Influence of antigen density and TLR ligands on preclinical efficacy of a VLP-based vaccine against peanut allergy.

BACKGROUND Virus-like particle (VLP) Peanut is a novel immunotherapeutic vaccine candidate for the treatment of peanut allergy. The active pharmaceutical ingredient represents cucumber mosaic VLPs (CuMVTT -VLPs) that are genetically fused with one of the major peanut allergens, Ara h 2 (CuMVTT -Ara h 2). We previously demonstrated the immunogenicity and the protective capacity of VLP Peanut-based immunization in a murine model for peanut allergy. Moreover, a Phase I clinical trial has been initiated using VLP Peanut material manufactured following a GMP-compliant manufacturing process. Key product characterization studies were undertaken here to understand the role and contribution of critical quality attributes that translate as predictive markers of immunogenicity and protective efficacy for clinical vaccine development. METHOD The role of prokaryotic RNA encapsulated within VLP Peanut on vaccine immunogenicity was assessed by producing a VLP Peanut batch with a reduced RNA content (VLP Peanut low RNA). Immunogenicity and peanut allergen challenge studies were conducted with VLP Peanut low RNA, as well as with VLP Peanut in WT and TLR 7 KO mice. Furthermore, mass spectrometry and SDS-PAGE based methods were used to determine Ara h 2 antigen density on the surface of VLP Peanut particles. This methodology was subsequently applied to investigate the relationship between Ara h 2 antigen density and immunogenicity of VLP Peanut. RESULTS A TLR 7 dependent formation of Ara h 2 specific high-avidity IgG antibodies, as well as a TLR 7 dependent change in the dominant IgG subclass, was observed following VLP Peanut vaccination, while total allergen-specific IgG remained relatively unaffected. Consistently, a missing TLR 7 signal caused only a weak decrease in allergen tolerability after vaccination. In contrast, a reduced RNA content for VLP Peanut resulted in diminished total Ara h 2 specific IgG responses, followed by a significant impairment in peanut allergen tolerability. The discrepant effect on allergen tolerance caused by an absent TLR 7 signal versus a reduced RNA content is explained by the observation that VLP Peanut-derived RNA not only stimulates TLR 7 but also TLR 3. Additionally, a strong correlation was observed between the number of Ara h 2 antigens displayed on the surface of VLP Peanut particles and the vaccine's immunogenicity and protective capacity. CONCLUSIONS Our findings demonstrate that prokaryotic RNA encapsulated within VLP Peanut, including antigen density of Ara h 2 on viral particles, are key contributors to the immunogenicity and protective capacity of the vaccine. Thus, antigenicity and RNA content are two critical quality attributes that need to be determined at the stage of manufacturing, providing robust information regarding the immunogenicity and protective capacity of VLP Peanut in the mouse which has translational relevance to the human setting

Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with theSanger Zebrafish Mutation Project revealed thatapproximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, afterwhich Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses

Fiscal decentralisation, efficiency, and growth

Much of the recent worldwide trend towards devolution has been driven by the belief that fiscal decentralisation is likely to have a positive effect on government efficiency and economic growth. It is generally assumed that the transfer of powers and resources to lower tiers of government allows for a better matching of public policies to local needs and thus for a better allocation of resources. These factors, in turn, are expected to lead to an improvement in regional economic performance, if subnational authorities shift resources from current to capital expenditures in search of a better response to local needs. In this paper we test these assumptions empirically by analysing the evolution of subnational expenditure categories and regional growth in Germany, India, Mexico, Spain, and the USA. We find that, contrary to expectations, decentralisation has coincided in the sample countries with a relative increase in current expenditures at the expense of capital expenditures, which has been associated with lower levels of economic growth in countries where devolution has been driven from above (India and Mexico), but not in Spain, where it has been driven from below. We hypothesise that the differences in legitimacy between the central or federal government and subnational governments in top-down and bottom-up processes of devolution may be at the origin of the diverse capacity to deliver greater allocative and productive efficiency and, eventually, greater economic growth by devolved governments