Variant Surface Glycoprotein Molecules Research Articles

Processing and Presentation of Variant Surface Glycoprotein Molecules to T Cells in African Trypanosomiasis

Th1 cell responses to the variant surface glycoprotein (VSG) of African trypanosomes play a critical role in controlling infection through the production of IFN-gamma, but the role of APCs in the induction and regulation of T cell-mediated protection is poorly understood. In this study, we have investigated the Ag presentation capabilities of dendritic cells (DCs) and macrophages during early trypanosome infection in relatively resistant responder and susceptible nonresponder mouse strains. Splenic DCs appeared to be the primary cell responsible for activating naive VSG-specific Th cell responses in resistant responder animals through the coordinated up-regulation of costimulatory molecules, secretion of IL-12, and presentation of VSG peptides to T cells in vivo. Splenic DC depletion and the down-regulation of costimulatory markers on splenic macrophages were observed in susceptible animals and may be associated with the inability of these animals to elicit a significant VSG-specific T cell response. In contrast to splenic APCs, peritoneal macrophages secreted NO, failed to activate naive Th cells in vitro, and presented relatively low levels of VSG peptides to T cells in vivo. Thus, VSG-specific Th1 cell responses may be determined by tissue- and cell-specific differences in Ag presentation. Additionally, all APCs from resistant and susceptible strains displayed a reduced ability to process and present newly encountered exogenous Ag, including new VSG molecules, during high parasitemia. Thus, initial uptake of VSG (or other trypanosome factors) may interfere with Ag presentation and have dramatic consequences for subsequent T cell responses to other proteins.

T-cell responses to the trypanosome variant surface glycoprotein are not limited to hypervariable subregions.

Variable subregions within the variant surface glycoprotein (VSG) coat displayed by African trypanosomes are predicted sites for T- and B-cell recognition. Hypervariable subregion 1 (HV-1) is localized to an internal amphipathic alpha helix in VSG monomers and may have evolved due to selective pressure by host T-cell responses to epitopes within this subregion. The prediction of T-cell receptor-reactive sites and major histocompatibility complex class II binding motifs within the HV-1 subregion, coupled with the conservation of amino acid residues in other regions of the molecule sufficient to maintain secondary and tertiary VSG structure, prompted us to test the hypothesis that Th cells may preferentially recognize HV-1 subregion peptides. Thus, we examined the fine specificity of VSG-specific T-cell lines, T-cell hybridomas, and Th cells activated during infection. Our results demonstrate that T-cell epitopes are distributed throughout the N-terminal domain of VSG but are not clustered exclusively within HV-1 or other hypervariable subregions. In contrast, T-cell-reactive sites were not detected within the relatively conserved C-terminal domain of VSG. Overall, this study is the first to dissect the fine specificity of T-cell responses to the trypanosome VSG and suggests that evolution of a conserved HV-1 region may be unrelated to selective pressures exerted by host T-cell responses. This study also demonstrates that T cells do not recognize the relatively invariant C-terminal region of the VSG molecule during infection, suggesting that it could serve as a potential subunit vaccine to provide variant cross-specific immunity for African trypanosomiasis.

Regulation of innate and acquired immunity in African trypanosomiasis

African trypanosomes are well known for their ability to avoid immune elimination by switching the immunodominant variant surface glycoprotein (VSG) coat during infection. However, antigenic variation is only one of several means by which trypanosomes manipulate the immune system of their hosts. In this article, the role of parasite factors such as GPI anchor residues of the shed VSG molecule and the release of CpG DNA, in addition to host factors such as IFN-gamma, in regulating key aspects of innate and acquired immunity during infection is examined. The biological relevance of these immunoregulatory events is discussed in the context of host and parasite survival.

Trypanosomes Expressing a Mosaic Variant Surface Glycoprotein Coat Escape Early Detection by the Immune System

Host resistance to African trypanosomiasis is partially dependent on an early and strong T-independent B-cell response against the variant surface glycoprotein (VSG) coat expressed by trypanosomes. The repetitive array of surface epitopes displayed by a monotypic surface coat, in which identical VSG molecules are closely packed together in a uniform architectural display, cross-links cognate B-cell receptors and initiates T-independent B-cell activation events. However, this repetitive array of identical VSG epitopes is altered during the process of antigenic variation, when former and nascent VSG proteins are transiently expressed together in a mosaic surface coat. Thus, T-independent B-cell recognition of the trypanosome surface coat may be disrupted by the introduction of heterologous VSG molecules into the coat structure. To address this hypothesis, we transformed Trypanosoma brucei rhodesiense LouTat 1 with the 117 VSG gene from Trypanosoma brucei brucei MiTat 1.4 in order to produce VSG double expressers; coexpression of the exogenous 117 gene along with the endogenous LouTat 1 VSG gene resulted in the display of a mosaic VSG coat. Results presented here demonstrate that the host's ability to produce VSG-specific antibodies and activate B cells during early infection with VSG double expressers is compromised relative to that during infection with the parental strain, which displays a monotypic coat. These findings suggest a previously unrecognized mechanism of immune response evasion in which coat-switching trypanosomes fail to directly activate B cells until coat VSG homogeneity is achieved. This process affords an immunological advantage to trypanosomes during the process of antigenic variation.

Structure of the C-terminal Domain from Trypanosoma brucei Variant Surface Glycoprotein MITat1.2

The variant surface glycoprotein (VSG) of African trypanosomes has a structural role in protecting other cell surface proteins from effector molecules of the mammalian immune system and also undergoes antigenic variation necessary for a persistent infection in a host. Here we have reported the solution structure of a VSG type 2 C-terminal domain from MITat1.2, completing the first structure of both domains of a VSG. The isolated C-terminal domain is a monomer in solution and forms a novel fold, which commences with a short alpha-helix followed by a single turn of 3(10)-helix and connected by a short loop to a small anti-parallel beta-sheet and then a longer alpha-helix at the C terminus. This compact domain is flanked by two unstructured regions. The structured part of the domain contains 42 residues, and the core comprises 2 disulfide bonds and 2 hydrophobic residues. These cysteines and hydrophobic residues are conserved in other VSGs, and we have modeled the structures of two further VSG C-terminal domains using the structure of MITat1.2. The models suggest that the overall structure of the core is conserved in the different VSGs but that the C-terminal alpha-helix is of variable length and depends on the presence of charged residues. The results provided evidence for a conserved tertiary structure for all the type 2 VSG C-terminal domains, indicated that VSG dimers form through interactions between N-terminal domains, and showed that the selection pressure for sequence variation within a conserved tertiary structure acts on the whole of the VSG molecule.

The surface coat of procyclic Trypanosoma brucei: Programmed expression and proteolytic cleavage of procyclin in the tsetse fly

Trypanosoma brucei, the protozoan parasite causing sleeping sickness, is transmitted by a tsetse fly vector. When the tsetse takes a blood meal from an infected human, it ingests bloodstream form trypanosomes that quickly differentiate into procyclic forms within the fly's midgut. During this process, the parasite loses the 10(7) molecules of variant surface glycoprotein that formed its surface coat, and it develops a new coat composed of several million procyclin molecules. Procyclins, the products of a small multigene family, are glycosyl phosphatidylinositol-anchored proteins containing characteristic amino acid repeats at the C terminus [either EP (EP procyclin, a form of procyclin rich in Glu-Pro repeats) or GPEET (GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats)]. We have used a sensitive and accurate mass spectrometry method to analyze the appearance of different procyclins during the establishment of midgut infections in tsetse flies. We found that different procyclin gene products are expressed in an orderly manner. Early in the infection (day 3), GPEET2 is the only procyclin detected. By day 7, however, GPEET2 disappears and is replaced by several isoforms of glycosylated EP, but not the unglycosylated isoform EP2. Unexpectedly, we discovered that the N-terminal domains of all procyclins are quantitatively removed by proteolysis in the fly, but not in culture. These findings suggest that one function of the protease-resistant C-terminal domain, containing the amino acid repeats, is to protect the parasite surface from digestive enzymes in the tsetse fly gut.

Cloning, Expression, and Characterization of the acyl-CoA-binding Protein in African Trypanosomes

African trypanosomes are shielded from their hosts' defenses by a coat of variant surface glycoprotein molecules, each of which is attached to the plasma membrane by a glycosylphosphatidylinositol anchor. During the later stages of glycosylphosphatidylinositol biosynthesis, myristic acid is incorporated into the anchor from the donor myristoyl-CoA by a series of unique fatty acid remodeling and exchange reactions. We have cloned and expressed a recombinant trypanosome acyl-CoA-binding protein that has a preference for binding relatively short chain acyl-CoAs and that has a high affinity for binding myristoyl-CoA (K(d) = 3.5 x 10(-10) M). This protein enhances fatty acid remodeling of glycosylphosphatidylinositol precursors in the trypanosome cell-free system. We speculate that the trypanosome acyl-CoA-binding protein plays an active role in supplying myristoyl-CoA to the fatty acid remodeling machinery in the parasite.

A Cell-free Assay for Glycosylphosphatidylinositol Anchoring in African Trypanosomes: DEMONSTRATION OF A TRANSAMIDATION REACTION MECHANISM

We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the membrane anchor of a GPI-anchored protein or pro-protein causing release of the protein into the aqueous medium. Cell membranes containing pulse-radiolabeled VSG were incubated with hydrazine, and the VSG released from the membranes was measured by carbonate extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis/fluorography. Release of VSG was time- and temperature-dependent, was stimulated by hydrazine, and occurred only for VSG molecules situated in early compartments of the secretory pathway. No nucleophile-induced VSG release was seen in membranes prepared from cells expressing a VSG variant with a conventional transmembrane anchor (i.e. a nonfunctional GPI signal sequence). Pro-VSG was shown to be a substrate in the reaction by assaying membranes prepared from cells treated with mannosamine, a GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine was used instead of hydrazine, the released VSG could be precipitated with streptavidin-agarose, indicating that the biotin moiety was covalently incorporated into the protein. Hydrazine was shown to block the C terminus of the released VSG hydrazide because the released material, unlike a truncated form of VSG lacking a GPI signal sequence, was not susceptible to proteolysis by carboxypeptidases. These results firmly establish that the released material in our assay is VSG hydrazide and strengthen the proof that GPI anchoring proceeds via a transamidation reaction mechanism. The reaction could be inhibited with sulfhydryl alkylating reagents, suggesting that the transamidase enzyme contains a functionally important sulfhydryl residue.

The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei

Trypanosoma brucei, in common with the other African trypanosomes, exhibits unusual cell-surface molecular architecture. The bloodstream form of the parasite is coated with a continuous layer of approximately five million variant surface glycoprotein (VSG) dimers that provide the parasite with a macromolecular diffusion barrier to guard against lysis by the alternative complement pathway. The procyclic form of the parasite has a more diffuse cell-surface coat made up of approximately 2.5 million copies of procyclic acidic repetitive protein (PARP). Within the VSG and PARP coats exist lower-abundance surface glycoproteins such as receptors and nutrient transporters. Both the VSG molecules and the PARP molecules are attached to the membrane via glycosylphosphatidylinositol (GPI) membrane anchors and the VSGs and one form of PARP are N-glycosylated. In this article, the structures of the N-glycans and the GPI anchors of T. brucei VSGs and PARPs are reviewed and simple models of the surfaces of bloodstream and procyclic trypomastigotes are presented.

The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei

The compound diisopropylfluorophosphate (DFP) selectively inhibits an inositol deacylase activity in living trypanosomes that, together with the previously described phenylmethylsulfonyl fluoride (PMSF)-sensitive inositol acyltransferase, maintains a dynamic equilibrium between the glycosylphosphatidylinositol (GPI) anchor precursor, glycolipid A [NH2(CH2)2PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-sn-1,2-dimyristoylglycerol], and its inositol acylated form, glycolipid C. Experiments using DFP in living trypanosomes and a trypanosome cell-free system suggest that earlier GPI intermediates are also in equilibrium between their inositol acylated and nonacylated forms. However, unlike mammalian and yeast cells, bloodstream form trypanosomes do not appear to produce an inositol acylated form of glucosaminylphosphatidylinositol (GlcN-PI). A specific function of inositol acylation in trypanosomes may be to enhance the efficiency of ethanolamine phosphate addition to the Man3GlcN-(acyl)PI intermediate. Inositol deacylation appears to be a prerequisite for fatty acid remodelling of GPI intermediates that leads to the exclusive presence of myristic acid in glycolipid A and, ultimately, in the variant surface glycoprotein (VSG). In the presence of DFP, the de novo synthesis of GPI precursors cannot proceed beyond glycolipid C' (the unremodelled version of glycolipid C) and lyso-glycolipid C'. Under these conditions glycolipid C'-type GPI anchors appear on newly synthesized VSG molecules. However, the efficiencies of both anchor addition to VSG and N-glycosylation of VSG were significantly reduced. A modified model of the GPI biosynthetic pathway in bloodstream form African trypanosomes incorporating these findings is presented.

The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei.

The compound diisopropylfluorophosphate (DFP) selectively inhibits an inositol deacylase activity in living trypanosomes that, together with the previously described phenylmethylsulfonyl fluoride (PMSF)-sensitive inositol acyltransferase, maintains a dynamic equilibrium between the glycosylphosphatidylinositol (GPI) anchor precursor, glycolipid A [NH2(CH2)2PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-sn-1,2-dimyristoylglycerol], and its inositol acylated form, glycolipid C. Experiments using DFP in living trypanosomes and a trypanosome cell-free system suggest that earlier GPI intermediates are also in equilibrium between their inositol acylated and nonacylated forms. However, unlike mammalian and yeast cells, bloodstream form trypanosomes do not appear to produce an inositol acylated form of glucosaminylphosphatidylinositol (GlcN-PI). A specific function of inositol acylation in trypanosomes may be to enhance the efficiency of ethanolamine phosphate addition to the Man3GlcN-(acyl)PI intermediate. Inositol deacylation appears to be a prerequisite for fatty acid remodelling of GPI intermediates that leads to the exclusive presence of myristic acid in glycolipid A and, ultimately, in the variant surface glycoprotein (VSG). In the presence of DFP, the de novo synthesis of GPI precursors cannot proceed beyond glycolipid C' (the unremodelled version of glycolipid C) and lyso-glycolipid C'. Under these conditions glycolipid C'-type GPI anchors appear on newly synthesized VSG molecules. However, the efficiencies of both anchor addition to VSG and N-glycosylation of VSG were significantly reduced. A modified model of the GPI biosynthetic pathway in bloodstream form African trypanosomes incorporating these findings is presented.

Transport of a lysosomal membrane glycoprotein from the Golgi to endosomes and lysosomes via the cell surface in African trypanosomes

gp57/42 is a membrane glycoprotein localized in the trans-Golgi, flagellar pocket region of the cell surface, endosomes and lysosomes of bloodstream forms of Trypanosoma brucei rhodesiense. Pulse-chase immunoprecipitation experiments revealed that gp57/42 acquires a unique N-linked oligosaccharide recognized by the CB1 monoclonal antibody 20-30 minutes after protein synthesis, probably in the trans-Golgi. We refer to gp57/42 molecules that carry the CB1 epitope as CB1-gp. Pulse labeled CB1-gp contained only one core protein, p57, when chase times were 30 minutes or less. As time of chase increased from 30 to 60 minutes, a new polypeptide, p42, appeared in N-glycanase-treated CB1 immunoprecipitates. Since p57 and p42 share 10 of 13 methionyl peptides, we conclude that p42 is a fragment of p57. Cleavage of p57 to p42 was not inhibited when cells were chased in two thiol protease inhibitors or in 3,4-diisocoumarin, but was inhibited by leupeptin. Cell surface biotinylation was used to determine if newly synthesized CB1-gp was transported from the Golgi to the surface. When cells were pulse labeled and chased for 30 minutes, as much as 40% of the radiolabeled CB1-gp could be biotinylated on the cell surface. The amount of CB1-gp that could be biotinylated decreased when chases were extended from 30 to 60 minutes, suggesting that pulse labeled CB1-gp left the surface. In contrast, pulse labeled variant surface glycoprotein molecules continued to accumulate on the surface where they could be biotinylated between 30 and 60 minutes of chase. Biotinylated CB1-gp derived from cells chased for 30 minutes contained p57 but no p42. However, when labeled cells were biotinylated after a 30 minute chase and then incubated another 30 minutes at 37 degrees C, the biotinylated CB1-gp contained both p57 and p42. The p57 in biotinylated CB1-gp was not cleaved to p42 if the additional incubation was done at 4 or 12 degrees C. This suggests that transport to a compartment where processing occurs and/or the processing enzymes are inhibited by low temperature. When surface biotinylation was done after a 60 minute chase, p42 was detected in biotinylated CB1-gp, suggesting that CB1-gp molecules had passed through the processing compartment and then appeared on the cell surface. Thus, a major portion of the newly synthesized CB1-gp is routed from the Golgi to endocytic compartments via the cell surface. In trypanosomes this process involves a unique surface domain, the flagellar pocket.(ABSTRACT TRUNCATED AT 400 WORDS)

Bovine T cells specific for Trypanosoma brucei brucei variant surface glycoprotein recognize nonconserved areas of the molecule.

The specificity of bovine CD4+ T-cell responses to Trypanosoma brucei variant surface glycoprotein (VSG) has been examined by using a panel of seven T-cell clones and nested deletions of the ILTat 1.3 VSG gene expressed in Escherichia coli. All clones recognized the polymorphic N-terminal domain of the antigen, and the recognition sites of five of the clones were resolved to three areas with lengths of 14, 18, and 21 amino acids. Comparison of these regions with corresponding areas of other VSG molecules, including those derived from the same trypanosomal serodeme, has shown that the sites are not conserved. In the light of recent observations that VSG-specific T-cell responses are induced in mice infected with T. brucei, these results confirm with the belief that immune pressure from T cells may contribute to the generation of antigenic diversity on the surface of African trypanosomes.

A novel method for mean cell volume estimation

SummaryA novel method is described for the estimation of the mean cell volume of cell populations grown in suspension. The cells are filtered onto a nitrocellulose filter to form a cylindrical pellet which is embedded in epoxy resin. Using estimates of pellet height and radius, the number of cells in the pellet and of the volume density of the cells in the pellet, it is possible to produce an unbiased estimate of the mean cell volume. This method is compared, using cell suspensions of the blood parasite Trypanosoma brucei, with mean cell volume estimation using a Coulter channellizer. A Coulter channellizer was also used to compare the mean cell volume of living trypanosomes with that of aldehyde‐fixed populations, and the values obtained were compared with those obtained using the new method. The estimated mean cell volume of a T. brucei clone was used to derive values from volume densities obtained by point and intersection counts for the absolute volumes of the flagellar pocket, the nucleus, and endocytic organelles containing internalized horseradish peroxidase and transferrin‐gold after 30‐min incubations at 310 K. Estimated values for the surface area of the flagellar pocket and the surface area of a cell were also obtained. From known data on the total amount of variant surface glycoprotein molecules per cell and the known packing density of membrane proteins, it was estimated that approximately 80% of the molecules must reside in intracellular compartments. It was estimated that the equivalent of 5% of the surface membrane may be internalized per minute, an amount which is almost the size of the entire flagellar pocket membrane.

The mechanism of inhibition of glycosylphosphatidylinositol anchor biosynthesis in Trypanosoma brucei by mannosamine.

The inhibition of glycosylphosphatidylinositol anchor biosynthesis by mannosamine has been described previously in the procyclic forms of Trypanosoma brucei and in mammalian cells (Lisanti, M. P., Field, M. C., Caras, I. W. J., Menon, A. K., and Rodriguez-Boulan, E. (1991) EMBO J. 10, 1969-1977). A recent report has suggested that mannosamine exerts these effects by becoming incorporated into glycosylphosphatidylinositol anchor intermediates (Pan, Y-T., Kamitani, T., Bhuvaneswaran, C., Hallaq, Y., Warren, C. D., Yeh, E. T. H., and Elbein, A. D. (1992) J. Biol. Chem. 267, 21250-21255). In this paper we have analyzed the effects of mannosamine on glycosylphosphatidylinositol anchor and variant surface glycoprotein biosynthesis in the blood-stream form of T. brucei. Trypanosomes were biosynthetically labeled with [3H]mannosamine, and [3H]glucosamine in the presence of mannosamine, and the structures of the labeled glycolipids which accumulated were determined. The main glycolipid metabolite of mannosamine was shown to be ManN-Man-GlcN-PI. A trypanosome cell-free system preloaded with this compound was significantly impaired in its ability to synthesize glycosylphosphatidylinositol anchor intermediates beyond Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI. This compound is therefore proposed to be an inhibitor of the Dol-P-Man:Man alpha 1-6Man alpha 1-4GlcNa alpha 1-6PI alpha 1-2-mannosyltransferase of the GPI biosynthetic pathway. In living trypanosomes, 4 mM mannosamine had no effect on protein synthesis but reduced the rate of formation of mature glycosylphosphatidylinositol anchor precursors by 80%. This reduction in anchor precursor synthesis was insufficient to prevent the attachment of glycosylphosphatidylinositol anchors to newly synthesized variant surface glycoprotein molecules. These data suggest that the rate of anchor precursor synthesis in the bloodstream form of T. brucei, in contrast to mammalian cells and the procyclic form of T. brucei, is in large excess of the cellular requirements for protein anchorage.

