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Immunomodulatory Effects of Cpg-Containing Oligonucleotides

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Immunomodulatory Effects of Cpg-Containing Oligonucleotides Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2008 Immunomodulatory effects of CpG-containing oligonucleotides and their potential to induce resistance to the feline immunodeficiency virus (FIV) infection of the domestic cat Robert-Tissot, Céline Abstract: This project was designed to study the in vivo immunomodulatory effects of CpG-containing oligonucleotides in the domestic cat and to determine their potential to induce short-term protection against FIV infection. The prophylactic treatment of the cats was carried out with dSLIM™, a non- coding DNA molecule containing several unmethylated CpG motifs, previously shown to induce strong cellular immune responses and production of IFN-฀, both highly desirable in the combat against viral infections. A group of 10 spf cats was treated during 5 consecutive days; a second group, equally of 10 spf cats, was simultaneously treated with a placebo. Both groups were challenge infected with FIV 24 hours after the end of the treatment. Clinical and immunological parameters were closely monitored in the cats throughout the experiment. When compared to the cats of the control group, 2 individuals treated with dSLIM™ indicated a 3-week delay in detection of proviral and viral loads in blood as well as isolation of infectious virus from plasma. A cytokine pattern representative of deviation to Th1 immune responses after treatment, as well as comparatively low antibody production after challenge enabled to attribute the partial resistance observed in these cats to cellular mechanisms. Despite promising results in this study, further investigations are necessary to determine the feasibility of stimulation of innate immunity as alternative to the development of a vaccine against FIV. Sowohl die immunmodulatorische Wirkungen von CpG Oligonukleotiden als auch ihr Potenzial einen kurzzeitigen Schutz gegen die FIV Infektion der Hauskatze zu erzeugen, wurden in dieser Studie in vivo untersucht. Die prophylaktische Behandlung der Katzen erfolgte mit dSLIM™, ein nicht-kodierendes DNA Molekül, das mehrere un- methylierte CpG Motive enthält und antivirale Eigenschaften besitzt, u.a. die Induktion einer zellulären Immunantwort und die Förderung der Produktion von IFN฀. Eine Gruppe von 10 SPF Katzen wurde während 5 Tagen mit dSLIM ™ behandelt. Eine weitere Gruppe von 10 Katzen wurde gleichzeitig mit einem Placebo behandelt. 24 Stunden nach Ende der Behandlung wurden alle Katzen einer Testinfek- tion mit FIV unterzogen. Klinische und immunologische Parameter wurden über einen Zeitraum von 12 Wochen regelmässig gemessen. In zwei mit dSLIM™ behandelten Katzen konnte im Blut provirale DNA, virale RNA und infektiöses Virus erst 3 Wochen später nachgewiesen werden als in den Katzen der Kontrollgruppe. Der beobachtete partielle Schutz konnte anhand des Th1 Zytokinmusters und des tiefen Antikörpertiters im Blut dieser zwei Katzen in den ersten Tagen nach der Testinfektion auf eine zelluläre Immunantwort zurückgeführt werden. Obwohl die Stimulierung des angeborenen Immunsys- tems im Kontext einer FIV Infektion versprechende Ergebnisse zeigte, sind weitere Studien erforderlich um diese Methode als Alternative zur Impfung zu erwägen. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-163761 Dissertation Published Version Originally published at: Robert-Tissot, Céline. Immunomodulatory effects of CpG-containing oligonucleotides and their potential to induce resistance to the feline immunodeficiency virus (FIV) infection of the domestic cat. 2008, University of Zurich, Vetsuisse Faculty. 2 Veterinärmedizinisches Labor der Vetsuisse Fakultät Universität Zürich Leiter: Prof. Dr. Hans Lutz Immunomodulatory Effects of CpG-containing Oligonucleotides and their Potential to Induce Resistance to the Feline Immunodeficiency Virus (FIV) Infection of the Domestic Cat INAUGURAL DISSERTATON zur Erlangung der Doktorwürde der Vetsuisse Fakultät Universität Zürich vorgelegt von Céline Robert-Tissot Tierärztin von Le Locle (NE) genehmigt auf Antrag von Prof. Dr. H. Lutz, Referent Prof. Dr. M. Ackermann, Korreferent Zürich 2008 Table of contents 1 Summary 1 2 Introduction 3 3 Literature 7 3.1 FIV Literature Overview 7 3.1.1 Viral Morphology and Genetic Structure 7 3.1.2 Viral Life Cycle 10 3.1.2.1 Receptor Usage and Cell Entry 10 3.1.2.2 Cellular Tropism 11 3.1.2.3 Replication and Production of Infectious Virions 12 3.1.3 Pathogenesis 14 3.1.3.1 Viral Transmission 14 3.1.3.2 Course of Disease 15 3.1.3.3 Immunological Aspects 17 3.1.3.3.1 CD4+ T Lymphocyte Depletion 17 3.1.3.3.2 CD8+ T Lymphocyte Anti-Viral Activity 18 3.1.3.3.3 T Cell Dysfunction, Anergy and Apoptosis 19 3.1.3.3.4 Cytokine Production 20 3.1.3.3.5 Antibody Responses 22 3.1.4 Epidemiology 23 3.1.4.1 Viral Clades 23 3.1.4.2 Host Range 24 3.1.4.3 Occurrence and Prevalence Studies 25 3.1.5 Clinical Aspects 26 3.1.6 Diagnostic Approach 30 I Table of contents 3.1.7 Treatment 33 3.1.8 Prevention 39 3.1.8.1 General Considerations 39 3.1.8.2 Vaccination 40 3.1.8.2.1 Choice of Adjuvant 41 3.1.8.2.2 Importance of Challenge Methods 44 3.1.8.2.3 Immune Correlates of Protection and Passive Immunity 46 3.1.8.2.4 FIV Vaccine Trials 47 3.1.8.2.4.1 Generalities 47 3.1.8.2.4.2 Conventional Vaccines 49 3.1.8.2.4.3 Subunit Vaccines 54 3.1.8.2.4.4 DNA Vaccines 59 3.1.8.2.5 Enhancement Problem 62 3.2 CpG Oligonucleotide Literature Overview 66 3.2.1 Insight on Innate Immunity and Toll-like Receptors 67 3.2.2 Definition of CpGs and Natural Occurrence 70 3.2.3 Uptake Mechanisms and Signal Transduction 71 3.2.4 Immunological Effects of CpGs 75 3.2.4.1 Cellular Immunomodulation of CpG DNA 76 3.2.4.1.1 B Cells 77 3.2.4.1.2 Dendritic Cells 78 3.2.4.1.3 Monocytes and Macrophages 79 3.2.4.1.4 Natural Killer Cells 80 3.2.4.1.5 T Cells 81 3.2.4.1.6 Neutrophils 82 3.2.4.2 Creation of a Th1-like Cytokine Milieu 82 3.2.4.3 Production of IFN-α 83 3.2.5 Species Specificity in CpG DNA Recognition 85 3.2.6 Production of Synthetic Oligonucleotides 87 3.2.7 Immunotherapeutic Possibilities 89 3.2.7.1 Protection of Immunocomprimised Hosts 89 II Table of contents 3.2.7.2 Cancer 91 3.2.7.3 Allergy 94 3.2.7.4 Infectious Diseases 96 3.2.7.4.1 Bacterial and Parasitical Infections 97 3.2.7.4.2 Viral Infections 98 3.2.7.5 Use of CpG ODN as Vaccine Adjuvants 102 3.2.7.6 Safety Concerns 105 4 Material and Methods 109 4.1 Objectives 109 4.2 General Information and Important Methods 110 4.2.1 Enzyme-linked Immunosorbent Assay 110 4.2.2 Nucleic Acid Extractions 111 4.2.3 Real-Time Fluorogenic Polymerase Chain Reaction 112 4.2.4 Peripheral Blood Mononuclear Cell Isolation 114 4.3 Pre-experiment Formalities 116 4.3.1 Cats 116 4.3.2 Adaptation Period and Assessment of Health Status 117 4.4 Course of the Study 118 4.4.1 Treatment Protocol 119 4.4.2 Challenge Infection 120 4.4.3 Clinical Auscultations, Blood and Oropharyngeal Swab Collections 121 4.4.4 Laboratory Analyses of Relevant Host and Viral Parameters 121 4.4.4.1 Haematology and Clinical Chemistry 121 4.4.4.2 Evaluation of Host Immune Parameters 122 4.4.4.2.1 Seroconversion 122 4.4.4.2.2 Determination of Cytokine Expression 122 4.4.4.2.2.1 Cytokine Measurements in Whole Blood 123 4.4.4.2.2.2 Cytokine Measurements in Stimulated and Unstimulated PBMCs 124 4.4.4.3 Evolution of FIV Infection 125 4.4.4.3.1 Measurements of Proviral and Viral Loads in Blood 125 III Table of contents 4.4.4.3.2 Isolation of Virus from Plasma 126 4.4.4.3.3 Measurements of Viral Load in Saliva 127 4.5 Statistical Analysis 127 5 Results 129 5.1 Clinical Examination 130 5.1.1 Body Weight 130 5.1.2 Body Temperature 130 5.1.3 Clinical Symptoms 131 5.2 Haematology 132 5.2.1 Red Blood Cell Parameters 133 5.2.2 White Blood Cell Parameters 134 5.2.2.1 Absolute White Blood Cell Counts 134 5.2.2.2 Neutrophilic and Eosinophilic Granulocytes 135 5.2.2.3 Monocytes 136 5.2.2.4 Lymphocytes 137 5.3 Clinical Chemistry 138 5.3.1 Substrates 138 5.3.2 Enzymes 139 5.3.3 Electrolytes 140 5.4 Immunology 142 5.4.1 Antibodies to FIV TM 142 5.4.2 Cytokine Measurements 143 5.4.2.1 Cytokine Expression in Whole Blood 143 5.4.2.2 Stimulation of Cytokine Production in vitro 145 5.5 Evaluation of Viral Infection 148 5.5.1 FIV Proviral Loads in Whole Blood 148 5.5.2 FIV Viral Loads 149 5.5.2.1 FIV Viral Loads in Plasma 149 5.5.2.2 FIV Viral Loads in Saliva 150 5.5.3 Isolation of Virus from Plasma 152 IV Table of contents 6 Discussion 155 6.1 Background of the Study 155 6.2 Design of the Study 158 6.3 Relevant Results 162 6.3.1 Results of the Clinical Examinations 162 6.3.2 Blood Parameters 164 6.3.2.1 Haematology 164 6.3.2.1.1 Deviation in Red Blood Cell Parameters 164 6.3.2.1.1.1 Hematocrit 164 6.3.2.1.2 Deviation in White Blood Cell Parameters 165 6.3.2.1.2.1 Leucocytes 165 6.3.2.1.2.2 Neutrophilic Granulocytes 165 6.3.2.1.2.3 Eosinophilic Granulocytes 166 6.3.2.1.2.4 Monocytes 166 6.3.2.1.2.5 Lymphocytes 167 6.3.2.2 Clinical Chemistry 167 6.3.2.2.1 Substrates 167 6.3.2.2.1.1 Blood Glucose Levels 167 6.3.2.2.1.2 Blood Urea Levels 169 6.3.2.2.2 Enzymes 169 6.3.2.2.2.1 Lipase 169 6.3.2.2.2.2 ALAT 169 6.3.2.2.3 Electrolytes 170 6.3.2.2.3.1 Blood Chloride Levels 170 6.3.2.2.3.2 Blood Phosphate Levels 170 6.3.3 Evaluation of Viral Infection 170 6.3.3.1 FIV Proviral Loads in Whole Blood 171 6.3.3.2 FIV Viral Loads 172 6.3.3.2.1 FIV Viral Loads in Plasma 172 6.3.3.2.2 FIV Viral Loads in Saliva 173 6.3.3.3 Isolation of Virus from Plasma 174 V Table of contents 6.3.4 Immunological Measurements 176
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