Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea

Abstract

Porcine rotaviruses cause severe economic losses in the Korean swine industry due to G- and P-genotype mismatches between the predominant field and vaccine strains. Here, we developed a live attenuated trivalent porcine group A rotavirus vaccine using 80 cell culture passages of the representative Korean predominant strains G8P[7] 174-1, G9P[23] PRG942, and G5P[7] K71. Vaccination with the trivalent vaccine or its individual components induced no diarrhea during the first 2 weeks post-vaccination, i.e., the vaccines were attenuated. Challenge of trivalent-vaccinated or component-vaccinated piglets with homologous virulent strain(s) did not induce diarrhea for 2 weeks post-challenge. Immunization with the trivalent vaccine or its individual components also alleviated the histopathological lesions in the small intestines caused by challenge with the corresponding original virulent strain(s). Fecal secretory IgAs specific for each of vaccine strains were detected starting at 14 days post-vaccination (dpv), and IgA levels gradually increased up to 28 dpv. Oral immunization with the trivalent vaccine or its individual components induced high levels of serum virus-neutralizing antibody by 7 dpv. No diarrhea was observed in any experimental piglets during five consecutive passages of each vaccine strain. Our data indicated that the live attenuated trivalent vaccine was safe and effective at protecting piglets from diarrhea induced by challenge exposure of homologous virulent strains. This trivalent vaccine will potentially contribute toward controlling porcine rotavirus disease in South Korea and other countries where rotavirus infections with similar G and P genotypes are problematic.

Figure 1

Electropherogram (PAGE) of selected passages in MA104 cells of three porcine RVA strains. The RNA genomic segments of three vaccine strains from different passage numbers were separated by PAGE and visualized using silver staining. The original virulent strains and each passage of 174-1 (A), K71 (B), and PRG942 (C) demonstrated typical RVA’s RNA segment patterns of 4-2-3-2 and maintained their own patterns throughout the serial passages. Lane 1, original virulent strain; lane 2, 10^th passage; lane 3, 20^th passage; lane 4, 40^th passage; lane 5, 60^th passage; lane 6, 80^th passage.

Figure 2

Histopathological changes in piglets infected with each virulent RVA strain or after challenge following immunization. (A, E, I) Duodenum, jejunum, and ileum of mock-inoculated piglets showed villi of normal length and no crypt hyperplasia. (B–D, F–H, and J–L) Duodenum (B–D), jejunum (F–H), and ileum (J–L) sampled at 7 dpi from piglets inoculated with virulent strains 174-1, PRG942, or K71 showed severe villous atrophy, moderate crypt hyperplasia, and severe lymphoid cell infiltrations in the lamina propria. (M–X) Duodenum (M–P), jejunum (Q–T), and ileum (U–X) sampled at 28 dpv from piglets first immunized with one of the live attenuated monovalent or trivalent vaccines and then challenged with the corresponding original virulent strain(s) 174-1, K71 or PRG942 showed villi of normal length, mild crypt hyperplasia, and mild infiltrations of lymphoid cells in the lamina propria in a manner similar to that of the mock-inoculated group. Hematoxylin and eosin stain. Bars = 200 μm.

Figure 3

Serum neutralizing antibody titers in response to the monovalent or trivalent porcine rotavirus vaccination. A Serum samples obtained weekly from piglets immunized with monovalent 174-1V-80, PRG942V-80, or K71V80 vaccines and challenged by exposure with the corresponding virulent strain at 2 weeks post-vaccination. B Serum samples obtained weekly from piglets immunized with a trivalent porcine live attenuated rotavirus vaccine and challenged by exposure with its virulent strain 174-1 at 2 weeks post-vaccination. Serum neutralizing antibody titers were calculated as the geometric mean titers for each group (n = 5 per group).

Figure 4

Serum and fecal RV-specific IgM, IgG, and IgA antibody titers determined by ELISA. A, B Serum and fecal samples obtained weekly from piglets immunized with a live attenuated strain 174-1V-80 and challenged by exposure with its virulent strain 174-1 at 2 weeks post-vaccination. C, D Serum and fecal samples obtained weekly from piglets immunized with a live attenuated strain PRG942V-80 and challenged by exposure with its virulent strain PRG942 at 2 weeks post-vaccination. E, F Serum and fecal samples obtained weekly from piglets immunized with a live attenuated strain K71V-80 and challenged by exposure with its virulent strain K71 at 2 weeks post-vaccination. G, H Serum and fecal samples obtained weekly from piglets immunized with a live attenuated trivalent vaccine containing live attenuated strains 174-1V-80, PRG942V-80, and K71V-80 and challenged by exposure with its virulent strains 174-1, PRG942, and K71 at 2 weeks post-vaccination. Antibody titers are expressed as the geometric mean titers (GMTs) for each group (n = 5 piglets per each group).