Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps.

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.

Viral Fitness Landscapes Based on Self-organizing Maps.

The creation of fitness maps from viral populations especially in the case of RNA viruses, with high mutation rates producing quasispecies, is complex since the mutant spectrum is in a very high-dimensional space. In this work, a new approach is presented using a class of neural networks, Self-Organized Maps (SOM), to represent realistic fitness landscapes in two RNA viruses: Human Immunodeficiency Virus type 1 (HIV-1) and Hepatitis C Virus (HCV). This methodology has proven to be very effective in the classification of viral quasispecies, using as criterium the mutant sequences in the population. With HIV-1, the fitness landscapes are constructed by representing the experimentally determined fitness on the sequence map. This approach permitted the depiction of the evolutionary paths of the variants subjected to processes of fitness loss and gain in cell culture. In the case of HCV, the efficiency was measured as a function of the frequency of each haplotype in the population by ultra-deep sequencing. The fitness landscapes obtained provided information on the efficiency of each variant in the quasispecies environment, that is, in relation to the entire spectrum of mutants. With the SOM maps, it is possible to determine the evolutionary dynamics of the different haplotypes.

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 CD4+ 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 CD4+ 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 CD4+ and CD8+ T-cell activation, maturational phenotype, and expression of CCR5 and CXCR6 in CD4+ 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-DR+CD38+ CD4+ and CD8+ 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 CD4+ 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 CD4+ T cells as potential drivers of LoH in VNPs. These data bring novel insights into the correlates of nonpathogenic HIV-1 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 CD4+ 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 CD4+ 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.

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.

Characterizing the antiviral effect of an ATR inhibitor on human immunodeficiency virus type 1 replication.

In the search for new antiviral therapies against human immunodeficiency virus type 1 (HIV-1), several cellular targets are being investigated. Ataxia telangiectasia and Rad3-related protein (ATR) has been implicated in HIV-1 replication, namely during retroviral DNA integration. We studied the effect of the ATR inhibitor ETP-46464 on HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and in the persistently HIV-1-infected cell line H61-D. After treatment with ETP-46464, a significant decrease in virus production was observed in both cell systems. Quantification of viral DNA forms in the acutely infected PBMCs suggests that inhibition could take place in the early phase of the viral life cycle before viral DNA integration. Moreover, after treatment of H61-D cells with 3'-azido-3'-deoxythymidine (AZT), which blocks new reverse transcription events, ETP-46464 decreased viral production, suggesting that inhibition of viral replication occurred in the late phase of the life cycle after viral DNA integration. A decrease in virus production after transfection of 293T cells with an HIV-1 infectious molecular clone also suggested that the effect of ETP-46464 is exerted at a post-integration step. We propose that ETP-46464 produces its inhibitory effect on HIV-1 replication by acting in both the early and late phases of the retroviral replication cycle. Thus, ATR could represent a new target for inhibition of HIV-1 replication.

Factors Leading to the Loss of Natural Elite Control of HIV-1 Infection.

HIV-1 elite controllers (EC) maintain undetectable viral loads (VL) in the absence of antiretroviral treatment. However, these subjects have heterogeneous clinical outcomes, including a proportion that loses HIV-1 control over time. In this work, we compared, in a longitudinal design, transient EC, analyzed before and after the loss of virological control, with persistent EC. The aim was to identify factors leading to the loss of natural virological control of HIV-1 infection with a longitudinal retrospective study design. Gag-specific T-cell responses were assessed by in vitro intracellular polycytokine production quantified by flow cytometry. Viral diversity determinations and sequence dating were performed in proviral DNA by PCR amplification at limiting dilution of env and gag genes. The expression profile of 70 serum cytokines and chemokines was assessed by multiplex immunoassays. We identified transient EC as subjects with low Gag-specific T-cell polyfunctionality, high viral diversity, and high proinflammatory cytokine levels before the loss of control. Gag-specific T-cell polyfunctionality was inversely associated with viral diversity in transient controllers before the loss of control (r = -0.8; P = 0.02). RANTES was a potential biomarker of transient control. This study identified virological and immunological factors, including inflammatory biomarkers associated with two different phenotypes within EC. These results may allow a more accurate definition of EC, which could help in better clinical management of these individuals and in the development of future curative approaches.IMPORTANCE There is a rare group of HIV-infected patients who have the extraordinary capacity to maintain undetectable viral load levels in the absence of antiretroviral treatment, the so-called HIV-1 elite controllers (EC). However, there is a proportion within these subjects that eventually loses this capability. In this work, we found differences in virological and immune factors, including soluble inflammatory biomarkers, between subjects with persistent control of viral replication and EC that will lose virological control. The identification of these factors could be a key point for a right medical care of those EC who are going to lose natural control of viral replication and for the design of future immunotherapeutic strategies using as a model the natural persistent control of HIV infection.

Analysis of Non-AIDS-Defining Events in HIV Controllers.

BACKGROUND: Human immunodeficiency virus (HIV) controllers have the striking ability to maintain viremia at extremely low or undetectable levels without antiretroviral treatment. Even though these patients have been widely studied, information about clinical outcomes, especially concerning to non-AIDS-defining events (nADEs), is scarce. We have analyzed the frequency and rate of nADEs and their associated factors in a large multicenter HIV controller cohort. METHODS: Data on nADEs were recorded for 320 HIV controllers within the multicenter Spanish AIDS Research Network HIV Controllers Cohort (ECRIS). Percentages and crude incidence rates (CIRs) per 100 person-years of follow-up (PYFU) were calculated for the entire follow-up period and for 2 separate periods: the period under control and the period after loss of control. These rates were compared with those for 632 noncontrollers. Demographic and immunological data collected from the controllers were included in a multivariate model to assess factors that were independently associated with nADEs in HIV controllers. RESULTS: HIV controllers experience nADEs, albeit at lower rates than patients who do not spontaneously control the virus (1.252 [95% confidence interval {CI}, .974-1.586] per 100 PYFU and 2.481 [95% CI, 2.153-2.845] per 100 PYFU, respectively; P < .001). Hepatitis C virus (HCV) coinfection was the main factor associated with nADEs in all of the studied periods. Although hepatic events were the most prevalent, they represented only approximately 30% of the total events. CIRs of cardiovascular events increased in the post-loss-of-control period. CONCLUSIONS: HCV/HIV coinfection was the main factor associated with hepatic and extrahepatic nADEs in HIV controllers. The eradication of HCV infection may ameliorate the presence of comorbidities in these patients.

Influence of mutation and recombination on HIV-1 in vitro fitness recovery.

The understanding of the evolutionary processes underlying HIV-1 fitness recovery is fundamental for HIV-1 pathogenesis, antiretroviral treatment and vaccine design. It is known that HIV-1 can present very high mutation and recombination rates, however the specific contribution of these evolutionary forces in the "in vitro" viral fitness recovery has not been simultaneously quantified. To this aim, we analyzed substitution, recombination and molecular adaptation rates in a variety of HIV-1 biological clones derived from a viral isolate after severe population bottlenecks and a number of large population cell culture passages. These clones presented an overall but uneven fitness gain, mean of 3-fold, respect to the initial passage values. We found a significant relationship between the fitness increase and the appearance and fixation of mutations. In addition, these fixed mutations presented molecular signatures of positive selection through the accumulation of non-synonymous substitutions. Interestingly, viral recombination correlated with fitness recovery in most of studied viral quasispecies. The genetic diversity generated by these evolutionary processes was positively correlated with the viral fitness. We conclude that HIV-1 fitness recovery can be derived from the genetic heterogeneity generated through both mutation and recombination, and under diversifying molecular adaptation. The findings also suggest nonrandom evolutionary pathways for in vitro fitness recovery.

Evidence of ongoing replication in a human immunodeficiency virus type 1 persistently infected cell line.

Human immunodeficiency virus type 1 (HIV-1) persistently infected cell lines are characterized by the continuous viral production without cytopathic effect. However, it is not completely clear if this production is contributed only by viral transcription or also by new cycles of viral replication. We studied an HIV-1 persistently infected cell line, designated H61-D, providing evidence of new replication cycles as sustained by: (i) a decrease in viral production, measured by p24 protein, after treatment of the culture with 3'-azydo-3'-deoxythymydine; (ii) detection of new integration events in the course of cell culture, and (iii) finding of two-long-terminal repeat circles in the cells. H61-D cells were not infected by cell-free virus, but infection was possible by co-culture with another productive-infected cell line. In conclusion, ongoing viral replication is taking place in H61-D persistent cells and new infections are mediated by a cell-to-cell spread mechanism.

Dynamics of in vitro fitness recovery of HIV-1.

