Maternally derived antibody titer dynamics and risk of hospitalized infant dengue disease.

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.

Assessing the role of multiple mechanisms increasing the age of dengue cases in Thailand.

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 mosquito­human 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.

Protective Role of NS1-Specific Antibodies in the Immune Response to Dengue Virus through Antibody-Dependent Cellular Cytotoxicity.

BACKGROUND: Dengue virus (DENV) non-structural protein 1 (NS1) has multiple functions within infected cells, on the cell surface, and in secreted form, and is highly immunogenic. Immunity from previous DENV infections is known to exert both positive and negative effects on subsequent DENV infections, but the contribution of NS1-specific antibodies to these effects is incompletely understood. METHODS: We investigated the functions of NS1-specific antibodies and their significance in DENV infection. We analyzed plasma samples collected in a prospective cohort study prior to symptomatic or subclinical secondary DENV infection. We measured binding to purified recombinant NS1 protein and to NS1-expressing CEM cells, antibody-mediated NK cell activation by plate-bound NS1 protein, and antibody-dependent cellular cytotoxicity (ADCC) of NS1-expressing target cells. RESULTS: We found that antibody responses to NS1 were highly serotype-cross-reactive and that subjects who experienced subclinical DENV infection had significantly higher antibody responses to NS1 in pre-infection plasma than subjects who experienced symptomatic infection. We observed strong positive correlations between antibody binding and NK activation. CONCLUSIONS: These findings demonstrate the involvement of NS1-specific antibodies in ADCC and provide evidence for a protective effect of NS1-specific antibodies in secondary DENV infection.

Reconstruction of antibody dynamics and infection histories to evaluate dengue risk.

As with many pathogens, most dengue infections are subclinical and therefore unobserved 1 . Coupled with limited understanding of the dynamic behaviour of potential serological markers of infection, this observational problem has wide-ranging implications, including hampering our understanding of individual- and population-level correlates of infection and disease risk and how these change over time, between assay interpretations and with cohort design. Here we develop a framework that simultaneously characterizes antibody dynamics and identifies subclinical infections via Bayesian augmentation from detailed cohort data (3,451 individuals with blood draws every 91 days, 143,548 haemagglutination inhibition assay titre measurements)2,3. We identify 1,149 infections (95% confidence interval, 1,135-1,163) that were not detected by active surveillance and estimate that 65% of infections are subclinical. After infection, individuals develop a stable set point antibody load after one year that places them within or outside a risk window. Individuals with pre-existing titres of ≤1:40 develop haemorrhagic fever 7.4 (95% confidence interval, 2.5-8.2) times more often than naive individuals compared to 0.0 times for individuals with titres >1:40 (95% confidence interval: 0.0-1.3). Plaque reduction neutralization test titres ≤1:100 were similarly associated with severe disease. Across the population, variability in the size of epidemics results in large-scale temporal changes in infection and disease risk that correlate poorly with age.

Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections.

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.

Individual, Household, and Community Drivers of Dengue Virus Infection Risk in Kamphaeng Phet Province, Thailand.

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.

Disease-driven reduction in human mobility influences human-mosquito contacts and dengue transmission dynamics.

Heterogeneous exposure to mosquitoes determines an individual's contribution to vector-borne pathogen transmission. Particularly for dengue virus (DENV), there is a major difficulty in quantifying human-vector contacts due to the unknown coupled effect of key heterogeneities. To test the hypothesis that the reduction of human out-of-home mobility due to dengue illness will significantly influence population-level dynamics and the structure of DENV transmission chains, we extended an existing modeling framework to include social structure, disease-driven mobility reductions, and heterogeneous transmissibility from different infectious groups. Compared to a baseline model, naïve to human pre-symptomatic infectiousness and disease-driven mobility changes, a model including both parameters predicted an increase of 37% in the probability of a DENV outbreak occurring; a model including mobility change alone predicted a 15.5% increase compared to the baseline model. At the individual level, models including mobility change led to a reduction of the importance of out-of-home onward transmission (R, the fraction of secondary cases predicted to be generated by an individual) by symptomatic individuals (up to -62%) at the expense of an increase in the relevance of their home (up to +40%). An individual's positive contribution to R could be predicted by a GAM including a non-linear interaction between an individual's biting suitability and the number of mosquitoes in their home (>10 mosquitoes and 0.6 individual attractiveness significantly increased R). We conclude that the complex fabric of social relationships and differential behavioral response to dengue illness cause the fraction of symptomatic DENV infections to concentrate transmission in specific locations, whereas asymptomatic carriers (including individuals in their pre-symptomatic period) move the virus throughout the landscape. Our findings point to the difficulty of focusing vector control interventions reactively on the home of symptomatic individuals, as this approach will fail to contain virus propagation by visitors to their house and asymptomatic carriers.

Effect of low-passage number on dengue consensus genomes and intra-host variant frequencies.

Intra-host single nucleotide variants (iSNVs) have been increasingly used in genomic epidemiology to increase phylogenetic resolution and reconstruct fine-scale outbreak dynamics. These analyses are preferably done on sequence data from direct clinical samples, but in many cases due to low viral loads, there might not be enough genetic material for deep sequencing and iSNV determination. Isolation of the virus from clinical samples with low-passage number increases viral load, but few studies have investigated how dengue virus (DENV) culture isolation from a clinical sample impacts the consensus sequence and the intra-host virus population frequencies. In this study, we investigate consensus and iSNV frequency differences between DENV sequenced directly from clinical samples and their corresponding low-passage isolates. Twenty five DENV1 and DENV2 positive sera and their corresponding viral isolates (T. splendens inoculation and C6/36 passage) were obtained from a prospective cohort study in the Philippines. These were sequenced on MiSeq with minimum nucleotide depth of coverage of 500×, and iSNVs were detected using LoFreq. For both DENV1 and DENV2, we found a maximum of one consensus nucleotide difference between clinical sample and isolate. Interestingly, we found that iSNVs with frequencies ≥5 % were often preserved between the samples, and that the number of iSNV positions, and sample diversity, at this frequency cutoff did not differ significantly between the sample pairs (clinical sample and isolate) in either DENV1 or DENV2 data. Our results show that low-passage DENV isolate consensus genomes are largely representative of their direct sample parental viruses, and that low-passage isolates often mirror high frequency within-host variants from direct samples.

Major Histocompatibility Complex Class I Chain-Related A and B (MICA and MICB) Gene, Allele, and Haplotype Associations With Dengue Infections in Ethnic Thais.

BACKGROUND: Major histocompatibility complex class I chain-related (MIC) A and B (MICA and MICB) are polymorphic stress molecules recognized by natural killer cells. This study was performed to analyze MIC gene profiles in hospitalized Thai children with acute dengue illness. METHODS: MIC allele profiles were determined in a discovery cohort of patients with dengue fever or dengue hemorrhagic fever (DHF) (n = 166) and controls (n = 149). A replication cohort of patients with dengue (n = 222) was used to confirm specific MICB associations with disease. RESULTS: MICA*045 and MICB*004 associated with susceptibility to DHF in secondary dengue virus (DENV) infections (odds ratio [OR], 3.22; [95% confidence interval (CI), 1.18-8.84] and 1.99 [1.07-2.13], respectively), and MICB*002 with protection from DHF in secondary DENV infections (OR, 0.41; 95% CI, .21-.68). The protective effect of MICB*002 against secondary DHF was confirmed in the replication cohort (OR, 0.43; 95% CI, .22-.82) and was stronger when MICB*002 is present in individuals also carrying HLA-B*18, B*40, and B*44 alleles which form the B44 supertype of functionally related alleles (0.29, 95% CI, .14-.60). CONCLUSIONS: Given that MICB*002 is a low expresser of soluble proteins, these data indicate that surface expression of MICB*002 with B44 supertype alleles on DENV-infected cells confer a protective advantage in controlling DENV infection using natural killer cells.

Heterogeneity of Dengue Illness in Community-Based Prospective Study, Iquitos, Peru.

Measuring heterogeneity of dengue illness is necessary to define suitable endpoints in dengue vaccine and therapeutic trials and will help clarify behavioral responses to illness. To quantify heterogeneity in dengue illness, including milder cases, we developed the Dengue Illness Perceptions Response (IPR) survey, which captured detailed symptom data, including intensity, duration, and character, and change in routine activities caused by illness. During 2016-2019, we collected IPR data daily during the acute phase of illness for 79 persons with a positive reverse transcription PCR result for dengue virus RNA. Most participants had mild ambulatory disease. However, we measured substantial heterogeneity in illness experience, symptom duration, and maximum reported intensity of individual symptoms. Symptom intensity was a more valuable predicter of major activity change during dengue illness than symptom presence or absence alone. These data suggest that the IPR measures clinically useful heterogeneity in dengue illness experience and its relation to altered human behavior.

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.

Multiplexed FluoroSpot for the Analysis of Dengue Virus- and Zika Virus-Specific and Cross-Reactive Memory B Cells.

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections.

Activation of Peripheral T Follicular Helper Cells During Acute Dengue Virus Infection.

Background: Follicular helper T cells (TFH) are specialized CD4 T cells required for B-cell help and antibody production. Methods: Given the postulated role of immune activation in dengue disease, we measured the expansion and activation of TFH in the circulation (peripheral TFH [pTFH]) collected from Thai children with laboratory-confirmed acute dengue virus (DENV) infection. Results: We found significant expansion and activation of pTFH subsets during acute infection with the highest frequencies of activated pTFH (PD1hi pTFH and PD1+CD38+ pTFH) detected during the critical phase of illness. Numbers of activated pTFH were higher in patients with secondary compared with primary infections and in patients with more severe disease. We also found a positive correlation between the frequencies of activated pTFH and the frequencies of plasmablasts. Conclusions: To our knowledge, this is the first ex vivo analysis of pTFH activation during acute DENV infection. Overall, our study supports the model that pTFH contribute to disease evolution during the critical stage of illness.

Case Management of Dengue: Lessons Learned.

The global burden of dengue and its geographic distribution have increased over the past several decades. The introduction of dengue in new areas has often been accompanied by high case-fatality rates. Drawing on the experience in managing dengue cases at the Queen Sirikit National Institute of Child Health in Bangkok, Thailand, this article provides the authors' perspectives on key clinical lessons to improve dengue-related outcomes. Parallels between this clinical experience and outcomes reported in randomized controlled trials, results of efforts to disseminate practice recommendations, and suggestions for areas for further research are also discussed.

Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

BACKGROUND: The development of reagents to identify and characterize antigen-specific B cells has been challenging. METHODS: We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. RESULTS: In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. CONCLUSIONS: AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination.

Dengue Virus (DENV) Neutralizing Antibody Kinetics in Children After Symptomatic Primary and Postprimary DENV Infection.

The immune response to dengue virus (DENV) infection is complex and not fully understood. Using longitudinal data from 181 children with dengue in Thailand who were followed for up to 3 years, we describe neutralizing antibody kinetics following symptomatic DENV infection. We observed that antibody titers varied by serotype, homotypic vs heterotypic responses, and primary versus postprimary infections. The rates of change in antibody titers over time varied between primary and postprimary responses. For primary infections, titers increased from convalescence to 6 months. By comparing homotypic and heterotypic antibody titers, we saw an increase in type specificity from convalescence to 6 months for primary DENV3 infections but not primary DENV1 infections. In postprimary cases, there was a decrease in titers from convalescence up until 6 months after infection. Beginning 1 year after both primary and postprimary infections, there was evidence of increasing antibody titers, with greater increases in children with lower titers, suggesting that antibody titers were boosted due to infection and that higher levels of neutralizing antibody may be more likely to confer a sterilizing immune response. These findings may help to model virus transmission dynamics and provide baseline data to support the development of vaccines and therapeutics.

HLA Class I Supertype Associations With Clinical Outcome of Secondary Dengue Virus Infections in Ethnic Thais.

BACKGROUND: Human leukocyte antigen (HLA) supertypes are groups of functionally related alleles that present structurally similar antigens to the immune system. OBJECTIVES: To analyze HLA class I supertype associations with clinical outcome in hospitalized Thai children with acute dengue illness. METHODS: Seven hundred sixty-two patients and population-matched controls recruited predominantly in Bangkok were HLA-A and -B typed. HLA supertype frequencies were compared and tested for significant dengue disease associations using logistic regression analyses. Multivariable models were built by conducting forward stepwise selection procedures. RESULTS: In the final logistic regression model, the HLA-B44 supertype was protective against dengue hemorrhagic fever (DHF) in secondary infections (odds ratio [OR] = 0.46, 95% confidence interval [CI], .30-.72), while the HLA-A02 supertype (OR = 1.92, 95% CI, 1.30-2.83) and the HLA-A01/03 supertype (OR = 3.01, 95% CI, 1.01-8.92) were associated with susceptibility to secondary dengue fever. The B07 supertype was associated with susceptibility to secondary DHF in the univariate analysis (OR = 1.60, 95% CI, 1.05-2.46), whereas that was not retained in the final model. CONCLUSIONS: As the HLA-B44 supertype is predicted to target conserved epitopes in dengue, our results suggest that B44 supertype-restricted immune responses to highly conserved regions of the dengue proteome may protect against secondary DHF.

Sequential dengue virus infections detected in active and passive surveillance programs in Thailand, 1994-2010.

BACKGROUND: The effect of prior dengue virus (DENV) exposure on subsequent heterologous infection can be beneficial or detrimental depending on many factors including timing of infection. We sought to evaluate this effect by examining a large database of DENV infections captured by both active and passive surveillance encompassing a wide clinical spectrum of disease. METHODS: We evaluated datasets from 17 years of hospital-based passive surveillance and nine years of cohort studies, including clinical and subclinical DENV infections, to assess the outcomes of sequential heterologous infections. Chi square or Fisher's exact test was used to compare proportions of infection outcomes such as disease severity; ANOVA was used for continuous variables. Multivariate logistic regression was used to assess risk factors for infection outcomes. RESULTS: Of 38,740 DENV infections, two or more infections were detected in 502 individuals; 14 had three infections. The mean ages at the time of the first and second detected infections were 7.6 ± 3.0 and 11.2 ± 3.0 years. The shortest time between sequential infections was 66 days. A longer time interval between sequential infections was associated with dengue hemorrhagic fever (DHF) in the second detected infection (OR 1.3, 95% CI 1.2-1.4). All possible sequential serotype pairs were observed among 201 subjects with DHF at the second detected infection, except DENV-4 followed by DENV-3. Among DENV infections detected in cohort subjects by active study surveillance and subsequent non-study hospital-based passive surveillance, hospitalization at the first detected infection increased the likelihood of hospitalization at the second detected infection. CONCLUSIONS: Increasing time between sequential DENV infections was associated with greater severity of the second detected infection, supporting the role of heterotypic immunity in both protection and enhancement. Hospitalization was positively associated between the first and second detected infections, suggesting a possible predisposition in some individuals to more severe dengue disease.

DHIM supporting immunologic investigations and the identification of immune correlates of protection.

Evidence suggesting that immune responses to dengue virus (DENV) have the potential for both beneficial and detrimental effects on the outcome of infection is a concern for dengue vaccine development. There is thus a great need to define measures of DENV-specific immune responses that reliably indicate when immunity is protective. The existence of 4 main DENV serotypes and the difficulty in defining which individuals have been exposed and to which viruses present challenges to defining immune correlates of protective immunity against DENV in field efficacy studies; experimental infection studies in humans offer a pathway to address these challenges.

Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection.

Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation.