Enhancing protective immunity to malaria with a highly immunogenic virus-like particle vaccine

Abstract

The leading malaria vaccine in development is the circumsporozoite protein (CSP)-based particle vaccine, RTS,S, which targets the pre-erythrocytic stage of Plasmodium falciparum infection. It induces modest levels of protective efficacy, thought to be mediated primarily by CSP-specific antibodies. We aimed to enhance vaccine efficacy by generating a more immunogenic CSP-based particle vaccine and therefore developed a next-generation RTS,S-like vaccine, called R21. The major improvement is that in contrast to RTS,S, R21 particles are formed from a single CSP-hepatitis B surface antigen (HBsAg) fusion protein, and this leads to a vaccine composed of a much higher proportion of CSP than in RTS,S. We demonstrate that in BALB/c mice R21 is immunogenic at very low doses and when administered with the adjuvants Abisco-100 and Matrix-M it elicits sterile protection against transgenic sporozoite challenge. Concurrent induction of potent cellular and humoral immune responses was also achieved by combining R21 with TRAP-based viral vectors and protective efficacy was significantly enhanced. In addition, in contrast to RTS,S, only a minimal antibody response to the HBsAg carrier was induced. These studies identify an anti-sporozoite vaccine component that may improve upon the current leading malaria vaccine RTS,S. R21 is now under evaluation in Phase 1/2a clinical trials.

Figure 1. R21 design and characterization.

(a) Schematic representation of R21 particle design in comparison to RTS,S. (b) Transmission electron micrograph of R21 negatively stained with 2% uranyl acetate. (c) Assessment of NANP and HBsAg accessibility on the surface of the R21 particle using and CSP antigen ELISA and the Monolisa HBsAg ELISA kit. (d) Analysis of the R21 vaccine by analytical size exclusion chromatography and (e) reducing SDS-PAGE with silver staining and western blot analysis using anti-CSP antibody.

Figure 2. R21 Immunogenicity.

(a) BALB/c mice were immunised i.m. with 0.5 μg R21 formulated with adjuvant as detailed in the graphs. Three immunisations were given 3 weeks apart and NANP-specific IgG was assayed by ELISA 3 weeks after each immunisation. The dotted line indicates the limit of detection. (b) Spleens were taken 3 weeks after final vaccination and antigen-specific IFNγ secreting T cells assayed in an ex-vivo IFNγ ELISpot using the a pool of overlapping CSP peptides. The dotted line indicates the background response. (c) BALB/c mice were immunised i.m. with either 0.5 μg R21 formulated with Alhydrogel or Abisco-100, or 0.5 μg HBsAg+ Alhydrogel. Three immunisations were given 3 weeks apart and HBsAg-specific IgG and NANP-specific IgG were assayed by ELISA 3 weeks after each immunisation. The dotted line indicates the limit of detection. BALB/c mice were immunised i.m. with 0.5 μg R21 formulated with Abisco-100 with different immunisation regimens as detailed in Table 1. (d) NANP-specific IgG was assayed by ELISA 3 weeks after the final vaccination. The dotted line indicates the limit of detection. (e) Spleens were taken three weeks after final vaccination and antigen-specific IFNγ secreting T cells assayed in an ex-vivo IFNγ ELISpot using a pool of overlapping CSP peptides. The dotted line indicates the background response. Group median responses are shown with interquartile range and compared by Kruskal-Wallis with Dunn’s multiple comparison test. R = R21 and H = HBsAg.

Figure 3. Viral vector and adjuvant immunogenicity.

(a) BALB/c mice were immunised i.m. with ChAd63-MVA ME-TRAP 8 week prime-boost regimen, either alone or combined with adjuvant. Blood was taken 2 weeks after the prime vaccination and CD8+ cytokine secreting T cell frequencies were assessed by ICS. Cells were stimulated for six hours with the immunodominant H-2K^d CD8+ epitope Pb9 (SYIPSAEKI) present in the ME string of the viral vector insert and three different cytokines were assessed (IFNγ, TNF and IL2). Results are expressed as the percentage of CD8+ T cells expressing each cytokine. (b) Spleens were taken 3 weeks after the final vaccination and Pb9-specific IFNγ secreting T cells assayed in an ex-vivo IFNγ ELISpot. The dotted line indicates background response. (c) Blood was taken and assayed by TRAP ELISA 3 weeks and 8 weeks after the prime and 3 weeks after the boost. The dotted line indicates the limit of detection. Group medians with interquartile range are shown and compared by Kruskal-Wallis with Dunn’s multiple comparison test *p < 0.05. Ad TR = ChAd63 ME-TRAP, Ad-M TR = ChAd63-MVA ME-TRAP.

