In vivo evaluation of recombinant Vaccinia virus MVA delivering ancestral H9 hemagglutinin

106 Pages·2015·4.93 MB·English

by  Jens Michael Becker

IN VIVO EVALUATION OF RECOMBINANT VACCINIA VIRUS
MVA DELIVERING ANCESTRAL H9 HEMAGGLUTININ
ANTIGEN OF AVIAN INFLUENZA VIRUS

von Jens Michael Becker
Inaugural-Disseration zur Erlangung der Doktorwürde
der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität
München

IN VIVO EVALUATION OF RECOMBINANT VACCINIA VIRUS
MVA DELIVERING ANCESTRAL H9 HEMAGGLUTININ
ANTIGEN OF AVIAN INFLUENZA VIRUS

von Jens Michael Becker
aus Karlsruhe
München 2015
Aus dem Veterinärwissenschaftlichen Department der Tierärztlichen Fakultät
der Ludwigs-Maximilians-Universität München

Lehrstuhl für Virologie

Arbeit angefertigt unter der Leitung von Prof. Dr. med. vet. Dr. habil Gerd Sutter

Angefertigt an der Ecole Nationale Vétérinaire de Toulouse, Frankreich
Mentor: Dr. Mariette Ducatez
Gedruckt mit Genehmigung der Tierärztlichen Fakultät
der Ludwig-Maximilians Universität München

Dekan: Univ.-Prof. Dr. Joachim Braun

Berichterstatter: Univ.-Prof. Dr. Gerd Sutter

Korreferent/en: Univ.-Prof. Dr. Dušan Palić

Tag der Promotion: 18. Juli 2015

1. Introduction and Aim of the Study ......................................................... 10
2. Review of Literature ...................................................................................11
2.1 Influenza A Virus ..................................................................................................... 11
2.1.1 Viral Classification and Structure ..............................................................................................11
2.1.2 Major Influenza A Virus Proteins ............................................................................................. 12
2.1.3 Viral Reproduction Cycle ......................................................................................................... 16
2.1.4 Receptors and Host Spectrum .................................................................................................. 16
2.1.5 Evolution of Avian Influenza Viruses – Antigenic Drift and Antigenic Shift .......................... 17
2.1.6 Clinical Signs in Birds .............................................................................................................. 19
2.1.7 Approach to Epidemiological Processes using the example of HPAI H5N1 ........................... 20
2.2 Low Pathogenic Avian Influenza H9N2 Virus ....................................................... 24
2.2.1 Geographical Distribution and Prevalence in Birds ................................................................. 24
2.2.2 Human Cases and Prevalence in People ................................................................................... 25
2.2.3 Evolution .................................................................................................................................. 25
2.2.4 Specific Clinical Signs ............................................................................................................. 27
2.2.5 Pathogenesis in Different Species ............................................................................................ 27
2.2.6 Transmission ............................................................................................................................ 28
2.3 MVA Vector Vaccine ............................................................................................... 29
2.3.1 Ancestral HA strategy .............................................................................................................. 29
3. Material and Methods ............................................................................... 31
3.1 Vaccine Production ................................................................................................. 31
3.1.1 Provision of Vaccine Components ............................................................................................ 31
3.1.2 Dataset selection and Phylogenetic Analyses ........................................................................... 31
3.1.3 Generation of Ancestral Sequence ........................................................................................... 32
3.1.4 Generation of recombinant MVA vaccine ................................................................................ 32
3.1.5 Purification, Titration and Quality Control of the rMVA ......................................................... 34
3.2 Birds and Challenge Virus Strain ........................................................................... 34
3.2.1 Birds ......................................................................................................................................... 34
3.2.2 Challenge Strain A/chicken/Tunisia/12/2010 ........................................................................... 35
3.3 Immunization and Challenge .................................................................................. 35
3.3.1 Setup and Protocol Timeline of the Animal Experiment .......................................................... 35
3.3.2 Vaccine Dose and Route of Inoculation ................................................................................... 36
3.3.3 Observation of Clinical Signs................................................................................................... 36
3.3.4 Swab Sampling ......................................................................................................................... 36
3.3.5 Necropsy ................................................................................................................................... 36
3.3.6 Histopathology and Immunohistochemistry............................................................................. 39
3.4 Virus Shedding Titration ......................................................................................... 39
3.4.1 RNA extractions ....................................................................................................................... 39
3.4.2 Reverse Transcription Real-Time Polymerase Chain Reaction (rt-RT-PCR) .......................... 40
3.5 Serology .................................................................................................................. 42
3.5.1 Hemagglutination Inhibition (HI) Assay .................................................................................. 42
3.5.2 Seroneutralization ..................................................................................................................... 42
4. Results .......................................................................................................... 44
4.1 Vaccine design ........................................................................................................ 44
4.2 Clinical Appearance ................................................................................................ 47
4.2.1 Clinical Signs ........................................................................................................................... 47
4.2.2 Macroscopic Lesions ................................................................................................................ 47
4.2.3 Histopathology ......................................................................................................................... 47
4.3 Virus Shedding ....................................................................................................... 52
4.3.1 Group Mean Virus Titres .......................................................................................................... 52
4.3.2 Individual virus titers................................................................................................................ 53
4.4 Serology .................................................................................................................. 55
4.5 Cross-reactivity of ancestral MVA H9 sera with different H9N2 viruses .............. 64
5. Discussion and Conclusion ....................................................................... 65
6. Summary ..................................................................................................... 71
7. Résumé ......................................................................................................... 72
8. Zusammenfassung ..................................................................................... 73
9. Danksagung ................................................................................................ 75
10. List of Figures ............................................................................................. 76
11. List of Tables ............................................................................................... 80
12. Abbreviations .............................................................................................. 81
13. References ................................................................................................... 82
14. Appendix ..................................................................................................... 87
14.1 Poster « Low Pathogenic Influenza (H9N2) in chicken: Evaluation of ancestral
MVA vaccine » ........................................................................................................ 87
14.2 Application Document for Animal Experiment to the Ethical Committee ............ 87