Cancer susceptibility of beta HPV49 E6 and E7 transgenic mice to 4-nitroquinoline 1-oxide treatment correlates with mutational signatures of tobacco exposure.

We have previously showed that a transgenic (Tg) mouse model with cytokeratin 14 promoter (K14)-driven expression of E6 and E7 from beta-3 HPV49 in the basal layer of the epidermis and of the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7 Tg mice) are highly susceptible to upper digestive tract carcinogenesis upon exposure to 4-nitroquinoline 1-oxide (4NQO). Using whole-exome sequencing, we show that in K14 HPV49 E6/E7 Tg mice, development of 4NQO-induced cancers tightly correlates with the accumulation of somatic mutations in cancer-related genes. The mutational signature in 4NQO-treated mice was similar to the signature observed in humans exposed to tobacco smoking and tobacco chewing. Similar results were obtained with K14 Tg animals expressing mucosal high-risk HPV16 E6 and E7 oncogenes. Thus, beta-3 HPV49 share some functional similarities with HPV16 in Tg animals.

Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Human papillomaviruses and carcinogenesis: well-established and novel models.

Human papillomaviruses (HPVs) infect the cutaneous or mucosal epithelia and are classified phylogenetically as genera and species. Persistent infections by the mucosal high-risk (HR) HPV types from genus alpha are associated with cancer development of the genital and upper respiratory tracts. The products of two early genes, E6 and E7, are the major HR HPV oncoproteins, being essential in all steps of the carcinogenic process. Cutaneous beta HPV types are proposed, together with ultraviolet (UV) radiation, to promote non-melanoma skin cancer development. However, in contrast to the HR HPV types, beta HPV types appear to be required only at an early stage of carcinogenesis, facilitating the accumulation of UV-induced DNA mutations. Although findings in experimental models also suggest that beta HPV types and other carcinogens may synergize in the induction of malignancies, these possibilities need to be confirmed in human studies.

Correction: E6 and E7 from Beta Hpv38 Cooperate with Ultraviolet Light in the Development of Actinic Keratosis-Like Lesions and Squamous Cell Carcinoma in Mice.

[This corrects the article DOI: 10.1371/journal.ppat.1002125.].

Novel ß-HPV49 Transgenic Mouse Model of Upper Digestive Tract Cancer.

The beta genus of human papillomaviruses (ß-HPV) includes approximately 50 different viral types that are subdivided into five species (ß-1 through ß-5). Nonmelanoma cancers may involve some ß-1 and ß-2 HPV types, but the biology of most ß-HPV types and their possible connections to human disease are still little characterized. In this study, we studied the effects of ß-3 type HPV49 in a novel transgenic (Tg) mouse model, using a cytokeratin K14 promoter to drive expression of the E6 and E7 genes from this virus in the basal skin epidermis and the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7-Tg mice). Viral oncogene expression only marginally increased cellular proliferation in the epidermis of Tg animals, compared with wild-type littermates, and we observed no spontaneous tumor formation during their entire lifespan. However, we found that K14 HPV49 E6/E7-Tg mice were highly susceptible to upper digestive tract carcinogenesis upon initiation with 4-nitroquinoline 1-oxide (4NQO). This was a selective effect, as the same mice did not exhibit any skin lesions after chronic UV irradiation. Opposite results were observed in an analogous Tg model expressing the ß-2 HPV38 E6 and E7 oncogenes at the same anatomic sites. While these mice were highly susceptible to UV-induced skin carcinogenesis, as previously shown, they were little affected by 4NQO treatment. Overall, our findings highlight important differences in the biologic properties of certain ß-type HPV that affect their impact on carcinogenesis in an anatomic site-specific manner. Cancer Res; 76(14); 4216-25. ©2016 AACR.

Immunization with Human Papillomavirus 16 L1+E2 Chimeric Capsomers Elicits Cellular Immune Response and Antitumor Activity in a Mouse Model.

Development of cervical cancer is associated with persistent infections by high-risk human papillomavirus (HPV). Although current HPV L1-based prophylactic vaccines prevent infection, they do not help to eliminate prevalent infections or lesions. Our aims were (i) to generate a vaccine combining prophylactic and therapeutic properties by producing chimeric capsomers after fusion of the L1 protein to different fragments of E2 from HPV 16, and (ii) to evaluate their capacity to generate an antitumoral cellular response, while conserving L1 neutralizing epitopes. Chimeric proteins were produced in Escherichia coli and purified by glutathione S-transferase (GST)-affinity chromatography. Their structure was characterized using size exclusion chromatography, sucrose gradient centrifugation, electron microscopy, and anti-L1 enzyme-linked immunosorbent assay. All chimeric proteins form capsomers and heterogeneous aggregates. One, containing part of the carboxy-terminal domain of E2 and its hinge region (L1Δ+E2H/NC, aa 206-307), conserved the neutralizing epitope H16.V5. We then evaluated the capacity of this chimeric protein to induce a cytotoxic T-cell response against HPV 16 E2. In (51)Cr release cytotoxicity assays, splenocytes from C57BL/6 immunized mice recognized and lysed TC-1/E2 cells, which express and present endogenously processed E2 peptides. Moreover, this E2-specific cytotoxic response inhibited the growth of tumors of TC-1/E2 cells in mice. Finally, we identified an epitope (aa 292-301) of E2 involved in this cytotoxic response. We conclude that the L1Δ+E2H/NC chimeric protein produced in bacteria can be an effective and economically interesting candidate for a combined prophylactic and therapeutic vaccine that could help eliminating HPV16-positive low-grade cervical lesions and persistent viral infections, thus preventing the development of lesions and, at the same time, the establishment of new infections.

Identification and validation of immunogenic potential of India specific HPV-16 variant constructs: In-silico &in-vivo insight to vaccine development.

Cervical cancer is one of the most common gynecological cancers in the world but in India, it is the top most cancer among women. Persistent infection with high-risk human papillomaviruses (HR-HPVs) is the most important risk factor. The sequence variation(s) in the most common HR-HPV i.e. HPV type 16 leads to altered biological functions with possible clinical significance in the different geographical locations. Sixteen major variants (V1-V16) in full length L1 gene of HPV-16 were identified following analysis of 250 prospectively collected cervical cancer tissue biopsies and their effect on immunogenicity was studied. The effect of these major variations on the epitopes were predicted by in silico methods and the immunogenicity of variants and respective reference DNA vaccine constructs were evaluated by administration of prepared DNA vaccine constructs in female BALB/c mice to evaluate antibody titer. In the present study, L500F (V16) variation showed a significant ~2.7 fold (p < 0.002) increase in antibody titer, whereas T379P (V8) showed ~0.4 fold (p < 0.328) decrease after final injection. These results showed a promising roadmap for the development of DNA based vaccine and for the generation of effective response, though there is a need to study more prevalent variants of HPV in the Indian population.

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy.

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.

HPV16 RNA patterns defined by novel high-throughput RT-qPCR as triage marker in HPV-based cervical cancer precursor screening.

OBJECTIVE: Cervical cancer precursor screening by HPV testing has a low positive predictive value for advanced lesion. HPV16 RNA patterns characteristic for HPV16-transformed cells but based on laborious, cost-intensive singleplex NASBA reactions promised high value in triaging HPV16 DNA-positive women. METHODS: We developed two high-throughput reverse transcriptase quantitative (RT-q) PCR assays for the HPV16 transcripts E6*I, E1^E4 and E1C and the cellular transcript ubiquitin C and analysed RNA of 158 singly HPV16 DNA-positive cervical cell samples archived in PreservCyt buffer for the presence of transformation-associated HPV16 RNA patterns, i.e., upregulation of E6*I relative to E1^E4 and/or presence of E1C. RESULTS: HPV16 RNA pattern analyses classified 85% of 58 samples diagnosed ≤CIN1 (no cytologically and histologically detectable cervical lesion or CIN grade 1) as negative and 90% of 59 samples diagnosed as ≥CIN3 (CIN grade 3 or invasive cancer) as positive. Among 41 CIN grade 2 samples representing an intermediate lesion group, 49% were HPV16 RNA patterns-positive. Interestingly, 3 of 4 HPV16 RNA patterns-positive lesions initially diagnosed as ≤CIN1 at follow-up 5-24 months later had progressed to ≥CIN2. CONCLUSIONS: We successfully developed and validated a second generation of HPV16 RNA patterns assay by rapid RT-qPCR as triage marker for HPV16 DNA-positive women offering clinical utility to distinguish between the need for immediate colposcopy and continued observation. Limited follow-up data suggests that HPV16 RNA patterns-positivity in ≤CIN1 lesions can predict disease progression.

Identification of human papillomavirus-16 E6 variation in cervical cancer and their impact on T and B cell epitopes.

The infection with high-risk human papillomavirus (HR-HPV) is the most important risk factor for development of cervical cancer. The intra-type variations of HPV have different biological and pathological consequences with respect to disease progression. In the present study, six major Indian variants were experimentally identified in E6 gene of HPV-16 and showed their impact on immunogenicity by in silico methods. Four different phylogenetic lineages were observed in sequences including European (E) prototype, European variant, Asian and American Asian variant classes and complete absence of African phylogenetic lineages. On the prediction of B- and T-cell epitopes, 18 and 23 potent epitopes for MHC-II alleles, 10 potent MHC-I and 15 B-cell epitopes in each reference and variant sequence were identified. Interestingly, the presence of variation H78Y and L83V result in creation of four new epitopes for the HLA-DQA1*0101/DQB1*0501. Out of 15 B-cell predicted epitopes, three most potent epitopes were identified in both reference and variant sequence. Notably the amino acid stretch from amino acid 16-60 and 76-94 are very important for the immunological properties of E6 protein because these regions contain majority of the predicted epitopes. In future, this could control the cervical cancer by targeting these amino acid stretches for the development of HPV-16 vaccine.

Evaluation of specific humoral and cellular immune responses against the major capsid L1 protein of cutaneous wart-associated alpha-Papillomaviruses in solid organ transplant recipients.

BACKGROUND: Infection with different species of cutaneous human papillomaviruses (cHPV) of genus alpha (cαHPVs) and associated skin disease are highly prevalent in solid organ transplant recipients (OTR), documenting the importance of the immunological control of HPV infection. OBJECTIVES: To investigate the natural course of cαHPV-specific cellular and humoral immune responses during systemic long-term immunosuppression. METHODS: Integrating bead-based multiplex serology and flow cytometry we analyzed natural cαHPV-specific antibodies and T(H) cell responses against the major capsid protein L1 of HPV types 2, 27, 57 (species 4) and 3, 10 and 77 (species 2) in sera and blood of OTR before and after initiation of iatrogenic immunosuppression and in comparison to immunocompetent individuals (IC). RESULTS: Among OTR we observed an overall 42% decrease in humoral L1-specific immune responses during the course of iatrogenic immunosuppression, comparing median values 30 d before and 30 d after initiation of immunosuppressive therapy (p < 0.05). This difference disappeared after long-term (>1 year) immunosuppression. The predominant cellular L1-specific immune response was of type T(H)1 (CD4(+)CD40L(+)IL-2(+)IFN-γ(+)). Consistent with the detected L1-specific antibody titers, L1-specific T(H)1 responses were unchanged in long-term immunosuppressed OTR compared to IC. Notably, cαHPV-L1-specific IL-2(+)/CD40L(+)CD4(+) or IFN-γ(+)/CD40L(+) CD4(+) T(H) cell responses against any of the cαHPV-L1 types were significantly higher in OTR with clinically apparent common warts. CONCLUSION: The systemic humoral immune response against cαHPV may reflect the individual degree of iatrogenic immunosuppression indicating a higher susceptibility for cαHPV infection among OTR during the early phase after organ transplantation. Humoral cαHPV-specific immune responses may show a reconstitution to pre-transplantation levels despite continuous potent immunosuppression.

Design of a highly effective therapeutic HPV16 E6/E7-specific DNA vaccine: optimization by different ways of sequence rearrangements (shuffling).

Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism.

Pathogenic role of the eight probably/possibly carcinogenic HPV types 26, 53, 66, 67, 68, 70, 73 and 82 in cervical cancer.

Eight HPV types (HPV26, 53, 66, 67, 68, 70, 73 and 82) that are phylogenetically closely related to 12 WHO-defined high-risk (HR) HPV have been rarely but consistently identified as single HPV infections in about 3% of cervical cancer (CxCa) tissues. Due to lack of biological data, these types are referred to as probable/possible (p) HR-HPV. To analyse their biological activity in direct comparison to HR-HPV types, we selected 55 formalin-fixed, paraffin-embedded (FFPE) CxCa tissues harbouring single pHR-HPV infections (2-13 cases per type) and 266 tissues harbouring single HR-HPV (7-40 cases per type) from a worldwide, retrospective, cross-sectional study. Single HPV infection was verified by two genotyping methods. Presence of type-specific spliced E6*I mRNA transcripts and expression of cellular proteins indicative of HPV transformation were assessed in all cases. In 55 CxCa tissues with pHR-HPV, E6*I mRNA expression was 100%; high p16(INK4a) , 98%; low pRb, 96%; low CyD1, 93%; and low p53, 84%. Compared to HPV16 tissues as a reference, individual frequencies of these five markers did not differ significantly, either for any of the eight pHR-HPV and the 11 other HR types individually or for the groups of pHR and HR types without HPV16. We conclude that the eight pHR-HPV types, when present as a single infection in CxCa, are biologically active and affect the same cellular pathways as any of the fully recognized carcinogenic HR-HPV types. Therefore we have provided molecular evidence of carcinogenicity for types currently classified as probably/possibly carcinogenic. Although this evidence is crucial for HPV-type carcinogenicity classification, per se it is not sufficient for inclusion of these HPV types into population-wide primary and secondary prevention programmes. Such decisions have to include careful estimation of effectiveness and cost-benefit analyses.

Helicobacter pylori antibody patterns in Germany: a cross-sectional population study.

