Molecular and Biochemical Parasitology, 42 (1990) 125-132 Elsevier

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MOLBIO 01375

Variant specific epitopes of Giardia lamblia T h e o d o r e E. Nash, John T. Conrad, and James W. Merritt, Jr. Laboratory of Parasitic Diseases, National Instttute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A. (Received 14 February 1990; accepted 10 April 1990)

Giardia lamblia undergoes surface antigenic variation. The capability of different isolates to express certain epitopes on the surfaces of trophozoites from different isolates and clones was determined using 4 surface-reacting monoclonal antibodies (mAbs) to variants derived from WB or WB-like Giardia (mAbs 6E7, 5C1, and 3F6) and GS/M (mAb G10/4). Of 28 isolates, 11 possessed trophozoites reactive with mAbs 6E7, 5C1 and 3F6, 6 with mAb 3F6, 2 with Mab G10/4, 1 with mAb 6E7, and 8 showed no reactivity as determined by direct or indirect immunofluorescence. Newly established clones from different isolates generated small numbers of reactive trophozoites similar to their parents. Only one epitope was found on any single trophozoite. Southern blots hybridized to a probe encoding for the epitope recognized by mAb 6E7 revealed that the inability to express the antigen in most isolates was due to lack of the gene. Analysis of the surface antigens of mAb 6E7 reactive clones from 3 isolates revealed that mAb 6E7 reacted with surface antigens of different molecular masses. Key words: Giardia; Antigenic variation; Surface antigen; Cysteine-rich proteins

Introduction

The surface antigens of Giardia lamblia isolates show a remarkable diversity which is due in part to surface antigenic variation [1--4]. Antigenic variation is common to all isolates studied [1-3], involves cysteine-rich proteins [2,3], occurs in humans [5] and animals [4,6] experimentally infected with G. lamblia, and may involve genetic rearrangements [2]. The frequency of surface antigenic variation is isolate-dependent and occurred once in every 12-13 generations and 6-7 generations in two different isolates [7]. A major unanswered question is the nature and number of surface antigens among Giardia. Prior studies showed varying degrees of antigenic relatedness [8,9] among isolates which could be due to antigenic cross reactivity, quantitative differences such as the amount of a particular epitope on the Correspondence address" T.E. Nash, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A. Abbreviations: mAb, monoclonal antibody; PBS, phosphatebuffered saline.

surface of a trophozoite, or varying numbers of trophozoites expressing a particular epitope in a heterogenous population. To determine whether similar epitopes could be found in different isolates, monoclonal antibodies were produced to four different surface antigens, and the ability of these mAbs to react with the surfaces of 28 different isolates was evaluated. The presence of the gene encoding one of the epitopes was determined using Southern blots hybridized to a probe specific for one of the epitopes. Additionally, the surface antigens of different Giardia isolates which reacted with the same mAbs were analyzed. Materials and Methods Isolates. Twenty-eight isolates representing all but one of the isolates available in the laboratory were studied [8,9]. Pertinent information about these isolates is summarized in Table I. Maintenance. Giardia were cryopreserved until use. Trophozoites were maintained in TYI-S-33 medium with antibiotics usually for less than 1

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TABLE I Reactivity of isolates to monoclonal antibody Monoclonal

3F6

5C

6E7

G 10/4

Hybridization w/CRP- 170

Isolate (source) W B (NIH: 1179) Isr (NIH:1183) LTb RSb Be-2 ~ (IP-0583:1) CAT- 1a Dan (NIH:0984) G2M d G3M d P-la GP- 1a

++ ++ + + + + ++ ++ ++ ++ ++

++ + + + + ++ + ++++ ++ ++ +++

++++ ++++ ++++ ++++ ++++ ++++ ++++ + +++ + ++

0 0 0 0 0 0 0 0 0 0 0

+ + + + + + + + + + +

DH (NIH:0284-2) JH (NIH:1182) Sug (NIH:0784:1) Ac (NIH:0484:1) N (NIH:0782) AB (NIH:0883:l)

+ + + + ++ +

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

+ + + + +

E2/M (NIH:0285-2)

0

0

++

0

+

Be-1 c (IP-0482:1) Erin (NIH:0284-3) And~ (NIH: 1284-2) M a n o (NIH: 1284-1 ) G1M d E9/M (NIH:0285-9) PM (NIH:0988) E4/E (NIH0285-4)

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

GS/M-83 (NIH: 1083-2) CM (NIH:0883:2) GS/M-83-H7 GS/M-85 GS/M-85-B6

0 0 0 ++ ++++

0 0 0 ++ ++

0 0 0 ++ +

++++ + ++++ ++ 0

+ +

WB-A6-6E7S WB-A6-6E7R WB-69-6E7R, 5C1R

++ ++ +

++ ++++ +

++++ + +

0 0 0

+ + n.d.

