Supramolecular peptide hydrogel adjuvanted subunit vaccine elicits protective antibody responses against West Nile virus.

A crucial issue in vaccine development is to balance safety with immunogenicity. The low immunogenicity of most subunit antigens warrants a search for adjuvants able to stimulate both cell-mediated and humoral immunity. In recent years, successful applications of nanotechnology and bioengineering in the field of vaccine development have enabled the production of novel adjuvant technologies. In this work, we investigated totally synthetic and supramolecular peptide hydrogels as novel vaccine adjuvants in conjunction with the immunoprotective envelope protein domain III (EIII) of West Nile virus as an immunogen in a mouse model. Our results indicate that, compared to the clinically approved adjuvant alum, peptide hydrogel adjuvanted antigen elicited stronger antibody responses and conferred significant protection against mortality after virus challenge. The high chemical definition and biocompatibility of self-assembling peptide hydrogels makes them attractive as immune adjuvants for the production of subunit vaccines against viral and bacterial infections where antibody-mediated protection is desirable.

Plasticity of a critical antigenic determinant in the West Nile virus NY99 envelope protein domain III.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes febrile illness, encephalitis, and occasionally death in humans. The envelope protein is the main component of the WNV virion surface, and domain III of the envelope protein (EIII) is both a putative receptor binding domain and a target of highly specific, potently neutralizing antibodies. Envelope E-332 (E-332) is known to have naturally occurring variation and to be a key determinant of neutralization for anti-EIII antibodies. A panel of viruses containing all possible amino acid substitutions at E-332 was constructed. E-332 was found to be highly tolerant of mutation, and almost all of these changes had large impacts on antigenicity of EIII but only limited effects on growth or virulence phenotypes.

ELISA and Neutralization Methods to Measure Anti-West Nile Virus Antibody Responses.

Measurements of humoral immune responses to West Nile virus (WNV) infection in mouse or other animal models are valuable components of basic laboratory investigations to assess immunogenicity of candidate vaccines or to evaluate seroconversion following challenge with WNV. Here, we outline the steps for screening or titrating of total antibodies by indirect enzyme linked immunosorbent assay (ELISA) as well as assessment of neutralizing antibody titers by immunofocus detection.

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence.

UNLABELLED: Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, within vivo pathogenesis often not being correlated within vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. IMPORTANCE: The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a significant role for this sequence in determining the pathogenesis of the virus within the mammalian host. Our studies demonstrate that these chimeras are viable and suggest that such recombinant viruses may be useful for investigation of domain-specific antibody responses and the more extensive definition of the contributions of EIII to the tropism and pathogenesis of WNV or other flaviviruses.

Thermodynamic mechanism for the evasion of antibody neutralization in flaviviruses.

Mutations in the epitopes of antigenic proteins can confer viral resistance to antibody-mediated neutralization. However, the fundamental properties that characterize epitope residues and how mutations affect antibody binding to alter virus susceptibility to neutralization remain largely unknown. To address these questions, we used an ensemble-based algorithm to characterize the effects of mutations on the thermodynamics of protein conformational fluctuations. We applied this method to the envelope protein domain III (ED3) of two medically important flaviviruses: West Nile and dengue 2. We determined an intimate relationship between the susceptibility of a residue to thermodynamic perturbations and epitope location. This relationship allows the successful identification of the primary epitopes in each ED3, despite their high sequence and structural similarity. Mutations that allow the ED3 to evade detection by the antibody either increase or decrease conformational fluctuations of the epitopes through local effects or long-range interactions. Spatially distant interactions originate in the redistribution of conformations of the ED3 ensembles, not through a mechanically connected array of contiguous amino acids. These results reconcile previous observations of evasion of neutralization by mutations at a distance from the epitopes. Finally, we established a quantitative correlation between subtle changes in the conformational fluctuations of the epitope and large defects in antibody binding affinity. This correlation suggests that mutations that allow viral growth, while reducing neutralization, do not generate significant structural changes and underscores the importance of protein fluctuations and long-range interactions in the mechanism of antibody-mediated neutralization resistance.

Co-circulation of West Nile virus variants, Arizona, USA, 2010.

Molecular analysis of West Nile virus (WNV) isolates obtained during a 2010 outbreak in Maricopa County, Arizona, USA, demonstrated co-circulation of 3 distinct genetic variants, including strains with novel envelope protein mutations. These results highlight the continuing evolution of WNV in North America and the current complexity of WNV dispersal and transmission.

Adaptation of yellow fever virus 17D to Vero cells is associated with mutations in structural and non-structural protein genes.

Serial passaging of yellow fever virus 17D in Vero cells was employed to derive seed material for a novel inactivated vaccine, XRX-001. Two independent passaging series identified a novel lysine to arginine mutation at amino acid 160 of the envelope protein, a surface-exposed residue in structural domain I. A third passage series resulted in an isoleucine to methionine mutation at residue 113 of the NS4B protein, a central membrane spanning region of the protein which has previously been associated with Vero cell adaptation of other mosquito-borne flaviviruses. These studies confirm that flavivirus adaptation to growth in Vero cells can be mediated by structural or non-structural protein mutations.

Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate.

The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.

Role of BC loop residues in structure, function and antigenicity of the West Nile virus envelope protein receptor-binding domain III.

Site-directed mutagenesis of residues in the BC loop (residues 329-333) of the envelope (E) protein domain III in a West Nile virus (WNV) infectious clone and in plasmids encoding recombinant WNV and dengue type 2 virus domain III proteins demonstrated a critical role for residues in this loop in the function and antigenicity of the E protein. This included a strict requirement for the tyrosine at residue 329 of WNV for virus viability and E domain III folding. The absence of an equivalent residue in this region of yellow fever group viruses and most tick-borne flavivirus suggests there is an evolutionary divergence in the molecular mechanisms of domain III folding employed by different flaviviruses.

Development and characterization of non-glycosylated E and NS1 mutant viruses as a potential candidate vaccine for West Nile virus.

West Nile virus is an arthropod-borne flavivirus that has caused substantial morbidity and mortality to animals as well as humans since its introduction in to the New York area in 1999. Given that there are no antiviral drugs available for treatment of the disease, vaccines provide an efficacious alternative to control this disease. Herein we describe an attenuated WNV strain developed by the ablation of the glycosylation sites in the envelope (E) and non-structural 1 (NS1) proteins. This E(154S)/NS1(130A/175A/207A) strain showed modest reduction in multiplication kinetics in cell culture and small plaque phenotype compared to the parental NY99 strain yet displayed greater than a 200,000-fold attenuation for mouse neuroinvasiveness compared to the parental strain. Mice infected with 1000PFU of E(154S)/NS1(130A/175A/207A) showed undectable viremia at either two or three days post infection; nonetheless, high titer neutralizing antibodies were detected in mice inoculated with low doses of this virus and protected against lethal challenge with a 50% protective dose of 50PFU.

Structure of yellow fever virus envelope protein domain III.

The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism.

Development of resistance to passive therapy with a potently neutralizing humanized monoclonal antibody against West Nile virus.

Previous studies have established the therapeutic efficacy of humanized E16 (hE16) monoclonal antibody against West Nile virus in animals. Here, we assess the potential for West Nile virus strains encoding mutations in the hE16 epitope to resist passive immunotherapy and for the selection of neutralization escape variants during hE16 treatment. Resistance to hE16 in vivo was less common than expected, because several mutations that affected neutralization in vitro did not significantly affect protection in mice. Moreover, the emergence of resistant variants after infection with fully sensitive virus occurred but was relatively rare, even in highly immunocompromised B and T cell-deficient RAG mice.

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad.

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.

Genetic variation of St. Louis encephalitis virus.

St. Louis encephalitis virus (SLEV) has been regularly isolated throughout the Americas since 1933. Previous phylogenetic studies involving 62 isolates have defined seven major lineages (I-VII), further divided into 14 clades. In this study, 28 strains isolated in Texas in 1991 and 2001-2003, and three older, previously unsequenced strains from Jamaica and California were sequenced over the envelope protein gene. The inclusion of these new sequences, and others published since 2001, has allowed better delineation of the previously published SLEV lineages, in particular the clades of lineage II. Phylogenetic analysis of 106 isolates identified 13 clades. All 1991 and 2001-2003 isolates from Nueces, Jefferson and Harris Counties (Texas Gulf Coast) group in clade IIB with other isolates from these counties isolated during the 1980s and 1990s. This lack of evidence for introduction of novel strains into the Texas Gulf Coast over a long period of time is consistent with overwintering of SLEV in this region. Two El Paso isolates, both from 2002, group in clade VA with recent Californian isolates from 1998-2001 and some South American strains with a broad temporal range. Overall, these data are consistent with multiple introductions of SLEV from South America into North America, and provide support for the hypothesis that in most situations, SLEV circulates within a locality, with occasional incursions from other areas. Finally, SLEV has much lower nucleotide (10.1 %) and amino acid variation (2.8 %) than other members of the Japanese encephalitis virus complex (maximum variation 24.6 % nucleotide and 11.8 % amino acid).

NMR assignments of the sylvatic dengue 1 virus envelope protein domain III.

Nearly complete backbone and side chain resonance assignments have been obtained for the third domain, residues M289-K400, of the envelope protein from the sylvatic strain (P72-1244) of the dengue 1 virus, containing mutations N336S and E370K, using double- and triple-resonance spectroscopy.

Long range communication in the envelope protein domain III and its effect on the resistance of West Nile virus to antibody-mediated neutralization.

The envelope protein domain III (ED3) of West Nile virus is the major virus-specific neutralization domain and harbors most of the critical mutations that induce resistance against antibody-mediated neutralization. We investigated the molecular mechanisms of neutralization resistance by studying the biophysical perturbations of monoclonal antibody (mAb)-resistant mutations on ED3 wild type. Our results showed that although the solution structure between ED3 wild type and mutants was preserved, the mutations that confer the highest degree of resistance to mAbs showed low protein stability and high local dynamic motions. Interestingly, the latter was observed in regions outside the mutation sites, indicating long range communications within ED3. Thus, we hypothesized that the mechanisms involved in resistance to mAb neutralization may include, in addition to mutations in the epitope, long range effects among distant structural elements. This hypothesis is consistent with reported mutations in other flaviviruses whose surfaces are not exposed for the interaction with other macromolecules, yet they confer mAb neutralization resistance.

Solution structure of the envelope protein domain III of dengue-4 virus.

The disease dengue (DEN) is caused by four serologically related viruses termed DEN1, DEN2, DEN3 and DEN4. The structure of the ectodomain of the envelope protein has been determined previously for DEN2 and DEN3 viruses. Using NMR spectroscopic methods, we solved the solution structure of domain III (ED3), the receptor-binding domain, of the envelope protein of DEN4 virus, human strain 703-4. The structure shows that the nine amino acid changes in ED3 that separate the sylvatic and human DEN4 strains are surface exposed. Important structural differences between DEN4-rED3 and ED3 domains of DEN2, DEN3 and other flaviviruses are discussed.

Thioaptamer decoy targeting of AP-1 proteins influences cytokine expression and the outcome of arenavirus infections.

Viral haemorrhagic fever (VHF) is caused by a number of viruses, including arenaviruses. The pathogenesis is believed to involve dysregulation of cytokine production. The arenaviruses Lassa virus and Pichinde virus have a tropism for macrophages and other reticuloendothelial cells and both appear to suppress the normal macrophage response to virus infection. A decoy thioaptamer, XBY-S2, was developed and was found to bind to AP-1 transcription factor proteins. The P388D1 macrophage-like cell line contains members of the AP-1 family which may act as negative regulators of AP-1-controlled transcription. XBY-S2 was found to bind to Fra-2 and JunB, and enhance the induction of cytokines IL-6, IL-8 and TNF-alpha, while reducing the binding to AP-1 promoter elements. Administration of XBY-S2 to Pichinde virus-infected guinea pigs resulted in a significant reduction in Pichinde virus-induced mortality and enhanced the expression of cytokines from primary guinea pig macrophages, which may contribute to its ability to increase survival of Pichinde virus-infected guinea pigs. These data demonstrate a proof of concept that thioaptamers can be used to modulate the outcome of in vivo viral infections by arenaviruses by the manipulation of transcription factors involved in the regulation of the immune response.

A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus.

Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.

Structure of the envelope protein domain III of Omsk hemorrhagic fever virus.

We have solved the NMR solution structure of domain III from the Omsk hemorrhagic fever virus envelope protein and report the first sequencing of the Guriev strain of this virus. Important structural differences between tick-borne flaviviruses, such as OHFV and TBE, and mosquito-borne flaviviruses, such as West Nile virus, are discussed.