Gain without pain: adaptation and increased virulence of Zika virus in vertebrate host without fitness cost in mosquito vector.

IMPORTANCE: Previously, we modeled direct transmission chains of Zika virus (ZIKV) by serially passaging ZIKV in mice and mosquitoes and found that direct mouse transmission chains selected for viruses with increased virulence in mice and the acquisition of non-synonymous amino acid substitutions. Here, we show that these same mouse-passaged viruses also maintain fitness and transmission capacity in mosquitoes. We used infectious clone-derived viruses to demonstrate that the substitution in nonstructural protein 4A contributes to increased virulence in mice.

Design of a multi-epitope Zika virus vaccine candidate - an in-silico study.

Zika virus (ZIKV), an RNA virus, rapidly spreads Aedes mosquito-borne sickness. Currently, there are neither effective vaccines nor therapeutics available to prevent or treat ZIKV infection. In this study, to address these unmet medical needs, we aimed to design B- and T-cell candidate multi-epitope-based subunit against ZIKV using an in silico approach. In this study we applied immunoinformatics, molecular docking, and dynamic simulation assessments targeting the most immunogenic proteins; the capsid (C), envelope (E) proteins and the non-stuctural protein (NS1), described in our previous study, and which predicted immunodominant B and T cell epitopes. The final non-allergenic and highly antigenic multi-epitope was constituted of immunogenic screened-epitopes (3 CTL and 3 HTL) and the ß-defensin as an adjuvant that have been linked using EAAAK, AAY, and GPGPG linkers, respectively. The final construct containing 143 amino acids was characterized for its allergenicity, antigenicity, and physiochemical properties; and found to be safe and immunogenic with a good prediction of solubility. The existence of IFN-γ epitopes asserts the capacity to trigger strong immune responses. Subsequently, the molecular docking among vaccine and immune receptors (TLR2/TLR4) was revealed with a good binding affinity with and stable molecular interactions. Molecular dynamics simulation confirmed the stability of the complexes. Finally, the construct was subjected to in silico cloning demonstrating the efficiently of its expression in E.coli. However, this study needs the experimental validation to demonstrate vaccine safety and efficacy.Communicated by Ramaswamy H. Sarma.

Identification of potential inhibitors of Zika virus targeting NS3 helicase using molecular dynamics simulations and DFT studies.

Despite the various research efforts towards the drug discovery program for Zika virus treatment, no antiviral drugs or vaccines have yet been discovered. The spread of the mosquito vector and ZIKV infection exposure is expected to accelerate globally due to continuing global travel. The NS3-Hel is a non-structural protein part and involved in different functions such as polyprotein processing, genome replication, etc. It makes an NS3-Hel protein an attractive target for designing novel drugs for ZIKV treatment. This investigation identifies the novel, potent ZIKV inhibitors by virtual screening and elucidates the binding pattern using molecular docking and molecular dynamics simulation studies. The molecular dynamics simulation results indicate dynamic stability between protein and ligand complexes, and the structures keep significantly unchanged at the binding site during the simulation period. All inhibitors found within the acceptable range having drug-likeness properties. The synthetic feasibility score suggests that all screened inhibitors can be easily synthesizable. Therefore, possible inhibitors obtained from this study can be considered a potential inhibitor for NS3 Hel, and further, it could be provided as a lead for drug development.

Hidden symmetry of the flavivirus protein shell and pH-controlled reconstruction of the viral surface.

Using recent Zika virus structural data we reveal a hidden symmetry of protein order in immature and mature flavivirus shells, violating the Caspar-Klug paradigmatic model of capsid structures. We show that proteins of the outer immature shell layer exhibit trihexagonal tiling, while proteins from inner and outer layers conjointly form a double-shelled close-packed structure, based on a common triangular spherical lattice. Within the proposed structural model, we furthermore rationalize the structural organization of misassembled non-infectious subviral particles that have no inner capsid. We consider a pH-controlled structural reconstruction of the outer shell from the trimeric to the dimeric state, and demonstrate that this transition, occurring during the virus maturation, can be induced by changes in protein charges at lower pH, leading to a decrease in the electrostatic interaction free energy. This transition could also be assisted by electrostatic attraction of shell proteins to the interposed lipid membrane substrate separating the shells.

