Generation of Robust Antibody Responses to HIV-1 MPER Antigens in Mice Reconstituted with Cultured B cells

Transcription

1 Generation of Robust Antibody Responses to HIV-1 MPER Antigens in Mice Reconstituted with Cultured B cells T. Matt Holl 1, Masayuki Kuraoka 1, Dongmei Liao 1, Laurent Verkoczy 2, M. Anthony Moody 2, Munir Alam 2, Barton F. Haynes 1,2 and Garnett Kelsoe 1 Department of Immunology 1 and Human Vaccine Institute 2, Duke University Medical Center

2 Why are Broadly Reactive Neutralizing HIV-1 Antibodies Rare? Broadly neutralizing monoclonal antibodies have been generated; however, these antibodies are frequently polyreactive, bind self-antigens and have structural characteristics of autoantibodies. The tolerance hypothesis predicts that B cells that could make rare broadly HIV-1 neutralizing antibodies are deleted by tolerance mechanisms that limit self-reactivity.

3 Why Are Broadly Reactive Neutralizing HIV-1 Antibodies Rare? We have tested the tolerance hypothesis by determining whether MPER-specific B cells are generated, but then lost to immunological tolerance during B-cell development. In addition, we have asked if B-cell populations grown in vitro - in the absence of the bone marrow s tolerizing environment - are enriched for autoreactivity and the capacity to recognize and respond to MPER antigen.

4 B Lymphopoiesis: Tolerance Checkpoints Central B-cell Tolerance Small pre-b Immature T1 T2 Mature 60%-70% 20% Loss of Autoreactive B Cells during Maturation

5 The 2F5 mab Recognizes Conserved Cellular Antigens HEp-2 2F5 mab NIH-3T3 Haynes et al., Science 2004 Human Mouse

6 B-Cell Tetramers to Identify MPER-specific Cells gp41 MPER epitope QQEKNEQELLELDKWASLWN HIV-1 Gelderblom, HR, AIDS 5, 1991 MPER-tetramer + B cell Antibody:Tetramer Interaction

7 Methods B-cell tetramers were used to determine the frequency of MPER-specific cells. Stromal cell-independent, B-cell cultures were used to generate B cells outside the tolerizing environment of the bone marrow. C57BL/6 mice were used as controls in immunization studies. Lymphocyte-deficient, RAG1 -/- mice were reconstituted with cells that developed outside the bone marrow s tolerizing environment for immunization studies.

8 Specificity of B-Cell Tetramer Reagents

9 Specific Labeling of B-Cell Lines by MPER Tetramer Tetramer + Cells (%) Bare tetramer R4A tetramer APC Signal (M.F.I.) R4A tetramer Bare tetramer 0 Unst L 1:0.6 1:1.3 1:2.5 1:5 Tet. Unst L 1:0.6 1:1.3 1:2.5 1:5 Tet. 1:2.5 1:5 1:10 1:20 Pept. 1:2.5 1:5 1:10 1:20 Pept. Molar Ratio (Label:Inhibitor) unlabeled MPER tetramer unlabeled MPER peptide unlabeled R4A (dsdna) tetramer unlabeled R4A (dsdna) peptide P3 cell line

10 Tetramer Labeling of Bone Marrow B Cells: Inhibition by Homologous Peptide >80% inhibition Tetramer reagents bind specifically to B cells

11 Frequencies of MPER-reactive B cells Before and After Tolerance Checkpoints

12 Frequencies of HIV-1 MPER-Reactive B cells Fall After a Major Tolerance Checkpoint in Bone Marrow Fraction of Gated Population (%) * * 0 P/P Imm T1 T2 MF MZ MPER tetramer - BM MPER tetramer - Spl R4A (dsdna) tetramer - BM R4A (dsdna) tetramer - Spl

13 Generation of B cells In Vitro - Outside of the Tolerizing Environment of Bone Marrow - as a Source of MPER-reactive Lymphocytes

14 In Vitro B-Cell Development: Culture Derived B Cells Mature Normally B6 BM ex vivo IL-7 Cultured B-cells BAFF IgD IgM

15 Activated In Vitro, Culture Derived B Cells Secrete Autoantibody ELISpot IgD Culture with LPS/BAFF ELISA IgM Imm. Fluorescence C4 -/- C. luciliae Assay detection of dsdna-specific antibody RAG -/- B6 CD B cells

16 MPER-reactive B cells are Generated In Vitro Outside of the Tolerizing Environment of Bone Marrow 0.5 Fraction of Gated Population (%) * * P/P Imm T1 T2 * * MPER tetramer R4A (dsdna) tetramer Bare tetramer

17 Flow Cytometric Sorting of Tetramer + B Cells Enriches MPER-specific B cells 12-Fold Control ELISpot MPER-2F5 ELISpot Sorted Unsorted

18 Long Term Reconstitution of RAG1 -/- Mice by Culture Derived Cells: Persistence of Self-reactive B cells After Transfer

19 RAG-1 -/- Mice Reconstituted with CD cells have Elevated IgG Serum Auto-antibody C. luciliae Assay C4 -/- RAG -/- B6 CD-RAG C4 -/- HEp-2 Human Epithelial Cells RAG -/- B6 #1 CD-RAG #1 B6 #2 CD-RAG #2

