HPV-16 L1 Virus-like Particles Research Articles

Antibody response to human papillomavirus vaccine in subjects with inherited bone marrow failure syndromes

Patients with Fanconi anemia (FA) and other inherited bone marrow failure syndromes (IBMFS) have extremely high risks of squamous cell head and neck, and gynecologic carcinomas. In the general population, these sites are often associated with infection with human papillomaviruses (HPV), particularly HPV16 and HPV18. Our objectives were to measure the levels of HPV antibodies in unvaccinated patients, and to determine whether these patients developed and maintained similar levels of antibodies as those observed in healthy women, following vaccination with the currently licensed HPV L1 virus-like particles (VLP) vaccines. We measured anti-HPV IgG antibody levels in sera from patients using an HPV16 or HPV18 L1 VLP enzyme-linked immunoabsorbent assay. Most unvaccinated patients did not have detectable antibody levels, except for a few women above age 25. Both FA and other IBMFS patients developed antibody levels following vaccination that were similar to those previously described in healthy women, and those levels appeared to be sustained out to 5 years after immunization. Thus, antibody responses to the HPV L1 VLP vaccine in patients with FA and other IBMFS appeared to be similar to the responses reported in the general population, implying potential efficacy against future infections with the HPV types contained in the vaccine.

A simple and rapid method to monitor the disassembly and reassembly of virus-like particles

Protein fluorescence spectra (∼300–440nm) could be used as a simple and sensitive method to monitor the disassembly and reassembly of virus-like particles (VLPs). Insect cell expressed and purified HPV-16 L1 VLPs show significantly high fluorescence intensity, whereas the fluorescence is almost quenched after disassembly by adding the reducing agent. By removing the reducing agent, the fluorescence was restored to its original intensity, indicating the reassembly of VLPs. The data are consistent with enzyme-linked immunosorbent assay (ELISA) reactivity using conformation-specific mouse monoclonal antibody. The same method could be extended to VLPs of other viruses.

Kinetic and HPV infection effects on cross-type neutralizing antibody and avidity responses induced by Cervarix®

BackgroundWe previously demonstrated that Cervarix® elicits antibody responses against vaccine-related types for which clinical efficacy was demonstrated (HPV-31 and -45). Here, we evaluated the kinetics of neutralization titers and avidity of Cervarix®-induced antibodies up to 36 months of follow-up in unexposed and HPV infected women. MethodsA subset of women who participated in the Cost Rica HPV-16/18 Vaccine Trial had pre- and post-vaccination sera tested for antibody responses to HPV-16, -18, -31, -45, and -58 using a pseudovirion-based neutralization assay, and HPV-16 antibody avidity using an HPV-16 L1 VLP (virus-like particle)-based ELISA developed in our laboratory. ResultsIn uninfected women, neutralizing antibody titers did not reach significance until after the 3rd dose for HPV-31 (month 12, p=0.009) and HPV-45 (month 12, p=0.003), but then persisted up to month 36 (HPV-31, p=0.01; HPV-45, p=0.002). Individuals infected with HPV-16 or HPV-31 at enrollment developed a significantly higher median antibody response to the corresponding HPV type after one dose, but there was not a difference between median titers after three doses compared to the HPV negative group. Median HPV-16 antibody avidity and titer increased over time up to month 12; however, the HPV-16 avidity did not correlate well with HPV-16 neutralizing antibody titers at each time point examined, except for month 6. The median avidity levels were higher in HPV-16 infected women at month 1 (p=0.04) and lower in HPV-16 infected women at month 12 (p=0.006) compared to the HPV negative women. ConclusionsThe persistence of cross-neutralization titers at month 36 suggests cross-reactive antibody responses are likely to persist long-term and are not influenced by infection status at enrollment. However, the weak correlation between avidity and neutralization titers emphasizes the need for examining avidity in efficacy studies to determine if high avidity antibodies play a critical role in protection against infection.

