Protection of Rabbits against Challenge with Rabbit Papillomaviruses by Immunization with the N Terminus of Human Papillomavirus Type 16 Minor Capsid Antigen L2

Abstract

Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.

The recognition that persistent infection with high-risk human papillomavirus (HPV) types is a necessary cause of cervical cancer has driven the development of prophylactic vaccines based upon the capsid proteins L1 and L2 (41). Vaccination with L1 virus-like particles (VLPs) (19, 25, 36) or capsomers (37), but not denatured L1, elicits high-titer but type-restricted neutralizing antibodies (8, 19, 32, 33, 35). Studies in dogs challenged with canine oral papillomavirus and rabbits challenged with cottontail rabbit papillomavirus (CRPV) demonstrate that neutralizing antibody induced by L1 VLP vaccination provides immunity from infection against the cognate papillomavirus type from which the vaccinogen was derived (1, 38). Recent clinical studies showed protection against the acquisition of persistent infection and clinical disease related to the HPV types used to derive the monovalent (21, 23), bivalent (13, 14), and tetravalent L1 VLP vaccines (39). Papanicolaou (PAP) cytologic screening and intervention in the United States is estimated to have reduced the incidence of cervical cancer by ∼80% (31), but at a cost of >$6 billion annually. Elimination of these expensive screening programs necessitates vaccination efforts that would require long-term or lifetime protection against most of the common high-risk HPV genotypes. It has been suggested that if protection is truly type specific with L1 VLP vaccines, then a nonavalent vaccine is warranted to achieve >90% protection from cervical cancer (24). However, there is evidence of partial cross-protection against very closely related HPV types (L1 sequence identity of ∼90%) (14, 26, 33).

A single cross-reactive antigen based upon the minor capsid protein L2 represents a possible alternative to highly multivalent L1 VLP vaccines for broad protection against infection with high-risk HPV types (31). Vaccination with L2 as full-length protein, partial polypeptides, or synthetic peptides protects rabbits and cattle against cognate type viral challenges at both cutaneous and mucosal sites (2, 4, 7, 10, 22, 27). L2-dependent immunity is likely to be mediated by neutralizing antibodies (10). L2 of genital HPV types contains broadly cross-neutralizing epitopes that are subdominant in the context of L1/L2 VLPs (34). However, we have shown that antisera to bovine papillomavirus type 1 (BPV1) L2 protein (residues 1 to 88; produced in Escherichia coli) cross-neutralize both cutaneous and mucosal papillomavirus types (29). These findings suggest that neutralizing epitopes at the N terminus of L2 may be conserved across HPV types and species due to some critical viral function (18, 30, 42, 43). Furthermore, it raises the possibility that a single L2-based vaccine simply generated in E. coli might provide comprehensive protection against the HPV types associated with anogenital cancer, benign genital warts, and possibly even those associated with cutaneous warts, and Epidermodyplasia verruciformis (EV) (29). We explore here this possibility by testing whether vaccination of rabbits with HPV16, BPV1, or CRPV L2 polypeptide can protect against concurrent CRPV and rabbit oral papillomavirus (ROPV) challenge at cutaneous and mucosal sites and examining the immune correlates of protection.

MATERIALS AND METHODS

Antigen preparation.

The L2 region comprising residues 1 to 88 of CRPV, HPV16, and BPV1, as well as L2 residues 11 to 200 of CRPV and HPV16, were cloned into pET28a vector (Novagen), and hexahistidine-tagged recombinant polypeptides expressed in E. coli BL21 (Rosetta cells; Novagen) (29). The recombinant L2 polypeptides were affinity purified by binding to a nickel-nitrilotriacetic acid column (QIAGEN) in 8 M urea (using the QiaExpressionist standard purification protocol for denaturing conditions) and then dialyzed in cassettes (Pierce) against Dulbecco phosphate-buffered saline (PBS). Purity was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein concentration was determined by bicinchoninic acid test (Pierce) using a bovine serum albumin standard.

Animal studies.

