Immunotherapy of papillomavirus-induced equine tumours

Currently, there is no effective therapy for the treatment of papillomavirus (PV)-induced human malignancies or animal cancers such as the equine sarcoid. PV E5, E6 and E7 oncoproteins are considered major targets for immune-based therapeutic strategies as preclinical studies have shown the importance of T cell responses, particularly CTL, in clearing PV-induced lesions. Chimeric influenza viruses do express foreign epitopes and raise a vigorous immune response against the latter, as shown for HIV or tuberculosis epitopes. Brandt and others have generated life-attenuated influenza viruses lacking the NS1 gene (delNS1). They only replicate in interferon (IFN)- deficient systems and induce a high type I IFN response which enhances MHC I expression and thus the generation of T helper cells and an increase in CTL activity. We aim at investigating the therapeutic potential of delNS1-based vaccines against oncoproteins E5, E6 and E7 of sarcoid-inducing bovine papillomavirus 1 (BPV1) as a novel treatment strategy. For this purpose, we will generate NS1-deleted equine influenza vectors of two different subtype expressing BPV E5, E6 and E7 antigens alone or in combination for prime and boost immunization, determine their immunogenicity in the equine model, determine their individual and combined potencies to prevent, reduce/clear tumours in an immune-competent hamster model and assess the therapeutic potential of thus identified most promising candidate(s) in the naturally affected equine patient.

Papillomavirus-induced tumours are a common cause of death in humans and animals. Persistent infection with high-risk human papilloma virus (HPV) types, in particular HPV16 and 18, cause all cervical, most anal and a subset of other genital (penile, vulvar, vaginal) and oropharyngeal cancers. Bovine papillomaviruses types 1 and 2 (BPV1, BPV2), and equine papillomavirus type 2 are the causative agents of the most common tumours in equids, i.e. sarcoids and genital squamous cell carcinoma. Transformation of normal to tumour cells is accomplished by viral oncoproteins, notably E6 and E7. To date, no universally effective strategy for the treatment of papillomavirus induced cancers is available. In the frame of this project, we aimed at generating and studying the therapeutic potential of an influenza virus vector-based therapeutic BPV1 vaccine candidate in a horse model. Due to technical constraints, we switched to a well established mouse model and hence adapted the vaccine candidate accordingly. As antigen delivery vector, we used a human NS1-deleted (delNS1) and thus live-attenuated influenza virus system, which has the advantage of being highly immunogenic and safe. As target immunogen, we generated an HPV16 E6^E7 construct (16E6E7-FL), which was cloned into the vectors delNS1 locus. Then recombinant influenza viruses were generated (2 serotypes) by reverse genetics and transfection of Vero cells. For safety concerns, a second fusion of the E6 and E7 oncogenes was generated, where short deletions were introduced to abrogate their biological function (16E6E7mFL). E6^E7 mRNA and fusion protein expression was verified by RT-PCR and Western Blot. Following injection of C57BL/6 mice with recombinant virus, the immune response was characterized by HPV 16 E6 and E7 peptide ELISA, IFN-? ELISpot and MLR experiments. Immune characterization revealed peptide-specific IFN-? production by splenocytes and proliferation of antigen-specific CD4+T cells in vaccinated mice. The recombinant vaccines were well tolerated and mice showed no detectable signs of toxicity. The therapeutic and prophylactic potential of the vaccine candidates were analysed in a mouse challenge model using HPV 16-transformed TC-1 tumour cells. Animals vaccinated and then challenged with TC-1 cells did not develop tumours or showed significantly decreased tumour growth rates as compared to control. Similar results were obtained in a therapeutic setting, where animals were inoculated subcutaneously with TC-1, primed with H1N1 recombinant virus and then boosted with the recombinant H3N2 serotype 10 days later. Furthermore, alternative vaccine administration routes (intranasal and intratumoural) were analysed. Intratumoural application led to tumour regression in 2 mice. Overall, vaccination with recombinant 16E6E7 viruses showed the most promising effects. Obtained results are suggestive for recombinant influenza delNS1-based PV vaccines representing a promising new approach for the treatment of PV induced tumour disease.