Systemic Homologous Neutralizing Antibodies Are Inadequate for the Evaluation of Vaccine Protective Efficacy against Coinfection by High Virulent PEDV and PRRSV

Abstract

G2 porcine epidemic diarrhea virus (G2 PEDV) and highly pathogenic porcine reproductive and respiratory syndrome virus 2 (HP-PRRSV2) are two of the most prevalent swine pathogens in China's swine herds, and their coinfection occurs commonly. Several PED and PRRS vaccines have been utilized in China for decades, and systemic homologous neutralizing antibodies (shnAbs) in serum are frequently used to evaluate the protective efficacy of PED and PRRS vaccines. To develop a vaccine candidate against G2 PEDV and HP-PRRSV2 coinfection, in this study, we generated a chimeric virus (rJSTZ1712-12-S) expressing S protein of G2 PEDV using an avirulent HP-PRRSV2 rJSTZ1712-12 infectious clone as the viral vector. The rJSTZ1712-12-S strain has similar replication efficacies as the parental rJSTZ1712-12 virus. In addition, animal inoculation indicated that rJSTZ1712-12-S is not pathogenic to piglets and can induce shnAbs against both G2 PEDV and HP-PRRSV2 isolates after prime-boost immunization. However, passive transfer study in neonatal piglets deprived of sow colostrum showed that rJSTZ1712-12-S-induced shnAbs may only decrease PEDV and PRRSV viremia but cannot confer sufficient protection against dual challenge of high virulent G2 PEDV XJ1904-34 strain and HP-PRRSV2 XJ17-5 isolate. Overall, this study provides the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for the evaluation of protective efficacy of PED and PRRS bivalent vaccine (especially for the PED vaccine). IMPORTANCE Porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) coinfection occurs commonly and can synergistically reduce feed intake and pig growth. Vaccination is an effective strategy utilized for PED and PRRS control, and systemic homologous neutralizing antibodies (shnAbs) in serum are commonly used for protective efficacy evaluation of PED and PRRS vaccines. Currently, no commercial vaccine is available against PEDV and PRRSV coinfection. This study generated a chimeric vaccine candidate against the coinfection of prevalent PEDV and PRRSV in China. The chimeric strain can induce satisfied shnAbs against both PEDV and PRRSV after prime-boost inoculation in pigs. But the shnAbs cannot confer sufficient protection against PEDV and PRRSV coinfection in neonatal piglets. To the best of our knowledge, these findings provide the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for evaluating PED and PRRS bivalent vaccine protective efficacy.

Keywords: chimeric vaccine; neonatal piglet; porcine epidemic diarrhea virus (PEDV); porcine reproductive and respiratory syndrome virus (PRRSV); protective efficacy; systemic homologous neutralizing antibodies (shnAbs).

FIG 1

The chimeric rJSTZ1712-12-S virus was constructed using rJSTZ1712-12 infectious clone as the backbone. The amplicon of S gene of G2 PEDV XJ1904-34 strain is 4158 bp (A). The S gene of G2 PEDV XJ1904-34 strain was inserted between ORF1b and ORF2a genes of rJSTZ1712-12 (B). The rJSTZ1712-12 and rJSTZ1712-12-S recombinant plasmids were double digested with PacI+AflII, AflII+AscI, AscI+NotI to generate F1, F2 and F3 fragments, respectively. The size of rJSTZ1712-12-F3 (rJS-F3) is 3561 bp, while the size of rJSTZ1712-12-S-F3 (rJS-S-F3) with the inserted S gene is 7719 bp, indicating the successful construction of rJSTZ1712-12-S recombinant plasmid (C).

FIG 2

The chimeric rJSTZ1712-12-S virus was rescued in Marc-145 cells. IFA results showed that both PEDV S antigen and PRRSV N antigen could be detected in rJSTZ1712-12-S infected cells but not in mock infected cells (A). Multiple-step growth curves in Marc-145 cells within 120 hpi was determined by real-time RT-PCR, which showed that no significant difference was observed in the in vitro replication of parental rJSTZ1712-12 and chimeric rJSTZ1712-12-S viruses (B). The chimeric rJSTZ1712-12-S virus also has similar plaque morphology as the parental rJSTZ1712-12 virus (C).

FIG 3

The chimeric rJSTZ1712-12-S virus is not pathogenic to piglets. Viremia could be generated with peaks at 14 dpi for both prime and boost inoculations in piglets (A). The inoculation of rJSTZ1712-12-S did not affect the weight gain of piglets (B). The inoculation of rJSTZ1712-12-S did not induce fever during the entire experiment (C). Necropsy examination did not detect obvious lung gross lesion in either rJSTZ1712-12-S infected or mock infected pigs (D and E). Only mild disseminated intravascular congestion could be observed in the lungs from rJSTZ1712-12-S infected pigs during the histopathological examination (F and G).

FIG 4

The chimeric rJSTZ1712-12-S virus induces shnAbs against both G2 PEDV and HP-PRRSV2. Flow cytometry results showed that no red fluorescent signals could be detected in serum (from rJSTZ1712-12-S immunized pigs) treated and infected samples, which indicated that rJSTZ1712-12-S-induced shnAbs could completely neutralize rXJ17-5-dsRed (A). Meanwhile, green fluorescent signals were significantly lower in serum treatment and infection cells than in rPEDV-EGFP infected cells (B). No specific fluorescent signals could be detected in mock infected cells.

FIG 5

The chimeric rJSTZ1712-12-S virus induced shnAbs provide insufficient protection against dual challenge of high pathogenic G2 PEDV XJ1904-34 strain and HP-PRRSV2 XJ17-5 isolate in neonatal piglets. PEDV virus load in feces was significantly lower in the serum feeding/challenge group than in the challenge group at 12 hpi (A). PEDV viremia was relatively lower in the serum feeding/challenge group than in the challenge group at 1 dpi but no difference in the following days (B). PRRSV viremia was always relatively lower in the serum feeding/challenge group than in the challenge group from 2 dpi to 6 dpi but with no statistically significant difference (P > 0.05) (C). Dual challenge induced high fever in neonatal piglets of both the serum feeding/challenge group and the challenge group at 1 and 2 dpi but not in the mock infected group (D). Neonatal piglets in both the serum feeding/challenge group and the challenge group had a certain degree of weight loss at the end of this study (P > 0.05) (E). All neonatal piglets in the serum feeding/challenge group and the challenge group died from 3 dpi to 9 dpi (F).

FIG 6

Neonatal piglets in the serum feeding/challenge group seemed to die of PEDV infection rather than PRRSV infection. Obvious lung consolidation could be observed in the challenge group but not in the serum feeding/challenge group or the mock infected group (A–C). Mild to severe degrees of widened alveolar septum were observed in the sera-feeding/challenge group and the challenge group, respectively (D–F). PRRSV specific antigen could be detected in both the sera-feeding/challenge group and the challenge group (G–I). Obvious flatulence and thinning of intestinal walls could be observed from both the sera-feeding/challenge group and the challenge group (a–c). Exfoliation of mucosal epithelial cells and exposed lamina propria of jejunum could be detected in both the sera-feeding/challenge group and the challenge group (d–f). PEDV specific antigen could also be detected in jejunum of both the sera-feeding/challenge group and the challenge group (g-i).