Immunodampening to overcome diversity in the malarial vaccine candidate 1 apical membrane ...

29 Pages·2014·1.67 MB·English

IAI Accepts, published online ahead of print on 25 August 2014
Infect. Immun. doi:10.1128/IAI.02061-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 Immunodampening to overcome diversity in the malarial vaccine candidate
2 apical membrane antigen 1
3
4 Karen S. Harris1, Christopher G. Adda1, Madhavi Khore1, Damien R. Drew2,
5 Antonina Valentini-Gatt1, Freya J.I. Fowkes2, James G. Beeson2, Sheetij Dutta3,
6 Robin F. Anders1 and Michael Foley1,*
7
8 1Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe
9 University, Melbourne, Australia. 2Centre for Biomedical Research, Burnet Institute,
10 Melbourne, Australia. 3Department of Epitope Mapping, Walter Reed Army Institute
11 of Research, Silver Spring, USA. D
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12 *To whom correspondence may be addressed. Tel.: [phone]; Fax: [phone]- n
13 2467; E-mail: [email protected]. lo
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14 Running title: Immunodampening to overcome AMA1 diversity e
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15
f
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16 Abstract o
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17 Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate however

18 its polymorphic nature may limit its success in the field. This study aimed to h
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19 circumvent AMA1 diversity by dampening the antibody response to the highly tp
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20 polymorphic loop Id, previously identified as a major target of strain-specific, //
21 invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this ia
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22 loop were mutated to alanine, glycine or serine on a 3D7 and FVO AMA1 backbone. a
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23 Initially the corresponding antigens were displayed on the surface of bacteriophage m
24 where the alanine and serine but not glycine mutants folded correctly. The alanine and .
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25 serine AMA1 mutants were expressed in E. coli, refolded in vitro and used to r
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26 immunize rabbits. Serological analyses indicated that immunization with a single /

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27 mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody n
28 response. Targeting the corresponding residues in an FVO backbone did not achieve A
29 this outcome. The inclusion of at least one engineered form of AMA1 in a bi-allelic p
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30 formulation resulted in an antibody response with broader reactivity against different il
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31 AMA1 alleles than combining the wild type forms of 3D7 and FVO AMA1 alleles. 1
32 For one combination, this extended to enhanced relative growth inhibition of a ,
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33 heterologous parasite line, although this was at the cost of reduced overall inhibitory 0
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34 activity. These results suggest that targeted mutagenesis of AMA1 is a promising
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35 strategy for overcoming antigenic diversity in AMA1 and reducing the number of b
36 variants required to induce an antibody response that protects against a broad range of y

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37 P. falciparum AMA1 genotypes However, optimization of the immunization regime
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38 and mutation strategy will be required for this potential to be realized. e
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39 t
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47 Introduction
48 The apicomplexan parasite Plasmodium falciparum, the causative agent of the most
49 severe form of human malaria, is responsible for more than 0.5 million deaths
50 annually. There are currently no licensed vaccines for malaria; however the
51 development of clinical immunity in naturally-exposed individuals suggests a vaccine
52 that reduces the morbidity and mortality associated with malaria is likely to be
53 achievable (1). RTS,S/ASO1, a vaccine targeting the pre-erythrocytic stages of P.
54 falciparum, is showing partial efficacy in a large multi-centre phase III trial in Africa
55 (2) but ultimately an improved vaccine with higher efficacy and longer duration of
56 protection will be required (3). This may be achieved through new vaccine
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57 approaches or by incorporating one or more asexual blood-stage antigens into a o
58 vaccine containing RTS,S. w
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59 lo
60 Of the asexual blood-stage antigens assessed as potential vaccine components, apical a
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61 membrane antigen 1 (AMA1) has been considered particularly promising and a large
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62 body of preclinical data supported the decision by several groups to take AMA1 d