The studies in rabbits described here were approved by the Animal Care and Use Committees at the Johns Hopkins University and Penn State College of Medicine. Rabbits were individually housed, fed rodent chow ad libitum, and received water from a cage-mounted system. Thirty-six New Zealand White rabbits were divided into six equal groups, each animal receiving three immunizations as indicated. Immunizations consisted of ∼300 μg of L2 polypeptides (CRPV L2 1-88, CRPV L2 11-200, BPV1 L2 1-88, HPV16 L2 1-88, and HPV16 L2 11-200) in PBS with RIBI adjuvant (Corixa Corp., Hamilton, MT) delivered subcutaneously at 3- to 4-week intervals. Each experimental animal was challenged with both ROPV and CRPV 4 weeks after the last immunization. For CRPV infection, rabbits were lightly anesthetized with a mixture of ketamine-HCl (40 mg/kg) and xylazine (5 mg/kg), and their backs were shaved with electric clippers. Two sites on each side of the flanks of the mid-dorsum were scarified with a scalpel until abraded areas of approximately 1 by 1 cm were produced. CRPV papilloma extract (10% [wt/vol] in PBS with a biological 50% inhibitory concentration infectious titer of 10⁻³) was prepared by homogenization of cottontail papillomas obtained from Pennsylvania cottontail rabbit skin xenografts grown in athymic mice (6). Each scarified site received 0.05 ml of papilloma extract, rubbed gently into the wound, and allowed to air dry. The animals were observed, beginning at 3 weeks, for the development of papillomas. Papillomas were measured in three dimensions, and the geometric volume of each papilloma was calculated. Concurrent with the CRPV challenge, and while the rabbits were under anesthesia, ROPV infection was performed. The rabbits were placed on their backs, and each tongue was stretched out by using a pair of forceps. Several small needle punctures were made by using a tuberculin syringe with a 25-gauge needle held at a shallow angle. Nine punctures were made in an area ∼1-cm square onto the right underside midregion of the tongue. Then, 10 μl of ROPV viral stock prepared from homogenates of ROPV papillomas from ROPV-infected xenografts grown in athymic mice (5) was applied to the needle puncture region after blotting with sterile gauze to stop incidental bleeding. The tongue was then placed back into the mouth. The undersurface of the tongue was viewed at several time points beginning at 21 days after infection, and photographs obtained by using a digital camera.

ELISAs.

Immobilon plates (Nunc) were coated with 100 ng of CRPV L1/L2 pseudovirions produced in 293TT cells/well in PBS overnight at 4°C. Wells were then blocked with 1% bovine serum albumin-PBS for 1 h at room temperature and incubated with twofold dilutions of rabbit sera for 1 h at room temperature. After a wash step with PBS-0.01% (vol/vol) Tween 20, peroxidase-labeled goat anti-rabbit immunoglobulin G (KPL, Inc., Gaithersburg, MD) diluted 1:5,000 in 1% bovine serum albumin-PBS was added for 1 h. The plates were then washed and developed with ABTS [2,2′azinobis(3-ethylbenzthiazolinesulfonic acid)] solution (Roche) for 10 min as described previously (40). For measuring immune response against ROPV, nickel-nitrilotriacetic acid-coated microtiter plates were coated with 50 ng of purified ROPV L2 11-200 protein, and an enzyme-linked immunosorbent assay (ELISA) was carried out as recommended by the manufacturer (Sigma).

Neutralization assays.

