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

Abstract

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.

Fig 1. Characterising the expression of sexual-stage antigens on chimeric VLPs.

Total antibody binding to (A) Pfs230c-dS/dS, (B) Pfs230D1M-dS/dS, (C) Pfs25-dS/dS and (D) plain dS VLPs were measured by ELISA. VLPs were coated at varying concentrations (μg/ml) and probed with either a Pfs25 or Pfs230-specific polyclonal antibody (1μg/ml). In (D), plain dS VLPs were probed with anti-Pfs230 antibody. The level of antibody binding is expressed as optical density (OD) measured at 405nm; symbols represent the mean and error bars represent the range between samples tested in duplicate.

Fig 2. Immunogenicity of sexual-stage chimeric VLPs in rabbits.

The level of antibody binding to a titration of monomeric recombinant (A, C) Pfs230D1M and (E) Pfs25 was measured in whole rabbit serum (1/100). Rabbits were immunised with chimeric Pfs230c-dS/dS (A, B), Pfs230D1M-dS/dS (C, D) or Pfs25-dS/dS (E, F) VLPs. Monomeric recombinant proteins were serially diluted from 4μg/ml. The level of antibody binding to monomeric recombinant (B, D) Pfs230D1M and (F) Pfs25 (coated at 1μg/ml) was measured by titrating whole rabbit serum. Rabbits were immunised with chimeric Pfs230c-dS/dS (A, B), Pfs230D1M-dS/dS (C, D) or Pfs25-dS/dS (E, F) VLPs. Whole rabbit serum was serially diluted from 1/100. For all graphs, antibody binding is expressed as optical density (OD) measured at 405nm; symbols represent the mean and error bars represent the range between samples tested in duplicate (n = 8 for Pfs230c-dS/dS, n = 4 for Pfs230D1M-dS/dS, n = 2 for Pfs25-dS/dS). Rabbits R1864-R1871 received Pfs230c-dS/dS VLPs formulated with and without Alhydrogel (A, B; Alh). Rabbits R1917 and R1918 received Pfs230D1M-dS/dS VLPs formulated with Freund’s adjuvant (Fre), while R1919 and R1920 received Pfs230D1M-dS/dS VLPs formulated with Alhydrogel (C, D). Rabbits R1825 and R1826 received Pfs25-dS/dS VLPs formulated with Freund’s adjuvant (E, F). Adjuvants and total VLP protein content used for immunisations are also presented in S1–S3 Tables.

Fig 3. Characterising the antibody function of rabbits immunised with chimeric VLPs.

A. Total antibody binding to the surface of native P. falciparum gametocytes (stage V gametocyte-infected erythrocytes were permeabilised with saponin) measured by flow cytometry. Whole serum from rabbits immunised with Pfs230c-dS/dS VLPs were used (n = 8). Antibody levels are expressed as geometric mean fluorescence intensity (MFI); bars represent mean and range of samples tested in duplicate. B. Immunofluorescence microscopy demonstrates the recognition of the native gametocyte surface by serum antibodies from rabbits immunised with Pfs230c-dS/dS VLPs (R1870; green). Cells were fixed with 90% acetone and 10% methanol, and DAPI was used to stain nuclear DNA (blue). Representative images taken of gametocytes labelled with R1870 are shown.