63 vaccines into clinical development (recently reviewed in (4)). Additionally, AMA1 is fr
o
64 an important target of acquired immunity; antibodies to AMA1 have been associated m
65 with protection from malaria in different populations and settings (5, 6), and human
h
66 antibodies to AMA1 can inhibit blood stage replication of P. falciparum (7). AMA1 is tt
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67 one of very few candidate malaria vaccines that has shown efficacy in phase II :
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68 clinical trials (1). One of two AMA1 vaccines to reach Phase II testing in the field /ia
69 showed significant efficacy, but this was restricted to protection against parasites with i.
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70 vaccine-like alleles of AMA1, indicating the protective effect was allele-specific (8). s
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71 As molecular epidemiological studies showed that many of the polymorphic sites in
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72 AMA1 were under balancing selection, presumably by protective antibody responses o
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73 (9-12), it is not surprizing that a vaccine containing a single allelic form of AMA1 g
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74 failed to generate protection against the majority of P. falciparum AMA1 genotypes. o
75 This has highlighted the problem that polymorphisms in AMA1 and other asexual n

A
76 blood-stage antigens may limit the effectiveness of these antigens as vaccine
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77 components. r
78 il 1
79 Based on disulphide bond connectivity (13) and the three-dimensional crystal 1
,

80 structure (14), AMA1 has been divided into three domains. Domain I harbours the 2
0
81 majority of the polymorphic sites and these can be grouped into three clusters 1
82 according to their spatial distribution: C1, C2 and C3 (14-16). The C1 cluster was 9

83 shown to be largely responsible for allowing the FVO strain of P. falciparum to b
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84 escape inhibition by rabbit anti-3D7 AMA1 antibodies in vitro (15). Within this g
85 cluster, residues located in the highly polymorphic loop Id made the largest u
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86 contribution to escape. This group of polymorphisms, termed C1-L, forms a large part s
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87 of the epitope recognized by the strain-specific, inhibitory antibody mAb 1F9 and is a
88 target of naturally-acquired antibodies to AMA1 (17, 18). Human antibodies to this
89 epitope are acquired with increasing exposure to malaria and are associated with both
90 protective immunity and growth-inhibitory activity in vitro (18). Compelling evidence
91 of the importance of this polymorphic cluster has come from an analysis of the
92 breakthrough parasites in the Phase II trial of a 3D7 AMA1 vaccine in Mali; there was
93 no significant efficacy against all malarial episodes, but efficacy was 64% for malaria
94 episodes caused by parasites identical to the vaccine-strain AMA1 at polymorphic
95 sites within C1-L (residues 196, 197, 199, 200, 201, 204, 206 and 207) (8, 19). If the
96 development of AMA1 as a component of a malaria vaccine is to continue, strategies
97 to circumvent the problem posed by the polymorphisms must be a priority.
98
99 Although sequence diversity within AMA1 is large, genetic analyses suggest that
100 variants can be grouped into as few as six different populations and it is possible that
101 a vaccine containing representative alleles from each population, or broadly covering
102 the diversity in AMA1 may be an effective approach to cover the majority of parasite
103 genotypes (16, 20-24). However, AMA1 haplotype groups are only weakly predictive
104 of the cross-reactivity or cross-inhibitory activity of antibodies (21); this highlights
105 the need for further studies to understand key polymorphic epitopes and strategies to
106 overcome diversity in AMA1. Immunization of animals with combinations of D
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107 multiple AMA1 alleles has been shown by several groups to induce an antibody
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108 response more directed towards conserved epitopes (23, 25-28). If these antibodies n
109 are equally protective as allele-specific responses, as some data suggests, a lo
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110 combination of a relatively small number of alleles may be sufficient. However, it d
111 should be noted that immunizing with a combination of two forms of P. chabaudi e
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112 AMA1 did not protect mice from challenge with P. chabaudi expressing a third allelic
f
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113 form of AMA1 (29). Also, no efficacy was observed in a phase II trial using a o
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114 combination of 3D7 and FVO allelic forms of AMA1 (30). However, the lack of

115 protection in this trial has been attributed to insufficient immunogenicity rather than h
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116 an inability to control heterologous infections (31). tp
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117 /
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118 An alternative, or complementary, strategy to multi-allele vaccine approaches ia
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119 involves the generation of mutated forms of AMA1 with the aim of dampening the a
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120 antibody response to dominant strain-specific epitopes and the expectation that there m
121 will be an enhanced response to cross-reactive epitopes. Others have used this .
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122 strategy with little success (32) but here we have explored this approach using a r
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123 smaller subset of polymorphic residues in both FVO and 3D7 AMA1, which differ in /

