Bacterial expression of a VLP Sub-unit for rapid and cheap influenza vaccination
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1 Engineering Conferences International ECI Digital Archives Vaccine Technology IV Proceedings Spring Bacterial expression of a VLP Sub-unit for rapid and cheap influenza vaccination Anton Middelberg Australian Institute for Bioengineering and Nanotechnology The University of Queensland, Australia Follow this and additional works at: Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Anton Middelberg, "Bacterial expression of a VLP Sub-unit for rapid and cheap influenza vaccination" in "Vaccine Technology IV", B. Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves, ITQB/IBET; K. Jansen, Wyeth Vaccine Research Eds, ECI Symposium Series, (2013). This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Vaccine Technology IV by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.
2 Vaccine Technology IV May 20 25, 2012 Engineering VLP and Capsomere Vaccines Anton Middelberg Australian Institute for Bioengineering and Nanotechnology The University of Queensland, Australia
3 Introduction Vaccination enormously successful Smallpox eradicated, polio close More to do 15 M people still die annually, half children <5 yrs Emergent and re-emergent disease Technological gap in approaches Pasteur s Isolate-Inactivate-Inject dominates Opportunity to engineer better systems
4 Modular Vaccine Design &pagename=FSSA%2FDFYPage%2FF ord-default&c=dfypage&site=fssa BASE with sites for insertion of optional modules + Front guard MODULE MODULAR DESIGN Viral antigen 3
5 Murine Polyomavirus X 72 VP1 Protein Capsomere Virus-like Particle (VLP or Capsid)
6 Antigen Insertion Sites on VLP Surface
7 Bioprocess Engineering pgex-4t-1-vp bp GST Thrombin Cleavage Site + GG BamHI (931) VP1 XhoI (2098) Best available expression in literature: 1 mg/l.od After factorial optimisation, Host selection and redesign: mg/l.od J. Biotechnol. (2008), 134(1-2): Ampicillin Resistance Gene Confirmed 2-4 g/l in fed-batch E. coli fermentation. J. Biotechnol. (2010), 150(2):
8 VP1 self-assembly in vitro
9 Process Flowsheet S-103 S-101 P-2 / HG-101 S-102 P-1 / V-101 Fermentation Homogenization P-3 / C-101 Expanded Bed P-4 / V-102 Tag Removal S-104 S-106 S-105 S-107 P-7 / V-103 Capsid Assembly P-6 / DE-101 Sterilee Filtration P-5 / C-102 Capsomere Purification
10 The UQ Microbial Vaccine Platform (MVP) Speed same process for different viruses time from DNA to purified antigen < 1 week processing can be automated Scale Makes protein using industrial biotechnology tools 100M doses per kl of bacterial culture in 24 h Safety we make protein, not virus we can sterile filter before virus assembly 9
11 The UQ Microbial Vaccine Platform (MVP) Purification and Assembly Water Glucose Salts Bacteria 2 g/l Soluble Antigen Within 24 h Purification And Assembly (48 h) >100,000 doses per litre 1000L pilot = 100M doses In 24 h Dose Excess regime. Everyone can cultivate bacteria.
12 Pre-existing immunity against the platform? 11
13 Pre-existing immunity Day Day Response against wt VLP J8i (Group A Streptoccocus) Day 21 Day 77 ns Endpoint titer (Anti-antigen spec cific IgG) wt primed PBS primed wt primed PBS primed ns = not significant 12
14 Application of the Platform: Influenza 13
15 Influenza Global Challenge When the virus changes, existing vaccine does not work. 14
16 H1N1 (2009): April 27 th 15
17 H1N1 (2009): May 27 th 16
18 H1N1 (2009): September 27 th 17
19 Influenza in a Connected World Share Control Identify People die while they wait for the new vaccine 18
20 Rapid response for emergent virus Laboratory Timeline (Days): Vaccine available for in vivo testing Microbial culture Modular capsomere Modular VLP 19
21 Initial Target Epitopes Completely Non-Universal HA1 receptor binding regions Other HA1 epitopes Assume Influenza Changes Somewhat Universal M2e of matrix protein 2 HA stalk regions Assume Influenza Behaves 20
22 Haemagglutinin Helix 190 Influenza A virus Receptor binding site. Biology 101 blocking the receptor binding site will block viral entry. Glycosylation? Structure? 2/influenza-virus-diagram.jpg 21
23 Modularize into VLP format Target epitope Capsomere presenting target epitope x72 x5 Viral protein VP1 VLP presenting target epitope 22
24 Structural analysis of helix 190 peptide MD simulation Gromacs In PBS solution 20 ns Helix 190 in native HA Peptide B1 Peptide B2 23
25 RMSD Structural deviation of designed he elix 190 peptide from native 30 Peptide B Peptide B2 Angstrom C-alpha Main-chain Side-chain Whole peptide 0 24
26 Module Structure Matters Peptide *** ns Glycosylated HA *** *** ns = not significant 25
27 Glycosylation Matters Less B2 VLPs B1 VLPs controls 26
28 Initial Target Epitopes Completely Non-Universal HA1 receptor binding regions Other HA1 epitopes Biology 101 Somewhat Universal M2e of matrix protein 2 HA stalk regions 27
29 Matrix Protein M2e Influenza A virus Immunogenic Broad cross protection Complementary mechanism Modularize into capsomere format x5 fluenza-virus-diagram.jpg VP1 28
30 Engineering VP1 for antigen modules Assembly incompetent Improved stability x5 Trypsin digestion site Antigen insertion: two surface loops N-terminus C-terminus 29
31 Modular capsomere 30
32 Screening of modularized capsomere Expression level Solubility level Downstream bioprocessing yield 1011 and
33 Capsomere format improves immunogenicity p = Endpoi int titre (Anti-M2e specific IgG) PBS 1011 Alhydrogel Alhydrogel 32
34 Modular capsomere 1011 vs 2022 Endpoint titre (Anti-M M2e specific IgG) Wild-type capsomere Alhydrogel 2022 Alhydrogel Excellent IgG titres Little sensitivity to more modules Adjuvant necessary for capsomere format Excellent epitope tolerance 33
35 Conclusions VLP and capsomere platform developed Remarkable productivity, protein not virus based Excellent developability and manufacturability Excellent end point titres Moving to protection studiess Multitude of insertions successfully handled Flexibility afforded by VLP and Capsomere formats 34
36 Influenza in a Connected World Share Control Identify 35
37 Acknowledgements Dr Linda Lua & UQ Protein Expression Facility Ms Melisa Anggraeni 36
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