Variable Antigen Types Research Articles

Experimental evidence that immune trypanolysis using the LiTat 1.3 and LiTat 1.5 variant antigen types is not specific to Trypanosoma brucei gambiense in pigs.

In the context of the human African trypanosomiasis elimination process, reliable and accurate diagnostic tools are crucial for exploring the role of a potential animal reservoir of Trypanosoma brucei gambiense. The immune trypanolysis test (TL) using the variant antigen types (VAT) LiTat 1.3 and LiTat 1.5, described as a specific serological method to detect people infected by T. b. gambiense, seems to be a promising tool. However, its specificity was recently questioned during field animal surveys. The present study evaluates the performance of TL during experimental T. b. brucei infection in pigs. Eight infected pigs and four uninfected pigs were followed up with blood and plasma collection. Blood was used for parasitological investigation. TL was performed on the plasma with the LiTat 1.3, LiTat 1.5 and LiTat 1.6 VATs. All control pigs remained negative to parasitological investigation and TL. Trypanosomes were detected in all the infected pigs and the first detection was between 10 and 14 days post infection (dpi). TL results showed that infected pigs developed antibodies against the three VATs. The first antibody detections by TL occurred between 14 and 21 dpi for antibodies directed against LiTat 1.6, 21 and 168 dpi for antibodies directed against LiTat 1.5 and 70, and 182 dpi for antibodies directed against LiTat 1.3. This study highlights for the first time that TL using LiTat 1.3 and LiTat 1.5 VATs is not specific to T. b. gambiense. Development of specific diagnostic tools for the detection of T. b. gambiense infections in animals, especially in pigs, is still needed.

A LiTat 1.5 variant surface glycoprotein‐derived peptide with diagnostic potential for Trypanosoma brucei gambiense

To evaluate the accuracy of a peptide, corresponding to the variant surface glycoprotein (VSG) LiTat 1.5 amino acid (AA) sequence 268-281 and identified through alignment of monoclonal antibody selected mimotopes, for diagnosis of Trypanosoma brucei gambiense sleeping sickness. A synthetic biotinylated peptide (peptide 1.5/268-281), native VSG LiTat 1.3 and VSG LiTat 1.5 were tested in an indirect ELISA with 102 sera from patients with HAT and 102 endemic HAT-negative controls. The area under the curve (AUC) of peptide 1.5/268-281 was 0.954 (95% confidence interval 0.918-0.980), indicating diagnostic potential. The areas under the curve of VSG LiTat 1.3 and LiTat 1.5 were 1.000 (0.982-1.000) and 0.997 (0.973-1.000), respectively, and significantly higher than the AUC of peptide 1.5/268-281. On a model of VSG LiTat 1.5, peptide 1.5/268-281 was mapped near the top of the VSG. A biotinylated peptide corresponding to AA 268-281 of VSG LiTat 1.5 may replace the native VSG in serodiagnostic tests, but the diagnostic accuracy is lower than for the full-length native VSG LiTat 1.3 and VSG LiTat 1.5.

A new format of the CATT test for the detection of Human African Trypanosomiasis, designed for use in peripheral health facilities

To test the reproducibility and thermostability of a new format of the Card-Agglutination Test for Trypanosomiasis (CATT) test for Human African Trypanosomiasis (HAT), designed for use at primary health care facility level in endemic countries. A population of 4217 from highly endemic villages was screened using the existing format of the CATT test (CATT-R250) on whole blood. All those testing positive (220) and a random sample of negatives (555) were retested in the field with the new format (CATT-D10). Inter-format reproducibility was assessed by calculating kappa. All samples testing positive on whole blood with either method were further evaluated in Belgium by CATT titration of serum by two observers, using both old and new format. CATT-D10 test kits were incubated under four temperature regimens (4, 37, 45 degrees C and fluctuating) with regular assessments of reactivity over 18 months. Inter-format reproducibility of CATT-D10 vs. CATT-R250 on whole blood performed by laboratory technicians in the field was excellent with kappa values of 0.83-0.89. Both inter- and intra-format reproducibility assessed by CATT titration were excellent, with 96.5-100% of all differences observed falling within the limits of +/-1 titration step. After 18 months, reactivity of test kits incubated under all four temperature regimens was still well above the minimum threshold considered acceptable. The CATT-D10 is thermostable and can be used interchangeably with the old format of the CATT test. It is highly suitable for use in peripheral health facilities in HAT-endemic countries.

