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author:("dallas, Esper")
1.  Human CD8+ T-Cell Responses Against the 4 Dengue Virus Serotypes Are Associated With Distinct Patterns of Protein Targets 
The Journal of Infectious Diseases  2015;212(11):1743-1751.
Background. All 4 dengue virus (DENV) serotypes are now simultaneously circulating worldwide and responsible for up to 400 million human infections each year. Previous studies of CD8+ T-cell responses in HLA-transgenic mice and human vaccinees demonstrated that the hierarchy of immunodominance among structural versus nonstructural proteins differs as a function of the infecting serotype. This led to the hypothesis that there are intrinsic differences in the serotype-specific reactivity of CD8+ T-cell responses.
Methods. We tested this hypothesis by analyzing serotype-specific CD8+ T-cell reactivity in naturally infected human donors from Sri Lanka and Nicaragua, using ex vivo interferon γ–specific enzyme-linked immunosorbent spot assays.
Results. Remarkably similar and clear serotype-specific patterns of immunodominance in both cohorts were identified. Pooling of epitopes that accounted for 90% of the interferon γ response in both cohorts resulted in a global epitope pool. Its reactivity was confirmed in naturally infected donors from Brazil, demonstrating its global applicability.
Conclusions. This study provides new insight into differential serotype-specific immunogenicity of DENV proteins. It further provides a potentially valuable tool for future investigations of CD8+ T-cell responses in the typically small sample volumes available from patients with acute fever and children without requiring prior knowledge of either infecting DENV serotype or HLA type.
PMCID: PMC4633759  PMID: 25980035
dengue virus; serotype specific; CD8+ T cells; Nicaragua; Sri Lanka; Brazil
2.  A Side-by-side Comparison of T cell Reactivity to Fifty-nine M. tuberculosis Antigens in Diverse Populations from five Continents 
We compared T cell recognition of 59 prevalently recognized Mycobacterium tuberculosis (MTB) antigens in individuals latently infected with MTB (LTBI), and uninfected individuals with previous BCG vaccination, from nine locations and populations with different HLA distribution, MTB exposure rates, and standards of TB care. This comparison revealed similar response magnitudes in diverse LTBI and BCG-vaccinated cohorts and significant correlation between responses in LTBIs from the USA and other locations. Many antigens were uniformly recognized, suggesting suitability for inclusion in vaccines targeting diverse populations. Several antigens were similarly immunodominant in LTBI and BCG cohorts, suggesting applicability for vaccines aimed at boosting BCG responses. The panel of MTB antigens will be valuable for characterizing MTB-specific CD4 T cell responses irrespective of ethnicity, infecting MTB strains and BCG vaccination status. Our results illustrate how a comparative analysis can provide insight into the relative immunogenicity of existing and novel vaccine candidates in LTBIs.
PMCID: PMC4666753  PMID: 26277695
Tuberculosis; T cell antigen; vaccine; CD4; LTBI; BCG
3.  HIV pre-exposure prophylaxis in transgender women: A subgroup analysis of the iPrEx trial 
The lancet. HIV  2015;2(12):e512-e519.
Oral emtricitabine-tenofovir disoproxil fumarate (FTC/TDF) pre-exposure prophylaxis (PrEP) is used to prevent the sexual acquisition of HIV. Transgender women (TGW) have unique characteristics that may relate to PrEP use, effectiveness, and safety.
The iPrEx trial was a randomized controlled trial (RCT) of oral FTC/TDF PrEP versus placebo among men who have sex with men (MSM) and TGW, followed by an open label extension (OLE). Drug concentrations were measured in blood by liquid chromatography and tandem mass spectroscopy.
Of the 2499 participants enrolled in the RCT, 29 (1%) identified as women, 296 (12%) identified as “trans”, 14 (1%) identified as men but reported use of feminizing hormones, such that 339 (14%) reported one or more of these characteristics (TGW). Compared with MSM, TGW more frequently reported transactional sex, receptive anal intercourse without a condom, or more than 5 partners in the past 3 months. Among TGW, there were 11 HIV infections in the active arm and 10 in the placebo arm, representing a hazard ratio of 1.1 (95% CI: 0.5 to 2.7). Among active arm participants, drug was detected in none of the TGW at the seroconversion visit, 18% (6/37) of seronegative TGW (P=0.31), and 52% (58/111) of seronegative MSM (P < 0.0001). PrEP use was not linked to behavioral indicators of HIV risk among TGW, while MSM at highest risk were more adherent.
There were no HIV infections among TGW having drug concentrations commensurate with use of 4 or more FTC/TDF tablets per week. TGW receiving PrEP had low drug concentrations, especially at times of potential HIV exposure, leading to no PrEP effectiveness among this subgroup.
U.S. National Institutes of Health and the Bill and Melinda Gates Foundation; study medication was donated by Gilead Sciences.
PMCID: PMC5111857  PMID: 26614965
4.  Persistence of Yellow Fever vaccine-induced antibodies after cord blood stem cell transplant 
We report the case of a cord blood haematopoietic stem cell transplant recipient who was vaccinated for Yellow Fever (YF) 7 days before initiating chemotherapy and had persistent YF antibodies more than 3 years after vaccination. Since the stem cell donor was never exposed to wild YF or to the YF vaccine, and our patient was not exposed to YF or revaccinated, this finding strongly suggests the persistence of recipient immunity. We briefly discuss potential consequences of incomplete elimination of recipient's leukocytes following existing haematopoietic cancer treatments.
PMCID: PMC4962932  PMID: 26618995
cord blood stem cell transplant; immunosuppression; vaccine; yellow fever
5.  p16INK4a Expression and Immunologic Aging in Chronic HIV Infection 
PLoS ONE  2016;11(11):e0166759.
Chronic HIV infection is characterized by increased immune activation and immunosenescence. p16 INK4a (p16) is a member of the cyclin-dependent kinase antagonist family that inhibits cellular proliferation, and its protein expression increases during normal chronological aging. However, some infectious diseases can increase the expression of this anti-proliferative protein, potentially accelerating immunological aging and dysfunction. In order to investigate the immunological aging in HIV patients, p16 protein expression was evaluated by flow cytometry, in T cell subsets in a cohort of chronically HIV-infected patients on and off ART as well as age-matched healthy controls. Results showed that untreated HIV-infected subjects exhibited increased per-cell p16 protein expression that was discordant with chronological aging. ART restored p16 protein expression to levels comparable with HIV-negative subjects in the CD4 compartment, but not in CD8 T cells, which can be an indicative of an irreversible activation/exhaustion status on these cells. Additionally, the frequency of activated CD4+ and CD8+ T cells was positively correlated with p16 expression in CD4+ and CD8+ T cells in untreated subjects. In contrast to healthy controls, untreated HIV-infected individuals had increased p16 levels within the effector memory (TEM) subset, indicating a possible role for this marker in impaired clonal expansion during antiviral effector function. Taken together, these data demonstrate that chronic HIV infection is associated with elevated expression of the cellular aging marker p16 in T cells. ART restored normal p16 levels in the CD4+ T cell compartment, indicating that use of therapy can be of fundamental importance to normal cell cycling and maintaining immune homeostasis.
PMCID: PMC5115792  PMID: 27861555
6.  Circulating Follicular Helper-Like T Cells in Systemic Lupus Erythematosus: Association with Disease Activity 
To assess circulating follicular helper-like CD4+ T (cTfh-like) cells in systemic lupus erythematosus (SLE) and determine their relationship to disease activity.
We analyzed blood samples from SLE patients, and as controls, Behçet’s disease (BD) patients and healthy individuals. We used flow cytometry to enumerate cTfh-like cells using as markers the C-X-C chemokine receptor type 5 (CXCR5), inducible T-cell costimulator (ICOS), programmed cell death protein-1 (PCDC1, PD-1), and secretion of interleukin-21 (IL-21). We compared the frequency of cTfh-like cells with that of circulating plasmablasts (CD19+IgD−CD38+) and evaluated their possible association with disease activity.
cTfh-like T cells, identified as CXCR5hiICOShiPD-1hi, were expanded in the blood of SLE patients compared to BD and healthy controls. Such cells produced IL-21 with lower expression of CCR7, compared to circulating CXCR5hi central memory (Tcm) cells, enabling their distinction. PD-1, not ICOS or CXCR5, expression was significantly elevated in cTfh-like cells from SLE patients compared to controls. PD-1 expression among CXCR5hi cTfh-like cells correlated with disease activity, circulating plasmablasts, and anti-dsDNA antibody positivity, but not disease duration nor past organ injury; rather, it reflected current active disease.
