Experimental infection and protection against ... - TI Pharma

More documents

Recommendations

Info

Safety and Immunogenicity of a Recombinant Plasmodium falciparum AMA1 Malaria vaccine Adjuvanted with Alhydrogel TM, Montanide ISA 720 or AS02 ELISPOT for IFNy and IL-5 ELISPOT was performed according to manufacturer’s instructions (Becton and Dickinson Elispot Set Human IFNγ or IL-5). In summary, plates provided in the set were coated with either IFNγ or IL-5 capture antibody (5 µg/ml, overnight, 4°C). After blocking with complete medium solution (RPMI 1640 (Invitrogen) containing 10% Fetal Bovine Serum (FBS Invitrogen, Breda, The Netherlands), 1% Glutamax (Invitrogen), 1% Penicillin-Streptomycin (GIBCO-BRL, Invitrogen), 1% MEM) 100 µl of 105 PBMC suspension and 100 µl of PfAMA1 containing either 60 µg, 12 µg or 2.4 µg was added per well. Positive controls were stimulated with Tetanus Toxoid 10 µg/ml (RIVM, Bilthoven, The Netherlands) and phytohaemagglutinin 5 µg/ml (PHA-L Sigma-Aldrich) end concentration. Negative controls were incubated with complete medium solution (mean SFC/10 5 cells 31±17 for IFNγ and 9±7 for IL-5). After incubation (40 hours, 37°C in humidified 5% CO2), biotinylated anti human IFNγ and IL-5 (0.25 µg/ml and 2 µg/ml, respectively), containing 10% FBS was added (two hours at room temperature). Streptavidin-HRP was used as an enzyme conjugate. Detection was performed with the Becton and Dickinson AEC Substrate Reagent Set, according to manufacturer’s instruction. Spot-forming cell numbers were counted by ELISPOT reader (4 Microtitre Plate Reader, AELVIS, Sanquin, Amsterdam) and analysed by the ELISPOT Analysis Software Version 4.0 (Sanquin, Amsterdam). All measurements were performed in triplo. Variation between triplicates was set to a maximum of 20%. Lymphocyte Stimulation Assay Lymphocyte stimulation assays were performed as described previously [31]. Peripheral blood mononuclear cell suspension (PBMC) was diluted to 1 x 10 6 PBMC per ml in Dulbecco’s MEM (DMEM) with Glutamax-I, 2 mM pyruvate and high Glucose (GIBCO BRL, Invitrogen) supplemented with 10 mM HEPES buffer (GIBCO BRL, Invitrogen), 100 IU/mL Penicillin-Streptomycin (GIBCO BRL, Invitrogen), 100 µM non-essential amino acids (GIBCO BRL, Invitrogen) and 2.5% human AB serum (AB) (Bodinco BV, Alkmaar, The Netherlands). 100 µl of PBMC was added to 100 µl PfAMA1 (30, 6 or 1.2 µg/ml in PBS) in 96 well Nunclon surface flat plates (Life Technology). Plates were incubated (six days, +37°C, humidified 0.5%CO2) before labelling (10 µl 3H thymidine, 0.25 uCi per well, 24 hours) and harvested onto Wallac filter mats using the Wallac Beta plate harvester. Incorporated 3H-thimidine was determined using a Wallac Beta Plate counter. Stimulation indices (SI) were calculated relative to control wells to which no PfAMA1 had been added. PBMC were tested in parallel for their 39
40 Chapter 2 Figure 1. Study flow chart showing number of volunteers randomised, withdrawn and completing follow-up. Coding for adjuvant as follows: Alum = Alhydrogel, Mon = Montanide. Reasons for withdrawal are given: “rash” = allergic rash unrelated to study procedure, “erythema” = grade 3 injection site erythema leading to withdrawal, “other vac.” = concomitant Hepatitis B vaccination leading to exclusion. ability to be stimulated with Tetanus Toxoid (Purified Tetanus Toxoid 150 Lf/ml, RIVM, Bilthoven, The Netherlands) and phytohaemagglutinin 5 µg/ml (PHA-L, Sigma-Aldrich). In vitro parasite growth inhibition Antibodies to be used for parasite inhibition assays were purified on protein A columns (Immunopure Plus Pierce, St Louis, MO, USA) using standard protocols, exchanged into RPMI 1640 using Amicon Ultra-15 concentrators (30 kDa cut-off, Millipore, Ireland), filter-sterilised and stored at -20°C until use. IgG concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). P. falciparum strain FCR3 was cultured in vitro using standard P. falciparum culture techniques in an atmosphere of 5% CO2, 5% O2 and 90% N2. FCR3 AMA1 (accession no. M34553) differs by one amino acid in the pro-sequence from FVO AMA1 (accession no. AJ277646).
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 58 and 59: Chapter 3 Humoral immune responses
Page 60 and 61: Humoral immune responses to a singl
Page 80: Humoral immune responses to a singl
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:
Page 144 and 145:
Page 146 and 147:
Page 148 and 149:
Page 150 and 151:
Page 152 and 153:
Chapter 8 Induction of malaria in v
Page 154 and 155:
Induction of malaria in volunteers
Page 156 and 157:
Page 158 and 159:
Page 160 and 161:
Page 162 and 163:
Page 164 and 165:
Page 166 and 167:
Page 168 and 169:
Page 170 and 171:
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 and 192:
190 Chapter 9 27. Orjih AU. Acute m
Page 193 and 194:
192 Chapter 9 medium A (Caltag Labo
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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?