Natalia Laufer

Magnitude, Breadth, and Functional Profile of T-Cell Responses during Human Immunodeficiency Virus Primary Infection with B and BF Viral Variants

ABSTRACT The molecular pattern of the human immunodeficiency virus (HIV) epidemic in Argentina provides an appropriate scenario to study cellular immune responses in patients with non-clade B infection. We aimed to map T-cell responses in patients infected with BF recombinant variants and compare them with those of clade B patients. Sixteen recently infected patients were enrolled and grouped by viral subtype. Nef-specific responses were evaluated with a peptide matrix-based gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay using B and BF overlapping peptides. Cross-clade and clade-specific responses were found. A correlation between B versus BF Nef-specific responses was identified. Detailed analysis at the single-peptide level revealed that BF patients show a narrower response but greater magnitude. Nef immunodominant responses agreed with previous publications, although the B loop was targeted at an unexpectedly high frequency. The putative HLA allele(s) restricting each positive response was determined. Single-peptide level screening with two different peptide sets uncovered discordant responses (mostly caused by peptide offsetting) and allowed detection of increased breadth. Positive responses identified by ELISPOT assay were further studied by intracellular cytokine staining. These were almost exclusively mediated by CD8 T cells. Characterization of concordant responses revealed that cells show distinct functional profiles, depending on the peptide presented. Last, quality (in terms of polyfunctionality) of T cells was associated with better viral replication containment. Overall, interclade differences in the frequency of epitopes recognized, structural domains targeted, and magnitude of responses were identified. Screening T-cell responses with multiple sets increased sensitivity. Further support for the notion of polyfunctional CD8+ T-cell requirement to better control viral replication is also provided.

MicroRNAs differentially present in the plasma of HIV elite controllers reduce HIV infection in vitro

Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

ABSTRACT The important role of the CD8+ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8+ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8+ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4+ T-cell set points were observed in PHI subjects with higher HIV-specific CD8+ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8+ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.

Acute retroviral syndrome and high baseline viral load are predictors of rapid HIV progression among untreated Argentinean seroconverters

BackgroundDiagnosis of primary HIV infection (PHI) has important clinical and public health implications. HAART initiation at this stage remains controversial.MethodsOur objective was to identify predictors of disease progression among Argentinean seroconverters during the first year of infection, within a multicentre registry of PHI-patients diagnosed between 1997 and 2008. Cox regression was used to analyze predictors of progression (LT-CD4 < 350 cells/mm3, B, C events or death) at 12 months among untreated patients.ResultsAmong 134 subjects, 74% presented with acute retroviral syndrome (ARS). Seven opportunistic infections (one death), nine B events, and 10 non-AIDS defining serious events were observed. Among the 92 untreated patients, 24 (26%) progressed at 12 months versus three (7%) in the treated group (p = 0.01). The 12-month progression rate among untreated patients with ARS was 34% (95% CI 22.5-46.3) versus 13% (95% CI 1.1-24.7) in asymptomatic patients (p = 0.04). In univariate analysis, ARS, baseline LT-CD4 < 350 cells/mm3, and baseline and six-month viral load (VL) > 100,000 copies/mL were associated with progression. In multivariate analysis, only ARS and baseline VL > 100,000 copies/mL remained independently associated; HR: 8.44 (95% CI 0.97-73.42) and 9.44 (95% CI 1.38-64.68), respectively.ConclusionsIn Argentina, PHI is associated with significant morbidity. HAART should be considered in PHI patients with ARS and high baseline VL to prevent disease progression.

Th17 and Th17/Treg ratio at early HIV infection associate with protective HIV-specific CD8 + T-cell responses and disease progression

The aim of this study was to analyze Th17 and Treg subsets and their correlation with anti-HIV T-cell responses and clinical parameters during (acute/early) primary HIV infection (PHI) and up to one year post-infection (p.i). Samples from 14 healthy donors (HDs), 40 PHI patients, 17 Chronics, and 13 Elite controllers (ECs) were studied. The percentages of Th17 and Treg subsets were severely altered in Chronics, whereas all HIV-infected individuals (including ECs) showed Th17/Treg imbalance compared to HDs, in concordance with higher frequencies of activated CD8+ T-cells (HLA-DR+/CD38+). Better clinical status (higher CD4 counts, lower viral loads and activation) was associated with higher Th17 and lower Treg levels. We found positive correlations between Th17 at baseline and anti-HIV CD8+ T-cell functionality: viral inhibitory activity (VIA) and key polyfunctions (IFN-γ+/CD107A/B+) at both early and later times p.i, highlighting the prognostic value of Th17 cells to preserve an effective HIV T-cell immunity. Th17/Treg ratio and the IL-17 relative mean fluorescence intensity (rMFI of IL-17) were also positively correlated with VIA. Taken together, our results suggested a potential link between Th17 and Th17/Treg ratio with key HIV-specific CD8+ T-cell responses against the infection.

