J Vet Sci.  2018 May;19(3):393-405. 10.4142/jvs.2018.19.3.393.

Toward the development of a one-dose classical swine fever subunit vaccine: antigen titration, immunity onset, and duration of immunity

• Madera RF
• Wang L
• Gong W
• Burakova Y
• Buist S
• Nietfeld J
• Henningson J
• Cino-Ozuna AG
• Tu C
• Shi J

• KMID: 2412130
• DOI: http://doi.org/10.4142/jvs.2018.19.3.393

Abstract

Highly contagious classical swine fever (CSF) remains a major trade and health problem in the pig industry, resulting in large economic losses worldwide. In CSF-endemic countries, attenuated CSF virus (CSFV) vaccines have been routinely used to control the disease. However, eradication of CSFV in a geographical area would require permanent reduction to zero presence of the virus. It is therefore of paramount importance to develop a safe, potent, and non-infectious CSF vaccine. We have previously reported on a cost-effective CSF E2 subunit vaccine, KNB-E2, which can protect against CSF symptoms in a single dose containing 75 µg of recombinant CSFV glycoprotein E2. In this study, we report on a series of animal studies undertaken to elucidate further the efficacy of KNB-E2. We found that pigs vaccinated with a single KNB-E2 dose containing 25 µg of recombinant CSFV glycoprotein E2 were protected from clinical symptoms of CSF. In addition, KNB-E2-mediated reduction of CSF symptoms was observed at two weeks post-vaccination and the vaccinated pigs continued to exhibit reduced CSF clinical signs when virus challenged at two months and four months post-vaccination. These results suggest that KNB-E2 effectively reduces CSF clinical signs, indicating the potential of this vaccine for safely minimizing CSF-related losses.

Keyword

adjuvants; classical swine virus; subunit; vaccines

MeSH Terms

Animals
Classical Swine Fever*
Glycoproteins
Swine
Vaccines
Glycoproteins
Vaccines

Figure

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  Fig. 1 Pigs vaccinated with KNB-E2 containing 25 µg E2 antigen per pig exhibited decreased classical swine fever virus (CSFV) symptoms. Pigs were immunized with KNB-E2 containing 75 µg, 50 µg, or 25 µg of E2 antigen on day 0. Four weeks after vaccination (28 days post-vaccination), pigs were challenged with 1 × 105 50% tissue culture infective dose (TCID50) of CSFV Alfort. (A) All KNB-E2-immunized pigs appeared healthy and survived the CSFV challenge. (B) KNB-E2-vaccinated pigs did not have body temperatures higher than 40.5℃. (C) PBS-vaccinated CSFV-challenged (−/+) control pigs developed a marked reduction of white blood cells (WBC) in the blood with the greatest reduction at 6 days post-challenge (DPC). Lower levels of WBC were transiently observed in KNB-E2-vaccinated pigs at 3 DPC, but the level quickly recovered. Data are presented as mean ± SEM values for five pigs per group. *p < 0.05.

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  Fig. 2 Decreased viremia and high levels of E2-specific antibodies were detected in pigs immunized with KNB-E2 containing 25 µg, 50 µg, or 75 µg E2 antigen. Classical swine fever virus (CSFV) RNA was detected in serum (A) and nasal cavity (B) by real-time reverse transcriptase polymerase chain reaction analyses in which threshold cycle values equal to or less than 40 were considered CSFV positive. KNB-E2-vaccinated pigs exhibited transient viremia only at 6 days post-challenge compared with the marked viremia in the phosphate-buffered saline–vaccinated CSFV-challenged (−/+) control pigs. E2-specific antibodies were determined in the serum during the vaccination phase (dilution 1/1,000) (C) and the challenge phase (dilution 1/10,000) (D) by enzyme-linked immunosorbent assay. KNB-E2-vaccinated pigs produced significantly high levels of E2-specific antibodies after CSFV challenge. Data are presented as mean ± SEM values for five pigs per group. *p < 0.05.

