Article Figures & Data
Figures
- FIG 1
CPS immunization induces broad and variable humoral responses against P. falciparum antigens. (A) Plasma samples from 38 CPS-immunized volunteers from 3 clinical trials (n = 9 from study 1, n = 5 from study 2, and n = 24 from study 3) were probed on whole P. falciparum proteome microarrays. Thirty CPS-immunized volunteers were protected and eight were not protected from malaria challenge. RBC, red blood cells. (B) Plasma samples collected preimmunization (I1-7) and postimmunization/prechallenge (C-1) were probed, and signals were quantified. The heat map represents the signal intensity at time point C-1, corrected for background reactivity by subtracting the signal intensity at I1-7, against 548 immunogenic P. falciparum features (or 483 unique proteins) printed on proteome microarrays. For an antigen to be considered immunogenic, (i) its levels had to have increased 100% at C-1 compared to I1-7 and (ii) its seroprevalence had to be >15%. The specimens are ordered in clinical groups, nonprotected (n = 8) and protected (n = 30). Samples are arranged with increasing reactivity from left to right, while the antigens are arranged with decreasing reactivity from top to bottom. A color gradient is used to display the signal intensity detected for each antigen, as shown by the key. (C) Comparison of aggregated C-1 reactivity of the 548 immunogenic antigens among the 3 different immunization dose groups. During the 3 rounds of immunization, 10, 9, and 19 volunteers received 5, 10, and 15 mosquito bites, respectively. Dots show individual data points, center lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5 times the interquartile range (IQR). Aggregate antibody levels were compared using the Kruskal-Wallis rank sum test. To detect only malaria-specific responses, the I1-7 responses were subtracted from the C-1 responses.
- FIG 2
CPS immunization induces trimodal IgG responses in previously malaria-naive adults. (A) Three-component mixture model for breadth counts observed in CPS-immunized individuals (n = 38). The solid curved lines show the fitted breadth count distributions in the mixture model. The dashed curve line shows the mixture-model fit for components 1 (orange), 2 (blue), and 3 (green). The two dashed straight lines show the estimated cutoff points at 180 and 681 antigens, which distinguish the different responder groups (low, medium, and high responders). The histogram represents the frequency of the breadth counts in the different bins. Breadth counts are ranked from the lowest to the highest (range, 9 to 881). (B) Box plots of low-, medium-, and high-responder groups showing protected and nonprotected volunteers. Comparison of breadth counts within the medium-responder group was performed by a Wilcoxon rank sum test. Gray dotted lines show the cutoff points between responder groups. Dots show individual data points, center lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend to show the minimum and maximum breadth counts. (C) Humoral reactivity was compared prechallenge (C-1) and post-CHMI challenge (C+35). Mean antibody signal intensities measured against the top 50 most reactive antigens after CHMI challenge are shown, comparing low, medium, and high responders. The medium-responder group is further stratified into protected (“Med-P”) and nonprotected (“Med-NP”) volunteers. The mean reactivities of the top 5 most seroprevalent antigens, CSP, LSA1, LISP2, EXP1, and MSP2, are shown. Black dotted lines indicate the other boosted antigens after challenge (see Table S2 in the supplemental material).
- FIG 3
Stage of expression of proteins printed on the microarray. Publicly available tandem mass spectrometry (MS/MS) data on protein expression were obtained from PlasmoDB. (A) Venn diagram showing stage-specific profiles of the proportions of proteins expressed by sporozoite and blood stages with MS/MS evidence of expression and printed on the microarray. (B and C) Bar plots showing numbers of P. falciparum proteins uniquely or commonly expressed at sporozoite and/or blood stages and recognized by at least 25% of nonprotected or protected volunteers (B) or low, medium, and high responders (C). The names and gene annotations of these proteins are indicated in Table S1 in the supplemental material.
