Ultra-low dose immunization and multi-component vaccination strategies enhance protection against malaria in mice

Abstract

An effective vaccine would be a valuable tool for malaria control and elimination; however, the leading malaria vaccine in development, RTS,S/AS01, provided only partial protection in a Phase 3 trial. R21 is a next-generation RTS,S-like vaccine. We have previously shown in mice that R21 administered in Matrix-M is highly immunogenic, able to elicit complete protection against sporozoite challenge, and can be successfully administered with TRAP based viral-vectors resulting in enhanced protection. In this study, we developed a novel, GMP-compatible purification process for R21, and evaluated the immunogenicity and protective efficacy of ultra-low doses of both R21 and RTS,S when formulated in AS01. We demonstrated that both vaccines are highly immunogenic and also elicit comparable high levels of protection against transgenic parasites in BALB/c mice. By lowering the vaccine dose there was a trend for increased immunogenicity and sterile protection, with the highest dose vaccine groups achieving the lowest efficacy (50% sterile protection). We also evaluated the ability to combine RTS,S/AS01 with TRAP based viral-vectors and observed concurrent induction of immune responses to both antigens with minimal interference when mixing the vaccines prior to administration. These studies suggest that R21 or RTS,S could be combined with viral-vectors for a multi-component vaccination approach and indicate that low dose vaccination should be fully explored in humans to maximize potential efficacy.

Figure 1

Minimal immunological interference when RTS,S/AS01 and ME.TRAP based viral vectors are co-administered at different sites or mixed together. BALB/c mice were immunized with RTS,S/AS01 and/or ChAd63 ME.TRAP—MVA ME.TRAP either alone, staggered or co-administered in different limbs, as indicated in Table 1. Two weeks after the final immunization (A) NANP-specific IgG responses and (B) TRAP-specific IgG responses were measured by ELISA, lines indicate the medians. T cell responses were measured in the blood ~ 2 weeks after the final immunization by ICS for (C) CSP-specific cytokine secreting CD4 + T cells or (D) Pb9-specific cytokine secreting CD8 + T cells, bars indicate the medians. BALB/c mice were immunized with RTS,S/AS01 and/or ChAd63 ME.TRAP—MVA ME.TRAP either alone, co-administered or mixed before immunization, as indicated in Table 2 using two doses of RTS,S/AS01 (5 µg or 1.6 µg) . Two weeks after the final immunization (E) NANP-specific IgG responses and (F) TRAP-specific IgG responses were measured by ELISA, lines indicate the medians. T cell responses were measured in the blood ~ 2 weeks after the final immunization by ICS for (G) CSP-specific cytokine secreting CD4 + T cells or (H) Pb9-specific cytokine secreting CD8 + T cells. Open bars and circles represent the 5 µg dose groups and grey bars and circles represent the 1.6 µg dose groups. Bars indicate the medians. Dotted lines indicate average background response. R = RTS,S/AS01, A = ChAd63 ME.TRAP, M = MVA ME.TRAP. All groups compared by Kruskal–Wallis with Dunn’s multiple comparison post-test comparing selected groups (*P < 0.05, **p < 0.01 and ***p < 0.0001).

Figure 2

Immunogenicity and efficacy of RTS,S/AS01 and PbTRAP based viral-vector combinations. C57BL/6 mice were immunized with RTS,S/AS01 and/or ChAd63 PbTRAP—MVA PbTRAP either alone, co-administered or mixed as indicated in Table 3. Two weeks after the final immunization (A) NANP-specific IgG responses and (B) TRAP-specific IgG responses were measured by ELISA. Lines indicate the mean and groups compared by One-way ANOVA with Dunnett’s multiple comparison post-test comparing all pairs of groups. T cell responses were measured in the blood ~ 2 weeks after the final immunization by ICS for (C) CSP-specific cytokine secreting CD4 + T cells or (D) TRAP-specific cytokine secreting CD8 + T cells. Lines indicate the median. Dotted lines indicate average background response. Groups compared by Kruskal–Wallis test with Dunn’s multiple comparison post-test comparing all pairs of groups. Two weeks after the final immunization all immunized mice and 10 naïve mice were challenged with 1000 transgenic P. berghei parasites expressing P. falciparum CSP. Blood-stage parasitemia was monitored from day 5 after challenge by thin-film blood smear, and (E) time to 1% parasitemia was calculated using linear regression. Lines indicate the median and groups compared by Kruskal–Wallis test with Dunn’s multiple comparison post-test comparing all pairs of groups. The results are presented in Kaplan–Meier survival graphs and survival curves compared by Log-rank (Mantel–Cox) Test for (F) individual vaccinations, (G) combination and mixture vaccination, and (H) comparison of individual vaccination to all combinations. R = RTS,S/AS01, A = ChAd63 ME.TRAP, M = MVA ME.TRAP. *P < 0.05, **p < 0.01 and ***p < 0.0001.

Figure 3

C-tagged R21 design and characterization. (A) The composition of fusion proteins making up the monomeric subunits of the particle vaccine products shown as bars and diagrammatic representation of the particle structure (R21c = C-tagged). (B) C-tagged R21 fusion proteins can be visualized by silver staining (lanes I and II) and anti-HBsAg western blot (lanes III and IV) after purification by C-Tag affinity chromatography (lanes I and III) followed by size exclusion chromatography (lanes II and IV). Transmission electron micrographs of (C) C-tagged R21 and (D) HBsAg particles after staining with 2% uranyl acetate. Scale bar = 50 µm. (E) NANP-specific IgG responses measured by ELISA in BALB/c mice 3 weeks after immunization with one dose of 0.5 µg C-tagged R21 (R21c) or untagged R21. (F) C-tag-specific and NANP-specific IgG responses measured by ELISA in BALB/c mice (n = 6 per group) immunized with 5 µg C-tagged R21 formulated either in Matrix-M or AS01 or without adjuvant (3 immunizations, 2 weeks apart). Individual data points shown with the group medians. Groups were compared by Kruskal–Wallis test with Dunn’s multiple comparison post-test. *P < 0.05, **p < 0.01 and ***p < 0.0001 Dotted line shows assay detection limit.

