Bluetongue virus (BTV) is the type species of the genus Orbivirus and causative agent of bluetongue (BT), a hemorrhagic ruminant disease with a broad host range, although mortality and morbidity varies among species [153,154]. This disease is mainly appreciated in sheep and some wild ruminants such as white-tailed deer (Odocoileus virginianus), with significant mortality rates. On the contrary, goat, cattle and most wild ruminants usually display either subclinical or asymptomatic BT signs [155,156,157]. Traditionally, BTV global distribution is enclosed to regions between approximately 50° N and 35° S. Nonetheless, northwards expansion has occurred during recent years, with it being identified in all continents except Antarctica [5]. The worldwide economic impact of BT is estimated to be 3 billion US dollars per year [158]. Such expenses are the result of productivity losses, animal death, animal trade restrictions and the implementation of surveillance measures [158,159]. Thus, it is included in the notifiable disease list of the World Organization for Animal Health (WOAH, former OIE) because of its detrimental consequences in the livestock industry. More than 29 serotypes have been identified so far [17], based on extensive phylogenetic studies, sequencing data and cross-neutralization assays [18,160]. The global distribution of individual BTV serotypes is highly heterogenous [161]. Conventional approaches based on inactivated or live attenuated vaccines have been permitted to contain BTV. However, they present several pitfalls, highlighting their serotype specificity and lack of DIVA properties. A huge effort has been made to develop next-generation vaccines that could solve drawbacks of these marketed classical vaccines, including subunit, viral vector, DISC (disabled infectious single cycle) and DISA (disabled infectious single animal) vaccines [17,18].

VLPs and CLPs (core-like particles) have been extensively evaluated as potential nanoparticle-based vaccines against BTV. Homogenous BTV CLPs and VLPs were first assembled in baculovirus–insect cell expression systems. CLPs consist on the simultaneous expression of VP3 and VP7 in absence of the polymerase complex (VP1, VP4 and VP6) and any non-structural protein, being identical in size and shape to authentic BTV cores [162]. VLPs are composed of the core particle (VP3 and VP7) and the outer capsid layer (VP2 and VP5), and resembled BTV in size, appearance and biochemical constitution, lacking the double-stranded RNA and the RNA polymerase complex [138]. Importantly, VP3 and VP7 are relatively well conserved between serotypes so that outer capsid proteins from different serotypes can be coated onto heterologous inner core proteins, which implies an advantage of this VLP system to generate vaccines to new serotypes by replacing VP2, the most variable BTV protein [163]. Apart from a baculovirus expression system, a plant-based high-level expression system was also implemented to produce assembled subcore-, core- and virus-like particles of BTV [164,165].

Regarding their evaluation as vaccine candidates, CLPs showed immunogenicity in vivo but were incompletely protective against BTV [138,163,164,166]. The major outer core protein and cross-reactive antigen VP7 has been shown to be protective against BTV [167,168]. So, despite CLPs lack of major immunogenicity determinant VP2, the main inductor of neutralizing antibodies (NAbs), the induction of a cell-mediated immune response specific of VP7 could explain the mild protective capacity observed [166]. Conversely, VLPs were able to confer serotype-specific protection from clinical disease and viral replication in sheep, one of the natural BTV hosts, even in the long-term [72,166,169,170,171,172]. Nonetheless, complete protection against BTV was dependent on the utilization of adjuvants, as adjuvant-free VLPs were demonstrated to be only partially protective in sheep, inducing low levels of NAbs [172]. As stated above, one of the major drawbacks of BTV vaccination is the serotype-specificity. Cross-protection was only partially achieved against phylogenetically related serotypes after immunization with VLPs [169]. In consequence, the rationale would be the design of a cocktail of VLPs of different serotypes that could work as a multivalent vaccine. This strategy has been successfully explored as multivalent vaccine candidates protected against homologous serotypes [72,169,171]. Considering that development of multivalent vaccines could induce immune interferences between different antigens present within the same vaccine formulation [173,174,175], it is important to note that the combination of VLPs containing VP2 from different serotypes did not lead to interferences of any kind, which places cocktails of VLPs as potential DIVA, multiserotype and inherently safe vaccines against BTV. Nevertheless, these vaccine candidates have not been marketed yet, despite being proven to be effective, safe and DIVA candidates, something that could be explained by a low affordability. In a similar way, plant-produced VLPs offered protective immunity against homologous serotypes [164,165] so that VLP production in plants may work as a more cost-effective alternative with the advantage of a feasible scalability. Leastways, a plausible and more likely affordable alternative to BTV VLPs is the 60-meric self-assembled enzymatic complexes made of lumazine synthase from the hyperthermophile Aquifex aeolicus carrying the immunodominant VP2 [176]. These virus-like nanoparticles have been applied for host receptor identification during virus entry, but their potential as vaccines candidates against BTV has not been characterized so far.

One of the exploitations of VLPs is their use as carriers for foreign epitopes [177,178,179,180,181,182]. The rabbit hemorrhagic disease virus VP60 VLPs carrying a six-residue epitope from BTV virus capsid protein VP7 is an example. This vaccine candidate was not further evaluated as a vaccine candidate [183]. BTV CLPs have been also explored as a delivery platform. These were loaded on their surface with heterologous antigens such as preS2 of the hepatitis B virus [184], an immunogenic epitope of the rabies virus glycoprotein [185], a T-cell epitope of the influenza A virus matrix (M1) protein [186] or a sequence of the bovine leukemia virus (BLV) gp51 protein [187]. The EIII ectodomain of dengue virus serotypes 1 (DENV1) and 4 (DENV4), and Zika virus, have been also displayed on the inner surface of BTV CLPs fused to the amino terminal end of VP3 [188]. Although these strategies showed some mild immunogenicity to foreign antigens, the length of the aminoacidic inserted sequence dramatically conditioned the assembly and stability of chimeric CLPs in some cases. The interior of empty plant-based BTV CLPs has also been labeled with GFP molecules [189].

Recently, different studies have noted the capability of the conserved non-structural protein NS1 of BTV to promote a specific CD8+ T-cell response able to protect immunized animals against several non-related serotypes of BTV [124,127]. The combination of both virus-neutralizing antibodies and cytotoxic T lymphocytes (CTLs) is crucial for the development of a long-lasting immunity in animals infected with BTV [120,190]. The immune response induced by BTV VLPs is eminently based on the induction of NAbs, although the presence of VP7 may also stimulate a cross-reactive cell-mediated response. Immunization with NS1, VP2 and VP7 loaded on avian reovirus muNS-microspheres fostered both arms of the adaptive immune response, providing sterile immunity against homologous BTV serotype in IFNAR(-/-) mice [191], mainly mediated by triggering neutralizing antibodies and CD4+ T-cell activation. Heterologous combination with a modified vaccina Ankara (MVA) viral vector expressing these BTV antigens led to complete heterologous protection against BTV, which could rely on a more potent BTV-specific cell response influenced by the viral vector used [192]. In addition, BTV-muNS-MS presented an intrinsic adjuvant capacity, which differs from VLPs adjuvant-dependent response. Nonetheless, results on the efficacy of BTV-muNS-MS in the mouse model must be assayed in a BTV natural host. In any case, due to the plasticity and easy method of production implied in this technology, additional BTV antigens could be included in this subunit vaccine formulation, as could be the case of NS2 or its amino-terminal end (NS2-Nt), where combination with NS1 optimizes the multiserotype protection elicited by NS1 alone, avoiding the progression of clinical disease in sheep [125].

A feature of BTV infection is the generation of tubular structures of ~52.3 nm in diameter and ~100 nm in length within the cytoplasm of infected cells [193], originated from the non-structural protein 1 (NS1) multimerization. The 64 kDa NS1 protein, which acts as a positive regulator of viral protein synthesis [194], is the most abundant viral protein expressed in the cytoplasm of BTV-infected cells and is the only one responsible for the formation of these characteristic dynamic helical tubules [193,195,196,197]. Akin to the coat of a filamentous phage or tobacco mosaic virus displaying foreign antigens on their surface [198,199,200,201,202,203], these NS1 multimeric structures have been proposed as an efficient particulate delivery system as long as they are easily purified, highly stable at high temperatures (between 30 and 45 °C) and at a pH of 7 to 8.5, can accommodate bigger polypeptides than VLPs (up to 552 amino acids [204]) and are composed of thousands of NS1 monomers, which would ensure a high exposure of the foreign antigen [193,204,205,206]. The C-terminus of the NS1 protein is regularly disposed on the surface of the tubules [197,207,208]. The addition of foreign epitope sequences (as fusion proteins or by cross-linking [209]) in the C-terminal end of NS1 did not interfere with tubule formation and allowed the exposure of antigenic sequences in the surface of NS1 tubules [207,208]. Interestingly, these chimeric structures showed immunogenicity against inserted antigenic sequences, successfully stimulating both cellular and humoral arms of the immune system in vivo [204,209,210,211,212] and showing a significant degree of protection against the pathogen of interest in some cases [210,211].

Considering recent studies that corroborate the capacity of NS1 to efficiently protect against BTV [124,125,127], it must be noted that the NS1-based VLP-like carrier has not been evaluated yet as a vaccine candidate against BTV. In view of the advantages of this technology, it is a question that needs to be addressed. If so, taking into consideration the proficiency of these NS1 tubules to simultaneously carry a mix of different foreign antigenic sequences [208], it would be feasible to delineate multivalent vaccines against different ruminant viral diseases. This is an important issue when considering a reduction in the time and costs of production, and faster and more effective vaccination campaigns. Alike to some evaluated multivalent viral vector vaccines [213,214,215,216,217], delivery of antigens from the Rift Valley fever virus (RVFV), lumpy skin disease virus (LSDV), peste des petits ruminants virus (PPRV) or rinderpest virus could lead to multivalent vaccine candidates against some of these ruminant pathogens including BTV.

African horse sickness is on the WOAH list of notifiable viral diseases and is one of the most lethal viral diseases that affect equids [4]. Mortality rates can reach up to 90%, which depends on the form of the disease and the presence of naturally resistant and/or previously infected horses [218]. Asiatic and European donkeys also suffer severe forms of AHS [218]. Other equids species, such as some zebra species and the African donkey, suffer the less severe form and act as primary maintenance reservoirs [219,220]. AHSV is endemic in regions of Africa, and causes epidemics in Europe and Asia [218]. Without forgetting working equids in low-income countries, AHS is a major economic threat to the high-performance horse production industry [4]. AHSV outbreaks are controlled by the quarantine of animals coming from endemic areas, stabling and vaccination [4]. Vaccination with live attenuated strains of AHSV is the unique available prophylactic measure to control this viral disease. The currently used LAV contains serotypes 1, 2, 3, 4, 6, 7 and 8. However, drawbacks shared with BTV LAVs, such as documented gene segment re-assortment between outbreak and vaccine strains and reversion to virulence of attenuated vaccine strains [221], plus the need of multiple vaccination doses over the years [222], urge the evaluation of alternatives. In this sense, inactivated, DNA, subunit, viral-vectored and reverse genetics-based vaccines, have been explored [19,223].

Analogous to BTV, co-expression of major core structural proteins VP3 and VP7 by recombinant baculovirus expression systems led to the generation of AHSV CLPs that resembled in size and appearance genuine AHSV cores, as demonstrated by electron microscopy [224]. Later, Maree et al. [225] reported the production of AHSV-9 VLPs by baculovirus-mediated co-expression of the four major structural proteins (VP2, VP5, VP7 and VP3) in insect cells. Importantly, the authors also showed the molecular interaction of VP2 or VP5 with CLPs, demonstrating that the simultaneous expression of both proteins is not imperative for individual attachment of these proteins to CLPs, resulting in what could be called as partial VLPs, composed of VP2 or VP5 along with VP7 and VP3. Nonetheless, partial VLPs, carrying either VP2 or VP5, have not been evaluated yet for their possible immunogenic potential. The authors also reported the formation of crystalline structures made of VP7, a phenomenon also observed during AHSV infection [226], and hypothesized them as the cause of a low VLP formation yield. To address this problem, rational design of a mutant of the highly hydrophobic VP7 resulted in an increased solubility and improved efficiency of CLPs (and VLPs as a consequence) production. Similarly, a mutated version of VP7 (seven amino acid substitutions in the N-terminal region) was designed to increase VLP concentration in Nicotiana benthamiana plant expression systems, in which fully assembled AHSV-5 VLPs were successfully expressed. Density gradient-purified VLPs showed immunogenicity in guinea pigs -VP2, VP5 and VP7 were recognized by sera from immunized animals- with NAbs titers ranging from 1:640 to 1:5120 against the homologous AHSV-5 and, importantly, with titers between 1:56 and 1:160 against the heterologous AHSV-8 [227]. Surprisingly, VP7-mutant VLPs were excluded for immunization of horses as authors exposed that wild-type-VP7 VLPs were purified in a similar yield to that of the mutated protein. In any case, AHSV-5 VLPs did induce high titers of NAbs in the high (200 µg; 1:320) and low (100 µg; 1:160) dose immunization groups against the homologous AHSV-5. Like guinea pigs, immunized horses also developed a strong heterologous neutralizing response against AHSV-8 (between 1:160 and 1:320). Two horses also reached a AHSV-4 neutralizing antibody titer of 1:112 after the booster dose although it might be a product of an anamnestic response due to previous exposure to the LAV [228]. Agroinfiltration of Nicotiana benthamiana dXT/FT plants was also exploited for production of immunogenic chimeric AHSV VLPs by exchanging VP2 proteins of different serotypes [70].

Presumably, the former reviewed strategies are serotype-specific and would require the combination of a cocktail of VLPs for design of multivalent vaccines as for BTV. One of the virus-encoded nonstructural proteins, NS1, is highly conserved between the different serotypes. In addition, this protein, which forms tubular structures in the cytoplasm of infected cells similar to BTV NS1 [229], contains T CD8+ epitopes within its aminoacidic sequence [230]. For those reasons, ARV muNS-MS were loaded with AHSV-4 NS1. Immunization of IFNAR(-/-) mice with two doses of muNS-Mi-NS1 elicited a T-cell-mediated immune response that partially protected against the lethal challenge with the homologous AHSV-4. Much better, heterologous combination with MVA expressing NS1 conferred sterile protection in IFNAR(-/-) mice. Despite AHSV-9 was not lethal in IFNAR(-/-) mice, the heterologous strategy also prevented mice from developing viraemia and weight loss after infection, indicating the multiserotype protective potential of NS1 [128]. Certainly, further research should be carried out on AHSV natural hosts to confirm the results observed in the mouse model.

EHD, caused by EHDV, is a non-contagious hemorrhagic disease included in the WOAH list of multispecies/transboundary diseases. EHD is historically associated with wild and domestic ungulates such as white-tailed deer and cattle, showing important mortality rates in white-tail deer[20]. Notably, EHDV infection in cattle has led to clinical cases, causing morbidity rates varying from 1% to 18% whereas sheep and goats suffer a subclinical infection and may play a role in EHDV epidemiology [20,231,232]. This virus has been detected in regions of North, Central and South America, Australia, Asia and Africa [231,232,233,234,235,236,237,238,239,240]. To date, there is no evidence of the presence of EHDV in Europe. However, its presence in this continent is feasible as EHDV has been detected in countries of the Mediterranean Basin such as Morocco, Algeria, Tunisia, Israel, Jordan and Turkey and this virus shares epidemiologic similarities with BTV [20]. The economic losses derived from EHDV infection of dairy cattle has been estimated in 2.5 million US dollars due to reduced milk and meat production during an outbreak in 2006 in Israel [241]. Thus, the presence of susceptible and naïve cattle in Europe makes an outbreak of EHDV a likely high-risk upcoming event.

The effort to develop next-generation vaccines against EHDV is not comparable to those made for the related orbiviruses BTV and AHSV. Up to seven distinct serotypes of EHDV have been identified so far, and, with the exception of autogenous vaccines used in the US, just a monovalent live attenuated vaccine and a bivalent (Ibaraki virus (EHDV-2)/bovine ephemeral fever virus) inactivated vaccine are commercialized and used in Japan [242]. Together with them, a recombinant subunit vaccine is the unique experimental candidate that has demonstrated efficacy in the natural host. Baculovirus-expressed VP2 induced a NAbs response in cattle and white-tail deer, and conferred sterile serotype-specific protection against EHDV challenge [243]. Recently, a reverse genetic system has been developed for EHDV, which could serve for novel EHDV vaccine generation [244,245]. In parallel, the knowledge gathered for BTV VLPs design and production was applied for the generation of EHDV CLPs and VLPs. As previously mentioned for BTV and AHSV, CLPs of EHDV are composed of the intermediate VP7 and the innermost VP3 proteins that act as scaffolds for the assembly of VP2 and VP5, the most variable proteins of EHDV, to generate VLPs. Alshaikhahmed and Roy [246] described the generation of CLPs and VLPs of serotype 1 of EHDV by using the baculovirus expression system. Rabbit immunization with these VLPs demonstrated their capacity to promote the induction of strong titers of homologous NAbs in presence of adjuvants. Cell-mediated immune responses were not evaluated. Low levels of NAbs against EHDV-1 related heterologous serotypes (EHDV-2 and EHDV-6) were also detected. They also showed that EHDV-1 CLPs can serve as scaffolds for the generation of VLPs of other EHDV serotypes, which relies on the high conservation degree of VP3 and VP7 [233,247]. Similarly, Forzan et al. [248] successfully explored the usage of the baculovirus expression system for the generation of field isolate EHDV-6 VLPs, although there is a lack of data on related immunogenicity and protection. Importantly, recombinant baculovirus EHDV VLPs production may be restrained by the same conditionings previously discussed for BTV VLPs. Therefore, plant-based production would be highly desirable.