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1.  Subsets of Memory CD4+ T Cell and Bactericidal Antibody Response to Neisseria meningitidis Serogroup C after Immunization of HIV-Infected Children and Adolescents 
PLoS ONE  2014;9(12):e115887.
Meningococcal disease is endemic in Brazil, with periodic outbreaks and case fatality rates reach as high as 18 to 20% of cases. Conjugate vaccines against meningococci are immunogenic in healthy children. However, we have previously shown a poor bactericidal antibody response to a Men C conjugate vaccine in Brazilian HIV-infected children and adolescents after a single vaccine administration. The goal of the present work was to investigate associations between bactericidal antibody response induced by MenC vaccine and the frequency and activation profile (expression of CD38, HLA-DR and CCR5 molecules) of total CD4+ memory T cell sub-populations in HIV-1-infected children and adolescents. Responders to vaccination against MenC had a predominance (about 44%) of CD4+ TINTERMEDIATE subset followed by TTRANSITIONAL memory subset (23 to 26%). Importantly, CD4+ TINT frequency was positively associated with bactericidal antibody response induced by vaccination. The positive correlation persisted despite the observation that the frequency TINT CD38+HLA-DR+ was higher in responders. In contrast, CD4+ TCENTRAL MEMORY (TCM) subset negatively correlated with bactericidal antibodies. In conclusion, these data indicate that less differentiated CD+ T cells, like TCM may be constantly differentiating into intermediate and later differentiated CD4+ T cell subsets. These include CD4 TINT subset which showed a positive association with bactericidal antibodies.
doi:10.1371/journal.pone.0115887
PMCID: PMC4274125  PMID: 25532028
2.  Vaccine-Induced Gag-Specific T Cells Are Associated With Reduced Viremia After HIV-1 Infection 
The Journal of Infectious Diseases  2013;208(8):1231-1239.
The contribution of host T-cell immunity and HLA class I alleles to the control of human immunodeficiency virus (HIV-1) replication in natural infection is widely recognized. We assessed whether vaccine-induced T-cell immunity, or expression of certain HLA alleles, impacted HIV-1 control after infection in the Step MRKAd5/HIV-1 gag/pol/nef study. Vaccine-induced T cells were associated with reduced plasma viremia, with subjects targeting ≥3 gag peptides presenting with half-log lower mean viral loads than subjects without Gag responses. This effect was stronger in participants infected proximal to vaccination and was independent of our observed association of HLA-B*27, –B*57 and –B*58:01 alleles with lower HIV-1 viremia. These findings support the ability of vaccine-induced T-cell responses to influence postinfection outcome and provide a rationale for the generation of T-cell responses by vaccination to reduce viremia if protection from acquisition is not achieved. Clinical trials identifier: NCT00095576.
doi:10.1093/infdis/jit322
PMCID: PMC3778967  PMID: 23878319
HIV-1 vaccine; Step study; Gag-specific T cells; HLA class I alleles
3.  Hyperferritinaemia in Dengue Virus Infected Patients Is Associated with Immune Activation and Coagulation Disturbances 
Background
During a dengue outbreak on the Caribbean island Aruba, highly elevated levels of ferritin were detected in dengue virus infected patients. Ferritin is an acute-phase reactant and hyperferritinaemia is a hallmark of diseases caused by extensive immune activation, such as haemophagocytic lymphohistiocytosis. The aim of this study was to investigate whether hyperferritinaemia in dengue patients was associated with clinical markers of extensive immune activation and coagulation disturbances.
Methodology/Principal Findings
Levels of ferritin, standard laboratory markers, sIL-2R, IL-18 and coagulation and fibrinolytic markers were determined in samples from patients with uncomplicated dengue in Aruba. Levels of ferritin were significantly increased in dengue patients compared to patients with other febrile illnesses. Moreover, levels of ferritin associated significantly with the occurrence of viraemia. Hyperferritinaemia was also significantly associated with thrombocytopenia, elevated liver enzymes and coagulation disturbances. The results were validated in a cohort of dengue virus infected patients in Brazil. In this cohort levels of ferritin and cytokine profiles were determined. Increased levels of ferritin in dengue virus infected patients in Brazil were associated with disease severity and a pro-inflammatory cytokine profile.
