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.     

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.     

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 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.     

Trypanosome variant surface glycoproteins are recognized by self-reactive antibodies in uninfected hosts.

The variant surface glycoproteins (VSGs) of African trypanosomes form a dense surface coat on the bloodstream parasites. VSGs are immunodominant antigens that stimulate a rapid antibody response in trypanosome-infected individuals. In the present study, we examined VSG-specific antibodies present not only in sera from infected individuals but also in sera from individuals that had never been exposed to trypanosomes. Native antibodies against different VSGs were detected in sera from uninfected mice, bovines, and humans; the antibodies were revealed to be exclusively directed against variable determinants of the antigens. Further experimentation demonstrated that such native antibodies immunoreact with cellular components of mice and thus are most likely produced by the self-reactive B-cell compartment of the murine immune system.     

In Vitro Effects of Cyclosporin A on Human B-Cell Responses

The in vitro effects of cyclosporin A (CsA) on T-cell-dependent and T-cell-independent mitogen responses of human B cells were studied. T-cell-dependent, pokeweed mitogen (PWM)-induced B-cell proliferation and B-cell differentiation to Ig-secreting cells were significantly inhibited by CsA, when purified B cells were cultured with T-cell helper factor containing supernatants instead of T cells. This indicates that the inhibitory effect of CsA on T-cell-dependent, PWM-induced B-cell proliferation and differentiation is not exclusively due to direct effects on helper T cells. B-cell proliferations induced by anti-IgM antibodies and by Staphylococcus aureus bacteria were also found to be sensitive to CsA. Since both types of reactions are T-cell-independent, the concept that responses of human B cells can also be affected by CsA in ways that seem to be independent of the well-documented direct effects of CsA on T cells is further supported. This seems not to be a general phenomenon, however. Epstein-Barr-virus-induced activation of human B cells, as reported previously and also observed by us, is completely insensitive to CsA. It seems, therefore, that certain B-cell activation mechanisms are sensitive to CsA while others remain unaltered. The difference between these two reaction patterns cannot be exclusively explained by a T-cell dependence or T-cell independence of these responses. CsA effects on certain functional B-cell subsets or interference with accessory cell mechanisms might be responsible.     

MHC-restricted B-cell antigen presentation in memory B-cell maintenance and differentiation

Memory B-cell development, maintenance, and differentiation have been believed to be tightly regulated by T cells through major histocompatibility complex (MHC)-II-restricted cognate interaction. However, recent studies have indicated that memory B cells are a heterogeneous population with various ontogeny and with derivations from T-cell-independent as well as T-cell-dependent response. In addition, several studies highlight T-cell-independent maintenance and differentiation of memory B cells by innate polyclonal stimuli associated with Toll-like-receptors (TLRs). Thus, both T-cell-dependent and T-cell-independent mechanisms with possibly different physiological functions are required for maximal memory B-cell maintenance and differentiation. In this review, we discuss the mechanisms of memory B-cell maintenance and differentiation by re-examining the requirement for MHC-II-restricted B-cell antigen presentation based on our recent study in a mouse model that lacks MHC-II on memory B cells. We propose that MHC-II-restricted memory B-cell maintenance and differentiation are the indispensable mechanisms by which we achieve health and the maximum quality of memory response with specific antibody production, while preventing plasma cell differentiation from self-reactive memory B cells under the control of T-helper cells and possibly regulatory T cells.     

The Effects of Mouse Alpha-Fetoprotein on T-Cell-Dependent and T-Cell-Independent Immune Responses In Vitro

Mouse alpha-fetoprotein (AFP) suppressed the specific antibody response to the T-cell-dependent antigen sheep erythrocytes (SRBC) and the phytohemagglutinin- and concanavalin-A-stimulated DNA synthesis of purified T lymphocytes but failed to inhibit the T-cell-independent antibody response to dinitrophenyl-substituted Ficoll (DNP-Ficoll) and the lipopolysaccharide-stimulated polyclonal B-cell antibody synthesis. Mouse amniotic fluid (MAF) suppressed antibody responses to both T-cell-dependent and T-cell-independent antigens; however, the effects could be differentiated since dialysis of the MAF removed most of the suppressive effect on the DNP-Ficoll response but did not diminish the inhibitory action on the anti-SRBC response. The results indicate that AFP suppresses certain T-lymphocyte functions in vitro and does not act by directly inhibiting B-cell functions.     

