Protective immunity against malaria by a nanoparticle CIS43-based junctional vaccine alone or in combination with R21

Abstract

Repetitive display of the major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) is the basis for two WHO-recommended vaccines: RTS,S/AS01 and R21/Matrix-M. Recently, however, the CIS43 monoclonal antibody that preferentially targets the junctional region of PfCSP has been shown to be highly protective in humans, highlighting its junctional epitope as a key vaccine target. Here, we develop a vaccine based on the tandem repeats of the junctional epitope displayed on a self-assembling nanoparticle, and compare this CIS43-based junctional vaccine alone or in combination with the benchmark R21 vaccine, using both B cell analysis and monoclonal antibody isolation to define targeting of the immune response. Comparable reduction in liver burden was observed following vaccination with junctional and R21 vaccines at a dose of 1 μg. At a dose of 0.25 μg, a modest reduction of malaria-liver burden with the junctional vaccine was observed compared to R21. Further, combining junctional and R21 vaccines induced modestly enhanced protection compared to either vaccine alone. While the R21 vaccine elicited antibodies primarily against the major repeats, the junctional vaccine elicited antibodies against both junctional and major repeat regions. In vivo-B cell analysis and isolation of monoclonal antibodies confirmed differences in vaccine-induced antibody specificities. Altogether, these data suggest the nanoparticle-formatted tandem-repeated CIS43-junctional vaccine to be a promising approach to broaden immunity against malaria, either as a standalone intervention or in combination with R21.

Keywords: CIS43; PfCSP; R21; antibodies; malaria; self-assembling nanoparticle; structure-based design; vaccine.

Figure 1.. Design and characterization of nanoparticle immunogens incorporating the CIS43 epitope.

a Schematic of plasmodium falciparum circumsporozoite protein (PfCSP) highlighting the CIS43 epitope. b Structure of CIS43 bound to peptide 21 of PfCSP. c Design of nanoparticle immunogens. d Characterization of immunogens by size exclusion chromatography and SDS-PAGE. e Negative stain EM shows properly assembled nanoparticle immunogens. f Antigenicity of nanoparticle immunogens to junction, minor, and major-directed antibodies assessed by biolayer interferometry.

Figure 2.. Malaria protection by vaccination with tandem-displayed CIS43 epitope on LuS nanoparticle.

a Immunization schema comprising of 2 immunizations 4 weeks apart followed by sporozoite challenge at week 8. b Serum antibody titers at weeks 2 and 6 assessed by biolayer interferometry (BLI) against biotinylated PfCSP, NANP9 (major repeat), and NPDP19 (junction). The average titer of each group is listed. c Liver burden assessment of immunized mice at day 2 post Pb-PfCSP-Luc sporozoite challenge is shown with the horizontal lines denoting the median flux; error bars show 95 % confidence interval; N=10 mice per group. The percent reduction is calculated based on geometric mean of each group and values above 50 % are highlighted in red. * p < 0.05, ** p < 0.01 as calculated by two-tailed Mann–Whitney test.

Figure 3.. Tandem LuS-displayed CIS43 epitope vaccine provides protection equivalent to R21 at 1 ug dose.

a Immunization schema comprising of 2 immunizations 4 weeks apart followed by sporozoite challenge at week 8. b Serum antibody titers at weeks 2 and 6 assessed by biolayer interferometry (BLI) against biotinylated PfCSP, NANP9 (major repeat), and NPDP19 (junction). The average titer of each group is listed. c Liver burden assessment of immunized mice at day 2 post Pb-PfCSP-Luc sporozoite challenge is shown with the horizontal lines denoting the median flux; error bars show 95 % confidence interval; N=10 mice per group. The percent reduction is calculated based on geometric mean of each group and values above 50 % are highlighted in red. * p < 0.05, ** p < 0.01 as calculated by two-tailed Mann–Whitney test.

Figure 4.. Tandem LuS-displayed CIS43 epitope vaccine provides increased protection relative to R21 at 0.25 ug dose.

a Immunization schema comprising of 2 immunizations 4 weeks apart followed by sporozoite challenge at week 8. b Serum antibody titers at weeks 2 and 6 assessed by biolayer interferometry (BLI) against biotinylated PfCSP, NANP9 (major repeat), and NPDP19 (junction). The average titer of each group is listed. c Liver burden assessment of immunized mice at day 2 post Pb-PfCSP-Luc sporozoite challenge is shown (top panel) with the horizontal lines denoting the median flux; error bars show 95 % confidence interval; N=10 mice per group. The repeat experiment is shown for R21 and PADRE-LuS-3T at 0.25 ug; N=10 mice per group (bottom left panel). The pooled results of two independent experiments is shown with N=20 mice per group (bottom right panel). The percent reduction is calculated based on geometric mean of each group and values above 50 % are highlighted in red. * p < 0.05, ** p < 0.01 as calculated by two-tailed Mann–Whitney test.

Figure 5.. PADRE-LuS-3T elicits robust germinal center response in CIS43 KI-mice.

a Schematic of adoptive transfer model to evaluate iGL_CIS43 responses to PADRE-LuS-3T immunogen. b Representative FACS plot of GC response and CD45.2+ B cells in GC. c Quantification of GC formation, CD45.2+ in GC, CSP+ in CD45.2+GCB and CSP+CD45.2+GCB frequency in total B cells. N = 10 mice in each group. The data is pooled from two independent experiments. Each symbol represents a different mouse. One-way ANOVA was applied, and the bars indicate mean ± SD. p>0.05 represents no significance (ns), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. d Representative FACS plot of antibody secreting cell (ASC) response and CD45.2+ B cells in ASC. e Quantification of ASC frequency, and CD45.2+ of ASC. N = 10 mice in each group. The data is pooled from two independent experiments. Each symbol represents a different mouse. One-way ANOVA was applied, and the bars indicate mean ± SD. p>0.05 represents no significance (ns), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6.. B-cell sorting and antibody antigenic analysis reveal PADRE-LuS-3T elicits cross-reactive responses in contrast to R21 in mice.

a Percentages of PfCSP+/P21+, PfCSP+/P29+ and PfCSP+/P21+/P29+ (+++) B cells among all IgG+ B cells in R21-immunized mouse (5116; from group 5 Fig. 4c) and two PADRE-LuS-3T immunized mice (5147, 5149; from group 3 Fig. 4c), analyzed by flowcytometry. b ELISA screening of RATPIg supernatants in 96-well plates containing expressed IgGs from R21 or PADRE-LuS-3T immunized mice. Each well corresponds to an index sorted B cell except the gray-shaded wells (no cell). The binding (OD450) of the supernatants to PfCSP, P21 and P29 is plotted in heatmaps. c List of antigen-specific antibodies identified by RATPIg from sorted splenic B cells of R21 (mouse 5116)- or PADRE-LuS-3T (mice 5147 and 5149)-immunized mice. Antibodies of similar clonal families are highlighted in blue or green.