Showing papers by "Eva Harris published in 2018"
TL;DR: The identification of a CD4-CTL precursor population may allow further investigation of how CD4+ cytotoxic T lymphocytes arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effectiveCD4- CTL immunity.
Abstract: CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the TEMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4+ T cells in the central memory (TCM) and effector memory (TEM) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared with TCM and TEM cells and that most of CD4-TEMRA were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across donors, with four distinct clusters identified by the single-cell analysis. We identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effective CD4-CTL immunity.
193 citations
TL;DR: Investigating the effect of obesity on the duration of viral shedding within household transmission studies in Managua, Nicaragua, over 3 seasons suggests that obesity may play an important role in influenza transmission.
Abstract: Epidemiologic studies indicate that obesity increases the risk of severe complications and death from influenza virus infections, especially in elderly individuals. This work investigates the effect of obesity on the duration of viral shedding within household transmission studies in Managua, Nicaragua, over 3 seasons (2015-2017). Symptomatic obese adults were shown to shed influenza A virus 42% longer than nonobese adults (adjusted event time ratio [ETR], 1.42; 95% confidence interval [CI], 1.06-1.89); no association was observed with influenza B virus shedding duration. Even among paucisymptomatic and asymptomatic adults, obesity increased the influenza A shedding duration by 104% (adjusted ETR, 2.04; 95% CI, 1.35-3.09). These findings suggest that obesity may play an important role in influenza transmission.
168 citations
TL;DR: The adaptive immune response generates a robust response against NS1, and its potential contribution to dengue vaccines is also discussed.
Abstract: Dengue virus (DENV) is the most prevalent medically important mosquito-borne virus in the world. Upon DENV infection of a host cell, DENV nonstructural protein 1 (NS1) can be found intracellularly as a monomer, associated with the cell surface as a dimer, and secreted as a hexamer into the bloodstream. NS1 plays a variety of roles in the viral life cycle, particularly in RNA replication and immune evasion of the complement pathway. Over the past several years, key roles for NS1 in the pathogenesis of severe dengue disease have emerged, including direct action of the protein on the vascular endothelium and triggering release of vasoactive cytokines from immune cells, both of which result in endothelial hyperpermeability and vascular leak. Importantly, the adaptive immune response generates a robust response against NS1, and its potential contribution to dengue vaccines is also discussed.
118 citations
TL;DR: Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex, which has implications for understanding natural immunity and vaccines.
Abstract: Background The 4 dengue virus serotypes (DENV1-4) and Zika virus (ZIKV) are related mosquito-borne flaviviruses of major importance globally. While monoclonal antibodies and plasma from DENV-immune donors can neutralize or enhance ZIKV in vitro and in small-animal models, and vice versa, the extent, duration, and significance of cross-reactivity in humans remains unknown, particularly in flavivirus-endemic regions. Methods We studied neutralizing antibodies to ZIKV and DENV1-4 in longitudinal serologic specimens collected through 3 years after infection from people in Latin America and Asia with laboratory-confirmed DENV infections. We also evaluated neutralizing antibodies to ZIKV and DENV1-4 in patients with Zika through 6 months after infection. Results In patients with Zika, the highest neutralizing antibody titers were to ZIKV, with low-level cross-reactivity to DENV1-4 that was greater in DENV-immune individuals. We found that, in primary and secondary DENV infections, neutralizing antibody titers to ZIKV were markedly lower than to the infecting DENV and heterologous DENV serotypes. Cross-neutralization was greatest in early convalescence, then ZIKV neutralization decreased, remaining at low levels over time. Conclusions Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex. Neutralizing antibody titers can distinguish ZIKV from DENV infections when all viruses are analyzed simultaneously. These findings have implications for understanding natural immunity and vaccines.
