10th International Rotavirus Symposium Bangkok, Thailand

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1 Rotavirus Host Range Restriction and Innate Immunity: Mechanisms of Vaccine Attenuation Harry Greenberg MD Stanford University 10th International Rotavirus Symposium Bangkok, Thailand 09/19/12

B dsrna Segment 1 2 3 4 A Protein VP1 VP2 VP3 VP4 polymerase inner shell capping

interferon antagonist VP6 middle shell 7 8 9 NSP3 NSP2 VP7 systemic spread viroplasm

2 B dsrna Segment A Protein VP1 VP2 VP3 VP4 polymerase inner shell capping enzyme neutralization Rotavirus Structure, Genome, Proteome C VP1 & VP3 5 6 NSP1 interferon antagonist VP6 middle shell NSP3 NSP2 VP7 systemic spread viroplasm neutralization VP4 VP7 VP6 VP2 10 NSP4 enterotoxin 11 NSP5 NSP6 viroplasm Courtesy Dr. M.K. Estes

3 Reassortment: genetic process by which a new rotavirus strain with a different phenotype can arise Strain A Strain B Reassortant with distinct antigenicity/ virulence from either parent VP4 NSP1 This general approach forms the basis of several effective RV vaccines

4 Two Currently Licensed Rotavirus Vaccines (Global) Rotateq TM (Merck) Rotarix TM (GSK) G2 G3 G1 P1A[8] G4 G1,P1A[8] Heterologous bovine rotavirus Host range restriction Homologous human rotavirus

5 B cell immunity to VP4 or VP7 is sufficient for protection. CD8+ T cells enhance resolution of disease, not protection from disease

6 Rota Innate Immunity and Rotavirus Infection TLR7/9? (in pdc) RIG-I 1 PAMPs 2 Lgp2 Virus replication MDA-5 6 NSP1 IPS-1/MAVS 3 IFN-α/β 15 TBK-1 9 TAK-1? IFNα/γ-R 8 IRF7 IRF3 p-irf IKK-β IκB-α NF-κB Viroplasm sequestered 13 β-trcp NF-κB 11 IκB-α (Ub)n 1 10 NIK-1 PI3-K STAT1 JAK/STAT IRF9, etc STAT2 Antiviral state ISGs, IFNs VSIGs, ISGs, type III IFN, etc Type I IFNs IFN mrna VSIGs and ISGs Cell fate and survival PKR 14 IFN-α/β protein

Infected enterocytes 10,000,000,000 1,000,000,000 100,000,000 10,000,000

7 PFU equivalent/g intestinal tissue Replication of murine and simian RV in the suckling mouse intestine EW (murine RV ) RRV (simian RV ) RV Infected enterocytes 10,000,000,000 1,000,000, ,000,000 10,000,000 1,000, ,000 10,000 1, EW RRV Days post infection

8 Experimental design EW (or UK) X RRV Reassortant 5 day old wt. or STAT1 KO mice orally inoculated with 10 4 PFU of parental/reassortant RV. 2 or 3 days post infection, mice sacrificed, intestine collected and virus titers determined.

9 Intestinal replication of EWxRRV reassortants in wt. mice Reassortants Rotavirus Genes Wild type pfu/g B7/2 R R E R R R R R R R R 30 (0/4) B2/1 R E E R R R E R R R E 30 (0/6) B4/1 R R E R R R E E E R R 30 (0/7) B6/1 R E E R R R E R R R E 30 (0/4) E14/2 E E E R R E E R E E R 30 (0/9) H5/1 E E E R R E E E R E R 52 (1/7) E9/1 E E E R R E E E E E R 77 (1/4) D4/3 E E E R R E E E E E E 70 (2/8) D10/2 R R R R E R R R R R R 900 (3/4) E4/1 R R E R E R E E R R R 224 (4/7) A/11 R R R R E R R R E R R 194 (5/10) EA R R E R E R R R E R R 253 (6/9) DEA R R E R E R R R R R R 6,162 (6/6) DEA R R R R E R R E E R R 4,531 (8/8) D6/2 E E E R E E E E E E E 127,487 (11/11) D1/5 R E E R E R R E E R E 1,274,569 (10/10) C3/2 R E E R E R R E E R E 185,580 (4/4) EA R R E R E R R E E R R 112,383 (5/5) BA R E E R E R E E E R R 98,762 (4/4) RRV R R R R R R R R R R R 39 (1/9) EW E E E E E E E E E E E 13,774,839* (5/5)

