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Proton low-resolution NMR, exploiting orientation-dependent dipole-dipole couplings within the monomer units, provides quantitative information on anisotropic chain motion in polymeric systems. Based on this observable, the crosslink density, related inhomogeneity and elastically inactive defects can be quantified for the case of many technologically relevant elastomers. In case of polymer melts, the entanglement spacing and the timescales of the tube/reputation model can be assessed. This contribution summarizes recent results extending our multiple-quantum NMR methodology to validating microscopic elasticity theories by studying the NMR response of swollen and deformed samples, and to detecting timescales of “sticky” chain motion in supramolecular elastomers for application in self-healing rubbers, Proton low-resolution NMR, exploiting orientation-dependent dipole-dipole couplings within the monomer units, provides quantitative information on anisotropic chain motion in polymeric systems. Based on this observable, the crosslink density, related inhomogeneity and elastically inactive defects can be quantified for the case of many technologically relevant elastomers. In case of polymer melts, the entanglement spacing and the timescales of the tube/reputation model can be assessed. This contribution summarizes recent results extending our multiple-quantum NMR methodology to validating microscopic elasticity theories by studying the NMR response of swollen and deformed samples, and to detecting timescales of “sticky” chain motion in supramolecular elastomers for application in self-healing rubbers

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity.

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. METHODS: We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. RESULTS: In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90% on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. CONCLUSION: The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.

BACKGROUND: Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field. RESULTS: In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C. CONCLUSIONS: The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the

An Arxula adeninivorans integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris. The vector harbours a conserved A. adeninivorans-derived 25S rDNA sequence for targeting, the A. adeninivorans-derived TEF1 promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants. Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene. The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level.