Experimental Rhodococcus equi and equine infectious anemia virus DNA vaccination in adult and neonatal horses: effect of IL-12, dose, and route

Abstract

Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates.

Fig. 1

(A) Immunoblot of purified rEqIL12 reacted with Anti-V5 MAb. pBudCE4 control reduced (Lane 1), pBudCE4 non-reduced (Lane 2), pIL12 reduced (Lane 3), pIL12 non-reduced (Lane 4). Migration of molecular weight markers in kilodaltons is indicated by arrows on right side of panel. (B) Functional activity of rEqIL12 was measured using a thymidine uptake proliferation assay. Serial dilutions of media from pIL12 or pBudCE4-transduced 3T3 cells were added to PHA-P stimulated PBMC then harvested 3 days later and counted. rEqIL12, pBudCE4, Con A.

Fig. 2

Tetramer analysis: Immunization with VR-p15/p26 with or without EqIL-12 induced Gag-GW12-specific CD8+ T cells. PBMC obtained from vaccinated horses A2185, A2201, A2190, and A2192 at the indicated time-points were stimulated for 1 week with Gag peptide pool 1, then labeled with the 7–6/Gag-GW12 tetramer, and percent tetramer positive CD8+ cells were determined on CD3+ gated lymphocytes using flow cytometry. A2185 and A2201 were immunized with VR-p15/p26 + EqIL-12; A2190 and A2192 were immunized with VR-p15/p26 without Eq-IL12.

Fig. 3

Cytotoxic T lymphocytes: Immunization with VR-p15/p26 with or without EqIL-12 induced Gag-specific CTL in adult horses. PBMC obtained at the indicated time-points were stimulated for 1 week with Gag peptide pools 1–7 and CTL activity determined on Gag peptide pool-pulsed EK targets for vaccinated horses (a) A2185, (b) A2201, (c) A2190, and (d) A2192. Error bars are S.E. E:T ratio, 50:1. Significant specific lysis is indicated with an asterisk. A2185 and A2201 were immunized with VR-p15/p26 + EqIL-12; A2190 and A2192 were immunized with VR-p15/p26 without Eq-IL12.

Fig. 4

Lymphoproliferation: Immunization of adult horses with VR-p15/p26 with or without rEqIL-12 and immunization of foals with VR-p15/p26 alone induced EIAV antigen-specific lymphoproliferative responses. Peripheral blood mononuclear cells obtained at the indicated time points were stimulated for 5 days with Gag peptide pool and p15 peptide. (A) Horses A2185 and A2201 were co-immunized with rEqIL-12 DNA and horses A2190 and A2192 were co-immunized with the pBudCE4 vector control. (B) Foals were immunized with VR-p15/p26 alone. An asterisk (*) indicates an SI of at least 3× the pre-immunization SI for that antigen and a corresponding PWM SI of 10 or greater.