Participants and Samples

Figure 2. Figure 2. Data Generation and Sample and Data Filtration for the End Point of Primary Clinical Malaria among Children 5 to 17 Months of Age. Vaccine recipients were considered to be out of interval for the dose regimen if they did not receive booster vaccinations according to the schedule specified by the trial protocol. The sample cards used were Whatman FTA cards. PCR denotes polymerase chain reaction.

Figure 2, and Fig. S2 in the Supplementary Appendix, summarize sample size and follow-up information for the per-protocol group of participants who were 5 to 17 months of age; in this cohort, P. falciparum genetic data were analyzed from 1181 RTS,S/AS01-vaccine recipients and 909 control-vaccine recipients in whom the primary clinical malaria end point was confirmed, as well as from 284 RTS,S/AS01-vaccine recipients and 208 control-vaccine recipients in whom the parasite-positivity end point was confirmed. Fig. S3 and S4 in the Supplementary Appendix show this information for the per-protocol group of infants who were 6 to 12 weeks of age, and Fig. S5 through S8 in the Supplementary Appendix provide information on the samples studied for both end points and age categories for the NANP–NVDP repeat amplicon.

Complexity of Infection

Figure 3. Figure 3. Complexity of Infection and Frequencies of 3D7-Matched Malaria among Children 5 to 17 Months of Age with the Primary Clinical Malaria End Point. The denominator for the frequencies in Panels B through D is the number of samples representing the primary clinical malaria end point that had sequence data available. Ag denotes Agogo, Ba Bagamoyo, Kil Kilifi, Kin Kintampo, Kom Kombewa, Kor Korogwe, La Lambaréné, Li Lilongwe, Na Nanoro, and Si Siaya.

The majority of samples from participants with primary clinical malaria in both age categories (68% of samples from infants 6 to 12 weeks of age and 65% of samples from children 5 to 17 months of age) had complex infections, defined as being founded by two or more distinct parasite lineages. In the older age category, the distribution of complexity of infection was shifted toward fewer parasite lineages in RTS,S/AS01-vaccine recipients than in control-vaccine recipients (complex infections in 61% vs. 71% of samples, P<0.001 by Wald test) (Figure 3A), whereas in the younger age category there was no evidence of a different distribution between the RTS,S/AS01 and control groups (67% and 70%, respectively; P=0.43) (Fig. S10 in the Supplementary Appendix). This observation for the older age category is concordant with findings in two phase 2 trials of the related RTS,S/AS02 vaccine,31,32 and there were fewer 3D7-matching full-amplicon haplotypes in infections of high complexity in the RTS,S/AS01-vaccinated group than in the control group (Fig. S11 in the Supplementary Appendix).

Population Variation Profile

We searched for a sieve effect based on perfect 3D7 match–mismatch in the C-terminal of the circumsporozoite protein at three scales: the full-amplicon haplotype (95 amino acids), four described epitopes or polymorphism cluster haplotypes (10 to 17 amino acids apiece), and 25 individual polymorphic positions. In the category of children 5 to 17 months of age, the frequency of haplotypes with an exact match to the 3D7 vaccine strain across all polymorphic positions varied considerably among study sites (Figure 3B). In addition, there was a lower frequency of 3D7-matching haplotypes among RTS,S/AS01-vaccine recipients than among control vaccine recipients, especially at geographic sites with at least a 5% frequency of 3D7-matching haplotypes in the control-vaccine group. Similar differences were evident with respect to the epitope haplotype frequencies (Figure 3C). The frequency of alleles matching the 3D7 vaccine strain at individual polymorphic positions was variable (Figure 3D). In the category of infants 6 to 12 weeks of age, the frequencies of 3D7 matching at all three scales in the RTS,S/AS01-vaccinated group were similar to those in the control-vaccinated group (Fig. S10 in the Supplementary Appendix).

C-Terminal Region Sieve Effects

Figure 4. Figure 4. Cumulative Incidences and Vaccine Efficacy against the Primary Clinical Malaria End Point among Children 5 to 17 Months of Age. Shown is the cumulative incidence of 3D7-matched malaria (Panel A) and 3D7-mismatched malaria (Panel B) among RTS,S/AS01-vaccine recipients and control-vaccine recipients during 12 months of post-vaccination follow-up, the cumulative vaccine efficacy (one minus the ratio [RTS,S/AS01 vs. control] of cumulative incidences of the first or only episode of clinical malaria with a specific haplotype) against 3D7-matched and 3D7-mismatched malaria over the entire post-vaccination follow-up period (Panel C), and the cumulative vaccine efficacy and hazard-ratio vaccine efficacy (one minus the ratio [RTS,S/AS01 vs. control] of instantaneous incidences of the end point under the assumption that incidences are proportional over time) against 3D7-matched and 3D7-mismatched malaria at 12 months after vaccination, with tests for differential haplotype-specific vaccine efficacy (Panel D). The I bars in Panel D indicate 95% confidence intervals.

Figure 5. Figure 5. Vaccine Efficacy against the Primary Clinical Malaria End Point among Children 5 to 17 Months of Age. Shown are the cumulative vaccine efficacy (Panel A) and hazard-ratio vaccine efficacy (Panel B) for the prevention of clinical malaria in which parasites were matches or mismatches with the RTS,S/AS01 vaccine strain (3D7) at each haplotype locus. Estimates were stratified according to study site. For each haplotype locus, the calculation of haplotype-matched vaccine efficacy included only clinical malaria end-point events with samples in which parasites matched 3D7 at the given locus; the calculation of haplotype-mismatched vaccine efficacy included only clinical malaria end-point events with samples in which parasites mismatched 3D7 at the given locus. Asterisks indicate that the difference in efficacy was significant (Q value ≤0.2 for all 28 multiply compared haplotype loci and unadjusted P≤0.05 for the full circumsporozoite protein C-terminal amplicon). FWER denotes family-wise error rate.

