Molecular and Biochemical Parasitology, 41 (1990) 53-64 Elsevier
53
MOLBIO 01342
Identification and characterization of a target antigen of a m o n o c l o n a l antibody directed against Eimeria tenella m e r o z o i t e s Christine Ko 1, Charles K. Smith 112 and Michael McDonell 1. lSynergen, Inc., Boulder, CO, U.S.A., and ~Eli Lilly and Company, Lilly Research Laboratories, Greenfield, IN, U.S.A.
(Received 4 September 1989; accepted 25 January 1990)
Monoclonal antibodies (Mab) were produced against Eimeria tenella merozoites. A single Mab, LPMC-61, was selected because of its ability to bind to merozoites by indirect immunofluorescence assay (IFA) and to inhibit in vitro sporozoite development. Mab LPMC-61 reacts with an approx. 10-12-kDa merozoite polypeptide in reduced SDS-PAGE, but with an approx. 80-kDa protein in non-reduced SDS-PAGE. The monoclonal recognizes similarly sized polypeptides in E. tenella sporozoites, oocysts and schizonts. A partial cDNA (LPMC-61f) encoding the LPMC-61 antigen was identified from an E. tenella sporozoite cDNA library in bacteriophage hgtll. In addition to Mab LPMC-61, the recombinant 13-galactosidase/LPMC-61ffusion protein is recognized by hyperimmune rabbit anti-E, tenella sporogoite serum, rabbit anti-E, tenella merozoite serum, and E. tenella-infected and immune chicken sera. DNA sequencing of LPMC-61f cDNA showed that the putative protein has an unusual tandem, non-perfect repeated sequence, with glutamine comprising about 48% of the predicted amino acids. A hydropathicity plot of the predicted amino acid sequence shows a central hydrophilic region, consisting of the repeated sequences, surrounded by hydrophobic regions on both sides. Since the merozoite ~tage of avian Eimeria has been implicated in the induction of a protective immune response in chickens, LPMC-61 may be an important immunogen for use as a vaccine against E. tenella. Key words: Eimeria tenella; Merozoite; Sporozoite; Repeat sequence; Monoclonal antibody.
Introduction Eimeria a n d P l a s m o d i u m a r e r e l a t e d p r o t o z o a of t h e p h y l u m A p i c o m p l e x a [1]. T h e fact t h a t m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t Plasmodium surface c i r c u m s p o r o z o i t e p r o t e i n s will inhibit s p o r o z o i t e m o t i l i t y a n d cell p e n e t r a t i o n in vitro [2-5] h a s s t i m u l a t e d s i m i l a r efforts in the s t u d y o f Eimeria. M o n o c l o n a l s d i r e c t e d against Correspondence address: Christine Ko, Synergen, Inc., 1885, 33rd Street, Boulder, CO 80301, U.S.A. "Present address: Syntro Corporation, San Diego, CA, U.S.A. Abbreviations: IPTG, isopropyl 13-o-thiogalactopyranoside; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HBSS, Hanks' balanced salts solution; IFA, indirect immunofluorescence assay. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M30933.
Eimeria tenella s p o r o z o i t e surface p r o t e i n s h a v e b e e n f o u n d by D a n f o r t h to inhibit s p o r o z o i t e p e n e t r a t i o n of c u l t u r e d cells a n d to i n h i b i t develo p m e n t of the p a r a s i t e in cell c u l t u r e , p r o v i d e d t h a t the a n t i b o d y was p r e s e n t c o n t i n u o u s l y in t h e cell c u l t u r e m e d i u m [6]. G o r e a n d N e w m a n [7] described m o n o c l o n a l antibodies Ptn 7.2A4/4 and Ptn 9.9D12 which w e r e c a p a b l e o f n e u t r a l i z i n g E. tenella s p o r o z o i t e s in an in vitro assay a n d b o t h r e a c t e d with t h e surface of s p o r o z o i t e s in i n d i r e c t immunofluorescent a n t i b o d y tests. H o w e v e r , n o n e of t h e f o u r m e r o z o i t e - e l i c i t e d m o n o c l o n a l antib o d i e s t h a t c r o s s - r e a c t e d with s p o r o z o i t e surface antigens, as d e s c r i b e d b y A u g u s t i n e a n d D a n forth, i n h i b i t e d the i n v a s i o n o f cells b y t h e spor o z o i t e s [8]. R e s e a r c h into t h e Eimeria p a r a s i t i c stages t h a t i n d u c e i m m u n i t y in chickens suggest t h a t different stages o f t h e life cycle differ in t h e i r abilities to i n d u c e p r o t e c t i v e i m m u n e r e s p o n s e s . T h e sexual stages have little immunizing potential [9], and
0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
54 the sporozoites and the first generation schizonts are probably not very immunogenic [10]. The second generation schizont or merozoite stages of avian Eimeria have been implicated as the most immunogenic of all stages of coccidia [11]. Therefore, an effective vaccine to protect poultry against coccidiosis might include merozoite antigens or common antigens shared by most of the life cycle stages. In this report we describe a monoclonal antibody, designated LPMC-61, which was raised against E. tenella merozoites. Mab LPMC-61 reacts with polypeptides present in several developmental stages and inhibits sporozoite development in vitro, suggesting that the target antigen for this monoclonal may be an important immunogen. This report also describes the cDNA cloning and characterization of a target antigen of Mab LPMC-61. The target antigen for LPMC-61 has an unusual repeated sequence similar to other repeated sequences previously identified [12].
jection with purified second generation merozoites of E. tenella, Eli Lilly strain 65. This antibody-producing cell line was then cloned by limiting dilution and ascites were produced in pristane-primed mice.
Materials and Methods
In vitro anti-coccidial activity. Bovine turbinate cells (American Type Tissue Culture, CRL 1390) were seeded into 8-chamber Lab-Tek tissue culture slides (Nunc) in DMEM plus 10% fetal bovine serum (FBS). The cultures were permitted to reach 70-80% confluency and then 4.5 x 10 4 E. tenella sporozoites were added to each well in cell culture medium containing dilutions of mouse ascites generated by the hybridoma LPMC-61 (1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280). One infected control and one non-infected control well was maintained on each slide. Contemporary cultures were prepared exactly as described except that the ascites was generated by the parent myeloma (P3X-ascites), which produced no antibody reactive with E. tenella. A second series of cultures was prepared as above, except that the sporozoites were preincubated in medium containing the various dilutions of ascites for 1 h at 37°C and washed 2 x with HBSS prior to addition to cell cultures. All cultures were incubated at 37°C and representatives fixed and stained at 24, 48 and 72 h. The slides were examined microscopically for the presence of intracellular parasites.
Preparation of sporozoites,, schizonts and merozoites. Sporozoites and schizonts were prepared according to published procedures [13]. Purified E. tenella merozoites were prepared essentially as described by Fernando et al. [14] with modifications. Briefly, the cecae were removed from chickens 96 h following inoculation with E. tenella, washed with Hanks' balanced salt solution (HBSS), the mucosa scraped with a glass microscope slide, and collected in HBSS. This suspension was then homogenized with 8-10 strokes of a Dounce tissue grinder (Wheaton Scientific), filtered through a Leuko-pak column (Travenol Laboratories, Inc.), centrifuged at 850 × g for 10 min and the supernatant discarded. The merozoites were then purified by centrifugation in a Percoll gradient as described. Monoclonal antibody. Monoclonal antibody designated LPMC-61 was produced by a murine hybridoma resulting from the fusion of mouse spleen cells and mouse myeloma cells of the line P3-X63AG8, according to the method of Danforth [15]. Briefly, the spleen cells were obtained from a mouse which had been immunized by tail vein in-
Immunofluorescence localization of antibody reactivity. Spent media and ascites generated by the clone producing the antibody LPMC-61 was reacted with air-dried E. tenella merozoites and the antibody localized by indirect immunofluorescence assay essentially as described by Danforth [15]. In order to determine reactivity with intracellular stages of the parasite, cultures of bovine turbinate cells, prepared as described below, were infected with E. tenella sporozoites. At 4 h post-infection, cultures were washed 2 x with fresh medium to remove sporozoites that had not invaded. Cultures were removed at 24, 48 and 72 h, fixed with cold acetone, washed 2 x with IFA buffer, air-dried and stored at -80°C until use.
Polyacrylamide gel electrophoresis and Western blotting. Polyacrylamide gel electrophoresis was
55 performed according to the method of Laemmli [16]. Sporozoites, schizonts and merozoites were prepared for electrophoresis by diluting in an equal volume of 2 x sample buffer (0.05% bromophenol blue, 20% glycerol, 2% [3-mercaptoethanol, 4% SDS and 0.125 M Tris-HCl, pH 6.8) for reducing gels. Non-reducing gel sample buffer is the same, except no [3-mercaptoethanol is present. Samples were boiled immediately prior to electrophoresis. Prestained high molecular weight standards were run on each gel. When sporulated oocysts were used as sampies, the oocysts were disrupted by vortexing with glass beads in sample buffer and boiled. The material was centrifuged and proteins in the soluble extracts separated by electrophoresis. Polypeptides were transferred electrophoretically from polyacrylamide gels to nitrocellulose paper according to Towbin et al. [17]. After blocking in TBS containing 5% (w/v) Carnation instant dry milk, proteins impregnated onto nitrocellulose paper were reacted with monoclonal antibody (1:200 dilution in TBS containing 0.2% Tween 20) for 2 h at room temperature. The nitrocellulose blot was washed with TBS and incubated for another hour at room temperature in 1:500 dilution of rabbit anti-mouse IgG conjugated to horseradish peroxidase (Cappel Worthington Biochemical). Substrates used in color development were 4-chloro-l-naphthol and 0.01% H202 in TBS buffer.
