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

Abstract

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. 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.

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

“…L2 is the minor capsid protein of HPV, and we have previously shown that an Nterminal region comprising amino acids 20-38 can induce broadly cross-neutralizing antibody responses when inserted into, and displayed on, the surface of P. furiosus thioredoxin (Pf Trx) (12,19,37). We have also documented a sizeable increase in immunogenicity upon fusion of Pf Trx-L2 to a selfassembling protein module (named OVX313) derived from the complement-binding protein C4bp and conversion of the antigen into a heptameric nanoparticle form (24,25,43). Therefore, we asked whether further multimerization of the basic Pf TrxL2 antigen would promote an additional increase in immunogenicity.…”

“…A major step in Pf TrxL2 antigen development has been the construction of a Pf Trx-displayed polytope comprising tandemly repeated L2 epitopes from seven different mucosal oncogenic and one cutaneous HPV types. This was followed by genetic fusion of the PfTrx-L2(8x) (Pf Trx8mer) polytope with OVX313, a selfassembling polypeptide that promoted covalent heptamerization of the antigen into a super-scaffolded form, leading to a 5-to 10-fold increase in neutralizing antibody titers compared to the corresponding monomeric antigen (24,25). The resulting nanoparticle vaccine afforded protection against 26 mucosal and cutaneous HPV types.…”

“…20-38 peptide from minor capsid protein L2, which is not organized in a trimeric form on the surface of HPV virions. An additional point of interest was to ask whether higher-order multimerization, i.e., an increase in valency from seven [as in our previous, OVX-313-based nanoparticle vaccine (24,25)] to 24 surface-exposed epitopes might further enhance the immune-performance of our HPV Trx-L2 vaccine.…”

Virus-like Particle-Based L2 Vaccines against HPVs: Where Are We Today?

“…For example, immunizations with an L2 peptide conjugated to thioredoxin [44,45], immunizations with concatemers of L2 protein fused to a self-adjuvanting protein (flagellin) [46,47] as well as immunizations with a concatemer of L2 proteins derived from different HPV types [48,49] have been used. More recently, an L2 polypeptide and a heptamerizing coiled-coil polypeptide OVX313 fused to a nanoparticle derived from a thermostable thioredoxin has been used [50][51][52]. These approaches have enhanced the immunogenicity of L2 peptides, especially immunization with the nanoparticle thioredoxin-L2-OVX313 candidate vaccine.…”

“…These approaches have enhanced the immunogenicity of L2 peptides, especially immunization with the nanoparticle thioredoxin-L2-OVX313 candidate vaccine. The nanoparticle candidate vaccine offers protection against more than 14 HPV types [50]. Although the aforementioned strategies enhanced immune responses, the responses were observed mostly when large doses (up to 25 µg) of antigens, coupled with large amount of adjuvant with multiple immunizations regimens were used.…”