Sterile protective immunity to malaria is associated with a panel of novel P. falciparum antigens

Abstract

The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized.

Fig. 1.

Antibody profiles of irradiated sporozoite immunized and malaria naïve individuals. The antibody reactivities of plasma samples are depicted as a heatmap, with the most immunoreactive 1000 Pf fragments represented in rows in descending order of immunoreactivity. A, Average signal intensity for each individual ordered left to right by increasing signal intensity for each time point, and (1B) average group signal intensity, are depicted. The data are clustered by clinical groups: protected (n = 6), not-protected (n = 5), infectivity controls (n = 3), mock immunized (n = 5), and malaria naïve (n = 10). Samples collected pre-immunization (Pre-I), post-third immunization (Post-I), post-last immunization = pre-challenge (Pre-C), and post-challenge (Post-C) for irradiated sporozoite immunized volunteers, or corresponding time points for infectivity control and mock immunized subjects, were assayed. Red indicates high reactivity, black intermediate reactivity, and green low/no reactivity.

Fig. 2.

Summary of selection criteria and AUC profile. A, Analyses were carried out to identify antigens that were putatively associated with sporozoite-induced protection. After spot quantification, the signal intensity was asinh/vsn log transformed to control variance, scaled by the negative control, post-immunization (post-I) and pre-challenge (pre-C) time points combined, and then analyzed according to defined statistical (Bayes-regularized t-tests, area under the receiver operating characteristics curve (AUC) analysis) and biological criteria. A total of 86 fragments (77 Pf proteins) were identified by either approach (global irradiated sporozoite list) and 20 were in common to both approaches (Top 20). B, AUC values of all antigens for the not-protected and protected cohorts were determined by R statistical environment software (www.r-project.org). Antigen rank is plotted relative to their AUC value. An AUC value approaching 1.0 suggests a very strong association of an antigen to protection induced by irradiated sporozoite immunization; an AUC value of 0.5 indicates pure chance. An AUC value of 0.7 was chosen as a threshold for positivity.

Fig. 3.

Magnitude and frequency of recognition of the Top 20 antigens putatively associated with irradiated sporozoite induced protection. A, Average signal intensities of the Top 20 antigens for each clinical group (protected, white bar; not-protected, black bar) are presented as histograms, with antigen IDs listed on the x-axis. The average signal intensity (± S.E.) for each antigen is shown. Antigens are ordered by decreasing AUC value. B, For the Top 20 antigens (ranked by AUC), the cumulative signal intensity representing the sum of signal intensities for each antigen by all subjects from protected or not-protected groups are presented. *** p < 0.0055. C, Frequency of recognition of Top 20 individuals by protected (P) and not-protected (NP) individuals. # represents current clinical candidates: AMA1 (PF11_0344); CSP (PFC0210c); SSP2/TRAP (PF13_0201).

Fig. 4.

Distribution of functional categories. A, The functional categories for the Top 20 antigens putatively associated with irradiated sporozoite-induced protection and (4B) 1200 antigens present on the Pf protein microarray as annotated in PlasmoDB (www.plasmodb.org) are shown above.

Fig. 5.

Antigens putatively associated with blood stage malaria. Twenty-one novel antigens and two current vaccine candidates (MSP1 and LSA3) were recognized by individuals who developed blood stage infection postsporozoite challenge. A, Average signal intensity for protected or not-protected individuals. Group (1) putative blood stage specific antigens; Group (2) antigens highly expressed at blood stage that might also be expressed in the liver stage. B, Presence in independent blood stage specific proteomic or transcriptomic data sets: blood stage mass spectrometry, published (4, 7) and unpublished (Leiden Malaria group, www.PlasmoDB.org); blood stage EST (13); and experimentally infected or naturally exposed protein microarray data sets (3, 14).

Fig. 6.

Network interactions of PF11_0404 to other Top 20 antigens. Map of predicted protein interactions of PF11_0404 and associated score threshold for each interaction link is shown (PlasmoDB, http://www.cbil.upenn.edu/plasmoMAP/index-v1.html, (62)).