We found that cTfh-like cells are associated with disease activity in SLE, suggesting that their presence indicates abnormal homeostasis of T-B cell collaboration with a causal relationship central to disease pathogenesis. These findings also suggest that cTfh-like cells provide a surrogate for aberrant GC activity in SLE, and that their PD-1 expression offers a tool for following disease activity and response to therapies.
PMCID: PMC4450082  PMID: 25581113
7.  Durable sequence stability and bone marrow tropism in a macaque model of human pegivirus infection 
Science translational medicine  2015;7(305):305ra144.
Human Pegivirus (HPgV) – formerly known as GB virus C and hepatitis G virus – is a poorly characterized RNA virus that infects approximately one-sixth of the global human population and is transmitted frequently in the blood supply. Here, we create an animal model of HPgV infection by infecting macaque monkeys with a new simian pegivirus (SPgV) discovered in wild baboons. Using this model, we provide a high-resolution, longitudinal picture of SPgV viremia where the dose, route, and timing of infection are known. We detail the highly-variable acute-phase of SPgV infection, showing that the viral load trajectory early in infection is dependent upon the infecting dose, whereas the chronic-phase viremic set-point is not. We also show that SPgV has an extremely low propensity for accumulating sequence variation, with no consensus-level variants detected during the acute phase of infection and an average of only 1.5 variants generated per 100 infection days. Finally, we show that SPgV RNA is highly concentrated in only two tissues: spleen and bone marrow, with bone marrow likely producing the majority of virus detected in plasma. Together, these results reconcile several paradoxical observations from cross-sectional analyses of HPgV in humans and provide an animal model for studying pegivirus biology.
PMCID: PMC4605385  PMID: 26378244
8.  Patterns and Correlates of PrEP Drug Detection among MSM and Transgender Women in the Global iPrEx Study 
Adherence to pre-exposure prophylaxis (PrEP) is critical for efficacy. Antiretroviral concentrations are an objective measure of PrEP use and correlate with efficacy. Understanding patterns and correlates of drug detection can identify populations at risk for non-adherence and inform design of PrEP adherence interventions.
Blood antiretroviral concentrations were assessed among active-arm participants in iPrEx, a randomized, placebo-controlled trial of emtricitabine/tenofovir in men who have sex with men (MSM) and transgender women in 6 countries. We evaluated rates and correlates of drug detection among a random sample of 470 participants at week 8 and a longitudinal cohort of 303 participants through 72 weeks of follow-up.
Overall, 55% (95% CI 49–60%) of participants tested at week 8 had drug detected. Drug detection was associated with older age and varied by study site. In longitudinal analysis, 31% never had drug detected, 30% always had drug detected, and 39% had an inconsistent pattern. Overall detection rates declined over time. Drug detection at some or all visits was associated with older age; indices of sexual risk, including condomless receptive anal sex; and responding "don't know" to a question about belief of PrEP efficacy (0–10 scale).
Distinct patterns of study-product use were identified, with a significant proportion demonstrating no drug detection at any visit. Research literacy may explain greater drug detection among populations having greater research experience, such as older MSM in the US. Greater drug detection among those reporting highest-risk sexual practices is expected to increase the impact and cost-effectiveness of PrEP.
PMCID: PMC4229454  PMID: 25230290
pre-exposure prophylaxis; drug detection; emtricitabine/tenofovir; adherence; men who have sex with men
9.  Efficient Identification of HIV Serodiscordant Couples by Existing HIV Testing Programs in South Brazil 
PLoS ONE  2015;10(11):e0142638.
To examine the feasibility of identifying HIV negative at risk individuals in HIV serodiscordant couples, during voluntary HIV testing in South Brazil.
We surveyed HIV testers at 4 public testing sites in Rio Grande do Sul. We obtained information on risk behaviors and sexual partnerships. HIV testing and testing for recent infection were performed; HIV prevalence and risk behaviors were assessed among subjects who reported having a steady partner who was HIV positive (serodiscordant group) and compared with the general testing population.
