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.

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

Repurposing of Zika virus live-attenuated vaccine (ZIKV-LAV) strains as oncolytic viruses targeting human glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain cancer affecting the adult population. Median overall survival for GBM patients is poor (15 months), primarily due to high rates of tumour recurrence and the paucity of treatment options. Oncolytic virotherapy is a promising treatment alternative for GBM patients, where engineered viruses selectively infect and eradicate cancer cells by inducing cell lysis and eliciting robust anti-tumour immune response. In this study, we evaluated the oncolytic potency of live-attenuated vaccine strains of Zika virus (ZIKV-LAV) against human GBM cells in vitro. Our findings revealed that Axl and integrin αvß5 function as cellular receptors mediating ZIKV-LAV infection in GBM cells. ZIKV-LAV strains productively infected and lysed human GBM cells but not primary endothelia and terminally differentiated neurons. Upon infection, ZIKV-LAV mediated GBM cell death via apoptosis and pyroptosis. This is the first in-depth molecular dissection of how oncolytic ZIKV infects and induces death in tumour cells.

Race against dengue.

Understanding the kinetics of dengue viruses in the bloodstream can provide insights into the clinical outcomes of the disease.

Dengue and Zika RNA-RNA interactomes reveal pro- and anti-viral RNA in human cells.

BACKGROUND: Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication. RESULTS: Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection. CONCLUSIONS: This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis.

Minding the "T"s beyond the "B"s: Shaping vaccines for future pandemics.

The COVID-19 pandemic has accelerated the development of vaccines for viral infections. However, a failure to integrate T cell immunity as a determinant of vaccine efficacy could curtail advancement of newer vaccines for pandemic preparedness.

Insights into dengue immunity from vaccine trials.

The quest for an effective dengue vaccine has culminated in two approved vaccines and another that has completed phase 3 clinical trials. However, shortcomings exist in each, suggesting that the knowledge on dengue immunity used to develop these vaccines was incomplete. Vaccine trial findings could refine our understanding of dengue immunity, because these are experimentally derived, placebo-controlled data. Results from these trials suggest that neutralizing antibody titers alone are insufficient to inform protection against symptomatic infection, implicating a role for cellular immunity in protection. These findings have relevance for both future dengue vaccine development and application of current vaccines for maximal public health benefit.

Proceedings of the 5th Asia Dengue Summit.

The 5th Asia Dengue Summit, themed "Roll Back Dengue", was held in Singapore from 13 to 15 June 2022. The summit was co-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), and the Fondation Mérieux (FMx). 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 270 speakers and delegates from over 14 countries, 12 symposiums, and 3 full days, the 5th ADS highlighted the growing threat of dengue, shared innovations and strategies for successful dengue control, and emphasized the need for multi-sectoral collaboration to control dengue.

RNase2 is a possible trigger of acute-on-chronic inflammation leading to mRNA vaccine-associated cardiac complication.

BACKGROUND: Post-mRNA vaccination-associated cardiac complication is a rare but life-threatening adverse event. Its risk has been well balanced by the benefit of vaccination-induced protection against severe COVID-19. As the rate of severe COVID-19 has consequently declined, future booster vaccination to sustain immunity, especially against infection with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, may encounter benefit-risk ratios that are less favorable than at the start of the COVID-19 vaccination campaign. Understanding the pathogenesis of rare but severe vaccine-associated adverse events to minimize its risk is thus urgent. METHODS: Here, we report a serendipitous finding of a case of cardiac complication following a third shot of COVID-19 mRNA vaccine. As this case was enrolled in a cohort study, pre-vaccination and pre-symptomatic blood samples were available for genomic and multiplex cytokine analyses. FINDINGS: These analyses revealed the presence of subclinical chronic inflammation, with an elevated expression of RNASE2 at pre-booster baseline as a possible trigger of an acute-on-chronic inflammation that resulted in the cardiac complication. RNASE2 encodes for the ribonuclease RNase2, which cleaves RNA at the 3' side of uridine, which may thus remove the only Toll-like receptor (TLR)-avoidance safety feature of current mRNA vaccines. CONCLUSIONS: These pre-booster and pre-symptomatic gene and cytokine expression data provide unique insights into the possible pathogenesis of vaccine-associated cardiac complication and suggest the incorporation of additional nucleoside modification for an added safety margin. FUNDING: This work was funded by the NMRC Open Fund-Large Collaborative Grant on Integrated Innovations on Infectious Diseases (OFLCG19May-0034).

