Dengue virus (DENV) is an increasingly important human pathogen, with already half of the globe's population living in environments with transmission potential. Since many cases are missed by direct detection methods (RT-PCR or antigen tests), serological assays play an important role in the diagnostic process. However, individual assays can suffer from low sensitivity and specificity and interpreting results from multiple assays remains challenging, particularly because interpretations from multiple assays may differ, creating uncertainty over how to generate finalized interpretations. We develop a Bayesian mixture model that can jointly model data from multiple paired serological assays, to infer infection events. We first test the performance of our model using simulated data. We then apply our model to 677 pairs of acute and convalescent serum collected as a part of illness and household investigations across two longitudinal cohort studies in Kamphaeng Phet, Thailand, including data from 232 RT-PCR confirmed infections (gold standard). We compare the classification of the new model to prior standard interpretations that independently utilize information from either the hemagglutination inhibition assay (HAI) or the enzyme-linked immunosorbent assay (EIA). We find that additional serological assays improve accuracy of infection detection for both simulated and real world data. Models incorporating paired IgG and IgM data as well as those incorporating IgG, IgM, and HAI data consistently have higher accuracy when using PCR confirmed infections as a gold standard (87-90% F1 scores, a combined metric of sensitivity and specificity) than currently implemented cut-point approaches (82-84% F1 scores). Our results provide a probabilistic framework through which multiple serological assays across different platforms can be leveraged across sequential serum samples to provide insight into whether individuals have recently experienced a DENV infection. These methods are applicable to other pathogen systems where multiple serological assays can be leveraged to quantify infection history.

Uncovering rates at which susceptible individuals become infected with a pathogen, i.e., the force of infection (FOI), is essential for assessing transmission risk and reconstructing distribution of immunity in a population. For dengue, reconstructing exposure and susceptibility statuses from the measured FOI is of particular significance as prior exposure is a strong risk factor for severe disease. FOI can be measured via many study designs. Longitudinal serology is considered gold standard measurements, as they directly track the transition of seronegative individuals to seropositive due to incident infections (seroincidence). Cross-sectional serology can provide estimates of FOI by contrasting seroprevalence across ages. Age of reported cases can also be used to infer FOI. Agreement of these measurements, however, has not been assessed. Using 26 y of data from cohort studies and hospital-attended cases from Kamphaeng Phet province, Thailand, we found FOI estimates from the three sources to be highly inconsistent. Annual FOI estimates from seroincidence were 1.75 to 4.05 times higher than case-derived FOI. Seroprevalence-derived was moderately correlated with case-derived FOI (correlation coefficient = 0.47) with slightly lower estimates. Through extensive simulations and theoretical analysis, we show that incongruences between methods can result from failing to account for dengue antibody kinetics, assay noise, and heterogeneity in FOI across ages. Extending standard inference models to include these processes reconciled the FOI and susceptibility estimates. Our results highlight the importance of comparing inferences across multiple data types to uncover additional insights not attainable through a single data type/analysis.

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. Virological, clinical and immunological characterization of a dengue virus 3 human challenge model could help with identifying vaccine and drug candidates.

Rapid diagnostic tests (RDTs) are critical for preparedness and response against an outbreak or pandemic and have been highlighted in the 100 Days Mission, a global initiative that aims to prepare the world for the next epidemic/pandemic by driving the development of diagnostics, vaccines and therapeutics within 100 days of recognition of a novel Disease X threat.RDTs play a pivotal role in early case identification, surveillance and case management, and are critical for initiating deployment of vaccine and monoclonal antibodies. Currently available RDTs, however, have limited clinical sensitivity and specificity and inadequate validation. The development, validation and implementation of RDTs require adequate and sustained financing from both public and private sources. While the World Health Assembly recently passed a resolution on diagnostic capacity strengthening that urges individual Member States to commit resources towards this, the resolution is not binding and implementation will likely be impeded by limited financial resources and other competing priorities, particularly in low-income countries. Meanwhile, the diagnostic industry has not sufficiently invested in RDT development for high priority pathogens.Currently, vaccine development projects are getting the largest funding support among medical countermeasures. Yet vaccines are insufficient tools in isolation, and pandemic preparedness will be incomplete without parallel investment in diagnostics and therapeutics.The Pandemic Fund, a global financing mechanism recently established for strengthening pandemic prevention, preparedness and response, may be a future avenue for supporting diagnostic development.In this paper, we discuss why RDTs are critical for preparedness and response. We also discuss RDT investment challenges and reflect on the way forward.

Infants less than 1 y of age experience high rates of dengue disease in dengue virus (DENV) endemic countries. This burden is commonly attributed to antibody-dependent enhancement (ADE), whereby concentrations of maternally derived DENV antibodies become subneutralizing, and infection-enhancing. Understanding antibody-related mechanisms of enhanced infant dengue disease risk represents a significant challenge due to the dynamic nature of antibodies and their imperfect measurement processes. Further, key uncertainties exist regarding the impact of long-term shifts in birth rates, population-level infection risks, and maternal ages on the DENV immune landscape of newborns and their subsequent risks of severe dengue disease in infancy. Here, we analyze DENV antibody data from two infant cohorts (N = 142 infants with 605 blood draws) and 40 y of infant dengue hospitalization data from Thailand. We use mathematical models to reconstruct maternally derived antibody dynamics, accounting for discretized measurement processes and limits of assay detection. We then explore possible antibody-related mechanisms of enhanced infant dengue disease risk and their ability to reconstruct the observed age distribution of hospitalized infant dengue cases. We find that ADE mechanisms are best able to reconstruct the observed data. Finally, we describe how the shifting epidemiology of dengue in Thailand, combined with declining birth rates, have decreased the absolute risk of infant dengue disease by 88% over a 40-y period while having minimal impact on the mean age of infant hospitalized dengue disease.

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.

The Flaviviruses comprise a group of related viruses that are highly diverse in their ecological niches, life-cycles, evolution and disease manifestations in humans. Common among them is their importance in producing human disease and as emerging or emerged global health threats. Yellow fever virus, the prototypical Flavivirus, a historical example of an arbovirus maintained in a sylvatic cycle in the jungles and through the slave trade and transportation of infected individuals and its mosquito vector caused millions of deaths throughout the Americas. The dengue viruses a modern day example of the rapid evolution of an arbovirus that circulates in a mosquito to human to mosquito cycle and is now an emerged global health problem and the most common arboviral infection in the tropical and subtropical regions of the world causing millions of hospitalizations and death from hemorrhagic fever. In this chapter we hope to review the unique viral characteristics of the Flaviviruses and their importance as human pathogens.

Dengue virus (DENV) infection risk is known to vary substantially, even across small communities, with infections in and around the home driving transmission. However, It remains unclear how the immune status of an individual or household dictate this risk in part due to transmission being dominated by subclinical infections. In this study, we used demographic, household characteristic, and serological data from a multigenerational cohort study of 2860 individuals from 470 households in Kamphaeng Phet, Thailand, to determine the incidence and risk factors for DENV infections. We used hemagglutination inhibition (HAI) antibody titers measured in sequential serum samples to identify subclinical infections through a gradient boosted regression model. This approach identified ∼10% more cases than commonly used methods with approximately 90% of all infections being subclinical. As expected, we found that having higher DENV antibody titers was protective against infection. Individuals were additionally protected if other household members had higher titers suggesting that there are indirect effects of household immunity on the individuals found within a household. Our study provides a framework for inferring subclinical infections and characterizing the epidemiology of DENV infection in households.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe NIH Grant P01 AI034533 and R35 GM138361 supported the work.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This study was approved by Thailand Ministry of Public Health Ethical Research Committee, Siriraj Ethics Committee on Research Involving Human Subjects, Institutional Review Board for the Protection of Human Subjects State University of New York Upstate Medical University, and Walter Reed Army Institute of Research Institutional Review Board (protocol #2119).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data and code associated with the work can be found at https://github.com/marcohamins/role-of-HH-immunity.

Although it is known that household infections drive the transmission of dengue virus (DENV), it is unclear how household composition and the immune status of inhabitants affect the individual risk of infection. Most population-based studies to date have focused on paediatric cohorts because more severe forms of dengue mainly occur in children, and the role of adults in dengue transmission is understudied. Here we analysed data from a multigenerational cohort study of 470 households, comprising 2,860 individuals, in Kamphaeng Phet, Thailand, to evaluate risk factors for DENV infection. Using a gradient-boosted regression model trained on annual haemagglutination inhibition antibody titre inputs, we identified 1,049 infections, 90% of which were subclinical. By analysing imputed infections, we found that individual antibody titres, household composition and antibody titres of other members in the same household affect an individual’s risk of DENV infection. Those individuals living in households with high average antibody titres, or households with more adults, had a reduced risk of infection. We propose that herd immunity to dengue acts at the household level and may provide insight into the drivers of the recent change in the shifting age distribution of dengue cases in Thailand.