Comparison of the immune responses to the CIA06-adjuvanted human papillomavirus L1 VLP vaccine with those against the licensed HPV vaccine Cervarix™ in mice

CIA05 is a toll-like receptor (TLR) 4 agonist derived from an Escherichia coli lipopolysaccharide (LPS) mutant and has been shown to have potential as a vaccine adjuvant. In this study, we investigated the immunopotentiating activity of the adjuvant system CIA06, which is comprised of CIA05 and aluminum hydroxide (alum), when used with the human papillomavirus (HPV) L1 virus-like particles (VLPs) vaccine. BALB/c mice were immunized intramuscularly three times at 2-week intervals with HPV16 L1 VLPs alone or in the presence of various combinations of CIA05 and alum, and the immune responses were assessed. We found that the combination of CIA05 and alum at a ratio of 1:50 (designated CIA06B) yielded the highest immune response in terms of serum anti-HPV L1 VLP IgG antibody titers, splenocyte interferon (IFN)-γ secretion, and antigen-specific memory B cell responses. The immunogenicity of the CIA06B-adjuvanted HPV16/18 L1 VLP vaccine was compared with that of the currently licensed HPV vaccine Cervarix™. The CIA06B-adjuvanted vaccine was similar to Cervarix™ with regard to eliciting serum antigen-specific IgG antibodies and virus-neutralizing antibodies but more effective at inducing splenic cytokine production and memory B cells. We also observed that the antigen-specific IgG antibody titers, splenic IFN-γ secretion and memory B cells induced by the CIA06B-adjuvanted HPV vaccine remained high up to 24 weeks post-immunization. Based on these data, we concluded that CIA06B may have potential as an adjuvant in a potent prophylactic vaccine against HPV infection.

Oral administration of HPV-16 L2 displayed on Lactobacillus casei induces systematic and mucosal cross-neutralizing effects in Balb/c mice

The human papillomavirus (HPV) minor capsid protein, L2, is a good candidate for prophylactic vaccine development because L2-specific antibodies have cross-neutralizing activity against diverse HPV types. Here, we developed a HPV mucosal vaccine candidate using the poly-γ-glutamic acid synthetase A (pgsA) protein to display a partial HPV-16 L2 protein (N-terminal 1–224 amino acid) on the surface of Lactobacillus casei (L. casei). The oral immunization with L. casei-L2 induced productions of L2-specific serum IgG and vaginal IgG and IgA in Balb/c mice. To examine cross-neutralizing activity, we used a sensitive high-throughput neutralization assay based on HPV-16, -18, -45, -58, and bovine papillomavirus 1 (BPV1) pseudovirions. Our results revealed that mice vaccinated with L. casei-L2 not only generated neutralizing antibodies against HPV-16, but they also produced antibodies capable of cross-neutralizing the HPV-18, -45, and -58 pseudovirions. Consistent with previous reports, vaccination with HPV-16 L1 virus-like particles (VLPs) failed to show cross-neutralizing activity. Finally, we found that oral administration of L. casei-L2 induced significant neutralizing activities against genital infection by HPV-16, -18, -45, and -58 pseudovirions encoding a fluorescence reporter gene. These results collectively indicate that oral administration of L2 displayed on L. casei induces systemic and mucosal cross-neutralizing effects in mice.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity

BackgroundHuman papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs).ResultsVLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced.Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entireHPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres.ConclusionsD/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

Characterization of the HPV-specific memory B cell and systemic antibody responses in women receiving an unadjuvanted HPV16 L1 VLP vaccine

Human papillomavirus (HPV)-specific antibodies are proposed to be the correlate of protection afforded by HPV L1 virus-like particle (VLP) vaccines. Previous studies have characterized the systemic antibody response to immunization in terms of both the quantity and the ability to neutralize HPV. Here, we have adapted a generalized memory B cell ELISPOT to the HPV16 system and expanded the analysis of the systemic antibody response to include an avidity measurement of HPV L1 VLP-specific antibodies. We show the results of the memory B cell ELISPOT significantly correlated with IgG and neutralizing antibody titers, but not with the avidity measurement. This is the first comprehensive study to correlate a variety of humoral aspects potentially associated with protective immunity following vaccination with a HPV16 L1 VLP vaccine.

