Variable Antigen Types Research Articles

The membrane form of variant surface glycoproteins of Trypanosoma brucei.

African trypanosomes are parasitic protozoa which are enveloped by a surface coat consisting of a matrix of identical glycoprotein molecules. Variations in the composition of these variant surface glycoproteins (VSGs) allow the parasite to escape the host's immune system and render effective immunoprophylaxis improbable. However, underlying the surface coat, all variant antigen types contain common membrane components, some of which can activate complement by the alternative pathway, leading to lysis of uncoated trypanosomes. Hence, stimulation of VSG release in vivo should be a potential form of chemotherapy, and we have therefore investigated the mode of attachment of VSG to the plasma membrane. Biochemical characterization of VSGs from several species has been performed on material purified after release from the cell surface following rupture of the trypanosome. We demonstrate here that VSGs of Trypanosoma brucei when bound to the membrane exist in a form which differs both biochemically and immunochemically from VSGs purified in the conventional manner. After rupture of the cell, membrane-form VSG (mfVSG) is enzymatically transformed into the commonly isolated water-soluble released form (sVSG). In conditions in which this modification does not take place, purified VSGs have amphiphilic properties and behave as integral membrane proteins by the criterion of charge-shift electrophoresis. The difference between the two forms lies in the C-terminal domain, which is phosphorylated in both forms. This domain in sVSGs contains an immunogenic oligosaccharide known as the cross-reacting determinant (CRD), attached to the C-terminal amino acid. Recognition of this determinant by anti-CRD antibodies is impaired in the membrane form.

Lymphocyte function in experimental African trypanosomiasis. V. Role of antibody and the mononuclear phagocyte system in variant-specific immunity.

The role of parasite-specific antibody and the mononuclear phagocyte system (MPS) in immunity to the African trypanosomes was examined. For this study C57BL/10SnJ mice were infected with Trypanosoma rhodesiense clone LouTat 1.0. Infected mice were injected with 75Se-labeled LouTat 1.0 trypanosomes, and clearance from the blood upon reexposure was measured throughout the course of infection. Clearance of labeled organisms occurred only on or after day 5, which was the day of natural elimination of LouTat 1.0 from the blood. Clearance was dependent on a functional immune system and correlated with the appearance of antibody to the variant-specific surface antigen (VSSA) of the trypanosomes. The ability to clear trypanosomes was transferred to normal, uninfected mice by immune serum. Both the IgM and IgG fractions of immune serum mediated the clearance, and VSSA-specific IgM fractions were as efficient in clearing LouTat 1.0 as the IgG fractions. Normal levels of complement (C3) were not required for clearance. The liver was the primary organ of clearance, and the ability of the liver to sequester radiolabeled trypanosomes was not impaired in the terminal phase of the disease or by large numbers of circulating trypanosomes present representing different variant antigenic types (VAT). We conclude that in African trypanosomiasis the MPS is not depressed in its ability to clear trypanosomes of the infecting VAT at any time during the course of infection. The observed clearance function requires parasite-specific antibody but normal levels of C3.

Comparative analysis of Trypanosoma brucei gambiense antigen gene family and its potential use in epidemiology of sleeping sickness

The identification of African trypanosomes infective for man and the characterization of their repertoire of variable antigen types (VATs) is a major challenge to epidemiologists. The present work, based on previous studies, brings evidence that DNA analysis may provide a molecular approach to these problems. Two cloned cDNAs coding for Trypanosoma brucei brucei variant-specific antigens have been used as probes to evaluate, by hybridization on DNA blots, the relatedness of different trypanosome clone stocks, mainly of T. b. gambiense. It appears that, in all T. b. gambiense stocks, the AnTat 1.8-like gene family produces a typical pattern of bands which is discussed in terms of gene evolution and of possible use as a means to discriminate T. b. gambiense from the other sub-species. A deviation from this common pattern concerns the basic copy of the gene, the presence or absence of which is correlated with the ability of the stocks to express AnTat 1.8 isotypes: this observation suggests that only one member of the AnTat 1.8 DNA sequence family might be involved in the expression of the AnTat 1.8 isotype, and that useful information on serodemes and their VAT repertoires could be obtained from DNA analysis. Among the silent sequences of this family, one appears remarkably similar to the basic copy. Its apparent inability to be expressed could be linked to alterations of the repetitive sequences flanking the transposable element. This observation supports the hypothesis that these repeats are involved in the transposition leading to expression of the gene.

Genomic organization of variant surface glycoprotein genes in Trypanosoma brucei procyclic culture forms.