Elution of antibodies from plaque lift filters. The elution of antibodies from plaque lift filters to characterize the specific antigen of a phage clone was based on the technique of Smith et al. [18]. Bacteriophage hgtll clones were plated in the same manner as for immunological screening of the E. tenella library. Approximately 10000 plaques were plated per 100-mm plate, hgtll nonrecombinants were plated in same manner for use as control in the experiment. When plaques were visible on the plate, nitrocellulose circles soaked in 10 mM IPTG and air-dried were placed on the plaques and plates returned to 37°C overnight. The following day, filters were removed, washed in TBS, blocked in TBS containing 10% BSA (w/v) for 1 h at room temperature, and washed again in TBS.
A strip approximately 5 mm wide was cut from the middle of the filter, incubated with polyclonal rabbit anti-sporozoite serum (1:100 dilution in TBS + 0.2% Tween 20), and rocked overnight at room temperature. The nitrocellulose strip was washed three times for 10 min each, in TBS + 0.2% Tween, and blotted slightly to remove excess liquid. The strip was then placed into a plastic test tube with the plaque side down. Antibodies bound to each strip were eluted with three 30s washes with 200 I~1 each of 5 mM glycine-HCl, pH 2.3,500 mM NaC1, 0.5% Tween 20 (v/v), 100 p~g m1-1 of BSA. The three eluates were combined and immediately neutralized with 1/20th volume of 1 M Tris-HC1, pH 7.4. The combined neutralized eluates were diluted three times with TBS + 0.2% Tween and used to probe a strip of a sporozoite Western blot overnight at room temperature. Color visualization of the Western blot was performed as described above.
Expression of ~-galactosidase/LPMC-61f fusion proteins. The EcoRI insert from LPMC-61f was subcloned into a plasmid expression vector, pSEV4, for expression as [3-galactosidase/LPMC61f fusion proteins. This vector is modified from pLG2, a pBR322 derived plasmid [19]. The vector (pSEV4) has the wild-type Escherichia coli lac operon elements (repressor, operator and promoter) in front of the [3-galactosidase gene containing the identical unique EcoRI site 57 bp from the carboxy terminus of the lacZ gene used for cloning in hgtll. Thus, by a subcloning procedure, we can move insert DNAs from the hgtll screening system to an identical region in this plasmid that is capable of overproducing the [3galactosidase/antigen fusion polypeptide. Because the plasmid has a copy of the wild-type lac repressor, the [3-galactosidase gene can be efficiently turned off, preventing expression of any cloned sequences that may be deleterious to the E. coli host cell. When the vector was induced with 2 mM IPTG for 2 h, [3-galactosidase activity was increased by approximately 300-fold. Cultures of E. coli strain AMA1004 [20] carrying the plasmid pSEV4 or pSEV4 containing the LPMC-61f gene were grown in L broth supplemented with 50 ixg m1-1 ampillicin to an A600 of
56 0.2 at 37°C. I P T G was added to a final concentration of 2 m M and cultures were shaken at 37°C for another 2 h. Cells were harvested by centrifugation and washed in PBS. Cell pellets were solubilized in Laemmli gel sample buffer for gel electrophoresis and Western blot analysis as described above. Results
Monoclonal antibody, designated LPMC-61 (class IgG3), was obtained from fusion of spleen cells from mice h y p e r i m m u n e to E. tenella merozoites with mouse m y e l o m a cells. This monoclonal LPMC-61 was shown by I F A to bind to merozoites. It also cross-reacted with E. tenella sporozoites and both immature and mature schizonts although the localization of its binding varied among the different life-cycle stages (Fig. 1). In vitro functionality assay. Sporozoite invasion of bovine turbinate cells was not significantly influenced by either continuous exposure to or pretreatment with LPMC-61 ascites or P3X-ascites as c o m p a r e d to infected controls. However, continuous exposure to LPMC-61 ascites did have a marked effect on first generation schizogony. A 1:640 dilution reduced the n u m b e r of developing schizonts by about 90% and a 1:1280 dilution resuited in an approximate 75% reduction as compared to infected controls (Table I). This result was apparent even by a cursory microscopic examination of the slides fixed at 48 h. No such effect was observed f r o m either the P3X-ascites or from LPMC-61 pretreatment. The n u m b e r of intracellular schizonts was also apparently reduced at 72 h post-infection. Even those schizonts that began to develop did not mature as evidenced by an absence of merozoites which are numerous in the infected controls. Western blot analysis. Western blot analysis of monoclonal antibody LPMC-61 demonstrated a strong reactivity with an immunogenic region of approx. 10-12-kDa merozoite polypeptides in reduced S D S - P A G E (Fig. 2, panel A, lane 4 in reduced S D S - P A G E ) . It also cross-reacted with a similarly sized immunogenic region from E. tenella oocysts, sporozoites and schizonts although
C Fig. 1. Indirect immunofluorescencelocalization of the monoclonal antibody LPMC-61 on: (a) Eimeria tenella merozoite; (b) sporozoites 24 h after inoculation into cultures of bovine turbinate cells; and (c) intracellular schizonts 72 h post-inoculation. Arrows point to representative cells. the reactivities are not as strong as with merozoites (Fig. 2A, lanes 1-3) and the exact polypeptide sizes may be slightly different for different life-cycle stages. However, in the absence of the reducing agent 13-mercaptoethanol, the monoclonal LPMC-61 does not react with the 10-12-
57
TABLE I
A
Influence of LPMC-61 ascites on penetration and development of E. tenella sporozoites in cell culture Treatment
Intracellular sporozoites (24 h)
Schizonts (48 h)
LPMC-61 ascites (continuous) 1:40 1:80 1:160 1:320 1:640 1:1280
83 87 90 95 92 93
0.0 0.0 5.4 5.4 11.3 25.0
LPMC-61 ascites (pretreatment) 1:40 1:80 1:160 1:320 1:640 1:1280
87 89 94 97 93 95
83.3 87.4 95.9 96.0 93.7 93.3
P3X ascites (continuous) 1:40 1:80 1:160 1:320 1:640 1:1280
kD
1
2
B 3
4
1
2
3
4
200 -97-68-43--
26--
18-14 m
iii~iiiii~i
93 93 95 94 92 94
89.4 93.4 92.9 87.5 97.6 93.0
Values are percent of infected controls. k D a polypeptide; rather, the monoclonal recognizes a new approx. 80-kDa sporozoite or merozoite protein (Fig. 2A, lanes 1-4). Developmental synthesis o f the LPMC-61 gene product during sporulation. Unsporulated E. tenella oocysts were allowed to sporulate and develop in the presence of oxygen. Samples were taken every four hours for 72 h. Protein extracts were separated on reduced S D S - P A G E , blotted to nitrocellulose, and p r o b e d with LPMC-61 hybridoma antibodies. The Western blot (Fig. 3a) shows that the antigen recognized by LPMC-61 under reducing conditions is present as an approx. 10-12 k D a polypeptide in all stages throughout the sporulation of the oocysts, and is also present in the sporozoites following excystation. The difference in the immunological reactivities at various time points is probably due to the different amounts of proteins loaded on the polyacrylamide gel as evidence by silver staining of the gel after Western blotting (Fig. 3B).
~!i~i~ i~
~i i l ~i ~
Fig. 2. Western blot analysis of SDS-solubilized samples (panel A, reduced; panel B, non-reduced samples) with monoclonal antibody LPMC-61. Lanes 1, E. tenella sporozoites. Lanes 2, E. tenella oocysts. Lanes 3, E. tenella schizonts. Lanes 4, E. tenella merozoites. The molecular weights (kDa) of size standards are indicated. The arrow indicates the LPMC-61 immunoreactive region. Identification and characterization o f a c D N A encoding the LPMC-61 antigen. Since Western blot analysis showed that the LPMC-61 antigen is present throughout oocyst sporulation and present in sporozoites following excystation, a sporozoite c D N A library was screened [12] to try to identify a specific antigenic clone reactive with the LPMC-61 monoclonal antibody. F r o m approximately 60000 individual recombinants screened, two monoclonal antibody-reactive phages, LPMC61e and LPMC-61f, were identified and plaquepurified. LPMC-61e and LPMC-61f cross-hybridized to each other at the D N A level and therefore represent the same gene. Since LPMC-61f has a larger c D N A insert than LPMC-61e, and LPMC-61e has a defective E c o R I site (probably and artifact during the c D N A library construction), LPMC-61f was chosen for the studies described in this report. Phage D N A prepared from LPMC-61f contained a c D N A insert of about 770 bp.
58
A 0 4
12 20 24
2 8 3 2 3 6 4 0 4 4 4 8 5 5 72
S
kD 200-97-68-43--
26-18--
14--
!
I
I
I
I
I
I
I
l
|
l
l
\
2oo-
lected antibodies that reacted with an approx. 10-12-kDa sporozoite polypeptide on reducing SDS-PAGE and an approx. 80-kDa sporozoite polypeptide on non-reducing SDS-PAGE (Fig. 4A and B, lanes 1). Lanes 2 in Fig. 4A and B show that bacteriophage kgtll containing no E. tenella DNA insert did not select any antibodies from the rabbit anti-E, tenella sporozoite serum. Fig. 4C shows the Western blot pattern of the polyclonal serum used in the antibody elution experiments. As seen in Fig. 4A, lane 1, eluted antibodies from LPMC-61f also reacted weakly with a few high-molecular-weight sporozoite polypeptides. These polypeptides may contain similar epitopes to LPMC-61f. However, under non-reducing conditions, LPMC-61f specifically selected the approx. 80-kDa polypeptide.
