Background The discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite's flagellar pocket. Methodology/Principal Findings Here we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (KD), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the KD remained unaffected. Conclusions/Significance This study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity.
References
[1]
Beschin A, Brys L, Magez S, Radwanska M, De Baetselier P (1998) Trypanosoma brucei infection elicits nitric oxide-dependent and nitric oxide-independent suppressive mechanisms. J Leukoc Biol 63: 429–439. doi: 10.1194/jlr.r800084-jlr200
[2]
Darji A, Beschin A, Sileghem M, Heremans H, Brys L, et al. (1996) In vitro simulation of immunosuppression caused by Trypanosoma brucei: active involvement of gamma interferon and tumor necrosis factor in the pathway of suppression. Infect Immun 64: 1937–1943. doi: 10.1194/jlr.r800084-jlr200
[3]
Radwanska M, Guirnalda P, De Trez C, Ryffel B, Black S, et al. (2008) Trypanosomiasis-induced B cell apoptosis results in loss of protective anti-parasite antibody responses and abolishment of vaccine-induced memory responses. PLoS Pathog 4: e1000078. doi: 10.1371/journal.ppat.1000078
[4]
Bockstal V, Guirnalda P, Caljon G, Goenka R, Telfer JC, et al. (2011) T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis. PLoS Pathog 7: e1002089. doi: 10.1371/journal.ppat.1002089
[5]
Donelson JE (2003) Antigenic variation and the African trypanosome genome. Acta Trop 85: 391–404. doi: 10.1016/S0001-706X(02)00237-1
[6]
Pays E, Vanhamme L, Perez-Morga D (2004) Antigenic variation in Trypanosoma brucei: facts, challenges and mysteries. Curr Opin Microbiol 7: 369–374. doi: 10.1016/j.mib.2004.05.001
[7]
Vanhamme L, Lecordier L, Pays E (2001) Control and function of the bloodstream variant surface glycoprotein expression sites in Trypanosoma brucei. Int J Parasitol 31: 523–531. doi: 10.1016/S0020-7519(01)00143-6
[8]
Barry JD, McCulloch R (2001) Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite. Adv Parasitol 49: 1–70. doi: 10.1016/s0065-308x(01)49037-3
[9]
Auffret CA, Turner MJ (1981) Variant specific antigens of Trypanosoma brucei exist in solution as glycoprotein dimers. Biochem J 193: 647–650. doi: 10.1016/s0065-308x(01)49037-3
Nguyen VK, Desmyter A, Muyldermans S (2001) Functional heavy-chain antibodies in Camelidae. Adv Immunol 79: 261–296. doi: 10.1016/s0065-2776(01)79006-2
[12]
Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Holzer W, et al. (1998) Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J 17: 3512–3520. doi: 10.1093/emboj/17.13.3512
[13]
Engstler M, Thilo L, Weise F, Grünfelder CG, Schwarz H, et al. (2004) Kinetics of endocytosis and recycling of the GPI-anchored variant surface glycoprotein in Trypanosoma brucei. J Cell Sci 117: 1105–1115. doi: 10.1242/jcs.00938
[14]
Balber AE, Bangs JD, Jones SM, Proia RL (1979) Inactivation or elimination of potentially trypanolytic, complement-activating immune complexes by pathogenic trypanosomes. Infect Immun 24: 617–627. doi: 10.1016/s0065-2776(01)79006-2
[15]
O'Beirne C, Lowry CM, Voorheis HP (1998) Both IgM and IgG anti-VSG antibodies initiate a cycle of aggregation-disaggregation of bloodstream forms of Trypanosoma brucei without damage to the parasite. Mol Biochem Parasitol 91: 165–193. doi: 10.1016/S0166-6851(97)00191-6
[16]
Stijlemans B, Caljon G, Natesan SK, Saerens D, Conrath K, et al. (2011) High affinity nanobodies against the Trypanosoma brucei VSG are potent trypanolytic agents that block endocytosis. PLoS Pathog 7: e1002072. doi: 10.1371/journal.ppat.1002072
[17]
Vu KB, Ghahroudi MA, Wyns L, Muyldermans S (1997) Comparison of llama VH sequences from conventional and heavy chain antibodies. Mol Immunol 34: 1121–1131. doi: 10.1016/S0161-5890(97)00146-6
[18]
Conrath K, Vincke C, Stijlemans B, Schymkowitz J, Decanniere K, et al. (2005) Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH. J Mol Biol 350: 112–125. doi: 10.1016/j.jmb.2005.04.050
[19]
Cross GA (1975) Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitol 71: 393–417. doi: 10.1017/s003118200004717x
[20]
Lanham SM, Godfrey DG (1970) Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp Parasitol 28: 521–534. doi: 10.1016/0014-4894(70)90120-7
[21]
Saerens D, Kinne J, Bosmans E, Wernery U, Muyldermans S, et al. (2004) Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J Biol Chem 279: 51965–51972. doi: 10.1074/jbc.M409292200
[22]
Saerens D, Stijlemans B, Baral TN, Nguyen Thi GT, Wernery U, et al. (2008) Parallel selection of multiple anti-infectome Nanobodies without access to purified antigens. J Immunol Methods 329: 138–150. doi: 10.1016/j.jim.2007.10.005
[23]
Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS lett 414: 521–526. doi: 10.1016/S0014-5793(97)01062-4
[24]
Stijlemans B, Conrath K, Cortez-Retamozo V, Van Xong H, Wyns L, et al. (2004) Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies. African trypanosomes as paradigm. J Biol Chem 279: 1256–1261. doi: 10.1074/jbc.M307341200
[25]
Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, et al. (2001) Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob Agents Chemother 45: 2807–2812. doi: 10.1128/AAC.45.10.2807-2812.2001
[26]
Allen CL, Goulding D, Field MC (2003) Clathrin-mediated endocytosis is essential in Trypanosoma brucei. EMBO J 22: 4991–5002. doi: 10.1093/emboj/cdg481
[27]
Garcia-Salcedo JA, Perez-Morga D, Gijon P, Dilbeck V, Pays E, et al. (2004) A differential role for actin during the life cycle of Trypanosoma brucei. EMBO J 23: 780–789. doi: 10.1038/sj.emboj.7600094
[28]
Hall BS, Smith E, Langer W, Jacobs LA, Goulding D, et al. (2005) Developmental variation in Rab11-dependent trafficking in Trypanosoma brucei. Eukaryot Cell 4: 971–980. doi: 10.1128/EC.4.5.971-980.2005
[29]
Spitznagel D, O'Rourke JF, Leddy N, Hanrahan O, Nolan DP (2010) Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei. PLoS One 5: e12282. doi: 10.1371/journal.pone.0012282
[30]
Teeling JL, French RR, Cragg MS, van den Brakel J, Pluyter M, et al. (2004) Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 104: 1793–1800. doi: 10.1182/blood-2004-01-0039
[31]
Teeling JL, Mackus WJ, Wiegman LJ, van den Brakel JH, Beers SA, et al. (2006) The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20. J Immunol 177: 362–371.
[32]
Zamze SE, Ashford DA, Wooten EW, Rademacher TW, Dwek RA (1991) Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins. J Biol Chem 266: 20244–20261.
[33]
Leal S, Acosta-Serrano A, Morris J, Cross GA (2004) Transposon mutagenesis of Trypanosoma brucei identifies glycosylation mutants resistant to concanavalin A. J Biol Chem 279: 28979–28988. doi: 10.1074/jbc.M403479200
[34]
Pearson TW, Beecroft RP, Welburn SC, Ruepp S, Roditi I, et al. (2000) The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro. Mol Biochem Parasitol 111: 333–349. doi: 10.1016/S0166-6851(00)00327-3
[35]
De Vooght L, Caljon G, Stijlemans B, De Baetselier P, Coosemans M, et al. (2012) Expression and extracellular release of a functional anti-trypanosome Nanobody(R) in Sodalis glossinidius, a bacterial symbiont of the tsetse fly. Microb Cell Fact 11: 23. doi: 10.1186/1475-2859-11-23
