Making Babies Bluish Mechanisms of Respiratory Syncytial Virus Pathogenesis
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Making Babies Bluish Mechanisms of Respiratory Syncytial Virus Pathogenesis Marty Moore lab, Emory Dept of Pediatrics, Infectious Diseases No relevant financial conflicts of interest RSV: Paramyxoviridae, Pneumovirinae NS1 NS2 3’ SH M2 5’ Marty Moore, Vanderbilt RSV Significance •Leading cause of bronchiolitis, viral pneumonia, respiratory failure, and viral death in infants in USA & worldwide RSV 100% URI 1% LRI Bronchiolitis Pneumonia ✴Approx 100,000 infant hospitalizations per year in USA ✴Estimated 200K - 600K children die of RSV infection per year worldwide No vaccines or therapies in use Hospitalization + ED Cases RSV Seasonality and Clinical Impact CHOA “Virometer” Week Feb 2010 12/12/10 2/21/11 CHOA one of largest pediatric systems in US Data from Dr. Bob Jerris, CHOA Director of Microbiology Fatal RSV Bronchiolitis PAS Stain Intrabronchiolar cell/mucus mixture Johnson et al. Mod. Pathol. (2007) 20, 108-119 Is there a useful animal model of RSV bronchiolitis? RSV-Induced Airway Mucus Expression Depends on the RSV Strain Intranasal RSV infection of BALB/c mice (semi-permissive) PAS A2 Line 19 Lukacs et al. (2006) Am J. Path. 169: 977-986. Collaboration with Stokes Peebles (Vanderbilt) RSV Line 19 BALB/c Mouse Model of RSV-Induced Airway Mucus Moore et al. J. Virol. (2009) 83: 4185-94 ★Lukacs group RSV line 19 papers on IL-13 and STAT6, RANTES, CXCR2, pDC, Notch, MIP-1alpha, IL-17 Viral Determinants of Mucus Expression: Fusion Protein Residues differing between line 19 and Long strains IL-13 and mucus Moore et al. J. Virol. (2009) 83: 4185-94 A2-Line19F Pathogenesis: What Residues in Line 19 F are Involved? What is the Mechanism? Annie Hotard Emory Microbiology and Molecular Genetics (MMG) graduate student Starting 3rd year F Annie generated a novel BAC-RSV Recombineering System to make RSV point mutants Annie has so far generated 6 full-length mutant BAC-RSV plasmids and recovered and confirmed sequence for rA2-line19F(M79I) and rA2-line19F(K357T) Hypothesis: Line 19 Fusion Protein Has High Fusion Activity in Mouse Cells Model of pre-fusion RSV F highlighting residues differing between Line 19 and Long Modeled by Richard Plemper, Emory Equivalent Growth in BEAS-2B Cells ✤ Annie Hotard Similar Results in HEp-2 cells A2-Line19F Replicates Efficienty in BHK-21 Cells But M79I and K357T do not affect A2-line19F Growth in BHK-21 Annie Hotard ✤ Annie is currently analyzing growth of these viruses in mouse cells In Vitro Fusion Assay Use synthesized codon-optimized RSV cDNAs F + Transfect Cells With F and DSP1-7 Plasmids Overlay with DSP8-11-Transfected Cells Fusion and Content Mixing: GFP and Luciferase Readout Naoyuki Kondo et al. JBC (2010) 285: 14681-14688 Twist on classic paramyxovirus glycoprotein fusion assays - Bob Lamb Cell-cell fusion is surrogate measure of paramyxovirus-cell fusion Lab Pirate Party ARRRR! Michael Currier Annie Hotard Line 19 F More Fusogenic Than Long F in 293T Cells 293T cells 8 hr post-overlay Effector Cells: Overlay Cells: Annie Hotard DSP1-7 + DSP8-11 DSP1-7 + pcDNA DSP1-7 + Line19F DSP1-7 + LongF - DSP8-11 DSP8-11 DSP8-11 Line 19 F More Fusogenic Than Long F in BHK Cells Effector Cells: Overlay Cells: DSP1-7 + DSP8-11 DSP1-7 + pcDNA DSP1-7 + Line19F DSP1-7 + LongF - DSP8-11 DSP8-11 DSP8-11 Line 19 F summary: ✦Line 19 F contains unique residues ✦Line 19 F confers more efficient growth in BHK cells than Long F ✦Line 19 F more fusogenic than Long F in 293T and BHK cells ✦Annie: Complete in vitro analysis of all the mutants ✦Characterize pathogenesis: mucus ✦Does fusion in vitro relate to mucus in vivo? Annie Hotard Simple Hypothesis: RSV Strain Variation Impacts Pathogenesis Infected BALB/c mice with A2, Long, Line 19, and 6 different low-passage clinical isolates Mock infection or 105 PFU/mouse Day 8 post-infection Lung homogenate ELISA mm, Vanderbilt Michael Chi (Vanderbilt) Stokes et al. J. Virol. 