Molecular and structural characterization of the L1 virus-like particles that are used as vaccine antigens in Cervarix™, the AS04-adjuvanted HPV-16 and -18 cervical cancer vaccine

Cervarix™ is a prophylactic human papillomavirus (HPV)-16/18 vaccine developed for the prevention of cervical cancer. The vaccine antigens are HPV-16 and HPV-18 L1 virus-like particles (VLPs) made from baculovirus expression vector system (BEVS)-produced HPV-16 and HPV-18 L1 proteins, respectively. In this study, we demonstrate that truncation of the nuclear/DNA binding signals at the C-terminus of the HPV-16 and HPV-18 L1 proteins prevented intra-nuclear formation of the VLPs in the host cells and led to cytoplasmic localization of the L1 proteins as shown by in situ immunogold detection and electron microscopy. Following purification, these L1 proteins were able to form VLPs. The characteristics of these HPV-16 and HPV-18 L1 VLPs were studied using various physicochemical and immunological techniques. Amino acid analysis, SDS-PAGE and western blotting demonstrated the high purity of the L1 proteins and batch-to-batch consistency. The structure of the VLPs was shown to be similar to that reported for the native virions, as evaluated by microscopic observations, protein tomography and disc centrifugation experiments. The presence of important conformation-dependent neutralizing epitopes, such as U4, V5 and J4, was confirmed by ELISA and surface plasmon resonance. Structural robustness and consistency among batches was also observed by differential scanning calorimetry and electron microscopy. Moreover, adsorption to aluminum was shown not to impair VLP structure. In conclusion, the BEVS-produced HPV-16 and HPV-18 L1 VLPs display key structural and immunological features, which contribute to the efficacy of Cervarix™ vaccination.

Abstract A143: Vaccination of a concatenated L2 fusion protein with L1 VLP or capsomeres for broad protection against HPV infection

Abstract Background: Immunization with L1 major capsid protein in the form of the pentavalent capsomeres or 360mer virus-like particles (VLPs) generates high and long-lived titers of neutralizing antibodies and protection primarily against the human papillomavirus (HPV) type from which the vaccine was derived. Consequently, multivalent L1 VLP vaccines have been developed to expand the breadth of coverage, e.g. Cervarix which contains L1 VLPs that are derived from HPV16 and HPV18 and formulated in alum and monophosphoryl lipid A (MPL) adjuvant. Vaccination with L2 minor capsid protein induces lower titer, but more broadly neutralizing and protective antibody responses in animals. To enhance cross-protection we generated multi-type L2 proteins by fusing cross-neutralizing epitopes of divergent HPV types, e.g. 11-88×5 that is a concatamer of L2 residues 11–88 of HPV1, HPV5, HPV6, HPV16 and HPV18. Objectives: A low cost HPV vaccine that yields cross-protective antibodies and broad protection against more than a dozen oncogenic HPV types is urgently needed. We sought to combine the advantages of each protective antigen by immunization with titrated doses of multi-type L2 11-88×5 in alum+MPL, or with either HPV16 L1 capsomeres or Cervarix and to assess the importance of adjuvant for L2 dose sparing. Methods: Mice were vaccinated three times every alternate week and bled two weeks later. Serum antibody titers were determined by ELISA using HPV16 L1 VLP or L2 11-88×5antigen, as well as in vitro HPV pseudovirion neutralization assays. Results: The absolute titers and dose response to 11-88×5 were similar in mice vaccinated with 11-88×5 alone or in combination with either L1 capsomeres or VLPs. HPV16 L1 VLP generated significantly higher HPV16 neutralization titers than L1 capsomeres, and the capsomeres induce higher titers than 11-88×5. Importantly, the serum IgG titers specific to HPV16 L1 VLP were not influenced by vaccination with L1 capsomeres or VLP in the presence of L2 11-88×5. The in vitro neutralization titers elicited by vaccination with 11-88×5 in the combinations tested were not significantly different at two weeks after immunization but there was a trend at four months for reduced cross-neutralization when 11-88×5 was combined with L1 VLP but not capsomeres. Conclusions: There is no evidence of interference between the L1 and L2 serum antibody response to co-administration of L1 capsomeres, but not VLP, with multi-type L2 vaccines based on neutralization titers. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A143.

Combined prophylactic and therapeutic intranasal vaccination against human papillomavirus type-16 using different adeno-associated virus serotype vectors