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124 the extent to which they induce a strain-specific antibody response. Furthermore, we n
125 substituted each target site with alanine, glycine and serine, all of which are likely to A
126 reduce immunogenicity, and used phage-display to rapidly assess the integrity of p
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127 important conformational epitopes. We report that serine substitution of 5 residues il
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128 (196, 197, 200, 201 and 204) within 3D7 C1-L can generate a more cross-reactive
1
129 antibody response and increase relative growth inhibition of a heterologous parasite ,
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130 line. 0
131 1
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132
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133 Methods
134
135 Monoclonal antibodies
136 This study utilised the reduction-sensitive, growth-inhibitory MAbs 1F9 (17, 33) and
137 4G2 (34) and the reduction-insensitive, non-inhibitory MAb 5G8 (33). Generation of
138 MAbs 1F9 and 5G8 were as previously described (33). MAb 4G2 was a kind gift
139 from Dr. Clemens Kocken, Biomedical Primate Research Centre, Rijswijk, The
140 Netherlands.
141
142 Mutagenesis of AMA1
143 Five residues in loop Id (196, 197, 200, 201, 206) of AMA1 were mutated to alanine, D
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144 glycine or serine essentially as described previously (35). Briefly, codon-optimized
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145 3D7 and FVO AMA1 genes were inserted into the phage display vector pHENH6 and n
146 single-stranded, uracilated DNA was produced. Oligonucleotides (GeneWorks) lo
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147 incorporating the desired substitutions were annealed to this single-stranded DNA and d
148 used to prime synthesis of covalently closed, double-stranded DNA. Following e
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149 transformation of TG1 Escherichia coli cells, colonies harbouring AMA1 mutants
f
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150 were identified by DNA sequencing (Australian Genome Research Facility). o
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151

152 Phage display h
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153 Phage displaying AMA1 (residues 25-546) with a C-terminal myc tag were prepared tp
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154 essentially as described previously (36). Briefly, TG1 cells harbouring the pHENH6 /
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155 vector with the desired ama1 gene inserted were cultured to turbidity. Following the ia
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156 addition of M13K07 helper phage (GE Healthcare), the starter culture was transferred a
157 to 200 ml of media and incubated at 37oC for approximately 16 h. Phage were then sm
158 precipitated with polyethylene glycol/NaCl solution (30% (w/v) polyethylene glycol .
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159 8000 (Sigma), 2.6 M NaCl) and harvested by centrifugation. The phage pellet was r
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160 then resuspended in phosphate-buffered saline (PBS). /

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161 n
162 Phage ELISA A
163 MAbs were diluted to 2 µg/ml in coating buffer (15 mM Na CO , 34 mM NaHCO , p
2 3 3 r
164 pH 9.6) and immobilized on a 96-well microtiter plate (Maxisorp, Nunc). Plates were il
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165 washed with PBS to remove unbound protein and blocked with 10% skim milk
1
166 powder in PBS. Phage-displaying AMA1 (residues 25-546) were diluted in PBS ,
2
167 0.05% Tween 20 (PBST; Sigma) and applied to the wells in duplicate. After 1 h 0
168 incubation, unbound phage were removed by washing with PBST and bound phage 1
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169 were detected with peroxidase-conjugated anti-M13 antibodies (GE Healthcare;
b
170 1:5000). Binding was visualized using 3,3’,5,5’-tetramethylbenzidine (Sigma) and the y

