Immune Response to Dengue and Zika.

Flaviviruses such as dengue (DENV), yellow fever (YFV), West Nile (WNV), and Zika (ZIKV) are human pathogens of global significance. In particular, DENV causes the most prevalent mosquito-borne viral diseases in humans, and ZIKV emerged from obscurity into the spotlight in 2016 as the etiologic agent of congenital Zika syndrome. Owing to the recent emergence of ZIKV as a global pandemic threat, the roles of the immune system during ZIKV infections are as yet unclear. In contrast, decades of DENV research implicate a dual role for the immune system in protection against and pathogenesis of DENV infection. As DENV and ZIKV are closely related, knowledge based on DENV studies has been used to prioritize investigation of ZIKV immunity and pathogenesis, and to accelerate ZIKV diagnostic, therapeutic, and vaccine design. This review discusses the following topics related to innate and adaptive immune responses to DENV and ZIKV: the interferon system as the key mechanism of host defense and viral target for immune evasion, antibody-mediated protection versus antibody-dependent enhancement, and T cell-mediated protection versus original T cell antigenic sin. Understanding the mechanisms that regulate the balance between immune-mediated protection and pathogenesis during DENV and ZIKV infections is critical toward development of safe and effective DENV and ZIKV therapeutics and vaccines.

Protective Zika vaccines engineered to eliminate enhancement of dengue infection via immunodominance switch.

Antibody-dependent enhancement (ADE) is an important safety concern for vaccine development against dengue virus (DENV) and its antigenically related Zika virus (ZIKV) because vaccine may prime deleterious antibodies to enhance natural infections. Cross-reactive antibodies targeting the conserved fusion loop epitope (FLE) are known as the main sources of ADE. We design ZIKV immunogens engineered to change the FLE conformation but preserve neutralizing epitopes. Single vaccination conferred sterilizing immunity against ZIKV without ADE of DENV-serotype 1-4 infections and abrogated maternal-neonatal transmission in mice. Unlike the wild-type-based vaccine inducing predominately cross-reactive ADE-prone antibodies, B cell profiling revealed that the engineered vaccines switched immunodominance to dispersed patterns without DENV enhancement. The crystal structure of the engineered immunogen showed the dimeric conformation of the envelope protein with FLE disruption. We provide vaccine candidates that will prevent both ZIKV infection and infection-/vaccination-induced DENV ADE.

Immune response to dengue virus and prospects for a vaccine.

Dengue virus (DENV) is a mosquito-borne member of the Flavivirus genus and includes four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), each of which is capable of causing dengue fever and dengue hemorrhagic fever/dengue shock syndrome. Serious disease can be seen during primary infection but is more frequent following second infection with a serotype different from that of a previous infection. Infection with wild-type DENV induces high-titered neutralizing antibody that can provide long-term immunity to the homotypic virus and can provide short-term immunity (only several months duration) to a heterotypic DENV. The high level of virus replication seen during both secondary infection with a heterotypic virus and during primary DENV infection in late infancy is a direct consequence of antibody-dependent enhancement of replication. This enhanced virus replication is mediated primarily by preexisting, nonneutralizing, or subneutralizing antibodies to the virion surface antigens that enhance access of the virion-antibody complex to FcγR-bearing cells. Vaccines will need to provide long-term protection against each of the four DENV serotypes by inducing neutralizing antibodies, and live, attenuated and various nonliving virus vaccines are in development.

A Dengue Vaccine.

Denvaxia is the first licensed vaccine for the prevention of dengue. It is a live vaccine developed using recombinant DNA technology. The vaccine is given as three doses over the course of a year and has the potential to prevent hundreds of thousands of hospitalizations each year.

Molecular Insight into Dengue Virus Pathogenesis and Its Implications for Disease Control.

Dengue virus (DENV) is a mosquito-transmitted RNA virus that infects an estimated 390 million humans each year. Here, we review recent advances in our understanding of the biology of DENV and describe knowledge gaps that have impacted the development of effective vaccines and therapeutics.

Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement.

Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions.

Live, Attenuated, Tetravalent Butantan-Dengue Vaccine in Children and Adults.

