Analysis Of Immune Response In Swine Within The “Grey Zone” In Antibody Response After Immunization With The Inactivated Foot-and-Mouth Disease Vaccine

McCullough et al have revealed that the level of protection in vaccinated animals after challenge with foot and mouth disease virus(FMDV) is closely related to the antibody titer, which can be classified into three zones: a “white zone”, a “grey zone”, and a “black zone”. Animals in the white zone have high antibody levels and are likely to be fully protected against infection with FMDV; animals in the black zone have very low antibody levels and are susceptible to infection; and animals of the grey zone have intermediate antibody titers and unpredictable protection against FMDV infection. The aim of the present study was to clarify the immune mechanisms operating in the grey zone, in which vaccinated animals have intermediate antibody titers, making it difficult to predict the level of protection.To illuminate the mechanism responsible for the grey zone, thirty-three healthy pigs were immunized with inactivated FMD vaccine and challenged with homologous virus at 28 days post vaccination and then followed by observation for 10 days. Anticoagulated and whole blood were collected at 0 and 28 days post vaccination(dpv) and used for the following studies. Serum antibody titers were determined by liquid phase blocking-ELISA(LPB-ELISA) of serotype O, the distribution of lymphocyte subpopulations in whole blood was analyzed by the flow cytometry analysis and the level of cytokines were measured with Quantibody array kits. The antibody titer scopes of grey zone were ascertained according to the relationship between serum antibody titer at 28 days post vaccination and the incidence of animals after challenge. The results indicated that the antibody titer in pigs in the grey zone ranged from 1:6–1:45. The distribution of Cytotoxic T lymphocyte subpopulations, expression levels of Th1 cytokines such as TNF-α, IFN-γ, interleukin IL-12, IL-15, IL-18, MIG, and of GM-CSF, IL-1α, TGF-α, and TWEAK R varied after immunization between protected and unprotected animals in the grey zone. The percentage of CTLs increased after vaccination in protected pigs within the grey zone, but decreased significantly in the unprotected group. Compared with before vaccination, concentrations of IFN-γ, IL-12, GM-CSF, IL-1α, IL-15, and TNF-α increased at 28 dpv in protected pigs in the grey zone, but remained unchanged or decreased in unprotected animals. MIG and TWEAK R levels remained largely unchanged in the protected pigs but were reduced in the unprotected animals. The contents of IL-18 and TGF-α reduced in protected pigs at 28 dpv, but remained unchanged or declined, respectively, in the unprotected group. The results of this study suggest that Cytotoxic T lymphocyte subpopulations, expression levels of Th1 cytokines such as TNF-α, IFN-γ, IL-12 and IL-15 that represent the cellular immune response can increase anti-virus ability of animals, indicating that the cellular immune response is a key factor responsible for immunoprotection in vaccinated animals with antibody titers within the grey zone.Another thirty-three pigs were used to investigate the changing trend of lymphocyte subpopulations in the peripheral blood after vaccination. Blood samples were collected at 0, 1, 3, 5, 7, 10, 14, 21 and 35 day after vaccination and assayed with flow cytometry. Serum antibody titers were also determined with LPB-ELISA assay kits of serotype O. The results showed the changing trend of B cells proportion was highly relevant with Th cells and a same correlation also appeared in the antibody titers. This phenomenon revealed that the interaction between B cells and Th cells have significant effects on humoral immune response. The proportion of dendritic cells(DCs) increased significantly in a short time after vaccination indicating this kind of cell can be activated quickly to exert its function. The results showed that the innate immune system could be activated rapidly after vaccination with the inactivated vaccine, and the adaptive immune response including cellular immune response and humoral immune response could be invoked subsequently. The increased proportion of B cells and Th cells suggested that the humoral immune response was activated and the high level and high affinity of neutralizing antibodies was of great importance in evading FMDV. The proportion of CTLs increased after vaccination indicating the cellular immune response also exerted a crucial role in evading FMDV.