prM Protein Research Articles

A prM mutation that attenuates dengue virus replication in human cells enhances midgut infection in mosquitoes.

Dengue viruses (DENVs), like all viruses, evolve to perpetuate transmission of their species in their hosts. However, how DENV genetics influences dengue disease outbreaks remains poorly understood. Here, we examined isolates of the South Pacific dengue virus type 2 (DENV-2) that emerged in the 1970s and caused major dengue outbreaks in islands in this region until it reached Tonga, where only a few mild cases were reported. Phylogenetically, the DENV-2 strain isolated in Tonga segregated into a clade different from those clades infecting populations in other South Pacific islands. We found that this epidemiological observation could be explained by a single histidine-to-arginine substitution in position 86 of the premembrane (prM) protein of the Tonga DENV-2 strain. This mutation attenuated viral protein translation in mammalian cells but not in midgut cells of the mosquito vector Aedes aegypti. In mammalian cells, the prM mutation resulted in reduced translation of the viral genome and subsequent reduced virus replication. In contrast, in mosquito midgut cells, the prM mutation conferred a selective infection advantage, possibly because of the positively charged arginine residue introduced by the mutation. These findings provide molecular insights into the year-long silent transmission of attenuated DENV-2 in Tonga during the 1970s dengue outbreak in the South Pacific.

The structure of immature tick-borne encephalitis virus supports the collapse model of flavivirus maturation.

We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation.

Characterization of a Small Plaque Variant Derived from Genotype V Japanese Encephalitis Virus Clinical Isolate K15P38.

Genotype V (GV) Japanese encephalitis virus (JEV) has been predominantly reported in the Republic of Korea (ROK) since 2010. GV JEV exhibits higher virulence and distinct antigenicity compared to other genotypes, which results in reduced efficacy of existing vaccines. Research on GV JEV is essential to minimize its clinical impact, but the only available clinical strain in the ROK is K15P38, isolated from the cerebrospinal fluid of a patient in 2015. We obtained this virus from National Culture Collection for Pathogens (NCCP) and isolated a variant forming small plaques during our research. We identified that this variant has one amino acid substitution each in the PrM and NS5 proteins compared to the reported K15P38. Additionally, we confirmed that this virus exhibits delayed propagation in vitro and an attenuated phenotype in mice. The isolation of this variant is a critical reference for researchers intending to study K15P38 obtained from NCCP, and the mutations in the small plaque-forming virus are expected to be useful for studying the pathology of GV JEV.

Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

Mutation of conserved histidine residues of dengue virus envelope protein impairs viral like particle maturation and secretion

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development.

Detection of Zika Virus Antibodies in Retrospective Serum Samples from Suspected Dengue Cases in Sarawak, Malaysian Borneo

Sarawak, a state in Malaysian Borneo, has never reported a case of Zika virus (ZIKV) infection nor conducted any seroprevalence studies on the virus. This study aimed to provide a serological insight into ZIKV in Sarawak. 212 sera samples collected from a dengue surveillance study conducted from 2007 to 2011 were retrospectively analyzed. Samples negative for both dengue and Japanese encephalitis virus by RT-PCR were screened by immunoblots assay for ZIKV prM protein. Positive samples were further subjected to 50% plaque reduction neutralization test (PRNT50) for confirmation. 20 (9.4%) sera samples were positive for ZIKV prM protein but not against prM of dengue and Japanese encephalitis virus. These 20 samples were further subjected to PRNT50, and six samples (2.8%) showed possible seropositivity for ZIKV. This preliminary investigation provides serological evidence of ZIKV infection in Sarawak and highlights the importance of conducting surveillance programs for ZIKV in this dengue-endemic state.

Ferroptosis contributes to JEV-induced neuronal damage and neuroinflammation

Ferroptosis is a newly discovered prototype of programmed cell death (PCD) driven by iron-dependent phospholipid peroxidation ​accumulation, and it has been linked to numerous organ injuries and degenerative pathologies. Although studies have shown that a variety of cell death processes contribute to JEV-induced neuroinflammation and neuronal injury, there is currently limited research on the specific involvement of ferroptosis. In this study, we explored the neuronal ferroptosis induced by JEV infection in vitro and in vivo. Our results indicated that JEV infection induces neuronal ferroptosis through inhibiting the function of the antioxidant system mediated by glutathione (GSH)/glutathione peroxidase 4 (GPX4), as well as by promoting lipid peroxidation mediated by yes-associated protein 1 (YAP1)/long-chain acyl-CoA synthetase 4 (ACSL4). Further analyses revealed that JEV E and prM proteins function as agonists, inducing ferroptosis. Moreover, we found that treatment with a ferroptosis inhibitor in JEV-infected mice reduces the viral titers and inflammation in the mouse brains, ultimately improving the survival rate of infected mice. In conclusion, our study unveils a critical role of ferroptosis in the pathogenesis of JEV, providing new ideas for the prevention and treatment of viral encephalitis.

Exploring structural antigens of yellow fever virus to design multi-epitope subunit vaccine candidate by utilizing an immuno-informatics approach

BackgroundYellow fever is a mosquito-borne viral hemorrhagic disease transmitted by several species of virus-infected mosquitoes endemic to tropical regions of Central and South America and Africa. Earlier in the twentieth century, mass vaccination integrated with mosquito control was implemented to eradicate the yellow fever virus. However, regular outbreaks occur in these regions which pose a threat to travelers and residents of Africa and South America. There is no specific antiviral therapy, but there can be an effective peptide-based vaccine candidate to combat infection caused by the virus. Therefore, the study aims to design a multi-epitope-based subunit vaccine (MESV) construct against the yellow fever virus to reduce the time and cost using reverse vaccinology (RV) approach. MethodsYellow fever virus contains 10,233 nucleotides that encode for 10 proteins (C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) including 3 structural and 7 non-structural proteins. Structural proteins—precursor membrane protein (prM) and envelope protein (E)—were taken as a target for B cell and T cell epitope screening. Further, various immunoinformatics approaches were employed to FASTA sequences of structural proteins to retrieve B cell and T cell epitopes. MESV was constructed from these epitopes based on allergenicity, antigenicity and immunogenicity, toxicity, conservancy, and population coverage followed by structure prediction. The efficacy of the MESV construct to bind with human TLR-3, TLR-4, and TLR-8 were evaluated using molecular docking and simulation studies. Finally, in-silico cloning of vaccine construct was performed withpBR322 Escherichia coli expression system using codon optimization. ResultsPredicted epitopes evaluated and selected for MESV construction were found stable, non-allergenic, highly antigenic, and global population coverage of 68.03% according to in-silico analysis. However, this can be further tested in in-vitro and in-vivo investigations. Epitopes were sequentially merged to construct a MESV consisting of 393 amino acids using adjuvant and linkers. Molecular docking and simulation studies revealed stable and high-affinity interactions. Furthermore, in-silico immune response graphs showed effective immune response generation. Finally, higher CAI value ensured high gene expression of vaccine in the host cell. ConclusionThe designed MESV construct in the present in-silico study can be effective in generating an immune response against the yellow fever virus. Therefore, to prevent yellow fever, it can be an effective vaccine candidate. However, further downstream, in-vitro study is required.

Development of a tetravalent subunit vaccine against dengue virus through a vaccinomics approach.

Dengue virus infection (DVI) is a mosquito-borne disease that can lead to serious morbidity and mortality. Dengue fever (DF) is a major public health concern that affects approximately 3.9 billion people each year globally. However, there is no vaccine or drug available to deal with DVI. Dengue virus consists of four distinct serotypes (DENV1-4), each raising a different immunological response. In the present study, we designed a tetravalent subunit multi-epitope vaccine, targeting proteins including the structural protein envelope domain III (EDIII), precursor membrane proteins (prM), and a non-structural protein (NS1) from each serotype by employing an immunoinformatic approach. Only conserved sequences obtained through a multiple sequence alignment were used for epitope mapping to ensure efficacy against all serotypes. The epitopes were shortlisted based on an IC50 value <50, antigenicity, allergenicity, and a toxicity analysis. In the final vaccine construct, overall, 11 B-cell epitopes, 10 HTL epitopes, and 10 CTL epitopes from EDIII, prM, and NS1 proteins targeting all serotypes were selected and joined via KK, AAY, and GGGS linkers, respectively. We incorporated a 45-amino-acid-long B-defensins adjuvant in the final vaccine construct for a better immunogenic response. The vaccine construct has an antigenic score of 0.79 via VaxiJen and is non-toxic and non-allergenic. Our refined vaccine structure has a Ramachandran score of 96.4%. The vaccine has shown stable interaction with TLR3, which has been validated by 50 ns of molecular dynamics (MD) simulation. Our findings propose that a designed multi-epitope vaccine has substantial potential to elicit a strong immune response against all dengue serotypes without causing any adverse effects. Furthermore, the proposed vaccine can be experimentally validated as a probable vaccine, suggesting it may serve as an effective preventative measure against dengue virus infection.

Zika virus prM protein contains cholesterol binding motifs required for virus entry and assembly

For successful infection of host cells and virion production, enveloped viruses, including Zika virus (ZIKV), extensively rely on cellular lipids. However, how virus protein–lipid interactions contribute to the viral life cycle remains unclear. Here, we employ a chemo-proteomics approach with a bifunctional cholesterol probe and show that cholesterol is closely associated with the ZIKV structural protein prM. Bioinformatic analyses, reverse genetics alongside with photoaffinity labeling assays, and atomistic molecular dynamics simulations identified two functional cholesterol binding motifs within the prM transmembrane domain. Loss of prM–cholesterol association has a bipartite effect reducing ZIKV entry and leading to assembly defects. We propose a model in which membrane-resident M facilitates cholesterol-supported lipid exchange during endosomal entry and, together with cholesterol, creates a platform promoting virion assembly. In summary, we identify a bifunctional role of prM in the ZIKV life cycle by mediating viral entry and virus assembly in a cholesterol-dependent manner.

Zika virus M protein latches and locks the E protein from transitioning to an immature state after prM cleavage

During flavivirus maturation, the structural proteins prM (pre-membrane) and E (envelope) undergo extensive low pH-mediated conformational changes, transitioning from spiky trimeric to smooth dimeric prM/E heterodimers which allow for furin cleavage of prM into pr and M and forms the irreversible mature conformation of smooth M/E heterodimers. The mechanisms of irreversible conformational changes to E protein following the pr cleavage are not understood. Utilizing cryo-EM structures of immature virus and structure-based mutagenesis of Zika virus, we identified two critical “latching and locking” interactions mediated by M protein residues Arg38 and Trp19, respectively, that stabilize the E protein structure in the smooth mature stage. M protein thus latches and locks the E protein in an irreversible mature structure, preventing premature fusion in the secretory pathway. Our studies provide mechanistic insights into the reversible structural transition of immature trimeric spikes and the irreversible transition of smooth dimeric M/E heterodimers critical for virus infectivity.

Dengue virus 4/2 envelope domain chimeric virus panel maps type-specific responses against dengue serotype 2.

The four dengue virus (DENV) serotypes infect several hundred million people each year. Although primary infection is generally mild, subsequent infection by differing serotypes increases the risk for symptomatic disease ranging from fever to life-threatening shock. Despite the availability of licensed vaccines, a comprehensive understanding of antibodies that target the viral envelope protein and protect from infection remains incomplete. In this manuscript, we develop a panel of recombinant viruses that graft each envelope domain of DENV2 onto the DENV4 envelope glycoprotein, revealing protein interactions important for virus viability. Furthermore, we map neutralizing antibody responses after primary DENV2 natural infection and a human challenge model to distinct domains on the viral envelope protein. The panel of recombinant viruses provides a new tool for dissecting the E domain-specific targeting of protective antibody responses, informing future DENV vaccine design.

