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.

Broad Neutralization Responses Against Oncogenic Human Papillomaviruses Induced by a Minor Capsid L2 Polytope Genetically Incorporated Into Bacterial Ferritin Nanoparticles.

Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.

Minor Capsid Protein L2 Polytope Induces Broad Protection against Oncogenic and Mucosal Human Papillomaviruses.

The amino terminus of the human papillomavirus (HPV) minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. A wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1-based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV16, -31, and -51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here, we report the development of a thermostable thioredoxin-based single-peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types that we have analyzed plus the low-risk HPVs 6 and 11 and a number of cutaneous HPVs.IMPORTANCE Infections by a large number of human papillomaviruses lead to malignant and nonmalignant disease. Current commercial vaccines based on virus-like particles (VLPs) effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so-called neutralization epitopes within its N terminus. We designed polytopes comprising the L2 epitope amino acids 20 to 38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile archaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising a heptamerized L2 8-mer thioredoxin single-peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.