The study on the evolutionary consequences of an RNA viral population's fluctuations can be approached by in vitro experiments. This work describes the fitness recovery of HIV-1 after 20 large-population passages in 10 debilitated clones. The serial passages promoted an increase in viral fitness. In addition, we detected a significant number of mutations fixed in the complete genome consensus sequence of the final viral populations. Among the mutations, events of convergent evolution with important phenotypic characteristics occurred in several independent clones. One common change, V35I, in the nuclear localization signal of the p17 protein appeared in four viruses of three different lineages. Other common alterations mapped in position E196K of the reverse transcriptase or in position S316K of the V3 loop of the gp120 residue that is associated with the X4/R5 phenotype. Together with this mutational analysis, we studied the quasispecies heterogeneity of the initial and final viruses, revealing that fitness increase correlated with an augmentation in the genetic heterogeneity of viral quasispecies. However, while heterogeneity was mostly composed of synonymous (dS) mutations in the first 10 passages performed, at passage 21 it switched to nonsynonymous (dN) substitutions, with significant differences in dN - dS values between passages 11 and 21. In summary, the HIV-1 in vitro fitness recovery depicts a multiphase process occurring first by generation of mutations followed by fixation of the beneficial ones, depicting a classical Darwinian process.

The Characteristics of the HIV-1 Env Glycoprotein Are Linked With Viral Pathogenesis.

The understanding of HIV-1 pathogenesis and clinical progression is incomplete due to the variable contribution of host, immune, and viral factors. The involvement of viral factors has been investigated in extreme clinical phenotypes from rapid progressors to long-term non-progressors (LTNPs). Among HIV-1 proteins, the envelope glycoprotein complex (Env) has been concentrated on in many studies for its important role in the immune response and in the first steps of viral replication. In this study, we analyzed the contribution of 41 Envs from 24 patients with different clinical progression rates and viral loads (VLs), LTNP-Elite Controllers (LTNP-ECs); Viremic LTNPs (vLTNPs), and non-controller individuals contemporary to LTNPs or recent, named Old and Modern progressors. We studied the Env expression, the fusion and cell-to-cell transfer capacities, as well as viral infectivity. The sequence and phylogenetic analysis of Envs were also performed. In every functional characteristic, the Envs from subjects with viral control (LTNP-ECs and vLTNPs) showed significant lower performance compared to those from the progressor individuals (Old and Modern). Regarding sequence analysis, the variable loops of the gp120 subunit of the Env (i.e., V2, V4, and mainly V5) of the progressor individuals showed longer and more glycosylated sequences than controller subjects. Therefore, HIV-1 Envs from virus of patients presenting viremic control and the non-progressor clinical phenotype showed poor viral functions and shorter sequences, whereas functional Envs were associated with virus of patients lacking virological control and with progressor clinical phenotypes. These correlations support the role of Env genotypic and phenotypic characteristics in the in vivo HIV-1 infection and pathogenesis.

Contribution of the HIV-1 Envelope Glycoprotein to AIDS Pathogenesis and Clinical Progression.

In the absence of antiviral therapy, HIV-1 infection progresses to a wide spectrum of clinical manifestations that are the result of an entangled contribution of host, immune and viral factors. The contribution of these factors is not completely established. Several investigations have described the involvement of the immune system in the viral control. In addition, distinct HLA-B alleles, HLA-B27, -B57-58, were associated with infection control. The combination of these elements and antiviral host restriction factors results in different clinical outcomes. The role of the viral proteins in HIV-1 infection has been, however, less investigated. We will review contributions dedicated to the pathogenesis of HIV-1 infection focusing on studies identifying the function of the viral envelope glycoprotein (Env) in the clinical progression because of its essential role in the initial events of the virus life-cycle. Some analysis showed that inefficient viral Envs were dominant in non-progressor individuals. These poorly-functional viral proteins resulted in lower cellular activation, viral replication and minor viral loads. This limited viral antigenic production allows a better immune response and a lower immune exhaustion. Thus, the properties of HIV-1 Env are significant in the clinical outcome of the HIV-1 infection and AIDS pathogenesis.

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

A Two-Level, Intramutant Spectrum Haplotype Profile of Hepatitis C Virus Revealed by Self-Organized Maps.

