Simultaneous analysis of antigen-specific B and T cells after SARS-CoV-2 infection and vaccination.

Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B and T cell tandem lymphocyte evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an activation induced marker (AIM) T cell assay and dual-color B cell antigen probes. Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection.

Immunogenicity of a Live-Attenuated Dengue Vaccine Using a Heterologous Prime-Boost Strategy in a Phase 1 Randomized Clinical Trial.

BACKGROUND: Dengue is a global health problem and the development of a tetravalent dengue vaccine with durable protection is a high priority. A heterologous prime-boost strategy has the advantage of eliciting immune responses through different mechanisms and therefore may be superior to homologous prime-boost strategies for generating durable tetravalent immunity. METHODS: In this phase 1 first-in-human heterologous prime-boost study, 80 volunteers were assigned to 4 groups and received a tetravalent dengue virus (DENV-1-4) purified inactivated vaccine (TDENV-PIV) with alum adjuvant and a tetravalent dengue virus (DENV-1-4) live attenuated vaccine (TDENV-LAV) in different orders and dosing schedules (28 or 180 days apart). RESULTS: All vaccination regimens had acceptable safety profiles and there were no vaccine-related serious adverse events. TDEN-PIV followed by TDEN-LAV induced higher neutralizing antibody titers and a higher rate of tetravalent seroconversions compared to TDEN-LAV followed by TDEN-PIV. Both TDEN-PIV followed by TDEN-LAV groups demonstrated 100% tetravalent seroconversion 28 days following the booster dose, which was maintained for most of these subjects through the day 180 measurement. CONCLUSIONS: A heterologous prime-boost vaccination strategy for dengue merits additional evaluation for safety, immunogenicity, and potential for clinical benefit. CLINICAL TRIALS REGISTRATION: NCT02239614.

A Phase 1, Open-Label Assessment of a Dengue Virus-1 Live Virus Human Challenge Strain.

BACKGROUND: Dengue human infection models (DHIM) have been used as a safe means to test the viability of prophylaxis and therapeutics. METHODS: A phase 1 study of 12 healthy adult volunteers using a challenge virus, DENV-1-LVHC strain 45AZ5, was performed. A dose escalating design was used to determine the safety and performance profile of the challenge virus. Subjects were evaluated extensively until 28 days and then out to 6 months. RESULTS: Twelve subjects received the challenge virus: 6 with 0.5 mL of 6.5 × 103 plaque-forming units (PFU)/mL (low-dose group) and 6 with 0.5 mL of 6.5 × 104 PFU/mL (mid-dose group). All except 1 in the low-dose group developed detectable viremia. For all subjects the mean incubation period was 5.9 days (range 5-9 days) and mean time of viremia was 6.8 days (range 3-9 days). Mean peak for all subjects was 1.6 × 107 genome equivalents (GE)/mL (range 4.6 × 103 to 5 × 107 GE/mL). There were no serious adverse events or long-term safety signals noted. CONCLUSIONS: We conclude that DENV-1-LVHC was well-tolerated, resulted in an uncomplicated dengue illness, and may be a suitable DHIM for therapeutic and prophylactic product testing. CLINICAL TRIALS REGISTRATION: NCT02372175.

Effect of Antimalarial Drugs on the Immune Response to Intramuscular Rabies Vaccination Using a Postexposure Prophylaxis Regimen.