113 citations
University of Oxford1, University of California, San Francisco2, Harvard University3, Wellcome Trust Centre for Human Genetics4, University of California, Berkeley5, California Department of Public Health6, Systems Research Institute7, Johns Hopkins University8, Mexican Social Security Institute9, Northwestern University10, National Autonomous University of Mexico11
TL;DR: Multiple lines of evidence indicate biannual peaks of ZIKV transmission in the region, likely driven by varying local environmental conditions for mosquito vectors and herd immunity and challenges arbovirus surveillance and disease control measures.
82 citations
TL;DR: A high level of immunity against ZIKV in Managua as a result of the 2016 Zika epidemic is revealed, making a second large Zika epidemic unlikely in the near future and suggesting that ZikV infection risk varies at small spatial scales.
Abstract: In 2015, a Zika epidemic in Brazil began spreading throughout the Americas. Zika virus (ZIKV) entered Managua, Nicaragua, in January 2016 and caused an epidemic that peaked in July–September 2016. ZIKV seropositivity was estimated among participants of pediatric (n = 3,740) and household (n = 2,147) cohort studies, including an adult-only subset from the household cohort (n = 1,074), in Managua. Seropositivity was based on a highly sensitive and specific assay, the Zika NS1 blockade-of-binding ELISA, which can be used in dengue-endemic populations. Overall seropositivity for the pediatric (ages 2–14), household (ages 2–80), and adult (ages 15–80) cohorts was 36, 46, and 56%, respectively. Trend, risk factor, and contour mapping analyses demonstrated that ZIKV seroprevalence increased nonlinearly with age and that body surface area was statistically associated with increasing seroprevalence in children. ZIKV seropositivity was higher in females than in males across almost all ages, with adjusted prevalence ratios in children and adults of 1.11 (95% CI: 1.02–1.21) and 1.14 (95% CI: 1.01–1.28), respectively. No household-level risk factors were statistically significant in multivariate analyses. A spatial analysis revealed a 10–15% difference in the risk of ZIKV infections across our 3-km-wide study site, suggesting that ZIKV infection risk varies at small spatial scales. To our knowledge, this is the largest ZIKV seroprevalence study reported in the Americas, and the only one in Central America and in children to date. It reveals a high level of immunity against ZIKV in Managua as a result of the 2016 epidemic, making a second large Zika epidemic unlikely in the near future.
67 citations
TL;DR: A high-density microarray comprising nonredundant 12-mer peptides that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide is built and two assays that enable sensitive and specific diagnosis of exposure to ZikV are established that may be useful in guiding clinical management of mothers at risk for potential exposure to the virus.
Abstract: Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection). IMPORTANCE The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.
66 citations
TL;DR: The ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%.
Abstract: Zika virus (ZIKV) is a mosquito-borne flavivirus that is responsible for recent explosive epidemics in the Americas. Notably, ZIKV infection during pregnancy has been found to cause congenital birth defects, including microcephaly, and ZIKV has been associated with Guillain-Barre syndrome in adults. Diagnosis and surveillance of Zika in the Americas have been challenging due to similar clinical manifestations and extensive antibody cross-reactivity with endemic flaviviral diseases, such as dengue. We evaluated four serological and two reverse transcription-PCR (RT-PCR) methods in acute-phase (mean day, 1.8), early-convalescent-phase (mean day, 16.7), and late-convalescent-phase (mean, ~7 months) samples from the same individuals in a long-term pediatric cohort study in Nicaragua. Well-characterized samples from 301 cases of Zika, dengue, or non-Zika, nondengue febrile illnesses were tested. Compared to a composite reference, an in-house IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the NIAID-Biodefense and Emerging Infections (BEI) MAC-ELISA measuring IgM yielded sensitivities of 94.5% and 70.1% and specificities of 85.6% and 82.8%, respectively. The NS1 blockade-of-binding ELISA measuring anti-ZIKV NS1 antibody levels yielded sensitivities of 85.0% and 96.5% and specificities of 91.4% and 92.6% at early and late convalescence, respectively. An inhibition ELISA detecting total anti-ZIKV antibodies had sensitivity and specificity values of 68.3% and 58.3% for diagnosis and 94.0% and 98.6% for measuring annual infection incidence. Finally, the ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%. Together, these assays resolve the urgent need for diagnostic and surveillance tools for countries affected by Zika virus infections.