Figure 4. R21 and viral vector immunogenicity in combination regimen.

BALB/c mice were immunised i.m. with ChAd63-MVA ME-TRAP (Ad-M TR) or 0.5 μg R21+ adjuvant (Abisco-100 or MF59) or the particle and viral vectors combined together (combination). Blood was taken 3 weeks and 8 weeks after the first immunisation and 3 weeks after the second immunisation. (a) NANP-specific IgG and (b) TRAP-specific IgG were assayed by ELISA and the dotted line indicates the limit of detection. Spleens were taken three weeks after final vaccination and antigen-specific, IFNγ secreting T cells assayed in an ex-vivo IFNγ ELISpot using (c) a pool of overlapping CSP peptides or (e) Pb9 peptide stimulation. The dotted line indicates background response. (d) Blood was taken 2 weeks after the prime and 1 week after boost vaccination and frequencies of Pb9-specific CD8+ T cell secreting IFNγ, TNF and IL2 were assayed by ICS. Results are displayed as the percentage of CD8+ T cells expressing each cytokine and the dotted line indicates background response. Group medians with interquartile range are shown and groups compared by Mann-Whitney test *p < 0.05, **p < 0.01.

Figure 5. Protective efficacy of R21.

BALB/c mice were immunised i.m. with 0.5 μg R21+ adjuvant, twice 8 weeks apart n = 8/group (except for R21+ AddaVax n = 7). Mice were challenged 3 weeks after the final vaccination by i.v. injection of 1000 sporozoites (Tg Pb+ PfCSP) along with 8 naïve mice. Two groups of adjuvant control mice (n = 5/group) were also challenged 3 weeks after receiving two shots of adjuvant (Abisco-100 or AddaVax) i.m. 8 weeks apart. Blood stage parasitaemia was monitored from day 5 after challenge by thin film blood smear, and time to 1% parasitaemia was calculated using linear regression. The results are presented in the Kaplan-Meier survival graphs and survival curves were compared by Log-rank (Mantel-Cox) Test. (a) Protective efficacy of R21 with ISCOM adjuvants and R21 with squalene based oil-in-water (o/w) emulsions. (b) NANP specific IgG assayed by ELISA 3 weeks after each immunisation (Day 21 and Day 77). Comparison between R21+ Matrix-M and R21+ MF59 (n = 8/group) (c) protective efficacy and (d) NANP-specific IgG titres. For all ELISAs the dotted line indicates the limit of detection, the group mean responses are shown and groups are compared by One-way ANOVA with Bonferroni’s multiple comparison test. (e) Blood was taken three weeks after the final vaccination to assess CSP-specific CD4+ and CD8+ cytokine secreting T cell frequencies by ICS (IFNγ, TNF and IL2). Results are expressed as the percentage of CD4+ or CD8+ T cells expressing each cytokine. Group medians with interquartile range are shown and the groups compared by Mann Whitney test **p < 0.01. The dotted line indicates background response.

Figure 6. Immunogenicity and protective efficacy in the combination regimen.

BALB/c mice were immunised i.m. with ChAd63-MVA PbTRAP 8 week prime-boost regimen, either alone (Ad-M PbTR) combined with MF59 (Ad-M PbTR+ MF59) or combined with 0.5 μg R21+ MF59 (Ad-M PbTR+ R21+ MF59). (a) Blood was taken one week after the final vaccination to assess PbTRAP-specific CD4+ and CD8+ cytokine secreting T cell frequencies by ICS (IFNγ, TNF and IL2). Results are expressed as the percentage of CD8+ or CD4+ T cells expressing the cytokine with box plots indicating the median and whiskers showing the minimum and maximum response. Three weeks after the final vaccination (b) PbTRAP-specific IgG and (c) NANP-specific IgG were assayed by ELISA. Group means are shown and the dotted line indicates limit of detection. Groups compared by One-way ANOVA with Bonferroni’s multiple comparison test. (d) Mice were challenged 3 weeks after the final vaccination by i.v. injection of 1000 transgenic sporozoites (Tg Pb+ PfCSP) along with 8 naïve mice. Blood stage parasitemia was monitored from day 5 after challenge by thin film blood smear, and time to 1% parasitemia was calculated using linear regression. The results are presented in the Kaplan-Meier survival graphs and survival curves were compared by Log-rank (Mantel-Cox) Test.