BACKGROUND: Chloroquine can impair the immune responses to intradermal rabies vaccination. Current guidelines recommend an extra intramuscular dose be given for postexposure prophylaxis in previously unvaccinated persons taking any antimalarial drug. METHODS: We conducted a randomized, open-label, single-site study in 103 previously unvaccinated healthy adults age ≥18 to ≤60 years old to evaluate the effects of chloroquine, atovaquone/proguanil (Malarone), and doxycycline on the antibody response to a purified chick embryo cell vaccine, given on a postexposure prophylaxis schedule. All treatment groups received antimalarials 14 days prior to and during vaccination. RESULTS: All subjects achieved accepted neutralizing antibody titers of ≥0.5 IU/mL following the second rabies vaccination dose and maintained this protection through the duration of the study. We observed a reduction in rabies antibody geometric mean titer in the chloroquine versus control groups 28 days after vaccination: 2.3 versus 6.87 IU/mL, respectively (P < .001, t test). A significant difference was not observed for those taking Malarone or doxycycline. CONCLUSIONS: We conclude that there is no reduction of rabies antibody response in subjects taking Malarone or doxycycline, but a significant reduction in those taking chloroquine; however, accepted antibody levels were achieved for all 3 antimalarials. CLINICAL TRIALS REGISTRATION: NCT02564471.

Serologic Response of 2 Versus 3 Doses and Intradermal Versus Intramuscular Administration of a Licensed Rabies Vaccine for Preexposure Prophylaxis.

BACKGROUND: The World Health Organization recommends intradermal (ID) administration of rabies vaccine for preexposure prophylaxis. METHODS: In a randomized trial in adults assigned to 1 of 6 treatment groups (ID vs intramuscular [IM], 2 vs 3 doses, and controls), rabies neutralizing antibody titers were measured to 1 year postvaccination. RESULTS: ID vaccination produced acceptable antibody levels in all subjects (2- and 3-dose groups). At day 365, acceptable levels were 40% for IM and 50% for ID 2-dose schedule, and 70% for IM and 60% for ID 3-dose schedule. CONCLUSIONS: ID rabies vaccination induces acceptable antibody titers at a fraction of the dose. CLINICAL TRIALS REGISTRATION: NCT02374814.

The Effects of Japanese Encephalitis Vaccine and Accelerated Dosing Scheduling on the Immunogenicity of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine: A Phase II, Randomized, Open-Label, Single-Center Trial in Adults Aged 18 to 45 Years in the United States.

BACKGROUND: Dengue is a global health problem requiring an effective, safe dengue vaccine. METHODS: We report the results of a phase II, randomized, open-label, single-center trial in adults aged 18 to 45 years in the United States designed to explore the effects of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine (CYD-TDV, Dengvaxia) when administered on its designated schedule (months 0, 6, and 12) or on an accelerated dosing schedule (months 0, 2, and 6) and/or given before, or concomitantly with, a vaccine against Japanese encephalitis (JE). RESULTS: Based on dengue virus serotype-specific neutralizing antibody (NAb), the accelerated dosing schedule was comparable to the 0, 6, and 12-month schedule. Giving JE vaccine concurrently with CYD-TDV did not result in an increase in overall NAb titers. Immunophenotyping of peripheral blood mononuclear cells revealed an increase in activated CD8+ T cells after CYD-TDV vaccination, a phenomenon that was greatest for the JE vaccine primed. CONCLUSIONS: We conclude that an accelerated dosing schedule of CYD-TDV results in essentially equivalent dengue serotype-specific NAb titers as the currently used schedule, and there may be an early benefit in antibody titers and activated CD8+ T cells by the administration of the JE vaccine before CYD-TDV vaccination.

An Innovative, Prospective, Hybrid Cohort-Cluster Study Design to Characterize Dengue Virus Transmission in Multigenerational Households in Kamphaeng Phet, Thailand.

Difficulties inherent in the identification of immune correlates of protection or severe disease have challenged the development and evaluation of dengue vaccines. There persist substantial gaps in knowledge about the complex effects of age and sequential dengue virus (DENV) exposures on these correlations. To address these gaps, we were conducting a novel family-based cohort-cluster study for DENV transmission in Kamphaeng Phet, Thailand. The study began in 2015 and is funded until at least 2023. As of May 2019, 2,870 individuals in 485 families were actively enrolled. The families comprise at least 1 child born into the study as a newborn, 1 other child, a parent, and a grandparent. The median age of enrolled participants is 21 years (range 0-93 years). Active surveillance is performed to detect acute dengue illnesses, and annual blood testing identifies subclinical seroconversions. Extended follow-up of this cohort will detect sequential infections and correlate antibody kinetics and sequence of infections with disease outcomes. The central goal of this prospective study is to characterize how different DENV exposure histories within multigenerational family units, from DENV-naive infants to grandparents with multiple prior DENV exposures, affect transmission, disease, and protection at the level of the individual, household, and community.

