Current Status of Zika Virus Vaccines: Successes and Challenges

The recently emerged Zika virus (ZIKV) spread to the Americas, causing a spectrum of congenital diseases including microcephaly in newborn and Guillain-Barré syndrome (GBS) in adults. The unprecedented nature of the epidemic and serious diseases associated with the viral infections prompted the global research community to understand the immunopathogenic mechanisms of the virus and rapidly develop safe and efficacious vaccines. This has led to a number of ZIKV vaccine candidates that have shown significant promise in human clinical trials. These candidates include nucleic acid vaccines, inactivated vaccines, viral-vectored vaccines, and attenuated vaccines. Additionally, a number of vaccine candidates have been shown to protect animals in preclinical studies. However, as the epidemic has waned in the last three years, further development of the most promising vaccine candidates faces challenges in clinical efficacy trials, which is needed before a vaccine is brought to licensure. It is important that a coalition of government funding agencies and private sector companies is established to move forward with a safe and effective vaccine ready for deployment when the next ZIKV epidemic occurs

Current Status of Zika Virus Vaccines: Successes and Challenges

The recently emerged Zika virus (ZIKV) spread to the Americas, causing a spectrum of congenital diseases including microcephaly in newborn and Guillain-Barré syndrome (GBS) in adults. The unprecedented nature of the epidemic and serious diseases associated with the viral infections prompted the global research community to understand the immunopathogenic mechanisms of the virus and rapidly develop safe and efficacious vaccines. This has led to a number of ZIKV vaccine candidates that have shown significant promise in human clinical trials. These candidates include nucleic acid vaccines, inactivated vaccines, viral-vectored vaccines, and attenuated vaccines. Additionally, a number of vaccine candidates have been shown to protect animals in preclinical studies. However, as the epidemic has waned in the last three years, further development of the most promising vaccine candidates faces challenges in clinical efficacy trials, which is needed before a vaccine is brought to licensure. It is important that a coalition of government funding agencies and private sector companies is established to move forward with a safe and effective vaccine ready for deployment when the next ZIKV epidemic occurs

An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

Zika virus (ZIKV), a mosquito-transmitted flavivirus, emerged in the last decade causing serious human diseases, including congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Although many vaccine platforms are at various stages of development, no licensed vaccines are currently available. Previously, we described a mutant MR766 ZIKV (m2MR) bearing an E protein mutation (N154A) that prevented its glycosylation, resulting in attenuation and defective neuroinvasion. To further attenuate m2MR for its potential use as a live viral vaccine, we incorporated additional mutations into m2MR by substituting the asparagine residues in the glycosylation sites (N130 and N207) of NS1 with alanine residues. Examination of pathogenic properties revealed that the virus (m5MR) carrying mutations in E (N154A) and NS1 (N130A and N207A) was fully attenuated with no disease signs in infected mice, inducing high levels of humoral and cell-mediated immune responses, and protecting mice from subsequent lethal virus challenge. Furthermore, passive transfer of sera from m5MR-infected mice into naïve animals resulted in complete protection from lethal challenge. The immune sera from m5MR-infected animals neutralized both African and Asian lineage viruses equally well, suggesting that m5MR virus could be developed as a potentially broad live virus vaccine candidate

An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

Zika virus (ZIKV), a mosquito-transmitted flavivirus, emerged in the last decade causing serious human diseases, including congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Although many vaccine platforms are at various stages of development, no licensed vaccines are currently available. Previously, we described a mutant MR766 ZIKV (m2MR) bearing an E protein mutation (N154A) that prevented its glycosylation, resulting in attenuation and defective neuroinvasion. To further attenuate m2MR for its potential use as a live viral vaccine, we incorporated additional mutations into m2MR by substituting the asparagine residues in the glycosylation sites (N130 and N207) of NS1 with alanine residues. Examination of pathogenic properties revealed that the virus (m5MR) carrying mutations in E (N154A) and NS1 (N130A and N207A) was fully attenuated with no disease signs in infected mice, inducing high levels of humoral and cell-mediated immune responses, and protecting mice from subsequent lethal virus challenge. Furthermore, passive transfer of sera from m5MR-infected mice into naïve animals resulted in complete protection from lethal challenge. The immune sera from m5MR-infected animals neutralized both African and Asian lineage viruses equally well, suggesting that m5MR virus could be developed as a potentially broad live virus vaccine candidate