- FIG 4
Novel antigens associated with CPS immunization-induced sterile protection. (A) Differential signal intensities of 229 antigens at C-1 in plasma samples of protected (n = 7) and nonprotected (n = 12) medium responders. The red dotted line indicates the threshold P value of <0.05; dots in the green area and dots in the orange area represent antigens significantly more reactive in protected and nonprotected subjects, respectively. (B) Gene identifications of the antigens associated with protection and susceptibility in medium responders. Wilcoxon rank sum test P values (Wilcox Pval), Wilcoxon rank sum test P values with Benjamini-Hochberg correction for multiple comparisons (Wilcox Pval BH), area under the receiver operating characteristic curve (AUC) values, and leave-one-out cross-validation (LOOCV) AUC values for protected and nonprotected volunteers are shown. (C and D) Specific antigens associated with protection (C) or susceptibility (D). Boxes represent individual antigens in nonprotected (orange) (n = 7) and protected (green) (n = 12) subjects. Dots show individual data points, center lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5 times the IQR. Comparisons were tested by a Wilcoxon rank sum test.
- FIG 5
Breadth of antigen response as a correlate of protection. (A and B) Breadth counts of protective (A) and nonprotective (B) antigens in protected (n = 30) and nonprotected volunteers (n = 8). Dots show individual data points, center lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5 times the IQR. Antibody intensities were compared using the Wilcoxon rank sum test. (C) The difference between protective and susceptible seropositivity counts was used to construct an ROC curve. LOOCV analysis was also performed. An AUC value of 0.5 indicates that there is no difference between the groups.
Supplemental Material
TABLE S1
Plasmodium falciparum proteins printed on the microarrays. Shown is a list of all proteins printed on the microarray. Gene identifications, gene product descriptions, protein lengths, and the presence of transmembrane domains and signal peptides according to PlasmoDB are shown. Additionally, the numbers of nonprotected and protected subjects reactive and the seroprevalence of a specific feature are indicated. Proteins with a seropositive response in at least 25% of the subjects in a group and with mass spectrometry evidence of protein expression from PlasmoDB are shown. Download Table S1, XLSX file, 0.7 MB.
Copyright © 2019 Obiero et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
Raw microarray antibody reactivity. (A) Bar plots showing representative samples at the C-1 time point. Humoral reactivity against 7,455 P. falciparum antigens on the microarray is sorted from most reactive to least reactive. The black dotted line shows the cutoff point obtained using a mixture-model analysis. (B) Each subject’s humoral response data from all time points were analyzed using a mixture model. This allowed for identification of positive (blue curved line) and negative (yellow curved line) signal intensity distributions in an overall population of signals. The mean of the negative signal distribution plus 3 standard deviations is used as a cutoff point (shown here as a dotted black line). This individualized cutoff point is applied to data from each time point (C-1 shown in panel A) to identify reactive antigens in each of the samples. For each individual sample, the number of seropositive antigens was counted to determine the breadth count. For better visualization, the horizontal axis scale was transformed using the function sqrt_trans() in the R package “scales.” Download FIG S1, TIF file, 0.5 MB.
Copyright © 2019 Obiero et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
Antigens boosted after challenge in nonprotected volunteers. Download Table S2, TIF file, 1.9 MB.
Copyright © 2019 Obiero et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Antibodies to top immunogenic antigens are not associated with protection. (A) Box plots showing comparisons of these antigens between protected (green) (n = 12) and nonprotected (red) (n = 7) subjects. Dots show individual data points, center lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5 times the IQR. (B) Receiver operating characteristic (ROC) curves were used to test individual antigens for association with protection. The performances of the top five most immunogenic antigens with an area under the receiving operating characteristic curve (AUC) are shown. LOOCV analysis was also performed for these antigens. An AUC value of 0.5 indicates that there is no difference between the groups. Download FIG S2, TIF file, 0.5 MB.
Copyright © 2019 Obiero et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S3
Breadth counts and protective and susceptible seropositivity counts. Download Table S3, TIF file, 0.9 MB.
Copyright © 2019 Obiero et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Reactivity against representative antigens of early- and mid/late-liver-stage development of parasites. Antibody reactivity in low responders (light gray) (n = 16) was compared to that in medium/high responders (dark gray) (n = 25) by a Wilcoxon rank sum test. Dots show individual data points, center lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5 times the IQR. Download FIG S3, TIF file, 0.3 MB.
Copyright © 2019 Obiero et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