Dengue virus non-structural protein 1 activates the p38 MAPK pathway to decrease barrier integrity in primary human endothelial cells.

Dengue virus (DENV) causes an estimated 390 million infections worldwide annually, with severe forms of disease marked by vascular leakage. Endothelial cells (EC) are directly responsible for vascular homeostasis and are highly responsive to circulating mediators but are not commonly infected. DENV encodes seven non-structural (NS) proteins; with only one of those, NS1, secreted from infected cells and accumulating in the blood of patients. NS1 has been implicated in the pathogenesis of vascular permeability, but the mechanism is not completely understood. Here we used primary endothelial cells and an array of in vitro approaches to study the effect of NS1 in disease-relevant human ECs. Confocal microscopy demonstrated rapid NS1 internalization by ECs into endosomes with accumulation over time. Transcriptomic and pathway analysis showed significant changes in functions associated with EC homeostasis and vascular permeability. Functional significance of this activation was assessed by trans-endothelial electrical resistance and showed that NS1 induced rapid and transient loss in EC barrier function within 3 h post-treatment. To understand the molecular mechanism by which NS1 induced EC activation, we evaluated the stress-sensing p38 MAPK pathway known to be directly involved in EC permeability and inflammation. WB analysis of NS1-stimulated ECs showed clear activation of p38 MAPK and downstream effectors MAPKAPK-2 and HSP27 with chemical inhibition of the p38 MAP kinase pathway restoring barrier function. Our results suggest that DENV NS1 may be involved in the pathogenesis of severe dengue by activating the p38 MAPK in ECs, promoting increased permeability that characterizes severe disease.

A comparison of passive surveillance and active cluster-based surveillance for dengue fever in southern coastal Ecuador.

BACKGROUND: Dengue is a major emerging infectious disease, endemic throughout the tropics and subtropics, with approximately 2.5 billion people at risk globally. Active (AS) and passive surveillance (PS), when combined, can improve our understanding of dengue's complex disease dynamics to guide effective, targeted public health interventions. The objective of this study was to compare findings from the Ministry of Health (MoH) PS to a prospective AS arbovirus research study in Machala, Ecuador in 2014 and 2015. METHODS: Dengue cases in the PS system were compared to laboratory confirmed acute dengue illness cases that entered the AS study during the study period. Variables of interest included age class and sex. Outbreak detection curves by epidemiologic week, overall cumulative incidence and age-specific incidence proportions were calculated. Descriptive statistics were tabulated for all variables of interest. Chi-square tests were performed to compare demographic characteristics between the AS and PS data sets in 2014 and 2015. RESULTS: 177 and 245 cases were identified from 1/1/2014 to 12/31/2015 by PS and AS, respectively; nine cases appeared in both systems. AS identified a greater number of laboratory-confirmed cases in 2014, accounting for more than 60% of dengue cases in the study area. In 2015, the opposite trend was observed with PS identifying 60% of the dengue cases in the study area. Peak transmission time in laboratory confirmed dengue illness, as noted by AS and PS was similar in 2014, whereas earlier detection (7 weeks) was observed by AS in 2015. Younger patients were more frequently identified by PS, while older patients were identified more frequently by AS. The cumulative incidence proportion for laboratory confirmed dengue illness reported via PS to the MoH was 4.12 cases per 10,000 residents in 2014, and 2.21 cases per 10,000 residents in 2015. CONCLUSIONS: Each surveillance system captured distinct demographic subgroups within the Machala population, possibly due to differences in healthcare seeking behaviors, access to care, emerging threats of other viruses transmitted by the same mosquito vector and/or differences in clinical presentation. Integrating AS with pre-existing PS can aid in identifying additional cases in previously underdiagnosed subpopulations, improving our understanding of disease dynamics, and facilitating the implementation of timely public health interventions.

The Emergence of Zika Virus: A Narrative Review.

Zika virus (ZIKV) is yet another arbovirus that is rapidly emerging on a global scale, on the heels of a chikungunya epidemic in the Americas that began in 2013. A ZIKV epidemic that began in Brazil in 2015 has now spread rapidly to more than 30 countries in the Americas and the Caribbean, infecting more than 2 million inhabitants. This epidemic currently continues unabated. The explosive nature of recent outbreaks and concerning links to Guillain-Barré syndrome and microcephaly are incompletely understood. Also unknown is the relative importance of sexual transmission of ZIKV and asymptomatic ZIKV infections to the overall burden of transmission. The limited understanding of ZIKV presents an enormous challenge for responses to this rapidly emerging threat to human health. This article reviews the existing literature on ZIKV and proposes critical questions for vaccine development and other areas of needed research.

Revealing the microscale spatial signature of dengue transmission and immunity in an urban population.

It is well-known that the distribution of immunity in a population dictates the future incidence of infectious disease, but this process is generally understood at individual or macroscales. For example, herd immunity to multiple pathogens has been observed at national and city levels. However, the effects of population immunity have not previously been shown at scales smaller than the city (e.g., neighborhoods). In particular, no study has shown long-term effects of population immunity at scales consistent with the spatial scale of person-to-person transmission. Here, we use the location of dengue patients' homes in Bangkok with the serotype of the infecting pathogen to investigate the spatiotemporal distribution of disease risk at small spatial scales over a 5-y period. We find evidence for localized transmission at distances of under 1 km. We also observe patterns of spatiotemporal dependence consistent with the expected impacts of homotypic immunity, heterotypic immunity, and immune enhancement of disease at these distances. Our observations indicate that immunological memory of dengue serotypes occurs at the neighborhood level in this large urban setting. These methods have broad applications to studying the spatiotemporal structure of disease risk where pathogen serotype or genetic information is known.

Dengue human infection model performance parameters.

Dengue is a global health problem and of concern to travelers and deploying military personnel with development and licensure of an effective tetravalent dengue vaccine a public health priority. The dengue viruses (DENVs) are mosquito-borne flaviviruses transmitted by infected Aedes mosquitoes. Illness manifests across a clinical spectrum with severe disease characterized by intravascular volume depletion and hemorrhage. DENV illness results from a complex interaction of viral properties and host immune responses. Dengue vaccine development efforts are challenged by immunologic complexity, lack of an adequate animal model of disease, absence of an immune correlate of protection, and only partially informative immunogenicity assays. A dengue human infection model (DHIM) will be an essential tool in developing potential dengue vaccines or antivirals. The potential performance parameters needed for a DHIM to support vaccine or antiviral candidates are discussed.

A shorter time interval between first and second dengue infections is associated with protection from clinical illness in a school-based cohort in Thailand.

BACKGROUND: Despite the strong association between secondary dengue virus (DENV) infections and dengue hemorrhagic fever (DHF), the majority of secondary infections are subclinical or mild. The determinants of clinical severity remain unclear, though studies indicate a titer-dependent and time-dependent role of cross-protective anti-DENV antibodies. METHODS: Data from 2 sequential prospective cohort studies were analyzed for subclinical and symptomatic DENV infections in schoolchildren in Kamphaeng Phet, Thailand (1998-2002 and 2004-2007). Children experiencing ≥ 1 DENV infection were selected as the population for analysis (contributing 2169 person-years of follow-up). RESULTS: In total, 1696 children had ≥ 1 DENV infection detected during their enrollment; 268 experienced 2 or more infections. A shorter time interval between infections was associated with subclinical infection in children seronegative for DENV at enrollment, for whom a second-detected DENV infection is more likely to reflect a true second infection (average of 2.6 years between infections for DHF, 1.9 for DF, and 1.6 for subclinical infections). CONCLUSIONS: These findings support a pathogenesis model where cross-reactive antibodies wane from higher-titer, protective levels to lower-titer, detrimental levels. This is one of the first studies of human subjects to suggest a window of cross-protection following DENV infection since Sabin's challenge studies in the 1940s.