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  Fig. 3 KNB-E2-immunized pigs challenged as early as two weeks post-vaccination have decreased classical swine fever virus (CSFV) symptoms. Pigs were immunized with KNB-E2 containing 40 µg of E2 antigen on day 0. Pigs were challenged with 1 × 105 50% tissue culture infective dose (TCID50) of CSFV Alfort at 2-, 3-, and 4 weeks post-vaccination. (A) All KNB-E2-immunized pigs appeared healthy and survived the CSFV challenge. (B) The CSFV-challenged pigs developed fever starting at 3 days post-challenge (DPC) with the phosphate-buffered saline–vaccinated CSFV-challenged (−/+) control pigs exhibiting high fever. Transient fever was observed in KNB-E2-vaccinated pigs, but no other CSF symptoms were observed. (C) All KNB-E2-vaccinated pigs continued to gain weight after CSFV challenge. Fold values for total body weight gain during the study were calculated by considering the weight of the pig on 0 DPC as 1. At 8 DPC, only 1 of the 6 (−/+) control pigs remained alive and this pig exhibited recovery from CSFV challenge with a normal body temperature (B) and weight gain (C). (D) White blood cell (WBC) numbers of CSFV-challenged pigs decreased after CSFV challenge. The WBC numbers in KNB-E2-vaccinated pigs decreased in the first six days post-challenge and recovered by 9 DPC. Data are presented as mean ± SEM values for five pigs per group. *p < 0.05.

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  Fig. 4 Decreased viremia and high levels of E2-specific antibodies were detected in pigs challenged with classical swine fever virus (CSFV) at 2-week, 3-week, and 4-week post KNB-E2 vaccination. CSFV RNA was detected in the serum (A) and nasal cavity (B) by real-time reverse transcriptase polymerase chain reaction analyses in which threshold cycle values equal to or less than 40 were considered CSFV positive. The KNB-E2-vaccinated pigs challenged at 3 weeks and 4 weeks post-vaccination had little or no CSFV RNA detected. CSFV RNA was detected in KNB-E2-vaccinated pigs challenged at 2 weeks post-vaccination but the level had recovered by 12 days post-challenge (DPC). E2-specific antibodies were determined in the serum during the challenge phase (dilution 1/10,000) (C) by enzyme-linked immunosorbent assay. KNB-E2-vaccinated pigs produced significantly high levels of E2-specific antibodies after CSFV challenge. At 3 weeks and 4 weeks post-vaccination, KNB-E2 pigs produced higher E2-specific antibodies than the pigs challenged at 2 weeks post-vaccination. Data are presented as mean ± SEM values for five pigs per group. *p < 0.05.

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  Fig. 5 KNB-E2-immunized pigs challenged at 2 months and 4 months post-vaccination have decreased classical swine fever virus (CSFV) symptoms. Pigs were immunized with KNB-E2 containing 50 µg of E2 antigen on day 0. Pigs were challenged with 1 × 105 50% tissue culture infective dose (TCID50) of CSFV Alfort at 2 months and 4 months post-vaccination. (A) All KNB-E2-immunized pigs appeared healthy and survived the CSFV challenge. (B) The older CSFV-challenged pigs developed fever starting at 4 days post-challenge (DPC). The KNB-E2-vaccinated pigs developed a one-day fever but quickly recovered and exhibited normal temperatures throughout the remainder of the study. (C) All KNB-E2-vaccinated pigs continued to gain weight (kg) after CSFV challenge but exhibited a slight decrease in weight gain at 6 DPC. At 10 DPC, only 2 of the 4 phosphate-buffered saline–vaccinated CSFV-challenged (−/+) control pigs remained alive and these pigs exhibited recovery from CSFV challenge (A–C). (D) A slight decrease in the WBC numbers was observed in KNB-E2-vaccinated pigs after CSFV challenge and the WBC levels had recovered by 9 DPC. Data are presented as mean ± SEM values for five pigs per group. *p < 0.05.

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  Fig. 6 Decreased viremia and high levels of E2-specific antibodies were detected in pigs challenged with classical swine fever virus (CSFV) at 2 months and 4 months post-vaccination. CSFV RNA was detected in the serum (A) and nasal cavity (B) by real-time reverse transcriptase polymerase chain reaction analyses in which threshold cycle values equal to or less than 40 were considered CSFV positive. Both 2 months and 4 months post-vaccination KNB E2-vaccinated pig groups exhibited decreased viremia and produced a significantly higher level of E2-specific antibodies post-CSFV challenge (dilution 1/10,000) (C) compared with the level in the phosphate-buffered saline–vaccinated CSFV-challenged (−/+) control group. Data are presented as mean ± SEM values for five pigs per group. *p < 0.05.

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