Lethal West Nile Virus Challenge Research Articles

A Chimeric Classical Insect-Specific Flavivirus Provides Complete Protection Against West Nile Virus Lethal Challenge in Mice.

West Nile virus (WNV), an arthropod-borne flavivirus, can cause severe symptoms, including encephalitis, and death, posing a threat to public health and the economy. However, there is still no approved treatment or vaccine available for humans. Here, we developed a novel vaccine platform based on a classical insect-specific flavivirus (cISF) YN15-283-02, which was derived from Culicoides. The cISF-WNV chimera was constructed by replacing prME structural genes of the infectious YN15-283-02 cDNA clone with those of WNV and successfully rescued in Aedes albopictus cells. cISF-WNV was nonreplicable in vertebrate cells and nonpathogenic in type I interferon receptor (IFNAR)-deficient mice. A single-dose immunization of cISF-WNV elicited considerable Th1-biased antibody responses in C57BL/6 mice, which was sufficient to offer complete protection against lethal WNV challenge with no symptoms. Our studies demonstrated the potential of the insect-specific cISF-WNV as a prophylactic vaccine candidate to prevent infection with WNV.

Antibody-Dependent Enhancement Activity of a Plant-Made Vaccine against West Nile Virus.

West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue (DENV) virus. Specifically, a plant-produced virus-like particle (VLP) that displays the WNV Envelope protein domain III (wDIII) elicited both high neutralizing antibody titers and antigen-specific cellular immune responses in mice. Passive transfer of serum from VLP-vaccinated mice protected recipient mice from a lethal challenge of WNV infection. Notably, VLP-induced antibodies did not enhance the infection of Fc gamma receptor-expressing K562 cells by ZIKV or DENV through ADE. Thus, a plant-made wDIII-displaying VLP presents a promising WNV vaccine candidate that induces protective immunity and minimizes the concern of inducing ADE-prone antibodies to predispose vaccinees to severe infection by DENV or ZIKV.

Identification of clinical candidates against West Nile virus by activity screening in vitro and effect evaluation in vivo.

The West Nile virus (WNV) is a member of the flavivirus and is known to causeencephalitis. There is currently no specific treatment for WNV infection. Repurposing of clinically approved drugs appeared promising for rapidly identifying effective, safe, and readily available candidates for antiviral drugs. Here, we screened the small-molecule compounds with anti-WNV activity from 978 Food Drug Administration-approved drugs. Four compounds, including cilnidipine, mycophenolate mofetil, nitazoxanide, and teriflunomide, were found to efficiently abrogate WNV infection in Vero cells and human neuroblastoma SH-SY5Y cells. The four compounds also exert broad-spectrum antiviral activity against the Zika virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and chikungunya virus. Furthermore, nitazoxanide (a synthetic benzamide) and teriflunomide (an inhibitor of dihydroorotate dehydrogenase, DHODH) protected 20% and 40% of mice from lethal WNV challenge, respectively. Both drugs, which are orally bioavailable and have been approved clinically for many years, may be promising therapeutics for WNV infection. Moreover, the other two DHODH inhibitors,ML390 and vidofludimus, also displayed potent activity against WNV infection in vitro and in vivo.

Lifelong cytomegalovirus and early-LIFE irradiation synergistically potentiate age-related defects in response to vaccination and infection.

While whole‐body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early‐life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non‐lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub‐lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV‐infected animals experiencing 4Gy, but not lower, dose of sub‐lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV‐infected, but not irradiated, mice were both protected to the same high level as the old non‐irradiated, uninfected controls. Analysis of the quality and quantity of anti‐WNV immunity showed that higher mortality in MCMV‐positive WBI mice correlated with increased levels of MCMV‐specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late‐life mortality following early‐life irradiation and late‐life acute infection.

A protective human monoclonal antibody targeting the West Nile virus E protein preferentially recognizes mature virions

West Nile virus (WNV), a member of the Flavivirus genus, is a leading cause of viral encephalitis in the United States1. The development of neutralizing antibodies against the flavivirus envelope (E) protein is critical for immunity and vaccine protection2. Previously identified candidate therapeutic mouse and human neutralizing monoclonal antibodies (mAbs) target epitopes within the E domain III lateral ridge and the domain I-II hinge region, respectively3. To explore the neutralizing antibody repertoire elicited by WNV infection for potential therapeutic application, we isolated 10 mAbs from WNV-infected individuals. MAb WNV-86 neutralized WNV with a 50% inhibitory concentration (IC50) of 2 ng/mL, one of the most potently neutralizing flavivirus-specific antibodies ever isolated. WNV-86 targets an epitope in E domain II, and preferentially recognizes mature virions lacking an uncleaved form of the chaperone protein prM, unlike most flavivirus-specific antibodies4. In vitro selection experiments revealed a neutralization escape mechanism involving a glycan addition to E domain II. Finally, a single dose of WNV-86 administered two days post-infection protected mice from lethal WNV challenge. This study identifies a highly potent human neutralizing mAb with therapeutic potential that targets an epitope preferentially displayed on mature virions.

