A Human Papillomavirus (HPV)In VitroNeutralization Assay That Recapitulates theIn VitroProcess of Infection Provides a Sensitive Measure of HPV L2 Infection-Inhibiting Antibodies

Abstract

ABSTRACTPapillomavirus L2-based vaccines have generally induced low-level or undetectable neutralizing antibodies in standardin vitroassays yet typically protect well againstin vivoexperimental 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 standardin vitroneutralization assay. To address this discrepancy, we developed a neutralization assay based on anin vitroinfectivity mechanism that more closely mimics thein vivoinfectious 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 vitroneutralizing 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 vitroneutralization assay should prove useful in critically evaluating the immunogenicity of L2 vaccine candidates in preclinical studies and future clinical trials.