Boosting PRRSV-Specific Cellular Immunity: The Immunological Profiling of an Fc-Fused Multi-CTL Epitope Vaccine in Mice

Abstract

The continuously evolving PRRSV has been plaguing pig farms worldwide for over 30 years, with conventional vaccines suffering from insufficient protection and biosecurity risks. To address these challenges, we identified 10 PRRSV-specific CTL epitopes through enzyme-linked immunospot assay (ELISPOT) and constructed a multi-epitope peptide (PTE) by linking them in tandem. This PTE was then fused with a modified porcine Fc molecule to create the recombinant protein pFc-PTE. Our findings indicate that pFc-PTE effectively stimulates PRRSV-infected specific splenic lymphocytes to secrete high levels of interferon-gamma (IFN-γ) and is predicted to be non-toxic and non-allergenic. Compared to PTE alone, pFc-PTE not only induced a comparable cellular immune response in mice but also extended the duration of the immune response to at least 10 weeks post-immunization. Additionally, pFc-PTE predominantly induced a Th1 immune response, suggesting its potential advantage in enhancing cellular immunity. Consequently, pFc-PTE holds promise as a novel, safe, and potent candidate vaccine for PRRSV and may also provide new perspectives for vaccine design against other viral diseases.

Keywords: CTL epitope; Fc; cellular immunity; porcine reproductive and respiratory syndrome virus (PRRSV); vaccine.

Figure 1

Schematic representation of animal grouping and immunization protocol.

Figure 2

Construction, evaluation, and purification of recombination proteins: (A) Assessment of the immunostimulatory efficiency of the 22 CTL epitopes. After synthesis, these epitopes were used to stimulate PBMCs from experimental pigs (n = 5) that had been immunized with the JXA1-R vaccine and subsequently challenged with the JXA1 wild-type strain. The secretion of INF-γ was quantified by ELISPOT assay. (B) Schematic diagram of the genetic structures of the recombinant proteins. (C) Prediction of antigenicity, immunogenicity, allergenicity, and toxicity. (D) SDS-PAGE of purified proteins. Sup: supernatant after ultrasonic disruption and centrifugation. FT: flow-through (containing unbound proteins), E1–E4: eluted proteins, 1 mL/tube. The red wireframe indicates the location of the purified proteins.

Figure 3

Recombinant proteins induce IFN-γ and IL-4 secretion from porcine splenocytes and mice: (A,B) ELISPOT analysis reveals IFN-γ and IL-4 secretion by PRRSV-specific splenocytes induced by PTE, pFc, and pFc-PTE. (C,D) Sustained cytokine production induced by recombinant proteins in mice. Bars show mean ± SD. ** p < 0.01, and *** p < 0.001, n.s. = not significant.

Figure 4

pFc-PTE induces a Th1-biased immune response: (A–C) Concentration of Th1 cytokines, IFN-γ, IL-2, and IL-12 induced by recombinant proteins in mice sera. (D–F) Concentration of Th2 cytokines, IL-4, IL-5, and IL-10 induced by recombinant proteins in mice sera. The tested sera were collected 21 days post-immunization. Bars show mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s. = not significant.