58 citations
TL;DR: A multiscale network is presented that summarizes all observed modulations across cellular and transcriptomic levels and their interactions with clinical outcomes, providing a uniquely global view of the biomolecular landscape of human CHIKV infection.
Abstract: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes global epidemics of debilitating disease worldwide. To gain functional insight into the host cellular genes required for virus infection, we performed whole-blood RNA-seq, 37-plex mass cytometry of peripheral blood mononuclear cells (PBMCs), and serum cytokine measurements of acute- and convalescent-phase samples obtained from 42 children naturally infected with CHIKV Semi-supervised classification and clustering of single-cell events into 57 sub-communities of canonical leukocyte phenotypes revealed a monocyte-driven response to acute infection, with the greatest expansions in "intermediate" CD14++CD16+ monocytes and an activated subpopulation of CD14+ monocytes. Increases in acute-phase CHIKV envelope protein E2 expression were highest for monocytes and dendritic cells. Serum cytokine measurements confirmed significant acute-phase upregulation of monocyte chemoattractants. Distinct transcriptomic signatures were associated with infection timepoint, as well as convalescent-phase anti-CHIKV antibody titer, acute-phase viremia, and symptom severity. We present a multiscale network that summarizes all observed modulations across cellular and transcriptomic levels and their interactions with clinical outcomes, providing a uniquely global view of the biomolecular landscape of human CHIKV infection.
57 citations
TL;DR: ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.
Abstract: Background Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs). Methods Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified. Results In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells. Conclusions ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.
55 citations
TL;DR: An international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation is suggested, and access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building.
Abstract: Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building.
La Jolla Institute for Allergy and Immunology1, University of São Paulo2, Genentech3, University of North Carolina at Chapel Hill4, Emory University5, Systems Research Institute6, Universidad del Norte, Colombia7, University of Vermont8, University of Miami9, Vaccine Research Center10, University of California, San Diego11, University of California, Berkeley12
TL;DR: This work elucidates the first in-depth characterization of human CD8+ T cells responding to ZIKV infection, characterized by a polyfunctional IFN-γ signature with upregulation of TNF-α, TNF receptors, and related activation markers, such as CD69, as well as a cytotoxic signature characterized by strong up regulation of GZMB and CRTAM.
Abstract: Zika virus (ZIKV) constitutes an increasing public health problem. Previous studies have shown that CD8 + T cells play an important role in ZIKV-specific protective immunity. We have previously defined antigenic targets of the ZIKV-specific CD8 + T cell response in humans. In this study, we characterized the quality and phenotypes of these responses by a combined use of flow cytometry and transcriptomic methods, using PBMCs from donors deriving from different geographical locations collected in the convalescent phase of infection. We show that ZIKV-specific CD8 + T cells are characterized by a polyfunctional IFN-γ signature with upregulation of TNF-α, TNF receptors, and related activation markers, such as CD69, as well as a cytotoxic signature characterized by strong upregulation of GZMB and CRTAM. The signature is stable and not influenced by previous dengue virus exposure, geographical location, or time of sample collection postinfection. To our knowledge, this work elucidates the first in-depth characterization of human CD8 + T cells responding to ZIKV infection.
World Health Organization1, University of Texas Medical Branch2, National Health Surveillance Agency3, European Medicines Agency4, University of North Carolina at Chapel Hill5, Johns Hopkins University6, State University of New York Upstate Medical University7, University of California, Berkeley8, University of London9, Food and Drug Administration10
TL;DR: Clinical and regulatory points for consideration that may guide vaccine developers on some aspects of trial design and facilitate regulatory review to enable broader public health recommendations for second-generation dengue vaccines are summarized.
TL;DR: The latest findings regarding ZIKV-induced adaptive immunity, such as monoclonal and polyclonal antibody responses, structural immunology, and T cell-mediated responses are discussed.
TL;DR: It is shown that the age at which children acquired DENV-specific immunity more than doubled during the observational period of a pediatric cohort study in Managua, Nicaragua, and that the overall gradual decline in the FoI can be attributed to demographic shifts.
Abstract: Dengue virus (DENV) is the most prevalent human vector-borne viral disease. The force of infection (FoI), the rate at which susceptible individuals are infected in a population, is an important metric for infectious disease modeling. Understanding how and why the FoI of DENV changes over time is critical for developing immunization and vector control policies. We used age-stratified seroprevalence data from 12 years of the Pediatric Dengue Cohort Study in Nicaragua to estimate the annual FoI of DENV from 1994 to 2015. Seroprevalence data revealed a change in the rate at which children acquire DENV-specific immunity: in 2004, 50% of children age >4 years were seropositive, but by 2015, 50% seropositivity was reached only by age 11 years. We estimated a spike in the FoI in 1997-1998 and 1998-1999 and a gradual decline thereafter, and children age <4 years experienced a lower FoI. Two hypotheses to explain the change in the FoI were tested: (i) a transition from introduction of specific DENV serotypes to their endemic transmission and (ii) a population demographic transition due to declining birth rates and increasing life expectancy. We used mathematical models to simulate these hypotheses. We show that the initial high FoI can be explained by the introduction of DENV-3 in 1994-1998, and that the overall gradual decline in the FoI can be attributed to demographic shifts. Changes in immunity and demographics strongly impacted DENV transmission in Nicaragua. Population-level measures of transmission intensity are dynamic and thus challenging to use to guide vaccine implementation locally and globally.
TL;DR: Recent discoveries about the human antibody response to DENV are highlighted and guidelines for advancing development of safe and effective dengue vaccines are proposed.
Abstract: Dengue virus (DENV) is the most common arthropod-borne viral disease of humans. Although effective vaccines exist against other flaviviral diseases like yellow fever and Japanese encephalitis, dengue vaccine development is complicated by the presence of four virus serotypes and the possibility of partial immunity enhancing dengue disease severity. Several live attenuated dengue vaccines are being tested in human clinical trials. Initial results are mixed, with variable efficacy depending on DENV serotype and previous DENV exposure. Here, we highlight recent discoveries about the human antibody response to DENV and propose guidelines for advancing development of safe and effective dengue vaccines.
TL;DR: In this low-income country setting, ≈16% of household contacts acquired infections from index patients, despite high oseltamivir use.
Abstract: During August 2012-November 2014, we conducted a case ascertainment study to investigate household transmission of influenza virus in Managua, Nicaragua. We collected up to 5 respiratory swab samples from each of 536 household contacts of 133 influenza virus-infected persons and assessed for evidence of influenza virus transmission. The overall risk for influenza virus infection of household contacts was 15.7% (95% CI 12.7%-19.0%). Oseltamivir treatment of index patients did not appear to reduce household transmission. The mean serial interval for within-household transmission was 3.1 (95% CI 1.6-8.4) days. We found the transmissibility of influenza B virus to be higher than that of influenza A virus among children. Compared with households with 4 household contacts appeared to have a reduced risk for infection. Further research is needed to model household influenza virus transmission and design interventions for these settings.
TL;DR: This study sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014–2015 Caribbean/Americas epidemic and significantly expands the understanding of the emergence and evolution of CHikV CO clade in the Americas.