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.

Dengue human infection models present an opportunity to explore a vaccine, antiviral, or immuno-compound’s potential for clinical benefit in a controlled setting. Herein, we report the outcome of a phase 1, open-label assessment of a DENV-3 challenge model. In this study, 9 participants received a subcutaneous inoculation with 0.5ml of a 1.4×103 PFU/ml suspension of the DENV-3 strain CH53489. All subjects developed RNAemia within 7 days of inoculation, with peak titers ranging from 3.13×104 to 7.02×108 GE/ml. Symptoms and clinical lab abnormalities consistent with mild dengue infection were observed in all subjects. DENV-3 specific seroconversion was observed by 14 days after inoculation, along with DENV-3 specific memory T cell responses. RNAseq and serum cytokine analysis revealed the presence of an antiviral transcriptional and cytokine response to infection that overlapped with the period of viremia. The magnitude and frequency of clinical and immunologic endpoints correlated with an individual’s peak viral titer.### Competing Interest StatementSJ Thomas is an academic and executes clinical trials and other research related to dengue. The funding for these is provided by federal and sponsored funding. These relationships are between his University and the funder. He currently has funding related to dengue from Janssen, Merck, Atea, NIH, and CDMRP. He also sits on advisory boards and review committees related to dengue countermeasure development. In some cases, he is compensated for his time and effort with monies, equity, or both. Current personal relationships of this variety include Merck (chair, scientific ad committee, dengue vaccines, financial comp.), PrimeVax (member, scientific ad board, dengue use in immuno-oncology, equity), Island Pharma (member, scientific ad board, dengue drug, equity), and Takeda (member, advisory board, dengue vaccines, financial comp; chair, dengue case adjudication committee, dengue vaccine, financial comp). He also participates in similar advisory and committee positions for non-dengue related countermeasure development work. He states that none of the work above relates to the submitted manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.### Clinical TrialNCT04298138### Funding StatementFunding for this research was provided by the Department of Defense, Medical Research and Material Command, the Military Infectious Disease Research Program, and the State of New York.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethical approval for the Dengue Human Infection Model and associated analysis was approved by the State University of New York Upstate Medical University (SUNY-UMU) and the Department of Defense's Human Research Protection OfficeI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesThe authors declare that all data supporting the findings of this study are available within this article and its Supplementary Information files, or from the corresponding author upon reasonable request. RNAseq gene expression data have been deposited in the NCBI Gene Expression Omnibus (GEO GSE216328).

The development of effective vaccines and therapies against viral diseases requires reliable approaches for assessing durable immunological memory. Although a wealth of information is available from peripheral blood mononuclear cell samples, 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 optimized Activation Induced Marker (AIM) T cell assay and a 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. While spike-specific B cell and CD8+ T cell responses were higher following mRNA vaccination than natural infection alone, the quantity of CD4+ T cells recognizing spike was enhanced only in subjects with low responses to natural infection. Our findings suggest divergent patterns of B and T cell subset mobilization with repeated antigen encounter and highlight the utility of examining these populations in concert. Supported by the State of New York.

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.

MIRM is an uncommon entity characterized by prominent mucositis, usually with sparse cutaneous involvement. The diagnosis of MIRM can be challenging due to the lack of awareness amongst clinicians. Patients with Behçet's disease usually present with recurrent and painful mucocutaneous ulcers, while other clinical manifestations of the disease are more variable. Here, we describe an interesting case of MIRM mimicking Behçet's disease and PNP highlighting the overlapping manifestations and diagnostic challenges.

We explored the drivers of dengue infection in an endemic setting testing blood taken from multigenerational households and looking for antibodies against dengue virus. We showed that most of the variance in seropositivity was explained by unmeasured house-level effects. Background Dengue virus (DENV) often circulates endemically. In such settings with high levels of transmission, it remains unclear whether there are risk factors that alter individual infection risk. Methods We tested blood taken from individuals living in multigenerational households in Kamphaeng Phet province, Thailand for DENV antibodies (N = 2364, mean age 31 years). Seropositivity ranged from 45.4% among those 1-5 years old to 99.5% for those >30 years. Using spatially explicit catalytic models, we estimated that 11.8% of the susceptible population gets infected annually. Results We found that 37.5% of the variance in seropositivity was explained by unmeasured household-level effects with only 4.2% explained by spatial differences between households. The serostatus of individuals from the same household remained significantly correlated even when separated by up to 15 years in age. Conclusions These findings show that despite highly endemic transmission, persistent differences in infection risk exist across households, the reasons for which remain unclear.

