DNA-immunisation with dengue virus E protein domains I/II, but not domain III, enhances Zika, West Nile and Yellow Fever virus infection

Dengue virus (DENV), the causative agent of dengue disease, is among the most important mosquito-borne pathogens worldwide. DENV is composed of four closely related serotypes and belongs to the Flaviviridae family alongside other important arthropod-borne viral pathogens such as Zika virus (ZIKV), West Nile virus (WNV) and Yellow Fever virus (YFV). After infection, the antibody response is mostly directed to the viral E glycoprotein which is composed of three structural domains named DI, DII and DIII that share variable degrees of homology among different viruses. Recent evidence supports a close serological interaction between ZIKV and DENV. The possibility of worse clinical outcomes as a consequence of antibody-dependent enhancement of infection (ADE) due to cross-reactive antibodies with poor neutralisation activity is a matter of concern. We tested polyclonal sera from groups of female Balb/C mice vaccinated with DNA constructs expressing DI/DII, DIII or the whole sE from different DENV serotypes and compared their activity in terms of cross-reactivity, neutralisation of virus infection and ADE. Our results indicate that the polyclonal antibody responses against the whole sE protein are highly cross-reactive with strong ADE and poor neutralisation activities due to DI/DII immunodominance. Conversely, anti-DIII polyclonal antibodies are type-specific, with no ADE towards ZIKV, WNV and YFV, and strong neutralisation activity restricted only to DENV

Long-term stability of antibody responses elicited by Dengue virus envelope DIII-based DNA vaccines

Dengue virus (DENV) is one the most important viral pathogens worldwide. Currently there is an imperative need for a reliable vaccine capable of inducing durable protection against all four serotypes. We have previously reported strongly neutralizing and highly specific antibody responses from all four serotypes to a DNA vaccine based on an engineered version of DENV E protein's domain III (DIII). Here, we show that monovalent and tetravalent immunizations with the DIII-based DNA vaccines are also capable of inducing highly stable antibody responses that remain strongly neutralizing over long periods of time. Our results demonstrate that DNA-vaccinated mice maintain a strong antibody response in terms of titre, avidity and virus-neutralizing capability 1 year after immunization

Role of N-glycosylation on Zika virus E protein secretion, viral assembly and infectivity

Zika virus has rapidly spread reaching a global distribution pattern similar to that of dengue virus, and has been associated with serious neurological and developmental pathologies, like congenital malformation during pregnancy and Guillain-Barré syndrome. Sequence analysis of different clinical and laboratory isolates has shown the existence of mutants with loss of the conserved N-glycosylation motif on domain I of protein E that is common to all flaviviruses. We found that loss of E N-linked glycosylation leads to compromised expression and secretion of E ectodomain from mammalian cells. For both, wild type and glycosylation-negative mutant, secretion was independent of co-expression of the PrM viral protein, but highly dependent on temperature. Low temperature (28 °C) favoured secretion, although the glycosylation mutant E ectodomain showed impaired secretion and membrane display compared to the wild type. Production of pseudoviral particles with a West Nile virus replicon packaged with the Zika virus structural proteins C-PrM-E was significantly reduced with the non-glycosylated E. Similarly, glycosylation-negative pseudoviral particles showed impaired infectivity of Vero cells and reduced ability to infect K562 cells upon particles opsonisation with anti-E antibodies

Role of N-glycosylation on Zika virus E protein secretion, viral assembly and infectivity

Zika virus has rapidly spread reaching a global distribution pattern similar to that of dengue virus, and has been associated with serious neurological and developmental pathologies, like congenital malformation during pregnancy and Guillain-Barré syndrome. Sequence analysis of different clinical and laboratory isolates has shown the existence of mutants with loss of the conserved N-glycosylation motif on domain I of protein E that is common to all flaviviruses. We found that loss of E N-linked glycosylation leads to compromised expression and secretion of E ectodomain from mammalian cells. For both, wild type and glycosylation-negative mutant, secretion was independent of co-expression of the PrM viral protein, but highly dependent on temperature. Low temperature (28 °C) favoured secretion, although the glycosylation mutant E ectodomain showed impaired secretion and membrane display compared to the wild type. Production of pseudoviral particles with a West Nile virus replicon packaged with the Zika virus structural proteins C-PrM-E was significantly reduced with the non-glycosylated E. Similarly, glycosylation-negative pseudoviral particles showed impaired infectivity of Vero cells and reduced ability to infect K562 cells upon particles opsonisation with anti-E antibodies

Temperature-dependent folding allows stable dimerization of secretory and virus-associated E proteins of Dengue and Zika viruses in mammalian cells

Dengue and Zika are two of the most important human viral pathogens worldwide. In both cases, the envelope glycoprotein E is the main target of the antibody response. Recently, new complex quaternary epitopes were identified which are the consequence of the arrangement of the antiparallel E dimers on the viral surface. Such epitopes can be exploited to develop more efficient cross-neutralizing vaccines. Here we describe a successful covalent stabilization of E dimers from Dengue and Zika viruses in mammalian cells. Folding and dimerization of secretory E was found to be strongly dependent on temperature but independent of PrM co-expression. In addition, we found that, due to the close relationship between flaviviruses, Dengue and Zika viruses E proteins can form heterodimers and assemble into mosaic viral particles. Finally, we present new virus-free analytical platforms to study and screen antibody responses against Dengue and Zika, which allow for differentiation of epitopes restricted to specific domains, dimers and higher order arrangements of E

Temperature-dependent folding allows stable dimerization of secretory and virus-associated E proteins of Dengue and Zika viruses in mammalian cells

Dengue and Zika are two of the most important human viral pathogens worldwide. In both cases, the envelope glycoprotein E is the main target of the antibody response. Recently, new complex quaternary epitopes were identified which are the consequence of the arrangement of the antiparallel E dimers on the viral surface. Such epitopes can be exploited to develop more efficient cross-neutralizing vaccines. Here we describe a successful covalent stabilization of E dimers from Dengue and Zika viruses in mammalian cells. Folding and dimerization of secretory E was found to be strongly dependent on temperature but independent of PrM co-expression. In addition, we found that, due to the close relationship between flaviviruses, Dengue and Zika viruses E proteins can form heterodimers and assemble into mosaic viral particles. Finally, we present new virus-free analytical platforms to study and screen antibody responses against Dengue and Zika, which allow for differentiation of epitopes restricted to specific domains, dimers and higher order arrangements of E

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