventions aimed at HIV-1 cure. Author summary The infection with the human immunodeficiency virus (HIV), as for other viral infections, induce global DNA Methylation changes in the host genome. Herein, we identified for first time the methylation impact on host genome in untreated HIV-1 infection with different degrees ofin vivovirus control. Specifically, we observed that individuals with a better HIV-1 control showed a hypermethylation of genes associated with antiviral and interferon pathways and the hypomethylation of genes associated with the differentiation process of T follicular helper cells. Interestingly, these epigenetic imprints in host genome were strongly correlated with virus content and HIV-specific T cell responses. Therefore, we propose DNA Methylation as the regulation mechanism of host genes involved in immune HIV-1 control that could interfere in the efficacy of cure strategies. We also highlight the importance of DNA Methylation to regulate immune responses not only in HIV-1 but also in chronic infections or other pathologic situations associated with a sustained activation of the immune system

Methylation regulation of Antiviral host factors, Interferon Stimulated Genes (ISGs) and T-cell responses associated with natural HIV control

GWAS, immune analyses and biomarker screenings have identified host factors associated with in vivo HIV-1 control. However, there is a gap in the knowledge about the mechanisms that regulate the expression of such host factors. Here, we aimed to assess DNA methylation impact on host genome in natural HIV-1 control. To this end, whole DNA methylome in 70 untreated HIV-1 infected individuals with either high (>50,000 HIV-1-RNA copies/ml, n = 29) or low (<10,000 HIV-1-RNA copies/ml, n = 41) plasma viral load (pVL) levels were compared and identified 2,649 differentially methylated positions (DMPs). Of these, a classification random forest model selected 55 DMPs that correlated with virologic (pVL and proviral levels) and HIV-1 specific adaptive immunity parameters (IFNg-T cell responses and neutralizing antibodies capacity). Then, cluster and functional analyses identified two DMP clusters: cluster 1 contained hypo-methylated genes involved in antiviral and interferon response (e.g. PARP9, MX1, and USP18) in individuals with high viral loads while in cluster 2, genes related to T follicular helper cell (Tfh) commitment (e.g. CXCR5 and TCF7) were hyper-methylated in the same group of individuals with uncontrolled infection. For selected genes, mRNA levels negatively correlated with DNA methylation, confirming an epigenetic regulation of gene expression. Further, these gene expression signatures were also confirmed in early and chronic stages of infection, including untreated, cART treated and elite controllers HIV-1 infected individuals (n = 37). These data provide the first evidence that host genes critically involved in immune control of the virus are under methylation regulation in HIV-1 infection. These insights may offer new opportunities to identify novel mechanisms of in vivo virus control and may prove crucial for the development of future therapeutic interventions aimed at HIV-1 cure

Gut microbiome signatures linked to HIV-1 reservoir size and viremia control

Background: The potential role of the gut microbiome as a predictor of immune-mediated HIV-1 control in the absence of antiretroviral therapy (ART) is still unknown. In the BCN02 clinical trial, which combined the MVA.HIVconsv immunogen with the latency-reversing agent romidepsin in early-ART treated HIV-1 infected individuals, 23% (3/13) of participants showed sustained low-levels of plasma viremia during 32 weeks of a monitored ART pause (MAP). Here, we present a multi-omics analysis to identify compositional and functional gut microbiome patterns associated with HIV-1 control in the BCN02 trial. Results: Viremic controllers during the MAP (controllers) exhibited higher Bacteroidales/Clostridiales ratio and lower microbial gene richness before vaccination and throughout the study intervention when compared to non-controllers. Longitudinal assessment indicated that the gut microbiome of controllers was enriched in pro-inflammatory bacteria and depleted in butyrate-producing bacteria and methanogenic archaea. Functional profiling also showed that metabolic pathways related to fatty acid and lipid biosynthesis were significantly increased in controllers. Fecal metaproteome analyses confirmed that baseline functional differences were mainly driven by Clostridiales. Participants with high baseline Bacteroidales/Clostridiales ratio had increased pre-existing immune activation-related transcripts. The Bacteroidales/Clostridiales ratio as well as host immune-activation signatures inversely correlated with HIV-1 reservoir size. Conclusions: The present proof-of-concept study suggests the Bacteroidales/Clostridiales ratio as a novel gut microbiome signature associated with HIV-1 reservoir size and immune-mediated viral control after ART interruption. Video abstract

