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.

Pouyanfard S ，Spagnoli G ，Bulli L ，Balz K ，Yang F ，Odenwald C ，Seitz H ，Mariz FC ，Bolchi A ，Ottonello S ，Müller M ... -  《-》

Broad Cross-Protection Is Induced in Preclinical Models by a Human Papillomavirus Vaccine Composed of L1/L2 Chimeric Virus-Like Particles.

At least 15 high-risk human papillomaviruses (HPVs) are linked to anogenital preneoplastic lesions and cancer. Currently, there are three licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of the L1 major capsid protein from HPV-2, -4, or -9, including the AS04-adjuvanted HPV-16/18 L1 vaccine. The L2 minor capsid protein contains HPV-neutralizing epitopes that are well conserved across numerous high-risk HPVs. Therefore, the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 with one or two L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C terminus of L1. Based on the shape, yield, size, and immunogenicity, one of seven chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 with insertions of HPV-33 L2 (amino acid residues 17 to 36; L1 DE loop) and HPV-58 L2 (amino acid residues 56 to 75; L1 C terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPVs evaluated (HPV-6, -11, -16, -31, -35, -39, -45, -58, and -59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 L1 vaccine. Therefore, the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16 and HPV-18 L1 VLPs) formulated with AS04 has the potential to provide broad protective efficacy in human subjects. From evaluations in human papillomavirus (HPV) protection models in rabbits and mice, our study has identified a prophylactic vaccine with the potential to target a wide range of HPVs linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of the L1 major capsid protein from two, four, or nine different HPVs. Rather than increasing the diversity of L1 VLPs, this vaccine contains VLPs based on a recombinant chimera of two highly conserved neutralizing epitopes from the L2 capsid protein inserted into L1. Our study demonstrated that the chimeric L1/L2 VLP is an effective vehicle for displaying two different L2 epitopes and can be used in a quantity equivalent to what is used in the licensed vaccines. Hence, using the chimeric L1/L2 VLP may be a more cost-effective approach for vaccine formulation than adding different VLPs for each HPV.

Boxus M ，Fochesato M ，Miseur A ，Mertens E ，Dendouga N ，Brendle S ，Balogh KK ，Christensen ND ，Giannini SL ... -  《-》

A three component mix of thioredoxin-L2 antigens elicits broadly neutralizing responses against oncogenic human papillomaviruses.

Current human papillomavirus (HPV) vaccines based on major capsid protein L1 virus-like particles (VLP) provide potent type-specific protection against vaccine-type viruses (mainly HPV16 and 18), but cross-protect against only a small subset of the approximately 15 oncogenic HPV types. It is estimated that L1-VLP vaccines, which require a fairly complex production system and are still quite costly, fail to cover 20-30% of HPV cervical cancers worldwide, especially in low-resource countries. Alternative antigens relying on the N-terminal region of minor capsid protein L2 are intrinsically less immunogenic but capable of eliciting broadly neutralizing responses. We previously demonstrated the enhanced immunogenicity and cross-neutralization potential of an easily produced recombinant L2 antigen bearing the HPV16 L2(20-38) peptide epitope internally fused to bacterial thioredoxin (Trx). However, antibodies induced by Trx-HPV16 L2(20-38) failed to cross-neutralize notable high-risk HPV types such as HPV31. In the present work, the Trx-L2 design was modified to include L2 sequence information from the highly divergent HPV31 and HPV51 types in addition to HPV16, with the aim of extending cross-neutralization. Multivalent antigens comprising L2(20-38) peptides from all three HPV types on a single Trx scaffold molecule were compared to a mixture of the three type-specific monovalent Trx-L2 antigens. While multivalent antigens as well as the mixed antigens elicited similar anti-HPV16 neutralization titers, cross-reactive responses against HPV31 and HPV51 were of higher magnitude and more robust for the latter formulation. A mixture of monovalent Trx-L2 antigens thus represents a candidate lead for the development of a broadly cross-protective, low-cost second-generation anti-HPV vaccine.

Seitz H ，Canali E ，Ribeiro-Müller L ，Pàlfi A ，Bolchi A ，Tommasino M ，Ottonello S ，Müller M ... -  《-》

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.

Yang F ，Mariz FC ，Zhao X ，Spagnoli G ，Ottonello S ，Müller M ... -  《-》

Lipidated L2 epitope repeats fused with a single-chain antibody fragment targeting human FcγRI elicited cross-neutralizing antibodies against a broad spectrum of human papillomavirus types.

Numerous types of human papillomaviruses (HPVs) have been identified, and the global burden of diseases associated with HPV infection is remarkable, especially in developing regions. Thus a low-cost broad-spectrum prophylactic vaccine is urgently needed. The N-terminal amino acid 17-36 of HPV 16 L2 protein is confirmed to be a major cross-neutralizing epitope (RG-1 epitope). Monomeric proteins containing RG-1 epitopes and scaffold proteins, such as bacterial thioredoxin or modified IgG1 Fc fragment and L2 epitope fusion protein, induced cross-neutralizing antibodies, arousing the possibility of the development of low-cost monomeric vaccine in bacterial expression system. Here we show that a novel immunogen-scaffold protein containing a lipidated triple-repeat HPV 16RG-1 epitope and a hFcγRI specific single-chain antibody fragment (H22scFv), named LpE3H22, elicited high titers of cross-neutralizing antibodies against a broad range of mucosal and cutaneous HPV types when adjuvanted with MF59 and poly I:C. LpE3H22 was produced in E. coli expression system. In contrast to three repeats of RG-1 epitope (E3) and unlipidated fusion protein E3H22, vaccination of LpE3H22 induced robust cross-neutralizing antibody responses in hFcγRI transgenic mice. Furthermore, the neutralizing antibody response induced by LpE3H22 was significantly weaker in WT mice than in the Tg mice. The cross-neutralizing antibodies induced by LpE3H22 sustained for at least 10months in Tg mice. Our results demonstrate that hFcγRI targeting and lipidation both contribute to the enhancement of immunogenicity of L2 antigen. Therefore, delivering the lipidated L2 antigen with H22scFv opens a new avenue for low-cost pan-HPV vaccine development.

Zhang T ，Liu H ，Chen X ，Wang Z ，Wang S ，Qu C ，Zhang J ，Xu X ... -  《-》