Incorporation of RG1 epitope concatemers into a self-adjuvanting Flagellin-L2 vaccine broaden durable protection against cutaneous challenge with diverse human papillomavirus genotypes.

AIM: To achieve durable and broad protection against human papillomaviruses by vaccination with multimers of minor capsid antigen L2 using self-adjuvanting fusions with the toll-like receptor-5 (TLR5) ligand bacterial flagellin (Fla) instead of co-formulation with alum. METHODS: Fla fusions with L2 protective epitopes comprising residues 11-200, 11-88 and/or 17-38 of a single or multiple HPV types were produced in E. coli and their capacity to activate TLR5 signaling was assessed. Immunogenicity was evaluated serially following administration of 3 intramuscular doses of Fla-L2 multimer without exogenous adjuvant, followed by challenge 1, 3, 6 or 12months later, and efficacy compared to vaccination with human doses of L1 VLP vaccines (Gardasil and Cervarix) or L2 multimer formulated in alum. Serum antibody responses were assessed by peptide ELISA, in vitro neutralization assays and passive transfer to naïve rabbits in which End-Point Protection Titers (EPPT) were determined using serial dilutions of pooled immune sera collected 1, 3, 6 or 12months after completing active immunization. Efficacy was assessed by determining wart volume following concurrent challenge at different sites with HPV6/16/18/31/45/58 'quasivirions' containing cottontail rabbit papillomavirus (CRPV) genomes. RESULTS: Vaccination in the absence of exogenous adjuvant with Fla-HPV16 L2 11-200 fusion protein elicited durable protection against HPV16, but limited cross-protection against other HPV types. Peptide mapping data suggested the importance of the 17-38 aa region in conferring immunity. Indeed, addition of L2 residues 17-38 of HPV6/18/31/39/52 to a Fla-HPV16 L2 11-200 or 11-88 elicited broader protection via active or passive immunization, similar to that seen with vaccination with an alum-adjuvanted L2 multimer comprising the aa 11-88 peptides of five or eight genital HPV types. CONCLUSIONS: Vaccination with flagellin fused L2 multimers provided lasting (>1year) immunity without the need for an exogenous adjuvant. Inclusion of the L2 amino acid 17-38 region in such multi-HPV type fusions expanded the spectrum of protection.

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

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.

High-pressure induced conformational and phase transformations of 1,2-dichloroethane probed by Raman spectroscopy.

1,2-Dichloroethane (DCE) was loaded into diamond anvil cells and compressed up to 30 GPa at room temperature. Pressure-induced transformations were probed using Raman spectroscopy. At pressures below 0.6 GPa, fluid DCE exists in two conformations, gauche and trans in equilibrium, which is shifted to gauche on compression. DCE transforms to a solid phase with exclusive trans conformation upon further compression. All the characteristic Raman shifts remain constant in fluid phase and move to higher frequencies in the solid phase with increasing pressure. At about 4-5 GPa, DCE transforms from a possible disordered phase into a crystalline phase as evidenced by the observation of several lattice modes and peak narrowing. At 8-9 GPa, dramatic changes in Raman patterns of DCE were observed. The splitting of the C-C-Cl bending mode at 325 cm-1, together with the observation of inactive internal mode at 684 cm-1 as well as new lattice modes indicates another pressure-induced phase transformation. All Raman modes exhibit significant changes in pressure dependence at the transformation pressure. The new phase remains crystalline, but likely with a lower symmetry. The observed transformations are reversible in the entire pressure region upon decompression.