Establishing RTS,S/AS01 as a benchmark for comparison to next-generation malaria vaccines in a mouse model

Abstract

New strategies are needed to reduce the incidence of malaria, and promising approaches include vaccines targeting the circumsporozoite protein (CSP). To improve upon the malaria vaccine, RTS,S/AS01, it is essential to standardize preclinical assays to measure the potency of next-generation vaccines against this benchmark. We focus on RTS,S/AS01-induced antibody responses and functional activity in conjunction with robust statistical analyses. Transgenic Plasmodium berghei sporozoites containing full-length P. falciparum CSP (tgPb-PfCSP) allow two assessments of efficacy: quantitative reduction in liver infection following intravenous challenge, and sterile protection from mosquito bite challenge. Two or three doses of RTS,S/AS01 were given intramuscularly at 3-week intervals, with challenge 2-weeks after the last vaccination. Minimal inter- and intra-assay variability indicates the reproducibility of the methods. Importantly, the range of this model is suitable for screening more potent vaccines. Levels of induced anti-CSP antibody 2A10 equivalency were also associated with activity: 105 μg/mL (95% CI: 68.8, 141) reduced liver infection by 50%, whereas 285 μg/mL (95% CI: 166, 404) is required for 50% sterile protection from mosquito bite challenge. Additionally, the liver burden model was able to differentiate between protected and non-protected human plasma samples from a controlled human malaria infection study, supporting these models' relevance and predictive capability. Comparison in animal models of CSP-based vaccine candidates to RTS,S/AS01 is now possible under well controlled conditions. Assessment of the quality of induced antibodies, likely a determinant of durability of protection in humans, should be possible using these methods.

Fig. 1. Liver infection measured as Log10 flux among RTS,S dose response (0.05, 0.2, 1, 5, or 10 μg RTS,S) and naïve, infected controls (0 μg RTS,S injected with PBS), by number of doses.

Green box plots represent experiments with 2x vaccinations and blue box plots represent experiments with 3x vaccinations. Points are individual animals with shapes used to indicate experiment ID. The mid-line of the boxes denotes the median and the ends of the box denote the 25^th and 75^th percentiles. The whiskers denote the most extreme data points that were no more than 1.5 times the interquartile range (i.e., height of the box).

Fig. 2. Distribution of RTS,S-induced sera antibody concentrations (2A10 equivalence, μg/mL) by total doses administered and dose levels.

Points indicate measurements from individual animals. The mid-line of the boxes denotes the median and the ends of the box denote the 25^th and 75^th percentiles. The whiskers denote the most extreme data points that were no more than 1.5 times the interquartile range (i.e., height of the box).

Fig. 3. Predicted liver infection measured as log₁₀ flux and 95% CI (shaded area) from the 4PL model by total administrations (2 and 3, color), overlaid on observed log₁₀ flux values (x’s).

Each EC₅₀ and its 95% CI are shown with a white point and horizontal line on the 4PL curve. The large points on the curve depict the mean sera 2A10 equivalence (μg/mL) by dose-level (shape) with corresponding predicted flux. The EC₅₀ values for the 2x and 3x dose groups were significantly different (p < 0.001, two-sided t-test). There was a slight vertical difference in EC₅₀ log₁₀ flux values by dose groups due to differences in experiment-matched infected controls that determine the upper asymptote. Log₁₀ flux values below the mean assay background measurements were truncated at 5.37 (dotted line).

Fig. 4. Minimum detectable flux fold-changes with 80% power, using a t-test to compare log₁₀ flux between RTS,S/AS01 treatment groups, with 5 to 15 mice.

Colors denote different input SDs for log₁₀ flux.

Fig. 5. Distributions of antibody concentration measured as sera 2A10 equivalence (μg/mL), measured 2 days prior to challenge, by infection status, by two and three administration experiments.

The mid-line of the boxes denotes the median and the ends of the box denote the 25^th and 75^th percentiles. The whiskers denote the most extreme data points that were no more than 1.5 times the interquartile range (i.e., height of the box).

Fig. 6. Predicted probability of infection and 95% CI (shaded area) for varying range of sera antibody concentration measured as 2A10 equivalence (μg/mL) based on the logistic model, overlaid on observed infection status (x’s) for the three administration experiments (black curve with gray shaded area).

The EC₅₀ and its 95% CI are shown with a white point and horizontal line on the 2PL curve. The blue points on the curve depict the mean sera 2A10 equivalence (μg/mL) by dose-level (shape) with corresponding predicted infection probability. The symbols for the 5 and 10 μg dosages are overlapping on the curve.

Fig. 7. Statistical power from Barnard’s test comparing superiority of test vaccine with protection levels ranging from 65% to 100% to three administrations of 5 μg RTS,S protection of 60% by sample size (N = 7–15).

For exact tests comparing binary outcomes, power may decrease at increments of sample size increases.

Fig. 8. Percent inhibition of liver infection conferred in mice receiving via passive transfer 500 μL of individual day of challenge human plasma samples from protected (n = 24) and non-protected (n = 12) subjects in the AduFx and 2PedFx cohorts receiving the adult dose or a double pediatric dose, thus the same amount of antigen and adjuvant, on a 0, 1, 7 month schedule of the MAL092 CHMI study.

Plasma from protected subjects conferred significantly greater inhibition of liver infection than plasma from non-protected subjects. (Mann-Whitney U test).

Fig. 9. Total flux (photons/second) plotted against sera 2A10 equivalence (μg/mL) and fit to a 4PL regression curve.

The 4PL fit is shown in the solid black line, with 95% CIs depicted by the dotted lines. The modeled three-fold improvement is shown in the dotted blue line, also with 95% CIs.