Among 3100 patients, 490 (15.8%) reported being in a steady relationship with an HIV positive partner. New HIV infections were diagnosed in 23% of the serodiscordant group (vs. 13% in the general population, p = 0.01); among newly positive subjects, recent HIV infections were more frequent (23/86, 26.7%) among testers with positive partners than among the general testing group (52/334; 15.6%; p = 0.016). Less than half of the serodiscordant testers reported having used a condom during the last sexual intercourse with their HIV-positive partner. Participants with inconsistent condom use with steady partner were four times more likely to test positive for HIV compared to those who reported always using condoms with the steady partner (OR: 4.2; 95% CI: 2.3 to 7.5).
It is highly feasible to identify large numbers of HIV susceptible individuals who are in HIV serodiscordant relationships in South Brazil testing sites. Condom use within HIV serodiscordant couples is low in this setting, suggesting urgent need for biomedical prevention strategies to reduce HIV transmission.
PMCID: PMC4642994  PMID: 26562436
10.  New Players in the Same Old Game: Disturbance of Group 2 Innate Lymphoid Cells in HIV-1 and Mycobacterium leprae Co-infected Patients 
PLoS Neglected Tropical Diseases  2015;9(9):e0004030.
Leprosy control is achieved through a fine-tuning of TH1 and TH2 immune response pattern balance. Given the increasing epidemiological overlay of HIV and M. leprae infections, immune response in co-infected patients consists in an important contemporary issue. Here we describe for the first time the innate lymphoid cells compartment in peripheral blood of leprosy and HIV/M. leprae co-infected patients, and show that co-infection increases group 2 innate lymphoid whilst decreasing group 1 innate lymphoid cells frequencies and function.
Author Summary
Mycobacterium leprae is a clinical relevant pathogen that can lead to leprosy upon infection. This chronic infectious disease is characterized by the appearance of skin and peripheral nerve lesions. Normally, a healthy immune system is able to control the infection and impede the generation of lesions. However, immunosuppressive conditions may reduce the fitness of host’s immune system, with consequences of disease progression and pathogen spreading. HIV is the major cause of pathogen-associated immunodeficiency, and during the past decades, an epidemiological overlay of M. leprae and HIV infections spectra has been observed. Thus, the increase of co-infection cases raised the interest of the scientific community in this subject. Accordingly, our group has previously shown that co-infected patients presented a shift in the type of cellular immune response. Nevertheless, in the past few years a new group of innate lymphoid cells (ILCs) was shown to play an important role in the initiation and maintenance of effective immune responses. Here we describe for the first time the circulating ILCs subsets in leprosy, HIV and M. leprae/HIV co-infected patients. Additionally, we found that the co-infection scenario leads to a shift in different ILCs population, concordant with those observed previously for T lymphocytes and innate immunity cells.
PMCID: PMC4559394  PMID: 26335023
11.  Streamlining HIV Testing for HIV Preexposure Prophylaxis 
Journal of Clinical Microbiology  2014;53(1):179-183.
HIV-testing algorithms for preexposure prophylaxis (PrEP) should be optimized to minimize the risk of drug resistance, the time off PrEP required to evaluate false-positive screening results, and costs and to expedite the start of therapy for those confirmed to be infected. HIV rapid tests (RTs) for anti-HIV antibodies provide results in less than 1 h and can be conducted by nonlicensed staff at the point of care. In many regions, Western blot (WB) testing is required to confirm reactive RT results. WB testing, however, causes delays in diagnosis and adds expense. The iPrEx study evaluated the safety and efficacy of daily oral emtricitabine-tenofovir disoproxil fumarate among HIV-seronegative men and transgender women who have sex with men: HIV infection was assessed with two RTs plus WB confirmation, followed by HIV-1 plasma viral load testing. During the iPrEx study, there were 51,260 HIV status evaluations among 2,499 volunteers using RTs: 142 (0.28%) had concordant positive results (100% were eventually confirmed) and 19 (0.04%) had discordant results among 14 participants; 11 were eventually determined to be HIV infected. A streamlined approach using only one RT to screen and a second RT to confirm (without WB) would have had nearly the same accuracy. Discrepant RT results are best evaluated with nucleic acid testing, which would also increase sensitivity.
