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Rapid and systemic innate immune responses to an adenoviral HIV vaccine Erica Andersen-Nissen McElrath Lab Fred Hutchinson Cancer Research Center Developing an HIV vaccine • Very challenging task! • Two potential scenarios for an HIV vaccine: -slow clinical disease progression -reduce transmission DH Barouch Nature 455, 613-619 (2008) • Regardless of the type of HIV vaccine developed, we need to better understand how to induce a protective adaptive response – > innate immune response Arms of the Immune System When a microbe invades, the immune system: • Recognizes it • Mobilizes and eliminates it • Remembers it • Returns to baseline Innate immune system Adaptive immune system Response in minutes to hours Response in days to weeks Recognizes broad classes of pathogens Pathogen-specific response No immunological memory Exposure leads to immunological memory Found in nearly all forms of life Found only in jawed vertebrates Key Players in Innate Immunity Barriers (Skin, epithelia) Enzymes, Anti-microbial peptides Microbial Recognition Receptors Complement proteins IMM/MICROM 441 Lecture #4 October 1/08 Kaja Page 4 of 26 What influences adaptive immune response to a vaccine? - Innate immune responses shape the quantity, quality and longevity of the adaptive response (Pulendran and Ahmed, Cell, 2006) -Engineer a vaccine to induce desired innate responses Vaccine vector or adjuvant Vaccine antigen http://research.dfci.harvard.edu/innate/images/innate/immunities.gif Innate Immune Responses to Vaccination • Few studies have examined innate responses in humans after vaccination (Querec et al., NI, 2009; Gaucher et al., JEM, 2008) • We implemented a clinical trial of an adenoviral vector HIV vaccine (Merck Ad5): 1. Which systemic innate immune responses can be measured in blood after vaccination by intramuscular injection? 2. What are the optimal time points to identify these innate responses in humans? 3. Which innate responses correlate with adaptive responses? Adenoviral vectors for T-cell-based HIV vaccines • Adenoviral vectors containing HIV inserts found to elicit high-magnitude CD8+ T cell responses ~80% of vaccinees demonstrate T cell immunogenicity by ELISpot ~300 SFU/106 PBMC -responses were durable (Priddy et al., CID, 2008) (Corey et al., AIDS 2009) • Step Study Innate Immune Responses: HVTN 071 (n=35) 1.5 x 1010 genomes MrkAd5 gag/pol/nef Ad5 injection (IM): Sampling points: Day: Samples: 0 6h 1 3 7 Whole blood (Flow cytometry) Serum (Luminex) PBMC RNA (Microarray) n=11 28 PBMC (ICS) Innate Immune Responses: HVTN 071 1.5 x 1010 genomes MrkAd5 gag/pol/nef Ad5 injection (IM): Sampling points: Day: Samples: 0 6h 1 3 n=24 (serum only) 7 28 PBMC (ICS) PTID: 071-008 071-048 071-056 071-035 071-044 071-050 071-025 071-032 071-052 071-046 071-015 071-054 071-018 071-042 071-024 071-028 071-057 071-006 071-022 071-047 071-012 071-059 071-033 071-053 071-058 071-031 071-029 071-017 071-036 071-016 071-055 071-040 071-051 071-014 071-026 Ad5 serum neutralizing antibody titer Baseline Serum Ad5 Neutralizing Antibody Titers 10000 Baseline Ad5 Titer 1000 High, >1000 Moderate, 200-999 Low, 19-199 Negative, ≤18 100 10 Group A Group B (pre-vaccine and day 1) (n=11) (n=24) Rapid Changes in Peripheral Blood Cell Populations after MRKAd5 gag/pol/nef Vaccination Lymphocytes 4000 3000 1000 * 250 0 0 24 48 72 96 120 144 168 Granulocytes 6000 * 500 1000 0 * 0 24 48 72 96 120 144 168 Baseline Ad5 Titer Moderate, 200-999 Low, 19-199 Negative, ≤18 4000 Cells/ml Blood Monocytes 750 * 2000 1250 2000 p<0.05, * Timepoint vs. 0 0 24 48 72 96 120 144 168 Hours Post Vaccination pre-vaccine (0 hrs.), Wilcoxon signed rank test Multiple Cell Types Exit the Blood CD8 T cells 1000 * 500 CD4 cells CD4+ TTcells cells CD19+B Lymphocytes 2000 400 1500 300 1000 24 48 72 168 NK cells 800 600 Cells/ml Blood 100 0 0 400 * 200 0 0 24 48 72 Hours Post Vaccination 168 0 0 14 12 10 8 6 4 2 0 * 200 * 500 0 * 24 48 72 168 Plasmacytoid DC 0 24 48 72 168 0 24 48 72 168 Myeloid DC 35 30 25 20 15 10 5 0 0 24 48 72 168 Serum Proinflammatory Cytokines & Chemokines Increase Rapidly after Vaccination IFN-g * 100 35 4 10 0 24 48 72 5 2 0 0 0 96 120 144 168 IP-10/CXCL-10 2000 1500 24 48 72 96 120 144 168 MCP-1/CCL2 2000 * * 0 24 48 72 96 120 144 168 Baseline Ad5 Titer Moderate, 200-999 Low, 19-199 Negative, ≤18 1500 1000 * 6 15 0 * 12 8 20 25 TNF-a 10 25 50 Serum pg/ml * 30 75 500 IL-6 1000 * * * 0 p<0.05, * Timepoint vs. 500 0 0 24 48 72 96 120 144 168 Hours Post Vaccination 0 24 48 72 96 120 144 168 pre-vaccine (0 hrs.), Wilcoxon signed rank test Serum Anti-inflammatory Cytokines Also Increase after MRKAd5 gag/pol/nef Vaccination * IL-1Ra 5000 4000 IL-10 100 * 75 3000 * Serum pg/ml 50 2000 25 1000 0 0 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 Hours Post Vaccination Baseline Ad5 Titer Moderate, 200-999 Low, 19-199 Negative, ≤18 p<0.05, * Timepoint vs. pre-vaccine (0 hrs.), Wilcoxon signed rank test Summary of Significant Changes in Serum Cytokines/Chemokines at 24 Hours Cytokine/ Chemokine IP-10/CXCL10 IL-1Ra IL-10 IFN-γ MCP-1/CCL2 IL-6 IL-7 TNFα MIP-1β Median Serum Cytokine Levels (pg/ml) n=35 Pre-vaccine 24 hours 70 350 5.4 0.1 180 4.3 7.1 5.1 120 630 1700 12 4.4 380 6.7 8.0 7.1 160 Median Fold Change n=35 11 4.7 2.4 2.4 2.0 1.7 1.4 1.3 1.2 Other serum analytes that were measured, but did not change at 24 hours: EGF, Eotaxin, Fractalkine, G-CSF, GM-CSF, IFN-a, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-8, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, MIP-1a, sCD40L, TGF-a, VEGF Ad5 Negative/Low Titer Individuals Show Significantly Greater Changes in Serum Cytokines/Chemokines at 24 Hours Cytokine/ Chemokine MCP-1 IP-10 IL-1Ra Median Serum Cytokine Levels (pg/ml) At 24 Hours Post Vaccination Ad5 negative/low n=27 Ad5 medium/high n=6 p-value (Wilcoxon Rank Sum Test on log fold change relative to prevaccine) 420 720 2100 260 180 870 0.002 0.02 0.02 PBMC Gene Expression PBMC Isolate RNA Affymetrix human exon arrays (10 people; 5 timepoints Analysis (collaboration with Dan Zak, ISB) • Discover other innate immune responses to Ad5 vaccination • Identify additional differences in responses between Ad5 naïve and Ad5 immune individuals PBMC Microarrays Show Many Genes with Significant* Temporal Expression Responses Med Ad5 Titer Low Med Ad5 Titer Ad5 Titer volunteer ID: timepoint: *p<10-6, 1 way ANOVA Up-regulated Down-regulated What do the gene expression changes mean? • 2 sources of changes in gene expression: – Changes in the PBMC composition – Changes in the response of cells that are in the blood to the vaccine • Attenuated gene expression changes in Ad5 medium titer subjects – are these individuals effectively exposed to a lower vaccine dose? 6hrs: 1 gene down-regulated (p<0.001, |log2(FC)| > 0.5) Innate DB: www.innatedb.ca/ 6hrs: 1 gene down-regulated (p<0.001, |log2(FC)| > 0.5) CCL3 MIP-1a 24hrs: 1065 genes up/down-regulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.01) 24hrs: TLR pathway up-regulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.01) 24hrs: STAT1 targets/interactors upregulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.01) 24hrs: TCR pathway down-regulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.01) 72hrs: 116 genes up/down-regulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.02) 72hrs: 116 genes up/down-regulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.02) CSF1R CSF1R 168hrs: 11 genes up/down-regulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.2) ENDO-PDI TXNDC5 TXNDC5 = EndoPDI: a protein preferentially expressed in endothelial cells that acts as a stress survival factor during hypoxia (Sullivan et al., 2003) Summary • MRK Ad5 gag/pol/nef administered IM induces robust innate immune responses in the blood: – rapid influx of monocytes/mDC into the blood and efflux of lymphocytes/pDC from the blood • trafficking to and from the site of vaccination? – changes in serum chemokines and PBMC gene expression indicate PBMC are the source of many serum factors – maximal responses occur at 24hrs post-vaccination • Pre-existing neutralizing antibodies to Ad5 attenuate the innate response – reduced production of MCP-1, IP-10 and IL-1Ra – muted gene expression changes in PBMC • Potential correlates for magnitude of CD8 response are being identified Future Directions • Compare innate responses to Mrk rAd5 vaccine with responses to other vaccines – Other candidate HIV vaccines (e.g. MVA: HVTN 908) – Vaccines currently in use (e.g. YFV) • Evaluate innate immune responses to vaccines at mucosal sites to determine effects at the site of HIV acquisition • Develop an in vitro system to test effects of identified innate response genes on the adaptive response Acknowledgements Julie McElrath Steve De Rosa Eric Peterson Joanne Chang Sam Pine Greg Spies Don Carter Olivier Defawe Mingchao Shen Reneé Ireton Phyllis Stegall HVTN Laboratory Program HVTU (Seattle, Rochester, Nashville, Birmingham) Ann Duerr Larry Corey Jim Kublin Volunteers Merck Michael Robertson NIAID/DAIDS Margaret Johnston Dan Zak Alan Aderem UW STD/AIDS Research Training Fellowship CAVD Investigators Joleen Borgerding Nidhi Kochar Liza Noonan Yunda Huang Li Qin Steve Self Innate immune detection of adenovirus (Gilliet et al., 2008) The Step Study • 3000-person HIV vaccine trial started in late 2004 • Phase IIB test-of-concept study to establish whether MRKAd5 expressing HIV-1 gag/pol/nef could lower: – HIV-1 infection rates – Plasma viremia • Interim analysis performed when 30 per-protocol HIV infections occurred HIV Incidence (% per year) Step Study Results Overall • No effect of the vaccine on viral load HIV Incidence (% per year) 0-26 26-52 52-78 • Increased HIV infection in vaccinees that had preexisting immunity to Ad5 Ad5≤18 HIV Incidence (% per year) 0-26 26-52 52-78 • All further vaccinations were halted Ad5>18 0-26 26-52 52-78 Time Interval (weeks) (Buchbinder et al., 2008) • Mechanisms under investigation (Buchbinder et al., 2008; McElrath et al., 2008)