Abstract: Chikungunya virus (CHIKV) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes. In late 2013, the Asian genotype of CHIKV was responsible for the Caribbean outbreak (CO) that rapidly became an epidemic throughout the Americas. There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic. We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014–2015 Caribbean/Americas epidemic. Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak (CO) clade, revealed considerable genetic diversity within the CO clade, and described different epidemiological dynamics of CHIKV in the Americas. Specifically, we identified multiple introductions in both Nicaragua and Florida, with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US. Our phylogenetic analysis also showed phylogeographic clustering of the CO clade. In addition, we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade. Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels. Finally, we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua. To date, this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas.
TL;DR: This review describes how longitudinal prospective community-based, school- based, and household-based cohort studies contribute to improving knowledge of viral disease, focusing specifically on contributions to understanding and preventing dengue.
TL;DR: It is found that urea wash in a Zika virus nonstructural protein 1 IgG ELISA distinguishes secondary dengue virus infection from Zika virus infection with previous d Dengue with specificity of 93.8%.
Abstract: Serologic testing remains crucial for Zika virus diagnosis. We found that urea wash in a Zika virus nonstructural protein 1 IgG ELISA distinguishes secondary dengue virus infection from Zika virus infection with previous dengue (sensitivity 87.5%, specificity 93.8%). This test will aid serodiagnosis, serosurveillance, and monitoring of Zika complications in dengue-endemic regions.
TL;DR: Finding the majority of ZIKV infection in Nicaraguan households to be asymptomatic emphasizes the importance of silent ZikV transmission and helps inform public health interventions in the region and globally.
Abstract: Zika virus (ZIKV) infection recently caused major epidemics in the Americas and is linked to congenital birth defects and Guillain-Barre Syndrome. A pilot study of ZIKV infection in Nicaraguan households was conducted from August 31 to October 21, 2016, in Managua, Nicaragua. We enrolled 33 laboratory-confirmed Zika index cases and their household members (109 contacts) and followed them on days 3–4, 6–7, 9–10, and 21, collecting serum/plasma, urine, and saliva specimens along with clinical, demographic, and socio-economic status information. Collected samples were processed by rRT-PCR to determine viral load (VL) and duration of detectable ZIKV RNA in human bodily fluids. At enrollment, 11 (10%) contacts were ZIKV rRT-PCR-positive and 23 (21%) were positive by IgM antibodies; 3 incident cases were detected during the study period. Twenty of 33 (61%) index households had contacts with ZIKV infection, with an average of 1.9 (range 1–6) positive contacts per household, and in 60% of these households, ≥50% of the members were positive for ZIKV infection. Analysis of clinical information allowed us to estimate the symptomatic to asymptomatic (S:A) ratio of 14:23 (1:1.6) among the contacts, finding 62% of the infections to be asymptomatic. The maximum number of days during which ZIKV RNA was detected was 7 days post-symptom onset in saliva and serum/plasma and 22 days in urine. Overall, VL levels in serum/plasma, saliva, and urine specimens were comparable, with means of 5.6, 5.3 and 4.5 log10 copies/ml respectively, with serum attaining the highest VL peak at 8.1 log10 copies/ml. Detecting ZIKV RNA in saliva over a similar time-period and level as in serum/plasma indicates that saliva could potentially serve as a more accessible diagnostic sample. Finding the majority of infections to be asymptomatic emphasizes the importance of silent ZIKV transmission and helps inform public health interventions in the region and globally.
TL;DR: An internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of dengue virus and yellow fever virus and demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/μL of eluate for each DENV serotype and YFV.
Abstract: The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/μL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.
TL;DR: DNA sequence-based typing was performed on anonymized samples provided by 339 healthy adult blood bank donors in Managua, Nicaragua to characterize allele frequencies in the local population to support studies of T cell immunity against pathogens.
TL;DR: Live attenuated dengue vaccination elicits early transcriptional responses that mirror those found in symptomatic natural infection and provide candidate early markers of protection against DENV infection.