Cervical cancer is the second most frequent cancer among woman worldwide and is considered to be caused by infection with high-risk papilloma viruses. Genetic immunization using recombinant adeno-associated virus (rAAV) vectors has shown great promise for vaccination against human papillomavirus (HPV) infections. rAAV5, -8 and -9 vectors expressing an HPV16 L1/E7 fusion gene were generated and applied intranasally for combined prophylactic and therapeutic vaccination of mice. The rAAV5 and the rAAV9 vectors showed efficient induction of both humoral and cellular immune responses, whereas rAAV8 failed to immunize mice by the intranasal route. The L1-specific immune response evoked by expression of the L1/E7 fusion gene, however, was lower than that evoked by expression of the L1 antigen alone. This deficiency could be compensated by application of Escherichia coli heat-labile enterotoxin or monophsphoryl lipid as adjuvant upon vaccination with rAAV5-L1/E7. Coimmunization of rAAV9-L1/E7 with rAAV5-L1 or boosting of rAAV9-L1/E7 with rAAV5-L1 strongly increased L1-specific neutralizing antibody titres to levels above those achieved by vaccination with vectors expressing L1 alone. Both vectors elicited a vibrant cytotoxic T-lymphocyte response against L1 or E7. Nasal immunization with rAAV5 or rAAV9 was superior to vaccination with HPV16-L1 virus-like particles (VLPs) or HPV16-L1/E7 CVLPs with respect to humoral and cellular immune responses. Vaccination with the rAAV vectors led to a significant protection of animals against a challenge with different HPV tumour cell lines. Our results show that rAAV5 and rAAV9 vectors are promising candidates for a non-invasive nasal vaccination strategy.

A Major Role for the Minor Capsid Protein of Human Papillomavirus Type 16 in Immune Escape

High-risk human papillomavirus (HPV) infection of the cervical epithelium is causally linked with the generation of cervical cancer. HPV does not activate Langerhans cells (LC), the APC at the site of infection, leading to immune evasion. The HPV protein responsible for inducing this immune escape has not been determined. We demonstrate that LC exposed to the minor capsid protein L2 in HPV16L1L2 virus-like particles do not phenotypically or functionally mature. However, HPV16L1 virus-like particles significantly induce activation of LC. Our data suggest that the L2 protein plays a specific role in the induction of this immune escape of HPV16 through the manipulation of LC. This novel function is the first immune modulating action attributed to the L2 protein and adds significantly to our understanding of the mechanism of HPV immune escape.

Concatenated Multitype L2 Fusion Proteins as Candidate Prophylactic Pan-Human Papillomavirus Vaccines

BackgroundVaccination with minor capsid protein L2 induces antibodies that cross-neutralize diverse papillomavirus types. However, neutralizing antibody titers against the papillomavirus type from which the L2 vaccine was derived are generally higher than the titers against heterologous types, which could limit effectiveness against heterologous types. We hypothesized that vaccination with concatenated multitype L2 fusion proteins derived from known cross-protective epitopes of several divergent human papillomavirus (HPV) types might enhance immunity across clinically relevant HPV genotypes.MethodsAntibody responses of mice (n = 120) and rabbits (n = 23) to vaccination with HPV-16 amino-terminal L2 polypeptides or multitype L2 fusion proteins, namely, 11-200 × 3 (HPV types 6, 16, 18), 11-88 × 5 (HPV types 1, 5, 6, 16, 18), or 17-36 × 22 (five cutaneous, two mucosal low-risk, and 15 oncogenic types), that were formulated alone or in GPI-0100, alum, or 1018 ISS adjuvants were compared with vaccination with L1 virus-like particles (VLPs), including Gardasil, a licensed quadrivalent HPV L1 vaccine, and a negative control. Mice were challenged with HPV-16 pseudovirions 4 months after vaccination. Statistical tests were two-sided.ResultsThe HPV-16 L2 polypeptides generated robust HPV-16–neutralizing antibody responses, albeit lower than those to HPV-16 L1 VLPs, and lower responses against other HPVs. In contrast, vaccination with the multitype L2 fusion proteins 11-200 x 3 and 11-88 x 5 induced high serum neutralizing antibody titers against all heterologous HPVs tested. 11-200 × 3 formulated in GPI-0100 adjuvant or alum with 1018 ISS protected mice against HPV-16 challenge (reduction in HPV-16 infection vs phosphate-buffered saline control, P < .001) 4 months after vaccination as well as HPV-16 L1 VLPs, but 11-200 × 3 alone or formulated with either alum or 1018 ISS was less effective (reduction in HPV-16 infection, P < .001).ConclusionConcatenated multitype L2 proteins in adjuvant have potential as pan-oncogenic HPV vaccines.

