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. 2022 Dec 20:12:1049065.
doi: 10.3389/fcimb.2022.1049065. eCollection 2022.

RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting

Julie Healer  1   2 , Jennifer K Thompson  1 , Karen L Mackwell  1 , Cecille D Browne  3 , Benjamin A Seager  1   2 , Anna Ngo  1 , Kym N Lowes  1   2 , Sarah E Silk  4 , David Pulido  4 , Lloyd D W King  4 , Jayne M Christen  3 , Amy R Noe  3 , Vinayaka Kotraiah  3 , Paul J Masendycz  1 , Rajkannan Rajagopalan  5 , Leanne Lucas  5 , Marianne M Stanford  5 , Lorraine Soisson  6 , Carter Diggs  6 , Robin Miller  6 , Susan Youll  6 , Kaye Wycherley  1 , Simon J Draper  4 , Alan F Cowman  1   2
Affiliations

RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting

Julie Healer et al. Front Cell Infect Microbiol. .

Abstract

Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro.

Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays.

Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr.

Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.

Keywords: CyRPA; Plasmodium falciparum; RH5 complex; Ripr; malaria; merozoite invasion; vaccine.

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Conflict of interest statement

The authors LL and RR are employed, and MS was employed by IMV Inc. JC, AN and VK are employed by Leidos Life Sciences. SD is a named inventor on patent applications relating to RH5-based malaria vaccines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Strategy and antigens in the RCR complex used in single, double, and triple combinations to determine immunogenicity and ability to block P. falciparum growth. (A) Workflow of experiments to determine immunogenicity and development of growth inhibitory antibodies for the RCR complex. Adjuvants and antigen dose were determined prior to immunisation experiments using combinations of RH5.1, CyRPA and Ripr. (B) Schematic of recombinantly expressed proteins expressed with C-terminal affinity tags. RH5.1 (RH5) and CyRPA were expressed with a C-tag (EPEA) (purple) and Ripr was expressed with a StrepII tag (green) for purification. CyRPA and RH5 sequences were modified from the 3D7 wildtype sequence (at numbered positions) to remove potential N-linked glycosylation sites. RH5.1 and Ripr were expressed in S2 Drosophila cells, whereas CyRPA was expressed in mammalian HEK293 cells. (C) RH5.1, CyRPA and Ripr and RCR complex purified antigens analysed by SDS-PAGE and Coomassie staining to visualize the bands.
Figure 2
Figure 2
DPX-formulated candidates induce P. falciparum growth inhibitory antibodies against RH5, CyRPA and Ripr in mice and rats. (A) A GIA assay was performed with IgG pooled from 6 mice immunized with 20 μg antigen formulated with DPX - RH5 (red squares), CyRPA (green triangles) or Ripr (blue circles). Intra-cohort variability in GIA of IgG from six individual rats immunized with 2 μg antigen - RH5.1 (B) CyRPA (C) and Ripr (D) antigens were formulated with DPX. Error bars represent SEM of triplicate wells.
Figure 3
Figure 3
Functional activity is not correlated with immunogenicity in RCR multiple antigen combination immunisations. (A) Antibody reactivity in terminal bleed sera from individual rats was tested in ELISA against individual antigen components and the results are shown as OD1 titers calculated from 4PL curve fits based on the dilution curves of individual samples. Circles represent the OD1 titer from an individual rat serum titration and bars show the median of the six serum OD1 values. (B) GIA in IgG purified from individual rat sera. Bars show mean % GIA in each cohort of 6 rats at an IgG concentration of 2 mg/mL. Error bars show SEM.
Figure 4
Figure 4
Antibody titers reveal differences in responses to individual and RCR-complexed antigens. (A) Antibody responses against the RCR complex in sera from RH5.1, CyRPA, Ripr, RH5.1+Ripr, RH5.1+CyRPA, Ripr+CyRPA, RCR (equimolar ratio) and RCR (mass ratio) immunized rats. The immunogens are shown on the x-axis, and OD1 titers of each cohort against the RCR complex are shown on the y-axis. Only significant associations are shown. **** p<0.0001, ** p<0.005, * p<0.05. (B) Antibody titers in rats immunized with single antigens RH5.1, CyRPA and Ripr are shown against the RCR complex (in black circles) and the immunogen (in grey circles). Circles show individual serum OD1 values and group medians shown by lines. Significant differences between cohort responses to the single antigen and RCR calculated using Mann-Whitney U test with Prism 9 software to calculate p values. ns, not significant; ** p = 0.01. (C) Immunization with RCR antigens in a 1:1:1 mass ratio induced significantly higher anti-CyRPA titers than the equimolar ratio immunization. RCR equimolar responses are shown in grey circles and RCR mass responses in black circles. Circles show individual serum OD1 values and group means are shown by lines. Data was tested for normality and significant differences between groups was tested by unpaired t-test ** p<0.005. (D) Pairwise comparison of antibody titers against RH5, CyRPA, Ripr and RCR in cohorts immunized with RCR in an equimolar ratio. *** p<0.001 (E) Pairwise comparison of antibody titers against RH5.1, CyRPA, Ripr and RCR in cohorts immunized with RCR in a mass ratio. For (A, D, E): Circles show individual serum OD1 values and group means are shown by lines. Data was tested for normality and testing for significant differences between groups was conducted by one way ANOVA with Tukey’s test for multiple comparisons using Prism software to calculate p values.
Figure 5
Figure 5
Depletion of specific antibodies to determine effect on inhibition of P. falciparum growth. IgG pools were made from the RCR equimolar antigen cohort IgG (A) and RCR mass cohort IgG (B) and incubated with antigens to deplete specific antibodies. Black bars - before depletion with antigen and others after depletion with specific antigen as indicated. Open bars – single antigen treatment (RCR complex is counted here as a single antigen). Hatched bars - antigen combinations. (C) Data extracted from panels (A, B) showing that GIA of RCR equimolar IgG and RCR mass IgG following CyRPA-depletion are not significantly different from each other. (A–C) Histograms show mean ± SD of two independent experiments (each performed in triplicate wells). ns, not significant.
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