Probing novel epitopes on the Plasmodium falciparum circumsporozoite protein for vaccine development

Abstract

RTS,S and R21 are the only vaccines recommended by the WHO to protect children from Plasmodium falciparum (Pf) clinical malaria. Both vaccines target the Pf sporozoite surface protein circumsporozoite protein (CSP). Recent studies showed that human antibodies neutralize Pf sporozoites most efficiently when simultaneously binding to the PfCSP NANP repeat and the NPDP junction domain. However, neither RTS,S nor R21 targets this junction domain. To test the potential of the NPDP junction domain and other sites of PfCSP as innovative vaccine targets, we developed multiple vaccine candidates based on cucumber mosaic virus-like particles (CuMV_TT-VLPs). These candidates vary in several aspects: the number of targeted NANP repeats, the presence or absence of the junction domain, the cleavage site, and up to three NVDP repeats within the target sequence. Immunogenicity and efficacy studies were conducted in BALB/c mice, utilizing chimeric Plasmodium berghei (Pb) sporozoites, in which the endogenous CSP has been replaced by PfCSP (Pb/PfCSP). We observed a positive association between the number of targeted NANP repeats and the induction of specific IgM/IgG antibodies. Elevated humoral responses led to enhanced protection against parasitemia after Pb/PfCSP sporozoite challenge. Especially high-avidity/affinity antibody formation and vaccine protection were NANP repeat-dependent. Intriguingly, vaccine efficacy was not enhanced by targeting sites on PfCSP other than the NANP repeats. Our data emphasize the dominant role of the NANP repeat region for induction of protective antibodies. Furthermore, we present here novel malaria vaccine candidates with an excellent immunogenic profile that confer sterile protection in mice, even in absence of adjuvants.

Fig. 1. Design and characterization of CuMV_TT-based malaria vaccine candidates targeting different sites of PfCSP.

a Schematic representation of the vaccine candidate designs. Vaccine candidates are based on CuMV_TT-VLPs genetically fused to different amino acid sequences of PfCSP (PlasmoDB: PF3D7_0304600). The sequences include 19 NANP repeats (CuMV_TT-NANP₁₉), KQPGDGNDPDNANPN (CuMV_TT-J1-NANP₁), and KQPGDGNDPDNANPNVDPNANPNVDPNANPNANPNANPNANP (CuMVTT-J2-NANP₆). Particles are expressed as mosaic VLPs, comprising CuMV_TT subunits fused to parts of PfCSP and subunits that remain unfused. The scheme was created with BioRender.com. b TEM and DLS of vaccine candidates after expression in E. coli, scale bar 200 nm. c 12% SDS–PAGE of C. CuMV_TT, 1. CuMV_TT-NANP₁₉, 2. CuMV_TT-J1-NANP₁, 3. CuMV_TT-J2-NANP₆, and M. Protein Marker after Coomassie staining. Protein bands derived from the fusion product of CuMV_TT to different amino acid sequences of PfCSP are circled in orange, and CuMV_TT-derived protein bands are circled in violet. d Antigen density of PfCSP-derived amino acid sequences on the surface of the particles: 1. CuMV_TT-NANP₁₉, 2. CuMV_TT-J1-NANP₁, 3. CuMV_TT-J2-NANP₆, and C. CuMV_TT. Antigen density was determined based on FIJI image J software analysis of protein band intensities from SDS–PAGE depicted in Fig. 1c. e Agarose gel analysis of 1. CuMV_TT-NANP₁₉, 2. CuMV_TT-J1-NANP₁, 3. CuMV_TT-J2-NANP₆, and M_N. nucleic acid marker depicting the VLP encapsulated RNA derived from the expression host. Statistical analyses were performed as follows: data are presented as mean ± SEM. In (d), one-way ANOVA with Tukey correction was used for comparison. Significance levels are denoted as follows: p < 0.001 (***), p < 0.0001 (****). N = 2.

Fig. 2. Immunogenicity and protective efficacy of CuMV_TT-based malaria vaccine candidates.

a Mouse model to evaluate the immunogenicity and protective efficacy of the different malaria vaccine candidates. 8-week-old female BALB/cOlaHsd mice were immunized on days 0 and 28 with 30 µg of the vaccine candidate, formulated in 100 µL PBS per mouse and administered s.c. On day 38, the mice were infected with 5000 Pb/PfCSP sporozoites, delivered i.d. in the ear. Parasitemia was assessed daily by flow cytometry, checking for GFP-positive erythrocytes (Pb/PfCSP infected). Blood samples were collected from the tail vein on days 0, 28, and 38, and serum was isolated for immunological analysis. The scheme was created with BioRender.com. b Pb/PfCSP sporozoite-specific serum IgG measured by ELISA, Log₁₀ OD₅₀ is shown. c rPfCSP-specific serum IgG measured by ELISA, Log₁₀ OD₅₀ is shown. d rPfCSP-specific serum IgM measured by ELISA, Log₁₀ OD₅₀ is shown. e Avidity Index (proportion of high-avidity antibodies) of rPfCSP-specific serum IgG measured by an adapted ELISA protocol using a 7 M urea washing step to remove low-avidity antibodies from the plate. f Association between vaccine-induced rPfCSP-specific IgG or IgM titers and the number of NANP repeats targeted by the vaccine approximated using a saturation curve model (left and middle). Linear correlation between rPfCSP-specific high-avidity IgG antibodies and the number of NANP repeats targeted by the vaccine (right). g Parasite-free mice after i.d. injection of 5000 Pb/PfCSP sporozoites. Statistical analyses were performed as follows: data are presented as mean ± SEM. In (b–e), one-way ANOVA with Tukey correction was used for comparisons. In (g), statistical significance was assessed using the Log-rank (Mantel–Cox) test. Significance levels are denoted as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). N = 9. One representative of two comparable experiments is shown.

