Merozoite surface protein-1 from P. falciparum is a major target of opsonizing antibodies in

53 Pages·2017·2.72 MB·English

CVI Accepted Manuscript Posted Online 6 September 2017
Clin. Vaccine Immunol. doi:10.1128/CVI.00155-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Merozoite surface protein-1 from P. falciparum is a major target of
2 opsonizing antibodies in individuals with acquired immunity against
3 malaria
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5 Anja Jäschke a, Boubacar Coulibaly b, Edmond J. Remarque c, Hermann Bujard d #, Christian Epp a* n
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7 a Center for Infectious Diseases – Parasitology, University Hospital Heidelberg, Germany; b o
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8 Centre de Recherche en Santé de Nouna (CRSN), Nouna, Burkina Faso, Africa; c Biomedical tt
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9 Primate Research Center (BPRC), Department of Virology, Rijswijk, The Netherlands; d Center for c
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10 Molecular Biology of University of Heidelberg (ZMBH), Heidelberg, Germany s
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13 Running title: Opsonizing antibodies target MSP-1 from P. falciparum 1
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14 Key words: P. falciparum MSP-1, opsonizing antibodies, ADRB, respiratory burst, neutrophil 1
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16 # Address correspondence to Hermann Bujard, [email protected] t
17 * Present address: Christian Epp, Laborgesellschaft für Bauanalytik mbH, Hockenheim, Germany

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18 Abstract
19 Naturally acquired immunity against malaria is largely mediated by serum antibodies controlling
20 levels of blood stage parasites. A limited understanding of the antigenic targets and functional
21 mechanisms of protective antibodies has hampered so far the development of efficient malaria
22 vaccines. Besides directly inhibiting growth of Plasmodium parasites, antibodies can opsonize D
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23 merozoites and recruit immune effector cells such as monocytes or neutrophils. Antibodies n
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24 against the vaccine candidate merozoite surface protein-1 (MSP-1) are acquired during natural d
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25 infection and have been associated with protection against malaria in several epidemiological r
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26 studies. h
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27 Here we analyzed serum antibodies from semi-immune individuals from Burkina Faso for their c
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28 potential (i) to directly inhibit the growth of P. falciparum blood stages in vitro and (ii) to s
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29 opsonize merozoites and induce antibody-dependent respiratory burst (ADRB) of neutrophils. r
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30 While a few sera were identified, which directly inhibited growth of P. falciparum blood stages,
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31 IgG from all individuals clearly mediated the activation of neutrophils. The level of neutrophil il
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32 activation correlated with antibody levels to MSP-1 and affinity-purified MSP-1 specific 2
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33 antibodies elicited ADRB activity. Furthermore, immunization of non-human primates with
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34 recombinant full-size MSP-1 induced antibodies, which efficiently opsonized P. falciparum u
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35 merozoites. Reversing the function by pre-incubation with recombinant antigens allowed us to
36 quantify the contribution of MSP-1 to the anti-parasitic effect of serum antibodies. Our data
37 suggest that MSP-1 – especially the partially conserved subunit MSP-1 - is a major target of
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38 opsonizing antibodies acquired during natural exposure to malaria. Induction of opsonizing
39 antibodies might be a crucial effector mechanism for MSP-1-based malaria vaccines.
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41 Number of words: 250
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43 Introduction
44 Despite remarkable progress over the last years, malaria remains a major global health issue
45 with approximately 438 000 deaths and 214 million clinical cases worldwide in 2015, of which
46 most occur in Africa due to infection with Plasmodium falciparum (1). Emerging multi-drug
47 resistant parasites emphasize the need for effective vaccines, which are currently not available D
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48 (2). For vaccine development it is important to understand protective immune mechanisms, to n
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49 identify antigenic targets and to establish robust and reliable assays measuring correlates of d
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50 protection. Individuals living in malaria-endemic regions naturally acquire immunity against the r
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51 disease with increasing numbers of survived infections (3-6). This protection appears largely h
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52 mediated by serum antibodies controlling levels of blood stage parasites (7). //
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53 Merozoites are key targets of naturally acquired antibodies (8-10) and for several merozoite s
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54 antigens an association between antibody levels and protective human immunity has been r
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55 reported (10-13). Antibodies targeting the merozoite can function via different pathways such n