The production of the variant surface glycoprotein coat of bloodstream form African trypanosomes ceases after conversion to the procyclic form. In the bloodstream stage alternate expression of different variant surface glycoprotein genes is responsible for the antigenic variation that occurs during relapse infections in the mammalian host. We have examined procyclic stage populations, derived from different bloodstream variant antigen types, for the two types of genomic alterations associated with variant surface glycoprotein genes in the bloodstream stage. Transcriptional activation of some variant antigen genes is accompanied by the generation of a new copy of the gene, the expression-linked copy. We find that the expression-linked copy is maintained after conversion to procyclic form, indicating that the presence of an expression-linked copy is not sufficient for the expression of a surface coat. Sequences 3' to other variant surface glycoprotein genes show expression-independent variation in bloodstream stage trypanosomes. The same genes showed variation between procyclic populations of different origin, and between procyclics and their bloodstream parent. These data are discussed in light of observations on the sequence of variant antigen expression after cyclic transmission.

Immunization of mice against African trypanosomiasis using anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised against three protective monoclonal antibodies, each with specificity for the variable antigen type (VAT) of a clone of Trypanosoma rhodesiense. The IgG1 fractions of each were pooled and administered to BALB/c mice 3-4 wk before homologous challenge. The course of primary parasitemia was altered in 19 of 30 anti-Id-treated animals. The immunity was manifested as either: (a) complete protection, (b) reduced parasitemia, or (c) selection against parasites bearing the original VAT. The three idiotypes (Id) were found in variable levels in serum during the course of infection in control animals. However, in all anti-Id-treated mice that displayed immunity, one Id in particular (7H11) was detectable much earlier in infection and in higher levels than in control mice or anti-Id-treated, nonimmune mice. Six of nine mice treated with the anti-7H11 Id alone also displayed immunity, manifested in this case exclusively as selection against parasites bearing the original VAT. The effect was again associated with the more rapid appearance of the Id after infection. Specificity of the anti-Id-induced immunity was supported by the failure of anti-7H11 Id treatment to alter the course of infection with a heterologous clone of T. rhodesiense. To our knowledge, this is the first report of the antigen-independent induction of antimicrobial immunity using anti-Id antibodies.

Biochemistry of the Variant Surface Glycoproteins of Salivarian Trypanosomes

This chapter describes the progress made over the past years in characterizing antigens of the principal pathogenic salivarian trypanosomes. Although bulk of information comes from work on the variant antigens of T. b. brucei in particular, much interesting work has been done on the variant antigens of other species and is discussed in the chapter. Future perspectives for immunoprophylaxis, serodiagnosis, or chemotherapy of trypanosome infections are also presented. The mammalian trypanosomes have been divided into seven subgenera that are grouped into two sections based on the site of the production of infective metacyclic trypanosomes in the invertebrate host. The subgenera are Trypanosoma brucei brucei , Trypanosoma brucei gambiense , Trypanosoma brucei rhodesiense , Trypanosoma evansi , Trypanosoma equiperdum Trypanosoma ( Nannomonas ) congolense , and Trypanosoma ( Duttonella ) vivax . The chapter presents the systematic position of the genus Trypanosoma and the seven subgenera that infect mammals and summarizes the main features of each subgenus. Antigenic variation in the mammalian host is the most striking characteristic of salivarian trypanosomes. Variant surface glycoproteins (VSGs) are synthesized with a hydrophobic C-terminus absent from purified VSG. Little work has been done on the biochemistry of antigens expressed by metacyclic trypanosomes of any species, because the amount of material available is limited. There is interest in the antigenic composition of metacyclic trypanosomes because if the repertoire is restricted, vaccination could be a possibility. The serological evidence suggests that the repertoire of metacyclic variant antigen types (VATS) is restricted, and there is limited overlap between VATS present in metacyclic and in bloodstream populations.

Host Susceptibility to African Trypanosomiasis: Trypanotolerance

Publisher Summary The exploitation of genetic resistance to disease is an important consideration in livestock development programs, where conventional disease control measures are not effective or are too costly. Such an approach may be directly applicable to African animal trypanosomiasis. In African trypanosomiasis, the control measures currently in use include diagnosis and treatment, chemoprophylaxis, and control or eradication of tsetse with insecticides. At present, there is no effective field vaccine available against African trypanosomiasis. The major constraints to the development of a vaccine include the existence of the different species of trypanosomes and of different serodemes within the same species, all with the capability of producing different repertoires of variable antigen types (VATS). This chapter discusses the available information on the aspects of trypanotolerance, with regard to cattle, sheep, goats, wildlife, and man. It describes experimental results derived from mouse models and the relevance of these models to trypanosomiasis of livestock. The genetic resistance to trypanosomiasis is not necessarily associated with low productivity. Trypanotolerance is not a stable characteristic, and evidence suggests that it can be supplemented or reduced by a number of factors affecting the host and its environment, which include age and sex; maternally-derived immunity, intensity of challenge, virulence, previous exposure, stress, and susceptibility to other diseases. Investigations of trypanotolerance also represent an exciting approach to the important interactions between host and parasite. Trypanotolerance exists as an innate characteristic and that it is probably inherited as a dominant trait. While the level of trypanotolerance can be reduced under certain circumstances, it can also be enhanced, for example, by previous exposure. Therefore, there is hope that it may be possible in the future to supplement the level of trypanotolerance both by genetic selection and by immunological or therapeutic procedures.

Cyclical transmission of in vitro cultivated bloodstream forms and procyclic trypomastigotes of trypanosoma brucei brucei by Glossina morsitans morsitans.

In vitro cultivated bloodstream and procyclic forms of Trypanosoma b. brucei STIB 247 were cyclically transmitted by Glossina m. morsitans. The tsetse flies were infected artificially on a silicon membrane. Metacyclic trypanosomes from mature salivary gland infections were used to initiate bloodstream form cultures. They transformed into slender bloodstream forms and gave rise to established cultures that proved to be infective for the vector. The metacyclic forms retained the strain-specific basic set of variable antigen types.

Antigenic variation in cyclically transmittedTrypanosoma brucei. Variable antigen type composition of the first parasitaemia in mice bitten by trypanosome-infectedGlossina morsitans

SUMMARYTsetse flies were infected with 5 different variable antigen types (VATs) or with a mixture of VATs of the AnTAR 1 serodeme ofTrypanosoma brucei. Metacyclic forms from the salivary glands of infected flies were used to initiate infections in mice. Immunofluorescence and trypanolysis reactions employing 24 monospecific antisera were used to analyse the VATs present in the mice following cyclical transmission. Regardless of the VAT used to infect tsetse flies, the first VATs detectable in the bloodstream were those previously identified as metacyclic VATs (M-VATs). These were present until at least 5 days after infection, at which time lytic antibodies against at least 2 of the M-VATs were detectable in the blood of infected mice. In mice immunosuppressed by X-irradiation the M-VATs were detectable in the bloodstream for longer periods, but the percentage of the population labelled with anti-metacyclic sera showed a decrease on day 5 as in non-irradiated animals. The VAT ingested by the tsetse was always detectable early during the first parasitaemia following cyclical transmission and was usually the first VAT detected after the M-VATs. Neutralization of selected M-VATs before infecting mice resulted in elimination of the neutralized M-VAT from the first parasitaemia but had no effect on the expression of other VATs in the early infection.

Antigenic variation in cyclically transmittedTrypanosoma brucei. Variable antigen type composition of metacyclic trypanosome populations from the salivary glands ofGlossina morsitans

SUMMARYTsetse flies (Glossina morsitans) were fed on the blood of mice containing any one of 5 variable antigen types (VATs) ofTrypanosoma bruceiAnTAR 1 serodeme. The VATs of the metacyclic trypanosomes subsequently detected in the flies' saliva probes were investigated using monospecific antisera to AnTAR 1 VATs in indirect immunofluorescence and trypanolysis reactions; these sera included 3 raised against AnTats 1.6, 1.30 and 1.45, previously identified as components of the metacyclic population (M-VATs), and against the 5 VATs originally ingested by the flies. The percentage of metacyclics reacting with a particular M-VAT antiserum remained more or less constant (AnTat 1.6, 6·0–8·3%; AnTat 1.30, 13·7–18·2%; AnTat 1.45, 2·0–8·0%), regardless of the age of the fly or the ingested VAT. As these 3 VATs accounted for no more than 30% of the metacyclic population, the existence of at least one more VAT is envisaged. The ingested VAT could not be detected among the AnTAR 1 metacyclic trypanosomes.

The expression-linked copy of a surface antigen gene in Trypanosoma is probably the one transcribed.

The antigenic specificity of the living trypanosome seems to be determined by the protein component of a unique glycoprotein species covering the whole surface of the parasite. During chronic infection, a single clone of trypanosomes may successively express a large repertoire of different variable antigen types (VATs). There are probably as many genes as variant-specific antigens (VSAs) (see refs 1-3 for reviews). The expression of the genes coding for the synthesis of these antigens is linked to genomic rearrangements involving duplication of the coding sequence and transposition of the additional copy. The regulation of the expression of the VSA genes is operated at the transcriptional level. It can thus be supposed that their transcription depends on the presence of the additional, transposed copy. We report here that this additional copy is in a chromatin configuration highly sensitive to pancreatic deoxyribonuclease, suggesting that it is the transcribed one.

Variation in the sensitivity of successive variable antigen types in a Trypanosoma (Trypanozoon) brucei subspecies clone to some African game animal sera.

A Trypanosoma brucei subspecies clone was passaged in rats at ten-day intervals and the sensitivity/resistance to a variety of mammalian sera, of the successive variable antigen types (VATs) produced, was examined sequentially in a modified version of the blood incubation infectivity test (BIIT). A proven homogeneous VAT was used to initiate this series of tests, in which seven successive and different VATs were each exposed in vitro at 38.5 degrees C for 2 h to standard samples of pooled rat serum (PRS), normal human serum (NHS) and to sera from two different eland and three different hippopotami. Samples were then inoculated into susceptible rats to determine the effects of the individual sera on the subsequent infectivity of the trypanosomes. The seven VATs were found to vary widely in their sensitivity to the different game sera, though all remained strongly resistant to the pooled rat and the normal human serum samples. BII testing of isolates from positive test rats in the sequential study showed that their resistance to normal human serum was unaffected by their exposure in vitro to the different game sera.

Trypanosoma vivax: Host influence on appearance of variable antigen types

The infectivity of a single stabilate of Trypanosoma vivax Zaria Y486 differed in mice, rats, and cattle. The variable antigen types present in the first bloodstream population of mice and cattle also differed. In rats infected with different isolates of this stock, the same VATs always appeared in the first relapse populations despite antigenic differences in the isolates. The infectivity of certain variable antigen types of T. vivax Zaria Y486 for rodents could be enhanced by either preincubation of the parasites in ruminant serum or simultaneous supplementation of the rodents with ruminant serum. Incubation of variable antigen types, which did not usually infect rats, in rat serum, did not subsequently alter the infectivity of such variable antigen types of T. vivax for mice.

Bovine immune response to African ;trypanosomes: specific antibodies to variable surface glycoproteins of Trypanosoma brucei.

Cattle were infected with two different clones of Trypanosoma brucei (MITat 1.2 and ILTat 1.3) and antibody response was followed by radioimmunoassay. In four of the seven animals there were at least two peaks of antibody activity to the infecting clones, with the second peak much higher than the first. Specific antibodies (IgG1 and IgM but not IgG2) were eluted from the immunoabsorbent columns on which the variant surface glycoproteins (VSGs) were coupled. By neutralization of infectivity tests, IgM antibodies from the first peak of antibody activity were more efficient in neutralizing trypanosomes than IgG1 but the reverse was true of the antibodies isolated from the second peak. By absorption with multiple variable antigen types isolated during the course of infection, all the IgM and IgG1 in the first 3 weeks of infection were shown to be specific. It is suggested that polyclonal B cell stimulation leading to dysfunction in the control of IgM and IgG production may not be responsible for the high levels of these immunoglobulins in bovine trypanosomiasis.

Use of monoclonal antibody to immunochemically characterize variant-specific surface coat glycoprotein from Trypanosoma rhodesiense.

Monoclonal antibodies were employed to study the molecular basis for charge heterogeneity in variant-specific surface coat glycoprotein prepared from clone CP3B4 of the Wellcome strain of Trypanosoma rhodesiense. Thirteen hybridomas secreting monoclonal antibodies specific for CP3B4 were obtained by fusing murine plasmacytoma cells to spleen cells from mice immunized with purified surface coat glycoprotein. The clone population of CP3B4 trypanosomes was shown to be homogeneous by means of immunofluorescent assays using culture supernatants from each of the 13 hybridomas. No cross-reactivity was found with other variant antigenic types of the same serodeme. Ascitic fluids were generated from 4 of the hybridomas and th molecular and epitopic specificities of the fluids or their IgG fractions were determined isoelectrofocusing of immunoprecipitates of radioiodinated glycoprotein antigen followed by autoradiography revealed that all 3 major components of the charge heterogeneous CP3B4 surface-coat glycoprotein were immunoprecipitated by each of the 4 monoclonal IgG fractions. Immunofluorescent staining of live trypanosomes was obtained with only one of the 4 ascitic fluids. The results show that charge heterogeneity does not derive from a heterogeneous population of parasites. Furthermore, the data indicate that there are at least 2 different epitopic specificities exhibited by the monoclonal antibodies tested and that each of the 3 charge heterogeneous components of the surface coat glycoprotein contains these epitopes. Charge heterogeneity of CP3B4 surface coat glycoprotein may be attributed to post-translational modification or to limited proteolysis.