Human African trypanosomes: challenges posed to the human immune system.

Human infection by either Trypanosoma brucei gambiense (Tbg) or Trypanosoma brucei rhodesiense (Tbr) and the establishment of disease is made possible by the intermittent switching of their variant surfaceglycoprotein (VSG) and expression of serum resistance associated (SRA) protein (by Tbr) which nullifies the lytic action of the normal human serum. The ability to switch expression from one VSG to the other is recognized to be the major mechanism that permits the parasite to evade the otherwise efficient host antibody, hence preventing parasite elimination and allowing the establishment of a chronic infection. These changes were reported to: disable the host’s capacity to mount a protective anti-parasite antibody response and prevent the development of effective B-cell memory against encountered variant antigenic parasite types (VATs). Both B cell-mediated antibody response and the Th1 cell responses leading to the production of interferon-gamma (IFN-ϒ) are required for maximum host resistance to trypanosomes, with IFN-ϒ acting to induce macrophage trypanolytic and trypanostatic activities. High levels of both tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10) have been associated with trypanosomal infection. Trypanosomal genetics (including the parasite’s intrinsic characteristics), human immune response polymorphisms and geographical locations are important elements that describe the severity or mildness of HAT. As the parasite devices ways to evade thehuman immune system, and in the absence of a suitable vaccine, surveillance, prompt diagnosis and treatment with available drugs and vector control efforts will go along way in reducing the incidence of HAT.Keywords: African trypanosomes, Variant surface glycoprotein, Serum resistance associated (SRA) protein, Trypanosomal genetic polymorphisms, Immune response polymorphisms

Towards a New Reference Test for Surra in Camels

Current serological diagnosis of Trypanosoma evansi infection in camels is based on the native variable antigen type RoTat 1.2. The goal of this study was to develop a novel serological diagnostic test based on a nonvariable protein and freed from the use of rats or mice for its production. An enzyme-linked immunosorbent assay using a recombinant extracellular domain of invariant surface glycoprotein 75 (ELISA/rISG75) was developed and tested on a collection of 184 camel sera. The results were compared to those obtained from three established antibody detection tests based on variable surface glycoprotein RoTat 1.2: an ELISA for T. evansi (ELISA/T. evansi), a card agglutination test for trypanosomiasis (CATT/T. evansi), and an immune trypanolysis (TL) assay. The ELISA/rISG75 and the ELISA/T. evansi showed a sensitivity of 94.6% (95% confidence interval [CI], 87.8 to 98.2%, at 19% positivity cutoff value) and 98.9% (95% CI, 94.1 to 99.8, at 12% positivity cutoff value), respectively. The ELISA/rISG75 had 100% specificity (CI, 95.9 to 100%), while the ELISA/T. evansi showed 98.9% specificity (CI, 95.9 to 100%). The ELISA/rISG75 demonstrated an almost perfect agreement with the TL assay, the CATT/T. evansi, and the ELISA/T. evansi, with kappa scores of at least 0.94. The ELISA/rISG75, having a performance comparable to that of the gold standard (the TL assay) and being independent of antigenic variation, may become a new reference test for surra in camels. It opens avenues for the diagnosis of T. evansi infections in other hosts as well as for the development of a pan-Trypanozoon test for detection of Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, and T. equiperdum.