Acute systemic DNA damage in youth does not impair immune defense with aging.

SummaryAging‐related decline in immunity is believed to be the main driver behind decreased vaccine efficacy and reduced resistance to infections in older adults. Unrepaired DNA damage is known to precipitate cellular senescence, which was hypothesized to be the underlying cause of certain age‐related phenotypes. Consistent with this, some hallmarks of immune aging were more prevalent in individuals exposed to whole‐body irradiation (WBI), which leaves no anatomical repository of undamaged hematopoietic cells. To decisively test whether and to what extent WBI in youth will leave a mark on the immune system as it ages, we exposed young male C57BL/6 mice to sublethal WBI (0.5–4 Gy), mimicking human survivor exposure during nuclear catastrophe. We followed lymphocyte homeostasis thorough the lifespan, response to vaccination, and ability to resist lethal viral challenge in the old age. None of the irradiated groups showed significant differences compared with mock‐irradiated (0 Gy) animals for the parameters measured. Even the mice that received the highest dose of sublethal WBI in youth (4 Gy) exhibited equilibrated lymphocyte homeostasis, robust T‐ and B‐cell responses to live attenuated West Nile virus (WNV) vaccine and full survival following vaccination upon lethal WNV challenge. Therefore, a single dose of nonlethal WBI in youth, resulting in widespread DNA damage and repopulation stress in hematopoietic cells, leaves no significant trace of increased immune aging in a lethal vaccine challenge model.

Targeting DNA vaccines to dendritic cells using a small peptide to enhance immune response (VAC4P.1110)

Abstract Dendritic cell targeting of antigens greatly enhances immunogenicity. Although there are several reagents (DEC205, Dectin-1, Langerin, Clec9, nanoparticle, etc) to target protein antigens to DCs, there is still no method to target DNA vaccines directly to DCs. Here we show that a small peptide derived from the rabies virus glycoprotein, fused to protamine residues (RVG-P) can target DNA to DCs resulting in enhanced T-cell and humoral responses. Targeted delivery of DNA vaccine encoding the immunodominant Vaccinia virus B8R gene to DCs with RVG-P was able to re-stimulate Vaccinia-specific memory T cells in vitro. Moreover, a single iv injection of B8R gene bound to RVG-P without any adjuvants was able prime a Vaccinia-specific T-cell response that was able to rapidly clear a subsequent Vaccinia virus challenge in mice. Finally, immunization of mice with a DNA vaccine encoding with West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WN neutralizing antibodies that protected mice from lethal WNV challenge. Thus, RVG-P provides a tool to target DNA vaccines to DCs to elicit robust T-cell and humoral immune responses.

Targeting DNA vaccines to myeloid cells using a small peptide.

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

A Recombinant Novirhabdovirus Presenting at the Surface the E Glycoprotein from West Nile Virus (WNV) Is Immunogenic and Provides Partial Protection against Lethal WNV Challenge in BALB/c Mice

West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.

A West Nile virus CD4 T cell epitope improves the immunogenicity of dengue virus serotype 2 vaccines

Flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are among the most prevalent human disease-causing arboviruses world-wide. As they continue to expand their geographic range, multivalent flavivirus vaccines may become an important public health tool. Here we describe the immune kinetics of WNV DNA vaccination and the identification of a CD4 epitope that increases heterologous flavivirus vaccine immunogenicity. Lethal WNV challenge two days post-vaccination resulted in 90% protection with complete protection by four days, and was temporally associated with a rapid influx of activated CD4 T cells. CD4 T cells from WNV vaccinated mice could be stimulated from epitopic regions in the envelope protein transmembrane domain. Incorporation of this WNV epitope into DENV-2 DNA and virus-like particle vaccines significantly increased neutralizing antibody titers. Incorporating such potent epitopes into multivalent flavivirus vaccines could improve their immunogenicity and may help alleviate concerns of imbalanced immunity in multivalent vaccine approaches.

Cross protection against lethal West Nile virus challenge in mice immunized with recombinant E protein domain III of Japanese encephalitis virus

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that cause severe encephalitic diseases with global impact. Cross protection among JEV and WNV has been previously described, and most cross reactive epitopes were identified within the domain II of E protein (EDII). In this study, the E protein domain III (EDIII) of JEV was successfully expressed in Escherichia coli, purified by a Ni-NTA column and characterized by Western blotting assay. Competitive inhibition assay showed that this recombinant JEV EDIII blocks the entry of JEV into BHK-21 cells. Mice immunized with the recombinant JEV EDIII developed high IgG and neutralizing antibodies titers against JEV. Most importantly, antibodies induced by JEV EDIII could neutralize WNV in vitro and partially protected mice against lethal WNV challenge. These results demonstrate that immunization with JEV EDIII induces cross-protective immunity against WNV infection, indicating a possible role of EDIII for the cross-protection among flavivirus.

