Short communicationIdentification of potential SLA-I-restricted CTL epitopes within the M protein of porcine reproductive and respiratory syndrome virus
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.
Section snippets
Virus
PRRSV live attenuated vaccine strain JXA1-R (Pulike Biological Engineering, Inc.) was cultured and titrated in MARC-145 cells. The JXA1-R (GenBank accession no. FJ548853.1) vaccine was obtained via 82 serial passages during continuous culture of HP-PRRSV JXA1 (GenBank accession no. EF112445) in MARC-145 cells (Yu et al., 2015).
Animal immunization, antibody detection and viremia detection
Eight 5-week-old Large White piglets from a PRRSV-free farm were confirmed via laboratory testing to be free of PRV, CSFV, PCV2 and seronegative for anti-PRRSV
Animal immunization and T-cell immunodominant epitopes analysis
After immunization, antibodies specific for PRRSV-2 N protein were analyzed via ELISA while RNA copy numbers were detected via quantitative real-time RT-PCR analysis. Eight pigs were seronegative for anti-PRRSV antibodies prior to challenge. ELISA results of antibody responses of the four piglets (pig1#, 3#, 4#, 5#) indicated they seroconverted on DPI 7, while the remaining pig (pig2#) seroconverted on DPI 14. No anti-PRRSV-N protein antibodies were detected in negative control pigs (pig6#, 7#,
Discussion
Pathogen-derived CTL epitopes that can initiate a CTL immune response to kill cells infected with pathogens and eliminate infection-associated antigenic reservoirs are key to limiting severity disease severity (Tungatt et al., 2018; Abbas et al., 2017). In our study, three 9-mer peptides were identified as potential CTL epitopes using an approach involving ELISPOT, ELISA, in-silico predictive methods and protein complex formation between antigen-presenting molecules and CTL epitope candidate
Conclusions
In conclusion, three 9-mer CTL epitopes and SLA molecules they could bind to were identified (M2−5, M6−1 could bind SLA-2*0502; M9−1 could bind SLA-2*1201). These CTL epitopes were confirmed via ELISPOT to efficiently stimulate PBMCs with specific SLA genetic backgrounds to secrete IFN-γ. These findings provide new insights into potential strategies for identifying CTL epitopes toward the future goal of designing a broad-spectrum CD8+ T-cell vaccine against multiple PRRSV strains.
Ethics approval
All experiments were performed in accordance with good animal practices as required by the International Guiding Principles for Biomedical Research Involving Animals from the Council for the International Organizations of Medical Sciences. Groups of pigs were housed in a single wide room with separate indoor and outdoor enclosures. Pigs were fed fruits, vegetables and feed. Pigs received care and treatment from a licensed veterinarian.
Funding
This study was supported by the National Natural Science Foundation of China (grant no. 31472177); National Natural Science Foundation of China (grant no. 31702246); and National Natural Science Foundation of China (grant no. 31672526).
Declaration of Competing Interest
The authors report no declarations of interest.
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