As Zika virus (ZIKV) emerges into Dengue virus (DENV)-endemic areas, cases of ZIKV infection in DENV-immune pregnant women may rise. Here we show that prior DENV immunity affects maternal and fetal ZIKV infection in pregnancy using sequential DENV and ZIKV infection models. Fetuses in ZIKV-infected DENV-immune dams were normal sized, whereas fetal demise occurred in non-immune dams. Moreover, reduced ZIKV RNA is present in the placenta and fetuses of ZIKV-infected DENV-immune dams. DENV cross-reactive CD8+ T cells expand in the maternal spleen and decidua of ZIKV-infected dams, their depletion increases ZIKV infection in the placenta and fetus, and results in fetal demise. The inducement of cross-reactive CD8+ T cells via peptide immunization or adoptive transfer results in decreased ZIKV infection in the placenta. Prior DENV immunity can protect against ZIKV infection during pregnancy in mice, and CD8+ T cells are sufficient for this cross-protection. This has implications for understanding the natural history of ZIKV in DENV-endemic areas and the development of optimal ZIKV vaccines.

Dengue virus (DENV) currently circulates in more than 100 countries and causes an estimated 390 million infections per year. While most cases manifest as a self-resolving fever, ∼1.5% of infections develop into a more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which causes ∼20,000 deaths annually. The underlying pathological feature of DHF/DSS, also known as Severe Dengue, is an acute increase in vascular permeability leading to hypovolemia and shock. Angiogenic factors and cytokines, such as vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF), have been implicated in the increased vascular permeability, suggesting a potential therapeutic strategy for Severe Dengue. Here, we employed a mouse model of antibody-dependent enhancement of DENV infection, which recapitulates the fatal capillary leakage and shock of human Severe Dengue, to investigate the effects of approved VEGF- and TNF-targeting drugs. DENV infection caused a significant increase in serum VEGF levels within 2 days and resulted in ∼80% mortality within 8 days of infection. Treatment of mice with sunitinib, a VEGF receptor tyrosine kinase inhibitor, once (day 2) or twice (days 1 and 2) post-infection reduced mortality by 50-80% compared with untreated mice. Notably, sunitinib treatment decreased serum TNF levels, white blood cell counts, and hematocrit levels relative to untreated mice, but had only marginal effects on tissue viral burden. Combination therapy with anti-TNF antibody and sunitinib significantly reduced vascular leakage and synergized to provide superior protection from lethal DENV infection compared with either agent alone. These data suggest that a two-pronged anti-angiogenic and anti-inflammatory approach may be useful for the rapid treatment of DHF/DSS.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that represents a major threat to global health. ZIKV infections in adults are generally asymptomatic or present with mild symptoms. However, recent outbreaks of ZIKV have revealed that it can cause Congenital Zika Syndrome in neonates and Guillain-Barré syndrome in adults. Currently, no ZIKV-specific vaccines or antiviral treatments are available. In this study, we tested the efficacy of convalescent plasma IgG hyperimmune product (ZIKV-IG) isolated from individuals with high neutralizing anti-ZIKV titers as a therapeutic candidate against ZIKV infection using a model of ZIKV infection in Ifnar1 −/− mice. ZIKV-IG successfully protected mice from lethal ZIKV challenge. In particular, ZIKV-IG treatment at 24 hours after lethal ZIKV infection improved survival by reducing weight loss and tissue viral burden and improving clinical score. Additionally, ZIKV-IG eliminated ZIKV-induced tissue damage and inflammation in the brain and liver. These results indicate that ZIKV-IG is efficacious against ZIKV, suggesting this human polyclonal antibody is a viable candidate for further development as a treatment against human ZIKV infection.

Background: Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus. Previous studies have identified neutralizing antibodies from Zika patients that bind to quaternary epitopes across neighboring envelope (E) proteins, called E dimer epitopes (EDE). An asparagine-linked glycan on the "glycan loop" (GL) of the ZIKV envelope protein protects the functionally important "fusion loop" on the opposite E subunit in the dimer, and EDE antibodies have been shown to bind to both of these loops. Human EDE antibodies have been divided into two subclasses based on how they bind to the glycan loop region: EDE1 antibodies do not require glycosylation for binding, while EDE2 antibodies strongly rely on the glycan for binding. Methods: ZIKV GL was expressed on tobacco mosaic virus nanoparticles. Mice were immunized with GL or fulllength monomeric E and the immune response was analyzed by testing the ability of sera and monoclonal antibodies to bind to GL and to neutralize ZIKV in in vitro cellular assay. Results: We report here the existence of ZIKV moderately neutralizing antibodies that bind to E monomers through epitopes that include the glycan loop. We show that sera from human Zika patients contain antibodies capable of binding to the unglycosylated glycan loop in the absence of the rest of the envelope protein. Furthermore, mice were inoculated with recombinant E monomers and produced neutralizing antibodies that either recognize unglycosylated glycan loop or require glycan for their binding to monomeric E. We demonstrate that both types of antibodies neutralize ZIKV to some extent in a cellular virus neutralization assay. Conclusions: Analogous to the existing EDE antibody nomenclature, we propose a new classification for antibodies that bind to E monomer epitopes (EME): EME1 and EME2 for those that do not require and those that do require glycan for binding to E, respectively.

Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1−/− transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine–induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.