Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Intra-serotypic antigenic diversity of dengue virus serotype 3 in Thailand during 2004-2015.

In addition to the well-known differences among the four dengue serotypes, intra-serotypic antigenic diversity has been proposed to play a role in viral evolution and epidemic fluctuation. A replacement of genotype II by genotype III of dengue virus serotype 3 (DENV3) occurred in Thailand during 2007-2014, raising questions about the role of intra-serotypic antigenic differences in this genotype shift. We characterized the antigenic difference of DENV3 of genotypes II and III in Thailand, utilizing a neutralizing antibody assay with DENV3 vaccine sera and monotypic DENV3 sera. Although there was significant antigenic diversity among the DENV3, it did not clearly associate with the genotype. Our data therefore do not support the role of intra-serotypic antigenic difference in the genotype replacement. Amino acid alignment showed that eight positions are potentially associated with diversity between distinct antigenic subgroups. Most of these amino acids were found in envelope domain II. Some positions (aa81, aa124, and aa172) were located on the surface of virus particles, probably involving the neutralization sensitivity. Notably, the strains of both genotypes II and III showed clear antigenic differences from the vaccine genotype I strain. Whether this differencewill affect vaccine efficacy requires further studies.

Survey on neutralizing antibodies against Zika virus eighteen months post-outbreak in two southern Thailand communities.

BACKGROUND: In 2016 and 2017, Zika virus (ZIKV) infection outbreaks occurred in two communities in southern Thailand. This re-immerging infection can widely spread by mosquito bites and cause serious complications in a central nervous system among children born to infected mothers. Thus, they should be protected. This study aims to (1) To determine the prevalence of neutralizing ZIKV antibodies in the post-outbreak areas among the general population and pregnancy women residing at various distances from the houses of the nearest index patients; (2) To examine the cross-neutralizing capacity of antibodies against ZIKV on other flaviviruses commonly found in the study areas; (3) To identify factors associated with the presence of neutralizing ZIKV antibodies. METHODS: The two post-outbreak communities were visited at 18 months after the outbreaks. We enrolled (1) 18 confirmed ZIKV infected (index) cases, (2) sample of 554 neighbors in the outbreak areas who lived at various distances from the index patients' houses, (3) 190 residents of non-outbreak areas, and (4) all pregnant women regardless of gestational age residing in the study areas (n = 805). All serum specimens underwent the plaque reduction neutralization test (PRNT). Ten randomly selected ZIKV seropositive and ten randomly selected seronegative specimens were tested for dengue virus serotypes 1-4 (DENV1-4) and Japanese encephalitis virus (JEV) antibodies using PRNT90. Serum titer above 1:10 was considered positive. Multiple logistic regression was used to assess factors associated with seropositivity. RESULTS: Out of all 18 index cases, 9 remained seropositive. The seroprevalence (95% CI) in the two outbreak areas were 43.7% (35.9-51.6%) and 29.7% (23.3-36.0%) in general population, and 24.3% (20.1-28.8%) and 12.8% (9.7-16.5%) in pregnant women. Multivariate analysis showed that seropositivity was independent of the distance gradient from the index's houses. However, being elderly was associated with seropositivity. DENV1-4 and JEV neutralizing antibodies were present in most ZIKV-positive and negative subsamples. CONCLUSION: Protective herd immunity for ZIKV infection is inadequate, especially among pregnant women in the two post-outbreak areas in southern Thailand.

Serological Evidence of Japanese Encephalitis Virus Circulation in Asian Children From Dengue-Endemic Countries.

Background: Japanese encephalitis virus (JEV) is a zoonotic, mosquito-borne flavivirus, distributed across Asia. Infections are mostly mild or asymptomatic, but symptoms include neurological disorders, sequelae, and fatalities. Data to inform control strategies are limited due to incomplete case reporting. Methods: We used JEV serological data from a multicountry Asian dengue vaccine study in children aged 2-14 years to describe JEV endemicity, measuring antibodies by plaque reduction neutralization test (PRNT50). Results: A total 1479 unvaccinated subjects were included. A minimal estimate of pediatric JEV seroprevalence in dengue-naive individuals was 8.1% in Indonesia, 5.8% in Malaysia, 10.8% in the Philippines, and 30.7% in Vietnam, translating to annual infection risks varying from 0.8% (in Malaysia) to 5.2% (in Vietnam). JEV seroprevalence and annual infection estimates were much higher in children with history of dengue infection, indicating cross-neutralization within the JEV PRNT50 assay. Conclusions: These data confirm JEV transmission across predominantly urban areas and support a greater emphasis on JEV case finding, diagnosis, and prevention.

