Full-length P. Falciparum Circumsporozoite Protein Administered with Poly-ICLC or GLA/SE Elicits ...

71 Pages·2012·1.04 MB·English

IAI Accepts, published online ahead of print on 28 December 2012
Infect. Immun. doi:10.1128/IAI.01108-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 Title: Full-length P. Falciparum Circumsporozoite Protein
2 Administered with Poly-ICLC or GLA/SE Elicits Potent Antibody and
3 CD4+ T Cell Immunity and Protection in Mice
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5 Kathrin Kastenmüller1‡, Diego A. Espinosa2‡, Lauren Trager1,6, Cristina D
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6 Stoyanov1,2, Andres M. Salazar3, Santosh Pokalwar4, Sanjay Singh4, Sheetij nlo
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7 Dutta5, Christian F. Ockenhouse5, Fidel Zavala2, Robert A. Seder1,§ e
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9 1Vaccine Research Center and Cellular Immunology Section, National ttp
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10 Institute of Allergy and Infectious Diseases, National Institutes of Health, i.
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11 Bethesda, MD 20892 .
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12 2Department of Molecular Microbiology and Immunology, Bloomberg o/
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13 School of Public Health, The Johns Hopkins University, Baltimore, MD p
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14 21205 ,
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15 3Oncovir, Washington, DC 20008 9

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16 4Gennova Biopharmaceuticals Ltd, Pune 411057, India g
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17 5Division of Malaria Vaccine Development, Walter Reed Army Institute of t
18 Research, Silver Spring, MD 20910
19 6Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg,
20 VA 24061
21 Running title: CSP and Adjuvants Induce Antibody Mediated Protection
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23 Footnote:
24 ‡ Authorship note: Kathrin Kastenmüller and Diego A. Espinosa contributed
25 equally to this work D
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27 §Address correspondence to: Robert A. Seder, Cellular Immunology e
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28 Section, Vaccine Research Center, NIAID, NIH, 40 Convent Drive MSC m

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29 3025, Building 40, Room 3512, Bethesda, Maryland 20892-3005, USA. tp
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30 Phone: [phone]; Fax: [phone]; E-mail: [email protected]. i.
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40
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41 ABSTRACT
42 The Plasmodium falciparum (P. falciparum) circumsporozoite (CS)
43 protein (CSP) is a major vaccine target for preventing malaria infection.
44 Thus, developing strong and durable antibody and T cell responses
45 against CSP with novel immunogens and potent adjuvants may improve D
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46 upon the success of current approaches. Here we compared four distinct lo
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47 full-length P. falciparum CS proteins expressed in Escherichia coli e
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48 (E.coli) or yeast (Pichia pastoris) for their ability to induce immunity m

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49 and protection in mice when administered with poly-ICLC as an tp
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50 adjuvant. CS proteins expressed in E.coli induced high titer antibody i.
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51 responses against the NANP repeat region and potent CSP-specific .
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52 CD4+ T cell responses. Moreover, E.coli derived CS proteins in o
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53 combination with poly-ICLC induced potent multi-functional (IL- p
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54 2+/TNFα+/IFN-γ+) CD4+ effector T-cell responses in blood, spleen and ,
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55 particularly in the liver. Using transgenic Plasmodium berghei 9

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56 expressing the repeat region of P. falciparum CSP (Pb-CS(Pf)) we u
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57 showed that there was a 1-4 log decrease in malaria ribosomal RNA in
58 the liver following a high dose challenge and ~ 50% sterilizing
59 protection with a low dose challenge compared to the controls.
60 Protection was directly correlated with high-level antibody titers but not
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61 CD4+ T cell responses. Finally, protective immunity was also induced
62 using GLA/SE as adjuvant, which also correlated with high antibody
63 titers yet significantly less potent CD4+ T cell immunity than poly-
64 ICLC. Overall, these data suggest that full-length CS proteins and poly-
65 ICLC or GLA/SE offer a simple vaccine formulation to be used alone or D
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66 in combination with other vaccines for preventing malaria infection. lo
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80 INTRODUCTION
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82 Malaria infection with Plasmodium falciparum (P. falciparum) causes more
83 than 600,000 deaths annually as well as significant morbidity worldwide (1).
84 A range of efforts to control and treat malaria include public health measures
85 such as insecticide-treated bed nets, indoor residual spraying and widespread D
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86 usage of antimalarial drugs. Despite the impact of these approaches the most lo
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87 cost-effective solution to prevent infection and to ultimately control the e
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88 malaria endemic is to develop a vaccine. m