Characterization of T helper cell responses to the trypanosome variant surface glycoprotein.

T cell responses to the variant surface glycoprotein (VSG) previously have not been detected in animals infected with the African trypanosomes despite the fact that such animals make strong T-dependent B cell responses to VSG molecules displayed by the parasites. In the present study, we have examined B 10.BR mice for VSG-specific Th cell responses at different times after infection with Trypanosoma brucei rhodesiense clone LouTat 1. T cell populations derived from different tissues were tested for their ability to proliferate and secrete cytokines when stimulated with purified LouTat 1 VSG. Furthermore, VSG-specific T cell lines and clones were derived from immunized mice and examined for their phenotypic and functional profiles in comparison with T cell responses of infected mice. The results of this study show that VSG-specific T cells were not consistently detected in the peripheral lymphoid tissues such as spleen or lymph nodes of infected animals. In contrast, VSG Ag-specific T cells were detectable principally in the peritoneal T cell populations of infected mice. Peritoneal T cells did not proliferate in response to VSG, yet produced substantial cytokine responses when stimulated; the cytokines produced were IFN-gamma and IL-2, without detectable IL-4. The cellular phenotype of VSG-responsive T cells was that of classical Th cells in that all cells were CD4-positive and expressed the CD3 alpha/beta TCR membrane complex. Thus, the VSG appears to preferentially stimulate a Th1 cell subset response during infection. Intrinsic molecular characteristics of the VSG molecule did not induce mice to make this response, however, since VSG-specific T cell lines derived from VSG-immunized mice displayed cytokine profiles characteristic of both Th1 and Th2 cells. Isolation of Th1 clones from selected lines demonstrated that these cells displayed the same membrane-phenotypic characteristics and cytokine profiles as the T cells from infected mice. Furthermore, all Th clones were VSG type-specific, APC-dependent, and I-Ak-restricted in their responses. In summary, these experiments provide the first direct evidence for VSG-specific responses at the T cell level. T cell responses to the VSG molecule during infection appear to be anatomically compartmentalized and exhibit evidence of clonal maturation (cytokine production) but not clonal expansion (proliferation) after antigenic stimulation. The cellular phenotype and cytokine profiles predict that infection predisposes the animals to mount Th1 cell subset responses to VSG. The results of this study, including the T clones generated, provide an experimental basis for examining the regulation of VSG-specific immune responses during infection.

Variable and conserved structural elements of trypanosome variant surface glycoproteins

The characterization of B cell epitopes on the trypanosome variant surface glycoprotein (VSG) rests on elucidation of variant specific amino acid sequences that may be exposed or buried as a result of the natural conformation of these molecules in the surface coat. Despite the fact that different VSGs have heterogeneous primary sequences and unique antigenic characteristics, recent high resolution X-ray crystallographic analyses of VSGs have revealed a conserved 3-dimensional structure common to these surface proteins [19]. We took advantage of this conserved structural conformation to help predict which variant subregions of VSG molecules may contain exposed or buried variant specific B cell epitopes. Using Staden data tables, we aligned the deduced amino acid sequence of Trypanosoma brucei rhodesiense LouTat 1 VSG, a molecule that has been characterized immunologically in this laboratory, with 12 other complete VSG sequences including the T. b. brucei MiTat 1.2 VSG that has been characterized in crystallographic studies. Results of this analysis predict that there are eight defined clusters of variant amino acids which may contribute to exposed B cell epitopes, and ten defined clusters of variant amino acids which may contribute to buried B cell epitopes, on all VSG molecules. Interestingly, this analysis also revealed a VSG consensus sequence in which certain conserved motifs are present in all VSGs. The shared elements of VSG sequences corresponded to known secondary structures present in MiTat 1.2, and included groups of conserved amino acids responsible for turns in subregions of the protein, for structural positioning of the variable residues on the exposed surface, and for the dimerization of VSG monomers. Overall, these observations may aid in the targeting and mapping of exposed and buried VSG specific B cell epitopes, and also may offer clues as to elements of the primary sequence that are important for the conserved 3-dimensional structure of antigenically distinct VSG molecules.

T-cell-independent and T-cell-dependent B-cell responses to exposed variant surface glycoprotein epitopes in trypanosome-infected mice

The T-cell dependency of B-cell responses to variant surface glycoprotein (VSG) epitopes exposed in their native surface conformation on Trypanosoma brucei rhodesiense clone LouTat 1 was investigated. T-cell requirements were examined by analyses of gamma globulin preparations derived from trypanosome-infected BALB/c nude (nu/nu) and thymus-intact (nu/+) mice. A radioimmunoassay was used to selectively quantitate antibody binding to native VSG 1 epitopes present on the surface of viable trypanosomes. Such analyses of VSG-specific antibody in infected mice demonstrated that in the absence of T cells there was a significant B-cell response to exposed VSG epitopes; however, in the presence of T cells these surface epitope-specific responses were greatly enhanced. In contrast to infection, immunization of mice with purified VSG 1 or paraformaldehyde-fixed parasites elicited significant VSG surface epitope-specific responses only in the presence of T cells (i.e., in nu/+ mice only). VSG-specific antibody responses in mice infected with three other clonal T. brucei rhodesiense populations (LouTat 1.2, 1.5, and 1.9) were found to be similar in this pattern, although not identical, to the anti-LouTat 1 responses. An important exception was that mice infected with LouTat 1.8 required T cells to produce VSG surface-specific antibody. Thus, the VSG surface epitope-specific B-cell responses in trypanosome-infected mice represent composite T-cell-independent and T-cell-dependent processes, and a significantly stronger response is made in the presence of T cells. However, immunization with VSG in the absence of infection elicited only T-cell-dependent responses. Since the relative contribution of T-cell-independent and T-cell-dependent processes to the total VSG-specific antibody produced during infection was variable (as seen with the absence of a T-cell-independent response to LouTat 1.8), this may reflect differences in the primary structure or display of VSG molecules on the trypanosome membrane or may represent active parasite interference with some epitope-specific B-cell responses.

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.

Regulation of B cell responses to the variant surface glycoprotein molecule in trypanosomiasis. II. Down-regulation of idiotype expression is associated with the appearance of lymphocytes expressing antiidiotypic receptors.

The current study examines the idiotypic expression and regulation of variant surface glycoprotein (VSG)-specific B cell responses during African trypanosomiasis. Utilizing competitive inhibition RIA analysis, we detected antibodies in the serum of BALB/cByJ mice infected with Trypanosoma brucei rhodesiense clone LouTat 1.5 that recognized the same VSG epitopes as three VSG 1.5-specific mAb. These epitope-specific antibody responses were detectable by day 5 of infection, peaked by day 10, and then declined slowly through day 15 of infection. VSG-specific antibodies detectable in the serum of infected BALB/cByJ mice included those that were idiotypically cross-reactive with the VSG 1.5-specific mAb. These idiotypically defined, VSG-specific antibody responses appeared to peak around day 7 of infection, but then declined to near preimmune levels by day 15 of infection, demonstrating that the aggregate epitope-specific response was composed only in part by the idiotypically cross-reactive responses. Although corresponding antiidiotypic antibodies could not be detected in infected sera during periods of up- or down-regulation of idiotypically defined antibodies, flow cytometry analysis of lymphocytes isolated from the spleens of LouTat 1.5-infected BALB/cByJ mice revealed the presence of antiidiotypic receptor-bearing cells. These cells were detectable primarily during days 10 to 12 of infection and subsequently down-regulated their receptors, or declined in numbers, to near preimmune levels by day 15 of infection. The appearance of these antiidiotypic receptor-bearing cells coincides with the decline of idiotypic antibody present in the serum of the LouTat 1.5-infected mice and may represent nascent evidence for idiotypic regulation of trypanosome-specific immune responses in infected animals.