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.Human papillomaviruses (HPVs) are a family of doublestranded DNA viruses that infect epithelial cells in a tissuespecific fashion. Infection with certain "high-risk" types that infect the genital mucosa, such as types 16 (HPV16), 18, and 31, has been shown to be a necessary step in the progression to cervical cancer (1). In nations without effective cervical cancer screening programs, HPVs are the cause of considerable morbidity and mortality (25). In the United States, more than $6 billion is spent annually on evaluation and management of low-grade lesions caused by HPV infection (9). For these reasons, the newly developed vaccines that can prevent HPV infection and that hold promise for eradication of cervical cancer have been greeted with enthusiasm. Currently, vaccines for types 16 and 18 and for the "low-risk" types 6 and 11 (which cause genital warts) are in phase 3 clinical trials (11, 24).The vaccines that are now being evaluated are composed of the HPV major late protein (L1) for each type. This protein self-assembles into empty capsids, also referred to as virus-like particles (VLPs) (10, 13). Early indications are that VLP vaccines are safe and provide protection from persistent HPV infection in a type-specific fashion (11,24). Animal studies suggest that protection from papillomavirus infection is mediated by antibodies (2, 23).Type-specific antibodies recognize conformation-dependent epitopes involving the surface-exposed loops of L1 proteins that exhibit considerable amino acid sequence variation between t...

The humoral response to Gardasil over four years as defined by Total IgG and competitive Luminex immunoassay

“…Vaccination with L1 VLPs elicits a polyclonal response that includes neutralizing antibodies directed against numerous epitopes displayed on the VLP surface. 15,29,40,41 Various assays are available to measure antibodies elicited by HPV L1 VLP vaccines. Some immunoassays are designed to measure a broad Titers are recorded in arbitrary units specific for the assay (i.e., IgG GMT mMU/mL and cLIa GMT mMU/mL).…”

“…It is known that immunization with HPV or VLPs elicits a broad, polyclonal response that recognizes many epitopes found on the capsid surface. 15,29,40,41 In natural infection, however, it is not known if a single neutralizing antibody provides complete protection, or if in fact, many antibodies may be required, working singly or in combination, to achieve complete protection. For HPV 16, the most thoroughly studied HPV type with regard to antibodymediated neutralization, an immunodominant epitope (V5) within L1 has been described, which elicits antibody responses in most naturally infected women.…”

“…15,44 In addition, the most immunodominant epitopes on a VLP may or may not be type-specific. These kinds of differences may explain why antibody levels in the cLIA declined further for HPV 18 in relation to peak levels than antibody levels for the other three vaccine types.…”

“…35,36 On each VLP there are neutralizing epitopes that may or may not be HPV type-specific, as well as epitopes that are non-neutralizing. 15,29,40,41,[52][53][54][55][56] The cLIA is a multiplex assay that involves the displacement of a phycoerythrin (PE)-labeled HPV type-specific, VLP-conformation dependent, neutralizing monoclonal antibody (mAb): H6.M48, K11.B2, H16.V5 and H18.J4 for the HPV 6, 11, 16 and 18 assays, respectively. 35,36 Luminex microspheres are coated with HPV type 6, 11, 16 and 18 L1 VLPs and incubated with the PE-labeled mAbs.…”

Development of a human papillomavirus competitive luminex immunoassay for 9 HPV types

“…[2][3][4] In the clinical trials of the qHPV vaccine, antibodies to the L1 VLPs were measured by an HPV-4 competitive Luminex immunoassay (cLIA). 5,6 This type-specific multiplexed assay measures the competition for antibody binding to a single neutralizing epitope for each VLP.…”