20 The 2F5 mab Recognizes Conserved Cellular Antigens HEp-2 2F5 mab NIH-3T3 Haynes et al., Science 2004 HEp-2 CD-RAG Serum NIH-3T3 Human Mouse

21 RAG1 -/- Mice Reconstituted with Culture Derived Cells: Strong Humoral Immune Responses to HIV-1 Env gp41 MPER Antigens

22 CD-RAG Mice Generate Robust GC Responses after MPER Peptide Immunization B220 hi GL-7 hi cells d16 (%) * * B6 B-cell follicle CD-RAG T-cell zone 0 B6 Naive CD-RAG B6 CD-RAG B6 CD-RAG primary secondary Immune B-cell follicle B220 TCRβ GL-7 T-cell zone

23 MPER IgG Responses by RAG1 -/- Mice Reconstituted with Culture Derived Cells: Higher (3- to 10-fold) than Controls NP-specific serum IgG (μg/ml) B6 Control Antigen CD-RAG B6 CD-RAG MPER-specific serum IgG (μg/ml) B6 CD-RAG B6 MPER Antigen * * CD-RAG ** * B6 CD-RAG Naive Immune Naive primary secondary Immune

24 Summary HIV-1 MPER-reactive B cells are frequent in bone marrow, but rare in the spleen. Loss of MPER-reactive B cells coincides with a major checkpoint of B-cell tolerance and is correlated with poor antibody responses of C57BL/6 mice after MPER immunizations. B-cell populations that develop in vitro are enriched for cells that generate self- and MPER-reactive antibodies after stimulation with a TLR-ligand (LPS). RAG-1 -/- mice reconstituted with culture derived cells constitutively express high levels of serum autoantibody, and respond to MPER immunization with 3- to 10-fold higher quantities of MPER-specific IgG serum antibody compared to C57BL/6 controls.

25 Conclusions The i) significant decrease in the frequency of MPERspecific B cells during B-cell maturation, and ii) enhanced MPER serum IgG responses in mice reconstituted with B cells that have developed away from the bone marrow s tolerizing environment, are consistent with the hypothesis that immunological tolerance limits humoral immune responses to HIV-1 Env gp41 MPER antigens.

26 Future Studies Determine HIV-1 neutralization titers in MPER serum antibody from immunized C57BL/6 and reconstituted RAG1 -/- mice. (Limitations: neutralization in mouse serum requires IgG concentrations 100 μg/ml and it may be necessary to concentrate serum IgG fractions before testing.) Generate MPER-specific hybridomas from immunized C57BL/6 and reconstituted RAG1 -/- mice for genetic and neutralization studies.

27 Acknowledgements Kelsoe Lab Yoshihiro Ueda Huaiyong Chen HVI Flow Facility John Whitesides Patti McDermott Derek Cain Pilar Harvey Kathleen O Hara

28 2F5 mab Competitively Inhibits MPER Antigen Binding of CD-RAG Sera 1.6 Absorbance (450nm) F5 (ng/ml) CD-RAG Serum 13H11 mab

29 RAG-1 -/- Mice Reconstituted with CD cells have Elevated Igκ Serum Auto-antibody C. luciliae Assay C4 -/- RAG -/- B6 CD-RAG C4 -/- #1 HEp-2 Human Epithelial Cells RAG -/- #1 B6 #1 CD-RAG #1 C4 -/- #2 RAG -/- #2 B6 #2 CD-RAG #2

30 Achilles Heels of the HIV-1 Trimeric Envelope Self Carbohydrates 2G12 Anti- Carbohydrate Ab Uncommonly Made Abs CD4bs 1b12 CD4 Binding Site Ab Uncommonly Made Abs dsdna Commonly Made Abs Non-neutralizing Immunodominant gp41 region N C' C' N Membrane Proximal Region 2F5 4E10 Z13 lipids C C C Uncommonly Made Abs

31 Definition of B-cell Compartments B220 + BM B220 + Spl IgM Mature T2 T1 Mature Imm T2 Pro/Pre T1/MZ IgD IgD IgM CD23 CD21 CD93 CD23 CD21 CD93

32 Paratopic Structure of the 2F5 Antibody: A. Distinct Sites for Lipid and Peptide Interaction 2F5 h L100 a A: 2F5 h F100 b A: 2F5 h LF100 ab AA: RRGPTTAFGVPIARGPVNAMDV RRGPTTLAGVPIARGPVNAMDV RRGPTTAAGVPIARGPVNAMDV B. 2F5 14 x 10-9 M 2F5 h L100 a A 55 x 10-9 M 2F5 h L100 b A 52 x 10-9 M 2F5 h LF100 ab AA 69 x 10-9 M C. 2F5 h R95A 472 x 10-9 M 600 2F5 h R95A: ARGPTTLFGVPIARGPVNAMDV 500 2F F5 h R95A Response F5 h LF100 ab AA M. Alam, DHVI 0 2F5 h L100 a A Time (s)

33 B Lymphopoiesis: Tolerance Checkpoints Igh Igκ/λ CLP Pro-B Pre-B Immature Mature Induction of tolerance T1 T2 Pre-BCR Transitional B Cell Stages: T1 and T2 IgM IgD