INFECTION AND IMMUNITY, Aug. 1994, p. 3441-3446 Vol. 62, No. 80019-9567/94/$04.00+0

Mice Infected with Trypanosoma cruzi Produce Antibodiesagainst the Enzymatic Domain of trans-Sialidase

That Inhibit Its ActivityMARIA S. LEGUIZAMON,' OSCAR E. CAMPETELLA,2 STELLA M. GONZALEZ CAPPA,I

AND ALBERTO C. C. FRASCH2*Departamento de Microbiologia, Facultad de Medicina, Universidad de Buenos Aires,' and Instituto

de Investigaciones Bioquimicas, "Fundaci6n Campomar,"2 Buenos Aires, Argentina

Received 7 October 1993/Returned for modification 20 December 1993/Accepted 1 June 1994

trans-Sialidase (TS) is an enzymatic activity described only for trypanosomes that is involved in the invasionof host cells by Trypanosoma cruzi. The enzyme that is shed by the parasite is made of two domains, theC-terminal region containing immunodominant amino acid repeats that define the SAPA antigen and theN-terminal domain that contains the putative region for enzymatic activity. The SAPA antigen induces a stronghumoral response detected shortly after infection, both in humans and in mice. This response is directed to theimmunodominant domain but is irrelevant in terms of neutralization of TS activity. We now show that TSactivity can be detected in sera from acutely infected mice. However, mice infected with a T. cruzi strain whosegrowth can be controlled by the host did not have detectable levels ofTS activity in sera. In fact, sera from thesemice were able to abolish TS activity. This inhibition was due to the presence of specific antibodies directedagainst the enzymatic domain of the protein since they also abolish the activity of a recombinant moleculelacking the immunodominant amino acid repeats. The neutralizing antibodies were present from day 30 afterthe infection, while antibodies to the immunodominant repeats were detected by day 8 postinoculation,suggesting that the in vivo role of these repeats is to defect the humoral response to the repeat domain untilthe infection is established.

Trypanosoma cruzi is the protozoan parasite agent of Cha-gas' disease. The parasite possesses a digenetic life cycle,involving a hematophagous insect vector (kissing bugs) and amammalian host. In natural infections, metacyclic trypomas-tigotes present in the insect feces invade the mammalian hostthrough mucose or microlesions in the skin. Metacyclic trypo-mastigotes are unable to duplicate. They must invade cells inorder to differentiate into intracellular amastigotes that, afterseveral duplicating cycles, give rise to nonduplicative blood-stream trypomastigotes that invade other cells.

SAPA-trans-sialidase (TS)-neuraminidase (NA) was inde-pendently described as an antigenic molecule named SAPA forshed-acute-phase-antigen (1, 18) and as the NA of the parasite(16) and was recently found to be the TS of T. cruzi (15, 28). TSis an enzyme described so far only for trypanosomes, whichdiffers from sialyl transferases (15, 20, 25, 29). TS uses glyco-lipids and glycoproteins as sialic acid donors, instead ofCMP-sialic acid (15, 25, 29), and it is located in the surfacemembrane of trypomastigotes of T. cruzi (27) and shed into themedium (1). Sialic acid can also be hydrolyzed from a sialy-lated compound by this enzyme (NA activity). However, thishappens when no suitable acceptor molecules are present (15).

Recent results suggest that TS is one of the factors producedby T. cruzi that is involved in the invasion of host cells. Itsialylates epitopes present on the surface of the trypomastig-ote, named Ssp-3, that are required for invasion (2, 24, 25).Monoclonal antibodies against the Ssp-3 epitope inhibit at-tachment and invasion by trypomastigotes (24, 26), and incu-bation of trypomastigotes with molecules containing sialic acid

* Corresponding author. Mailing address: Instituto de Investigacio-nes Bioquimicas, "Fundaci6n Campomar," Av. Patricias Argentinas435, 1405 Buenos Aires, Argentina. Fax: 54-1-8652246 and 54-1-881916.

enhances invasion (17). Sialylated molecules in the host cellsmight also be important for invasion since mutant cell linesthat express less sialic acid are poorly invaded and this functionis restored by resialylation of the host cells (12, 23). TS has alsobeen suggested to be a counterreceptor from trypomastigotesbinding to a-(2,3)-sialyl receptors on host cells as a prelude toT. cruzi invasion (15).TS seems to be a naturally chimeric protein having function-

ally independent enzymatic and antigenic domains (6). Theenzymatic domain was suggested to be located on the aminoside of the molecule, but it remains to be demonstratedwhether this is the only region of the molecule required toconfer enzymatic activity. The antigenic domain is located onthe carboxy terminus of the molecule and is made up oftandemly repeated 12-amino-acid units which constitute theSAPA epitopes. These epitopes induce an early and strongantibody response in acute and congenital human infections (1,22) as well as in mouse infections (11). Consequently, theseamino acid repeats were suggested to constitute the immuno-dominant portion of the molecule (6). A similar conclusion wasreached recently through the mapping of B-cell epitopes inanother molecule belonging to the SAPA-TS protein family(21). However, the anti-SAPA repeat response is unable toneutralize the TS activity (15), suggesting that the immuno-dominant domain plays a distractive role to delay the inductionof a neutralizing humoral response against the catalytic do-main.

After the acute period of the infection, T. cruzi growth iscontrolled by the host and parasites are no longer detectable inlarge amounts in blood. Given the suggested importance of TSin the process of the infection, generation of antibodies thatinhibit the enzyme should be expected in order to control theinfection. With this hypothesis in mind, we decided to assay TSactivity and anti-TS antibodies in serum samples obtained from

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3442 LEGUIZAMON ET AL.

mice infected with two T. cruzi populations that show differentbiological behaviors, as do K-98 and RA. The former is a cloneobtained from a myotropic strain (CA-I) which hardly infectsphagocytes and is not lethal for mice (3). In contrast, RA is apantropic strain, which successfully invades phagocytes and ishighly lethal for mice (10). In this paper, we show thatantibodies able to neutralize TS activity were induced only inT. cruzi-infected mice able to survive the acute phase. Anenzymatically active recombinant molecule that lacks theamino acid repeats was used to show that antibodies thatinhibited TS are directed against the enzymatic domain of themolecule.

MATERIALS AND METHODS

Mice. Rockland and C3H/HeN mice were bred at theDepartamento de Microbiologia, Facultad de Medicina, Uni-versidad de Buenos Aires, Buenos Aires, Argentina. NIH nude(nulnu) mice were obtained from the Comision Nacional deEnergia Atomica, Ezeiza, Buenos Aires.

Parasites. The RA strain and the K-98 clone of T. cruzi weremaintained by serial passages in Rockland mice. Twenty-day-old Rockland mice were injected with 3 x 105 bloodstreamtrypomastigotes and were bled at day 7 postinoculation (p.i.)when all animals died displaying maximum parasitemia values.Three-month-old C3H/HeN male mice were inoculated intra-peritoneally with 50 parasites of the RA strain or 50,000 fromthe K-98 clone for the acute and the chronic model, respec-tively (7). Serum samples and parasitemia were evaluated fromdays 10 to 18 after inoculation with the RA parasites. In theacute model, 100% mortality is observed at day 18 p.i. (10). Inthe chronic model, serum samples and parasitemia wereevaluated from days 18 till 113 p.i. Mortality in this model isusually only 10% (7). Some serum samples were taken at day8 p.i. To obtain parasites free of seric antibodies, NIH nudemice were inoculated intraperitoneally with 50 parasites of theRA strain or with 100,000 from the K-98 clone.TS assays. Serum or parasite lysate samples (20 RI) were

assayed for TS activity in a final volume of 30 pLI containing(final concentrations) 1 mM sialyllactose (N-acetylneuramin-lactose, a mixture of 72% 2-3 and 28% 2-6 isomers [Sigma, St.Louis, Mo.]), 50 mM PIPES buffer [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 7.0), and 0.4 nmol (about 40,000cpm) of 14C-labelled lactose (54.3 mCi/mmol [Amersham,Little Chalfont, United Kingdom]). After 1 h of incubation at37°C, 1 ml of water, followed by 100 ,ul of a dense slurry ofquaternary aminoethyl-Sephadex A-25 (Sigma) in water, wasadded. After being vortexed briefly, beads were pelleted bycentrifugation and washed three times with 1 ml of water, thebound material was eluted with 1 ml of 1 M ammoniumformate, and the counts per minute were quantitated. To assayfor the presence of TS in bloodstream parasite lysates, infectedNIH nude mice were bled and the heparinized blood sampleswere centrifuged at low speed to pellet the erythrocytes.Supernatants were spun at 10,000 x g for 30 min, and pelletedparasites were washed with phosphate-buffered saline (PBS)plus 5% bovine serum albumin. Parasites were resuspended inPBS and lysed by addition of (final concentrations) 1% TritonX-100-1 mM phenylmethylsulfonyl fluoride-0.5 mM Noa-p-tosyl-L-lysine chloro-methyl ketone-0.5 mM trans-epoxysucci-nyl-L-leucylamido-(4-guanidino)butane-5 mM O-phenanthro-line (all from Sigma). Lysates were centrifuged at 10,000 x gfor 10 min, and 20 RI of the supernatants (equivalent to 2 x 106parasites) were assayed for TS activity.

Clostridium perfringens NA treatment. TS reactions wereperformed exactly as described above, but after the 1-h incu-

bation, 0.01 U of C. perfringens NA (type X [Sigma]) was addedand incubation was continued for 30 min. Controls wereincubated for 90 min without NA addition. Reactions werestopped with 1 ml of water and processed as described above.Paper electrophoresis. After the TS reaction was stopped

with 1 ml of water, samples were subjected to Sephadex G-25chromatography (NAP-10 columns [Pharmacia-LKB, Uppsala,Sweden]) to deplete serum proteins. Included volume wasdried, resuspended in 20 RI of water, and electrophoresed onWhatman no. 1 filter paper at 25 V/cm for 2 h. The bufferemployed was pyridine-acetic acid-water (1:0.04:9 [vol/vol]),pH 6.5. Lactose and sialyllactose (Sigma) were employed asmigration markers.

Purification of antibodies. Sera were diluted 1/40 with PBS,and an equal volume of a 50% suspension of protein A-Sepharose (Sigma) in PBS was added. After 30 min of incu-bation with gentle rocking at room temperature, the superna-tants were harvested and the beads were washed three timeswith 1 ml of PBS. Antibodies were eluted by adding 50 [lI of 0.1M glycine-HCI (pH 2.6) to the pelleted beads, and after a5-min incubation at room temperature, the tubes were centri-fuged briefly. The supernatants containing the antibodies wereneutralized with 10 RI of 1 M Tris-HCI (pH 9.5).Dot spot assay. A fusion recombinant protein containing the

SAPA repeats (1) in the pGEX vector (Pharmacia-LKB) wasaffinity purified through a glutathione-agarose column (Sigma)and employed to analyze the presence of antibodies against theTS amino acids repeats in sera from mice. Spots (1 RI)containing 4 ,ug of protein obtained either from pGEX-SAPAor from pGEX-transformed bacteria were made on nitrocel-lulose sheets. After being blocked with 3% nonfat milk in PBS,filters were incubated for 1 h with a 1/20 to 1/50 dilution in PBSof each serum to be assayed. After being washed with PBS,filters were treated with alkaline phosphatase-conjugated goatanti-mouse immunoglobulin G (IgG) (Promega, Chicago, Ill.)diluted 1/2,000 in PBS, and the reaction was developed color-imetrically.TS activity-inhibition assays. Sera or antibodies obtained by

purification with protein A were mixed with affinity-purified TS(2 RI) obtained from RA strain-infected Vero cell cultures (19)or with 10 ,lI from the soluble fraction of bacteria containing arecombinant DNA clone (Tc-TS1N1) expressing the enzymaticdomain of TS (see below). After 10 min at room temperature,remanent TS activity was assayed as described before.

Identification of the recombinant TS clone. Details concern-ing the construction of Tc-TS1N1 have been published else-where (5). Briefly, genomic fragments from T. cruzi RA DNAwere cloned in pBluescript (Stratagene, La Jolla, Calif.) afterdigestion with SacII. Colonies were screened for the presenceof SAPA repeat-codifying sequences (1). After that, PCR wasperformed on the positive recombinant clones with a pair ofoligonucleotides designed according to the mature proteinsequence (19) and to the sequences just before the tandemamino acid repeats, which include a third of the first repeat.PCR products were cloned in pKK 223-3 (Pharmacia-LKB),and protein was expressed as described by Uemura et al. (28).

RESULTS

TS activity can be detected in sera from mice infected withT. cruzi. Enzymatic activity was assayed in sera from infectedmice under conditions that were shown to be specific to detectTS (15). Sera from six 20-day-old Rockland mice infected withthe RA strain of T. cruzi were assayed independently andfound to transfer sialic acid from a donor to a labelled acceptormolecule (Table 1). The product of the reaction was sensitive

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TABLE 1. Parasitemia and TS activity (in counts per minute)in sera from 20-day-old Rockland mice infected

with the RA strain of T. cnziP

Serum No. of parasite/ml cpm

1 8.8 X 106 1,2282 1.3 x 107 1,6643 2.4 x 107 1,8854 3.5 x 107 2,8135 6 x 106 1,3546 6 X 106 902Normalb 65

" Twenty microliters of serum was assayed for TS activity.b A pool of sera from five uninfected mice used as control.

800

- 600

> 400

I 200CD

0

to treatment with C. perfringens NA, and paper electrophoresisconfirmed that it was sialyllactose (Fig. 1). The sera used forthese assays were collected at day 7 p.i., when all the animalsdied displaying high parasitemia values. Sera from controlanimals tested under identical conditions did not transfer sialicacid to a labelled acceptor molecule (Fig. 1 and Table 1). We

0O RA+ K-98

0

0

0

2 4 6

1 06 parasites/mlFIG. 2. TS activity in sera taken from 3-month-old C3H/HeN male

mice infected with either RA or K-98 populations of T cruzi. Datawere plotted and lineal curves were fitted by using the KaleidaGraph2.1 program for the Macintosh computer. The 0* means that para-sitemia values were found to be lower than the detection level (10,000parasites per ml).

cpm 1

1 onn-4

1000

1000

2

A

15

FIG. 1. Paper electrophoresis of products obtained from the TSreaction performed with 20-day-old Rockland mouse sera. (A) Resultsobtained with a pool of five noninfected mouse serum samples; (B)products obtained with serum taken from a heavily infected 20-day-oldRockland mouse; (C) same as panel B, after treatment with C.perfringens NA. The markers used were lactose (arrow 1) and sialyllac-tose (arrow 2).

conclude that TS activity is present in sera from mice heavilyinfected with T. cruzi.TS activity in sera from acute and chronic mouse experi-

mental models. To study TS activity in the murine acute andchronic models of the infection, the RA strain and the K-98clone of T. cruzi were used to infect 3-month-old C3H/HeNmale mice. RA parasites are lethal within 20 days afterreceiving an inoculum of 50 parasites. In contrast, an inoculumof 50,000 parasites of the K-98 clone induces a chronicinfection in which animals survive after controlling the para-sitemia peak (7). Therefore, RA and K-98 parasite populationswere used as acute and chronic infection models, respectively.Sera from mice infected with RA were collected at days 10 to17 p.i., with day 10 being the earliest day p.i. that parasitescould be detected by microhematocrite analysis. TS activitywas detected in sera only when parasitemia values were higherthan 105 parasites per ml (Fig. 2, circles). TS activity was higherwith increasing parasite concentration in blood. In contrast, noTS activity was detected in sera from mice infected with K-98(chronic model) from day 18 (when parasites became detect-able) to day 113 p.i. (Fig. 2, crosses), even in serum samplestaken when parasitemia values were similar to those present inRA-infected mice. Sera from control C3H/HeN mice did nothave TS activity.A neutralizing activity of TS is detectable in sera from

chronically infected mice. The absence of detectable TS inK-98-infected mice might be due to a neutralizing activitypresent in their sera. To test this hypothesis, undiluted serumsamples from K-98-infected mice collected from days 18 to 113p.i. were mixed with partially purified TS obtained frominfected Vero cell supernatant, and after 10 min of incubation,the remanent TS activity was assayed. Parallel assays wereperformed with undiluted normal mice sera whose inhibitoryeffect on the TS activity was found to range between 10 and23%. Therefore, count per minute values in the TS inhibitionassay obtained with a pool of normal mouse sera were consid-ered as 0% inhibition. An inhibitory TS activity that increasedwith the postinfection time was observed in sera from K-98-infected mice, reaching values of almost 90% inhibition (Fig.

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3444 LEGUIZAMON ET AL.

TABLE 2. Neutralization of TS activity by sera and IgG fractionsfrom C3H/HeN mice infected with T. cruzi K-98 parasites'

Inhibition of TS activity*

Protein A-SepharoseMouse Days p.i. Serum

Unbounded Eluted

% cpm % cpm % cpm

6258 113 88.3 172 0 1,476 76 3536264 60 86.4 200 10.2 1,321 69.4 4506267 113 79.7 298 0 1,510 73.1 3956276 86 74.1 380 30.2 1,026 41.1 8666284 60 75.8 355 7.5 1,361 44.3 819Normal 0 1,471 8.7 1,342 0 1,580

a Partially purified TS obtained from infected Vero cell cultures was mixedeither with sera diluted 1/80, with the unbounded fraction, or with the IgGfraction (8 p,g) eluted at pH 2.6 from protein A-Sepharose.

b The 0% inhibtion value corresponds to that obtained with a pool of sera fromfive control animals (Normal).

10-21 22-40 41-113

Days piFIG. 3. TS-inhibitory activity present in sera from C3H/HeN mice

infected with the K-98 clone of T. cruzi. Undiluted sera were mixedwith purified TS, and after 10 min of incubation, remanent TS activitywas assayed. Percentage of inhibition is based on values obtained witha pool of normal sera being considered 0% inhibition.

3). Serial dilutions of these highly neutralizing sera wereperformed in order to determine the neutralization titersreached, which were found to range between 1/160 and 1/2,560.Interference with the TS assay due to normal serum compo-nents was no longer detectable after 1/40 dilution (data notshown).The absence of TS activity in sera from K-98-infected mice

might also be due to a low TS expression by the parasite. Toanalyze this alternative, athymic mice were inoculated with RAor K-98 parasites to prevent the humoral response against TS.Bloodstream forms were purified and assayed for TS activity.No differences were observed when lysates from 2 x 106parasites were tested (2,920 and 3,158 cpm for RA strain andK-98 clone, respectively). Therefore, the absence of TS activityis due not to the absence of expression by the parasite but tothe presence of humoral response-neutralizing factors.

Detection of TS activity in sera from RA-infected mice(acute model) suggests that the TS-neutralizing response iseither present in low amounts or not present in these animals.In fact, sera taken from K-98-infected mice, which containsTS-neutralizing activity, were able to abolish the enzymaticactivity present in sera from RA-infected mice (data notshown).

TS-inhibitory activity in sera from mice infected with T.cruzi K-98 is due to antibodies. In order to know whether theneutralizing activity detected in sera from mice infected withthe K-98 clone of T. cruzi was due to the presence ofantibodies, serum samples obtained at different periods afterthe infection were diluted 1/40 and fractionated on proteinA-Sepharose. The unbounded fraction and the IgG fractioneluted at low pH were tested in the TS inhibition assay. Asshown in Table 2, unbound material loses the ability toneutralize TS activity. The inhibitory activity was recovered, todifferent extents, in the eluted material. These results showedthat antibodies were responsible for the inhibition of TS. In

spite of the absence of neutralizing activity in the acute model,dot spot assays performed with a recombinant protein havingthe tandem amino acid repeats showed that IgG anti-SAPAwas present from days 8 to 10 p.i. in both K-98- and RA-infected mice, lasting throughout the experimental period(data not shown) as described in previous reports (11).

Neutralizing antibodies are directed to the enzymatic do-main of the TS. To know whether the inhibitory antibodiespresent in sera from murine experimental infections weredirected against the enzymatic domain of TS, a recombinantmolecule lacking the amino acid tandem repeats was em-ployed. This recombinant clone (Tc-TS1N1) expresses a pro-tein with TS activity that is identical to the one displayed by thenative TS (5).As shown in Table 3, Tc-TSlNl activity was abolished by

sera taken at different times p.i. from K-98-infected mice. Anantibody fraction obtained by protein A-Sepharose from twoof these mice gave the same results (Table 3). Control exper-iments performed with sera from normal mice or a monoclonalantibody directed against the amino acid repeats (11) failed toinhibit the Tc-TSlNl activity (Table 3).

TABLE 3. Neutralization of Tc-TSlN1 recombinant TS by sera andIgG fractions of T. cruzi-infected C3H/HeN mice'

Tc-TSIN1 neutralization

Mouse Days p.i. Serum Protein A-purifiedIgG

So cpm % cpm

6258 113 92 140 71 3716262 63 86 235 NDb ND6264 60 74 434 85.7 2426267 113 82 310 ND ND6284 60 64 608 ND NDMAbc 2.47 1,695Normal 0 1,695 ND ND

a A soluble fraction from bacteria transformed with Tc-TSlN1 containing TSactivity was mixed either with sera diluted 1/80 or with an IgG fraction purifiedby protein A-Sepharose (8 ,ug). The results obtained with a pool of normal serafrom five animals were considered as 0% inhibition.

b ND, not determined.c MAb, anti-SAPA amino acid repeat mouse monoclonal antibody (11).

100

o 80'._

-Clc 601

_A 40''I-0a20

0

0

0

0

0

0

8

0

0

0

0

8

0

00

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DISCUSSION

TS was suggested to be a chimeric molecule evolutionarilyselected to have two functionally independent domains (seereference 6 for a review). The antigenic domain is located onthe carboxy side of the molecule and contains the immuno-dominant tandem repeats (SAPA antigen) (1). This antigenicdomain was suggested to lack any influence on the enzymaticactivity, which was suggested to be located on the amino side ofthe molecule (6). Preliminary evidence on this comes fromexperiments in which different regions of inactive and activerecombinant molecules were combined (6). We have recentlyobtained a recombinant protein lacking the repeats (5) whichretains the enzymatic activity. Thus, it is now clear that theamino acid repeats are not required for the TS activity.TS is present on the parasite surface and shed into the

medium (18, 27). It is one of the first molecules from theparasite detected by the immune system of the host, and mostantibodies to TS generated by immunization with parasites orpurified proteins or during natural or experimental infectionsare directed to the tandem amino acid repeats (1, 11, 22).These antibodies do not abolish TS activity (6, 15). We havenow demonstrated that antibodies produced during experi-mental infections against the enzymatic domain of the mole-cule are able to neutralize TS activity. This neutralizationactivity is genuine since it is able to abolish the TS activityassayed on small substrates. Polyclonal and monoclonal anti-bodies to T. cruzi NA, directed against the repeats, were ableto inhibit enzymatic activity when assayed on erythrocytes (21).However, these antibodies were unable to inhibit the activitywhen assayed on small substrates. Therefore, as suggested bythe authors, the inhibition of the erythrocyte assay may bebased only on a steric impediment of access to the target (21).Previous works showing NA-inhibitory activity in one case ofan acute human infection or in experimental mice infectionsevaluated on erythrocytes should also be analyzed in thiscontext (8, 9).TS is considered one of the factors involved in the establish-

ment of the infection caused by T. cruzi. A possible mode ofaction proposed is through the generation of a sialylatedepitope required for the invasion of host cells (25, 26). Wehave now shown that mice able to control the parasitemia peakhave serum antibodies able to neutralize TS activity. It isinteresting to note that C3H/HeN mice infected with a parasitestrain such as RA whose growth is not controlled by the host donot generate detectable levels of antibodies inhibiting TSactivity. Consequently, TS is detectable in sera from RA-infected mice. The interesting hypothesis that the inability toblock TS activity is hampering the control of parasite dissem-ination and death has to be analyzed in the future. In supportof this hypothesis, sera taken from the few survivors of theinfection with T. cruzi RA in less susceptible mouse strains likeBALB/c or Rockland contain TS-neutralizing antibodies (datanot shown). The RA survivors are reported to display ahumoral response against the bloodstream form of the parasitethat can be functionally evaluated as opsonins and lysins andalso as antibodies able to neutralize parasite infectivity; incontrast, in mice infected with K-98, or its mother strain CA-I,these humoral responses are seldom induced, although anti-bodies against the parasite are clearly present (7, 13, 14).However, both models are surviving a T. cruzi infection andanimals from both groups have TS-neutralizing antibodies.

Antibodies to repeats were induced early (8 to 10 days p.i.),in both the acute (RA-infected) and the chronic (K-98-infected) models, and they lasted for all the experimentalperiod, consistent with previous reports (11). On the other

hand, TS-neutralizing antibodies were detected at about day 30p.i. only in the chronic model since mice from the acute modeldied before displaying detectable neutralizing activity. It hasbeen shown that there is a superfamily of TS-related antigenslacking enzymatic activity (see reference 4 for a recent review).Therefore, RA-infected mice may be trapped in a network ofTS-related antigens, being unable to induce high enoughamounts of the effectively neutralizing antibodies. Altogether,these results might suggest the following picture for theinterrelation between SAPA-TS and the immune response ofthe host during the acute phase. After the infection, TS eitheracts on the parasite surface or free in the milieu after shedding,to generate the sialylated epitopes required for cell invasion.Early after the infection (at about day 8 in mouse infections),antibodies against the immunodominant domain, the aminoacid repeats, are generated. They do not block enzymaticactivity, and consequently, parasites are not hampered indisseminating the infection to other cells. At about day 30 afterthe infection, antibodies against the enzymatic domain of themolecule are produced by the immune system of the host. Theyinhibit the TS activity, helping to control the parasitemia. Ifthis model can be confirmed, SAPA-TS might become aconcrete example of a bifunctional molecule from a protozoanparasite in which two important functions for T cruzi survival,an enzymatic activity and an immunodominant epitope, arepresent in two functionally independent domains from thesame molecule.

ACKNOWLEDGMENTS

We are indebted to A. Uttaro for his valuable help in the paperelectrophoresis assays. The technical skills of M. Perri are alsoappreciated.

This work was supported by grants from the World Bank/UNDP/WHO Special Programme for Research and Training in TropicalDiseases (TDR), the Swedish Agency for Research Cooperation withDeveloping Countries (SAREC), the University of Buenos Aires,the Consejo Nacional de Investigaciones Cientificas y Tecnicas(CONICET), and the Fundaci6n Antorchas, Buenos Aires, Argentina.

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