Identification of human papillomavirus type 16 L1 surface loops required for neutralization by human sera

Abstract

The variable surface loops on human papillomavirus (HPV) virions required for type-specific neutralization by human sera remain poorly defined. To determine which loops are required for neutralization, a series of hybrid virus-like particles (VLPs) were used to adsorb neutralizing activity from HPV type 16 (HPV16)-reactive human sera before being tested in an HPV16 pseudovirion neutralization assay. The hybrid VLPs used were composed of L1 sequences of either HPV16 or HPV31, on which one or two regions were replaced with homologous sequences from the other type. The regions chosen for substitution were the five known loops that form surface epitopes recognized by monoclonal antibodies and two additional variable regions between residues 400 and 450. Pretreatment of human sera, previously found to react to HPV16 VLPs in enzyme-linked immunosorbent assays, with wild-type HPV16 VLPs and hybrid VLPs that retained the neutralizing epitopes reduced or eliminated the ability of sera to inhibit pseudovirus infection in vitro. Surprisingly, substitution of a single loop often ablated the ability of VLPs to adsorb neutralizing antibodies from human sera. However, for all sera tested, multiple surface loops were found to be important for neutralizing activity. Three regions, defined by loops DE, FG, and HI, were most frequently identified as being essential for binding by neutralizing antibodies. These observations are consistent with the existence of multiple neutralizing epitopes on the HPV virion surface.

FIG. 1.

Diagram of the method used for identification of neutralizing epitopes. Antibodies and VLPs were incubated together overnight as described in Materials and Methods. In the presence of wild-type HPV16 VLPs or hybrid VLPs that retained neutralizing epitopes, the effective concentrations of neutralizing antibodies are reduced. Pseudovirus was then added, the cells were infected, and secreted alkaline phosphatase activity was measured 3 days postinfection. If pretreatment of sera failed to adsorb neutralizing activity, low alkaline phosphatase activity was detected. Conversely, high alkaline phosphatase activity indicated a low concentration of neutralizing antibodies and that the VLPs used for pretreatment retained neutralizing epitopes.

FIG. 2.

(A) Identifying surfaces recognized by antibodies and required for neutralizing activity by H16.H5. To determine if the VLPs were correctly folded, they were tested in direct binding assays (ELISAs) using MAbs known to recognize the various hybrids. H16.V5 and H16.U4 are HPV16 MAbs that recognize epitopes known to depend on the native conformation of the HPV16 VLPs. H31.A4 is a specific HPV31 MAb that recognizes a conformation-dependent epitope on HPV31 VLPs. (B) H16.V5 was used to inhibit pseudovirus infection following incubation with VLPs. H16.V5 was titrated across a plate, and hybrid or wild-type VLPs were added. The following day, HPV16 pseudovirions were added to each well and incubated on ice for 1 h. Those samples were transferred to a 96-well tissue culture dish seeded with 293TT cells. After 3 days, 30 μl was removed from each well and tested for alkaline phosphatase activity.

FIG. 3.

Identification of VLP surface loops important for neutralizing activity in human sera by titration of VLPs. Wild-type and hybrid VLPs were titrated and incubated with one of four human sera overnight. These samples were then tested for residual activity in a pseudovirus neutralization assay. Lower optical density readings indicated that neutralization activity was not adsorbed by pretreatment with VLPs. Higher optical density readings indicated that neutralization activity was adsorbed or that there was no neutralizing activity in the serum. Serum A was known to be nonreactive with HPV16 VLPs (not shown). A control for each experiment was serum that was not pretreated with VLPs and that was used to neutralize pseudovirus infection (on the right side of each graph). The symbols represent the same VLP preparations as in Fig. 2.

FIG. 4.

Identification of surface loops required for neutralizing activity in human sera by titration of sera. Human sera were titrated, mixed with wild-type or hybrid VLPs, and incubated overnight. These samples were then tested for residual activity in a pseudovirus neutralization assay. Lower optical density readings indicated that neutralization activity was not adsorbed by pretreatment with VLPs. Relatively high optical density readings indicated that neutralization activity was adsorbed or that there was no neutralizing activity in the serum. The bar graphs represent the optical density values from the graphs on the left that have been normalized for HPV16 wild-type activity. Only the three dilutions of VLPs that showed the greatest difference between HPV16 wild type and HPV31 wild type were used, except for serum J, where only the highest concentration of VLPs was used. The error bars are standard deviations for the three values.