Structural characterization and serological specificities of lipopolysaccharides from Salmonella enterica serovar Gallinarum biovar Pullorum standard, intermediate and variant antigenic type strains

The structure and serological specificities of the lipopolysaccharides (LPSs) from Salmonella enterica serovar Gallinarum biovar Pullorum were studied to provide an improved basis for the distinction between antigenic types and the development of improved diagnostic tests. The structure of the LPS O-polysaccharide (O-PS) from S. Pullorum standard, intermediate and variant antigenic type strains was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and chemical analysis. The LPS of the three strains shared a common structural repeating oligosaccharide unit containing d-mannose, l-rhamnose, d-galactose and d-tyvelose (1:1:1:1). The O-PS of the variant type LPS contained an additional d-glucose residue linked to the O-4 position of the d-galactose residue. The O-PS of the intermediate type LPS was partially the same as that of the variant LPS, however, the molar ratio of the d-glucose component was lower with respect to the other glycose components. Serological specificities of the three antigenic type LPSs were examined with anti- S. Pullorum LPS monoclonal antibodies (Mabs). On immunoblots, Mabs to the standard type O-PS reacted with high molecular mass (HMM) and low molecular mass (LMM) LPS from the standard strain, and with LMM but not HMM LPS from the variant strain. Monoclonal antibodies to the variant type O-PS reacted with HMM but not LMM LPS from the variant strain, and did not react with HMM or LMM LPS from the standard strain. On ELISA, the standard, intermediate and variant antigenic type strains were differentiated by the relative reactivity with the anti-LPS O-PS Mabs. Several of the anti-LPS O-PS Mabs were specific for S. Pullorum and other serogroup D1 Salmonella, and are potentially useful for the development of improved diagnostic tests for these organisms.

Protection of mice against homologous or heterologous infections with antiserum mixture to the predominant variable antigen type repertoire of Trypanosoma evansi YNB stock

The objective of this study was to test a hypothesis that the predominant variable antigen type (VAT) repertoire of a single stock of Trypanosoma evansi was limited and small. It was further assumed that six rabbits could produce all antibodies against the predominant VAT repertoire of a stock of T. evansi and the antiserum mixture from the six rabbits containing all the antibodies could completely protect mice against any homologous stock infections and partially protect mice against some heterologous stock infections. Mice were each intraperitoneally infected with 100 parasites of clone-derived and non-clone-derived populations of the YNB stock, Kazakhstan strain or Vietnam strain of T. evansi, and treated with the antiserum mixture when trypanosomes had been detected in the blood. All of the 10 mice infected with either non-clone-derived or clone-derived populations of the YNB stock survived, and some (4/10) of mice infected with the heterologous Kazakhstan strain survived, while all those (10/10) infected with the heterologous Vietnam strain died. These results support the hypothesis that the predominant VAT repertoire of a single stock of T. evansi was limited and small, and have important implications in the consideration of treating human trypanosomosis due to drug resistant strains with antiserum mixture.

Probabilistic order in antigenic variation of Trypanosoma brucei

Antigenic variation in African trypanosomes displays a degree of order that is usually described as ‘semi-predictable’ but which has not been analysed in statistical detail. It has been proposed that, during switching, the variable antigen type (VAT) being inactivated can influence which VAT is subsequently activated. Antigenic variation proceeds by the differential activation of members of the large archive of distinct variable surface glycoprotein ( VSG) genes. The most popular model for ordered expression of VATs invokes differential activation probabilities for individual VSG genes, dictated in part by which of the four types of genetic locus they occupy. We have shown, in pilot experiments in cattle, correlation between the timing of appearance of VSG-specific mRNA and of lytic antibodies corresponding to seven VSGs encoded by single-copy genes. We have then determined the times of appearance of VAT-specific antibodies, as a measure of appearance of the VATs, in a statistically significant number of mouse infections ( n=22). There is a surprisingly high degree of order in temporal appearance of the VATs, indicating that antigenic variation proceeds through order in the probability of activation of each VAT. In addition, for the few examples of each available, the locus type inhabited by the silent ‘donor’ VSG plays a significant role in determination of order. We have analysed in detail previously published data on VATs appearing in first relapse peaks, and find that the variant being switched off does not influence which one is being switched on. This differs from what has been reported for Plasmodium falciparum var antigenic variation. All these features of trypanosome antigenic variation can be explained by a one-step model in which, following an initial deactivation event, the switch process and the imposition of order early in infection arise from the inherent activation probabilities of the specific VSG being switched on.