The HPV, BPV1, and CRPV pseudovirion in vitro neutralization assays were performed as described earlier (28), the secreted alkaline phosphatase content in the clarified supernatant was determined using the p-nitrophenyl phosphate tablets (Sigma, St. Louis, MO) dissolved in diethanolamine, and the absorbance was measured at 405 nm. Constructs and detailed protocols for the preparation of the pseudovirions can be found online (http://home.ccr.cancer.gov/lco/). Titers were defined as the reciprocal of the highest dilution that caused a 50% reduction in A₄₀₅, and a titer of <15 was not considered significant. For assay of ROPV neutralization, authentic ROPV virions isolated from infected xenografts were incubated with diluted sera (1:50) for 2 h at 37°C and then used to inoculate monolayers of RK13 cells (rabbit epithelial cells) in fresh Eagle medium (10% fetal bovine serum) (final concentration of 1:100). Medium was exchanged 2 days postinfection. At 4 days postinfection, total RNA was harvested by using TRIzol (Invitrogen), and isolated RNAs were analyzed for UV absorbance (260/280). Reverse transcription-PCR was performed with 800 ng of input RNA with a GeneAmp RNA PCR kit (Applied Biosystems). Viral E1̂E4 cDNA was produced by using the transcript specific downstream primer 5′-AGGAGTAGGTGGTCTAAAGGCG-3′. The master mix for the first round of PCR for E1̂E4 amplicons included the upstream primer 5′-GCTACGCTTTACCTGTGTGAGC-3′. Second-round nested PCR was performed with 2.5 μl of the first-round product with a master mix containing upstream primer 5′-TTACTTTGCAAGGACTGCGCC-3′ and downstream primer 5′-ATCTCCTCCGTCCGTAACGAGA-3′ for a 196-bp amplicon containing the E1̂E4 splice junction. β-Actin was amplified by reverse transcription using random hexamers (RT) and a single round of the PCR using primers 5′-GATGACCCAGATCATGTTTG-3′ and 5′-GGAGCAATGATCTTGATCTTC-3′.

Statistical analysis.

We tested for differences in protection from cutaneous CRPV challenge by CRPV seroconversion status (determined by ELISA and neutralization assays) and by vaccine type using Fisher exact tests. Differences in numbers of oral papillomas after ROPV challenge by vaccine type were tested by using the Mann-Whitney test. The results were considered statistically significant at a P value of <0.05. All analyses were performed by using STATA 9.0 (College Station, TX).

RESULTS

Mounting evidence suggests that immunization with several different L2 polypeptides induces very broadly cross-neutralizing antibodies compared to the type-restricted neutralizing serum antibodies and immunity generated by L1 VLP vaccines (16, 17, 29, 34). Rabbits provide the opportunity to study protective immunity to both cutaneous and mucosotropic papillomavirus types because they can be simultaneously challenged with both CRPV and ROPV (10). To examine the in vivo protective and cross-protective potential of L2 vaccines and to determine which polypeptide is most effective, we generated recombinant polypeptides from the L2 of CRPV (of genus kappa like ROPV), HPV16 and BPV1 (alpha and delta papillomaviruses, respectively), representing highly divergent papillomavirus types from different genera (9). Since prior studies had demonstrated that protective epitopes reside within residues 11 to 200 of L2 (4) and that broadly cross-neutralizing epitopes are present within residues 1 to 88 (29), we tested whether vaccination with these two polypeptides generates immunity against cognate and heterologous type papillomavirus challenge (Tables 1 and 2).

TABLE 1.

Summary of L2 vaccine study at 35 days after CRPV challenge^a

Immunogen	No. of papillomas/no. of sites	No. of rabbits with papilloma(s)/no. of rabbits examined	Approximate papilloma size (mm)
HPV16 L2 1-88	7/20	3/5b	1-4
BPV1 L2 1-88	3/24	2/6	∼1
CRPV L2 11-200	1/24	1/6	3
HPV16 L2 11-200	2/24	1/6	1
CRPV L2 1-88	1/24	1/6	1
Control (none)	15/16	4/4c	3-8

^aFour cutaneous sites per rabbit were infected with CRPV virions.

^bOne animal died.

^cTwo animals died.

TABLE 2.

Summary of L2 vaccine study at 35 days after ROPV challenge^a

Immunogen	No. of papillomas per animal (six rabbits/group)	Total no. of papillomas	Approximate papilloma size(s)
HPV16 L2 1-88	1, 0, 2, 1, 2, 0	6	All very small
BPV1 L2 1-88	1, 2, 1, 2, 0, 0	6	All very small
CRPV L2 11-200	1, 0, 1, 1, 0, 0	3	All very small
HPV16 L2 11-200	0, 1, 1, 0, 0, 1	3	All very small
CRPV L2 1-88	0, 1, 1, 4, 0, 0	6	Very small to small
Control (none)	1, 2, 5, 7, 1, 3	19	All large

^aEach animal received nine tongue punctures, but not all sites became papillomas. The numbers of papillomas per animal and the approximate size(s) are presented. Two animals died in the control group, but we were able to obtain ROPV data from them.

L2 vaccination effects on CRPV challenge.