Sirtuin-2, NAD-Dependent Deacetylase, is a new potential therapeutic target for HIV-1 infection and HIV-related neurological dysfunction

The implementation and access to combined antiretroviral treatment (cART) have dramatically improved the quality of life of people living with HIV (PLWH). However, some comorbidities, such as neurological disorders associated with HIV infection still represent a serious clinical challenge. Soluble factors in plasma that are associated with control of HIV replication and neurological dysfunction could serve as early biomarkers and as new therapeutic targets for this comorbidity. We used a customized antibody array for determination of blood plasma factors in 40 untreated PLWH with different levels of viremia and found sirtuin-2 (SIRT2), an NAD-dependent deacetylase, to be strongly associated with elevated viral loads and HIV provirus levels, as well as with markers of neurological damage (a-synuclein [SNCA], brain-derived neurotrophic factor [BDNF], microtubule-associated protein tau [MAPT], and neurofilament light protein [NFL]). Also, longitudinal analysis in HIV-infected individuals with immediate (n = 9) or delayed initiation (n = 10) of cART revealed that after 1 year on cART, SIRT2 plasma levels differed between both groups and correlated inversely with brain orbitofrontal cortex involution. Furthermore, targeting SIRT2 with specific small-molecule inhibitors in in vitro systems using J-LAT A2 and primary glial cells led to diminished HIV replication and virus reactivation from latency. Our data thus identify SIRT2 as a novel biomarker of uncontrolled HIV infection, with potential impact on neurological dysfunction and offers a new therapeutic target for HIV treatment and cure. IMPORTANCE Neurocognitive disorders are frequently reported in people living with HIV (PLWH) even with the introduction of combined antiretroviral treatment (cART). To identify biomarkers and potential therapeutic tools to target HIV infection in peripheral blood and in the central nervous system (CNS), plasma proteomics were applied in untreated chronic HIV-infected individuals with different levels of virus control. High plasma levels of sirtuin-2 (SIRT2), an NAD+ deacetylase, were detected in uncontrolled HIV infection and were strongly associated with plasma viral load and proviral levels. In parallel, SIRT2 levels in the peripheral blood and CNS were associated with markers of neurological damage and brain involution and were more pronounced in individuals who initiated cART later in infection. In vitro infection experiments using specific SIRT2 inhibitors suggest that specific targeting of SIRT2 could offer new therapeutic treatment options for HIV infections and their associated neurological dysfunction

Systems Biology for the identification of epigenetic biomarkers and host factors associated with HIV-1 control

