A PRRSV GP5-Mosaic vaccine: Protection of pigs from challenge and ex vivo detection of IFNγ responses against several genotype 2 strains

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV), is a highly mutable RNA virus that affects swine worldwide and its control is very challenging due to its formidable heterogeneity in the field. In the present study, DNA vaccines constructed with PRRSV GP5-Mosaic sequences were complexed to cationic liposomes and administered to experimental pigs by intradermal and intramuscular injection, followed by three boosters 14, 28 and 42 days later. The GP5-Mosaic vaccine thus formulated was immunogenic and induced protection from challenge in vaccinated pigs comparable to that induced by a wild type (VR2332) GP5 DNA vaccine (GP5-WT). Periodic sampling of blood and testing of vaccine-induced responses followed. Interferon-γ (IFN-γ) mRNA expression by virus-stimulated peripheral blood mononuclear cells (PBMCs) of GP5-Mosaic-vaccinated pigs was significantly higher compared to pigs vaccinated with either GP5-WT or empty vector at 21, 35 and 48 days after vaccination. Cross-reactive cellular responses were also demonstrated in GP5-Mosaic vaccinated pigs after stimulation of PBMCs with divergent strains of PRRSV. Thus, significantly higher levels of IFN-γ mRNA were detected when PBMCs from GP5-Mosaic-vaccinated pigs were stimulated by four Genotype 2 strains (VR2332, NADC9, NADC30 and SDSU73), which have at least 10% difference in GP5 amino acid sequences, while such responses were recorded only upon VR2332 stimulation in GP5-WT-vaccinated pigs. In addition, the levels of virus-specific neutralizing antibodies were higher in GP5-Mosaic or GP5-WT vaccinated pigs than those in vector-control pigs. The experimental pigs vaccinated with either the GP5-Mosaic vaccine or the GP5-WT vaccine were partially protected from challenge with VR2332, as measured by significantly lower viral loads in sera and tissues and lower lung lesion scores than the vector control group. These data demonstrate that the GP5-Mosaic vaccine can induce cross-reactive cellular responses to diverse strains, neutralizing antibodies, and protection in pigs.

Broad Protection of Pigs against Heterologous PRRSV Strains by a GP5-Mosaic DNA Vaccine Prime/GP5-Mosaic rVaccinia (VACV) Vaccine Boost

“…It has also been demonstrated that Mosaic T-cell vaccines have great potential for other viruses with extraordinary diversity, such as HIV [33][34][35][36][37][38]. In an earlier study, we demonstrated the immunogenicity of GP5-Mosaic vaccines in swine and their ability to induce cross-reactivity, as shown by their broad recall responses ex-vivo, and conferred the partial protection they provide in pigs [29,30]. The ability of the GP5-Mosaic vaccines to induce cross-protection in pigs against heterologous PRRSV strains was confirmed in the present study.…”

“…A combination of gene synthesis (DNA 2.0, Menlo Park, CA, USA) and standard sub-cloning was utilized to generate three transfer vectors. Each transfer vector contained a cassette with the tetR gene (based on GenBank: X00694), and either a natural or Mosaic version of Genotype 2 PRRSV ORF 5 [29,30], followed by the EMCV IRES (based on GenBank: NC_001479.1) and the EGFP gene (based on plasmid pEGFP-1, GenBank: U55761). The tetR gene and the ORF 5-IRES-EGFP genetic segment were placed under back-to-back synthetic VACV early/late promoters [41].…”

“…Virus neutralization testing was carried out as previously reported [30]. Briefly, each serum was mixed with equal volumes of DMEM containing 100 TCID 50 of VR2332 or MN184C and the mixtures were incubated at 37 • C for 1 h. The serum-virus mixtures were then added to 96-well plates containing 80-90% confluent MARC-145 cells and incubated for 48 h at 37 • C in a 5% CO 2 atmosphere (final serum dilution 1:4).…”

“…Thus, the development of vaccines that can induce protection against different subgroups and lineages is highly desirable. Multi-subunit vaccines [26], consensus vaccines [27], molecular breeding of different viral structural proteins [28], Mosaic T-cell vaccines [29,30], a polyvalent vaccine containing five different live-attenuated PRRSV strains [31] and DNA prime-MLV boost [32] are just some of the vaccines that are or have been under evaluation recently.…”

“…Mosaic vaccines have been shown to elicit broader immune responses successfully and to confer cross protection in non-human primates and these Mosaic HIV vaccines have been trialed in humans [34][35][36][37][38]. We previously reported that a PRRSV Mosaic vaccine based on 748 GP5 sequences of the Genotype 2 PRRSV strain was immunogenic [29] and induced reactivity to four divergent Genotype-2 PRRSV strains that had at least 10% or more difference in their GP5 sequence-as shown by the expression of interferon gamma (IFN-γ) by peripheral blood mononuclear cells-and, further, that vaccinated pigs were protected against challenge with VR2332 [30]. The present study demonstrates further that vaccination of pigs with the GP5-Mosaic by using a DNA prime and rVaccinia boost approach protects them against strains that are more than 10% divergent in their sequences (VR2332 and MN184C).…”