Vrc01-class Precursor Frequency in an African Population is Influenced by Ighv1-2 Allelic Composition and Not Malaria Exposure

Technical Summary: VRC01-Class Precursor Frequency in an African Population is Influenced by IGHV1-2 Allelic Composition and Not Malaria Exposure

Problem Statement
Germline-targeting (GT) vaccine strategies aim to elicit broadly neutralizing antibodies (bnAbs) against HIV by using engineered immunogens to selectively expand rare naïve B cell precursors. The success of this approach depends on the presence of these precursors at sufficient frequencies and affinities within target populations. While VRC01-class bnAb precursors have been characterized in North American populations, there is limited data regarding their frequency and properties in African populations, which bear the highest burden of HIV. Furthermore, it is unknown whether chronic exposure to endemic pathogens, specifically malaria, alters the naïve B cell repertoire or impacts the frequency of these specific precursors. Additionally, the high genetic heterogeneity in African populations suggests that allelic variations in the immunoglobulin heavy chain variable gene 1-2 (IGHV1-2) may differ from reference databases, potentially affecting the efficacy of GT immunogens designed based on Western reference alleles.

Methodology
The study recruited 60 healthy Kenyan adults from four regions with varying malaria transmission intensities: Ahero and Junju (high endemicity) versus Nairobi and Ngerenya (low endemicity). Malaria exposure levels were confirmed via anti-P. falciparum schizont IgG ELISA.

• Cell Sorting and Sequencing: Peripheral blood mononuclear cells (PBMCs) were enriched for B cells. Epitope-specific naïve B cells were identified and sorted using fluorescence-activated cell sorting (FACS) based on binding to the germline-targeting immunogen eOD-GT8 (double-positive) and lack of binding to a knockout control (eOD-GT8-KO11).
• Single-Cell Analysis: Sorted cells underwent high-throughput droplet-based single-cell B cell receptor (BCR) sequencing to identify paired heavy and light chain sequences.
• Genotyping: Participant IGHV1-2 genotypes were inferred using the IgDiscover pipeline on bulk RNAseq data from enriched B cells. Novel alleles were identified and validated.
• Functional Assays: A subset of identified sequences was expressed as monoclonal antibodies (mAbs) to measure binding affinity to eOD-GT8 and eOD-GT8-KO11 using surface plasmon resonance (SPR).
• Analysis: Precursor frequencies were estimated by calculating the ratio of VRC01-class sequences (defined by IGHV1-2 usage and 5-amino acid light chain CDR3) to total naïve B cells. Statistical comparisons were made between malaria endemicity groups and against historical North American data.

Key Contributions and Results

1. Precursor Frequency and Malaria Exposure: The study detected VRC01-class precursors in approximately 59% of participants with permissive genotypes. The overall median precursor frequency was 1 in 345,557 naïve B cells. Crucially, there was no statistically significant difference in precursor frequencies between participants from high and low malaria endemicity settings. This suggests that chronic malaria exposure does not negatively impact the frequency of these specific naïve precursors in this population.
2. Genetic Influence (IGHV1-2 Alleles): Precursor frequency was strongly influenced by IGHV1-2 allelic composition. Participants homozygous for the IGHV1-202 allele exhibited significantly higher precursor frequencies (~2.7-fold higher than *02/*04 and ~4.2-fold higher than *02/*06) compared to other genotypes. Approximately 82% of participants possessed at least one 02 or 04 allele, supporting the feasibility of GT strategies in this population.
3. Novel Allelic Variants: The study identified four novel IGHV1-2 allelic variants (*02_S4303, *02_S4953, *06_S5416, and 06_S5931). Notably, the novel IGHV1-206_S5416 allele, which encodes a Glutamine (Q) instead of the canonical Arginine (R) at position 50 (R50Q), was found to produce a precursor capable of binding eOD-GT8. This contrasts with the reference 06 allele, which typically lacks the critical tryptophan residue required for binding.
4. Sequence Characteristics and Affinity: Identified VRC01-class precursors shared key germline-encoded features with mature bnAbs, including specific light chain gene usage and a short 5-amino acid LCDR3. The median binding affinity of these precursors to eOD-GT8 was 2.01 µM. A positive correlation was observed between a "bnAb characteristic score" (based on the presence of key germline features) and binding affinity.
5. Non-VRC01-Class Repertoire: The study also characterized non-VRC01-class CD4bs-specific precursors, identifying a public light chain clonotype (QQYGSSPWTF) associated with IGκ3-20 and Jκ1-01 that was present in multiple individuals across both endemicity settings.

Significance
The paper concludes that the frequency of VRC01-class bnAb precursors in this Kenyan population is comparable to estimates from North American populations and is not diminished by chronic malaria exposure. This finding is reassuring for the implementation of GT HIV vaccine trials in malaria-endemic regions. However, the study emphasizes that IGHV1-2 allelic composition is a critical determinant of precursor frequency and binding capability. The discovery of novel alleles, particularly IGHV1-206_S5416, which can engage the immunogen despite deviating from the reference sequence, underscores the necessity of participant genotyping in GT vaccine trials. The authors assert that these data support the continued development of GT vaccines for this population but highlight the need to account for high genetic diversity and rare, function-restoring variants in immunogen design and trial analysis.