Evaluation of Zika virus DNA vaccines based on NS1 and domain III of E.

BACKGROUND: A large-scale outbreak of Zika virus (ZIKV) has occurred in Brazil and other South American countries, and has rapidly spread to 60 countries and regions worldwide since 2015, but no approved anti-ZIKV vaccines are available as of 2021. METHODS: We developed four types of anti-ZIKV DNA vaccine candidates: VPC-NS1, VPC-prME, VPC-prME-NS1, and VPC-EIII-NS1. They were developed against the structural proteins prM and E, and non-structural protein 1 (NS1) of ZIKV using the mammalian cell expression vector pcDNA3.1(+) as the backbone. For immunization, we intramuscularly injected mice with each vaccine candidate (n = 12 to 15 per group) on day 0 and day 14, with mice injected with phosphate-buffered saline (PBS) and pcDNA3.1(+) backbone vector as controls. On day 7, 21, and 35 after initial immunization, the effect of DNA vaccines was evaluated by ZIKV-specific humoral immunity determined by enzyme-linked immunosorbent assay (ELISA), ZIKV-specific T cell immunity determined by intracellular cytokine staining by flow cytometry and serum neutralization capacity determined by plaque reduction neutralization test (PRNT50) assay. RESULTS: The sequencing results showed that DNA vaccine vectors were successfully constructed. Western blotting and immunofluorescence results demonstrated the successful expression of immunogens carried by the DNA vaccines. On day 21 and 35 after the initial immunization, the levels of serum total immunoglobulin (Ig)G in all vaccine-given groups were slightly higher (approximately 1.5- to 2-fold) than those in the control groups. By contrast, ZIKV-specific IgG levels of all vaccine-given groups were significantly higher (approximately 10- to 1000- fold) than those of the control groups. The PRNT50 assay showed that the average serum dilution factors for neutralizing half ZIKV virions from vaccine-given groups were at least 32-fold (highest, 93-fold), while the sera from control group showed no protection. For cellular immunity, the proportions of CD11b+ myeloid cells, CD19+ B lymphocytes and CD3+ T lymphocytes in the mouse spleens as well as the percentages of CD4+ and CD8+ subsets of T cell were not changed 35 days after initial immunization. By contrast, the proportions of ZIKV-specific CD4+T cell and CD8+T cell in all vaccine-given groups were 2- to 10-folds and 2- to 30-fold than those in the control groups, respectively. CONCLUSION: All four DNA vaccines designed for the ZIKV induced neutralizing IgGs and cellular immune responses against ZIKV. Particularly, VPC-EIII-NS1 induced high level of humoral response comparable to the vaccine candidate containing prM, E and NS1 polyprotein, suggesting a potent reduced ADE effect and reserved neutralizing activity. Our findings may provide guidance for improving safety of anti-ZIKV vaccines in the future.

Developing a robust method integrating with selective membrane-based preconcentration and signal amplification for field virus detection.

Infectious diseases caused by viruses have attracted global concern owing to their rapid spread and catastrophic consequences. Therefore, developing fast and reliable on-site virus detection methods is essential for the prevention and treatment of virus-related diseases. In this study, immunoassays on a membrane, combining virus preconcentration with nanoparticle-based signal amplification, were used to realize the rapid and accurate visual detection of viruses. The biotin-streptavidin scaffolds for target virus preconcentration were established on a membrane, and subsequently a Zika aptamer (Apt) immobilized on the membrane recognized and captured the nonstructural protein 1 of Zika virus (ZIKV-NS1). The probe for detection was synthesized by conjugating the Zika Apt with a high level of horseradish peroxidase on gold nanoparticles. The ZIKV-loaded membrane was incubated with the probes, and the viral signal was amplified as the signal of horseradish peroxidase. In the presence of 3,3,5',5'-tetramethyl benzidine and hydrogen peroxide, the green color of the probe-coated membrane indicated the level of ZIKV-NS1. Our developed method could reach a detection limit of 5 ng mL-1, and the whole procedure could be completed within 1 h. Analyses of rabbit serum and environmental water samples demonstrated that an immunoassay-based approach on the membrane could accurately determine the level of ZIKV-NS1 against the complicated matrix. Our results suggest that this virus detection method has a high potential for application in clinical and environmental settings.

