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

Acta Tropwa, 49(1991)193-199 Elsevier

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Trypanosoma congolense: Re-expression of a deleted metacyclic variable antigen type in vivo and in vitro D.V. Sutherland, C.A Ross and A G. Luckins Centrefor Tropical Veterinary Medicine, Easter Bush, Roshn, MMlothtan, U K

(Recewed 22 November 1990, accepted 3 January 1991) The expression of varmble antigen types (VATs) was determined among &wdlng populations of T congolensegrowing m wvo in rabbit chancres and m vitro on bowne aorta endothehal cell monolayers Experiments were performed m which a single metacychc VAT (M-VAT) was deleted from a cultured metacychc population by neutrahsatlon with a monoclonal antibody and complement Subsequent expression of the deleted M-VAT and two unrelated M-VATs was determined by an m&rect lmmunofluorescent antibody test The deleted M-VAT was re-expressed both In VlVOand m wtro and the proportions of unrelated M-VATs were not markedly affected by the neutrahsatlon of this single M-VAT In ad&tlon, an overall similarity was observed between M-VAT expression and re-expression m VlVOand in vitro Key words Trypanosoma congolense, Chancres, Neutrahsatlon

Metacychc varmble antigen

types (M-VATs),

Introduction The early stage o f a T congolense infection occurs e x t r a v a s c u l a r l y in the skin o f the v e r t e b r a t e host, following m o c u l a t l o n with m e t a c y c h c f o r m s by infected tsetse files d u r i n g feeding ( L u c k l n s a n d G r a y , 1978, A k o l a n d M u r r a y , 1982) T h e p a r a s i t e s m u l t i p l y at the bite site causing a local skin r e a c t i o n o r c h a n c r e T r y p a n o s o m e s at this stage o f infection are o f p a r t i c u l a r interest as they r e p r e s e n t the first e x p o s u r e o f the p a r a s i t e to the v e r t e b r a t e h o s t T congolense m e t a c y c h c s express a limited, c o n s e r v e d a n d s e r o d e m e specific subset o f v a r i a b l e a n t i g e n types (VATs) k n o w n as the M - V A T r e p e r t o t r e O n differentiation to d i v i d i n g f o r m s in the m a m m a l , new VATs are expressed in t r y p a n o s o m e p o p u l a t i o n s m the c h a n c r e ( L u c k m s et a l , 1990) a l t h o u g h their levels r e m a i n low until M - V A T s have been cleared A similar s i t u a t i o n has been o b s e r v e d m t r y p a n o s o m e p o p u l a t i o n s g r o w n in vitro by i n o c u l a t i o n o f m e t a c y c h c t r y p a n o s o m e s o n t o b o v i n e a o r t a e n d o t h e h a l ( B A E ) cell m o n o l a y e r s ( L u c k m s et al., 1986), p r o v i d i n g a m o d e l system for the s t u d y o f M - V A T expression early in infection a n d m the d e v e l o p m e n t o f infection In a p r e v i o u s study, P r a l n a n d R o s s (1988) used M-VAT-specific m o n o c l o n a l a n t i b o d i e s ( M c A b s ) for selective n e u t r a l l s a t l o n o f d i v i d i n g p o p u l a t i o n s o f t r y p a n o somes at 28°C m culture T h e y f o u n d t h a t deleted M - V A T s were re-expressed a n d Correspondence address D V Sutherland, Centre for Tropical Veterinary Medicine, Easter Bush, Roshn EH25 9RG, Midlothlan, U K

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that deletion of one M-VAT had no effect on the expression and growth of other unrelated M-VATs In the follow~eng experiments, metacychc trypanosomes were neutrahsed w~th an M-VAT-specific McAb and subsequent expression of this M-VAT and selected unrelated M-VATs was determined both m VlVO m the skin of infected rabbits and m v~tro on BAE cell monolayers at the higher temperature of 34~C

Experimental procedure Trypanosomes T congolense T R E U 1457, a cloned derivative of a stock originally isolated in Nigeria (Zarla/67/LUMP/69), was used in this study Insect Jorm culture~ These cultures, which provided the metacychc trypanosomes used m this study, were maintained at 28°C as described by Gray et al (1984) Metacychc trypanosomes were separated on a dlethylammoethyl cellulose column (DE52 Whatman Blochemlcals) (Gray et al, 1984) and eluted with phosphate sahne glucose (PSG =PBS, pH 8 0, containing 1% glucose) supplemented with thloglycerol (100 ~tM), sodium pyruvate ( 2 m M ) and hypoxanthlne/thymldme ( 0 1 m M / 0 0 1 6 m M ) (all Sigma Chemical Company Lid ), to minlmlse the effects of elutton on the metabolism of the trypanosomes (Lonsdale-Eccles and Grab, 1987) Suspensions of these metacycllcs provided the starting material for the refection of rabbits and inoculation of BAE cell monolayers

Monclonal antibodies The McAbs used m these experiments - - designated 12 4, 12 5 and 12 11 - - were prepared against cultured metacychc trypanosomes of stock T R E U 1457 (Crowe et al, 1983) McAbs 12 4 and 12 5 recognlse an M-VAT normally present in relatively high proportions ( > 20%) among T R E U 1457 metacychcs whereas 12 11 recogmses an M-VAT normally present in relatively low p r o p o m o n s ( < 10%) McAb 12 5 was used as the neutrahsmg antibody in this study

Neutrahsatton test The metacychc trypanosomes collected from the Insect form cultures were divided into three fractions and before inoculation were either (1) neutrahsed by incubation in mammahan form culture medium containing McAb 12 5 (to a final dilution of 1 20) and guinea pig complement (GPC') (1 20) at 28°C for 2 h (n) set up as the neutrahsatlon control in medium containing GPC' but no McAb and incubated as for (t) (in) left untreated as a further control

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Mammahan form cultures Metacychc trypanosomes were introduced onto BAE cell monolayers (Gray et al, 1985) at a seeding of approximately 2 × 10~ per 25 cm 3 flask (Falcon, Becton Dickinson and Company) and maintained in sealed flasks at 34°C m Eagles minimal essentml medium containing 10% (v/v) calf serum, 2 mM glutamlne, 4 mM sodmm pyruvate, 100 ~tM thloglycerol and 0 1 mM hypoxanthlne/0 016 mM thymldlne (all Sigma Chemical Company Ltd ) (Baltz et al, 1985) Three flasks were set up for each experiment, one flask for each of the neutrallsed (1) and control [(n) & (nl)] populations. Suspensions of mammalian form trypanosomes were prepared by washlng the cells vigorously with culture me&urn to remove adherent trypanosomes Trypanosomes reached sufficient numbers for harvesting by Day 5 but decreased after Day 9

Experimental chancres Chancres were produced on adult New Zealand white (NZW) rabbits by lntradermai inoculation, at five separate sites per rabbit, with 1 x 105 metacychcs per site For each experiment, three rabbits were inoculated, one with the parasites neutrallsed with McAb 12.5 (1) and the other two with the un-neutrahsed controls (11 & hi) Trypanosomes were collected from the chancres 6 days after inoculation as described by Luckms et al (1990)

Indtrect fluorescent anttbo@ tests ( IFAT) Metacychc trypanosomes from suspensions used for inoculation of rabbits and BAE cell monolayers (Day 0) and trypanosomes collected from chancres (Day 6) and from monolayers (Days 5, 6 & 9) were washed in PSG, concentrated by centrlfugatlon and dried onto 15-well antigen slides (Flow Labs) Shdes were fixed in acetone for 10 rain, dried and stored at - 20°C untd reqmred The test was performed as described by Luckins et al (1986) using the three McAbs at optimal dilutions (I 640-1 1280) and rabbit anti-mouse IgG conjugated with fluoresceln lsothlocyanate (FITC Nor&c Immunochemlcals) To determine the proportaon of parasites labelled with each antibody, 300-500 trypanosomes were counted using phase-contrast and fluorescence microscopy

