Display of malaria transmission-blocking antigens on chimeric duck hepatitis B virus-derived virus-like particles produced inHansenula polymorpha

Abstract

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...

Malaria vaccine candidates displayed on novel virus-like particles are immunogenic and induce transmission-blocking activity

“…Chimeric VLPs expressing sexual-stage P . falciparum antigens, Pfs230 and Pfs25, were produced and purified as described [25]. Antigens were incorporated into the VLP scaffold through genetic fusion of Pfs230 and Pfs25 with the dS protein.…”

“…The production and purification of the three different kinds of chimeric VLPs (Pfs230c-dS/dS, Pfs230D1M-dS/dS and Pfs25-dS/dS) was conducted as previously described [16,25]. Genes encoding the three fusion proteins Pfs230c-dS, Pfs230D1M-dS and Pfs25-dS were designed and codon-optimized for expression in Hansenula polymorpha (Genbank ref.…”

“…The purification of the chimeric VLPs was based on a downstream process approved for human VLP vaccine production from yeast [26] including adaptations for the purification of VLPs containing the P . falciparum antigens [25]. The relative incorporation of the Pfs230-dS/dS or Pfs25-dS/dS fusion proteins into VLPs, compared to total protein, was estimated by densitometry analysis of Coomassie stained gels of VLPs, as described in the accompanying manuscript [25].…”

“…falciparum antigens [25]. The relative incorporation of the Pfs230-dS/dS or Pfs25-dS/dS fusion proteins into VLPs, compared to total protein, was estimated by densitometry analysis of Coomassie stained gels of VLPs, as described in the accompanying manuscript [25]. The relative incorporation rates were ~30% for Pfs230c-dS/dS VLPs, ~24% for Pfs230D1M-dS/dS VLPs, and ~3% for Pfs25-dS/dS VLPs.…”

“…falciparum sexual-stage antigens, Pfs25 and the domains Pfs230c [23] and Pfs230D1M [24] derived from the full-length Pfs230 protein, were genetically fused to the dS and the resulting fusion proteins were co-expressed with wild-type dS in methylotrophic yeast Hansenula polymorpha [16]. The successful display of antigens on the chimeric VLPs (referred to as Pfs25-dS/dS, Pfs230c-dS/dS and Pfs230D1M-dS/dS) was confirmed using Western blot analyses and visualized by electron microscopy and super resolution microscopy (presented in the accompanying manuscript by Wetzel D et al 2019, submitted) [25]. In this manuscript, we report the immunogenicity of these sexual-stage chimeric VLPs through animal immunisations and further characterized their functional antibody response through mosquito feeding assays.…”