BACKGROUND: Butantan-Dengue Vaccine (Butantan-DV) is an investigational, single-dose, live, attenuated, tetravalent vaccine against dengue disease, but data on its overall efficacy are needed. METHODS: In an ongoing phase 3, double-blind trial in Brazil, we randomly assigned participants to receive Butantan-DV or placebo, with stratification according to age (2 to 6 years, 7 to 17 years, and 18 to 59 years); 5 years of follow-up is planned. The objectives of the trial were to evaluate overall vaccine efficacy against symptomatic, virologically confirmed dengue of any serotype occurring more than 28 days after vaccination (the primary efficacy end point), regardless of serostatus at baseline, and to describe safety up to day 21 (the primary safety end point). Here, vaccine efficacy was assessed on the basis of 2 years of follow-up for each participant, and safety as solicited vaccine-related adverse events reported up to day 21 after injection. Key secondary objectives were to assess vaccine efficacy among participants according to dengue serostatus at baseline and according to the dengue viral serotype; efficacy according to age was also assessed. RESULTS: Over a 3-year enrollment period, 16,235 participants received either Butantan-DV (10,259 participants) or placebo (5976 participants). The overall 2-year vaccine efficacy was 79.6% (95% confidence interval [CI], 70.0 to 86.3) - 73.6% (95% CI, 57.6 to 83.7) among participants with no evidence of previous dengue exposure and 89.2% (95% CI, 77.6 to 95.6) among those with a history of exposure. Vaccine efficacy was 80.1% (95% CI, 66.0 to 88.4) among participants 2 to 6 years of age, 77.8% (95% CI, 55.6 to 89.6) among those 7 to 17 years of age, and 90.0% (95% CI, 68.2 to 97.5) among those 18 to 59 years of age. Efficacy against DENV-1 was 89.5% (95% CI, 78.7 to 95.0) and against DENV-2 was 69.6% (95% CI, 50.8 to 81.5). DENV-3 and DENV-4 were not detected during the follow-up period. Solicited systemic vaccine- or placebo-related adverse events within 21 days after injection were more common with Butantan-DV than with placebo (58.3% of participants, vs. 45.6%). CONCLUSIONS: A single dose of Butantan-DV prevented symptomatic DENV-1 and DENV-2, regardless of dengue serostatus at baseline, through 2 years of follow-up. (Funded by Instituto Butantan and others; DEN-03-IB ClinicalTrials.gov number, NCT02406729, and WHO ICTRP number, U1111-1168-8679.).

Dengue.

Dengue, caused by four closely related viruses, is a growing global public health concern, with outbreaks capable of overwhelming health-care systems and disrupting economies. Dengue is endemic in more than 100 countries across tropical and subtropical regions worldwide, and the expanding range of the mosquito vector, affected in part by climate change, increases risk in new areas such as Spain, Portugal, and the southern USA, while emerging evidence points to silent epidemics in Africa. Substantial advances in our understanding of the virus, immune responses, and disease progression have been made within the past decade. Novel interventions have emerged, including partially effective vaccines and innovative mosquito control strategies, although a reliable immune correlate of protection remains a challenge for the assessment of vaccines. These developments mark the beginning of a new era in dengue prevention and control, offering promise in addressing this pressing global health issue.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos, Peru.

Over half the world's population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito-human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = -9.11) and 12.4% (95% CI 4.2%, ∞); z = -2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Efficacy of Wolbachia-Infected Mosquito Deployments for the Control of Dengue.

BACKGROUND: Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia pipientis are less susceptible than wild-type A. aegypti to dengue virus infection. METHODS: We conducted a cluster-randomized trial involving releases of wMel-infected A. aegypti mosquitoes for the control of dengue in Yogyakarta, Indonesia. We randomly assigned 12 geographic clusters to receive deployments of wMel-infected A. aegypti (intervention clusters) and 12 clusters to receive no deployments (control clusters). All clusters practiced local mosquito-control measures as usual. A test-negative design was used to assess the efficacy of the intervention. Patients with acute undifferentiated fever who presented to local primary care clinics and were 3 to 45 years of age were recruited. Laboratory testing was used to identify participants who had virologically confirmed dengue (VCD) and those who were test-negative controls. The primary end point was symptomatic VCD of any severity caused by any dengue virus serotype. RESULTS: After successful introgression of wMel into the intervention clusters, 8144 participants were enrolled; 3721 lived in intervention clusters, and 4423 lived in control clusters. In the intention-to-treat analysis, VCD occurred in 67 of 2905 participants (2.3%) in the intervention clusters and in 318 of 3401 (9.4%) in the control clusters (aggregate odds ratio for VCD, 0.23; 95% confidence interval [CI], 0.15 to 0.35; P = 0.004). The protective efficacy of the intervention was 77.1% (95% CI, 65.3 to 84.9) and was similar against the four dengue virus serotypes. The incidence of hospitalization for VCD was lower among participants who lived in intervention clusters (13 of 2905 participants [0.4%]) than among those who lived in control clusters (102 of 3401 [3.0%]) (protective efficacy, 86.2%; 95% CI, 66.2 to 94.3). CONCLUSIONS: Introgression of wMel into A. aegypti populations was effective in reducing the incidence of symptomatic dengue and resulted in fewer hospitalizations for dengue among the participants. (Funded by the Tahija Foundation and others; AWED ClinicalTrials.gov number, NCT03055585; Indonesia Registry number, INA-A7OB6TW.).