Low-dose dengue virus 3 human challenge model: a phase 1 open-label study.

Dengue human infection models present an opportunity to explore the potential of a vaccine, anti-viral or immuno-compound for clinical benefit in a controlled setting. Here we report the outcome of a phase 1 open-label assessment of a low-dose dengue virus 3 (DENV-3) challenge model (NCT04298138), in which nine participants received a subcutaneous inoculation with 0.5 ml of a 1.4 × 103 plaque-forming unit per ml suspension of the attenuated DENV-3 strain CH53489. The primary and secondary endpoints of the study were to assess the safety of this DENV-3 strain in healthy flavivirus-seronegative individuals. All participants developed RNAaemia within 7 days after inoculation with peak titre ranging from 3.13 × 104 to 7.02 × 108 genome equivalents per ml. Solicited symptoms such as fever and rash, clinical laboratory abnormalities such as lymphopenia and thrombocytopenia, and self-reported symptoms such as myalgia were consistent with mild-to-moderate dengue in all volunteers. DENV-3-specific seroconversion and memory T cell responses were observed within 14 days after inoculation as assessed by enzyme-linked immunosorbent assay and interferon-gamma-based enzyme-linked immunospot. RNA sequencing and serum cytokine analysis revealed anti-viral responses that overlapped with the period of viraemia. The magnitude and frequency of clinical and immunologic endpoints correlated with an individual's peak viral titre.

Current issues in dengue vaccination.

PURPOSE OF REVIEW: Dengue is a global health problem and of concern to travelers and deploying military personnel, with development and licensure of an effective tetravalent dengue vaccine a public health priority. The recent performance of the lead dengue vaccine in a phase 2b efficacy trial underscores dengue vaccine development challenges. This review focuses on current issues in dengue vaccination. RECENT FINDINGS: The dengue viruses (DENVs) are mosquito-borne flaviviruses transmitted by infected Aedes mosquitoes. Illness manifests across a clinical spectrum with severe disease characterized by intravascular volume depletion and hemorrhage. Recent estimates on the burden of DENV infection determined that there are 390 million dengue infections per year, three times the current estimate by the WHO. There are no licensed antivirals or vaccines to treat or prevent dengue though many are in preclinical or clinical development. DENV illness results from a complex interaction of viral properties and host immune responses. Immunologic complexity, lack of an adequate animal model of disease, absence of an immune correlate of protection, and only partially informative immunogenicity assays are challenging dengue vaccine development efforts. SUMMARY: Dengue vaccine development efforts have numerous complex challenges to overcome before a well tolerated and effective vaccine is licensed and available. In this review, the authors discuss the current issues in dengue vaccination.

Using Google Trends to Estimate the Geographic Distribution of Soil-Transmitted Helminthiasis in the United States from 2016 to 2021.

Soil-transmitted helminth infections are assumed to be uncommon in the US, despite numerous studies in the past few decades showing high burdens in Appalachia and the southern states. We assessed trends of interest in the Google search engine to gauge spatiotemporal patterns of potential soil-transmitted helminth transmission. We conducted a further ecological study comparing Google search trends to risk factors for soil-transmitted helminth transmission. Google search trends for terms related to soil-transmitted helminths were clustered in Appalachia and the south, with seasonal surges suggestive of endemic transmission for hookworm, roundworm (Ascaris), and threadworm. Furthermore, lower access to plumbing, increased septic tank use, and more rural environments were associated with increased soil-transmitted helminth-related Google search terms. Together, these results suggest that soil-transmitted helminthiasis remains endemic in parts of Appalachia and the south.