An accidental laboratory infection with trypanosomes of a defined stock II. Studies on the serological response of the patient and the identity of the infecting organism

Summary We describe the serological investigation of a student who developed acute trypanosomiasis whilst working with the ETat variable antigen types of a stock of Trypanosoma brucei ssp which was thought to be non-infective for man. The patient exhibited the extreme elevation of IgM typical in this disease but no serological evidence was obtained to show that this represented anything but a specific response to antigens of the infecting organism. The IgM deteriorated rapidly during storage. Immunoglobulin E was also considerably increased above normal. Serological evidence was obtained that the infection was initiated by variable antigen type ETat 10, which organism appears to be the only one from the ETat serodeme that, when grown in rodents, is infective for man.

An accidental laboratory infection with African trypanosomes of a defined stock I. The clinical course of the infection

Summary The case history and successful treatment of a patient accidentally infected with Trypanosoma brucei rhodesiense in a Scottish laboratory are reported. As the experimental use of trypanosomes is increasing it seems probable that other accidents may occur in the laboratory. Preparations that might be made in anticipation of these are suggested. The accidental infection provided the opportunity for an investigation which led to the discovery that only one of the variable antigen types derived from this stock was infective for man after passage in rodents. This valuable piece of epidemiological information might not have been discovered in any other way. The aetiologically important variable antigen type involved is one of a group or ‘serodeme' which has been accurately defined immunologically and it is likely that this is the first case of an accidental infection with trypanosomes from a well defined stock, although this serodeme and others are held in many laboratories.

Antigenic variation in clones of animal-infective Trypanosoma brucei derived and maintained in vitro.

Eighteen clones of variable antigen type 052 of Trypanosoma brucei stock S.427 were derived and maintained as animal-infective bloodstream forms in vitro for up to 60 days of cultivation. The antigenic composition of such clones was monitored weekly by immunofluorescent analysis of viable trypanosomes, using antisera raised to isolated variant-specific surface glycoproteins of both 052 and variable antigen type (221) which consistently appeared in the first relapse population of type 052 in vitro. The appearance of new variants was detected in 9 of the 18 clones 18--46 days following initiation of the clone and variable antigen type 221 was found in all 9 clones. On one or more occasions in 8 of such clones, viable trypanosomes were found which did not react with either antiserum but were mouse-infective on the 4 occasions tested and probably represent other variable antigen types. The process of antigen variation in vitro appears to resemble the process in vivo except that new variant types are detected earlier in vivo. This possibly results from different growth rates of the trypanosomes in vivo and in vitro, together with the fact that elimination of the initial variant population by the host's immune response facilitates the detection of newly arising variable antigen types in vivo.

In vitro cloning of animal-infective bloodstream forms of Trypanosoma brucei.

Clones of animal-infective bloodstream forms of Trypanosoma brucei (stocks S.427 and LUMP 227) were made by transferring a single organism from bloodstream-form cultures into each well of Microtest II Tissue Culture Plates containing bovine fibroblast-like feeder cells. When the number of trypanosomes increased to 10(2)--10(3)/well on days 4--16, they were transferred into plastic T-25 culture flasks also containing feeder cells and fresh medium. Cultures were thereafter maintained by partially replacing the trypanosome suspension with the same volume of fresh medium (diluting the density to 2--5 x 15(5) trypanosomes/ml) every 24 h. Sub-cultivations could be made by transferring 1--2 ml of the trypanosome suspension to a new culture flask at 4--5 day intervals. A total of 42 clones in the 3 series TC221, TC52 and TC227, carrying variable antigen types (VATs) 221, 052 and ILTat 1.4, respectively, have been established. Average population doubling times for clones of TC221, TC52 and TC227 were 8.7, 14.5 and 15.5 h respectively. Of 35 populations examined, 34 clones retained the original specificity of their VATs for at least 8--32 days from cloning. One cloned population of TC52 consisted of 99.8% VAT 052 and 0.2% VAT 221 at the time when the first VAT test was made on day 18.

Antigenic changes in Trypanosoma brucei on transmission by tsetse fly

Summary Antigenic variation has been demonstrated throughout a chronic infection of Trypanosoma brucei (derived from TREU 667) in the mouse. The variant antigenic types (VATs) detected were compared with those appearing on transmission from various time points through the infection. Reversion of VAT was found to occur on transmission through Glossina regardless of the VAT(s) present at the time of ingestion. The first patent parasitaemia after transmission is heterogeneous with regard to VAT; three predominant VATs appearing consistently. Indirect evidence suggests that this VAT heterogeneity is most likely not reflected in the metacyclic population but comes about immediately on transmission and can occur in the absence of antibody.