Approaches to genetic vaccines

1. Approaches to genetic vaccines Britta Wahren Karolinska Institutet Riga 2013

2. Selection of immunogen DNA, RNA or (glyco)protein Whole genes to cover polymorphic MHC in humans All genes to cover structural and regulatory genes Several subtypes for virus variability and polymorphic MHC Vectors like vaccinia or adeno Physical approach Small total vector size Intracellular delivery (skin)- electroporation Delivery devices Total cell targets – repeated injections Divided delivery-avoid immunocompetition Additional adjuvant like cytokine gene, carrier compound

3. 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Minicircle1x 650 bp % nicked Minicircle3x 1 900 bp sc sc sc nicked nicked Plasmid 3 579 bp 1: untreated 2: linearized 3: UV-nicked 4: shot to empty vial 5: shot through mouse hide Small minicircles were more robust when exposed to shearing forces than the corresponding plasmid. Also as splice correcting vectors, they exhibited a more long-term expression as compared to plasmids in vivo.

4. Electroporation after needle/jet stream injection Needle injection Jet stream injection Intracellular delivery Dermis

5. Electroporation after needle/jet stream injection Needle injection Jet stream injection Dermis Secreted antigens 1. Transfection of somatic and professional antigen presenting cells (APCs) Somatic cell APC MHC I MHC II MHC I APC 2. Antigen presentation to and activation of lymphocytes in the draining lymph nodes CD4 T cell APC T cell receptor Co- stimmulatory signals MHC II MHC I Y T cell receptor CD8 T cell Y B cell 3. Activated lymphocytes

6. Delivery device Biojector im id spacer Left arm: Env/rev, 3 inj +/- GMCSF sc (needle) Left arm: Env/rev, 1 inj +/- GMCSF im Right arm: Gag/RT, 1 inj Right arm: Gag/RT, 2 inj

7. 2x450V Voltage 8x110V Time Total length of pulse-train: 0,27 seconds Delivery device electroporator

8. Repeated injection of DNA, dispersed delivery

9. Volume and concentration • Comparison of different immunization protocols using ID EP • BALB/c mice • Imm w 0, 4 and 8 with GagB • ID+EP (15ug) or IM (50ug) • IFNg ELISpot • Comparison of different immunization methods • BALB/c mice • 1 imm with GagB • IFNg ELISpot

10. Divided delivery of env and gag genes, subtypes A, B, C Vial 1 Envelope and rev plasmids Vial 2 gag and RT plasmids Vaccine intramuscularly delivered Vial 1 in the left arm, Vial 2 in the right

11. DNA vaccines, licenced and clinical Licenced • West Nile virus for horses • Hematopoietic necrosis (rhabdovirus) for salmon • Melanoma (tyrosine kinase) for dogs, humans? • Pig hormonal treatment Clinical small trials • HCV for chronic disease, Sällberg et al, SE • Prostate heterologous antigen, Pisa et al, SE • CEA heterologous and homologous antigen for gi cancer Mellstedt, Wahren et al, SE • HIV for healthy individuals, Ho et al, USA and China, • HIV for healthy or chronic disease, Wahren et al, SE, IT, TZ, MC; Pantaleo et al, CH, Robinson et al US

12. Acknowledgements: The volunteers Walter Reed Army Institute of Research Josephine Cox Mary Marovich Nelson Michael Merlin Robb Deborah Birx SMI/KI Britta Wahren Andreas Bråve Margaret Liu Karl Ljungberg Gunnel Biberfeld Jorma Hinkula Erik Rollman Gunnel Engström Kristian Hallermalm Lindvi Gudmundsdotter Andreas Boberg Susanne Johansson Anne Kjerrström Maria Isaguliants Charlotta Nilsson Katarina Karlén Reinhold Benthin Pontus Blomberg Jenny Enger Sofia Stenler Nils Carlin NIH/NIAID Bernard Moss Patricia Earl Richard Stout Members of the AVIP Consortium University of Munich Volker Erle Georg Gasteiger ISS, Rome Barbara Ensoli Bambino Gesú Paolo Rossi Paolo Palma CytoPulse Richard Walters Anna-Karin Roos Cellectis Karolinska/SöS Eric Sandström Bo Hejdeman Lars Eriksson Viveca Holmberg MUHAS, Dar es Salaam Fred Mhalu Muhammad Bakari Eligius Lyuamuya Staffan Pauli Bartek Zuber