Ellagic Acid as a Potential Inhibitor against the Nonstructural Protein NS3 Helicase of Zika Virus: A Molecular Modelling Study.

Zika virus is a member of the Flaviviridae family and genus Flavivirus, which has a phylogenetic relationship with spondweni virus. It spreads to humans through a mosquito bite. To identify potential inhibitors for the Zika virus with biosafety, we selected natural antiviral compounds isolated from plant sources and screened against NS3 helicase of the Zika virus. The enzymatic activity of the NS3 helicase is associated with the C-terminal region and is concerned with RNA synthesis and genome replication. It serves as a crucial target for the Zika virus. We carried out molecular docking for the target NS3 helicase against the selected 25 phytochemicals using AutoDock Vina software. Among the 25 plant compounds, we identified NS3 helicase-ellagic acid (-9.9 kcal/mol), NS3 helicase-hypericin (-9.8 kcal/mol), and NS3 helicase-pentagalloylglucose (-9.5 kcal/mol) as the best binding affinity compounds based on their binding energies. To understand the stability of these complexes, molecular dynamic simulations were executed and the trajectory analysis exposed that the NS3 helicase-ellagic acid complex possesses greater stability than the other two complexes such as NS3 helicase-hypericin and NS3 helicase-pentagalloylglucose. The ADMET property prediction of these compounds resulted in nontoxicity and noncarcinogenicity.

Investigating the conformational dynamics of Zika virus NS4B protein.

Zika virus (ZIKV) NS4B protein is a membranotropic multifunctional protein. Despite its versatile functioning, its topology and dynamics are not entirely understood. There is no X-ray or cryo-EM structure available for any flaviviral NS4B full-length protein. In this study, we have investigated the structural dynamics of full-length ZIKV NS4B protein through 3D structure models using molecular dynamics simulations and experimental techniques. Also, we employed a reductionist approach to understand the dynamics of NS4B protein where we studied its N-terminal (residues 1-38), C-terminal (residues 194-251), and cytosolic (residues 131-169) regions in isolation in addition to the full-length protein. Further, using a series of circular dichroism spectroscopic experiments, we validate the cytosolic region as an intrinsically disordered protein region. The microsecond-long all atoms molecular dynamics and replica-exchange simulations complement the experimental observations. Furthermore, we have also studied the NS4B proteins C-terminal regions of four other flaviviruses viz. DENV2, JEV, WNV, and YFV through microsecond simulations to characterize their behaviour in presence and absence of lipid membranes. There are significant differences observed in the conformations of other flavivirus NS4B C-terminal regions in comparison to ZIKV NS4B. Lastly, we have proposed a ZIKV NS4B protein model illustrating its putative topology consisting of various membrane-spanning and non-membranous regions.

Identification and characterization of key residues in Zika virus envelope protein for virus assembly and entry.

Zika virus (ZIKV), a family member in the Flavivirus genus, has re-emerged as a global public health concern. The envelope (E) proteins of flaviviruses play a dual role in viral assembly and entry. To identify the key residues of E in virus entry, we generated a ZIKV trans-complemented particle (ZIKVTCP) system, in which a subgenomic reporter replicon was packaged by trans-complementation with expression of CprME. We performed mutagenesis studies of the loop regions that protrude from the surface of the virion in the E ectodomains (DI, DII, DIII). Most mutated ZIKVTCPs exhibited deficient egress. Mutations in DII and in the hinge region of DI and DIII affected prM expression. With a bioorthogonal system, photocrosslinking experiments identified crosslinked intracellular E trimers and demonstrated that egress-deficient mutants in DIII impaired E trimerization. Of these mutants, an E-trimerization-dead mutation D389A that nears the E-E interface between two neighbouring spikes in the immature virion completely abolished viral egress. Several mutations abolished ZIKVTCPs' entry, without severely affecting viral egress. Further virus binding experiments demonstrated a deficiency of the mutated ZIKVTCPs in virus attachment. Strikingly, synthesized peptide containing residues of two mutants (268-273aa in DII) could bind to host cells and significantly compete for viral attachment and interfere with viral infection, suggesting an important role of these resides in virus entry. Our findings uncovered the requirement for DIII mediated-E trimerization in viral egress, and discovered a key residue group in DII that participates in virus entry.

