Metacyclic Variable Antigen Types Research Articles

Early stages of infection with Trypanosoma congolense: Parasite kinetics and expression of metacyclic variable antigen types

Trypanosoma congolense develops in the skin of sheep at the site of inoculation of metacyclic trypanosomes, forming a chancre containing large numbers of parasites. By cannulating the afferent and efferent lymphatic ducts draining the skin and regional lymph node, the progressive development and migration of trypanosomes from the chancre was monitored and the expression of metacyclic antigen types (M-VATs) was determined. The kinetics of development of parasitosis in the afferent and efferent lymph was similar. Trypanosomes were detected in lymph 5 to 6 days after the inoculation of cultured metacyclic trypanosomes, at the same time as the chancre first appeared in the skin. The numbers of trypanosomes in the lymph reached their peak levels 8 to 10 days post infection and thereafter numbers fell, although there were still many parasites in the lymph after the chancre had regressed. Trypanosomes in the afferent lymph expressed mainly M-VATs and the absolute numbers of four M-VATs which were monitored increased up to 9 days post infection. There was a fall in numbers by day 10, but 92% of the trypanosomes in the afferent lymph continued to express M-VATs. In contrast, trypanosomes from the efferent lymph were found not to express M-VATs suggesting that a major switch in VAT expression occurs in the lymph node. Specific antibody responses, measured by neutralization tests, were evident 16 to 20 days after infection in afferent lymph but only low levels of antibodies were found in efferent lymph.

Trypanosoma congolense: Re-expression of a deleted metacyclic variable antigen type in vivo and in vitro

The expression of variable antigen types (VATs) was determined among dividing populations of T. congolense growing in vivo in rabbit chancres and in vitro on bovine aorta endothelial cell monolayers. Experiments were performed in which a single metacyclic VAT (M-VAT) was deleted from a cultured metacyclic population by neutralisation with a monoclonal antibody and complement. Subsequent expression of the deleted M-VAT and two unrelated M-VATs was determined by an indirect immunofluorescent antibody test. The deleted M-VAT was re-expressed both in vivo and in vitro and the proportions of unrelated M-VATs were not markedly affected by the neutralisation of this single M-VAT. In addition, an overall similarity was observed between M-VAT expression and re-expression in vivo and in vitro.

Characterization ofTrypanosoma congolenseserodemes in stocks isolated from Chipata District, Zambia

Six stocks of Trypanosoma congolense were cloned from 17 stocks isolated from Eastern Zambia and used to initiate insect-form in vitro cultures producing metacyclic trypanosomes. Serological assays were then developed using these in vitro-derived metacyclics as a reference collection of antigens. Monoclonal antibodies recognized 8 metacyclic variable antigen types (M-VATs) of one stock, T. congolense TREU 1885, representing 70-80% of that stock's M-VAT repertoire, and in an indirect fluorescent antibody test (IFAT) there were no cross-reactions between them and the metacyclic trypanosomes of the other 5 stocks. Cross-protection assays between the 6 stocks in mice showed that the stocks cultured in vitro were serologically distinct. In order to facilitate serological typing for serodeme characterization, an IFAT was developed using formalin-fixed metacyclic trypanosomes to identify VAT specific immune responses using 21 day post-infection antisera. The cultured stocks reacted only with their homologous antisera thus confirming the results obtained in the cross-protection assays. No cross-reactions were observed with the 6 cloned stocks and antisera against the 11 stocks of T. congolense isolated in the same area at the same time suggesting that these stocks were different from the reference collection of cultured metacyclics. Hence, at least 7 serodemes of T. congolense have been identified from the 17 stocks isolated.

Stability of metacyclic variable antigen types (M-VATs) during the early stages of infection with Trypanosoma congolense

Expression of nine metacyclic variable antigen types (M-VATs) of Trypanosoma congolense in chancres from infected rabbits was determined using monoclonal antibodies raised against metacyclic forms of trypanosomes. Trypanosomes present in chancres 7–9 days post infection expressed M-VATs present in metacyclic populations of the parasites. The majority of M-VATs expressed showed little proportional change from those observed on metacyclic trypanosomes during this period although expression of one M-VAT icnreased, and another decreased. Although trypanosomes in chancres continued to express M-VATs, other VATs, not present in the M-VAT repertoire were also expressed and neutralization tests showed that new VATs appeared by 7 days after infection. In infected sheep neutralizing antibodies against M-VATs were detected by day 14 in lymph from efferent lymphatics draining lymph nodes in the region of chancres. Neutralizing antibodies directed against metacyclics were also present in the serum by day 14 and were still detectable for up to 35 days post infection. Hence, it is likely that in the vertebrate host the trypanosomes multiplying in the skin at the site of tsetse bite express all M-VATs characteristic of that particular serodeme, enabling the host to develop immunity to all antigen types present in the M-VAT repertoire.

Trypanosoma congolense: Host responses following tsetse transmitted infection of Kilifi isolates in goats

East African x Galla goats, when infected with Trypanosoma congolense isolates from the Kilifi area of Kenya by Glossina morsitans centralis, did not develop the characteristic chancre reaction at the bite sites, whereas bites of tsetse infected with the cloned T. congolense IL.1180 from Serengeti, Tanzania, resulted in chancres in the same goats. Histological changes could not be observed in skin biopsies collected 8 or 9 days after infection with Kilifi isolates. However, all goats became parasitemic about 10 days after challenge. It is concluded that the absence of chancre development is a characteristic feature of T. congolense parasites from Kilifi. The isoenzyme analysis of clones of two T. congolense Kilifi isolates and the T. congolense clone IL.1180 indicated that they belong to different zymodemes. Neutralizing antibodies to homologous metacyclic variable antigen types were detected in six out of seven (86%) of the sera from goats infected with a clone or stock of a T. congolense Kilifi isolate, 20 days after infection. Goats primed by tsetse transmitted infection with a stock or clone of T. congolense from Kilifi and treated with Berenil were, in three out of eight cases (37%), not immune to homologous challenge. It is suggested that the reduced immune response to metacyclic variable antigen types could be a result of the absence of cellular infiltration, i.e., chancre development in the skin at the tsetse bite site. It is concluded that the use of the chancre reaction as a marker for serodeme analysis of recently isolated stocks of T. congolense from Kilifi was not feasible.

Onset of expression of the variant surface glycoproteins of Trypanosoma brucei in the tsetse fly studied using immunoelectron microscopy.

The acquisition of the variant surface glycoprotein (variable antigen) coat by metacyclic stage Trypanosoma brucei in the salivary glands of the tsetse fly, Glossina morsitans, has been studied in situ by transmission and scanning electron microscopy using monoclonal antibodies raised against metacyclic variable antigen types and complexed with horseradish peroxidase or colloidal gold. The coat is acquired after binary fission has ceased but while the parasite is still attached to the gland epithelium, i.e. before the mature metacyclic is released into the gland lumen. The variable antigen type heterogeneity previously observed in discharged mature metacyclics is here demonstrated in the nascent (attached) metacyclic population. The variant surface glycoprotein genes are thus not expressed in a fixed sequence since different metacyclic variable antigen types are present ab initio. The distribution of immunogold-marked nascent metacyclics of a particular variable antigen type, as shown by quadrat analysis of a scanning electron micrograph montage of the infected salivary gland epithelium, conforms to a Poisson series. This provides evidence that individual variant surface glycoprotein genes are stochastically activated and suggests that selective activation occurs after trypanosome division has ceased.

Characterization of genes coding for two major metacyclic surface antigens in Trypanosoma brucei.

In African trypanosomes, only a very small fraction of the total repertoire of variable antigen types (VATs) is expressed by the metacyclic form. In Trypanosoma brucei stock EATRO 1125, the VATs AnTat 1.30 and 1.45 are reproducibly present in about 15% and 4% of the metacyclic population, respectively. The genes encoding the corresponding antigens or variant surface glycoproteins (VSGs) are in telomeres of large chromosomes, as are some non-metacyclic VSG genes from the same stock. Their activation mechanism has been studied in seven independent clones, 3 of which, referred to as 'first wave' metacyclic VATs (M-VATs), have been cloned from the first wave of parasitemia after cyclic transmission. In all these clones, activation of the antigen gene was linked to the transposition of an expression linked copy (ELC) of the gene to a telomeric expression site. For first wave M-VATs, this site seems variable, although restricted to large chromosomes, and it can be re-used for VSG gene expression in the bloodstream form. In 'late bloodstream' M-VATs, isolated from established chronic infections, the active expression site, at the end of a 200 kb chromosome, is the one preferred for the expression of late antigen types. It can be concluded that no characteristic feature in the genomic location and expression mechanism can distinguish metacyclic antigen genes from those expressed in the bloodstream forms, although the control of their expression must clearly be different.

Apparent exhaustion of the variable antigen repertoires of Trypanosoma vivax in infected cattle.

Three groups of cattle, each group comprising six animals, were inoculated intravenously with populations of bloodstream forms of Trypanosoma vivax. The first group received T. vivax clone ILDat 1.3 derived from an isolate from Nigeria, while the other two received T. vivax stocks IL 1875 or IL 2133 isolated from Coast Province, Kenya. One animal from the group that was infected with IL 1875 died 8 weeks postinfection. The remaining 17 animals became aparasitemic in 8 to 12 weeks without intervention by drug therapy. The recovered animals developed serodeme-specific immunity against Glossina morsitans subsp. centralis-transmitted challenge. There was complete cross-protection between the two East African T. vivax stocks, although they were isolated from areas 80 to 90 km apart, indicating that they belong to the same serodeme. Antibodies to the homologous metacyclic variable antigen types (VATs) were not detected in sera from recovered animals, suggesting that the immunity displayed by the recovered animals was directed at the bloodstream and not the metacyclic VATs. It is thus suggested that recovery in these animals is due to exhaustion of the repertoire of bloodstream VATs expressed in the animals by the infecting T. vivax clone or stocks.

Analysis of trypanosome variable antigen types in cultures of metacyclic and mammalian forms of Trypanosoma congolense.

Cultured metacyclic forms of Trypanosoma congolense display a characteristic repertoire of metacyclic variable antigen types (M-VATs) similar to that exhibited in vitro in the tsetse fly. There appeared to be no change in expression of M-VATs in cultures of two stocks of T. congolense even after several passages, cryopreservation or long-term cultivation in vitro. Metacyclic forms transformed into mammalian forms when transferred to cultures of bovine aorta endothelial cells and whilst one stock retained expression of M-VATs without change even after 4 months, the other stock underwent antigenic variation within 14 days of transfer. Analysis of the M-VAT composition of mammalian forms of this stock using monoclonal antibodies showed that although the proportion of mammalian forms expressing certain M-VATs declined considerably, trypanosomes expressing one M-VAT increased proportionally to comprise 50% of the population. In contrast, only small changes were seen in antigen expression in cultures of metacyclic trypanosomes from which mammalian-form cultures were derived. It was possible to produce in vitro, loss and reacquisition of variable antigen surface coat, similar to the differentiation process occurring when bloodstream trypanosomes are ingested by the tsetse fly and eventually develop into metacyclic forms.

Neutralization of individual variable antigen types in metacyclic populations of Trypanosoma brucei does not prevent their subsequent expression in mice.

The Trypanosoma brucei metacyclic population in the salivary glands of the tsetse fly displays a characteristic set of variable antigen types (VATs) which represents only a restricted part of the parasite's total VAT repertoire. After introduction into the mammalian host by fly bite, the metacyclics transform into bloodstream forms which retain expression of the metacyclic VATs. Specific antibodies, both polyvalent and monoclonal, have been used to neutralize separately 4 individual VATs from metacyclic populations. Control experiments and visual observation confirmed lysis of each VAT. On injection of the surviving trypanosomes, after washing, into mice each neutralized VAT was nevertheless expressed within a few days. Simultaneous neutralization of 2 metacyclic VATs which usually switch to one another in bloodstream infections did not prevent expression of either on subsequent injection into mice. Expression of neutralized VATs was not influenced by the antigenic composition of the population originally ingested by the tsetse fly. Metacyclic forms and their immediate successors thus appear to switch rapidly to expression of other metacyclic VATs in bloodstream populations.

Characteristics of trypanosome variant antigen genes active in the tsetse fly.

Trypanosoma brucei contains a repertoire of more than 100 different genes for Variant Surface Glycoproteins (VSGs). A small and strain-specific fraction of these genes is expressed in the salivary glands of the tsetse fly (M-genes), giving rise to metacyclic Variable Antigen Types (M-VATs). Antibodies produced in a chronic trypanosome infection initiated by syringe inoculation of bloodstream forms into mammals (i.e. against B-VATs), will react with most of the M-VATs suggesting that these B-VATs express VSG genes that are similar or identical to M-genes. We have cloned DNA complementary to the VSG mRNA of four of such B-VATs and used this to characterize the corresponding VSG genes. In three of the four VATs we find a single VSG gene hybridizing with the cDNA probe and we provide supporting evidence that this gene is expressed as an M-gene. In the bloodstream repertoire these genes appear to be activated by duplicative translocation to another telomere. In all four variants the putative M-genes are telomeric and in the three cases where the location of the genes on chromosome-sized DNA molecules could be determined, the genes were located in large DNA, whereas the majority of the telomeric VSG genes are in chromosomes less than 1000 kb. Our results are best explained by models for M-gene activation involving telomeric expression sites for these genes which are separate from those used by bloodstream forms. The implications of these results for vaccination are discussed.

