Mastomys Natalensis Papillomavirus Research Articles

Next generation L2-based HPV vaccines cross-protect against cutaneous papillomavirus infection and tumor development.

Licensed L1-VLP-based immunizations against high-risk mucosal human papillomavirus (HPV) types have been a great success in reducing anogenital cancers, although they are limited in their cross-protection against HPV types not covered by the vaccine. Further, their utility in protection against cutaneous HPV types, of which some contribute to non-melanoma skin cancer (NMSC) development, is rather low. Next generation vaccines achieve broadly cross-protective immunity against highly conserved sequences of L2. In this exploratory study, we tested two novel HPV vaccine candidates, HPV16 RG1-VLP and CUT-PANHPVAX, in the preclinical natural infection model Mastomys coucha. After immunization with either vaccines, a mock control or MnPV L1-VLPs, the animals were experimentally infected and monitored. Besides vaccine-specific seroconversion against HPV L2 peptides, the animals also developed cross-reactive antibodies against the cutaneous Mastomys natalensis papillomavirus (MnPV) L2, which were cross-neutralizing MnPV pseudovirions in vitro. Further, both L2-based vaccines also conferred in vivo protection as the viral loads in plucked hair after experimental infection were lower compared to mock-vaccinated control animals. Importantly, the formation of neutralizing antibodies, whether directed against L1-VLPs or L2, was able to prevent skin tumor formation and even microscopical signs of MnPV infection in the skin. For the first time, our study shows the proof-of-principle of next generation L2-based vaccines even across different PV genera in an infection animal model with its genuine PV. It provides fundamental insights into the humoral immunity elicited by L2-based vaccines against PV-induced skin tumors, with important implications to the design of next generation HPV vaccines.

Isoforms of the Papillomavirus Major Capsid Protein Differ in Their Ability to Block Viral Spread and Tumor Formation.

Notably, the majority of papillomaviruses associated with a high cancer risk have the potential to translate different isoforms of the L1 major capsid protein. In an infection model, the cutaneous Mastomys natalensis papillomavirus (MnPV) circumvents the humoral immune response of its natural host by first expressing a 30 amino acid extended L1 isoform (L1LONG). Although inducing a robust seroconversion, the raised antibodies are not neutralizing in vitro. In contrast, neutralizing antibodies induced by the capsid-forming isoform (L1SHORT) appear delayed by several months. We now provide evidence that, although L1LONG vaccination showed a strong seroconversion, these antibodies were not protective. As a consequence, virus-free animals subsequently infected with MnPV still accumulated high numbers of transcriptionally active viral genomes, ultimately leading to skin tumor formation. In contrast, vaccination with L1SHORT was completely protective. This shows that papillomavirus L1LONG expression is a unique strategy to escape from antiviral immune surveillance.

Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity.

Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse Mastomys coucha as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing. Only after a delay of around 4 months, protecting antibodies against the short L1 appeared, enabling the virus to successfully establish an infection. This argues for a novel humoral immune escape mechanism that may also have important implications on the interpretation of epidemiological data in terms of seropositivity and protection of PV infections in general.

The interplay of UV and cutaneous papillomavirus infection in skin cancer development.

Cutaneous human papillomaviruses (HPVs) are considered as cofactors for non-melanoma skin cancer (NMSC) development, especially in association with UVB. Extensively studied transgenic mouse models failed to mimic all aspects of virus-host interactions starting from primary infection to the appearance of a tumor. Using the natural model Mastomys coucha, which reflects the human situation in many aspects, we provide the first evidence that only UVB and Mastomys natalensis papillomavirus (MnPV) infection strongly promote NMSC formation. Using UVB exposures that correspond to UV indices of different geographical regions, irradiated animals developed either well-differentiated keratinizing squamous cell carcinomas (SCCs), still supporting productive infections with high viral loads and transcriptional activity, or poorly differentiated non-keratinizing SCCs almost lacking MnPV DNA and in turn, early and late viral transcription. Intriguingly, animals with the latter phenotype, however, still showed strong seropositivity, clearly verifying a preceding MnPV infection. Of note, the mere presence of MnPV could induce γH2AX foci, indicating that viral infection without prior UVB exposure can already perturb genome stability of the host cell. Moreover, as shown both under in vitro and in vivo conditions, MnPV E6/E7 expression also attenuates the excision repair of cyclobutane pyrimidine dimers upon UVB irradiation, suggesting a viral impact on the DNA damage response. While mutations of Ras family members (e.g. Hras, Kras, and Nras) were absent, the majority of SCCs harbored—like in humans—Trp53 mutations especially at two hot-spots in the DNA-binding domain, resulting in a loss of function that favored tumor dedifferentiation, counter-selective for viral maintenance. Such a constellation provides a reasonable explanation for making continuous viral presence dispensable during skin carcinogenesis as observed in patients with NMSC.

Transcriptome analysis of Mastomys natalensis papillomavirus in productive lesions after natural infection.