10 PFU equivalent/g intestinal tissue Replication of wild type EW, RRV and D6/2 (EW/RRV VP4) in mouse intestine 10,000,000,000 1,000,000,000 * ** ** * ** 100,000,000 10,000,000 ** 1,000, ,000 10,000 1,000 EW D6/2 RRV Days post infection titers by qrt PCR and converted to PFU equivalents based on titer of control RRV.

11 Intestinal viral titer (pfu/g of tissue) RRV NSP1 confers RRV-like low intestinal growth EW NSP1 is necessary but not sufficient for EW-like high intestinal growth 100,000,000 10,000,000 1,000, ,000 10,000 1, RRV EW RRV-like reassortants, RRV NSP1 RRV-like reassortants, EW NSP1 EW-like reassortants, EW NSP1

12 Conclusions I Homologous murine RV replication is 10,000 to 100,000 fold greater than the heterologous simian RV in suckling mouse intestine. RRV NSP1 confers the simian-like restricted replication phenotype. EW NSP1 is necessary but not sufficient for the murine-like permissive replication phenotype. RRV VP4 efficiently mediates viral entry to mouse intestine epithelial cells but virus replication is reduced approximately 10X compared to murine VP4. In addition to murine VP 4, other proteins (VP3, NSP2 and NSP3) are also required for the murine-like intestinal replication phenotype. What is the mechanism by which NSP1 affects host range?

13 Intestinal replication of EWxRRV reassortant RV in STAT1 KO mice Reassortants Rotavirus Genes Wild type STAT1 KO pfu/g D3 pfu/g B7/2 R R E R R R R R R R R 30 (0/4) 179,055 (6/6) A15/1 R R R R R R E R R R R 30 (0/7) 16,502 (4/4) B2/1 R E E R R R E R R R E 30 (0/6) 51,667 (5/5) B4/1 R R E R R R E E E R R 30 (0/7) 37,251 (7/7) B6/1 R E E R R R E R R R E 30 (0/4) 160,707 (8/8) E14/2 E E E R R E E R E E R 30 (0/9) 118,166 (7/7) H5/1 E E E R R E E E R E R 52 (1/7) 40,907 (6/6) E9/1 E E E R R E E E E E R 77 (1/4) 155,672 (6/6) D4/3 E E E R R E E E E E E 70 (2/8) 26,889 (6/6) D10/2 R R R R E R R R R R R 88 (1/5) 18,736 (12/12) E4/1 R R E R E R E E R R R 224 (4/7) 7,167 (7/7) A/11 R R R R E R R R E R R 194 (5/10) 130,731 (7/7) E4A11-11 R R E R E R R R E R R 253 (6/9) 44,344 (4/4) DE4A11-24 R R E R E R R R R R R 6,162 (6/6) 39,719 (4/4) DE4A11-3 R R R R E R R E E R R 4,531 (8/8) 138,792 (3/3) D6/2 E E E R E E E E E E E 127,487 (11/11) 134,217 (5/5) D1/5 R E E R E R R E E R E 1,274,569 (10/10) 11,687,256 (5/5) C3/2 R E E R E R R E E R E 185,580 (4/4) 4,365,849 (6/6) E4A11-4 R R E R E R R E E R R 74,944 (9/9) 862,379 (4/4) BA1-1-3 R E E R E R E E E R R 26,923 (4/4) 41,918 (4/4) RRV R R R R R R R R R R R 39 (1/9) 33,568 (14/14) EW E E E E E E E E E E E 13,774,839* (5/5) 28,786,099* (5/5)

14 Fold changes of intestinal replication STAT1 KO/wild type mice Replication Increase in Intestine of STAT1 KO vs. Wt. Mice after Infection with RRV, EW or EWxRRV Reassortants 10,000 1, RRV EW RRV-llike reassortants, RRV NSP1 RRV-like reassortants, EW NSP1 EW-like reassortants, EW NSP1