Monoclonal antibody-based capture ELISA in the diagnosis of previous dengue infection.

BACKGROUND: Dengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand. METHODS: The MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3-11 years in Ratchaburi Province, Thailand. RESULTS: The sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects. CONCLUSIONS: This research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.

Differences in activation and tissue homing markers of natural killer cell subsets during acute dengue infection.

Dengue virus (DENV) infection is considered one of the most important mosquito-borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV-infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56hi CD16- and CD56lo CD16+ NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56hi CD16- subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56lo CD16+ subset compared with those in the CD56hi CD16- subset. Moreover, although the CD56lo CD16+ subset contained a high frequency of cells expressing skin-homing markers, the CD56hi CD16- subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection.

The synergistic effect of nsP2-L618, nsP3-R117, and E2-K187 on the large plaque phenotype of chikungunya virus.

Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is the etiological agent of chikungunya fever. CHIKV re-emerged from 2004 onwards, and subsequently caused major outbreaks in many parts of the world including the Indian Ocean islands, Asia, and the Americas. In this study, a large plaque variant of CHIKV isolated from patient in Thailand was subjected to repeated cycles of plaque-purification in Vero cells. The resulting virus produced homogenous large plaques and showed a more pathogenic phenotype than the parental wild-type CHIKV. Whole genome analysis of the large plaque virus in comparison to parental isolate revealed a number of mutations, leading to the following amino acid changes: nsP2 (P618→L), nsP3 (G117→R), and E2 (N187→K). Eight recombinant CHIKVs were constructed to determine which amino acids mediated the large plaque phenotype. The results showed the recombinant virus which contains all three mutations, rCHK-L, produced significantly larger plaques than the other recombinant viruses (p < 0.01). Moreover, the plaque size of the other recombinant virus tended to be smaller if they contained only one or two of the large plaque associated mutations in the viral genome. In conclusion, the combination of all three residues (nsP2-L618, nsP3-R117, and E2-K187) is required to produce the large plaque phenotype of CHIKV.

Small plaque size variant of chikungunya primary isolate showed reduced virulence in mice.

BACKGROUND: Plaque size is a common feature of viral characterization. Small plaque size is used as a marker of attenuation for live-attenuated vaccine development. OBJECTIVE: To investigate whether the naturally occurring plaque size variation reflects virulence of the variants of chikungunya virus (CHIKV). METHODS: We selected and purified a variant with small plaque size from the primary isolate. The viral variant was tested for the plaque morphology, in vitro growth kinetics and mouse neurovirulence in comparison with the parental wild type. RESULTS: The small plaque size variant showed stable homogenous small plaques after 4 plaque purifications. The small plaque virus grew slower and to the lower titer when compared with wild type virus. After 21 days of infection, mice that received small plaque virus showed 98% survival rate while 74% of mice survived after infected with wild type virus. CONCLUSION: The small plaque size variant of CHIKV can be obtained by plaque purification and the virus displays decreased virulence.

Persistence of Wild-Type Japanese Encephalitis Virus Strains Cross-Neutralization 5 Years After JE-CV Immunization.

