Zanotti Cinzia, Martinelli Nicola, Lelli Davide, Amadori Massimo
1 Laboratory of Cellular Immunology, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna , Brescia, Italy .
2 Laboratory Animal Medicine, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna , Brescia, Italy .
Viral Immunol. 2015 Dec;28(10):600-8. doi: 10.1089/vim.2015.0021. Epub 2015 Sep 24.
Porcine circovirus type 2 (PCV2) is associated with a number of diseases and syndromes, collectively referred to as porcine circovirus-associated disease. The main objective of this study was to define some in vitro correlates of protection after injection of inactivated PCV2 vaccines with a defined antigen mass. Twelve pigs were vaccinated with three different doses of inactivated, whole-virus antigen (211-844 ng), while four animals were injected with a commercial vaccine (positive control) and four other pigs were mock-vaccinated with phosphate-buffered saline (PBS) in the same oil emulsion. Four weeks later, they were intranasally challenged with 2 × 10(5) TCID50 of a PCV2a strain. Antibody was measured in blood and oral fluids by enzyme-linked immunosorbent assay (ELISA) and a neutralization assay. PCV2 was quantified in serum by real-time polymerase chain reaction for ORF2 gene. PCV2-specific cell-mediated responses were investigated by an IFN-γ release assay in whole blood, IFN-γ ELISPOT, and lymphocyte proliferation (Ki-67 and BrDU assays). All the vaccines under study but mock provided complete or incomplete protection from PCV2 infection in terms of post-challenge viremia. Serum antibody titers (ELISA and neutralizing) after vaccination were not correlated with protection, as opposed to the early neutralizing antibody levels of vaccinated pigs at day 7 after infection. Cell-mediated immune parameters showed a good correlation with vaccine efficacy. In particular, the IFN-γ release assay at 3 weeks after vaccination was an effective marker for predicting protection. All control pigs always tested negative in assays of cell-mediated immunity. Our results outline in vitro testing procedures toward reduced animal usage in the control of PCV2 vaccine batches.
猪圆环病毒2型（PCV2）与多种疾病和综合征相关，统称为猪圆环病毒相关疾病。本研究的主要目的是确定在注射具有特定抗原量的灭活PCV2疫苗后，一些体外保护相关性指标。给12头猪接种三种不同剂量的灭活全病毒抗原（211 - 844纳克），同时给4头动物注射商业疫苗（阳性对照），另外4头猪用相同油乳剂的磷酸盐缓冲盐水（PBS）进行 mock 接种。4周后，给它们经鼻接种2×10⁵ TCID₅₀的PCV2a毒株。通过酶联免疫吸附测定（ELISA）和中和试验检测血液和口腔液中的抗体。通过实时聚合酶链反应对血清中的ORF2基因进行PCV2定量。通过全血中的IFN - γ释放试验、IFN - γ ELISPOT和淋巴细胞增殖（Ki - 67和BrDU试验）研究PCV2特异性细胞介导的反应。就攻毒后的病毒血症而言，除mock组外，所有研究中的疫苗都提供了完全或不完全的PCV2感染保护。接种疫苗后的血清抗体滴度（ELISA和中和试验）与保护无关，这与感染后第7天接种疫苗猪的早期中和抗体水平相反。细胞介导的免疫参数与疫苗效力显示出良好的相关性。特别是，接种疫苗3周后的IFN - γ释放试验是预测保护的有效标志物。所有对照猪在细胞介导免疫试验中始终检测为阴性。我们的结果概述了得在控制PCV2疫苗批次时减少动物使用的体外检测程序。
