Fan Jie, Fan Fangcheng, Chen Zhixiong, Chen Ping, Zhu Yanli, Li Xin, Liu Tiantian, Li Runcheng, Dong Wei, Ge Meng
College of Veterinary Medicine, Hunan Agricultural University, Changsha, 410125, China.
Porcine Health Manag. 2025 Jul 10;11(1):38. doi: 10.1186/s40813-025-00452-7.
Porcine Circovirus 2 (PCV2) vaccination plays a crucial role in preventing porcine circovirus-associated disease (PCVAD). Nevertheless, pig farms face significant challenges in evaluating vaccination efficacy due to the inability of PCV2 vaccines to achieve sterilizing immunity and the variability among vaccine manufacturers. These challenges are further compounded by the limitations of conventional antibody detection methods, which fail to distinguish between maternally-derived antibodies (MDAs) and vaccine-induced antibodies. The accurate evaluation and selection of PCV2 vaccines is critical for the swine industry. The present study aimed to develop an Enzyme-linked immunospot (ELISpot) assay for directly detecting PCV2-specific memory B cells. This approach was used to assess the presence of PCV2-specific memory B cells in piglets with high levels of MDA vaccinated with different PCV2 vaccines, thus enabling the evaluation of vaccine immunogenicity at the cellular level. Furthermore, antibody levels and the viremia status were analyzed using Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) respectively to provide a comprehensive assessment of the ELISpot assay potential for evaluating the vaccine immunogenicity of PCV2 vaccines.
The findings revealed that the optimal conditions for the developed ELISpot assay included stimulation with R848 at a final concentration of 1 µg·mL⁻¹ for three days, a PCV2 Cap protein coating concentration of 1.25 µg·mL⁻¹, a biotinylated goat anti-pig IgG antibody concentration of 5 µg·mL⁻¹, and an HRP-streptavidin concentration of 0.25 µg·mL⁻¹. In high MDA piglets immunized with different vaccines, serum antibody detection showed that PCV2 antibody levels declined continuously over time in all vaccinated and saline-injected control groups, demonstrating similar trends. In contrast, ELISpot analysis demonstrated a significant increase in PCV2-specific memory B cell levels in all three vaccinated groups compared to the saline-injected group. Among the vaccines tested, Vaccine A induced the highest levels of specific memory B cells, followed by Vaccine B. This was consistent with the lower PCV2 infection rates and viremia levels observed in Vaccine A and Vaccine B groups, compared to Vaccine C and saline-injected control groups.
We established an ELISpot assay to quantify PCV2-specific memory B cells, revealing that vaccinated piglets with high MDA levels developed robust memory B cell responses. However, levels of PCV2 IgG antibodies in vaccinated piglets remained statistically indistinguishable from control piglets. These findings demonstrate that ELISpot-based profiling of PCV2-specific memory B cells overcomes the confounding effects of MDA in vaccine efficacy assessments. This approach reliably reflects the humoral immune response induced by vaccination and its relevance in combating natural PCV2 infection, providing valuable guidance for preventing and controlling PCVAD.
猪圆环病毒2型（PCV2）疫苗接种在预防猪圆环病毒相关疾病（PCVAD）中起着关键作用。然而，由于PCV2疫苗无法实现无菌免疫以及疫苗生产商之间的差异，养猪场在评估疫苗接种效果方面面临重大挑战。传统抗体检测方法的局限性进一步加剧了这些挑战，这些方法无法区分母源抗体（MDA）和疫苗诱导的抗体。准确评估和选择PCV2疫苗对养猪业至关重要。本研究旨在开发一种酶联免疫斑点（ELISpot）检测方法，用于直接检测PCV2特异性记忆B细胞。该方法用于评估接种不同PCV2疫苗且MDA水平较高的仔猪中PCV2特异性记忆B细胞的存在情况，从而能够在细胞水平上评估疫苗的免疫原性。此外，分别使用酶联免疫吸附测定（ELISA）和定量实时聚合酶链反应（qPCR）分析抗体水平和病毒血症状态，以全面评估ELISpot检测方法评估PCV2疫苗免疫原性的潜力。
研究结果表明，所开发的ELISpot检测方法的最佳条件包括用终浓度为1 μg·mL⁻¹的R848刺激三天，PCV2 Cap蛋白包被浓度为1.25 μg·mL⁻¹，生物素化山羊抗猪IgG抗体浓度为5 μg·mL⁻¹，以及HRP-链霉亲和素浓度为0.25 μg·mL⁻¹。在接种不同疫苗的高MDA仔猪中，血清抗体检测表明，所有接种疫苗组和注射生理盐水的对照组中PCV2抗体水平均随时间持续下降，呈现相似趋势。相比之下，ELISpot分析表明，与注射生理盐水组相比，所有三个接种疫苗组中PCV2特异性记忆B细胞水平均显著增加。在所测试的疫苗中，疫苗A诱导的特异性记忆B细胞水平最高，其次是疫苗B。这与在疫苗A组和疫苗B组中观察到的PCV2感染率和病毒血症水平低于疫苗C组和注射生理盐水对照组一致。
我们建立了一种ELISpot检测方法来定量PCV2特异性记忆B细胞，发现MDA水平较高的接种疫苗仔猪产生了强大的记忆B细胞反应。然而，接种疫苗仔猪中PCV2 IgG抗体水平在统计学上与对照仔猪仍无差异。这些发现表明，基于ELISpot的PCV2特异性记忆B细胞分析克服了MDA在疫苗效果评估中的混杂效应。这种方法可靠地反映了疫苗接种诱导的体液免疫反应及其在对抗自然PCV2感染中的相关性，为预防和控制PCVAD提供了有价值的指导。