Fig. 3. Design and characterization of optimized CuMV_TT-based malaria vaccine candidates.

a Schematic illustration of the design of optimized vaccine candidates. Vaccine candidates are based on CuMV_TT-VLPs genetically fused to different amino acid sequences of PfCSP (PlasmoDB: PF3D7_0304600). The sequences include KHKKLKQPADGNPDP followed by 19 NANP repeats (CuMV_TT-L1-NANP₁₉) and EDNEKLRKPKHKKLKQPADGNPDP followed by 19 NANP repeats interspersed with three single NVDP repeats (CuMV_TT-L2-NANP₁₉). Particles are expressed as mosaic VLPs, comprising CuMV_TT subunits fused to parts of PfCSP and subunits that remain unfused. The scheme was created with BioRender.com. b TEM and DLS of vaccine candidates after expression in E. coli, scale bar 200 nm. c 12% SDS–PAGE of M. Protein Marker, C. CuMV_TT, 1. CuMV_TT-19 NANP, 2. CuMV_TT-L1-NANP₁₉, and 3. CuMV_TT-L2-NANP₁₉ after staining with coomassie. Protein bands resulting from the fusion of CuMV_TT to different amino acid sequences of PfCSP are highlighted in orange, protein bands derived from CuMV_TT are marked in violet. d Antigen density of PfCSP-derived amino acid sequences on the surface of the particles: 1. CuMV_TT-NANP₁₉, 2. CuMV_TT-L1-NANP₁₉, 3. CuMV_TT-L2-NANP₁₉, and C. CuMV_TT. Antigen density was calculated by analyzing protein band intensities from SDS–PAGE depicted in (c) using FIJI image J software. e Agarose gel analysis of 1. CuMV_TT-NANP₁₉, 2. CuMV_TT-L1-NANP₁₉, 3. CuMV_TT-L2-NANP₁₉, and M_N. nucleic acid marker depicting the VLP encapsulated prokaryotic RNA. Statistical analyses were performed as follows: data are presented as mean ± SEM. In (d) one-way ANOVA with Tukey correction was used for comparison. Significance levels are denoted as follows: p < 0.0001 (****). N = 2.

Fig. 4. Immune responses and protective efficacy after vaccination with optimized CuMV_TT-based malaria vaccine candidates.

a Mouse model to evaluate the immunogenicity and protective efficacy of the optimized malaria vaccine candidates. 8-week-old female BALB/cOlaHsd mice were immunized on days 0 and 28 with 30 µg of the vaccine candidate, formulated in 100 µL PBS per mouse and administered s.c. On day 38, the mice were infected with 5000 Pb/PfCSP sporozoites, delivered i.d. in the ear. Parasitemia was assessed daily by flow cytometry, checking for GFP-positive erythrocytes (Pb/PfCSP infected). Blood samples were collected from the tail vein on days 0, 28, and 38, and serum was isolated for immunological analysis. The scheme was created with BioRender.com. b Pb/PfCSP sporozoite-specific serum IgG measured by ELISA, Log₁₀ OD₅₀ is shown. c rPfCSP-specific serum IgG measured by ELISA, Log₁₀ OD₅₀ is shown. d Avidity Index (proportion of high-avidity antibodies) of rPfCSP-specific serum IgG measured by an adapted ELISA protocol using a 7 M urea washing step to remove low-avidity antibodies from the plate. e rPfCSP-specific serum IgG1, IgG2a, IgG2b, and IgG3 measured by ELISA, Log₁₀ OD₅₀ is shown. f rPfCSP-specific serum IgM measured by ELISA, Log₁₀ OD₅₀ is shown. g Parasite-free mice after i.d. injection of 5000 Pb/PfCSP sporozoites. Statistical analyses were performed as follows: data are presented as mean ± SEM. In (b–f), one-way ANOVA with Tukey correction was used for comparisons. In (g), statistical significance was assessed using the Log-rank (Mantel–Cox) test. Significance levels are denoted as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). N = 6. One representative of two similar experiments is shown. Data of both experiments were combined in (g) (N = 12).

Fig. 5. Binding capacity of vaccine candidate-induced IgG antibodies to Pb/PfCSP sporozoites.

Immunofluorescence assay showing the reactivity of serum from vaccinated mice (D38) with Pb/PfCSP sporozoites. Pb sporozoites expressing endogenous PbCSP (676 cl1 parental line) serve as a control. DAPI nucleic acid stain is depicted in blue, vaccine-induced IgG antibodies are depicted in red, and PbTRAP-specific antibodies are depicted in purple. Scale bar 5 µm. One representative of two similar experiments is shown.

Fig. 6. Epitope mapping of IgG antibodies induced by vaccine candidates.

a Serum samples collected on day 38 analyzed by ELISA for epitope-specific IgG, Log₁₀ OD₅₀ is shown. Binding specificity to the following epitopes was assessed: 1. NANPNANP, 2. NANPNVDP, 3. GNNPDPNA, 4. KHKKLKQP. b Average epitope-specific serum IgG titer (Log₁₀ OD₅₀) displayed in a heat map. Data are presented as mean ± SEM. N = 6.