Breadth and Magnitude of Antibody Responses to Multiple Plasmodium falciparum Merozoite Antigens Are Associated with Protection from Clinical Malaria

Faith H. A. Osier; Gregory Fegan; Spencer D. Polley; Linda Murungi; Federica Verra; Kevin K. A. Tetteh; Brett Lowe; Tabitha Mwangi; Peter C. Bull; Alan W. Thomas; David R. Cavanagh; Jana S. McBride; David E. Lanar; Margaret J. Mackinnon; David J. Conway; Kevin Marsh

doi:10.1128/IAI.01585-07

DOI: 10.1128/IAI.01585-07

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FIG. 1.
The predicted probability of an episode of malaria in children decreases with increasing antibody titer for most antigens (n = 119). Each panel represents the allelic antigens tested at each locus, as well as parasite schizont extract. The red horizontal line represents the risk of an episode without taking antibody responses to any antigen into account. The final panel combines antibodies to one allelic form of each antigen (and one antigen from each of the three main allelic families of MSP-1 block 2). The lines, from top to bottom, represent MSP-1_B2_Wellcome, MSP-3_K1, MSP-2_Dd2, schizont extract, AMA1_3D7, EBA-175_F2_3D7, MSP-1_B2_3D7, MSP-1_B2_RO33, and MSP-1₁₉.
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FIG. 2.
Protective efficacy increased with increasing breadth of response in children from the Chonyi (parasitemic children, n = 119) (a) and hospital cohorts (all children, n = 387) (b). Each bar represents the comparison between individuals making high-titer responses to n number of antigens with those who make no responses to any antigen. Proportions above each bar are the percentage of individuals making high-titer responses to n antigens. The effect of high-titer responses to P. falciparum schizont extract is also shown.
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FIG. 3.
The breadth of antibody specificity increased with age in both the Chonyi cohort (n = 119) (a) and the case-control study (n = 387) (b).

TABLE 1.

Protective effects^a of high levels of antibodies to individual antigens

Antigen	% of children with high-titer response	Univariate analysis^b		Age-adjusted analysis^b		Age and schizont adjusted^b
Antigen	% of children with high-titer response	Risk ratio (95% CI)	P value	Risk ratio (95% CI)	P value	Risk ratio (95% CI)	P value
AMA1_FVO	49	0.45 (0.25-0.80)	0.007*	0.60 (0.33-1.08)	0.093	0.65 (0.36-1.21)	0.178
AMA1_3D7	51	0.40 (0.22-0.72)	0.002*	0.50 (0.28-0.90)	0.021*	0.54 (0.29-1.00)	0.052
MSP-2_CH150/9	54	0.32 (0.17-0.59)	0.000*	0.39 (0.21-0.70)	0.002*	0.41 (0.22-0.74)	0.004*
MSP-2_Dd2	54	0.28 (0.15-0.53)	0.000*	0.35 (0.18-0.65)	0.001*	0.36 (0.19-0.70)	0.003*
MSP-3_K1	39	0.39 (0.20-0.78)	0.008*	0.50 (0.26-0.95)	0.037*	0.52 (0.27-1.01)	0.055
MSP-3_3D7	40	0.58 (0.32-1.04)	0.072	0.67 (0.38-1.17)	0.166	0.70 (0.40-1.22)	0.216
EBA-175_F2_CAMP	34	0.72 (0.40-1.29)	0.274	1.05 (0.60-1.82)	0.858	1.25 (0.71-2.19)	0.437
EBA-175_F2_3D7	41	0.41 (0.21-0.79)	0.008*	0.53 (0.27-1.04)	0.067	0.57 (0.29-1.14)	0.114
MSP-1_B2_3D7	20	0.43 (0.12-1.12)	0.085	0.56 (0.20-1.50)	0.252	0.60 (0.22-1.64)	0.328
MSP-1_B2_PaloAlto	18	0.93 (0.47-1.83)	0.846	0.91 (0.48-1.71)	0.774	0.95 (0.50-1.81)	0.895
MSP-1_B2_Wellcome	19	0.33 (0.11-1.00)	0.051	0.50 (0.16-1.51)	0.222	0.54 (0.18-1.65)	0.286
MSP-1_B2_MAD20	23	0.48 (0.21-1.12)	0.092	0.73 (0.32-1.70)	0.478	0.76 (0.33-1.78)	0.543
MSP-1_B2_RO33	15	1.40 (0.77-2.53)	0.263	1.20 (0.74-1.93)	0.443	1.43 (0.86-2.38)	0.162
MSP-1₁₉	36	1.44 (0.87-2.38)	0.148	1.14 (0.74-1.76)	0.544	1.59 (0.93-2.74)	0.089

↵ a Risk of developing clinical malaria associated with high titers compared to low/undetectable titers of antibodies to individual antigens in a subset of the Chonyi cohort (n = 119). Antigens are designated by their locus name and P. falciparum strain ([locus]_[strain]).
↵ b Risk ratios (with 95% confidence intervals [CI]) are presented for univariate and multivariate analyses (adjusted initially for age and subsequently for both age and reactivity to P. falciparum parasite schizont extract as a proxy for exposure). *, P < 0.05.

TABLE 2.

