Trypanosomes expressing a mosaic variant surface glycoprotein coat escape early detection by the immune system

Abstract

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.

FIG. 1.

pXS5 expression vector for ectopic VSG gene expression from the ribosomal locus of trypanosomes. From 5′ to 3′, the vector contains a nontranscribed targeting sequence from the 3′ end of a ribosomal repeat (Ribo 3′); the rRNA promoter (Ribo Prom); the 3′ end of the procyclic acidic repetitive protein (PARP) B2α splice acceptor site; the 117 VSG gene inserted into the multiple cloning site; the aldolase intergenic region (Aldo Int), containing a polyadenylation site and a 3′ splice acceptor site; the neomycin phosphotransferase gene (neo^r); and the tubulin αβ intergenic region (Tub αβ Int), also containing polyadenylation and slice acceptor sites. Linearization of the vector with XhoI targets the construct to replace a ribosomal repeat. Expression of neomycin phosphotransferase results in resistance to the neomycin analog G418 to allow for selection of transformants in vivo or in vitro. Asterisk, splice acceptor site; A_n, polyadenylation site.

FIG. 2.

Amplification of LouTat 1 and 117 VSG mRNAs from LT1:117 VSG double expressers. LT1:117 total RNA was isolated using the Ultraspec-II RNA isolation system. The T-lymphocyte-triggering factor LouTat 1 VSG, and 117 VSG genes were amplified using gene-specific primers. A reaction control was provided with the Access RT-PCR core kit (Promega, Madison, WI). Appropriate negative controls were also run (not shown), and trypanosomes expressing monotypic coats expressed only the relevant VSG gene transcripts (not shown).

FIG. 3.

LouTat 1 and LT1:117 trypanosomes exhibit similar infectivity and growth patterns. C57BL/6 wt (left) and C57BL/6 scid (right) mice were infected with 10⁵ viable trypanosomes and the early parasitemia profiles monitored. The data presented are from individual mouse infections but are representative of parasitemia profiles for 10 mice infected with each strain. Parasitemia scores: 4 = >100 trypanosomes per 400× field; 1 = 1 to 5 trypanosomes per 10 400× microscope fields. Symbols: ▴, LouTat 1; ▾, LT1:117.

FIG. 4.

LouTat 1 VSG and 117 VSG are stably coexpressed for up to 1 week by LT1:117 VSG gene double expressers in the absence of immune selection. C57BL/6 scid mice were infected with 10⁵ LouTat 1 or LT1:117 trypanosomes in the presence or absence of G418 drug selection. VSG expression was monitored on trypanosomes by immunofluorescence staining with LouTat 1 VSG- and 117 VSG-specific antibodies at days 7 and 14 postinfection.

FIG. 5.

LouTat 1 VSG and 117 VSG colocalize to the endoplasmic reticulum, flagellar pocket, and cell surface in LT1:117 VSG gene double expressers. G418-selected trypanosomes were fixed with 1% formaldehyde/0.05% glutaraldehyde and attached to slides by acetone/methanol fixation. Slides were stained with a mouse anti-LouTat 1 VSG monoclonal antibody and a rabbit anti-117 polyclonal serum. Positive cells were detected using Alexa 633-conjugated anti-mouse IgM (red) and Alexa 488-conjugated anti-rabbit IgM (green) and viewed by fluorescent microscopy. Immunostaining: (A) mouse anti-LouTat 1 VSG; (B) rabbit anti-117 VSG; (C) merged image; (D) differential interference contrast with DAPI counterstain. Control stains demonstrated that these antibodies do not cross-react with the heterologous VSG in this assay. Images were pseudocolored and merged using OpenLabs 3.0 software (Improvision, Inc., Lexington, MA).

FIG. 6.

Similar amounts of LouTat 1 VSG and 117 VSG are present in LT1:117 VSG double-expresser trypanosomes. Trypanosomes were metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine in Met/Cys-depleted medium for 4 h. Cells were lysed, and the lysates were incubated with rabbit anti-LouTat 1 VSG or anti-117 VSG antibodies coupled to Sepharose A beads. Equivalent amounts of immunoprecipitated material were separated by SDS-PAGE and the bands detected by autoradiography. Band density was measured using GeneTools imaging software (Hitachi Genetic Systems; Hitachi Software Engineering Co., Ltd.). Scan pixel densities for precipitated LouTat 1 and 117 VSGs in autoradiograms were 1,422,050 and 1,036,078, respectively.

FIG. 7.

VSG-specific B-cell responses are depressed during infection with VSG double expressers relative to those for infection with the wt strain. C57BL/6 nu/nu mice were infected with 10⁵ trypanosomes expressing either a homogeneous LouTat 1 VSG coat or the mosaic LT1:117 VSG coat. (Top) Serum was collected on the days indicated and analyzed by VSG-specific ELISAs using LouTat 1 VSG (left) or 117 VSG (right) as the capture molecule. Data are presented as relative change in optical density (OD) ± standard deviation from five separate experiments over an 8-month period. (Bottom) ELISPOT assays were performed with spleen cells from infected mice. Data are presented as mean number of antibody-forming cells (AFC)/10⁵ spleen cells from a single experiment. Symbols: ▴, LouTat 1-infected mice; ▾, LT1:117-infected mice; ○, uninfected mice.