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56 as direct growth inhibition or recruitment of immune effector cells (reviewed in (14)). However, r
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57 it remains elusive which antibody mechanisms determine protection against malaria. The direct 2
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58 growth inhibition assay (GIA) of Plasmodium blood stages still remains the most commonly used 9

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59 functional assay for blood stage vaccine candidates and merozoite antigens (15, 16) although it g
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60 is controversial whether direct growth inhibitory activity in vitro correlates with protection t
61 against clinical malaria (16).
62 Increasing evidence points towards an important role of opsonizing antibodies, which bind to
63 merozoites and recruit effector cells such as monocytes (17, 18) or neutrophils (19) via their Fc
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64 receptors. These eliminate the parasite, either by phagocytosis (18) or by secretion of reactive
65 oxygen species (19). Indeed, merozoites are mainly targeted by the cytophilic antibodies IgG1
66 and IgG3 (20-22), which can bind to the Fc receptor of immune cells (18) or fix complement
67 factors (23). The acquisition of opsonizing antibodies increases with age and malaria exposition
68 (18) and correlates with protection (18, 19). Four functional assays have been developed to
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69 measure opsonizing antibodies in vitro: (i) the antibody-dependent cellular inhibition assay w
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70 (ADCI) (17), (ii) the opsonic phagocytosis assay (OPA) (18, 24), (iii) the antibody-dependent a
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71 complement inhibition (Ab-C) assay (23) and (iv) the antibody-dependent respiratory burst
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72 (ADRB) assay (19). Importantly, several studies using these assays show a correlation between

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73 opsonizing antibodies and protection against malaria (18, 19, 23-27). :
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74 In the ADRB assay antibodies opsonize P. falciparum merozoites and interact with neutrophils i.a
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75 via their Fc receptor resulting in the production of reactive oxygen species (ROS) (19) .o
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76 (Supplementary figure 1). Oxidant damage mediated by ROS can kill the parasite (28-32) and is o
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77 associated with protection against malaria (19, 33, 34). Interestingly, the antimalarial drugs p
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78 mefloquine and artesunate cause parasite death by generation of ROS (29, 35) and ROS also 1
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79 protects against severe malaria in sickle and fetal erythrocytes (36) as well as in thalassemic and 1
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80 G6PD deficient red blood cells (33). In malaria endemic regions higher age (37) and a more y

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81 diverse anti-merozoite antibody repertoire (38) promote ADRB activity suggesting that it plays a
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82 role in naturally acquired immunity. Indeed, ADRB activity correlates with protection from
83 clinical malaria in meso- and holoendemic areas in Senegal (19).
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84 A number of merozoite antigens have been identified which elicit opsonizing antibodies such as
85 MSP-1 (23, 39), MSP-1 (40), MSP-2 (18, 23), MSP-3 (18, 41, 42), MSP-5 (37), MSP-6 (43),
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86 MSPDBL-1/-2 (27, 44) and GLURP (45). However, the antigenic targets of antibodies inducing
87 complement fixation, opsonic phagocytosis or neutrophil respiratory burst might differ. So far,
88 only MSP-1 , the small conserved C-terminal part of MSP-1, has been identified as antigenic
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89 target which contributes to neutrophil respiratory burst activity (39). w
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90 Merozoite surface protein-1 (MSP-1) is the major protein at the surface of Plasmodium d
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91 merozoites (46). The approximately 190 kDa GPI anchored precursor protein is processed into 4 fr
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92 major subunits – MSP-183, MSP-130, MSP-138, MSP-142 - by PfSUB1 prior to invasion (47-49); h
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93 MSP-142 is further processed into MSP-133 and MSP-119 by PfSUB2. MSP-1 appears mainly ://
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94 dimorphic and all P. falciparum strains can be assigned to one of the two allelic types K1 (e.g. i.a
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95 FCB1 strain) and Mad20 (e.g. 3d7 strain) – corresponding to MSP-1F and MSP-1D, respectively. .o
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96 MSP-1 is essential for Plasmodium blood stages (50) and plays an important role during o
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97 erythrocyte invasion (51), especially for the initial interaction between merozoite and RBC (52, p
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98 53) as well as RBC rupture and parasite egress (54). Indeed, it was shown recently that MSP-1 1
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99 acts as a platform for several peripheral MSPs such as MSP-3/-6/-7 and MSPDBL-1/-2 and 1
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100 interestingly, all these complexes could be inhibited by targeting MSP-1 with antibodies (55). y
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101 Antibodies to MSP-1 can inhibit parasite growth in vitro (51, 56) and have been associated with
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102 protection against malaria in several epidemiological studies (57-60) and in immunization
103 experiments in animals (46, 61-63).
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104 In this study we used heterologously produced MSP-1 spanning the entire amino acid sequence
105 of the 191 kDa molecule from the P. falciparum strain 3d7, henceforth called MSP-1D. Sera from
106 semi-immune individuals from Burkina Faso were employed to analyze the role of naturally
107 acquired MSP-1-specific antibodies in mediating direct growth inhibition and neutrophil
108 respiratory burst activity. Besides correlation analyses between antibody levels and functional
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109 activity, affinity purified MSP-1D antibodies were examined for their activity in GIA and ADRB w
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110 assays. Furthermore, an antigen-reversal GIA and antigen-reversal ADRB assay was established a
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111 in order to quantify the contribution of single antigens to functional activity. Results show that
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112 MSP-1D and its subunit MSP-1 are major targets of opsonizing antibodies, which induce
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113 neutrophil respiratory burst in individuals with naturally acquired immunity. We demonstrate :
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114 furthermore, that immunization with MSP-1D induces opsonizing antibodies in rhesus monkeys. i.
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115 .o
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116 Results
117 Growth inhibitory potential of MSP-1 specific antibodies
118 Sera from eleven healthy young adults from Nouna, Burkina Faso - an area with high seasonal
119 malaria transmission – were analyzed for (i) antibodies directed towards P. falciparum specific
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120 antigens and (ii) for their potential to inhibit parasite growth in vitro. Sera of malaria-naïve o
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121 Europeans served as controls. There was a high correlation between antibody levels to MSP-1 lo
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122 and to P. falciparum schizont lysate or merozoites (Spearman`s correlation coefficients r=0.899
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123 (***) and r=0.886 (***), respectively) (Figure 1). Antibodies from four semi-immune individuals m

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124 directly inhibited the growth of P. falciparum blood stage parasites in vitro (Figure 2A). Total IgG tt
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125 from the two donors with highest GI activity were analyzed for the contribution of MSP-1 c
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126 specific antibodies to growth inhibitory activity using an Antigen-Reversal GIA. Here, growth s
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127 inhibition assays were performed in presence of increasing concentrations of MSP-1D. While rg
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128 MSP-1D had no effect on IgG from donor 8, the growth inhibitory activity of IgG from donor 6 n

