Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

Abstract

Parasite drug resistance and difficulties in developing effective vaccines have precipitated the search for alternative therapies for malaria. The success of passive immunization suggests that immunoglobulin (Ig)-based therapies are effective. To further explore the mechanism(s) by which antibody mediates its protective effect, we generated human chimeric IgG1 IntroductionMalaria continues to kill approximately 2 million people each year. 1 The success of passive immunization in humans and animals suggests that immunoglobulin (Ig)-based therapies could be effective. 2,3 Manipulating antibody (Ab) genes allows the design of Ig with defined class and specificity, targeting protective epitopes on the parasite surface. An appropriate target is the 19-kDa C-terminal region of merozoite surface protein 1 (MSP1 19 ). This polypeptide displays limited sequence polymorphism, 4 is expressed by all vertebrate life-cycle stages, 5 and acts as a major target of the erythrocyte invasion-inhibitory Ab response in individuals immune to Plasmodium falciparum malaria. 6 The mechanisms whereby Ig mediates protective immunity in malaria remain unclear. 7 A primary role of Ab appears to involve blocking of merozoite invasion of erythrocytes. 8 However, studies using human Ab suggest that the protective effects are mediated through interaction with specific receptors for the Ig Fc region (FcR) expressed on various effector cells. 9,10 Using Plasmodium yoelii in a murine model to address these possibilities, we generated human chimeric IgG1 and IgA1 Abs recognizing an epitope on P yoelii MSP1 19 . We have investigated the potential of these intact Abs, and a novel diabody with specificity for both MSP1 19 and a human receptor for IgG Fc, Fc␥RI, as novel malaria therapeutics. Materials and methods Construction of expression vectorsVariable heavy (VH) and light (VL) complementary DNAs (cDNAs) were cloned from mouse hybridomas B10 (anti-MSP1 19 ) and M22 (anti-human Fc␥RI) by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers (5Ј-SARGTNMARCTGCAGSAGTC-3Ј and 5Ј-TGAGGAGACGGTGACCGTGGTYCCTTGGCCCC-3Ј for VH and 5Ј-GAYATTGAGCTCACMCARWCTMCA-3Ј and 5Ј-CCGTTTBAKCTC-GAGCTTKGTSCC-3Ј for VL). 11,12 An alternative primer incorporating a BssHII restriction enzyme site was used to reamplify B10 VH to facilitate subcloning. The IgA1 heavy-chain constant region was subcloned as a BamHI-SalI fragment from an ␣ chain gene-containing plasmid 13 into pAD-Gal4-2.1 (Stratagene, La Jolla, CA) and then as a BamHI-XbaI fragment into pVHExpress (a kind gift from Andrew Bradbury, Los Alamos National Laboratory, Los Alamos, NM), 14 replacing the ␥1 constant domains to yield pVHExpress ␣1H. The VH genes were inserted into pVHExpress and pVHExpress ␣1H, upstream of the ␥1 or ␣1 constant regions, respectively, and the VL genes into pVKExpress upstream of the human gene. 14 To generate the Fc␥RI x MSP1 19 diabody, an overlapping PCR-based strategy was used. First, the VH and VL regions of B10 and M22 were amplified. The 5Ј VH primers inco...

Growth-Inhibitory Antibodies Are Not Necessary for Protective Immunity to Malaria Infection

“…The in vitro inhibitory assay measures the functional activity of antibodies independent of complement or other cellular mediators, and our results suggest they are important in this model system. Evidence has been accumulating to suggest that antibody action via the Fc interaction with monocytes/macrophages plays an important role in protective immunity (2,26,31,40). Such a mechanism is compatible with the results we have obtained in this experiment, and the anti-MSP1 19 antibodies we induced may act in vivo by cooperating with monocytes and/or macrophages.…”

Inhibition of Erythrocyte Invasion andPlasmodium falciparumMerozoite Surface Protein 1 Processing by Human Immunoglobulin G1 (IgG1) and IgG3 Antibodies

“…The human IgG3 constant gene was amplified from human genomic DNA by overlap PCR using two sets of primers (G3FOR1, 5Ј-CCTGGATCCTCGTGGATAGACAAG-3Ј; G3REV1, 5Ј-GAA GATATCGAGTTACTCAGATCTGGG-3Ј; G3FOR2, 5Ј-CTCAGCTCAGAC ACCTTCTC-3Ј; G3REV2, 5Ј-TCCCTTCTAGAACTCAGGCCTCAGAC-3Ј), cloned into the pCR2.1 TOPO vector (17), sequenced, and subcloned as a BamHI-XbaI fragment into pVH-C1-␥1 to create pVH-C1-␥3. The V L gene was subcloned as an ApaLI-XhoI fragment into pVK-C1-Express upstream of the human gene as previously described (21,29). Chinese hamster ovary (CHO-K1) cells were transfected with corresponding heavy-and light-chain constructs, and positive clones were selected (21,29).…”

“…The V L gene was subcloned as an ApaLI-XhoI fragment into pVK-C1-Express upstream of the human gene as previously described (21,29). Chinese hamster ovary (CHO-K1) cells were transfected with corresponding heavy-and light-chain constructs, and positive clones were selected (21,29). Clones secreting MSP1 19 -specific IgG1 or IgG3 were detected by enzyme-linked immunosorbent assay (ELISA) with recombinant MSP1 19 coated plates and by immunoblotting with goat antihuman IgG Abs conjugated to horseradish peroxidase.…”

Control of Malaria by Bio-Therapeutics and Drug Delivery Systems

“…This perception has sustained their candidacy to combat malaria by, for instance, curtailing the damage associated with any inappropriate host inflammatory responses [77]. The role of antibodies in malaria protection can also be attributed to inhibition of merozoite invasion of erythrocytes [78], antibody-mediated phagocytosis through FcR and complement pathways [79], and antibody-dependent cellular inhibition [80,81]. Both autoantibodies and antibody immune complexes can drive B-cell responses, through the PRR toll-like receptor-9, and support their potential in malaria [82].…”