Trypanosoma congolense infections: antibody-mediated phagocytosis by Kupffer cells.

Immunohistochemical double-label technique was used to detect trypanosomal antigen in macrophages. Immunoglobulin (Ig)M as well as IgG2a monoclonal antibodies (mAb) specific for the variant surface glycoprotein (VSG) mediated phagocytosis of Trypanosoma congolense variant antigenic type (VAT) TC13 by macrophages [bone marrow-derived macrophage cell line from BALB/c (BALB.BM)] in vitro. Administration of these IgM or IgG2a antibodies to BALB/c mice 30 min after injection of 3 x 10(8) T. congolense mediated phagocytosis of trypanosomes by Kupffer cells of the liver within 1 h. Plasma levels of the monokines interleukin (IL)-1beta, IL-10, and IL-12p40 were significantly increased 6-48 h after phagocytosis. In BALB/c mice infected with 10(3) T. congolense, a small degree of phagocytosis of trypanosomes by Kupffer cells, mediated by actively synthesized antibodies, was detected as early as 5 days after infection. Phagocytosis of trypanosomes was dramatically enhanced on day 6. Concomitantly, the Kupffer cells trippled in size. In BALB/c mice infected for 6 days, treatment with IgM or IgG2a mAb specific for T. congolense VSG led to clearance of VAT TC13 parasitemia but did not prevent death at the second parasitemia of a different VAT. We conclude that IgM as well as IgG antibody mediate phagocytosis of trypanosomes by Kupffer cells.

Evaluation of LATEX/ T.b.gambiense for mass screening of Trypanosoma brucei gambiense sleeping sickness in Central Africa

We compared the Card Agglutination Test for Trypanosomiasis (CATT), which consists of lyophilized bloodstream form trypomastigotes of Trypanosoma brucei gambiense ( T.b.g.) variable antigen type LiTat 1.3, with LATEX/ T.b.g., which consists of a lyophilized suspension of latex particles coated with variable surface glycoproteins of T.b.g. variable antigen types LiTat 1.3, 1.5 and 1.6. This study was carried out during two mass screening surveys in 1998 in Campo, a sleeping sickness focus in Cameroon, with a low prevalence (0.3%) and in 1999 in Batangafo which belongs to the Central African focus of Ouham which has a higher prevalence (3%). In Campo, we compared the CATT performed on whole blood with the LATEX/ T.b.g. on diluted blood. In Batangafo, both tests were performed on diluted blood. In all circumstances, the specificity of the LATEX/ T.b.g. was higher than of CATT. The use of LATEX/ T.b.g. on diluted blood instead of CATT results in an important decrease of workload and as a consequence, of costs related to parasitological examinations. In the case of Campo the workload was up to 12 times less than when using CATT 1.3 on whole blood and the cost divided by 3. In Batangafo the workload was decreased by nearly 20% with the LATEX/ T.b.g. Finally, it should be noted that in Batangafo, one of the parasitologically confirmed sleeping sickness patients was negative in CATT and positive in LATEX/ T.b.g. and that the reading of the test result in LATEX/ T.b.g. is easier than in CATT.

Expression of RoTat 1.2 cross-reactive variable antigen type in Trypanosoma evansi and T. equiperdum.

The variable antigen type (VAT) RoTat 1.2 has been cloned from a T. evansi strain, isolated in 1982 from a water buffalo in Indonesia. All T. evansi isolates hitherto tested express this VAT. In a study on the differential diagnosis of T. equiperdum and T. evansi in horses, we investigated serological evidence for the expression of RoTat 1.2 in 11 T. evansi and six T. equiperdum populations originating from Asia, Europe, Africa, and the Americas. Preinfection sera and sera of days 7, 14, 25, and 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the experiment, all rabbits infected with T. evansi became positive in the three serological tests. Five out of six rabbits infected with T. equiperdum also became positive in the three tests. Only one T. equiperdum strain (the OVI strain from South Africa) did not induce the production of antibodies reactive with RoTat 1.2 and thus might not contain or express a VSG that shares epitopes similar to those on the RoTat 1.2 VSG. The data lead to the conclusion that T. equiperdum can express VSGs containing epitopes serologically similar to those in the T. evansi RoTat 1.2 VAT. This explains, in part, why the antibody detection tests based on Ro Tat 1.2 VSG cannot reliably distinguish between the infections caused by T. evansi and those caused by T. equiperdum. There are no data that contradict the possibility that the putative T. equiperdum strains, which express VSGs with epitopes similar to those on RoTat 1.2, are actually T. evansi.

