Intracellular colocalization of variant surface glycoprotein and transferrin-gold in Trypanosoma brucei

Endocytosis and intracellular transport has been studied in the bloodstream forms of Trypanosoma brucei by light and electron microscopy, using colloidal gold coupled to bovine transferrin (transferrin-gold). The endocytosed transferrin-gold, visualized by silver intensification for light microscopy, was present in vesicular structures between the cell nucleus and flagellar pocket of the organism. At the ultrastructural level, transferrin-gold was present after a 10-min incubation in the flagellar pocket, coated vesicles, cisternal networks, and lysosomelike structures. Endocytosis and intracellular processing of T. brucei variable surface glycoprotein (VSG) was studied using two preparations of affinity-purified rabbit IgG directed against different parts of the VSG. One preparation of IgG was directed against the cross-reacting determinant (CRD): a complex glycolipid side chain covalently linked to the COOH-terminus of the VSG molecule. The other was directed against determinants on the rest of the VSG molecule. When the two IgG preparations were used on thawed, thin cryosections of trypanosomes that had been incubated in transferrin-gold before fixation, the organelles involved with transferrin-gold endocytosis labeled with both antibodies, as well as many vesicular, tubular, and vacuolar structures that did not contain endocytosed transferrin-gold. Both antibodies also labeled the cell surface. In double-labeling experiments both antibodies were closely associated except that IgG directed against the VSG molecule labeled all the cisternae of the Golgi apparatus, whereas anti-CRD IgG was shown to label only half of the Golgi apparatus. Evidence for sorting of VSG molecules from endocytosed transferrin-gold was found. Double-labeling experiments also showed some tubular profiles which labeled on one side with anti-CRD IgG and on the other side with anti-VSG IgG, suggesting a possible segregation of parts of the VSG molecule.     

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.     

Cell surface interactions between Trypanosoma congolense and macrophages during phagocytosis in vitro.

Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

Cell Surface Interactions between Trypanosoma congolense and Macrophages during Phagocytosis In Vitro

ABSTRACT. Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

Identification of a tryptophan-like epitope borne by the variable surface glycoprotein (VSG) of African trypanosomes

Antibodies (Ab) directed against a tryptophan-like epitope (WE) were previously detected in patients with human African trypanosomiasis (HAT). We investigated whether or not these Ab resulted from immunization against trypanosome antigen(s) expressing a WE. By Western blotting, we identified an antigen having an apparent molecular weight ranging from 60 to 65 kDa, recognized by purified rabbit anti-WE Ab. This antigen, present in trypomastigote forms, was absent in procyclic forms and Trypanosoma cruzi trypomastigotes. Using purified variable surface glycoproteins (VSG) from various trypanosomes, we showed that VSG was the parasite antigen recognized by these rabbit Ab. Anti-WE and anti-VSG Ab were purified from HAT sera by affinity chromatography. Immunoreactivity of purified antibodies eluted from affinity columns and of depleted fractions showed that WE was one of the epitopes borne by VSG. These data underline the existence of an invariant WE in the structure of VSG from several species of African trypanosomes.     

Coated vesicles from the protozoan parasite Trypanosoma brucei: purification and characterization

A procedure was developed to purify a coated vesicle fraction from the protozoan parasite Trypanosoma brucei. Electron microscopy revealed a difference between T. brucei coated vesicles and clathrin-coated vesicles from other eukaryotes: trypanosome vesicles were larger (100 to 150 nm in diameter) and contained an inner coat of electron-dense material in addition to the external coat. Evidence suggests that the internal coat is the parasite's variant surface glycoprotein (VSG) coat. The SDS-PAGE analysis shows the major protein of T. brucei coated vesicles has a molecular mass of 61 kD, similar to VSG; this protein was recognized in an immunoblot by anti-VSG serum. Trypanosome coated vesicles also contain a protein which comigrates with the major protein (clathrin) of coated vesicles purified from rat brains. However, this protein is a minor component and it is not serologically cross-reactive with mammalian clathrin. Immunoblot analysis demonstrated that the parasite vesicles contained host IgG, IgM, and serum albumin.     

High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis

The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.     

Coated Vesicles from the Protozoan Parasite Trypanosoma brucei: Purification and Characterization

ABSTRACT. A procedure was developed to purify a coated vesicle fraction from the protozoan parasite Trypanosoma brucei. Electron microscopy revealed a difference between T. brucei coated vesicles and clathrin-coated vesicles from other eukaryotes: trypanosome vesicles were larger (100 to ISO nm in diameter) and contained an inner coat of electron-dense material in addition to the external coat. Evidence suggests that the internal coat is the parasite's variant surface glycoprotein (VSG) coat. The SDS-PAGE analysis shows the major protein of T. brucei coated vesicles has a molecular mass of 61 kD, similar to VSG; this protein was recognized in an immunoblot by anti-VSG serum. Trypanosome coated vesicles also contain a protein which comigrates with the major protein (clathrin) of coated vesicles purified from rat brains. However, this protein is a minor component and it is not serologically cross-reactive with mammalian clathrin. Immunoblot analysis demonstrated that the parasite vesicles contained host IgG, IgM, and serum albumin.     

Expression of antigenic regions of a trypanosome variable surface glycoprotein in E. coli using Bal-31 nuclease digestion.

We have used the expression of a trypanosome variable surface glycoprotein (VSG) in E. coli to produce VSG serotype-specific antisera which have none of the cross-reacting specificities characteristic of antisera prepared against purified VSGs. This was accomplished by treating restriction fragments of VSG cDNAs with Bal-31 nuclease to facilitate expression of their open reading frames in the E. coli expression vector, pMR100 (4). The resultant VSG-beta-galactosidase fusion proteins possess various antigenic regions of the original VSG. This provides a rapid means for producing VSG-specific antisera for reagent use and has the capability of large scale production of antigen for immunological investigation.     

In Vitro Studies on the Biosynthesis of the Surface Glycoprotein of Trypanosoma congolense1,2

Tritiated leucine, glucosamine, mannose, and galactose were incorporated into the variant specific surface glycoprotein (VSG) of Trypanosoma congolense in vitro. The uptake of the precursors is shown by SDS-polyacrylamide electrophoresis and fluorography, by assay of the radioactivity in immunoprecipitates obtained with specific antisera, and by the isolation of the labeled antigens by affinity chromatography on concanavalin A-sepharose and isoelectric focusing. The in vitro labeled VSG exhibits the same degree of microheterogeneity as that observed in the VSG isolated from trypanosomes grown in animals. Analysis of the incorporated sugars after hydrolysis of the glycoprotein showed that glucosamine and mannose were utilized in biosynthesis of the carbohydrate moiety directly whereas galactose was converted possibly to other intermediates before being incorporated into the antigen. Tunicamycin completely prevented the incorporation of the radiolabeled sugars into the surface glycoprotein. The unglycosylated VSG with a molecular weight of 47 kDa had completely lost its size heterogeneity.     

A conserved flagellar pocket exposed high mannose moiety is used by African trypanosomes as a host cytokine binding molecule

Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.     

Purification of major variable-surface glycoproteins from African trypanosomes by reverse-phase high-performance liquid chromatography

Reverse-phase high-performance liquid chromatography (RP-HPLC) was used in a one-step procedure to purify and analyze several different major variable-surface glycoproteins (VSGs) from lysates of African trypanosomes. RP-HPLC was used to fractionate lysates of trypanosomes and the VSG localized to the major peak of the elution profile using a rabbit antiserum to the cross-reacting determinant of the VSG. Polyacrylamide gel electrophoresis of HPLC fractions showed that the purity of isolated VSGs was equivalent to or better than that attained using conventional purification procedures. The elution positions of purified VSGs from a variety of cloned trypanosomes were identical, indicating the presence of a common hydrophobic feature on the surface of these highly polymorphic antigens. Preliminary experiments have shown that purification of VSG from trypanosome lysates may be scaled up to preparative levels. The results show that RP-HPLC is a useful procedure for rapid preparation of highly purified trypanosome VSGs and that analysis of their various molecular forms will be facilitated by the application of HPLC methods.     

Phaseolus vulgaris phytohemagglutinin contains high-mannose and modified oligosaccharide chains

Phytohemagglutinin, the major lectin in the seeds of the common bean Phaseolus vulgaris L., was isolated by affinity chromatography from cotyledons of nearly mature seeds and from developing cotyledons labeled with [³H]glucosamine, [³H]mannose or [³H]fucose. The protein was subjected to exhaustive proteolysis and the carbohydrate composition of the resulting glycopeptides examined. Two classes of oligosaccharide side-chains were found. The sidechains of the first class are of the high-mannose type, containing two residues of N-acetylglucosamine and 8 or 9 mannose residues. The sidechains of the second class are of the modified type containing N-acetylglucosamine, mannose, fucose, xylose in molar ratios of 2:3.8:0.6:0.5. Two-dimensional gel electrophoresis shows that phytohemagglutinin can be fractionated into seven different glycosylated polypeptides, and that each one contains at least one modified oligosaccharide chain. The results indicate that most glycosylated polypeptides probably contain one chain of each class. The carbohydrate composition of the two types of chains is similar to that found in other plant glycoproteins, but this is the first report of a plant glycoprotein with both highmannose and modified oligosaccharides on the same polypeptide chain.Abbreviations endo H endo--N-acetylglucosaminidase H - GlcN glucosamine - GlcNAc N-acetylglucosamine - Man mannose - PHA phytohemagglutinin This work was done while A.V. was on leave from the Istituto Biosintesi Vegetali, C.N.R., via Bassini 15, I-20133 Milano, Italy     

Regulation of B cell responses to the variant surface glycoprotein (VSG) molecule in trypanosomiasis. I. Epitope specificity and idiotypic profile of monoclonal antibodies to the VSG of Trypanosoma brucei rhodesiense.

Regulatory mechanisms governing B cell responses to the trypanosome variant surface glycoprotein (VSG) molecule currently are being studied. As a fundamental basis for examining such regulation, the epitope specificities and idiotypic profiles of murine mAb produced to the VSG of Trypanosoma brucei rhodesiense clone LouTat 1.5 were determined. Variant specific mAb were used to probe VSG proteolytic peptides in Western blot analysis, to serve as competitive inhibitors in RIA analyses with purified VSG molecules, and to examine membrane-binding patterns of labeled trypanosome cells in order to evaluate epitope specificities. By using these approaches, a conformational epitope expressed only on the VSG 1.5 surface coat of viable trypanosomes was detected, and two nonconformationally determined epitope clusters were recognized within the subsurface V region of the VSG 1.5 molecule. The subsurface epitope clusters may be repeated on the VSG molecule because each was present on more than one proteolytic VSG peptide fragment. Idiotypic profiles of selected VSG-specific mAb subsequently were determined with xenogeneic antiidiotypic typing sera. Results from competitive inhibition RIA analyses using these reagents demonstrated that varying levels of idiotypic cross-reactivity exist among the subsurface VSG epitope-specific mAb; this cross-reactivity extended to idiotope(s) expressed by a mAb recognizing a surface conformational epitope of the VSG 1.5 molecule. Analysis of complementary idiotypic/antiidiotypic antibody pairs revealed that these specific interactions were inhibited by purified VSG 1.5 but not by purified VSG 1.9, which was derived from a heterologous variant antigenic type. The model mAb described here, and reagents recognizing their idiotypic markers, comprise a foundation for analysis of idiotypic regulation of VSG-specific B cell responses during infection.     

Characterization of a mannan-like oligosaccharide from Mycobacterium smegmatis

A nitrogen-free neutral mannooligosaccharide, similar in structure to the polysaccharide component of yeast mannoproteins, has been isolated from Mycobacterium smegmatis ATCC-356. It has a molecular weight of 3200 and is terminated at the reducing end by mannose. nuclear magentic resonance spectroscopy, methylation analysis, selective enzymic degradation and acetolysis indicates that the molecule consists of an alpha1 --> 6-linked backbone to which single mannose units are attached in alpha1 --> 2 linkage as sidechains.     

Developmental variation in Rab11-dependent trafficking in Trypanosoma brucei

In Trypanosoma brucei, endocytosis is developmentally regulated and is substantially more active in the mammalian infective stage, where it likely plays a role in immune evasion. The small GTPase TbRAB11 is highly expressed in the mammalian stage and mediates recycling of glycosylphosphatidylinositol-anchored proteins, including the variant surface glycoprotein (VSG) and the transferrin receptor, plus trafficking of internalized anti-VSG antibody and transferrin. No function has been assigned to TbRAB11 in the procyclic (insect) stage trypanosome. The importance of TbRAB11 to both bloodstream and procyclic form viability was assessed by RNA interference (RNAi). Suppression of TbRAB11 in the bloodstream form was rapidly lethal and led to cells with round morphology and an enlarged flagellar pocket. TbRAB11 RNAi was also lethal in procyclic forms, which also became rounded, but progression to cell death was significantly slower and the flagellar pocket remained normal. In bloodstream forms, silencing of TbRAB11 had no effect on exocytosis of newly synthesized VSG, fluid-phase endocytosis, or transferrin uptake, but export of internalized transferrin was inhibited. Lectin endocytosis assays revealed a block to postendosomal transport mediated by suppressing TbRAB11. By contrast, in procyclic forms, depletion of TbRAB11 blocks both fluid-phase endocytosis and internalization of surface proteins. In normal bloodstream forms, most VSG is recycled, but in procyclics, internalized surface proteins accumulated in the lysosome. These data demonstrate that TbRAB11 controls recycling and is essential in both life stages of T. brucei but that its primary role is subject to developmental variation.     

Disulfide bond involvement in the maintenance of the cryptic nature of the cross-reacting determinant of metacyclic forms of Trypanosoma congolense

The variable surface glycoprotein (VSG) of African trypanosomes possesses a 1,2-dimyristoylglycosylphosphatidylinositol at the carboxy terminus. Cleavage of the 1,2-dimyristoylglycerol (1,2-DMG) moiety from the VSG reportedly results in a higher apparent molecular mass and an increased binding of antibodies against the "cross-reacting determinant" (CRD), a cryptic epitope present on most VSGs. Using metacyclic forms of Trypanosoma congolense, we show that the processes involved are more complex than heretofore presumed and that the removal of the 1,2-DMG moiety may not be necessary for binding of anti-CRD antibodies (RxCRD). Among other findings, we observe the following: (1) in sonicated samples of trypanosomes metabolically labeled with [3H]myristate, the binding of RxCRD on Western blots is coincident with bands containing labeled (membrane form) VSGs; (2) disulfide reduction of trypanosome sonicates suffices to promote RxCRD binding in the presence or absence of inhibitors of a glycosylphosphatidylinositol-specific phospholipase C; (3) trypanosomes directly solubilized in detergents show quantitative and qualitative differences in RxCRD binding which depend upon the detergent used and the order of addition of disulfide reducing agents. We conclude that the binding of RxCRD to T. congolense metacyclic VSGs depends upon the degree of unfolding of the molecule and is clearly a complex, multistep process in which structural changes and disulfide reduction play pivotal roles.     

Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides

The variant surface glycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysis of the intact protein and of glycopeptides. Processing has been examined by comparing glycan structures purified from an immature intracellular form (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAc2) at Asn-432 in both the immature 58-kDa and mature 59-kDa forms. In contrast, the "core" oligosaccharide of Asn-419 (Man3-GlcNAc2) appears to be nearly quantitatively processed to a complex biantennary structure [Gal-GlcNAc-Man)2-Man-GlcNAc2) during VSG maturation. The asparagine-linked structures at Asn-419, but not those at Asn-432, are resistant to endo-beta-N-acetylglucosaminidase H within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This report provides a direct demonstration of the processing of such a glycolipid anchor following its attachment to protein.     

Independent regulation of B cell responses to surface and subsurface epitopes of African trypanosome variable surface glycoproteins

Regulation of B cell responses to the trypanosome surface Ag was examined in H-2k compatible "responder" B10.BR and "nonresponder" C3H mice after infection with two variant clones of Trypanosoma brucei rhodesiense. Development of a selective RIA for independent detection of antibody binding to surface (exposed) and subsurface (buried) epitopes of the trypanosome variable surface glycoprotein (VSG) molecule permitted sensitive quantitation and kinetic characterization of immune responses to these epitopes. The infected B10.BR mice responded to both exposed and buried VSG epitopes of clone LouTat 1 trypanosomes, whereas a B cell response by C3H mice to exposed VSG epitopes was not detected by RIA analyses at any time. However, VSG-specific IgM and IgG responses were produced to buried VSG epitopes, demonstrating that LouTat 1 induced immunoregulation was specific only for the B cell responses to exposed VSG epitopes. Subsequently, comparisons of B10.BR and C3H B cell responses to a heterologous variant, LouTat 1.5, were made. The results revealed that both infected mouse strains produced VSG 1.5-specific antibody to exposed and buried epitopes with different kinetics and maximal sera concentrations, showing, therefore, that these responses are not coordinately regulated. In addition, it was clear that the observed immunosuppression to exposed LouTat 1 VSG epitopes in C3H mice could be regulated by the parasite since functional C3H B cell responses were mounted against exposed VSG epitopes of a closely related variant (LouTat 1.5) after infection.