Gp96-Ig-SIV VACCINES INDUCE PREDOMINANT IMMUNE RESPONSES AT MUCOSAL SITES
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1 Gp96-Ig-SIV VACCINES INDUCE PREDOMINANT IMMUNE RESPONSES AT MUCOSAL SITES Natasa Strbo M.D. Ph.D. Paris, AIDS Vaccine 29 October 19 22
2 Why is cell secreted gp96 Ig vaccine approach innovative and unconventional? Within one molecule strong adjuvant properties for activation of innate immunity and antigen specificity for cross priming T cells Induces predominant immune responses at mucosal sites
3 Mechanisms of immunogenicity of gp96-peptide complexes Cell necrosis (virus infected cell, tumor cell) SP PC Gp96-peptide complex LOX-1 TLR2 TLR4 NF-kb CD91 Cross-presentation 98 2 Presentation of peptides Up-regulation of co-stimulatory molecules (, 6, CD4) and MHC II 8 2 OT1-gfp APC Cytokines (TNFα,IL1β,IL-12,IL-1,GM-CSF) Chemokines (MCP-1, MIP-1, RANTES) and NO release
4 Generation of secreted gp96 Ig 4 3 1ng/ml 5ng/ml higg1 293-gp96-Ig Gp96-Ig Gp96-Ig transfected transfected Gp96-Ig Mean OD ng/ml 125ng/ml 62.5ng/ml 31.25ng/ml 15.6ng/ml 7ng/ml dilutions 96 kda higg1 Gp96-Ig MHC I Gp96 KDEL GOLGI Gp96- Ig MHC I ER gp96 Gp96 KDEL TAP ANTIGEN A Gp96-Ig transfected cell Strbo N et al. AJRI 28.
5 Secreted gp96 Ig Vaccines Secreted gp96 Ig Vaccines Strbo, N., Oizumi, S.,et al. Immunity 18, (23): Robust expansion of CTL SP 98 2 PC 8 2 Oizumi, S., Strbo N, et al. J Immunol 179, (27): Independent of CD4 help (CD4 L) Subject to regulation by T regs OT1-gfp Recruit and activate DC and NK Local CTL expansion independent of LN Cell number x1 3NK1.1 F4/8 dim CD11c + DC EG7-gp96-Ig EG More than million-fold enhanced CTL cross priming over protein Day post immunization
6 NIH-3T3-OVA-gp96-Ig cells administered i.p. i.p. mediate strong systemic and and mucosal OT-I OT-I expansion Intraepithelial lymphocyte NIH-3T3-OVA -gp96-ig PPL IEL LPL Villus CCR9 CCL25 NIH-3T3-OVA CD13 E-cadherin 4.1 M cell 54.4 Gfp-OT-1 Peyer s patch MAdCAM-1 HEV 6.7 Gfp-OT-I (%-/+SEM of -gate) Brush border PPL* 3T3-OVA 3T3-OVA-gp96-Ig * IEL ** LPL CCL25 Crypt Folicle CD62L α4β7 Basement membrane CCR9 HEV MAdCAM-1 α4β7 Afferent lymphatics Lamina propria lymphocyte Afferent lymphatics Mesenteric lymph node Strbo et al. Mucosal Immunol 29
7 Gp96-Ig-induced activation/proliferation and and migration of of OT-I OT-I cells to to the the intestinal lamina propria after intra peritoneal immunization #CD11c+MHCII+ CD13+ cells (x1 4 ) in PEC CD11c high+ MHCII high+ CD13+ 1 *** 3T3-OVA DC 7 3T3-OVA-gp96-Ig PBS * Days after immunization 3T3-OVA-gp96-Ig i.p. CCR9 DC CD13 OT-I Peritoneal Cavity CCR9 α4β7 OT-I, medium CFSE Lymphatic vessels CD Blood circulation OT-I CD13- Lamina propria MAdCAM1- Expressing Endothelial cell Strbo et al. Mucosal Immunol 29 Summary I: Cell secreted gp96-ig induces predominant mucosal -CTL based immunity. Gp96-Ig induces migration of effector memory cells equipped with cytotoxic molecules (granzyme B) into the mucosal compartment by inducing selective expression of gut-homing markers (CCR9). % CCR9+cells * 3T3-OVA -gp96 ** CD13 + DC CD13 - DC PBS
8 Can gp96 induce HIV specific CTL? Yes, in HLA A2 transgenic mice; in SCID hu mice (P3 2) H2d HLA-A2 Vaccine: 293-HIVgag-gfp-gp96-Ig Day Day 14 Day 28 Day HIVgaggfp-gp96-Ig i.p. FACS HLA A2 transgenic mice HLA-A2 HIV-gag pentamer SPL 1.3% PEC 6.6% LPL 1.5% m
