A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis

The structural flexibility or 'breathing' of the envelope (E) protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F) of the West Nile virus (WNV) E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV), but not Zika virus (ZIKV), E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing

Structural Insights into the Mechanisms of Antibody-Mediated Neutralization of Flavivirus Infection: Implications for Vaccine Development

Flaviviruses are a group of small RNA viruses that cause severe disease in humans worldwide and are the target of several vaccine development programs. A primary goal of these efforts is to elicit a protective humoral response directed against the envelope proteins arrayed on the surface of the flavivirus virion. Advances in the structural biology of these viruses has catalyzed rapid progress toward understanding the complexity of the flavivirus immunogen and the molecular basis of antibody-mediated neutralization. These insights have identified factors that govern the potency of neutralizing antibodies and will inform the design and evaluation of novel vaccines

Characterization of a Speciese Adenovirus Vector as a Zika virus vaccine

The development of a safe and efficacious Zika virus (ZIKV) vaccine remains a global health priority. In our previous work, we developed an Adenovirus vectored ZIKV vaccine using a low-seroprevalent human Adenovirus type 4 (Ad4-prM-E) and compared it to an Ad5 vector (Ad5-prM-E). We found that vaccination with Ad4-prM-E leads to the development of a strong anti-ZIKV T-cell response without eliciting significant anti-ZIKV antibodies, while vaccination with Ad5-prM-E leads to the development of both anti-ZIKV antibody and T-cell responses in C57BL/6 mice. However, both vectors conferred protection against ZIKV infection in a lethal challenge model. Here we continued to characterize the T-cell biased immune response observed in Ad4 immunized mice. Vaccination of BALB/c mice resulted in immune correlates similar to C57BL/6 mice, confirming that this response is not mouse strain-specific. Vaccination with an Ad4 expressing an influenza hemagglutinin (HA) protein resulted in anti-HA T-cell responses without the development of significant anti-HA antibodies, indicating this unique response is specific to the Ad4 serotype rather than the transgene expressed. Co-administration of a UV inactivated Ad4 vector with the Ad5-prM-E vaccine led to a significant reduction in anti-ZIKV antibody development suggesting that this serotype-specific immune profile is capsid-dependent. These results highlight the serotype-specific immune profiles elicited by different Adenovirus vector types and emphasize the importance of continued characterization of these alternative Ad serotype

Characterization of a Species E Adenovirus Vector as a Zika virus vaccine

The development of a safe and efficacious Zika virus (ZIKV) vaccine remains a global health priority. In our previous work, we developed an Adenovirus vectored ZIKV vaccine using a low-seroprevalent human Adenovirus type 4 (Ad4-prM-E) and compared it to an Ad5 vector (Ad5-prM-E). We found that vaccination with Ad4-prM-E leads to the development of a strong anti-ZIKV T-cell response without eliciting significant anti-ZIKV antibodies, while vaccination with Ad5-prM-E leads to the development of both anti-ZIKV antibody and T-cell responses in C57BL/6 mice. However, both vectors conferred protection against ZIKV infection in a lethal challenge model. Here we continued to characterize the T-cell biased immune response observed in Ad4 immunized mice. Vaccination of BALB/c mice resulted in immune correlates similar to C57BL/6 mice, confirming that this response is not mouse strain-specific. Vaccination with an Ad4 expressing an influenza hemagglutinin (HA) protein resulted in anti-HA T-cell responses without the development of significant anti-HA antibodies, indicating this unique response is specific to the Ad4 serotype rather than the transgene expressed. Co-administration of a UV inactivated Ad4 vector with the Ad5-prM-E vaccine led to a significant reduction in anti-ZIKV antibody development suggesting that this serotype-specific immune profile is capsid-dependent. These results highlight the serotype-specific immune profiles elicited by different Adenovirus vector types and emphasize the importance of continued characterization of these alternative Ad serotypes

Ago-2-Mediated Slicer Activity Is Essential for Anti-Flaviviral Efficacy of RNAi

RNA interference can be mediated by fully complementary siRNA or partially complementary miRNA. siRNAs are widely used to suppress viral replication and the fully complementary siRNA bound Ago-2 in the RISC is known to degrade the target RNA. Although other argonaute proteins lacking slicer activity can also bind oligonucleotides with both si and miRNA structures, whether they can also contribute to antiviral effects is not entirely clear. We tested si and miRNA structured oligos for target repression in dual luciferase assays as well as for inhibition of Dengue and West Nile virus replication in ES cells expressing individual Ago proteins. In luciferase assays, both fully complementary and partially complementary oligos effectively repressed their targets in all individual Ago expressing cell lines, although the efficacy with fully complementary oligos was higher in Ago-2+ cells. However, partially complementary oligos had no effect on virus replication in any cell line, while fully complementary siRNAs were highly effective in Ago-2 expressing, but not in cells expressing other Ago proteins. This occurred irrespective of whether the target sequences were located in the coding region or 3′UTR of the virus. We conclude that Ago-2 slicer activity is essential for anti-viral efficacy of siRNAs and miRNA-mediated translational repression/transcript destabilization is too weak to suppress the abundantly expressed flaviviral proteins

Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization

West Nile virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. The E proteins of newly synthesized West Nile virus (WNV) are organized into trimeric spikes composed of pre-membrane (prM) and E protein heterodimers. During egress, immature virions undergo a protease-mediated cleavage of prM that results in a reorganization of E protein into the pseudo-icosahedral arrangement characteristic of mature virions. While cleavage of prM is a required step in the virus life cycle, complete maturation is not required for infectivity and infectious virions may be heterogeneous with respect to the extent of prM cleavage. In this study, we demonstrate that virion maturation impacts the sensitivity of WNV to antibody-mediated neutralization. Complete maturation results in a significant reduction in sensitivity to neutralization by antibodies specific for poorly accessible epitopes that comprise a major component of the human antibody response following WNV infection or vaccination. This reduction in neutralization sensitivity reflects a decrease in the accessibility of epitopes on virions to levels that fall below a threshold required for neutralization. Thus, in addition to a role in facilitating viral entry, changes in E protein arrangement associated with maturation modulate neutralization sensitivity and introduce an additional layer of complexity into humoral immunity against WNV

Structural basis of differential neutralization of DENV-1 genotypes by an antibody that recognizes a cryptic epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC' loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-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' 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

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

Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity