Immunoinformatics assisted profiling of West Nile virus proteome to determine immunodominant epitopes for the development of next-generation multi-peptide vaccine.

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.

Febre do Nilo Ocidental / West Nile fever

A Febre do Nilo Ocidental (FNO) é uma infecção viral transmitida por meio da picada de mosquitos, principalmente do gênero Culex (pernilongo) infectados pelo agente etiológico, cujos hospedeiros naturais são algumas espécies de aves silvestres, que atuam como amplificadoras do vírus e como fonte de infecção para os vetores. Tal doença pode também infectar humanos, equinos, primatas e outros mamíferos sendo que, homem e equídeos são considerados hospedeiros acidentais e terminais, uma vez que a contaminação pelo vírus se dá por um curto período de tempo e em níveis insuficientes para infectar mosquitos, encerrando o ciclo de transmissão (WHO, 2017; ECDC , 2022a; CDC, 2017; BRASIL, 2021)

West Nile Fever (WNF) is a viral infection transmitted through the bite of mosquitoes, mainly of the Culex genus (legged mosquito) infected by the etiological agent, whose natural hosts are some species of wild birds, which act as amplifiers of the virus and as source of infection for the vectors. Such a disease can also infect humans, horses, primates and other mammals, and humans and horses are considered accidental and terminal hosts, since contamination by the virus occurs for a short period of time and at levels insufficient to infect mosquitoes, ending the transmission cycle (WHO, 2017; ECDC, 2022a; CDC, 2017). ; BRAZIL, 2021)

Development of peptides targeting receptor binding site of the envelope glycoprotein to contain the West Nile virus infection.

West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood-brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.

Epidemiology, management, and graft outcomes after West Nile virus encephalitis in kidney transplant recipients.

BACKGROUND: Minimal data exist describing the epidemiology, management, and long-term graft outcomes after West Nile viral disease in kidney transplant recipients (KTRs). METHODS: Single-center observational cohort study of patients who received a kidney transplant between 1/1/1994 and 12/31/2018 and developed WNV at any time point after transplantation. RESULTS: During the 24-year study period, 11 patients had documented WNV infection. Seven patients were recipients of a kidney transplant alone, and four had a simultaneous kidney and pancreas transplant. The mean age at the time of transplant was 44.7 ± 17.1 years, and the mean age at the time of WNV infection was 48 ± 17.2 years. All patients received lymphocyte depleting induction at transplant (alemtuzumab (n = 2), OKT3 (n = 1), or anti-thymocyte globulin (n = 8)). The mean time from transplant to WNV infection was 3.4 ± 5.4 years, and none was suspected of having a donor-derived infection. Three patients were treated for rejection in the 6 months before infection. The most common presenting symptom was altered mental status (n = 7), followed by a combination of fever and headache (n = 4). All patients had detectable serum WNV IgM antibodies at the time of diagnosis. All patients had a reduction in their immunosuppression and received supportive care; only two patients were treated with intravenous immunoglobulins. Nine patients recovered with no residual deficit; however, two suffered permanent neurologic damage. The mean estimated glomerular filtration rate drop at 1 year after the infection was 8.4 ± 13 mL/min/1.73 m2 . Three patients suffered acute rejection within 1 year after the infection episode, likely attributable to aggressive immunosuppressive reduction. The mean follow-up after the infection was 5.1 ± 4.3 years. At last follow-up, two patients lost their kidney allograft, and five patients died. None of the graft losses or deaths occurred within a year of the WNV or were directly attributable to WNV. CONCLUSION: The majority of patients with WNV infection after KTR recovered fully with supportive care and immunosuppressive adjustment without residual neurologic sequelae. Additionally, WNV infection was associated with relatively small reductions in eGFR at 1 year.

West Nile virus vaccines - current situation and future directions.

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.

Recombinant influenza A viruses as vaccine vectors.

INTRODUCTION: Various viruses, including poxviruses, adenoviruses and vesicular stomatitis virus, have been considered as vaccine vectors for the delivery of antigens of interest in the development of vaccines against newly emerging pathogens. AREAS COVERED: Here, we review results that have been obtained with influenza A viruses (IAV) as vaccine vectors. With the advent of reverse genetics technology, IAV-based recombinant vaccine candidates have been constructed that induce protective immunity to a variety of different pathogens of interest, including West Nile virus, Plasmodium falciparum and respiratory syncytial virus. The various cloning strategies to produce effective and attenuated, safe to use IAV-based viral vectors are discussed. EXPERT COMMENTARY: It was concluded that IAV-based vector system has several advantages and holds promise for further development.

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 ten mAbs from WNV-infected individuals. mAb WNV-86 neutralized WNV with a 50% inhibitory concentration of 2 ng ml-1, 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.

Re-emergence of severe West Nile virus neuroinvasive disease in humans in Romania, 2012 to 2017-implications for travel medicine.

BACKGROUND: In Romania, after a major outbreak in 1996, West Nile neuroinvasive disease (WNND) was reported only in a limited number of cases annually. During 2016-2017, a significant increase in the number of WNND cases was reported at the national level, associated with high mortality rates. METHODS: A retrospective analysis of all cases confirmed with WNND, hospitalized during 2012-2017 in a single tertiary facility from Bucharest was performed in order to determine the annual prevalence and mortality rate and the risk factors associated with a severe outcome. RESULTS: 47 cases were confirmed as WNND. The mortality rate was 25.5%, all death occurred during 2016-2017. Coma, confusion, obtundation, sleepiness and depressed deep tendon reflexes were symptoms predicting a severe outcome. In a univariate analysis age (p < 0.001), associated cancers (p = 0.012) and low levels of chloride in the CSF (p = 0.008) were risk factors for mortality. In a multinomial logistic analysis, age older than 75 years remained the only independent predictor of death in WNND. CONCLUSIONS: The increase in both the number and the mortality rate of WNND cases suggest a changing pattern of WNV infection in Romania. Public health authorities and clinicians should be aware of the risk of severe WNV infection in travelers returning from Romania.

A plant-produced vaccine protects mice against lethal West Nile virus infection without enhancing Zika or dengue virus infectivity.

West Nile virus (WNV) has caused multiple global outbreaks with increased frequency of neuroinvasive disease in recent years. Despite many years of research, there are no licensed therapeutics or vaccines available for human use. One of the major impediments of vaccine development against WNV is the potential enhancement of infection by related flaviviruses in vaccinated subjects through the mechanism of antibody-dependent enhancement of infection (ADE). For instance, the recent finding of enhancement of Zika virus (ZIKV) infection by pre-exposure to WNV further complicates the development of WNV vaccines. Epidemics of WNV and the potential risk of ADE by current vaccine candidates demand the development of effective and safe vaccines. We have previously reported that the domain III (DIII) of the WNV envelope protein can be readily expressed in Nicotiana benthamiana leaves, purified to homogeneity, and promote antigen-specific antibody response in mice. Herein, we further investigated the in vivo potency of a plant-made DIII (plant-DIII) in providing protective immunity against WNV infection. Furthermore, we examined if vaccination with plant-DIII would enhance the risk of a subsequent infection by ZIKV and Dengue virus (DENV). Plant-DIII vaccination evoked antigen-specific cellular immune responses as well as humoral responses. DIII-specific antibodies were neutralizing and the neutralization titers met the threshold correlated with protective immunity by vaccines against multiple flaviviruses. Furthermore, passive administration of anti-plant DIII mouse serum provided full protection against a lethal challenge of WNV infection in mice. Notably, plant DIII-induced antibodies did not enhance ZIKV and DENV infection in Fc gamma receptor-expressing cells, addressing the concern of WNV vaccines in inducing cross-reactive antibodies and sensitizing subjects to subsequent infection by heterologous flavivirus. This study provides the first report of a WNV subunit vaccine that induces protective immunity, while circumventing induction of antibodies with enhancing activity for ZIKV and DENV infection.

Protection of mice deficient in mature B cells from West Nile virus infection by passive and active immunization.

B cell activating factor receptor (BAFFR)-/- mice have a profound reduction in mature B cells, but unlike µMT mice, they have normal numbers of newly formed, immature B cells. Using a West Nile virus (WNV) challenge model that requires antibodies (Abs) for protection, we found that unlike wild-type (WT) mice, BAFFR-/- mice were highly susceptible to WNV and succumbed to infection within 8 to 12 days after subcutaneous virus challenge. Although mature B cells were required to protect against lethal infection, infected BAFFR-/- mice had reduced WNV E-specific IgG responses and neutralizing Abs. Passive transfer of immune sera from previously infected WT mice rescued BAFFR-/- and fully B cell-deficient µMT mice, but unlike µMT mice that died around 30 days post-infection, BAFFR-/- mice survived, developed WNV-specific IgG Abs and overcame a second WNV challenge. Remarkably, protective immunity could be induced in mature B cell-deficient mice. Administration of a WNV E-anti-CD180 conjugate vaccine 30 days prior to WNV infection induced Ab responses that protected against lethal infection in BAFFR-/- mice but not in µMT mice. Thus, the immature B cells present in BAFFR-/- and not µMT mice contribute to protective antiviral immunity. A CD180-based vaccine may promote immunity in immunocompromised individuals.

State of the art: Vest Nile Virus circulation surveillance in Italy and transfusion risk early prevention methods.

After the Chikungunya outbreak in 2007 in Italy, a national Plan for the surveillance of human vector-borne diseases has been implemented and annually updated on the basis of the epidemiological changes based-evidences. The transfusion Authorities cooperates, since 2008, with public health services and veterinary (entomological and ornithological) surveillance systems. In some Italian regions, a common protocol for exchanging data is in place to identify the West Nile Virus (WNV) circulation in birds and mosquitoes: the goal is to anticipate the introduction of WNV-NAT screening in blood donors and, on the other hand, to limit testing only in geographic areas where the virus circulation is actual. The integration of surveillance activities and a multidisciplinary approach made it possible to introduce efficient and preventive measures for reducing the risk of of transmission of WNV trough blood, tissues and organ donation.

A recombinant fusion protein consisting of West Nile virus envelope domain III fused in-frame with equine CD40 ligand induces antiviral immune responses in horses.

West Nile Virus (WNV) is endemic in the US and causes severe neurologic disease in horses since its introduction in 1999. There is no effective pharmaceutical treatment for WNV infection rendering vaccination as the only approach to prevention and control of disease. The purpose of this study was to evaluate a recombinant vaccine containing domain III (DIII) of the WNV envelope glycoprotein with and without a natural adjuvant equine (CD40L) in producing virus neutralizing antibodies in horses. Serum IgG1 concentration in the groups of horses vaccinated with the DIII-CD40L+TiterMax and DIII-CD40L proteins were significantly increased (p<0.05) after the second booster vaccination compared to other groups. Serum IgG4 and IgG7, IgG3 and IgG5 concentrations were not significantly increased among all groups. Western blot results showed that animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited the highest specific anti-DIII antibody activities after vaccinations. Moreover, animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited significantly stronger neutralization activity (p<0.05) compared to other groups starting at week eight. The DIII-CD40L protein (with or without TiterMax) stimulated more CD8+T cells, but not CD4+T cells in equine PMBCs. The results demonstrated that vaccination with recombinant WNV E DIII-CD40L protein induced superior humoral and cellular immune response in healthy horses that may be protective against WNV-associated disease in infected animals. CD40L could be utilized as a non-toxic, alternative adjuvant to boost the immunogenicity of subunit vaccines in horses.

Development of a human live attenuated West Nile infectious DNA vaccine: Identification of a minimal mutation set conferring the attenuation level acceptable for a human vaccine.

For the development of a human West Nile (WN) infectious DNA (iDNA) vaccine, we created highly attenuated chimeric virus W1806 with the serological identity of highly virulent WN-NY99. Earlier, we attempted to utilize mutations found in the E protein of the SA14-14-2 vaccine to bring safety of W1806 to the level acceptable for human use (Yamshchikov et al., 2016). Here, we analyzed effects of the SA14-14-2 changes on growth properties and neurovirulence of W1806. A set including the E138K, K279M, K439R and G447D changes was identified as the perspective subset for satisfying the target safety profile without compromising immunogenicity of the vaccine candidate. The genetic stability of the attenuated phenotype was found to be unsatisfactory being dependent on a subset of attenuating changes incorporated in W1806. Elucidation of underlying mechanisms influencing selection of pathways for restoration of the envelope protein functionality will facilitate resolution of the emerged genetic stability issue.

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.

Newcastle disease virus-vectored West Nile fever vaccine is immunogenic in mammals and poultry.

BACKGROUND: West Nile virus (WNV) is an emerging zoonotic pathogen which is harmful to human and animal health. Effective vaccination in susceptible hosts should protect against WNV infection and significantly reduce viral transmission between animals and from animals to humans. A versatile vaccine suitable for different species that can be delivered via flexible routes remains an essential unmet medical need. In this study, we developed a recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing WNV premembrane/envelope (PrM/E) proteins (designated rLa-WNV-PrM/E) and evaluated its immunogenicity in mice, horses, chickens, ducks and geese. RESULTS: Mouse immunization experiments disclosed that rLa-WNV-PrM/E induces significant levels of WNV-neutralizing antibodies and E protein-specific CD4+ and CD8+ T-cell responses. Moreover, recombinant rLa-WNV-PrM/E elicited significant levels of WNV-specific IgG in horses upon delivery via intramuscular immunization, and in chickens, ducks and geese via intramuscular, oral or intranasal immunization. CONCLUSIONS: Our results collectively support the utility of rLa-WNV-PrM/E as a promising WNV veterinary vaccine candidate for mammals and poultry.

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.

Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans.

The E glycoprotein plays an essential role in the high pathogenicity of European-Mediterranean IS98 strain of West Nile virus.

West Nile virus (WNV) is the most widespread arbovirus in the world. Several recent outbreaks and epizootics have been reported in Europe and the Mediterranean basin with increased virulence. In contrast to the well-characterized American and Australian strains, little is known about the virulence determinants of the WNV European-Mediterranean strains. To investigate the viral factors involved in the virulence of these strains, we generated chimeras between the highly neuropathogenic Israel 1998 (IS-98-ST1, IS98) strain and the non-pathogenic Malaysian Kunjin virus (KJMP-502). In vivo analyses in a mouse model of WNV pathogenesis shows that chimeric virus where KJMP-502 E glycoprotein was replaced by that of IS98 is neuropathogenic, demonstrating that this protein is a major virulence determinant. Presence of the N-glycosylation site had limited impact on virus virulence and the 5'UTR does not seem to influence pathogenesis. Finally, mice inoculated with KJMP-502 virus were protected against lethal IS98 infection.