A Human Papillomavirus (HPV) In Vitro Neutralization Assay That Recapitulates the In Vitro Process of Infection Provides a Sensitive Measure of HPV L2 Infection-Inhibiting Antibodies
A Human Papillomavirus (HPV) In Vitro Neutralization Assay That Recapitulates the In Vitro Process of Infection Provides a Sensitive Measure of HPV L2 Infection-Inhibiting Antibodies
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DOI：
10.1128/cvi.00139-12
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发表时间：
2012-07-01
影响因子：
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通讯作者：
Schiller, John T.
中科院分区：
文献类型：
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作者：
Day, Patricia M.;Pang, Yuk-Ying S.;Schiller, John T.
基于乳头瘤病毒L2的疫苗在标准的体外检测中通常诱导出低水平或无法检测到的中和抗体，但在动物模型中通常能很好地抵御体内实验挑战。在此，我们记录了L2疫苗免疫小鼠，该疫苗包含HPV16、HPV18、HPV1、HPV5和HPV6的L2氨基酸11到88的融合蛋白，可以均匀地保护小鼠免受HPV16假病毒的宫颈阴道攻击，但使用标准的体外中和试验在它们的血清中很少检测到针对HPV16、-31、-33、-45或-58的中和抗体。为了解决这一差异，我们开发了一种基于体外感染性机制的中和试验，该机制更接近体内感染过程，特别是通过时空分离主要和次要受体的参与，并相应地通过改变主要的L2交叉中和表位与抗体接触的时间。使用新的检测方法，大多数血清的滴度在100到10,000的范围内被测量，而用标准的检测方法观察到无法检测到的中和活性。被动转移兔L2免疫血清后小鼠血清中和效价的测定与保护小鼠的宫颈阴道攻击有关。在临床前研究和未来的临床试验中，这种“基于L2的”体外中和试验在关键评估L2候选疫苗的免疫原性方面应该被证明是有用的。
Papillomavirus L2-based vaccines have generally induced low-level or undetectable neutralizing antibodies in standard in vitro assays yet typically protect well against in vivo experimental challenge in animal models. Herein we document that mice vaccinated with an L2 vaccine comprising a fusion protein of the L2 amino acids 11 to 88 of human papillomavirus type 16 (HPV16), HPV18, HPV1, HPV5, and HPV6 were uniformly protected from cervicovaginal challenge with HPV16 pseudovirus, but neutralizing antibodies against HPV16, -31, -33, -45, or -58 were rarely detected in their sera using a standard in vitro neutralization assay. To address this discrepancy, we developed a neutralization assay based on an in vitro infectivity mechanism that more closely mimics the in vivo infectious process, specifically by spaciotemporally separating primary and secondary receptor engagement and correspondingly by altering the timing of exposure of the dominant L2 cross-neutralizing epitopes to the antibodies. With the new assay, titers in the 100 to 10,000 range were measured for most sera, whereas undetectable neutralizing activities were observed with the standard assay. In vitro neutralizing titers measured in the serum of mice after passive transfer of rabbit L2 immune serum correlated with protection from cervicovaginal challenge of the mice. This "L2-based" in vitro neutralization assay should prove useful in critically evaluating the immunogenicity of L2 vaccine candidates in preclinical studies and future clinical trials.