Supplementary Material for: A Porcine Reproductive and Respiratory Syndrome Virus Vaccine Candidate Based on PRRSV Glycoprotein 5 and the Toll-Like Receptor 5 Agonist Salmonella typhimurium Flagellin

Glycoprotein 5 (GP5) from porcine reproductive and respiratory
syndrome virus (PRRSV) is a key inducer of neutralizing antibodies. A
truncated GP5 gene lacking the signal peptide and transmembrane
sequences was amplified via an overlap PCR method and inserted into
prokaryotic expression vectors, pET32a or pGEX-6p-1, to add an His or
GST tag, respectively. His-tagged GP5 was induced with IPTG, verified by
SDS-PAGE and Western blotting, and purified to serve as an immunogen
accompanied with the Salmonella typhimurium flagellin (FliC), a
Toll-like receptor 5 (TLR5) agonist. Levels of TLR5 and cytokine mRNAs
in spleens of mice following injection with FliC were detected by
qRT-PCR to verify the activation of innate immunity. FliC was used as an
adjuvant and administered with the GP5 to C57BL/6 mice via
intraperitoneal injection. Coadministration of GP5 with FliC induced a
significantly enhanced GP5-specific IgG and IFN-γ response compared with
administration of GP5 alone, and the GP5-specific titer in the GP5 +
FliC coadministration group was elevated almost twofold after the third
immunization. These results indicate that FliC is an effective adjuvant,
increasing the induction of antibodies against GP5 with the induction
of both humoral and cellular immune responses.