97--
A
68--
B
C
,3-
kD
1
2
1
2
200--
26-- ~
97-68 --
~
14--
43--
B Fig. 3. Developmental stages of E. tenella oocysts. Number on top of each lane is the time (in h) during development of the oocysts and 'S' is the sporozoite stage. Panel A shows the Western blot analysis of the developmental stages with monoclonal antibody LPMC-61. The arrow indicates the LPMC61 immunoreactive region. Panel B shows the silver staining pattern of the gel after Western blot transfer. The molecular weights (kDa) of size standards are indicated.
In order to obtain independent confirmation that LPMC-61f encodes the approx. 10-12-kDa and the approx. 80-kDa antigens recognized by Mab LPMC-61 under reducing and non-reducing SDS-PAGE conditions, respectively, we bound polyclonal hyperimmune rabbit anti-sporozoite serum to the phage plaques. The selected phagespecific antibodies were eluted from the nitrocellulose paper and used to probe a Western blot of both reduced and non-reduced sporozoite polypeptides. The recombinant LPMC-61f phage se-
26--
18-14--
Fig. 4. Western blot analysis of SDS-solubilized E. tenella sporozoites with eluted antibodies from hyperimmune rabbit anti-E, tenella sporozoite serum. Panels A and B are reduced and non-reduced samples, respectively. Lanes 1, clone LPMC61f; lanes 2, kgtll control. Panel C, Western blot analysis of E. tenella sporozoites with hyperimmune rabbit anti-sporozoite serum. The molecular weights (kDa) of size standards are indicated. The arrow indicates the LPMC-61 immuno-reactive region.
59 this 142-kDa 13-galactosidase fusion protein is estimated from S D S - P A G E to be about 31 kDa. The approx. 770 bp insert of LPMC-61f could encode an approx. 31 k D a protein (see below), therefore suggesting that the entire approx. 770bp insert encodes the parasite protein. The fusion protein is recognized by the LPMC-61 monoclonal (Fig. 5B), the rabbit anti-sporozoite serum (Fig. 5C), rabbit anti-merozoite serum (Fig. 5D), infected chicken serum (Fig. 5E), and immune chicken serum (Fig. 5F), but not recognized by normal chicken serum and normal rabbit serum (data not shown). 13-Galactosidase reacted very weakly with the antibodies used. The rabbit and chicken sera also recognized additional E. coli proteins. The same E. coli proteins were recognized by the pre-immune sera but did not react with the 13-Gal fusion protein (data not shown).
~-galactosidase-LPMC-61f fusion proteins. The E c o R I insert from LPMC-61f was subcloned into the [~-galactosidase gene of a plasmid expression vector (pSEV4) for overproduction of the fusion protein in E. coli strain AMA1004 as described in Materials and Methods. E. coli cells AMA1004 containing the pSEV4 or the pSEV4/LPMC-61f plasmids were treated with I P T G to induce production of the 13-galactosidase LPMC-61 fusion protein. Uninduced cells were processed as controls. The recombinant LPMC-61f fusion proteins were analyzed by S D S - P A G E , and Western blot analysis with monoclonal LPMC-61, rabbit anti-E, tenella sporozoite serum, rabbit anti-merozoite serum, infected and immune chicken serum. The fusion protein is synthesized in E. coli at high levels (about 5% of protein visible on silver-stained gels) and is approximately 142 k D a in size (Fig. 5). The LPMC-61f-encoded portion of
A 1
2
B 34
1
234
C 123
D 4
1 2
E 3 4
1 2 34
F 1 2 3 4
kD 200
.....
92--
69--
46--
Fig. 5. Silverstaining pattern and Western blot analysis of E. coli protein extracts. Proteins from an approximately equal number of cells are electrophoresed on each gel lane. Lanes 1 and 2 of each panel are extracts from E. coli containing the parent plasmid pSEV4 without the LPMC-61f insert, uninduced and induced with IPTG, respectively. Lanes 3 and 4 of each panel are protein extracts from E. coli containing the plasmid pSEV4 with LPMC-61f insert, uninduced and IPTG-induced, respectively. Panel A is the silver staining pattern. Panel B is probed with monoclonal antibody LPMC-61. Panel C is probed with hyperimmune rabbit anti-E, tenella sporozoite serum. Panel D is probed with hyperimmune rabbit anti-E, tenella merozoite serum. Panel E is probed with E. tenella infected chicken serum. Panel F is probed with immune chicken serum. The heavily staining bands in the IPTGinduced tracks (double arrow), which is larger than 13-galactosidase (single arrow), is the fusion protein. The molecular weights (kDa) of size standards are indicated.
60 DNA sequencing of LPMC-61f. The approx. 770 bp EcoRI insert of LPMC-61f was subcloned into M13 for D N A sequencing. The sequence determined (Fig. 6) revealed several interesting features. The sequence has a single open reading frame throughout the insert, has no poly(A) tail, and contains long stretches of CAG repeats similar to those found in other repeated E. tenella genes [12]. The deduced amino acid sequence predicts a protein of approx. 31.3 kDa. The amino acid sequence revealed that the gene can be divided into segments of tandem, non-perfect repeats, each segment beginning with the amino acid doublet tryptophan-proline or tryptophan-serine, followed by stretches of glutamine residues interspersed with other amino acids. Of the 255 amino acid residues encoded by the DNA sequence, 123 (48%) are glutamine. Amino acid analysis of unfused LPMC-61f protein produced
in E. coli agrees with the amino acid content predicted from the cDNA sequence (R. Hageman, Synergen, Inc., personal communication). There are two cysteine residues in the sequence available for disulfide bonding. We have examined the protein-encoding region of this gene for evidence of codon usage bias. Of the 123 glutamines, 114 are coded by CAG, with the remaining 9 coded by CAA; 15 of 21 leucines are encoded by CTG. Similar codon usage bias has been observed in the family of repeat E. tenella genes [12]. Analysis of the predicted amino acid sequence by hydropathicity plot using the Hopps and Woods program [21] showed that the protein has a large central hydrophilic area surrounded by two hydrophobic regions. The central hydrophilic area is made up of the tandem, non-perfect repeat region, with the 5'-region of the sequence consisting of hydrophobic stretches of leucine. No region resembling a
CTG CGG CTG CTG CTG AAG CTA CTG CTG CTG CTG CTG GGG CAG CAG AAA CAT TGG CCT GAG Leu Arg Leu Leu Leu Lys Leu Leu Leu Leu Leu Leu Gly Gin Gin Lys His Trp Pro Glu 61 AGA CAG CAG CAG CAG CAA CCG CAG CCG TGG CTA GAT CGA CAA CAG CAG CAG CAG CAG CAC Ar~ Gln Gln Gln Gln Gln Pro Gln Pro Trp Leu Asp Ar~ Gin Gln Gln Gln Gln Gin His 121 AAC CAA CAG CTG CAG AAG CAG CAG TGG CCT GAG GGC CAG CGG CAG CAG CTG TGG CCA GAG Asn Gin Gln Leu Gln Lys Gin Gln Trp Pro Glu Gly Gln Ar$ Gln Gln Leu Trp Pro Glu 181 CAA CAG CAG CAG CAG TGG CCT GAG CAG CAC CAG CAA GCA CAG CAG CAG CAG CAG TGG CCT Gln Gln Gln Gln Gln Trp Pro Glu Gln His Gln Gln Ala Gln Gln Gln Gln Gln Trp Pro 241 CAG CAG CAG CCA CAG ATG CAG CAG GAG CAG TGG CCT CAG CAG CAG CCA CAG GTG CAG CAG Gln Gin Gin Pro Gln Met Gin Gln Glu Gln Trp Pro Gln Gln Gln Pro Gln Val Gln Gln 301 CAG CAG CAG TGG CCT CAG CAG CAG CAC AGG CGC CAG CAC GGC CAG CAG CAG CAG TGC ATG Gln Gln Gln Trp Pro Gln Gln Gln His Ar$ Ar$ Gln His Gly Gln Gln Gln Gln Cys Met 361 AAC AGC CAG CAG CAG CTG CAG CAG TGC GGG CAG CAG CAG CAG CAG CAG CTG CAG CAG CAG Asn Ser Gln Gln Gln Leu Gln Gln Cys Gly Gin Gln Gln Gln Gln Gln Leu Gln Gin Gln 421 ITGG TCT GAG CAG CAG CAA CAG CAG CAG CAG CAG CAG TGG CCT GAG CAG CCA GAG CAG CAG [Trp Ser Glu Gln Gin Gln Gln Gin Gln Gin Gln Gin Trp Pro Glu Gin Pro Glu Gin Gin ]
481 CAG CAG CAA CAGITGG CCT GAG CAG CAG CAG CAG CAG TGG TCT GAT CAG AAC CAG CAG CAG Gln Gln Gln Gln]Trp Pro Glu Gln Gin Gln Gln Gln Trp Ser Asp Gln 'Asn Gln Gln Gln 541 CAA GCG CAA CAG TGG CAG GCG CAG CAG CAG CAG CAG TGG CCG CAG CAG CAG CAG CAG CCG Gln Ala Gln Gln Trp Gln Ala Gln Gln Gln Gln Gln Trp Pro Gln Gln Gln Gln Gln Pro 601 CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG IGAC TTG GGG CCC GAT GGA GTT GGG ATC GTC Gln Gln Gln Gin Gin Gln Gin Gln Gln Gln~Asp Leu Gly Pro Asp Gly Val Gly lle Val 661 GTT CCC TAC CTG GGA TCG TCT CCA GCA GAC TTC GAA GTC CAG AAC CTC GGG GTG TCT GTA Val Pro Tyr Leu Gly Ser Ser Pro Ala Asp Phe Glu Val Gln Ash Leu Gly Val Ser Val 721 CTC CCC AGA ATC AAG CTG CCC CTC CCC AAG TTC GAC AAC GAC GAC GG Leu Pro Arg Ile Lys Leu Pro Leu Pro Lys Phe Asp Ash Asp Asp
Fig. 6. DNA sequence and predicted amino acid sequence of LPMC-61f. Sequence displayed in 5' --9 3' direction. The tandem, non-perfect repeats are shown as boxed regions.
61 signal sequence was found in this partial cDNA sequence. Discussion
In this report we describe the development of a monoclonal antibody against purified second generation E. tenella merozoites and the identification and characterization of a target antigen of this monoclonal antibody. The monoclonal antibody LPMC-61 binds to air-dried merozoites by immunofluorescence assay. LPMC-61 inhibits parasite development when cultures are maintained in its continuous presence, but not when sporozoites are pre-treated with the antibody. No inhibitory activity is seen if the antibody is added after parasite invasion of the host cell. The LPMC-61 target antigen is not stage-specific. Mab LPMC-61 recognized an approx. 80-kDa sporozoite polypeptide in non-reducing SDS-PAGE. In the presence of 13-mercaptoethanol, LPMC-61 recognized an approx. 10-12-kDa sporozoite polypeptide. This finding suggests that the 10012kDa antigen is covalently linked by disulfide bonds to other polypeptides to form the 80-kDa antigen, since there are two cysteine residues within the cDNA encoding the LPMC-61 antigen. The other polypeptides to which the 10-12kDa protein is attached may be distinct gene products or portions of a precursor protein. In the presence of the reducing agent 13-mercaptoethanol, the 80-kDa precursor is processed to the 10-12-kDa subunits. Files et al. [22] have previously shown that the protein target (TA4) of two sporozoite-neutralizing monoclonal antibodies is synthesized as a 25-kDa precursor that is proteolytically processed to give the 17-kDa and 8-kDa subunits held together by disulfide bonds. A partial cDNA clone, designated LPMC-61f, containing the gene corresponding to this Mab was identified from an E. tenella sporozoite cDNA library. Antibody elution experiments confirmed that cDNA LPMC-61f indeed contains a target antigen to the Mab. The 13-galactosidase/LPMC61f fusion protein reacted with the LPMC-61 monoclonal antibody, and is also recognized by rabbit anti-E, tenella sporozoite serum, rabbit anti-merozoite serum, E. tenella infected and immune chicken serum. Therefore the LPMC-61f
antigen appears to be immunogenic in both rabbits and chickens and stimulates B cell production during a natural infection. The 13-galactosidase-LPMC-61f fusion protein is about 31 kDa larger than 13-galactosidase, and is therefore coded for by the entire LPMC-61f cDNA sequence. The larger size of the protein, predicted by the LPMC61 cDNA (31 kDa) than the 10o12 kDa polypeptide recognized by LPMC-61 Mab, is consistent with the 10012 kDa protein being encoded by a precursor protein, possibly the 80-kDa protein. Examination of the LPMC-61f DNA sequence revealed that it encodes stretches of glutamine repeats as have been found with some other repeated sequences in E. tenella [12]. The LPMC61f encoded protein sequence may be divided into segments of tandem, non-perfect repeats as shown in Fig. 5. Recently, Jenkins [23] reported the isolation of a cDNA (cMZ8) encoding an approx. 150 kDa merozoite surface protein in Eimeria acervulina containing tandem-repeated amino acid sequences very different from the LPMC-61f repeat sequences described here. Protozoan proteins with repeating amino acid sequences, either as stretches of single amino acids [24,25] or as tandem repeats of several amino acids [26] have been described. The LPMC-61f sequence contains long stretches of the amino acid glutamine within the tandem repeats and is different from the serine-rich [25] or asparagine-rich [24] plasmodium proteins. It is unclear at the present time as to what roles these glutamine-rich tandem repeated sequences play, just as no known functions have been known for the SERA or CARP [24,25]. Their preponderance in Eimeria [12] suggest that they may be important for an as yet unknown aspect of the parasite life cycle. The fact that infected and immune chicken sera recognize the target antigen of Mab LPMC-61 suggest that the Eirneria repeating amino acid sequences are immunodominant antigens in their hosts, as have been found for the tandem repeats of amino acids in the related Apicomplexa protozoa, the malaria parasite Plasmodium [27,28]. Eimeria merozoite has been implicated as the most immunogenic stage in the coccidia life cycle [11], studies have begun on the antigenic makeup of the merozoites. Although the function of this LPMC-61 antigen in E. tenella is unknown at the
62
present time, the fact that it cross-reacts with different stages in the life cycle of the coccidia shows that it is a common shared epitope. More importantly, the fact that Mab LPMC-61 inhibits the development of the parasite in vitro suggests that the LPMC-61 antigen may be an important immunogen. We have preliminary evidence that Mab LPMC-61 cross-reacts with an approx. 10-12kDa E. acervulina polypeptide under reducing conditions. Therefore LPMC-61 may also be present in other Eimeria species. Since LPMC-61f only encodes about 31 kDa of the approx. 80 kDa polypeptide recognized by the Mab, further work will be directed towards identifying a full-length cDNA that encodes the entire target antigen in order to study the complete protein sequence and structure. Further experiments also will be required to determine whether this antigen will elicit a protective immune response in chickens against
Acknowledgements This work was funded by Eli Lilly and Company. The authors would like to thank Dr. Jerry Kuner for the sporulation samples and the antibody elution technique, Dr. Robert Hageman for communicating the amino acid analysis data of LPMC-61f polypeptide, Dr. Leonard Guarente for the pLG2 plasmid, Dr. M.J. Casadaban for the E. coli strain, AMA1004 and Dr. W. Current for providing the E. acervulina sporozoites, Dr. M. Stiff for the chicken sera, and Dr. George Cox for the E. tenella sporozoite cDNA library and for the critical evaluation of this manuscript and to the reviewers for suggesting the non-reduced SDSPAGE experiments. We also thank Carla Worland for the preparation of the manuscript.
Eimeria tenella.
References 1 Long, P.L. (1982) The Biology of the Coccidia. Baltimore University Park Press. 2 Yoshida, N., Nussenzweig, R.S., Poloenjak, P., Nussenzweig, V. and Aikawa, M. (1979) Hybridoma produces protective antibodies directed against the sporozoite stage of malarial parasites. Science 207, 71-73. 3 Stewart, M.J., Nawrot, R.J., Schulman, S. and Vandenberg, J.P. (1986) Plasmodium berghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies. Infect. Immun. 51,859-864. 4 Cochrane, A.H., Santoro, F., Nussenzweig, V., Gwadz, R.W. and Nussenzweig, R.S. (1982) Monoclonal antibodies identify the protective antigens of sporozoites of Plasmodium knowlesi. Proc. Natl. Acad. Sci. USA 79, 5651-5655. 5 Cochrane, A.H., Collins, W.E. and Nussenzweig, R.S. (1984) Monoclonal antibody identifies circumsporozoite protein of Plasmodium malarial and detects a common epitope on Plasmodium brasilianum sporozoites. Infect. Immun. 45, 592-595. 6 Danforth, H.D. (1983) Use of monoclonal antibodies directed against Eimeria tenella sporozoites to determine stage specificity and in vitro effect on parasite penetration and development. Am. J. Vet. Res. 44, 1722-1727. 7 Gore, T.C. and Neuman, K.Z. (1988) In vitro and in vivo neutralization of Eimeria tenella Railliet and Lucet, 1891 and Eimeria necatrix, Johnson, 1930 (Apicomplexa, Eimeriina) sporozoites with monoclonal antibodies. J. Protozool. 8 Augustine, P.C. and Danforth, H.D. (1985) Effects of hybridoma antibodies on invasion of cultured cells by sporozoites of Eimeria. Avian Dis. 29, 1212-1223.
9 Rose, M.E. and Hesketh, P. (1976) Immunity to coccidiosis: Stages of the life cycle of Eimeria maxima which induce, and are affected by, the response of the host. Parasitology 73, 25-37. 10 Long, P.L. and Millard, B.J. (1968) Eimeria: effect of metichlorpindol and methylbenzoquate on endogenous stages in the chicken. Exp. Parasitol. 23, 331-338. 11 Rose, M.E. (1982) Host immune responses. In: The Biology of the Coccidia. (P.L. Long, ed.), pp. 329-371, University Park Press, Baltimore. 12 Ko, C., Cox, G.N., Smith II, C.K., Stiff, M. and McDonnell, M. (1989) Eimeria tenella: identification of a highly repeated DNA sequence. Submitted for publication. 13 Danforth, H.D. and McAndrew, S.J. (1987) Hybridoma antibody characterization of stage-specific and stage-crossreactive antigens of Eimeria tenella. J. Parasitol. 73, 985-992. 14 Fernando, M.A., AI-Attar, M.A. and Bowles, G.H. (1984) Preparation of a second generation merozoites of Eimeria necatrix free of host tissue. J. Parasitol. 70, 154-155. 15 Danforth, H.D. (1982) Development of hybridoma produced antibodies against Eimeria tenella and E. mitis. J. Parasitol. 68,392-397. 16 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. 17 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.
63 18 Smith, D.E. and Fisher, P.A. (1984) Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. Cell Biol. 99, 20-28. 19 Guarente, L. and Lauer, G. (1980) Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit 13-globin. Cell 20, 543-553. 20 Casadaban, M.J., Martinez-Arias, A., Shapira, S.K. and Chou, J. (1983) I~-Galactosidase gene fusions for analyzing gene expression in E. coli and yeast. Methods Enzymol. 100,293-308. 21 Hopp, T.P. and Woods, K.R. (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78, 3824-3828. 22 File, J.G., Paul, L.S. and Gabe, J.D. (1987) Identification and characterization of the gene for a major surface antigen of Eirneria tenella. In: Molecular Strategies of Parasitic Invasion, pp. 713-723. Alan R. Liss, New York.