Conclusions/Significance
Altogether, we provide evidence that ferritin can be used as a clinical marker to discriminate between dengue and other febrile illnesses. The occurrence of hyperferritinaemia in dengue virus infected patients is indicative for highly active disease resulting in immune activation and coagulation disturbances. Therefore, we recommend that patients with hyperferritinaemia are monitored carefully.
Author Summary
Ferritin is an acute-phase reactant and produced by reticulo-endothelial cells in response to inflammation and infection. In general, ferritin levels are increased in inflammatory conditions, but in this study we found that ferritin levels were much higher in dengue virus infected patients than in patients with other febrile illnesses. This indicates that ferritin could be used as a marker to discriminate between dengue and other febrile diseases. Moreover, the presence of hyperferritinaemia (ferritin levels≥500 µg/L) was associated with markers of immune activation and coagulation disturbances and clinical disease severity, suggesting that it could serve as a marker of activity of disease. Clinical markers to determine the presence and severity of dengue virus infection are important for diagnostic and treatment purposes. Our results indicate that increased ferritin levels could be used to increase the likelihood on a positive dengue diagnosis. Moreover, patients with hyperferritinaemia should be monitored carefully, because they are at risk to develop severe disease due to extensive immune activation.
doi:10.1371/journal.pntd.0003214
PMCID: PMC4191960  PMID: 25299654
4.  IVIg Immune Reconstitution Treatment Alleviates the State of Persistent Immune Activation and Suppressed CD4 T Cell Counts in CVID 
PLoS ONE  2013;8(10):e75199.
Common variable immunodeficiency (CVID) is characterized by defective B cell function, impaired antibody production, and increased susceptibility to bacterial infections. Here, we addressed the hypothesis that poor antibody-mediated immune control of infections may result in substantial perturbations in the T cell compartment. Newly diagnosed CVID patients were sampled before, and 6–12 months after, initiation of intravenous immunoglobulin (IVIg) therapy. Treatment-naïve CVID patients displayed suppressed CD4 T cell counts and myeloid dendritic cell (mDC) levels, as well as high levels of immune activation in CD8 T cells, CD4 T cells, and invariant natural killer T (iNKT) cells. Expression of co-stimulatory receptors CD80 and CD83 was elevated in mDCs and correlated with T cell activation. Levels of both FoxP3+ T regulatory (Treg) cells and iNKT cells were low, whereas soluble CD14 (sCD14), indicative of monocyte activation, was elevated. Importantly, immune reconstitution treatment with IVIg partially restored the CD4 T cell and mDC compartments. Treatment furthermore reduced the levels of CD8 T cell activation and mDC activation, whereas levels of Treg cells and iNKT cells remained low. Thus, primary deficiency in humoral immunity with impaired control of microbial infections is associated with significant pathological changes in cell-mediated immunity. Furthermore, therapeutic enhancement of humoral immunity with IVIg infusions alleviates several of these defects, indicating a relationship between poor antibody-mediated immune control of infections and the occurrence of abnormalities in the T cell and mDC compartments. These findings help our understanding of the immunopathogenesis of primary immunodeficiency, as well as acquired immunodeficiency caused by HIV-1 infection.
doi:10.1371/journal.pone.0075199
PMCID: PMC3793979  PMID: 24130688
6.  Nonhuman primate models and the failure of the Merck HIV-1 vaccine in humans 
Nature medicine  2008;14(6):617-621.
The recent announcement that a replication defective adenovirus-type 5 Gag-Pol-Nef HIV-1 vaccine developed by Merck failed in the STEP human Phase IIb efficacy trial to either prevent HIV-1 infection or to suppress viral load in subjects who subsequently became infected, was predicted by studies that had evaluated analogous vaccines in the simian immunodeficiency virus (SIV) challenge/rhesus macaque model. In contrast, vaccine protection studies in macaques that used a chimeric simian-human immunodeficiency virus (SHIV89.6P) challenge failed to predict the human trial results. Adenovirus-vector based vaccines did not protect animals from infection after SHIV89.6P challenge but did cause a substantial reduction in viral load and a preservation of CD4+ T-cell counts post-infection, findings that were not reproduced in the human trials. While disappointing for the clinical development of Merck’s vaccine candidate, these studies now enable vaccine designers to utilize the SIV-challenged macaque model with more confidence, thus facilitating the future prioritization of candidate vaccines. Vaccine designers must now develop T-cell vaccine strategies that reduce viral load after heterologous challenge.
doi:10.1038/nm.f.1759
PMCID: PMC3697853  PMID: 18535579
7.  Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease 
Background
Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection.
