Anti-malaria RTS,S/AS01 vaccine generates a limited pool of memory B cells expressing specificities associated with protection

Technical Summary: Anti-malaria RTS,S/AS01 vaccine generates a limited pool of memory B cells expressing specificities associated with protection

Problem Statement
The RTS,S/AS01 subunit malaria vaccine induces protective but short-lived antibody titers against the Plasmodium falciparum circumsporozoite protein (CSP). While antibodies targeting the NANP-repeat region of CSP are known to be protective, vaccine efficacy wanes within months, correlating with a loss of these protective serum antibodies. The mechanisms driving this limited durability and the inability to sustain protective responses following booster doses or re-exposure remain unclear. Specifically, it is unknown how the composition and specificity of the CSP-specific B cell memory pool evolve over time following vaccination in malaria-naïve individuals.

Methodology
The authors conducted a longitudinal analysis of CSP-specific B cells in malaria-naïve volunteers from two clinical trials: MAL071 and MAL092.

• Sample Collection: Peripheral blood mononuclear cells (PBMCs) were collected at multiple timepoints: pre-vaccination, 7 days post-third vaccination (acute response), and various memory timepoints (30 days post-vaccination, day of challenge, and months post-challenge).
• Cell Isolation and Phenotyping: CSP-specific B cells were isolated using a rigorous dual-tetramer flow cytometry approach. Cells were stained with a decoy tetramer followed by PE-conjugated CSP tetramers (full-length or C-terminus specific) to distinguish between NANP-repeat-specific and C-terminus-specific B cells. Cells were index-sorted based on surface markers (CD19, CD21, CD27, CD38, Ig isotypes) to identify plasmablasts, memory B cells (classical, activated, atypical), and naïve cells.
• Single-Cell Sequencing and Cloning: Single-cell BCR sequencing was performed on sorted cells to capture paired heavy and light chain sequences. A subset of these BCRs was cloned and recombinantly expressed as monoclonal antibodies (mAbs), standardized to an IgG1 constant region where necessary.
• Functional Characterization:
  • Specificity: mAbs were tested via ELISA against full-length CSP, NANP-repeat peptides, and C-terminus peptides.
  • Epitope Mapping: Competitive ELISAs were used to map C-terminus epitopes against known reference antibodies.
  • Binding: Biolayer interferometry (BLI) measured binding affinities. Immunofluorescence assays (IFA) assessed binding to whole sporozoites.
  • Protection: Passive transfer of mAbs into C57BL/6 mice followed by intravenous challenge with P. berghei expressing P. falciparum CSP (Pb-PfCSP-GFP-luc) was used to assess protective capacity. Liver parasite burden was quantified via luciferase activity.

Key Results

1. Shift in Specificity Over Time: Immediately following the third vaccination (Day 7), the CSP-specific B cell response was dominated by plasmablasts and class-switched memory B cells specific to the NANP-repeat region. However, at later memory timepoints (30 days post-vaccination and beyond), the frequency of NANP-repeat-specific memory B cells declined significantly.
2. Dominance of C-Terminal Memory: In contrast to the acute phase, the long-term memory B cell pool became dominated by class-switched (IgG), affinity-matured B cells specific to the C-terminus of CSP. This shift was observed across multiple subjects in both the MAL071 and MAL092 trials, regardless of protection status.
3. Limited NANP-Repeat Memory: While NANP-repeat-specific B cells remained detectable at memory timepoints, they were predominantly naïve (CD27-CD21+) or lacked the class-switched, somatically mutated phenotype required for robust secondary responses.
4. Lack of Protective Function in C-Terminal Antibodies: Despite being class-switched and affinity-matured, monoclonal antibodies derived from the long-lived C-terminus-specific memory B cells failed to protect mice against a transgenic parasite challenge in an intravenous model, even at high doses (up to 300 µg). In contrast, control NANP-repeat-specific antibodies (e.g., CIS43) provided significant protection.
5. Clonality and Gene Usage: The NANP-repeat-specific response showed a bias toward specific VH genes (e.g., VH3-33) but was largely composed of unique clones. The C-terminus-specific response exhibited greater diversity in VH gene usage but maintained a consistent phenotype of class-switched memory cells.

Significance and Claims
The paper claims that the limited long-term efficacy of the RTS,S/AS01 vaccine is likely due to the transient nature of the protective NANP-repeat-specific B cell response. The authors posit that the vaccine induces a robust but short-lived plasmablast response against the repetitive NANP region, which fails to generate a sufficient pool of long-lived memory B cells. Consequently, the memory compartment is eventually repopulated by C-terminus-specific memory B cells. While these C-terminus cells persist, the study demonstrates that the antibodies they produce do not confer protection in the tested murine model.

The authors suggest that this dynamic—where protective specificities are replaced by non-protective, persistent memory—may explain the vaccine's limited response to boosters and its waning efficacy. They conclude that future vaccine strategies may need to move away from high-copy repeat antigens that drive short-lived plasmablast differentiation and instead focus on inducing long-lived, NANP-repeat-specific memory B cells, potentially by altering antigen design or adjuvantation. The study does not claim to have identified a new vaccine but rather provides a mechanistic explanation for the observed immunological limitations of the current RTS,S formulation.