A chimeric porcine reproductive and respiratory syndrome virus 1 strain containing synthetic ORF2-6 genes can trigger T follicular helper cell and heterologous neutralizing antibody responses and confer enhanced cross-protection

Abstract

The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.

Keywords: IFNγ-secreting cells; ORF2-6 chimeric virus; T follicular helper (Tfh) cells; cross-protection; neutralizing antibodies (nAbs); porcine reproductive and respiratory syndrome virus 1 (PRRSV1).

Figure 1

Design, construction and rescue of the rHLJB1 and ORF2-6-CON infectious clones. This strategy was adopted from our previous studies [19, 28]. A The pACYC177-CMV-stuffer fragment containing five unique restriction enzymes was utilized for the construction of Chinese PRRSV1 full-length cDNA clones. B Four overlapping fragments were used to construct full-length rHLJB1 and ORF2-6-CON infectious clones. C The successful rescue of rHLJB1 and ORF2-6-CON was identified by IFA in PAMs and Marc-145 cells at 3 dpi, as determined by IFA. D The plaque morphology of ORF2-6-CON was determined in Marc-145 cells at 5 dpi.

Figure 2

Dynamics of viral load, antibody level, rectal temperature and weight gain during animal inoculation and challenge study. A Viremia was detected via a PRRSV1 real-time RT‒PCR assay [31]. B PRRSV-specific antibodies were detected with an IDEXX HerdCheck PRRS × 3 ELISA Kit. C Rectal temperature was determined daily for 14 dpi after inoculation and challenge and weekly thereafter. D Body weight was recorded weekly for all groups of pigs. E SD1291 viral loads in the lungs, lymph nodes and tonsils among the distinct groups were determined using the SD1291 strain-specific primers and probes shown in Table 2. F The gross pathology of the lungs was scored according to methods described previously [–35]. The data shown in the bar graphs are the means and SDs. Statistical significance is denoted by *, p < 0.05; **, p < 0.01; and ***, p < 0.001 (n = 5).

Figure 3

Post-mortem and histopathological examination. A A representative lung from four groups of pigs collected at 21 dpc. Lung gross lesions and pathological lesions (HEs) in each group were enlarged. B Representative hilar lymph nodes from four groups of pigs collected at 21 dpc. Hilar lymph node gross lesions and pathological lesions (HEs) were also enlarged. The enlarged regions of the lungs and hilar lymph nodes (both gross and HE) are shown as red dashed line boxes.

Figure 4

Generation of nAbs and porcine Tfh cells. A Generation of nAbs against the HLJB1 strain at 42 dpi and 21 dpc. B Generation of nAbs against SD1291 strains at 42 dpi and 21 dpc. C The percentages of porcine Tfh cells in blood samples among the four groups at different time points. D The percentages of porcine Tfh cells in different tissues among the four groups. * p < 0.05. E Representative dot plots depicting the percentages of porcine Tfh cells among CD4+ T cells in TBLNs upon SD1291 stimulation (MOI = 0.1). The data are shown as the mean ± SD from 5 pigs per group.

Figure 5

Frequencies of SD1291-specific IFNγ-secreting T lymphocytes. A Histograms showing IFNγ expression in CD3 + T cells from PBMCs from the four groups after RPMI-1640 or SD1291 stimulation at different time points. B Histograms showing IFNγ expression in CD3 + T cells from different tissues in the three groups upon RPMI-1640 or SD1291 stimulation. C, D Dot plots depicting the percentages of IFNγ-secreting cells among CD3 + T cells from spleens and TBLNs upon stimulation with SD1291. RPMI-1640-incubated cultures were utilized as negative controls. The numbers indicate the percentages of IFNγ-secreting CD3 + T cells. The data are shown as the mean ± SD from 5 pigs per group.