Identification of potential SLA-I-restricted CTL epitopes within the M protein of porcine reproductive and respiratory syndrome virus

Highlights

• Eight T-cell immunodominant epitopes of PRRSV-2 M protein were validated.

• 3 potential CTL peptides and their SLA-2 binding domain residues were identified.

• PRRSV-2 M protein peptides M₂₋₅ & M₆₋₁ bound SLA-2*0502; M₉₋₁ bound SLA-2*1201.

• PRRSV-2 M protein peptides were tested for SLA binding via rapid in vitro assays.

• ELISPOT, in-silico prediction, ELISA and peptide complex refolding tests were used.

Abstract

CD8+ cytotoxic T lymphocytes (CTLs), are essential for clearance of porcine reproductive and respiratory syndrome virus (PRRSV) infection and regulation of host immune responses. Identification of SLA I-restricted CD8+ CTL epitopes would facilitate PRRSV vaccine development. Here, cells isolated from peripheral blood mononuclear cells (PBMCs) of PRRSV-immunized Large White pigs (JXA1-R strain) were screened for immunodominant PRRSV-2 M protein T cell epitopes via ELISPOT assay. Of nine immunodominant epitopes detected, eight elicited significant IFN-γ secretion responses that varied among individual pigs and according to epitope. To predict which epitopes harbored potential CTL epitopes, swine leukocyte antigen (SLA) class I genes of Large White pigs were cloned and sequenced, yielding fourteen distinct SLA class I gene sequences. Based on ELISPOT and SLA genotyping results, SLA-restricted binding of the fourteen predicted class I proteins to peptides derived from the eight immunodominant epitopes were predicted in-silico. After evaluation of 42 pET-peptide-SLA-I-β2m complexes containing predicted restricted peptides, extracellular SLA class I domains and β2m, ELISA testing of 33 peptide-SLA-I-β2m complexes detected four complexed peptides. These four peptides were evaluated using in vitro complex refolding assays that confirmed that M₂₋₅ and M₆₋₁ peptides each formed complexes with SLA-2*0502 and sβ2m, while M₉₋₁ formed a complex with SLA-2*1201 and sβ2m. ELISPOT results confirmed these three 9-mer potential CTL epitopes efficiently stimulated IFN-γ secretion when presented by SLA class I molecules specified here. This study describes effective CTL epitope identification methods for use in future investigations of swine cellular immunity toward T cell-based vaccine development.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) has economically devastated the swine industry worldwide for nearly 30 years (Lunney et al., 2016). The virus that causes PRRS, PRRSV, is known to induce immunosuppressive respiratory disease in pigs that can lead to severe morbidity, secondary microbial infections and PRDC. Development of efficacious PRRSV vaccines has been a challenge, owing to the fact that the virus mutates rapidly, leading to poor vaccine performance against heterologous challenge (Montaner-Tarbes et al., 2019). Therefore, failed PRRS prevention and control efforts stemming from complexities of PRRS disease have permitted continual spread of this disease (Du et al., 2017).

PRRSV can cause prolonged infection of pigs with meager innate immune function, inadequate T cell responses and sluggish, inconsistent PRRSV neutralizing antibody (NAb) responses (Meier et al., 2003). Therefore, boosting of both antibody- and cell-mediated anti-PRRSV immune responses are essential for controlling PRRSV spread, with cell-mediated immunity (CMI) playing a critical role in development and regulation of antigen-specific immune responses (Loving et al., 2015). Importantly, efficient cellular immunity has been shown to effectively eliminate PRRS viremia by inducing local immunity and clearance of lung lesions (Kick et al., 2019; Madapong et al., 2020). Thus, it is essential that researchers gain better understanding of T cell-based immune responses.

The non-glycosylated membrane protein (M protein) encoded by PRRSV ORF6 is highly conserved structural protein with high homology among diverse PRRSV strains (Meng et al., 1995; Kappes and Faaberg, 2015). The M protein has been shown to play a crucial part in virus assembly, budding and viral neutralization (Lunney et al., 2016; Du et al., 2017). Additionally, PRRSV M protein has been shown to induce T cell proliferation and IFN-γ synthesis when used to immunize pigs (Jeong et al., 2010). Accordingly, M protein is considered to be a vital candidate target antigen for inducing cell-mediated immunity and for developing an efficacious vaccine.

As CTLs play a significant functional role in controlling host infection, it is plausible that triggering of an effective CTL response might improve the degree of protection afforded by a given vaccine. In this study, we describe a systematic strategy for identifying CTL epitopes. We first identified eight T cell immunodominant epitopes within the PRRSV-2 M protein. Next, SLA class I alleles of five Large White pigs used to obtain immunodominant epitope data were cloned and sequenced for in-silico prediction of CTL epitopes. Based on results of SLA genotyping and ELISPOT assays, SLA-I genes and their restricted peptides derived from the eight confirmed immunodominant epitopes were predicted using a rational bioinformatics-based approach. Next, peptide-SLA-I-β2m complexes were generated and tested via ELISA to identify which peptides were present within the complexes. Next, four peptides detected within complexes were further tested via refolding assays that subsequently confirmed that M₂₋₅ and M₆₋₁ could each form complexes with SLA-2*0502 and sβ2m, while M₉₋₁ formed a complex with SLA-2*1201 and sβ2m. ELISPOT results confirmed that these 9-mer potential CTL epitopes could efficiently elicit IFN-γ responses when presented by SLA molecules belonging to the specific SLA background tested here. Methods used here led to identification of useful CTL epitopes toward the development of next-generation PRRSV vaccines.