4. Discussion
Current vaccines against FMD consist in inactivated FMDV plus an oil-adjuvant. The virus is produced in suspension-growing BHK-21 cells and then inactivated with binary ethyleneimine (BEI) (Doel, 2003; OIE - World Organisation for Animal Health Act N° 22, 2018). This process needs high biosafety production facilities (BSL-4 OIE or higher), but requires strict protocols of production and constant investments in manufacturing plant up-grades and personnel qualifications. In addition, oil adjuvants as imports increase the costs of vaccines, especially in developing countries. Because of this, a new formulation using recombinant VLPs and a low-cost adjuvant that can be locally prepared are a good alternative to the marketed vaccine.
In this work, we examined the capacity of ISPA, a new cage-like particle adjuvant to elicit a protective and specific immune response against FMDV when used in VLPs-based vaccines. For that purpose, we used a mouse model developed in our laboratory, in which the humoral and protective immune responses against FMDV in mice correlate with those elicited in cattle (Batista et al., 2010; Bidart 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 the murine model, the use of ISPA in VLPs-based vaccines allowed to achieve 100% protection against viral challenge while the use of VLPs alone induced a protective response in only 40% of the animals. VLPs induced similar protection levels when using ISPA as adjuvant or CA. Animals vaccinated with adjuvant alone were not protected against viral challenge, showing that the protective response corresponded to an adaptive response against the virus and was not due to innate immune mechanisms elicited by the adjuvant.
Ab titers against FMDV were significantly elevated in mice that received VLPs-ISPA as compared to the group vaccinated with VLPs alone and similar to the Ab titers induced by the VLPs-CA vaccine. These results correlate with the protection induced upon challenge It is important to mention that the levels of protection achieved were the same as those induced by the Commercial Vaccine (positive control) based on inactivated virus. It is noteworthy that neutralizing antibody titers showed a good correlation with protection levels, substantiating the notion that they are an in vitro reflection of the immune response that occurs in vivo (Mattion et al., 2009; McCullough et al., 1992; OIE - World Organisation for Animal Health, 2012). Also, Ab titers against FMDV and VNT values induced in mice by VLPs-ISPA and VLPs-CA were higher than the response induced by VLPs alone. Furthermore, these humoral responses elicited by VLPs-ISPA and VLPs-CA vaccines were enough to induce a 100% protection against viral challenge, in a similar way to the protection levels achieved with the commercial vaccine containing the inactivated virus. Noteworthy, the commercial vaccine contains FMDV serotype O1/Campos/Brazil/58, A24/Cruzeiro/Brazil/55, C3/Indaial/Brazil/71 and A/Argentina/2001, all of which bear epitopes that participate in the immune response against FMDV (Mattion et al., 2004)
When isotype profiles were characterized, increases in IgG1, IgG2a, IgGb and IgG3 isotype levels were observed in the VLPs-ISPA, VLP-CA and Commercial vaccine groups as compared to the group inoculated with VLPs alone. Interestingly, a prevalence of IgG1 and IgG2b isotypes was detected in animals vaccinated with VLPs-ISPA. These are promising results because Gnazzo et al (2020) reported that in the mouse model, IgG1 and IgG2b isotypes are important in the protection against FMDV O1 Campos\sout. VLPs-ISPA-vaccinated animals showed higher IgG3 isotype levels than the VLPs-CA group. It is well described that IgG1 and IgG3-FMDV immune complexes are recognized by FcγRI receptors, present in macrophages, monocytes and dendritic cells, leading to a virus clearance (van der Poel et al., 2011). Thus, this mechanism could be involved in VLPs-ISPA protection. Also, IgG2a, IgG2b and IgG3 isotypes play a significant role in FMDV clearance trough complement-mediated phagocytosis (McCullough et al., 1988; Pérez Filgueira et al., 1995). Moreover, it has been reported that mice inoculated with inactivated FMDV plus some adjuvants generate a complement-fixing IgG profile that correlates with protection upon FMDV challenge (Batista et al., 2010; Pérez Filgueira et al., 1995). The different isotype profiles elicited by inoculation with these vaccines indicate a balanced Th1/Th2 response.
Regarding the cellular immune response obtained in mice using the experimental vaccines, both VLPs-ISPA and VLPs-CA induced an increased response compared to the VLPs group. These results suggest that ISPA and the Commercial Adjuvant improve the adaptive immune response against FMDV.
The VLPs-ISPA vaccine triggered proliferation and IFNγ production in FMDV-specific CD8+ T lymphocytes, as occurred in mice vaccinated with inactivated FMDV (C Langellotti et al., 2012; Ostrowski et al., 2005). Similar results were obtained in the VLPs-CA group. Surprisingly, in the group immunized with Commercial Vaccine, although lymphoprolferation was observed when splenocytes were stimulated with inactivated virus, IFNγ secretion only by CD4+ cells, but not by CD8+ cells, was registered (Supplementary Figure 1).
Previous studies described that ISCOMs improve the dendritic cell cross-presentation (den Brok et al., 2016; Maraskovsky et al., 2009; Wilson et al., 2012, 2014). The results described in the present work indicate that the VLPs- ISPA formulation generates a strong cellular response in agreement with previous reports that used cage like particles (Bertona et al., 2017; Prochetto et al., 2017).
The results of the humoral immune response profiles obtained in the murine model were further confirmed in calves, as was reported by Gnazzo et al. (2020) (Gnazzo et al., 2020).
Numerous studies performed in cattle show a correlation between Ab titers against FMDV elicited by vaccination and in vitro andin vivo protection upon experimental viral challenge. These correlations have allowed to estimate the Expected Percentage of Protection (EPP) to the homologous infection using titers of systemic α-FMDV Ab measured by lpELISA or viral seroneutralization (Maradei et al., 2008; Mattion et al., 2009; OIE - World Organisation for Animal Health, 2012).
In calves, one dose of the VLPs-ISPA formulation elicited total and neutralizing anti-FMDV Ab titers corresponding to an Expected Percentage of Protection (EPP) above 90%, similar to those obtained with inactivated FMDV and CA (Maradei et al., 2008; Robiolo et al., 2010; Servicio Nacional de Sanidad y Calidad Agroalimentaria Res 609/2017. CABA, Argentina, 2017). Importantly, an acceptable inactivated vaccine should induce 75% protection in cattle (OIE - World Organisation for Animal Health, 2012).
Moreover, IgG2 titers were higher in VLPs plus adjuvant formulations which is related to pathogen opsonization. Bovine macrophages and neutrophils possess an immunoglobulin receptor to which IgG2 can bind (Tizard, 1998). On the other hand, we detected a significant increase in IFN-γ secretion in cattle vaccinated with VLPs-ISPA as compared to cattle vaccinated with VLPs alone or VLPs-CA. IFN-γ has been reported to display activity against FMDV (Summerfield et al., 2009), by controlling viral replication and spreading within the host through natural killer cell and macrophage activation (Zhang et al., 2002). Our data of increased IFN-γ and increased presence of IgG2 in animals vaccinated with VLPs-ISPA, correlate with what was found by other researchers. Thus, a positive correlation between IFN-γ response and vaccine-induced protection as well as reduction of long-term persistence of FMDV has been observed in cattle (Oh et al., 2012). In addition, IFN-γ production is associated with a Th1 profile and an increased IgG2 production (Estes & Brown, 2002). The numbers of cattle included in this pilot study were as those used in other preliminary studies on vaccine candidates (Bachmann & Zinkernagel, 1997; Lee et al., 2005), although they were insufficient for statistical analysis (Soria et al., 2017). However, the results obtained serve as a proof of concept of the usefulness of VLPs as antigen and ISPA as adjuvant in FMDV vaccines. Although in calves, the iFMDV-CA group is too small to make any statistical comparisons, the neutralizing Ab titters induced by the vaccine containing the complete inactivated FMDV and commercial adjuvant were similar to those induced by VLPs with ISPA or CA.
The action of ISPA was similar in terms of levels, kinetics and profiles of humoral responses than the commercial oil adjuvant used in this study. ISPA has also been reported to have a better performance than ISCOMATRIX™ (Bertona et al., 2017). Importantly, particle size and adjuvant performance were conserved during a six-month period after preparation (Bertona et al., 2017). Future work will be devoted to examine whether ISPA exerts its action of enhancement of the immune response and protection levels by promoting the virus presentation to the immune effectors. Some authors have reported that ISCOMs induce local recruitment, activation and maturation of immune cells, such as dendritic cells, granulocytes, F4/80 int cells, T-, B- and NK-cells (Reed et al., 2009; Reimer et al., 2012; Sun et al., 2009), increasing in this way the chances of the antigen to come in contact with immune cells. In addition, Brok et al. (2016) proved that saponin-based adjuvants enhance antigen cross-presentation by dendritic cells and T-cell activation (den Brok et al., 2016). Immunization with a formulation based on Tripanosoma cruzi recombinant transialidase (mTS) and ISPA elicited high levels of protection upon challenge (Bertona et al., 2017). Moreover, this vaccine favorably modulates the regulatory arm of the immune system to reach immune protection against the parasite (Prochetto et al., 2017).
Noteworthy, VLPs-ISPA and VLPs-CA elicit high FMDV Ab titers. In cattle, the immune response elicited by FMDV (A22 Iraq strain) VLPs produced using the baculovirus technology and oil adjuvant was analyzed in a study by Porta et al. (2013) (Porta et al., 2013). Two vaccinations with 12 µg/dose elicited neutralizing Abs and 50% protection against challenge. When the capsids were mutated to increase their stability, the protection levels induced were of 75%. In another study, Li et al (2012) showed 80% protection with one dose of A/WH/CHA/09 VLPs-ISA206 formulation (Li et al., 2012). Our results show a comparable or higher performance of the VLPs/ISPA and VLPs/CA formulations since with one 25 µg dose, the levels of induced neutralizing Abs correlate with 90% of protection.
The recombinant VLPs used in this study were produced by transient transfection of suspension-growing mammalian cell cultures in serum free medium using a pTT5-based plasmid. This technology is a safe and cost-effective method of antigen production to develop a new non-infectious vaccine for controlling FMD in vaccination-free countries, as well as in FMD-endemic countries and in the event of an outbreak.
In conclusion, ISPA is a new cage-like particle adjuvant that can be used in combination with recombinant VLPs to develop a novel vaccine against FMD. Results showed ISPA is useful in increasing and modulating the humoral and cellular responses in vaccinated mice and cattle, yielding enhanced protection against challenge. Taken together, these results are promising for the development of a novel FMD vaccine with a recombinant, non-infectious antigen and a new, cost-effective and innovative adjuvant.