Volume 22, Issue 3, Pages 668-674 (March 2014) Clinical Assessment of a Novel Recombinant Simian Adenovirus ChAdOx1 as a Vectored Vaccine Expressing Conserved Influenza A Antigens  Richard D Antrobus, Lynda Coughlan, Tamara K Berthoud, Matthew D Dicks, Adrian VS Hill, Teresa Lambe, Sarah C Gilbert  Molecular Therapy  Volume 22, Issue 3, Pages 668-674 (March 2014) DOI: 10.1038/mt.2013.284 Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Safety data for ChAdOx1 NP+M1: the frequency of adverse reactions following vaccination with ChAdOx1 NP+M1 is shown, with severity indicated by shading. (a) Local adverse reactions and (b) systemic adverse reactions. Data represent adverse reactions from all 15 volunteers across all four doses. A breakdown of adverse reactions by dose is provided in the supplementary information. M1, matrix protein 1; NP, nucleoprotein. Molecular Therapy 2014 22, 668-674DOI: (10.1038/mt.2013.284) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Ex vivo interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) responses to influenza vaccine antigen NP+M1 following vaccination with increasing doses of ChAdOx1 NP+M1. Individual ex vivo IFN-γ ELISpot responses from vaccinated volunteers at baseline day (D) 0, D14, and D21. Volunteers were vaccinated with a single dose of ChAdOx1 NP+M1 intramuscularly at a dose of (a) 5 × 108, (b) 5 × 109, (c) 2.5 × 1010, or (d) 5 × 1010 viral particles (vp). Controls included cells stimulated with PHA/SEB, PPD, or irrelevant peptide TRAP33 (data not shown). Negative control was cells stimulated with media alone (data not shown). M1, matrix protein 1; NP, nucleoprotein; PBMC, peripheral blood mononuclear cell; PHA, phytohaemagglutinin; PPD, tuberculin purified protein derivative; SEB, staphylococcal enterotoxin B; SFU, spot forming unit. Molecular Therapy 2014 22, 668-674DOI: (10.1038/mt.2013.284) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Breadth of ex vivo interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) responses to NP+M1 peptide pools following vaccination with ChAdOx1NP+M1. Median and ex vivo IFN-γ ELISpot responses from all vaccinated volunteers at day (D) 0, D14, and D21. P1–8 indicate NP+M1 peptide pools 1–8, composed of 10 peptides per pool. M1, matrix protein 1; NP, nucleoprotein. Molecular Therapy 2014 22, 668-674DOI: (10.1038/mt.2013.284) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Neutralizing antibody (NAb) responses to adenoviral vectors in ChAdOx1 NP+M1 vaccinated cohorts. NAb titers in volunteer sera against ChAdOx1 NP+M1 (a–d) were determined pre- and postvaccination (day (D) 0, D14, and D21) for each group in the dose-escalation vaccination study. NAb titers were expressed as the reciprocal of the serum dilution required to reduce secreted alkaline phosphatase expression by 50% 24 hours posttransduction. 1/X, 1/dilution; M1, matrix protein 1; NP, nucleoprotein. Molecular Therapy 2014 22, 668-674DOI: (10.1038/mt.2013.284) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Median ex vivo interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) responses to influenza vaccine antigen NP+M1 in a ChAdOx1 NPM1 prime, MVA-NP+M1 boost regime. Individual ex vivo summed IFN-γ ELISpot responses to NP+M1 at baseline day (D) 0, D14, and D21 in vaccinated volunteers receiving a single dose of 5 × 1010 viral particles (vp) of ChAdOx1 NP+M1. Arrows indicate the time points for prime (D0) and MVA-NP+M1 boost (B) vaccinations. Three volunteers were boosted by intramuscular injection of 1.5 × 108 PFUs MVA-NP+M1 at 7 or 14 weeks (B) post-ChAdOx1 NP+M1 prime and followed up on D7, D21, and D56 post-boost (B+7, B+21, and B+56). Two volunteers who did not receive the MVA-NP+M1 boost were followed at B and B+56 time points. Vector names are abbreviated to ChAdOx1 and MVA on the graph. M1, matrix protein 1; MVA, modified vaccinia virus Ankara; NP, nucleoprotein. Molecular Therapy 2014 22, 668-674DOI: (10.1038/mt.2013.284) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions