Application of systems biology to identify predictors of HIV vaccine immunogenicity

Transcription

1 Application of systems biology to identify predictors of HIV vaccine immunogenicity Daniel Zak 1, Erica Andersen-Nissen 2, Haesun Park 3, Scott Hansen 3, Karl Mullen 3, Kristi Hamilton 1, Kathleen Kennedy 1, Robert Seder 4, Louis Picker 3, M. Juliana McElrath 2, Alan Aderem 1 1: Institute for Systems Biology, Seattle, WA, USA 2: Fred Hutchinson Cancer Research Center, Seattle, WA, USA 3: Oregon Health and Science University, Beaverton, OR, USA 4: National Institute of Health Vaccine Research Center, Bethesda, MD, USA

2 Overview Objective: identify innate immune response genes that are associated with enhanced HIV vaccine immunogenicity Approach: Seek correlations between expression profiles (microarray data) and functional T cell assays Human studies: HVTN 071 subjects (trivalent MRKAd5 gag/pol/nef) Non-human primate (NHP) studies: SIV gag protein + TLR ligand adjuvants See Alan Aderem s talk for an overview of his lab s Systems Immunology efforts (Today, 5:40pm, Room 2.40)

3 Human studies: HVTN 071 Trial See Erica Andersen-Nissen s talk for more detail (#3 this session) 10 subjects vaccinated with trivalent MRKAd5 HIV-1 gag/pol/nef from the Step trial (Erica Andersen-Nissen, Julie McElrath, FHCRC) Subjects had varying pre-vaccination immunity to vaccine vector (Ad5) Microarray profiling PBMC innate immune responses 6, 24, 72, AND 168hr after primary vaccination (ISB) Affymetrix GeneChip Human Exon 1.0 ST Array Intracellular Cytokine Staining (ICS) profiling HIV-specific T cell responses (Erica Andersen-Nissen, Julie McElrath, FHCRC)

4 Objectives Expression signatures for pre-existing immunity to vector (Ad5): Can Ad5 + subjects be predicted directly from microarray analysis of vaccine response? What is the innate immune gene expression signature for Ad5 + subjects? Expression signatures for vaccine-induced HIV-specific CD8 + T cell polyfunctionality

5 Identifying Ad5 + subjects by microarray analysis Assume Ad5 immune subjects will have attenuated responses to the vaccine, as measured by PBMC microarray response Microarray score (AU) Score subjects by enhancement in signal/noise observed when they are excluded from the analysis PTID

6 Identifying Ad5 + subjects by microarray analysis Assume Ad5 immune subjects will have attenuated responses to the vaccine, as measured by PBMC microarray response 800 Ad5 titer (MERCK) Ad5 Med Ad5 Low Ad5 Med Microarray score (AU) Score subjects by enhancement in signal/noise observed when they are excluded from the analysis PTID

7 Ad5 - profiles enriched for innate antiviral response Ad5 med Ad5 Low Ad5 Med hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr 6hr 24hr 72hr 168hr Gene ontology enrichments for up-regulated genes in Ad5 - subjects Up-regulation in response to vaccine Down-regulation in response to vaccine 490 genes at p < 1x10-6 (one-way ANOVA) p-value Description 6.85E-26 immune response 3.70E-19 response to virus

8 Ad5 Med Ad5 Med Ad5 Low Pre-vaccination expression signatures for Ad5 immunity Ad5 - Ad5 Ad5 titers (MERCK)

9 Ad5 Med Ad5 Med Ad5 Low Pre-vaccination expression signatures for Ad5 immunity Ad5 - Ad5 Ad5 titers (MERCK) Genes Subjects Relative pre-vaccination expression levels (213 genes, p<0.05 Spearman rank correlation)

10 Ad5 Med Ad5 Med Ad5 Low Pre-vaccination expression signatures for Ad5 immunity Ad5 - Ad5 Ad5 titers (MERCK) Genes enhanced in Ad5 immune subjects pre-vaccination are enriched for: -NF-κB pathway (NFKBIA, NFKBIE, RIPK2; p<0.0001) -Cell death pathways (PRF1, CASP5, CD38; p<0.005) -IRF1 expression and IRF1 binding sites (NFKBIA, NFKBIE, CASP5, SMAD1; p<0.005) Genes Subjects Relative pre-vaccination expression levels (213 genes, p<0.05 Spearman rank correlation)

