Minor Capsid Protein of Human Genital Papillomaviruses Contains Subdominant, Cross-Neutralizing Epitopes

Virology 270, 254–257 (2000) doi:10.1006/viro.2000.0272, available online at http://www.idealibrary.com on

RAPID COMMUNICATION Minor Capsid Protein of Human Genital Papillomaviruses Contains Subdominant, Cross-Neutralizing Epitopes Richard B. S. Roden,* ,† ,1 William H. Yutzy IV,† Rosemary Fallon,‡ Stephen Inglis,‡ Douglas R. Lowy,* and John T. Schiller* *Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892-4040; †Department of Pathology, The Johns Hopkins University, Baltimore, Maryland 21205-2196; and ‡Cantab Pharmaceuticals Research Limited, 310 Cambridge Science Park, Milton Road, Cambridge CB4 4GN, United Kingdom Received January 14, 2000; accepted February 23, 2000 Vaccination with virus-like particles (VLP), comprising both L1 and L2 of human papillomavirus (HPV) genital types 6, 16, and 18, induces predominantly type-specific neutralizing antibodies. L2 polypeptide vaccines protect animals against experimental challenge with homologous papillomavirus and cross-reactive epitopes are present in HPV L2. To assess L2-specific cross-neutralization of HPV genotypes, sheep were immunized with purified, bacterially expressed HPV6, 16, or 18 L2. In addition to neutralizing the homologous HPV type in vitro, antisera to each HPV L2 also cross-neutralized both heterologous HPV types. This suggests that unlike VLP-based prophylactic HPV vaccines, an L2 polypeptide vaccine may provide broad-spectrum protection. © 2000 Academic Press

like particles (VLPs) that are morphologically and immunologically very similar to virion (12). Vaccination with L1 VLPs induces high titers of conformationally dependent neutralizing antibodies and is able to protect animals from homologous experimental infection at both cutaneous and mucosal challenge sites (1, 11, 19). Passive transfer of purified immunoglobulin is sufficient to confer protection to na¨ive animals, consistent with the hypothesis that high levels of neutralizing antibodies in serum are sufficient for protection (1, 19). However, the neutralizing antibodies to L1 are predominantly type specific with the exception of the closely related types (⬎85% L1 amino acid identity) which show weak cross-reactivity (4, 17). Furthermore, vaccination with VLPs or virion derived from one papillomavirus type does not protect against experimental infection with heterologous types (1). L2 is incorporated into VLPs, probably at the capsid vertices, when coexpressed with L1 (12). Much of L2 is internal to the capsid and is required for genome encapsidation (16). However, a small portion of L2 is exposed on the capsid surface and accessible to antibodies (15), a subset of which are neutralizing (5, 6, 18). L2 is not required for binding of virions to cell surfaces and neutralizing antiserum to L2 does not affect this process (6, 18). Immunization of rabbits with CRPV L2 amino acids 259–492 fused with ␭ cII protein induced a serum neutralizing titer of 32 (64-fold lower than that obtained by immunization with CRPV virions) and was protective against experimental cutaneous infection (5). In a sec-

Over one hundred human papillomavirus (HPV) genotypes have been identified (http://hpv-web.lanl.gov/) and are grouped according to the location and type of disease they cause; cutaneous types that induce benign plantar warts, those types associated with the rare inherited disorder epidermodysplasia verruciformis, and the genital-tropic HPV types. These genital-tropic human papillomaviruses are responsible for considerable suffering and morbidity worldwide. They may be further classified as either “lowrisk” or “high-risk” genotypes by the disease they cause. The low-risk genotypes, such as HPV6 and 11, cause benign genital warts, condyloma accuminata, whereas highrisk genital HPVs are the etiologic agent of cervical and a number of other cancers (7). The high-risk HPV types are frequently detected in cancers and exhibit transforming and immortalizing properties in vitro. They are typified by HPV types 16 and 18, which are detected worldwide in 51 and 12% of intraepithelial neoplasia cases, respectively. Unlike other HPV types, the genital-tropic HPV types are primarily sexually transmitted (7). The papillomavirus capsid comprises two genetically unrelated proteins called L1 and L2 at an estimated ratio of 30:1 (20), which represent potential targets for a prophylactic vaccine. L1 is able to self-assemble into virus-

1 To whom correspondence and reprint requests should be addressed at Department of Pathology, Room 656, Ross Research Building, 720 Rutland Ave, Baltimore, MD, 21205-2196. Fax: (410) 614-3548. E-mail: [email protected].