Wolbachia strain wAlbB shows favourable characteristics for dengue control use in Aedes aegypti from Burkina Faso.

Dengue represents an increasing public health burden worldwide. In Africa, underreporting and misdiagnosis often mask its true epidemiology, and dengue is likely to be both more widespread than reported data suggest and increasing in incidence and distribution. Wolbachia-based dengue control is underway in Asia and the Americas but has not to date been deployed in Africa. Due to the genetic heterogeneity of African Aedes aegypti populations and the complexity of the host-symbiont interactions, characterization of key parameters of Wolbachia-carrying mosquitoes is paramount for determining the potential of the system as a control tool for dengue in Africa. The wAlbB Wolbachia strain was stably introduced into an African Ae. aegypti population by introgression, and showed high intracellular density in whole bodies and different mosquito tissues; high intracellular density was also maintained following larval rearing at high temperatures. No effect on the adult lifespan induced by Wolbachia presence was detected. Moreover, the ability of this strain to strongly inhibit DENV-2 dissemination and transmission in the host was also demonstrated in the African background. Our findings suggest the potential of harnessing Wolbachia for dengue control for African populations of Ae. aegypti.

Comparison of the sensitivity and specificity of commercial anti-dengue virus IgG tests to identify persons eligible for dengue vaccination.

The Advisory Committee on Immunization Practices (ACIP) recommended that dengue pre-vaccination screening tests for Dengvaxia administration have at least 98% specificity and 75% sensitivity. This study evaluates the performance of commercial anti-DENV IgG tests to identify tests that could be used for pre-vaccination screening. First, for seven tests, we evaluated sensitivity and specificity in early convalescent dengue virus (DENV) infection, using 44 samples collected 7-30 days after symptom onset and confirmed by RT-PCR. Next, for the five best-performing tests and two additional tests (with and without an external test reader) that became available later, we evaluated performance to detect past dengue infection among a panel of 44 specimens collected in 2018-2019 from healthy 9- to 16-year-old children from Puerto Rico. Finally, a full-scale evaluation was done with the four best-performing tests using 400 specimens from the same population. We used virus focus reduction neutralization test and an in-house DENV IgG ELISA as reference standards. Of seven tests, five showed ≥75% sensitivity in detecting anti-DENV IgG in early convalescent specimens with low cross-reactivity to the Zika virus. For the detection of previous DENV infections, the tests with the highest performance were the Euroimmun NS1 IgG ELISA (sensitivity 84.5%, specificity 97.1%) and CTK Dengue IgG rapid test R0065C with the test reader (sensitivity 76.2% specificity 98.1%). There are IgG tests available that can be used to accurately classify individuals with previous DENV infection as eligible for dengue vaccination to support safe vaccine implementation. IMPORTANCE: The Advisory Committee on Immunization Practices (ACIP) has set forth recommendations that dengue pre-vaccination screening tests must exhibit at least 98% specificity and 75% sensitivity. Our research rigorously assesses the performance of various commercial tests against these benchmarks using well-characterized specimens from Puerto Rico. The findings from our study are particularly relevant given FDA approval and ACIP recommendation of Sanofi Pasteur's Dengvaxia vaccine, highlighting the need for accurate pre-vaccination screening tools.

Pre-vaccination frequency of circulatory Tfh is associated with robust immune response to TV003 dengue vaccine.

It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees' ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.

Therapeutic efficacy of humanized monoclonal antibodies targeting dengue virus nonstructural protein 1 in the mouse model.

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection.

Evaluation of assays for nucleic acid testing for the prevention of chikungunya and dengue virus transmission by blood transfusion.

