Monitoring Anti-NS1 Antibodies in West Nile Virus-Infected and Vaccinated Horses.

West Nile virus (WNV) is a zoonotic arboviral pathogen affecting humans, birds, and horses. Vaccines are available for veterinary use, which efficiently prevent the infection in horses. Most common diagnostic tools rely on the identification of the agent (RT-PCR, virus isolation), or on the detection of antibodies (IgM and IgG) recognizing structural proteins of the virus or neutralizing virus infection in cell cultures (virus-neutralization tests). The recent emergence of WNV in different parts of the world has resulted in an increase in the vaccination of horses in many countries. Methods for differentiation between infected and vaccinated animals ("DIVA" assays) would be useful for surveillance and control purposes but are still not available. A usual approach in this regard is the use of antibodies to nonstructural proteins as markers of nonvaccinated, infected animals, and the nonstructural NS1 protein of WNV has been proposed as a candidate for such a marker. The aim of this study was to test the hypothesis that NS1 can be a useful antigen in DIVA assays for differentiating WNV vaccinated and infected horses in field conditions. For that, we examined serum samples from either vaccinated and infected horses both from experimental infections/vaccinations (under controlled conditions) and from the field, exposed to natural infection or vaccinated in response to a risk of infection. The overall conclusion of the study is that NS1 antigen can effectively differentiate WNV infected from vaccinated horses in experimental (controlled) conditions, but this differentiation might be difficult depending on the conditions prevailing in the field.

An Overview of Current Approaches Toward the Treatment and Prevention of West Nile Virus Infection.

The persistence of West Nile virus (WNV) infections throughout the USA since its inception in 1999 and its continuous spread throughout the globe calls for an urgent need of effective treatments and prevention measures. Although the licensing of several WNV vaccines for veterinary use provides a proof of concept, similar efforts on the development of an effective vaccine for humans remain still unsuccessful. Increased understanding of biology and pathogenesis of WNV together with recent technological advancements have raised hope that an effective WNV vaccine may be available in the near future. In addition, rapid progress in the structural and functional characterization of WNV and other flaviviral proteins have provided a solid base for the design and development of several classes of inhibitors as potential WNV therapeutics. Moreover, the therapeutic monoclonal antibodies demonstrate an excellent efficacy against WNV in animal models and represent a promising class of WNV therapeutics. However, there are some challenges as to the design and development of a safe and efficient WNV vaccine or therapeutic. In this chapter, we discuss the current approaches, progress, and challenges toward the development of WNV vaccines, therapeutic antibodies, and antiviral drugs.

A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations.

West Nile virus (WNV) is a mosquito-transmitted member of the Flaviviridae family that has emerged in recent years to become a serious public health threat. Given the sporadic nature of WNV epidemics both temporally and geographically, there is an urgent need for a vaccine that can rapidly provide effective immunity. Protection from WNV infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. Despite many promising E-protein vaccine candidates, there are currently none licensed for use in humans. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) by combining it with a novel synthetic TLR-4 agonist adjuvant. Using the murine model of WNV disease, we find that inclusion of a TLR-4 agonist in either a stable oil-in-water emulsion (SE) or aluminum hydroxide (Alum) formulation provides both dose and dosage sparing functions, whereby protection can be induced after a single immunization containing only 100 ng of WN-80E. Additionally, we find that inclusion of adjuvant with a single immunization reduced viral titers in sera to levels undetectable by viral plaque assay. The enhanced protection provided by adjuvanted immunization correlated with induction of a Th1 T-cell response and the resultant shaping of the IgG response. These findings suggest that inclusion of a next generation adjuvant may greatly enhance the protective capacity of WNV recombinant subunit vaccines, and establish a baseline for future development.

DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly, via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.).

As West Nile virus (WNV) can cause lethal diseases in raptors, a vaccination prophylaxis of free-living and captive populations is desirable. In the absence of vaccines approved for birds, equine vaccines have been used in falcons, but full protection against WNV infection was not achieved. Therefore, two DNA vaccines encoding the ectodomain of the envelope protein of WNV lineages 1 and 2, respectively, were evaluated in 28 large falcons. Four different vaccination protocols were used, including electroporation and booster-injections of recombinant WNV domain III protein, before challenge with the live WNV lineage 1 strain NY99. Drug safety, plasmid shedding and antibody production were monitored during the vaccination period. Serological, virological, histological, immunohistochemical and molecular biological investigations were performed during the challenge trials. Antibody response following vaccination was low overall and lasted for a maximum of three weeks. Plasmid shedding was not detected at any time. Viremia, mortality and levels, but not duration, of oral virus shedding were reduced in all of the groups during the challenge trial compared to the non-vaccinated control group. Likewise, clinical scoring, levels of cloacal virus shedding and viral load in organs were significantly reduced in three vaccination groups. Histopathological findings associated with WNV infections (meningo-encephalitis, myocarditis, and arteritis) were present in all groups, but immunohistochemical detection of the viral antigen was reduced. In conclusion, the vaccines can be used safely in falcons to reduce mortality and clinical signs and to lower the risk of virus transmission due to decreased levels of virus shedding and viremia, but full protection was not achieved in all groups.

