Ricardo Sobhie Diaz

Strong HIV-1-specific T cell responses in HIV-1-exposed uninfected infants and neonates revealed after regulatory T cell removal.

Background In utero transmission of HIV-1 occurs on average in only 3%–15% of HIV-1-exposed neonates born to mothers not on antiretroviral drug therapy. Thus, despite potential exposure, the majority of infants remain uninfected. Weak HIV-1-specific T-cell responses have been detected in children exposed to HIV-1, and potentially contribute to protection against infection. We, and others, have recently shown that the removal of CD4+CD25+ T-regulatory (Treg) cells can reveal strong HIV-1 specific T-cell responses in some HIV-1 infected adults. Here, we hypothesized that Treg cells could suppress HIV-1-specific immune responses in young children. Methodology/Principal Findings We studied two cohorts of children. The first group included HIV-1-exposed-uninfected (EU) as well as unexposed (UNEX) neonates. The second group comprised HIV-1-infected and HIV-1-EU children. We quantified the frequency of Treg cells, T-cell activation, and cell-mediated immune responses. We detected high levels of CD4+CD25+CD127− Treg cells and low levels of CD4+ and CD8+ T cell activation in the cord blood of the EU neonates. We observed HIV-1-specific T cell immune responses in all of the children exposed to the virus. These T-cell responses were not seen in the cord blood of control HIV-1 unexposed neonates. Moreover, the depletion of CD4+CD25+ Treg cells from the cord blood of EU newborns strikingly augmented both CD4+ and CD8+ HIV-1-specific immune responses. Conclusions/Significance This study provides new evidence that EU infants can mount strong HIV-1-specific T cell responses, and that in utero CD4+CD25+ T-regulatory cells may be contributing to the lack of vertical transmission by reducing T cell activation.

Differential Persistence of Transmitted HIV-1 Drug Resistance Mutation Classes

BACKGROUND Transmitted human immunodeficiency virus type 1 (HIV-1) drug resistance (TDR) mutations can become replaced over time by emerging wild-type viral variants with improved fitness. The impact of class-specific mutations on this rate of mutation replacement is uncertain. METHODS We studied participants with acute and/or early HIV infection and TDR in 2 cohorts (San Francisco, California, and São Paulo, Brazil). We followed baseline mutations longitudinally and compared replacement rates between mutation classes with use of a parametric proportional hazards model. RESULTS Among 75 individuals with 195 TDR mutations, M184V/I became undetectable markedly faster than did nonnucleoside reverse-transcriptase inhibitor (NNRTI) mutations (hazard ratio, 77.5; 95% confidence interval [CI], 14.7-408.2; P<.0001), while protease inhibitor and NNRTI replacement rates were similar. Higher plasma HIV-1 RNA level predicted faster mutation replacement, but this was not statistically significant (hazard ratio, 1.71 log(10) copies/mL; 95% CI, .90-3.25 log(10) copies/mL; P=.11). We found substantial person-to-person variability in mutation replacement rates not accounted for by viral load or mutation class (P<.0001). CONCLUSIONS The rapid replacement of M184V/I mutations is consistent with known fitness costs. The long-term persistence of NNRTI and protease inhibitor mutations suggests a risk for person-to-person propagation. Host and/or viral factors not accounted for by viral load or mutation class are likely influencing mutation replacement and warrant further study.

distribution of Hiv-1 subtypes seen in an Aids clinic in Sao Paulo City, Brazil

Objective: To determine the distribution of HIV‐1 subtypes in Sao Paulo, Brazil. Methods: Samples were obtained from 80 consecutive HIV‐1‐infected individuals attending the Immunodeficiency Clinic at the University of Sao Paulo in 1993. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll‐Hypaque gradient and a portion was used for routine CD4 counts; the remainder were frozen. PBMC were proteinase‐K‐digested and DNA‐purified by organic extraction. Samples were amplified for the env region of HIV, and envelope sequence subtypes determined by heteroduplex mobility analysis using prototypic subtypes as references. A subset of these were also sequenced through the C2‐V3 region of env. Results: A total 69 of 80 samples yielded env polymerase chain reaction product enabling subtype determination; samples that did not amplify were those with low DNA yields. Among 12 injecting drug users (IDU) or sexual partners of IDU, four were typed as clade F and eight as clade B. Forty‐three homosexual men or female sexual partners of bisexual men were typed as clade B. The 14 additional cases without known risk factors were typed as clade B. Conclusion: These data suggest that subtype F is related to injecting drug use in Brazil.

