Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

Abstract

Background: Development of an effective vaccine against the pathogenic blood-stage infection of human malaria has proved challenging, and no candidate vaccine has affected blood-stage parasitemia following controlled human malaria infection (CHMI) with blood-stage Plasmodium falciparum.

Methods: We undertook a phase I/IIa clinical trial in healthy adults in the United Kingdom of the RH5.1 recombinant protein vaccine, targeting the P. falciparum reticulocyte-binding protein homolog 5 (RH5), formulated in AS01_B adjuvant. We assessed safety, immunogenicity, and efficacy against blood-stage CHMI. Trial registered at ClinicalTrials.gov, NCT02927145.

Findings: The RH5.1/AS01_B formulation was administered using a range of RH5.1 protein vaccine doses (2, 10, and 50 μg) and was found to be safe and well tolerated. A regimen using a delayed and fractional third dose, in contrast to three doses given at monthly intervals, led to significantly improved antibody response longevity over ∼2 years of follow-up. Following primary and secondary CHMI of vaccinees with blood-stage P. falciparum, a significant reduction in parasite growth rate was observed, defining a milestone for the blood-stage malaria vaccine field. We show that growth inhibition activity measured in vitro using purified immunoglobulin G (IgG) antibody strongly correlates with in vivo reduction of the parasite growth rate and also identify other antibody feature sets by systems serology, including the plasma anti-RH5 IgA1 response, that are associated with challenge outcome.

Conclusions: Our data provide a new framework to guide rational design and delivery of next-generation vaccines to protect against malaria disease.

Funding: This study was supported by USAID, UK MRC, Wellcome Trust, NIAID, and the NIHR Oxford-BRC.

Keywords: CHMI; Plasmodium falciparum; RH5; blood-stage; clinical trial; malaria; systems serology; vaccine.

Graphical abstract

Figure 1

Antibody immunogenicity of RH5.1/AS01_B (A) Timing of immunizations and follow-up in groups 1–4. All antigen doses were formulated in 0.5 mL AS01_B. (B and C) Median and individual anti-RH5_FL serum total IgG responses 14 days after two vaccinations (Vacs; day 42, B) and after three Vacs (day 70 or day 196, C). Both datasets were analyzed separately by Kruskal-Wallis test with Dunn’s multiple comparisons test; ∗∗p < 0.01. Historical data for the VV-RH5 vaccine were not included in the analysis and are shown for comparison only. (D) In vitro GIA of purified IgG assessed at 10 mg/mL against 3D7 clone P. falciparum parasites. Individual data and medians are shown for each group at the stated time-point; pooled sera were used for each group at baseline (day 0). Historical data for VV-RH5 were included as before. (E) Dilution series of purified IgG for all group 1–4 samples starting from 10 mg/mL. (F) Relationship between GIA data from the dilution series shown in (E) and concentration of anti-RH5_FL purified IgG used in the assay as measured by ELISA. A non-linear regression curve is shown for all samples combined (solid line, r² = 0.96, n = 279). The EC₅₀ (concentration of anti-RH5_FL polyclonal IgG that gives 50% GIA, dashed line) was calculated.

Figure 2

Assessment of the DFx regimen (A) Median anti-RH5_FL serum total IgG responses for groups 1–4 over time. Individual responses are shown in Figure S4B. (B) Median and individual anti-RH5_FL serum total IgG responses at the time of the late bleed. Statistical analysis was performed using a Mann-Whitney test, ∗∗∗∗p < 0.0001. (C) Avidity of serum total IgG responses 14 days after three immunizations (day 70 or day 196) was assessed by NaSCN displacement RH5_FL ELISA and is reported as the molar concentration of NaSCN required to reduce the starting optical density (OD) in the ELISA by 50% (IC₅₀). Individual responses over time are shown in Figure S4C. Historical data for the VV-RH5 vaccine are shown for comparison. Kruskal-Wallis test with Dunn’s multiple comparison test, ∗∗∗p < 0.001 for group 3 versus groups 1, 2, and 4. (D) Estimated proportion of antibodies generated from LLPCs; for group 3, it was possible to provide separate estimates of the proportion following the first and second doses (purple) and following the third dose (pink). The long-lived response following the third dose in group 3 is significantly greater than in other groups; ∗∗∗∗p < 1 × 10⁻⁶ in all cases by one-sided t test. (E) Anti-RH5_FL antibody levels at peak and 1 and 4 years following the third vaccine dose. Peak antibody levels are based on the maximum measured values (day 70 or day 84 for groups 1, 2, and 4 and on day 196 or day 210 for group 3). Antibody levels at 1 and 4 years are based on model estimates and are presented with 95% CI.