RNA viruses replicate as complex mutant spectra termed viral quasispecies. The frequency of each individual genome in a mutant spectrum depends on its rate of generation and its relative fitness in the replicating population ensemble. The advent of deep sequencing methodologies allows for the first-time quantification of haplotype abundances within mutant spectra. There is no information on the haplotype profile of the resident genomes and how the landscape evolves when a virus replicates in a controlled cell culture environment. Here, we report the construction of intramutant spectrum haplotype landscapes of three amplicons of the NS5A-NS5B coding region of hepatitis C virus (HCV). Two-dimensional (2D) neural networks were constructed for 44 related HCV populations derived from a common clonal ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells in the absence of external selective pressures. The haplotype profiles consisted of an extended dense basal platform, from which a lower number of protruding higher peaks emerged. As HCV increased its adaptation to the cells, the number of haplotype peaks within each mutant spectrum expanded, and their distribution shifted in the 2D network. The results show that extensive HCV replication in a monotonous cell culture environment does not limit HCV exploration of sequence space through haplotype peak movements. The landscapes reflect dynamic variation in the intramutant spectrum haplotype profile and may serve as a reference to interpret the modifications produced by external selective pressures or to compare with the landscapes of mutant spectra in complex in vivo environments. IMPORTANCE The study provides for the first time the haplotype profile and its variation in the course of virus adaptation to a cell culture environment in the absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer haplotype distribution with an ample basal platform and a lower number of protruding peaks. The results suggest an inferred intramutant spectrum fitness landscape structure that offers potential benefits for virus resilience to mutational inputs.

Phylodynamics of HIV-1 circulating recombinant forms 12_BF and 38_BF in Argentina and Uruguay.

BACKGROUND: Although HIV-1 CRF12_BF and CRF38_BF are two epidemiologically important recombinant lineages circulating in Argentina and Uruguay, little is known about their population dynamics. METHODS: A total of 120 "CRF12_BF-like" and 20 "CRF38_BF-like" pol recombinant sequences collected in Argentina and Uruguay from 1997 to 2009 were subjected to phylogenetic and Bayesian coalescent-based analyses to estimate evolutionary and demographic parameters. RESULTS: Phylogenetic analyses revealed that CRF12_BF viruses from Argentina and Uruguay constitute a single epidemic with multiple genetic exchanges among countries; whereas circulation of the CRF38_BF seems to be confined to Uruguay. The mean estimated substitution rate of CRF12_BF at pol gene (2.5 x 10-3 substitutions/site/year) was similar to that previously described for subtype B. According to our estimates, CRF12_BF and CRF38_BF originated at 1983 (1978-1988) and 1986 (1981-1990), respectively. After their emergence, the CRF12_BF and CRF38_BF epidemics seem to have experienced a period of rapid expansion with initial growth rates of around 1.2 year-1 and 0.9 year-1, respectively. Later, the rate of spread of these CRFs_BF seems to have slowed down since the mid-1990s. CONCLUSIONS: Our results suggest that CRF12_BF and CRF38_BF viruses were generated during the 1980s, shortly after the estimated introduction of subtype F1 in South America (~1975-1980). After an initial phase of fast exponential expansion, the rate of spread of both CRFs_BF epidemics seems to have slowed down, thereby following a demographic pattern that resembles those previously reported for the HIV-1 epidemics in Brazil, USA, and Western Europe.

High-Risk Sexual Practices Contribute to HIV-1 Double Infection Among Men Who Have Sex with Men in Madrid.

Data on the prevalence of double infection (DI) in HIV individuals are lacking in Spain. To fill this gap, we analyzed the prevalence of DI in a cohort of men who have sex with men (MSM) and examined factors contributing to DI. We selected 81 MSM attending Centro Sanitario Sandoval, a sexually transmitted diseases clinic in Madrid. We obtained by ultra-deep sequencing the proviral sequences in gag and env genes and performed a phylogenetic analysis for the identification of DI. Clinical, behavioral, host, and viral factors were studied for its association with DI. We detected six individuals with DI and one case of superinfection with a global prevalence of 8.6%. The genetic distance among the subtype B viruses in monoinfected individuals (24.4%) was lower than the distance between the two viruses in subtype B DI individuals (29.5%). Individuals with a high number of sexual contacts (>25 partners/year) had an 8.66 times higher risk of DI (p = .017). In this MSM cohort the prevalence of HIV DI was estimated at 8.6%. DI was strongly associated with the number of sexual partners. Because of the pathogenic consequences of HIV DI, this high prevalence should promote public health programs targeted at high-risk population such as MSM for the control of HIV infection and DI. HIV DI should be considered for a better clinical management of these individuals.

Permanent control of HIV-1 pathogenesis in exceptional elite controllers: a model of spontaneous cure.

