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Vaccination Studies with the Mper of Hiv-1 Gp41 Grafted Into the Transmembrane Protein of a Gammaretrovirus

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Vaccination Studies with the Mper of Hiv-1 Gp41 Grafted Into the Transmembrane Protein of a Gammaretrovirus VACCINATION STUDIES WITH THE MPER OF HIV-1 GP41 GRAFTED INTO THE TRANSMEMBRANE PROTEIN OF A GAMMARETROVIRUS Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von NICOLA STRASZ aus Wien Juni 2013 Aus dem Robert Koch-Institut, Berlin, Deutschland. Angefertigt unter der Betreuung von Dr. Joachim Denner Februar 2009 bis Mai 2013 Erster Gutachter: Prof. Dr. Reinhard Kurth, Robert Koch-Institut, Berlin Zweiter Gutachter: Prof. Dr. Rupert Mutzel, Freie Universität Berlin Tag der Disputation: Die dieser Dissertation zugrunde liegenden Arbeiten wurden am Robert Koch-Institut in Berlin, in der Zeit vom 01. Februar 2009 bis 31. Mai 2012 durchgeführt. Hiermit erkläre ich, dass ich die vorliegende Dissertation selbstständig verfasst und keine anderen als die angegebenen Hilfsmittel verwendet habe. Wien, am ________ Nicola Strasz Table of Contents Abbreviations ...................................................................................................................................... 1 1. Introduction ......................................................................................................................... 3 1.1 The discovery of HIV ............................................................................................................ 3 1.2 HIV transmission and pathogenesis .................................................................................... 3 1.3 Genome structure of HIV-1 .................................................................................................. 5 1.4 Morphology of HIV-1 ............................................................................................................ 5 1.5 The replication cycle of HIV-1 .............................................................................................. 6 1.6 The envelope proteins ......................................................................................................... 7 1.6.1 The role of the envelope proteins gp120 and gp41 in the attachment and membrane fusion process ............................................................................................................................. 9 1.7 Blocking the infection process by neutralizing antibodies against envelope proteins ....... 10 1.7.1 Broadly neutralizing antibodies binding to gp120 ....................................................... 12 1.7.2 Neutralizing antibodies binding to gp41 ..................................................................... 13 1.8 Passive immunization using neutralizing antibodies ......................................................... 14 1.9 Additional characteristics of vaccine induced antibodies ................................................... 15 1.10 Vaccine development against HIV ..................................................................................... 16 1.10.1 Live-attenuated and inactivated vaccines .................................................................. 16 1.10.2 Subunit immunogen design ........................................................................................ 16 1.10.3 Lipopeptides and liposomes ....................................................................................... 17 1.10.4 Candidate HIV vaccines and clinical trials .................................................................. 19 1.11 The porcine endogenous retrovirus ................................................................................... 19 1.11.1 Phylogeny and genome organization ......................................................................... 19 1.11.2 Morphology, replication and pathogenic potential ...................................................... 20 1.12 The rationale of designed hybrid proteins based on gammaretroviruses .......................... 21 1.13 Aim of the thesis ................................................................................................................ 22 2. Materials and Methods ...................................................................................................... 23 2.1 Materials............................................................................................................................. 23 2.1.1 Primers ....................................................................................................................... 23 2.1.2 Plasmids ..................................................................................................................... 23 2.1.3 Enzymes ..................................................................................................................... 24 2.1.4 Bacterial strains .......................................................................................................... 24 2.1.5 Bacterial media ........................................................................................................... 24 2.1.6 Peptides, Pepspot-Membranes and Peptide-Microarrays .......................................... 25 2.1.7 Antibodies ................................................................................................................... 25 2.1.8 Cell lines ..................................................................................................................... 25 2.1.9 Viruses ........................................................................................................................ 25 i 2.2 Methods ............................................................................................................................. 26 2.2.1 Molecular biology ........................................................................................................ 26 2.2.2 Working with proteins and peptides ............................................................................ 28 2.2.3 Immunological methods .............................................................................................. 30 2.2.4 Animal experiments .................................................................................................... 33 2.2.5 Application of antigens ............................................................................................... 34 2.2.6 Cell culture .................................................................................................................. 35 2.2.7 DNA vaccination with the Semliki Forest Virus vector system ................................... 36 3. Results .............................................................................................................................. 39 3.1 Titer assessment of virus stocks ........................................................................................ 39 3.2 Design of hybrid proteins ................................................................................................... 39 3.3 N1 and N2 hybrid proteins ................................................................................................. 41 3.3.1 Design, expression and characterization of recombinant hybrid proteins N1 and N2 41 3.3.2 Immune responses induced by immunizations with N1 and N2 ................................. 43 3.4 N3 and N4 hybrid proteins ................................................................................................. 45 3.4.1 Design, cloning, expression and characterization of recombinant hybrid protein N3 45 3.4.2 Immunization of 4 guinea pigs and 4 rats and 1 rabbit with N3 ................................. 46 3.4.3 Design, expression and characterization of recombinant hybrid protein N4 .............. 49 3.4.4 Immunization of rats with N3 and N4 .......................................................................... 51 3.4.5 Immunization study of four rats and one goat with N4 ............................................... 54 3.5 Immunization with recombinant N1 in liposomes .............................................................. 58 3.6 Immunization with recombinant N3 and N4 in liposomes .................................................. 59 3.7 DNA immunization with recombinant hybrid antigens in 2 different expression vector systems ............................................................................................................................. 62 3.8 Testing of sera for reactivity to lipids ................................................................................. 67 4. Discussion ......................................................................................................................... 70 4.1 Protection against HIV infection ......................................................................................... 70 4.2 Hybrid proteins used as immunogens ............................................................................... 70 4.3 DNA vaccination of hybrid proteins .................................................................................... 76 4.4 Structure-based antigen design ......................................................................................... 78 4.5 Are MPER-specific bNAb autoreactive? ............................................................................ 79 4.6 Liposomes as vaccine carrier ............................................................................................ 81 4.7 Measuring neutralization with in-vitro assays .................................................................... 83 4.8 Prevention from HIV-1 infection ........................................................................................
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