Estimating primate effector T cell responses to DNA vaccination Oct 22 nd, 21 Devon J. Shedlock, PhD th Vaccine Renaissance Conference Estimating vaccineinduced effector T cell responses Vaccineinduced immunity is evaluated using standard immunological assays Polychromatic flow cytometry (PFC) ELISpot assay Proliferation assay Etc YF Vaccine These assays require antigenic restimulation during ex vivo primary cell culture May introduce bias May underestimate Simple phenotypic analysis may offer additional information when examining an effector response Tetramer staining Activation markers Dryvax Miller et al., Ahmed; 28; Immunity 1
Potential bias from standard, ex vivo immunological assays Assay Type Ag Restimu lation? Chemicals required Potential Chemical Bias Ex vivo Culture Potential Culture Bias Effector Readout Potential Readout Bias Proliferation Assay CFSE Cytotoxicity 96 1 h (6 days) Longterm culture/division affects phenotype/function CFSE dilution NL IFNg ELISpot Functional Y 16 2 h IFNg secretion Not all effectors make IFNg NL Polychromatic Flow Cytometry Golgi poisons Cytotoxicity 6 h Multiple cytokine production limited by which cytokines are assessed IFNg responses alone underestimate effector response Percentage of IFNg + response: 6% 77% 88% % Average functional response profile of the 2 donors and stratified by virus. PBMC from each subject were simultaneously assayed for perforin, IFNγ, IL2, TNFα, and CD17a upregulation. Distribution of responding CD8+ T cells across 16 different functional subsets. Possible functional combinations that were not observed are omitted for clarity. EBV responses are illustrated with blue bars, flu responses with black bars, CMV responses with red bars, Ad responses with orange bars, and SEB responses with green bars. P = Perforin, 2 = IL2, 7 = CD17a, G = IFNγ, T = TNFα. The yaxis denotes the proportion of the total CD8+ T cell response to the given virus that produces a specific profile of functions. Makedonas et al., Betts; 21; PLoS Path During some human infections, up to % of effector may not make IFNg Standard ELISpot assay measuring IFNg alone may underestimate 2
Reduction of potential bias by simple phenotypic analysis Assay Type Ag Restimu lation? Chemicals required Potential Chemical Bias Ex vivo Culture Potential Culture Bias Effector Readout Potential Readout Bias Proliferation Assay CFSE Cytotoxicity 96 1 h (6 days) Longterm culture may bias phenotype/function CFSE dilution NL IFNg ELISpot Polychromatic Flow Cytometry Functional Y Golgi poisons Cytotoxicity 16 2 h 6 h NL IFNg secretion Multiple cytokine production Not all effectors make IFNg limited by which cytokines are assessed Tetramer staining Activation marker staining Physical N Direct staining Activation marker detection Reagent limited Specificity and background (longitudinal) Activation marker staining: is not Agspecific (no evidence detected of bystander activation (Miller et al., Ahmed; 28; Immunity)) is affected by nonvaccineinduced responses (longitudinal assessment required) Underestimation of vaccineinduced effector T cell responses Stim conditions Peptides Estimated % Response.1 1.% YF Vaccine Infected cells..% Activation markers 1% Dryvax Activation marker staining may be a more comprehensive assessment than ex vivo restimulation assays alone Miller etal. Ahmed; 28; Immunity
Reagents required for evaluating immunity Assay Type Typical form Cost Antigenic processing required? Epitopic Display Potential Functional Bias Peptides Overlapping xmers $$$ N (fluid phase exchange) Limited (xmer length) [Peptide] must be saturating (functional avidity) Proteins/pathogen Infected cells Functional Whole/ Full length/ Truncated Whole/ Irradiated/ Lysed $$ $ Y (Primary APCs) Full/Ag Full (HLArestricted) Requires time and APCs for processing and display Tetramer staining Activation marker staining Physical un/labeled mono/tetramers $$ Reagent limited Activation marker staining: is simple, cheap, and not likely affected by potential biases associated w/antigenic restimulation DNA vaccine development and evaluation Our lab has helped develop and study DNA vaccines Ongoing efforts to increase expression and vaccine immunogenicity Currently several ways to do this: Transcriptional elements Protein expression Nextgeneration methods Kutzler and Weiner; 28, Nat Rev Genetics
Optimized delivery of DNA vaccines using In vivo EP EP enhancement of DNA vaccine expression DNA alone Light + UV pgfp UV DNA + EP +
IFNgproducing SFC/1 6 splenocytes Can level of T cell immunity from LCMV infection be replicated by DNA vaccination w/ep? plcmvnp i.m. or plcmvnp i.m. + EP or 1X LCMV (2x1 i.p.) #1 #2 # B6 (n=/group) Weeks 1 2 6 7 ELISPOT ELISPOT ELISPOT EP enhancement of DNA vaccine immunogenicity 7, 6,, plcmvnp +,, 2, 1, X 1X 2X X X 1X 2X X LCMV pvax plcmvnp ( ug) pvax plcmvnp ( ug) Day 7 p.i. DNA alone DNA + EP 2x1 PFU i.p. Shedlock et al., Weiner; 21; Submitted T cell responses from X DNA vaccination w/ep are ~8% of acute LCMV response 6
Percent survival Percent survival Are immune responses from DNA vaccination w/ep protective against lethal LCMV challenge? plcmvnp i.m. or plcmvnp i.m. + EP or 1X LCMV (2x1 i.p.) B6 (n=6/group) Weeks 2 6 8 1 12 1 8 weeks p.v. Lethal LCMV challenge Shedlock et al., Weiner; 21; Submitted Can DNA vaccination protect against lethal LCMV challenge? i.m. + EP i.m. alone 1 9 8 1X 7 6 2 1 % 2 6 8 1 12 1 16 8% 1 9 8 7 X 6 2 1 2 6 8 1 12 1 16 1% 6% 1 9 8 2X 7 6 2 1 2 6 8 1 12 1 16 Time in Days 1% % 1 9 8 X 7 6 2 1 2 6 8 1 12 1 16 Time in Days 1% % Shedlock et al., Weiner; 21; Submitted DNA + EP is 1% protective after 2 immunizations 7
SSCH LIVE/DEAD CD HLADR Gating Strategy for flowbased activation analysis in NHP DNA Immunization + EP Rhesus macaques (n=18) #1 #2 # # Month 1 2. Prebleed Postimmunization (2 wks p.imm.) Belisle et al., Boyer; 21; Submitted Gating Strategy for flowbased activation analysis in NHP Lymphocytes Live CD+ T cell activation (Effectors) CD+ FSCA T cells CD CD8+ CD8 CD9 Belisle et al., Boyer; 21; Submitted 8
AVE % Activation (Postimm. Prebleed) Control DNA Group #1 DNA Group #2 IFNg+ SFU/1 6 PBMCs HLADR activation correlates with ELISpot data Activation Staining ELISpot 1 12 1 8 6 2 ** CD8+ CD+ 2, 2, 1, 1,, Peptide set #1 Peptide set #2 Peptide set # n=6/group Control DNA Group #1 DNA Group #2 Total AVE ~1, spots = 1.% Agspecific x2. (T cells are ~% of PBMC) x2 (best assume for IFNg) ~ increase in T activation = (.8 7.%) ~ increase in CD8 T activation = (7.6 18.8%) Belisle et al., Boyer; 21; Submitted Gating Strategy for flowbased activation analysis in Humans DNA Immunization + EP Humans (n=16) #1 #2 # Month 1 2 6 7 8 9 bleeds 9
SSCH LIVE/DEAD <APCCy7A>: HLADR <APCCy7A>: HLADR <APCCy7A>: HLADR CD 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 <APCCy7A>: HLADR <APCCy7A>: HLADR <APCH7A>: HLADR 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 <APCCy7A>: HLADR <APCCy7A>: HLADR <APCCy7A>: HLADR <APCH7A>: HLADR 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 HLADR <APCCy7A>: HLADR <APCCy7A>: HLADR <APCCy7A>: HLADR <APCH7A>: HLADR 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 <APCCy7A>: HLADR <APCCy7A>: HLADR <APCCy7A>: HLADR 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 1 1 1 <AF 7A>: CD8 Gating Strategy for flowbased activation analysis in Humans Lymphocytes Live CD+ T cell activation (Effectors) CD+ FSCA T cells CD CD8+ CD8 CD8 HLADR/CD8 activation correlates with ELISpot data IFNgproducing SFC/1 6 6,,,, 2, 1, 126 1 11 17 Prebleed #2+7d #+7d #+1m #+6m Total AVE ~1, spots =.1% Agspecific x2. (T cells are ~% of PBMC) x2 (assume for IFNg) ~increase in activation =.% 2.% #1 #2 # 1 1 1 1 1 1 1 1 1 HLADR Prebleed.6.82.17 # CD8 1 1 1 1 1 1 1 1 1 7 days p.i.#2.6.26.286 1 1 1 1 1 1 1 1 1 1 1 1 7 days p.i.#.82 Measurable in blood if >1, SFC Population gating may enhance phenotype 1.16.9 2.62 1 1 1 1 1 1 1 1 1 1 month p.i.# 1 1 1 1..792 1.1 1.6 1 1 1 1 1 1 6 months p.i.# 1 1 1 1..769.6 1
CD28 <PECyA>: GranzB <PECyA>: Granz B <PECyA>: Granz B 1 1 1 1 1 1 1 1 1 1 1 <PEA>: Tbet <PEA>: Tbet 1 <PEA>: Tbet 1 1 1 1 1 1 <PECyA>: Granz B <PECyA>: GranzB <PECyA>: GranzB 1 1 1 1 1 1 1 1 1 1 1 1 <PEA>: Tbet <PEA>: Tbet <AF 67A>: Tbet 1 1 1 1 1 1 <PECyA>: GranzB <PECyA>: Granz B <PECyA>: Granz B <PECyA>: GranzB 1 1 1 1 1 1 1 1 1 1 1 1 <PETxRedA>: CD28 <PETxRedA>: CD28 1 1 1 1 1 1 1 1 1 1 <PETxRedA>: CD28 <PEA>: Tbet 1 <PEA>: Tbet 1 1 <PEA>: Tbet <AF 67A>: Tbet 1 1 <QD 6A>: CDRA 1 1 1 <QD 6A>: CDRA 1 1 <QD 6A>: CDRA 1 1 1 1 <PECyA>: GranzB <PECyA>: Granz B <PECyA>: Granz B <PECyA>: GranzB 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 <PETxRedA>: CD28 <PETxRedA>: CD28 <PETxRedA>: CD28 <PEA>: Tbet 1 1 1 1 1 1 1 <PEA>: Tbet 1 1 1 1 1 <PEA>: Tbet <AF 67A>: Tbet 1 1 1 1 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 1 1 1 <QD 6A>: CDRA 1 <PECyA>: GranzB <PECyA>: Granz B <PECyA>: Granz B 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 <PEA>: Tbet 1 1 <PEA>: Tbet <PEA>: Tbet <PETxRedA>: CD28 <PETxRedA>: CD28 <PETxRedA>: CD28 <PETxRedA>: CD28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 <QD 6A>: CDRA 1 1 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 1 1 1 1 1 1 1 <PETxRedA>: CD28 <PETxRedA>: CD28 <PETxRedA>: CD28 <PETxRedA>: CD28 1 1 1 1 1 1 1 1 1 <PECyA>: GranzB B 1 1 1 1 1 1 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 1 1 1 1 1 1 1 1 <PETxRedA>: CD28 <AF 67A>: Tbet <PETxRedA>: CD28 <PETxRedA>: CD28 1 1 1 1 1 1 1 1 1 1 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 <QD 6A>: CDRA 1 1 1 1 1 1 1 Phenotype of DNAinduced T cell effectors Total CD8+ Activated CD8+ (HLADR+CD28+) Prebleed 7 days p.i.#2 7 days p.i.# 1 month p.i.# 6 months p.i.# T CM N #1 T E T EM #2 CDRA # # Activated cells have a memory (T CM and T EM ) and effector (T E ) phenotype Lytic potential of DNAinduced T cell effectors #1 Prebleed 7 days p.i.#2 7 days p.i.# 1 month p.i.# 6 months p.i.# Total CD8+ Activated CD8+ (HLADR+CD28+) #2 # Tbet # Lytic effector = Granzyme B+ Tbet+ Perforin+ Activated cells exhibit lytic potential as measured by granzyme B, Tbet, and perforin Lytic granule loading of CD8 T cells is correlated w/immune control of HIV (Migueles, et al., Connors, 28, Immunity) 11
Summary DNA vaccination w/ep is immunogenic in RMs and humans Primate bloodderived, activated T effectors can be estimated by direct HLADR staining (if > 1, SFC by ELISpot) activation data correlates with standard immunological assays Activated T cells have a memory and effectorlike phenotype Lytic potential of effectors can be assessed directly restim triggers degranulation this is first study to demonstrate DNAinduced lytic potential This technique may provide a less biased assessment of global vaccineinduced activation should be combined w/standard immunological assays Acknowledgements David B Weiner Lab Jian Yan, PhD Panyupa Pankhong, PhD Thomas H Shin Kendra Talbott Stephan Wu Mathew Morrow, PhD Bernadette Ferraro, PhD Karuppiah Muthumani, PhD Jean D Boyer Lab Jiangmei Yin Anlan Dai Inovio Pharmaceuticals, Inc. Amir Khan, PhD Niranjan Y. Sardesai. PhD 12