Methods
Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of São Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles.
Results
Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease.
Conclusions
The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.
Author Summary
The pathogenesis of severe dengue virus (DENV) infection is still not fully understood. It is hypothesized that it is caused by a cytokine storm as is described in severe sepsis. In the sepsis field, the potent immunostimulator lipopolysaccharide (LPS) is proposed to play an important role in the development of a cytokine storm. In a previous study we have found elevated levels of LPS in children with severe DENV infection. In this study we have investigated if we could confirm that microbial translocation occurs in DENV-infected patients. Moreover, we have determined the levels of thirty cytokines to get more insight in the cytokine storm during DENV infections and we have investigated whether microbial translocation is associated with immune activation. The patients in this cohort were classified according to their clinical presentation. Furthermore, a cluster analysis based on the expression of the determined cytokines was applied to identify patients with similar cytokine profiles. With these two techniques, we identified cytokines that may contribute significantly to the cytokine storm, and we could relate elevated levels of LPS to patients with a pro-inflammatory cytokine profile.
doi:10.1371/journal.pntd.0002236
PMCID: PMC3662706  PMID: 23717702
8.  CD57 Expression and Cytokine Production by T Cells in Lesional and Unaffected Skin from Patients with Psoriasis 
PLoS ONE  2013;8(2):e52144.
Background
The immunopathogenic mechanisms leading to psoriasis remain unresolved. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and the proportion of CD57 expressing CD8+ T cells is increased in a number of inflammatory conditions.
Methodology
We examined the expression of CD57 on T cells in the skin of patients affected with psoriasis, comparing lesional and unaffected skin. We also assessed functionality of the T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-gamma, IL-2, IL-33, TNF-alpha, IL-21, IL-22, and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin biopsies of psoriasis patients.
Principal Findings
We observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly higher in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriatic lesional skin produced higher levels of IL-17A, IL-22, and IFN-gamma compared to unaffected skin, while sorted CD8+ T cells from lesional skin produced higher levels of IL-17, IL-22, IFN-gamma, TNF-alpha, and IL-2 compared to unaffected skin.
Conclusions/Significance
These findings suggest that T cells in unaffected skin from psoriasis patients exhibit a phenotype compatible with replicative inability. As they have a lower replicative capacity, CD57+ T cells are less frequent in lesional tissue due to the high cellular turnover.
doi:10.1371/journal.pone.0052144
PMCID: PMC3585296  PMID: 23468834
9.  Expansion in CD39+ CD4+ Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications 
HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4+ T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. The CD39 ectonucleotidase receptor is expressed on CD4+ T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39+CD25+) and effector (CD39+CD25−) function. Here, we investigated the expression of CD39 on CD4+ T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. The frequency of CD39+ CD4+ T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39+CD25− CD4+ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39+CD25+ regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39−CD25+ T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4+ T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4+ T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.
Author Summary
Human T-lymphotropic virus type 1 (HTLV-1) has been estimated to infect 10–20 million worldwide. The majority of infected individuals are asymptomatic, however, 2% to 3% develop a neurodegenerative disorder called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The reasons why persons with HTLV-1 develop these complications appear to be multiple and complex. Cellular immune response has been implicated in the development of inflammatory alterations in these patients, however the pathogenic mechanisms for disease progression remain unclear. Regulatory CD4+ T cells (Treg) and Th17 cells derive from a common progenitor and conflicting results regarding frequency and function are found in the development of HAM/TSP. The expression of the CD39 ectoenzyme, a molecule that can mediate immunostimulatory and inhibitory effects, is useful to define IL-17 secreting cell populations, suppressive CD4+ T cells and CD4+ T cells with immunostimulatory properties. The interplay of these T-cell subsets may reveal important aspects of HAM/TSP pathogenesis. In this study, we performed an evaluation of the immunoregulatory CD4+ T-cell subsets defined by CD39 expression including Th17 cells. Our results present phenotypic and functional alterations in the CD4+ T cell profile that could account for the transition from asymptomatic status to HAM/TSP, predicting clinical disease risk and tracking disease progression.
doi:10.1371/journal.pntd.0002028
PMCID: PMC3566991  PMID: 23409198
10.  Human Endogenous Retrovirus K(HML-2) Gag and Env specific T-cell responses are not detected in HTLV-I-infected subjects using standard peptide screening methods 
Background
An estimated 10–20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial.
Results
Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets.
