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Measles Virus: Genetics and Applications

Measles Virus: Genetics and Applications. Mononegavirales Superfamily. Filoviridae. Paramyxoviridae. Rhabdoviridae. Genus. Genus. Genus. Paramyxovirus. Rubulavirus. Vesiculovirus. Lyssavirus. Morbillivirus. Pneumovirus. Filovirus. Rabies virus. Marburg virus. Mumps virus.

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Measles Virus: Genetics and Applications

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  1. Measles Virus: Genetics and Applications

  2. Mononegavirales Superfamily Filoviridae Paramyxoviridae Rhabdoviridae Genus Genus Genus Paramyxovirus Rubulavirus Vesiculovirus Lyssavirus Morbillivirus Pneumovirus Filovirus Rabies virus Marburg virus Mumps virus Respiratory Syncytial virus Measles virus Vesicular Stomatitis virus Sendai virus Human parainfluenza Virus 1, 3 Human Parainfluenza Virus 2,4 Ebola virus Rinderpest virus ? Simian Parainfluenza Virus 5-41 Nepha virus Lettus Peste des Petits Ruminants virus necrotic ? yellows Plant Newcastle Disease virus Potato yellow Canine Distemper virus dwarf Avian Paramyxo- Viruses 2-9 Dolphin morbillivirus ? Equine morbillivirus ?

  3. Structure of Measles Virus ENVELOPE GLYCOPROTEINS • H, receptor attachment & • Helper F-function. • F, fusion at the host cell surface. MATRIX protein (M) • Regulates cell-cell fusion, • Virus release, • Env. protein sorting. GENOME • Helical (>1) • ssRNA, -ve sense, • N, nucleoprotein • P, phosphoprotein • L, polymerase Pleomorphic Diameter 200-350nm 100nm

  4. Attachment and release of measles virus Entry Attachment Release cell-cell fusion RNP CD46 or SLAM RNP Replication/transcription M, F, H interaction L, P depend mRNA Cytosolic: M, N, P, L F H protein synthesis Membrane bound: F, H

  5. Infection and Transmission Respiratory tract Transmission by aerosole 2 Apical virus infection and release 1 Basolateral fusion with Macrophages and uptake of infectious viral RNP 3 macrophages Spread to local lymph node Local lymph node Transport to the blood stream via Macrophages and virions Ductus thoracicus 4 Systemic infection

  6. Measles Typical symptoms Koplik spots Rash • 2-3 days prodormal stage: • Running eyes and nose • Cough • Fever • Koplik spots • 5 days later... • Rash appears on the face, • spreads to limbs, trunk then • the whole body. • Rash lasts 4-7 days.

  7. Measles (Robeola;red measles; nine day measles) • An ancient and highly contagious childhood disease. • First described by the Persian scientist El Rhazi, 840 AD. • The greatest killer of children in the history. • Affects about 50 million individuals annually and, causes up to one million deaths • primarily in developing world. • The infection spreads by aerosole. • Complications: bacterial infection (immunosuppression at the onset • of disease 3-7 days). SSPE (1:10000 cases) is a rare but deadly consequence. • Since mid 1960's a live attenuated vaccine became available.

  8. Attenuation of Wild Type Measles Virus Isolate from David Edmonston Mid 50's

  9. Break-through: Reverse genetics Rescue of Negative Strand RNA Viruses from cDNA Measles virus 1993-95 Rhabdoviruses 1995-96 Respiratory syncytial virus 1996-97 Parainfluenza virus 1996-97

  10. 14265 ClaI 11230 ScaI 15894 bp Cloning of full-length MV cDNA Virus stock Ultracentrifugation Isolation of genomic RNA Generation of cDNA RNA cDNA PCR Leader-SacII SacII-PacI PacI-ScaI ScaI-ClaI ClaI-Trailer

  11. Topo-Leader-SacII Topo-SacII-PacI Topo-PacI-ScaI Topo-ScaI-ClaI Topo-ClaI-Trailer Cloning of full-length MV cDNA Cloning of PCR fragements into intermediate plasmids Generation of plasmid encoding the full-length antigenome of MV

  12. Rescue of Measles virus from cDNA The technology: T7 EMC IRES T7 p(+)MV pEMCLa L Cotransfection • 293 cell line with • T7 RNA pol • N and P Syncytia formation Virus rescue