BACKGROUND: The live-attenuated Japanese encephalitis (JE) vaccine (JE-CV; IMOJEV) induces a protective response in children. A shift in circulating JE virus strains suggests that a genotype shift phenomenon may occur throughout Southeast Asia. We assessed the neutralization of wild-type (WT) JE virus isolates at distal time points after vaccination. METHODS: We analyzed serum samples from a subset of 47 children who had received a JE-CV booster after an inactivated JE vaccine primary immunization. We measured antibody titers (50% plaque reduction neutralization test) using a panel of WT JE strains at baseline, then after the booster at 28 days and 6 months in all subjects present at the time points and in a subset at year 5. Three additional recent isolates were tested at year 5. RESULTS: Of 47 subjects, 43 (91.5%) subjects had JE neutralizing antibody titers ≥10 (reciprocal serum dilution) against the homologous strain before JE-CV boost; all were seroprotected up to year 5 after the JE-CV boost. Baseline WT seroprotection ranged between 78.7% and 87.2%; all subjects were seroprotected against the 4 WT strains at 28 days and 6 months; year 5 seroprotection ranged between 95.7% and 97.9%. Similar rates of protection against 3 additional WT isolates were observed at year 5. CONCLUSIONS: The long-term immune responses induced after a JE-CV booster dose in toddlers were able to neutralize WT viruses from various genotypes circulating in Southeast Asia and India. CLINICAL TRIALS REGISTRATION: NCT00621764.

Marmosets (Callithrix jacchus) as a non-human primate model for evaluation of candidate dengue vaccines: induction and maintenance of specific protective immunity against challenges with clinical isolates.

Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.

Involvement of fatty acid synthase in dengue virus infection.

BACKGROUND: The mosquito transmitted Dengue virus (DENV) remains a significant public health problem in many tropical and subtropical countries. Increasing evidence has suggested that during the infection process cellular lipids play important roles at several stages of the replication cycle. This study sought to characterize the changes in lipid metabolism gene expression and investigated the role of one enzyme, fatty acid synthase, in DENV infection. METHODS: Transcriptional profiles of genes associated with lipid metabolism were evaluated by real-time PCR after infection of different cell lines (HepG2 and HEK293T/17) and with different DENVs (laboratory adapted and low passage). Expression profiles of genes were evaluated by western blotting. A critical lipid metabolism protein, fatty acid synthase was down-regulated through siRNA and inhibited with orlistat and the effect on DENV infection determined by flow cytometry, plaque assay, western blotting and confocal microscopy. RESULTS: The results showed alterations of gene transcription and expression were seen in genes variously associated with lipogenesis, lipolysis and fatty acid ß-oxidation during DENV infection. Interference of fatty acid synthase with either siRNA or orlistat had marked effects on virus production, with orlistat having an EC50 value of 10.07 µM at 24 h post infection. However, non-structural protein expression was largely unaffected. CONCLUSIONS: While drug treatment reduced virus titer by up to 3Log10, no significant effect on DENV non-structural protein expression was observed, suggesting that fatty acid synthase acts through an effect on virion formation.

High Anti-Dengue Virus Activity of the OAS Gene Family Is Associated With Increased Severity of Dengue.

Dengue is a mosquito-borne viral disease that afflicts millions of individuals worldwide every year. Infection by any of the 4 dengue virus (DENV) serotypes can result in a spectrum of disease severity. We investigated the impact of variants of interferon-regulated innate immunity genes with a potent antiviral effect on the outcome of DENV infection. We compared the effect of OAS gene family variants on 2 DENV serotypes in cell culture. While both OAS1-p42 and p46 showed antiviral activity against DENV-2, only OAS1-p42 presented anti-DENV-1 activity. Conversely, whereas both OAS3_S381 and R381 variants were able to block DENV-1 infection, the anti-DENV-2 activity observed for OAS3_S381 was largely lost for the R381 variant. By means of an allelic association study of a cohort of 740 patients with dengue, we found a protective effect of OAS3_R381 against shock (odds ratio [OR], 0.37; P < .001). This effect was due to DENV-2 infections (OR, 0.13; P = .007) but was absent for DENV-1, in accordance with the serotype-dependent OAS3 activity found in the functional study. Severe dengue has long been associated with a cytokine storm of unclear origin. This work identifies an early innate immunity process that could lead to the immune overreaction observed in severe dengue and could be triggered by a specific host genotype-pathogen genotype interaction.

Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.

Evidence of plasticity in the dengue virus: Host cell interaction.