PMCID: PMC4290918  PMID: 25378570
12.  The CD8+ Memory Stem T Cell (TSCM) Subset Is Associated with Improved Prognosis in Chronic HIV-1 Infection 
Journal of Virology  2014;88(23):13836-13844.
Memory stem T cells (TSCM) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. The hallmarks of HIV-1 pathogenesis are CD4+ T cell depletion and abnormal cellular activation. We investigated the impact of HIV-1 infection on the TSCM compartment, as well as any protective role these cells may have in disease progression, by characterizing this subset in a cohort of 113 subjects with various degrees of viral control on and off highly active antiretroviral therapy (HAART). We observed that the frequency of CD8+ TSCM was decreased in all individuals with chronic, untreated HIV-1 infection and that HAART had a restorative effect on this subset. In contrast, natural controllers of HIV-1 had the highest absolute number of CD4+ TSCM cells among all of the infected groups. The frequency of CD4+ TSCM predicted higher CD8+ TSCM frequencies, consistent with a role for the CD4+ subset in helping to maintain CD8+ memory T cells. In addition, TSCM appeared to be progenitors for effector T cells (TEM), as these two compartments were inversely correlated. Increased frequencies of CD8+ TSCM predicted lower viral loads, higher CD4+ counts, and less CD8+ T cell activation. Finally, we found that TSCM express the mucosal homing integrin α4β7 and can be identified in gut-associated lymphoid tissue (GALT). The frequency of mucosal CD4+ TSCM was inversely correlated with that in the blood, potentially reflecting the ability of these self-renewing cells to migrate to a crucial site of ongoing viral replication and CD4+ T cell depletion.
IMPORTANCE HIV-1 infection leads to profound impairment of the immune system. TSCM constitute a recently identified lymphocyte subset with stem cell-like qualities, including the ability to generate other memory T cell subtypes, and are therefore likely to play an important role in controlling viral infection. We investigated the relationship between the size of the CD8+ TSCM compartment and HIV-1 disease progression in a cohort of chronically infected individuals. Our results suggest that HAART restores a normal frequency of CD8+ TSCM and that the natural preservation of this subset in the setting of untreated HIV-1 infection is associated with improved viral control and immunity. Therefore, the CD8+ TSCM population may represent a correlate of protection in chronic HIV-1 infection that is directly relevant to the design of T cell-based vaccines, adoptive immunotherapy approaches, or the pharmacologic induction of TSCM.
PMCID: PMC4248986  PMID: 25253339
13.  CD8+ T Lymphocyte Expansion, Proliferation and Activation in Dengue Fever 
PLoS Neglected Tropical Diseases  2015;9(2):e0003520.
Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood mononuclear cells from 30 cases were thawed and evaluated using expanded phenotyping. The expansion of the CD8+ T cells was coupled with increased Ki67 expression. Cell activation was observed later in the course of disease, as determined by the expression of the activation markers CD38 and HLA-DR. This increased CD8+ T lymphocyte activation was observed in all memory subsets, but was more pronounced in the effector memory subset, as defined by higher CD38 expression. Our results show that most CD8+ T cell subsets are expanded during DENV2 infection and that the effector memory subset is the predominantly affected sub population.
Author Summary
Dengue is a disease affecting approximately 400 million people annually, especially in tropical and subtropical areas of the globe. The immune response against the dengue virus is still under investigation and it is important to understand why the disease can be fatal in a small proportion of cases. In this work, we explored how an important cell type of the immune system, namely the CD8+ T cell, reacts during dengue infection. Using a method known as flow cytometry, we demonstrated that these cells expand and become highly activated, during the days following the onset of dengue fever symptoms. This expansion is associated with a decreased dengue virus load in the patients’ blood, suggesting that CD8+ T cells play an important role in viral control. Interestingly, we found that a subset of CD8+ T cells, called effector memory, is greatly expanded during dengue infection. Our results are important because they might contribute to the understanding of disease mechanisms during dengue infection and may help in the development of a novel vaccine against dengue.
PMCID: PMC4326415  PMID: 25675375
14.  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.
PMCID: PMC4274125  PMID: 25532028
15.  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.
PMCID: PMC3778967  PMID: 23878319
HIV-1 vaccine; Step study; Gag-specific T cells; HLA class I alleles
16.  Hyperferritinaemia in Dengue Virus Infected Patients Is Associated with Immune Activation and Coagulation Disturbances 
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.
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.
PMCID: PMC4191960  PMID: 25299654
17.  Syphilis Predicts HIV Incidence Among Men and Transgender Women Who Have Sex With Men in a Preexposure Prophylaxis Trial 
Syphilis infection was associated with HIV incidence in an HIV-prevention trial that randomized participants to once-daily emtricitabine/tenofovir (FTC/TDF) vs placebo. FTC/TDF had no effect on the association between incident syphilis and HIV acquisition; syphilis infection did not decrease FTC/TDF efficacy.
Background. Syphilis infection may potentiate transmission of human immunodeficiency virus (HIV). We sought to determine the extent to which HIV acquisition was associated with syphilis infection within an HIV preexposure prophylaxis (PrEP) trial and whether emtricitabine/tenofovir (FTC/TDF) modified that association.
Methods. The Preexposure Prophylaxis Initiative (iPrEx) study randomly assigned 2499 HIV-seronegative men and transgender women who have sex with men (MSM) to receive oral daily FTC/TDF or placebo. Syphilis prevalence at screening and incidence during follow-up were measured. Hazard ratios for the effect of incident syphilis on HIV acquisition were calculated. The effect of FTC/TDF on incident syphilis and HIV acquisition was assessed.
Results. Of 2499 individuals, 360 (14.4%) had a positive rapid plasma reagin test at screening; 333 (92.5%) had a positive confirmatory test, which did not differ between the arms (FTC/TDF vs placebo, P = .81). The overall syphilis incidence during the trial was 7.3 cases per 100 person-years. There was no difference in syphilis incidence between the study arms (7.8 cases per 100 person-years for FTC/TDF vs 6.8 cases per 100 person-years for placebo, P = .304). HIV incidence varied by incident syphilis (2.8 cases per 100 person-years for no syphilis vs 8.0 cases per 100 person-years for incident syphilis), reflecting a hazard ratio of 2.6 (95% confidence interval, 1.6–4.4; P < .001). There was no evidence for interaction between randomization to the FTC/TDF arm and incident syphilis on HIV incidence.
Conclusions. In HIV-seronegative MSM, syphilis infection was associated with HIV acquisition in this PrEP trial; a syphilis diagnosis should prompt providers to offer PrEP unless otherwise contraindicated.
PMCID: PMC4166980  PMID: 24928295
chemoprophylaxis; HIV prevention; MSM; preexposure prophylaxis; syphilis
18.  No Evidence of Sexual Risk Compensation in the iPrEx Trial of Daily Oral HIV Preexposure Prophylaxis 
PLoS ONE  2013;8(12):e81997.
Preexposure prophylaxis (PrEP) with emtricitabine/tenofovir disoproxil fumarate (FTC/TDF) reduced HIV acquisition in the iPrEx trial among men who have sex with men and transgender women. Self-reported sexual risk behavior decreased overall, but may be affected by reporting bias. We evaluated potential risk compensation using biomarkers of sexual risk behavior.
Design and methods
Sexual practices were assessed at baseline and quarterly thereafter; perceived treatment assignment and PrEP efficacy beliefs were assessed at 12 weeks. Among participants with ≥1 follow-up behavioral assessment, sexual behavior, syphilis, and HIV infection were compared by perceived treatment assignment, actual treatment assignment, and perceived PrEP efficacy.
Overall, acute HIV infection and syphilis decreased during follow-up. Compared with participants believing they were receiving placebo, participants believing they were receiving FTC/TDF reported more receptive anal intercourse partners prior to initiating drug (12.8 vs. 7.7, P = 0.04). Belief in receiving FTC/TDF was not associated with an increase in receptive anal intercourse with no condom (ncRAI) from baseline through follow-up (risk ratio [RR] 0.9, 95% confidence interval [CI]: 0.6–1.4; P = 0.75), nor with a decrease after stopping study drug (RR 0.8, 95% CI: 0.5–1.3; P = 0.46). In the placebo arm, there were trends toward lower HIV incidence among participants believing they were receiving FTC/TDF (incidence rate ratio [IRR] 0.8, 95% CI: 0.4–1.8; P = 0.26) and also believing it was highly effective (IRR 0.5, 95% CI: 0.1–1.7; P = 0.12).