Development and biochemical characteristics of a monoclonal antibody against prM protein of Tembusu virus

Tembusu virus (TMUV), a pathogenic member of the Flavivirus family, is an infectious diseases that seriously jeopardize duck health in 2010 in China. TMUV disease causes significant economic losses to the duck industry. This study aimed to prepare monoclonal antibodies against TMUV prM protein and to identify their epitopes. The 501bp prM gene was amplified to the pET-32a prokaryotic expression vector and expressed as a recombinant protein of size 38 KD in Escherichia coli. The purified recombinant proteins were inoculated into BALB/c mice to generate splenic lymphocytes capable of secreting anti-prM antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named B27, which stably secreted IgG1-antibody against TMUV prM with high antibody titers up to 1:1:3,276,800 was screened. This monoclonal antibody (mAb) is well specific and can be used for ELISA/Western-blot(WB)/indirect fluorescence assay (IFA) etc. The mAb B27 has poor neutralization ability and concentration dependence, with a maximum neutralization degree of 23.87% at antibody dilution 10-6. Next, we truncated prM gene and expressed the truncated protein to screen antigen epitopes. The mAb's linear antigen epitope of the TMUV prM protein was first identified and was accurate to 6 consecutive amino acids 59GYEPED64, which located in the pr protein. Bioinformatic analysis showed that this antigenic epitope was located on the surface of the antigen, which was conducive to the direct contact of antigen antibody and conformed to the properties of antigenic epitopes. In addition, its 6 amino acids are highly homologous among 27 published TMUV strains, indicating that its epitope is stable. This study will help to further understand the protein structure and the function of prM, and lay the foundation for establishing specific prM detection methods and the mechanistic study of TMUV prM protein.

Immunoinformatics-Based Identification of the Conserved Immunogenic Peptides Targeting of Zika Virus Precursor Membrane Protein.

Zika virus infections lead to neurological complications such as congenital Zika syndrome and Guillain-Barré syndrome. Rising Zika infections in newborns and adults have triggered the need for vaccine development. In the current study, the precursor membrane (prM) protein of the Zika virus is explored for its functional importance and design of epitopes enriched conserved peptides with the usage of different immunoinformatics approach. Phylogenetic and mutational analyses inferred that the prM protein is highly conserved. Three conserved peptides containing multiple T and B cell epitopes were designed by employing different epitope prediction algorithms. IEDB population coverage analysis of selected peptides in six different continents has shown the population coverage of 60-99.8% (class I HLA) and 80-100% (class II HLA). Molecular docking of selected peptides/epitopes was carried out with each of class I and II HLA alleles using HADDOCK. A majority of peptide-HLA complex (pHLA) have HADDOCK scores found to be comparable and more than native-HLA complex representing the good binding interaction of peptides to HLA. Molecular dynamics simulation with best docked pHLA complexes revealed that pHLA complexes are stable with RMSD <5.5Å. Current work highlights the importance of prM as a strong antigenic protein and selected peptides have the potential to elicit humoral and cell-mediated immune responses.

Dengue virus M and E proteins belonging to genotype II (Cosmopolitan) of serotype 2 are influenced by the nature of M residue 36.