BACKGROUND: Helicobacter pylori infection that is usually acquired in childhood and lasts for lifetime is mostly asymptomatic but associated with severe gastrointestinal disease including cancer. During chronic infection, the gastric mucosa is histologically changing. This forces H. pylori to permanent adaptation in its gastric habitat by expression of different proteins which might be reflected in distinctive antibody patterns. METHODS: To characterize dynamics of the immune response to H. pylori we analysed 1797 sera of a cross-sectional study representative for the German population (age range 1-82 years) with multiplex serology, a fluorescent bead-based antibody binding assay that allows simultaneous and quantitative detection of antibodies. Fifteen recombinant, affinity-purified H. pylori proteins (UreA, GroEL, Catalase, NapA, CagA, CagM, Cagδ, HP0231, VacA, HpaA, Cad, HyuA, Omp, HcpC and HP0305) were used as antigens. RESULTS: H. pylori seroprevalence (positivity for at least three antigens) was 48% and increased with age from 12% in children <15 years to 69% in females and 90% in males >65 years. Prevalences were highest (>83%) for Omp, VacA and GroEL. For 11 proteins, seroprevalence was higher in males than females (P < 0.05) from age 55 onwards. For all antigens, the median prevalence increase per age decade was stronger in males (8.4%, range 3.8-12.9%) than females (6.1%, range 3.4-10.8%). However, among seropositives the median number of antigens recognized increased from children <15 years to individuals >65 years stronger in females (P = 0.02). Antibody reactivities to GroEL, HyuA, CagM, Catalase, NapA and UreA also increased stronger in females (average 1.7-fold/decade, SD 0.5) than in males (1.5-fold/decade, SD 0.4). CONCLUSION: H. pylori antibody response accumulates qualitatively and quantitatively with age. This may reflect a lifelong stimulation of the immune response by chronically active infection.

Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination.

The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

Biological activity of probable/possible high-risk human papillomavirus types in cervical cancer.

Judging the carcinogenicity of human papillomavirus (HPV) types rarely found in cervical cancer (CxCa) is hindered by lack of studies of their biological activity in cancer tissues. To asses transcriptional activity of HPV types, we have developed ultra-short amplimer, splice-site specific, E6*I mRNA RT-PCR assays for 12 high-risk (HR)-HPV (IARC Group 1) and eight probable/possible high-risk (pHR)-HPV types (IARC Group 2A/B carcinogens). Previously unreported E6*I splice sites of the six pHR-HPV types 26, 53, 67, 70, 73 and 82 were identified by cloning and sequencing. We analyzed 97 formalin-fixed paraffin-embedded (FFPE) Mongolian CxCa biopsies for presence of HPV DNA by two sensitive genotyping assays, for E6*I transcripts of all HR-/pHR-HPV types identified and for expression of HPV surrogate markers p16(INK4a), pRb and p53. E6*I of at least one HR-/pHR-HPV was expressed in 94 (98%) of cancer tissues including seven with pHR-HPV types 26, 66, 70 or 82 as single transcribed types. Fifty-eight of E6*I mRNA transcribing cases were analyzable by immunohistochemistry and displayed p16(INK4a) overexpression in 57 (98%), pRb downregulation in 56 (97%) and p53 downregulation in 36 (62%) tissues. The newly developed E6*I mRNA RT-PCR assays appeared to be highly sensitive method to analyze HPV transcription in FFPE materials. Our finding of viral oncogene transcription of pHR-HPV types 26, 66, 70 and 82 in cervical tumors, in the absence of any other transcriptionally active HR-type and with p16(INK4a) overexpression and pRb downregulation, may support a reassessment of the carcinogenicity classification of these pHR-HPV types.

Human papillomavirus type 38 E6 and E7 act as tumour promoters during chemically induced skin carcinogenesis.

Many findings support a possible involvement of a subgroup of human papillomaviruses (HPVs), called cutaneous beta HPV types, in the development of non-melanoma skin cancer. The skin of transgenic (Tg) mice expressing viral oncoproteins E6 and E7 from different cutaneous beta HPV types, including HPV38, showed an increased susceptibility to UV-induced and/or chemically induced skin carcinogenesis compared with wild-type animals. In this study, we show that beta HPV38 E6 and E7 oncoproteins act as promoter and progression factors in multi-stage skin carcinogenesis, strongly cooperating with the initiator and DNA damage agent 7,12-dimethylbenz[a]anthracene. In contrast, exposure of HPV38 E6/E7 Tg mice to the promoter 12-O-tetradecanoylphorbol-13-acetate did not significantly result in the development of skin lesions. These findings further support the role of beta HPV types in skin carcinogenesis, providing additional insight into their precise contribution to the multi-step process.

Development of AAVLP(HPV16/31L2) particles as broadly protective HPV vaccine candidate.

The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17-36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2--simulating the high prevalence of AAV2 antibodies in the population--even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.