Low doses of flagellin-L2 multimer vaccines protect against challenge with diverse papillomavirus genotypes

Abstract

Genetically modified bacterial flagellin (Fla), a Toll-like receptor-5 (TLR5) ligand, was evaluated as a fusion partner for human papillomavirus (HPV) L2-based immunogens in two animal challenge models; either cutaneous inoculation of rabbits with HPV ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes that induce warts, or intra-vaginal inoculation of mice with HPV ‘pseudovirions’ encapsidating a luciferase reporter plasmid and measurement of bioluminescence to determine infectivity. An Escherichia coli production system was developed for flagellin-L2 (Fla-L2) fusions containing either monomeric HPV-16 L2 a.a. 11(× 11–200) or oligomeric L2 comprising a fusion of the a.a. 11–88 peptides of five (Fla~5 × 11–88) or eight (Fla~8 × 11–88) genital HPV types. Immunogenicity and bioactivity of Fla-L2 constructs were assessed using an in vitro neutralization and cell-based TLR-5 binding assay, respectively. Efficacy was evaluated following active immunization of rabbits or mice administered 3 intramuscular doses of Fla-L2 recombinants without exogenous adjuvant, followed by challenge. In addition, passive immunization studies of naïve rabbits with serial dilutions of pooled immune sera were used to determine End-Point Protection Titers (EPPT) for each formulation against a broader spectrum of HPV quasivirions. Efficacy was assessed for up to 10 weeks on the basis of wart volume induced following challenge and results compared to licensed L1-VLP vaccines (Gardasil and Cervarix). Following active immunization at doses as low as 1 μg, Fla-L2 fusions afforded complete protection against infection (mice) and disease (rabbits) following either homologous or heterologous HPV challenge. Passive immunization with anti-L2 immune sera discriminated between the different vaccine candidates under evaluation, demonstrated the protective role of antibody and suggested the superiority of this oligomeric L2-TLR5 agonist fusion approach compared to L1-based vaccines in its ability to cross-protect against non-vaccine HPV types.

Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17–36 epitopes

“…These latter modifications are needed because of the sub-dominance of L2 within native HPV particles and the reduced immunity of L2 antigens presented separately [21,49,50]. Additional strategies to enhance the antigenicity of L2 have included L2 sequences fused to TLR5 activators [33], IgG1 Fc [51] and thioredoxin [52]. In some of these studies, the antigenicity of the L2 sequences remained low [53,54] and required potent and non-clinically relevant adjuvants to induce strong protection.…”

“…Three days after challenge, the mice were again anesthetized and 20 μL of luciferin (7.8 mg/mL) was deposited in the vaginal vault. Luciferase signals were acquired for 10 min with a Xenogen IVIS 100 imager, and analysis was performed with Living Image 2.5 software [32,33]. …”

Human Papilloma Virus Immunogen Creation on the Base of Chimeric Recombinant Protein L2e7

“…Однако N-конец белка L2 содержит консервативные эпитопы, к которым возможно образование нейтрализующих антител [14,20,33]. В литера-туре описаны успешные попытки повышения иммуногенности N-конца молекулы L2 путем слияния в одном белке N-концов белка L2 сразу нескольких штаммов [20,21,22,24].…”

“…Та-ким образом, при создании вакцины на основе L2 необходимо повысить его иммуногенность. Одним из методов решения этой задачи являет-ся конкатомеризация N-концов L2 различных штаммов ВПЧ [20,21,22,24]. Повышение имму-ногенности в этом случае объясняется наличием в одном белке множества одинаковых эпитопов, что вызывает интернализацию B-клеточных рецепторов на поверхности B-клетки и индук-цию ее пролиферации [1].…”

Preclinical refinements of a broadly protective VLP-based HPV vaccine targeting the minor capsid protein, L2

“…As an alternative to vaccines based on L1 VLPs, we and others have focused on developing broadly protective cost-effective next-generation HPV vaccines targeting highly conserved epitopes in the HPV minor capsid protein, L2 [9, 16, 17, 25-29]. Our group has developed candidate next-generation HPV vaccines based on bacteriophage VLPs displaying conserved L2 epitopes from HPVs and has shown that these vaccines provide broad protection from infection by diverse HPV types [9, 15-18].…”