R21/Matrix-M malaria vaccine drives diverse immune responses in pre-exposed adults: insights from a phase IIb controlled human malaria infection trial

Abstract

Introduction: The recently licenced R21/Matrix-M vaccine induces a protective antibody response. In this study, we examined vaccine-induced responses in semi-immune adults in a controlled human malaria infection (CHMI) Phase IIb clinical trial.

Methods: Plasma and peripheral blood mononuclear cells from healthy adult volunteers living in coastal Kenya were analysed following vaccination with R21/Matrix-M (n = 19) and CHMI challenge with Plasmodium falciparum (PfSPZ NF54) sporozoites (n = 17). Humoral immunity was evaluated by quantifying antigen specific antibody subtypes and subclasses via ELISA, alongside functional antibody properties including avidity and complement fixation elicited by vaccination and challenge. Antigen-specific memory B cells were characterised using FluoroSpot assays to detect concurrent secretion of multiple antibody isotypes and the frequency and phenotypes of circulating Tfh (cTfh) cells were assessed using multiparametric flow cytometry.

Results: Vaccination increased antibody titres across IgA, IgM, and IgG isotypes and IgG1 and IgG3 subclasses but not IgG2 or IgG4 subclasses, targeting different vaccine antigens (full-length R21, NANP, and C-terminus), indicating a broad and heterogeneous response. The responses were maintained over time and, importantly, they demonstrated complement-fixing capabilities. IgG+ and IgA+ antigen-specific memory B cells were boosted but were short-lived for IgA. We observed an increase in total CXCR5+/PD1+ cTfh cells following vaccination and challenge with the predominant Th2/Th17 population.

Discussion: We provide insights into the diverse immune responses induced by R21/Matrix-M vaccination and their potential contribution to protection against malaria. These findings highlight the potential of the R21/Matrix-M vaccination and protection in adults with varying levels of prior malaria exposure.

Keywords: R21/Matrix-M; T follicular helper cells; antibodies; complement fixing antibodies; controlled human malaria infection; malaria; memory B cells.

Figure 1

Anti-IgG responses measured by a single-plex ELISA assay following vaccination with R21/Matrix-M and sporozoite challenge. The kinetics of the total IgG responses were analysed over time from pre-vaccination (day 0) to day 84 (i.e. one month after the 3^rd booster dose) using standardised single-plex ELISAs. Following CHMI, antibody responses were monitored on the day of diagnosis (DoD), at C+35, (35 days post-challenge), and C+90, (90 days post-challenge) post-CHMI. (A) Anti-full-length R21 responses, (B) anti-NANP responses, and (C) anti-C-terminus. (D) Antibody durability measured by the fold change between days 84 and C+35 and C+90. Geometric mean antibody titres (GMT ±95% CI) are presented as log10 AU. The positivity threshold, indicated by a horizontal dashed line, represents the mean + 3x standard deviations of U.K. malaria-naïve (negative controls). Samples that exceeded this threshold were classified as positive samples. The vaccination time points are marked by vertical black dashed lines (A-C). Sporozoite challenge is marked by a red vertical dashed line. The ELISA detection limit was OD 0.2; values below this limit were assigned as AU values of 11 for R21, 14 for NANP, and 41 for the C-terminus. Open symbols represent vaccinees (n = 19 or 17 (from day 84)) and filled symbols represent unvaccinated controls (n = 9) (A-C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Analysis of antibody isotype profiles to full-length R21 and NANP following vaccination and sporozoite challenge. Vaccine induced antibody isotype profiles were analysed using single-plex ELISA at baseline, day 84 (one-month post the third dose), and 35 (C+35) and 90 (C+90) days post-CHMI. (A) Full-length R21 and (B) NANP responses. Antibody subclass (IgG1-4) levels are presented as raw ODs at 405 nm, shown as individual responses along with the median [± IQR] values, whereas IgM and IgA subclasses were converted to arbitrary units (AU) and are shown as GMT (± 95% CI) in log10 AU. The seropositivity threshold, indicated by a horizontal dashed line, represents the mean + 3x standard deviations of U.K. malaria-naïve negative controls, with values above this threshold classified as positive. Sporozoite challenge is marked by a red vertical dashed line. PHIS, pooled adult hyperimmune sera (positive controls). **P < 0.01.

Figure 3

Assessment of complement fixation activity and IgG avidity post-R21/Matrix-M vaccination and sporozoite challenge. Semi-immune adults vaccinated with R21/Matrix-M were tested for C1q fixation by NANP and C-terminal antigens (A and B respectively). Individual and median [IQR] OD_450nm responses are shown. The positivity cutoff was the mean plus three standard deviations of day 0 (horizontal dashed line). The avidity index was determined as the molar concentration of NaSCN required to reduce OD405 to 50% of the value observed in the absence of NaSCN. (C) Avidity to full-length R21, (D) avidity to NANP, and (E) avidity to the C-terminus. Samples with negative antigen-specific total IgG responses by ELISA or insufficient antibody levels for avidity testing at a given time point were excluded from the analysis. Individual avidity responses are displayed alongside the group median and ±95% CI to illustrate variability and overall trends in avidity over time. The Kruskal-Wallis non-parametric test with Dunn’s multiple comparisons was used to assess differences between time points. The challenge day is represented by the red dashed vertical line. *P < 0.05.

Figure 4

Memory B cell responses following vaccination and sporozoite challenge. The magnitude of MBC responses to full-length R21 and the C-terminus was measured using B cell FluoroSpot in pre-exposed adults vaccinated with R21/Matrix-M. Responses were measured after vaccination and post-CHMI. Top panel: Proportion of full-length R21-specific MBCs relative to total IgG-, IgM-, and IgA-producing cells. Bottom panel: Proportion of C-terminus-specific MBCs relative to total IgG-, IgM-, and IgA-producing cells. The magnitude of MBC responses was expressed as the proportion of antigen-specific spots per total IgG/A/M, expressed as a percentage (% of antigen-specific MBC/total IgG, IgA, or IgM). Individual and group medians [± IQR]) are shown. The Kruskal-Wallis non-parametric test with Dunn’s multiple comparisons was used to assess differences between time points. Challenge day is represented by a red dashed vertical line. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Analysis of circulating Tfh cells and subsets following vaccination and sporozoite challenge. Cryopreserved PBMCs from days 0, 14, 42, and 70 were stained ex vivo and analysed using flow cytometry. Circulating Tfh cells are defined by the expression of (A) CXCR5+/PD1+. Tfh were further analysed into subsets (B) Th1 (CXCR3+/CCR6-), (C) Th2 (CXCR3-/CCR6-), and (D) Th17 (CXCR3-/CCR6+). Data are presented as individual points and grouped median [ ± IQR]. The Kruskal-Wallis non-parametric test with Dunn’s multiple comparisons was used to assess differences between time points. *P < 0.05.