0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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ond study, rabbits immunized with amino acids 67–488 of CRPV L2 fused to trpE produced low levels of neutralizing antibodies (a titer of 10) but were completely protected from experimental infection (14). Rabbit antiserum to His 6 fusions of full length or amino acids 45–173 of BPV1 L2 had in vitro neutralization titers of 33 and 1000, respectively (18). However the relative contributions of the humoral and cellular immunity in L2 prophylaxis are unclear (8). While the ability of L2 vaccination to protect against natural (genital) transmission remains to be determined, L2 is a component of a candidate therapeutic vaccine for genital warts that is currently in clinical testing (13). Perhaps the L2 vaccination studies most relevant to genital HPV infection involve BPV4 that is associated with alimentary cancer in cattle (3). Immunization of cattle with BPV4 L2 fused to glutathione S-transferase (GST) elicits in vitro neutralizing antibodies and provides longlasting immunity to BPV4 infection (3). Prophylactic vaccination with BPV4 L2 was effective at low doses, over long periods, and using denatured material in alum adjuvant, suggesting that this approach might also be feasible against HPVs. The neutralizing epitopes have been mapped to within polypeptide L2a (amino acids 11–200) (6), including three immunodominant B cell epitopes represented by peptides 101–120, 131–151, and 151–170 (3), consistent with data for BPV1 L2 (18). Furthermore, immunization with L2a (3) or these three peptides in combination coupled to KLH was protective, with peptide 131–151 exhibiting a therapeutic effect, but no single neutralizing epitope was defined (2). A number of non-neutralizing epitopes on BPV1 L2 (15) and HPV33 L2 have been defined using monoclonal antibodies (Mabs). Recently, Kawana et al. (10) generated three Mabs to HPV16 L2 that neutralized HPV16 pseudovirus infection, and two of these Mab also neutralized HPV6. The epitope recognized by the Mab that neutralized only HPV16 comprises residues 69–81 of HPV16 L2, a region that is recognized by antibodies of patients with genital HPV infections (9, 10). The two monoclonal antibodies that neutralized both HPV6 and 16 recognized epitopes in residues 108–120 of HPV16 L2, a region of homology among the genital HPV genotypes (10). This epitope might be used for development of a broadly protective prophylactic HPV vaccine. Published studies of L2 vaccination have only addressed challenge by homologous papillomavirus type. At least 15 different high-risk genital HPV types have been identified in intraepithelial neoplasia which represent many serotypes (7). Vaccination with VLPs derived from each serotype would likely be required for complete protection, making formulation both cumbersome and expensive. Although neutralizing activity was not tested, both HPV type-specific and cross-reactive linear epitopes in L2 have been identified by Western blotting and ELISA (7). Cross-reactive epitopes on HPV11, 16, 18,