Abstract: Background Several promising live attenuated dengue vaccines are in development, but information about innate immune responses and early correlates of protection is lacking. Methods We characterized human genome-wide transcripts in whole blood from 10 volunteers at 11 time points after immunization with the dengue virus type 3 (DENV-3) component of the National Institutes of Health dengue vaccine candidate TV003 and from 30 hospitalized children with acute primary DENV-3 infection. We compared day-specific gene expression patterns with subsequent neutralizing antibody (NAb) titers. Results The transcriptional response to vaccination was largely confined to days 5-20 and was dominated by an interferon-associated signature and a cell cycle signature that peaked on days 8 and 14, respectively. Changes in transcript abundance were much greater in magnitude and scope in symptomatic natural infection than following vaccination (maximum fold-change >200 vs 21 postvaccination; 3210 vs 286 transcripts with significant fold-change), but shared gene modules were induced in the same sequence. The abundances of 131 transcripts on days 8 and 9 postvaccination were strongly correlated with NAb titers measured 6 weeks postvaccination. Conclusions Live attenuated dengue vaccination elicits early transcriptional responses that mirror those found in symptomatic natural infection and provide candidate early markers of protection against DENV infection. Clinical Trials Registration NCT00831012.
TL;DR: This novel assay allows us to analyze unique human samples from long-term studies of d Dengue and Zika in Nicaragua to investigate the nature of B cell/antibody responses and their role in pathogenesis and/or protection in secondary flavivirus infections and could have important implications for vaccine development for Zika and dengue.
Abstract: Co-circulation and re-emergence of antigenically related viruses such as dengue (DENV), Zika (ZIKV), and yellow fever (YF) in the Americas has brought a sense of urgency in the field to further define the genesis and to more fully describe the immune response. The recent explosive epidemics of Zika in the Americas and the co-circulation of ZIKV with the phylogenetically similar DENV has raised important questions and concerns regarding the role of cross-reactive immunity in protection and potential enhancement of severity of subsequent ZIKV or DENV infections in pre-immune individuals and the safety of vaccines against both viruses in endemic populations. Antibodies are a critical part of the immune response for clearing flavivirus infections, but the role of pre-existing antibodies in protection or enhancement of subsequent infection and disease with closely related viral species and strains is still not fully understood. We have developed a novel Multi-Color FluoroSpot (MCF) assay based on our ELISPOT-derived assay, previously designated the Quad-color FluoroSpot (QCF), in order to study the development of type-specific versus cross-reactive responses within the B cell pool of Zika virus (ZIKV)- and/or dengue virus (DENV)-infected patients. The QCF is based on a panel of four fluorescent Qdots, each conjugated to a monoclonal antibody specific to one of the four DENV serotypes; now we have included a fifth color (Qdot) for ZIKV to enable analysis of the specificity versus cross-reactivity of B cell populations at a single-cell level for all four DENV serotypes and ZIKV. This novel assay allows us to analyze unique human samples from long-term studies of dengue and Zika in Nicaragua to investigate the nature of B cell/antibody responses and their role in pathogenesis and/or protection in secondary flavivirus infections and could have important implications for vaccine development for Zika and dengue.
Broad Institute1, Harvard University2, Ragon Institute of MGH, MIT and Harvard3, Massachusetts Department of Public Health4, Oswaldo Cruz Foundation5, Florida Gulf Coast University6, Universidad Nacional Autónoma de Honduras7, Redeemer's University8, Pasteur Institute9, National Institutes of Health10, University of California, Berkeley11, Tufts University12, Massachusetts Institute of Technology13, Colorado State University14, University of Sierra Leone15
TL;DR: CATCH (Compact Aggregation of Targets for Comprehensive Hybridization), a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa, is developed and used to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of viral infections in samples with unknown content.
