Inactivated porcine reproductive and respiratory syndrome virus vaccine adjuvanted with Montanide™ Gel 01 ST elicits virus-specific cross-protective inter-genotypic response in piglets

Abstract

The efficacy of a novel BEI-inactivated porcine reproductive and respiratory syndrome virus (PRRSV) candidate vaccine in pigs, developed at RIBSP Republic of Kazakhstan and delivered with an adjuvant Montanide™ Gel 01 ST (D/KV/ADJ) was compared with a commercial killed PRRSV vaccine (NVDC-JXA1, C/KV/ADJ) used widely in swine herds of the Republic of Kazakhstan. Clinical parameters (body temperature and respiratory disease scores), virological and immunological profiles [ELISA and virus neutralizing (VN) antibody titers], macroscopic lung lesions and viral load in the lungs (quantitative real-time PCR and cell culture assay) were assessed in vaccinated and both genotype 1 and 2 PRRSV challenged pigs. Our results showed that the commercial vaccine failed to protect pigs adequately against the clinical disease, viremia and lung lesions caused by the challenged field isolates, Kazakh strains of PRRSV type 1 and type 2 genotypes. In contrast, clinical protection, absence of viremia and lung lesions in D/KV/ADJ vaccinated pigs was associated with generation of VN antibodies in both homologous vaccine strain LKZ/2010 (PRRSV type 2) and a heterogeneous type 1 PRRSV strain (CM/08) challenged pigs. Thus, our data indicated the induction of cross-protective VN antibodies by D/KV/ADJ vaccine, and importantly demonstrated that an inactivated PRRSV vaccine could also induce cross-protective response across the viral genotype.

Keywords: Binary ethylenimine; Cross-protection; Killed vaccines; Neutralizing antibodies; PRRSV; Polymeric adjuvant.

Fig. 1

Body temperature (A) and respiratory disease scores (B) in pigs post-challenge. The body temperature ≥40 °C was considered as fever (solid blue line). Severity of respiratory disease was scored as described in Methods. Data shown are mean ± SEM of 5 or 10 pigs in each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Detection of PRRSV-specific antibodies in serum by ELISA. Positive responses were classified as the S/P ratio of ≥0.4 (horizontal red line). Serum samples were collected from the pigs at DPFV 0 (A), 7 (B), 14 (C), 21 (D), 28 (E), 35 (F), 42 (G), 49 (H) 51 (I), 53 (J), 55 (K), 57 (L), 59 (M), 61 (N) and 63 (O). *First vaccination; **second vaccination for groups 3 and 4; ***second vaccination for groups 5 and 6; ****third vaccination for groups 3 and 4; *****challenge. Data are mean ± SEM titer of 5 or 10 pigs in each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Serum virus neutralizing antibody titers post-vaccination and challenge. # = first vaccination; ## = second vaccination for groups 1–4; ### = second vaccination for groups 5 and 6; #### = third vaccination for groups 1–4. Serum samples were titrated on MARC-145 cells and the levels of PRRSV neutralizing antibodies to LKZ/2010 (A) and CM/08 (B) were determined as the reciprocal of the highest dilution that inhibited virus induced immunofluorescence (CPE). The solid red line indicates the detection limit for the VN assay. Data are geometric mean ± SEM of 5 or 10 pigs in each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Macroscopic lung lesions (A) and quantification of PRRSV load by qRT-PCR (B). Lungs of pigs vaccinated and challenged with the PRRSV strains LKZ/2010 or CM/08 were collected at necropsy (DPC 14/63 DPFA). The viral RNA copy numbers in the lung homogenates were quantified by qRT-PCR and its limit of detection was 10³ RNA copies/ml. Data is shown as mean ± SEM viral RNA copy numbers of 5 or 10 pigs in each group; ****P < 0.0001; analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 5

PRRS viremia levels were measured by qPCR (A) and by assessing viral titers on Marc-145 cells (B). Data in (A) indicates the mean PRRSV ± SEM RNA copy numbers of 5 or 10 pigs in each group, and data in (B) were expressed as the geometric mean ± SEM log₁₀TCID₅₀/ml of 5 or 10 pigs in each group. Serum samples of pigs were collected up to DPC 14 (63 DPFA) challenged with PRRSV (LKZ/2010 or CM/08). The limit of detection by the assay kit was 10³ RNA copies per ml. The limit of detection for the virus titration assay was 10^0.75 TCID₅₀/ml.