Evaluation of a Tetravalent DNA Vaccine against Dengue: Integrating Biochemical Studies on Dengue Virus Envelope Protein to a Domain-Based Antigen Design.

Dengue virus (DENV) is among the most important mosquito-borne human pathogens worldwide. Concerns regarding the effectiveness of Dengue vaccines together with emergence of Zika virus (ZIKV) have reignited the interest for new vaccines using alternative approaches. Given that the envelope glycoprotein (E) is the main target of neutralizing antibodies, it has been used as the antigen of choice for vaccine development efforts. Here we present a detailed analysis of factors involved in the expression, secretion and folding of the E ectodomain from all four DENV serotypes and ZIKV in mammalian cells. Our data demonstrate that E domains II and III (DII and DIII) are important for proper E folding and stabilization of soluble dimers, respectively. In addition, we show that successful covalent stabilisation of E dimers, and E folding in general is strongly dependent on temperature but not on PrM co-expression, and that DENV and ZIKV E proteins can form heterodimers and assemble into mosaic viral particles. Our findings also show that antigen secretion determines the efficiency of DNA vaccines. Based on this, we developed a novel DNA gene-gun immunisation strategy using an engineered version of DIII fused to the CH3 domain of the IgG H chain, which is efficiently secreted from transfected cells and induced strong antibody responses that neutralise all DENV serotypes. The antibody responses were stable over long periods of time and different tetravalent formulations of the vaccine showed induction of neutralising antibodies against all four dengue serotypes as well. Finally, our results also indicate that the polyclonal antibody responses against DI/DII are highly cross-reactive, poorly neutralising and promote ADE towards all DENV serotypes, ZIKV, WNV and YFV. Conversely, anti-DIII antibodies are type-specific, with no ADE towards related flaviviruses, and with strong neutralisation activity restricted only to DENV

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal coronavirus disease (COVID-19) outcomes is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses, and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to intensive care units (ICU) with fatal COVID-19 outcomes, but not in individuals with non-fatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to ICU with fatal and non-fatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an original antigenic sin type-response

Broad and strong memory CD4(+)and CD8(+)T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design

DIII vaccination induces DENV-specific neutralising antibodies.

<p>A) Neutralisation and ADE of all four DENV serotypes, ZIKV, WNV and YFV, with DENV anti-DIII sera. To prove anti-DIII reactivity, DENV viruses were tested using pooled sera against the homologous serotypes, while non-DENV viruses were tested against all four anti-DIII sera (in all cases, n = 3 for each virus-sera combination). B) FRNT₅₀ titres, as determined in (A), * indicates FRNT₅₀ titres below detection limit of the assay (≥25).</p

DENV-DIII tetravalent vaccine formulation elicits DENV-specific neutralising antibodies.

<p>A) Neutralisation and ADE of all four DENV serotypes, ZIKV, WNV and YFV, with pooled sera from mice vaccinated with the Tetra-DIII formulation (in all cases, for each virus-sera combination n = 3). B) FRNT₅₀, as determined in (A), * indicates FRNT₅₀ titres below detection limit of the assay (≥25).</p

Reactivity profiles of anti-DENV sera in DENV-infected cells.

<p>Immunofluorescence of Vero cells infected with all four DENV serotypes and reacted with antibodies elicited with 3sE and 4sE or the corresponding 3DI/DII and 4DI/DII. mAb 4G2, the homologous anti-DIII and the tetravalent anti-DIII sera were included as controls to show reactivity towards each serotype. N.I., non-infected cells; P.I., pre-immune serum. Bar, 30 μm. Representative images from independent experiments are shown.</p

DENV DI/DII induces poorly-neutralising, highly-enhancing cross-reactive antibodies.

<p>A) Neutralisation and ADE of all four DENV serotypes, ZIKV, WNV and YFV, with DENV anti-3DI/DII and anti-4DI/DII pooled sera (in all cases, n = 3 for each virus-sera combination). B) FRNT₅₀ titres, as determined in (A), * indicates FRNT₅₀ titres below detection limit of the assay (≥25).</p

Immunogenicity and efficacy of Zika virus Envelope Domain III in DNA, protein and ChAdOx1 adenoviral-vectored vaccines

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV

A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection

Infections with dengue (DENV) and Zika (ZIKV) viruses can induce cross-reactive antibody responses. Two immunodominant epitopes, to precursor membrane protein (prM) or the fusion loop epitope (FLE) on envelope (E) protein are recognized by cross-reactive antibodies1, 2, 3 that are not only poorly neutralizing, but can also promote increased viral replication and disease seerity via Fc-gamma receptor mediated infection of myeloid cells, a process termed antibody-dependent enhancement (ADE)1, 4, 5 . ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly-neutralizing cross-reactive antibodies may prime an individual for ADE upon natural infection. In this report, we describe the design and production of covalently-stabilized ZIKV E-dimers, which lack prM and do not expose the immunodominant FLE. Immunization of mice with ZIKV E-dimers induces dimer-specific antibodies, which protected against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection