**1. Introduction**

Pathogen inoculation as a preventive measure against infectious diseases (namely smallpox) had been used for centuries in China and other parts of the world. However, and although other researchers had applied similar principles before, the credit for the first e ffective vaccine goes to Edward Jenner who, in 1798, published the first evidence that supported its e fficacy in preventing smallpox [1]. Since then, vaccine development and implementation have been continuously growing until became a breakthrough for animal and human health. Nevertheless, and even though vaccination campaigns are saving millions of lives yearly, nowadays, significant challenges still remain in the vaccinology field, as is exemplified by the devastating current SARS-CoV-2 pandemic. For example, the need: (i) for new technologies and adjuvants that allow faster development of more e ffective, stable, and low-cost vaccines; (ii) that the population is duly informed about the benefits of vaccines as important for their well-being and health, counteracting the increase in the dissemination of opinions of anti-vaccine groups and deniers observed in recent years with data and arguments easily understood by the population to, in this way, obtain their informed consent; (iii) to maintain investment in this field, regardless of the perception of its relevance for the global health of society as a whole and pharmaceutical concerns for the return on investment, thus avoiding situations such as those experienced in the case of Ebola vaccines, for which several candidates have demonstrated their e fficacy in animal models, but none has been authorized, which will hamper its control against possible new outbreaks; and (iv) to establish a global vaccine development fund since the current business model only prioritizes those with grea<sup>t</sup> market potential [2].

Among the main targets of viral vaccination are (re) emerging pathogens that have caused recent epidemics/pandemics around the world, such as avian influenza, Ebola, SARS, MERS, Chikungunya, Zika, Dengue, and West Nile viruses, as well as those that may pose a threat in the future. This review describes the status of West Nile virus (WNV) vaccines for horses, birds, and humans, summarizing and discussing the challenges they face in their clinical advance and introduction to the market.

### **2. West Nile Virus**

WNV is an enveloped flavivirus (family *Flaviviridae*) transmitted by mosquitoes, mainly of the *Culex pipiens* L. complex, whose natural hosts are birds [3]. The virus occasionally infects other vertebrates, mainly equids and humans, which are accidental "dead-end" hosts because viremias achieved in mammals are usually inadequate to maintain the virus cycle, being not enough high to infect a naïve mosquito while feeding on them [4]. Nevertheless, WNV infections in humans and horses have grea<sup>t</sup> economic and health repercussions. Although most WNV infections in humans are asymptomatic, around 20% may cause West Nile fever and less than one percent West Nile neuroinvasive disease, which may result in febrile illness, meningitis, encephalitis, flaccid paralysis, and even death, which can occur in around 10% of severe cases [5,6]. In fact, WNV is the arthropod-borne human pathogenic virus with the largest distribution and one of the major causes of human viral encephalitis worldwide [5,6].

WNV is classified into several lineages that do not consistently correlate with its geographical distribution, but only lineages 1 and 2 have been involved in human outbreaks [7]. Early reports suggested that both lineages had di fferences in pathogenicity, virulence, viremia, the clinical course of infection, and mortality. This initial hypothesis was based mainly on the lack of clinical incidence of WNV in Africa, where only mild diseases have been reported and no deaths have been documented in humans, and where lineage 2 was restricted until it colonized Europe [8]. However, later data in humans and in naturally or experimentally infected animals dismantled this hypothesis. Thus, a study of 644 Greek individuals, which provided a suitable blood sample and lived in an area su ffering a WNV lineage 2 epidemic, showed that 5.8% were seropositive for WNV-specific IgG and approximately 18% of them presented clinical manifestations of WNV disease, figures similar to those of patients infected with lineage 1 [9]. Likewise, falcons and magpies experimentally infected with strains of lineage 1 or 2 showed similar mortality rates [10,11]. Even more, studies performed in vaccinated animals showed a high degree of cross-protection between both lineages. Mice immunized with the inactivated Duravaxyn WNV vaccine or with an experimental RSP (Recombinant Subviral Particle) candidate, both based on lineage 1 strains, were protected against exposure to lineage 2 strains [12,13]. Similarly, horses experimentally vaccinated with the ALVAC ( ®)-WNV vaccine [14], or vaccinated under field conditions with the inactivated Equip vaccine [15], both based on lineage 1 strains, were also protected against challenge with heterologous strains of lineage 2.

WNV genome is a single-stranded RNA molecule of positive polarity that encodes three structural (E, prM/M, and C) and seven non-structural (NS1, 2A, 2B, 3, 4A, 4B, and 5) proteins [3]. Among the structural proteins, the E glycoprotein, which is involved in receptor binding, viral entry, and membrane fusion, is the most immunogenic one [16]. This protein has three domains (DI, DII, and DIII), being DIII an immunoglobulin-like structure that contains multiple epitopes recognized by neutralizing antibodies [3]. In fact, several flaviviruses share common epitopes recognized by cross-reactive neutralizing antibodies [17], which may have consequences for the implementation of vaccines, mainly in regions where several of them co-circulate.
