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Giardia-specific antibodies: their in-vitro anti-parasitic effects and a preliminary characterization… Ryan, Paula Patricia 1991

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GIARDIA-SPECIFIC ANTIBODIES: THEIR IN-VITRO ANTI-PARASITIC EFFECTS AND A PRELIMINARY CHARACTERIZATION OF THEIR TARGET ANTIGENS by Paula P a t r i c i a Ryan B.Sc, McGill University, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 12, 1991 © Paula P a t r i c i a Ryan, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Microbiology The .University of British Columbia Vancouver, Canada Date June 25, 19 91 DE-6 (2/88) Abstract In order to evaluate host defense mechanisms against the i n t e s t i n a l parasite G.lamblia, assays have been established which measure the a b i l i t y of both immune sera and f e c a l extracts to immobilize Giardia trophozoites. The immobilizing a c t i v i t y of immune serum against two stra i n s of Giardia was shown to be dependent on complement. When immune serum was absorbed against l i v e trophozoites, the immobilizing a c t i v i t y was abolished. These data, i n conjunction with r e s u l t s from immunoblots and immunofluorescense assays, indicate that a complement dependent, a n t i - g i a r d i a antibody response renders trophozoites immobilized i n - v i t r o . In an attempt to use these a n t i - p a r a s i t i c antibodies to i d e n t i f y g i a r d i a surface antigens, an immunoabsorption technique was developed to i s o l a t e immobilizing antibodies from immune sera by absorbing them to the surface of trophozoites. Surface-bound antibodies were recovered using a low pH glycine buffer. Antibodies which were eluted from the surface of trophozoites could not be shown to possess immobilizing a c t i v i t y . However, the sur f a c e - s p e c i f i c antibodies which were is o l a t e d were used to i d e n t i f y a number of Giardia surface antigens from preparations of whole trophozoite antigens as well as enriched membrane preparations. TABLE OF CONTENTS ABSTRACT p i i TABLE OF CONTENTS p i i i - i v LIST OF TABLES pv LIST OF FIGURES pvi ACKNOWLEDGEMENTS p v i i INTRODUCTION I. Description of G.lamblia pl-2 I I . Course of i n f e c t i o n p2 I I I . Pathogenesis p2-7 IV Mechanisms of host defense p8-10 V. Antigenic determinants plO-12 VI. Membrane extraction pl3-14 VII. Purpose of t h i s study pl4-15 MATERIALS AND METHODS pl6-29 I. Isolates pl6 I I . Culture media pl6-17 I I I . Bulk Harvest pl7-18 IV. Antigen preparation pl8-19 V. Preparation of rabbit antisera p20 VI. Gerbil model p21 VII. Inoculation of g e r b i l s p21 VIII. Preparation of g e r b i l f e c a l extracts p22 X. Immunoabsorption study p22-2 3 XI. In-vitro m o t i l i t y assay p24-25 i i i XII. In-vitro v i a b i l i t y assay XIII. Indirect Immunofluorescense assay XIV. Electrophoresis and immunoblot technique p25 p26 p27 -26 -27 -29 RESULTS I. I n - v i t r o e f f e c t of antisera against trophozoite m o t i l i t y p30,3 3 I I . Mechanisms of a n t i - m o t i l i t y e f f e c t p33,35 I I I . I n - v i t r o k i l l i n g assay p35,38 IV. Correlation between immobilization and k i l l i n g p38,41 V. S e n s i t i v i t y of trophozoites to immune serum p41 VI. S t r a i n v a r i a b i l i t y p41,45 VII. E f f e c t of fe c a l extracts on trophozoite m o t i l i t y p45 VIII. Absorption of immune sera p48,51 IX. Functional a c t i v i t y of depleted and eluted fractions p51,52 X. Indirect immunofluoresence assay p52 XI. Antigen s p e c i f i c i t y of depleted and eluted fract i o n s p56,59 XII. Extraction of Giardia membrane proteins p59,62 DISCUSSION AND SUMMARY p69-84 REFERENCES p85-99 i v LIST OF FIGURES f i g 1. In-vitro m o t i l i t y assay p31 2. M o t i l i t y assay using heat inactivated immune sera p36 3. In-vitro v i a b i l i t y assay p39 4. Immobilization of G.lamblia with d i l u t i o n s of immune serum p42 5. In-vitro assay using fecal extracts p46 6a. Protein composition of antibody f r a c t i o n p49 6b. Detection of surface-bound antibodies a f t e r e l u t i o n procedure p53 7. Detection of Giardia surface antigens using antibody f r a c t i o n (TCA antigens preparations) p57 8. Detection of Giardia surface antigens (1:8 d i l u t i o n of whole antibody fraction) p60 9a. Composition of membrane enriched preparation of 501 st a i n p63 9b. Detection of Giardia surface antigens using whole antibody f r a c t i o n (enriched membrane preparation) p65 9c. Detection of Giardia surface antigens using eluted antibody f r a c t i o n (enriched membrane preparation) p67 v LIST OF TABLES Table I. In-vitro immobilization of 501 s t r a i n p34 I I . Immobilization of L and DEL st r a i n s of G.lamblia trophozoites by pre-immune sera p44 I I I . E f f e c t of depleted and eluted antibody fractions on trophozoite (501 strain) m o t i l i t y p53 v i ACKNOWLEDGEMENTS Special thanks to my family, the coffee gang and e s p e c i a l l y M.L.B. for moral support. I would also l i k e to thank my supervisor W.R.Bowie for providing patient and t a c t f u l guidance, M.Krystal f o r h i s technical assistance, J.Isaac-Renton for allowing free reign i n her lab, and my supervisory committee members B.C.McBride and R.E.W.Hancock for t h e i r invaluable advice and d i r e c t i o n throughout t h i s project. The funding for t h i s project was provided by the Medical Research Council of Canada. INTRODUCTION I. DESCRIPTION OF GIARDIA Giardia i s an i n t e s t i n a l protozoan responsible for endemic and epidemic diarrhea throughout the world (38). Infection with t h i s parasite i s usually transmitted v i a f e c a l l y contaminated water or food although i t can also be transmitted from person to person (41). Giardia exists i n two stages, the i n f e c t i v e cyst stage and the r e p l i c a t i v e , motile trophozoite stage. Giardia cysts are oval shaped and range i n si z e from 8-12 urn i n length and 7-10 urn i n width. They're encapsulated by a cyst wall containing c h i t i n . The trophozoite form i s pear shaped with a f l a t ventral surface and a convex dorsal surface. It's size ranges from 9-21 um i n length by 5-15 um i n width (36). Internally, trophozoites are composed of axonemes, microtubules, vacuoles and a p a i r of median bodies. They are also binucleated. A d i s t i n c t i v e feature of Giardia trophozoites i s the adhesive or ventral d i s c . This d i s c i s composed of a cytoskeleton which i s made up of an ordered monolayer of c o i l e d microtubules. The microtubules are held adjacent to the plasma membrane by a network of microribbons and electron dense cross-bridges (11-14,37). Giardia cytoskeletons also contain four pairs of f l a g e l l a which are attached to the adhesive d i s c . The 1 axonemes of these f l a g e l l a are the 9+2 arrangement of microtubules found i n most eukaryotic f l a g e l l a (36). Three Giardia species have been i d e n t i f i e d based on the morphology of t h e i r median bodies: G.lamblia (also c a l l e d G . i n t e s t i n a l i s or duodenalis), i n f e c t i n g humans and other mammals, G. muris, found i n rodents, and G . a g i l i s , found i n amphibians (77). I I . COURSE OF INFECTION I n i t i a t i o n of i n f e c t i o n involves oral ingestion of as l i t t l e as 10-25 cysts (38). After ingestion, the cysts pass through the stomach into the upper in t e s t i n e . Here, the parasite excysts, a process whereby cysts change to the trophozoite form. The trophozoites then attach to the i n t e s t i n a l epithelium v i a the adhesive disc and multiply by binary f i s s i o n . Eventually, the trophozoites pass into the proximal colon where they encyst or revert back into the cyst form. The cysts are shed v i a the feces and can p e r s i s t i n the environment for several weeks (38). The duration of i n f e c t i o n i s normally 6 to 8 weeks and i s s e l f - l i m i t e d . In some instances, the course of i n f e c t i o n may be prolonged for months or even years (17). I I I . PATHOGENESIS G.lamblia e l i c i t s a wide spectrum of symptoms. Some indi v i d u a l s are asymptomatic while others exhibit severe 2 diarrhea with malabsorption. Although the actual cause of symptoms has not been elucidated, i t i s most l i k e l y due to a combination of factors such as s t r a i n pathogenicity and host response to the parasite (36). a. s t r a i n virulence The influence of parasite factors i n the outcome of i n f e c t i o n i s s t i l l speculative. I t has been suggested that there are d i f f e r e n t s t r a i n s of Giardia, some pathogenic and others non-pathogenic. While differences among Giardia i s o l a t e s have been detected at the antigenic (52,68,84), enzymatic (5), and genomic (52) l e v e l s , no s p e c i f i c v i r u l e n t markers or c h a r a c t e r i s t i c s have been discovered (23). b. host immune responses Several studies have demonstrated that G.lamblia e l i c i t s an host immune response. Epidemiological evidence indicates that an increased incidence of i n f e c t i o n occurs i n v i s i t o r s to an endemic area compared to the l o c a l population, suggesting developed resistance to r e - i n f e c t i o n (50). In areas endemic for Giardia, younger age groups tend to be more susceptible to i n f e c t i o n (25). C l i n i c a l evidence indicates an increased prevalence of g i a r d i a s i s i n immunocompromised indi v i d u a l s , e s p e c i a l l y those with hypogammaglobulinaemia (46) and HIV inf e c t i o n s (2). Animals which have been experimentally immunocompromised also show chronic infections and are susceptible to r e - i n f e c t i o n with G.muris (64,72,75). 3 c. humoral immune response Numerous studies have detected c i r c u l a t i n g antibodies i n patients with g i a r d i a s i s . Techniques such as immunofluorescence assays (IFA) and enzyme-linked immunosorbant assays (ELISA) have been used to detect predominant isotypes of antibody (60, 62). Anti-Giardia IgG (66), IgM (26) and IgA (63) have a l l been detected. IgG has been detected at the highest t i t e r i n serum anywhere from a few months to more than a year post-infection (66). Both anti-Giardia IgA and IgM responses appear to be r e l a t i v e l y short i n duration (26, 63). These s e r o l o g i c a l studies have been useful i n diagnosis and characterization of trophozoite antigens (36). d. c e l l u l a r immune response C e l l u l a r immune responses i n human g i a r d i a s i s haven't been studied i n d e t a i l . There have been reports showing an increase i n i n t r a e p i t h e l i a l lymphocytes (IEL) i n patients. The numbers of IEL have also been shown to decrease following treatment and eradication of the parasite (85). The r o l e of c e l l u l a r immunity i n the clearance of G.muris i n infected mice has been studied i n some d e t a i l . The role of T-helper c e l l s i n determining the course of i n f e c t i o n was shown when mice s e l e c t i v e l y depleted of T-helper c e l l s developed chronic g i a r d i a s i s (32). These mice also generated a d e f i c i e n t l o c a l antibody response (29). Based on t h i s , i t ' s been suggested that T-helper c e l l s are responsible for 4 coordinating the production of s p e c i f i c i n t e s t i n a l IgA antibodies (33) . The mechanism of T-helper induced antibody production may be i n the switching of peyer's patch surface IgM+ B c e l l s to IgA+ c e l l s (9,17,21). The generation of g i a r d i a - s p e c i f i c antibodies would, i n turn, contribute to the clearance of the parasite (9). Heyworth used mice depleted of Ly2- c e l l s (cytotoxic T lymphocytes) to show that cytotoxic T c e l l s are not responsible for the elimination of trophozoites (32). He also used mice depleted of natural k i l l e r (NK) c e l l s (beige mice) to show that NK c e l l s are also not responsible for parasite elimination (28). The role of intraluminal macrophages i n clearance of i n f e c t i o n i s controversial. There have been reports on the involvement of macrophages (both peritoneal and mucosal) i n antibody dependent c e l l u l a r c y t o t o x i c i t y (ADCC) reactions as well as d i r e c t c y t o t o x i c i t y against G.lamblia (67,70). However, the f e a s i b i l i t y of intraluminal macrophages playing an important e f f e c t o r role in-vivo has been questioned for two reasons: f i r s t l y , only small numbers of phagocytes are present i n the i n t e s t i n e of infected mice, and secondly, there i s no difference between the number of phagocytes i n the inte s t i n e s of infected normal mice and nude mice (32). Generally, macrophages residing i n Peyer's patches are considered important as antigen presenting c e l l s (32). There has been l i t t l e more than a suggestion that mucosal mast c e l l s may be important i n g i a r d i a s i s . Supporting 5 evidence includes reports that mast c e l l d e f i c i e n t mice develop chronic g i a r d i a s i s (17), and that immunocompetent mice treated with the antihistamine drug, cyproheptadine, develop chronic G.muris infections (17). e. l o c a l antibody response Since Giardia i s an i n t e s t i n a l parasite, a l o c a l secretory antibody response i s a l i k e l y mechanism for parasite elimination. Due to the d i f f i c u l t i e s involved i n analyzing human i n t e s t i n a l f l u i d s (4), l i t t l e i s known of the importance of the secretory immune response i n human g i a r d i a s i s . Most of the information on l o c a l antibody responses comes from animal models. Techniques such as solid-phase radioimmunoassays (3,70), IFA (29), and ELISA (31) have been used to show that immunocompetent mice infected with G.muris generate a s p e c i f i c i n t e s t i n a l response. The production of s p e c i f i c secretory b i l e IgA antibodies to surface antigens of G.lamblia was detected i n the rat model using both IFA (45) and ELISA (82). S p e c i f i c IgA antibodies i n human g i a r d i a s i s have been i d e n t i f i e d i n secretions such as s a l i v a (74) and milk (49). Since i t ' s l i k e l y that the intestine stimulates the production of antibodies at distant mucosal s i t e s , the presence of IgA i n these secretions may r e f l e c t the existence of s i m i l a r antibodies i n the intestine (17). While the predominance of an t i - g i a r d i a secretory IgA i s well documented (3,31,73), g i a r d i a s p e c i f i c IgG has also been detected i n secretions (29, 43) . 6 The r o l e of secretory IgA as an important e f f e c t o r antibody has been shown i n animal models using G.muris. A temporal association was demonstrated between the l e v e l s of g i a r d i a s p e c i f i c IgA i n gut secretions i n mice and the expulsion of G.muris (73) . In addition, mice treated from b i r t h with a n t i -IgM developed chronic G.muris infections associated with f a i l u r e to synthesize s p e c i f i c i n t e s t i n a l IgA antibodies (72). T-lymphocyte d e f i c i e n t (nude) mice, which become c h r o n i c a l l y infected with G.muris, showed l i t t l e evidence of surface-bound antibodies on trophozoites c o l l e c t e d from the i n t e s t i n a l lumen. In comparison, IgA was detected on trophozoites taken from normal mice which are able to clear i n f e c t i o n (29). Protection against i n f e c t i o n with G.muris was shown to be transferred from immune mothers to suckling mice v i a breast milk and was associated with s p e c i f i c anti-Giardia IgA (42). While more i s known about the possible roles of secretory IgA against G.muris, c l i n i c a l studies have supported a role for secretory IgA i n patients infected with G. lamblia. IFA's have been used to detect IgA on trophozoites present on the e p i t h e l i a l surface of human jejunal biopsy specimens (7). I t has been suggested (87), disproved (48), and then suggested one again (59) that patients with secretory IgA d e f i c i e n c i e s are more susceptible to chronic symptomatic g i a r d i a s i s . 7 VI. MECHANISMS OF HOST DEFENSE a. s p e c i f i c host immunity The mechanisms by which g i a r d i a - s p e c i f i c antibodies a l t e r the course of i n f e c t i o n i s unknown. A v a r i e t y of a n t i -p a r a s i t i c e f f e c t s have been postulated i n the human as well as the murine model (17,45). Secretory antibodies could block the attachment of trophozoites to i n t e s t i n a l epithelium by binding to antigens of the adhesive di s c . This mechanism of host defense was supported by Kaplan who reported that both immune mouse milk and immune rabbit serum reduced the adherence of trophozoites to the adult mouse epithelium (40). Secretory antibodies could also eliminate or cl e a r i n f e c t i o n by d i r e c t k i l l i n g or immobilization, by trapping the trophozoites i n the mucous coat or by preventing c e l l r e p l i c a t i o n or d i f f e r e n t i a t i o n (45). There have been a number of studies which examined the possible r o l e of serum antibodies and complement against the m o t i l i t y and c e l l v i a b i l i t y of G.lamblia and G.muris. Recently, H i l l et a l (35) demonstrated the a b i l i t y of human serum containing a n t i - p a r a s i t i c antibodies to k i l l G.lamblia trophozoites i n - v i t r o . This g i a r d i a c i d a l a c t i v i t y of d i f f e r e n t sera correlated with the t i t e r of serum IgG antibodies to trophozoites. Furthermore, the a c t i v i t y was shown to be dependent on the a c t i v a t i o n of the c l a s s i c a l complement pathway. Complement induced l y s i s of trophozoites 8 has also been demonstrated using IgM antibodies i s o l a t e d from the sera of symptomatic g i a r d i a s i s patients (18). In t h i s instance, i t was suggested that the l y t i c a c t i v i t y was dependent on both the c l a s s i c a l complement pathway as well as a unique pathway which required CI and factor B but was independent of C4 and C2. In murine g i a r d i a s i s , immune serum from Giardia infected mice was shown to immobilize and lyse trophozoites i n - v i t r o (6). Both a n t i - p a r a s i t i c e f f e c t s were complement dependent. Complement independent c i d a l a c t i v i t y and immobilizing a c t i v i t y has been demonstrated using monoclonal antibodies directed against G.muris and G.lamblia trophozoites i n - v i t r o (8,54) . Gi a r d i a - s p e c i f i c antibodies could also mediate antibody dependent c e l l u l a r c y t o t o x i c i t y (ADCC). Smith et a l demonstrated that g i a r d i a - s p e c i f i c serum IgG could p a r t i c i p a t e i n ADCC with human peripheral polymorphonuclear leukocytes i n -v i t r o (69). Whether ADCC eff e c t s trophozoites i n the int e s t i n e i s unknown (17). I n t r a e p i t h e l i a l T lymphocytes (IEL) have demonstrated d i r e c t c y t o t o x i c i t y against G.lamblia trophozoites (39). This, along with reports of increased IEL i n mice infected with G.lamblia (82), indicates another possible mechanism of mucosal defense against g i a r d i a i n f e c t i o n s . b. non-specific e f f e c t o r mechanisms 9 Resistance to g i a r d i a s i s could also involve non-specific host factors. Non-immune mechanisms of parasite elimination include g a s t r i c a c i d i t y , mucous secretion, i n t e s t i n a l m o t i l i t y , b i l e constituents and enzymatic a c t i v i t i e s (4). Recent work with G.lamblia indicates that products of l i p o l y s i s such as cis-unsaturated f a t t y acids, monoglycerides and lysophosphatidylcholines may be toxic to the parasite (24,61). On the other hand, i t ' s been demonstrated that mucous and possibly b i l e s a l t s protect Giardia from t o x i c l i p o l y t i c products i n the intestine and, i n the case of i n t e s t i n a l mucous, may promote attachment (86). Although non-s p e c i f i c mechanisms probably play an important role i n the course of i n f e c t i o n , t h e i r involvement needs to be defined and quantified. Furthermore the interaction between non-specific and s p e c i f i c immune factors should be assessed (4). V. ANTIGENIC DETERMINANTS Host defense mechanisms against Giardia are c l e a r l y an important aspect i n the host-parasite r e l a t i o n s h i p . However, the relevance of the host response against Giardia can't be f u l l y appreciated without a concurrent understanding of the biochemical composition of the parasite. With t h i s knowledge, target antigens of a n t i - p a r a s i t i c antibodies can be i d e n t i f i e d and characterized, and the role of s t r a i n pathogenicity i n i n f e c t i o n can be put into perspective. 10 a. biochemical composition of G.lamblia In- v i t r o c u l t i v a t i o n of G.lamblia trophozoites i n 1976 (47) allowed a more detailed analysis of t h e i r biochemical composition. Holberton and his collegues have contributed greatly to the knowledge of the biochemical composition of the cytoskeleton of Giardia (11-14,37). The axonemes and disc cytoskeleton were isola t e d using subcellular f r a c t i o n a t i o n procedures (37). Tubulin, a protein of 55 kd, was i s o l a t e d and characterized as a major constituent of microtubules and microribbons. Another group of cytoskeletal proteins of 28-36 kd c a l l e d giardins, were characterized as i n t e g r a l , tubulin-associated proteins of disc microribbons (11,13). Giardins have been l o c a l i z e d to the ventral dis c but not within the f l a g e l l a (57). Aside from work done on the cytoskeleton of G.lamblia, very l i t t l e i s known about the biochemical composition of t h i s parasite. N-acetyl-d-glucosamine, a surface carbohydrate residue of trophozoite membranes has been characterized using l e c t i n binding techniques (34) . Another study characterized and p a r t i a l l y p u r i f i e d a mannose-glucose binding l e c t i n derived from the surface of G.lamblia (22). b. antigenic v a r i a t i o n between strains Antigenic differences between i s o l a t e s of Giardia have been demonstrated but the significance of heterogeneity varies according to the technique used. Some of these techniques include ELISA (79), isoenzyme studies (5), SDS-PAGE of 11 proteins (69), and antigenic analysis by immunoblotting (52,67,84). I t has been demonstrated that some stra i n s of G.lamblia are capable of undergoing antigenic v a r i a t i o n (51,52). These varying antigens are major, cysteine-rich proteins located on the surface of trophozoites (1). c. characterization of surface immunogens A number of surface parasite immunogens have been i d e n t i f i e d using antibodies i n peripheral blood samples. However, the data c o n f l i c t regarding the number, molecular weights and immunogenicity of trophozoite antigens. For instance, the following antigens have a l l been i d e n t i f i e d as major surface antigens i n separate studies using human immune sera: 31-32 kd (76,80), 82-88 kd (19,20), 85, 63, and 55 kd, (55) and 94-200 kd (51). Despite these inconsistencies, the above studies support two theories; the f i r s t being that host antibodies are directed against surface components of trophozoites, the second i s that host-protective immune responses are directed against a se l e c t i v e number of t o t a l parasite antigens. VI. MEMBRANE EXTRACTION To f a c i l i t a t e a f i n e r analysis of surface antigenic determinants, attempts have been made to i s o l a t e membrane fraction s from the t o t a l repertoire of trophozoite antigens. One study used various detergents to i s o l a t e plasma membranes 12 from G.lamblia (10). Four protein size classes of 75 kd, 58/54 kd 32-38 kd and 15-22 kd were i d e n t i f i e d . Another technique using a membrane s t a b i l i z i n g agent followed by osmotic stress and manual sheer isol a t e d plasma membranes from PI s t r a i n s of G.lamblia trophozoites. From t h i s preparation, two major surface antigens of 82 kd and 56 kd were i d e n t i f i e d (44). A t h i r d study used u l t r a c e n t r i f u g a t i o n and extraction with aqueous, non-ionic and i o n i c detergent buffers to i s o l a t e subcellular fractions of WB s t r a i n of G.lamblia (17) . The aqueous soluble f r a c t i o n was enriched for c y t o s o l i c components, the non-ionic ( t r i t o n X-100) soluble f r a c t i o n was enriched for membrane-associated components and the i o n i c (SDS) soluble f r a c t i o n was enriched for cytoskeletal components. Each of these studies have employed d i f f e r e n t s t r a i n s of Giardia as well as d i f f e r e n t membrane extraction techniques. This has resulted i n heterogeneous membrane protein patterns. Clearly, more work needs to be done to e s t a b l i s h a standard technique of membrane i s o l a t i o n and p u r i f i c a t i o n . In the meantime, these techniques are useful i n simplifying analysis of the biochemical composition of g i a r d i a trophozoites. 13 VII. PURPOSE OF THIS STUDY Recently, much of the G i a r d i a research has gone into exploring the mechanisms of host defense against G i a r d i a , p a r t i c u l a r l y the role of g i a r d i a - s p e c i f i c antibody responses. S i g n i f i c a n t progress has also been made i n understanding the biology of G i a r d i a . However, not much i s known about the interactions between host and parasite. For instance, what are the pathogenic mechanisms whereby G i a r d i a causes symptoms, and how does the host immune response a l t e r the course of infection? There were two main objectives to t h i s study. The f i r s t was to further develop our understanding of host humoral immune defense against G i a r d i a infections by exploring the i n - v i t r o a n t i - p a r a s i t i c e f f e c t s of g i a r d i a - s p e c i f i c antibodies. To accomplish t h i s , i n - v i t r o m o t i l i t y assays were developed to assess the immobilizing effects of immune sera against G i a r d i a trophozoites. The second objective of t h i s study was to i s o l a t e the immobilizing antibodies from immune sera and use them to i d e n t i f y parasite antigens. In order to i s o l a t e immobilizing antibodies, an immunoabsorption technique was developed based on the assumption that the host defense mechanisms are directed i n i t i a l l y against the surface of the parasite. I t was thought that i f we could develop a technique to i s o l a t e s u r face-specific a n t i - g i a r d i a antibodies from rabbit sera, we would be one step closer to i s o l a t i n g 14 immobilizing antibodies. The is o l a t e d s u r f a c e - s p e c i f i c antibodies could then be used to target trophozoite antigens. This would help to elucidate which parasite antigens are relevant i n the host-parasite interaction. 15 MATERIALS AND METHODS I. ISOLATES Strains Vanc/87/UBC/22 (DEL), a s t r a i n i s o l a t e d from a symptomatic patient; Vanc/87/UBC/28 (501), a beaver s t r a i n ; and Vanc/87/UBC/8 (L), a s t r a i n i s o l a t e d from an asymptomatic patient, were used i n most of these experiments. L was is o l a t e d by i n v i t r o excystation (Isaac-Renton), and 501 was is o l a t e d by excystation i n g e r b i l s . A l l three s t r a i n s came from southwestern B.C. I I . CULTURE MEDIA Isolates were maintained i n f i l t e r - s t e r i l i z e d b i l e supplemented TYI-S-33 medium with casein digest (BBL, Microbiology Systems, Cockeysville, MD) rather than trypticase and L-cysteine hydrochloride i n concentrations of 200mg/100ml (43). For growth i n broth, approximately 15 ml of TYI-S-3 3 was added to 16 x 125 mm disposable b o r o s i l i c a t e glass screw-topped tubes with t e f l o n l i n e d caps (Kimble Company, Toledo, Ohio, U.S.A.). After inoculation, tubes were incubated at 37° at a 45° angle. When used for preparation of t r i c h l o r o a c e t i c acid (TCA) 16 p r e c i p i t a t e s , 48 hour growth phase trophozoites were u t i l i z e d . Culture tubes were placed on ice for ten minutes to remove adherent trophozoites and were then centrifuged for f i v e minutes at 850 x g. The trophozoite p e l l e t s were then washed three times with cold phosphate buffered s a l i n e (pH 7.2-7.4) (PBS). The p e l l e t was then resuspended i n 1 0 mis of PBS and counted with a haemocytometer. The trophozoites were then d i l u t e d or concentrated to the appropriate numbers. I I I . BULK HARVEST The following bulk harvest i s according to the procedure of denHollander ( 1 6 ) . In order to get 1-4 x 10 8 trophozoites needed for the immunoabsorption study and the membrane extraction procedure, the trophozoites were grown i n polystyrene t i s s u e culture flasks (Corning) ( 5 0 ml capacity) for approximately 48 hours or u n t i l there was a confluent monolayer of trophozoites adhering to the sides of the flask. Culture media and non-adherent trophozoites were discarded and the flasks were f i l l e d with ice cold PBS and were placed on ice for 1 5 minutes to dislodge the adherent trophozoites. The contents of each f l a s k were transferred to 50 ml conical tubes and were centrifuged at 350 x g for 1 5 minutes at 4°C. The supernatants were discarded and the p e l l e t s pooled. The wash step was repeated to make a t o t a l of 3 washes. The f i n a l p e l l e t was resuspended i n 50 ml of PBS and enumerated using a 17 haemacytometer. After a f i n a l spin, the p e l l e t was resuspended i n a volume of buffer appropriate to either the immunoabsorption study or the membrane extraction procedure. IV. ANTIGEN PREPARATION TCA p r e c i p i t a t i o n TCA p r e c i p i t a t e s (43) of trophozoites were prepared as follows: trophozoites (4 x 106) were grown and washed as described above. The trophozoite p e l l e t was resuspended i n 10 ml PBS, added to an equal volume of 2 0% TCA and then incubated at 4°C for 4-5 hours. The sample was centrifuged at 850 x g for 5 min at 4°C, washed twice with 10 ml cold PBS and resuspended i n 300ul PBS. Protein concentrations were determined by the modified method of Lowry (65). Samples usually contained 5-15 mg/ml. Aliquots were stored at -2 0°C. enriched membrane extraction procedure The membrane f r a c t i o n of Giardia trophozoites was p a r t i a l l y p u r i f i e d using a subcellular fractionation procedure by denHollander (17). Trophozoites were grown i n bulk as described above. Instead of washing trophozoites i n PBS, a HEPES supplemented Hanks buffered saline solution (HBSS) pH 18 7.0 was used. After a t o t a l of f i v e washes, the trophozoites were counted and the f i n a l p e l l e t was resuspended at a f i n a l concentration of 3 x 10 6in HBSS-A (HBSS, 25mM HEPES, 0.02% NaN3, ImM PMSF, 100 KlU/ml aprotinin, pH 7.0). The trophozoites were stored i n cryotubes and kept i n l i q u i d nitrogen u n t i l used. Subcellular f r a c t i o n a t i o n of s t r a i n 501 of G.lamblia was carr i e d out by ul t r a c e n t r i f u g a t i o n and extraction with detergent buffer as follows: trophozoites i n HBSS-A were i n i t i a l l y fractionated by 3 minute immersions i n l i q u i d nitrogen, followed by 3 minute incubations i n a 3 7oc water bath and 20 seconds of vigerous vortexing. This freeze-thaw cycle was repeated 4 times. The sample was centrifuged at 38 OOOx g for 70 minutes at 4°C. The supernatant (cytosolic fraction) was aliquoted and stored at -20°C. The following steps a l l occurred at room temperature. The p e l l e t was resuspended i n -300 u l HBSS-B (10 x HBSS without Ca" and Mg" 0.2% NaN3, 2 5mM HEPES, ImM PMSF, 100 KlU/ml Aprotinin, 0.5mM EDTA, ImM ATP, pH7.0) plus 100 u l t r i t o n X-100. This was l e f t overnight. The sample was mixed again and incubated a few hours. I t was then centrifuged at 38,000 x g for 70 minutes. The supernatant (membrane enriched fraction) was col l e c t e d and stored at -20°C. The p e l l e t was resuspended i n -300 u l HBSS-B plus 20% SDS and l e f t overnight. The sample was mixed and allowed to s i t a few hours before being centrifuged. The supernatant (cytoskeletal fraction) was co l l e c t e d and stored at -2 0°C. 19 V. PREPARATION OF RABBIT ANTISERA immunization Three New Zealand White rabbits were each immunized with approximately 7 x 106 trophozoites of either L, DEL, or 501 s t r a i n i n emulsified complete Freund's adjuvant (43). The rabbits received subsequent injections of 4 x 106 trophozoites i n incomplete Freund's adjuvant every week for four weeks. A sample (10ml) of blood was taken during the f i f t h week. Then the i n j e c t i o n s of 4 x 106 trophozoites i n incomplete adjuvant were continued for another four weeks. A l l injectons were given subcutaneously at multiple s i t e s . The rabbit was exsanguinated on the 9th week and the blood was incubated overnight at 37°C. The serum was collected, aliquoted and stored at -20°C. ammonium sulphate p r e c i p i t a t i o n of antisera Approximately 2-3 mis of rabbit antisera was d i l u t e d to 10 mis i n d i s t i l l e d water. This was added to an equal volume of saturated ammonium sulphate pH 7.8, was vortexed and was l e f t at room temperature for 3 0-60 minutes. The sample was centrifuged for 5 minutes at 7500 x g. The p e l l e t was 20 resuspended i n 2-3 mis of 1/2 PBS and then dialyzed against 1/2 PBS for 48 hours. Aliquots were stored at -20°C. VI. 6ERBIL MODEL Three to four week old male Mongolian g e r b i l s (approximately 2 0-2 5g i n weight) were obtained from Tumblebrook Farms, Inc, West Brookfield, Massachusetts. Animals s a c r i f i c e d randomly exhibited no sign of contamination with Giardia but some arrived with i n t e s t i n a l trichomonads. Gerbils were allowed to rest for one week and then were gavage fed with 2 0 mg of Metronizadole d a i l y on three consecutive days i n the manner of Be l o s i v i c et a l (6). After another week's rest, stools were co l l e c t e d over a three day period and were examined for cysts using the sucrose gradient method. Giardia were never documented i n post treatment stools, but i f the trichomonads were present i n i t i a l l y , they generally persisted. VII. INOCULATION OF GERBILS Gerbils were l i g h t l y anaesthetized with ether. They were then inoculated o r a l l y with a gavage needle i n either one of two ways; with 1-2 x 105 cysts obtained by the sucrose gradient method from the feces of infected g e r b i l s , or with 1 x 107 trophozoites i n 0.5ml aliquots. 21 VIII. PREPARATION OF GERBIL FECAL EXTRACTS The preparation of g e r b i l f e c a l extract was developed by Krystal (43) . Using metabolic chambers (Canlab S c i e n t i f i c ) , approximately 20 g of stool was c o l l e c t e d from each of two groups of g e r b i l s (at day 26 post i n f e c t i o n ) . The f i r s t group was the control and consisted of s i x non-infected g e r b i l s . The second group had been inoculated with i s o l a t e 501. At room temperature, stool was mixed with 45 mis of PBS containing 2 mM PMSF, 5 mM EDTA, and 0.05 U/ml aprotinin (Sigma) (Snider&U), and f i l t e r e d through two layers of cheesecloth. After centrifugation for f i v e minutes at 1100 X g, the supernatant was removed and centrifuged a second time for ten minutes at 7500 X g. The supernatant was removed and f i l t e r e d through a 1.2 um n i t r o c e l l u l o s e f i l t e r ( M i l l i p o r e ) . I t was then mixed with an equal volume of saturated ammonium sul f a t e f o r one hour at room temperature, and centrifuged for 10 minutes at 7500 X g. The p r e c i p i t a t e was dissolved i n two ml of 1/2 PBS and dialysed at 4°C for 48 hours against 1/2 PBS with several changes of buffer. The extracts were then frozen at -70°C u n t i l use. X. IMMUNOABSORPTION Po l y s p e c i f i c rabbit antisera was absorbed by incubation with whole, l i v i n g trophozoites as follows: adherent trophozoites were c o l l e c t e d at 48 hours, washed three times 22 i n PBS and counted. Approximately 1-4 x 10s trophozoites were pe l l e t e d and immediately incubated with 100-800 u l of ammonium sulphate p r e c i p i t a t e d antisera (whole antibody fraction) d i l u t e d with an equal volume of PBS. This incubation step was c a r r i e d out on i c e for 40 minutes on a shaker platform. The trophozoites were removed by centrifugation at 850 x g for 5 min. The supernatant (depleted antibody fraction) was f i l t e r e d and the absorbance was measured at 280 nm to determine the concentration of immunoglobulin. The trophozoites were washed once with PBS to further remove unbound antibodies. In order to elute antibodies which bound to the surface of the trophozoites, the c e l l s were washed i n 500-800 u l of 0.1M glycine HC1, pH 2.6 for 10 min on i c e . The trophozoites were removed by centrifugation and the supernatant (eluted antibody fraction) was neutralized with 0.5M NaOH, d i l u t e d with an equal volume of PBS, and dialyzed against PBS. The t o t a l volume of the eluted antibody f r a c t i o n was approximately 2-3 mis. For the m o t i l i t y assays, t h i s f r a c t i o n was concentrated to 100 u l using an amicon microconcentrator. For the immunoblots, approximately 1-1.5 mis (50%) of the eluted f r a c t i o n was d i l u t e d i n an equal volume of BLOTTO. 23 XI. IN-VITRO MOTILITY ASSAY The i n v i t r o m o t i l i t y assay was adapted from the procedure used by H i l l et a l (35). Growth media and non-attached Giardia trophozoites (strains DEL, L, and 501) of a c t i v e l y growing cultures were discarded and the adherent trophozoites were dislodged from the walls of the culture tubes by immersion i n ice cold PBS. After two washes i n PBS, the trophozoites were resuspended and counted. Approximately 1-2 x 105 Giardia trophozoites were added to wells i n 9 6 well Linbro t i s s u e culture plates and were incubated for up to 2 hours with a 1:5 d i l u t i o n of pre-immune serum and various d i l u t i o n s of immune serum. Control wells contained trophozoites i n PBS alone. At various i n t e r v a l s the contents of i n d i v i d u a l wells were agitated and the percent of motile trophozoites out of 100 trophozoites counted was determined using the 4OX objective of a phase contrast microscope. e f f e c t of complement on m o t i l i t y assay To determine whether the immobilization of trophozoites was mediated by complement, the assay was performed using complement inactivated immune serum. In t h i s experiment, 1-2 x 105 trophozoites i n 2 00 u l PBS were incubated with either 100 u l heat-inactivated (56°, 30 min) serum or 100 u l 24 heat-inactivated serum reconstituted with 50 u l rabbit complement. ro l e of s u r f a c e - s p e c i f i c anti-Giardia antibodies To determine whether sur f a c e - s p e c i f i c a n t i - g i a r d i a antibodies were required for immobilization of trophozoites i n - v i t r o , the assay was performed using 1:4 d i l u t i o n s of the whole and depleted antibody fractions, and the e n t i r e eluted antibody f r a c t i o n . In t h i s experiment, 1-2 x 105 trophozoites i n 200 u l PBS were incubated with 100 u l of antibody and 50 u l rabbit complement. e f f e c t of f e c a l extracts on m o t i l i t y In order to t e s t the immobilizing e f f e c t s of g i a r d i a - s p e c i f i c antibodies i s o l a t e d from experimentally infected g e r b i l s , trophozoites were also incubated with a 1:2 d i l u t i o n of f e c a l extracts taken from infected g e r b i l s . Control wells contained trophozoites with e i t h e r PBS or fecal extracts taken from non-infected g e r b i l s . XII. IN-VITRO VIABILITY ASSAYS This procedure was as described for the m o t i l i t y assay except that a f t e r the incubation of trophozoites and serum 25 samples for various i n t e r v a l s , the samples were incubated with neutral red (0.0015%) for 15 minutes at room temperature and counted using the 4OX objective (35). Trophozoites which retained the dye were counted as viable and those which appeared transparent were counted as non-viable. In order to determine whether g i a r d i a - s p e c i f i c antibodies i n rabbit antisera could lyse trophozoites i n v i t r o , the assay was again performed i n the same manner as described above except that the number of trophozoites was counted at time 0 and a f t e r a 60 and 120 minute incubation time. A trophozoite was considered lysed only i f the i n t e g r i t y of the c e l l was completely disrupted and could not be d i f f e r e n t i a t e d from debris. The percent l y s i s of trophozoites was calculated by subtracting the mean number of trophozoites counted at 60 or 12 0 minutes, from the mean number of trophozoites at time 0; divided by the mean number of trophozoites at time 0, x 100. XIII. INDIRECT IMMUNOFLUORESCENCE ASSAY To detect antibodies bound to the surface of Giardia trophozoites, 5 X 105 trophozoites, which had been used i n the immunoabsorption experiment, were incubated with 1:80 d i l u t i o n of FITC goat ant i - r a b b i t IgG (BHL) at 4°C for 3 0 minutes. After washing three times with PBS, the trophozoites were observed under fluorescence microscopy. Positive controls contained a fresh sample of trophozoites incubated with 1:50 26 d i l u t i o n of immune rabbit sera at 4°C for 3 0 minutes previous to the addition of FITC. Negative controls contained trophozoites incubated with a 1:50 d i l u t i o n of pre-immune serum or PBS. XIV. ELECTROPHORESIS AND IMMUNOBLOT TECHNIQUE SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed using acrylamide (Bio-Rad) stacking (4.0%) and separating (12.0%) gels. The separating gels measured 12.5 X 14 cm and were 1.5 mm thick. A Dual Protean 16 cm v e r t i c a l slab c e l l apparatus (Bio-Rad) was used for electrophoresis. Twenty-five u l (-50 ug protein of TCA samples) was applied to each lane. Samples included low molecular weight markers, G.lamblia trophozoites precipitated by TCA, and enriched membrane preparations. The gel was subjected to constant current with 20 mA through the stacking gel, followed by 30 mA u n t i l the dye front reached the bottom of the g e l . A f t e r electrophoresis, the proteins on the gels were eithe r transferred to n i t r o c e l l u l o s e for immunoblotting or were s i l v e r stained (43). Protein transfer to n i t r o c e l l u l o s e paper was done with the ANCOS semi-dry e l e c t r o b l o t t i n g method (Dimension Laboratories, Mississauga, Ontario). N i t r o c e l l u l o s e was cut to gel si z e , rehydrated i n deionized water, and placed on a f i l t e r paper stack. Six pieces of f i l t e r paper were soaked i n anode buffer 27 #1 (0.3M T r i s Base, 20% methanol). Three pieces were soaked i n anode buffer #2 (25 mM T r i s Base, 20% methanol). The gel was positioned on the n i t r o c e l l u l o s e and covered by 9 layers of f i l t e r paper soaked i n cathode buffer (25 mM T r i s , 40 mM 6-amino-n-hexanoic acid, 20% methanol). Transfer time was 1 hour at constant current of 0.8 mA/cm2 gel (-150 mA) . Protein transfer was assessed by Amido Black staining of a sample lane (43). Subsequent procedures involved probing and development of the n i t r o c e l l u l o s e and were done at room temperature with use of a rota t i n g platform. The unstained n i t r o c e l l u l o s e was placed i n a blocking solution (5% skim milk) for 2 hours. The whole, depleted, eluted antibody fractions and pre-immune sera were d i l u t e d i n blocking solution (1:40 for whole f r a c t i o n , depleted f r a c t i o n and pre-immune sera and 1:5 for eluted fraction) and incubated overnight with the n i t r o c e l l u l o s e . Three 10 minute washes with Tris-buffered saline-Tween 0.1% (TBS-T, pH 7.4) were followed by addition of b i o t i n labeled a n t i - r a b b i t IgG (Sigma) d i l u t e d 1:500 i n TBS-bovine serum albumin (TBS-BSA), and incubated for 2 hr. After three 10 minute washes with TBS-T-BSA, the n i t r o c e l l u l o s e was incubated for 3 0 minutes i n streptavidin horseradish peroxidase conjugate (Bio-Rad) d i l u t e d 1:500 with TBS-BSA. Three 10 minute washes with TBS-T (twice) and TBS (once) followed. The n i t r o c e l l u l o s e was developed with 4-chloro-l-naphthol and 28 0.06% hydrogen peroxide, negative control. Pre-immune rabbit sera served as the 29 RESULTS IN-VITRO EFFECT OF ANTISERA AGAINST TROPHOZOITE MOTILITY To evaluate the importance of host immune response i n g i a r d i a s i s , the i n - v i t r o e f f e c t of rabbit sera on trophozoite m o t i l i t y was assessed using three st r a i n s of G.lamblia trophozoites; 501, DEL and L. Figure 1 shows the immobilizing e f f e c t of sera against DEL s t r a i n . Sera obtained from non-infected rabbits immobilized trophozoites s l i g h t l y a f t e r one hour of incubation. However, t h i s e f f e c t was no more pronounced than the e f f e c t seen with controls containing trophozoites i n PBS. In comparison, sera obtained from hyperimmunized rabbits had a s i g n i f i c a n t l y greater e f f e c t on trophozoite m o t i l i t y . By the end of the one hour incubation, close to 100% of trophozoites were immobilized. The m o t i l i t y assay was repeated using 501 s t r a i n trophozoites incubated with either pre-immune sera or a n t i -501 immune sera. The res u l t s were almost i d e n t i c a l to those for DEL s t r a i n (data not shown). I t was our intention to include a t h i r d s t r a i n , L, i n these experiments. However, unlike 501 and DEL, L trophozoites were immobilized by pre-immune sera. Since i t was our goal to assess the a n t i p a r a s i t i c e f f e c t s of g i a r d i a - s p e c i f i c antibodies, L s t r a i n was excluded. As w i l l be described shortly, some preliminary work was done to further 30 FIGURE 1 : IN-VITRO MOTILITY ASSAY Eff e c t s of immune (B) and pre-immune (^ .) rabbit sera against trophozoite m o t i l i t y . Controls ( £ ) contain trophozoites i n PBS. Parasites were incubated with 15% serum or RPMI media at 37°C and the percent m o t i l i t y of trophozoites was determined a f t e r various time i n t e r v a l s . Each point on the graph represents the percent of motile trophozoites. Mean values (± s.e.m) of 3 experiments performed i n duplicate are shown. 31 IN-VITRO M O T I L I T Y A S S A Y 120 i Ql ! 1 0 30 60 90 TIME (min) 32 characterize the immobilizing e f f e c t s of pre-immune sera against L s t r a i n . Results indicate that for at least two st r a i n s of trophozoites, immune sera contains immobilizing a c t i v i t y that was not present i n pre-immune sera. The fact that t h i s a c t i v i t y was mediated by g i a r d i a - s p e c i f i c antibodies was supported by immunofluorescense assays which detected a n t i -g i a r d i a antibodies i n immune sera but not i n pre-immune sera (data not shown). A number of experiments including Western blot s and absorption studies also support the role of a n t i -g i a r d i a antibodies i n mediating t h i s a n t i - p a r a s i t i c e f f e c t . These experiments w i l l be described. MECHANISMS OF ANTI-MOTILITY EFFECT Current evidence suggests that g i a r d i a - s p e c i f i c antibodies play an important role i n preventing or cle a r i n g i n f e c t i o n . This has been supported by our i n - v i t r o work. However, the mechanisms by which antibodies mediate t h e i r e f f e c t i s unknown. To determine whether antibodies were capable of immobilizing trophozoites d i r e c t l y or whether additional serum components were required, the complement dependency of the a n t i - m o t i l i t y e f f e c t was assayed. Results show (table I) that heat inactivated immune sera (56oc, 30 min.) l o s t any immobilizing a c t i v i t y against 501 s t r a i n trophozoites. Addition of rabbit complement to the heat inactivated sera restored the immobilizing a c t i v i t y to the extent seen with 33 TABLE I : IN-VITRO IMMOBILIZATION OF 501 STRAIN Percent Motility Incubation conditions * (mean • s.e.m.) PBS 81.67 • 2.6 Rabbit serum (complement) # 84.75 ± 1.4 Immune rabbit serum 0.75 ± N/A Heat-inactivated immune serum # 83.83 + 1.4 Heat-inactivated immune serum & rabbit complement 2.17 + N/A * approximately 1-2 x 105 trophozoites i n 200 u l PBS were incubated for 60 minutes with eit h e r 100 u l PBS, or 50 u l of rabbit serum, heat inactivated antisera, or complement. The f i n a l volume i n each well was adjusted to 350 u l with PBS. # Trophozoites incubated with eit h e r rabbit complement or heat inactivated serum had a s l i g h t tendency to aggregate. Therefore, the values for these conditions represent the percent m o t i l i t y of non aggregated trophozoites. N/A : not applicable 34 whole, immune sera. These results indicate that the immobilizing e f f e c t was dependent on the presence of complement. Since rabbit complement alone did not a f f e c t trophozoite m o t i l i t y (table I ) , the most probable explanation i s that a n t i - g i a r d i a antibodies require complement for immobilization. Whereas heat inactivated anti-501 immune sera was unable to immobilize 501 s t r a i n trophozoites, t h i s wasn't the case with heat inactivated antisera against DEL s t r a i n . Figure 2 shows how heat inactivated immune sera was able to immobilize 58% of DEL trophozoites. This suggests that the immobilizing e f f e c t of DEL antisera was pa r t l y complement independent. The reasons why one s t r a i n , such as 501, was completely susceptible to a complement dependent a n t i - m o t i l i t y response i n - v i t r o whereas another s t r a i n , DEL, was only p a r t l y susceptible are unknown. IN-VITRO KILLING ASSAY In addition to the i n - v i t r o e f f e c t s of immune sera against trophozoite m o t i l i t y , we also assessed the a b i l i t y of serum to k i l l DEL trophozoites. The intent was to determine whether a c o r r e l a t i o n existed between immobilization and c e l l death. A number of methods were used to measure the l e t h a l e f f e c t s of rabbit sera against DEL s t r a i n v i a b i l i t y , including trypan blue exclusion, neutral red retention and percent l y s i s . 35 FIGURE 2 : MOTILITY ASSAY USING HEAT INACTIVATED SERA Immobilization of DEL s t r a i n trophozoites by immune sera (•) and heat inactivated immune sera ( A ) . Control wells (O) contain trophozoites i n RPMI media. Mean values (± s.e.m) of 3 experiments performed i n duplicate are shown. 36 M O T I L I T Y A S S A Y USING H E A T INACTIVATED IMMUNE S E R U M 120 0 ' ' ' J 0 30 60 90 TIME (min) 37 Repeated experiments t e s t i n g for the l y t i c a c t i v i t y of immune sera against DEL s t r a i n trophozoites i n - v i t r o indicated that a f t e r a one hour incubation period, there was no s i g n i f i c a n t l y s i s . The e f f e c t s of immune sera on l y s i s were not s i g n i f i c a n t l y d i f f e r e n t from those of pre-immune sera and controls containing trophozoites i n PBS alone (data not shown). The l e t h a l e f f e c t s of sera against trophozoites were also measured using trypan blue as an indicator of c e l l v i a b i l i t y . Due to the s u b j e c t i v i t y and inconsistency of the r e s u l t s , t h i s procedure was deemed unreliable (data not shown). The use of the supravital s t a i n neutral red as an indicator of c e l l v i a b i l i t y gave the most consistent r e s u l t s and was used i n the v i a b i l i t y assay to measure c e l l death. The re s u l t s of the v i a b i l i t y assay ( f i g . 3) show that i n comparison to the ef f e c t s of pre-immune sera and controls containing trophozoites i n RPMI media, immune sera had a much greater e f f e c t on trophozoite v i a b i l i t y . At the end of 2 hours, less than 40% of trophozoites remained v i a b l e . In the presence of pre-immune sera, over 80% of trophozoites were viab l e a f t e r 2 hours. CORRELATION BETWEEN IMMOBILIZATION AND KILLING Two a n t i - p a r a s i t i c e f f e c t s of rabbit antisera were measured i n - v i t r o ; immobilization and k i l l i n g . Although the r e s u l t s of these assays show that immune sera contributed to both a n t i -38 FIGURE 3: IN-VITRO VIABILITY ASSAY Effects of immune (B) a n d pre-immune (^) rabbit sera against trophozoite v i a b i l i t y . Control ( £) consists of trophozoits i n RPMI media. Each point represents the percent (mean ± s.e.m) of trophozoites which r e t a i n the supravital s t a i n neutral red. Mean values of 3 experiments performed i n t r i p l i c a t e are shown. 39 I N - V I T R O VIABILITY A S S A Y I20r z 0 30 60 90 120 150 TIME (min) 40 p a r a s i t i c e f f e c t s , there was i n s u f f i c i e n t c o r r e l a t i o n to assume that immobilization and k i l l i n g were the same. Since the m o t i l i t y assay gave the most consistent r e s u l t s and was the easiest and quickest to perform, t h i s was the a n t i -p a r a s i t i c e f f e c t we chose to pursue to assess the e f f e c t of antibody against g i a r d i a . SENSITIVITY OF TROPHOZOITES TO IMMUNE SERUM To determine the minimum concentration of immune serum necessary to immobilize trophozoites, serum was d i l u t e d 1:2, 1:8, 1:32. Figure 4 shows how the immobilizing e f f e c t was l o s t a f t e r only a 1:8 d i l u t i o n of immune serum. Thus, the immobilizing e f f e c t was highly dependent on the concentration of serum. The i n a b i l i t y of d i l u t e d immune sera to immobilize trophozoites could have been caused by inadequate concentrations of complement or g i a r d i a - s p e c i f i c antibodies. STRAIN VARIABILITY A number of d i f f e r e n t strains were used i n the i n - v i t r o m o t i l i t y assay and i t was observed that L s t r a i n , unlike 501 and DEL, was immobilized by pre-immune rabbit serum. Table II shows that the m o t i l i t y of DEL s t r a i n was only s l i g h t l y decreased i n the presence of pre-immune sera. In comparison, L s t r a i n m o t i l i t y was s i g n i f i c a n t l y decreased by pre-immune serum as compared to controls. I t ' s not known whether the 41 FIGURE 4: IMMOBILIZATION OF G.LAMBLIA WITH DILUTIONS OF IMMUNE SERUM 1-2 x 105 trophozoites i n 200ul PBS were incubated for 60 minutes with a constant volume (lOOul) of d i l u t e d serum. Each point on the graph represents the percent immobilized trophozoites (mean ± standard deviation). Mean values of data pooled from three separate experiments performed i n duplicate are shown. 42 IMMOBILIZATION O F G . L A M B L I A W I T H DILUTIONS O F I M M U N E S E R U M 100 -80 -60 -40 -20 -Dilutions of whole serum 43 TABLE II : IMMOBILIZATION OF L AND DEL STRAINS O F G.LAMBLIA TROPHOZOITES BY PRE-IMMUNE SERA strain Incubation Percent motility conditions (mean + s.e.m) L PBS 89.5 * 1.58 pre-immune serum 38.7 + 4.84 DEL PBS 88.67 • 2.33 pre-immune serum 69.00 <- 1.78 * trophozoites (1 x 105 /well) were incubated for 60 minutes with e i t h e r PBS or 20% pre-immune rabbit serum. Duplicate samples were counted from each well. Mean values of 2 separate experiments performed i n duplicate are shown. 44 s e n s i t i v i t y of L s t r a i n was caused by non-specific or immune components within the pre-immune serum. To determine whether complement was responsible for the decreased m o t i l i t y of L s t r a i n , the e f f e c t of rabbit complement against DEL and L s t r a i n m o t i l i t y was tested. Results (not shown) indicate that more concentrated rabbit complement (up to 1:8 dilution) caused both s t r a i n s of trophozoites to aggregate, making i t d i f f i c u l t to assay for f l a g e l l a r movement. Once d i l u t e d out 1:16, complement no longer caused aggregation. I t also had no s i g n i f i c a n t immobilizing e f f e c t on either s t r a i n (data not shown). EFFECT OF FECAL EXTRACTS ON TROPHOZOITE MOTILITY Using immunoblotting techniques, i t has been shown i n our lab that f e c a l extracts taken from g e r b i l s infected with G.lamblia contain g i a r d i a s p e c i f i c antibodies (Krystal et a l . Manuscript submitted for publication). To determine whether these antibodies had some functional role i n - v i t r o , the e f f e c t of f e c a l extracts against trophozoite m o t i l i t y was tested. Figure 5 shows the percent m o t i l i t y of s t r a i n 501 trophozoites a f t e r being incubated i n either RPMI media alone or with f e c a l extracts taken from infected or non-infected g e r b i l s . Fecal extracts prepared from infected g e r b i l s demonstrated the most s i g n i f i c a n t immobilizing e f f e c t . By 60 minutes, more than 80% of trophozoites were immotile, and a f t e r a 2 hour incubation, close to 100% of trophozoites were immotile. Fecal extracts 45 FIGURE 5: IN-VlTRO ASSAY USING FECAL EXTRACTS Effects of g e r b i l fe c a l extract preparations against trophozoite (501 strain) m o t i l i t y . 1 x 105 trophozoites i n 150ul RPMI media were incubated with either 150 u l of a f e c a l extract preparation taken 24 days post i n f e c t i o n (•) or an equal volume of fecal extracts taken from non-infected g e r b i l s ( # ) . Control ( A ) consisted of trophozoites i n RPMI media. Mean values (± standard deviation) of 3 separate experiments performed i n t r i p l i c a t e are shown. 46 IN-VITRO A S S A Y USING F E C A L E X T R A C T S 100 0 30 60 90 120 TIME (min) 47 prepared from non-infected g e r b i l s were able to immobilize trophozoites but the e f f e c t was less rapid and not as pronounced compared to immune fe c a l extracts. ABSORPTION OF IMMUNE SERA In a preliminary attempt to i s o l a t e immobilizing antibodies from immune sera, s u r f a c e - s p e c i f i c a n t i - g i a r d i a antibodies were absorbed from antisera using whole, l i v i n g trophozoites. For a l l the immunoabsorption experiments, antiserum was f i r s t treated with ammonium sulphate to p r e c i p i t a t e out immunoglobulins. The y i e l d from t h i s p u r i f i c a t i o n step has been referred to as the whole antibody f r a c t i o n . The absorption of the whole antibody f r a c t i o n against trophozoites yielded two fractions of interest; (1) the depleted f r a c t i o n which was depleted of s u r f a c e - s p e c i f i c antibodies, and (2) the eluted f r a c t i o n containing s u r f a c e - s p e c i f i c antibodies which had been eluted from the trophozoites. To determine whether the e l u t i o n step was s t r i p p i n g trophozoite antigens as well as g i a r d i a - s p e c i f i c antibodies, samples from the fractions were analyzed on an SDS g e l . Figure 6a shows the difference between the proteins found i n the whole antibody f r a c t i o n (lane 1), the depleted f r a c t i o n (lane 2), and the eluted f r a c t i o n (lane 3). Since the whole antibody f r a c t i o n was an ammonium sulphate p r e c i p i t a t e of whole serum, i t was expected that serum components other than antibody would be detected. The same res u l t s were expected 48 FIGURE 6a: PROTEIN COMPOSITION OF ANTIBODY FRACTIONS SDS-PAGE ( s i l v e r stained) showing the composition of the whole (lane 1), depleted (lane 2), and eluted (lane 3) antibody f r a c t i o n s . Protein concentrations of each of the fraction s was determined through t h e i r absorbance (280 nm) and are as follows: whole f r a c t i o n contained approximately 43 ng protein/well, depleted f r a c t i o n contained 22 ng/well and eluted f r a c t i o n contained 3 ng/well. 49 P R O T E I N C O M P O S I T I O N O F A N T I B O D Y F R A C T I O N S 50 f o r the depleted f r a c t i o n since t h e o r e t i c a l l y , the only difference between the whole antibody f r a c t i o n and the depleted f r a c t i o n i s the absence of s u r f a c e - s p e c i f i c antibodies. However, the absence of bands around 60 kd suggests that serum components may also be absorbed on to the trophozoites. In the eluted f r a c t i o n , there were a number of unique bands which did not correlate with the molecular weights of reduced antibody molecules and were not o r i g i n a l l y i n the whole antibody f r a c t i o n . Even though the whole antibody and depleted antibody fractions were more concentrated than the eluted antibody f r a c t i o n , these bands were c l e a r l y not present i n the two f r a c t i o n s . Controls containing trophozoites absorbed with PBS alone were not stripped of any antigens during the e l u t i o n process as indicated by an absence of bands on the gel (data not shown). It ' s l i k e l y that the incubation of trophozoites with g i a r d i a -s p e c i f i c antibodies rendered them more susceptible to s t r i p p i n g of antigens during the elu t i o n process than when they were incubated i n PBS alone. I t ' s also possible that the unique bands found i n the eluted antibody f r a c t i o n were degraded antibody molecules. Surface l a b e l l i n g of trophozoites would help to d i s t i n g u i s h between p a r a s i t i c components and serum components. Once the depleted and eluted antibody fract i o n s were isol a t e d , t h e i r functional a c t i v i t y against trophozoites was 51 determined using the m o t i l i t y assay, and t h e i r antigen s p e c i f i c i t y was determined using immunoblotting techniques. FUNCTIONAL ACTIVITY OF DEPLETED AND ELUTED FRACTIONS T h e o r e t i c a l l y , i f one or more sur f a c e - s p e c i f i c antibodies were responsible for the immobilizing a c t i v i t y seen with immune serum, i t would be fea s i b l e to demonstrate that the depleted antibody f r a c t i o n has l o s t t h i s e f f e c t to the eluted antibody f r a c t i o n . Table III shows the r e s u l t s of the i n -v i t r o m o t i l i t y assay using these two antibody f r a c t i o n s . As expected, the depleted antibody f r a c t i o n completely l o s t i t ' s immobilizing e f f e c t and was s i m i l a r to the control where over 80% of the trophozoites were motile a f t e r a 60 minute incubation period. Unfortunately, we were unable to show any s i g n i f i c a n t immobilizing e f f e c t for the eluted antibody f r a c t i o n . INDIRECT IMMUNOFLUORESCENCE ASSAY To determine i f the elu t i o n step recovered a l l surface bound antibodies from the trophozoites, i n d i r e c t immunofluorescence assays were performed a f t e r the elution procedure, to detect residual antibodies bound to the trophozoites. Trophozoites used i n the immunoabsorption study were incubated with a 1:80 d i l u t i o n of FITC conjugated goat a n t i -rabbit IgG. Figure 6b shows that the trophozoites were la b e l l e d , i n d i c a t i n g the presence of s u r f a c e - s p e c i f i c 52 Table I I I : EFFECT OF DEPLETED AND ELUTED ANTIBODY FRACTIONS ON TROPHOZOITE (STRAIN 501) MOTILITY Percent motility Incubation conditions * (mean ± s.e.m) P B S 90.0 ± 1.5 pre-immune sera 85.0 ± 2.0 whole antibody fraction // 36.8 ± 1.5 depleted antibody fraction 93.5 ± 2 . 8 eluted antibody fraction 87.0 ± 0.0 * 1-2 x 10s trophozoites i n 200 u l PBS were incubated for 60 minutes with lOOul of each of the antibody fractions l i s t e d above. The f i n a l volume i n each well was adjusted to 350 u l . A l l antibody fractions were supplemented with a source of complement (50ul). Control wells contain trophozoites i n PBS or 28% normal sera. Each value represents data pooled from three separate experiments. # ammonium sulphate pre c i p i t a t e d immune serum 53 FIGURE 6b: DETECTION OF SURFACE-BOUND ANTIBODIES AFTER ELUTION PROCEDURE Following the elut i o n procedure, trophozoites were washed once i n PBS to remove any remaining glycine buffer. They were then incubated with a 1:80 d i l u t i o n of a flu o r e s c e i n -conjugated goat anti-rabbit IgG. Trophozoites incubated i n PBS showed no fluorescence (data not shown). 54 D E T E C T I O N O F S U R F A C E - B O U N D A N T I B O D I E S A F T E R T H E E L U T I O N P R O C E D U R E t 55 antibodies. The e l u t i o n procedure was c l e a r l y not stringent enough to remove a l l the antibodies bound to the trophozoites. Therefore, i t ' s l i k e l y that the antibodies responsible for the immobilizing e f f e c t were not present i n the eluted antibody f r a c t i o n but remained bound to the trophozoites. ANTIGEN SPECIFICITY OF DEPLETED AND ELUTED FRACTIONS One of the aims of t h i s study was to characterize surface antigens of G i a r d i a using the s u r f a c e - s p e c i f i c antibodies we i s o l a t e d . Figure 7 shows immunoblots containing DEL s t r a i n antigen preparations probed with either whole (lane 1), depleted (lane 2) or eluted (lane 3) antibody f r a c t i o n s . In comparison to the whole antibody f r a c t i o n , there was an absence of a 40 kd band from the depleted f r a c t i o n . This band was consistently absent from t h i s f r a c t i o n i n 5 out of 6 experiments. This indicates that antibodies s p e c i f i c to a 40 kd surface antigen of g i a r d i a trophozoites were being absorbed from the whole antibody f r a c t i o n . In lane 3, the eluted f r a c t i o n recognized a major band of approximately 45 kd and a number of minor bands with approximate molecular weights of 33, 38, 40-42, 49, 57, 60, 73, 80 kd i n addition to two large molecular weight bands. These bands were consistently recognized by the eluted f r a c t i o n i n 6 experiments. However, the i n t e n s i t i e s of these bands varied between experiments, making i t d i f f i c u l t to consistently i d e n t i f y major bands. FIGURE 7: DETECTION OF GIARDIA SURFACE ANTIGENS USING DEPLETED AND ELUTED ANTIBODY FRACTIONS (TCA preparation) SDS-PAGE immunoblot of proteins of G.lamblia trophozoites (DEL s t r a i n ) . Lane 1 was reacted with a 1:40 d i l u t i o n of whole antibody against DEL s t r a i n . Lane 2 was reacted with a 1:40 d i l u t i o n of the depleted antibody f r a c t i o n and Lane 3 was reacted with one half of the eluted antibody f r a c t i o n . The numbers to the l e f t of the immunoblot represent the molecular weights (x 105) of the standards. 57 D E T E C T I O N O F GIARDIA S U R F A C E A N T I G E N S USING A N T I B O D Y F R A C T I O N S (TCA antigen preparation) 58 In our experiments, only one prominent band (40 kd) was consistently absent from the depleted f r a c t i o n ( f i g . 7). This band did not always appear i n the eluted f r a c t i o n . Also, few of the bands that appeared i n the eluted f r a c t i o n were absent from the depleted f r a c t i o n . These re s u l t s suggest that the immunoabsorption technique was i s o l a t i n g only a portion of the s u r f a c e - s p e c i f i c antibodies from immune serum. I t ' s possible that t h i s resulted from our adding an excess of the whole antibody f r a c t i o n to the p e l l e t of trophozoites. In an attempt to achieve an optimal antisera:trophozoite r a t i o , the absorption technique was repeated using a constant volume of trophozoites and varied concentrations of antisera. Figure 8 i s an immunoblot showing the antigen s p e c i f i c i t i e s of the antibody fractions where the i n i t i a l volume of antisera absorbed against trophozoites was 1:8 of that used previously. In comparison to the whole antibody f r a c t i o n (lane 1), there were a number of antibodies absent from the depleted f r a c t i o n (lane 2) r e s u l t i n g i n the absence of bands with approximate molecular weights of 93, 82, 79, 75, 60, 55, 49-40, 34, 32, 24-26 kd. In addition, a decrease i n the i n t e n s i t y of a major 33 kd band was evident. Also, a number of antibodies that were absent from the depleted f r a c t i o n were present i n the eluted f r a c t i o n . These antibodies recognized antigens with molecular weights of 49, 47, and 45 kd. There was also a major band of 3 3 kd that was present i n the eluted f r a c t i o n and was s i g n i f i c a n t l y decreased i n the depleted 59 FIGURE 8: DETECTION OF GIARDIA SURFACE ANTIGENS (1:8 d i l u t i o n of whole antibody fraction) SDS-PAGE immunoblot of trophozoite proteins showing the antigen s p e c i f i c i t y of the whole antibody f r a c t i o n (lane 1), the depleted antibody f r a c t i o n (lane 2), wash step (lane 3), and the undiluted eluted antibody f r a c t i o n (lane 4). The i n i t i a l volume of antisera used i n the absorbance study was only 1/8 of that used previously. 60 D E T E C T I O N O F GIARDIA S U R F A C E A N T I G E N S (1/8 dilution of whole antibody fraction) 1 2 3 4 97.4 -66.2 -42.7 -31.6 -21.5 -14.4 ~ j u 61 f r a c t i o n . These re s u l t s indicate that a more complete separation of s u r f a c e - s p e c i f i c antibodies from antisera was accomplished. EXTRACTION OP GIARDIA MEMBRANE PROTEINS To f a c i l i t a t e the detection of surface antigens by the eluted antibody f r a c t i o n , membrane enriched preparations were obtained from Giardia. Figure 9a shows the composition of t h i s preparation on an SDS/gel. Present were three major bands with approximate molecular weights of 32, 33 and 34 kd, as well as a number of minor bands; 21-31, 35, 37, 39, 50, and 70 kd. This membrane enriched preparation considerably reduced the complexity of antigens found i n whole trophozoite TCA preparations. This i s evident i n figure 9b where an immunoblot compares the antigen s p e c i f i c i t y of the whole antibody f r a c t i o n reacted against the TCA preparation (lane 2) and the membrane enriched preparation (lane 3). To determine whether the proteins i n the membrane enriched preparation were recognized by the eluted antibody f r a c t i o n , an immunoblot was performed. Figure 9c i s an SDS/PAGE immunoblot showing both TCA preparations (lane 2) and membrane preparations (lane 3) treated with the eluted f r a c t i o n . Whereas the eluted f r a c t i o n recognized a range of antigens i n the TCA preparation, there were only four antigens (33, 34, 51, 68 kd) detected i n the membrane preparation. 62 FIGURE 9a: COMPOSITION OF MEMBRANE ENRICHED PREPARATION OF 501 STRAIN Composition of a membrane enriched preparation of 501 by SDS/gel electrophoresis on a 12% polyacrylamide g e l . A 50 u l sample containing 3.7 ug of protein was added to the well. The gel was s i l v e r stained. Molecular weights of standards are indicated by numbers on the righ t . 63 C O M P O S I T I O N O F M E M B R A N E E N R I C H E D P R E P A R A T I O N O F 501 S T R A I N 97.4 -66.2 -i 42.7 -31.0 -21.5 -14.4 -64 FIGURE 9b: DETECTION OF GIARDIA SURFACE ANTIGENS USING WHOLE ANTIBODY FRACTION (enriched membrane preparation) SDS-PAGE immunoblot of TCA precipitated trophozoite (501 strain) antigens (lane 2) and enriched membrane preparations (lane 3) reacted against the whole antibody f r a c t i o n (1:40 d i l u t i o n ) . Lane 1 represents low molecular weight b i o t i n y l a t e d standards. 65 D E T E C T I O N O F GIARDIA S U R F A C E A N T I G E N S USING W H O L E A N T I B O D Y F R A C T I O N (enriched membrane preparation) 1 2 3 66 FIGURE 9c: DETECTION OF GIARDIA SURFACE ANTIGENS USING ELUTED ANTIBODY FRACTION (enriched membrane preparations) SDS-PAGE immunoblot of TCA preci p i t a t e d trophozoite (501 strain) antigens (lane 2) and enriched membrane preparations (lane 3) reacted against the eluted antibody f r a c t i o n (1:4 d i l u t i o n ) . Lane 1 represents low molecular weight b i o t i n y l a t e d standards. 67 D E T E C T I O N O F GIARDIA S U R F A C E A N T I G E N S USING E L U T E D A N T I B O D Y F R A C T I O N (enriched membrane preparations) 68 DISCUSSION There were two basic objectives to t h i s study. The f i r s t objective was to i d e n t i f y a n t i - p a r a s i t i c antibodies using i n -v i t r o m o t i l i t y assays and to i s o l a t e then from immune serum using an immunoabsorption technique. The second objective was a preliminary attempt to i d e n t i f y relevant surface antigens from the complex repertoire of u n i d e n t i f i e d trophozoite components using the is o l a t e d s u r f a c e - s p e c i f i c antibodies i n immunoblots. IN-VITRO ASSAYS MEASURE ANTI-PARASITIC EFFECTS OF ANTISERA In order to define the role of host antibody response against g i a r d i a s i s , i n - v i t r o assays were established to measure a n t i - p a r a s i t i c e f f e c t s of immune rabbit sera against s t r a i n s of G.lamblia. Two a n t i - p a r a s i t i c e f f e c t s , immobilization and c e l l death, consistently showed that immune serum was able to immobilize and k i l l two s t r a i n s of Giardia trophozoites (501 and DEL) whereas pre-immune serum was not. We also found that one p a r t i c u l a r s t r a i n , L, was immobilized by pre-immune sera. F l a g e l l a r movement was assessed using a phase contrast microscope. Healthy, viable trophozoites swim f r e e l y i n the media i n a r o t a t i o n a l movement and resemble a f a l l e n l e a f . Trophozoites may also attach to the surface of the culture tube v i a t h e i r d i s c . In t h i s case, wave-like movements of the 69 caudal f l a g e l l a can be c l e a r l y v i s u a l i z e d . In the presence of immune sera (501, DEL strains) or pre-immune sera (L s t r a i n ) , trophozoites become motionless and there i s no movement of the f l a g e l l a . By phase contrast microscopy, immobilized trophozoites also lose t h e i r refractory q u a l i t y and n u c l e i became v i s i b l e . The e f f e c t of immune sera on trophozoite m o t i l i t y was thought to be mediated by a n t i - g i a r d i a antibodies. This was suggested by (a) the a b i l i t y of immune sera but not pre-immune sera to immobilize DEL and 501 s t r a i n trophozoites (b) the absence of immobilization using absorbed immune sera, and (c) immunofluorescense assays (IFA) which detected the presence of g i a r d i a - s p e c i f i c antibodies i n immune but not pre-immune sera. Immobilization of 501 and DEL s t r a i n trophozoites by immune sera could occur v i a a number of d i f f e r e n t mechanisms. For example, antibodies could d i r e c t l y bind and agglutinate trophozoite f l a g e l l a . However, immobilizing a c t i v i t y was shown to be dependent, to varying a extent, on complement. Complement ac t i v a t i o n may r e s u l t i n a number of b i o l o g i c a l e f f e c t s such as (i) changes i n vascular permeability, ( i i ) the a t t r a c t i o n of leukocytes into the area of immunological reaction, ( i i i ) an enhanced a c t i v a t i o n of phagocytes, and (iv) membrane damage (34). Although trophozoite i n t e g r i t y appeared i n t a c t throughout the m o t i l i t y assay, membrane disruption could occur v i a small r i n g - l i k e lesions caused by l a t e phase complement components. These lesions could disrupt the io n i c 70 balance ( i . e . Ca") or proton motive force required for f l a g e l l a r movement. Whereas the r o l e of complement-fixing antibodies against DEL and 501 s t r a i n trophozoite m o t i l i t y i s f e a s i b l e , t h i s does not account for the immobilizing e f f e c t s of pre-immune sera against L s t r a i n trophozoites. I t seems more l i k e l y that i n t h i s case, an antibody independent mechanism i s involved. Since the a l t e r n a t i v e complement pathway can be activated i n the absence of antibodies, i t ' s s t i l l possible that L s t r a i n , l i k e 501 and DEL, could loose f l a g e l l a r m o t i l i t y from the small lesions i n the membrane caused by late phase complement components. To determine the role of complement as a mechanism of defense against Giardia, i t would be necessary to e s t a b l i s h whether the c l a s s i c a l or alternative complement pathway i s activated. Furthermore, the mechanism of complement action at the molecular l e v e l should be assessed; are lesions made in the membrane and what i s the r e s u l t . F i n a l l y , the relevancy of complement components in-vivo should be addressed ( i . e . what i s the r o l e of complement i n i n t e s t i n a l p a r a s i t i c infections?) The a b i l i t y of immune sera to immobilize trophozoites i n -v i t r o was concentration dependent. It's possible that the l i m i t i n g factors i n the assay were either non-immune (i.e complement) or due to g i a r d i a - s p e c i f i c antibodies. I t w i l l be 71 important for future work to determine which a c t i v i t y was l o s t when immune sera was di l u t e d . I t was recently reported that G.lamblia trophozoites were lysed i n the presence of g i a r d i a - s p e c i f i c antibodies as well as by the a c t i v a t i o n of the c l a s s i c a l complement pathway (34). Another study indicated that the l y t i c e f f e c t s of g i a r d i a -s p e c i f i c antibodies was dependent on a unique complement pathway which required Cl and Ca++ but was independent of the c l a s s i c a l pathway components C2 and C4 (18). Which complement pathway was involved i n our m o t i l i t y assays remains to be determined. We were unable to demonstrate s u f f i c i e n t c o r r e l a t i o n between immobilization and c e l l death to use f l a g e l l a r movement as a c r i t e r i o n for c e l l death. H i l l (34) demonstrated a high c o r r e l a t i o n between the immobilizing a c t i v i t y and l e t h a l e f f e c t s of human sera against G.lamblia trophozoites. In our experiments, there wasn't s u f f i c i e n t c o r r e l a t i o n between the results of the m o t i l i t y assay and those of the v i a b i l i t y assay to assume that immobilized trophozoites were non-viable. The lack of c o r r e l a t i o n we have observed between f l a g e l l a r movement and c e l l v i a b i l i t y may be due to the s u b j e c t i v i t y of the neutral red staining procedure. Although i t was not necessary for our purposes, reculture experiments could have been used i n conjunction with neutral red staining to give a more accurate assessment of c e l l v i a b i l i t y . Since we did not do the studies, for our purposes, 72 immobilized trophozoites were not considered synonymous with non-viable trophozoites. We were unable to show that immune serum lysed trophozoites i n - v i t r o . B e l o s i v i c reported that serum from mice infected with G.muris was able to lyse trophozoites i n - v i t r o (6). I t ' s possible that the absence of l y t i c a c t i v i t y i n our experiments was due to our method of preparing immune serum. L y t i c antibodies against T.crusi are generated as the r e s u l t of an active i n f e c t i o n , and are not e l i c i t e d a f t e r immunization with fix e d parasites (54). In our experiments, the serum was generated by hyperimmunizing rabbits so i t may not have contained the class of antibodies capable of l y s i n g trophozoites. This also raises the question of how many other functional antibodies may not be detected when using a model that does not represent a true active i n f e c t i o n . ROLE OF STRAIN VARIABILITY IN COURSE OF INFECTION The spectrum of c l i n i c a l symptoms encountered with Giardia may r e s u l t from host or parasite v a r i a b i l i t y alone or together (17). I t ' s been suggested, but not proven, that some stra i n s of Giardia may be more pathogenic than others and that t h i s may also account for the variety of c l i n i c a l symptoms encountered. Most of our i n - v i t r o assays support the r o l e of host antibody responses i n influencing the course of i n f e c t i o n . However, we have also shown s t r a i n v a r i a b i l i t y with differences i n the responses of i s o l a t e s of G.lamblia to 73 various host components such as complement and pre-immune serum. The degree of complement dependent immobilization of immune sera d i f f e r e d between two stra i n s , 501 and DEL. With 501 s t r a i n , the immobilizing e f f e c t was completely dependent on the presence of complement. With DEL s t r a i n , trophozoites were p a r t i a l l y immobilized i n the absence of complement. This suggests that d i f f e r e n t s t r a i n s of trophozoites may be susceptible to d i f f e r e n t mechanisms of host defense. We have also shown that one s t r a i n of G.lamblia, L s t r a i n , was immobilized by pre-immune serum. This s e n s i t i v i t y was not observed with either 501 or DEL. It i s of p a r t i c u l a r i n t e r e s t that L s t r a i n was iso l a t e d from an asymptomatic patient. I t ' s possible that L could be an example of a less pathogenic s t r a i n . Whether the s e n s i t i v i t y of L to pre-immune sera was due to non-specific or immune components remains to be determined. Our data are consistent with the p o s s i b i l i t y that s t r a i n v a r i a b i l i t y may play a role in the course of i n f e c t i o n . IN-VITRO ASSAYS MEASURE FUNCTIONAL ACTIVITY OF FECAL EXTRACTS Recently we have shown, using immunoblotting techniques, that f e c a l extracts from experimentally infected g e r b i l s contained g i a r d i a - s p e c i f i c antibodies (43). These same fecal extracts s i g n i f i c a n t l y decreased trophozoite m o t i l i t y i n -v i t r o . In comparison, fecal extracts from non-infected g e r b i l s had a decreased e f f e c t on trophozoite m o t i l i t y and showed eithe r a very low l e v e l or no Giardia s p e c i f i c responses i n immunoblots. Although pre-immune f e c a l extracts didn't have as great an e f f e c t on trophozoite m o t i l i t y as immune extracts, these pre-immune samples contained a s i g n i f i c a n t l y greater immobilizing e f f e c t than controls containing trophozoites i n RPMI media. This raises the p o s s i b i l i t y that non-immune host components contain a n t i -p a r a s i t i c a c t i v i t y which a f f e c t s parasite elimination. I t ' s been shown that trophozoites were k i l l e d i n - v i t r o by non-immune mechanisms such as free f a t t y acids from non-immune human milk (24,61) and l i p o l y t i c products i n i n t e s t i n a l f l u i d s from non-infected humans (86). Whether the immobilizing a c t i v i t y we have observed i n pre-immune fe c a l extracts was attri b u t e d to non-specific fecal components or immune components such as cross reactive antibodies remains to be determined. Because fe c a l extracts contain non-specific and immune components secreted from the intestine, i t may represent the closest approximation of mucosal host defense mechanisms. I t i s also an easier alternative to working with i n t e s t i n a l f l u i d s (4). Since G.lamblia i s an i n t e s t i n a l parasite, working with f e c a l extracts offers opportunities to i) determine the importance of the non-immune mucosal response in parasite elimination and i i ) to i d e n t i f y components i n immune fe c a l samples which are responsible for a n t i - p a r a s i t i c e f f e c t s seen i n - v i t r o . 75 IMMUNOABSORPTION The i n - v i t r o m o t i l i t y assay has consistently measured the immobilizing e f f e c t s of antisera and fe c a l extracts against d i f f e r e n t s t r a i n s of Giardia. Since we ultimately hoped to use a n t i - p a r a s i t i c antibodies to i d e n t i f y trophozoite antigens, we attempted to i s o l a t e immobilizing antibodies from antisera using an immunoabsorption technique. The immunoabsorption technique i s based on the assumption that a n t i - p a r a s i t i c antibodies important i n the host response are directed against surface components of trophozoites. Since surface antigens of giard i a have not been well characterized, whole, l i v i n g trophozoites were used to i s o l a t e s u r f a c e - s p e c i f i c antibodies from a n t i - g i a r d i a immune sera. The absorption technique yielded two antibody fractions of int e r e s t . The f i r s t f r a c t i o n consisted of antisera depleted of surface bound antibodies (depleted antibody fraction) and the second f r a c t i o n contained s u r f a c e - s p e c i f i c antibodies which had been eluted from trophozoites (eluted antibody f r a c t i o n ) . This was the f i r s t attempt made to d i r e c t l y i s o l a t e s u r f a c e - s p e c i f i c antibodies bound to trophozoites. LOCALIZATION OF IMMOBILIZING ACTIVITY To determine whether immobilizing antibodies were i s o l a t e d i n the eluted f r a c t i o n , the m o t i l i t y assay was performed using the depleted and eluted antibody fract i o n s . The re s u l t s show that the depleted antibody f r a c t i o n was unable to immobilize trophozoites. This indicates that antibodies responsible for 76 the immobilizing e f f e c t were absorbed from the antisera. Loss of functional a c t i v i t y by 'absorbed* sera (corresponding to our depleted fraction) has been demonstrated using antisera against G.muris where absorption of g i a r d i a - s p e c i f i c antibodies with l i v i n g trophozoites resulted i n a loss of l y t i c a c t i v i t y i n - v i t r o (6). Unfortunately, we were unable to show an e f f e c t of the eluted antibody f r a c t i o n on trophozoite m o t i l i t y . Controls confirmed the a c t i v i t y of whole antisera and the v i a b i l i t y of trophozoites incubated in PBS. We were l e f t with the p o s s i b i l i t y that the immunoabsorption technique had not successfully i s o l a t e d functional, immobilizing antibodies from the surface of trophozoites. Since i n d i r e c t immunofluorescence assays detected antibodies on the trophozoites following the elut i o n procedure, we concluded that the e l u t i o n procedure had not removed a l l surface-s p e c i f i c antibodies from the trophozoites. The proportion of antibodies remaining bound i s not known. I f immobilizing antibodies were directed against surface components, i t ' s l i k e l y that at least some of the immobilizing antibodies were not eluted from the surface of the trophozoites. Since we've shown that the immobilizing a c t i v i t y was dependent on the concentration of antisera, i f enough antibodies were l e f t on the trophozoites, i t may have been s u f f i c i e n t to abolish the i n - v i t r o a n t i - m o t i l i t y e f f e c t . 77 I t ' s also possible that the immobilizing antibodies remained bound to released trophozoite products. Trophozoites i n culture release antigenic, surface derived material into the surrounding culture media (51). I f antigens spontaneously released from parasites or stripped during the e l u t i o n procedure bound immobilizing antibodies, i t would r e s u l t i n a loss of functional a c t i v i t y i n the m o t i l i t y assay. To ensure complete separation of s u r f a c e - s p e c i f i c antibodies from trophozoites, methods need to be devised which w i l l i s o l a t e and p u r i f y the antibodies from t h e i r antigen binding s i t e s without disrupting the i n t e g r i t y of the antibody molecules. Once t h i s has been accomplished, the eluted f r a c t i o n should contain a l l s u r f a c e - s p e c i f i c antibodies and i t ' s immobilizing a c t i v i t y could be re-assessed. I t ' s also possible that the eluted antibody f r a c t i o n did contain a s u f f i c i e n t quantity of immobilizing antibodies but our m o t i l i t y assay wasn't sensitive enough to detect them. We have showed that the immobilizing a c t i v i t y of immune sera was quickly l o s t when the sera was d i l u t e d . At t h i s point, i t s t i l l hasn't been determined whether t h i s loss of a c t i v i t y was due to complement or antibodies. Therefore, the s e n s i t i v i t y of the m o t i l i t y assay needs to be improved and i t ' s l i m i t a t i o n s c l e a r l y defined. CHARACTERIZATION OF GIARDIA SURFACE ANTIGENS Since we couldn't i s o l a t e immobilizing antibodies s u f f i c i e n t l y , we were unable to s p e c i f i c a l l y characterize 78 t h e i r p o t e n t i a l target antigens. However, we had managed to i s o l a t e a f r a c t i o n of surface-specific antibodies from antisera which we used to characterize surface antigens of Giardia trophozoites by immunoblotting. Using immunoblotting techniques, we were able to consistently demonstrate that the depleted antibody f r a c t i o n had l o s t antibodies recognizing an antigen with a molecular weight of 40 kd. Occasionally, other minor bands were also absent on immunoblots using the depleted f r a c t i o n . The eluted f r a c t i o n recognized a number of bands on immunoblots with molecular weights of 33, 38, 40-42, 45, 49, 53, 57, 60, 73-75, 80 and 89 kd. There was too much v a r i a t i o n i n band i n t e n s i t i e s between experiments to make any quantitative statements. The major bands recognized by the eluted antibody f r a c t i o n s were at 33, 45, 49, 53 and 72 kd. Although the i n t e n s i t y of bands varied, the bands were consistently recognized by the eluted f r a c t i o n over a number of experiments. This indicates that q u a l i t a t i v e l y , the immunoabsorption technique was consistent. By combining information on the antibodies which were absent from the depleted f r a c t i o n and those present i n the eluted f r a c t i o n , we have i d e n t i f i e d approximately a dozen antigens recognized by s u r f a c e - s p e c i f i c antibodies. The key to complete separation of s u r f a c e - s p e c i f i c antibodies from antisera l i e s i n the optimal combination of antisera to a given volume of trophozoites. If t h i s antisera:trophozoite r a t i o i s not optimal, not a l l surface-s p e c i f i c antibodies w i l l be absorbed out of the antisera. This could occur for instance, i f a p a r t i c u l a r surface antigen ex i s t s i n small quantities but i s highly immunogenic. There w i l l be high concentrations of s p e c i f i c antibody where only a small number of binding s i t e s e x i s t . In our experiments, the sera:trophozoite r a t i o was not optimal since, i n immunoblots, there wasn't a complete c o r r e l a t i o n between bands that were absent from the depleted antibody f r a c t i o n and those present i n the eluted antibody f r a c t i o n . Complete separation of sur f a c e - s p e c i f i c antibodies from antisera could be confirmed by using IFA to detect s u r f a c e - s p e c i f i c antibodies remaining i n the depleted f r a c t i o n . Some of the bands observed i n t h i s study were detected i n previous studies. E i n f e l d (20) previously i d e n t i f i e d and characterized an 82 kd major surface antigen of G.lamblia using crossed immuno-electrophoretic analysis of trophozoites with g i a r d i a s p e c i f i c hyperimmunized rabbit sera. Minor surface l a b e l l e d proteins of 180, 105, 63, 55, 37, 30 and 24 kd were also i d e n t i f i e d . Taylor (76) used human a n t i -G.lamblia sera to p r e c i p i t a t e a major surface antigen of 31 kd. Upcroft (80) used immune sera from both symptomatic and asymptomatic patients to i d e n t i f y a 32 kd surface antigen of G.lamblia that was associated with f l a g e l l a and surface components. In another study, sera obtained from symptomatic and asymptomatic patients precipitated surface antigens from 80 l a b e l l e d trophozoites with molecular weights of 85, 63 and 55 kd (55). Human immune sera was also shown to p r e c i p i t a t e an 88 kd surface protein from radio-iodinated trophozoites (19). Monoclonal antibodies have also been used to characterize a 170 kd surface antigen of Giardia (54). ENRICHED MEMBRANE EXTRACTION OF GIARDIA To f a c i l i t a t e the detection of surface antigens by the eluted antibody f r a c t i o n , the repertoire of trophozoite antigens was considerably reduced by preparing membrane enriched extractions of trophozoites. Since we were not able to measure the purity of membrane markers of Giardia, and since recent work on membrane extraction has yielded varied r e s u l t s (10,17,44,), we can only suggest that t h i s membrane preparation i s enriched although possibly contaminated by c y t o s o l i c or cytoskeletal components. Analysis of the enriched membrane preparation by SDS/PAGE revealed 3 major bands shown as a t r i p l e t of 32, 3 3 and 3 4 kd. These bands resemble re s u l t s obtained by Clark (10) who i s o l a t e d membrane preparations of G.lamblia and found a group of polypeptides ranging from 32-38 kd. Others have also found l a b e l l i n g of these polypeptides at the surface of G.lamblia (20) and G.muris (21). These antigens were reported to be a n t i g e n i c a l l y d i s t i n c t from the 3 0 kd g i a r d i n proteins which were found i n the cytoskeleton (57). Proteins of approximately 30 kd have also been l o c a l i z e d by IFA i n the ventral f l a g e l l a of G.lamblia (14). I t ' s therefore possible 81 that the proteins we have isol a t e d i n membrane preparations were derived from the ventral f l a g e l l a . We can't exclude the p o s s i b i l i t y that they are cytoskeletal contaminants. I t would be necessary to surface label trophozoites or locate a membrane marker to esta b l i s h the purity of the membrane enriched preparation. Immunoblots of membrane enriched preparations reacted with the eluted antibody f r a c t i o n showed four bands with molecular weights of 33, 34, 51, and 68 kd. This r e s u l t suggests that these four immunogens are a l l potential targets for a n t i -p a r a s i t i c antibodies and should be considered for i s o l a t i o n and characterization. Since the eluted f r a c t i o n did not contain a l l surface-s p e c i f i c antibodies, there are probably some surface antigens that have gone undetected. I t i s also possible that the immunoblotting technique caused conformational changes i n trophozoite antigens and loss of antibody recognition s i t e s . To ensure that a l l antibody-antigen reactions are detected, immunoblots should be probed with ant i - r a b b i t IgM and IgA as well as IgG. So far, anti-rabbit IgG was the only secondary antibody used. Alternate methods such as immunoprecipitation should also be considered. SUMMARY 82 I n - v i t r o m o t i l i t y assays have consistently measured the immobilizing e f f e c t s of antisera and fe c a l extracts against d i f f e r e n t s t r a i n s of Giardia. We have manipulated the conditions of t h i s assay to determine the r o l e that a n t i -g i a r d i a antibodies, complement and parasite v a r i a t i o n may play i n the s u s c e p t i b i l i t y of trophozoites to host a n t i - p a r a s i t i c e f f e c t s . We've shown, through an immunoabsorption technique we've developed, that i t ' s possible to i s o l a t e surface-s p e c i f i c antibodies from immune sera using whole, l i v i n g trophozoites. Furthermore, these surface bound antibodies, upon e l u t i o n from trophozoites, retained t h e i r antigen binding cap a c i t i e s . By using both the depleted and eluted f r a c t i o n s i n immunoblots, we have i d e n t i f i e d , i n d i r e c t l y , antigens which are located on the surface of trophozoites. The use of enriched membrane preparations i n immunoblots was e s p e c i a l l y i n t e r e s t i n g . When incubated with the eluted f r a c t i o n , four bands of 33, 34, 51 and 68 kd were present. This confirmed that eluted antibodies were directed against surface antigens. The detection of the 33-34 kd bands by eluted antibodies was of p a r t i c u l a r i n t e r e s t since these may well be the same polypeptides that were derived from the ventral f l a g e l l a (57). Since our ultimate goal i s to i d e n t i f y antigenic targets of a n t i - p a r a s i t i c antibodies, the next l o g i c a l step would be to l o c a l i z e the immobilizing a c t i v i t y of g i a r d i a - s p e c i f i c antibodies to the eluted antibody f r a c t i o n , use t h i s f r a c t i o n to target antigens within the membrane enriched preparation, 83 p u r i f y the antigens and use them to generate monospecific antibodies. The advantage of using monospecific over polyclonal antibodies in the m o t i l i t y assay i s that the former may contain higher quantities of immobilizing antibodies compared to the l a t t e r . 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