Conclusions
Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.
doi:10.1186/1477-5751-12-3
PMCID: PMC3560086  PMID: 23305161
HTLV-I; Human endogenous retrovirus; T-cells; HTLV-1-associated myelopathy/tropical spastic paraparesis
11.  Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children 
PLoS ONE  2012;7(9):e45733.
As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an “immune exhaustion”, with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28−CD57+CD8+ T cells between the groups. However, the frequency of Tim-3+CD8+ and Tim-3+CD4+ exhausted T cells, but not PD-1+ T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1+CD8+ T cells were directly associated with T cell immune activation in children. The frequency of Tim-3+CD8+ T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.
doi:10.1371/journal.pone.0045733
PMCID: PMC3454343  PMID: 23029209
12.  Influence of HAART on Alternative Reading Frame Immune Responses over the Course of HIV-1 Infection 
PLoS ONE  2012;7(6):e39311.
Background
Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8+ T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo.
Methodology/Principal findings
In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased.
Conclusions/Significance
These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.
doi:10.1371/journal.pone.0039311
PMCID: PMC3387156  PMID: 22768072
13.  Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance 
PLoS ONE  2012;7(5):e36494.
Background
Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance.
Methods/Results
We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples.
Conclusion
The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3–5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.
doi:10.1371/journal.pone.0036494
PMCID: PMC3344889  PMID: 22574170
14.  Differential Persistence of Transmitted HIV-1 Drug Resistance Mutation Classes 
The Journal of Infectious Diseases  2011;203(8):1174-1181.
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 log10 copies/mL; 95% CI, .90–3.25 log10 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.
doi:10.1093/infdis/jiq167
PMCID: PMC3107558  PMID: 21451005
16.  Whole-Genome Characterization of Human and Simian Immunodeficiency Virus Intrahost Diversity by Ultradeep Pyrosequencing▿  
Journal of Virology  2010;84(22):12087-12092.
Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.
doi:10.1128/JVI.01378-10
PMCID: PMC2977871  PMID: 20844037
17.  HTLV-1 Tax Specific CD8+ T Cells Express Low Levels of Tim-3 in HTLV-1 Infection: Implications for Progression to Neurological Complications 
The T cell immunoglobulin mucin 3 (Tim-3) receptor is highly expressed on HIV-1-specific T cells, rendering them partially “exhausted” and unable to contribute to the effective immune mediated control of viral replication. To elucidate novel mechanisms contributing to the HTLV-1 neurological complex and its classic neurological presentation called HAM/TSP (HTLV-1 associated myelopathy/tropical spastic paraparesis), we investigated the expression of the Tim-3 receptor on CD8+ T cells from a cohort of HTLV-1 seropositive asymptomatic and symptomatic patients. Patients diagnosed with HAM/TSP down-regulated Tim-3 expression on both CD8+ and CD4+ T cells compared to asymptomatic patients and HTLV-1 seronegative controls. HTLV-1 Tax-specific, HLA-A*02 restricted CD8+ T cells among HAM/TSP individuals expressed markedly lower levels of Tim-3. We observed Tax expressing cells in both Tim-3+ and Tim-3− fractions. Taken together, these data indicate that there is a systematic downregulation of Tim-3 levels on T cells in HTLV-1 infection, sustaining a profoundly highly active population of potentially pathogenic T cells that may allow for the development of HTLV-1 complications.
Author Summary
The retrovirus, Human T lymphotropic virus type 1 (HTLV-1) infects 10–20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV-1 infection. The precise role of CD8+ killer T cells in the control or contribution of HTLV-1 disease progression remains unclear. The T-cell immunoglobulin mucin domain-containing (Tim) proteins are type 1 transmembrane proteins. Three human Tim proteins (Tim-1, -3, and -4) exist and display markedly diverse expression patterns and functions. Tim-3 is upregulated on CD8+ T cells during chronic viral infections leading to a population of poorly functioning T cells. We investigated the expression of Tim-3 on T cells from patients with asymptomatic and symptomatic HTLV-1 infection and compared this with HTLV-1 uninfected donors. Patients diagnosed with HAM/TSP down-regulated Tim-3 expression on T cells when compared to asymptomatic patients and uninfected controls. Our study provides evidence of a novel mechanism for the persistent inflammation observed in HTLV-1 infected patients with neurological deficits and significantly advances our understanding of how the Tim-3 pathway functions.
doi:10.1371/journal.pntd.0001030
PMCID: PMC3082508  PMID: 21541358
18.  Prevalence, Incidence Density, and Genotype Distribution of GB Virus C Infection in a Cohort of Recently HIV-1-Infected Subjects in Sao Paulo, Brazil 
PLoS ONE  2011;6(4):e18407.
