Secretory Immune Response to Membrane Antigens during Giardia ...

INFECTION AND IMMUNITY, Feb. 1998, p. 756–759 0019-9567/98/$04.0010 Copyright © 1998, American Society for Microbiology

Vol. 66, No. 2

Secretory Immune Response to Membrane Antigens during Giardia lamblia Infection in Humans ˜ A-LEYVA,3 DISNEY M. ROSALES-BORJAS,1,2 JUAN DI´AZ-RIVADENEYRA,1 ANTONIO DON ´ ,1 ANTONIO OSUNA,1 SERGIO A. ZAMBRANO-VILLA,4 CARMEN MASCARO 1,5 AND LIBRADO ORTIZ-ORTIZ * Grupo de Bioquı´mica y Parasitologı´a Molecular, Instituto de Biotecnologı´a, Universidad de Granada,1 and EMASAGRA,3 Granada, Spain; Hospital Universitario Dr. Miguel Oraa ´, Guanare, Edo. Portuguesa, Venezuela2; and Centro Universitario de Ciencias Exactas e Ingenierias, Universidad de Guadalajara, Guadalajara, Jalisco,4 and Department of Immunology, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto ´noma de Me´xico, Mexico, D.F.,5 Mexico Received 11 July 1997/Returned for modification 12 August 1997/Accepted 13 November 1997

The secretory immune response in humans infected with Giardia lamblia was studied by using saliva samples and a membrane-rich protein fraction. The membrane fraction, studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed 24 antigen bands, ranging from 170 to 14 kDa. Saliva samples from giardiasis patients showed a heterogeneous response against the membrane fraction when they were assayed by immunoblotting. Among the antigens recognized by patient saliva samples, those of 170, 105, 92, 66, 32, 29, and 14 kDa stood out. These antigens were not recognized by saliva samples from healthy individuals. They may be of importance in future studies of protection from or diagnosis of G. lamblia infections. Giardia lamblia is an important human pathogen of worldwide distribution (17, 22). G. lamblia transmission can be from person to person (12) but is more commonly waterborne, a result of the relative resistance of G. lamblia cysts to chlorination (9). Manifestations of the disease vary from asymptomatic carriage to severe diarrhea and malabsorption. Host factors are thought to be important in determining the severity of the response to this parasite. Immune responses to this protozoan pathogen play a major role in determining the natural history of this infection and in the eventual development of protective immunity (3, 15). Since trophozoites do not appear to invade tissues, mucosal surfaces remain stimulated by Giardia antigens during the entire life span of the parasite. In this case, immunity to G. lamblia is closely associated with the type of immune response generated by mucosa-associated lymphoid tissue (6). Knowledge of the antigenic composition of the parasite and the role that these antigens play in the immune response during infection is important for understanding the pathogenesis of the disease. Likewise, the identification of antigens recognized by the host immune system is of interest for understanding the modulation of G. lamblia infection. In this regard, surface membrane or plasma membrane antigens of G. lamblia seem to be more important because they very likely interact first with the host immune system. In this study, we examined the secretory immune response (SIR) during natural infection to G. lamblia membrane fractions by using saliva samples from patients with giardiasis and immunoblot techniques.

count of viable trophozoites was made with a hemocytometer and 0.2% trypan blue in saline solution. G. lamblia MRPF antigen. Viable G. lamblia trophozoites (9 3 109) were treated with the protease inhibitors phenylmethylsulfonyl fluoride (2 mM), phydroxymercuribenzoate (1 mM), and iodoacetamide (1 mM) (Sigma Chemical Co., St. Louis, Mo.) and then lysed by snap freeze-thaw four times in a dry ice-alcohol slurry and in a 37°C water bath. The membrane-rich protein fraction (MRPF) antigen was prepared by the method of Moss et al. (19). Protein in MRPF was determined by the method of Bradford (2) and stored at 280°C until needed. Saliva samples. Saliva samples were collected from 24 patients for whom a diagnosis of giardiasis had been confirmed by stool examination. In addition, anti-G. lamblia secretory immunoglobulin A (IgA) detection was carried out as described below. The ages of patients ranged from 3 to 53 years; there were 11 males and 13 females. All patients had diarrhea with Giardia cysts and/or trophozoites in their feces at the time saliva samples were obtained. Three of the patients also had Trichuris trichiura organisms, and one patient also had Hymenolepis nana organisms. Control saliva samples were obtained from 19 individuals with no Giardia cysts and/or trophozoites in their feces and no history of giardiasis or symptomatic gastrointestinal disease for the preceding 12 months. Patients and control individuals were from Guanare, Edo. Portuguesa, Venezuela. Saliva samples were centrifuged at 2,500 3 g for 30 min, and the supernatant was frozen at 220°C until used. At testing, the sample was thawed at 4°C and clarified by centrifugation at 14,000 3 g. ELISA. The search for sIgA antibodies to G. lamblia by enzyme-linked immunosorbent assay (ELISA) was performed essentially as previously described (4) with the MRPF antigen (1 mg/50 ml) from G. lamblia and 50 ml of serial dilutions of saliva samples under examination, followed by peroxidase-conjugated affinity-purified goat anti-human IgA (a-chain specific; Sigma) at a 1:1,600 dilution and finally O-phenylenediamine dihydrochloride (Sigma) and 10 ml of 30% H2O2 per 25 ml. Wells were scanned with an STL 210 ELISA reader (Kontron, S.A., Madrid, Spain). Optical densities were read at 492 nm after 15 min. The normal range of each assay was defined as the mean 1 3 standard deviations for the 19 saliva samples from healthy individuals. Electrophoresis and immunoblotting. Trophozoites (3 3 108) or MRPF antigens (10 mg/ml) were boiled (5 min) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer under nonreducing conditions and electrophoresed on SDS–12.5% PAGE gels prepared by the method of Laemmli (13). Western blots with nitrocellulose were performed by the method of Towbin et al. (24). Blots were exposed to saliva samples (tested at 1:8 dilutions), followed by peroxidase-conjugated affinity-purified goat anti-human IgA (a-chain specific; Sigma). After digitization and processing by Gel Scan (K. M. Allaim and M. L. Metcker, Department of Molecular and Human Genetics, Baylor College, Houston, Tex.) the molecular masses of relevant antigens were calculated according to their Rfs. Immunoplot. The molecular masses of antigens in relation to the band frequencies in patient and control saliva samples were analyzed by simple immunoplotting (14). In brief, the immunoplot (see Fig. 5) depicts the frequency with which each G. lamblia antigenic fraction on an immunoblot reacted with saliva

MATERIALS AND METHODS Culture of G. lamblia trophozoites. G. lamblia WB trophozoites (ATCC 30957) were cultured axenically at 37°C in TY1-S-33 medium with 10% bovine serum by the procedure of Keister (11). Parasites were harvested at 72 h and washed three times with cold 19 mM phosphate buffer (pH 7.2) containing 0.27 M NaCl. A

* Corresponding author. Mailing address: Department of Immunology, Instituto de Investigaciones Biome´dicas, UNAM, Apartado Postal 70228, 04510 Me´xico, D.F., Mexico. Phone: (52 5) 622 3890. Fax: (52 5) 550 0048. 756

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FIG. 1. Presence of anti-G. lamblia sIgA in saliva samples from patients with giardiasis (F) or healthy control individuals (‚) with MRPF as the antigen, as determined by ELISA. The horizontal line at 0.106 represents the mean optical density 1 3 standard deviations for saliva samples from healthy individuals.

FIG. 2. SDS-PAGE of Giardia antigens under nonreducing conditions. (A) MRPF antigen; (B) total Giardia antigens. To the right of the lanes is shown the densitogram of the SDS-PAGE gels for MRPF (dotted line) and total (solid line) antigens. Molecular mass standards are also indicated.

samples of infected individuals against the frequency with which the same antigenic fraction reacted with saliva samples from healthy controls. The order of antigenic bands was determined according to their molecular masses, and their presence or absence on each immunoblot was recorded. The frequency value for each band was then determined by dividing the number of saliva samples of a single group (patient or control) which reacted with that particular band by the total number of saliva samples tested in that group. Thus, troublesome bands which reacted frequently with control saliva samples could be identified immediately. Statistical analysis. Fisher’s test was used to determine the homogeneity of variance between groups. When the variance was found to be homogeneous, Student’s t test (7) was applied to estimate the significance of the difference between means. When the variance was heterogeneous, the Mann-Whitney U test (16) was used to estimate the significance of differences.

with molecular masses of 170, 110, 92, 88, 72, 66, 42, 40, 38, 32, 29, 27, 22, and 14 kDa. Bands of 92, 88, 70, 66, 42, 40, and 32 kDa were prominent. On the other hand, control saliva samples reacted with polypeptides of 154, 138, 125, 110, 88, 82, 72, 70, 58, 52, 42, 40, 38, 34, 27, and 22 kDa; however, the intensities of these bands were weaker than those with patient saliva samples. No appreciable reactivity of control saliva samples was observed with bands of molecular masses of 170, 105, 92, 66, 32, 29, and 14 kDa (Fig. 4). Immunoplot analysis. The pattern of reactivity observed with patient saliva samples was very complex; however, reactions occurred specifically with bands of 170, 105, 92, 66, 32, 29, and 14 kDa, with frequencies ranging from 37.5 to 91.4%. On the other hand, control saliva samples (n 5 19) recognized MRPF antigens at the lowest frequencies (Fig. 5).

RESULTS Antibody determination. Saliva samples from individuals infected with G. lamblia showed the presence of antibodies to the MRPF antigen from G. lamblia, as determined by microELISAs. The titers were usually higher than 1:8 and differed significantly (P , 0.001) from those of saliva samples obtained from healthy control individuals (Fig. 1). Electrophoretic analysis. Figure 2 shows the results for SDSPAGE performed under nonreducing conditions with Giardia antigens. Trophozoites showed the presence of approximately 36 bands, ranging from 180 to 14 kDa. When gels were analyzed, the densitogram showed at least 14 well-defined antigens, with molecular masses of 145, 138, 110, 92, 88, 82, 68, 66, 40, 38, 34, 31, 24, and 14 kDa. On the other hand, the MRPF antigen showed only 24 antigens, with molecular masses ranging from 180 to 14 kDa. The densitogram of the MRPF antigen showed 13 well-defined antigens of 170, 138, 92, 88, 72, 70, 66, 58, 42, 40, 38, 32, and 29 kDa, some of which formed doublets. As shown in Fig. 2, some membrane proteins were enriched after obtaining the MRPF antigen. Immunoblot analysis. Figure 3 shows blots of the MRPF antigen analyzed with saliva samples from 24 patients. Patient saliva samples gave very complex patterns of reactivity; collectively, they recognized up to 23 G. lamblia polypeptides of 170 to 14 kDa. Most saliva samples (.50%) recognized antigens

DISCUSSION The data here presented represent one of the first attempts to characterize the SIR in patients with natural Giardia infection by using saliva as a source of sIgA and MRPF from G. lamblia as the antigen. The SIR was studied in saliva samples

FIG. 3. Immunoblot analysis of the Giardia MRPF antigen with saliva samples from patients with giardiasis. Molecular mass standards (in kilodaltons) are shown on the left.

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FIG. 4. Immunoblot analysis of the Giardia MRPF antigen with saliva samples from healthy control individuals. Note that the intensity and number of bands in each blot are lower than those observed with saliva samples from giardiasis patients (Fig. 3). Molecular mass standards (in kilodaltons) are shown on the left.

by immunoblotting, a highly sensitive method that permitted us to assess the importance of protozoan antigenic epitopes in the induction of anti-G. lamblia sIgA during infection. Even though G. lamblia has not often been considered an invasive organism, the antigenic components of the parasite apparently reach mucosa-associated lymphoid tissue to cause a detectable IgA SIR. In this respect, the evidence to date suggests that sIgA in the intestinal lumen is likely to be involved in parasite clearance (5); therefore, identification of the antigenic determinants of the sIgA response may lead us to the discovery of protective antigens. In humans, limited studies have previously detected antibodies to G. lamblia in breast milk and intestinal specimens (1, 8, 10). sIgA antibodies to G. lamblia were found in milk samples from lactating women; Miotti et al. found a relationship between the levels of antibody to G. lamblia and the rate of exposure to this flagellate parasite in the population studied (18). Reiner and Gillin (23) have shown that serum and secretory antibodies recognize many Giardia antigens whose expression is induced by exposure to selected intestinal conditions. Nash et al. (22) showed increases in anti-Giardia serum IgM,

FIG. 5. Immunoplot of simple frequencies of the populations studied patients with acute giardiasis (x axis) versus healthy controls (y axis). The numbers in the immunoplot represent the molecular mass (in kilodaltons) of each Giardia antigen. The 170-, 105-, 92-, 66-, 32-, 29-, and 14-kDa antigens were recognized solely by patient saliva samples (specific bands) and therefore fall on the x axis. Antigens which reacted with saliva samples from both patients and controls (cross-reacting bands) fall between the two axes.

INFECT. IMMUN.

IgG, and IgA and intestinal fluid IgA antibodies after experimental infection of humans. Another interesting finding was that of Giardia antigenic variation in human infections, showing that humoral responses are in part isolated and surface antigen specific (21). Although available evidence suggests that humoral responses are important in antigenic variation in vivo, the actual effector mechanism(s) involved is not known. So far, no direct evidence links sIgA production to Giardia antigenic variation (20). In summary, sIgA from saliva samples of patients with giardiasis recognized various antigens in the MRPF antigen of G. lamblia. The response of these patients was characterized by the presence of anti-G. lamblia sIgA that recognized several membrane antigens; among them were antigens with molecular masses of 170, 105, 92, 66, 32, 29, and 14 kDa which were not recognized by the sIgA from the healthy control population. Although our results were obtained from a limited number of saliva samples, if they are substantiated by further analysis, such results would have implications for the subsequent isolation of important protective or diagnostic G. lamblia antigens. ACKNOWLEDGMENTS This study was supported by CICYT (PB-95-1200.0). L.O.-O. was a recipient of fellowships from the Direccio ´n General de Asuntos del Personal Acade´mico de la Universidad Nacional Auto ´noma de Me´xico and the Direccio ´n General de Ensen ˜anza Superior (Spain). We thank Isabel Pe´rez-Montfort for editing the manuscript. REFERENCES 1. Akerlund, A. S., L. A. Hanson, S. Ahlstedt, and B. Carlsson. 1977. A sensitive method for specific quantitation of secretory IgA. Scand. J. Immunol. 6:1275–1282. 2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 3. Char, S., N. Shetty, M. Narasimha, E. Elliot, R. Macaden, and M. J. G. Farthing. 1991. Serum antibody response in children with Giardia lamblia infection and identification of an immunodominant 57-kilodalton antigen. Parasite Immunol. 13:329–337. 4. Del Muro, R., E. Acosta, E. Merino, W. Glender, and L. Ortiz-Ortiz. 1990. Diagnosis of intestinal amebiasis using salivary IgA antibody detection. J. Infect. Dis. 162:1360–1364. 5. Farthing, M. J. G. 1989. Host-parasite interactions in human giardiasis. Q. J. Med. 70:191–204. 6. Faubert, G. M. 1996. The immune response to Giardia. Parasitol. Today 12:140–145. 7. Fisher, R. A. 1934. Statistical methods for research workers, 5th ed. Oliver and Boyd, Edinburgh, United Kingdom. 8. Islam, A., B. J. Stoll, I. Lungstroem, J. Biswas, H. Nazrul, and G. Huldt. 1983. Giardia lamblia infections in a cohort of Bangladesh mothers and infants followed for one year. J. Pediatr. 103:996–1000. 9. Jarroll, E. L., A. K. Bingham, and E. A. Meyer. 1981. Effect of chlorine on Giardia lamblia cyst viability. Appl. Environ. Microbiol. 41:483–487. 10. Jones, E. G., and W. R. Brown. 1974. Serum and intestinal fluid immunoglobulins in patients with giardiasis. Am. J. Dig. Dis. 19:791–796. 11. Keister, D. B. 1983. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans. R. Soc. Trop. Med. Hyg. 77:487–488. 12. Keystone, J. S., S. Krajden, and M. R. Warren. 1978. Person-to-person transmission of Giardia lamblia in day-care nurseries. Can. Med. Assoc. J. 119:241–248. 13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. 14. Larralde, C., R. M. Montoya, E. Sciutto, M. L. Dı´az, T. Govezensky, and E. Coltor. 1989. Deciphering Western blots of tapeworm antigens (Taenia solium, Echinococcus granulosus, and Taenia crassiceps) reacting with sera from neurocysticercosis and hydatid disease patients. Am. J. Trop. Med. Hyg. 40:282–290. 15. LoGalbo, P. R., H. A. Sampson, and R. H. Buckley. 1982. Symptomatic giardiasis in three patients with X-linked agammaglobulinemia. J. Pediatr. 101:78–80. 16. Mann, H. B., and D. R. Whitney. 1947. On a test of whether one of two variables is stochastically larger than the other. Ann. Math. Stat. 18:50–60. 17. Meyer, E. A., and E. L. Jarroll. 1980. Giardiasis. Am. J. Epidemiol. 111:1–12. 18. Miotti, P. G., R. H. Gilman, L. K. Pickering, G. Ruiz-Palacios, H. S. Park,

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