171 absorbance was read at 450 nm. g
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172 e
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173 Recombinant protein expression and purification t
174 AMA1 was expressed and purified essentially as described previously (7). Briefly, the
175 codon-optimized genes encoding wt and mutant forms of 3D7 and FVO AMA1
176 (residues 25-546) were inserted into the expression vector pQE9 (QIAgen) and
177 introduced into SG13009 E. coli cells. AMA1 expression was induced with IPTG and
178 after 3 h cells were harvested by centrifugation. Pellets were solubilised in guanidine
179 buffer. An N-terminal 6xHis tag facilitated capture of AMA1 by Ni-nitrilotriacetic
180 acid coupled agarose (Qiagen). Eluted AMA1 was refolded in vitro and further
181 purified by anion-exchange and size-exclusion chromatography.
182
183 Preparation of rabbit antisera
184 New Zealand white rabbits were immunized intramuscularly with 500 µl of AMA1
185 formulated in Montanide ISA-720 (3 parts antigen in PBS to 7 parts adjuvant (v/v);
186 Seppic) to a final concentration of 100-200 µg/ml. Rabbits were immunized on days
187 0, 28 and 70 and sera were collected on days 42, 84 (bleeds 1 and 2) and either 98 or
188 140 (kill-bleed). Three rabbits per group were immunized with individual forms of
189 AMA1 and four to six rabbits per group were immunized with combined forms of
190 AMA1.
191
192 IgG was purified from whole sera using protein G Sepharose (GE Healthcare). A total
193 of 4 ml of sera diluted 1 in 5 in binding buffer (20 mM phosphate pH 7) was twice D
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194 passed over 3 ml of resin. The beads were washed with 5 column volumes of binding
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195 buffer and bound IgG was eluted with low pH (0.1 M glycine, pH 2.7). The pH was n
196 immediately neutralized with phosphate buffer. The sample was buffer exchanged lo
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197 into PBS using either dialysis or Amicon Ultra-15 centrifugal filter units (30 MWCO; d
198 Millipore) and was concentrated using Amicon Ultra-15 centrifugal filter units. The e
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199 IgG was then sterile filtered and stored at 4oC until use.
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200 o
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201 Antibody ELISA

202 AMA1 was diluted to 2 µg/ml in PBS and immobilized on a 96-well microtiter plate h
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203 (Maxisorp, Nunc). Coating was allowed to occur overnight at 4oC. Unbound protein tp
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204 was removed by washing with PBST. Sera or MAbs were diluted to the appropriate /
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205 concentration in 5% skim milk powder in PBS and applied to the wells in duplicate. ia
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206 Following a 1.5 h incubation, unbound antibodies were removed by washing with a
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207 PBST. The appropriate peroxidase-conjugated secondary antibody (anti-rabbit (GE m
208 Healthcare; 1:1000), anti-mouse (Jackson Immunoresearch; 1:1000), or anti-rat .
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209 (Jackson Immunoresearch; 1:2500)) was diluted in 5% skim milk powder in PBS and r
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210 added to the wells. Following a 1 h incubation, excess secondary conjugate was /

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211 removed by washing. Binding was visualised by the addition of 150 µl/well of ABTS n
212 substrate (0.98 mM 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.98 mM A
213 citric acid and 0.003% H O ). After 45 min the absorbance was read at 414 nm. p
2 2 r
214 il
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215 Competition ELISA
1
216 Competition ELISAs were carried out to more sensitively assess the fine-specificity ,
2
217 of the antibody response. Rabbit sera were initially titrated on AMA1, as detailed in 0
218 the antibody ELISA section, and the serum dilution resulting in an OD of 3.5 - 3.8 1
9
219 (i.e. the upper section of the linear part of the curve) was determined. Sera were then
b
220 prepared at two-fold the desired dilution and mixed 1:1 with increasing concentrations y