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and 33 were identified by Western blot and enzymelinked immunosorbent assays (21). These cross-reactive epitopes occur in a region of L2 likely to be on the surface of the virion, to contain epitopes recognized by humans, and to be neutralizing (6, 9, 10, 15, 18). Therefore, although L2 is less well conserved than L1, we have examined L2 for potential as a broad specificity prophylactic genital HPV vaccine able to induce neutralizing antibody to many HPV genotypes. We chose HPV types 6, 16, and 18 for study since they represent both low- and high-risk genital HPVs, and they are the most common genital types and are each from distinct phylogenetic clades, groups A11, A9, and A7, respectively (http:// hpv-web.lanl.gov/). In Vitro Neutralization of HPV6, 16, and 18 Pseudovirions. To generate hyperimmune sera, sheep were immunized monthly with 0.1 mg of gel-purified L2 peptides derived from HPV types 6, 16, and 18, with Freund’s complete adjuvant to prime, and incomplete adjuvant for all boosts. HPV16 L2 protein was expressed as two separate GST-fusion proteins (incorporating aa 1–251 and aa 240–473, respectively), which were gel-purified. HPV18 L2 protein was also expressed as a GST fusion, but using the complete sequence. HPV6 L2 protein was expressed as a TrpE fusion, once again incorporating the complete L2 sequence. As a negative control, a sheep was immunized with crude extract of Escherichia coli that do not express L2 (null). The in vitro neutralization activity of these sera for BPV1 virions was determined as described previously (16). Because of difficulties in obtaining HPV virion, and consistently and quantitatively assaying infectivity we adopted a pseudovirion strategy, in which HPV capsid proteins are used to package the BPV1 genome that may be detected in vitro by focal transformation of mouse fibroblasts. We have previously applied this methodology to HPV16, to generate HPV16{BPV1} pseudovirions (16). HPV6 L1 and L2 were PCR-amplified from HPV6 virus DNA (a kind gift of Dr. William Bonnez, Rochester) and cloned into pSFV4.2 (generously donated by Dr. Joel Jessie, Life Technologies, Gaithersburg, MD) and pSFV1 expression vectors, respectively, from which recombinant SFV were produced and used to generate HPV6{BPV1} pseudovirions, as for HPV16{BPV1} (16). The same procedure was also applied to generate HPV18{BPV1} pseudovirions. The ability of antisera from rabbits immunized three times with 300 ␮g of L1/L2 VLPs derived from HPV types 6b, 11, 16, 18, 31 (L1 only), 33, and 45 (17) to neutralize these pseudovirion preparations in vitro was determined (Table 1). High neutralization titers toward viruses of the same type as that used to generate the VLP-specific sera, weak neutralization by very closely related types from within the same phylogenetic clade, e.g. HPV6 and 11, HPV18, and 45 (http://hpv-web.lanl.gov/), and absence of neutralization of heterologous types were detected. This

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In Vitro Neutralization of HPV Pseudovirions and BPV1 by Antisera to Papillomavirus Capsid Proteins Antiserum Null E. coli extract† Null E. coli extract* ,† HPV6 L2* ,† HPV16 L2† HPV16 L2* ,† HPV18 L2† HPV6 L1 ⫹ L2 VLP HPV11 L1 ⫹ L2 VLP HPV16 L1 ⫹ L2 VLP HPV18 L1 ⫹ L2 VLP HPV31 L1 VLP HPV33 L1 ⫹ L2 VLP HPV45 L1 ⫹ L2 VLP

HPV6{BPV1} HPV16{BPV1} HPV18{BPV1} BPV1 ⬍10

⬍10

⬍10

⬍10

⬍10 1000 100 100 1000 33,000

⬍10 33 1000 330 330 ⬍10

⬍10 33 33 33 1000 ⬍10

⬍10 ⬍10 ⬍10 ⬍10 10 ND

1000

⬍10

⬍10

ND

10

33,000

⬍10

ND

⬍10 ⬍10

⬍10 ⬍10

33,000 ⬍10

ND ND

⬍10

⬍10

⬍10

ND

⬍10

⬍10

10

ND

Note. HPV pseudovirions or BPV1 were incubated with titrated antisera for 1 h on ice and then incubated with mouse C127 cell monolayers for 1 h at 37°C. The C127 cells were maintained for 3 weeks and stained, and the foci counted. Titers indicate the reciprocal dilution that inhibits focus formation by approximately 50%. No neutralization of HPV6, 16, or 18 pseudovirion or BPV1 was observed with a 1:10 dilution of preimmunization sera for each antiserum described. Rabbit antiserum was used except for samples (†) in which sheep antisera were used. *Indicates that antibody purified from serum by capryllic acid precipitation and DEAE ion-exchange chromatography was used at 8 mg/ml for the in vitro neutralization assay.

neutralization between genital HPV types, but not of BPV1, was also observed (Table 1). This suggests that immunization with L2 derived from one genital HPV type may be sufficient to confer significant protection from infection by other genital HPVs, but not other papillomavirus types, consistent with serologic data (22) and Mab studies (10). It is surprising that immunization with VLPs containing L2 does not also provide cross-neutralizing antibodies. Perhaps the relevant epitopes of L2 are masked by the capsid except during infection, resulting in poor presentation to the immune system (3). Alternatively, the highly immunogenic nature of the regularly spaced L1 epitopes might dominate the humoral response. Poor antibody response to L2 was also observed upon infection with BPV, CRPV, and HPV (3, 22). Finally, the lack of crossneutralizing antibody may result from a lower dose of immunogen; 300 ␮g of L1/L2 VLP, containing ⬃10 ␮g of L2, was injected each immunization as compared to 100 ␮g of L2 gel-purified polypeptide. In conclusion, we have demonstrated that L2 of genital HPV types 6, 16, and 18 contains subdominant crossneutralizing epitopes. This is the first study to demon-

is consistent with previous hemagglutination inhibition and neutralization data (4, 16, 17). Note that even antisera to VLPs containing L2 are not cross-neutralizing between HPV phylogenetic clades. L2 Antibodies Cross-Neutralize Genital HPVs. The ability of sheep antiserum to HPV6, 16, and 18 L2 to neutralize these types in vitro was determined (Table 1). Consistent with studies of animal papillomaviruses, in each case the L2 antiserum was able to most strongly neutralize the homologous HPV virion type. The titers were 30-fold lower than those obtained with rabbit antiserum to the homologous VLPs; nevertheless animal studies indicate that this is sufficient to confer protection (6). Cross-neutralizing titers were 2–3 orders of magnitude lower than those induced by the homologous VLP type. The inability of HPV L2 antisera to neutralize BPV1 (except a 10-fold dilution of HPV18 L2 antiserum) or to neutralize HPV pseudovirions when preincubated with purified L2 protein (Fig. 1) demonstrates that inhibition of infection is L2 antibody-mediated. Further, neither preimmune serum nor immune serum to control E. coli extract neutralized at 1:10 dilution (Table 1). Interestingly, cross-

FIG. 1. Anti-HPV6 L2 serum-mediated neutralization of HPV18 pseudovirions is prevented by preincubation with purified HPV6 L2E7 protein. Anti-HPV6 L2 antibody was incubated with 100 ␮g of purified HPV6 L2-E7-fusion protein or buffer alone for 1 h on ice and then mixed with HPV18{BPV1} pseudovirions for a further hour on ice. The samples were incubated with monolayers of mouse C127 cells for 1 h at 37°C, the cells were washed and maintained for 3 weeks in complete medium, and the foci were stained. (A) HPV18{BPV1} pseudovirions alone. (B) HPV18{BPV1} pseudovirions preincubated with 1:100 rabbit antiserum to HPV18 L1/L2 VLPs, (C) HPV18{BPV1} pseudovirions preincubated with 1:10 sheep antibody to HPV6 L2, (D) HPV18{BPV1} pseudovirions preincubated with 1:10 sheep antibody to HPV6 L2 plus 100 ␮g of purified HPV6 L2-E7-fusion protein.

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strate generation of broadly cross-neutralizing antisera after vaccination with a specific papillomavirus immunogen derived from a single HPV genotype. It is particularly noteworthy that cross-neutralization was detected for the representatives of two major clades of high-risk genitaltropic HPV. While cross-neutralizing titers were between 33 and 330, this may be sufficient for protection. Immunization with bacterially expressed CRPV L2 elicited neutralizing titers of 10 (14) and 32 (5), yet rabbits were protected from experimental CRPV infection. Similarly, vaccination with bacterially expressed GST-BPV4 L2 elicited serum antibody neutralizing at a titer of 5 in cattle protected from BPV4 infection of the soft palate (6). Evidence that L2 vaccination protects animals from experimental infection (3, 14) and that cross-neutralizing epitopes exist in HPV L2 (our studies and (10)) suggest that further study of L2 as a broad spectrum, prophylactic genital HPV vaccine is warranted. Such a vaccine has several theoretical advantages; it should be easy and cheap to produce and could be safely used with alum adjuvant since ongoing studies have proven bacterially expressed HPV6 L2-E7 well tolerated in humans (13). We intend to further define the nature of the cross-reactive epitopes of L2 using patient sera (13) and refine them to an HPV L2 consensus sequence that might be even more broadly cross-neutralizing and immunogenic. ACKNOWLEDGMENTS This work was supported by the intramural program of the National Institutes of Health, and by the Richard TeLinde Fund. We thank Dr. Robert J. Kurman for his advice and support. Due to space limitations the referencing is not exhaustive and the authors apologize for any omissions.

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