Emerging infectious diseases represent a significant threat to global health, with West Nile virus (WNV) being a prominent example due to its potential to cause severe neurological disorders alongside mild feverish conditions. Particularly prevalent in the continental United States, WNV has emerged as a global concern, with outbreaks indicating the urgent need for effective prophylactic measures. The current problem is that the absence of a commercial vaccine against WNV highlights a critical gap in preventive strategies against WNV. This study aims to address this gap by proposing a novel, multivalent vaccine designed using immunoinformatics approaches to elicit comprehensive humoral and cellular immune responses against WNV. The objective of the study is to provide a theoretical framework for experimental scientists to formulate of vaccine against WNV and tackle the current problem by generating an immune response inside the host. The research employs reverse vaccinology and subtractive proteomics methodologies to identify NP_041724.2 polyprotein and YP_009164950.1 truncated flavivirus polyprotein NS1 as the prime antigens. The selection process for epitopes focused on B and T-cell reactivity, antigenicity, water solubility, and non-allergenic properties, prioritizing candidates with the potential for broad immunogenicity and safety. The designed vaccine construct integrates these epitopes, connected via GPGPG linkers, and supplemented with an adjuvant with the help of another linker EAAAK, to enhance immunogenicity. Preliminary computational analyses suggest that the proposed vaccine could achieve near-universal coverage, effectively targeting approximately 99.74% of the global population, with perfect coverage in specific regions such as Sweden and Finland. Molecular docking and immune simulation studies further validate the potential efficacy of the vaccine, indicating strong binding affinity with toll-like receptor 3 (TLR-3) and promising immune response profiles, including significant antibody-mediated and cellular responses. These findings present the vaccine construct as a viable candidate for further development and testing. While the theoretical and computational results are promising, advancing from in-silico predictions to a tangible vaccine requires comprehensive laboratory validation. This next step is essential to confirm the vaccine\'s efficacy and safety in eliciting an immune response against WNV. Through this study, we propose a novel approach to vaccine development against WNV and contribute to the broader field of immunoinformatics, showcasing the potential to accelerate the design of effective vaccines against emerging viral threats. The journey from hypothesis to practical solution embodies the interdisciplinary collaboration essential for modern infectious disease management and prevention strategies.

Humans and equines are two dead-end hosts of the mosquito-borne West Nile virus (WNV) with similar susceptibility and pathogenesis. Since the introduction of WNV vaccines into equine populations of the United States of America (USA) in late 2002, there have been only sporadic cases of WNV infection in equines. These cases are generally attributed to unvaccinated and under-vaccinated equines. In contrast, due to the lack of a human WNV vaccine, WNV cases in humans have remained steadily high. An average of 115 deaths have been reported per year in the USA since the first reported case in 1999. Therefore, the characterization of protective immune responses to WNV and the identification of immune correlates of protection in vaccinated equines will provide new fundamental information about the successful development and evaluation of WNV vaccines in humans. This review discusses the comparative epidemiology, transmission, susceptibility to infection and disease, clinical manifestation and pathogenesis, and immune responses of WNV in humans and equines. Furthermore, prophylactic and therapeutic strategies that are currently available and under development are described. In addition, the successful vaccination of equines against WNV and the potential lessons for human vaccine development are discussed.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1-/- mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1-/- model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.

Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, small molecule mast cell activators may be a more cost-efficient adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. To identify novel mast cell activating compounds that could be evaluated for mucosal vaccine adjuvant activity, we employed high-throughput screening to assess over 55,000 small molecules for mast cell degranulation activity. Fifteen mast cell activating compounds were down-selected to five compounds based on in vitro immune activation activities including cytokine production and cellular cytotoxicity, synthesis feasibility, and selection for functional diversity. These small molecule mast cell activators were evaluated for in vivo adjuvant activity and induction of protective immunity against West Nile Virus infection in BALB/c mice when combined with West Nile Virus envelope domain III (EDIII) protein in a nasal vaccine. We found that three of the five mast cell activators, ST101036, ST048871, and R529877, evoked high levels of EDIII-specific antibody and conferred comparable levels of protection against WNV challenge. The level of protection provided by these small molecule mast cell activators was comparable to the protection evoked by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Thus, novel small molecule mast cell activators identified by high throughput screening are as efficacious as previously described mast cell activators when used as nasal vaccine adjuvants and represent next-generation mast cell activators for evaluation in mucosal vaccine studies.

The genus Flavivirus comprises numerous emerging and re-emerging arboviruses causing human illness. Vaccines are the best approach to prevent flavivirus diseases. But pathogen diversities are always one of the major hindrances for timely development of new vaccines when confronting unpredicted flavivirus outbreaks. We used West Nile virus (WNV) as a model to develop a new live-attenuated vaccine (LAV), WNV-poly(A), by replacing 5\' portion (corresponding to SL and DB domains in WNV) of 3\'-UTR with internal poly(A) tract. WNV-poly(A) not only propagated efficiently in Vero cells, but also was highly attenuated in mouse model. A single-dose vaccination elicited robust and long-lasting immune responses, conferring full protection against WNV challenge. Such \"poly(A)\" vaccine strategy may be promising for wide application in the development of flavivirus LAVs because of its general target regions in flaviviruses.