Rectal and vaginal immunization of mice with human papillomavirus L1 virus-like particles

Human papillomavirus (HPV) vaccines based on L1 virus-like particle (VLP) can prevent genital HPV infection and associated lesions after three intramuscular injections. Needle-free administration might facilitate vaccine implementation, especially in developing countries. Here we have investigated rectal and vaginal administration of HPV16 L1 VLPs in mice and their ability to induce anti-VLP and HPV16-neutralizing antibodies in serum and in genital, rectal and oral secretions. Rectal and vaginal immunizations were not effective in the absence of adjuvant. Cholera toxin was able to enhance systemic and mucosal anti-VLPs responses after rectal immunization, but not after vaginal immunization. Rectal immunization with Resiquimod and to a lesser extent Imiquimod, but not monophosphoryl lipid A, induced anti-HPV16 VLP antibodies in serum and secretions. Vaginal immunization was immunogenic only if administered in mice treated with nonoxynol-9, a disrupter of the cervico-vaginal epithelium. Our findings show that rectal and vaginal administration of VLPs can induce significant HPV16-neutralizing antibody levels in secretions, despite the fact that low titers are induced in serum. Imidazoquinolines, largely used to treat genital and anal warts, and nonoxonol-9, used as genital microbicide/spermicide were identified as adjuvants that could be safely used by the rectal or vaginal route, respectively.

Gene Expression Patterns Induced by HPV-16 L1 Virus-Like Particles in Leukocytes from Vaccine Recipients

Human papillomavirus (HPV) virus-like particle (VLP) vaccines were recently licensed. Although neutralizing Ab titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMCs from 17 vaccine and 4 placebo recipients before vaccination and 1 mo after receiving the second immunization. Vaccination with a monovalent HPV-16 L1 VLP vaccine was associated with modulation of genes involved in the inflammatory/defense response, cytokine, IFN, and cell cycle pathways in VLP-stimulated PBMCs. Additionally, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing Ab titers were found for cyclin D2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with microarray data was seen for more than two-thirds of these probesets. Up-regulation of immune/defense response genes by HPV-16 L1 VLP, in particular, IFN-induced genes, was observed in PBMCs collected before vaccination, with many of these genes being further induced following vaccination. In conclusion, we identified important innate and adaptive response-related genes induced by vaccination with HPV-16 L1 VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials.

Abstract A138: Vaccination with HPV 16 L2E6E7 with GPI-0100 adjuvant elicits protective humoral as well as cell-mediated immune responses

Abstract A138 L1 virus-like particle (VLP) vaccines provide effective protection against Human papillomavirus (HPV) type from which the L1 was derived via the induction of neutralizing antibodies. While partial protection can occur against very closely related types, it is generally type-restricted and necessitates highly multivalent L1 VLP vaccines to obtain broad coverage. The minor capsid protein, L2, is emerging as an attractive alternative cross-protective antigen. Unfortunately, neither L1 VLP nor L2-based vaccines are effective against pre-existing disease, suggesting the need to develop therapeutic vaccines. Unlike the capsid proteins, the E6 and E7 oncoproteins are both required to maintain and expressed throughout cervical intraepithelial neoplasia (CIN) and cancer. TA-CIN, a vaccine comprising the HPV16 L2, E6 and E7 in a single tandem fusion protein, attempts to combine the advantages of broad cross-protection against HPV transmission with therapeutic responses targeting HPV16 early proteins. Here we test TA-CIN formulated along with GPI-0100, a semi-synthetic quillaja saponin analog, that was developed to promote both humoral and cellular immune responses. TA-CIN administered subcutaneously to mice three times at monthly intervals (125μg of clinical-grade protein) with 50μg GMP-grade GPI-0100 was found to elicit high titer antibodies that effectively neutralized not only HPV16 but also other oncogenic HPV types including HPV18, HPV31, HPV45, HPV58. Similarly, vaccination of pigtail macaques (Macaca nemestrina) with TA-CIN (three doses of 125μg of TA-CIN with 1000μg GMP-grade GPI-0100 at monthly intervals) was well tolerated and induced serum antibodies that neutralized HPV16, HPV18, and HPV31 in vitro. Notably, vaccination of mice with TA-CIN protected them from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP. Combination of TA-CIN along with adjuvant GPI-0100 enhanced production of E7-specific, interferon gamma producing CD8+ T cell precursors by 20-fold. Vaccination with TA-CIN in adjuvant GPI-0100 completely prevented tumor growth after challenge with 5 x 104 HPV16-transformed TC-1 tumor cells. Protection of the mice immunized with TA-CIN plus GPI-0100 against TC-1 in a prophylactic setting was significantly more effective than TA-CIN alone or GPI-0100 alone. Patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. GPI-0100 has been used safely as an adjuvant in several human trials suggesting that the combination of TA-CIN and GPI-0100 warrants further study. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A138.