Figure 4

Immunogenicity and protective efficacy of low dose R21/AS01 and RTS,S/AS01. BALB/c mice (n = 8 per group, except 0.5 µg R21/AS01: n = 7) received 2 immunizations, 2 weeks apart of either R21/AS01 or RTS,S/AS01 at range of doses (0.5 µg, 1.6 µg or 5 µg). Two weeks after the final immunization (A) NANP-specific IgG was measured by ELISA, and (B) CSP-specific, cytokine secreting T cells (IFNγ + , TNF + or IL2 +) were measured by ICS and background responses subtracted. Lines indicate the mean and groups compared by One-way ANOVA with Turkey’s multiple comparison post-test comparing all pairs of groups. Dotted lines indicate average background response. Two weeks after the final immunization all immunized mice and 8 naïve mice were challenged with 1000 transgenic P. berghei parasites expressing P. falciparum CSP. Blood-stage parasitemia was monitored from day 5 after challenge by thin-film blood smear, and time to 1% parasitemia calculated using linear regression. Results are presented in Kaplan–Meier survival graphs and survival curves compared by Log-rank (Mantel–Cox) Test. (C) Comparison of protective efficacy between all mice vaccinated with either RTS,S/AS01 (n = 24) or R21/AS01 (n = 23). (D) Protective efficacy compared between RTS,S/AS01 and R21/AS01 at the different doses. Six months after the first challenge the surviving mice were re-challenged as before along with 6 naïve mice. (5 µg RTS,S/AS01: n = 4, 1.6 µg RTS,S/AS01: n = 7, 0.5 µg RTS,S/AS01: n = 6, 5 µg R21/AS01: n = 4, 1.6 µg R21/AS01: n = 6, 0.5 µg R21/AS01: n = 6). (E) Comparison of protective efficacy after re-challenge between all mice vaccinated with either RTS,S/AS01 (n = 17) or R21/AS01 (n = 16). (F) Protective efficacy after re-challenge compared between RTS,S/AS01 and R21/AS01 at the different doses. (*P < 0.05, **p < 0.01 and ***p < 0.0001).

Figure 5

Immunogenicity and protective efficacy of ultra-low dose R21/AS01 and RTS,S/AS01. BALB/c mice (n = 10 per group) received 3 immunizations, 2 weeks apart of either R21/AS01 or RTS,S/AS01 at range of doses (0.05 µg, 0.16 µg or 0.5 µg). (A) Two weeks after the final immunization NANP-specific IgG was measured by ELISA. Lines indicate the medians and groups compared by Kruskal-Wallis test with Dunn’s multiple comparison post-test comparing all pairs of groups. At this time all immunized mice and 9 naïve mice were challenged with 1000 transgenic P. berghei parasites expressing P. falciparum CSP. Blood-stage parasitemia was monitored from day 5 after challenge by thin-film blood smear, and time to 1% parasitemia calculated using linear regression. The results are presented in Kaplan–Meier survival graphs and survival curves compared by Log-rank (Mantel–Cox) Test. (B) Comparison of protective efficacy between all mice vaccinated with either RTS,S/AS01 (n = 30) or R21/AS01 (n = 30). (C) Protective efficacy compared between RTS,S/AS01 and R21/AS01 at the different doses. Four months after the first challenge the surviving mice were re-challenged as before along with 8 naïve mice. (0.5 µg RTS,S/AS01: n = 10, 0.16 µg RTS,S/AS01: n = 8, 0.05 µg RTS,S/AS01: n = 7, 0.5 µg R21/AS01: n = 8, 0.16 µg R21/AS01: n = 10, 0.05 µg R21/AS01: n = 10). (D) Comparison of protective efficacy after re-challenge between all mice vaccinated with either RTS,S/AS01 (n = 25) or R21/AS01 (n = 28). (E) Protective efficacy after re-challenge compared between RTS,S/AS01 and R21/AS01 at the different doses. (F) Four months after the second challenge the surviving mice were re-challenged a second time as before along with 6 naïve mice RTS,S/AS01: n = 18 or R21/AS01: n = 21.

Figure 6

Durability of R21/AS01 and RTS,S/AS01 induced antibodies. BALB/c mice received 2 immunizations, 8 weeks apart of 1.6 µg of either R21/AS01 or RTS,S/AS01. (A) NANP-specific IgG was measured by ELISA every month after vaccination for one year. Bars represent mean with individual data points shown. One-way ANOVA with Dunnetts multiple comparison post-test comparing the titres at each time point to the titre at 1 month was used to evaluate reduction over time, and One-way ANOVA with Bonferroni multiple comparison post-test was used to compare R21 to RTS at each time point. (B) One year after the final immunization all surviving mice (R21: n = 8, RTS,S: n = 10) and 8 naïve mice were challenged with transgenic P. berghei parasites expressing P. falciparum CSP. Blood-stage parasitemia was monitored from day 5 after challenge by thin-film blood smear and time to 1% parasitemia calculated using linear regression. The results are presented in Kaplan–Meier survival graph and survival curves compared by Log-rank (Mantel–Cox) Test.