Protective effects^a of combinations of high-titer antibody responses

Antigen	Main effects and interaction^b		Combination effect^c
Antigen	Risk ratio (95% CI)^d	P value	Risk ratio (95% CI)^d	P value
AMA1_3D7	0.59 (0.32-1.08)	0.091	0.21 (0.05-0.88)	0.033*
MSP-3_K1	0.56 (0.29-1.08)	0.085
AMA1_3D7 + MSP-3_K1	0.28 (0.06-1.29)	0.104
AMA1_3D7	0.66 (0.35-1.25)	0.211	0.24 (0.09-0.64)	0.004*
MSP-2_Dd2	0.40 (0.20-0.78)	0.008*
AMA1_3D7 + MSP-2_Dd2	0.33 (0.09-1.17)	0.087
AMA1_3D7	0.62 (0.32-1.18)	0.147	0.61 (0.29-1.29)	0.199
EBA-175_F2_3D7	0.69 (0.33-1.42)	0.320
AMA1_3D7 + EBA-175_F2_3D7	1.90 (0.34-10.46)	0.459
AMA1_3D7	0.58 (0.31-1.08)	0.088	0.48 (0.17-1.38)	0.178
MSP-1₁₉	1.44 (0.82-2.54)	0.199
AMA1_3D7 + MSP-1₁₉	0.39 (0.12-1.30)	0.129
MSP-2_Dd2	0.40 (0.20-0.78)	0.008*	Total protection (n = 33)
MSP-3_K1	0.62 (0.34-1.13)	0.121
MSP-2_Dd2 + MSP-3_K1	Total protection (n = 33)
MSP-2_Dd2	0.38 (0.20-0.73)	0.004*	0.17 (0.04-0.73)	0.017*
EBA-175_F2_3D7	0.64 (0.33-1.22)	0.178
MSP-2_Dd2 + EBA-175_F2_3D7	0.26 (0.05-1.27)	0.098
MSP-2_Dd2	0.36 (0.18-0.71)	0.003*	0.52 (0.21-1.30)	0.166
MSP-1₁₉	1.55 (0.92-2.61)	0.097
MSP-2_Dd2 + MSP-1₁₉	0.62 (0.20-1.88)	0.402
MSP-3_K1	0.57 (0.29-1.11)	0.101	0.39 (0.12-1.20)	0.103
EBA-175_F2_3D7	0.66 (0.33-1.31)	0.240
MSP-3_K1 + EBA-175_F2_3D7	0.71 (0.17-2.97)	0.643
MSP-3_K1	0.48 (0.25-0.93)	0.030*	0.57 (0.25-1.31)	0.191
MSP-1₁₉	1.74 (1.07-2.84)	0.024
MSP-3_K1 + MSP-1₁₉	0.65 (0.19-2.27)	0.509
EBA-175_F2_3D7	0.56 (0.28-1.11)	0.101	0.69 (0.26-1.78)	0.445
MSP-1₁₉	1.61 (0.97-2.68)	0.062
EBA-175_F2_3D7 + MSP-1₁₉	0.70 (0.19-2.52)	0.586
AMA1_3D7	0.55 (0.30-1.01)	0.055	0.66 (0.29-1.52)	0.336
MSP-1_B2	0.87 (0.51-1.50)	0.636
AMA1_3D7 + MSP-1_B2	1.13 (0.36-3.56)	0.829
MSP-2_Dd2	0.36 (0.18-0.70)	0.008*	0.40 (0.15-1.05)	0.064
MSP-1_B2	1.04 (0.62-1.72)	0.875
MSP-2_Dd2 + MSP-1_B2	0.57 (0.17-1.90)	0.365
MSP-3_K1	0.52 (0.27-1.02)	0.059	0.16 (0.02-1.13)	0.067
MSP-1_B2	0.87 (0.51-1.49)	0.634
MSP-3_K1 + MSP-1_B2	0.15 (0.02-1.14)	0.068
MSP-1₁₉	1.61 (0.95-2.74)	0.073	0.98 (0.48-2.01)	0.972
MSP-1_B2	0.80 (0.45-1.41)	0.456
MSP-1₁₉ + MSP-1_B2	0.83 (0.27-2.46)	0.737
EBA-175_F2_3D7	0.58 (0.30-1.14)	0.119	0.85 (0.36-2.00)	0.720
MSP-1_B2	0.88 (0.51-1.51)	0.656
EBA-175_F2_3D7 + MSP-1_B2	2.11 (0.56-7.94)	0.265

↵ a Risk of developing clinical malaria associated with combinations of high titers compared to low/undetectable titers of antibodies to individual antigens in a subset of the Chonyi cohort (n = 119). In the majority of cases, significantly more protection was obtained with high-level antibody responses to pairs of antigens, compared to single antigens (Table 1). No strong evidence of statistical interaction between pairs of antibodies was observed.
↵ b The main effects of antibodies to each antigen were adjusted for each other. Interaction effects (combinations of two antigens) are those over and above the main effects.
↵ c Effects of combinations of high-titer responses (combines the main effects and interaction effects).
↵ d Risk ratios (with 95% confidence intervals [CI]) are from multivariate analyses (adjusted for both age and reactivity to P. falciparum schizont extract). *, P < 0.05.

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Supplemental file 1 - Fig. S1. Antibodies to MSP-1 block 2, the F2 subdomain of EBA-175, and MSP-1₁₉ in the Chonyi cohort.
Table S1. Details of recombinant merozoite antigens.
Table S2. ELISA OD cutoff levels used to define high versus low responders to merozoite antigens in the Chonyi cohort and the case-control study.
Table S3. Correlations between antibodies to the panel of merozoite antigens.
Supplemental text. Selection of a single allelic form of each antigen for analysis of combined antibody responses.
Table S4. Effects of high-titer antibodies to allelic combinations of antigens on the probability of a clinical episode in the Chonyi cohort.
Table S5. Effects of combinations of high-titer antibodies on admission to hospital with malaria.
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