Through 1 year after vaccination, we detected 139 clinical malaria cases with a perfect full-amplicon 3D7 match (Figure 4A) and 1951 cases that were mismatched (Figure 4B). During this period, the cumulative vaccine efficacy against clinical malaria with a perfect full-amplicon 3D7 match was 50.3% (95% confidence interval [CI], 34.6 to 62.3), and that against mismatched clinical malaria was 33.4% (95% CI, 29.3 to 37.2), with vaccine efficacy significantly higher against matched malaria (P=0.04 by Wald test for the sieve effect) (Figure 4D and Figure 5A). The covariate-adjusted analysis gave almost identical results (Table S5 in the Supplementary Appendix). The cumulative vaccine efficacy was higher against matched malaria than against mismatched malaria throughout the follow-up period; for example, during the period through month 6, vaccine efficacy against matched malaria was 70.2% (95% CI, 56.1 to 79.8), and that against mismatched malaria was 56.3% (95% CI, 51.1 to 60.9) (P=0.05 for the sieve effect) (Figure 4C). Cumulative vaccine efficacy and sieve effects for the circumsporozoite protein C-terminal also varied in magnitude among study sites when the sites were analyzed individually in the older age category (Table S6 in the Supplementary Appendix).

The hazard-ratio vaccine efficacy over the 12 months of post-vaccination follow-up was also higher against 3D7-matched malaria than against 3D7-mismatched malaria (62.7% [95% CI, 51.6 to 71.3] vs. 54.2% [95% CI, 49.9 to 58.1]; P=0.06 for the sieve effect) (Figure 5B, and Table S7 in the Supplementary Appendix). The overall vaccine efficacy was similar to the efficacy against mismatched malaria, because more than 90% of the infections were mismatched (Figure 4D). Post hoc analysis defining the haplotypes of a malaria case as “any match” or “no match” to 3D7 also identified a sieve effect for the circumsporozoite protein C-terminal (P<0.001 for the cumulative vaccine efficacy sieve effect; P=0.002 for the hazard-ratio vaccine efficacy sieve effect) (Tables S8 and S9 in the Supplementary Appendix). In contrast, the cumulative and hazard-ratio vaccine efficacies were similar against full-amplicon–matched malaria and full-amplicon–mismatched malaria in the category of infants 6 to 12 weeks of age (P=0.58 for the sieve effect) (Tables S10 and S11 and Fig. S12 in the Supplementary Appendix), as well as against SERA-2–matched malaria and SERA-2–mismatched malaria in both age categories (P values for the sieve effect >0.30) (Tables S12 through S15 and Fig. S13 and S14 in the Supplementary Appendix).

Table 1. Table 1. Cumulative Vaccine Efficacy against Primary Clinical Malaria.

Among the participants who were 5 to 17 months of age, there were also significant cumulative vaccine efficacy sieve effects (Q value ≤0.2) for the Th2R and Th3R epitopes, the Th2R–Th3R LD haplotype, and the DV10 region (Figure 5A), as well as for the individual amino acid positions 299, 301, 317, 354, 356, 359, and 361 (Table 1). The cumulative vaccine efficacy tended to decrease with the number of mismatches with 3D7 at these seven amino acid positions (Fig. S15 in the Supplementary Appendix). Hazard-ratio analyses of epitopes and regions (Figure 5B) and individual amino acid positions (Table S7 in the Supplementary Appendix) yielded differential vaccine efficacy results that were consistent with those from the cumulative vaccine efficacy analysis, at reduced levels of significance. In the younger age category, vaccine efficacy against malaria that matched 3D7 at individual circumsporozoite protein C-terminal amino acid positions was similar to that against malaria with mismatches (all Q values >0.20) (Tables S10 and S11 in the Supplementary Appendix). No evidence of sieve effects was found for individual positions in either age category for the SERA-2 locus (Table S12 through S15 in the Supplementary Appendix).

For the parasite-positivity end point at 18 months after dose 3, in the older age category, the estimates of vaccine efficacy tended to be higher for circumsporozoite protein C-terminal 3D7-matched malaria than for 3D7-mismatched malaria (e.g., vaccine efficacy, 53% vs. 30%; P=0.19 for the full amplicon), although none of the differences were significant (Table S16 in the Supplementary Appendix). In contrast, there was no evidence at all of a sieve effect for the circumsporozoite protein C-terminal in the younger age category with regard to this end point (Table S17 in the Supplementary Appendix), and there was no evidence of a sieve effect for SERA-2 in either age category (Tables S18 and S19 in the Supplementary Appendix).

NANP–NVDP Repeat Region

In 3137 samples representing the clinical malaria end point with sequence data that could be evaluated from the B-cell epitope repeat region, the NANP–NVDP repeat count ranged from 37 to 44, with a mode of 40 repeats. There was a nonsignificant trend toward declining cumulative vaccine efficacy with increasing NANP–NVDP repeat count in the older age category (P=0.07) (Fig. S16 in the Supplementary Appendix) and no significant differential vaccine efficacy according to repeat count in the younger age category (P=0.89) (Fig. S17 in the Supplementary Appendix). We did not assess the dependence of vaccine efficacy on NANP–NVDP repeat amino acid sequences because the vaccine construct contains a truncated repeat region (18.5 NANP–NVDP repeats).