23 Jenkins, M.C. (1988) A cDNA encoding a merozoite surface protein of the protozoan Eimeria acervulina contains tandem-repeated sequences. Nucleic Acids Res. 16, 9863. 24 Wahlgren, M., ,~slund, L., Franz6, L., Sundvall, M., Wahlin, B., Berzines, K., McNicol, L.A., Bj6rkman, A., Wiszell, H., Perlmann, P. and Pettersson, U. (1986) A Plasmodium falciparum antigen containing clusters of asparagine residues. Proc. Natl. Acad. Sci. USA 83, 2677-2681. 25 Li, W.-B., Bzik, D.J., Horii, T. and Inselburg, J. (1989) Structure and expression of the Plasmodiurn falciparum SERA gene. Mol. Biochem. Parasitol. 33, 13-26. 26 Ozaki, L.S., Svec, P., Nussenzweig, R.S., Nussenzweig, V. and Godson, G.N. (1983) Structure of the Plasmodium knowlesi gene coding for the circumsporozoite protein. Cell 34, 815-822. 27 Nussenzweig, V. and Nussenzweig, R.S. (1985) Circumsporozoite proteins of malarial parasite. Cell 42, 401-403. 28 Kemp, D.J., Coppel, R.L. and Anders, R.F. (1987) Repetitive proteins and genes of malaria. Annu. Rev. Microbiol. 41, 181-208.
53
MOLBIO 01342
Identification and characterization of a target antigen of a m o n o c l o n a l antibody directed against Eimeria tenella m e r o z o i t e s Christine Ko 1, Charles K. Smith 112 and Michael McDonell 1. lSynergen, Inc., Boulder, CO, U.S.A., and ~Eli Lilly and Company, Lilly Research Laboratories, Greenfield, IN, U.S.A.
(Received 4 September 1989; accepted 25 January 1990)
Monoclonal antibodies (Mab) were produced against Eimeria tenella merozoites. A single Mab, LPMC-61, was selected because of its ability to bind to merozoites by indirect immunofluorescence assay (IFA) and to inhibit in vitro sporozoite development. Mab LPMC-61 reacts with an approx. 10-12-kDa merozoite polypeptide in reduced SDS-PAGE, but with an approx. 80-kDa protein in non-reduced SDS-PAGE. The monoclonal recognizes similarly sized polypeptides in E. tenella sporozoites, oocysts and schizonts. A partial cDNA (LPMC-61f) encoding the LPMC-61 antigen was identified from an E. tenella sporozoite cDNA library in bacteriophage hgtll. In addition to Mab LPMC-61, the recombinant 13-galactosidase/LPMC-61ffusion protein is recognized by hyperimmune rabbit anti-E, tenella sporogoite serum, rabbit anti-E, tenella merozoite serum, and E. tenella-infected and immune chicken sera. DNA sequencing of LPMC-61f cDNA showed that the putative protein has an unusual tandem, non-perfect repeated sequence, with glutamine comprising about 48% of the predicted amino acids. A hydropathicity plot of the predicted amino acid sequence shows a central hydrophilic region, consisting of the repeated sequences, surrounded by hydrophobic regions on both sides. Since the merozoite ~tage of avian Eimeria has been implicated in the induction of a protective immune response in chickens, LPMC-61 may be an important immunogen for use as a vaccine against E. tenella. Key words: Eimeria tenella; Merozoite; Sporozoite; Repeat sequence; Monoclonal antibody.
Introduction Eimeria a n d P l a s m o d i u m a r e r e l a t e d p r o t o z o a of t h e p h y l u m A p i c o m p l e x a [1]. T h e fact t h a t m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t Plasmodium surface c i r c u m s p o r o z o i t e p r o t e i n s will inhibit s p o r o z o i t e m o t i l i t y a n d cell p e n e t r a t i o n in vitro [2-5] h a s s t i m u l a t e d s i m i l a r efforts in the s t u d y o f Eimeria. M o n o c l o n a l s d i r e c t e d against Correspondence address: Christine Ko, Synergen, Inc., 1885, 33rd Street, Boulder, CO 80301, U.S.A. "Present address: Syntro Corporation, San Diego, CA, U.S.A. Abbreviations: IPTG, isopropyl 13-o-thiogalactopyranoside; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HBSS, Hanks' balanced salts solution; IFA, indirect immunofluorescence assay. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M30933.
Eimeria tenella s p o r o z o i t e surface p r o t e i n s h a v e b e e n f o u n d by D a n f o r t h to inhibit s p o r o z o i t e p e n e t r a t i o n of c u l t u r e d cells a n d to i n h i b i t develo p m e n t of the p a r a s i t e in cell c u l t u r e , p r o v i d e d t h a t the a n t i b o d y was p r e s e n t c o n t i n u o u s l y in t h e cell c u l t u r e m e d i u m [6]. G o r e a n d N e w m a n [7] described m o n o c l o n a l antibodies Ptn 7.2A4/4 and Ptn 9.9D12 which w e r e c a p a b l e o f n e u t r a l i z i n g E. tenella s p o r o z o i t e s in an in vitro assay a n d b o t h r e a c t e d with t h e surface of s p o r o z o i t e s in i n d i r e c t immunofluorescent a n t i b o d y tests. H o w e v e r , n o n e of t h e f o u r m e r o z o i t e - e l i c i t e d m o n o c l o n a l antib o d i e s t h a t c r o s s - r e a c t e d with s p o r o z o i t e surface antigens, as d e s c r i b e d b y A u g u s t i n e a n d D a n forth, i n h i b i t e d the i n v a s i o n o f cells b y t h e spor o z o i t e s [8]. R e s e a r c h into t h e Eimeria p a r a s i t i c stages t h a t i n d u c e i m m u n i t y in chickens suggest t h a t different stages o f t h e life cycle differ in t h e i r abilities to i n d u c e p r o t e c t i v e i m m u n e r e s p o n s e s . T h e sexual stages have little immunizing potential [9], and
0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
54 the sporozoites and the first generation schizonts are probably not very immunogenic [10]. The second generation schizont or merozoite stages of avian Eimeria have been implicated as the most immunogenic of all stages of coccidia [11]. Therefore, an effective vaccine to protect poultry against coccidiosis might include merozoite antigens or common antigens shared by most of the life cycle stages. In this report we describe a monoclonal antibody, designated LPMC-61, which was raised against E. tenella merozoites. Mab LPMC-61 reacts with polypeptides present in several developmental stages and inhibits sporozoite development in vitro, suggesting that the target antigen for this monoclonal may be an important immunogen. This report also describes the cDNA cloning and characterization of a target antigen of Mab LPMC-61. The target antigen for LPMC-61 has an unusual repeated sequence similar to other repeated sequences previously identified [12].
jection with purified second generation merozoites of E. tenella, Eli Lilly strain 65. This antibody-producing cell line was then cloned by limiting dilution and ascites were produced in pristane-primed mice.
Materials and Methods
In vitro anti-coccidial activity. Bovine turbinate cells (American Type Tissue Culture, CRL 1390) were seeded into 8-chamber Lab-Tek tissue culture slides (Nunc) in DMEM plus 10% fetal bovine serum (FBS). The cultures were permitted to reach 70-80% confluency and then 4.5 x 10 4 E. tenella sporozoites were added to each well in cell culture medium containing dilutions of mouse ascites generated by the hybridoma LPMC-61 (1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280). One infected control and one non-infected control well was maintained on each slide. Contemporary cultures were prepared exactly as described except that the ascites was generated by the parent myeloma (P3X-ascites), which produced no antibody reactive with E. tenella. A second series of cultures was prepared as above, except that the sporozoites were preincubated in medium containing the various dilutions of ascites for 1 h at 37°C and washed 2 x with HBSS prior to addition to cell cultures. All cultures were incubated at 37°C and representatives fixed and stained at 24, 48 and 72 h. The slides were examined microscopically for the presence of intracellular parasites.
Preparation of sporozoites,, schizonts and merozoites. Sporozoites and schizonts were prepared according to published procedures [13]. Purified E. tenella merozoites were prepared essentially as described by Fernando et al. [14] with modifications. Briefly, the cecae were removed from chickens 96 h following inoculation with E. tenella, washed with Hanks' balanced salt solution (HBSS), the mucosa scraped with a glass microscope slide, and collected in HBSS. This suspension was then homogenized with 8-10 strokes of a Dounce tissue grinder (Wheaton Scientific), filtered through a Leuko-pak column (Travenol Laboratories, Inc.), centrifuged at 850 × g for 10 min and the supernatant discarded. The merozoites were then purified by centrifugation in a Percoll gradient as described. Monoclonal antibody. Monoclonal antibody designated LPMC-61 was produced by a murine hybridoma resulting from the fusion of mouse spleen cells and mouse myeloma cells of the line P3-X63AG8, according to the method of Danforth [15]. Briefly, the spleen cells were obtained from a mouse which had been immunized by tail vein in-
Immunofluorescence localization of antibody reactivity. Spent media and ascites generated by the clone producing the antibody LPMC-61 was reacted with air-dried E. tenella merozoites and the antibody localized by indirect immunofluorescence assay essentially as described by Danforth [15]. In order to determine reactivity with intracellular stages of the parasite, cultures of bovine turbinate cells, prepared as described below, were infected with E. tenella sporozoites. At 4 h post-infection, cultures were washed 2 x with fresh medium to remove sporozoites that had not invaded. Cultures were removed at 24, 48 and 72 h, fixed with cold acetone, washed 2 x with IFA buffer, air-dried and stored at -80°C until use.