221 of the appropriate forms of AMA1 (competitor antigens). The typical final g
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222 concentration range of the competing antigen was 0.316 - 31.6 µg/ml. This mixture e
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223 was incubated for 1 h before being applied to wells coated with AMA1 (2 µg/ml). t
224 Binding of sera to immobilized AMA1 was then detected as indicated in the antibody
225 ELISA section. Data was displayed as a percentage of maximal binding, with
226 maximal binding being indicated by the no inhibitor control wells. To enable
227 quantitative comparison of the competition curves, GraphPad Prism software was
228 used to plot a 3-parameter sigmoidal curve through the data and the predicted minimal
229 value was determined (26). This “residual binding” reflects the proportion of
230 antibodies captured by the immobilized target protein that do not react with a given
231 competitor antigen.
232
233 Parasite growth-inhibition assays
234 P. falciparum growth-inhibition assays (GIA) were performed as previously described
235 (21, 37). Briefly, synchronised early trophozoite stage parasites were adjusted to 0.1%
236 parasitemia and 2% hematocrit. Five μl of stock antibody or peptide was added to 45
237 μl of infected red blood cells and mixed to generate a final culture volume of 50 μl.
238 Parasites were allowed to develop through two cycles of erythrocyte invasion for 72 h
239 at 37oC. Early trophozoite-stage parasites formed after the second round of invasion
240 were fixed for 1 h at room temperature by the addition of glutaraldehyde
241 (ProSciTech) to a final concentration of 0.25% (v/v). After fixation, parasites were
242 washed in HTPBS, stained with 10x SYBR green dye (Invitrogen) and 5x105 red
243 blood cells counted per well using a BD FACSCantoII flow-cytometer. FACS counts D
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244 were analysed using FlowJoTM(Ver 6.4.7) software (Treestar). Percent growth w
245 inhibition = (1-(parasitemia in test well/mean parasitemia in pre-immunization rabbit n
246 controls wells))x100. All GIA were run in a 96-well plate format, with each IgG lo
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247 tested in duplicate wells. Parasite growth inhibition is represented as the combined d
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248 mean of duplicate wells from a single assay or as a percentage inhibition relative to d
249 another parasite line. f
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250 o
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251 To calculate parasite inhibition of the heterologous parasite line (W2mef) as a

h
252 percentage of the homologous parasite line (3D7 or FVO), we used an IgG
t
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253 concentration that could induce between 40-90% inhibition of the homologous p
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254 parasite lines. This was to avoid the loss of linearity and greater error associated with //
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255 levels of inhibition outside this range. We also set 20% as the threshold for
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256 meaningful parasite inhibition of the heterologous parasite line. Failure of an IgG a
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257 preparation to reach the minimum levels of inhibition at any of the concentrations m
258 tested led to exclusion from the analysis. .o
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259 /

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260 Results n
261 Analysis of phage-displayed AMA1 mutants A
262 This study aimed to decrease the immunogenicity of loop Id by replacing five surface p
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263 exposed polymorphic residues (at positions 196, 197, 200, 201 and 204) with either il
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264 alanine, glycine or serine (Fig. 1). To avoid lengthy protein synthesis and purification
1
265 of mutants with impaired folding ability, wt and mutant forms of 3D7 and FVO ,
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266 AMA1 were first expressed on the surface of phage. Protein expression at the phage 0
267 surface was standardized by equalizing MAb binding to a C-terminal myc tag (Fig. 1
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268 2A). Binding to MAb 5G8, which recognizes a linear epitope in the prodomain of
b
269 AMA1 (33), indicated that some N-terminal degradation of the 3D7 wt and FVO ser y

