Dengue virus (DENV) is a rapidly spreading pathogen with unusual pathogenesis, and correlates of protection from severe dengue disease and vaccine efficacy have not yet been established. Although ...DENV-specific CD8⁺ T-cell responses have been extensively studied, the breadth and specificity of CD4⁺ T-cell responses remains to be defined. Here we define HLA-restricted CD4⁺ T-cell epitopes resulting from natural infection with dengue virus in a hyperepidemic setting. Ex vivo flow-cytometric analysis of DENV-specific CD4⁺ T cells revealed that the virus-specific cells were highly polarized, with a strong bias toward a CX3CR1⁺ Eomesodermin⁺ perforin⁺ granzyme B⁺ CD45RA⁺ CD4 CTL phenotype. Importantly, these cells correlated with a protective HLA DR allele, and we demonstrate that these cells have direct ex vivo DENV-specific cytolytic activity. We speculate that cytotoxic dengue-specific CD4⁺ T cells may play a role in the control of dengue infection in vivo, and this immune correlate may be a key target for dengue virus vaccine development.
Dengue viruses (DENV) are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that ...bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE) is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE) protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to the development of safe, non-enhancing vaccines against dengue.
We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing ...human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.
Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using ...HLA class I and II predicted peptide “megapools,” circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%–27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%–60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating “common cold” coronaviruses and SARS-CoV-2.
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•Measuring immunity to SARS-CoV-2 is key for understanding COVID-19 and vaccine development•Epitope pools detect CD4+ and CD8+ T cells in 100% and 70% of convalescent COVID patients•T cell responses are focused not only on spike but also on M, N, and other ORFs•T cell reactivity to SARS-CoV-2 epitopes is also detected in non-exposed individuals
An analysis of immune cell responses to SARS-CoV-2 from recovered patients identifies the regions of the virus that is targeted and also reveals cross-reactivity with other common circulating coronaviruses
The Zika pandemic sparked intense interest in whether immune interactions among dengue virus serotypes 1 to 4 (DENV1 to -4) extend to the closely related Zika virus (ZIKV). We investigated ...prospective pediatric cohorts in Nicaragua that experienced sequential DENV1 to -3 (2004 to 2015), Zika (2016 to 2017), and DENV2 (2018 to 2020) epidemics. Risk of symptomatic DENV2 infection and severe disease was elevated by one prior ZIKV infection, one prior DENV infection, or one prior DENV infection followed by one ZIKV infection, compared with being flavivirus-naïve. By contrast, multiple prior DENV infections reduced dengue risk. Further, although high preexisting anti-DENV antibody titers protected against DENV1, DENV3, and ZIKV disease, intermediate titers induced by previous ZIKV or DENV infection enhanced future risk of DENV2 disease and severity, as well as DENV3 severity. The observation that prior ZIKV infection can modulate dengue disease severity like a DENV serotype poses challenges to development of dengue and Zika vaccines.
The 4 dengue virus serotypes (DENV1-4) and Zika virus (ZIKV) are related mosquito-borne flaviviruses of major importance globally. While monoclonal antibodies and plasma from DENV-immune donors can ...neutralize or enhance ZIKV in vitro and in small-animal models, and vice versa, the extent, duration, and significance of cross-reactivity in humans remains unknown, particularly in flavivirus-endemic regions.
We studied neutralizing antibodies to ZIKV and DENV1-4 in longitudinal serologic specimens collected through 3 years after infection from people in Latin America and Asia with laboratory-confirmed DENV infections. We also evaluated neutralizing antibodies to ZIKV and DENV1-4 in patients with Zika through 6 months after infection.
In patients with Zika, the highest neutralizing antibody titers were to ZIKV, with low-level cross-reactivity to DENV1-4 that was greater in DENV-immune individuals. We found that, in primary and secondary DENV infections, neutralizing antibody titers to ZIKV were markedly lower than to the infecting DENV and heterologous DENV serotypes. Cross-neutralization was greatest in early convalescence, then ZIKV neutralization decreased, remaining at low levels over time.
Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex. Neutralizing antibody titers can distinguish ZIKV from DENV infections when all viruses are analyzed simultaneously. These findings have implications for understanding natural immunity and vaccines.
The role of CD8 ⁺ T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced ...by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8 ⁺ responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8 ⁺ T cells is associated with protection from dengue virus disease.
Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal ...antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence (>6 months after infection) is unknown. We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus-specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity.
The four-dengue virus (DENV) serotypes infect several hundred million people annually. For the greatest safety and efficacy, tetravalent DENV vaccines are designed to stimulate balanced protective ...immunity to all four serotypes. However, this has been difficult to achieve. Clinical trials with a leading vaccine demonstrated that unbalanced replication and immunodominance of one vaccine component over others can lead to low efficacy and vaccine enhanced severe disease. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a live attenuated tetravalent DENV vaccine (TV003), which is currently being tested in phase 3 clinical trials. Here we report, our study to determine if TV003 stimulate balanced and serotype-specific (TS) neutralizing antibody (nAb) responses to each serotype. Serum samples from twenty-one dengue-naive individuals participated under study protocol CIR287 (ClinicalTrials.gov NCT02021968) are analyzed 6 months after vaccination. Most subjects (76%) develop TS nAbs to 3 or 4 DENV serotypes, indicating immunity is induced by each vaccine component. Vaccine-induced TS nAbs map to epitopes known to be targets of nAbs in people infected with wild type DENVs. Following challenge with a partially attenuated strain of DENV2, all 21 subjects are protected from the efficacy endpoints. However, some vaccinated individuals develop post challenge nAb boost, while others mount post-challenge antibody responses that are consistent with sterilizing immunity. TV003 vaccine induced DENV2 TS nAbs are associated with sterilizing immunity. Our results indicate that nAbs to TS epitopes on each serotype may be a better correlate than total levels of nAbs currently used for guiding DENV vaccine development.
The four serotypes of dengue virus (DENV1-4) are mosquito-borne flaviviruses that infect humans. Live attenuated tetravalent DENV vaccines are at different phases of clinical testing. DENV vaccine ...developers have relied on neutralizing antibodies (NAbs) as a correlate of protection. A leading tetravalent vaccine (Dengvaxia) stimulated NAbs to the 4 DENV serotypes, yet overall vaccine efficacy was low in children who were DENV seronegative at baseline before vaccination. We compared the properties of 1) NAbs induced by wild type DENV1 or 3 infections, which are strongly correlated with protection from repeat infections, and 2) NAbs induced by Dengvaxia in individuals who subsequently experienced DENV1 or DENV3 breakthrough infections. Wild type infections induced NAbs that recognized epitopes unique (type-specific) to each serotype, whereas the vaccine stimulated qualitatively different NAbs that recognized epitopes conserved (cross-reactive) between serotypes. Our results indicate that among children who were DENV seronegative at baseline, unbalanced replication of the DENV type 4 vaccine component in the tetravalent vaccine stimulates Abs capable of cross neutralizing DENV1 and 3 in vitro but not protect in vivo. In DENV seronegative individuals who are vaccinated, we propose that type specific NAbs are a better correlate of protection than total levels of NAbs.
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