Brazilian Network for HIV Drug Resistance Surveillance: a survey of individuals recently diagnosed with HIV

Use of antiretrovirals is widespread in Brazil, where more than 200,000 individuals are under treatment. Although general prevalence of primary antiretroviral resistance in Brazil is low, systematic sampling in large metropolitan areas has not being performed.The HIV Threshold Survey methodology (HIV-THS, WHO) was utilized, targeting Brazils four major regions and selecting the six most populated state capitals: Sao Paulo, Rio de Janeiro, Salvador, Porto Alegre, Brasilia and Belem. We were able to sequence samples from 210 individuals with recent HIV diagnosis, 17 of them (8.1%) carrying HIV isolates with primary antiretroviral resistance mutations. Five, nine and four isolates showed mutations related to resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs), respectively. Using HIV-THS, we could find an intermediate level of transmitted resistance (5% to 15%) in Belem/Brasilia, Sao Paulo and Rio de Janeiro. Lower level of transmitted resistance (<5%) were observed in the other areas. Despite the extensive antiretroviral exposure and high rates of virologic antiretroviral failure in Brazil, the general prevalence of primary resistance is still low. However, an intermediate level of primary resistance was found in the four major Brazilian cities, confirming the critical need to start larger sampling surveys to better define the risk factors associated with transmission of resistant HIV.

Low Accumulation of L90M in Protease from Subtype F HIV-1 with Resistance to Protease Inhibitors Is Caused by the L89M Polymorphism

BACKGROUND This work evaluates the role of subtype F human immunodeficiency virus type 1 (HIV-1) protease (PR) substitutions L89M and L90M in viral replication and resistance to PR inhibitors (PIs). METHODS Subtype B and F PR genes were subjected to site-directed mutagenesis, to create and reverse the methionine at positions 89 and 90. Viruses were re-created in cell culture, and their replicative capacity was assessed by fitness assay. Generated viruses were also phenotyped for PI resistance. RESULTS The subtype F clone (89M90L) showed a replicative capacity comparable to that of the PI-susceptible subtype B clone (89L90L) and was more fit than the L89M mutated subtype B clone (89M90L). Both 89M90M subtype B and F clones presented the lowest fitness s values. The L89M mutation impacted phenotypic resistance to all PIs in half of the subtype F isolates but not in the subtype B isolates. Subtype F isolates presented a phenotypic profile similar to that of subtype B isolates when the M89L mutation was introduced. CONCLUSION The L89M mutation in subtype F viruses is a high genetic barrier to the accumulation of the L90M resistance mutation and can function as a resistance mutation, depending on the presence of other polymorphisms in the subtype F PR backbone.

GB virus type C infection modulates T-cell activation independently of HIV-1 viral load

Background:Many clinical studies have suggested a beneficial effect of GB virus type C (GBV-C) on the course of HIV-1 infection, but the mechanisms involved in such amelioration are not clear. As recent evidence has implicated cellular activation in HIV-1 pathogenesis, we investigated the effect of GBV-C viremia on T-cell activation in early HIV-1 infection. Methods:Forty-eight recently infected HIV-1 patients (23 GBV-C viremic) were evaluated for T-cell counts, expanded immunophenotyping GBV-C RNA detection, and HIV-1 viral load. Nonparametric univariate and multivariate analyses were carried out to identify variables associated with cellular activation, including GBV-C status, HIV-1 viral load, T lymphocyte counts, and CD38 and chemokine (C–C motif) receptor 5 (CCR5) surface expression. Finding:We not only confirmed the positive correlation between HIV-1 viral load and the percentage of T cells positive for CD38+CD8+ but also observed that GBV-C viremic patients had a lower percentage of T cells positive for CD38+CD4+, CD38+CD8+, CCR5+CD4+, and CCR5+CD8+ compared with HIV-1-infected patients who were not GBV-C viremic. In regression models, GBV-C RNA+ status was associated with a reduction in the CD38 on CD4+ or CD8+ T cells and CCR5+ on CD8+ T cells, independent of the HIV-1 viral load or CD4+ and CD8+ T-cell counts. These results were also supported by the lower expression of CD69 and CD25 in GBV-C viremic patients. Interpretation:The association between GBV-C replication and lower T-cell activation may be a key mechanism involved in the protection conferred by this virus against HIV-1 disease progression to immunodeficiency in HIV-1-infected patients.

Prevalence of mutations related to HIV-1 antiretroviral resistance in Brazilian patients failing HAART

BACKGROUND Current guidelines for antiretroviral (ARV) therapy recommend at least triple-drug combination, the so-called highly active antiretroviral therapy (HAART). Not all patients respond to HAART and the development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV. OBJECTIVE In an attempt to correlate the HIV therapeutic failure with reverse transcriptase (RT) and protease resistance mutations, we describe the ARV resistance profile in patients failing HAART in Brazil. We studied 267 Brazilian HIV-1 infected patients failing HAART looking for mutations in RT and protease genes. The mutation profile of the viruses infecting these individuals were deduced and correlated to laboratorial parameters. STUDY DESIGN Two different HIV-1 genomic regions were targeted for PCR amplification, the protease (pro) and pol RT (palm finger region) genes. The mutations related to drug resistance in RT gene was analyzed using a line probe assay (LIPA(R)) and pro amino acids positions 82 and 90 were screened through RFLP using HincII restriction digestion. RESULTS There was strong correlation between the mutation in the pro and RT genes and therapeutic failure. The main mutation found in RT gene was the M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). In the pro gene the main mutation found was L90M (26%) followed by dual substitution in L90M and V82A (6%). All mutations profiles matched very well with the patients drug regimen. CONCLUSIONS This study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance.