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89 Currently, the most advanced vaccine tested in humans against P. tp
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90 falciparum infection is RTS,S which targets the CS protein (CSP), the major i.
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91 and most abundant antigen expressed on the surface of infectious .
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92 sporozoites. RTS,S given with the AS01 adjuvant (RTS,S/AS01) show ~ o
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93 30% protection against clinical disease and severe malaria (2, 3). Thus, p
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94 while these first results in phase III trials with RTS,S/AS01 are encouraging, ,
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95 there may be additional approaches for further optimizing the breadth, 9

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96 potency and duration of immunity against the CSP using different g
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97 immunogens or more potent adjuvants. In terms of antigen design, RTS,S is t
98 comprised of a truncated form of CSP containing the central repeat region,
99 NANP, that is a target for antibody mediated neutralization, as well as
100 CD8+ and CD4+ T-cell epitopes at the C-terminal end. This truncated CS
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101 protein is then fused to the Hepatitis B surface antigen creating an
102 immunogenic particle. Therefore, using a more full-length CSP, including
103 the N-terminal end and the R1 region of CSP as well as the minor repeat
104 region (NVDP), might favor broader antibody responses than against the
105 NANP repeat region alone (4-8). Moreover, a full-length CSP may provide D
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106 additional T cell epitopes leading to increased breadth of cellular immunity, lo
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107 which could also enhance protection. Another approach is to enhance the e
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108 humoral and cellular immune responses by altering the type of adjuvant m

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109 given with the full-length CSP-based protein vaccine. tp
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110 Early studies in mice showed that protection was associated with high i.
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111 antibody titers (9-11). The next generation of malaria vaccines combined .
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112 CSP with more potent adjuvants like Pseudomonas aeruginosa exotoxin A, o
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113 Monophospholipid A (MPL), mycobacterial cell wall skeleton or squalene p
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114 (Detox®, Ribi Immunochem) (12-14), which resulted in higher antibody ,
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115 titers, however they failed to confer sufficient protective efficacy (15-17). In 9

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116 contrast, studies using irradiated sporozoites for vaccination have shown a g
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117 critical requirement for IFN-γ and cellular immunity in mediating protection t
118 against malaria (18, 19). Accordingly, optimizing CD4+ T cell-derived IFN-
119 γ production after RTS,S vaccination by altering adjuvant formulation
120 enhanced protection (20-22). As such, using the TLR4 ligand MPL and
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121 saponin (QS-21) in an oil/water emulsion (AS02) or liposome (AS01B or E)
122 formulation led to strong antibody and increased CD4+ T cell immunity
123 compared to older formulations with alum and MPL (21, 23, 24).
124 Collectively, these data highlight the importance of adjuvant formulations in
125 optimizing immunity and protection. D
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126 In this study, poly-ICLC and GLA/SE were compared as adjuvants lo
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127 when administered with a number of CS proteins expressed in yeast or E. e
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128 coli which we refer to as full-length but lack the GPI signal region and m

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129 contained various numbers of the NANP repeat region. Poly-ICLC is a tp
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130 unique formulation of long-chain double stranded RNA, polyinosinic- i.
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131 polycytidylic acid and carboxymethylcellulose that has extended in vivo .
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132 activity compared to poly-IC. Poly-ICLC mediates innate signaling through o
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133 TLR3 and MDA-5 leading to activation of dendritic cells and induction of p
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134 IL-12 and type I IFNs (25-27). In addition, poly-IC has been shown to ,
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135 promote T cell survival and enhance germinal center formation, through the 9