Biologics for dengue prevention: up-to-date.

INTRODUCTION: Dengue is a worsening global public health problem. The vector-viral-host interactions driving the pathogenesis of dengue are multi-dimensional. Sequential dengue virus (DENV) infections with different DENV types significantly increase the risk of severe disease. Treatment is supportive in nature as there are no licensed anti-DENV antivirals or immuno-therapeutics. A single dengue vaccine has widely been licensed with two others in advanced clinical development. Dengvaxia® has been licensed in numerous countries but uptake has been slow as a result of safety signals noted in the youngest recipients and those who were dengue naïve at the time of vaccination. AREAS COVERED: In this review, the current state of dengue vaccine and antiviral drug development will be discussed as well as new developments in controlled human infection models to support product development. EXPERT OPINION: The world needs a safe and efficacious tetravalent dengue vaccine capable of protecting multiple different populations across a broad age range and different flavivirus immunologic backgrounds. Safe and effective antivirals are also needed to prevent or attenuate dengue disease in the unvaccinated, in cases of vaccine failure, or in high-risk populations.

Evolution of inflammation and immunity in a dengue virus 1 human infection model.

Dengue virus (DENV) infections are major causes of morbidity and mortality throughout the tropics and subtropics. More than 400 million infections are estimated to occur every year, resulting in nearly 100 million symptomatic infections and more than 20,000 deaths. Early immune response kinetics to infection remain unclear, in large part due to the variable incubation period exhibited by the DENVs after introduction into a susceptible host. To fill this knowledge gap, we performed a comprehensive virologic and immunologic analysis of individuals experimentally infected with the underattenuated DENV-1 strain 45AZ5. This analysis captured both the kinetics and composition of the innate, humoral, and cellular immune responses elicited by experimental DENV-1 infection, as well as virologic and clinical features. We observed a robust DENV-specific immunoglobulin A (IgA) antibody response that manifested between the appearance of DENV-specific IgM and IgG in all challenged individuals, as well as the presence of a non-neutralizing/NS1-specific antibody response that was delayed relative to the appearance of DENV virion-specific humoral immunity. RNA sequencing analysis revealed discrete and temporally restricted gene modules that correlated with acute viremia and the induction of adaptive immunity. Our analysis provides a detailed description, in time and space, of the evolving matrix of DENV-elicited human inflammation and immunity and reveals several previously unappreciated immunological aspects of primary DENV-1 infection that can inform countermeasure development and evaluation.

Memory CD8+ T cells from naturally acquired primary dengue virus infection are highly cross-reactive.

Cross-reactive memory T cells induced by primary infection with one of the four serotypes of dengue virus (DENV) are hypothesized to have an immunopathological function in secondary heterologous DENV infection. To define the T-cell response to heterologous serotypes, we isolated HLA-A(*)1101-restricted epitope-specific CD8(+) T-cell lines from primary DENV-immune donors. Cell lines exhibited marked cross-reactivity toward peptide variants representing the four DENV serotypes in tetramer binding and functional assays. Many clones responded similarly to homologous and heterologous serotypes with striking cross-reactivity between the DENV-1 and DENV-3 epitope variants. In vitro-stimulated T-cell lines consistently revealed a hierarchical induction of MIP-1ß>degranulation>tumor necrosis factor α (TNFα)>interferon-γ (IFNγ), which depended on the concentration of agonistic peptide. Phosphoflow assays showed peptide dose-dependent phosphorylation of ERK1/2, which correlated with cytolysis, degranulation, and induction of TNFα and IFNγ, but not MIP-1ß production. This is the first study to show significant DENV serotype-cross-reactivity of CD8(+) T cells after naturally acquired primary infection. We also show qualitatively different T-cell receptor signaling after stimulation with homologous and heterologous peptides. Our data support a model whereby the order of sequential DENV infections influences the immune response to secondary heterologous DENV infection, contributing to varying disease outcomes.