Pasupuleti Dedeepya, Bagwe Priyal, Ferguson Amarae, Uddin Mohammad N, D'Souza Martin J, Zughaier Susu M
Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University, Atlanta, GA 30341, USA.
College of Medicine, QU Health, Qatar University, Doha P.O. Box 2731, Qatar.
Vaccines (Basel). 2024 Sep 13;12(9):1049. doi: 10.3390/vaccines12091049.
Inducing T lymphocyte (T-cell) activation and proliferation with specificity against a pathogen is crucial in vaccine formulation. Assessing vaccine candidates' ability to induce T-cell proliferation helps optimize formulation for its safety, immunogenicity, and efficacy. Our in-house vaccine candidates use microparticles (MPs) and nanoparticles (NPs) to enhance antigen stability and target delivery to antigen-presenting cells (APCs), providing improved immunogenicity. Typically, vaccine formulations are screened for safety and immunostimulatory effects using in vitro methods, but extensive animal testing is often required to assess immunogenic responses. We identified the need for a rapid, intermediate screening process to select promising candidates before advancing to expensive and time-consuming in vivo evaluations. In this study, an in vitro overlay assay system was demonstrated as an effective high-throughput preclinical testing method to evaluate the immunogenic properties of early-stage vaccine formulations. The overlay assay's effectiveness in testing particulate vaccine candidates for immunogenic responses has been evaluated by optimizing the carboxyfluorescein succinimidyl ester (CFSE) T-cell proliferation assay. DCs were overlaid with T-cells, allowing vaccine-stimulated DCs to present antigens to CFSE-stained T-cells. T-cell proliferation was quantified using flow cytometry on days 0, 1, 2, 4, and 6 upon successful antigen presentation. The assay was tested with nanoparticulate vaccine formulations targeting (CDC F62, FA19, FA1090), measles, H1N1 flu prototype, canine coronavirus, and Zika, with adjuvants including Alhydrogel (Alum) and AddaVax™. The assay revealed robust T-cell proliferation in the vaccine treatment groups, with variations between bacterial and viral vaccine candidates. A dose-dependent study indicated immune stimulation varied with antigen dose. These findings highlight the assay's potential to differentiate and quantify effective antigen presentation, providing valuable insights for developing and optimizing vaccine formulations.
诱导针对病原体具有特异性的T淋巴细胞（T细胞）活化和增殖在疫苗制剂中至关重要。评估候选疫苗诱导T细胞增殖的能力有助于优化其安全性、免疫原性和效力的制剂。我们内部的候选疫苗使用微粒（MPs）和纳米颗粒（NPs）来增强抗原稳定性并将其靶向递送至抗原呈递细胞（APC），从而提高免疫原性。通常，使用体外方法筛选疫苗制剂的安全性和免疫刺激作用，但往往需要进行广泛的动物试验来评估免疫反应。我们认识到需要一种快速的中间筛选过程，以便在推进到昂贵且耗时的体内评估之前选择有前景的候选疫苗。在本研究中，体外覆盖测定系统被证明是一种有效的高通量临床前测试方法，用于评估早期疫苗制剂的免疫原性特性。通过优化羧基荧光素琥珀酰亚胺酯（CFSE）T细胞增殖测定，评估了覆盖测定在测试颗粒状候选疫苗免疫反应方面的有效性。将树突状细胞（DCs）与T细胞重叠，使疫苗刺激的DCs将抗原呈递给CFSE染色的T细胞。在成功进行抗原呈递后的第0、1、2、4和6天，使用流式细胞术对T细胞增殖进行定量。该测定用靶向（疾病控制与预防中心F62、FA19、FA1090）、麻疹、H1N1流感原型、犬冠状病毒和寨卡病毒的纳米颗粒疫苗制剂进行测试，佐剂包括氢氧化铝凝胶（明矾）和AddaVax™。该测定显示疫苗治疗组中有强劲的T细胞增殖，细菌和病毒候选疫苗之间存在差异。剂量依赖性研究表明免疫刺激随抗原剂量而变化。这些发现突出了该测定在区分和量化有效抗原呈递方面的潜力，为开发和优化疫苗制剂提供了有价值的见解。