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129 was diminished by about 50% upon addition of MSP-1 (Figure 2B). Interestingly, donor 6 had the ril
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130 highest antibody titer to MSP-1 (Figure 1) suggesting that high antibody concentrations are 2
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131 required in GI assays. Thus, naturally acquired MSP-1 specific antibodies can contribute to
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132 growth inhibitory activity in vitro. g
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133 Correlation of MSP-1 antibody titer with neutrophil respiratory burst activity
134 Antibody-dependent cellular immunity was analyzed via respiratory burst (ADRB) assay (19),
135 based on killing of parasites by reactive oxygen species (ROS) from recruited neutrophils
136 (Supplementary figure 1). The ADRB assay, in which oxygen radicals are measured by
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137 chemiluminescence using Isoluminol, was established in our lab. A clear chemiluminescence
138 signal was only detected if all assay components - P. falciparum merozoites, purified IgG from
139 malaria exposed individuals, freshly purified PMNs and Isoluminol - were present. Only
140 background activity was obtained with IgG from malaria-naïve individuals (Supplementary figure
141 2). The ADRB Index was calculated as previously described (19, 37, 39) using the
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142 chemiluminescence maximum of the curve. Interestingly, this readout correlated perfectly with w
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143 the total area under curve within the first 60 min (Pearson correlation coefficient R = 0.996) a
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144 (Supplementary figure 3). Furthermore, the ADRB assay showed a remarkable reproducibility
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145 even if different P. falciparum merozoite and PMN preparations were used (intra-assay CV < 6

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146 %, inter-assay CV < 12 %; Supplementary figure 4). :
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147 Purified IgG from all eleven semi-immune individuals from Burkina Faso can mediate the i.a
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148 activation of neutrophils and release of ROS (Figure 3A) and, remarkably, ADRB activity of these .o
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149 donors was higher than the activity measured with the WHO IgG Pool prepared from malaria- o
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150 exposed Kenyan adults (NIBSC code: 10/198; (64); Supplementary figure 5A). The level of ADRB p
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151 activity correlated well with antibody levels to MSP-1D (Spearman`s rho r= 0.758, P=0.001) 1
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152 (Figure 3B), MSP-1F (r=0.615, P=0.024) (Supplementary table 1) and also to P. falciparum 1
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153 schizont lysate (r=0.767, P<0.001) or merozoites (r=0.855, P<0.001) (Figure 3C). Thus, MSP-1 y

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154 specific antibodies may opsonize merozoites and activate neutrophils.
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155 Affinity-purified MSP-1 antibodies opsonize P. falciparum merozoites
156 To further examine opsonizing MSP-1 specific antibodies, sera from two semi-immune
157 individuals (Donor 2 & 8) were fractionated by antigen affinity purification with MSP-1D. A
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158 preparation of MSP-1 specific antibodies was obtained (Figure 4A and Figure 4B) while the flow-
159 through (FT) material was efficiently depleted, as shown by ELISA (Figure 4B). Importantly, ADRB
160 activity was reduced substantially in the FT fraction compared to total IgG and MSP-1 specific
161 antibodies could opsonize merozoites and activate neutrophils (Figure 4D). Interestingly, the
162 same eluate fractions containing MSP-1 specific antibodies were not active in the GI assay while
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163 MSP-1 depleted sera showed undiminished activity (Figure 4C). Thus, MSP-1 specific antibodies w
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164 from Donor 2 and 8 - though not active in the GI assay – were capable of activating neutrophils a
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165 via opsonized merozoites.
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166 MSP-1 and its processing fragment MSP-1 are major targets of opsonizing h
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167 antibodies //
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168 An antigen-reversal ADRB assay was established in order to (i) assess if MSP-1 specific s
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169 antibodies play a role in opsonization in all eleven semi-immune individuals from Burkina Faso, r
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170 (ii) examine potential cross-reactivity of the opsonizing antibodies between different P. n

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171 falciparum strains and (iii) identify the MSP-1 processing fragments eliciting opsonizing r
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172 antibodies. By pre-incubation of serum antibodies with recombinant antigens such as MSP-1 the ,
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173 contribution of antigen-specific antibodies to ADRB activity could be quantified. Prior to their 9

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174 use in antigen-reversal ADRB, all recombinant antigens were pre-tested for their g
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175 chemiluminescence activity in presence of PMNs and isoluminol but in absence of merozoites; t
176 all competitors showed only background chemiluminescence activity (Supplementary figure 5B).
177 Furthermore, pre-incubation of human IgG from semi-immune individuals together with control
178 proteins such as BSA did not reduce ADRB activity (Supplementary figure 5A) indicating that a
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