Vaccine Product Development, CSL Seqirus Ltd, Parkville, VIC, Australia.
Collaborating Centre for Reference and Research on Influenza, World Health Organisation, Melbourne, VIC, Australia.
Front Immunol. 2023 Mar 22;14:1147028. doi: 10.3389/fimmu.2023.1147028. eCollection 2023.
The current compendial assay for haemagglutinin antigen potency in influenza vaccine is the single radial immunodiffusion (SRID) which is time consuming and can lead to delays in release of vaccine. We previously described an alternate capture and detection enzyme linked immunoassay (ELISA) that utilizes sub-type specific, sub-clade cross-reactive monoclonal antibodies (mAbs) that are haemagglutination inhibiting (HAI) and correlate with SRID. The aim of this study is to determine the applicability of ELISA across current platforms for quantitation of seasonal quadrivalent vaccine.
A single mAb capture and detection ELISA was employed to quantitate hemagglutinin (HA) derived from different vaccine platforms and host organisms and compared to SRID and a polyclonal antibody based ELISA.
We selected mAbs that displayed appropriate characteristics for a stability indicating potency assay which reacted to avian, insect and mammalian derived HA. Qualification of the homologous mAb assay against egg and cell derived HA demonstrated performance similar to that of the SRID however, superiority in sensitivity and specificity against strains from both influenza B/Victoria and B/Yamagata lineages. Analysis of drifted strains across multiple seasons demonstrated continued utility of this approach, reducing the need to develop reagents each season. With modification of the assay, we were able to accurately measure HA from different platforms and process stages using a single calibrated reference standard. We demonstrated the accuracy of ELISA when testing vaccine formulations containing selected adjuvants at standard and higher concentrations. Accelerated stability analysis indicated a strong correlation in the rate of degradation between the homologous mAb ELISA and SRID but not with ELISA utilizing polyclonal antisera. Further, we demonstrated specificity was restricted to the trimeric and oligomeric forms of HA but not monomeric HA.
We believe this homologous mAb ELISA is a suitable replacement for the SRID compendial assay for HA antigen quantitation and stability assessment. Identification of suitable mAbs that are applicable across multiple vaccine platforms with extended sub-type reactivity across a number of influenza seasons, indicate that this assay has broad applicability, leading to earlier availability of seasonal and pandemic vaccines without frequent replacement of polyclonal antisera that is required with SRID.
目前用于流感疫苗血凝素抗原效力的法定检测方法是单扩免疫扩散法（SRID），该方法耗时且可能导致疫苗放行延迟。我们之前描述了一种替代的捕获和检测酶联免疫吸附测定（ELISA）方法，该方法利用亚型特异性、亚分支交叉反应性单克隆抗体（mAb），这些 mAb 具有血凝抑制（HAI）活性，并且与 SRID 相关。本研究的目的是确定 ELISA 在当前平台上用于定量季节性四价疫苗的适用性。
采用单 mAb 捕获和检测 ELISA 定量不同疫苗平台和宿主来源的血凝素（HA），并与 SRID 和基于多克隆抗体的 ELISA 进行比较。
我们选择了具有稳定性指示效力测定特性的 mAb，这些 mAb 能与禽类、昆虫和哺乳动物来源的 HA 发生反应。针对鸡蛋和细胞来源的 HA 对同源 mAb 测定的鉴定表明，其性能与 SRID 相似，但在灵敏度和特异性方面优于流感 B/Victoria 和 B/Yamagata 谱系的菌株。对多个季节漂移菌株的分析表明，该方法仍具有实用性，减少了每个季节开发试剂的需求。通过对测定方法的修改，我们能够使用单个校准参考标准准确测量来自不同平台和处理阶段的 HA。我们证明了 ELISA 在测试含有选定佐剂的疫苗配方时的准确性，包括标准浓度和更高浓度。加速稳定性分析表明，同源 mAb ELISA 与 SRID 的降解速率之间存在很强的相关性，但与利用多克隆抗血清的 ELISA 则没有。此外，我们证明该方法的特异性仅限于 HA 的三聚体和寡聚体形式，而不是单体 HA。
我们认为这种同源 mAb ELISA 是替代 SRID 法定检测方法用于 HA 抗原定量和稳定性评估的合适方法。鉴定适用于多种疫苗平台且具有广泛流感季节亚型反应性的合适 mAb，表明该测定方法具有广泛的适用性，无需频繁更换与 SRID 一起使用的多克隆抗血清，从而更早地获得季节性和大流行疫苗。