Mastomys coucha, an African rodent, is a useful animal model of papillomavirus infection, as it develops both premalignant and malignant skin tumors as a consequence of a persistent infection with Mastomys natalensis papillomavirus (MnPV). In this study, we mapped the MnPV transcriptome in productive lesions by both classical molecular techniques and high-throughput RNA sequencing. Combination of these methods revealed a complex and comprehensive transcription map, with novel splicing events not described in other papillomaviruses. Furthermore, these splicing occurrences could potentially lead to the expression of novel E2, E1∧E4, E7 and L2 isoforms. Expression level estimation of each transcript showed that late-region mRNAs considerably outnumber early transcripts, with species coding for L1 and E1∧E4 being the most abundant. In summary, the full transcription map assembled in this study will allow us to further understand MnPV gene expression and the mechanisms that lead to natural tumour development.

Protective Vaccination against Papillomavirus-Induced Skin Tumors under Immunocompetent and Immunosuppressive Conditions: A Preclinical Study Using a Natural Outbred Animal Model

Certain cutaneous human papillomaviruses (HPVs), which are ubiquitous and acquired early during childhood, can cause a variety of skin tumors and are likely involved in the development of non-melanoma skin cancer, especially in immunosuppressed patients. Hence, the burden of these clinical manifestations demands for a prophylactic approach. To evaluate whether protective efficacy of a vaccine is potentially translatable to patients, we used the rodent Mastomys coucha that is naturally infected with Mastomys natalensis papillomavirus (MnPV). This skin type papillomavirus induces not only benign skin tumours, such as papillomas and keratoacanthomas, but also squamous cell carcinomas, thereby allowing a straightforward read-out for successful vaccination in a small immunocompetent laboratory animal. Here, we examined the efficacy of a virus-like particle (VLP)-based vaccine on either previously or newly established infections. VLPs raise a strong and long-lasting neutralizing antibody response that confers protection even under systemic long-term cyclosporine A treatment. Remarkably, the vaccine completely prevents the appearance of benign as well as malignant skin tumors. Protection involves the maintenance of a low viral load in the skin by an antibody-dependent prevention of virus spread. Our results provide first evidence that VLPs elicit an effective immune response in the skin under immunocompetent and immunosuppressed conditions in an outbred animal model, irrespective of the infection status at the time of vaccination. These findings provide the basis for the clinical development of potent vaccination strategies against cutaneous HPV infections and HPV-induced tumors, especially in patients awaiting organ transplantation.

Serological markers for papillomavirus infection and skin tumour development in the rodent model Mastomys coucha

This study used the rodent Mastomys coucha latently infected with Mastomys natalensis papillomavirus (MnPV) and Mastomys coucha PV2 (McPV2), which induce skin papillomas and anogenital condylomas, respectively, to investigate PV antibody responses as serological markers during pathogenesis. In a case-control study (137 animals), virus and tumour prevalence correlated with the seroresponse against the early E2 and late L1 viral proteins. A prospective study (53 animals) revealed for the first time the course of these antibody responses during all stages of a natural PV infection. Numerous tumour entities were observed on the eyelid and in the oral cavity. DNA analyses indicated that McPV2 was not restricted to condylomas but was also present in these mucosa-associated papillomas. The serological survey using a recently established glutathione S-transferase-capture ELISA detected a strong correlation between MnPV L1-specific antibodies and the presence of papillomas on the skin, eye and ear (P<0.001). Notably, extensive antibody responses to MnPV E2 were also detected in these cases. A prospective study revealed that E2 reactivity occurred by the age of 1 month. MnPV L1 antibodies were found at 2.5 months, indicating the initiation of productive viral infection. Thirty-one out of 34 L1-seropositive animals at the age of 4.5 months developed MnPV-associated tumours (positive predictive value=77 %), and none of the seronegative animals developed skin papillomas (negative predictive value=100 %). MnPV E2 and L1 serology thus provides a powerful tool for monitoring early infection and skin tumour progression in M. coucha.

A capture ELISA for monitoring papillomavirus-induced antibodies in Mastomys coucha

The present report describes the development of an enzyme-linked immunosorbent assay (ELISA), whereby the first insights have been obtained into the humoral immune response to papillomavirus (PV) infections of the rodent Mastomys coucha, a natural model for papillomavirus-induced skin carcinogenesis. The established glutathione S-transferase (GST)-capture ELISA is based on a one-step purification of bacterially expressed antigens and was designed to detect serum antibodies against L1 capsid proteins of Mastomys natalensis papillomavirus (MnPV) and M. coucha papillomavirus 2 (McPV2). Both viruses are spread widely within the colony at the German Cancer Research Center. The animals are unique in that they spontaneously develop multiple epithelial tumors such as MnPV-induced papillomas and McPV2-induced condylomas. The humoral immune response of a cohort of 98 Mastomys was analysed and revealed a high prevalence of antibodies to L1 (MnPV: 36.7%, McPV2: 52.0%). Furthermore, the seroreactivity to both viruses was significantly increased in animals with the respective tumors (22 papillomas, 21 condylomas) as compared to 55 tumor-free controls (MnPV: p < 0.0001; McPV2: p = 0.0022). The identified assay conditions showed a high level of sensitivity (MnPV: 75.7%; McPV2: 85.7%) and reproducibility (MnPV: R 2 = 0.83; McPV2: R 2 = 0.79), validating the GST-capture ELISA as a powerful method for monitoring papillomavirus serology in M. coucha.