Abstract: Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. We developed CATCH (Compact Aggregation of Targets for Comprehensive Hybridization), a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs compact probe sets that achieve full coverage of known sequence diversity and that scale well with this diversity. To illustrate applications of CATCH, we focused on capturing viral genomes. We designed, synthesized, and validated multiple probe sets, including one that targets whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriched unique viral content on average 18-fold and allowed us to assemble genomes that we could not otherwise recover, while accurately preserving within-sample diversity. We used this approach to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of viral infections in samples with unknown content. Together, this work demonstrates a path toward more sensitive, cost-effective metagenomic sequencing.
TL;DR: Interferon- and cell cycle-associated gene transcript abundance levels in the peripheral blood of dengue vaccine recipients on days 8 and 9 post-vaccination were associated with d Dengue neutralizing antibody titers on day 42, and mirrored responses in primary dengued infection, suggesting the possibility of predicting protective immunity.
Abstract: Background: Several promising live attenuated virus (LAV) dengue vaccines are in development, but information about innate immune responses and early correlates of protection are lacking. Methods: We characterized human genome-wide transcripts in whole blood from 10 volunteers at 11 time-points after immunization with the dengue virus type 3 (DENV-3) component of the NIH dengue vaccine candidate TV003 and from 30 hospitalized children with acute primary DENV-3 infection. We compared day-specific gene expression patterns with subsequent neutralizing antibody (NAb) titers. Results: The transcriptional response to vaccination was largely confined to days 5-20 and was dominated by an interferon-associated signature and a cell cycle signature that peaked on days 8 and 14, respectively. Changes in transcript abundance were much greater in magnitude and scope in symptomatic natural infection than following vaccination (maximum fold-change >200 versus 21 post-vaccination; 3,210 versus 286 transcripts with significant fold-change), but shared gene modules were induced in the same sequence. The abundance of 131 transcripts on days 8 and 9 post-vaccination was strongly correlated with NAb titers measured 6 weeks post-vaccination. Conclusions: LAV dengue vaccination elicits early transcriptional responses that mirror those found in symptomatic natural infection and provide candidate early markers of protection against DENV infection.
TL;DR: It is suggested that virus and host-derived MHC ligands have under-exploited potential for describing the cell biology of DENV infection, and as candidates for designing effective DENV vaccines.
Abstract: Broadly effective vaccines against dengue virus (DENV) infection have remained elusive, despite rising infection rates in the developing world. Infection-specific peptide ligands presented on Major Histocompatibility Complexes (MHC) open new avenues for developing T-cell-based interventions. Past efforts towards mapping viral MHC epitopes were based on computational predictions that only partially reflected actual antigen presentation. To empirically identify DENV-specific MHC ligands, we developed an immuno-proteomics approach for interrogating DENV- and self-derived MHC ligands from infected B-lymphocytes. Here, we report four fundamental findings: First, over 700 infection-specific MHC-ligands reflected host cellular responses to DENV that were not apparent from the proteome. Second, we report 121 viral MHC-I ligands (108 novel) which clustered into discrete hotspots across the DENV polyprotein, some of which spanned DENV polyprotein components, described here as MHC ligands for the first time. Third, we found DENV ligands which were distinctly presented by MHC alleles previously associated with either high or low anti-DENV response. Fourth, we demonstrate that while our in vitro assay only overlapped with a small fraction of previously described DENV T-cell epitopes, several novel MHC ligands identified here were recognized by T-cells from DENV-infected patients despite having low binding affinities. Together, these discoveries suggest that virus and host-derived MHC ligands have under-exploited potential for describing the cell biology of DENV infection, and as candidates for designing effective DENV vaccines.
TL;DR: The whole-genome sequence of 11 Zika virus (ZIKV) samples from six pediatric patients in Nicaragua shows that the consensus ZIKV genomes are greater than 99% identical to each other.
Abstract: We report here the whole-genome sequence of 11 Zika virus (ZIKV) samples from six pediatric patients in Nicaragua. Serum samples were collected, and ZIKV was isolated in tissue culture. Both serum and virus isolates were sequenced. The consensus ZIKV genomes are greater than 99% identical to each other.