Hepatitis B precore/core promoter mutations in isolates from HBV-monoinfected and HBV-HIV coinfected patients: a 3-yr prospective study.

BACKGROUND The course of chronic HBV infection is modified by HIV-coexistence. OBJECTIVE To analyze the role of HBV genomic heterogeneity in basal core promoter (BCP) and precore (Pc) genomic regions. STUDY DESIGN In a 3-yr prospective study, 39 HBV infected patients (20 monoinfected and 19 HIV-coinfected) were included. Eighty-two HBV isolates were studied at quasispecies level in the BCP/Pc genomic region. Clinical records obtained include data on lamivudine therapy and resistance mutations, HBV and HIV-viral load. RESULTS HBV isolates were predominantly ascribed to genotype (Gt) A2 among HBV-monoinfected and HIV-coinfected patients. BCP mutations in isolates from monoinfected patients were significantly more frequent than in those from coinfected ones, irrespective of the HBe expression pattern (p<0.0001). Regardless of the HIV-coexistence, the Pc mutation at G1896A only barely appeared among clone-derived sequences of GtF1 isolates, mainly from HBe(-) HBV-monoinfected patients. CONCLUSIONS HBV isolates characterized from HIV-coinfected patients seem to be more prone to exhibit a wild type genomic pattern at BCP regulatory region with respect to those from HBV-monoinfected ones. Besides, mutations at Pc region might be genotype-dependent in their frequency but not on HIV co-presence related.

Early Skewed Distribution of Total and HIV-Specific CD8+ T-Cell Memory Phenotypes during Primary HIV Infection Is Related to Reduced Antiviral Activity and Faster Disease Progression

The important role of the CD8+ T-cells on HIV control is well established. However, correlates of immune protection remain elusive. Although the importance of CD8+ T-cell specificity and functionality in virus control has been underscored, further unraveling the link between CD8+ T-cell differentiation and viral control is needed. Here, an immunophenotypic analysis (in terms of memory markers and Programmed cell death 1 (PD-1) expression) of the CD8+ T-cell subset found in primary HIV infection (PHI) was performed. The aim was to seek for associations with functional properties of the CD8+ T-cell subsets, viral control and subsequent disease progression. Also, results were compared with samples from Chronics and Elite Controllers. It was found that normal maturation of total and HIV-specific CD8+ T-cells into memory subsets is skewed in PHI, but not at the dramatic level observed in Chronics. Within the HIV-specific compartment, this alteration was evidenced by an accumulation of effector memory CD8+ T (TEM) cells over fully differentiated terminal effector CD8+ T (TTE) cells. Furthermore, higher proportions of total and HIV-specific CD8+ TEM cells and higher HIV-specific TEM/(TEM+TTE) ratio correlated with markers of faster progression. Analysis of PD-1 expression on total and HIV-specific CD8+ T-cells from PHI subjects revealed not only an association with disease progression but also with skewed memory CD8+ T-cell differentiation. Most notably, significant direct correlations were obtained between the functional capacity of CD8+ T-cells to inhibit viral replication in vitro with higher proportions of fully-differentiated HIV-specific CD8+ TTE cells, both at baseline and at 12 months post-infection. Thus, a relationship between preservation of CD8+ T-cell differentiation pathway and cell functionality was established. This report presents evidence concerning the link among CD8+ T-cell function, phenotype and virus control, hence supporting the instauration of early interventions to prevent irreversible immune damage.

Hepatitis B virus, hepatitis C virus and HIV coinfection among people living with HIV/AIDS in Buenos Aires, Argentina.