Dengue virus (DENV) is the most important mosquito transmitted human viral pathogen. There are four different dengue viruses (DENV 1 to DENV 4) with multiple genotypes and strains. Whether there are significant differences in how these DENVs interact with and modulate the host cell proteome remains unclear. Using a panel of 12 DENVs representative of one isolate for each DENV from three different origins (lab adapted, low passage isolates from dengue fever patients, low passage isolates from dengue hemorrhagic fever patients) LLC-MK2 cells were equally infected and proteomic alterations compared by MALDI-TOF and principal component analysis and a sub-10 kDa peptidome analysis. There was no clear segregation of data with respect to either virus origin or serotype in either the MALDI-TOF or the peptidome analysis. The two isolates with the greatest variation from the other isolates in the MALDI-TOF analysis were a low passage DENV 3 dengue fever isolate and a low passage DENV 4 dengue hemorrhagic fever isolate. Analysis of the sub-10 kda protein fraction by LC-MS/MS identified 128 proteins of which only 28 (20%) were constantly expressed in all infections, while 80% showed variable expression, with no clear relationship with either serotype or virus origin. These results suggest that the interaction between DENV and the host cell is characterized by a degree of plasticity, whereby the end biological processes are not rigorously determined by specific proteome alterations, and that virus strain plays a role in determining the specific proteome changes.

Plaque reduction neutralization antibody test does not accurately predict protection against dengue infection in Ratchaburi cohort, Thailand.

BACKGROUND: The plaque reduction neutralization test (PRNT) is currently the best and most widely accepted approach to measuring virus-neutralizing and protective antibodies to dengue virus, and in assessing the immunogenicity of a dengue vaccine. However, the correlation between presence of dengue-neutralizing antibody and protection from infection is not absolute. FINDINGS: In a cohort study in Ratchaburi Province, Thailand, 48 subjects with serologically confirmed symptomatic dengue infection were tested for pre-existing dengue neutralizing antibody using PRNT. Nine subjects had quite high pre-existing PRNT50 titers (titer >90) to subsequent infecting dengue serotypes, but still had symptomatic infections. CONCLUSION: This report provides evidence that PRNT may not be a good test for predicting protection against subsequent dengue infection.

Long-term persistence of Chikungunya virus neutralizing antibodies in human populations of North Eastern Thailand.

BACKGROUND: Chikungunya virus (CHIKV) outbreak recurrences in Thailand are unpredictable and separated by unexplained and often long silent epidemiological periods that can last for several years. These silent periods could be explained in part by the fact that infection with one CHIKV strain confers lasting natural immunity, even against other CHIKV strains. In this study we evaluated the persistence of CHIKV-specific neutralizing antibodies in the population of Chumpae District, Khon Kaen Province, nineteen years after a CHIKV outbreak occurred in the same area in 1991. FINDINGS: Overall 39% (44/111) of 111 former patients had neutralizing antibodies reacting against CHIKV ECSA strain. Consistently high titers of neutralizing antibodies were found in 75% (33/44) of all positively-reacting sera, 70% of which (23/33) were collected from individuals amongst the >60 years old age group. Although the prevalence found in Pong Haeng village (70%) was significantly higher than the prevalence detected in the Nong Thum village (14%), control study villages without known previous Chikungunya epidemics had a high Chikungunya neutralizing antibody prevalence (65%). CONCLUSIONS: More than one-third of the pre-exposed population had persisting natural immunity that was more likely boosted by recent and repetitive exposure to the emerging ECSA CHIKV in Thailand. Also, Chikungunya virus appears to largely circulate in the country with a great variability appears between villages or area probably associated with the vector abundance and efficiency. Altogether these results show a potential for a lifelong immunity against CHIKV. Given the rapid spread of the highly pathogenic ECSA strain in Southern Thailand, the development of CHIK vaccine is strongly recommended.

Immune response to live-attenuated Japanese encephalitis vaccine (JE-CV) neutralizes Japanese encephalitis virus isolates from south-east Asia and India.