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136 generation of CD4+ T follicular helper cells (Tfh) (28). As an immune g
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137 adjuvant, poly-ICLC has been shown to elicit strong humoral and cellular t
138 immunity when administered with a variety of protein or dendritic cell
139 targeting vaccines in a number of mouse and NHP studies (29-35).
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140 Glucopyranosyl lipid A (GLA) is a synthetic and therefore homogeneous
141 variant of the TLR4 agonist lipid A, formulated in a stable oil-in-water
142 emulsion (SE) (36). Strong Th1 immune responses induced by protein
143 antigens in combination with GLA/SE have been observed in mouse models
144 of tuberculosis (37), leishmaniasis (38) and influenza (39). Additionally, D
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145 GLA/SE showed a similar or enhanced adjuvant activity as MPL-SE (one of lo
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146 the adjuvant component in AS01) in various animal models such as mice, e
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147 guinea pigs and nonhuman primates (40, 41). m

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148 Here we compare four full-length P. falciparum CS proteins expressed in E. tp
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149 coli or in the yeast Pichia pastoris (P. pastoris) given with poly-ICLC or i.
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150 GLA/SE as adjuvants and determine their relative immunogenicity and .
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151 protection using an in vivo mouse challenge model. Furthermore, one of the o
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152 CS proteins and poly-ICLC was tested in NHPs. Together, these data p
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153 provide a potential simple vaccine formulation for inducing potent and ,
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154 protective P. falciparum CSP responses. 9

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155 g
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160 MATERIALS AND METHODS
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162 Animals. 6-8 weeks old C57BL/6 mice were obtained from The Jackson
163 Laboratories (Bar Harbor, Maine) and maintained in the Vaccine Research
164 Center Animal Care Facility (Bethesda, MD) under pathogen-free D
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165 conditions. Male, Indian rhesus macaques were stratified into comparable lo
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166 groups on the basis of age and weight. The animals were housed at the e
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167 School of Medicine Comparative Medicine and Veterinary Resources, m

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168 University of Maryland. All experiments were conducted according to the tp
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169 guidelines of the National Research Council, under protocols approved by i.
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170 the Institutional Animal Care and Use Committee at the National Institutes .
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171 of Health. o
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172 p
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173 Reagents. Four P. falciparum CS proteins were provided by PATH- Malaria ,
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174 Vaccine Initiative (MVI; Washington, DC) and provided to us in a blinded 9

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175 manner by designating them as CSP1, CSP2, CSP3 and CSP4. These four g
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176 proteins were designated as full-length in the text, but lack the GPI signal t
177 region and contained various numbers of the repeat region. After the
178 immune studies were completed it was revealed to us that two proteins were
179 expressed in E.coli (CSP1 and CSP2) and two were expressed in P. pastoris
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180 (CSP3 and CSP4). CSP1 and CSP2 were provided to MVI from Gennova
181 (Pune, India) and WRAIR (Silver Spring, USA), respectively. Protein
182 concentration was measured using a BCA protein assay (Pierce, Rockford,
183 IL). Endotoxin contaminants were determined using Limulus amebocyte
184 lysate (LAL) assay (Lonza, Walkersville, MD). If endotoxin levels exceeded D
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185 2 EU/ml, endotoxin was removed using by two-phase extraction with Triton lo
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186 X-114. Small amounts of endotoxin was detected in the E.coli derived e
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187 proteins. Thus all four proteins including the two expressed in yeast which m

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188 had no detectable endotoxin were all treated with TritonX-114. All proteins tp
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189 used for immunization had final endotoxin levels below 1EU/ml. After i.
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190 removal of endotoxin protein concentration was determined to confirm that .
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191 all groups of mice received the same amount of protein. P. falciparum o
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192 CSP15-mer peptides overlapping by 11 amino acids and spanning the entire p
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193 length of the protein (P. falciparum 3D7 strain) were synthesized by ,
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194 GenScript. Each batch of CSP was tested for accurate size using SDS page. 9

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195 g
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196 Antibodies. The following anti-mouse antibodies for flow cytometry were t
197 purchased from BD Pharmingen: purified anti-CD28 (37.51), PerCP-Cy55-
198 anti-CD3 (145-2C11), and Alexa700-anti-CD4 (RM4-5). The following
199 antibodies were purchased from BioLegend: APC-Cy7-anti-CD8 (53-6.7).
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