The importance of human FcgammaRI in mediating protection to malaria - PubMed (original) (raw)

doi: 10.1371/journal.ppat.0030072.

Jianguo Shi, Richard M Jennings, Jonathan C Chappel, Tania F de Koning-Ward, Tim Smith, Judith Green, Marjolein van Egmond, Jeanette H W Leusen, Maria Lazarou, Jan van de Winkel, Tarran S Jones, Brendan S Crabb, Anthony A Holder, Richard J Pleass

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The importance of human FcgammaRI in mediating protection to malaria

Richard S McIntosh et al. PLoS Pathog. 2007.

Abstract

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Characterization of scFvs Derived from Phage Libraries

Estimation of diversity of library scFv inserts by BstNI digestion. Figure shows restriction digest patterns of 50 randomly selected scFvs from the October library. (A) 50 inserts of the VH–VK scFvs amplified by PCR screen from 50 randomly selected colonies. (B) The same VH–VK scFv inserts after digestion with BstNI. A large number of different digestion patterns are seen, suggesting a high degree of diversity among the library. (C–E) Binding of polyclonal scFv from panning rounds 1–4 to P. falciparum by IFA [20]. IFA was carried out using acetone-fixed parasites, and the polyclonal scFv tested against the T9–96 and FCB-1 strains of P. falciparum at 20 μg/ml [20]. MAb 12.8 was used as a positive control and polyclonal scFv prepared from the unpanned libraries used as negative controls. Immunoblots of polyclonal scFv from fourth round of panning binding to P. falciparum strain T9/96 (D) or FCB-1 (E) merozoites [20]. Lane 1, scFv March library unpanned; lane 2, scFv October library unpanned; lane 3–6, scFv from fourth round of panning with unprocessed merozoites; lane 7–8, scFv from fourth round of panning with processed merozoites; lane 9, scFv from fourth round of panning with recombinant MSP119-GST; lane 10, scFv D1.3 (anti-hen's egg lysozyme); lane 11, scFv X509 (anti-MSP133); lane 12, scFv 89.1 (anti-MSP183); lane 13, mAb 12.8 (anti-MSP142 and MSP119). (F) Inhibition of binding of mAbs 12.8 and 12.10 to MSP119 by scFvs derived from the fourth round of panning by competition ELISA [20].

Figure 2

Figure 2. Sequencing of V Genes and SPR Analysis

(A) Amino acid sequences of MSP119-binding scFvs. Sequences of six selected scFvs obtained by panning phage display libraries with recombinant MSP119 (C1) or P. falciparum merozoites in which secondary processing had been allowed to proceed (E9). Amino acids in bold represent residues in the E9 sequence differing to C1. (B) SPR association and dissociation curves of Ab binding to MSP119 immobilized on a CM5 sensor chip. Abs were injected into flow at time 0 and replaced with buffer at the point indicated by vertical arrow [24].

Figure 3

Figure 3. Characterization of Purified Abs

(A) 5 μg purified anti-MSP119 human IgG1 (JS1) was subjected to SDS-PAGE under nonreducing (lane 1) or reducing (lane 2) conditions on 4%–15% polyacrylamide gradient gels and stained with Simply Blue or immunoblotted with anti-human IgG-HRP. (B) Under nonreducing conditions and after transfer to nitrocellulose, the human anti-MSP119 IgG1 (JS1) detects the recombinant GST-MSP119 fusion protein (lane 3) but not the GST alone control (lane 4). Localization of MSP119 by IFA. (C) Schizont- and merozoite-stage parasites from the transgenic PbPbM19 and PbPfM19 lines were incubated with human Abs JS1 or JS2 (1:100), rabbit αPbM19 (1:1,000), or αPfM19 (1:1,000). After incubation with goat anti-rabbit Alexa-conjugated Ig (1:1,000) and FITC-conjugated anti-human IgG Fc (1:200), slides were washed and mounted in Vectrashield anti-fade. Parasites were visualized by fluorescence microscopy ×100 magnification, with the same fields photographed using filters to detect Alexa and FITC. (D) JS1 reactive with MSP119 on methanol-acetone-fixed smears of merozoites and erythrocytes infected with P. falciparum (strain 3D7) ×40 magnification. JS2 gave similar results. No specific fluorescence was detected with an irrelevant human IgG1 (B10) recognizing MSP119 from P. yoelii [24].

