Measurement of neutralizing serum antibodies of patients vaccinated with human papillomavirus L1 or L2-based immunogens using furin-cleaved HPV Pseudovirions

Abstract

Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant.

Figure 1. Correlation of neutralization assays.

The estimated EC50 values of each patient sample from the respective assays were log2 re-transformed and plotted using R package mcr. Values for Person's r, slope and intercepts were rounded to 2 decimal places. The 0.95-confidence bounds are calculated with the bootstrap (quantile) method. Comparison of HPV16 VLP vaccinated patient sera (n = 70) in FC-PBNA versus L1-PBNA (A). Comparison of L1-PBNA (B) and FC-PBNA (C) with previous findings (n = 12) by Pastrana and colleagues using SEAP-based L1-PBNA . Comparison of n = 30 Gardasil vaccinated patient sera in FC-PBNA and L1-PBNA against HPV16 (D), HPV18 (E) and HPV6(F).

Figure 2. Assessment of TA-CIN sera (n = 17).

HPV16 full length L2 ELISA using sera of patients vaccinated with TA-CIN was performed in triplicate and is presented as mean ± Standard deviation (A). Results of in vivo passive transfer studies with patient sera. Asterisk indicates significant difference in mean infection of the five mice per group ± standard error against intra vaginal HPV58 challenge observed between pre- and post-vaccinated sera (100 µL per mouse) (B). For both figures, white bars indicate pre-vaccinated serum and hashed bars indicate post-vaccinated serum.

Figure 3. FC-PBNA correlates well with both in vivo murine challenge model and L2-PBNA.

Comparison of FC-PBNA EC50 fitted in vitro neutralization titers curve (black line) with passive transfer studies using titrated dilutions (10 µL, 33 µL, 100 µL) of TA-CIN patient sera (white bars) where IT-9 (A), IT-10 (B), IT-13 (C), IT-15 (D), IT-16(E). For IT-13, sera did not cross 50% inhibition at 10 µL and thus, the experiment with a further 3 µL dilution was repeated to assess in vivo inhibition. Comparison of serum titers of patients vaccinated with TA-CIN as detected by FC-PBNA and the L2-PBNA and plotted using R (see text for Pearson's r, slope and intercept) (F).