11 Enhanced pre-vaccination IL-2RB expression and enhanced IL-2 in serum of Ad5 + subjects Log2(Expression) IL2RB 8 0 6hr 1d 3d 7d Time post-vaccination Ad5 Med Ad5 Low Ad5 - Log2(Luminex) Cytokine data: Erica Andersen-Nissen, Julie McElrath, FHCRC IL-2 0 6hr 1d 3d 7d Time post-vaccination

12 CD8 + T cell response polyfunctionality: % triple-positive (TNF + IFNγ + IL-2 +, tpcd8 ) (data from Erica Andersen-Nissen, Julie McElrath, FHCRC) Non-progressors consistently maintain higher levels of polyfunctional HIV-specific CD8 + T cells (Betts et al., 2006) 50 tpcd8 High % of responding CD8 + T cells (TNF + IFNγ + IL-2 + ) tpcd8 Med tpcd8 Low 0 PTID ICS of %HIV-gag responding CD8 + T cells (TNF +, IFNγ +, or IL-2 + ) that are triplepositive ( tpcd8 ) in PBMCs, 28 days post-primary vaccination

13 Signatures for CD8 + T cell polyfunctionality tpcd8 High tpcd8 Med tpcd8 Med tpcd8 Med tpcd8 Low tpcd8 Low %tpcd8 + %Triple-positive responding CD8 + T cells

14 Signatures for CD8 + T cell polyfunctionality tpcd8 High tpcd8 Med tpcd8 Med tpcd8 Med tpcd8 Low tpcd8 Low %tpcd8 + %Triple-positive responding CD8+ T cells Vaccine-induced gene expression responses (PBMC, 24hrs Fold-change, 255 genes at p<0.05, Spearman rank correlation) Genes Subjects 24hr Anticorrelated genes enriched for: -Immune system (ex: IDO, CCL3, TLR7, ICOSLG; p<5x10-4 ) -NF-κB regulation (ex: TNF, CARD9, CD40; p<1x10-3 )

15 CD8 + T cell polyfunctionality signature: IDO/INDO T cell inhibitor log2(fold-change) INDO tpcd8 High tpcd8 Med tpcd8 Low 0 6hr 1d 3d 7d Time post-vaccination INDO Log2(FC), Day 1 IDO/INDO activity inhibits T cells, particularly CD8 + (Forouzandeh et al., 2008) T cell proliferation

16 CD8 + T cell polyfunctionality signature: IDO/INDO T cell inhibitor log2(fold-change) INDO tpcd8 High tpcd8 Med tpcd8 Low %tpcd8 + is negatively correlated with INDO induction, 24 hrs post-vac hr 1d 3d 7d Time post-vaccination % tpcd8 + INDO Log2(FC), Day 1 INDO Log2(FC), 24hrs IDO/INDO activity inhibits T cells, particularly CD8 + T cell proliferation (Forouzandeh et al., 2008)

17 pdc CD8 + T cell polyfunctionality signature: BDCA2 (plasmacytoid DC marker, Ag uptake/ inhibitory receptor) Log2(expression) 7 6 CLEC4C/BDCA2 tpcd8 High tpcd8 Med tpcd8 Low Corr. to Pre-vac. % tpcd hr 1d 3d 7d Time post-vaccination CLEC4C Pre-vac expression

18 NHP studies: TLR ligands as adjuvants See Bob Seder s talk for more details (Today, 5:20pm, Room 1.40) NHP immunized with SIV Gag protein + Montanide + TLR ligands (Haesun Park & Louis Picker-OHSU, Bob Seder-NIH/VRC) Profile SIVgag-specific CD4 + & CD8 + T cell responses (Haesun Park & Louis Picker-OHSU, Bob Seder-NIH/VRC) Profile gene expression responses in PBMC and LN (ISB) Myeloid DC (cdc/mdc) (PolyIC) TLR3 Mda5 TLR3 + TLR7/8 Mda5 (3M-012) TLR7/8 Myeloid DC Plasmacytoid DC (cdc/mdc) + (pdc) TLR3 + TLR9 Mda5 SIVgag-specific T cell responses? TLR4 + TLR7/8 Myeloid DC (cdc/mdc) TLR4 (MPL) TLR4 + TLR9 TLR9 (CpG) Plasmacytoid DC (pdc)

19 PolyIC-containing adjuvants (TLR3) induce strongest CD4 + responses (data from Haesun Park & Louis Picker-OHSU, Bob Seder-NIH/VRC) %CD4+ responding to SIV gag with TNF or IFNγ PolyIC-containing adjuvants induce strongest CD4 + CD4 + Median-scaled values over time for %CD4 + T cells responding to SIV gag with TNF or IFNγ in BAL in response to primary vaccination. Up to 60% responding.

20 TLR7/8 more potently induces Interferon Stimulated Genes than TLR3/PolyIC (Day 2 post-vaccination PBMC, qrt-pcr) DDX58/RIG-I Gag Gag+TLR7/8 Gag+TLR3 Gag Gag Gag Gag Gag+TLR7/ Gag+TLR7/ Gag+TLR7/ Gag+TLR7/ Gag+TLR Gag+TLR Gag+TLR Gag+TLR Log2(Fold change) Equivalent results obtained for IFIH1/MDA5 and RSAD2/Viperin

21 Identify novel predictors of immunity: Correlate T cell response magnitudes with gene expression responses Gag-specific CD4 + T cell responses (TNF or IFNγ)

22 Identify novel predictors of immunity: Correlate T cell response magnitudes with gene expression responses Correlated genes Gag-specific CD4 + T cell responses (TNF or IFNγ) Anti-correlated genes Vaccine-induced gene expression responses (PBMC, 14 days FC) Genes Monkeys Red: Up-regulation compared to pre-vaccination Green: Down-regulation compared to pre-vaccination

23 Signature for CD4 + T cell response Vaccine-induced CCR2B expression (PBMC, 14 days) Gag-specific CD4 + T cell response (%responding TNF or IFNγ) (p< 0.002, Spearman Rank) CCR2: -MCP-1 receptor, minor HIV-1 coreceptor -CCR2-64I SNP associated with protection from early AIDS progression (Ioannidis et al., 2001; Mulherin et al., 2003) -Important for CD4 + T cell recruitment to lung during influenza infection (Wareing et al., 2007) CCR2B expression log2(fold-change) compared to pre-vaccination Stronger induction of CCR2B is associated with stronger CD4 + T cell responses

24 Signature for CD4 + T cell response Vaccine-induced KLF5 expression (PBMC, 14 days) Gag-specific CD4 + T cell response (%responding TNF or IFNγ) (p< 1x10-5, Spearman Rank) KLF5: Transcription factor that regulates TNF and LPSinduced pro-inflammatory cytokines in various cell lines (Changevalap et al., 2006; Kumekawa et al., 2008) KLF5 expression log2(fold-change) compared to pre-vaccination Stronger repression of KLF5 is associated with stronger CD4 + T cell responses

25 Summary We have integrated expression profiling and functional measurements to identify novel signatures of immunogenicity Attenuated response to MRKAd5 gag/pol/nef in Ad5 + subjects CD8 + T cell polyfunctionality signature: ex, IDO, pdcs Signatures for CD4 + T cell response: ex, CCR2B and KLF5 We made some surprising observations Baseline Ad5 + expression signature: pre-existing altered expression of immune relevant genes, potential roles for IL-2, IRF1 CD8 + polyfunctionality: negative association with inflammatory gene expression responses NHP: most immunogenic adjuvant (PolyIC) does not induce the strongest expression responses

26 Next steps Incorporate additional functional assays CD8 + magnitude, CD4 + polyfunctionality, viral load after challenge (NHP), etc. Validate baseline Ad5 + signatures by profiling additional Ad5 + and Ad5 - individuals Identify signatures that are consistent across vaccine platforms (and species) Profiling expression and T cell responses to successful vaccines in humans (ex: YFV, HBV) and alternative HIV vaccine candidates Seek global correlations across multiple platforms Identify cell-type specific signatures by integrating with expression profiles of sorted cellular subsets CD8 + signatures, CD4 + signatures, DC subsets, NK-cells, etc.

27 Thank you! Research supported by Fred Hutchinson Cancer Research Center as part of the Collaboration for AIDS Vaccine Discovery with support from the Bill & Melinda Gates Foundation Aderem lab (ISB): Alan Aderem Katy Kennedy Bruz Marzolf Pamela Troisch Kristi Hamilton Picker lab (OHSU): Louis Picker Haesun Park Scott Hansen Andrew Sylwester Karl Mullen Tom Rolf Tae Ha Seder lab (NIH): Bob Seder Barbara Flynn Kelly Rausch Merck Michael Robertson McElrath CAVD (FHCRC): Julie McElrath Erica Andersen- Nissen Mingchao Shen Greg Spies