Polyacrylamide gel electrophoresis and Western blotting. Polyacrylamide gel electrophoresis was
55 performed according to the method of Laemmli [16]. Sporozoites, schizonts and merozoites were prepared for electrophoresis by diluting in an equal volume of 2 x sample buffer (0.05% bromophenol blue, 20% glycerol, 2% [3-mercaptoethanol, 4% SDS and 0.125 M Tris-HCl, pH 6.8) for reducing gels. Non-reducing gel sample buffer is the same, except no [3-mercaptoethanol is present. Samples were boiled immediately prior to electrophoresis. Prestained high molecular weight standards were run on each gel. When sporulated oocysts were used as sampies, the oocysts were disrupted by vortexing with glass beads in sample buffer and boiled. The material was centrifuged and proteins in the soluble extracts separated by electrophoresis. Polypeptides were transferred electrophoretically from polyacrylamide gels to nitrocellulose paper according to Towbin et al. [17]. After blocking in TBS containing 5% (w/v) Carnation instant dry milk, proteins impregnated onto nitrocellulose paper were reacted with monoclonal antibody (1:200 dilution in TBS containing 0.2% Tween 20) for 2 h at room temperature. The nitrocellulose blot was washed with TBS and incubated for another hour at room temperature in 1:500 dilution of rabbit anti-mouse IgG conjugated to horseradish peroxidase (Cappel Worthington Biochemical). Substrates used in color development were 4-chloro-l-naphthol and 0.01% H202 in TBS buffer.
Elution of antibodies from plaque lift filters. The elution of antibodies from plaque lift filters to characterize the specific antigen of a phage clone was based on the technique of Smith et al. [18]. Bacteriophage hgtll clones were plated in the same manner as for immunological screening of the E. tenella library. Approximately 10000 plaques were plated per 100-mm plate, hgtll nonrecombinants were plated in same manner for use as control in the experiment. When plaques were visible on the plate, nitrocellulose circles soaked in 10 mM IPTG and air-dried were placed on the plaques and plates returned to 37°C overnight. The following day, filters were removed, washed in TBS, blocked in TBS containing 10% BSA (w/v) for 1 h at room temperature, and washed again in TBS.
A strip approximately 5 mm wide was cut from the middle of the filter, incubated with polyclonal rabbit anti-sporozoite serum (1:100 dilution in TBS + 0.2% Tween 20), and rocked overnight at room temperature. The nitrocellulose strip was washed three times for 10 min each, in TBS + 0.2% Tween, and blotted slightly to remove excess liquid. The strip was then placed into a plastic test tube with the plaque side down. Antibodies bound to each strip were eluted with three 30s washes with 200 I~1 each of 5 mM glycine-HCl, pH 2.3,500 mM NaC1, 0.5% Tween 20 (v/v), 100 p~g m1-1 of BSA. The three eluates were combined and immediately neutralized with 1/20th volume of 1 M Tris-HC1, pH 7.4. The combined neutralized eluates were diluted three times with TBS + 0.2% Tween and used to probe a strip of a sporozoite Western blot overnight at room temperature. Color visualization of the Western blot was performed as described above.
Expression of ~-galactosidase/LPMC-61f fusion proteins. The EcoRI insert from LPMC-61f was subcloned into a plasmid expression vector, pSEV4, for expression as [3-galactosidase/LPMC61f fusion proteins. This vector is modified from pLG2, a pBR322 derived plasmid [19]. The vector (pSEV4) has the wild-type Escherichia coli lac operon elements (repressor, operator and promoter) in front of the [3-galactosidase gene containing the identical unique EcoRI site 57 bp from the carboxy terminus of the lacZ gene used for cloning in hgtll. Thus, by a subcloning procedure, we can move insert DNAs from the hgtll screening system to an identical region in this plasmid that is capable of overproducing the [3galactosidase/antigen fusion polypeptide. Because the plasmid has a copy of the wild-type lac repressor, the [3-galactosidase gene can be efficiently turned off, preventing expression of any cloned sequences that may be deleterious to the E. coli host cell. When the vector was induced with 2 mM IPTG for 2 h, [3-galactosidase activity was increased by approximately 300-fold. Cultures of E. coli strain AMA1004 [20] carrying the plasmid pSEV4 or pSEV4 containing the LPMC-61f gene were grown in L broth supplemented with 50 ixg m1-1 ampillicin to an A600 of
56 0.2 at 37°C. I P T G was added to a final concentration of 2 m M and cultures were shaken at 37°C for another 2 h. Cells were harvested by centrifugation and washed in PBS. Cell pellets were solubilized in Laemmli gel sample buffer for gel electrophoresis and Western blot analysis as described above. Results
Monoclonal antibody, designated LPMC-61 (class IgG3), was obtained from fusion of spleen cells from mice h y p e r i m m u n e to E. tenella merozoites with mouse m y e l o m a cells. This monoclonal LPMC-61 was shown by I F A to bind to merozoites. It also cross-reacted with E. tenella sporozoites and both immature and mature schizonts although the localization of its binding varied among the different life-cycle stages (Fig. 1). In vitro functionality assay. Sporozoite invasion of bovine turbinate cells was not significantly influenced by either continuous exposure to or pretreatment with LPMC-61 ascites or P3X-ascites as c o m p a r e d to infected controls. However, continuous exposure to LPMC-61 ascites did have a marked effect on first generation schizogony. A 1:640 dilution reduced the n u m b e r of developing schizonts by about 90% and a 1:1280 dilution resuited in an approximate 75% reduction as compared to infected controls (Table I). This result was apparent even by a cursory microscopic examination of the slides fixed at 48 h. No such effect was observed f r o m either the P3X-ascites or from LPMC-61 pretreatment. The n u m b e r of intracellular schizonts was also apparently reduced at 72 h post-infection. Even those schizonts that began to develop did not mature as evidenced by an absence of merozoites which are numerous in the infected controls. Western blot analysis. Western blot analysis of monoclonal antibody LPMC-61 demonstrated a strong reactivity with an immunogenic region of approx. 10-12-kDa merozoite polypeptides in reduced S D S - P A G E (Fig. 2, panel A, lane 4 in reduced S D S - P A G E ) . It also cross-reacted with a similarly sized immunogenic region from E. tenella oocysts, sporozoites and schizonts although
C Fig. 1. Indirect immunofluorescencelocalization of the monoclonal antibody LPMC-61 on: (a) Eimeria tenella merozoite; (b) sporozoites 24 h after inoculation into cultures of bovine turbinate cells; and (c) intracellular schizonts 72 h post-inoculation. Arrows point to representative cells. the reactivities are not as strong as with merozoites (Fig. 2A, lanes 1-3) and the exact polypeptide sizes may be slightly different for different life-cycle stages. However, in the absence of the reducing agent 13-mercaptoethanol, the monoclonal LPMC-61 does not react with the 10-12-
57
TABLE I
A
Influence of LPMC-61 ascites on penetration and development of E. tenella sporozoites in cell culture Treatment
Intracellular sporozoites (24 h)
Schizonts (48 h)
LPMC-61 ascites (continuous) 1:40 1:80 1:160 1:320 1:640 1:1280
83 87 90 95 92 93
0.0 0.0 5.4 5.4 11.3 25.0
LPMC-61 ascites (pretreatment) 1:40 1:80 1:160 1:320 1:640 1:1280
87 89 94 97 93 95
83.3 87.4 95.9 96.0 93.7 93.3
P3X ascites (continuous) 1:40 1:80 1:160 1:320 1:640 1:1280
kD
1
2
B 3
4
1
2
3
4
200 -97-68-43--
26--
18-14 m
iii~iiiii~i
93 93 95 94 92 94
89.4 93.4 92.9 87.5 97.6 93.0
Values are percent of infected controls. k D a polypeptide; rather, the monoclonal recognizes a new approx. 80-kDa sporozoite or merozoite protein (Fig. 2A, lanes 1-4). Developmental synthesis o f the LPMC-61 gene product during sporulation. Unsporulated E. tenella oocysts were allowed to sporulate and develop in the presence of oxygen. Samples were taken every four hours for 72 h. Protein extracts were separated on reduced S D S - P A G E , blotted to nitrocellulose, and p r o b e d with LPMC-61 hybridoma antibodies. The Western blot (Fig. 3a) shows that the antigen recognized by LPMC-61 under reducing conditions is present as an approx. 10-12 k D a polypeptide in all stages throughout the sporulation of the oocysts, and is also present in the sporozoites following excystation. The difference in the immunological reactivities at various time points is probably due to the different amounts of proteins loaded on the polyacrylamide gel as evidence by silver staining of the gel after Western blotting (Fig. 3B).
~!i~i~ i~
~i i l ~i ~
Fig. 2. Western blot analysis of SDS-solubilized samples (panel A, reduced; panel B, non-reduced samples) with monoclonal antibody LPMC-61. Lanes 1, E. tenella sporozoites. Lanes 2, E. tenella oocysts. Lanes 3, E. tenella schizonts. Lanes 4, E. tenella merozoites. The molecular weights (kDa) of size standards are indicated. The arrow indicates the LPMC-61 immunoreactive region. Identification and characterization o f a c D N A encoding the LPMC-61 antigen. Since Western blot analysis showed that the LPMC-61 antigen is present throughout oocyst sporulation and present in sporozoites following excystation, a sporozoite c D N A library was screened [12] to try to identify a specific antigenic clone reactive with the LPMC-61 monoclonal antibody. F r o m approximately 60000 individual recombinants screened, two monoclonal antibody-reactive phages, LPMC61e and LPMC-61f, were identified and plaquepurified. LPMC-61e and LPMC-61f cross-hybridized to each other at the D N A level and therefore represent the same gene. Since LPMC-61f has a larger c D N A insert than LPMC-61e, and LPMC-61e has a defective E c o R I site (probably and artifact during the c D N A library construction), LPMC-61f was chosen for the studies described in this report. Phage D N A prepared from LPMC-61f contained a c D N A insert of about 770 bp.
58
A 0 4
12 20 24
2 8 3 2 3 6 4 0 4 4 4 8 5 5 72
S
kD 200-97-68-43--
26-18--
14--
!
I
I
I
I
I
I
I
l
|
l
l
\
2oo-
lected antibodies that reacted with an approx. 10-12-kDa sporozoite polypeptide on reducing SDS-PAGE and an approx. 80-kDa sporozoite polypeptide on non-reducing SDS-PAGE (Fig. 4A and B, lanes 1). Lanes 2 in Fig. 4A and B show that bacteriophage kgtll containing no E. tenella DNA insert did not select any antibodies from the rabbit anti-E, tenella sporozoite serum. Fig. 4C shows the Western blot pattern of the polyclonal serum used in the antibody elution experiments. As seen in Fig. 4A, lane 1, eluted antibodies from LPMC-61f also reacted weakly with a few high-molecular-weight sporozoite polypeptides. These polypeptides may contain similar epitopes to LPMC-61f. However, under non-reducing conditions, LPMC-61f specifically selected the approx. 80-kDa polypeptide.
97--
A
68--
B
C
,3-
kD
1
2
1
2
200--
26-- ~
97-68 --
~
14--
43--
B Fig. 3. Developmental stages of E. tenella oocysts. Number on top of each lane is the time (in h) during development of the oocysts and 'S' is the sporozoite stage. Panel A shows the Western blot analysis of the developmental stages with monoclonal antibody LPMC-61. The arrow indicates the LPMC61 immunoreactive region. Panel B shows the silver staining pattern of the gel after Western blot transfer. The molecular weights (kDa) of size standards are indicated.
In order to obtain independent confirmation that LPMC-61f encodes the approx. 10-12-kDa and the approx. 80-kDa antigens recognized by Mab LPMC-61 under reducing and non-reducing SDS-PAGE conditions, respectively, we bound polyclonal hyperimmune rabbit anti-sporozoite serum to the phage plaques. The selected phagespecific antibodies were eluted from the nitrocellulose paper and used to probe a Western blot of both reduced and non-reduced sporozoite polypeptides. The recombinant LPMC-61f phage se-
26--
18-14--
Fig. 4. Western blot analysis of SDS-solubilized E. tenella sporozoites with eluted antibodies from hyperimmune rabbit anti-E, tenella sporozoite serum. Panels A and B are reduced and non-reduced samples, respectively. Lanes 1, clone LPMC61f; lanes 2, kgtll control. Panel C, Western blot analysis of E. tenella sporozoites with hyperimmune rabbit anti-sporozoite serum. The molecular weights (kDa) of size standards are indicated. The arrow indicates the LPMC-61 immuno-reactive region.
59 this 142-kDa 13-galactosidase fusion protein is estimated from S D S - P A G E to be about 31 kDa. The approx. 770 bp insert of LPMC-61f could encode an approx. 31 k D a protein (see below), therefore suggesting that the entire approx. 770bp insert encodes the parasite protein. The fusion protein is recognized by the LPMC-61 monoclonal (Fig. 5B), the rabbit anti-sporozoite serum (Fig. 5C), rabbit anti-merozoite serum (Fig. 5D), infected chicken serum (Fig. 5E), and immune chicken serum (Fig. 5F), but not recognized by normal chicken serum and normal rabbit serum (data not shown). 13-Galactosidase reacted very weakly with the antibodies used. The rabbit and chicken sera also recognized additional E. coli proteins. The same E. coli proteins were recognized by the pre-immune sera but did not react with the 13-Gal fusion protein (data not shown).
~-galactosidase-LPMC-61f fusion proteins. The E c o R I insert from LPMC-61f was subcloned into the [~-galactosidase gene of a plasmid expression vector (pSEV4) for overproduction of the fusion protein in E. coli strain AMA1004 as described in Materials and Methods. E. coli cells AMA1004 containing the pSEV4 or the pSEV4/LPMC-61f plasmids were treated with I P T G to induce production of the 13-galactosidase LPMC-61 fusion protein. Uninduced cells were processed as controls. The recombinant LPMC-61f fusion proteins were analyzed by S D S - P A G E , and Western blot analysis with monoclonal LPMC-61, rabbit anti-E, tenella sporozoite serum, rabbit anti-merozoite serum, infected and immune chicken serum. The fusion protein is synthesized in E. coli at high levels (about 5% of protein visible on silver-stained gels) and is approximately 142 k D a in size (Fig. 5). The LPMC-61f-encoded portion of
A 1
2
B 34
1
234
C 123
D 4
1 2
E 3 4
1 2 34
F 1 2 3 4
kD 200
.....
92--
69--
46--
Fig. 5. Silverstaining pattern and Western blot analysis of E. coli protein extracts. Proteins from an approximately equal number of cells are electrophoresed on each gel lane. Lanes 1 and 2 of each panel are extracts from E. coli containing the parent plasmid pSEV4 without the LPMC-61f insert, uninduced and induced with IPTG, respectively. Lanes 3 and 4 of each panel are protein extracts from E. coli containing the plasmid pSEV4 with LPMC-61f insert, uninduced and IPTG-induced, respectively. Panel A is the silver staining pattern. Panel B is probed with monoclonal antibody LPMC-61. Panel C is probed with hyperimmune rabbit anti-E, tenella sporozoite serum. Panel D is probed with hyperimmune rabbit anti-E, tenella merozoite serum. Panel E is probed with E. tenella infected chicken serum. Panel F is probed with immune chicken serum. The heavily staining bands in the IPTGinduced tracks (double arrow), which is larger than 13-galactosidase (single arrow), is the fusion protein. The molecular weights (kDa) of size standards are indicated.
60 DNA sequencing of LPMC-61f. The approx. 770 bp EcoRI insert of LPMC-61f was subcloned into M13 for D N A sequencing. The sequence determined (Fig. 6) revealed several interesting features. The sequence has a single open reading frame throughout the insert, has no poly(A) tail, and contains long stretches of CAG repeats similar to those found in other repeated E. tenella genes [12]. The deduced amino acid sequence predicts a protein of approx. 31.3 kDa. The amino acid sequence revealed that the gene can be divided into segments of tandem, non-perfect repeats, each segment beginning with the amino acid doublet tryptophan-proline or tryptophan-serine, followed by stretches of glutamine residues interspersed with other amino acids. Of the 255 amino acid residues encoded by the DNA sequence, 123 (48%) are glutamine. Amino acid analysis of unfused LPMC-61f protein produced
in E. coli agrees with the amino acid content predicted from the cDNA sequence (R. Hageman, Synergen, Inc., personal communication). There are two cysteine residues in the sequence available for disulfide bonding. We have examined the protein-encoding region of this gene for evidence of codon usage bias. Of the 123 glutamines, 114 are coded by CAG, with the remaining 9 coded by CAA; 15 of 21 leucines are encoded by CTG. Similar codon usage bias has been observed in the family of repeat E. tenella genes [12]. Analysis of the predicted amino acid sequence by hydropathicity plot using the Hopps and Woods program [21] showed that the protein has a large central hydrophilic area surrounded by two hydrophobic regions. The central hydrophilic area is made up of the tandem, non-perfect repeat region, with the 5'-region of the sequence consisting of hydrophobic stretches of leucine. No region resembling a
CTG CGG CTG CTG CTG AAG CTA CTG CTG CTG CTG CTG GGG CAG CAG AAA CAT TGG CCT GAG Leu Arg Leu Leu Leu Lys Leu Leu Leu Leu Leu Leu Gly Gin Gin Lys His Trp Pro Glu 61 AGA CAG CAG CAG CAG CAA CCG CAG CCG TGG CTA GAT CGA CAA CAG CAG CAG CAG CAG CAC Ar~ Gln Gln Gln Gln Gln Pro Gln Pro Trp Leu Asp Ar~ Gin Gln Gln Gln Gln Gin His 121 AAC CAA CAG CTG CAG AAG CAG CAG TGG CCT GAG GGC CAG CGG CAG CAG CTG TGG CCA GAG Asn Gin Gln Leu Gln Lys Gin Gln Trp Pro Glu Gly Gln Ar$ Gln Gln Leu Trp Pro Glu 181 CAA CAG CAG CAG CAG TGG CCT GAG CAG CAC CAG CAA GCA CAG CAG CAG CAG CAG TGG CCT Gln Gln Gln Gln Gln Trp Pro Glu Gln His Gln Gln Ala Gln Gln Gln Gln Gln Trp Pro 241 CAG CAG CAG CCA CAG ATG CAG CAG GAG CAG TGG CCT CAG CAG CAG CCA CAG GTG CAG CAG Gln Gin Gin Pro Gln Met Gin Gln Glu Gln Trp Pro Gln Gln Gln Pro Gln Val Gln Gln 301 CAG CAG CAG TGG CCT CAG CAG CAG CAC AGG CGC CAG CAC GGC CAG CAG CAG CAG TGC ATG Gln Gln Gln Trp Pro Gln Gln Gln His Ar$ Ar$ Gln His Gly Gln Gln Gln Gln Cys Met 361 AAC AGC CAG CAG CAG CTG CAG CAG TGC GGG CAG CAG CAG CAG CAG CAG CTG CAG CAG CAG Asn Ser Gln Gln Gln Leu Gln Gln Cys Gly Gin Gln Gln Gln Gln Gln Leu Gln Gin Gln 421 ITGG TCT GAG CAG CAG CAA CAG CAG CAG CAG CAG CAG TGG CCT GAG CAG CCA GAG CAG CAG [Trp Ser Glu Gln Gin Gln Gln Gin Gln Gin Gln Gin Trp Pro Glu Gin Pro Glu Gin Gin ]
481 CAG CAG CAA CAGITGG CCT GAG CAG CAG CAG CAG CAG TGG TCT GAT CAG AAC CAG CAG CAG Gln Gln Gln Gln]Trp Pro Glu Gln Gin Gln Gln Gln Trp Ser Asp Gln 'Asn Gln Gln Gln 541 CAA GCG CAA CAG TGG CAG GCG CAG CAG CAG CAG CAG TGG CCG CAG CAG CAG CAG CAG CCG Gln Ala Gln Gln Trp Gln Ala Gln Gln Gln Gln Gln Trp Pro Gln Gln Gln Gln Gln Pro 601 CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG IGAC TTG GGG CCC GAT GGA GTT GGG ATC GTC Gln Gln Gln Gin Gin Gln Gin Gln Gln Gln~Asp Leu Gly Pro Asp Gly Val Gly lle Val 661 GTT CCC TAC CTG GGA TCG TCT CCA GCA GAC TTC GAA GTC CAG AAC CTC GGG GTG TCT GTA Val Pro Tyr Leu Gly Ser Ser Pro Ala Asp Phe Glu Val Gln Ash Leu Gly Val Ser Val 721 CTC CCC AGA ATC AAG CTG CCC CTC CCC AAG TTC GAC AAC GAC GAC GG Leu Pro Arg Ile Lys Leu Pro Leu Pro Lys Phe Asp Ash Asp Asp
Fig. 6. DNA sequence and predicted amino acid sequence of LPMC-61f. Sequence displayed in 5' --9 3' direction. The tandem, non-perfect repeats are shown as boxed regions.
61 signal sequence was found in this partial cDNA sequence. Discussion
In this report we describe the development of a monoclonal antibody against purified second generation E. tenella merozoites and the identification and characterization of a target antigen of this monoclonal antibody. The monoclonal antibody LPMC-61 binds to air-dried merozoites by immunofluorescence assay. LPMC-61 inhibits parasite development when cultures are maintained in its continuous presence, but not when sporozoites are pre-treated with the antibody. No inhibitory activity is seen if the antibody is added after parasite invasion of the host cell. The LPMC-61 target antigen is not stage-specific. Mab LPMC-61 recognized an approx. 80-kDa sporozoite polypeptide in non-reducing SDS-PAGE. In the presence of 13-mercaptoethanol, LPMC-61 recognized an approx. 10-12-kDa sporozoite polypeptide. This finding suggests that the 10012kDa antigen is covalently linked by disulfide bonds to other polypeptides to form the 80-kDa antigen, since there are two cysteine residues within the cDNA encoding the LPMC-61 antigen. The other polypeptides to which the 10-12kDa protein is attached may be distinct gene products or portions of a precursor protein. In the presence of the reducing agent 13-mercaptoethanol, the 80-kDa precursor is processed to the 10-12-kDa subunits. Files et al. [22] have previously shown that the protein target (TA4) of two sporozoite-neutralizing monoclonal antibodies is synthesized as a 25-kDa precursor that is proteolytically processed to give the 17-kDa and 8-kDa subunits held together by disulfide bonds. A partial cDNA clone, designated LPMC-61f, containing the gene corresponding to this Mab was identified from an E. tenella sporozoite cDNA library. Antibody elution experiments confirmed that cDNA LPMC-61f indeed contains a target antigen to the Mab. The 13-galactosidase/LPMC61f fusion protein reacted with the LPMC-61 monoclonal antibody, and is also recognized by rabbit anti-E, tenella sporozoite serum, rabbit anti-merozoite serum, E. tenella infected and immune chicken serum. Therefore the LPMC-61f
antigen appears to be immunogenic in both rabbits and chickens and stimulates B cell production during a natural infection. The 13-galactosidase-LPMC-61f fusion protein is about 31 kDa larger than 13-galactosidase, and is therefore coded for by the entire LPMC-61f cDNA sequence. The larger size of the protein, predicted by the LPMC61 cDNA (31 kDa) than the 10o12 kDa polypeptide recognized by LPMC-61 Mab, is consistent with the 10012 kDa protein being encoded by a precursor protein, possibly the 80-kDa protein. Examination of the LPMC-61f DNA sequence revealed that it encodes stretches of glutamine repeats as have been found with some other repeated sequences in E. tenella [12]. The LPMC61f encoded protein sequence may be divided into segments of tandem, non-perfect repeats as shown in Fig. 5. Recently, Jenkins [23] reported the isolation of a cDNA (cMZ8) encoding an approx. 150 kDa merozoite surface protein in Eimeria acervulina containing tandem-repeated amino acid sequences very different from the LPMC-61f repeat sequences described here. Protozoan proteins with repeating amino acid sequences, either as stretches of single amino acids [24,25] or as tandem repeats of several amino acids [26] have been described. The LPMC-61f sequence contains long stretches of the amino acid glutamine within the tandem repeats and is different from the serine-rich [25] or asparagine-rich [24] plasmodium proteins. It is unclear at the present time as to what roles these glutamine-rich tandem repeated sequences play, just as no known functions have been known for the SERA or CARP [24,25]. Their preponderance in Eimeria [12] suggest that they may be important for an as yet unknown aspect of the parasite life cycle. The fact that infected and immune chicken sera recognize the target antigen of Mab LPMC-61 suggest that the Eirneria repeating amino acid sequences are immunodominant antigens in their hosts, as have been found for the tandem repeats of amino acids in the related Apicomplexa protozoa, the malaria parasite Plasmodium [27,28]. Eimeria merozoite has been implicated as the most immunogenic stage in the coccidia life cycle [11], studies have begun on the antigenic makeup of the merozoites. Although the function of this LPMC-61 antigen in E. tenella is unknown at the
62
present time, the fact that it cross-reacts with different stages in the life cycle of the coccidia shows that it is a common shared epitope. More importantly, the fact that Mab LPMC-61 inhibits the development of the parasite in vitro suggests that the LPMC-61 antigen may be an important immunogen. We have preliminary evidence that Mab LPMC-61 cross-reacts with an approx. 10-12kDa E. acervulina polypeptide under reducing conditions. Therefore LPMC-61 may also be present in other Eimeria species. Since LPMC-61f only encodes about 31 kDa of the approx. 80 kDa polypeptide recognized by the Mab, further work will be directed towards identifying a full-length cDNA that encodes the entire target antigen in order to study the complete protein sequence and structure. Further experiments also will be required to determine whether this antigen will elicit a protective immune response in chickens against
Acknowledgements This work was funded by Eli Lilly and Company. The authors would like to thank Dr. Jerry Kuner for the sporulation samples and the antibody elution technique, Dr. Robert Hageman for communicating the amino acid analysis data of LPMC-61f polypeptide, Dr. Leonard Guarente for the pLG2 plasmid, Dr. M.J. Casadaban for the E. coli strain, AMA1004 and Dr. W. Current for providing the E. acervulina sporozoites, Dr. M. Stiff for the chicken sera, and Dr. George Cox for the E. tenella sporozoite cDNA library and for the critical evaluation of this manuscript and to the reviewers for suggesting the non-reduced SDSPAGE experiments. We also thank Carla Worland for the preparation of the manuscript.
Eimeria tenella.
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63 18 Smith, D.E. and Fisher, P.A. (1984) Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. Cell Biol. 99, 20-28. 19 Guarente, L. and Lauer, G. (1980) Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit 13-globin. Cell 20, 543-553. 20 Casadaban, M.J., Martinez-Arias, A., Shapira, S.K. and Chou, J. (1983) I~-Galactosidase gene fusions for analyzing gene expression in E. coli and yeast. Methods Enzymol. 100,293-308. 21 Hopp, T.P. and Woods, K.R. (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78, 3824-3828. 22 File, J.G., Paul, L.S. and Gabe, J.D. (1987) Identification and characterization of the gene for a major surface antigen of Eirneria tenella. In: Molecular Strategies of Parasitic Invasion, pp. 713-723. Alan R. Liss, New York.
23 Jenkins, M.C. (1988) A cDNA encoding a merozoite surface protein of the protozoan Eimeria acervulina contains tandem-repeated sequences. Nucleic Acids Res. 16, 9863. 24 Wahlgren, M., ,~slund, L., Franz6, L., Sundvall, M., Wahlin, B., Berzines, K., McNicol, L.A., Bj6rkman, A., Wiszell, H., Perlmann, P. and Pettersson, U. (1986) A Plasmodium falciparum antigen containing clusters of asparagine residues. Proc. Natl. Acad. Sci. USA 83, 2677-2681. 25 Li, W.-B., Bzik, D.J., Horii, T. and Inselburg, J. (1989) Structure and expression of the Plasmodiurn falciparum SERA gene. Mol. Biochem. Parasitol. 33, 13-26. 26 Ozaki, L.S., Svec, P., Nussenzweig, R.S., Nussenzweig, V. and Godson, G.N. (1983) Structure of the Plasmodium knowlesi gene coding for the circumsporozoite protein. Cell 34, 815-822. 27 Nussenzweig, V. and Nussenzweig, R.S. (1985) Circumsporozoite proteins of malarial parasite. Cell 42, 401-403. 28 Kemp, D.J., Coppel, R.L. and Anders, R.F. (1987) Repetitive proteins and genes of malaria. Annu. Rev. Microbiol. 41, 181-208.
