A gorilla adenovirus-based vaccine against Zika virus induces durable immunity and confers protection in pregnancy

The teratogenic potential of Zika virus (ZIKV) has made the development of an effective vaccine a global health priority. Here, we generate two gorilla adenovirus-based ZIKV vaccines that encode for pre-membrane (prM) and envelope (E) proteins (GAd-Zvp) or prM and the ectodomain of E protein (GAd-Eecto). Both vaccines induce humoral and cell-mediated immune responses and prevent lethality after ZIKV challenge in mice. Protection is antibody dependent, CD

Lipid Metabolism and HCV Infection

Chronic infection by hepatitis C virus (HCV) can lead to severe liver disease and is a global healthcare problem. The liver is highly metabolically active and one of its key functions is to control the balance of lipid throughout the body. A number of pathologies have been linked to the impact of HCV infection on liver metabolism. However, there is also growing evidence that hepatic metabolic processes contribute to the HCV life cycle. This review summarizes the relationship between lipid metabolism and key stages in the production of infectious HCV

The Fc region of an antibody impacts the neutralization of West Nile viruses in different maturation states

Flavivirus-infected cells secrete a structurally heterogeneous population of viruses because of an inefficient virion maturation process. Flaviviruses assemble as noninfectious, immature virions composed of trimers of envelope (E) and precursor membrane (prM) protein heterodimers. Cleavage of prM is a required process during virion maturation, although this often remains incomplete for infectious virus particles. Previous work demonstrated that the efficiency of virion maturation could impact antibody neutralization through changes in the accessibility of otherwise cryptic epitopes on the virion. In this study, we show that the neutralization potency of monoclonal antibody (MAb) E33 is sensitive to the maturation state of West Nile virus (WNV), despite its recognition of an accessible epitope, the domain III lateral ridge (DIII-LR). Comprehensive epitope mapping studies with 166 E protein DIII-LR variants revealed that the functional footprint of MAb E33 on the E protein differs subtly from that of the well-characterized DIII-LR MAb E16. Remarkably, aromatic substitutions at E protein residue 306 ablated the maturation state sensitivity of E33 IgG, and the neutralization efficacy of E33 Fab fragments was not affected by changes in the virion maturation state. We propose that E33 IgG binding on mature virions orients the Fc region in a manner that impacts subsequent antibody binding to nearby sites. This Fc-mediated steric constraint is a novel mechanism by which the maturation state of a virion modulates the efficacy of the humoral immune response to flavivirus infection

A Structural Perspective of Antibody Neutralization of Dengue Virus

The four dengue viruses: DENV) are mosquito-borne flaviviruses and are considered the world\u27s most significant arboviruses in terms of worldwide disease burden. Symptoms of dengue disease are classified into dengue fever, a mild, febrile illness, and the potentially fatal severe dengue, which can include hemorrhaging and shock. Antibody protection against DENV correlates with the production of neutralizing antibodies against the envelope: E) glycoprotein. To understand the role of antibodies in DENV infection, we sought to dissect the relationship between epitope and function. Virologic studies had identified that the most potently neutralizing antibodies are against domain III: DIII) of the E protein. We have identified five epitopes within DENV DIII. Our data suggests that the most potently neutralizing antibodies are specific for a single serotype, while cross-reactive antibodies are relatively poorly neutralizing. Additionally, we were surprised to define neutralizing epitopes that were shown to be inaccessible on the surface of the virion in cryo-electron microscopy studies. Fine epitope mapping was used to define the epitopes of a panel of existing DENV-2 antibodies. Antibodies against the lateral ridge were the most potently neutralizing antibodies and reacted only with the DENV-2 serotype. The second epitope was centered on the DIII A-strand, and antibodies against this epitope reacted with several serotypes of DENV. Several poorly neutralizing antibodies reacted to all four DENV serotypes, as well as West Nile virus, a related flavivirus, mapped to the highly conserved AB loop of DIII. We expanded our studies of DIII-specific antibodies to the DENV-1 serotype. One antibody, E106, potently neutralized the five DENV-1 strains representing the five genotypes, and bound a composite epitope of the lateral ridge and A-strand epitopes. Despite the potency of E106-mediated neutralization, a combination of structural, biophysical, virologic data suggest that potent DENV-1 neutralization by E106 is coincident with bivalent engagement of the virus. Additionally, we determined the crystal structures of E111 bound to a novel fifth CC\u27 loop epitope on domain III: DIII) of the E protein from two different DENV-1 genotypes. The available atomic models of DENV virions revealed that the E111 epitope was inaccessible, suggesting that it recognizes an uncharacterized virus conformation. While the affinity of binding between E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC\u27 loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development

Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2

Dengue virus (DENV) infection remains a serious health threat despite the availability of supportive care in modern medicine. Monoclonal antibodies (mAbs) of DENV would be powerful research tools for antiviral development, diagnosis and pathological investigations. Here we described generation and characterization of seventeen mAbs with high reactivity for E protein of DENV. Four of these mAbs showed high neutralizing activity against DENV-2 infection in mice. The monoclonal antibody mAb DB32-6 showed the strongest neutralizing activity against diverse DENV-2 and protected DENV-2-infected mice against mortality in therapeutic models. We identified neutralizing epitopes of DENV located at residues K310 and E311 of viral envelope protein domain III (E-DIII) through the combination of biological and molecular strategies. Comparing the strong neutralizing activity of mAbs targeting A-strand with mAbs targeting lateral ridge, we found that epitopes located in A-strand induced stronger neutralizing activity than those located on the lateral ridge. DB32-6 humanized version was successfully developed. Humanized DB32-6 variant retained neutralizing activity and prevented DENV infection. Understanding the epitope-based antibody-mediated neutralization is crucial to controlling dengue infection. Additionally, this study also introduces a novel humanized mAb as a candidate for therapy of dengue patients

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential

Defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection

With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents

The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change

The Structural Basis of Flaviviridae Interaction with Antibodies and Receptors

Flaviviridae are a family of enveloped, positive-stranded RNA viruses responsible for a variety of diseases including encephalitis, hemorrhagic fever and hepatocellular carcinoma. The envelope: E) proteins that coat the outer surface of these viruses provide the molecular machinery that drives receptor interaction and membrane fusion. The assignment of biological functions to specific structural elements of these E proteins has proven crucial to the understanding of viral entry into host cells. Clearance is dependent upon the presence of neutralizing antibodies that are able to disrupt several stages of this process. Given their fundamental role in the viral life cycle, we sought to determine the structural basis for envelope protein interaction with antibodies and receptors for human pathogens of the Flaviviridae family Japanese Encephalitis Virus, Hepatitis C Virus and St. Louis Encephalitis Virus. Viruses of the Flavivirus genus within Flaviviridae are grouped into serocomplexes with similar clinical manifestations that are defined by cross-neutralization tests with polysera from heterologous infections. Japanese Encephalitis Virus: JEV) is the leading cause of viral encephalitis and prototypical member of the JEV serocomplex. We determined the 2.1Ã… resolution crystal structure of the JEV E protein ectodomain to investigate whether structural features could contribute to our understanding of serocomplex-specific pathogenesis. JEV E possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies revealed residues localized to the domain I lateral ridge, fusion loop, domain III lateral ridge and domain I-II hinge. The dimer interface, however, is remarkably small and lacks several contacts present in other flavivirus E homodimers. Uniquely conserved histidines of the JEV serocomplex suggest that pH-mediated structural transitions may be assisted by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation of dimer structure and stability may influence the assembly, receptor interaction and uncoating of virions. St. Louis Encephalitis Virus: SLEV) is another member of the JEV serocomplex with similar pathogenesis to JEV. We determined the 4.0 Ã… structure of the SLEV E protein in the post-fusion trimer conformation to compare it with E trimer structures from other flavivirus serocomplexes. SLEV E crystallized as a trimer in the absence of lipids or detergents, requiring only low pH. However, its domain arrangement was nearly identical to other post-fusion structures. This suggests that viruses can alter dimer assembly but the structure of the activated, fusogenic conformation may be more strictly conserved. The only member of Flaviviridae known to chronically infect humans is Hepatitits C Virus: HCV). HCV is blood borne and carried by roughly 3 percent of the world\u27s population. Clinical manifestations include hepatitis, cirrhosis and hepatocellular carcinoma. HCV envelope protein E2 mediates interaction with host receptors CD81 and scavenger receptor BI: SR-BI) and is the primary target of neutralizing antibodies. To elucidate detailed biochemical roles for these receptors\u27 interactions with E2, we determined that the E2 ectodomain: sE2) interacts with soluble CD81 large extracellular loop: CD81-LEL) with 2:2 stoichiometry, and that this interaction inhibits subsequent engagement of SR-BI. We then evaluated the affinity and kinetics of sE2:CD81-LEL binding. Interaction between these proteins was enhanced by deletion of hypervariable region 1: HVR1) of E2 and modulated by the genotype from which sE2 was generated. Furthermore, neutralization of HVR1-deleted HCV by a cross-reactive antibody was enhanced in a genotype-specific manner that correlated with sE2:CD81-LEL affinity measurements. Our results suggest that E2 cannot engage CD81 and SR-BI simultaneously, that HVR1 obscures conserved CD81 and antibody binding sites, and that genotypic variation influences HCV host receptor preference

