Increased frequencies of highly activated regulatory T cells skewed to a T helper 1-like phenotype with reduced suppressive capacity in dengue patients.

The pathogenesis of dengue involves a complex interplay between the viral factor and the host immune response. A mismatch between the infecting serotype and the adaptive memory response is hypothesized to lead to exacerbated immune responses resulting in severe dengue. Here, we aim to define in detail the phenotype and function of different regulatory T cell (Treg) subsets and their association with disease severity in a cohort of acute dengue virus (DENV)-infected Cambodian children. Treg frequencies and proliferation of Tregs are increased in dengue patients compared to age-matched controls. Tregs from dengue patients are skewed to a Th1-type Treg phenotype. Interestingly, Tregs from severe dengue patients produce more interleukin-10 after in vitro stimulation compared to Tregs from classical dengue fever patients. Functionally, Tregs from dengue patients have reduced suppressive capacity, irrespective of disease severity. Taken together, these data suggest that even though Treg frequencies are increased in the blood of acute DENV-infected patients, Tregs fail to resolve inflammation and thereby could contribute to the immunopathology of dengue. IMPORTANCE: According to the World Health Organization, dengue is the fastest-spreading, epidemic-prone infectious disease. The extent of dengue virus infections increased over the years, mainly driven by globalization-including travel and trade-and environmental changes. Dengue is an immunopathology caused by an imbalanced immune response to a secondary heterotypic infection. As regulatory T cells (Tregs) are essential in maintaining immune homeostasis and dampening excessive immune activation, this study addressed the role of Tregs in dengue immunopathology. We show that Tregs from dengue patients are highly activated, skewed to a Th1-like Treg phenotype and less suppressive compared to healthy donor Tregs. Our data suggest that Tregs fail to resolve ongoing inflammation during dengue infection and hence contribute to the immunopathology of severe dengue disease. These data clarify the role of Tregs in dengue immunopathogenesis, emphasizing the need to develop T cell-based vaccines for dengue.

In-vitro assessment of cutaneous immune responses to aedes mosquito salivary gland extract and dengue virus in Cambodian individuals.

Dengue virus (DENV) poses a global health threat, affecting millions individuals annually with no specific therapy and limited vaccines. Mosquitoes, mainly Aedes aegypti and Aedes albopictus worldwide, transmit DENV through their saliva during blood meals. In this study, we aimed to understand how Aedes mosquito saliva modulate skin immune responses during DENV infection in individuals living in mosquito-endemic regions. To accomplish this, we dissociated skin cells from Cambodian volunteers and incubated them with salivary gland extract (SGE) from three different mosquito strains: Ae. aegypti USDA strain, Ae. aegypti and Ae. albopictus wild type (WT) in the presence/absence of DENV. We observed notable alterations in skin immune cell phenotypes subsequent to exposure to Aedes salivary gland extract (SGE). Specifically, exposure lead to an increase in the frequency of macrophages expressing chemokine receptor CCR2, and neutrophils expressing CD69. Additionally, we noted a substantial increase in the percentage of macrophages that became infected with DENV in the presence of Aedes SGE. Differences in cellular responses were observed when Aedes SGE of three distinct mosquito strains were compared. Our findings deepen the understanding of mosquito saliva's role in DENV infection and skin immune responses in individuals regularly exposed to mosquito bites. This study provides insights into skin immune cell dynamics that could guide strategies to mitigate DENV transmission and other arbovirus diseases.

Characterization of soluble TLR2 and CD14 levels during acute dengue virus infection.

Dengue virus infection results in a broad spectrum of diseases ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Hitherto, there is no consensus biomarker for the prediction of severe dengue disease in patients. Yet, early identification of patients who progress to severe dengue is pivotal for better clinical management. We have recently reported that an increased frequency of classical (CD14 ++CD16-) monocytes with sustained high TLR2 expression in acutely infected dengue patients correlates with severe dengue development. Here, we hypothesized that the relatively lower TLR2 and CD14 expression in mild dengue patients is due to the shedding of their soluble forms (sTLR2 and sCD14) and that these could be used as indicators of disease progression. Therefore, using commercial sandwich ELISAs, we evaluated the release of sTLR2 and sCD14 by peripheral blood mononuclear cells (PBMCs) in response to in vitro dengue virus (DENV) infection and assessed their levels in acute-phase plasma of 109 dengue patients. We show that while both sTLR2 and sCD14 are released by PBMCs in response to DENV infection in vitro, their co-circulation in an acute phase of the disease is not always apparent. In fact, sTLR2 was found only in 20% of patients irrespective of disease status. In contrast, sCD14 levels were detected in all patients and were significantly elevated in DF patients when compared to DHF patients and age-matched healthy donors. Altogether, our results suggest that sCD14 may help in identifying patients at risk of severe dengue at hospital admittance.

Evaluation of cutaneous immune response in a controlled human in vivo model of mosquito bites.

Mosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito 'bite', eliciting immune responses that shape the establishment of infection and pathogenesis. Here we assess the cutaneous innate and adaptive immune responses to controlled Aedes aegypti feedings in humans living in Aedes-endemic areas. In this single-arm, cross-sectional interventional study (trial registration #NCT04350905), we enroll 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We perform 3-mm skin biopsies at baseline as well as 30 min, 4 h, and 48 h after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints are measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. The results reveal induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2 macrophages. As the immune reaction progresses T cell priming and regulatory pathways are upregulated along with a shift to Th2-driven responses and CD8+ T cell activation. Stimulation of participants' bitten skin cells with Aedes aegypti salivary gland extract results in reduced pro-inflammatory cytokine production. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites and can be leveraged to inform and develop novel therapeutics and vector-targeted vaccine candidates to interfere with vector-mediated disease.

Temporal patterns of functional anti-dengue antibodies in dengue infected individuals with different disease outcome or infection history.

Heterotypic secondary dengue virus (DENV) infection is a risk factor for the development of severe disease. To assess the contribution of the developing polyclonal humoral immune response to the course of acute infection, we have determined anti-DENV IgG titers, neutralizing antibodies, percentages of antibodies binding to DENV-infected cells and antibody­dependent enhancement (ADE) to the infecting serotype in DENV-infected Cambodian children (n = 58), ranging from asymptomatic dengue to severe disease. The results showed that ADE titers are highest against the infecting serotype during heterotypic secondary DENV-2 infection. Moreover, IgG titers, neutralizing antibodies and ADE titers against the infecting serotype peak at D10 and are maintained until D60 after laboratory-confirmed secondary DENV infection. Anti-DENV IgG titers and the magnitude of the functional antibody response were higher in secondary DENV-infected patients compared to primary infected patients. No differences in antibody titers, neutralizing or enhancing antibodies could be observed between asymptomatic or hospitalized patients between 6 and 8 days after laboratory-confirmed DENV-1 infection. However, at this time point, the level of IgG bound to DENV-infected cells was associated with disease severity in hospitalized patients. Taken together, our data offer insights for more comprehensive interpretation of antibody response profile to natural infection and its correlation to disease outcome.

Rapid Generation of In-House Serological Assays Is Comparable to Commercial Kits Critical for Early Response to Pandemics: A Case With SARS-CoV-2.

Introduction: Accurate and sensitive measurement of antibodies is critical to assess the prevalence of infection, especially asymptomatic infection, and to analyze the immune response to vaccination during outbreaks and pandemics. A broad variety of commercial and in-house serological assays are available to cater to different laboratory requirements; however direct comparison is necessary to understand utility. Materials and Methods: We investigate the performance of six serological methods against SARS-CoV-2 to determine the antibody profile of 250 serum samples, including 234 RT-PCR-confirmed SARS-CoV-2 cases, the majority with asymptomatic presentation (87.2%) at 1-51 days post laboratory diagnosis. First, we compare to the performance of two in-house antibody assays: (i) an in-house IgG ELISA, utilizing UV-inactivated virus, and (ii) a live-virus neutralization assay (PRNT) using the same Cambodian isolate as the ELISA. In-house assays are then compared to standardized commercial anti-SARS-CoV-2 electrochemiluminescence immunoassays (Elecsys ECLIAs, Roche Diagnostics; targeting anti-N and anti-S antibodies) along with a flow cytometry based assay (FACS) that measures IgM and IgG against spike (S) protein and a multiplex microsphere-based immunoassay (MIA) determining the antibodies against various spike and nucleoprotein (N) antigens of SARS-CoV-2 and other coronaviruses (SARS-CoV-1, MERS-CoV, hCoVs 229E, NL63, HKU1). Results: Overall, specificity of assays was 100%, except for the anti-S IgM flow cytometry based assay (96.2%), and the in-house IgG ELISA (94.2%). Sensitivity ranged from 97.3% for the anti-S ECLIA down to 76.3% for the anti-S IgG flow cytometry based assay. PRNT and in-house IgG ELISA performed similarly well when compared to the commercial ECLIA: sensitivity of ELISA and PRNT was 94.7 and 91.1%, respectively, compared to S- and N-targeting ECLIA with 97.3 and 96.8%, respectively. The MIA revealed cross-reactivity of antibodies from SARS-CoV-2-infected patients to the nucleocapsid of SARS-CoV-1, and the spike S1 domain of HKU1. Conclusion: In-house serological assays, especially ELISA and PRNT, perform similarly to commercial assays, a critical factor in pandemic response. Selection of suitable immunoassays should be made based on available resources and diagnostic needs.

Robust and Functional Immune Memory Up to 9 Months After SARS-CoV-2 Infection: A Southeast Asian Longitudinal Cohort.

The duration of humoral and cellular immune memory following SARS-CoV-2 infection in populations in least developed countries remains understudied but is key to overcome the current SARS-CoV-2 pandemic. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for Spike (S)-binding and neutralizing antibodies and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-S antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immune memory was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased antibody-dependent cellular cytotoxicity (ADCC) and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immune memory. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection and in the absence of re-infection.

Comparison of Japanese Encephalitis Force of Infection in Pigs, Poultry and Dogs in Cambodian Villages.

Japanese encephalitis virus (JEV) is the main cause of human viral encephalitis in Asia, with a mortality rate reaching 30%, mostly affecting children. The traditionally described cycle involving wild birds as reservoirs, pigs as amplifying hosts and Culex mosquitoes as vectors is questioned, with increasing evidence of a more complex multi-host system involved in areas where densities of pigs are low, such as in Cambodia. In 2018, we examined pigs, chickens, ducks and dogs from Kandal province, Cambodia, for antibody response against JEV by hemagglutination inhibition and virus neutralization assays. Forces of infection (FOI) for flaviviruses and JEV were estimated per species and per unit of body surface area (BSA). JEV seroprevalence reached 31% (95% CI: 23-41%) in pigs, 1% (95% CI: 0.1-3%) in chickens, 12% (95% CI: 7-19%) in ducks and 35% (95% CI: 28-42%) in dogs. Pigs were most likely to be infected (FOI: 0.09 per month), but the FOI was higher in ducks than in pigs for a given BSA (ratio of 0.13). Dogs had a lower FOI than ducks but a higher FOI than chickens (0.01 per month). For a given BSA, dogs were less likely to be infected than pigs (ratio of 1.9). In Cambodia, the virus may be circulating between multiple hosts. Dogs live in close contact with humans, and estimating their exposure to JEV infection could be a relevant indicator of the risk for humans to get infected, which is poorly known due to underdiagnosis. Understanding the JEV cycle and developing tools to quantify the exposure of humans is essential to adapt and support control measures for this vaccine-preventable disease.

Decreased Type I Interferon Production by Plasmacytoid Dendritic Cells Contributes to Severe Dengue.

The clinical presentation of dengue virus (DENV) infection is variable. Severe complications mainly result from exacerbated immune responses. Type I interferons (IFN-I) are important in antiviral responses and form a crucial link between innate and adaptive immunity. Their contribution to host defense during DENV infection remains under-studied, as direct quantification of IFN-I is challenging. We combined ultra-sensitive single-molecule array (Simoa) digital ELISA with IFN-I gene expression to elucidate the role of IFN-I in a well-characterized cohort of hospitalized Cambodian children undergoing acute DENV infection. Higher concentrations of type I IFN proteins were observed in blood of DENV patients, compared to healthy donors, and correlated with viral load. Stratifying patients for disease severity, we found a decreased expression of IFN-I in patients with a more severe clinical outcome, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). This was seen in parallel to a correlation between low IFNα protein concentrations and decreased platelet counts. Type I IFNs concentrations were correlated to frequencies of plasmacytoid DCs, not DENV-infected myloid DCs and correlated inversely with neutralizing anti-DENV antibody titers. Hence, type I IFN produced in the acute phase of infection is associated with less severe outcome of dengue disease.