La cura del VIH-1 és un dels majors reptes científics i l'únic tractament és l'administració de fàrmacs antiretrovirals per mantenir una càrrega viral indetectable, i bloquejar el progrés de la malaltia associada a la infecció. Malgrat els múltiples beneficis dels antiretrovirals, aquests fàrmacs no arriben a tota la població mundial i requereixen una adherència al tractament a llarg termini, ja que per la capacitat del VIH-1 d'induir latència, si s'interromp el tractament, la càrrega viral augmenta ràpidament. L'estudi d'individus capaços de controlar la infecció del VIH-1 en absència de tractament antiretroviral ha estat rellevant en el disseny de les estratègies curatives del VIH-1, però encara es desconeixen tots els mecanismes que indueixen aquest control. En aquesta tesis, hem aplicat anàlisis de dades òmiques per identificar els factors de l'hoste i els mecanismes biològics associats al control natural de la infecció per VIH-1, o bé involucrats en el control induït per una teràpia kick-and-kill. En el primer capítol de la tesis, es descriuen els factors de l'hoste regulats epigenèticament associats al control del VIH-1. Breument, l'estudi de la metilació de l'ADN en PBMCs d'individus amb diferent capacitat de controlar la replicació viral, ha demostrat que la metilació en gens involucrats en la resposta antiviral i en la resposta de cèl·lula T, s'associa a la capacitat de controlar espontàniament la infecció. A més, els nivells de metilació d'aquests gens correlacionen amb la càrrega viral, els nivells de proviral, els marcadors de resposta T, i la capacitat neutralitzant d'anticossos. Aquestes observacions indiquen que les marques epigenètiques s'han de tenir en compte en futures estratègies d'eradicació i/o curació del VIH-1. L'objectiu del segon capítol de la tesis és la identificació de biomarcadors en plasma predictius del control del VIH-1. En aquest estudi, els nivells de les formes solubles del TL1A i el DR3 es troben associats al control del VIH-1 i a la resposta immune mediada per cèl·lules T. Aquests resultats, conjuntament amb experiments in vitro, han demostrat que la co-estimulació del DR3 millora la resposta T especifica contra el VIH-1, i ofereixen una possible eina per millorar la resposta immune cel·lular en futurs assaigs clínics. Finalment, el tercer capítol inclou una anàlisis de biologia de sistemes en els participants de l'assaig clínic BCN02 per identificar els desencadenants de control viral després d'una estratègia kick-and-kill. Als participants del BCN02 se'ls va administrar romidepsina per reactivar la latència del VIH-1, i se'ls va vacunar amb l'immunogen HIVconsv per induir una resposta cel·lular contra el VIH-1. Seguidament, en la fase MAP (de l'anglès, Monitored Antiretroviral Pause), es va interrompre el tractament antiretroviral i es va observar que 4 d'ells mostraven un rebot viral retardat amb una càrrega viral inferior a les 2,000 còpies d'ARN viral/ml durant, al menys, 8 setmanes. En l'estudi exploratori de la metilació de l'ADN i l'expressió gènica global en PBMCs dels participants del BCN02, s'ha observat com la intervenció clínica, impactava tan els patrons epigenètics com d'expressió gènica de l'hoste, incloent gens implicats en vies de senyalització relacionades amb la infecció del VIH-1 i la resposta immune cel·lular. També s'ha identificat una petjada epigenètica prèvia a la interrupció del tractament antiretroviral, que s'associa al rebot viral temprà o tardà. Aquests resultats suggereixen que els biomarcadors basats en la metilació de l'ADN podrien utilitzar-se com a variable subrogada del control viral post-tractament. Globalment, aquestes anàlisis han permès identificar gens i vies de senyalització diferencialment regulades tant en el control natural de la infecció del VIH-1, com en el control post-tractament. Per tant, els estudis inclosos en aquesta tesis han permès identificar nous candidats a biomarcadors de control viral i possibles dianes terapèutiques.La cura del VIH-1 es uno de los mayores retos científicos y el único tratamiento son los fármacos antirretrovirales que mantienen una carga viral indetectable y bloquean el progreso de la enfermedad asociada a la infección. A pesar de los múltiples beneficios de los antiretrovirales, éstos no llegan a toda la población mundial y requieren la adherencia al tratamiento a largo plazo, ya por la capacidad del VIH de establecer latencia, la interrupción del tratamiento desencadena un rápido rebote viral. El estudio de individuos capaces de controlar la infección del VIH-1 sin tratamiento antiretroviral, ha sido de especial relevancia en el diseño de estrategias curativas. Sin embargo, se desconocen todos los mecanismos asociados al control viral. En la presente tesis, aplicamos un análisis de datos ómicos para identificar factores del huésped y mecanismos biológicos asociados al control natural de la, o al control inducido después de la terapia kick-and-kill. En el primer capítulo de la tesis, describimos factores del huésped regulados epigenéticamente y asociados al control espontáneo del VIH. Brevemente, el estudio de la metilación del ADN en PBMCs de individuos con diferente capacidad de controlar la replicación viral, ha demostrado que la metilación de genes de respuesta antiviral y de respuesta celular, se asocia al control natural de la infección. Además, los niveles de metilación de estos genes correlacionan con la carga viral, los niveles de proviral, marcadores de respuesta celular T, y la capacidad neutralizante de los anticuerpos. Estas observaciones indican que la regulación epigenética de la respuesta inmune debería considerarse en futuras estrategias de curación y/o erradicación del VIH. El objetivo del segundo capítulo es la identificación de biomarcadores en plasma predictivos del control natural del VIH-1. En este estudio, los niveles de las formas solubles del TLA y el DR3 se asociaron al control viral y a la respuesta celular T. Estos resultados y los de