Elite controllers (EC) represent a small subset of HIV-1-infected people that spontaneously control viral replication. However, natural virological suppression and absence of immune dysfunction are not always long-term sustained. We define exceptional EC (EEC) as HIV-1 subjects who maintain the EC characteristics without disease progression for more than 25 years. We analyzed three EEC, diagnosed between 1988 and 1992, who never showed signs of clinical disease progression in absence of any antiretroviral treatment. A comprehensive clinical, virological, and immunological study was performed. The individuals simultaneously exhibited ≥3 described host protective alleles, low levels of total HIV-1 DNA (<20 copies/106 CD4+ T-cells) without evidence of replication-competent viruses (<0.025 IUPM), consistent with high levels of defective genomes, strong cellular HIV-1-specific immune response, and a high poly-functionality index (>0.50). Inflammation levels of EEC were similar to HIV-1 negative donors. Remarkably, they showed an exceptional lack of viral evolution and 8-fold lower genetic diversity (<0.01 s/n) in env gene than other EC. We postulate that these EEC represent cases of spontaneous functional HIV-1 cure. A non-functional and non-genetically evolving viral reservoir along with an HIV-1-specific immune response seems to be key for the spontaneous functional cure.

Persistent HIV-controllers are more prone to spontaneously clear HCV: a retrospective cohort study.

INTRODUCTION: HIV-controllers have the ability to spontaneously maintain viraemia at low or undetectable levels in the absence of antiretroviral treatment. Furthermore, HIV-controllers seem to have a superior capacity to spontaneously clear hepatitis C virus (HCV) compared to non HIV-controllers. Some of these subjects eventually lose HIV-controller status (transient controllers), whereas some HIV-controllers show a persistent natural HIV control (persistent controllers). We aimed to analyse whether persistent controllers have superior capacity to spontaneously clear HCV compared to transient controllers. METHODS: We recruited HIV-controllers from January 1981 up to October 2016 with available antibodies to HCV (anti-HCV) data (n = 744). Factors associated with HIV spontaneous control in relation to HCV status were analysed in persistent and transient HIV-controllers with anti-HCV positive (n = 202 and n = 138 respectively) in comparison with 1700 HCV positive non HIV-controllers recruited from January 1981 up to March 2018, bivariate and multivariate analyses, following a logistic regression model, were applied. In addition, the factors related to the loss and time to lose HIV-controller status were explored (n = 744) using Log rank test and Kaplan-Meier curves, in this case the multivariate analysis consisted in a Cox regression model. RESULTS: A higher frequency of HCV spontaneous clearance was found in persistent HIV-controllers (25.5%) compared to non-controllers (10.2%). After adjusting for potential confounders, as sex, age, HIV transmission risk, CD4+ T-cell nadir and time of follow-up, HCV clearance was independently associated with persistent HIV spontaneous control (p = 0.002; OR (95% CI) = 2.573 (1.428 to 4.633)), but not with transient spontaneous control (p = 0.119; 1.589 (0.888 to 2.845)). Furthermore, persistent HIV-controllers were more likely to spontaneously clear the HCV in comparison with transient controllers (p = 0.027; 0.377 (0.159 to 0.893). Finally, not to lose or lengthen the time of losing this control was independently associated with HCV spontaneous clearance (p = 0.010; 0.503 (0.297 to 0.850). CONCLUSIONS: This study shows an association between spontaneous persistent HIV-control and HCV spontaneous clearance. The study findings support the idea of preserved immune mechanisms in persistent HIV control implicated in HCV spontaneous clearance. These results highlight persistent HIV-controllers but not transient controllers as a good model of functional HIV cure.

Human immunodeficiency virus type 1 chronic infection is associated with different gene expression in MT-4, H9 and U937 cell lines.

To investigate cellular factors involved in HIV-1 chronic infection, three cell lines chronically infected with the same HIV-1 viral isolate (s61) were studied by cDNA microarray analysis. Two T cell lines, H61 and M61, showed the characteristics of a persistent infection whereas U61 cell line displayed a latent infection pattern. Analysis of genes with altered expression in the three cell lines revealed evidence of apoptosis control by up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes. In addition, cell cycle control was affected in the two persistent T cell lines particularly through the down-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Moreover, each cell line showed specific characteristics, like in M61 cells, genes related with cellular activation and with cell migration and motility. In U61 cells, genes associated with immune response were activated. Genes with altered expression in our experiments, and not previously related with HIV such as ANXA 1 or CFLAR were detected and validated. This work revealed that different cell mechanism such as control of apoptosis and cell cycle are important for "in vitro" HIV-1 chronic infections, and discovered new genes previously not related with HIV-1 replication.