15 Conclusions II Replication of heterologous simian RRV was dramatically increased in STAT1 KO mice. Replication of homologous EW was minimally increased in STAT1 KO mice. The levels of replication increase were significantly greater in reassortants with RRV NSP1 than reassortants with EW NSP1. Both RRV and EW NSP1s effectively degrade mouse IRF3 and suppress host IFN in vitro but the effects of these two NSP1s on replication in vivo are very distinct. STAT1 dependent effects play a dominant, but not absolute, role in determining host-range restriction of RV replication in the intestine. VP4 likely plays a variable role How does homologous (murine) RV overcome the host innate immune response within small intestinal mature villous enterocytes?

Single Cell and Bulk Analysis of the Intestinal IFN

responses don t reveal heterogeneity in single cells.

levels of virus Sorted single cells (infected and

transcripts belonging to different categories measured

16 Single Cell and Bulk Analysis of the Intestinal IFN Response Bulk (averaged) measurements of antiviral responses don t reveal heterogeneity in single cells. Enterocytes from proximal small intestine had enriched levels of virus Sorted single cells (infected and uninfected mice) analyzed using fluidigm arrays 81 transcripts belonging to different categories measured per cell Does murine RV inhibit IRF3/NF-kBdependent IFN induction or signaling?

17 Early intestinal IFN induction at the bulk level is independent of RV replication efficiency Relative Expression (2 -DDCt ) Fold change over uninfected(/gapdh) 10 7 RRV = simian virus EW = murine virus NSP5 NSP3 VP6(+) At 16hpi, EW replicates better than RRV 10 0 IFN-α4 ISG20 IFI203 Both RRV and EW induce similar intestinal IFN responses Intestinal IFN induction is generally independent of RV replication efficiency Results demonstrate that murine RV grows efficiently despite substantial induction of IFN while simian RV does not grow

18 Categorical clustering of single cells from the villous epithelium Non- Infected Infected (CIAs) Cells Bystanders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ategory 1-based Enterocyte Hi/Low clusters Assay Category: Hi Low RV + Hi Low Hi Low Transcripts Low Ct value Hierarchical clustering of the data based on infection status and category 1 transcripts High

Selected Examples of Hierarchical Reconstruction of the Epithelial IFN Response to Homologous RV Cells from infected animals IRF3 Lgp2 NF B RV positive cells IRF3 Rsad2 Isg20 Mda5 Lgp2

19 Selected Examples of Hierarchical Reconstruction of the Epithelial IFN Response to Homologous RV Cells from infected animals IRF3 Lgp2 NF B RV positive cells IRF3 Rsad2 Isg20 Mda5 Lgp2 Isg15 Ifit1 NF B Rsad2 Isg20 Isg15 Ifit1 IFN-α4 IFN-α5 IFN-β Peli1* A20* Bystander cells IRF3 Lgp2 NF B Rsad2 Isg15 A20* Ifit2 Ifit1 Peli1* No change IFN-α4 IFN-α5 IFN-β IFN-α4 IFN-α5 IFN-β

20 Conclusions III Efficient homologous RV replication paradoxically induces type I interferons & antiviral genes in bulk intestinal tissues Intestinal type I IFN induction at bulk level occurs primarily in intestinal hematopoietic cells In villous epithelial cells, murine RV replicates despite the transcription of various antiviral genes (i.e. an antiviral state ) and occurs preferentially in cells expressing highest basal IFN levels RV-infected villous epithelium is unable to induce type I IFN genes; this defect correlates with perturbation of NF-kB (but not IRF3) activation Single cell analysis reveals RV inhibition of host type I IFN within intestinal villous epithelium and its induction in immune cells

Genetic and mechanistic Determinants of Rotavirus Host Range Restriction

Genetic and mechanistic Determinants of Rotavirus Host Range Restriction Harry Greenberg MD and the Greenberg Lab Stanford University School of Medicine Eleventh International Rotavirus Symposium New Delhi,