Efficient Targeting of Conserved Cryptic Epitopes of Infectious Agents by Single Domain Antibodies

Abstract

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::β-lactamase fusion activating a prodrug. Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::β-lactamase fusion activating a prodrug. Several infectious agents, e.g. human immunodeficiency virus, Hepatitis, Plasmodium, and Trypanosomes, have acquired a system of antigenic variation to circumvent the immune response of their host (1.Johnson W.E. Desrosiers R.C. Annu. Rev. Med. 2002; 53: 499-518Crossref PubMed Scopus (175) Google Scholar, 2.Staalsoe T. Hamad A.A. Hviid L. Elhassan I.M. Arnot D.E. Theander T.G. J. Infect. Dis. 2002; 186: 719-722Crossref PubMed Scopus (28) Google Scholar, 3.Wang H. Bian T. Merrill S.J. Eckels D.D. J. Mol. Evol. 2002; 54: 465-473Crossref PubMed Scopus (30) Google Scholar, 4.Cross G.A. Annu. Rev. Immunol. 1990; 8: 83-110Crossref PubMed Scopus (242) Google Scholar). The paradigm of this defense mechanism can be found in the African trypanosomes (4.Cross G.A. Annu. Rev. Immunol. 1990; 8: 83-110Crossref PubMed Scopus (242) Google Scholar, 5.Vanhamme L. Pays E. McCulloch R. Barry J.D. Trends Parasitol. 2001; 17: 338-343Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6.Donelson J.E. Hill K.L. El-Sayed N.M. Mol. Biochem. Parasitol. 1998; 91: 51-66Crossref PubMed Scopus (84) Google Scholar). The key player in their antigenic variation mechanism is the variant-specific surface glycoprotein (VSG) 1The abbreviations used are: VSG, variant-specific surface glycoprotein; CCM, 7-(4-carboxibutamido)-cephalosporin; FITC, fluorescein isothiocyanate; Fv, variable fragment; HCAb, heavy-chain antibody; PBS, phosphate-buffered saline; PDM, phenylenediamine mustard; TNF-α, tumor necrosis factor-α; VHH, variable fragment of heavy chain of HCAb; ELISA, enzyme-linked immunosorbent assay; DAPI, 4′,6-diamidino-2-phenylindole. dimer of which 107 identical copies form a dense layer covering the entire surface of the unicellular parasite (7.Cross G.A. Parasitology. 1975; 71: 393-417Crossref PubMed Scopus (638) Google Scholar, 8.Mehlert A. Zitzmann N. Richardson J.M. Treumann A. Ferguson M.A. Mol. Biochem. Parasitol. 1998; 91: 145-152Crossref PubMed Scopus (87) Google Scholar). At regular time intervals a new VSG type appears on the surface of the parasite, preventing recognition of the parasites by an upcoming anti-VSG response directed against the previous VSG type. Obviously, the trypanosomes possess a vast repertoire of VSG genes and multiple diversification mechanisms to express one VSG after the other so that only one type is expressed at the time (5.Vanhamme L. Pays E. McCulloch R. Barry J.D. Trends Parasitol. 2001; 17: 338-343Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 9.Borst P. Ulbert S. Mol. Biochem. Parasitol. 2001; 114: 17-27Crossref PubMed Scopus (102) Google Scholar, 10.Barry J.D. McCulloch R. Adv. Parasitol. 2001; 49: 1-70Crossref PubMed Google Scholar). The comparison of the primary structures of VSGs taking into account the position and number of conserved cysteine residues and N-glycosylation sites revealed the presence of three VSG subclasses represented by AnTat 1.1 (class I), MITat 1.1 (class II), and MITat 1.5 (class III) (11.Richards F.F. J. Protozool. 1984; 31: 60-64Crossref PubMed Scopus (5) Google Scholar, 12.Carrington M. Boothroyd J. Mol. Biochem. Parasitol. 1996; 81: 119-126Crossref PubMed Scopus (62) Google Scholar). However, even VSGs belonging to the same subclass expose only unique and immunogenic regions to the solvent when present on the coat of the parasite (13.Clarke M.W. Barbet A.F. Pearson T.W. Mol. Immunol. 1987; 24: 707-713Crossref PubMed Scopus (10) Google Scholar, 14.Miller E.N. Allan L.M. Turner M.J. Mol. Biochem. Parasitol. 1984; 13: 67-81Crossref PubMed Scopus (31) Google Scholar, 15.Reinitz D.M. Aizenstein B.D. Mansfield J.M. Mol. Biochem. Parasitol. 1992; 51: 119-132Crossref PubMed Scopus (19) Google Scholar). Therefore, trypanosomes have adopted a strategy of actively forcing the immune system to produce antibodies against regions that change continuously. Consequently, most monoclonal antibodies raised against VSG recognize a determinant with an accessible location on the parasite (13.Clarke M.W. Barbet A.F. Pearson T.W. Mol. Immunol. 1987; 24: 707-713Crossref PubMed Scopus (10) Google Scholar, 14.Miller E.N. Allan L.M. Turner M.J. Mol. Biochem. Parasitol. 1984; 13: 67-81Crossref PubMed Scopus (31) Google Scholar). Isolation of a monoclonal antibody with a pan-reactivity toward multiple VSGs on viable parasites appears to be far more challenging, because the conserved epitopes become cryptic upon the assembly of VSGs on the membrane of the parasite. To be effective as an anti-trypanosome agent it seems that molecules are required that infiltrate the dense VSG coat to reach a conserved epitope common to the majority of the VSG molecules. The VSGs contain such conserved epitopes, e.g. an Asn-linked high mannose oligosaccharide (GlnNAc2-Man5–9) is conserved among all VSG of Trypanosoma brucei brucei subspecies (16.Bangs J.D. Doering T.L. Englund P.T. Hart G.W. J. Biol. Chem. 1988; 263: 17697-17705Abstract Full Text PDF PubMed Google Scholar). This particular carbohydrate epitope should be a preferred target to design specific drugs because it is absent from glycoproteins of the parasite host (17.Carroll M. McCrorie P. Comp. Biochem. Physiol. B. 1987; 88: 7-12Crossref PubMed Scopus (2) Google Scholar). To circumvent the inaccessibility of the conserved epitopes we introduced dromedary antibodies. The serum of Camelidae contains a high proportion of functional antibodies devoid of light chains (18.Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2177) Google Scholar). The antigen-binding fragment of these so-called heavy-chain antibodies (HCAbs) is comprised in one single domain of only 15 kDa. This dedicated variable domain referred to as VHH is the smallest intact antigen-binding fragment derived from bona fide antibodies (19.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (132) Google Scholar). After immunization of dromedaries, or llamas, antigen-specific single domain can be isolated by the of the (19.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (132) Google Scholar, M. M. Desmyter A. J. E. L. Muyldermans S. J. 1998; 17: PubMed Scopus Google Scholar, M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). are conventional antibodies it is to that these antibody a more VSG and reach epitopes that are inaccessible for conventional antibodies. In it that the antigenic regions for or on a are from for the antigen-binding of conventional antibodies M. M. Desmyter A. J. E. L. Muyldermans S. J. 1998; 17: PubMed Scopus Google Scholar, M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). This with their that a of to target new epitopes such as on the surface of trypanosomes that are or inaccessible for conventional antibodies. The of in this should not only be to African trypanosomes but be to other infectious agents on an antigenic variation mechanism (1.Johnson W.E. Desrosiers R.C. Annu. Rev. Med. 2002; 53: 499-518Crossref PubMed Scopus (175) Google Scholar, 2.Staalsoe T. Hamad A.A. Hviid L. Elhassan I.M. Arnot D.E. Theander T.G. J. Infect. Dis. 2002; 186: 719-722Crossref PubMed Scopus (28) Google Scholar, 3.Wang H. Bian T. Merrill S.J. Eckels D.D. J. Mol. Evol. 2002; 54: 465-473Crossref PubMed Scopus (30) Google Scholar). we on the of a VHH, specific for the conserved carbohydrate epitope of larger it is that this VHH the dense VSG coat of the parasite and stains trypanosomes of the expressed VSG The of this antibody fragment as a small molecular recognition unit in a more to new diagnostic and to combat is of this be of VSG AnTat MITat MITat and of Trypanosoma brucei brucei parasites the VSG by of with and parasites from blood by VSG was isolated and (7.Cross G.A. Parasitology. 1975; 71: 393-417Crossref PubMed Scopus (638) Google Scholar, Parasitol. PubMed Scopus Google Scholar). was by VSG for at with of was from the of the concanavalin-A with and was by dromedary of VSG AnTat 1.1 at intervals M. M. Desmyter A. J. E. L. Muyldermans S. J. 1998; 17: PubMed Scopus Google Scholar). after the serum was and into subclasses by on and as M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). At the same blood was to with blood and of and at VSG MITat 1.1 or MITat was a membrane and and sites for with with serum The membrane was with one of the dromedary in a and a antibody to In the was a with after the in the was by the membrane with VSG AnTat MITat or MITat at to sites with in After with of dromedary dromedary was with a serum and a antibody to after of the the was at The carbohydrate specificity of was by of with was and the VHH was in at VHH was a antibody and a antibody to after the was with and the was at was used as the concanavalin-A we VSG AnTat 1.1 at on sites for at with in The VHH from in was in or and to the of VHH was as for the carbohydrate VHH and of of from VHH and into a was as M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). of VSG specific was by three of on at in M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). After the and of the VHH was induced with The VHH, from the was for recognition by of the was by and of the the and the by a the and sites of M. M. Desmyter A. J. E. L. Muyldermans S. J. 1998; 17: PubMed Scopus Google Scholar, M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). the particular was from Biochem. J. PubMed Scopus Google by and sites and as a fragment into the to the (19.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (132) Google was as a fragment and sites the The the as after the was used as The fragment was by and in the the by and of and genes of the that in into an as M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). and of antibody and of the was as M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar, M. L. Muyldermans S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). was as by The at of the and the as from the used to the and VHH, antibodies and 1975; PubMed Scopus Google and the of was The of T. brucei brucei MITat and MITat was used in and of 107 parasites for in an with to or concanavalin-A After the parasites and with serum to antibodies and in or by with was to to with the The same was with the and parasites with for The was in or presence of or to the parasite to The are from in of the phenylenediamine and the by T. brucei brucei AnTat 1.1 parasites in with or Chem. 1993; PubMed Scopus (62) Google Scholar). To the of the VHH to and we T. brucei brucei AnTat 1.1 or MITat 1.5 parasites with After of and the parasites This was in and of to of dromedary and a with VSG from T. brucei brucei AnTat The conventional antibody subclass the and the subclasses and from the dromedary serum (18.Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2177) Google Scholar, M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar). dromedary and the 1.1 serum used in a to their to recognize epitopes by the three VSG represented by AnTat MITat and MITat (11.Richards F.F. J. Protozool. 1984; 31: 60-64Crossref PubMed Scopus (5) Google Scholar, 12.Carrington M. Boothroyd J. Mol. Biochem. Parasitol. 1996; 81: 119-126Crossref PubMed Scopus (62) Google The conventional antibodies from and as as the of the all recognize epitopes on the VSG AnTat 1.1 and MITat the of the all three VSG to a other of the to be antigenic for the from the it is that the dromedary contains a of antibodies that with epitopes common to VSGs of classes. the antigen-binding fragment of a dromedary is comprised one single domain the it was to these small molecular recognition The VHH repertoire present in an of 107 was in a M. M. Desmyter A. J. E. L. Muyldermans S. J. 1998; 17: PubMed Scopus Google Scholar, M. M. A. J.M. J. L. Muyldermans S. 2001; PubMed Scopus Google Scholar, E. M.A. L. Muyldermans S. 1998; PubMed Scopus Google Scholar). of this VHH with AnTat 1.1 these VHH genes the that the variable domain of a heavy chain of a from that of a conventional antibody (19.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (132) Google Scholar). in and their specificity and with other VSGs was on AnTat MITat MITat and AnTat 1.1 recognize AnTat of but to the MITat 1.1 and MITat are directed against unique epitopes present in the of AnTat the and with all three VSG molecules. However, the antigen-binding of the is upon of the whereas the with the AnTat of of because it is expressed in ELISA, and seems to recognize the carbohydrate of VSG, a of the that is to be solvent accessible in the The carbohydrate specificity of was by with by of and by to VSG or the of a of the from to in a with AnTat 1.1 are for an in the The by the presence of concanavalin-A a to the carbohydrates to VSG M.J. J. Protozool. 1990; PubMed Scopus Google Scholar). a the and concanavalin-A not be in an by it is that the with the antibody fragment for epitope. The of other VHH with AnTat 1.1 is not by an the of and the carbohydrate of the VSG, that in the flagellar of parasites S. M. A. H. H. R. Pays E. P. J. Biol. PubMed Scopus Google Scholar, S. M. B. Pays E. P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This in T. brucei AnTat 1.1 is by the presence of is a specific because the of with of the other VHH, e.g. to the trypanosomes from is for a and so the antibody fragment and the the of with other for the same from all after AnTat 1.1 with S. M. B. Pays E. P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google only the carbohydrates by a in a with in a ELISA, only the to a carbohydrate the Asn-linked of trypanosomes (16.Bangs J.D. Doering T.L. Englund P.T. Hart G.W. J. Biol. Chem. 1988; 263: 17697-17705Abstract Full Text PDF PubMed Google Scholar). In all other to with this as by in of VSG with of to recognize epitope on trypanosomes was and that stains parasites of the three VSG not to the same However, the is larger that with In the variant specific VHH labeled with stains only parasites AnTat 1.1 The VSGs of other are not and the is from an of parasites, or trypanosomes with an antibody fragment of it seems that T. brucei parasites. to the same of the VSG from so to a as by The concanavalin-A stains only the flagellar pocket of parasites to the the Asn-linked carbohydrates of VSG are only accessible for the large when in the flagellar pocket M.J. J. Protozool. 1990; PubMed Scopus Google Scholar). In the stains the entire surface of the parasite the in carbohydrate epitope even when present in the dense VSG coat of parasites where for larger molecules is to the high and the high of for the it becomes to the trypanosomes in blood a can be achieved with concanavalin-A because recognition of the of the host of the parasite as and the of the flagellar pocket of as in a single domain of the it as into more M. L. Muyldermans S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google e.g. to an To this we the genes for this molecular recognition unit that trypanosomes with that of The of the for at 1.1 for the and the that the to the antibody fragment not the of the The antigen-binding of the in the was in a on and with as E. P. C. C. E. N. Med. Google Scholar). the of the VHH and the of the are in the fusion The can the into the highly Chem. 1993; PubMed Scopus (62) Google Scholar). of to to form T. brucei AnTat 1.1 parasites in revealed an of for this which is the The of become highly for the trypanosomes when to parasites after a with the This that by of the antibody to the more from was with trypanosomes that express the MITat that the trypanosome specificity of the is in the This is specific because exposing the parasites to a to is by trypanosomes is a VHH with specificity for E. M.A. L. Muyldermans S. 1998; PubMed Scopus Google Scholar, A. M.A. Hamers R. Muyldermans S. L. Biol. 1996; PubMed Scopus Google single domain with specificity for the Asn-linked carbohydrate of the VSG This the potential to trypanosome on blood and to become the molecular recognition unit in an design for trypanosomosis The to single domain antibody for this type of is the small size of the (19.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (132) Google and the of the VSG molecules on the surface of the African trypanosomes exposing their immunogenic and the conserved epitopes of VSG (13.Clarke M.W. Barbet A.F. Pearson T.W. Mol. Immunol. 1987; 24: 707-713Crossref PubMed Scopus (10) Google Scholar, 14.Miller E.N. Allan L.M. Turner M.J. Mol. Biochem. Parasitol. 1984; 13: 67-81Crossref PubMed Scopus (31) Google Scholar, 15.Reinitz D.M. Aizenstein B.D. Mansfield J.M. Mol. Biochem. Parasitol. 1992; 51: 119-132Crossref PubMed Scopus (19) Google of the number and size of VSG molecules and the surface of the parasite it that the VSG molecules should other A. Zitzmann N. Richardson J.M. Treumann A. Ferguson M.A. Mol. Biochem. Parasitol. 1998; 91: 145-152Crossref PubMed Scopus (87) Google Scholar, J. PubMed Google Scholar, J. M. Turner M. J. Mol. Biol. 1990; PubMed Scopus (84) Google Scholar, Ferguson M.A. T.W. PubMed Scopus Google Scholar). This of is in where the of the of the VSG dimer is or for the of which are we the of the of the VSG and it it to the of the VSG to a more of the VSG and VSG on the coat of the The of the VSG is at the of these VSG whereas the Asn-linked carbohydrate is to the of the the in the VSG molecules. The concanavalin-A to the VSG this can only with the carbohydrates in the flagellar pocket M.J. J. Protozool. 1990; PubMed Scopus Google Scholar). The same carbohydrates of the VSGs on the parasite surface are inaccessible for molecules of this as illustrated by In the same a monoclonal antibody against the carbohydrate of the VSG, or even a derived from such a is to be from the carbohydrate in the In a dromedary single domain antibody should an to epitopes to the cryptic for larger to size of and of be that the of conserved VSG epitopes on parasites can be as with a Fv, the smallest antigen-binding fragment of a conventional However, these molecules are as large with the VHH molecules. In the are that a the and to at a single chain or the single chain and the are more to express in large R. M. A. 1990; PubMed Scopus Google Scholar). these the less for the The to dromedary antibodies in their recognition of epitopes that are less immunogenic for conventional antibodies. This was by the large number of or present in serum of an dromedary or in the of M. M. Desmyter A. J. E. L. Muyldermans S. J. 1998; 17: PubMed Scopus Google Scholar). for this was by of to their A. M.A. Hamers R. Muyldermans S. L. Biol. 1996; PubMed Scopus Google Scholar, A. S. M. L. Muyldermans S. C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. L. Trends Biochem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of the VHH in the of the of the E. M.A. L. Muyldermans S. 1998; PubMed Scopus Google Scholar, A. M.A. Hamers R. Muyldermans S. L. Biol. 1996; PubMed Scopus Google Scholar). antigen-binding are in conventional whereas have an that in (19.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (132) Google Scholar). In a binder with was able to with the pocket of because the entire domain in this on the surface A. S. M. L. Muyldermans S. C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). epitopes are less antigenic for conventional antibodies. it seems that a or on the antigenic whereas conventional antibodies on (19.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (132) Google Scholar, S. C. L. Trends Biochem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The and conventional antibodies was with the serum of the dromedary with AnTat the AnTat MITat and MITat the presence of antibodies. these antibodies with an epitope that is common to AnTat 1.1 and at one other VSG The be the VHH of such a as it the conserved carbohydrate of the VSG. The specificity of was by concanavalin-A by of by recognition of the Asn-linked high mannose oligosaccharide by with a and by a with AnTat In with this binder to epitope present the parasites. the high specificity for the and the of this epitope on the of to trypanosomes in blood smears. the of small on the VHH to of the epitope parasites other VSG a of the from an VSG packing or from the presence of or VSG To a agent for of trypanosomosis it be to an to this small molecular recognition a of we the as fusion because of multiple it is a small of that is expressed in J. P. J.M. E. Biol. 2001; PubMed Scopus Google and it can be to the of a VHH, because the and the antigen-binding of the antibody fragment are on of the and the of the is from the the for as as a sensitive A. M. 2002; PubMed Scopus Google Scholar). the can the into a Chem. 1993; PubMed Scopus (62) Google so that the fusion can be used in an antibody directed antibody the is absent in host and sensitive are T. B. S. A. 2002; PubMed Scopus Google Scholar). The with on trypanosomes are the antibody of the is able to the parasites, the of the diagnostic this fusion can be used in with to as as a that is to the most detection for trypanosomosis M. L. Rev. Med. Pays 1996; 49: Google Scholar, M. S. H. H. J. J.M. P. J. 2002; PubMed Scopus Google Scholar). In the potential to the parasites with the same in a therapy with was We that the successful employment of single domain antibody to combat trypanosomosis be to other infectious agents form of antigenic variation (1.Johnson W.E. Desrosiers R.C. Annu. Rev. Med. 2002; 53: 499-518Crossref PubMed Scopus (175) Google Scholar, 2.Staalsoe T. Hamad A.A. Hviid L. Elhassan I.M. Arnot D.E. Theander T.G. J. Infect. Dis. 2002; 186: 719-722Crossref PubMed Scopus (28) Google Scholar, 3.Wang H. Bian T. Merrill S.J. Eckels D.D. J. Mol. Evol. 2002; 54: 465-473Crossref PubMed Scopus (30) Google Scholar, 4.Cross G.A. Annu. Rev. Immunol. 1990; 8: 83-110Crossref PubMed Scopus (242) Google because the for their on their the small size and the to recognize epitopes of the variable that become cryptic for larger antibodies when into the We P. for