Human papillomaviruses (HPVs) have a nonenveloped icosahedral capsid of 50 to 55 nm composed of the major L1 protein and the minor L2 protein. The capsid contains 72 pentamers of L1, centered on the vertices of a T=7 icosahedral lattice (1
). The number of L2 molecules per capsid has been estimated to be 12 (48
). The major capsid protein L1 of HPV can self-assemble into virus-like particles (VLPs) which have the size, shape, and conformational epitopes of virion capsids (25
). Progress has recently been made concerning the structure of papillomavirus capsids (9
), and significant progress has been made in the study of neutralizing antibodies, but limited information is available concerning the nature of L1 sequences corresponding to neutralizing epitopes.
Ninety-two HPVs have been identified to date. They induce benign epidermal and mucosal papillomas, and the development of cervical cancer is strongly associated with genital infection by specific types, such as HPV type 16 (HPV-16), HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-52, HPV-58, and HPV-59 (33
). Numerous serologic studies have demonstrated that infection with genital HPVs is followed by a serologic immune response to the major viral capsid protein, L1. This immune response persists for many years and is largely HPV type specific and directed against conformational epitopes (4
). Moreover, both linear and conformational epitopes have been identified on the surface of HPV L1 VLPs (11
Studies using canine papillomavirus and cottontail papillomavirus have shown that immunization with L1 VLPs can protect animals from subsequent challenge with infectious virus (3
). Moreover, protection can also be obtained by passive transfer of serum antibodies from vaccinated or naturally infected animals to naive animals, suggesting that the protection is mediated by neutralizing antibodies (3
). In addition, immunization of mice with HPV VLPs (but not unassembled L1) generates predominantly type-specific neutralizing antibodies (11
The first tests developed to determine neutralizing antibodies were based on the mouse xenograft system (2
). However, the number of HPV types that have been successfully grown in this model is very limited, and the technique is time-consuming. The second means to measure neutralizing antibodies is to generate pseudovirions in vitro and to measure the inhibition of focus formation or gene expression due to the pseudovirions. Several procedures have been developed to produce pseudovirions. It has been shown that HPV VLPs composed of L1 or L1/L2 have the ability to package the bovine papillomavirus genome or irrelevant plasmid DNA in cellular (36
) and acellular (23
) systems. The pseudovirions obtained have the ability to transfer the plasmid DNA into cells where the reporter gene is expressed. Moreover, it has been shown that the presence of L2 in HPV VLPs dramatically increases their gene transfer efficiency (23
It has been demonstrated that neutralization epitopes are present in the L1 major capsid protein (11
) and in the L2 minor capsid protein (24
). Both linear and conformational epitopes have been identified on the surface of HPV-16 L1 VLPs, and at least three L1 regions, i.e., amino acids 111 to 130, 174 to 185, and 261 to 280, contain linear epitopes (13
). The results suggest that conformational B-cell epitopes of HPV virions or VLPs induce neutralizing antibodies (10
). In contrast, cross-reactive epitopes are linear epitopes and mostly nonneutralizing (12
). It has been suggested that such linear epitopes are not surface exposed (14
The L1 protein sequences of certain genital HPVs share strong homology (8
), but the majority of anti-VLP antibodies are not cross-neutralizing (20
). Using in vitro infectivity assays, some cross-neutralization between HPV-31 and -33 and between HPV-18 and -45 has been observed (20
). Such cross-neutralization is in agreement with the cross-reactivity observed by Roden et al. (35
) using hemagglutination assays.
Recombinant HPV VLPs particles are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated in human subjects (18
). It is thus important to determine how many HPV types might be needed in a vaccine intended to protect against most genital HPV-associated cancers. This is related to the types associated with cancers (33
), their relative frequency, and the degree of cross-protection that could be induced by one particular type.
The aim of this study was to investigate the neutralizing activity of mouse monoclonal and polyclonal HPV L1 antibodies by using the pseudoinfection system for seven high-risk HPVs in order to obtain better definition of cross-neutralization between genital HPVs. Cross-neutralization between HPV-16, -18, -31, -33, -45, -58, and -59 was investigated, and we also characterized the neutralizing properties of 16 HPV L1 monoclonal antibodies (MAbs).