Tissue-specific expansion of Zika virus isogenic variants drive disease pathogenesis.

BACKGROUND: The Asian lineage Zika virus (ZIKV) emerged as a public health emergency in 2016 causing severe neurological pathologies with no apparent historical correlate to the mild, disease-causing innocuous member of the mosquito-borne flavivirus genus that was discovered in Africa in 1947. Replication error rate of RNA viruses combined with viral protein/RNA structural plasticity can lead to evolution of virus-induced pathogenicity that is critical to identify and validate. METHODS: Infection studies in cells and A129 interferon alpha/beta receptor deficient mice with ZIKV French Polynesian H/PF/2013 clinical isolate, plaque-purified isogenic clone derivatives as well as infectious cDNA clone derived wild-type and site-specific mutant viruses, were employed together with Next-Generation Sequencing (NGS) to pin-point the contributions of specific viral variants in neurovirulence recapitulated in our ZIKV mouse model. FINDINGS: NGS analysis of the low-passage inoculum virus as well as mouse serum, brain and testis derived virus, revealed specific enrichment in the mouse brain that were not found in the other tissues. Specifically, non-structural (NS) protein 2A variant at position 117 along with changes in NS1 and NS4B were uniquely associated with the mouse brain isolate. Mutational analysis of these variants in cDNA infectious clones identified the NS2A A117V as the lethal pathogenic determinant with potential epistatic contribution of NS1 and NS4B variants in ZIKV brain penetrance. INTERPRETATION: Our findings confirm that viral subpopulations drive ZIKV neuropathogenicity and identify specific sequence variants that expand in the mouse brain that associates with this phenotype which can serve as predictors of severe epidemics. FUNDING: Duke-NUS Khoo Post-doctoral Fellowship Award 2020 (KWKC) and National Medical Research Council of Singapore grants MOH-000524 (OFIRG) (SW) and MOH-OFIRG20nov-0002 (SGV).

An experimental medicine decipher of a minimum correlate of cellular immunity: Study protocol for a double-blind randomized controlled trial.

Vaccination induces an adaptive immune response that protects against infectious diseases. A defined magnitude of adaptive immune response that correlates with protection from the disease of interest, or correlates of protection (CoP), is useful for guiding vaccine development. Despite mounting evidence for the protective role of cellular immunity against viral diseases, studies on CoP have almost exclusively focused on humoral immune responses. Moreover, although studies have measured cellular immunity following vaccination, no study has defined if a "threshold" of T cells, both in frequency and functionality, is needed to reduce infection burden. We will thus conduct a double-blind, randomized clinical trial in 56 healthy adult volunteers, using the licensed live-attenuated yellow fever (YF17D) and chimeric Japanese encephalitis-YF17D (JE-YF17D) vaccines. These vaccines share the entire non-structural and capsid proteome where the majority of the T cell epitopes reside. The neutralizing antibody epitopes, in contrast, are found on the structural proteins which are not shared between the two vaccines and are thus distinct from one another. Study participants will receive JE-YF17D vaccination followed by YF17D challenge, or YF17D vaccination followed by JE-YF17D challenge. A separate cohort of 14 healthy adults will receive the inactivated Japanese Encephalitis virus (JEV) vaccine followed by YF17D challenge that controls for the effect of cross-reactive flaviviral antibodies. We hypothesize that a strong T cell response induced by YF17D vaccination will reduce JE-YF17D RNAemia upon challenge, as compared to JE-YF17D vaccination followed by YF17D challenge. The expected gradient of YF17D-specific T cell abundance and functionality would also allow us to gain insight into a T cell threshold for controlling acute viral infections. The knowledge gleaned from this study could guide the assessment of cellular immunity and vaccine development. Clinical trial registration: Clinicaltrials.gov, NCT05568953.