experimentación in vitro, han demostrado que la co-estimulación del DR3 mejora la respuesta T específica contra el VIH-1, y ofrecen una posible herramienta terapéutica para mejorar la respuesta inmune celular contra el virus. Finalmente, el tercer capítulo incluye un análisis de biología de sistemas en PBMCs de los participantes del ensayo clínico BCN02 para identificar los desencadenantes del control viral después de la estrategia kick-and-kill. A los participantes del BCN02 se les administró romidepsina para reactivar la latencia del VIH, y se los vacunó con el inmunógeno HIVconsv para inducir una respuesta inmune celular contra el VIH. En fase MAP (del inglés, Monitored Antiretroviral Pause), se interrumpió el tratamiento antirretroviral y se observó que 4 de ellos mostraban un rebote viral tardío, siendo capaces de mantener una carga viral por debajo las 2,000 copias de ARN viral/ml durante, al menos, 8 semanas. Mediante el estudio exploratorio de la metilación del ADN y la expresión génica global en PBMCs de los participantes, se observó que la intervención, tenía un gran impacto en los patrones transcripcionales y epigenéticos del huésped, incluyendo genes de vías de señalización involucradas en la replicación del VIH-1 y en la respuesta inmune. También se identificó una impronta epigenética, previa a la interrupción del tratamiento, que se asocia al rebote viral temprano o tardío durante la MAP. Estos resultados sugieren un posible uso de biomarcadores basados en la metilación del ADN cómo variables subrogadas del control viral post-tratamiento. Globalmente, estos análisis han permitido la identificación de genes y vías de señalización diferencialmente reguladas tanto en el control natural de la infección cómo en el control inducido posttratamiento. Por lo tanto, los estudios incluidos en esta tesis permiten identificar nuevos candidatos a biomarcadores y posibles dianas terapéuticas.An effective HIV-1 cure is still one of the major scientific challenges, and a global solution is not yet available beyond the administration of antiretroviral drugs, which maintain undetectable viral load and pause the progression of HIV associated disease. Albeit the evident benefits of antiretroviral therapy, it is not available worldwide, it requires life-long medication and, if stopped, a rapid viral rebound is observed due to HIV-1's capacity to establish latency and form an HIV-1 reservoir. In the development of current HIV-1 strategies, the study of a small subset of individuals with the capacity to spontaneously control HIV-1 infection has been relevant. However, the mechanisms of this control are not fully understood. In the present thesis, we have applied different omics-based analyses to gain insight into host factors and biological mechanisms associated with natural HIV-1 control. In parallel, these analyses are also applied in the context of the BCN02 clinical trial, designed to achieve a functional HIV-1 cure, in order to identify mechanisms of post-treatment control, which might be different from the ones observed in natural control. Chapter I describes the identification of epigenetically regulated host factors associated with spontaneous control of HIV-1. In brief, the genome-wide DNA methylation of PBMCs was studied in individuals with different levels of relative in vivo control of HIV-1 replication. Results indicated that differential DNA methylation on genes involved in the antiviral immune response and T-cell activation were associated with the capacity of natural control. Additionally, these methylation imprints were strongly associated with viral load, proviral DNA levels, markers of the T-cell response against HIV-1 and neutralizing antibody capacity. These observations indicate a crucial role of epigenetic imprints and have the potential to guide future HIV eradication and cure strategies. The objective of Chapter II was to identify plasma biomarkers predictive of HIV-1 disease control. Specifically, soluble forms of TL1A and DR3 were found to be associated with virus control but also with parameters of T-cell mediated immunity. These results, coupled with validation analysis and in vitro experiments, showed that DR3 stimulation enhances HIV-1 specific T cell-responses, providing a potential tool for boosting HIV- specific T-cell responses in therapeutic vaccination strategies. Finally, an integrated systems biology analysis was conducted in the setting of the kick-and-kill intervention applied in the BCN02 clinical trial, to understand the biological mechanisms impacted by the intervention and to identify potential drivers of post-treatment control (Chapter III). Participants from the BCN02 study were treated with romidepsin to disrupt HIV-1 latency, and vaccinated with HIVconsv immunogen to elicit T-cell responses against conserved regions of HIV-1. Subsequently, individuals interrupted their antiretroviral treatment in a "monitored antiretroviral pause" (MAP) during which 4 individuals showed a delayed viral rebound and maintained viral replication below 2,000 HIV RNA copies/ml for, at least, 8 weeks. This exploratory study demonstrated how the intervention impacted the host PBMCs' transcriptional and DNA methylation programs, especially after HIVconsv vaccination and romidepsin administration. Importantly multiple biological pathways, including the ones involved in HIV- 1 infection and T-cell immunity, were modulated at both the epigenetic and transcriptional level. Additionally, individuals with an early or late rebound during MAP showed differential epigenetic signatures prior to treatment interruption, suggesting the use of DNA methylation-based biomarkers as a surrogate of post-treatment control. Overall, omics-based systems biology analyses enabled the study of human plasma and PBMC samples to unveil different genes and pathways that are differentially modulated in natural and post-treatment HIV- 1 control, thus providing new potential biomarkers and therapeutic targets

