Assessment of Mycobacterium tuberculosis dodecin scaffold as a multimerization platform on the immunogenicity of HPV L2 antigens

Human papillomavirus infection, the primary cause of cervical cancer (the fourth most common cancer in women), is preventable through prophylactic HPV vaccines. As an alternative to current HPV vaccines, all based on a cocktail of L1 virus-like particles (VLPs) from multiple HPV types, the L2 capsid protein offers cross-protection via a conserved epitope residing at the N-terminus between residues 20-38. The Trx-L2_(20-38)-8mer (Trx-8mer) is an L2-based antigen composed of a polytope of L2_(20-38) epitopes from eight HPV types inserted into thioredoxin from the hyperthermophilic archaeon Pyrococcus furiosus as a scaffold. We designed multivalent nanoparticle forms of Trx-8mer by using a dodecin protein from Mycobacterium tuberculosis (mtDod) assembling into dodecameric nanoparticles as a multimerization platform. Here, we explored two approaches to form dodecameric Trx-8mer nanoparticles: (i) direct fusion of the Trx-8mer to mtDod at the DNA level and (ii) decoration of mtDod nanoparticles after assembly with the Trx-8mer via the protein glue DogTag/DogCatcher. The reaction between Tag and Catcher results in isopeptide bond formation; thus, the covalent decoration of mtDod particles with the cargo (Trx-8mer) occurs. Despite the formation of nanoparticles by direct genetic fusion, this approach did not offer superior immunogenicity compared with the reference antigen, a heptameric form of the Trx-8mer. However, we showed that the decoration of assembled DogTag-mtDod nanoparticles with the cargo yielded the final antigen with a significant increase in immunogenicity with the induction of high neutralizing and cross-neutralizing antibody titers upon injection to BALB/c mice. We think that the lower immunogenicity observed with the direct genetic fusion of Trx-8mer to mtDod may be attributed to structural constraints affecting the accessibility of L2 epitopes to B cell receptors. Taken together, our final antigen obtained by decoration of DogTag-mtDod with DogCatcher-Trx-8mer holds potential as a multivalent L2-based antigen candidate for next-generation prophylactic HPV vaccines.