Mosquito-borne dengue disease is caused by the dengue virus serotype-1 to serotype-4. The contemporary dengue outbreaks in the southwestern Indian ocean coincided with the widespread of dengue virus serotype 2 genotype II (Cosmopolitan), including epidemic viral strains DES-14 and RUN-18 isolated in Dar es Salaam (Tanzania) in 2014 and La Reunion Island (France) in 2018, respectively. Heterodimeric interaction between prM (intracellular precursor of surface structural M protein) and envelope E proteins is required during the initial stage of dengue virus assembly. Amino acid 127 of DES-14 prM protein (equivalent to M36) has been identified as an infrequent valine whereas RUN-18 has a common isoleucine. In the present study, we examined the effect of M-I36V mutation on the expression of a recombinant RUN-18 E protein co-expressed with prM in human epithelial A549 cells. The M ectodomain of dengue virus serotype 2 embeds a pro-apoptotic peptide referred as D2AMP. The impact of M-I36V mutation on the death-promoting capability of D2AMP was assessed in A549 cells. We showed that valine at position M36 affects expression of recombinant RUN-18 E protein and potentiates apoptosis-inducing activity of D2AMP. We propose that the nature of M residue 36 influences the virological characteristics of dengue 2 M and E proteins belonging to genotype II that contributes to global dengue burden.

Sterile 20-like kinase 3 promotes tick-borne encephalitis virus assembly by interacting with NS2A and prM and enhancing the NS2A-NS4A association.

Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection in humans. Investigating virus-host interaction is important for understanding the pathogenesis of TBEV and developing effective antiviral drugs against this virus. Here, we report that mammalian ste20-like kinase 3 (MST3) is involved in the regulation of TBEV infection. The knockdown or knockout of MST3, but not other mammalian ste20-like kinase family members, inhibited TBEV replication. The knockdown of MST3 also significantly reduced TBEV replication in mouse primary astrocytes. Life cycle analysis indicated that MST3 remarkably impaired virion assembly efficiency and specific infectivity by respectively 59% and 95% in MST3-knockout cells. We further found that MST3 interacts with the viral proteins NS2A and prM; and MST3 enhances the interaction of NS2A-NS4A. Thus, MST3-NS2A complex plays a major role in recruiting prM-E heterodimers and NS4A and mediates the virion assembly. Additionally, we found that MST3 was biotinylated and combined with other proteins (e.g., ATG5, Sec24A, and SNX4) that are associated with the cellular membrane required for TBEV infection. Overall, our study revealed a novel function for MST3 in TBEV infection and identified as a novel host factor supporting TBEV assembly.

Key Residue in the Precursor Region of M Protein Contributes to the Neurovirulence and Neuroinvasiveness of the African Lineage of Zika Virus.

The Zika virus (ZIKV) represents an important global health threat due to its unusual association with congenital Zika syndrome. ZIKV strains are phylogenetically grouped into the African and Asian lineages. However, the viral determinants underlying the phenotypic differences between the lineages remain unknown. Here, multiple sequence alignment revealed a highly conserved residue at position 21 of the premembrane (prM) protein, which is glutamic acid and lysine in the Asian and African lineages, respectively. Using reverse genetics, we generated a recombinant virus carrying an E21K mutation based on the genomic backbone of the Asian lineage strain FSS13025 (termed E21K). The E21K mutation significantly increased viral replication in multiple neural cell lines with a higher ratio of M to prM production. Animal studies showed E21K exhibited increased neurovirulence in suckling mice, leading to more severe defects in mouse brains by causing more neural cell death and destruction of hippocampus integrity. Moreover, the E21K substitution enhanced neuroinvasiveness in interferon alpha/beta (IFN-α/β) receptor knockout mice, as indicated by the increased mortality, and enhanced replication in mouse brains. The global transcriptional analysis showed E21K infection profoundly altered neuron development networks and induced stronger antiviral immune response than wild type (WT) in both neural cells and mouse brains. More importantly, the reverse K21E mutation based on the genomic backbone of the African strain MR766 caused less mouse neurovirulence. Overall, our findings support the 21st residue of prM functions as a determinant for neurovirulence and neuroinvasiveness of the African lineage of ZIKV. IMPORTANCE The suspected link of Zika virus (ZIKV) to birth defects led the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. ZIKV has been identified to have two dominant phylogenetic lineages, African and Asian. Significant differences exist between the two lineages in terms of neurovirulence and neuroinvasiveness in mice. However, the viral determinants underlying the phenotypic differences are still unknown. Here, combining reverse genetics, animal studies, and global transcriptional analysis, we provide evidence that a single E21K mutation of prM confers to the Asian lineage strain FSS130125 significantly enhanced replication in neural cell lines and more neurovirulent and neuroinvasiveness phenotypes in mice. Our findings support that the highly conserved residue at position 21 of prM functions as a determinant of neurovirulence and neuroinvasiveness of the African lineage of ZIKV in mice.

