Chanda Abhishek, Song Yujuan, Nazir Junaid, Lin Chenwei, Cheng Alicia, Sargent Jennifer, Sikora Aleksandra E
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97330, United States.
Department of Clinical Biochemistry, Lovely Professional University, Phagwara Punjab, India.
Res Sq. 2025 Apr 21:rs.3.rs-6241509. doi: 10.21203/rs.3.rs-6241509/v1.
Assessing antibody titers and functional responses is essential for evaluating vaccine efficacy, yet the impact of blood collection methods on these immunological assessments remains unclear. Retro-orbital (RO) blood collection is commonly used but significant complications can occur. Increasingly, investigators have adopted alternative blood collection approaches, such as saphenous vein (SV) sampling to improve laboratory animal welfare. This study compared RO and SV sampling in the development of a (Ng) vaccine, evaluating Adhesin Complex Protein (ACP) and multiple transferable resistance (Mtr) E protein (MtrE) as antigen candidates. Epitope mapping revealed that ACP and MtrE possess multiple, highly accessible B-cell and T-cell epitope clusters, reinforcing their immunological potential. Following intranasal immunization with rACP, rACP+CpG, and rMtrE+CpG, we assessed the specificity, magnitude, kinetics, and functional quality of immune responses elicited by the immunization regimens. Out of 45 comparisons, only eight significant differences were detected in antibody titers, while the human serum bactericidal assays revealed no differences between RO and SV in antigen-immunized groups. However, antibodies elicited by rACP alone or ACP+CpG in SV samples restored 30.05% and 75.2% of human lysozyme hydrolytic activity compared to 19.3 and 59.9 % in RO, respectively suggesting that SV sampling may be more reliable for assessing functional antibody responses. Beyond its immunological advantages, SV sampling reduces stress, minimizes ocular trauma, and improves animal welfare, making it a viable alternative to RO collection. Given its widespread use in vaccine research, standardizing SV sampling could improve data reliability, ethical compliance, and translational relevance in preclinical studies.
评估抗体滴度和功能反应对于评估疫苗效力至关重要，但采血方法对这些免疫学评估的影响仍不清楚。眶后（RO）采血是常用方法，但可能会出现严重并发症。越来越多的研究人员采用了替代采血方法，如隐静脉（SV）采样，以改善实验动物的福利。本研究在开发一种（Ng）疫苗时比较了RO和SV采样，评估粘附素复合蛋白（ACP）和多重可转移耐药性（Mtr）E蛋白（MtrE）作为抗原候选物。表位作图显示，ACP和MtrE具有多个高度易接近的B细胞和T细胞表位簇，增强了它们的免疫潜力。在用rACP、rACP+CpG和rMtrE+CpG进行鼻内免疫后，我们评估了免疫方案引发的免疫反应的特异性、强度、动力学和功能质量。在45项比较中，仅在抗体滴度上检测到8个显著差异，而人血清杀菌试验显示，抗原免疫组中RO和SV之间没有差异。然而，与RO中的19.3%和59.9%相比，SV样本中单独由rACP或ACP+CpG引发的抗体分别恢复了人溶菌酶水解活性的30.05%和75.2%，这表明SV采样在评估功能性抗体反应方面可能更可靠。除了其免疫学优势外，SV采样还能减轻压力、减少眼部创伤并改善动物福利，使其成为RO采血的可行替代方法。鉴于其在疫苗研究中的广泛应用，标准化SV采样可以提高临床前研究中数据的可靠性、伦理合规性和转化相关性。
