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

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.


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The expression of monomeric recombinant Pfs230D1M was performed in HEK293F cells as 155 previously described [27]. Briefly, a truncated form of Pfs230 containing the first 6-cys 156 domain of Pfs230 has previously been expressed as a monomeric recombinant protein in P. 7 or Pfs25). Secondary HRP-conjugated antibodies (polyclonal goat anti-mouse IgG at 1/1000 detection was developed using ABTS or TMB liquid substrate (Sigma-Aldrich), which was subsequently stopped using 1% SDS (for ABTS) or 1M sulphuric acid (for TMB). PBS was 172 used as a negative control and plates were washed thrice using PBS with 0.05% Tween in 173 between antibody incubation steps. The level of antibody binding was measured as optical 174 density at 405nm (for ABTS) or 450nm (for TMB).

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Thin blood smears of stage V 3D7 gametocyte-infected erythrocytes were fixed in 90% 188 acetone and 10% methanol for 5 min at -20C as previously described [30]

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Measuring transmission-blocking activity by standard membrane feeding assays

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IgG purification from individual rabbit serum samples was performed using Protein G 219 columns (GE Healthcare) according to the manufacturer instructions and adjusted to a final 220 concentration of 20 mg/ml in PBS. The standardized methodology for performing the 221 standard membrane feeding assays (SMFA) was described previously [31]. Briefly, [16][17][18] 222 days old gametocyte cultures of the P. falciparum NF54 line were mixed with purified test immediately fed to ~50 female Anopheles stephensi mosquitoes through a membrane-225 feeding apparatus. Mosquitoes were kept for 8 days and dissected to enumerate the oocysts 226 in the midgut (n=40 per group for the negative control group, and n=20 for test groups). As 227 the negative controls, total IgG purified from pre-immune rabbit sera were utilised. Only 228 midguts from mosquitoes with any eggs in their ovaries at the time of dissection were 229 analyzed. The human serum and red blood cells used for the SMFA were purchased from   includes the first two 6-cysteine domains of Pfs230 [23], was used to generate fusion 250 proteins for VLP formation. Pfs230c has been previously shown to elicit transmission-blocking antibodies [23]. The expression and purification of Pfs230c-dS VLPs was 252 challenging [25]. Therefore, a shorter construct of Pfs230, termed Pfs230D1M (based on the 253 construct described by [24]) which includes only the first 6-cysteine domain of Pfs230, was 254 designed and fused to the dS antigen. Further, Pfs230D1M is a leading transmission-255 blocking vaccine candidate currently in clinical trials. Here, we evaluated the display of these 256 sexual-stage antigens on the surface of native VLPs. We used specific antibodies generated 257 against Pfs230 and Pfs25 to detect the expression of these antigens on the chimeric VLPs 258 by ELISA (Fig 1). We found that a Pfs230-specific polyclonal antibody recognised the 259 surface of Pfs230c-dS/dS ( Fig 1A) and Pfs230D1M-dS/dS VLPs (Fig 1B). Similarly, a Pfs25-260 specific monoclonal antibody recognised the surface of Pfs25-dS VLPs ( Fig 1C). These 261 results confirm the expression of sexual-stage antigens on the surface of chimeric VLPs.

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There was no recognition of a plain dS VLP without a Pfs230 or Pfs25 fusion protein (Fig   263   1D).

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To understand whether these chimeric VLPs displaying sexual-stage antigens were capable 267 of eliciting an immune response, the VLP constructs were used to immunise rabbits (n=8 for 268 Pfs230c-dS/dS, n=4 for Pfs230D1M-dS/dS, n=2 for Pfs25-dS/dS). Serum from immunised 269 rabbits had significant antibody recognition of monomeric recombinant Pfs230D1M (

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The higher dose appeared to induce a higher antibody response in rabbits, and formulation 274 with the Alhydrogel adjuvant also contributed to higher antibody levels (Fig 2A, B

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To address the functional significance of rabbit antibodies generated against sexual-stage 297 chimeric VLPs, we examined the ability of these antibodies to inhibit mosquito infection 298 through standard membrane feeding assays (SMFA) in the presence of human complement 299 [31]. Functional transmission-blocking activity is defined as the reduction in oocyst count 300 compared to a negative control group. When antibodies against Pfs230c-dS/dS VLPs were 301 tested at 7.5mg/ml, none of them showed significant inhibition (Table 1). In contrast, 2 out of 302 4 rabbits immunized with Pfs230D1M-dS/dS VLPs demonstrated significant inhibitions in 303 SMFA (Table 2). Antibodies generated against Pfs25-dS/dS VLPs from one of the two 304 rabbits also successfully blocked the development of oocysts within the mosquito midgut inhibit transmission despite substantial induction of antibodies detected by ELISA, 307 suggesting that antibody titre and specificity may be important for functional activity.

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Recombinant hepatitis B vaccines-disease characterization and vaccine production.

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For all graphs, antibody binding is expressed as optical density (OD)