Early peripheral blood MCEMP1 and HLA-DRA expression predicts COVID-19 prognosis.

BACKGROUND: Mass vaccination has dramatically reduced the incidence of severe COVID-19, with most cases now presenting as self-limiting upper respiratory tract infections. However, those with co-morbidities, the elderly and immunocompromised, as well as the unvaccinated, remain disproportionately vulnerable to severe COVID-19 and its sequelae. Furthermore, as the effectiveness of vaccination wanes with time, immune escape SARS-CoV-2 variants could emerge to cause severe COVID-19. Reliable prognostic biomarkers for severe disease could be used as early indicator of re-emergence of severe COVID-19 as well as for triaging of patients for antiviral therapy. METHODS: We performed a systematic review and re-analysis of 7 publicly available datasets, analysing a total of 140 severe and 181 mild COVID-19 patients, to determine the most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients. In addition, we included an independent cohort where blood transcriptomics of COVID-19 patients were prospectively and longitudinally monitored previously, to track the time in which these gene expression changes occur before nadir of respiratory function. Single cell RNA-sequencing of peripheral blood mononuclear cells from publicly available datasets was then used to determine the immune cell subsets involved. FINDINGS: The most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients were MCEMP1, HLA-DRA and ETS1 across the 7 transcriptomics datasets. Moreover, we found significantly heightened MCEMP1 and reduced HLA-DRA expression as early as four days before the nadir of respiratory function, and the differential expression of MCEMP1 and HLA-DRA occurred predominantly in CD14+ cells. The online platform which we developed is publicly available at https://kuanrongchan-covid19-severity-app-t7l38g.streamlitapp.com/, for users to query gene expression differences between severe and mild COVID-19 patients in these datasets. INTERPRETATION: Elevated MCEMP1 and reduced HLA-DRA gene expression in CD14+ cells during the early phase of disease are prognostic of severe COVID-19. FUNDING: K.R.C is funded by the National Medical Research Council (NMRC) of Singapore under the Open Fund Individual Research Grant (MOH-000610). E.E.O. is funded by the NMRC Senior Clinician-Scientist Award (MOH-000135-00). J.G.H.L. is funded by the NMRC under the Clinician-Scientist Award (NMRC/CSAINV/013/2016-01). S.K. is funded by the NMRC under the Transition Award. This study was sponsored in part by a generous gift from The Hour Glass.

Durability of Vaccine-Induced and Natural Immunity Against COVID-19: A Narrative Review.

Vaccines developed against SARS-CoV-2 have proven to be highly effective in preventing symptomatic infection. Similarly, prior infection with SARS-CoV-2 has been shown to provide substantial protection against reinfection. However, it has become apparent that the protection provided to an individual after either vaccination or infection wanes over time. Waning protection is driven by both waning immunity over time since vaccination or initial infection, and the evolution of new variants of SARS-CoV-2. Both antibody and T/B-cells levels have been investigated as potential correlates of protection post-vaccination or post-infection. The activity of antibodies and T/B-cells provide some potential insight into the underlying causes of waning protection. This review seeks to summarise what is currently known about the waning of protection provided by both vaccination and/or prior infection, as well as the current information on the respective antibody and T/B-cell responses.

BNT162b2 mRNA Vaccine-Induced Immune Response in Oral Fluids and Serum.