Results The data presented are obtained from a single experiment. Although three experiments were carried out, the numbers of trypanosomes obtained from the m VlVO system were only sufficient for analysis m th~s one experiment However, s~mllar trends were observed m the other two experiments On examination by IFAT, with the exception of the pre-lnoculatlon population neutrahsed with McAb 12 5, each of the trypanosome populations analysed was found to contain trypanosomes which were recognlsed by the three M-VAT-specific McAbs 124, 12 5 and 12 11

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Fig 1 illustrates the proportion o f trypanosomes in the three populations in VlVO and in vitro labelled by each o f the three McAbs Examination o f the neutrahsed populations revealed that the deleted M-VAT reappeared both in VlVO and in vitro Six days after neutrahsatlon, 8% o f trypanosomes from the chancre and 2 5% o f trypanosomes from BAE cell monolayers at 34°C were expressing the deleted 12 5-specific M-VAT (Fig la) The proportion o f trypanosomes expressing this M-VAT in vitro &ffered from the in VlVO proportions over the period of the experiment In comparison with pre- and post-inoculation control populations, the neutrallsed population contained low proporttons of the deleted M-VAT Expression of this (a) 4O

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Fig 1 Histograms showing the proportions ofm vlvo and m vitro-derived trypanosomes from the neutralised population ((1) V1) and control populations ((n) [] and (m) III) of T congolense T R E U 1457 labelled by the M-VAT-specific monoclonal antibodies 12 5(a), 12 4(b) and 12 1 l(c)

197 12 5-specific M-VAT was 2-3 times greater in the control populations after 6 days incubation in VlVO and 8-10 times greater in the control populations after the corresponding 6 days incubation m vitro. To determine whether McAb neutrahsatlon allowed a compensatory greater expression of other M-VATs in one population, the in VlVO and m vitro-derived trypanosome samples examined for the expression of the deleted M-VAT were also examined by IFAT with two McAbs which recognlse other M-VATs - - 12 4 and 1211 The distribution of these two M-VATs is illustrated In Fig lb (12 4) and Fig lc (12 1 l) Although some variation was observed, no major alteration in the proportion of 12 11-specific M-VAT was seen when the neutralised population was compared with the control population in VlVO or In vitro A similar trend was observed with the 12 4 specific M-VAT, the only marked difference being when the neutrahsed population was compared with the control population in the in vitro samples of day 9, 28% of trypanosomes from this neutrahsed population expressed this M-VAT compared with 18% and 19% of trypanosomes in the control populations Temporal changes in the expression of the three M-VATs were monitored over 9 days in vitro The proportion of trypanosomes in the control populations expressing the M-VAT recognised by McAb 12 5, shown in Fig l(a), rose shghtly from 16-23% on Day 0 to 2 5 - 3 6 % on Day 5, and then fell to a level similar to pre-lnoculatlon levels The proportion of trypanosomes expressing the M-VAT recognlsed by McAb 12.11, shown in Fig l(c), fell from 6 - 1 0 % on D a y 0 to 2 - 3 % after 6 d a y s culture and remained at this level up to Day 9 whereas the proportion of trypanosomes expressing the M-VAT recognlsed by McAb 12 4, shown In Fig l(b), rose from 3 - 6 % on Day 0 to 17-24% on Day 5, remaining at this higher level over the 9 days of the study

Discussion

In order to understand the role which chancre forms of trypanosomes play in the induction of host immunity, it is necessary to obtain more information on antigenic expression a m o n g trypanosomes at this stage of an Infection In this study, in vivo and in vitro systems for these early stages of trypanosomal Infection were employed to investigate M-VAT expression a m o n g the T congolense T R E U 1457 stock M-VAT proportions were determined by IFAT using monoclonal antibodies When one M-VAT was deleted from the population of metacychc trypanosomes by neutrallsatlon using specific antibody, the reappearance of this deleted M-VAT was demonstrated in VlVO in chancres following Infection of rabbits and in vitro on BAE cell monolayer populations While re-expression of deleted M-VATs has been described in lmmunologlcally suppressed hosts for T bruce1 (Barry et a l , 1985) and T congolense (Praln and Ross, 1988), we have now shown that in the chancre of an lmmunologlcally competent host, re-expression of deleted M-VATs also occurs in the presence of a functional immune system Although the deleted 12 5-specific M-VAT was re-expressed in the chancre, It accounted for only 8% of the neutrahsed population at Day 6 compared with 21% of trypanosomes in chancres produced by the unneutrahsed control populations at Day 6 PraIn and Ross (1988) noted a

198 similar degree of re-expression of a deleted M-VAT in their cyclophosphamldetreated mice five days post-infection The results obtained in this study are similar to the in vitro results reported by Praln and Ross (1988), thus confirming the suggestion that metacychc trypanosomes differentiating into dividing populations in VlVO in chancres and in vitro on BAE cell monolayers have the ability to express any M-VAT within the specific repertoire Such antigen switching during the early stages of an infection would be advantageous for the survival of the parasites by increasing their heterogeneity Tsetse flies introduce only small numbers of metacychcs into the skin of the host (Harley and Wilson, 1968, Otleno and Darjl, 1979) therefore it is unlikely that all VATs of the infecting trypanosome's repertoire are represented at this stage In addition, Seed and Sechelskl (1988) found that early in infection with T brucel, host mice were able to recognlse VATs which represent only a small proportion of the heterogenous population Therefore, the subsequent multiplication and antigenic variation of trypanosomes in the chancre following inoculation might be important in the evasion of the host immune response The continued expression of M-VATs six days post-infection in the chancre is in agreement with the observations of Lucklns et al 0990) It was found that the proportions of two unrelated M-VATs were not significantly affected by the neutrahsatlon of a single M-VAT either in VlVO or in vitro Although the immune response does not appear to induce antigenic switching (Doyle et a l , 1980, Vlckerman et a l , 1980) it may affect the proportions of individual M-VATs In culture, however, no mechanisms exist for the removal of M-VATs Therefore in vitro switching in expression of M-VATs or differential growth are probably the mare determinants of the VAT composition of cultured mammalian forms (Lucklns et a l , 1986) However, the expression in VlVO and in vitro of the three M-VATs investigated in this study did not differ markedly, thus supporting suggestions that the in vitro culture system is a useful model for investigating trypanosomes at an early stage of infection The in vitro culture system has the advantage that it is easier to manipulate and to define conditions of growth and there are no host factors to complicate interpretation of results The slightly more rapid rate of regrowth of the deleted M-VAT in VlVO as compared with m vitro probably reflects a higher growth rate among parasites in the chancre The host immune response may, however, be affecting the rate of re-expression of the deleted M-VAT It is possible that selective pressures in VlVO favour the growth of a population not present at the time of infection However, it is unlikely that at such an early stage of infection - - six days - - specific humoral immune responses are operating Luckins et al (1990) found that antibodies against M-VATs of T congolen~e were not detectable in the lymph and serum of rabbits and sheep until day 14 post-infectmn It was noted that the predominant M-VAT of the three investigated differed depending on the trypanosome population The 12 5-specific M-VAT was the most c o m m o n a m o n g in VlVO and in vitro-derived control populations whereas the 12 4specific M-VAT was the most c o m m o n within the in vitro-derived neutralised population and the 12 ll-specific M-VAT the most c o m m o n within the in Vlvo-derived neutrahsed population. The difference in expression of M-VATs following removal of the normally predominant M-VAT from these populations may reflect differences In growth rate in the in VlVO and in vitro culture systems It may be necessary to relate a shorter time period in VlVO with a longer time period in vitro to make the two systems directly comparable

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Acknowledgements Thts work was supported by the Wellcome Trust A G L and C A R are funded by the O v e r s e a s D e v e l o p m e n t A d m t m s t r a t t o n We are grateful to M r C G D B r o w n for helpful comments

References Akol, G W O and Murray, M (1982) Early events following challenge of cattle injected with Trvpanosoma congoleme Development of local skin reaction Vet Rec 110, 295-302 Baltz, T , Baltz, D , Glroud, Ch and Crocket, J (1985) Cultivation in a semi-defined medium of ammal infective forms of Trvpano~oma bru¢et, T equtperdum, T evansz, T rhodeslense and T gambten~e Embo J 4, 1273-1277 Barry, J D , Crowe, J S and Vickerman, K (1985) Neutrallsatlon of individual variable antigen types In metacychc populations of Trvpanosoma bruce: does not prevent their subsequent expression in mice Parasitology 90, 79 88 Crowe, J S, Barry, J D , Lucklns, A G , Ross, C A and Vlckerman, K (1983) All metacychc variable antigen types of Tr)pano~oma congolenw identified using monoclonal antibodies Nature 306, 389-391 Doyle, J J , Hlruml, H , Hlruml, K , Lupton, E N and Cross, G A M (1980) Anhgenlc variation in clones of animal-infective Trvpano~oma brucet derived and maintained m vitro Parasitology 80, 356-369 Gray, M A , Ross, C A , Taylor, A M and Lucklns, A G (19841 In vitro cultivation of Trvpanosoma ~ongolense the production of mfectwe metacycllc trypanosomes m cultures lmt~ated from cloned stocks Acta Trop 41, 343-353 Gray, M A , Ross, C A , Taylor, A , Tetley, L and Lucklns, A G (1985) In vitro cultwatlon of Trvpanosoma congolense the production of infective forms from metacychc trypanosomes cultured on bovine endothehal cell monolayers Acta Trop 42, 99 111 Harley, J M B and Wilson, A J (1968) Comparison between Glossma morsttan~, G palhdtpes and G fus~tpes as vectors of trypanosomes of the Trypanosoma congolenw group the proportions infected experimentally and the numbers of mfectwe orgamsms extruded during feeding Ann Trop Med Parasltol 62, 178-187 Lonsdale-Eccles, J D and Grab, D J (1987) Purtficahon of African trypanosomes can cause biochemical changes In the parasites J Protozool 34, 405 408 Luckms, A G and Gray, A R (1978) An extravascular site of development of Trvpano~oma congolense Nature 272, 613-614 Luckms, A G , Frame, 1 A , Gray, M A , Crowe, J S and Ross, C A (1986) Analysis of trypanosome variable antigen types m cultures of metacycllc and mammahan forms of Tr)panosoma ~ongolense Parasitology 93, 99 109 Luckms, A G , Hopkins, J Rae, P F and Ross, C A (1990) Stability of metacychc variable antigen types (M-VATs) during the early stages of infection with Tr)pano~oma congolenw Acta Trop 47, 129-136 Otleno, L H and Darjl, N (1979) The abundance of pathogenic African trypanosomes m the sahvary secretions of wlld Glos~ma palhdtpes Ann Trop Med Parasltol 73, 583-588 Pram, C J and Ross, C A (1988) Tr)pano,soma congolen~e interactions between trypanosomes expressing different metacychc variable antigen types m vitro and m wvo Parasitology 97, 277-286 Seed, J R and Sechelskl, J B (1988) Immune response to minor variant antigen types (VATs) m a mixed VAT infection of the African trypanosomes Paraslt Immunol 10, 569-580 VJckerman, K , Barry, J D , Hajduk, S L and Tetley, L (1980) Antigenic variation in trypanosomes In H Van den Bossche (Ed), The Host-lnvader Interplay, Elsevier/North Holland Biomedical Press, Amsterdam, pp 179-190