Investigating the Hepatitis C Virus (HCV) RNA Translation Modulation by Non-Structural Protein 5A (NS5A)

Hepatitis C virus (HCV) non-structural protein NS5A is a multifunctional protein and despite lacking enzymatic activity has critical roles in viral replication and assembly. The role of NS5A in HCV RNA translation has not been well studied. In an attempt to better understand the role of HCV NS5A in RNA translation, our previous work showed that HCV-1b NS5A downregulates viral RNA translation by binding to the poly(U/UC) region in the 3’UTR. All three domains are capable of individually downregulating translation, albeit with a lesser effect than the full-length wild-type NS5A. There are multiple HCV genotypes and NS5A from different genotypes may or may not carry out the same function. Therefore, to determine whether the role of NS5A is conserved in other genotypes, we studied the effect of HCV-2a NS5A on monocistronic HCV-2a RNA reporters and replication defective genomic RNA with or without poly(U/UC) region at the 3’UTR. We found that although HCV-2a NS5A also downregulates viral translation, it does not require the poly(U/UC) region in 3’UTR. The translation downregulation by HCV-2a NS5A was predominantly mediated by domain I. Our results elucidated that HCV-2a NS5A modulates viral translation through a mechanism different from HCV-1b NS5A. NS5A is a phospho-protein and exists as hypo- and hyper-phosphorylated NS5A. The hyperphosphorylation of NS5A is mediated through the phosphorylation of the conserved serine residues cluster in the low complexity sequence LCS I. The serine residues are S222, S225, S229, S232, S235 and S238. Phosphorylation on these serine residues has been found to be important for HCV replication and viral assembly. To further understand the significance of NS5A hyperphosphorylation on HCV life cycle, we investigated the role of HCV-1b NS5A hyperphosphorylation on translation by analyzing the effects of phospho-ablative and phospho-mimetic mutants of the six serine residues on HCV-1b genomic RNA translation. We showed that phosphorylation of S222, S225, S235 is not involved in translation downregulation by NS5A. In contrast, alanine mutations at S229 or S238 can no longer downregulate translation, whereas S229D or S238D mutations have no effect. Interestingly, S232D, but not S232A, abrogates translation downregulation by NS5A. NS5A exists as a dimer and its dimerization is important for regulating its function. Therefore, we studied the effect of phospho-mutants of S229, S232, and S238 on dimerization in a protein-protein interaction assay and showed that phospho-mimetic S229D or S238D mutations enhance NS5A dimerization, whereas the phospho-ablative mutations of them have no effect. In contrast, neither phospho-ablative nor phospho-mimetic mutations of S232 affect dimerization. In conclusion, these results indicated that phosphorylation of NS5A at S229, S232, and S238 is involved in viral translation regulation and NS5A dimerization. In summary, these findings suggest that NS5A downregulates the translation of HCV RNA however, the mechanism may differ within the genotypes. In addition, hyperphosphorylation of NS5A is involved in regulation of HCV translation and NS5A dimerization. These results aid in the understanding the mechanism involved in regulation of viral translation by NS5A and may help in the development of pan-genotypic novel antiviral targets