Enhanced germinal center reaction by targeting vaccine antigen to major histocompatibility complex class II molecules

Abstract

Enhancing the germinal center (GC) reaction is a prime objective in vaccine development. Targeting of antigen to MHCII on APCs has previously been shown to increase antibody responses, but the underlying mechanism has been unclear. We have here investigated the GC reaction after targeting antigen to MHCII in (i) a defined model with T and B cells of known specificity using adjuvant-free vaccine proteins, and (ii) an infectious disease model using a DNA vaccine. MHCII-targeting enhanced presentation of peptide: MHCII on APCs, and increased the numbers of GC B cells, T_FH, and plasma cells. Antibodies appeared earlier and levels were increased. BCR of GC B cells and serum antibodies had increased avidity for antigen. The improved responses required cross-linking of BCR and MHCII in either cis or trans. The enhanced GC reaction induced by MHCII-targeting of antigen has clear implications for design of more efficient subunit vaccines.

Fig. 1

Vaccine proteins and model system. a Targeted vaccine proteins have a scFv^αI-Ed targeting unit (specific for I-E^d) linked to the scFv³¹⁵ antigen via a human C_H3 dimerization domain. In non-targeted vaccine proteins, the scFv^αI-Ed has been exchanged with a scFv^αNIP specific for the hapten NIP. b Purified vaccine proteins analyzed by SDS-PAGE, either with or without β-mercaptoethanol. The displayed bands are from the same gel and were processed in parallel. c Targeted vaccine proteins bind to I-E^d MHCII molecules on APC, followed by processing and presentation. The scFv³¹⁵ antigenic unit contains an idiotypic(Id)-peptide in the CDR3 loop of its Vλ2 region; this Id-peptide is presented on MHCII molecules (I-E^d) to Id-specific CD4⁺ T cells from TCR-transgenic mice. In addition, the pId³¹⁵:I-E^d complex can be physically recognized by a T-cell receptor mimetic (TCRm) in a scFv format. d Model system for testing vaccine proteins in T–B-cell collaboration experiments. B cells from anti-Id BCR knock-in (anti-Id^DKI) mice have V regions from an anti-Id mAb (Ab2-1.4) that binds to the antigen binding site of scFv³¹⁵. Anti-Id B cells can therefore bind the vaccine protein in two ways: (i) the scFv^αI-Ed part of targeted vaccine protein, or (ii) the scFv³¹⁵ antigenic part common to targeted and non-targeted vaccine proteins. Presentation of the Id-peptide on I-E^d can be detected either by Id-specific CD4⁺ T cells from TCR-transgenic mice, or by TCRm. e Binding of purified vaccine proteins to spleen B cells (CD19⁺), macrophages (F4/80⁺ CD64⁺), DCs (Lin⁻CD11c^hi), and T cells (CD3⁺) from BALB/c mice, and to anti-Id B cells of anti-Id^DKI mice

Fig. 2

MHCII-targeting enhances signaling and peptide:MHCII presentation. a Symbols. b, c Response of negatively enriched anti-Id B cells in the presence of 500 nM of the indicated vaccine proteins. b Calcium flux. The arrow indicates time point of added ligand. c Phosphotyrosine levels at indicated time points measured in western blot. Relative phosphotyrosine levels quantified from the blot are shown as the area under the peaks normalized to loading control. The blots are from the same experiment and were processed in parallel. d Anti-Id B cells were incubated with 1 nM of vaccine proteins for 20 h and analyzed for expression of the indicated surface markers in flow cytometry. e, f Splenocytes from anti-Id^DKI or BALB/c mice were incubated for 16 h with titrated doses of the indicated vaccine proteins. Surface expression of pId³¹⁵:I-E^d complex was detected with a TCRm on the indicated cell types. e Detection of pId³¹⁵:I-E^d on anti-Id B cells. f Detection of pId³¹⁵:I-E^d on BALB/c B cells, macrophages, and DCs. Flow panels in e, f show pId³¹⁵:I-E^d signal after incubation with 1 µM targeted protein and gating for cell subset (dashed) and pId³¹⁵:I-E^d positive signal (solid). All experiments are representative of two or three independent experiments. d–f n = 5 per group. Mean ± SEM. *p < 0.05 and **p < 0.01, (e–f; αMHCII-scFv³¹⁵ vs. αNIP-scFv³¹⁵) unpaired two-tailed Student’s t test

