Evaluation of immunogenicity and cross-reactive responses of vaccines prepared from two chimeric serotype O foot-and-mouth disease viruses in pigs and cattle

Abstract

Foot-and-mouth disease (FMD) remains a very serious barrier to agricultural development and the international trade of animals and animal products. Recently, serotype O has been the most prevalent FMDV serotype in China, and it has evolved into four different lineages: O/SEA/Mya-98, O/ME-SA/PanAsia, O/ME-SA/Ind-2001 and O/Cathay. PanAsia-2, belonging to the O/ME-SA topotype, is prevalent in neighbouring countries and poses the risk of cross-border spread in China. This study aimed to develop a promising vaccine candidate strain that can not only provide the best protection against all serotype O FMDVs circulating in China but also be used as an emergency vaccine for the prevention and control of transboundary incursion of PanAsia-2. Here, two chimeric FMDVs (rHN/TURVP1 and rHN/NXVP1) featuring substitution of VP1 genes of the O/TUR/5/2009 vaccine strain (PanAsia-2) and O/NXYCh/CHA/2018 epidemic strain (Mya98) were constructed and evaluated. The biological properties of the two chimeric FMDVs were similar to those of the wild-type (wt) virus despite slight differences in plaque sizes observed in BHK-21 cells. The structural protein-specific antibody titres induced by the rHN/TURVP1 and wt virus vaccines in pigs and cows were higher than those induced by the rHN/NXVP1 vaccine at 28-56 dpv. The vaccines prepared from the two chimeric viruses and wt virus all induced the production of protective cross-neutralizing antibodies against the viruses of the Mya-98, PanAsia and Ind-2001 lineages in pigs and cattle at 28 dpv; however, only the animals vaccinated with the rHN/TURVP1 vaccine produced a protective immune response to the field isolate of the Cathay lineage at 28 dpv, whereas the animals receiving the wt virus and the rHN/NXVP1 vaccines did not, although the wt virus and O/GXCX/CHA/2018 both belong to the Cathay topotype. This study will provide very useful information to help develop a potential vaccine candidate for the prevention and control of serotype O FMD in China.

Keywords: Immunogenicity; chimeric FMDV; cross-reactive responses; vaccines.

Figure 1

Schematic diagram of the exchange-cassette cloning strategy described in this study. The FMDV full-length cDNA clone pOFS was used for the construction of two chimeric genome-length cDNA copies. The specific genes (from nt 2924–4183) with the substitution of the VP1 gene of FMDV O/TUR/5/2009 and O/NXYCh/CHA/2018 were synthesized by a biotechnology company.

Figure 2

Growth characteristics of FMDVs in BHK-21 cells. A Plaque morphologies of the mutant viruses and wt virus. Monolayers were infected with tenfold serial dilutions of rHN/TURVP1, rHN/TURVP1 and rHN. After 1 h of adsorption, infected cells were overlaid with 0.6% gum tragacanth. The cells were fixed, stained and photographed at 48 h post-infection. B One-step growth curves for the mutant viruses and the wt virus. Monolayers were infected with rHN/TURVP1, rHN/TURVP1 and rHN (5 moi), and samples were analysed at 4, 8, 12, 16 and 20 hpi. The results were obtained from three individual experiments.

Figure 3

Electron microscopy of FMDVs. The mutant viruses and the wt virus (fifth passage) were cultured in adherent BHK-21 cells and harvested after the occurrence of 100% CPE. The virus particles were isolated by sucrose density gradient centrifugation and observed by negative-stain electron microscopy. A rHN/NXVP1, B rHN/TURVP1, C rHN. Bar = 100 nm.

Figure 4

The survival of suckling mice inoculated with FMDVs. Two-day-old BALB/c suckling mice were inoculated IP with tenfold dilutions of the mutant viruses and the wt virus and observed for 7 days. A The survival rate of suckling mice inoculated with rHN. B The survival rate of suckling mice inoculated with rHN/NXVP1. C The survival rate of suckling mice inoculated with rHN/TURVP1. (black filled circle) Dilution of 10^–5, (black filled square) dilution of 10^–6, (black filled triangle) dilution of 10^–7, (black filled inverted triangle) dilution of 10^–8.

Figure 5

The anti-structural protein antibodies in vaccinated pigs and cows. Groups of six pigs and six cows were vaccinated with the mutant virus (rHN/TURVP1 or rHN/NXVP1) or the wt virus vaccine at Days 0 and 28; blood samples were assayed by liquid-phase blocking ELISA (LPBE) on Days 0, 14, 21, 28, 35, 42, 49 and 56 post-vaccination. The results were obtained from three replicates of six animals. Error bars indicate SDs from the mean.

Figure 6

Comparisons of the virus neutralization antibody titres against homologous and heterologous viruses in immunized pigs and cattle. Groups of six pigs or six cattle were vaccinated with either the mutant virus (rHN/TURVP1 or rHN/NXVP1) or the wt virus vaccines on Days 0 and 28; blood samples were assayed by the virus neutralization test on Days 28 and 56 post-vaccination. The results were obtained from three replicates of six animals. Error bars indicate SDs from the mean. Statistical analysis was conducted using an unpaired t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant). A Mean virus-neutralizing antibody titre (VNT (log₁₀)) of six pigs vaccinated with three experimental vaccines at 28 dpv. B Mean VNT of six cows vaccinated with three experimental vaccines at 28 dpv. C Mean VNT of six pigs vaccinated with three experimental vaccines at 56 dpv. D Mean VNT of six cows vaccinated with three experimental vaccines at 56 dpv. The dotted line indicates the 1.65 log₁₀ VNT cut-off level.

Figure 7

Serological cross-reactive responses against heterologous FMDVs. A two-dimensional (2D) virus neutralization test was performed using sera from six cows vaccinated with the rHN, rHN/NXVP1 or rHN/TURVP1 vaccine at 28 dpv. The dotted line indicates the cut-off value of 0.3. Three experiments were performed independently, and the mean r₁ values were obtained from three replicates of six animals. Error bars indicate SDs from the mean.

Figure 8

Phylogenetic tree of FMDVs. A phylogenetic tree based on the VP1 nucleotide sequences was produced using MEGA 7.0. The VP1 sequences of FMDVs were obtained from GenBank. The viruses used in this study are marked with an inverted triangle (▼).

Figure 9

Comparison of amino acid sequences of the VP1 protein of FMDVs. Amino acid sequences of VP1 of O/HN/CHA/93, O/TUR/5/2009 and O/NXYCh/CHA/2018 were aligned using DNASTAR software package version 7.0. The VP1 sequences of these viruses were obtained from GenBank.