270 AMA1 phage preparations was present (Fig. 2B). However, this had minimal impact g
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271 on the conserved, conformational epitope recognized by the inhibitory MAb 4G2 e
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272 (Fig. 2C) (34, 38). In contrast, both the 3D7 gly and FVO gly mutants showed t
273 reduced reactivity with MAb 4G2 when compared with wt, indicating that the
274 introduction of glycine at these 5 positions hindered folding ability. For this reason,
275 the glycine mutants were not produced as purified recombinant proteins.
276
277 Synthesis and characterization of recombinant AMA1
278 Six recombinant forms of AMA1 (residues 25-546; N-terminal 6xHis tag) were
279 produced using our established procedure for synthesis, refolding and purification of
280 AMA1: 3D7, 3D7 ala, 3D7 ser, FVO, FVO ala and FVO ser. The final products were
281 highly pure, as indicated by silver staining after SDS-PAGE (Fig. 3A). Western
282 blotting showed that MAb 5G8, which reacts with a linear determinant in AMA1,
283 showed strong reactivity with both the reduced and non-reduced antigens (Fig. 3B).
284 All six proteins reacted with MAb 4G2 only under non-reducing conditions,
285 consistent with the conformational nature of the 4G2 epitope (34) (Fig. 3C).
286
287 The five polymorphic residues selected for mutagenesis make a large contribution to
288 the epitope recognized by the strain-specific, inhibitory MAb 1F9, and this epitope is
289 a target of naturally-acquired human antibodies (17, 18). Consistent with this, 1F9
290 bound well to wt 3D7 AMA1 but did not bind to the 3D7 mutants or any of the FVO
291 variants produced (Fig. S1A). In contrast, reactivity of all proteins with MAb 4G2
292 was almost identical indicating that the conserved, conformation-dependent epitope D
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293 recognized by this MAb was not significantly affected by mutagenesis of loop Id (Fig.
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294 S1B). n
295 lo
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296 To assess folding on a more global level, each protein was also tested for binding to d
297 conformation-sensitive polyclonal sera raised against 3D7 (Fig. S1C) or FVO AMA1 e
d
298 (Fig. S1D). Reactivity of the 3D7 mutants with the homologous polyclonal sera was
f
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299 slightly decreased compared with the wt antigen, however this can most likely be o
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300 attributed to disruption of epitopes incorporating the mutated residues rather than

301 aberrant folding. As expected, each conformation-specific sera showed reduced h
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302 reactivity with both wt and mutant forms of the heterologous antigen. tp
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303 /
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304 Generation of anti-AMA1 sera ia
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305 Since antibody reactivity data indicated that the AMA1 mutants were correctly folded, a
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306 all six forms of recombinant AMA1 were used to immunize rabbits. Blood was m
307 collected two weeks after the first boost (bleed 1), two weeks after the second boost .
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308 (bleed 2) and 10 weeks after the second boost (kill-bleed; day 140). The relative titer r
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309 of each bleed on the corresponding wt antigen was determined by ELISA (Fig. S2). In /

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310 most cases the titer of anti-AMA1 antibodies was similar in bleeds 1 and 2 whereas, n
311 surprisingly, lower antibody titers were seen in the kill-bleeds. All further analysis A
312 was carried out using bleed 2. There was no clear overall reduction in the response p
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313 generated against the wt antigen when rabbits were immunized with 3D7 ala, FVO ala il
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314 or FVO ser, compared with the antibody response induced by the wt antigen itself.
1
315 However, in 2/3 rabbits immunized with 3D7 ser, a reduced response to 3D7 wt was ,
2
316 observed. Immunization of further rabbits would be required to determine if this 0
317 difference is statistically significant. 1
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318
b
319 Analysis of the antibody response to loop Id y

320 To analyse the overall antibody response to loop Id in rabbits immunized with wt g
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321 AMA1, competition ELISAs were used. In these assays, we tested the ability of wt e
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322 and mutant recombinant AMA1 proteins to inhibit the binding of antibodies to t
323 immobilized wt AMA1. Since the mutant proteins contain five substitutions within
324 loop Id, subsequent loss of competitor efficacy indicates decreased binding of
325 antibodies that target this region and is reflective of the loop Id antibody response.
326 The competition ELISAs revealed that the corresponding AMA1 mutants were less
327 potent when inhibiting serum raised against 3D7 wt compared with FVO wt,
328 suggesting that loop Id is more dominant when 3D7 wt is the immunogen (Fig. 4A).
329 To enable quantitative comparison of the antibody response to this region, the
330 predicted minimal values (residual binding displayed as a percentage of the maximal
331 signal) for each competitor antigen were plotted for all rabbits immunized with AMA
332 wt (Fig. 4B). Higher residual binding (i.e. less competition) for a given competitor
333 suggests that it is reactive with a smaller subset of anti-AMA1 antibodies. The
334 response to loop Id was consistently higher in all three rabbits immunized with 3D7
335 AMA1, as evidenced by higher residual binding of 3D7 ala and ser, compared with
336 those immunized with FVO AMA1.
337
338 Generation of a mutant-specific antibody response
339 To determine if immunization of rabbits with mutated AMA1 generated mutant-
340 specific antibodies, we evaluated the ability of wt and mutant forms of AMA1 to
341 block each serum from binding to its immunogen and plotted the residual binding
342 (Fig. S3). Higher residual binding of AMA1 wt reflects the presence of a greater D
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343 proportion of antibodies specific for the mutant form. Although a mutant-specific
w
344 antibody response was usually present, in most rabbits this was lower than the n
345 response induced by the wt antigens to the wt loop Id. Interestingly, the mutant- lo
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346 specific antibody response appeared to be largely independent of the amino acid d
347 substitution, suggesting there may be a structural feature common to the mutants that e
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348 is absent in the wt antigen.
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349 o
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350 Analysis of the antibody response to cross-reactive epitopes