Inactivated West Nile Virus (WNV) vaccine, Duvaxyn WNV, protects against a highly neuroinvasive lineage 2 WNV strain in mice.

BACKGROUND: Lineage 2 West Nile Virus (WNV) is endemic to southern Africa and Madagascar, and has recently been associated with encephalitis outbreaks in humans and horses in South Africa, central Europe, Italy and Greece. Commercial vaccines have mostly been evaluated against WNV lineage 1 strains and their efficacy against lineage 2 strains rarely reported. METHODS: To evaluate protection of Duvaxyn WNV vaccine against lineage 2 strains associated with encephalitis in South Africa, mice were vaccinated twice intramuscularly three weeks apart, and challenged four weeks later with highly neuroinvasive lineage 1 strain NY385/99 or lineage 2 strain SPU93/01. Neutralising antibody titres were measured at the time of challenge and three weeks later. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were conducted on brains of mice that succumbed during the trial, on controls and on vaccinated mice that survived. RESULTS: Serum neutralising antibodies in vaccinated mice were detected but low three weeks after primovaccination. Three weeks post-challenge, vaccinated mice had significantly higher serum neutralising antibody titres against both lineages than unvaccinated controls. After challenge, all vaccinated mice remained healthy but all unvaccinated mice demonstrated severe neurological signs with 75% mortality rate. WNV was not detected in brains of vaccinated mice whereas virus replicated in most unvaccinated mice challenged with either lineage. Gross and microscopic lesions were found only in unvaccinated mice challenged with both lineages. CONCLUSION: Duvaxyn WNV vaccine provided complete protection against challenge with lineage 2 WNV and stimulated significant cross protective neutralising antibodies in mice against lineage 2.

Protective efficacy of a recombinant subunit West Nile virus vaccine in domestic geese (Anser anser).

Introduction of the West Nile virus (WNV) to Hawai'i will undoubtedly devastate many populations of critically endangered avian species indigenous to Hawai'i. The protective efficacy of a protein-based WNV subunit vaccine formulated with adjuvant was evaluated in domestic geese as a surrogate species for the endangered Nene, the state bird of Hawai'i. Prevention of viremia following viral infection of vaccinated birds was used as the clinical endpoint of protection. ELISA and plaque reduction neutralization tests demonstrate that significant levels of vaccine antigen-specific antibody were produced in groups of birds vaccinated with 5 or 10 microg of the WN-80E antigen formulated with ISA720 adjuvant. Moreover, after challenge with WNV, no viremia was detected in vaccinated birds, whereas viremia was detected up to 4 days after and virus was detected by oral swab for 6 days after infection among control groups. Safe and effective vaccination of managed or captive endangered bird populations will protect species with critically low numbers that could not survive the added mortality of introduced disease.

Co-immunization with West Nile DNA and inactivated vaccines provides synergistic increases in their immunogenicities in mice.

West Nile virus is now distributed throughout many temperate, subtropical and tropical areas: vaccines need to be developed that are affordable for developed and developing countries. Here, we constructed and evaluated a DNA vaccine expressing the premembrane and envelope proteins of West Nile virus (pcWNME). Mice immunized twice with 100 or 10 microg of pcWNME developed high or moderate levels of neutralizing antibodies, respectively. These mice were protected from viremia and death after lethal challenge. Mice immunized with a mixture of 1 microg of pcWNME and a small amount (1/10 dose) of a commercial inactivated vaccine developed moderate levels of neutralizing antibodies, whereas immunization with pcWNME or the inactivated vaccine alone induced only low or undetectable levels: co-immunization with the DNA and protein vaccines synergistically increased their own immunogenicities. The synergism reduced the amount of DNA sufficient to induce neutralizing antibodies: a single immunization with doses as low as 0.1 microg induced a titer of 1:40 at a 90% plaque reduction 6 or 9 weeks after immunization. Both IgG1 and IgG2a antibodies were induced in mice by co-immunization with the DNA and protein vaccines.