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.

The mean age of dengue hemorrhagic fever (DHF) cases increased considerably in Thailand from 8.1 to 24.3 y between 1981 and 2017 (mean annual increase of 0.45 y). Alternative proposed explanations for this trend, such as changes in surveillance practices, reduced mosquito–human contact, and shifts in population demographics, have different implications for global dengue epidemiology. To evaluate the contribution of each of these hypothesized mechanisms to the observed data, we developed 20 nested epidemiological models of dengue virus infection, allowing for variation over time in population demographics, infection hazards, and reporting rates. We also quantified the effect of removing or retaining each source of variation in simulations of the age trajectory. Shifts in the age structure of susceptibility explained 58% of the observed change in age. Adding heterogeneous reporting by age and reductions in per-serotype infection hazard to models with shifts in susceptibility explained an additional 42%. Reductions in infection hazards were mostly driven by changes in the number of infectious individuals at any time (another consequence of shifting age demographics) rather than changes in the transmissibility of individual infections. We conclude that the demographic transition drives the overwhelming majority of the observed change as it changes both the age structure of susceptibility and the number of infectious individuals. With the projected Thai population age structure, our results suggest a continuing increase in age of DHF cases, shifting the burden toward individuals with more comorbidity. These insights into dengue epidemiology may be relevant to many regions of the globe currently undergoing comparable changes in population demographics.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the pandemic human respiratory illness COVID-19, is a global health emergency. While severe acute disease has been linked to an expansion of antibody-secreting plasmablasts, we sought to identify B cell responses that correlated with positive clinical outcomes in convalescent patients. We characterized the peripheral blood B cell immunophenotype and plasma antibody responses in 40 recovered non-hospitalized COVID-19 subjects that were enrolled as donors in a convalescent plasma treatment study. We observed a significant negative correlation between the frequency of peripheral blood memory B cells and the duration of symptoms for convalescent subjects. Memory B cell subsets in convalescent subjects were composed of classical CD24+ class-switched memory B cells, but also activated CD24-negative and natural unswitched CD27+ IgD+ IgM+ subsets. Memory B cell frequency was significantly correlated with both IgG1 and IgM responses to the SARS-CoV-2 spike protein receptor binding domain (RBD) in most seropositive subjects. IgM+ memory, but not switched memory, directly correlated with virus-specific antibody responses, and remained stable over 3 months. Our findings suggest that the frequency of memory B cells is a critical indicator of disease resolution, and that IgM+ memory B cells may play an important role in SARS-CoV-2 immunity.

Memory T cells resulting from primary dengue virus (DENV) infection are hypothesized to influence the clinical outcome of subsequent DENV infection. However, the few studies involving prospectively collected blood samples have found weak and inconsistent associations with outcome and variable temporal trends in DENV-specific memory T cell responses between subjects. This study used both ex-vivo and cultured ELISPOT assays to further evaluate the associations between DENV serotype-cross-reactive memory T cells and severity of secondary infection. Using ex-vivo ELISPOT assays, frequencies of memory T cells secreting IFN-γ in response to DENV structural and non-structural peptide pools were low in PBMC from multiple time points prior to symptomatic secondary DENV infection and showed a variable response to infection. There were no differences in responses between subjects who were not hospitalized (NH, n=6) and those who were hospitalized with dengue hemorrhagic fever (hDHF, n=4). In contrast, responses in cultured ELISPOT assays were more reliably detectable prior to secondary infection and showed more consistent increases after infection. Responses in cultured ELISPOT assays were higher in individuals with hDHF (n=8) compared to NH (n=9) individuals before the secondary infection, with no difference between these groups after infection. These data demonstrate an association of pre-existing DENV-specific memory responses with the severity of illness in subsequent DENV infection, and suggest that frequencies of DENV-reactive T cells measured after short-term culture may be of particular importance for assessing the risk for more severe dengue disease.