Mambetaliyev Muratbay, Alieva Alina, Abduraimov Yergali, Rsaliyev Aralbek, Zhugunissov Kuandyk
Laboratory of Microorganism Collection, Research Institute for Biological Safety Problems, Guardeyskiy, Kazakhstan.
Department of Biological Safety, National Holding "QazBioPharm", Astana, Kazakhstan.
Front Immunol. 2025 Jul 21;16:1640056. doi: 10.3389/fimmu.2025.1640056. eCollection 2025.
Serological assessment of antibody levels is a crucial measure of immunity in vaccinated animals. Establishing the level of antibodies considered protective is essential for vaccine standardization and evaluation of efficacy. The virus neutralization test (VNT), recognized as the gold standard for detecting virus-specific antibodies able to neutralize virus.
This study evaluated the effect of viral dose on the detection of humoral immune responses in rabbits vaccinated with a cowpox virus-based vaccine. Blood serum samples were collected on days 14, 21, and 28 post-vaccination. VNT was conducted using viral doses of 100, 50, 25, and 10 TCID. Additionally, the infectious dose 50 (ID) of the challenge virus was determined based on the induction of skin necrosis in 50% of infected animals. This dose (316 ID per 0.1 mL) was then used to challenge vaccinated rabbits in order to determine the protective antibody titer threshold.
Lower viral doses (25 and 10 TCID) demonstrated higher sensitivity, with neutralizing antibody titers detected at 1:16 and above, significantly exceeding those obtained using 50 and 100 TCID. Based on these findings, 25 TCID was selected as the optimal dose for future VNT. Following cowpox virus challenge, rabbits with neutralizing titers ≥1:16 were protected from skin necrosis, while non-immunized animals developed characteristic lesions.
These results suggest that a low-dose (25 TCID) VNT improves the sensitivity and that a titer of 1:16 can be considered a protective threshold. This approach provides a reliable laboratory model for assessing the immunogenicity and efficacy of cowpox virus vaccines. The results obtained in this study allow for an objective assessment of the immunity elicited from a cowpox vaccine using a laboratory model.
抗体水平的血清学评估是疫苗接种动物免疫的关键指标。确定具有保护作用的抗体水平对于疫苗标准化和疗效评估至关重要。病毒中和试验（VNT）被认为是检测能够中和病毒的病毒特异性抗体的金标准。
本研究评估了病毒剂量对接种牛痘病毒疫苗的家兔体液免疫反应检测的影响。在接种后第14、21和28天采集血清样本。使用100、50、25和10 TCID的病毒剂量进行病毒中和试验。此外，根据50%受感染动物皮肤坏死的诱导情况确定攻毒病毒的半数感染剂量（ID）。然后使用该剂量（每0.1 mL含316 ID）对接种疫苗的家兔进行攻毒，以确定保护性抗体滴度阈值。
较低的病毒剂量（25和10 TCID）显示出更高的灵敏度，中和抗体滴度在1:16及以上被检测到，显著超过使用50和100 TCID时获得的滴度。基于这些发现，选择25 TCID作为未来病毒中和试验的最佳剂量。在用牛痘病毒攻毒后，中和滴度≥1:16的家兔免受皮肤坏死，而未免疫的动物出现特征性病变。
这些结果表明，低剂量（25 TCID）的病毒中和试验提高了灵敏度，1:16的滴度可被视为保护性阈值。这种方法为评估牛痘病毒疫苗的免疫原性和疗效提供了可靠的实验室模型。本研究获得的结果允许使用实验室模型客观评估牛痘疫苗引发的免疫力。