OBJECTIVES: The COVID-19 vaccine is currently being administered worldwide to address the ongoing pandemic. Although these vaccines have proven effective in preventing severe disease, the level of immunity required to prevent respiratory mucosal infection remains less well understood. Therefore, it is desirable to develop a noninvasive screening strategy such as oral fluid to monitor secreted antibodies longitudinally as potential surrogates of mucosal immunity. METHODS: We evaluated the anti-spike protein antibodies in gingival crevicular fluid (GCF) and saliva and compared them to immune responses in the blood of 50 healthy health care workers following 2 doses of intramuscular Pfizer/BioNTech-BNT162b2 vaccine. RESULTS: The antibodies to SARS-CoV-2 spike and subdomain proteins (RBD, S1, S2, and NTD) were significantly higher in serum than oral fluids but showed a greater detection rate and higher median titres in GCF than saliva. For all tested SARS-CoV-2 antigens, IgG in GCF (as opposed to saliva) showed a more significant and stronger correlation with IgG in serum. Serum-neutralising antibodies (Nab) titres also displayed a significant and stronger correlation with anti-spike protein and their subdomains in GCF than saliva. Interestingly, the time post-second dose of vaccine and sex had a similar influence on IgG in serum and GCF. However, interferon (IFN)-γ-producing T-cell responses showed no association with SARS-Cov-2 IgG antibodies in serum, GCF, or saliva and neutralisation antibodies in serum. The correlation matrix of all measured parameters grouped serum and GCF IgG parameters separately from salivary IgG parameters indicating that GCF better represents the humoural response in serum than saliva. CONCLUSIONS: Within limitations, we propose that GCF could be a less invasive alternative to serum and more appropriate than saliva to detect antibody responses by current COVID-19 vaccines if the GCF collection procedure could be standardised. Further research is needed to investigate the suitability of GCF for community immune surveillance for vaccines.

A phase I/II randomized, double-blinded, placebo-controlled trial of a self-amplifying Covid-19 mRNA vaccine.

Coronavirus disease-19 (Covid-19) pandemic have demonstrated the importantance of vaccines in disease prevention. Self-amplifying mRNA vaccines could be another option for disease prevention if demonstrated to be safe and immunogenic. Phase 1 of this randomized, double-blinded, placebo-controlled trial (N = 42) assessed the safety, tolerability, and immunogenicity in healthy young and older adults of ascending levels of one-dose ARCT-021, a self-amplifying mRNA vaccine against Covid-19. Phase 2 (N = 64) tested two-doses of ARCT-021 given 28 days apart. During phase 1, ARCT-021 was well tolerated up to one 7.5 µg dose and two 5.0 µg doses. Local solicited AEs, namely injection-site pain and tenderness were more common in ARCT-021vaccinated, while systemic solicited AEs, mainly fatigue, headache and myalgia were reported in 62.8% and 46.4% of ARCT-021 and placebo recipients, respectively. Seroconversion rate for anti-S IgG was 100% in all cohorts, except for the 1 µg one-dose in younger adults and the 7.5 µg one-dose in older adults. Anti-S IgG and neutralizing antibody titers showed a general increase with increasing dose, and overlapped with titers in Covid-19 convalescent patients. T-cell responses were also observed in response to stimulation with S-protein peptides. Taken collectively, ARCT-021 is immunogenic and has favorable safety profile for further development.

A fast-growing dengue virus mutant reveals a dual role of STING in response to infection.

The four dengue viruses (DENVs) have evolved multiple mechanisms to ensure its survival. Among these mechanisms is the ability to regulate its replication rate, which may contribute to avoiding premature immune activation that limit infection dissemination: DENVs associated with dengue epidemics have shown slower replication rate than pre-epidemic strains. Correspondingly, wild-type DENVs replicate more slowly than their clinically attenuated derivatives. To understand how DENVs 'make haste slowly', we generated and screened for DENV2 mutants with accelerated replication that also induced high type-I interferon (IFN) expression in infected cells. We chanced upon a single NS2B-I114T amino acid substitution, in an otherwise highly conserved amino acid residue. Accelerated DENV2 replication damaged host DNA as mutant infection was dependent on host DNA damage repair factors, namely RAD21, EID3 and NEK5. DNA damage induced cGAS/STING signalling and activated early type-I IFN response that inhibited infection dissemination. Unexpectedly, STING activation also supported mutant DENV replication in infected cells through STING-induced autophagy. Our findings thus show that DENV NS2B has multi-faceted role in controlling DENV replication rate and immune evasion and suggest that the dual role of STING in supporting virus replication within infected cells but inhibiting infection dissemination could be particularly advantageous for live attenuated vaccine development.