General expression of RoTat 1.2 variable antigen type in Trypanosoma evansi isolates from different origin

The variable surface glycoprotein of Trypanosoma evansi RoTat 1.2 variable antigen type (VAT) is used as an antigen in different antibody detection assays for T. evansi. To obtain more information on the predominant character of RoTat 1.2 and its diagnostic potential in antibody detection tests, we checked its expression in 10 different T. evansi stocks and clones from different parts of the world. Cryostabilates were injected into mice and the trypanosomes of the first peak parasitaemia were screened for the presence of RoTat 1.2 by VAT specific immunofluorescence. To monitor the appearance of RoTat 1.2 specific antibodies during infection, rabbits were infected and serologically tested at different time intervals with VAT specific immune trypanolysis, CATT/ T. evansi, LATEX/ T. evansi and ELISA/ T. evansi. Test results confirm the predominant character of RoTat 1.2.

Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam

In the present study, a collection of 415 water buffalo serum samples originating from the north of Vietnam was used for evaluation of different diagnostic antibody detection methods available to detect infections with Trypanosoma evansi. The diagnostic sensitivity and specificity of a direct card agglutination test (CATT/ T. evansi), an indirect card agglutination test (LATEX/ T. evansi) and a newly developed antibody detection ELISA (ELISA/ T. evansi) was calculated on the basis of parasitological results, obtained by mouse inoculation, and compared for all assays. The immume trypanolysis assay with the predominant T. evansi RoTat 1.2 variable antigen type was used as reference test for antibody presence. All parasitologically confirmed animals ( n=8) were positive in all tests. Diagnostic specificity was highest in CATT/ T. evansi (98%) followed by the ELISA/ T. evansi (95%) and the LATEX/ T. evansi (82%). Concordance of the variant specific immune trypanolysis test with the other tests was calculated and revealed that few (1–8%) false positive results were actually due to a specific reactions, and that LATEX/ T. evansi and ELISA/ T. evansi detected more immune trypanolysis positives than the CATT/ T. evansi. It was concluded that, apart from the immune trypanolysis test, which is not generally applicable, ELISA/ T. evansi with a 30% positivity cut-off and LATEX/ T. evansi, thanks to their superior capacity of detecting T. evansi specific antibodies, would be suitable as epidemiological tools detecting both active infections and persisting T. evansi specific antibodies. The ELISA/ T. evansi with a 50% positivity cut-off and the CATT/ T. evansi on the other hand, seem more appropriate to detect true infected water buffaloes.

Periodic appearance of a predominant variant antigen type during a chronic Giardia lamblia infection in a mouse model

Giardia lamblia (syn. Giardia duodenalis, Giardia intestinalis) infections are associated with continuous antigenic variation of the parasite which is mediated by the parasite's major surface antigen, named variant surface protein. Offspring mice and corresponding mothers were infected with G. lamblia clone GS/M-83-H7 (expressing variant surface protein H7) and various parameters of this infection were assessed in a long-term follow-up investigation. Our experimentation revealed that variant surface protein H7-type trophozoites were replaced by new variant-type trophozoites during the early stage of infection (around day 8 p.i.), but the original variant-type re-emerged at at least two time-points during the later stages of infection (at days 22 and 42 p.i.). Such periods of variant surface protein H7-type trophozoite re-expansion were accompanied by transient production of intestinal IgA against variant-specific epitopes on a 314-aa N-terminal region of variant surface protein H7. At late stages of infection (between days 42 and 200 p.i.), most mice produced intestinal IgA against both variant surface protein H7 and other antigens of the parasite. At these stages, infection seemed to be resolved in most mice, but occasional reappearance of relatively high (at day 64 p.i.) or at least detectable (at days 80 and 120 p.i.) amounts of intestinal parasites indicated that G. lamblia GS/M-83-H7 infections in mice may enter into a latent chronic phase which is interrupted by sporadic breakthroughs of parasite growth.

Antigenic variation in Trypanosoma brucei infections: an holistic view.

Trypanosoma brucei parasites undergo clonal phenotypic (antigenic) variation to promote their transmission between mammals and tsetse-fly vectors. This process is classically considered to be a mechanism for evading humoral immune responses, but such an explanation cannot account for the high rate of switching between variable antigens or for their hierarchical (i.e. non-random) expression. I suggest that these anomalies can be explained by a new model: that antigenic variation has evolved as a bifunctional, rather than as a unifunctional, strategy that not only evades humoral immune responses but also enables competition between parasite strains in concomitantly infected hosts. This competition causes a depression of cellular responses. My proposal gives rise to a number of testable predictions. First, low numbers of trypanosomes should express some variable antigen types (VATs) in infections several weeks before these VATs are detectable. Second, as an infection progresses, the number of VATs expressed simultaneously in the population should decrease. Third, immunisation to generate a T helper 1 response against those VATs that are expressed most frequently should lower parasitaemias and reduce virulence.

Predominance of duplicative VSG gene conversion in antigenic variation in African trypanosomes.

A number of mechanisms have been described by which African trypanosomes undergo the genetic switches that differentially activate their variant surface glycoprotein genes (VSGs) and bring about antigenic variation. These mechanisms have been observed mainly in trypanosome lines adapted, by rapid syringe passaging, to laboratory conditions. Such "monomorphic" lines, which routinely yield only the proliferative bloodstream form and do not develop through their life cycle, have VSG switch rates up to 4 or 5 orders of magnitude lower than those of nonadapted lines. We have proposed that nonadapted, or pleomorphic, trypanosomes normally have an active VSG switch mechanism, involving gene duplication, that is depressed, or from which a component is absent, in monomorphic lines. We have characterized 88 trypanosome clones from the first two relapse peaks of a single rabbit infection with pleomorphic trypanosomes and shown that they represent 11 different variable antigen types (VATs). The pattern of appearance in the first relapse peak was generally reproducible in three more rabbit infections. Nine of these VATs had activated VSGs by gene duplication, the tenth possibly also had done so, and only one had activated a VSG by the transcriptional switch mechanism that predominates in monomorphic lines. At least 10 of the donor genes have telomeric silent copies, and many reside on minichromosomes. It appears that trypanosome antigenic variation is dominated by one, relatively highly active, mechanism rather than by the plethora of pathways described before.

The Anatomy and Transcription of a Monocistronic Expression Site for a Metacyclic Variant Surface Glycoprotein Gene inTrypanosoma brucei

African trypanosomes evade the immune response of their mammalian hosts by switching the expression of their variant surface glycoprotein genes (vsg). The bloodstream trypanosome clone MVAT4 of Trypanosoma brucei rhodesiense expresses a metacyclic vsg as a monocistronic RNA from a promoter located 2 kilobases (kb) upstream of its start codon. Determination of 23 kb of sequence at the metacyclic variant antigen type 4 (MVAT) vsg expression site (ES) revealed an ES-associated gene (esag) 1 preceded by an ingi retroposon and an inverted region containing an unrelated vsg, short stretches of 70-bp repeats and a pseudo esag 3. Nuclear run-on experiments indicate that the 18-kb region upstream of the MVAT4 vsg promoter is transcriptionally silent. However, multiple members of different esag families are expressed from elsewhere in the genome. The MVAT4 vsg promoter is highly repressed in the procyclic stage, in contrast to the known polycistronic vsg ESs which undergo abortive transcription. Activation of the MVAT4 vsg ES occurs in situ without nucleotide sequence changes, although this monocistronic ES undergoes a pattern of base J modifications similar to that reported for the polycistronic ESs. The relative simplicity of the MVAT4 vsg ES and the uncoupled expression of the vsg and esags provide a unique opportunity for investigating the molecular mechanisms responsible for antigenic variation in African trypanosomes.