351 To establish whether limiting the induction of antibodies to loop Id would increase the h
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352 proportion of antibodies to more cross-reactive epitopes, competition assays using tp
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353 five different allelic forms of recombinant AMA1 (3D7, FVO, W2mef, HB3 and /
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354 7G8) as the competitor antigen were used. The residual binding was then plotted for ia
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355 all rabbits (Fig. 5). In rabbits immunized with 3D7 wt, heterologous forms of AMA1 a
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356 were poor inhibitors of antibody binding to the immobilized antigen, as evidenced by m
357 high residual binding (Fig. 5A). This indicates that anti-3D7 wt sera are highly strain- .
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358 specific. Following immunization with 3D7 ala and 3D7 ser mutants a trend towards r
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359 decreased residual binding values for heterologous antigens was observed, indicating /

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360 some broadening of antibody reactivity. Although the difference was not dramatic, n
361 this illustrates that the substitution of just five residues in 3D7 AMA1 can alter the A
362 specificity of the antibody response, leading to more cross-reactive antibodies. p
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363 Compared with 3D7 wt, anti-FVO wt sera were more cross-reactive with the allelic il
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364 forms tested, shown by the lower residual binding of heterologous antigens when
1
365 competing with these sera (Fig. 5B). Immunization with mutant forms of FVO AMA1 ,
2
366 did not further improve cross-strain recognition. 0
367 1
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368 Inhibition of parasite growth: individual antigen immunizations b
369 To determine if increased cross-reactivity corresponded to more effective inhibition of y

370 a heterologous parasite line, we tested the ability of purified IgG from each rabbit to g
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371 inhibit growth of 3D7 and FVO parasites (Fig. 6; Table S1). Although the overall e
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372 inhibition levels were higher for the anti-wt antibodies compared with the anti-mutant, t
373 we observed a trend towards improved relative inhibition of the heterologous parasite
374 line when a mutant form of 3D7 (but not FVO) AMA1 was the immunogen.
375 However, group sizes were insufficient for statistical analysis and low overall
376 inhibition (<40% inhibition of the homologous parasite line; <20% inhibition of the
377 heterologous parasite line) necessitated the exclusion of one rabbit from the 3D7 ala
378 group and two rabbits from the 3D7 ser group. To address this, and to determine the
379 best possible combination of wt and mutant antigens for generating a cross-reactive
380 antibody response, an expanded study incorporating larger group sizes, a modified
381 immunization regime and concurrent immunization with two antigens was carried out.
382
383 Immunization with combinations of 3D7 and FVO AMA1
384 Groups of six rabbits were immunized with one of five different antigen
385 combinations. Since the individual antigen immunizations showed that, in contrast to
386 3D7, mutagenesis of FVO did not achieve the desired outcome, mutated forms of 3D7
387 AMA1 were combined with wt FVO (3D7 ala/FVO and 3D7 ser/FVO immunization
388 groups). However, it was also possible that a synergistic effect could be achieved by
389 combining two engineered forms of AMA1. Therefore we also assessed formulations
390 that included mutant versions of both 3D7 and FVO AMA1 (3D7 ala/FVO ala and
391 3D7 ser/FVO ser immunization groups). The final group included both wt antigens
392 for comparison (3D7/FVO immunization group). One rabbit from each of the 3D7 D
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393 ala/FVO and 3D7 ala/FVO ala groups was excluded due to illness or injury. Rabbits
w
394 were immunized with a total of 100 µg of combined antigen per dose. Sera were n
395 collected at the same intervals outlined for the previous rabbit study, except the kill- lo
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396 bleed was taken on day 98. There was a trend towards a decreased overall response to d
397 AMA1 when a mutant was included in the formulation (data not shown). e
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398
f
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399 The residual levels of antibody binding measured in competition ELISAs were used to o
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400 compare responses to cross-reactive epitopes when rabbits were immunized with