Abstract B93: Concatenated multitype L2 fusion proteins as prophylactic human papillomavirus vaccines

Abstract B93 Vaccination with minor capsid protein L2, is known to induce antibodies that cross-neutralize diverse papillomavirus types.However, neutralizing titers against the homologous type are generally much higher than the titers against heterologous types. We hypothesized that vaccination with concatenated multi-type L2 fusion proteins derived from known cross-protective epitopes of several divergent HPV types might increase immunity across medically significant HPV genotypes. Here, the antibody responses of mice and rabbits to vaccination with HPV16 L2 polypeptides comprising residues 1-88 or 11-200, was compared with multi-type L2 fusion proteins, namely 11-200x3 types (HPV6, 16, 18), 11-88x5 types (HPV1, 5, 6,16, 18), or 17-36x22 types (5 cutaneous, 2 mucosal low risk and 15 oncogenic types), and with a licensed quadrivalent HPV L1 virus-like particle (VLP) vaccine, Gardasil. The monotype L2 polypeptides generated robust neutralization titers against HPV16 but lower titers against other HPV types. In contrast, the antisera to the multi-type L2 fusion proteins, notably 11-200x3 and 11-88x5, induced high neutralizing titers against all heterologous HPVs tested. However, in mice, these neutralizing titers were consistently lower than those to L1 VLP. The in vitro neutralization titers elicited by vaccination with 11-200x3 in different adjuvant combinations tested were not significantly different at two weeks after immunization. However, the vaccines formulated in GPI-0100 adjuvant or alum and ISS 1018 protected mice against HPV16 challenge at four months after immunization in the same manner as HPV16 L1 VLP, whereas 11-200x3 alone or formulated with either alum or ISS 1018 alone was significantly less effective. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B93.

Cervarix&amp;trade;: a vaccine for the prevention of HPV 16, 18-associated cervical cancer

Cervical cancer continues to be the second largest cause of cancer deaths in women worldwide. Persistent infection with high-risk types of human papillomavirus (HPV) is a necessary cause of cervical cancer. Thus, prophylactic vaccination against HPV is an attractive strategy to prevent cervical cancer. Current strategies for the development of safe and effective preventive vaccines are based on the induction of neutralizing antibodies against the major capsid protein, L1 of HPV. Cervarix() is one of the preventive HPV vaccines that has been approved in the Europe and Australia and is currently under review by the US Food and Drug Administration. Cervarix is composed of HPV16 and HPV18 L1 virus-like particles (VLPs) formulated in ASO4 adjuvant. Vaccination with Cervarix has been shown to protect women against a high proportion of precursor lesions of cervical cancer caused by these two HPV types. This review explores the various features of this new vaccine candidate and discusses the future directions in the field of HPV vaccine development.

Type-specific and cross-reactive antibodies induced by human papillomavirus 31 L1/L2 virus-like particles

The aim of this study was to determine whether antibodies induced by human papillomavirus (HPV) type 31 L1/L2 virus-like particles (VLPs) could cross-react with VLPs of the closely related HPV-16 and distantly related HPV-11, and to investigate the potential role of the L2 protein in L1/L2 VLPs in inducing cross-neutralizing antibodies. Antisera were prepared from rabbits immunized with intact or denatured HPV-31 L1/L2 VLPs. Cross-reaction and cross-neutralization were analysed by Western blotting and ELISA, and by haemagglutination inhibition, respectively. Western blotting results showed that H31 L1/L2 (D) antiserum (antiserum from a rabbit immunized with denatured HPV-31 L1/L2 VLPs) could cross-react with the L1 protein of HPV-11 and -16. HPV-31 L1/L2 VLP antiserum showed strong cross-reaction with and cross-neutralization of HPV-16 VLPs, but this was significantly less with HPV-11 VLPs. In addition, the cross-neutralizing activity against HPV-16 L1/L2 VLPs was slightly higher than that against HPV-16 L1 VLPs, although the difference was not statistically significant. Epitope-blocking ELISA showed that mAb H16.V5 could partially inhibit the cross-reaction of HPV-31 L1/L2 VLP antiserum with HPV-16 L1/L2 VLPs. These results suggested that (i) H31 L1/L2 (D) antiserum could cross-react with L1 protein from both closely related and distantly related HPV types, but HPV-31 L1/L2 VLP antiserum could only cross-neutralize closely related HPV types, (ii) surface-exposed epitopes of the L2 protein in L1/L2 VLPs may induce only low titres of cross-neutralizing antibodies and (iii) certain epitopes that cross-reacted with HPV-31 L1/L2 VLP antiserum are located close to the epitopes recognized by mAb H16.V5. These findings may provide suggestions for the design of multivalent VLP vaccines.