2011; 85: 5782 RSV Clinical Isolates’ Viral Load in Mice Kate Stokes Emory Immunology and Molecular Pathogenesis (IMP) graduate student Starting 5th year Stokes et al. J. Virol. 2011; 85: 5782 RSV Clinical Isolates: Viral Antigen In Bronchiolar Epithelium at One Day Post-Infection Kate Stokes Stokes et al. J. Virol. 2011; 85: 5782 RSV Clinical Isolates: Damage to Bronchiolar Epithelium One Day Post-Infection Kate Stokes Stokes et al. J. Virol. 2011; 85: 5782 RSV 2-20 Strain: Damage to Bronchiolar Epithelium One Day Post-Infection RSV Line 19: Damage to Bronchiolar Epithelium One Day Post-Infection Line 19 1 d p.i. Morphometric Quantification of Airway Mucin Expression Periodic Acid Schiff (PAS) Stain Kate Stokes Morphometric Quantification of Airway Mucin Expression Kate Stokes Stokes et al. J. Virol. 2011; 85: 5782 Like RSV Line 19, RSV 2-20 is “Mucogenic” in BALB/c Mice Kate Stokes Stokes et al. J. Virol. 2011; 85: 5782 Like Line19*, 2-20-Induced Airway Mucin Expression is IL13-Dependent Kate Stokes Stokes et al. J. Virol. 2011; 85: 5782 *Line 19-Induced Mucin is IL-13-dependent: Tekkanat et al. J. Immunol. 2001; 166: 3542-8 RSV 2-20 Causes Increased Breathing Effort in BALB/c Mice Stokes et al. J. Virol. 2011; 85: 5782 mm and Dawn Newcomb, Vanderbilt RSV 2-20 Causes Bronchiolitis in BALB/c Mice Hypothesis: RSV 2-20 F Protein Contributes to Virulence Kate Generated A2-220F RSV Chimeric Strain 2-20 F sufficient for higher viral load 1 day p.i. in A2 genetic background Kate Stokes Hypothesis: RSV 2-20 F Protein Contributes to Virulence Kate Stokes Hypothesis: 2-20 F is More Fusogenic Than A2 F Model of pre-fusion RSV F highlighting residues differing between 2-20 and A2 Modeled by Richard Plemper, Emory Hypothesis: 2-20 F is More Fusogenic Than A2 F BEAS-2B Cells BEAS-2B Cells Hypothesis Wrong Michael Currier Technician BEAS-2B Cells Michael Interested in G Interesting Sequence Differences in A2 G and 2-20 G 2-20 G (Attachment) Protein Boosts Fusion F 293T Cells MVA-T7 + T7-luc 2 X 107 Light Units 1.5 X 107 1 X 107 293T Cells 5 X 106 Michael Currier pcDNA A2F A2F A2G 220F 220F 220G Line19F Line19F Line19G Fusogenicity in vitro correlates with mucogenicity in vivo Line 19 and 2-20 Strains “A model is a lie that that helps you see the truth.” - Howard Skipper PhD, Lasker Awardee, self-professed “mouse doctor” F and G Variation Differences in Fusion More fusion akin to more CPE CPE in vivo, epithelial damage mock A2-LongF ? A2-Line19F Epithelial Damage Leads to Mucus Expression HEp-2, 72 hr p.i. “A model is a lie that that helps you see the truth.” - Howard Skipper PhD, Lasker Awardee, self-professed “mouse doctor” Kate Stokes: reading Jay Nadel, papers on neutrophils, TNF-alpha, EGFR in pulmonary mucus Shim et al. Am J. Physol. Lung Cell Mol. Physiol. 2001; 280: L134 CPE in vivo, epithelial damage IL-13 (CD4 T ?) Neutrophil Recruitment TNF-α upregulates MUC5AC TNF-α upregulates EGFR EGFRindependent MUC5AC EGFRdependent MUC5AC Neutrophil elsastase activates EGFR EGFR signaling: MUC5AC Hypothesis: Neutrophils Mediate RSV-Induced Mucus Kate Stokes Bronchoalveolar Lavage (BAL) Neutrophils Role of Neutrophils in RSV 2-20-Induced Bronchiolitis Anti-Ly6G treatment to deplete neutrophils in vivo Flow cytometry, peripheral blood leukocytes Kate Stokes Depletion of Neutrophils in the Lungs of RSV-Infected Mice Anti-Ly6G treatment to deplete neutrophils in vivo Kate Stokes Effect of Ly-6G Ab on RSV 2-20 Viral Load Anti-Ly6G treatment to deplete neutrophils in vivo Kate Stokes Neutrophil Depletion Had Minimal Effect on RSV 2-20 Viral Load Effect of Ly-6G Ab on Cytokines in RSV-Infected Mice Anti-Ly6G treatment to deplete neutrophils in vivo cytokine bead array cytokine bead array cytokine bead array Kate Stokes Day 6 p.i. i.c. flow Day 6 p.i. i.c. flow Kate Stokes Neutrophil Depletion: PAS Stain 8 Days Post-Infection mock isotype mock anti-Ly6G RSV 2-20 isotype RSV 2-20 anti-Ly6G Kate Stokes Neutrophil Depletion: PAS Stain 8 Days Post-Infection RSV 2-20 isotype Neutrophils important for RSV-induced airway mucus Kate Stokes Neutrophil Depletion: Effect on RSV Histopathology Role of Neutrophils RSV 2-20 anti-Ly6G ❖Contribute to RSV-induced TNF-α ❖Contribute to RSV-induced IL-4/IL-13 ❖Contribute to RSV-induced mucus ❖Promote CD8 T cell response to RSV ❖Little effect on viral load ❖important finding: neutrophils protect against RSV-induced lung pathology These RSV strains are tools for studying pathogenesis mechanisms F and G Variation Differences in Fusion CPE in vivo, epithelial damage Neutrophil function TNF-α, IL-13, mucus Are RSV strain differences significant for human disease? Prediction of this model: CPE correlates with disease We showed that six clinical isolates vary in pathogenesis in mice Do RSV genotypes correlate with disease severity in infants? Will investigate using many RSV clinical isolates from Tennessee and Argentina Relevant for vaccine strategies because variation in F and G affects nAb sites RSV vaccines: looking forward, thinking backwards Correlates of Protection in RSV Disease Ag: F, G Ag: ? IgG CD8 T cells? serum nAb titer 1:300 RSV G is a Major Target of Immune Responses Sujin Lee, PhD RSV G is a Major Target of Immune Responses BALB/c mice 25 i.m. Naive M1 only VLP RSV-G VLP RSV-F VlP A2 Strain RSV nAb 3 weeks post-boost RSV A2-coated ELISA plates serum 4 days post-challenge RSV G is a Major Target of Immune Responses BALB/c mice 25 i.m. Naive M1 only VLP RSV-G VLP RSV-F VlP A2 Strain RSV A2 Strain RSV RSV G is a Major Target of Immune Responses BALB/c mice 25 i.m. Naive M1 only VLP RSV-G VLP RSV-F VlP A2-line19F Strain RSV • A2-Line19F challenge is more informative than A2 • VLP provide a good system to evaluate vaccine Ags • We would like to do that with G of different strains • Although F has been the preferred Ag, Sujin Lee, PhD vaccination with G may have advantages over F •Need a better understanding of nAb epitopes in G and how strain variation affects Ab responses Thank You Notes Current Lab Members Kate Stokes Carla Pretto Nicolle Marshall Sujin Lee Michael Chi (Vanderbilt) Annie Hotard Carla Shoffeitt Michael Currier Collaborators •Kaori Sakamoto (UGA) •Stokes Peebles (Vanderbilt) •Jim Crowe (Vanderbilt) •John Williams (Vanderbilt) •Tina Hartert (Vanderbilt) •Nick Lukacs (Michigan) •Richard Plemper (Emory) •Larry Anderson (Emory) • Ultan Power (Queens U Belfast) •Teresa Peret (CDC) •Olen Kew & Jaume Jorba (CDC) •Kathy Spindler (Michigan) •Gregory Melikyan (Emory) Craig Shapiro Zoe Meng Funding ❖NIH NIAID ❖Emory University ❖CHOA ❖Georgia Research Alliance ❖Atlanta Clinical and Translational Science Institute BAC Mutagenesis of RSV pSynk-BAC-RSV Tennessee Children’s Respiratory Initiative (TCRI): 4 RSV Season Cohort Tina Hartert Lead PI of TCRI Vanderbilt RSV Clade Distribution in TCRI Cohort RSV one serotype Antigenic subgroup A Clades GA1-GA7, SAA1 Antigenic subgroup B Clades GB1-GB5, GB3/BA TCRI 209 Genotyped* RSV GA2 54 GA5 88 GB3/BA 67 *genotype based on 270 nt sequence in G gene RSV Clade Genotyping: Larry Anderson (then at CDC), Dean Erdman (CDC), Teresa Peret (CDC) RSV Clade Distribution in TCRI Cohort RSV one serotype Antigenic subgroup A Clades GA1-GA7, SAA1 Antigenic subgroup B Clades GB1-GB5, GB3/BA TCRI 209 Genotyped RSV GA2 54 GA5 88 GB3/BA 67 RSV Clade Genotyping: Larry Anderson (then at CDC), Dean Erdman (CDC), Teresa Peret (CDC) RSV Bronchiolitis Severity Score by Clade and Season in the TCRI Cohort Unfortunately not able to culture isolates from cohort Relevance to disease and to vaccines We need better understanding of RSV molecular (sero)epidemiology Abs to G are strain-specific; Abs to F generally X-neutralizing RNA viruses change; We should study extant strains We are doing this all again with ReSPIRA/U19 Genome of RSV Line 19: Surprisingly Similar to Long Strain Where Did It Come From? Residues differing between line 19 and Long strains Proc Soc Exp Biol Med. 1966; 121: 37-41 Equivalent Expression of F Proteins Equivalent Expression of G Proteins Equivalent F Expression by Western and flow Equivalent F Expression by Western and flow Equivalent G expression by Western and flow CPE of M79I and K357T Line19 F Mutant Viruses in HEp-2 Cells Mock 24 hr 48 hr 72 hr 96 hr rA2-LongF rA2-Line19F rA2-Line19F(M79I) rA2-Line19F(K357T) CPE of M79I and K357T Line19 F Mutant Viruses in BHK-21 Cells Mock 24 hr 48 hr 72 hr rA2-LongF rA2-Line19F rA2-Line19F(M79I) rA2-Line19F(K357T) Model System to Characterize Vaccine-Elicited RSV-Specific CD8 t cells J. Exp. Med. 2004; 199: 775-784 Sujin Lee, PhD “TriVax”: Peptide (epitope) + anti-CD40 + polyI:C Peptide: RSV M282-90 SYIGSINNI, MHC I-Kd RSV TriVax Induces Robust And Long-Lasting RSV-Specific CD8 T cells Naive CD8 M282-90 Tet Sujin Lee, PhD d0 d6 RSV TriVax: 200 g peptide 50 g anti-CD40 50 g polyI:C i.v. route isolate PBL TriVax RSV TriVax-Induced CD8 T Cells are Effective CTL d0 TriVax In Vivo CTL Assay Sujin Lee, PhD d6 1X TriVax CTL Assay d0 d 14 TriVax TriVax boost d 20 2X TriVax CTL Assay RSV TriVax Vaccination protects Mice Against RSV Infection Challenge Strain of Choice: A2-Line19F d0 d6 d 10 Measure CD8 T cells and viral load in lung TriVax d0 TriVax ✤ A2-Line19F d 14 TriVax boost d 20 d 24 A2-Line19F Measure CD8 T cells and viral load in lung Huge Number of RSV-Specific CD8 T cells in the lung! Surely The Lungs Are Wrecked? Sujin Lee, PhD RSV TriVax Protects Against A2-Line19 F Bronchiolitis and Inflammation Naive + Mock Naive + A2-line19F TriVax + A2-line19F Path data: day 8 post-challenge Surprising to me Sujin Lee, PhD TriVax-Induced T Cells Qualitatively Different From Infection-Induced T Cells RSV Infection Suppresses Lung CD8 T Cell Response Chang et al. Nat. Med. 2002; 8: 54-60 Sujin Lee, PhD Why? Hypothesis: priming site, lung vs. periphery ✴TriVax induces robust, protective RSV-specific cytotoxic CD8 T cells ✴Having large number of anti-viral CTL in lung not necessarily bad ✴Epitope-elicited T cells can be of higher quality than virus-elicited T cells ✴Major questions: MEMORY, duration of immunity? ✴Role of Ag-specific CD4 T cells? ✴What are the human CD8 T cell epitopes of RSV?