The discovery of Zika virus NS2B-NS3 inhibitors with antiviral activity via an integrated virtual screening approach.

With expanding recent outbreaks and a lack of treatment options, the Zika virus (ZIKV) poses a severe health concern. The availability of ZIKV NS2B-NS3 co-crystallized structures paved the way for rational drug discovery. A computer-aided structure-based approach was used to screen a diverse library of compounds against ZIKV NS2B-NS3 protease. The top hits were selected based on various binding free energy calculations followed by per-residue decomposition analysis. The selected hits were then evaluated for their biological potential with ZIKV protease inhibition assay and antiviral activity. Among 26 selected compounds, 8 compounds showed promising activity against ZIKV protease with a percentage inhibition of greater than 25 and 3 compounds displayed ∼50% at 10 µM, which indicates an enrichment rate of approximately 36% (threshold IC50 < 10 µM) in the ZIKV-NS2B-NS3 protease inhibition assay. Of these, only one compound (23) produced whole-cell anti-ZIKV activity, and the binding mode of 23 was extensively analyzed through long-run molecular dynamics simulations. The current study provides a promising starting point for the further development of novel compounds against ZIKV.

Multiple Salivary Proteins from Aedes aegypti Mosquito Bind to the Zika Virus Envelope Protein.

Aedes aegypti mosquitoes are important vectors of several debilitating and deadly arthropod-borne (arbo) viruses, including Yellow Fever virus, Dengue virus, West Nile virus and Zika virus (ZIKV). Arbovirus transmission occurs when an infected mosquito probes the host's skin in search of a blood meal. Salivary proteins from mosquitoes help to acquire blood and have also been shown to enhance pathogen transmission in vivo and in vitro. Here, we evaluated the interaction of mosquito salivary proteins with ZIKV by surface plasmon resonance and enzyme-linked immunosorbent assay. We found that three salivary proteins AAEL000793, AAEL007420, and AAEL006347 bind to the envelope protein of ZIKV with nanomolar affinities. Similar results were obtained using virus-like particles in binding assays. These interactions have no effect on viral replication in cultured endothelial cells and keratinocytes. Additionally, we found detectable antibody levels in ZIKV and DENV serum samples against the recombinant proteins that interact with ZIKV. These results highlight complex interactions between viruses, salivary proteins and antibodies that could be present during viral transmissions.

Dengue and Zika Virus Capsid Proteins Contain a Common PEX19-Binding Motif.

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.

In silico construction of a multiepitope Zika virus vaccine using immunoinformatics tools.

Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine's average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation.

Backbone 1H, 15N, and 13C resonance assignments of the non-structural protein NS2B of Zika virus.

Zika virus (ZIKV) emerged as a global public health concern due to its relationship with severe neurological disorders. Non-structural (NS) proteins of ZIKV are essential for viral replication, regulatory function, and subversion of host responses. NS2B is a membrane protein responsible for the regulation of viral protease activity. This protein has transmembrane domains critical for the localization of viral protease to the endoplasmic reticulum membrane and a hydrophilic domain essential for folding, recruitment, and protease activity. Therefore, NS2B is considered a cofactor of viral protease which processes viral polyprotein and is essential for virus replication, making it an attractive antiviral drug target. Here, we report the backbone 1H, 15N, 13C resonance assignments of the full-length NS2B by high-resolution NMR. The backbone assignment will be necessary for determining the three-dimensional structure and backbone dynamics of NS2B, interaction mapping and screening potential of antiviral drugs against ZIKV and related pathogenic flaviviruses.

INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation.

The evasion of the Interferon response has important implications in Zika virus (ZIKV) disease. Mutations in ZIKV viral protein NS4B, associated with modulation of the interferon (IFN) system, have been linked to increased pathogenicity in animal models. In this study, we unravel ZIKV NS4B as antagonist of the IFN signaling cascade. Firstly, we reported the genomic characterization of NS4B isolated from a strain of the 2016 outbreak, ZIKV Brazil/2016/INMI1, and we predicted its membrane topology. Secondly, we analyzed its phylogenetic correlation with other flaviviruses, finding a high similarity with dengue virus 2 (DEN2) strains; in particular, the highest conservation was found when NS4B was aligned with the IFN inhibitory domain of DEN2 NS4B. Hence, we asked whether ZIKV NS4B was also able to inhibit the IFN signaling cascade, as reported for DEN2 NS4B. Our results showed that ZIKV NS4B was able to strongly inhibit the IFN stimulated response element and the IFN-γ-activated site transcription, blocking IFN-I/-II responses. mRNA expression levels of the IFN stimulated genes ISG15 and OAS1 were also strongly reduced in presence of NS4B. We found that the viral protein was acting by suppressing the STAT1 phosphorylation and consequently blocking the nuclear transport of both STAT1 and STAT2.

Evaluation of ELISA-Based Multiplex Peptides for the Detection of Human Serum Antibodies Induced by Zika Virus Infection across Various Countries.

Zika virus (ZIKV) is a mosquito-borne Flavivirus with a positive-sense RNA genome, which are generally transmitted through the bite of an infected Aedes mosquito. ZIKV infections could be associated with neurological sequelae that, and otherwise produces similar clinical symptoms as other co-circulating pathogens. Past infection with one member of the Flavivirus genus often induces cross-reactive antibodies against other flaviruses. These attributes complicate the ability to differentially diagnose ZIKV infection from other endemic mosquito-borne viruses, making it both a public health issue as well as a diagnostic challenge. We report the results from serological analyses using arbovirus-specific peptides on 339 samples that were previously collected from 6 countries. Overall, we found that our multiplexed peptide-based ELISA was highly efficient for identifying ZIKV antibodies as early as 2 weeks post infection, and that it correlates with microneutralization, plaque reduction neutralization tests (PRNTs) and commercial tests for ZIKV in previously characterized samples. We observed that seropositivity varied by patient cohort, reflecting the sampling period in relation to the 2015-2016 ZIKV outbreak. This work evaluates the accuracy, specificity, and sensitivity of our peptide-based ELISA method for detecting ZIKV antibodies from geographically diverse regions. These findings can contribute to ongoing serological methods development and can be adapted for use in future studies.

Doxycycline Interferes with Zika Virus Serine Protease and Inhibits Virus Replication in Human Skin Fibroblasts.

Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (-7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.

Evaluation of potential MHC-I allele-specific epitopes in Zika virus proteins and the effects of mutations on peptide-MHC-I interaction studied using in silico approaches.

Zika virus (ZIKV) infection is a global health concern due to its association with microcephaly and neurological complications. The development of a T-cell vaccine is important to combat this disease. In this study, we propose ZIKV major histocompatibility complex I (MHC-I) epitopes based on in silico screening consensus followed by molecular docking, PRODIGY, and molecular dynamics (MD) simulation analyses. The effects of the reported mutations on peptide-MHC-I (pMHC-I) complexes were also evaluated. In general, our data indicate an allele-specific peptide-binding human leukocyte antigen (HLA) and potential epitopes. For HLA-B44, we showed that the absence of acidic residue Glu at P2, due to the loss of the electrostatic interaction with Lys45, has a negative impact on the pMHC-I complex stability and explains the low free energy estimated for the immunodominant peptide E-4 (IGVSNRDFV). Our MD data also suggest the deleterious effects of acidic residue Asp at P1 on the pMHC-I stability of HLA-B8 due to destabilization of the α-helix and ß-strand. Free energy estimation further indicated that the mutation from Val to Ala at P9 of peptide E-247 (DAHAKRQTV), which was found exclusively in microcephaly samples, did not reduce HLA-B8 affinity. In contrast, the mutation from Thr to Pro at P2 of the peptide NS5-832 (VTKWTDIPY) decreased the interaction energy, number of intermolecular interactions, and adversely affected its binding mode with HLA-A1. Overall, our findings are important with regard to the design of T-cell peptide vaccines and for understanding how ZIKV escapes recognition by CD8 + T-cells.

Conjugation of Hemoglobin and Mannan Markedly Improves the Immunogenicity of Domain III of the Zika Virus E Protein: Structural and Immunological Study.