Effect of Alternative Aluminum Adjuvants on the Absorption and Immunogenicity of HPV16 L1 VLPs in Mice

Aluminum adjuvants are commonly used in prophylactic vaccines to enhance antigen immunogenicity through induction of high-titer antibody responses. Three major forms of aluminum adjuvants with substantially different physical and chemical properties have been described: aluminum phosphate (AlPO4), aluminum hydroxide (AlOH) and amorphous aluminum hydroxyphosphate sulfate (AAHS). Here we describe the effect of these different aluminum adjuvants on the formulation and subsequent immunogenicity in mice of virus-like particles (VLPs) consisting of the L1 protein of Human Papillomavirus (HPV) Type 16. Electron microscopy demonstrated that the physical appearance of the phosphate-containing aluminum adjuvants was markedly different from that of aluminum hydroxide. All three aluminum adjuvants were found to display unique surface charge profiles over a range of pH, while AAHS demonstrated the greatest inherent capacity for adsorption of L1 VLPs. These differences were associated with differences in immunogenicity: anti-HPV L1 VLP responses from mice immunized with AAHS-formulated HPV16 vaccine were substantially greater than those produced by mice immunized with the same antigen formulated with aluminum hydroxide. In addition, HPV L1 VLPs formulated on AAHS also induced a substantial interferon-gamma secreting T cell response to L1 peptides indicating the potential for an enhanced memory response to this antigen. These results indicate that the chemical composition of aluminum adjuvants can have a profound influence on the magnitude and quality of the immune response to HPV VLP vaccines.

Incidence and Duration of Cervical Human Papillomavirus 6, 11, 16, and 18 Infections in Young Women: An Evaluation from Multiple Analytic Perspectives

To estimate the incidence and duration of cervical human papillomavirus (HPV)-6, HPV-11, HPV-16, and HPV-18 infections in a population of young American women. The study population consisted of U.S. women who at baseline were 16 to 23 years of age, reported zero to five lifetime sexual partners, never having been pregnant, and never having had a prior abnormal Papanicolaou test and were enrolled in the placebo arm of a randomized multicenter clinical trial of a HPV-16 L1 virus-like particle vaccine. Women underwent type-specific endocervical/ectocervical swab HPV DNA testing at approximately 6-month intervals for up to 48 months of follow-up. To contribute person-time in the analyses of type-specific HPV incidence, a woman must have had at least three satisfactory swab specimens available and been negative for the relevant HPV type (HPV-6, HPV-11, HPV-16, or HPV-18) on her first two trial swabs. The duration of incident HPV infections was estimated using Kaplan-Meier survival analysis methods. Person-years of exposure ranged by type-specific analysis from 2,645 to 3,188, with an incidence rate per 100 person-years of 3.6 for HPV-6, 0.4 for HPV-11, 5.4 for HPV-16, and 2.1 for HPV-18. With censoring at the time of treatment for cervical intraepithelial neoplasia, where done, the mean duration of incident infections was 9.3, 8.4, 18.2, and 16.4 months, respectively, for HPV-6 (n = 103), HPV-11 (n = 13), HPV-16 (n = 142), and HPV-18 (n = 62). When the duration of HPV infections was truncated at the time of cervical intraepithelial neoplasia detection (any grade), where applicable, mean duration figures were 8.4, 8.1, 14.0, and 15.1 months for HPV-6, HPV-11, HPV-16, and HPV-18 infections, respectively. Previous studies of the mean duration of cervical HPV infection have been based on prevalent infections and/or featured relatively short duration of follow-up. This study tested women for HPV infection over a period of up to 48 months and observed a mean duration of incident HPV-16/HPV-18 infections approximately twice that of HPV-6/HPV-11.