9 SIV Gp96 Vaccination of NHP SIV Gp96 Vaccination of NHP HYPOTHESIS SIV-gp96-Ig vaccine will be able to prime and boost systemic and mucosal immunity Vaccine Cell SIV Gp96 Ig SIV gag, 3T3 env,retanef ova gp96 HEK 293 Gp96 Ig Lysis SIV infected cells TLR2 CD91 TLR4 IFN γ SIV NK DC IL 12 SIV MAMUTCR B7.1 B7.2 CD28 OT I OT I OT I Clonal Expansion, Differentiation
10 Cell-based SIV-gp96-Ig vaccine Cell-based SIV-gp96-Ig vaccine a Anti Grp94 (gp96) Anti SIVnef Anti SIVgag Anti SIVgp b Mean OD 4 1ng/ml 3 5ng/ml ng/ml higg1 293-RTNgp96Ig /gag/gp RTNgp96Ig /gag/gp16 irradiated 125ng/ml 62.5ng/ml 31.25ng/ml dilutions 25
11 Animals and vaccination schedule Animals and vaccination schedule Group II Gp96-Ig-SIV Dose 5 ug M943 M944 Group I Gp96-Ig-SIV Dose 1ug M62 M94 Group III Gp96-Ig-SIV Dose 5 ug M945 M947 Group IV Control 293 (5x1 6 ) M96 M964 Vaccinated* M62 M94 M943 M944 M945 M947 Control* M96 M gp96-Ig-SIV (i.p.) Euthanize (i.p.) weeks Euthanize weeks * Mamu-A*1+ females macaques
12 a SIV-gp96 immunization-induced SIV-Gag- and and Tat-specific T cells in in the the rectal mucosa %Gag CM9+ and Tat SL8+ + cells Rectal mucosa LPL weeks post immunization b Gag CM9 Week 1 Week 2 Week c % Gag CM9+ + T cells *** Gag 5μg SIV-gp96 control (293) % Tat SL8+ + T cells ** Tat 5μg SIV-gp96 control (293) LPL InLN LPL InLN LPL InLN LPL InLN
13 SIV-gp96-Ig vaccine induces Gag- and and Tat-specific T cells in in the the lamina propria from jejunum, ileum and and colon. 6 a RECTUM 5 4 b % tetramer+ (within gate) GagCM9 TatSL control JEJUNUM control ILEUM control COLON ND control μg gp96-ig secreted/24h Gag CM9 Tat SL8 M964 (control) M945 (5μg) M964 (control) M945 (5μg) Rectal mucosa Jejunum Ileum Colon
14 SIV-gp96-Ig vaccine induces Gag- and and Tat-specific T cells in in the the intaepithelial compartment b a %Gag CM9+ and Tat SL8+ +cells Rectal mucosa IEL (control) SSC IEL rectal mucosa M945 (5μg) β weeks post immunization FCS Gag CM9 IEL rectal mucosa M964 (293).3 FCS β CD13 granzymeb
15 SIV SIV gag-, tat-, tat-, env-specific polyfunctional + and and CD4+ T-cell responses in in lamina propria and and intraepithelial compartment of of SIV-gp96-Ig vaccinated macaques. Lamina propria lymphocytes Intraepithelial lymphocytes M943 5μg M943 5μg SIV gag SIV tat SIV env %.4% 2.9% SIV gag SIV tat SIV env % 3.6% 12% CD % % % IFNγ IL
16 Summary SIV-gp96 vaccination induces strong mucosal immunity in LPL and IEL Mucosal immune response is poly-specific and multifunctional Mucosal immune response has memory character and is strongly boosted Cell secreted SIV-gp96 vaccines have great prophylactic and therapeutic potential
17 NCI/NIH Genoveffa Franchini Monica Vaccari Barbara Felber George Pavlakis Acknowledgments University of Miami Podack lab Advanced BioScience Laboratories, Inc Deborah Weiss Jim Treece Eckhard R Podack Savita Pahwa Michael Kolber D-CFAR Core: Laboratory Sciences (Flow cytometry core) NIH/NIAID R33AI NIH/NIAID P3AI Koichi Yamazaki Satoshi Oizumi Louis Gonzalez Vadim Deyev Taylor Schreiber Dietlinde Wolf Eva Fisher Emily Leibovitch Yangping Xiao Ernesto Luna Lesley De Armas Kirill Lyapichev Alejo Morales Matthew Tsai Samia Khan
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