BackgroundChimeric 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.ResultsIn 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.ConclusionsThe 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 dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

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.

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L(-1) when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L(-1). The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L(-1). Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.

DW) 33 34 35 36Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP 56 with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported 57 the surface display of the malaria antigens on the native VLP. 58 Conclusion: 59The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the 60 dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. 61Competitive processes for efficient production and purification were established in this study. 62 3 Background 63Malaria is one of the world's deadliest human diseases with nearly half of the global population 64 living at risk. There were an estimated 216 million cases and 445,000 deaths due to malaria in 65 2016 [1]. This life-threatening disease is caused by Plasmodium parasites and is transmitted 66 via the bite of infected female Anopheles mosquitoes. The majority of malaria is caused by P. 67 falciparum, with P. vivax being a second major cause of disease [1]. Despite substantial 68 financial investment, US$ 2.7 billion in 2016, and decades of intense research and 69 development, only one malaria vaccine has progressed through phase 3 clinical trials and is 70 now undergoing phase 4 implementation trials (RTS,S; Mosquirix TM ). However, vaccine 71 efficacy in phase III clinical trials was low in young children (up to 50% efficacy in the first year, 72 but waning over 18 months) [2]. The World Health Organization has set a strategic goal of 73 developing vaccines with at least 75% efficacy [3], including the development of vaccines that 74 block malaria transmission [1]. Various approaches are under investigation including whole 75 parasite vaccines and subunit vaccines that are composed of defined, purified antigens or their 76 sub-domains [4]. Subunit vaccines have the potential to use established technologies and 77 processes for low-cost production and distribution through existing vaccine delivery 78 mechanisms [5]. A variety of Plasmodium antigens are currently under investigation as 79 potential subunit vaccine components and can be classified into one of the following groups 80 based on Plasmodium lifecycle stages [6]: i) pre-erythrocytic antigens (e.g. CSP [7]); ii) blood-81 stage antigens [8]; iii) transmission-stage antigens (e.g. Pfs25, Pfs230 [9-11]). 82 83Unfortunately, subunit vaccine candidates often suffer from weak immunogenicity that has to 84 be compensated by smart formulation and/or delivery strategies [12] such as virus-like 85 particles (VLP [13,14]). Since the 1980`s, VLP have been approved for use as safe and 86 effective subunit vaccines against several pathogens [15]. They can also be used as a scaffold 87 for the incorporation of antigens derived from foreign pathogens to enhance their immunogenic 88 potential (chimeric VLP [16]). Accordingly, the RTS,S vaccine contains chimeric VLP with a 89 truncated construct of CSP, the major surface antigen expressed on sporozoites during the 90 pre-erythrocytic stage. However...

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