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A saturation of antibody avidity and concentration induced by malaria vaccine candidate Apical Membrane Antigen 1 Introduction The functional activity of antibodies depends on epitope specificity, titre and affinity. The avidity of antibodies defines the sum of the individual affinities between the antibody binding sites and single epitopes [1, 2] and is an important parameter defining antibody quality. Both concentration and avidity of antibodies should be addressed in vaccines that depend on humoral responses to convey protection. One candidate immunogen is Plasmodium falciparum Apical Membrane Antigen 1 (PfAMA1). Located at the surface of blood-stage malaria parasites, AMA1 plays an essential role in the process of red blood cell invasion [3-5]. Functional antibody binding has been shown to prevent parasite invasion [6], protecting mice and monkeys against parasite multiplication and disease [7]. The full-length ectodomain recombinant PfAMA1 (amino acids 25-545) of the Plasmodium falciparum FVO strain has been expressed under cGCP in Pichia pastoris [8]. The product was formulated with Alhydrogel, Montanide ISA 720 and AS02A and tested for safety and immunogenicity in humans [9]. We investigated the concentration and avidity of antibodies induced by recombinant AMA1 in rabbits and human immunizations studies by Surface Plasmon Resonance. We correlated these data with the capacity of sera to functionally inhibit parasite growth in vitro. Materials and Methods Antigens Six different recombinant PfAMA1 constructs were prepared as described previously [10]. Pf3mH comprises the prosequence, subdomain I and subdomain II, Pf8mH comprises subdomain II only, Pf9mH comprises subdomain II and subdomain III and Pf10mH comprises subdomain III only, covering amino-acid residues 25-442, 303-442, 303-544 and 419-544 of the FVO AMA1 antigen respectively (Figure 1). The Pf4mH construct comprises the prosequence, subdomain I, subdomain II and subdomain III, representing residues 25 to 544 from the Pf FVO strain as described by Kocken et al. [11]. Construct Pf11.0 comprises amino-acid residues 25-545, but lacks the additional Myc-His tag (Figure 1). Based on rabbit immunization studies, the latter construct was used for a human phase I trial [9]. 83
84 Chapter 4 Figure 1. Schematic representation of the different AMA1 antigen constructs used for the Rabbit immunizations. Construct Pf11.0 was used for the Phase I clinical trial. Sera Immunizations of rabbits were performed as described previously [11]. Briefly, injection of 100 μg of purified FVO strain AMA1 antigen with Freund's complete adjuvant was followed by three booster injections of 100 μg AMA1 at days 14, 28, and 56 with Freund's incomplete adjuvant. Rabbits were immunized with the different AMA1 constructs shown in Figure 1. A total number of 18 rabbits were immunized, with minimum sets of 2 rabbits receiving the same recombinant antigen. Antisera obtained 4 weeks after the last injection were used for antibody evaluation. Sera from rabbits immunized with the same antigen were pooled to obtain enough material for kinetic analysis. Pre-immunization sera were used as a negative control. Human sera were obtained from a previously published phase I trial [9]. In short, healthy malaria-naïve Dutch volunteers were immunized with either 10 or 50µg of PfAMA1-FVO [25-545] adjuvanted with either Alhydrogel TM , Montanide ISA 720 (SEPPIC, Paris, France) or AS02A (a proprietary Adjuvant System from GlaxoSmithKline Biologicals) at three occasions with monthly intervals. Sera were obtained one month after the third immunisation. For SPR analysis immunoglobulins (Ig) from a random selection of six human sera from every trial arm (18 total) were purified according to manufacturer’s instructions using HiTrap TM MabSelect SuRe TM (GE Healthcare, Diegem, Belgium) and concentrated to their original volume by Vivaspin® 20 (GE Healthcare, Diegem, Belgium). Sera from ten volunteers living in malaria endemic areas in Burkina Faso, Came-
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Experimental infection and protecti
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Experimental infection and protecti
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Contents Contents 5 Chapter 1 - Int
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Introduction 9 Malaria Malaria is o
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Introduction 11 Preclinical Clinica
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Introduction 13 pipeline of clinica
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Introduction 15 The division of ant
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Introduction 17 Figure 4. Populatio
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Introduction 19 candidates. Initial
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Introduction 21 - to identify param
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Introduction 23 20. Nussenzweig RS,
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Introduction 25 50. Ouedraogo AL, R
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Introduction 27 RTS,S/AS02 in malar
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Chapter 2 Safety and Immunogenicity
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Page 58 and 59: Chapter 3 Humoral immune responses
Page 60 and 61: Humoral immune responses to a singl
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Page 87 and 88: 86 Chapter 4 Concentration (mg/ml,
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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
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NF135.C10: a new Plasmodium falcipa
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Chapter 8 Induction of malaria in v
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Induction of malaria in volunteers
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Section 3 Whole parasite inoculatio
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174 Chapter 9 Abstract An effective
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176 Chapter 9 Figure 1. Study desig
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178 Chapter 9 followed by five iden
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180 Chapter 9 Figure 2. Parasitemia
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182 Chapter 9 Test Day I-1 Day C-1
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184 Chapter 9 strain P. falciparum-
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186 Chapter 9 protective role in ot
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188 Chapter 9 References 1. Greenwo
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190 Chapter 9 27. Orjih AU. Acute m
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192 Chapter 9 medium A (Caltag Labo
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194 Chapter 10 Abstract Induction o
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196 Chapter 10 Pre-erythrocytic sta
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198 Chapter 10 procedures described
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200 Chapter 10 by hand-dissection.
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202 Chapter 10 Figure 3. Mean numbe
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204 Chapter 10 Figure 4. Number of
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206 Chapter 10 temperature (Pearson
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208 Chapter 10 immune modulating ef
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210 Chapter 10 cytokine measurement
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212 Chapter 10 14. Hermsen CC, Telg
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Chapter 11 General Discussion
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General Discussion 217 occurrence o
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General Discussion 219 mediating th
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General Discussion 221 AMA1 express
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General Discussion 223 troponins ca
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General Discussion 225 and between
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General Discussion 227 immunologica
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General Discussion 229 to induce pr
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General Discussion 231 Conclusions
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General Discussion 233 References 1
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General Discussion 235 polymorphonu
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General Discussion 237 57. Church L
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General Discussion 239 85. Beier JC
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General Discussion 241 118. Mueller
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Chapter 12 Summary Samenvatting Lis
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246 Chapter 12 Controlled human mal
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Summary, Samenvatting, List of publ
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254 Chapter 12 Roestenberg M, Teirl
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