Experimental infection and protection against ... - TI Pharma

More documents

Recommendations

Info

Protection against a Malaria Challenge by Sporozoite Inoculation 191 Supplementary methods Serology To assess antibody titres, asexual ELISAs were performed as previously described [1]. CSP ELISAs were performed with (NANP)6 NA (kind gift from G. Corradin) according to published methods [2]. AMA-1 and GLURP ELISAs were performed according to a standard in-house protocol [2,3]. In all ELISAs a plasma pool of 8 healthy malaria-naïve Dutch volunteers was used as a negative control to define sample positivity (above mean+2SD of negative control). A plasma pool of Tanzanian adults (n=100) living in a highly malaria endemic area was used as reference positive control, defined to contain 100 arbitrary units (AU). Immunofluorescence assays were performed by incubating plasma at 1/320 dilution with air-dried whole sporozoites or at 1/40 dilution with asynchronous cultures of NF54 strain asexual P. falciparum parasites, as previously described [3]. Cellular immunology Cellular immune responses were assessed by in vitro stimulation assays. For use in these assays, Percoll-purified asynchronous asexual-stage cultures of NF54 strain parasites of 80-90% parasitemia, consisting of more than 95% schizonts/mature trophozoites, were washed, aliquoted and cryopreserved in advance. Mock-cultured uninfected erythrocytes (uRBC) were obtained similarly and served as control. Cryopreserved PBMCs were thawed immediately prior to use in in vitro stimulation assays, washed and resuspended in RPMI 1640 culture medium containing 2mM glutamine, 1mM pyruvate, 50μg/mL gentamicine and 10% pooled human AB+ serum (Sanquin, Nijmegen, NL), for a final concentration of 2.5x10 6 /mL. PBMCs were transferred into 96-well round-bottom plates (5x10 5 /well) and stimuli were added to duplicate wells. Stimuli included cryopreserved PfRBC (5x10 6 /mL), uRBC, PHA (10 μg/mL) or RPMI only. PBMCs were stimulated for 24 hours at 37°C/5%CO2. 4 hours prior to cell harvest, 100μL/well supernatant was collected and replaced with 100 μL/well fresh culture medium containing 20 μg/mL brefeldin A (Sigma). PBMCs from both time points per volunteer were tested simultaneously and cells from two vaccinees and one control were always tested together. Following 24 hour in vitro stimulation, PBMCs were harvested, washed once in FACS buffer (0.5% BSA/PBS) and incubated for 15’ with fluorescent mAbs against cell surface markers. Cells were washed again and incubated for 15’ in fixation
192 Chapter 9 medium A (Caltag Laboratories, Carlsbad, CA) according to the manufacturer’s instructions, washed again and incubated for 15’ with fluorescent mAbs against intracellular cytokines in permeabilization medium B. After a final wash step, cells were resuspended in FACS buffer and read on a FACScalibur flowcytometer. The following fluorescent mAbs were used: CD3-PerCP, CD4-PerCP, CD8-PE (BD, Breda, NL), IFNγ-FITC, TNFα-PE, IL-2-APC, CD45RO-PE, CD62L-PeCy7, mouse IgG1-FITC & IgG2a-PE and rat IgG2a-APC isotype controls (all Ebioscience, Uithoorn, NL). References 1. Bousema JT, Roeffen W, van der KM et al. Rapid onset of transmissionreducing antibodies in javanese migrants exposed to malaria in Papua, Indonesia. Am J Trop Med Hyg 2006; 74:425-31. 2. Remarque EJ, Faber BW, Kocken CH, Thomas AW. A diversity-covering approach to immunisation with Plasmodium falciparum AMA1 induces broader allelic recognition and growth inhibition responses in rabbits. Infect Immun 2008. 3. Hermsen CC, Verhage DF, Telgt DS et al. Glutamate-rich protein (GLURP) induces antibodies that inhibit in vitro growth of Plasmodium falciparum in a phase 1 malaria vaccine trial. Vaccine 2007; 25:2930-40.
Page 2 and 3:
Experimental infection and protecti
Page 4 and 5:
Experimental infection and protecti
Page 6:
Contents Contents 5 Chapter 1 - Int
Page 10 and 11:
Introduction 9 Malaria Malaria is o
Page 12 and 13:
Introduction 11 Preclinical Clinica
Page 14 and 15:
Introduction 13 pipeline of clinica
Page 16 and 17:
Introduction 15 The division of ant
Page 18 and 19:
Introduction 17 Figure 4. Populatio
Page 20 and 21:
Introduction 19 candidates. Initial
Page 22 and 23:
Introduction 21 - to identify param
Page 24 and 25:
Introduction 23 20. Nussenzweig RS,
Page 26 and 27:
Introduction 25 50. Ouedraogo AL, R
Page 28:
Introduction 27 RTS,S/AS02 in malar
Page 32 and 33:
Chapter 2 Safety and Immunogenicity
Page 34 and 35:
Safety and Immunogenicity of a Reco
Page 36 and 37:
Page 38 and 39:
Page 40 and 41:
Page 42 and 43:
Page 44 and 45:
Page 46 and 47:
Page 48 and 49:
Page 50 and 51:
Page 52 and 53:
Page 54 and 55:
Page 56 and 57:
Page 58 and 59:
Chapter 3 Humoral immune responses
Page 60 and 61:
Humoral immune responses to a singl
Page 62 and 63:
Page 64 and 65:
Page 66 and 67:
Page 68 and 69:
Page 70 and 71:
Page 72 and 73:
Page 74 and 75:
Page 76 and 77:
Page 78 and 79:
Page 80:
Page 83 and 84:
Chapter 4 82 Abstract The functiona
Page 85 and 86:
84 Chapter 4 Figure 1. Schematic re
Page 87 and 88:
86 Chapter 4 Concentration (mg/ml,
Page 89 and 90:
88 Chapter 4 Figure 4: Correlation
Page 91 and 92:
90 Chapter 4 positively correlated
Page 93 and 94:
92 Chapter 4 Acknowledgements The a
Page 95 and 96:
94 Chapter 4 determinant of subsequ
Page 98 and 99:
Chapter 5 Comparison of clinical an
Page 100 and 101:
Comparison of clinical and parasito
Page 102 and 103:
Page 104 and 105:
Page 106 and 107:
Page 108 and 109:
Page 110 and 111:
Page 112 and 113:
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
Chapter 6 Efficacy of pre-erythrocy
Page 120 and 121:
Efficacy of pre-erythrocytic and bl
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
Chapter 7 NF135.C10: a new Plasmodi
Page 132 and 133:
NF135.C10: a new Plasmodium falcipa
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
Page 140 and 141:
Page 142 and 143: NF135.C10: a new Plasmodium falcipa
Page 152 and 153: Chapter 8 Induction of malaria in v
Page 154 and 155: Induction of malaria in volunteers
Page 172: Section 3 Whole parasite inoculatio
Page 175 and 176: 174 Chapter 9 Abstract An effective
Page 177 and 178: 176 Chapter 9 Figure 1. Study desig
Page 179 and 180: 178 Chapter 9 followed by five iden
Page 181 and 182: 180 Chapter 9 Figure 2. Parasitemia
Page 183 and 184: 182 Chapter 9 Test Day I-1 Day C-1
Page 185 and 186: 184 Chapter 9 strain P. falciparum-
Page 187 and 188: 186 Chapter 9 protective role in ot
Page 189 and 190: 188 Chapter 9 References 1. Greenwo
Page 191: 190 Chapter 9 27. Orjih AU. Acute m
Page 195 and 196: 194 Chapter 10 Abstract Induction o
Page 197 and 198: 196 Chapter 10 Pre-erythrocytic sta
Page 199 and 200: 198 Chapter 10 procedures described
Page 201 and 202: 200 Chapter 10 by hand-dissection.
Page 203 and 204: 202 Chapter 10 Figure 3. Mean numbe
Page 205 and 206: 204 Chapter 10 Figure 4. Number of
Page 207 and 208: 206 Chapter 10 temperature (Pearson
Page 209 and 210: 208 Chapter 10 immune modulating ef
Page 211 and 212: 210 Chapter 10 cytokine measurement
Page 213 and 214: 212 Chapter 10 14. Hermsen CC, Telg
Page 216 and 217: Chapter 11 General Discussion
Page 218 and 219: General Discussion 217 occurrence o
Page 220 and 221: General Discussion 219 mediating th
Page 222 and 223: General Discussion 221 AMA1 express
Page 224 and 225: General Discussion 223 troponins ca
Page 226 and 227: General Discussion 225 and between
Page 228 and 229: General Discussion 227 immunologica
Page 230 and 231: General Discussion 229 to induce pr
Page 232 and 233: General Discussion 231 Conclusions
Page 234 and 235: General Discussion 233 References 1
Page 236 and 237: General Discussion 235 polymorphonu
Page 238 and 239: General Discussion 237 57. Church L
Page 240 and 241: General Discussion 239 85. Beier JC
Page 242 and 243:
General Discussion 241 118. Mueller
Page 244:
Chapter 12 Summary Samenvatting Lis
Page 247 and 248:
246 Chapter 12 Controlled human mal
Page 250 and 251:
Summary, Samenvatting, List of publ
Page 252:
Page 255 and 256:
254 Chapter 12 Roestenberg M, Teirl
Page 258 and 259:
Page 260 and 261:
Page 262:
show all

Experimental infection and protection against ... - TI Pharma

Create successful ePaper yourself

Delete template?

Save as template?