Resistance of cattle to tsetse-transmitted challenge with Trypanosoma brucei or Trypanosoma congolense after spontaneous recovery from syringe-passaged infections.

Groups of cattle were inoculated intravenously with cloned populations of bloodstream forms of Trypanosoma brucei or Trypanosoma congolense. All five steers infected with T. brucei ILTat 2.1 and six of the eight steers infected with T. congolense IL 13-E14 became aparasitemic within 16 and 32 weeks postinfection, respectively. Examination of sera from animals infected with T. brucei by indirect immunofluorescence and neutralization assays revealed the presence of antibodies against all the metacyclic variable antigen types (VATs) of the infecting clone. The neutralizing capacity of the sera increased with the course of infection from 1:10 at 2 months to 1:100 at 3 to 4 months postinfection. The recovered animals were completely immune to challenge by Glossina morsitans subsp. centralis infected with clone IL Tat 2.1, which had initiated the infection, as well as with another clone (IL Tat 2.2) belonging to the same serodeme, but they were susceptible to a tsetse-transmitted heterologous challenge with isolate STIB 367-H. Similar results were obtained with sera from T. congolense IL 13-E14-infected steers. The six steers infected with a different T. congolense ILNat 3.1 clone did not recover spontaneously; however, 2 months postinfection, sera from five of them also contained neutralizing antibodies against ILNat 3.1 metacyclic VATs. These results indicate that some of the bloodstream VATs that arise during the course of a chronic infection possess surface epitopes in their variable surface glycoproteins that are identical to those of the metacyclic VATs. It is suggested that in chronic infection, the infecting trypanosomes could exhaust their VAT repertoire, including those that cross-react with metacyclics, thereby leading to both "self-cure" and subsequent immunity to homologous cyclically transmitted challenge.

Cytotoxicity of monoclonal antibodies to Trypanosoma brucei

Monoclonal antibodies (McAbs) were raised against Metacyclic Variable Antigen Types (M-VATs) of the AnTAR 1 and ETAR 1 serodemes of Trypanosoma brucei. Two dominant M-VATs, one from each serodeme, were labelled by two of the McAbs using the indirect immunofluorescence technique. These McAbs were of the IgM class, and labelled exposed epitopes on living trypanosomes. They showed lytic activity in vitro towards their respective homologous VAT trypanosomes, both in the presence and absence of complement. In vivo, the McAbs promoted lysis and clearance of trypanosomes from the bloodstream of infected mice. Prevention of reinfection with trypanosomes expressing the same VAT was conferred by the McAbs.

Instability of the Trypanosoma brucei rhodesiense metacyclic variable antigen repertoire.

Trypanosoma brucei rhodesiense undergoes antigenic variation in its mammalian host by changing the glycoprotein composing its surface coat. Trypanosome clones which have the same repertoire of variable antigen types (VATs) are said to belong to the same serodeme. Tsetse flies infected with a particular serodeme extrude infective metacyclic trypanosomes which express only a restricted part of this repertoire. As the only known acquired immunity in African trypanosomiasis is VAT-specific this limitation of metacyclic VAT (M-VAT) repertoire could be important in devising a vaccine. This possibility of immunoprophylaxis could depend, however, on whether or not the M-VAT repertoire is conserved over long periods of repeated cyclical transmission and between epidemics. Studies reported here on isolates made from an East African focus of sleeping sickness over a 20-yr period suggest substantial changes in the M-VATs expressed during this time. Furthermore, we have detected change in expression of 3 M-VATs during sequential tsetse transmission of a clone in the laboratory indicating a possible instability in the organization of M-VAT genes.

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.

Trypanosoma congolense: interactions between trypanosomes expressing different metacyclic variable antigen types in vitro and in vivo.

In Trypanosome congolense, the surface antigens expressed by cultured metacyclic forms are a limited and serodeme-specific subset of variable antigen types (VATs). Experiments were carried out in mammalian-form (MF) cultures, comprising dividing trypanosomes which express metacyclic VATs (M-VATs) maintained in vitro with a mammalian cell feeder layer, or in mice following infection with cultured metacyclic populations. Selective neutralization experiments were performed by incubating populations of trypanosomes with one or a combination of McAbs to remove their respective M-VATs. Deletion of one M-VAT from the population had no effect on expression and growth of other unrelated M-VATs. Deleted M-VATs were detected in very small proportions of the total population 2-7 days following neutralization. Subsequent incubation of neutralized cultures resulted in growth of deleted M-VATs which, in most cases, reached pre-neutralization levels. It appears, therefore, that in conditions where expression of M-VATs on dividing trypanosomes occurs, removal of individual M-VATs results in their re-expression within the population.