+ + + -

+, 1_~ 10/slide; ++, >10/slide but 10 per slide but < 1%), or +++ (1% or greater positive) or ++++ (almost all the Giardia were positive). Appropriate negative and positive controls were used with each assay. Assays were performed by an observer unaware of the identity of the samples being tested; most isolates were tested on multiple occasions. At times indirect and direct assays were both performed on the same samples and gave comparable results. Two epitopes were tested in the same assay using two different mAbs (6E7 and 3F6), each conjugated to different dyes to determine if both epitopes were present on the same trophozoite. Assays were also performed in microtiter plates using fixed trophozoites. Giardia which adhered to the bottom of the wells were rinsed with warm PBS and then fixed with n-propanol for approximately 1 min at room temperature. Assays were performed using the same dilutions, reaction times, and reagents, except that the assay was carried out at room temperature; medium was used

128 as a diluent to diminish background fluorescence; and fluids were removed by aspiration. Trophozoites with surface reactivity were determined with an inverted Zeiss microscope IM equipped with Hoffman optics and epifluorescence. Surface radiolabeling, using 125I [9,13], 5-15% gradient polyacrylamide electrophoresis under non-reducing conditions [9,13], Western blots [1], and radioimmunoassays [ 1,9,11 ] were performed as previously outlined. Southern blots were performed as described [2,8] but electrophoresis was carried out overnight instead of 48 h and hybridized using a probe which encodes for the epitope recognized by mAb 6E7 (crp 170) [2]. The probe consists of a 1-kb portion consisting of two tandem repeats of 195 nucleotides and part of a third and a nonrepetitive portion. Filters were washed twice for 1 h in 0.2 x SSC (3 M NaC1, 0.3 M Na-citrate)/0.1% sodium dodecyl sulfate at 65°C. Autoradiography was performed with intensifying screens at - 7 0 ° C for between 2 h and 5 days. Results

Varying numbers of trophozoites reactive to mAbs were reproducibly seen in cultures from WB clones. Trophozoites reacting with mAbs 6E7, 3F6, and 5C1 (when tested) were detected in clones of WB or derived progeny possessing different variant surface antigens (WB-676E7R; WB-65-6E7R,; WB-69-6E7R,5C1R; WBC9-6E7R; and WB-A6-6E7R). To rule out contamination, a mAb 6E7S positive clone, WBC9-6E7S, as well as mAb 6E7 reacting isolates CAT-I, Be-2, and G3M, were recloned and these newly established clones tested for reactivity with mAbs 6E7, 3F6 and, at times, with 5C 1. Predominantly 6E7S clones or cultures yielded trophozoites that reacted almost exclusively with mAb 6E7 but also contained small numbers which reacted with mAbs 5C1 and 3F6. Small numbers of trophozoites in WB-C9-6E7R, a clone which does not react predominantly with any of our mAbs (a null clone), reacted with mAbs 6E7, 5C1, and 3F6. Similar results were obtained using isolate CM, which contained rare trophozoites reactive with mAb G10/4. Newly established clones of CM contained small numbers of

G10/4-reacting trophozoites. Direct immunofluorescence using rhodamine and fluorescein labeled mAbs 3F6 and 6E7 and an indirect assay for mAb 5C1 reactivity followed by direct immunofluorescence assays revealed only a single epitope on the surface of any reacting trophozoite. Therefore, individual trophozoites from different isolates generate variants that possess epitopes which were the predominant surface antigens in their parent cultures and certain other isolates and clones. The presence or absence of trophozoites capable of reacting with a panel of four mAbs (two from variants of isolate WB (6E7 and 5C1), one from isolate GS (G10/4), and the fourth (3F6) most likely from WB or a WB-like isolate) was determined in 28 isolates (Table I). Trophozoites with surface reactivity to each of the three mAb 6E7, 5C1, or 3F6 were observed in eleven isolates. Only one mAb reacted with any trophozoite. In 6 other isolates, trophozoites with surface reactivity to 3F6 alone were observed and one isolate reacted only with mAb 6E7. In addition, 2 reacted with mAb G10/4 and 8 showed no reactivity to any of the mAbs. mAb G10/4 reactive trophozoites were never seen in the same isolate with trophozoites reactive with mAb 6E7, 5C1, or 3F6. The presence of these epitopes was mutually exclusive. Southern blots of the DNA from 28 isolates restricted with HindIII and EcoRI and hybridized using a probe which encodes the epitope recognized by mAb 6E7 revealed all 12 isolates which expressed the epitope recognized by mAb 6E7, 5 of 6 which reacted with the epitope recognized by mAb 3F6, and none of 10 which reacted with mAb G 10]4 or failed to react with any of the other mAbs hybridized to the crp 170 probe (Fig. 1) (Table I). Hybridization to Giardin (14) showed a strong signal in the isolates which failed to hybridize to the crp 170 probe (data not shown). Although the hybridization patterns differed among the isolates and may further differentiate major groupings, the complete failure of some isolates to hybridize indicates that they lack the gene encoding for crp 170 antigen. Because the pattern of reactivity of GS isolates differed, a number of GS isolates cryopreserved at different times since axenization were tested for surface immunofluorescence. These included

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