BACKGROUND: During clinical development of the licensed Japanese encephalitis chimeric virus vaccine (JE-CV), the neutralization capacity of vaccine-induced antibodies was assessed against the vaccine virus and against well characterized wild-type (wt) viruses isolated between 1949-1991. We assessed whether JE-CV-induced antibodies can also neutralize more recent wt Japanese encephalitis virus (JEV) isolates including a genotype 1 isolate. METHODS: Sera from 12-18 month-old children who received a single dose of JE-CV in a phase III study in Thailand and the Philippines (ClinicalTrials.gov NCT00735644) were randomly selected and pooled according to neutralization titer against JE-CV into eight samples. Neutralization was assessed by plaque reduction neutralization tests (PRNT50) against three recent isolates from JEV genotypes 1 and 3 in addition to four JEV previously tested. RESULTS: Neutralization titers against the three recent JEV strains were comparable to those observed previously against other strains and the vaccine virus. The observed differences between responses to genotype 1 and 3 viruses were within assay variability for the PRNT50. CONCLUSIONS: The results were consistent with previously generated data on the neutralization of wt JEV isolates, immune responses induced by JE-CV neutralize recently isolated virus from southeast (SE) Asia and India.

Dengue, Japanese encephalitis and Chikungunya virus antibody prevalence among captive monkey (Macaca nemestrina) colonies of Northern Thailand.

The potential of macaque Macaca nemestrina leonina in Thailand to be infected by endemic arboviruses was assessed. The prevalence of antibodies of three arboviruses actively circulating in Thailand was determined by Plaque Reduction Neutralization assay procedures using samples from captive colonies in Northern Thailand. Out of 38 macaques, 9 (24%) presented reacting antibodies against dengue virus, 5 (13%) against Japanese encephalitis virus, and 4 (10%) against Chikungunya virus. Our results indicate that the northern pig-tailed macaque in Thailand can be infected by these arboviruses, inferring therefore that their virus specific vectors have bitten them. Given that, northern pig-tailed macaque represents an abundant population, living in close range to human or in peridomestic setting, they could play a role as potential reservoir host for arboviruses circulating in Thailand.

A variant in the CD209 promoter is associated with severity of dengue disease.

Dengue fever and dengue hemorrhagic fever are mosquito-borne viral diseases. Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN1, encoded by CD209), an attachment receptor of dengue virus, is essential for productive infection of dendritic cells. Here, we report strong association between a promoter variant of CD209, DCSIGN1-336, and risk of dengue fever compared with dengue hemorrhagic fever or population controls. The G allele of the variant DCSIGN1-336 was associated with strong protection against dengue fever in three independent cohorts from Thailand, with a carrier frequency of 4.7% in individuals with dengue fever compared with 22.4% in individuals with dengue hemorrhagic fever (odds ratio for risk of dengue hemorrhagic fever versus dengue fever: 5.84, P = 1.4 x 10(-7)) and 19.5% in controls (odds ratio for protection: 4.90, P = 2 x 10(-6)). This variant affects an Sp1-like binding site and transcriptional activity in vitro. These results indicate that CD209 has a crucial role in dengue pathogenesis, which discriminates between severe dengue fever and dengue hemorrhagic fever. This may have consequences for therapeutic and preventive strategies.

Proceedings of the 6th Asia Dengue Summit, June 2023.

The 6th Asia Dengue Summit (ADS) themed "Road Map to Zero Dengue Death" was held in Thailand from 15th-16th June 2023. The summit was hosted by Tropical Medicine Cluster, Chulalongkorn University, Bangkok, Thailand in conjunction with Queen Saovabha Memorial Institute, The Thai Red Cross Society; Faculty of Tropical Medicine, Mahidol University; and the Ministry of Public Health. The 6th ADS was convened by Asia Dengue Voice and Action (ADVA); Global Dengue and Aedes Transmitted Diseases Consortium (GDAC); Southeast Asian Ministers of Education Tropical Medicine and Public Health Network (SEAMEO TROPMED); Fondation Mérieux (FMx) and the International Society for Neglected Tropical Diseases (ISNTD). Dengue experts from academia and research, and representatives from the Ministries of Health, Regional and Global World Health Organization (WHO) and International Vaccine Institute (IVI) participated in the three-day summit. With more than 51 speakers and 451 delegates from over 24 countries, 10 symposiums, and 2 full days, the 6th ADS highlighted the growing threat of dengue and its antigenic evolution, flagged the urgent need to overcome vaccine hesitancy and misinformation crisis, and focused on dengue control policies, newer diagnostics, therapeutics and vaccines, travel-associated dengue, and strategies to improve community involvement.