Introduction

Foot-and-Mouth Disease (FMD) is a highly contagious viral infection of cloven-hoofed animals. FMD is endemic in many parts of the world and continues to pose a major threat to livestock industries. Its presence in a country results in severe restrictions to international trade and an outbreak in FMD-free countries causes billionaire losses (OIE - World Organisation for Animal Health Act N° 22, 2018; Thompson et al., 2002). Prevention and eradication of the disease requires sustained efforts at a significant cost. Vaccination is a major strategy in developing countries to control FMD (Parida, 2009).
The etiological agent is Foot-and-Mouth Disease Virus (FMDV), a member of the Aphthovirus genus within the Picornaviridae family. FMDV has a single-stranded, positive sense RNA genome that is enclosed within a protein shell comprised of 60 copies of 4 structural proteins, VP1, VP2, VP3 and VP4. VP1-VP3 are surface exposed on the virus particles,, whereas VP4 is internal (Acharya et al., 1989). The capsid proteins facilitate virus entry and delivery of the genome into the cytoplasm of the cell where translation and replication of the viral RNA takes place, and new virus particles are formed (Belsham, 2005; Jackson et al., 2000; Monaghan et al., 2005). The immune system of virus-infected animals produces neutralizing Abs against the surface-exposed capsid proteins, which is a key requirement for protection (Doel, 2003).
Seven FMDV serotypes (A, O, C, SAT1, SAT2, SAT3 and ASIA1) and several subtypes within each serotype have been described. Viral infection or vaccination with one serotype does not confer protection against other serotypes (Mattion et al., 2004). Thus, an update of the antigenic composition of the vaccine is required when new field strains appear. The vaccine currently used consists on chemically inactivated virus. To produce this vaccine, suspension-growing baby hamster kidney-21 (BHK-21) cells are infected with virus and binary ethyleneimine is used for the inactivation process (Doel, 2003; Grubman & Baxt, 2004; OIE - World Organisation for Animal Health, 2012). There are disadvantages with the use of this type of vaccine, including the need for high biosafety production facilities, the risk of incomplete inactivation of the virus, the need of an strict purification process that guaranties differentiation of infected from vaccinated animals and the fact that some serotypes and subtypes have problems to grow in cell cultures (Grubman, 2005; Rodriguez & Grubman, 2009). Thus, the development of new vaccines is desirable.
Recombinant VLPs may represent a good alternative to the conventional FMD vaccine since they are non-infectious and can be produced without the need of high containment facilities and may also be modified to enhance their stability (Caridi et al., 2015; Ellard et al., 1999; Kotecha et al., 2015; Mateo et al., 2008; Porta et al., 2013; Rincón et al., 2014). The use of suspension-growing mammalian cells, economic and efficient transfection reagents and optimized expression vectors has allowed transient gene expression (TGE) to become a simple, scalable and powerful technology to generate large amounts of recombinant VLPs within a short time period (Baldi et al., 2007; Mignaqui et al., 2013; Pham et al., 2006).
Although some reports have demonstrated the efficacy of FMD vaccines based on VLPs (Li et al., 2012; Porta et al., 2013; Xiao et al., 2016), a lower performance of a VLP based vaccine can be expected when compared with a traditional vaccine due to the lack of viral RNA, a well-known activator of the innate immune response (Medina et al., 2018). Moreover, vaccines based on these empty capsid particles may still be expected to suffer from some of the same shortcomings, (e.g. in terms of duration of immunity)(Gullberg et al., 2016). The use of novel adjuvants can be a promising tool to improve the performance of these vaccines.
In the present work, FMDV serotype A/Argentina/2001 VLPs were produced by transient gene expression in serum-free suspension-growing mammalian cells, using polyethylenimine (PEI) as transfection reagent (Mignaqui et al., 2013). Serotype A/Argentina/2001, isolated during an outbreak of FMD in Argentina in 2000 (Mattion et al., 2004), was used in the present study as proof of concept.
Our laboratory has ample experience in a murine model that proved useful to evaluate the potency of FMDV vaccines (Batista et al., 2010; Bidart et al., 2020; Gnazzo et al., 2020; C Langellotti et al., 2012; Cecilia Langellotti et al., 2015; V Quattrocchi et al., 2011, 2013; Valeria Quattrocchi et al., 2005; Romanutti et al., 2013; Zamorano et al., 2010). In this model, there is a correlation with the humoral and protective immune responses against infective FMDV that take place in cattle (Gnazzo et al., 2020).
Vaccine adjuvants improve the immune response elicited against antigens, direct the immune response to a particular profile, increase the number of responding individuals, reduce the amount of vaccine doses and/or allow to reach homogenous immune responses (Mohan et al., 2013). It is of great importance to find new adjuvants that allow reducing the amount of virus or antigens in vaccines and induce Th1/Th2 responses. Other desirable characteristics include low cost and stability. It has been reported that Immune Stimulating Complexes (ISCOMs) are capable of developing a Th1/Th2 balanced immune response, in addition to increasing cytotoxic responses (Bertona et al., 2017; Maraskovsky et al., 2009; Singh, 2006; Sun et al., 2009). ISCOMs are spherical particles of approximately 40 nm in diameter, composed of phospholipids, cholesterol and saponin, which can retain the antigen through hydrophobic interactions (Morein et al., 1984; Singh, 2006). They have been applied to the development of several registered vaccines for veterinary applications (Sun et al., 2009). Recently, the Immunostimulating Particle Adjuvant or ISPA, an empty cage-like particle formulation similar to ISCOMATRIX™, was described. It contains dipalmitoyl-phosphatidylcholine (DPPC), cholesterol (CHO), stearylamine (STEA), alpha-tocopherol (TOCO) and Quil A saponin (Bertona et al., 2017; Bidart et al., 2020; Prochetto et al., 2017). This adjuvant was shown to surpass conventional ones by improving humoral and cellular CD4+ / CD8+ responses (Bertona et al., 2017). Recently, we reported that an inactivated FMDV serotype A vaccine adjuvanted with ISPA was capable of inducing protection against challenge in a murine model and of improving the specific immune responses against FMDV in cattle (Bidart et al., 2020).
In this report, we demonstrate for first time the effect of ISPA as adjuvant for a subunit vaccine using VLPs from FMDV both in a murine model and in cattle.