Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease

Abstract

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.

Fig 1

Clinical outcome after FMDV A12-WT and A12-SAP inoculation. Groups of three pigs were i.d. inoculated with different doses of A12-WT (10⁵ or 10⁶ PFU/animal) or A12-SAP (10⁵, 10⁶, or 10⁷ PFU/animal), and temperature and clinical signs (A) and the presence of virus in serum and nasal swabs (B) were monitored daily for 7 dpi. Clinical score is expressed as the number of toes showing lesions plus one more point scored when lesions were present in either the mouth or snout, or both (the maximum score is 17). Virus levels are expressed as the number of PFU per ml in plasma or medium (in which the swabs were collected). Each data point represents the mean (±SD) of each group.

Fig 2

Presence of antibodies in serum of inoculated animals. (A) Serum neutralization titers. The neutralizing antibodies of swine inoculated with different doses of A12-WT (10⁵ or 10⁶ PFU/animal) or A12-SAP (10⁵, 10⁶, or 10⁷ PFU/animal) were determined at 0, 7, 14, and 21 dpi. Titers are expressed as the log₁₀ of the inverse dilution of serum yielding a 70% reduction in the number of plaques (PRN₇₀). (B) Immunoprecipitation of structural and NS viral proteins with serum of animals inoculated with different doses of FMDV A12-WT (10⁵ PFU/animal or 10⁶ PFU/animal) or A12-SAP (10⁵ PFU/animal, 10⁶ PFU/animal, or 10⁷ PFU/animal). Cytoplasmic extracts of FMDV A12-WT-infected BHK-21 cells labeled with [³⁵S]methionine/[³⁵S]cysteine were immunoprecipitated with serum from infected animals after 21 dpi and with convalescent-phase serum as a positive control (lane C). The products were examined by SDS-PAGE on a 15% gel. (C) Antibody isotype profiles in swine sera after infection. FMDV-specific IgM, IgG1, and IgG2 were detected by sandwich ELISA at 7, 14, and 21 dpi. Each data point represents the mean (±SD) of each group. OD, optical density.

Fig 3

Cytokine protein profiles in serum after FMDV infection. Levels of IFN-α (A) and IL-10 (B) in the serum of animals inoculated with FMDV A12-WT (10⁵ or 10⁶ PFU/animal) or A12-SAP (10⁵, 10⁶, or 10⁷ PFU/animal) during the first 4 days after infection were detected by sandwich ELISA. Amount of protein is expressed in relative levels for each individual animal at different times postinfection with respect to its own level at day 0. The gray areas represent time points at which a statistically significant difference from the amount at 0 dpi was observed (P < 0.05).

Fig 4

Cytokine protein profiles in serum after FMDV infection. Levels of IL-1β (A), IL-6 (B), and TNF-α (C) in the serum of animals inoculated with FMDV A12-WT (10⁵ or 10⁶ PFU/animal) or A12-SAP (10⁵, 10⁶, or 10⁷ PFU/animal) during the first 4 days after infection were detected by sandwich ELISA. Amount of protein is expressed in relative levels for each individual animal at different times postinfection with respect to its own level at day 0. The gray areas represent time points at which a statistically significant difference from the amount at 0 dpi was observed (P < 0.01).

Fig 5

Clinical outcome of animals vaccinated with attenuated A12-SAP after FMDV A12-WT challenge. Groups of three pigs were s.c. vaccinated with A12-SAP (10⁶ PFU/animal) and i.d. challenged at different times postvaccination (2, 4, 7, and 14 dpv) with 5 × 10⁵ PFU/animal of A12-WT, and clinical signs (bars) and the presence of virus in serum (solid lines) and nasal swabs (dashed lines) were monitored daily during various dpc. Clinical score and virus levels are expressed as described in the Fig. 1 legend. The error bars represent the variation within the three animals from each group.

Fig 6

Serum neutralization titers of vaccinated and control animals at the day of challenge and up to 14 dpc. The neutralizing antibodies of swine vaccinated at different time points (2, 4, 7, and 14 dpv) with attenuated A12-SAP (10⁶ PFU/animal) were measured at the day of challenge (arrow) with A12-WT (5 × 10⁵ PFU/animal) and at 4, 7, and 14 dpc. Titers are expressed as described in the Fig. 2 legend.

Fig 7

Cell-mediated immunity induced by A12-SAP vaccination. Specific cellular response was measured by intracellular cytokine staining (ICCS). PBMCs from A12-SAP-vaccinated and control animals were extracted at different times before (dpv) and after (dpc) challenge with A12-WT and stimulated with homologous FMDV A12-WT, and the capacity of CD8⁺ T cells to produce IFN-γ was evaluated by ICCS. The percentage of CD8⁺ T cells that produce IFN-γ is shown. A vertical dashed line separates the data between vaccination and challenge. The error bars represent the variation within the three animals from each group. *, P < 0.05; **, P < 0.01.

Fig 8

Cytokine profile in animals inoculated with FMDV A12-SAP or FMDV A12-WT. Pro- and anti-inflammatory cytokines were detected in serum by ELISA (A) or in PBMCs by rRT-PCR (B). (A) Levels of IFN-α, IL-10, IL-1β, IL-6, and TNF-α are expressed relative to the amount detected at day 0. (B) Relative mRNA levels of IL-10, IL-1β, IL-6, and TNF-α were determined by comparative cycle threshold analysis utilizing as a reference the samples at 0 dpi. Only values of ≥2 are considered upregulated. The error bars represent the variation within the three animals from each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.