Fig. 3

Targeting antigen to MHC class II molecules increases proliferation of T and B cells in vitro. a Symbols. Naive T and B cells were enriched by negative selection from the spleens of TCR Tg and anti-Id^DKI mice (Supplementary Fig. 2), or BALB/c mice. b–h Either T cells or B cells were irradiated (irr.), and indicated mixtures of 5 × 10⁴ T cells and 1 × 10⁵ B cells were seeded with titrated amounts of indicated vaccine proteins. Proliferation was assayed by ³HTdR incorporation. i, j Id-specific T cells and anti-Id B cells were CFSE-labeled and cultured (1:1, 5 × 10⁵) together with 1 nM of the indicated vaccine proteins for 5 days. i Flow cytometry analysis of CFSE signal and expression of CD69 on Id-specific T cells. j Anti-Id IgM levels in supernatant. All experiments are representatives from single experiments repeated two or three times. b–h n = 3 per group. j n = 4 per group. Mean ± SEM. *p < 0.05 and **p < 0.01, (b–h; αMHCII-scFv³¹⁵ vs. αNIP-scFv³¹⁵) unpaired two-tailed Student’s t test

Fig. 4

MHCII-targeting of antigen increases antibody responses in vivo independent of dendritic cells. a Experimental layout and symbols. b Anti-Id IgG levels in sera over time in groups receiving different amounts of vaccine proteins. c Anti-Id IgG1, IgG2a, and IgG2b levels in serum on day 14. d Experimental layout using either NSG or BALB/c mice as recipients. Anti-Id IgG levels in sera were measured on day 14. n = 4 mice per group. Mean ± SEM. *p < 0.05 and **p < 0.01, (b; αMHCII-scFv³¹⁵ vs. αNIP-scFv³¹⁵) unpaired two-tailed Student’s t test

Fig. 5

Increased proliferation of T and B cells in vivo after MHCII-targeting of antigen. a Experimental layout and symbols. Transfer of congenically marked T and B cells (Supplementary Fig. 6b). b Proliferation of Id-specific T cells in LNs and spleen. c Proliferation of anti-Id B cells in LNs and spleen. d Fractions and absolute numbers of anti-Id B cells with a GC phenotype (PNA^hi IgD^lo) in the spleen 7 and 14 days after immunization. n = 4 per group. Mean ± SEM. *p < 0.05; two-tailed Mann–Whitney test

Fig. 6

MHCII-targeting of antigen increases the GC response. a Experimental layout and symbols. b Gating strategy for GC B cells (left panel) and their quantification (right). c Identification of early plasma cells (CD45.2⁺ CD4⁻B220^lo MHCII^hi CXCR4⁺ CD138⁺) and their quantification. d Gating of T_FH cells and their quantification. e Upregulation of CD40L on Id-specific T cells. f Representative micrographs of immunostained cryosections of spleens. Scale bar is 200 µm. g Quantification of GCs with GL7⁺ cells and interspersed Id-specific T cells. 120 10x fields were counted in total per group and represented as number of GC per cryosection of spleen. n = 4 per group. Mean ± SEM. *p < 0.05 and **p < 0.01, two-tailed Mann–Whitney test

Fig. 7

MHCII-targeting of influenza hemagglutinin delivered by a DNA vaccine increases germinal center differentiation of B cells. a Experimental layout and symbols. BALB/c mice were vaccinated i.d. with the indicated plasmid DNA constructs followed by electroporation. Draining LNs were harvested at 3 and 5 weeks post vaccination. b Gated GC B cells were stained with recombinant rHA^Y98F (66 nM) (Supplementary Fig. 7). Representative data are shown. c Absolute numbers of HA-reactive GC B cells in draining LNs. d Gated GC B cells from week 3 and 5 were stained with serially diluted rHA^Y98F probe. e BCR avidity measurements at week 5 with affinity constant determined from non-linear curve fitting of binding curves in d. f Endpoint titers of HA-specific IgG in serum at week 5. g Avidity of week 5 serum anti-HA IgG determined by Urea-wash ELISA. h–i Lymphocytes collected at week 3 were stimulated with MHCII-(SVSSFERFEIFPK, HNTNGVTAACSHEG) or MHCI- (IYSTVASSL) restricted HA peptides or rHA, and the number of IL-4 h and IL-21 i secreting cells determined by ELISPOT. j Sera collected at week 3 were assayed for CXCL13 by ELISA. k Quantification of bone marrow HA-specific plasma cells at week 5. l–m Schematic representation of two possible mechanisms for enhanced B-cell responses observed by MHCII-targeting. l Cross-linking of BCR and MHC class II on a single B cell. m Vaccine proteins could form a bridge between an APC and a B cell, in an APC-B-cell synapse. c–e, k n = 3 per group, f–g, j n = 6 per group, h–i n = 4 per group. Mean ± SEM. *p < 0.05 and **p < 0.01 e extra sum-of-squares F test, c, f–k unpaired two-tailed student’s t test