The acquisition of humoral immune responses targeting Plasmodium falciparum sexual stages in controlled human malaria infections

Abstract

Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection.

Keywords: Plasmodium falciparum; antibody responses; controlled human malaria infection (CHMI); gametocyte antigens; malaria; sexual stage.

Figure 1

Participant parasite exposure and antibody response to crude parasite extracts. IgG antibody responses to crude asexual and gametocyte extracts. In all plots, red solid circles denote mosquito bite (sporozoite) infection cohorts (SPZ Control, SPZ Gct) and blue hollow circles denote asexual parasite infection (BS Control, BS Gct). (A) Anti-asexual antibody response pre- and post- (day 36) infection. (B) Anti-gametocyte antibody response pre- and post- (day 36) infection. p = p-values from paired t-tests on log-transformed data.

Figure 2

Antibody responses to purified recombinant proteins. IgG antibody responses to a selection of purified sporozoite, asexual and sexual stage antigens. Statistical analysis of response over time compared to baseline for all antigens is in Supplemental Table S3. In all plots, red solid circles denote mosquito bite (sporozoite) infection cohorts (SPZ Control, SPZ Gct) and blue hollow circles denote asexual parasite infection (BS Control, BS Gct). Data are presented as log2 median fluorescence intensity (MFI) ratios of response over baseline, each line representing a single individual.

Figure 3

Antibody breadth and magnitude. Data points for each individual within each cohort group are represented with dots in a beeswarm pattern. Overlayed boxplots represent the median (thick line), interquartile range (box limits) and the 25^th/75^th percentiles plus 1.5*IQR (whiskers). (A) Antibody breadth is the number of protein targets (out of a total of 943) with responses above background, for each individual. For SPZ Gct, the mean MFI ratio of antibody responses to all proteins for one individual was -0.47 at C-1; this data point was not included in the plot, but the parameters of the relevant box plot were calculated from all data points. (B) Antibody magnitude is shown as the mean magnitude of response by each individual in a cohort group/timepoint to all protein targets. Magnitude of response is shown as a log2 MFI ratio, where 0 represent no change relative to background, and 1 represents doubling with respect to background. (C) Antibody magnitude is shown as the mean magnitude of response to each protein target by all individuals in a cohort group/timepoint, with units as in (B) P-values are from paired two-sided t-tests for difference between C-1 and C+35/36, C-1 and C+51, or C+36 and C+51, as indicated. MFI: median fluorescence intensity; IQR: Interquartile range. P = P-value from paired t-tests. Ns = Not significant at p = 0.05.

Figure 4

Differences in antibody response to microarray targets associated with gametocyte exposure. (A) Array targets demonstrating minimal response (<7.5% increase in the mean magnitude of response between pre- [day 0] and post- [day 35] inoculation timepoints) in the control CHMI cohorts are shown in red for SPZ Control (sporozoite inoculum) and blue for BS Control (asexual inoculum). Array targets demonstrating a positive response (>7.5% increase in the mean magnitude of response between pre- [day 0] and post- [day 36] inoculation timepoints) in the control CHMI cohorts are shown in dashed circles for each cohort, with overlap representing those targets with minimal response in control and positive response in Gct cohorts. (B) Axes show pre- and post-inoculation mean magnitude of response (log2 MFI ratio) to each target on the array. Mean magnitude for each target is represented by a single marker. Grey markers are those with increased responses after inoculation in the Control cohorts. Red and blue markers are as in (A) Targets with responses only in the CHMI Gct cohorts are outlined in black.