Greater sage-grouse (Centrocercus urophasianus) are an endangered species in Canada and have been extirpated from five states in the United States. Infectious diseases can pose a challenge to conservation efforts, and the greater sage-grouse is susceptible to West Nile virus (WNV). This study measured the humoral response to a commercially available WNV vaccine in a zoo-based conservation breeding population of greater sage-grouse. Since the initiation of the conservation program in 2014, all sage-grouse at the Calgary Zoo\'s Wildlife Conservation Centre have been vaccinated against WNV. Juveniles received a series of three vaccines every 2-3 wk starting around 2 wk of age; adults had received the same vaccination series at hatch and had since been boostered annually. Antibody titers were measured on 60 serum samples from 36 juveniles and 18 adults collected in 2018 using either serum neutralization or plaque-reduction neutralization methods. No detectable antibody response was noted in juveniles after their second (n = 17) or third booster vaccinations (n = 23). Only 35% of 20 adult samples collected had positive titers even after multiple years of vaccination. Only two cases of adverse vaccine reaction have been noted in this species with over 800 doses being administered between 2014 and 2020. Despite a lack of antibody response in juveniles and most adults, there have been no WNV-associated deaths in this population even with confirmed cases of WNV in free-ranging greater sage-grouse and other species in the area during this time frame, suggesting that cell-mediated immunity may be of greater importance for protection against WNV infections in this species. The initial vaccination series was changed in 2019 to a series of only two doses of vaccine, and no clinical cases or mortality from WNV occurred in 2019 or 2020 with the new protocol.

West Nile virus (WNV) is the leading cause of arboviral disease in the United States and is associated with significant morbidity and mortality. A previous analysis found that a vaccination program targeting persons aged ≥60 years was more cost-effective than universal vaccination, but costs remained high.
We used a mathematical Markov model to evaluate cost-effectiveness of an age- and incidence-based WNV vaccination program. We grouped states and large counties (≥100 000 persons aged ≥60 years) by median annual WNV incidence rates from 2004 to 2017 for persons aged ≥60 years. We defined WNV incidence thresholds, in increments of 0.5 cases per 100 000 persons ≥60 years. We calculated potential cost per WNV vaccine-prevented case and per quality adjusted life-years (QALYs) saved.
Vaccinating persons aged ≥60 years in states with an annual incidence of WNV neuroinvasive disease of ≥0.5 per 100 000 resulted in approximately half the cost per health outcome averted compared to vaccinating persons aged ≥60 years in the contiguous United States. This approach could potentially prevent 37% of all neuroinvasive disease cases and 63% of WNV-related deaths nationally. Employing such a threshold at a county level further improved cost-effectiveness ratios while preventing 19% and 30% of WNV-related neuroinvasive disease cases and deaths, respectively.
An age- and incidence-based WNV vaccination program could be a more cost-effective strategy than an age-based program while still having a substantial impact on lowering WNV-related morbidity and mortality.

West Nile Virus (WNV) causes a debilitating and life-threatening neurological disease in humans. Since its emergence in Africa 50 years ago, new strains of WNV and an expanding geographical distribution have increased public health concerns. There are no licensed therapeutics against WNV, limiting effective infection control. Vaccines represent the most efficacious and efficient medical intervention known. Epitope-based vaccines against WNV remain significantly underexploited. Here, we use a selection protocol to identify a set of conserved prevalidated immunogenic T cell epitopes comprising a putative WNV vaccine. Experimentally validated immunogenic WNV epitopes and WNV sequences were retrieved from the IEDB and West Nile Virus Variation Database. Clustering and multiple sequence alignment identified a smaller subset of representative sequences. Protein variability analysis identified evolutionarily conserved sequences, which were used to select a diverse set of immunogenic candidate T cell epitopes. Cross-reactivity and human leukocyte antigen-binding affinities were assessed to eliminate unsuitable epitope candidates. Population protection coverage (PPC) quantified individual epitopes and epitope combinations against the world population. 3 CD8+ T cell epitopes (ITYTDVLRY, TLARGFPFV, and SYHDRRWCF) and 1 CD4+ epitope (VTVNPFVSVATANAKVLI) were selected as a putative WNV vaccine, with an estimated PPC of 97.14%.

Although West Nile virus (WNV) has been a prominent mosquito-transmitted infection in North America for twenty years, no human vaccine has been licensed. With a cumulative number of 24,714 neurological disease cases and 2314 deaths in the U.S. since 1999, plus a large outbreak in Europe in 2018 involving over 2000 human cases in 15 countries, a vaccine is essential to prevent continued morbidity, mortality, and economic burden. Currently, four veterinary vaccines are licensed, and six vaccines have progressed into clinical trials in humans. All four veterinary vaccines require multiple primary doses and annual boosters, but for a human vaccine to be protective and cost effective in the most vulnerable older age population, it is ideal that the vaccine be strongly immunogenic with only a single dose and without subsequent annual boosters. Of six human vaccine candidates, the two live, attenuated vaccines were the only ones that elicited strong immunity after a single dose. As none of these candidates have yet progressed beyond phase II clinical trials, development of new candidate vaccines and improvement of vaccination strategies remains an important area of research.

West Nile virus (WNV) is a widely spread human pathogenic arthropod-borne virus. It can lead to severe, sometimes fatal, neurological disease. Over the last two decades, several vaccine candidates for the protection of humans from WNV have been developed. Some technologies were transferred into clinical testing, but these approaches have not yet led to a licensed product. This review summarizes the current status of a human WNV vaccine and discusses reasons for the lack of clinically advanced product candidates. It also discusses the problem of immunological cross-reactivity between flaviviruses and how it can be addressed during vaccine development.