Mouse marginal zone B cells harbor specificities similar to human broadly neutralizing HIV antibodies

Abstract

A series of potent, broadly neutralizing HIV antibodies have been isolated from B cells of HIV-infected individuals. VRC01 represents a subset of these antibodies that mediate neutralization with a restricted set of IGHV genes. The memory B cells expressing these antibodies were isolated years after infection; thus, the B-cell subpopulation from which they originated and the extent of participation in the initial HIV antibody response, if any, are unclear. Here we evaluated the frequency of anti-gp120 B cells in follicular (FO) and marginal zone (MZ) B-cell compartments of naïve WT mice and comparable human populations in uninfected individuals. We found that in non-HIV-exposed humans and mice, the majority of gp120-reactive B cells are of naïve and FO phenotype, respectively. Murine FO B cells express a diverse antibody repertoire to recognize gp120. In contrast, mouse MZ B cells recognize gp120 less frequently but preferentially use IGHV1-53 to encode gp120-specific antibodies. Notably, IGHV1-53 shows high identity to human IGHV1-2*02, which has been repeatedly found to encode broadly neutralizing mutated HIV antibodies, such as VRC01. Finally, we show that human MZ-like B cells express IGHV1-2*02, and that IGHV1-53 expression is enriched in mouse MZ B cells. These data suggest that efforts toward developing an HIV vaccine might consider eliciting protective HIV antibody responses selectively from alternative B-cell populations harboring IGHV gene segments capable of producing protective antibodies.

Fig. 1.

Most gp120-reactive naïve B cells are of follicular origin. (A) Naïve splenic FO (CD21^intCD1d^int) and MZ B cells (CD21^hiCD1d^hi) were stimulated with 50 μg/mL LPS for 5 d. Supernatants were analyzed for total (Left) and gp120-reactive (Right) IgM production by ELISA. Results are from three independent experiments with between five and eight mice each. (B) Splenocytes from naïve mice were stained with gp120-biotin-Alexa Fluor 647 and enriched by magnetic cell sorting using antibiotin beads. Enriched B220⁺ gp120-binding B cells (Left) were then assessed for subset phenotype (Right). Representative plots of column-bound cells pregated on singlet live B220⁺ cells are shown. The gate for gp120 binding was set based on the fluorescence level of negative cells that did not bind the column. (C) Percentage of gp120-binding FO (open circles) and MZ (filled circles) B cells from individual mice. Results are from three independent experiments, each with two mice. (D) gp120-binding enrichment as described in B performed with three human spleen samples. Representative dot plot of gp120-binding by column-enriched CD20⁺ B cells (Left) and the distribution of these gp120-reactive B cells as IgD⁺CD27⁻ naïve and IgD⁺CD27⁺ MZ-like B cells (Right) of each spleen are shown. (E) The percent (Left) and normalized frequency (Right) ) of CD20⁺ gp120-binding B cells in the naïve (open circles) and MZ-like (filled circles) B-cell populations depicted for each individual. The percentage of gp120-reactive naïve and MZ-like B cells was normalized to the frequency of each population in unenriched spleen. P values are derived from the Student t test.

Fig. 2.

Mouse MZ B cells repeatedly use the same IGHV gene to encode antibodies that recognize gp120. Hybridomas were generated from LPS-stimulated naïve FO and MZ B cells (five independent fusions from B-cell populations sorted from four to eight mice each). Hybridomas were screened for gp120-reactivity by anti-gp120 ELISA. Reactivity against the blocking reagent was excluded by ELISA performed with BSA in parallel with fish skin gelatin. Only 24 FO and 16 MZ hybridomas showed reactivity to gp120 under both conditions and were considered positive. The Ig heavy chain of each hybridoma was identified using a PCR strategy that selectively amplifies most mouse VDJ rearrangements. (A) Heavy-chain V (Left), D (Center), and J (Right) gene families used by FO (solid) and MZ (hatched) B cells to recognize gp120. (B) IGHV1 family member use in gp120-reactive FO (solid; Upper) and MZ (hatched; Lower) hybridomas and antigen-unselected controls (open). Antigen-unselected controls were generated by sequencing Ig genes from bulk cDNA of LPS-stimulated FO and MZ B cells. *P = 0.0108, Fisher’s exact test.

Fig. 3.

Mouse IGHV1-53 is orthologous to human IGHV1-2*02. (A) Amino acid sequence alignment encoded by germ-line murine IGHV1-53*01 (Upper) and human IGHV1-02*02 (Lower) genes. Identities are indicated by a vertical line; similarities, by “+.” Identical CDRH2 residues are in red. Below the CDRH2 region, an asterisk indicates residues mutated in VRC01, and black circles indicate residues involved in interaction with gp120. (B) Mouse IGHV1-53*01 (Left), human IGHV1-2*02 (Center), and the mature mutated VRC01 VH (Right) regions. Black circles identify amino acids identical in all three, red circles indicate identity between murine IGHV1-53 and human IGHV1-2*02, gray circles indicate identity between IGHV1-2*02 and the mature VRC01, and white circles indicate amino acids not common with IGHV1-2*02.

Fig. 4.

Germ-line IGHV1-53 antibodies are polyreactive and do not neutralize HIV. (A) The IGHV1-53 MZ antibodies were tested for polyreactivity by binding to chromatin (Left) and cardiolipin (Center) by ELISA or to HEp-2 cells (Right) by indirect immunofluorescence staining. Mouse IgM hybridomas specific for NP (B1-8) or H-2^b (3-83) were used as isotype controls. (B) The IGHV1-53 MZ antibodies were tested for CD4 binding site recognition by ELISA against RSC3. VRC01 was used as a positive control. (C) Neutralization ability of IGHV1-53 MZ antibodies was determined using a TZM-bl assay. Mouse IGHV1-53 antibodies were incubated with JR-FL pseudovirus for 30 min before culturing with TZM-bl cells for 48 h. Infection was determined with the Beta-Glo assay and quantitated on a luminometer. Percent infection was calculated in relation to values from wells that received no antibody. Human b12 was used as a positive control for neutralization, and a mouse IgM specific for LPS served as a negative control.

Fig. 5.

IGHV1-53 is enriched in mouse MZ, whereas IGHV1-2*02 is comparably expressed by both naïve and MZ-like human B cells. (A) FO B cells and MZ B cells were sorted from between four and six pooled C57BL/6 mice. Gene expression of IGHV1-53 (Left) and the constant region of the Ig kappa chain (IgκC; Right) were assessed by RT-PCR. The results of one representative experiment of two experiments are shown. (B) IGHV1-53 expression was normalized to IgCκ to control for different amounts of Ig transcript in the different subsets. Circles represent individual experiments, and bars indicate the mean. (C) Naïve and MZ-like B cells were sorted from a human spleen. Gene expression of IGHV1-2*02 (Left) and human IgM (IGHM; Right) was assessed by RT-PCR. (D) IGHV1-2*02 expression by naïve and MZ-like B cells normalized to IgM to control for different amounts of Ig transcript in the different subsets.