A Plant-Produced Porcine Parvovirus 1-82 VP2 Subunit Vaccine Protects Pregnant Sows against Challenge with a Genetically Heterologous PPV1 Strain

Abstract

Porcine parvovirus (PPV) causes reproductive failure in sows, and vaccination remains the most effective means of preventing infection. The NADL-2 strain has been used as a vaccine for ~50 years; however, it does not protect animals against genetically heterologous PPV strains. Thus, new effective and safe vaccines are needed. In this study, we aimed to identify novel PPV1 strains, and to develop PPV1 subunit vaccines. We isolated and sequenced PPV1 VP2 genes from 926 pigs and identified ten PPV1 strains (belonging to Groups C, D and E). We selected the Group D PPV1-82 strain as a vaccine candidate because it was close to the highly pathogenic 27a strain. The PPV1-82 VP2 protein was produced in Nicotiana benthamiana. It formed virus-like particles and exhibited a 2¹¹ agglutination value. The PPV1-190313 strain (Group E), isolated from an aborted fetus, was used as the challenging strain because it was pathogenic. The unvaccinated sow miscarried at 8 days postchallenge, and mummified fetuses were all PPV1-positive. By contrast, pregnant sows vaccinated with PPV1-82 VP2 had 9-11 Log₂ antibody titers and produced normal fetuses after PPV1-190313 challenge. These results suggest the PPV1-82 VP2 subunit vaccine protects pregnant sows against a genetically heterologous PPV1 strain by inducing neutralizing antibodies.

Keywords: cross reactivation; plant-produced subunit vaccine; porcine parvovirus 1; viral protein 2; virus-like particle.

Figure 1

Phylogenetic analysis based on PPV1 VP2 gene sequences. The maximum-likelihood method (Tamura-Nei model of nucleotide substitution, gamma distributed with invariant sites) was used in molecular epidemiology and analysis of rapidly evolving heterochronos PPV1 VP2 sequences, with the 1000 bootstrap replicates setting of the MEGA v 6.0 program. VP2 amino acid sequences of the ten PPV1s isolated from Korean pig farms were compared with the reference sequences PVK2, T142, 27a, NADL-2 and VRI-1. GenBank Accession numbers are provided in the parentheses next to each strain name. The log likelihood (log L) value for the genome sequences was −28,543.99. Bootstrap values are indicated at the nodes. The scale bar indicates the number of nucleotide substitutions per site.

Figure 2

Plant-produced PPV1-82 VP2 protein purification and immunogenicity. (A) The PPV1-82 VP2 size-exclusion chromatography trace and an SDS-PAGE gel image of the collected fractions. The bidirectional red arrow indicates the elution fractions (32–36) that the purified PPV1-82 VP2 protein was collected from. (B) Transmission electron microscopy image of virus-like particles formed using the purified PPV1-82 VP2 protein (scale bar = 100 nm, HV = 75.0 kV, direct magnification: 100,000×). (C) HA results, sedimentary red blood cells represented non-agglutination. (D) Results from the HI assay performed on samples from three piglets vaccinated with PPV1-82 VP2; wpv, weeks postvaccination; * p < 0.05; ** p < 0.01.

Figure 2

Plant-produced PPV1-82 VP2 protein purification and immunogenicity. (A) The PPV1-82 VP2 size-exclusion chromatography trace and an SDS-PAGE gel image of the collected fractions. The bidirectional red arrow indicates the elution fractions (32–36) that the purified PPV1-82 VP2 protein was collected from. (B) Transmission electron microscopy image of virus-like particles formed using the purified PPV1-82 VP2 protein (scale bar = 100 nm, HV = 75.0 kV, direct magnification: 100,000×). (C) HA results, sedimentary red blood cells represented non-agglutination. (D) Results from the HI assay performed on samples from three piglets vaccinated with PPV1-82 VP2; wpv, weeks postvaccination; * p < 0.05; ** p < 0.01.

Figure 3

Pathogenicity of PPV1-190313 in three gestational Yukatan miniature sows. Sows were infected with 8 mL of the PPV1-190313 strain (at 4.0 × 10⁵ [TCID₅₀]/mL); 4 mL was administered by intramuscular injection into the neck, and the other 4 mL was delivered intranasally via a spray. (A) HA assay results obtained with PPV1-190313 after 40 passages in porcine kidney-15 cells. (B) Photographic and hematoxylin and eosin staining images of the uterus from the W19-090 sow, which miscarried during the experiment. Photographic images of the uterus and fetuses of sow W19-117 (C) and sow W19-118 (D).

Figure 4

The vaccination and challenge experiment. Sows were intramuscularly vaccinated with the PPV1-82 VP2 antigen (HA value = 2¹³) combined with the MONTANIDE IMS1313 adjuvant at 50% (v/v) at 4 and 2 weeks before conception. At 40 days after conception, vaccinated sows were challenged with 4 mL of the PPV1-190313 strain (at 4.0 × 10⁵ TCID₅₀/mL); 2 mL was administered by intramuscular injection into the neck, and the other 2 mL was delivered intranasally via a spray. (A) Schematic representation of the experimental design. (B) Fetuses from vaccinated sows. (C) The uterus and fetuses from the unvaccinated PPV1-190313-challenged control sow, which miscarried at 8 DPC. (D) Fetuses from the unvaccinated unchallenged negative control sow.