Consistent with earlier studies showing that vaccination of cows with BPV4 L2 11-200 protects from BPV4 challenge, vaccination with CRPV L2 11-200 effectively protected rabbits from CRPV challenge applied to scarified skin at four sites (one of six rabbits with papillomas observed in vaccinated versus four of four in placebo rabbits; P = 0.05) (Table 1 and Fig. 1A and B). Similarly, vaccination with the smaller polypeptide, CRPV L2 1-88, was also highly protective against CRPV-induced papillomas (two of six rabbits with papillomas observed in vaccinated versus four of four in placebo rabbits; P = 0.08). Vaccination with L2 1-88 derived from the divergent type BPV1, which induces broadly cross-neutralizing antibodies, partially protected against CRPV challenge, although there was more evidence of breakthrough infection. Although vaccination with HPV16 L2 1-88 was less protective than either CRPV or BPV1 L2 1-88, the HPV16 L2 11-200 polypeptide provided the same level of effective protection against CRPV challenge as that observed for the homologous CRPV L2 polypeptides (P = 0.05). Thus, the longer polypeptide comprising residues 11 to 200 might more effectively induce cross-PV genera protection in this model than the 1-88 polypeptide (Table 1 and Fig. 1A and B).

FIG. 1.

Protection of L2-vaccinated rabbits against CRPV but not CRPV DNA challenge. (A) Rabbits were vaccinated with the L2 peptides indicated and challenged (four sites/animal) with infectious CRPV at 3 weeks after the last immunization. The papillomas were measured beginning on day 14, and the percentage of challenge sites (four per animal) with papillomas and the standard deviation (SD) of sites with papillomas in each vaccine group is shown. (B) The mean size of the papillomas (mm³ ± the SD) on the rabbits at the number of days indicated after CRPV challenge for each vaccine group. (C) L2-vaccinated rabbits that were completely immune to CRPV virion challenge, and an additional three naive rabbits, were subsequently challenged with CRPV genomic DNA. The mean size of the papillomas (mm ± the SD) on the rabbits at the number of days indicated after CRPV genomic DNA challenge for each vaccine group is shown. The data for challenge with CRPV genomic DNA that was wild type or L2 deficient are combined since there was no significant difference in the growth rate of the papillomas that they induce.

Cutaneous challenge with CRPV genomic DNA, like native virions, induces papillomas in naive rabbits. To determine whether the protection was mediated by neutralization of the viral inoculum by antibody to L2 and/or L2-specific cell-mediated immunity, the L2-vaccinated experimental rabbits that failed to develop papillomas after CRPV challenge plus three additional naive rabbits were challenged with infectious CRPV genomic DNA instead of virions (Table 3 and Fig. 1C). CRPV genomic DNA was applied to scarified skin at two sites, and CRPV genomic DNA deficient for L2 expression (ATG mutant) was applied at two other sites on each animal. After CRPV DNA challenge, papilloma growth was evident in 3 of 3 naive rabbits and 16 of 17 L2 vaccinated rabbits that were immune to CRPV virion challenge. Furthermore, there was no significant difference in the rate of papilloma growth at sites inoculated with wild-type CRPV genomic DNA or CRPV DNA deficient for L2 expression in either the unvaccinated or the L2-vaccinated animals. The failure of L2 vaccination to protect against viral DNA challenge (despite protecting against challenge with CRPV virions) and the similarity of growth rates for papillomas induced by wild-type and L2 knockout genomes provides evidence that protection against cognate and heterologous viral challenge upon vaccination with L2 polypeptides is not elicited via cell-mediated immunity and suggests the importance of cross-neutralizing antibodies (Table 3).

TABLE 3.

Summary of papilloma growth after cutaneous challenge of L2-vaccinated rabbits with CRPV DNA^a

Immunogen	No. of rabbits in group	No. of sites with papillomas/no. of total sites inoculated
HPV16 L2 1-88	1	4/4
BPV1 L2 1-88	2	4/8b
CRPV L2 11-200	5	20/20
HPV16 L2 11-200	5	20/20
CRPV L2 1-88	4	16/16
Naive control	3	11/12

^aCRPV wild-type and L2-deficient DNA-induced papillomas are grouped together because they were not significantly different.

