Vaccine Delivery and TLR Ligands Influence the Quality of T cell Responses in NHP. Robert A. Seder, M.D. Vaccine Research Center NIAID, NIH

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1 Vaccine Delivery and TLR Ligands Influence the Quality of T cell Responses in NHP Robert A. Seder, M.D. Vaccine Research Center NIAID, NIH

2 Vaccines Against HIV, Malaria and Tuberculosis Will Require Neutralizing Antibody and CellMediated Immunity Design vaccines that elicit broadbased immunity Develop methods to characterize better T cell responses Define a T cell response that will be optimal for mediating protection

3 Rational for protein based vaccines Protein vaccines induce broadbased immune responses Antibody Th1 and CD8 T cell responses Protein vaccines are not limited by preexisting immunity Used to induce or enhance T cell responses Vaccine formulation and the type of TLR ligand are important variables for optimizing responses

4 Toll like receptor expression in antigen presenting cells Myeloid DCs Plasmacytoid DCs Monocytes Bcells IL12 IFNa TLR expression TLR 4 TLR 7 TLR 8

5 Signaling Pathways for TLR Synergy Trinchieri and Sher Nature Reviews Immunology 7, Napolatani et al. Nature Immunology 6, (25)

6 Vaccine Formulation Oil/water (SE), Liposomes, ISCOMS, Montanide Conjugation of protein to TLR 7/8 ligand HIV GagTLR 7/8 conjugate induces potent antibody and multifunctional Th1 and CD8 T cell responses in nonhuman primates Linker protein

7 Goal of Studies Compare the magnitude and quality of T cell responses in nonhuman primates varying the vaccine formulation and the type of TLR ligand Formulation Administer protein with TLR 7/8 ligand in oil/water emulsion (SE) Protein conjugated to the TLR 7/8 ligand Determine whether combining TLR ligands elicit higher responses Compare TLR 4 (MPL) TLR7/8 ligand vs MPL or TLR 7/8 alone

8 Immunization Protocol Vaccine Groups 1. MML proteintlr 7/8 conjugate 2. MML proteintlr 7/8 SE 3. MML protein MPL/SE 4. MML protein MPL/SETLR 7/8 Immunization:, 1 and 3 months Analysis of T cell responses: MagnitudeFrequency of IFNγ producing T cells by Elispot QualityAssess T cell response by multiparameter flow cytometry

9 MPLTLR 7/8 ligand induces more IL12 from monkey PBMCs than either TLR alone IL12 (pg/ml) MPL MPLTLR7/8 TLR7/8

10 MMLTLR 7/8 conjugate induces the highest frequency of IFNγ producing cells IFNγ SFC per 1 6 cells st vaccination 2nd vaccination 3rd vaccination MemoryTime of Infection TLR7/8 Conjugate TLR7/8/SE MPL/SE MPL/SE TLR7/8 MML Protein Formulation

11 Defining the quality of the response with multiparameter flow cytometry Staining panel Dead cells (ViViD) CD3 (PerCP Cy5.5) CD4 (Alexa 7) CD8 (APC Cy7) CCR7 (Biotin / QD 655 SA) IFNγ (APC) IL2 (PE) TNF (PE Cy7) CD3 CD4 FSCA % Singlets 1 2 FSCH % CD3 CD SSC CD FSCH 97.7 % CD4 T cells ViViD 9.6 % Live cells 1 2 FSCH Boolean gating IL2 TNF CD4 1 FSCH % % % 1 4 IFNγ 1 4 IL2 1 4 TNF % Lymphocytes IFNγ IL2 TNF IFNγ IL2 TNF IFNγ Fraction of response

12 MultiFunctional Th1 Responses (IFNγ IL2 TNF ) Correlate With Protection Protection Protection Percent of total response

13 Why are multifunctional cells better? Secretion of all three cytokines Increased effector function by secreting IFNγ and TNFα IL2 production leads to expansion of cells Secrete more cytokine per cell

14 Multifunctional Th1 cells produce more IFNγ and TNFα IFNγ, TNF, IL2 Triple Positive Double Positive Single Positive TNFα IFNγ Previously infected Mouse More TNFα More IFNγ The fluorescence intensity of the cells is related to how much cytokine they have produced. Triplepositive cells appear to produce considerably more IFNγ and TNF per cell. Cytokine production by a population can be estimated by the MFI (median fluorescence intensity) IFNγ MFI of Single Positives: ~2, IFNγ MFI of Triple Positives: ~2, Each triple positive cell is binding 1x as much antiifnγ antibody as each single positive indicating that they are capable of secreting 1x as much cytokine on a per cell basis.

15 MMLTLR 7/8 conjugate and MMLMPL/SE elicit distinctly different qualitative responses Frequency of cytokineproducing memory CD4 T cells (%) IFNγ IL2 TNF MMLTLR7/8 Conjugate MMLMPL/SE (Pies) MMLTLR7/8 Conjugate MMLMPL/SE

16 MMLTLR 7/8 conjugate vaccines elicits more potent IFNγ producing cells than MMLMPL/SE MMLTLR7/8 Conjugate IFNγ MFI (x1 3 ) MMLMPL/SE IFNγ IL2 TNF

17 Summary ProteinTLR 7/8 conjugate immunization elicits potent and durable T cell responses in nonhuman primates Magnitude and quality of T cell responses are better than vaccines using TLR 4 or TLR 7/8 alone or in combination using SE as a vehicle No synergy in vivo with MPL and TLR7/8 ligand Vaccine formulation and the type of TLR ligand are important variables for optimizing both the magnitude and quality of the T cell responses

18 How does the proteintlr 7/8 conjugate vaccine elicit multifunctional T cell responses? TLR7/8 agonist CD123 Plasmacytoid DCs CD11c Myeloid DCs Tcell IFNγ IL2 TNFα IFNα IL12 Which DC subsets are required for induction of Th1 and CD8 T cell responses and imprint the quality of the T cell response?

19 Acknowledgments Vaccine Research Center Tricia Darrah Ross Lindsay Dipti Patel Paula Deluca Kathy Foulds Ulli WilleReece Barbara Flynn Mario Roederer Infectious Disease Research Institute Steven Reed