Effect of plasmid size on the immune response to Japanese Encephalitis virus DNA vaccine in mice

Background: The envelope (E) protein, along with the pre-membrane (prM) protein of the Japanese encephalitis virus (JEV), are importantmediators of humoral immunity against the virus. Objective: To establish whether the plasmid size could influence the immunogenicityof this DNA vaccine candidate in terms of antibody inductive capacity. Methods: The prM and E genes of JEV were cloned in pCEP4 andpCDNA to produce the constructs pMEa and pcMEa, respectively. The former construct was approximately double the size of the latter.The plasmid constructs were used to immunize mice, both in terms of equal weight, as well as equal number of molecules. Results:The antibody-inductive capacity (both anti-JEV and neutralizing antibodies) of pCDNA was superior to that of the pCEP4. Importantly,the mice challenge studies indicated that the higher titer of neutralizing antibodies of the pCDNA group lead to higher survival in thisgroup. Conclusion: A smaller plasmid would be better for preparing a DNA vaccine construct.

Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling

BackgroundVector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive.ResultsWe showed that TBEV structural protein, pre-membrane (prM) protein, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeding the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKV and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKV prM significantly promoted the replication of TBEV and Sendai virus.ConclusionOur findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development.

Influence of Dosing Regimen and Adjuvant Type on the Immunogenicity of Novel Recombinant Zika Virus-Like Particles.

Zika virus (ZIKV) is a reemerging mosquito-borne flavivirus that causes febrile illness and is also linked to Guillain-Barré syndrome as well as to microcephaly in newborns. Due to the risk of fetuses developing microcephaly, ZIKV is a serious problem for pregnant women. Although different types of vaccine antigens have been investigated, there is still no approved vaccine that prevents ZIKV. The aim of this study was to produce a potential anti-Zika virus vaccine candidate based on virus-like particles (VLPs) in mammalian cells and to analyze the role of dosing regimen and adjuvant type on the immunogenicity of the obtained antigen. Novel recombinant VLPs (F2A) were designed by introducing the optimized signal sequence of prM protein and by adding a self-cleavage peptide 2A between proteins prM and E. These modifications improved the formation of the glycoprotein E dimer. It has been shown that the increasing dosing regimen generates a significantly higher titer of antibodies; however, the adjuvant type does not affect this process. Sera from mice immunized using an increasing dosing schedule also showed higher neutralization activity against both Zika strains (H/PAN/2016/BEI-259634, a pandemic strain belonging to Asian lineage, and MR766, a reference strain from African lineage). In summary, this is the first report showing the influence of vaccination schedules and adjuvants on the immunogenicity of ZIKV virus-like particles. IMPORTANCE Considering the transmission of ZIKV and the risk of another epidemic as well as the neurological complications that follow ZIKV infection, the virus remains a serious problem for the human population, especially pregnant women. Therefore, there is a great need to develop new effective vaccine candidates. Although different types of vaccine antigens have been used in preclinical studies worldwide, there is still no approved vaccine to prevent ZIKV. VLPs are among the most potent antigens, but to use VLPs, adjuvants must be added to the formulation and appropriate administration must be performed. In this study, we show for the first time the influence of vaccination schedules and adjuvants on the immunogenicity of recombinant ZIKV VLPs. The obtained results can be used in new vaccine designs not only against ZIKV but also against other important viral pathogens.