Toll-like receptor pathway signaling is differently regulated in neutrophils and peripheral mononuclear cells of patients with sepsis, severe sepsis, and septic shock.

Objectives:Up- and down-regulation of inflammatory response was described in blood cells from septic patients, according to the stage of sepsis and the cells evaluated. This study aimed to evaluate the Toll-like receptor (TLR) signaling pathway gene expression in peripheral blood mononuclear cells (PBMC) and neutrophils in patients throughout the different stages of sepsis. Design:Prospective, observational study. Settings:Two emergency rooms and two intensive care units in one university and one teaching hospital. Patients and Controls:A total of 15 septic patients, five with sepsis, five with severe sepsis, and five with septic shock, in addition to five healthy volunteers were enrolled. Interventions:None. Measurements and Main Results:The Human-TLR Signaling Pathway, which comprises 84 genes related to TLR-mediated signal transduction, was evaluated by real time polymerase chain reaction in PBMC and neutrophils obtained from patients and controls. The fold change for each gene (2(−&Dgr;&Dgr;Ct)) was compared between the groups. Genes with fold changes greater than 2 and significant changes in &Dgr;CT are reported as differently expressed. The fold change ratios in PBMC gene expression between septic patients and healthy controls revealed a dynamic process according to the stage of sepsis, tending toward down-regulation of the TLR signaling pathway in PBMC in the more severe forms of the disease. However, the differential gene expression was restricted to five down-regulated genes in septic shock patients, which are found in the effector and downstream pathways. Neutrophils showed a different pattern of adaptation. Patients with sepsis, severe sepsis, and septic shock presented a broad gene up-regulation, which included all functional groups evaluated and persisted throughout the stages of the disease. Conclusions:TLR-signaling pathway genes are differently regulated in PBMC and neutrophils of septic patients, and are dynamically modulated throughout the different stages of sepsis.

Genetic diversity and HIV-1 incidence estimation among cocaine users in Sao Paulo Brazil.

Summary: We describe HIV‐1 incidence and the prevalence of genetic subtypes among cocaine users in São Paulo, Brazil. A cross‐sectional HIV‐1 survey was carried out among 839 current cocaine users attending seven drug treatment units in the São Paulo metropolitan area from 1997 to 1998. HIV‐1 subtyping was performed among 41 positive individuals using the heteroduplex mobility assay and DNA sequencing. Participants were mainly male (95.7%) with a history of previous imprisonment (54%), and the mean age was 26.9 years (SD = 7.2). The majority (64.4%) were current crack cocaine users, and 82.1% of the total participants were noninjectors. HIV‐1 seroprevalence was 4.9% (95% confidence interval [CI], 3.6%‐6.6%), and the incidence (estimated by the sensitive/less‐sensitive immunoassay testing strategy) was 0.71% per year (95% CI, 0.07‐3.03). HIV‐1 subtype B was predominant (90.3%), followed by subtype F. There was no statistically significant association between HIV‐1 subtype and specific route of drug administration. Our incidence data show evidence of recent HIV‐1 transmission among cocaine users, mainly among noninjectors. Detection of recently infected HIV‐1 cases linked to genetic diversity analysis may provide baseline information for public health interventions in this sentinel group.

Influence of Hepatitis B Virus (HBV) Genotype on the Clinical Course of Disease in Patients Coinfected with HBV and Hepatitis Delta Virus

OBJECTIVE We evaluated the influence of hepatitis B virus (HBV) genotype on the course of disease in patients coinfected with HBV and hepatitis delta virus (HDV). METHODS We evaluated HBV genotypes in 190 patients, 140 of whom had chronic HBV monoinfection and 50 of whom had chronic HBV-HDV coinfection. Real-time polymerase chain reactions for the amplification of HBV DNA and HDV RNA were developed, and we compared the patient groups with respect to HBV genotype, viral load, alanine aminotransferase (ALT) and bilirubin levels, and disease severity. RESULTS Coinfected patients had higher ALT and bilirubin levels as well as a higher prevalence of liver cirrhosis and liver carcinoma. ALT levels were higher among individuals coinfected with HDV and HBV genotype F than among individuals infected only with HBV genotype F. Among HDV-HBV-coinfected patients, HDV load was lower among those infected with HBV genotype A than among those infected with HBV genotype D or genotype F. CONCLUSION Liver inflammation and HDV load are influenced by HBV genotype in individuals coinfected with HBV and HDV.