The VP1 G-H loop hypervariable epitope contributes to protective immunity against Foot and Mouth Disease Virus in swine

Abstract

Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV) possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization. Here, we investigate the effect of replacement of the G-H loop hypervariable epitope with a xenoepitope from PRRS virus on the immunogenicity and efficacy of an adenovirus vectored FMDV vaccine (Ad5-FMD). Pigs were vaccinated with Ad5-FMD, the modified Ad5-FMD_xeno, or PBS, followed by intradermal challenge with FDMV strain O₁ Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMD_xeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMD_xeno, when compared to those vaccinated with Ad5-FMD, prior to viral challenge, indicative of immune redirection away from VP1 towards non-neutralizing epitopes. As expected, animals vaccinated with unmodified Ad5-FMD were protected from lesions, fever, and viremia. In contrast, animals vaccinated with Ad5-FMD_xeno developed clinical signs and viremia, but at lower levels than that observed in PBS-treated controls. No significant difference was found in nasal shedding of virions between the two Ad5-FMD vaccinated groups. This data suggests that the hypervariable epitope of the VP1 G-H loop contributes to protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of immunogenicity.

Introduction

Foot-and-Mouth Disease is a highly transmissible viral disease that can result in enormous economic losses to the livestock industry worldwide [13], [14]. Although typically effective, prophylactic vaccination against FMDV is complicated in part due to the high rate of viral mutation, which results in year-to-year strain differences rendering some vaccination efforts unsuccessful [13], [15]. It has been speculated that viral evolution along with relatively easy adaptation to many susceptible hosts results in heterogeneity within the FMDV VP1 capsid protein which may be responsible for some vaccine failures [42], [44].

FMDV is a small RNA virus of the Aphthovirus genus in the Picornaviridae family [1], [21], [22], [26]. The FMDV genome comprises 8.4 kilobases and codes for 12 proteins, which must be proteolytically cleaved to form mature and functional proteins [21]. The virus forms an icosahedral capsid comprising 60 copies each of the structural viral proteins VP0, VP1 and VP3, and organized in the form of twelve pentamers. A final maturation cleavage of VP0 occurs in the presence of RNA to produce VP4 (the N-terminal 85 residues of VP0) and VP2. VP1, 2, and 3 are surface exposed, with VP1 surrounding the 5-fold axes of symmetry, and VP2 and VP3 alternating around the icosahedral 3-fold axes [1]. FMDV structural proteins are important facets of immune recognition of the virus [2]. Within VP1, the G-H loop plays a major role in the viral infection cycle, largely due to the highly conserved RGD amino acid motif [10]. This motif binds to αV family of integrin receptors expressed on host epithelial cells to mediate viral entry, and strains lacking this domain have diminished abilities to infect permissive cells [3], [6], [7], [18].

Immune recognition of RNA viruses can be complicated by rapid mutation of progeny virions during the course of infection. Low fidelity ‘error-prone’ RNA polymerases incorporate mutations that may affect immunologically relevant epitopes, increasing viral variance and thus presenting substantial hurdles for vaccine development. Within the type O VP1 G-H loop, an eight amino acid region has been identified as hypervariable (HV), and its proximity to the highly conserved and immunogenic RGD motif has suggested a potential role of this element in immune evasion by the virus [4], [11], [31], [32], [41], [43]. Some reports have shown that mutations in this epitope are well tolerated by the virus, but have a detrimental impact on antibody binding affinity and virus neutralization [2], [31], [43], [47], [49]. However, in some cases, related mutants induced significant levels of neutralizing antibodies in the natural host, comparable to those elicited by wild type (WT) virus [30], [40], [47], [48]. Additionally, it has been shown that the VP1 G-H loop is a highly immunogenic site of FMDV, with some reports indicating that more than 25% of neutralizing antibodies elicited during FMDV infection are directed towards this domain of VP1 [33], [45].

We have previously found that replacing the HV region of the VP1 G-H loop could broaden immunity in mice, wherein antibodies induced by a peptide vaccine were found to be capable of binding to heterologous isolates [43]. However, in other mouse studies, it was demonstrated that complete removal of the G-H loop did not induce sufficient protection against lethal challenge, suggesting a key role for this region in eliciting protective immunity [19]. For this reason, we aimed to identify whether stable replacement of the G-H loop HV region by an unrelated epitope could broaden immunity without affecting the immunogenicity of an Ad5-vectored FMD vaccine in swine, a natural host for FMDV. The 8 amino acid HV region of the VP1 G-H loop was replaced with a xenoepitope from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) gp5 protein, in the context of an Ad5-FMD Manisa capsid-based vaccine [16]. Similarly to other Ad5-FMD vaccines directed to serotype A [34], [35], [38], this construct has been demonstrated to be highly efficacious in swine and cattle [14], [16]. The modified vaccine, denoted Ad5-FMD_xeno, was assessed for protective efficacy in swine, as compared to an unmodified Ad5-FMD which delivers O₁ Manisa full capsid antigen.