Inhibition of Zika virus replication by G-quadruplex-binding ligands

Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco- 19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

ABSTRACT Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion. IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity

Regulation of Kir channels in bovine retinal pigment epithelial cells by phosphatidylinositol 4,5-bisphosphate

The inwardly rectifying K+ (Kir) current in mammalian retinal pigment epithelial (RPE) cells, which is largely mediated by Kir7.1 channels, is stable in cells dialyzed with MgATP but runs down when intracellular ATP is depleted. A potential mechanism for this rundown is a decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) regeneration by ATP-dependent lipid kinases. Here, we used the whole cell voltage-clamp technique to investigate the membrane PIP2 dependence of Kir channels in isolated bovine RPE cells. When RPE cells were dialyzed with ATP-free solution containing PIP2 (25–50 μM), rundown persisted but was markedly reduced. Removal of Mg2+ from the pipette solution also slowed rundown, indicating that elevated intracellular Mg2+ concentration contributes to rundown. Cell dialysis with the PIP2 scavenger neomycin in MgATP solution diminished Kir current in a voltage-dependent manner, suggesting that it acted at least in part by blocking the Kir channel. Kir current in MgATP-loaded cells was partially inhibited by bath application of quercetin (100 μM), phenylarsine oxide (100 μM), or wortmannin (50 μM), inhibitors of phosphatidylinositol (PI) kinases, and was completely inhibited by cell dialysis with 2 mM adenosine, a PI4 kinase inhibitor. Both LY-294002 (100 μM), an inhibitor of PI3 kinases, and its inactive analog LY-303511 (100 μM) rapidly and reversibly inhibited Kir current, suggesting that these compounds act as direct channel blockers. We conclude that the activity of Kir channels in the RPE is critically dependent on the regeneration of membrane PIP2 by PI4 kinases and that this may explain the dependence of these channels on hydrolyzable ATP

A Ferritin Nanoparticle-Based Zika Virus Vaccine Candidate Induces Robust Humoral and Cellular Immune Responses and Protects Mice from Lethal Virus Challenge

The severe consequences of the Zika virus (ZIKV) infections resulting in congenital Zika syndrome in infants and the autoimmune Guillain–Barre syndrome in adults warrant the development of safe and efficacious vaccines and therapeutics. Currently, there are no approved treatment options for ZIKV infection. Herein, we describe the development of a bacterial ferritin-based nanoparticle vaccine candidate for ZIKV. The viral envelope (E) protein domain III (DIII) was fused in-frame at the amino-terminus of ferritin. The resulting nanoparticle displaying the DIII was examined for its ability to induce immune responses and protect vaccinated animals upon lethal virus challenge. Our results show that immunization of mice with a single dose of the nanoparticle vaccine candidate (zDIII-F) resulted in the robust induction of neutralizing antibody responses that protected the animals from the lethal ZIKV challenge. The antibodies neutralized infectivity of other ZIKV lineages indicating that the zDIII-F can confer heterologous protection. The vaccine candidate also induced a significantly higher frequency of interferon (IFN)- γ positive CD4 T cells and CD8 T cells suggesting that both humoral and cell-mediated immune responses were induced by the vaccine candidate. Although our studies showed that a soluble DIII vaccine candidate could also induce humoral and cell-mediated immunity and protect from lethal ZIKV challenge, the immune responses and protection conferred by the nanoparticle vaccine candidate were superior. Further, passive transfer of neutralizing antibodies from the vaccinated animals to naïve animals protected against lethal ZIKV challenge. Since previous studies have shown that antibodies directed at the DIII region of the E protein do not to induce antibody-dependent enhancement (ADE) of ZIKV or other related flavivirus infections, our studies support the use of the zDIII-F nanoparticle vaccine candidate for safe and enhanced immunological responses against ZIKV