Enhancement of anti-DIII antibodies by the C3d derivative P28 results in lower viral titers and augments protection in mice

Antibodies generated against West Nile virus (WNV) during infection are essential for controlling dissemination. Recent studies have demonstrated that epitopes in all three domains of the flavivirus envelope protein (E) are targets for neutralizing antibodies, with determinants in domain III (DIII) eliciting antibodies with strong inhibitory properties. In order to increase the magnitude and quality of the antibody response against the WNV E protein, DNA vaccines with derivatives of the WNV E gene (full length E, truncated E, or DIII region, some in the context of the pre-membrane [prM] gene) were conjugated to the molecular adjuvant P28. The P28 region of the complement protein C3d is the minimum CR2-binding domain necessary for the adjuvant activity of C3d. Delivery of DNA-based vaccines by gene gun and intramuscular routes stimulated production of IgG antibodies against the WNV DIII region of the E protein. With the exception of the vaccine expressing prM/E given intramuscularly, only mice that received DNA vaccines by gene gun produced protective neutralizing antibody titers (FRNT80 titer >1/40). Correspondingly, mice vaccinated by the gene gun route were protected to a greater level from lethal WNV challenge. In general, mice vaccinated with P28-adjuvated vaccines produced higher IgG titers than mice vaccinated with non-adjuvanted vaccines.

Single-Chain HLA-A2 MHC Trimers That Incorporate an Immundominant Peptide Elicit Protective T Cell Immunity against Lethal West Nile Virus Infection

The generation of a robust CD8(+) T cell response is an ongoing challenge for the development of DNA vaccines. One problem encountered with classical DNA plasmid immunization is that peptides produced are noncovalently and transiently associated with MHC class I molecules and thus may not durably stimulate CD8(+) T cell responses. To address this and enhance the expression and presentation of the antigenic peptide/MHC complexes, we generated single-chain trimers (SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, beta(2)-microglobulin, and H chain connected by flexible linkers. In this study, we test whether the preassembled nature of the SCT makes them effective for eliciting protective CD8(+) T cell responses against pathogens. A DNA plasmid was constructed encoding an SCT incorporating the human MHC class I molecule HLA-A2 and the immunodominant peptide SVG9 derived from the envelope protein of West Nile virus (WNV). HLA-A2 transgenic mice vaccinated with the DNA encoding the SVG9/HLA-A2 SCT generated a robust epitope-specific CD8(+) T cell response and showed enhanced survival rate and lower viral burden in the brain after lethal WNV challenge. Inclusion of a CD4(+) Th cell epitope within the SCT did not increase the frequency of SVG9-specific CD8(+) T cells, but did enhance protection against WNV challenge. Overall, these findings demonstrate that the SCT platform can induce protective CD8(+) T cell responses against lethal virus infection and may be paired with immunogens that elicit robust neutralizing Ab responses to generate vaccines that optimally activate all facets of adaptive immunity.

Antigen‐specific cytotoxic T lymphocytes protect against lethal West Nile virus encephalitis

Infection with West Nile virus (WNV) causes fatal encephalitis in immunocompromised animals. Previous studies in mice have established that T cell protection is required for clearance of WNV infection from tissues and preventing viral persistence. The current study assessed whether specific WNV peptide epitopes could elicit a cytotoxic T lymphocyte (CTL) response capable of protecting against virus infection. Hidden Markov model analysis was used to identify WNV-encoded peptides that bound the MHC class I proteins K(b) or D(b). Of the 35 peptides predicted to bind MHC class I molecules, one immunodominant CTL recognition peptide was identified in each of the envelope and non-structural protein 4B genes. Addition of these but not control peptides to CD8(+) T cells from WNV-infected mice induced IFN-gamma production. CTL clones that were generated ex vivo lysed peptide-pulsed or WNV-infected target cells in an antigen-specific manner. Finally, adoptive transfer of a mixture of envelope- and non-structural protein 4B-specific CTL to recipient mice protected against lethal WNV challenge. Based on this, we conclude that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines.

A West Nile Virus Recombinant Protein Vaccine That Coactivates Innate and Adaptive Immunity

A chimeric protein West Nile virus (WNV) vaccine capable of delivering both innate and adaptive immune signals was designed by fusing a modified version of bacterial flagellin (STF2 Delta ) to the EIII domain of the WNV envelope protein. This fusion protein stimulated interleukin-8 production in a Toll-like receptor (TLR)-5-dependent fashion, confirming appropriate in vitro TLR5 bioactivity, and also retained critical WNV-E-specific conformation-dependent neutralizing epitopes as measured by enzyme-linked immunosorbent assay. When administered without adjuvant to C3H/HeN mice, the fusion protein elicited a strong WNV-E-specific immunoglobulin G antibody response that neutralized viral infectivity and conferred protection against a lethal WNV challenge. This potent EIII-specific immune response requires a direct linkage of EIII to STF2 Delta , given that a simple mixture of the 2 components failed to induce an antibody response or to provide protection against virus challenge. The presence of a functional TLR5 gene in vivo is also required--TLR5-deficient mice elicited only a minimal antigen-specific response. These results confirm that vaccines designed to coordinately regulate the innate and adaptive immune responses can induce protective immune responses without the need for potentially toxic adjuvants. They also support the further development of an effective WNV vaccine and novel monovalent and multivalent vaccines for related flaviviruses.

Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.