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.