  13. IRES d T F pdA H F M T7 L T7 P L N p(+)MV pEMC-La helper cell N 1. plasmid DNA transfection (293-3-46) P T7 2. cytoplasmic T7 transcription T7 3. simultaneous encapsidation (+) 5' 3' 5' 3' ACCA UGGU 4. MV N, P, and L-dependent replication AUG A(n) L 5. MV P/L-dependent transcription _ ( ) 3' 5' MV genome as RNP (15894 nt.) 6. MV proteins A(n) 7. virus assembly A(n) A(n) A(n) A(n) A(n) MV particle N P M F H L Rescue system • Transfection of two plasmids p(+)MV and pEMC-La the T7RNA polymerase produces MV antigenomes and the mRNA for the MV L protein. •The MV antigenome is tightly encapsidated by N and P proteins, stabily expressed in the helper cell line. • Together with the transiently expressed L, a RNP is formed  template for MV-N,P, L- dependent transcription of all MV mRNAs. • With increasing amounts of MV mRNAs  switch from transcription to replication  production of full-length RNPs  virus assembly  formation of progeny MV particles.

  14. H F L P L P N N M 3 1 2 RNA RNA d N P M F H L T7 T7t C V Generation of live recombinant vaccines Measles vaccine Recombinant vaccine Additional transgene

  15. HIV-nef N P M F H L Recombinant MVs with foreign genes Position 1: Position 2: HBV sAg s N P M F H L Mumps N P HN/F M F H L IL-12 N P M F H L p40/p35 SIV genes N P M F H L SIV/HIV gag/pol N P M F H L

  16. SIV/HIV gp160/gag N P M F H L Malaria gp190/gp42 H P M F L N HBV cAg N P M F H L HBV sAg HBV cAg N P M F H L gp160 N P M F H L gag/pol Recombinant MV with foreign genes Position 3: Multiple gene insertions:

  17. Applications Pathogenesis and disease Virus-Cell interaction Basic Research Molecular Biology of the virus • entry and • replication • assembly • budding • immunology • transmission • complication • disease • structure and function • attenuation Cell-culture model Animal models

  18. Applications • Prophylactic Vaccines: • AIDS • Malaria • Emerging diseases • Therapeutic Vaccines: • Cancer

  19. Prophylactic vaccine AIDS

  20. Expression of HIV proteins by rMV Env B3 Env B2, dV1,2 Env B3, dV1,2 gag B3 p17,p24 C2 p17,p24 C3

  21. 129-S-IFNAR/CD46 mice were injected with recombinant viruses (5 X106 pfu, ip) MV and HIV serology were determined by ELISA (1 month pi) (MV, Trinity Biotech, HIV, Sanofi Diagnostics) A (anti-MV) B (anti-HIV) 100000 100000 10000 10000 1000 1000 100 100 10 10 1 1 MV2 gp140 MV3 gp140dV3 MV2 gp160 MV2 gp160dV3 MV2 gp140 MV3 gp140dV3 MV2 gp160 MV2 gp160dV3 Immunization by recombinant MV-HIV89.6p env induces humoral responses in susceptible mice Titer (1/log dilution)

  22. Therapeutic vaccine Cancer

  23. 50 um Day 15 Day 0 Day 6 NP Regression of tumor grafts in nude mice during i.t. MV treatment Immunoreactivity of MV NP (peroxidase staining)

  24. after treatment before treatment Clinical study

  25. Immunoreactivity for MV NP (peroxidase staining for MV nucleoprotein (arrows))

  26. Conclusions • MV is certified, widely used, and has a good record of efficacy and safety. • Highly immunogenic. Replicates in macrophages, dendritic cells, and lymphocytes (long-lasting IgG, CTL and mucosal immunity). • Large number of vaccination doses can be produced, easily and economically. • Flexibility, and stability of MV-reverse genetics system. • rMV-HIV induce neutralizing and cellular anti-HIV antibodies • The recombinant vaccine works well in pre-immunized subjects. • MV induces local and systemic immunological and clinical responses. • Strategies for specific retargeting of MV to target cells are quite promising.

  27. Acknowledgements Berna Biotech AG Valérie Künzi Armando Zuniga Matthias Liniger Sara Weibel Hussein Naim University of Zürich Zili Wang Lars Hangertner Martin Billeter

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