^bOne rabbit had no papillomas.

To address the role of L2-specific antibody in protection against CRPV-induced papillomas, we measured L2-specific serum antibody titer after L2 vaccination. The antibody titer was assessed by ELISA reactivity to CRPV particles with L2 compared to CRPV particles without L2 (Fig. 2A). The presence of detectable CRPV L2 antibody (titer of >15 as determined by ELISA) correlated with protection against CRPV challenge (P = 0.01). Finally, detection of serum antibody that neutralizes CRPV pseudovirions in vitro (titer of >15) strongly correlated with protection of the same animals from CRPV-induced papillomas (P < 0.001) (Fig. 2B).

FIG. 2.

L2-specific serum antibody titers in vaccinated rabbits. (A) CRPV L1/L2 pseudovirus ELISA titers of individual rabbits and means in each vaccine group. No specific reactivity was observed in control animals. Titers of <15 were considered as not detected. (B) In vitro CRPV neutralization titers of the sera described in panel A.

L2 vaccination effects on ROPV challenge.

We explored the potential for protection against a mucosal papillomavirus by concurrently challenging the same rabbits orally with ROPV at the same time as the cutaneous CRPV challenge. For ROPV infection, crude stock (Hershey isolate) was applied to nine needle puncture sites on the underside of each rabbit's tongue, and oral papilloma growth was monitored by number and diameter (Table 2 and Fig. 3A). Immunization with the two longer polypeptides (HPV16 and CRPV L2 11-200) resulted in significantly fewer ROPV-induced oral papillomas (P = 0.012, Mann-Whitney test) and appeared to be more effective than any of the three shorter polypeptides (BPV1, HPV16, and CRPV L2 1-88 [P = 0.05, P = 0.11, and P = 0.05, respectively, as determined by the Mann-Whitney test]). Likewise, protection against ROPV-induced papillomas by these different L2 polypeptides (Table 3) was also consistent with the titer of serum antibody reactive with ROPV L2 11-200 by ELISA (Fig. 3B) and with the detection of in vitro ROPV neutralization (Fig. 3C).

FIG. 3.

Protection of rabbits against ROPV challenge and their antibody responses after vaccination with L2 polypeptides. (A) Each group of vaccinated rabbits was challenged with ROPV on the underside of the tongue. The data shows the mean number of challenge sites (+ the SD) per animal with oral papillomas in each vaccine group as determined 35 days after infection. (B) ROPV L2 11-200 ELISA titers of individual rabbits and means in each vaccine group (CRPV L2 1-88, CRPV L2 11-200, BPV1 L2 1-88, HPV16 L2 1-88, and HPV16 L2 11-200). No specific reactivity was observed in control animals. Titers of <15 were considered as not detected. (C) Rabbit sera with a range of ROPV L2 11-200 ELISA titers were tested in a semiquantitative authentic ROPV virion neutralization assay. The rabbit sera (as indicated at 1:50) were incubated with ROPV virions for 2 h at 37°C and then used to inoculate monolayers of RK13 cells. Four days later, total RNA was isolated and assayed for the presence of viral E1̂E4 transcripts by nested reverse transcription-PCR. Lanes show no virus, or no RNA, or rabbit sera with ROPV L2 11-200 (ELISA titers of <15), HPV 16 L2 1-88 serum (titer of 2,560), HPV16 L2 11-200 serum (titer of 40,960), CRPV L2 1-88 serum (titer of 1,280), CRPV L2 11-200 serum (titer of 10,240), and BPV L2 1-88 serum (titer of <20) and a no-RNA control.

Cross-neutralizing activity in serum of L2-vaccinated rabbits.

Since vaccination with L2 provides cross-protection via neutralizing antibodies, we addressed the breadth of cross-neutralization by L2-specific antibodies among HPV types found most often in human cancers as well as BPV1 and HPV5 (Fig. 4). Vaccination with CRPV L2 1-88 or 11-200 induced low and inconsistent levels of cross-neutralization of HPV types, whereas BPV L2 1-88 induced broadly cross-neutralizing serum antibodies. We attempted to map the epitopes in HPV16 L2 recognized by the antisera of cross-neutralizing sera obtained from two rabbits immunized with CRPV L2 11-200 using overlapping synthetic 15-mer peptides. However, reactivity across the full length of the first 200 residues of HPV16 L2, including several previously defined neutralizing epitopes (17-36, 65-81, 94-122, 131-151, and 151-170) that may contribute to protection (3, 4, 20), was observed (data not shown), suggesting a broad response. Importantly, vaccination with HPV16 L2 1-88 or 11-200 induced detectable titers of neutralizing antibodies against all of the HPV pseudovirions tested, although the titers were generally higher in the animals vaccinated with the longer polypeptide and against the homologous rather than against the heterologous papillomavirus types (Fig. 4).

FIG. 4.

In vitro pseudovirion neutralization titers of sera from rabbits vaccinated with L2 polypeptides. Serum titers of individual rabbits and means in each vaccine group (CRPV L2 1-88, CRPV L2 11-200, BPV1 L2 1-88, HPV16 L2 1-88, and HPV16 L2 11-200) are indicated for in vitro neutralization of pseudovirions derived from BPV1, HPV5, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 (A to H, respectively). No specific reactivity (titer of >15) was observed in control animals.

DISCUSSION

In the present study we show that vaccination with L2 induces cross-protection against divergent papillomavirus types from different genera after challenge at both cutaneous and mucosal sites in a relevant animal model. Furthermore, protection appears to be mediated by broadly cross-neutralizing serum antibodies and strongly correlates with the detection of even low antibody titers. We have recently shown that patients vaccinated with full-length HPV16 L2 induce low levels of cross-neutralizing antibodies (11), even without the use of adjuvant, suggesting that our observations in the rabbit model may be broadly generalizable to humans. Notably, vaccination with HPV16 L2 induced more effective cross-neutralization of other HPV types than vaccination with CRPV L2 polypeptides. This likely reflects the relative similarity among the genital HPV types compared to the divergent CRPV. Thus, vaccination with HPV16 L2 and challenge with a rabbit papillomavirus represents a stringent test of cross-protection, and vaccination with HPV16 L2 might provide better protection against natural acquisition of genital HPV than was seen for high-dose challenge with rabbit papillomavirus.

Determination of the relevant immune correlate and minimal level for protection is an important issue for clinical development of HPV vaccines and assessment of the likelihood that an individual is immune to a particular HPV strain. Clinical studies of Cervarix (HPV16 and HPV18 L1 VLPs formulated in alum plus monophosphoryl lipid A) suggest type-restricted protection with partial cross-protection against highly related HPV types (e.g., HPV45 and HPV31) (26). Since L1 VLP-specific cross-neutralization titers are likely >100-fold less than for the cognate type, this finding suggests that even low titers of neutralizing serum antibodies are protective against natural acquisition of oncogenic HPV. Earlier animal studies with L2 vaccines are consistent with this notion. Vaccination with residues 94 to 112 or 107 to 122 of CRPV L2 induced neutralization titers ranging from 1:5 to 1:10 but protected rabbits from experimental viral infection by CRPV (10). Immunization of calves with BPV4 L2 11-200 yielded ELISA titers of 1:100 (and neutralization at a dilution of 1:5) (12), and yet the calves were completely protected against BPV-4 challenge (4). Vaccination with three BPV4 L2 peptides (101-120, 131-151, and 151-170 combined, but not separately) also protected calves from experimental challenge, but generated ELISA titers of only 1:30 (3). Similarly, vaccination with L2 polypeptide induces both protection and low neutralization titers (7, 22). Although these studies suggest that even low titers of L2-specific neutralizing antibodies in serum are likely to confer protection, additional work to boost cross-neutralizing antibody titers with adjuvants or capsid display technologies may be necessary to prolong broad protection. Notably, we used RIBI as an adjuvant to stimulate L2-specific immunity. The RIBI analog adjuvant, monophosphoryl lipid A, is approved for use in Europe in the hepatitis B vaccine Engerix and is utilized in Cervarix. However, other adjuvants can elicit higher titers of neutralizing antibody when used with HPV16 L2 11-200 (data not shown), suggesting that this may not be the optimal formulation for L2.