Figure 4

Figure 4. JS1 and JS2 Are Fully Functional Human Antibodies

(A) Human neutrophil-mediated phagocytosis of GST-_Pf_MSP119 coated fluorescent 1-μm microspheres by JS1. A control human IgG1 recognizing the homologous GST-MSP119 from P. yoelii was unable to opsonize beads and no ingestion was observed. Phagocytosed beads (red) were visualized in the cytoplasm of neutrophils (arrowed) whose nuclear DNA was counterstained in blue by DAPI. (B) Stimulation of neutrophil NADPH oxidative bursts using JS1 and JS2 attached to GST-MSP119-coated microtiter plates. Chemiluminescence (CL; arbitary units) was induced by anti-_Pf_MSP119 human IgG1 JS1 (⋄), JS2 (○), human IgA1 (□), or no antibody (▵). All antibodies at 1 × 10−7 M. Data are presented as mean arbitary units from duplicate wells with neutrophils from a single donor.

Figure 5

Figure 5. Inhibition of Binding of mAbs 12.10 and 12.8 by Fully Human Anti-MSP119 IgG1s (JS1 and JS2) by Competition ELISA

The binding of 12.8 is reduced by over 60% and the binding by 12.10 reduced by 30% suggesting that JS1 and JS2 compete with mAbs 12.8 and 12.10 for similar or overlapping epitopes. mAb 12.8, JS1, and JS2 were used at 0.5 μg/ml and mAb 12.10 at 0.05 μg/ml. Similar results were also observed when saturating concentrations of antibodies were used.

Figure 6

Figure 6. Epitope Mapping of JS1 and JS2 Binding Sites

Shows the location in the three-dimensional model of P. falciparum MSP119 of residues in the first epidermal growth factor domain, which on mutation affect binding by JS1 or JS2. Mutation of Cys28 shown in red completely ablated binding of both mAbs (12.10 and 12.8) and JS1 or JS2. Mutation of the partnering Cys12, also shown in red, while ablating binding by the murine mAbs, had no effect on the binding by JS1 or JS2. Arg20 and Asn33 in salmon had intermediate effects on binding as determined by SPR analysis when mutated to more neutral or negatively charged side-chains (see Table 1). Three further substitutions at Lys40, Lys29, and Asn39 seen in brown had minor effects on binding when the interaction was studied by ELISA. The model of P. falciparum MSP119 was generated by PyMol using atomic coordinates available from NCBI under accession number PDB: 1CEJ.

Figure 7

Figure 7. Course of a P. falciparum MSP119 Transgenic P. berghei Infection in Mice

(A) Groups of 2–3 FcγRI transgenic (Tg) or nontransgenic (NTg) littermates were injected i.p. with a total dose of 1.5 mg fully human anti–P. falciparum MSP119 IgG1 (JS1), an irrelevant human IgG1 (B10) recognizing MSP119 from P. yoelii or PBS. Similar results were obtained in two independent experiments. **Only groups of mice given JS1 in the FcγRI Tg were significantly different to all the other control groups with a p < 0.01. †, death of mice. (B) Repeat experiment in FcγRI Tr animals using a lower total dose (0.75 mg) of JS1. Coadministration of the blocking mAb 10.1 specific for the IgG1 binding site on human FcγRI abrogates the protection mediated by the passively administered JS1 antibody. Each point represents the geometric mean parasitemia of mice in each group at the time after i.p. challenge with 5,000 parasitized erythrocytes. Only those animals receiving the fully human anti–P. falciparum MSP119 IgG1 Ab in a human FcγRI background survived an otherwise lethal infection; all the mice in the other groups with high parasitemias were killed on either day 7 or 8. Similar results were obtained in two independent experiments. **Only groups of mice given JS1 in the FcγRI Tg were significantly different to all the other control groups with a p < 0.01. †, death of mice. (C–E) ×100 magnification of Giemsa-stained smears of blood taken from control animals (C) and FcγRI Tg animals treated with JS1 (D and E). Note the presence of phagocytosed merozoites within the cytoplasm of cells displaying mononuclear morphology (arrow).

References

    1. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI, et al. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature. 2005;434:214–217. -PMC -PubMed
    1. Cohen S, McGregor IA, Carrington S. Gamma-globulin and acquired immunity to human malaria. Nature. 1961;192:733–737. -PubMed
    1. Good MF, Kaslow DC, Miller LH. Pathways and strategies for developing a malaria blood-stage vaccine. Annu Rev Immunol. 1998;16:57–87. -PubMed
    1. Pleass RJ, Holder AA. Antibody-based therapies for malaria. Nat Rev Micro. 2005;3:893–899. -PubMed
    1. Shi J, McIntosh RS, Pleass RJ. Antibody and Fc-receptor-based therapeutics for malaria. Clin Sci. 2005;110:11–19. -PubMed

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