Leah C. Katzelnick

Dengue is the canonical viral disease for which immune history is a determinant of protective versus pathogenic responses and immunogenicity. Yet, because of limitations in animal models and clinical presentation after peak viremia, how preinfection and early immune responses affect dengue outcome is not confirmed. We conducted a phase 1 clinical trial with 45 healthy adults to test whether secondary infection challenge with a heterotypic, full-length, attenuated virus increases viremia and immunogenicity compared with primary and tertiary infection. Viremia was associated with more, but still mild, clinical signs and symptoms, and secondary infection resulted in greater viremia, neutralizing antibodies, and protective T cell responses. However, those whose baseline antibodies most efficiently increased dengue virus replication in an in vitro assay experienced delayed inflammatory and adaptive immune activation, the highest viremia, strong acute immune responses, and waning of potent CD8 ⁺ T cells and antibodies. In contrast, higher baseline antibodies against the nonstructural protein 1 of multiple serotypes predicted early interferon and balanced immune activation, viremia control, and development of enduring, potent B and T cells, revealing how vaccines can induce broad, long-lasting protection. Last, individuals with potent and neutralizing antibodies and IFN-γ ⁺ T cells at baseline experienced minimal viral replication. Together, these findings demonstrate that controlled human challenge with dengue virus can delineate coordinated versus dysregulated acute immune responses and their effects on immunogenicity, informing therapeutic and vaccine strategies for dengue and other viral diseases.

Sequential dengue virus (DENV) infection is thought to induce long-lived protection against dengue. However, little is known about the specificity, phenotype, and repertoire of the enduring B cell response due to limitations in high-throughput methods. We used stabilized DENV envelope (E) dimers as probes for in-depth memory B cell repertoire characterization using two complementary sequencing strategies. Using Rapid Assembly, Transfection, and Production of Immunoglobulins (RATP-Ig), we isolated 227 DENV2 E dimer-specific memory B cells from an immune donor, of which 87% bound at least two serotypes, 68% bound the fusion loop (FL-sensitive), and 42% were neutralizing. Among the most neutralizing, cross-binding antibodies, the FL-sensitive antibodies had weaker but broad neutralization, including to other flaviviruses, whereas FL-agnostic antibodies mapped to E domain II and III and were potently neutralizing, but only against one or two serotypes. Competitive binding of the DENV1-4 E dimers distinguished the more potently neutralizing FL-agnostic antibodies and broad and potent anti (α)-envelope dimer epitope (EDE) antibodies from the FL-sensitive antibodies. Further, analysis of the heavy chain complementarity determining region 3 (CDRH3) showed FL-sensitive and α-EDE antibodies were more negatively charged, but anti-EDE antibodies were distinguished by longer and more aromatic CDRH3s. We then demonstrated how oligonucleotide-tagged DENV1-4 dimers and proteogenomic single-cell B cell receptor sequencing (LIBRA-seq) could identify memory B cells predicted to have potent neutralization based on DENV-binding and CDRH3 physiochemical properties. This study provides insight into the secondary DENV-specific B cell repertoire and approaches for identifying protective antibodies as part of large-screening efforts.

Dengue is the canonical viral disease for which immune history predicts protective versus pathogenic responses and immunogenicity. Yet, due to limitations in animal models and clinical presentation after peak viremia, how pre-infection and early immune responses affect dengue outcomes is not confirmed. We conducted a phase 1 clinical trial with 45 healthy adults to test if secondary infection challenge with a heterotypic, full-length, attenuated virus increases viremia and immunogenicity compared to primary and tertiary infection. Viremia was associated with more, but still mild, clinical signs and symptoms, and secondary infection predicted greater viremia and neutralizing antibodies. However, those with the highest baseline enhancing antibodies experienced delayed inflammatory and adaptive activation, the highest viremia, strong acute immune responses, but waning of potent CD8 ⁺ T cells and antibodies. Baseline antibodies to non-structural protein 1 of multiple serotypes predicted early interferon and balanced immune activation, viremia control, and development of enduring, potent B and T cells, revealing how vaccines can induce broad long-lasting protection. Finally, these antibody and T cell profiles at baseline predicted sterilization of infection. We demonstrate that controlled human challenge can delineate coordinated versus dysregulated acute responses and effects on immunogenicity, informing therapeutic and vaccine strategies for dengue and other viral diseases.

