Elicitation of liver-stage immunity by nanoparticle immunogens displaying P. falciparum CSP-derived antigens

Abstract

A vaccine that provides robust, durable protection against malaria remains a global health priority. Although a breakthrough in the fight against malaria has recently been achieved by the licensure of two vaccines based on the circumsporozoite protein (CSP), the effectiveness and durability of protection can still be improved. Both vaccines contain a portion of CSP that does not include epitopes targeted by recently identified, potently protective monoclonal antibodies, suggesting that newer immunogens can expand the breadth of immunity and potentially increase protection. Here we explored >100 alternative CSP-based immunogens and evaluated the immunogenicity and protection of a large number of candidates, comparing several to the licensed R21 vaccine. The data highlight several general features that improve the stability and immunogenicity of CSP-based vaccines, such as inclusion of the C-terminal domain and high-density display on protein nanoparticle scaffolds. We also identify antigen design strategies that do not warrant further exploration, such as synthetic repeat regions that include non-native repeat cadences. The benchmark R21 vaccine outperformed our best immunogen for immunogenicity and protection. Overall, our data provide valuable insights on the inclusion of junctional region epitopes that will guide the development of potent and durable vaccines against malaria.

Fig. 1. Design, biophysical characterization, antigenicity, and immunogenicity of SAmut-5/3 multimers.

a Primary structures of PfCSP variants, including the R21 immunogen. SP, signal peptide; RI, Region I; RIII, Region III; RII + , Region II; GPI, glycosylphosphatidylinositol anchor sequence. Red lines indicate sites of cysteine and PEXEL mutations. b Reducing SDS-PAGE of purified SAmut-5/3 immunogens. c nsEM of the SAmut-5/3 multimers. Micrographs and structural models of each multimer are shown. d Binding of multimers to PfCSP-directed mAbs measured by ELISA. NHP20 is an unpublished antibody isolated from a non-human primate immunized with WT PfCSP that binds to the NTD, CIS43 is a dual binder for the junctional epitope and major repeats, L9 is a dual binder for the minor epitope and major repeats, 311 binds the major repeats, and mAb15 binds the CTD. VRC01 is an anti-HIV-1 antibody used as a negative control. e Immunization regimen and details. IV, intravenous; SPZ, sporozoite. f Parasite burden in the liver after challenge with transgenic sporozoites. R21 was used as a benchmark immunogen. Naive refers to uninfected negative control mice and max burden to infected positive control mice. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 as calculated by Kruskal–Wallis test with multiple comparisons.

Fig. 2. Design, characterization, and immunogenicity of nanoparticle immunogens comprising the CSP junctional region.

a Models of the trimeric RT-I53-50A (RT in dark gray and blue, I53-50A in light gray) and pentameric I53-50B (orange) components, and an assembled RT-I53-50 nanoparticle. b Schematics of junctional region antigens. Each antigen was genetically fused to I53-50A. c Immunization regimen and details of the study. d Serum antibody titers against SAmut, determined by ELISA using sera obtained 1–2 weeks after the primary and third immunizations. Statistical significance was calculated by one-way ANOVA with multiple comparisons. e Peptide mapping ELISAs using pooled sera from each group, measured using mesoscale discovery (MSD) -multi-spot assay system. f Parasite burden in the liver after challenge with transgenic sporozoites. RT-I53-50 was used as the benchmark immunogen. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 as calculated by Kruskal–Wallis test with multiple comparisons.

Fig. 3. Design, characterization, and immunogenicity of non-native CSP-repeat nanoparticles.

a Models of the CSP X-I53-50A trimer (CSP X in green, dark gray, and blue; I53-50A in light gray), I53-50B (orange), and an assembled CSP X-I53-50 nanoparticle. b Schematics of non-native CSP-repeat antigens. Each antigen was genetically fused to I53-50A. c Immunization regimen and details of the study. d Serum antibody titers against SAmut, determined by ELISA using sera obtained after the second and third immunizations. Statistical significance was calculated by one-way ANOVA test with multiple comparisons. e Parasite burden in the liver after challenge with transgenic sporozoites. RT-I53-50 was used as the benchmark immunogen. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 as calculated by Kruskal–Wallis test with multiple comparisons.

Fig. 4. Immunogenicity and protection afforded by mosaic and cocktail nanoparticle immunogens.

a Immunization regimen and details of the study. b Schematics depicting the antigenic composition of each immunogen: either mosaic nanoparticles with 33 or 50% valency of each antigen, or groups (cocktails) of monovalent nanoparticles. c ELISA endpoint titer of each immunogen to SAmut-coated plates post-prime. Statistical significance was calculated by one-way ANOVA test with multiple comparisons. d Parasite burden in the liver after challenge with transgenic sporozoites. RT-I53-50 was used as the benchmark immunogen. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 as calculated by Kruskal–Wallis test with multiple comparisons.

Fig. 5. Comparison of R21 and RT-I53-50 adjuvanted with ALFQ.

a Groups and doses used. b Immunization regimens and details for the three-dose and two-dose experiments. c ELISA endpoint titer for each immunogen in the three-dose study to SAmut-coated plates after the second boost and before the challenge. Statistical significance was calculated by one-way ANOVA test with multiple comparisons. d Parasite burden in the liver after three immunizations followed by challenge with transgenic sporozoites. For the CIS43 group, 300 μg of CIS43 was administered 2 h prior to infection. *p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001 as calculated by Kruskal–Wallis with multiple comparisons test compared to infected (max burden) control. e ELISA endpoint titer for each immunogen in the two-dose titration study to SAmut-coated plates after the prime and boost immunizations. Statistical significance was calculated by one-way ANOVA test with multiple comparisons. f Parasite burden in the liver after two immunizations followed by challenge with transgenic sporozoites. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 as calculated by Kruskal–Wallis test with multiple comparisons compared to max burden control.