Zika virus (ZIKV) leads to congenital microcephaly and anomalies and severe neurological diseases such as Guillain-Barre syndrome. Safe and effective vaccines are necessitated to deal with these severe health threats. As an ideal antigen, the domain III of the envelope protein (EDIII) of ZIKV can evoke potent neutralizing antibodies without any antibody-dependent enhancement (ADE) effect. However, EDIII necessitates to be formulated with an antigen delivery system or adjuvants to improve its immunogenicity. Hemoglobin (Hb) regulates inflammation, cytokine levels, and activate macrophage. Mannan is a polysaccharide of the fungal cell wall with an immunomodulatory activity. In this study, EDIII was conjugated with Hb and mannan, using the disulfide bond as the linker. Hb and mannan both functioned as the adjuvants. Conjugation of Hb and mannan acted as the delivery system for EDIII. The structure of EDIII was essentially maintained upon conjugation of Hb and mannan. The intracellular release of EDIII from the conjugate (HM-EDIII-2) was achieved by reduction of the glutathione-sensitive disulfide bond. As compared with EDIII, HM-EDIII-2 elicited high EDIII-specific IgG titers and high levels of Th1-type cytokines (IFN-γ and IL-2) and Th2-type cytokines (IL-5 and IL-10), along with no apparent toxicity to the organs. Moreover, the pharmacokinetic study revealed a prolonged serum exposure of HM-EDIII-2 to the immune cells. Thus, HM-EDIII-2 could boost a strong humoral and cellular immune response to EDIII. Our study was expected to provide the feasibility necessary to develop a robust and potentially safe ZIKV vaccine.

Monoclonal Antibodies against Zika Virus NS1 Protein Confer Protection via Fcγ Receptor-Dependent and -Independent Pathways.

Zika virus (ZIKV) infection during pregnancy causes congenital defects such as fetal microcephaly. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) have the potential to suppress ZIKV pathogenicity without enhancement of disease, but the pathways through which they confer protection remain obscure. Here, we report two types of NS1-targeted human MAbs that inhibit ZIKV infection through distinct mechanisms. MAbs 3G2 and 4B8 show a better efficacy than MAb 4F10 in suppressing ZIKV infection in C57BL/6 neonatal mice. Unlike MAb 4F10 that mainly triggers antibody-dependent cell-mediated cytotoxicity (ADCC), MAbs 3G2 and 4B8 not only trigger ADCC but inhibit ZIKV infection without Fcγ receptor-bearing effector cells, possibly at postentry stages. Destroying the Fc-mediated effector function of MAbs 3G2 and 4B8 reduces but does not abolish their protective effects, whereas destroying the effector function of MAb 4F10 eliminates the protective effects, suggesting that MAbs 3G2 and 4B8 engage both Fcγ receptor-dependent and -independent pathways. Further analysis reveals that MAbs 3G2 and 4B8 target the N-terminal region of NS1 protein, whereas MAb 4F10 targets the C-terminal region, implying that the protective efficacy of an NS1-targeted MAb may be associated with its epitope recognition. Our results illustrate that NS1-targeted MAbs have multifaceted protective effects and provide insights for the development of NS1-based vaccines and therapeutics.IMPORTANCE Zika virus (ZIKV) is a mosquito-borne flavivirus that has been linked to congenital microcephaly during recent epidemics. No licensed antiviral drug or vaccine is available. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) inhibit ZIKV pathogenicity but do not enhance the disease as envelope protein-targeted MAbs do. However, the protection mechanisms are not fully understood. Here, we show that in the presence or absence of Fcγ receptor-bearing effector cells, NS1-targeted human MAbs 3G2 and 4B8 inhibit ZIKV infection. Compared to MAb 4F10 that has no inhibitory effects without effector cells, 3G2 and 4B8 confer better protection in ZIKV-infected neonatal mice. Destroying the Fc-mediated effector function reduces but does not abolish the protection of 3G2 and 4B8, suggesting that they engage both Fcγ receptor-dependent and -independent pathways. The protective efficacy of NS1-targeted MAbs may be associated with their epitope recognition. Our findings will help to develop NS1-based vaccines and therapeutics.