Exploring HLA class II allele associations with markers of HIV control

Curs 2015-2016Among HIV seropositive individuals, the progression of the HIV-related disease is quite heterogeneous, partially due to the genetic background of the patients. Different host genetic factors have been statistically associated with HIV disease control or progression, especially HLA class I (HLA-I) polymorphisms because of the direct role that these molecules play in the immune response against the virus. In recent years, the immune response against HIV based on CD4+ T helper cells and HLA class II (HLA-II) restricted responses has gained importance to refine current vaccine approaches. Therefore, the main objective of the present study was to explore potential statistical associations of HLA-II with markers of HIV control, in particular HIV viral load and CD4+ counts, in a Peruvian cohort of almost 400 individuals with existing HIV infection or at high risk for infection. Additionally, we also studied the associations of HLA-II with the presence of T cell responses to overlapping peptides (OLPs) covering the whole HIV proteome. In order to achieve such objectives, different statistical approaches using the R statistical software were applied. Mainly, the Fisher’s Exact Test was used to assess whether the expression of certain HLA-II alleles was associated with HIV infection or with the response to the different HIV epitopes. On the other hand, the Mann-Whitney Test was used to detect differences in viral loads or CD4+ counts between patients with or without a certain HLA-II allele. In our analyses, alleles HLA-DRB1*1201 and HLA-DRB1*1302 appeared to be significantly associated (p<0.05) with low and high viral loads, respectively. A number of additional alleles were significantly associated with CD4+ counts. Most of the identified associations included the HLA-DRB1 locus, the most polymorphic of the HLA class II loci. Additionally, expression of HLA-DRB1*1201 was associated with T cell response to OLP 41 in the Gag protein, and HLA-DRB1*1302, with the response to OLP 82 in Nef protein, identifying dominant targets of the T cell response restricted by these two alleles. Overall, the associations of HLA-DRB1*1201 and HLA-DRB1*1302 with viral load, support previous data suggesting a potential effect of the CD4+ T cell responses on HIV disease control