Background
The results of previous studies elsewhere have indicated that GB virus C (GBV-C) infection is frequent in patients infected with the human immunodeficiency virus type 1 (HIV-1) due to similar transmission routes of both viruses. The aim of this study was to determine the prevalence, incidence density and genotypic characteristics of GBV-C in this population.
Methodology/Principal Findings
The study population included 233 patients from a cohort primarily comprised of homosexual men recently infected with HIV-1 in São Paulo, Brazil. The presence of GBV-C RNA was determined in plasma samples by reverse transcriptase-nested polymerase chain reaction and quantified by real-time PCR. GBV-C genotypes were determined by direct sequencing. HIV viral load, CD4+ T lymphocyte and CD8+ T lymphocyte count were also tested in all patients. The overall prevalence of GBV-C infection was 0.23 (95% CI: 0.18 to 0.29) in the study group. There was no significant difference between patients with and without GBV-C infection and Glycoprotein E2 antibody presence regarding age, sex, HIV-1 viral load, CD4+ and CD8+T cell counts and treatment with antiretroviral drugs. An inverse correlation was observed between GBV-C and HIV-1 loads at enrollment and after one year. Also, a positive but not significant correlation was observed between GBV-C load and CD4+ T lymphocyte. Phylogenetic analysis of the GBV-C isolates revealed the presence of genotype 1 and genotype 2, these sub classified into subtype 2a and 2b.
Conclusion/Significance
GBV-C infection is common in recently HIV -1 infected patients in Sao Paulo, Brazil and the predominant genotype is 2b. This study provides the first report of the GBV-C prevalence at the time of diagnosis of HIV-1 and the incidence density of GBV-C infection in one year.
doi:10.1371/journal.pone.0018407
PMCID: PMC3071701  PMID: 21483671
19.  A Novel Human CD4+ T cell Inducer Subset with Potent Immunostimulatory Properties 
European journal of immunology  2010;40(1):134-141.
Summary
The complexity of immunoregulation has focused attention on the CD4+ T “suppressor” regulatory cell (Treg), which helps maintain balance between immunity and tolerance. An immunoregulatory T cell population that upon activation amplifies cellular immune responses was described in murine models more than thirty years ago. However, no study has yet identified a naturally occurring T “inducer” cell type. Here, we report that the ectoenzyme CD39/NTPDase1 (ecto-nucleoside triphosphate diphosphohydrolase 1) helps to delineate a novel population of human “inducer” CD4+ T cells (TInd) that significantly increases the proliferation and cytokine production of responder T cells in a dose-dependent manner. Furthermore, this unique TInd cell subset produces a distinct repertoire of cytokines in comparison to the other CD4+ T cell subsets. We propose that this novel CD4+ T cell population counterbalances the suppressive activity of suppressor Treg cells in peripheral blood and serves as a calibrator of immunoregulation.
doi:10.1002/eji.200939258
PMCID: PMC2902274  PMID: 19877008
CD4; CD39; Regulatory T cells; FOXP3; CD127; CD25; proliferation; Inducer; cytokine; IFN-γ; IL-6; IL-10; TNF-α; GM-CSF; ATP
20.  Decay Kinetics of HIV-1 Specific T Cell Responses in Vertically HIV-1 Exposed Seronegative Infants 
Objective: The majority of infants born, in developed countries, to HIV-1 positive women are exposed to the HIV-1 virus in utero or peri/post-partum, but are born uninfected. We, and others, have previously shown HIV-1 specific T cell responses in HIV-1 exposed seronegative (HESN) neonates/infants. Our objective in this study was to examine the rate of decay in their HIV-1 specific T cell response over time from birth. Design: Cross-sectional and longitudinal studies of HIV-1 specific T cell responses in HESN infants were performed. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 18 HIV-1 DNA PCR negative infants born to HIV-1 infected mothers receiving care at the Jacobi Medical Center, Bronx, NY, USA. PBMC were examined for T cell responses to HIV-1 antigens by interferon-gamma (IFN-γ) ELISPOT. Results: PBMC from 15 HESN neonates/infants were analyzed. We observed a decay of HIV-1 specific T cell responses from birth at a rate of −0.599 spot forming unit/106 cells per day, with a median half-life decay rate of 21.38 weeks (13.39–115.8). Conclusion: Our results support the dynamic nature of T cell immunity in the context of a developing immune system. The disparate rate of decay with studies of adults placed on antiretroviral drugs suggests that antigen specific T cell responses are driven by the natural rate of decay of the T cell sub-populations themselves.