Analysis of macrophage activation in African trypanosomiasis

African trypanosomes cause a fatal disease of man and animals that is characterized by extensive functional, histological, and pathological changes in the lymphoid tissues of infected hosts, including an increase in the numbers and activation state of macrophages. Macrophage activation during infection is the result of exposure of these cells to parasite components and host-derived IFN-gamma, produced in response to parasite antigens. The balance of these different activation signals may determine the outcome of infection. In the experiments described here, we assessed the ability of the variant surface glycoprotein (VSG) of the organism Trypanosoma brucei rhodesiense (T.b. rhodesiense) to activate macrophages directly. Our results demonstrate that macrophages bind and are activated by the VSG molecule. The resulting profile of activation differs from that stimulated by IFN-gamma. These results suggest that the interaction of host macrophages with VSG released during parasite infection may be a key component of trypanosomiasis.     

Role of expression site switching in the development of resistance to human Trypanosome Lytic Factor-1 in Trypanosoma brucei brucei

Human high-density lipoproteins (HDLs) play an important role in human innate immunity to infection by African trypanosomes with a minor subclass, Trypanosome Lytic Factor-1 (TLF-1), displaying highly selective cytotoxicity to the veterinary pathogen Trypanosoma brucei brucei but not against the human sleeping sickness pathogens Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. T. b. rhodesiense has evolved the serum resistance associated protein (SRA) that binds and confers resistance to TLF-1 while T. b. gambiense lacks the gene for SRA indicating that these parasites have diverse mechanisms of resistance to TLF-1. Recently, we have shown that T. b. gambiense (group 1) resistance to TLF-1 correlated with the loss of the haptoglobin/hemoglobin receptor (HpHbR) expression, the protein responsible for high affinity binding and uptake of TLF-1. In the course of these studies we also examined TLF-1 resistant T. b. brucei cell lines, generated by long-term in vitro selection. We found that changes in TLF-1 susceptibility in T. b. brucei correlated with changes in variant surface glycoprotein (VSG) expression in addition to reduced TLF-1 binding and uptake. To determine whether the expressed VSG or expression site associated genes (ESAGs) contribute to TLF-1 resistance we prepared a TLF-1 resistant T. b. brucei with a selectable marker in a silent bloodstream expression site (BES). Drug treatment allowed rapid selection of trypanosomes that activated the tagged BES. These studies show that TLF-1 resistance in T. b. brucei is largely independent of the expressed VSG or ESAGs further supporting the central role of HpHbR expression in TLF-1 susceptibility in these cells.     

B-cell proliferation initiated by Ia cross-linking and sustained by interleukins leads to class switching but not somatic mutation in vitro.

Somatic mutations that are acquired by antibody V genes of antigen-stimulated B cells ultimately provide the clonal diversity from which memory B cells are selected during immune responses to T-cell-dependent antigens. Somatic mutations apparently are not acquired when B cells are stimulated by mitogens nor when they participate in immune responses to T-cell-independent antigens. Since the basis of T-cell-dependent humoral immunity is T-cell recognition of processed antigen in the context of class II major histocompatibility glycoproteins (Ia) on the B-cell surface, we sought to determine whether the ligation of Ia on B cells induces somatic mutation. B cells were stimulated in vitro by a procedure in which their proliferation was dependent upon ligation of surface Ia with antibody. Sequences of hybridoma V genes derived from these B cells revealed no somatic mutations despite prolonged stimulation in vitro and the induction of immunoglobulin secretion and switching to isotypes characteristic of T cell-dependent humoral immunity. We infer that Ia-mediated signalling and isotype switching are not causally related to somatic mutation. The avenue of differentiation that leads to somatic mutation in memory B cells is apparently separable from that leading to proliferation, immunoglobulin secretion and switching.     

Antibody responses in resistant and susceptible inbred mice infected with Trypanosoma congolense.