Dengue virus (DENV) vaccines should be designed to induce balanced protective immunity to all 4 DENV serotypes to mitigate the risk of vaccine-enhanced dengue disease. The first tetravalent live DENV vaccine (Dengvaxia) tested in humans was efficacious in children who were partially immune to DENV at baseline. In DENV-naive children, the vaccine was not efficacious and placed some children at risk of more severe WT DENV breakthrough infections. To define dengue vaccine responses at the individual patient level and their relationship to mild and severe dengue infections, we prospectively studied a cohort of DENV-naive children who received 1 dose of Dengvaxia. The vaccine stimulated variable responses that neutralized 0, 1 (monotypic), or 2+ (multitypic) serotypes in individual children. Using a logistic regression model, we found that vaccinated children with neutralizing antibody (NAb) to 1 serotype only were at greater risk developing dengue compared with children who were not vaccinated (odds ratio 5.07). This risk was not observed in vaccinated children with no NAb or NAb to 2 or more serotypes. We propose that individuals with durable NAb to 1 serotype have an abundance of serotype cross-reactive, nonneutralizing antibodies implicated in the enhanced replication of heterologous serotypes.

COVID-19 was first reported in the Philippines in January 2020 and the country experienced several COVID-19 waves. Little is known on the seroprevalence of SARS-CoV-2 among adolescents in a developing country such as the Philippines. In this study, we incorporated testing for acute COVID-19 and SARS-CoV-2 seropositivity in an ongoing dengue study of 9- to 14-year-old participants enrolled in 2017. Acute febrile episodes were detected through active surveillance and a nasopharyngeal swab was obtained for SARS-CoV-2 RT-PCR. Before COVID-19 vaccination was offered, we tested a subset of sera obtained in 2021 for SARS-CoV-2 receptor binding domain (RBD) antibodies. Development of SARS-CoV-2 seropositivity acquired through natural infection and vaccination was conducted by measuring antibodies pre- (2018–2019), early- (2019–2020), and late-pandemic (2022–2023) in those with matched samples. From July 2021-October 2022, we recorded 29 acute febrile illness episodes in the cohort and 17.0% were SARS-CoV-2 RT-PCR positive. In the subset of 499 samples tested for SARS-CoV-2 RBD antibodies in 2021, 30.3% were seropositive. Among these, 4.0% were seropositive in 2018–2019, and 11.0% had seroconverted by 2020. Interestingly, 74.0% experienced antibody waning between 2020 and 2021 sampling. By 2022, 345/499 children received full COVID-19 vaccination and 13/499 was partially vaccinated, with 96.6% SARS-CoV-2 seropositive by late-pandemic sample in 2022–2023. Vaccination and site were predictors of SARS-CoV-2 RBD antibodies. These findings suggest both variation in local transmission over time as well as association of vaccination with the development of SARS-CoV-2 immunity in adolescents in the Philippines.

: Dengue fever, a vector-borne disease caused by four serotypes of dengue virus (DENV), ranges from asymptomatic to severe illness, including hemorrhagic fever and shock syndrome. A 2023 outbreak (August 2023–May 2024) in Mali affected six districts in Bamako, causing 1,422 confirmed cases with a 2.7% fatality rate. In this study, viral sequencing provided insights into the molecular epidemiology and transmission dynamics of this outbreak. From September to October 2023, 23 of 42 suspected cases detected through national dengue surveillance activities were tested using the pan-vertebrate virus metagenomics method (virome capture sequencing platform for vertebrate viruses [VirCapSeq-VERT]). Sequencing data were analyzed using the Rapid Identification of Microbes pipeline, and Bayesian phylogenetic inference with Monte Carlo methods was used to assess viral genomic evolution. Among 23 patients, 61% were male, and the median age was 37 years (range: 20–74). The most common symptoms were fever (93.1%), headache (56.5%), and asthenia (47.8%). The largest proportion experienced dengue with warning signs (65.2%), followed by dengue without warning signs (30.4%) and severe dengue (4.4%). The successful sequencing of 19 samples revealed dengue virus serotype 3 (DENV-3; genotype III) in 15 (65.21%) samples and dengue virus serotype 1 (DENV-1; genotype III) in 4 (17.39%) samples. The DENV-1 sequences were analogous to West African sequences, and the DENV-3 were clustered with West African, Asian, and Caribbean sequences. In this study of DENV in Mali, we offer insights into the molecular epidemiology of the virus and underscore the benefits of the genomic surveillance of arboviruses in West Africa.

Cross-reactive antibodies to epitopes that span envelope proteins on the virion surface are hypothesized to protect against dengue virus (DENV) infection and disease. Here, we measured antibodies targeting a quaternary epitope called the envelope dimer epitope (EDE) as well as neutralizing and binding antibodies and evaluated their association with DENV infection, vaccine response, and disease outcome in dengue-vaccinated ( n = 164) and dengue-unvaccinated children ( n = 88) within a longitudinal cohort in Cebu, Philippines ( n = 2996). Antibodies targeting EDE were prevalent and associated with broad neutralization of mature DENV1 to DENV4 virions in those with evidence of at least two prior DENV infections but were mostly absent in those with only one prior infection. Subsequent infection and vaccination boosted titers of EDE-like antibodies, neutralizing antibodies, and DENV-binding antibodies. EDE-like antibodies were associated with reduced risk of symptomatic dengue and more severe dengue and statistically explained the protective effect of binding and neutralizing antibodies on dengue. Thus, antibodies targeting quaternary epitopes help explain the broad cross-protection observed in those with multiple prior DENV exposures, making them useful for evaluation and development of future vaccines and therapeutics.

The standard dengue virus (DENV) neutralization assay inconsistently predicts dengue protection. We compare how IgG ELISA, envelope domain III (EDIII), or non-structural protein 1 (NS1) binding antibodies, and titers from plaque reduction neutralization tests (PRNTs) using standard and mature viruses are associated with dengue. The ELISA measures IgG antibodies that bind to inactivated DENV1-4. The EDIII and NS1 assays measure binding antibodies, and the PRNTs measure neutralizing antibodies to each specific DENV serotype. Healthy children (n = 1206) in Cebu, Philippines were followed for 5 years. ELISA IgG≥3 was associated with reduced dengue probability relative to naïve children (3% vs. 10%, p = 0.007). Serotype-specific antibodies binding EDIII or NS1 had no association with dengue risk. Standard virus PRNT geometric mean titers (GMT) > 200 and mature GMT > 100 were associated with reduced dengue disease overall (p < 0.01). High DENV2 and DENV3 titers against either standard or mature viruses protected against the matched serotype (p < 0.01). While 43% of dengue cases had standard virus PRNT titers>100, only 2% of cases had mature virus PRNT titers>100 (p < 0.001), indicating a lower, more consistent threshold for protection. These assays may serve as correlates of protection.

Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with fivefold to sixfold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a fivefold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen. IMPORTANCE: Updates to COVID-19 vaccine antigens depend on assessing how much vaccine antigens differ antigenically from newer SARS-CoV-2 variants. Human sera from single variant infections are ideal for discriminating antigenic differences among variants, but such primary infection sera are now rare due to high population immunity. It remains unclear whether sera from experimentally infected animals could substitute for human sera for antigenic assessments. This report shows that neutralization titers of variant-matched human and hamster primary infection sera correlate well and recognize variants similarly, indicating that hamster sera can be a proxy for human sera for antigenic assessments. We further show that human sera following an XBB.1.5 booster vaccine broadly neutralized XBB sub-lineage variants but titers were fivefold lower against the more recent JN.1 variant. These findings support updating the current COVID-19 vaccine variant composition and developing a framework for assessing antigenic differences in future variants using hamster primary infection sera.