doi:10.3389/fimmu.2011.00094
PMCID: PMC3341962  PMID: 22566883
HIV-1; exposed seronegative; vertically exposed; neonates; T cells
21.  Skewed Distribution of Circulating Activated Natural Killer T (NKT) Cells in Patients with Common Variable Immunodeficiency Disorders (CVID) 
PLoS ONE  2010;5(9):e12652.
Common variable immunodeficiency disorder (CVID) is the commonest cause of primary antibody failure in adults and children, and characterized clinically by recurrent bacterial infections and autoimmune manifestations. Several innate immune defects have been described in CVID, but no study has yet investigated the frequency, phenotype or function of the key regulatory cell population, natural killer T (NKT) cells. We measured the frequencies and subsets of NKT cells in patients with CVID and compared these to healthy controls. Our results show a skewing of NKT cell subsets, with CD4+ NKT cells at higher frequencies, and CD8+ NKT cells at lower frequencies. However, these cells were highly activated and expression CD161. The NKT cells had a higher expression of CCR5 and concomitantly expression of CCR5+CD69+CXCR6 suggesting a compensation of the remaining population of NKT cells for rapid effector action.
doi:10.1371/journal.pone.0012652
PMCID: PMC2936579  PMID: 20844745
22.  Characterization of Dengue Virus Type 2: New Insights on the 2010 Brazilian Epidemic 
PLoS ONE  2010;5(7):e11811.
Dengue viruses (DENV) serotypes 1, 2, and 3 have been causing yearly outbreaks in Brazil. In this study, we report the re-introduction of DENV2 in the coast of São Paulo State. Partial envelope viral genes were sequenced from eighteen patients with dengue fever during the 2010 epidemic. Phylogenetic analysis showed this strain belongs to the American/Asian genotype and was closely related to the virus that circulated in Rio de Janeiro in 2007 and 2008. The phylogeny also showed no clustering by clinical presentation, suggesting that the disease severity could not be explained by distinct variants or genotypes. The time of the most recent common ancestor of American/Asian genotype and the São Paulo and Rio de Janeiro (SP/RJ) monophyletic cluster was estimated to be around 40 and 10 years, respectively. Since this virus was first identified in Brazil in 2007, we suggest that it was already circulating in the country before causing the first documented outbreak. This is the first description of the 2010 outbreak in the State of São Paulo, Brazil, and should contribute to efforts to control and monitor the spread of DENVs in endemic areas.
doi:10.1371/journal.pone.0011811
PMCID: PMC2911371  PMID: 20676363
23.  Unexpected Diversity of Cellular Immune Responses against Nef and Vif in HIV-1-Infected Patients Who Spontaneously Control Viral Replication 
PLoS ONE  2010;5(7):e11436.
Background
HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus.
Methodology and Principal Findings
Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides.
Conclusion and Significance
This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.
doi:10.1371/journal.pone.0011436
PMCID: PMC2896403  PMID: 20625436
24.  Transcriptional Errors in Human Immunodeficiency Virus Type 1 Generate Targets for T-Cell Responses▿  
We measured T-cell responses to human immunodeficiency virus type 1 (HIV-1) cryptic epitopes encoded by regions of the viral genome not normally translated into viral proteins. T-cell responses to cryptic epitopes and to regions normally spliced out of the HIV-1 viral proteins Rev and Tat were detected in HIV-1-infected subjects.
doi:10.1128/CVI.00410-08
PMCID: PMC2745002  PMID: 19571107
25.  A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency 
PLoS ONE  2009;4(7):e6269.
Introduction
Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4+CD25high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.
Objective
In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.
Materials and Methods
Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).
Results
A significantly lower proportion of CD4+CD25highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8+ T cells from CVID patients expressing the activation markers, CD38+ and HLA-DR+ (P<0.05), we observed no significant correlation between Tregs and immune activation.
Conclusion
Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.
doi:10.1371/journal.pone.0006269
PMCID: PMC2715881  PMID: 19649263

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