Antigenic variation and the murine immune response to Giardia lamblia

The protozoan parasite Giardia lamblia is an important causative agent of acute or chronic diarrhoea in humans and various animals. During infection, the parasite survives the hosts reactions by undergoing continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). The VSPs form a unique family of cysteine-rich proteins that are extremely heterogeneous in size. The relevance of antigenic variation for the survival in the host has been most successfully studied by performing experimental infections in a combined mother/offspring mouse system and by using the G. lamblia clone GS/M-83-H7 (human isolate) as model parasite. In-vivo antigenic variation of G. lamblia clone GS/M-83-H7 is characterised by a diversification of the intestinal parasite population into a complex mixture of different variant antigen types. It could be shown that maternally transferred lactogenic anti-VSP IgA antibodies exhibit cytotoxic activity on the Giardia variant-specific trophozoites in suckling mice, and thus express a modulatory function on the proliferative parasite population characteristics. Complementarily, in-vitro as well as in-vivo experiments in adult animals indicated that non-immunological factors such as intestinal proteases may interfere into the process of antigen variation in that they favour proliferation of those variant antigen-type populations which resist the hostile physiological conditions within the intestine. These observations suggest that an interplay between immunological and physiological factors, rather than one of these two factor alone, modulates antigenic diversification of a G. lamblia population within an experimental murine host and thus influences the survival rate and strategy of the parasite.

Characterization of a Relatively Rare Class B, Type 2 Trypanosome Variant Surface Glycoprotein Gene

The variant surface glycoprotein (VSG) gene of Trypanosoma brucei rhodesiense LouTat 1.5, a defined African trypanosome variant antigenic type, was cloned and sequenced. Southern blot analysis revealed 2 DNA restriction fragments in both VSG 1.5 expressor and nonexpressor populations, suggesting that there are 2 genomic copies of the VSG 1.5 gene and no expression-linked copies. Pulsed-field gel electrophoresis followed by Southern blot analysis showed that each copy of the VSG 1.5 gene exists on a separate large chromosome in both the expressor (approximately 3.5- and 4-megabase (Mb) chromosomes) and nonexpressor (approximately 4- and 5.7-Mb chromosomes) populations. Thus, VSG genes may be present on larger chromosomes than previously reported. Sequence analysis and alignments revealed that the VSG 1.5 molecule is a class B VSG with 12 cysteine residues in the N-terminus and is classified as a type 2 VSG based on C-terminus motifs. This classification shows that the VSG 1.5 molecule represents a relatively rare VSG class and type. Taken together, these studies provide additional information on VSG genes and proteins and supply the foundation for structure-function analysis of the VSG 1.5 surface antigen expressed by trypanosomes of the LouTat 1 serodeme.

Antigenic variation during malaria infection--the contribution from the murine parasite Plasmodium chabaudi.

P. chabaudi AS strain in laboratory mice provides an accessible and useful model for investigating antigenic variation in malaria parasites. Evidence that P. chabaudi AS undergoes antigenic variation is summarized. A live indirect fluorescent test (IFAT) detects a variable antigen on the surface of schizont-infected erythrocytes. Five different variable antigen types (VATS) (detected using the live IFAT) are described from a cloned mosquito transmitted parent population. Even during the rising primary parasitaemia VATS switch at high and variable rates (1.2-1.6%). Work towards cloning genes coding for the variable antigen is briefly summarized. Acquired immunity to blood-stage P. chabaudi AS is initially mediated through Th1 CD4+ T cells and after the primary parasitaemia there is a switch to Th2 CD4+ T cells. Acquired immune effector mechanisms are discussed in the context of antigenic variation by the parasite.