401 different combinations of antigens. Relative to a single allelic form, immunization h
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402 with combinations of wt AMA1 resulted in dramatically improved cross-strain tp
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403 recognition (Fig. 7). However, it was of particular interest to determine if cross- /
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404 reactivity could be increased further by immunizing with combinations that included ia
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405 one or more AMA1 mutants. When assessing the antibody response to 3D7, decreased a
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406 residual binding values when heterologous competitor antigens were tested indicated m
407 an increased response to cross-reactive epitopes when the immunogen included one or .
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408 more loop Id mutant (Fig. 8A). This was most striking for the 3D7 ser/FVO group, r
g
409 which exhibited significantly greater cross-reactivity (when compared to the wt /

o
410 combination group) with all allelic forms tested (FVO, W2mef, HB3 and 7G8) n
411 (P<0.01). The additional inclusion of an FVO mutant did not further improve cross A
412 reactivity (P>0.05). When examining the anti-FVO antibody response (Fig. 8B), a p
r
413 significant improvement in cross-reactivity was observed on only one occasion (3D7 il
1
414 ser/FVO ser immunization group; HB3 competitor antigen). However, the inclusion
1
415 of a 3D7 mutant in some cases significantly decreased the cross-reactivity of the FVO ,
2
416 response (3D7 ala/FVO immunization group, 3D7 and W2mef competitor antigens; 0
417 3D7 ser/FVO immunization group, 3D7 competitor antigen). These data suggest that 1
9
418 the inclusion of a mutant form of 3D7 AMA1, in particular 3D7 ser, in a combined
b
419 formulation is sufficient to elicit a more cross-reactive response to 3D7 AMA1. y

420 However, the additional inclusion of FVO AMA1 mutated at loop Id does not g
u
421 consistently shift the antibody response to more cross-reactive epitopes. e
s
422 t
423 Inhibition of parasite growth: combined antigen immunizations
424 To determine whether including a mutant form of AMA1 in a dual antigen

425 formulation would increase the induction of cross-reactive inhibitory antibodies,
426 purified IgG from individual rabbits were tested for their ability to inhibit growth of
427 3D7, FVO and W2mef parasite lines. We were unable to generate reliable data from
428 further parasite lines due to insufficient amounts of IgG. When growth inhibition of
429 W2mef parasites was assessed relative to inhibition of the 3D7 parasite line, a
430 significant increase (P<0.01) in cross-strain inhibition in the 3D7 ser/FVO group was
431 consistently observed relative to the wt group (Fig. 9A; Table S2). This indicates that
432 the increased proportion of cross-reactive antibodies observed by ELISA corresponds,
433 at least partly, to cross-reactive inhibitory antibodies. In contrast, immunizing with
434 loop Id mutants did not result in enhanced inhibition of the W2mef parasite line
435 relative to FVO (Fig. 9B), consistent with the ELISA data. Since this study was
436 primarily concerned with assessing the vaccine potential of each antigen combination,
437 we did not test whether combining antibodies elicited by individual antigens would
438 enable the same level of cross-inhibition to be reached as that achieved when the
439 vaccine formulation incorporated two antigens.
440 It was evident that the overall inhibition of 3D7 parasites was impaired when 3D7 ala
441 or ser were included in the immunogen (Fig. S4A). Similarly, inclusion of FVO ala or D
442 ser resulted in decreased overall inhibition of FVO parasite growth (Fig. S4B). This o
w
443 suggests that the overall inhibitory response is reduced when a mutant is included in n
444 the formulation and this is consistent with the observed reduction in anti-AMA1 titer. lo
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445 e
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446
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447 ro
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