Persistence of Mastomys natalensis papillomavirus in multiple organs identifies novel targets for infection

The high incidence of multiple wart formation and skin cancer in organ-transplant recipients, as well as the question of an involvement of papillomaviruses in a variety of human cancers, require a model system for papillomavirus infections in immunocompetent animals. Such an in vivo model is represented by the multimammate rat Mastomys coucha, which is infected with Mastomys natalensis papillomavirus (MnPV). MnPV primarily induces benign skin tumours, such as papillomas and keratoacanthomas. Here, the incidence of MnPV infections in different skin areas and various organs is described. In situ hybridization showed that hair follicle cells were positive for viral DNA and that the amount of MnPV in normal skin may be considered a predictor for the development of skin tumours. MnPV infection is not restricted to the skin, but can also be detected in inner organs. As the blood and the lymphatic system were temporarily also found to be virus-positive, a haematogenic propagation of MnPV can be assumed. However, MnPV is apparently not transmitted through the germ line, as fetuses and newborns lack viral DNA, despite infection of their mothers. In conclusion, M. coucha is not only useful to study papillomavirus-induced skin carcinogenesis, but may also serve as a model to identify additional, still unknown target cells of papillomavirus infections and the potential pathological impact.

The Mastomys natalensis Papillomavirus: Nucleotide Sequence, Genome Organization, and Phylogenetic Relationship of a Rodent Papillomavirus Involved in Tumorigenesis of Cutaneous Epithelia

Mastomys natalensis is a rodent of African origin afflicted with a very high incidence of skin tumors (keratoacanthomas and squamous carcinomas), which are associated with a papillomavirus, M. natalensis papillomavirus (MnPV). We have determined the genomic sequence of MnPV, which has a size of 7687 bp. The genomic organization is similar to that of other papillomaviruses, with open reading frames E6, E7, E1, E2, and E4 in the early and L2 and L1 in the late region. Due to an unusually large hinge region, the transcriptional activator E2 has a size of 542 amino acids rather than 400 to 460 amino acids, as in other papillomaviruses. An open reading frame E5 coding for a small hydrophobic membrane protein is missing, as is the case for some cutaneous human papillomaviruses (HPV). This fact, together with the composition of cis -responsive elements in its long control region and phylogenetic evaluation of segments of its E6, E1, and L1 genes, indicates a relationship of MnPV to the cottontail rabbit papillomavirus and several HPV types found in lesions of cutaneous epithelia, in particular to those that are associated with epidermodysplasia verruciformis. MnPV may be a useful model system for tumorigenesis of cutaneous epithelia in humans.

Neoplasms of the skin in a wild-colored inbred strain of Mastomys natalensis (WSA Giessen)

This is the first report on spontaneous and experimentally induced skin neoplasms of a wild-colored, black-eyed inbred line of Mastomys natalensis (WSA Gießen). Seventeen spontaneous skin tumors from four animals and two experimentally induced tumors from two animals were examined. Routine histology and electron microscopy were done. For the transmission experiments, tumor suspension containing tumor cells and virus was applied to the depilated and scarified skin of the lumbar region of young Mastomys natalensis. The tumor suspension contained a mixture of squamous cell carcinoma and so-called keratoacanthoma cells, both taken from another inbred line of Mastomys natalensis (GRA Gießen). Depilation was done by application of aqueous suspension of equal parts of strontium sulfide and talcum powder. Narcosis was achieved by administration of 40 mg/kg body weight of sodium pentobarbital. The 17 spontaneous neoplasms included 13 so-called keratoacanthomas, 2 squamous cell carcinomas, I papilloma and 3 localized epithelial proliferations. The two experimentally induced neoplasms were so-called keratoacanthomas. All tumors grew slowly and neither spontaneous regression nor metastasis was seen. All neoplasms showed keratinizing epidermal proliferation, with the greatest increase in prickle cells and granulosum cells. Two spontaneous and two experimentally induced “keratoacanthomas” were observed under an electron microscope. Papilloma-like virus particles were seen in all four. The virus particles were spaced regularly throughout the nuclei of the upper part of the granulosum cells and between parts of disintegrated cells of the horny layers. The spontaneous and experimentally induced neoplasms of this WSA-line correspond to those of another inbred line (GRA Gießen) of Mastomys natalensis. The tumors are mutually transmissible. Both inbred lines are suitable for propagation of the Mastomys natalensis papilloma virus and for studying in greater detail the biological behaviour of this oncogenic agent in epithelial skin cells.