Although vaccination with L1 VLP and possibly with L2 induces a cellular immune response, this arm of the immune system is not likely to affect protection because of the unique biology of papillomavirus capsid antigen expression. Our studies with CRPV genome-induced papillomas suggest that vaccination with L2, as for L1 VLP vaccines, provided no therapeutic benefit. This likely reflects the failure of the basal epithelial cells that harbor CRPV genomes to express either capsid gene. Some caution should be taken in interpreting this experiment because CRPV produces only low levels of virions in domestic NZW rabbit papillomas, suggesting that L2 may not be an effective target for cell-mediated immunity; however, CRPV L1 vaccination via gene-gun can induce some protection (15), suggesting that at least L1 is expressed in CRPV papillomas of NZW rabbits. Further passive antibody transfer experiments need to be carried out to demonstrate that neutralizing antibodies are sufficient for protection in this model.

Current HPV vaccines target only 2 of the 15+ oncogenic HPV genotypes, all of which must be covered to eradicate cervical cancer. Here we have shown that HPV16 L2 11-200 vaccination in the rabbit papillomavirus model (that was successfully used to develop L1 VLP vaccines because there is no animal model for genital HPV infection) provides remarkably broad cross-genotype protection against two rabbit papillomaviruses. Further, in addition to neutralizing all of the oncogenic genotypes tested, L2 antibodies are also effective against low-risk HPV types and cutaneous HPV types, suggesting potential as a pan-HPV preventive vaccine.

The potential benefits of vaccination against low-risk HPV types should not be underestimated. HPV6 and HPV11 are the major types causing condyloma accuminata and also the less common conditions of recurrent respiratory papillomatosis and Buschke-Lowenstein tumors. In addition to morbidity suffered by patients, the physician visits associated with these conditions are costly. Protection against these types would provide an additional rationale for the vaccination of men. Oncogenic HPV, in particular HPV16, also causes a significant fraction of oral cancers, including the salivary gland, tonsils, and base of the tongue. Here we show that vaccination with HPV16 L2 11-200 provided significant protection against ROPV challenge on the underside of the tongue.

It is not clear whether cross-protection would extend to the types associated with plantar and planar warts, although comparison of sequence homology suggests that this is a possibility. Interestingly, we observed cross-neutralization of the EV-associated type HPV5 by sera of rabbits vaccinated with BPV1 or HPV16 L2 polypeptides. HPV5 is associated with non-melanoma skin cancer in patients with the rare heritable condition EV and in organ transplant patients. Furthermore, an association between EV-related HPV types and psoriasis has been proposed. Notably, CRPV challenge was performed in cutaneous skin (and frequently induces squamous cell cancer), and L2 vaccination provided protection at this site.

A theoretical concern is that by eliminating the two major oncogenic HPV genotypes through vaccination, the remaining oncogenic types will become more common to fill a niche previously occupied by HPV16 and HPV18, thereby reducing the impact of the current vaccines on cervical cancer. However, a highly multivalent HPV vaccine or potentially an L2-based broadly protective vaccine would negate this concern.

Unless highly multivalent formulations of L1 VLPs are produced screening programs must remain in place even if effective national vaccination programs were instituted, because current HPV vaccines target only two oncogenic types (albeit those responsible for ∼70% of cervical cancer). The formulation, testing, and manufacture of such complex vaccines is expensive (a course of the quadrivalent L1 VLP Gardasil vaccine costs $360) and comes with rapidly diminishing returns since many oncogenic HPV genotypes are responsible for only a small percentage of cervical cancers. Cost is particularly important because cervical cancer is a major problem in developing nations that lack the resources or are too remote for national screening programs. The United States spends >$6 billion per annum on cytologic screening and intervention; as a result, there are only 3,700 deaths/year in the United States due to cervical cancer. In contrast, cervical cancer is the second leading cancer killer of women in developing countries, after breast cancer. For example 100,000 women die from cervical cancer each year in India. A low-cost HPV vaccine eliciting protection against a broad spectrum of HPV types would be particularly useful where national screening programs are currently not instituted. L2 is produced simply as a single recombinant protein in bacteria and affinity purified under denaturing conditions. Local production of such a vaccine might achieve the goal of production at cents/dose in the developing countries with the most cases of cervical cancer.