There was no evidence of sexual risk compensation in iPrEx. Participants believing they were receiving FTC/TDF had more partners prior to initiating drug, suggesting that risk behavior was not a consequence of PrEP use.
PMCID: PMC3867330  PMID: 24367497
19.  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.
PMCID: PMC3793979  PMID: 24130688
21.  Inter- and Intra-Host Viral Diversity in a Large Seasonal DENV2 Outbreak 
PLoS ONE  2013;8(8):e70318.
High genetic diversity at both inter- and intra-host level are hallmarks of RNA viruses due to the error-prone nature of their genome replication. Several groups have evaluated the extent of viral variability using different RNA virus deep sequencing methods. Although much of this effort has been dedicated to pathogens that cause chronic infections in humans, few studies investigated arthropod-borne, acute viral infections.
Methods and Principal Findings
We deep sequenced the complete genome of ten DENV2 isolates from representative classical and severe cases sampled in a large outbreak in Brazil using two different approaches. Analysis of the consensus genomes confirmed the larger extent of the 2010 epidemic in comparison to a previous epidemic caused by the same viruses in another city two years before (genetic distance = 0.002 and 0.0008 respectively). Analysis of viral populations within the host revealed a high level of conservation. After excluding homopolymer regions of 454/Roche generated sequences, we found 10 to 44 variable sites per genome population at a frequency of >1%, resulting in very low intra-host genetic diversity. While up to 60% of all variable sites at intra-host level were non-synonymous changes, only 10% of inter-host variability resulted from non-synonymous mutations, indicative of purifying selection at the population level.
Conclusions and Significance
Despite the error-prone nature of RNA-dependent RNA-polymerase, dengue viruses maintain low levels of intra-host variability.
PMCID: PMC3732279  PMID: 23936406
22.  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.
PMCID: PMC3697853  PMID: 18535579
23.  Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease 
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.
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.
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.
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.
PMCID: PMC3662706  PMID: 23717702
24.  Variability of HIV-1 Genomes among Children and Adolescents from São Paulo, Brazil 
PLoS ONE  2013;8(5):e62552.
Genetic variability is a major feature of the human immunodeficiency virus type 1 (HIV-1) and considered the key factor to frustrating efforts to halt the virus epidemic. In this study, we aimed to investigate the genetic variability of HIV-1 strains among children and adolescents born from 1992 to 2009 in the state of Sao Paulo, Brazil.
Plasma and peripheral blood mononuclear cells (PBMC) were collected from 51 HIV-1-positive children and adolescents on ART followed between September 1992 and July 2009. After extraction, the genetic materials were used in a polymerase chain reaction (PCR) to amplify the viral near full length genomes (NFLGs) from 5 overlapped fragments. NFLGs and partial amplicons were directly sequenced and data were phylogenetically inferred.
Of the 51 samples studied, the NFLGs and partial fragments of HIV-1 from 42 PBMCs and 25 plasma were successfully subtyped. Results based on proviral DNA revealed that 22 (52.4%) patients were infected with subtype B, 16 (38.1%) were infected with BF1 mosaic variants and 4 (9.5%) were infected with sub-subtype F1. All the BF1 recombinants were unique and distinct from any previously identified unique or circulating recombinant forms in South America. Evidence of dual infections was detected in 3 patients coinfected with the same or distinct HIV-1 subtypes. Ten of the 31 (32.2%) and 12 of the 21 (57.1%) subjects with recovered proviral and plasma, respectively, protease sequences were infected with major mutants resistant to protease inhibitors. The V3 sequences of 14 patients with available sequences from PBMC/or plasma were predicted to be R5-tropic virus except for two patients who harbored an X4 strain.
The high proportion of HIV-1 BF1 recombinant, coinfection rate and vertical transmission in Brazil merits urgent attention and effective measures to reduce the transmission of HIV among spouses and sex partners.
PMCID: PMC3646872  PMID: 23667488
25.  From trials to the public health 
Colombia Médica : CM  null;44(1):64-65.
PMCID: PMC4002009  PMID: 24892325

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