Figure 3

Results of primary and secondary blood-stage CHMI (A) Timing of immunizations, CHMIs, and follow-up in groups 5–9. All antigen doses were formulated in 0.5 mL AS01_B. (B) qPCR data for the VAC063A phase IIa study; group 5 (n = 14) and group 6 (n = 15). Median parasitemia is shown over time for each group. The lower limit of quantification is indicated by the dotted line at 20 p/mL; values below this level are plotted for information only. Time = days after blood-stage CHMI. (C) Primary efficacy endpoint analysis of PMR, showing each individual plus the mean. Both datasets are normally distributed (D’Agostino-Pearson test); ∗p = 0.031 using two-tailed t test with Welch’s correction for non-equal variances (F test; p = 0.008). (D) Kaplan-Meier plot of time to diagnosis in days for the VAC063A study. Median time to patent parasitemia was 9.5 days for control individuals and 10.5 days for vaccinees. Secondary pre-specified efficacy analysis in the protocol compared time to diagnosis between the groups; p = 0.01, Mann-Whitney test. (E) Post hoc analysis combining the VAC063A dataset with the AMA1/AS01_B trial (VAC054) data. Mean PMR ± 95% CI is shown for control individuals (n = 15 from VAC063A and n = 15 from VAC054), AMA1 vaccinees (n = 12 from VAC054), and RH5 vaccinees (n = 14 from VAC063A). ∗p < 0.05 for RH5 versus AMA1 and control individuals, using one-way ANOVA with Bonferroni correction for multiple comparisons. (F) qPCR data for the VAC063B phase IIa study shown as in (B); group 7 (n = 9), group 8 (n = 8), and group 9 (n = 6). (G) Secondary efficacy endpoint analysis of PMR, showing each individual, plus the median. ∗p = 0.022; Kruskal-Wallis test with Dunn’s multiple comparison test.

Figure 4

Antibody responses after CHMI and the fourth booster immunization (A) Median and individual anti-RH5_FL serum total IgG responses shown for groups 5 and 7 over time (note that a subset of group 5 vaccinees became group 7; Figure S6). Gray shading indicates periods of CHMI, and arrows indicate Vacs. The legend indicates the sequence of 10-μg RH5.1/AS01_B Vacs as well as the first and second CHMIs, as relevant to each group. (B–E) Individual and median responses are shown for the indicated groups and time points. dC, day of challenge. ∗p < 0.05, ∗∗p < 0.01; Wilcoxon matched-pairs signed-rank test in (C) and (D). ∗p < 0.05, Mann-Whitney test in (E). (F) Avidity of serum total IgG responses in groups 5 and 7 at the indicated time points was assessed by NaSCN displacement RH5_FL ELISA. ∗p < 0.05, ∗∗p < 0.01; Friedman test for paired samples with Dunn’s multiple comparisons test.

Figure 5

Analysis of in vitro GIA versus IVGI (A) In vitro GIA of purified IgG assessed at 10 mg/mL against 3D7 clone P. falciparum parasites. Individual data and medians are shown for each group (Figure 3A) at the stated time points. (B) Relationship between GIA and concentration of anti-RH5_FL purified IgG used in the assay, as measured by ELISA. A non-linear regression curve is shown for all samples combined (solid line, r² = 0.97, n = 180). The EC₅₀ (dashed line) was calculated. (C) In vitro GIA as in (A), using purified IgG assessed at 2.5 mg/mL. Historical data from the AMA1/AS01_B (VAC054) trial are included. The median percent IVGI observed in each group following blood-stage CHMI is indicated below the graph. The red dashed line at 60% GIA indicates the threshold level required for protection in Aotus monkeys.^, ∗p < 0.05, ∗∗p < 0.01; Kruskal-Wallis test with Dunn’s multiple comparisons test. (D) Correlation of % IVGI observed in each individual following blood-stage CHMI versus their individual in vitro GIA measured at dC−1 using 2.5 mg/mL purified IgG. Spearman’s rank correlation coefficient and p value are shown; n = 35. Colored symbols are the same as those used in (C) for AMA1, group 5 (G5), and G7.

Figure 6

Systems serology analysis of CHMI outcome in RH5 vaccinees (A) Correlation heatmap showing the Spearman rank correlation coefficients (r_S) between Fc functions and titers as well as the rank correlation of the features to the CHMI readouts of DOD and IVGI. ∗q < 0.1, ∗∗q < 0.01 (Benjamini-Hochberg procedure for multiple testing correction; the correction was done within the groups of comparison; 54 comparisons for Fc functions/titer, 40 for features/DOD, and 40 for features/IVGI). (B) Scatterplot to show the relationship between ADNP score and IVGI. Each color corresponds to one volunteer, and the shape indicates the group: G5 (n = 13) or G7 (n = 7). (C) Prediction for the random forest regression model plotted against the data for DOD. The model was obtained using leave-one-volunteer-out cross-validation. The Pearson correlation coefficient (r_P) and p value are shown. (D) The antibody features in the predictive model for DOD are ranked according to how often they were chosen for 100 repetitions of recursive feature elimination (RFE) in the leave-one-volunteer-out cross-validation. (E) Graphs showing DOD versus systems serology assay data for the seven antibody features that were chosen in more than 10% of the elimination procedures (D). (F) The co-correlates network shows the pairwise correlation of features. The nodes correspond to features and the edges to Spearman rank correlations between the features. Only significant correlations (Benjamini-Hochberg q < 0.05) between features that are selected in more than 10% of the RFE procedures and all other features are shown. G2FB Fc glycan, fucosylated 2-galactose with bisecting N-acetylglucosamine.