BACKGROUND: The large dengue (DENV) and chikungunya (CHIKV) outbreaks observed during the last decade across the world, as well as local transmissions in non-endemic areas are a growing concern for blood safety. The aim of this study was to evaluate and compare the sensitivity of nucleic acid tests (NAT) detecting DENV and CHIKV RNA. MATERIALS AND METHODS: Using DENV 1 to 4 International Standards, the limits of detection (LODs) calculated by probit analysis of two NAT assays; the cobas CHIKV/DENV assay (Roche Diagnostics) and the Procleix Dengue Virus Assay (Grifols) were compared. In addition, CHIKV-RNA LOD of the cobas CHIKV/DENV assay was evaluated. RESULTS: For dengue, the 95% LOD of the cobas assay ranged between 4.10 [CI95%: 2.70-8.19] IU/mL (DENV-2) and 7.07 [CI95%: 4.34-14.89] IU/mL (DENV-4), and between 2.19 [CI95%: 1.53-3.83] IU/mL (DENV-3) and 5.84 [CI95%: 3.84-10.77] IU/mL (DENV-1) for Procleix assay. The Procleix assay had a significant lower LOD for DENV-3 (2.19 vs. 5.89 IU/mL) when compared to the cobas assay (p = 0.005). The 95% LOD for CHIKV-RNA detection of the cobas assay was 4.76 [CI95%: 3.08-8.94] IU/mL. DISCUSSION: The two NAT assays developed for blood donor screening evaluated in this study demonstrated high and similar analytical performance. Subject to an appropriate risk-benefit assessment, they can be used to support blood safety during outbreaks in endemic areas or in non-endemic areas as an alternative to deferring blood donors during local transmission likely to affect the blood supply. The development of multiplex assays is expected to optimize laboratory organization.

Optimising the surveillance of Aedes aegypti in Brazil by selecting smaller representative areas within an endemic city.

OBJECTIVES: Arboviruses, such as dengue (DENV), zika (ZIKV), and chikungunya (CHIKV), constitute a growing urban public health threat. Focusing on Aedes aegypti mosquitoes, their primary vectors, is crucial for mitigation. While traditional immature-stage mosquito surveillance has limitations, capturing adult mosquitoes through traps yields more accurate data on disease transmission. However, deploying traps presents logistical and financial challenges, demonstrating effective temporal predictions but lacking spatial accuracy. Our goal is to identify smaller representative areas within cities to enhance the early warning system for DENV outbreaks. METHODS: We created Sentinel Geographic Units (SGUs), smaller areas of 1 km2 within each stratum, larger areas, with the aim of aligning the Trap Positivity Index (TPI) and Adult Density Index (ADI) with their respective strata. We conducted a two-step evaluation of SGUs. First, we examined the equivalence of TPI and ADI between SGUs and strata from January 2017 to July 2022. Second, we assessed the ability of SGU's TPI and ADI to predict DENV outbreaks in comparison to Foz do Iguaçu's Early-Warning System, which forecasts outbreaks up to 4 weeks ahead. Spatial and temporal analyses were carried out, including data interpolation and model selection based on Akaike information criteria (AIC). RESULTS: Entomological indicators produced in small SGUs can effectively replace larger sentinel areas to access dengue outbreaks. Based on historical data, the best predictive capability is achieved 2 weeks after infestation verification. Implementing the SGU strategy with more frequent sampling can provide more precise space-time estimates and enhance dengue control. CONCLUSIONS: The implementation of SGUs offers an efficient way to monitor mosquito populations, reducing the need for extensive resources. This approach has the potential to improve dengue transmission management and enhance the public health response in endemic cities.

Two-step spatiotemporal anomaly detection corrected for lag reporting time with application to real-time dengue surveillance in Thailand.

BACKGROUND: Dengue infection ranges from asymptomatic to severe and life-threatening, with no specific treatment available. Vector control is crucial for interrupting its transmission cycle. Accurate estimation of outbreak timing and location is essential for efficient resource allocation. Timely and reliable notification systems are necessary to monitor dengue incidence, including spatial and temporal distributions, to detect outbreaks promptly and implement effective control measures. METHODS: We proposed an integrated two-step methodology for real-time spatiotemporal cluster detection, accounting for reporting delays. In the first step, we employed space-time nowcasting modeling to compensate for lags in the reporting system. Subsequently, anomaly detection methods were applied to assess adverse risks. To illustrate the effectiveness of these detection methods, we conducted a case study using weekly dengue surveillance data from Thailand. RESULTS: The developed methodology demonstrated robust surveillance effectiveness. By combining space-time nowcasting modeling and anomaly detection, we achieved enhanced detection capabilities, accounting for reporting delays and identifying clusters of elevated risk in real-time. The case study in Thailand showcased the practical application of our methodology, enabling timely initiation of disease control activities. CONCLUSION: Our integrated two-step methodology provides a valuable approach for real-time spatiotemporal cluster detection in dengue surveillance. By addressing reporting delays and incorporating anomaly detection, it complements existing surveillance systems and forecasting efforts. Implementing this methodology can facilitate the timely initiation of disease control activities, contributing to more effective prevention and control strategies for dengue in Thailand and potentially other regions facing similar challenges.