Antibody responses were evaluated in inbred mice previously shown to be susceptible (A/J) or resistant (C57BL/6J and B6AF1 hybrid) to infections with relatively avirulent Trypanosoma congolense. Titres and the isotype distribution antibodies specific for the trypanosome variant surface glycoprotein (VSG) were determined by indirect immunofluorescence in sera of mice after primary infections with Trypanosoma congolense and after challenge infections with the same variant following drug cure. The results of these investigations showed that, during active infection, resistant mice made relatively strong VSG-specific IgM antibodies. This isotype also predominated in challenge infections with the homologous variant following drug cure. In contrast, A/J mice made little or no VSG-specific antibody on first exposure to T. congolense. However, these animals were able to produce substantial amounts of protective VSG-specific IgG antibody after multiple-challenge infections with the homologous variant. Substantial titres of VSG-specific antibodies in resistant mice did not influence the numbers of trypanosomes in the first parasitaemic peak as initial parastiaemias were similar in both C57BL/6J and A/J mice. However, C57BL/6J mice cleared parasites in this peak, whereas A/J mice did not. Mice of both strains immunized by infection cure were equally effective in clearing parasites when challenged with homologous trypanosomes. It is clear from the results of this study that antibody is not the sole factor contributing to murine resistance to African trypanosomes.     

In vivo analysis of impaired macrophage bactericidal capacity during experimental African trypanosomiasis

Since innate resistance of mice to Salmonella typhimurium depends on an intact macrophage system, we have used this bacterium to investigate the effect of Trypanosoma brucei subsp. rhodesiense infection on macrophage phagocytic and cytolytic function. CBA/CaJ mice infected with T. brucei subsp. rhodesiense have decreased resistance to S. typhimurium, since doubly infected mice rapidly succumb to sublethal doses of S. typhimurium. Although trypanosomiasis is known to suppress antibody formation, such a suppression of antibody does not seem to play a role in trypanosome-induced sensitivity to S. typhimurium. A trypanosome-induced blockade of the reticuloendothelial system also does not occur, since parasitized and control mice clear S. typhimurium from the blood equally well. Early killing (0 to 48 h) of S. typhimurium in the liver and spleen is mainly macrophage mediated, and mice infected with trypanosomes kill S. typhimurium in the liver and spleen very poorly. Apparently trypanosomiasis inhibits macrophage bactericidal activity, but has no effect on phagocytosis.     

Comparative analysis of antibody responses against HSP60, invariant surface glycoprotein 70, and variant surface glycoprotein reveals a complex antigen-specific pattern of immunoglobulin isotype switching during infection by Trypanosoma brucei

During Trypanosoma brucei infections, the response against the variant surface glycoprotein (VSG) of the parasite represents a major interaction between the mammalian host immune system and the parasite surface. Since immune recognition of other parasite derived factors also occurs, we examined the humoral host response against trypanosome heat shock protein 60 (HSP60), a conserved antigen with an autoimmune character. During experimental T. brucei infection in BALB/c mice, the anti-HSP60 response was induced when parasites differentiated into stumpy forms. This response was characterized by a stage-specific immunoglobulin isotype switching as well as by the induction of an autoimmune response. Specific recognition of trypanosome HSP60 was found to occur during the entire course of infection. Immunoglobulin G2a (IgG2a) and IgG2b antibodies, induced mainly in a T-cell-independent manner, were observed during the first peak of parasitemia, whereas IgG1 and IgG3 antibodies were found at the end of the infection, due to a specific T-cell-mediated response. Comparative analysis of the kinetics of anti-HSP60, anti-invariant surface glycoprotein 70 (ISG70), and anti-VSG antibody responses indicated that the three trypanosome antigens give rise to specific and independent patterns of immunoglobulin isotype switching.     

Depletion of complement has distinct effects on the primary and secondary antibody responses to a conjugate of pneumococcal serotype 14 capsular polysaccharide and a T-cell-dependent protein carrier

Complement activation plays a critical role in the immune response to T-cell-dependent and T-cell-independent antigens. However, the effect of conjugation of T-cell-dependent protein carriers to T-cell-independent type 2 antigens on the requirement for complement in the humoral immune response to such antigens remains unknown. We studied the role of complement activation on the antibody response of BALB/c mice immunized with the T-cell-independent type 2 antigen serotype 14 pneumococcal capsular polysaccharide (PPS14), either in unmodified form or conjugated to ovalbumin (OVA). In mice immunized with either PPS14 or PPS14-OVA, depletion of endogenous complement at the time of primary immunization by treatment with cobra venom factor (CVF) diminished serum anti-PPS14 concentrations after primary immunization but enhanced antibody responses after secondary immunization. The secondary immunoglobulin G (IgG) anti-PPS14 antibody response after immunization with PPS14-OVA was especially enhanced by complement depletion, was observed at doses as low as 0.2 mug of antigen, and was maximal when CVF was administered within 2 days of immunization. The avidity and opsonophagocytic functions of IgG anti-PPS14 antibodies were comparable in mice immunized with PPS14-OVA with or without complement depletion. Serum anti-PPS14 antibody concentrations were near normal, and the enhancing effects of CVF treatment on the secondary anti-PPS14 antibody response were also apparent in splenectomized mice immunized with PPS14-OVA. These results demonstrate that complement activation can have distinct effects on the primary and secondary antibody responses to a T-cell-independent type 2 antigen, either unmodified or conjugated to a T-cell-dependent protein carrier. These differences should be taken into consideration when using complement to modulate the immune response to vaccines.     

Marginal zone B-cell depletion impairs murine host defense against Borrelia burgdorferi infection

Marginal zone B (MZB) cells are a B-cell subset that produces T-cell-independent antibodies to blood-borne antigens. In this study, we examined the effects of MZB cell depletion on the immune response to the Lyme disease spirochete Borrelia burgdorferi, an extracellular pathogen for which T-cell-independent antibody is an important host defense. MZB cell depletion of C3H/HeJ mice using monoclonal antibody to LFA-1 and alpha(4)beta(1) integrins reduced B. burgdorferi-specific immunoglobulin M (IgM) titers, enhanced pathogen burden, and led to more severe arthritis assessed within the first 2 weeks of infection. In addition, MZB cell-depleted mice had reduced levels of B. burgdorferi-specific IgG, which correlated with diminished splenic CD4+ T-cell-activation, proliferation, and cytokine production. Passive transfer of immune mouse serum from infected control mice into infected MZB cell-depleted mice reduced pathogen burden but did not alter the expression of T-cell activation markers on splenic CD4+ T cells. These findings demonstrate that MZB cells not only are a source of pathogen-specific IgM important for limiting spirochete burden and pathology but also play a prominent role in the priming of splenic T-cell responses to a blood-borne pathogen.     

Differential susceptibilities of mice genomically deleted of CD4 and CD8 to infections with Trypanosoma cruzi or Trypanosoma brucei.

The role of CD4+ and CD8+ T cells in the surveillance of Trypanosoma cruzi or Trypanosoma brucei brucei was studied in mice which lacked CD4 or CD8 molecules and which were generated by embryonic stem cell technology. Whereas wild-type mice infected with T. cruzi (Tulahuén strain) displayed low levels of parasitemia and no mortality, striking increases in parasite growth and mortality occurred in both CD8- and CD4- mice. On the contrary, CD8- and, to a lesser degree, CD4- mice showed enhanced resistance to T. b. brucei. T-cell-dependent immunoglobulin G-specific responses were produced in CD8- but not CD4- mice. Normal T-cell proliferative responses were measured in both CD4- and CD8- mice. Interleukin-4 production after concanavalin A or anti-CD3 monoclonal antibody stimulation was strikingly enhanced in CD8- but not CD4- spleen cells, whereas gamma interferon production was normal in both CD4- and CD8- spleen cells. Spleen and lymph node cells from CD8- (but not CD4-) mice at 20 days postinfection with T. cruzi had higher levels of interleukin-4 mRNA than the wild-type controls, as shown in a competitive polymerase chain reaction assay. On the other hand, CD4- (but not CD8-) mice at 20 days postinfection with T. cruzi had lower levels of gamma interferon mRNA than the wild-type mice.