Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

Geochemical and Sr, Nd, Pb isotope investigation of the New Caledonia ophiolite

The New Caledonia ophiolite hosts one of the largest obducted mantle section in the world, hence providing a unique insight for the study of upper mantle processes. These mantle rocks belong to an “atypical” ophiolitic sequence, which is dominated by refractory harzburgites but it also includes minor spinel and plagioclase lherzolites. Upper crust is notably absent in the ophiolite, with the exception of some mafic-ultramafic cumulates cropping out in the southern part of the island. Although the New Caledonia ophiolite has been under investigation for decades, its ultra-depleted nature has made its characterization an analytical challenge, so that few trace element data are available, while isotopic data are completely missing. In this thesis a comprehensive geochemical study (major, trace element and Sr-Nd-Pb isotopes) of the peridotites and the associated intrusive mafic rocks from the New Caledonia ophiolite has been carried out. The peridotites are low-strain tectonites showing porphyroclastic textures. Spinel lherzolites are undepleted lithotypes, as attested by the presence of 7-8 vol% of Na2O and Al2O3-rich clinopyroxene (up to 0.5 wt% Na2O; 6.5 wt% Al2O3), Fo content of olivine (88.5-90.0 mol%) and low Cr# of spinel (13-17). Conversely, harzburgites display a refractory nature, proven by the remarkable absence of primary clinopyroxene, very high Fo content in olivine (90.9-92.9 mol%), high Mg# in orthopyroxene (89.8-94.2) and Cr# in spinel (39-71). REE contents show abyssal-type patterns for spinel lherzolites, while harzburgites display U-shaped patterns, typical of fore-arc settings. Spinel lherzolites REE compositions are consistent with relatively low degree (8-9%) of fractional melting of a DMM source, starting in the garnet stability field. Conversely, REE models for harzburgites indicate high melting degrees (20-25%) of a DMM mantle source under spinel faies conditions, consistent with hydrous melting in forearc setting. Plagioclase lherzolites exhibit melt impregnation microtextures, Cr- and TiO2-enriched spinels and REE, Ti, Y, Zr progressive increase with respect to spinel lherzolites. Impregnation models indicate that plagioclase lherzolites may derive from spinel lherzolites by entrapment of highly depleted MORB melts in the shallow oceanic lithosphere. Mafic intrusives are olivine gabbronorites with a very refractory composition, as attested by high Fo content of olivine (87.3-88.9 mol.%), very high Mg# of clinopyroxene (87.7-92.2) and extreme anorthitic content of plagioclase (An = 90-96 mol%). The high Mg#, low TiO2 concentrations in pyroxenes and the anorthitic composition of plagioclase point out an origin from ultra-depleted primitive magmas in a convergent setting. Geochemical trace element models show that the parental melts of gabbronorites are primitive magmas with striking depleted compositions, bearing only in part similarities with the primitive boninitic melts of Bonin Islands. The first Sr, Nd and Pb isotope data obtained for the New Caledonia ophiolite highlight the presence of DM mantle source variably modified by different processes. Nd-Sr-Pb isotopic ratios for the lherzolites (+6.98≤epsilon Ndi≤+10.97) indicate a DM source that suffered low-temperature hydrothermal reactions. Harzburgites are characterized by a wide variation of Sr, Nd and Pb isotopic values, extending from DM-type to EM2 compositions (-0.82≤ epsilon Ndi≤+17.55), suggesting that harzburgite source was strongly affected by subduction-related processes. Conversely, combined trace element and Sr-Nd-Pb isotopic data for gabbronorites indicate a derivation from a source with composition similar to Indian-type mantle, but affected by fluid input in subduction environment. These geochemical features point out an evolution in a pre-Eocenic marginal basin setting, possibly in the proximity of a transform fault, for the lherzolites. Conversely, the harzburgites acquired their main geochemical and isotopic fingerprint in subduction zone setting.