Mixed Origins: HIV gp120-Specific Memory Develops from Pre-Existing Memory and Naive B Cells Following Vaccination in Humans

Abstract

The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naive B cell receptor, fail to bind Env, suggesting that the initial responding B cell population not only exclusively comprises a naive population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants in HVTN 105, an HIV vaccine trial. Using deep sequencing, focused on immunoglobulin G (IgG), IgA, and IgM, VH-lineage tracking, we identified four of these mAb lineages in pre-immune peripheral blood. We also looked through the ∼7 month postvaccination bone marrow, and interestingly, several of these lineages that were found in prevaccination blood were still persistent in the postvaccination bone marrow, including the CD138+ long-lived plasma cell compartment. The majority of the pre-immune lineage members included IgM, however, IgG and IgA members were also prevalent and exhibited somatic hypermutation. These results suggest that vaccine-induced gp120-specific antibody lineages originate from both naive and cross-reactive memory B cells. ClinicalTrials.gov NCT02207920.

Keywords: B cell; HIV-1; antibody repertoire; envelope; gp120; memory; naive; pre-immune.

FIG. 1.

Characterization of pre-immune B cell repertoire in blood. (A) Representative ELISpot showing Env-reactive pre-immune CD27pos (memory) and CD27neg B cells isolated by magnetic bead separation in HIV-negative healthy subjects (blue, IgM; red, IgG). (B) Time line showing the collection of peripheral blood at baseline (month 0) and 7 days after final immunization (M6) from HVTN 105 participants (n = 21) who received AIDSVAX B/E Protein and DNA-HIV-PT123 plasmid immunizations intramuscularly at months 0, 1, 3, and 6. (C–E) Frequency of (C) IgA, IgG, and IgM, (D) lineages with long HCDR3 (≥22 amino acid), (E) expanded lineages with ≥10% average mutation from germline in gp120pos and gp120neg B cell fractions. Each symbol represents an individual subject. Env, envelope; Ig, immunoglobulin.

FIG. 2.

Expansion of HIV-1 Env-specific pre-immune B cells following HIV Env vaccination. gp120pos pre-immune B cells that shared lineages with M6 peripheral blood that was obtained 7 days after final immunization were split into naive (≤1% nucleotide mutations from germline in the variable gene) and non-naive (>10% nucleotide mutations from germline in the variable gene) compartments and characterized. Each symbol represents an individual subject. (A) Number of lineages, (B) percentage of lineages, (C) VH and (D) JH gene usage.

FIG. 3.

HIV-1 Env reactive 1131A5 lineage in pre-immune blood. (A) Phylogenic analysis and alignments of 11131A5 lineage. Lineage members were defined as same heavy-chain V and J gene usage, HCDR3 length, and >80% HCDR3 similarity to the mAb sequence, and clustered based on Partis analysis. Individual nodes indicate identical nucleotide sequences (n = 1–6). Black nodes indicate undefined isotype, symbol shape indicates time point. (B) Alignments depict germline, pre-immune lineage members, and M6 mAb-derived sequences. mAb, monoclonal antibody.

FIG. 4.

HIV-1 Env reactive 1095A8 lineage in pre-immune blood. (A) Phylogenic analysis and alignments of 1095A8 lineage. Lineage members were defined as same heavy-chain V and J gene usage, HCDR3 length, and >80% HCDR3 similarity to the mAb sequence, and clustered based on Partis analysis. Individual nodes indicate identical nucleotide sequences (n = 1–37). Black nodes indicate undefined isotype, symbol shape indicates time point. Nodes with sequences from multiple time points are in black borders. (B) Alignments depict germline, pre-immune lineage members, and M6 mAb sequences.

FIG. 5.

HIV-1 Env reactive 1098B8 lineage in pre-immune blood. Phylogenic analysis of 1098B8 lineage. Lineage members were defined as same heavy-chain V and J gene usage, HCDR3 length, and >80% HCDR3 similarity to the mAb sequence, and clustered based on Partis analysis. M13 sequences were obtained from peripheral blood, total BM, and CD138+ BM PC. Individual nodes indicate identical nucleotide sequences (n = 1–653). Black nodes indicate undefined isotype, symbol shape indicates time point. BM, bone marrow; PC, plasma cells.

FIG. 6.

Sequence alignment of 1098B8 lineage members. Alignments depict germline, pre-immune lineage members, M6 mAb, and M13 BM PC-derived sequences for 1098B8 lineage.

FIG. 7.

gp120 binding by pre-immune antibodies. (A, B) Reactivity of germline and pre- and post-vaccination versions of representative mAbs to (A) MN.B and (B) A244.AE gp120 determined by ELISA (n = 3 replicates per dilution). A HIV-irrelevant hIgG1 was used as an isotype control. Each line represents the mean of an individual mAb. (C) Binding profile of germline and pre- and post-vaccination versions of 1098B8 lineage to MN.B gp120 determined by surface plasmon resonance analysis and (D) their dissociation constants (K_D).