Chimeric HP-PRRSV2 containing an ORF2-6 consensus sequence induces antibodies with broadly neutralizing activity and confers cross protection against virulent NADC30-like isolate

Abstract

Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.

Keywords: Broadly neutralizing antibodies; Cross protection; Genetic engineered vaccine; Infectious clone; ORF2-6 consensus sequence; PRRSV.

Figure 1

Design of the consensus sequence of ORF2-6 genes (ORF2-6-CON). The ORF2-6-CON was designed according to 30 representative Chinese PRRSV isolates. A phylogenetic tree based on ORF2-6 genes was constructed (A). The ORF2-6-CON shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates existing in China (B).

Figure 2

Strategy to construct the full-length cDNA clones of avirulent HP-PRRSV2 JSTZ1712-12 isolate and a chimeric virus containing ORF2-6-CON. The strategy was adopted from our previous study [25]. The pACYC177-CMV-Stuffer fragment including the unique restriction enzymes was shown in the upper part (A). The three overlapped fragments of the rJSTZ1712-12 (B) and rJS-ORF2-6-CON (C) genomes were produced by PCR amplification and are shown in the bottom part.

Figure 3

Identification of the rescued rJSTZ1712-12 and rJS-ORF2-6-CON in vitro. PRRSV-specific antigen was detected by the immunofluorescence assay (IFA) (A). The multiple-step growth curves in Marc-145 cells within 96 hpi were determined by real-time RT-PCR assay [32]. No significant difference was observed in the in vitro replication of the parental and cloned viruses (B). The JSTZ1712-12, rJSTZ1712-12 and rJS-ORF2-6-CON viruses have similar plaque morphology (C).

Figure 4

Dynamics of rectal temperature, weight gain, virus load, antibody and IFN-γ levels during inoculation and challenge studies. After the challenge of NADC30-like SD17-38 isolate, all positive control pigs and two of TJM-F92 vaccinated pigs showed high fever. However, the rectal temperature of rJS-ORF2-6-CON inoculated and mock-infected pigs did not reach 40 °C during the whole experiment (A). rJS-ORF2-6-CON inoculated pigs had significantly higher weight gain than TJM-F92 vaccinated pigs at 14 dpc (B). Dynamics of viremia were detected by real-time RT-PCR assay [32]. Compared with TJM-F92 vaccinated pigs, the virus load was significantly lower in rJS-ORF2-6-CON inoculated pigs since 7 dpc (C). PRRSV-specific antibody level was detected by IDEXX HerdCheck*PRRS×3 Antibody Detection ELISA Kit. The threshold for seroconversion was set at a sample-to-positive (s/p) ratio of 0.4. PRRSV-specific antibody could be detected in all PRRSV-infected pigs from 14 dpi to the end of the study (D). IFN-γ level was detected using a commercial Porcine IFN-gamma ELISA kit. No significant difference of IFN-γ amounts was detected in rJS-ORF2-6-CON inoculated and TJM-F92 vaccinated pigs at 42 dpi and 14 dpc (E). Each bar represents the average for all pigs in each group ± standard deviation (SD). The significant difference is marked with an asterisk.

Figure 5

Lung gross lesion, histopathological and immunohistochemical examinations. Lung consolidation was observed in positive control and TJM-F92 vaccinated pigs (B, C) but not in mock-infected and rJS-ORF2-6-CON inoculated pigs (A, D). Interstitial pneumonia with infiltration of mononuclear cells could be observed in positive control and TJM-F92 vaccinated pigs (F, G) but not in mock-infected and rJS-ORF2-6-CON inoculated pigs (E, H). PRRSV-specific antigen could be observed in all PRRSV-infected pigs (marked by the black arrows) (J, K, L) but not in mock-infected pigs (I).

Figure 6

Frequencies of PRRSV-specific IFN-γ secreting cells. PBMC were isolated from each groups of pigs at 0 dpc (42 dpi), 7 dpc (49 dpi), 14 dpc (56 dpi) and stimulated with or without HP-PRRSV2 JXA1-R strain at 0.01 MOI for 20 h. The intracellular IFN-γ expression on total CD3+ T cells was further analyzed by flow cytometry. Representative dot-plots from each group were shown with frequencies of PRRSV-specific IFN-γ secreting cell population (left panel). The overall frequencies of PRRSV-specific IFN-γ secreting cells in each group were compared and are shown as bar graphs at the indicated time points (right panel). No significant difference was detected between rJS-ORF2-6-CON inoculated and TJM-F92 vaccinated pigs.