The HIV epidemic in Argentina has changed since the first case was reported in 1982. Since the beginning of the 1990s, a decrease in the number parenterally acquired infections has been observed, with a marked increase in transmission through unprotected sexual contact (heterosexual and homosexual), and in the number of women living with HIV/AIDS [1]. Few prevalence studies have addressed the hepatitis B and C virus coinfection in Argentina. We performed this study in a large single clinic in Buenos Aires, taking care of more than 3,000 HIV patients. During a seven-month period (9/2004 to 3/2005), all HIV-positive patients ≥ 18 years old, who were followed up at our unit and who had their scheduled controls for HIV viral load (VL) at the Argentinean National Reference Centre for AIDS (CNRS), were invited to participate in the study. The study was approved by the Fernandez Hospital Ethics Committee. Patients gave their informed consent to be included in the study. Six hundred subjects were asked to enter the study, and 593 accepted. Studied population: 65.6% males, 64% young adults between 20 and 40 years old. The main route of HIV infection was through sexual contact (70%). Of the 593 samples, 52% (n=308) showed positive results for serological markers (HBcAb/HBsAg/anti-HCV) for hepatitis B or C coinfection. Coinfection rates and subjects characteristics are described in Table 1. Table 1 Demographics, rates of HCV/HBV coinfection, main routes of infection, median levels of CD4 cell count, HIV viral load, HAART, frequency of ALT level among HIV-positive patients studied HBV genotype A was identified in 85% of the samples with detectable HBV DNA, instead of genotype F, the most prevalent in our country among blood donors [2]. Among the 22 HBsAg-positive samples, four showed mutations in the rt domain of the pol gene. These samples belonged to patients who had a prolonged history of exposure to lamivudine (median: 25 months) and none of them had received tenofovir. This data reinforces the importance of adequate management of drugs with HBV/HIV dual activity when selecting antiretroviral therapies in coinfected patients, as stated in the last update of the European and North American guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents [3,4]. The main isolated HCV genotype was 1 (65%), followed by genotype 3 (16.5%). Our study shows a change in the prevalence of HCV and HBV coinfection comparing with a previous report of the same geographical area [5], among people living with HIV/AIDS in Buenos Aires, the city with the highest prevalence of HIV in Argentina. A reduction in prevalence rates of dual HIV/HCV (from 58.5% in 1999 to 21%, p 0.000) and HIV/HBV coinfection (from 14.5% in 1999 to 3.7%, p 0.000) was observed. This could be related to the decreasing number of injecting drug users in our country because of the introduction of non-injecting recreational drugs and the increasing transmission through unprotected sex [1]. Despite the decrease in the rates of HCV/HIV and HBV/HIV coinfections the prevalence of those coinfections continues to be high and it could lead to the increase in the morbidity and mortality associated with liver disease, especially in the context of expanded antirretroviral therapy in Argentina. The epidemiological data presented in this study provides information for the development of prevention campaigns for hepatitis C through harm reduction policies and for hepatitis B vaccination programs among people living with HIV/AIDS.

Env-Specific IgA from Viremic HIV-Infected Subjects Compromises Antibody-Dependent Cellular Cytotoxicity

ABSTRACT Elucidating the factors that modulate HIV-specific antibody-dependent cellular cytotoxicity (ADCC) will help in understanding its role in HIV immunity. The aim of this study was to determine whether IgA could modify the magnitude of ADCC in HIV infection, abrogating its protective role. Plasma samples from 20 HIV-positive (HIV+) subjects enrolled during primary HIV infection (PHI), 10 chronically infected subjects (chronic), and 7 elite controllers (EC) were used. ADCC was determined by using a fluorometric ADCC assay, before and after removal of plasma IgA. Data were analyzed by using nonparametric statistics. ADCC was documented in 80% of PHI enrollment samples and in 100% of PHI 12-month, chronic, and EC samples; it peaked after acute infection, reached a plateau in chronic infection, and decreased after initiation of antiretroviral treatment (ART). Significant associations between ADCC and disease progression were found only after removal of plasma IgA from 12-month PHI samples: the magnitude of ADCC not only increased after IgA removal but also correlated with CD4+ T-cell preservation. This work provides evidence that gp120-specific IgA was capable of modifying ADCC responses during natural HIV infection for the first time and adds to similar evidence provided in other settings. Furthermore, it underscores the complexity of the ADCC phenomenon and will help in an understanding of its underlying mechanisms. IMPORTANCE Although the induction of ADCC-mediating antibodies in HIV-infected subjects has been extensively documented, the association of these antibodies with protection from disease progression is poorly understood. Here, we demonstrate that plasma IgA is a factor capable of modifying the magnitude of IgG-mediated ADCC in HIV infection, mitigating its beneficial effect. These results help in understanding why previous studies failed to demonstrate correlations between ADCC and disease progression, and they also contribute to the notion that an HIV vaccine should stimulate the production of ADCC-mediating IgG antibodies but not IgA.

HIV-1 tropism dynamics and phylogenetic analysis from longitudinal ultra-deep sequencing data of CCR5- and CXCR4-using variants.

Objective Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships. Methods Here, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained. Results Although a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence. Conclusions CXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors.