Entamoeba histolytical immunogenic protein and cDNA clone

An Entamoeba histolytica specific cDNA clone which encodes an antigenic surface membrane protein possessing multiple tandem repeats and expression in E. coli is disclosed.

BACKGROUND OF THE INVENTION 
This invention relates to an immunogenic protein and cDNA which codes for 
said protein. More particularly, the invention is concerned with a surface 
membrane antigen of Entamoeba histolytica and a E. histolytica specific 
cDNA clone which encodes a serine rich E. histolytica protein. 
The protozoan pathogen Entamoeba histolytica is a major cause of 
debilitating illness and death worldwide, infecting more than 500,000,000 
people, and causing an estimated 50,000,000 cases of diarrhea, and 50,000 
deaths yearly [Walsh in Aembiasis, Human Infection by Entamoeba 
histolytica, ed. Ravdin, J. I., John Wiley & Sons, Inc. New York, N.Y., 
pp. 93-105 (1988)]. There is an urgent need for a vaccine which could 
prevent the establishment of E. histolytica infection, or the development 
of invasive disease. Previous studies in animal models have demonstrated 
that immunity to E. histolytica infection can be produced by immunization 
with E. histolytica lysates [Ghadirian et al., Am. J. Trop. Med. Hyg. 29, 
779-784 (1980); Krupp, Am. J. Trop. Med. Hyg. 23, 355-360 (1974); and 
Swartzwelder and Avant, Am. J. Trop. Med. Hyg. 1, 567-575 (1952)]. 
However, the difficulty in obtaining large quantities of trophozoites, and 
the relatively crude nature of the immunizing preparations have severely 
limited the scope of these prior studies. 
Recently, genomic differences between pathogenic and nonpathogenic E. 
histolytica have been reported by Tannich et al., Proc. Natl. Acad. Sci. 
USA 86, 5118- 5122 (1989). These scientists utilized antibody screening 
and reported an amino acid sequence derived from a partial cDNA clone. No 
putative initiator methionine was found and no nucleotide data was 
reported by them. Nor are any tandem repeats or other characterization of 
the partial amino acid sequence provided by Tannich et al. No biological 
role for the Tannich et al protein is found in their report; instead, the 
paper is completely directed to the use of their partial cDNA clone to 
detect genomic differences between E. histolytica strains. However, 
Southern blotting with actin (a conserved protein, found in almost all 
organisms, and originally isolated by another scientific group) shows the 
same ability to differentiate between strains of E. histolytica as their 
probe, thereby suggesting that their probe is not unique in its ability to 
differentiate between E. histolytica strains. 
BRIEF DESCRIPTION OF THE INVENTION 
In accordance with the present invention an Entamoeba histolytica specific 
cDNA clone which encodes an antigenic surface membrane protein possessing 
multiple tandem repeats has been isolated and expressed in E. coli. 
In particular, differential hybridization screening was used to isolate an 
illustrative E. histolytica specific cDNA clone, designated cl. The cDNA 
was found to encode a serine rich E. histolytica protein (hereinafter also 
referred to as SREHP) containing multiple tandem repeats. The structural 
motif of SREHP resembles some of the repetitive antigens of malarial 
species, especially the circumsporozoite proteins. A recombinant trpE 
fusion protein containing the tandem repeats of SREHP was recognized by 
immune serum from a patient with amebiasis, demonstrating that SREHP is a 
naturally immunogenic protein. An antiserum raised against the recombinant 
fusion protein specifically bound to two distinct bands with apparent 
molecular weights of 46 and 52 kd in a crude preparation of E. histolytica 
trophozoite membranes. This antiserum also inhibited E. histolytica 
trophozoite adhesion to Chinese Hamster Ovary cells in vitro. These 
properties suggest that SREHP plays a role in a key part (adhesion) of E. 
histolytica pathogenesis. SREHP also can be used as the target antigen in 
a serologic test for invasive amebiasis. 
The recombinant trpE fusion protein is constructed by fusing the SREHP or a 
fragment thereof which contains at least the tandem repeats to the 
N-terminal two-thirds of the trpE gene of E. coli. This fusion can be 
carried out by conventional procedures such as described by Hardy and 
Strauss, J. Virol. 62(3), 998-1007 (1988), for making a fusion of 
polypeptides of Sindbis virus and the N-terminal two-thirds of the trpE 
gene of E. coli. 
The ability to isolate E. histolytica specific genes, and to express those 
genes in E. coli, is important to medical science in studying the 
molecular basis of E. histolytica pathogenesis, and for development of 
vaccines and diagnostics. 
The illustrative cl cDNA sequence consists of 722 nucleotides and contains 
two open reading frames, designated ORF1 and ORF2. ORF1 contains a 
continuous open reading frame from a putative initiator methionine 
beginning at nucleotide 4 to a TAA termination at nucleotide 703. ORF2 has 
a most 5' methionine at nucleotide 188 and terminates at nucleotide 689. 
The cDNA sequence encodes a 25 kDa protein of 233 amino acids. Since 
serine constituted 52 of the 233 amino acids, the derived amino acid 
sequence of ORF1 is referred to as the serine rich E. histolytica protein 
(SREHP). By comparison, the derived 167 amino acid sequence of ORF2 is 
serine poor. The cl cDNA nucleotide sequence and the two respective amino 
acid sequences encoded by ORF1 and ORF2 are as follows. The nucleotides 
and amino acids are numbered on the right. 
##STR1## 
In order to demonstrate a practical utility of the novel SREHP of the 
invention, 90 sera from 88 patients were examined by Western blotting for 
the presence of antibodies to this serine rich E. histolytica protein. The 
target antigen was a recombinant trpE fusion protein containing most of 
the SREHP sequence, including the tandem repeats. Among patients with 
amebic liver abscess from Durban, San Diego, and Mexico, 49 of 61, (79%) 
had antibodies to SREHP. In contrast only 1 patient out of 24 (4%) without 
acute invasive amebiasis had antibodies to SREHP. The specificity of 
anti-SREHP antibodies in the detection of acute invasive amebiasis was 
most marked when analyzed in the patients from Durban, where 11 of 12 
(92%) patients seropositive for SREHP had acute invasive amebiasis, versus 
17 of the 26 (65%) patients positive by agar gel diffusion. The use of a 
serologic test based on the recombinant SREHP fusion protein thus is 
deemed to be a useful adjunct to the diagnosis of acute invasive amebiasis 
in endemic regions.

Standard biochemical nomenclature is used herein in which the nucleotide 
bases are designated as adenine (A); thymine (T); guanine (G); and 
cytosine (C). Corresponding nucleotides are, for example, 
deosyadenosine-5'-triphosphate ((dATP). As is conventional for convenience 
in the structural representation of a DNA nucleotide sequence, only one 
strand is usually shown in which A on one strand connotes T on its 
complement and G connotes C. 
Amino acids are shown either by their common three letter or one letter 
abbreviations as follows: 
______________________________________ 
Abbreviated Designation 
Amino Acid 
______________________________________ 
A Ala Alanine 
C Cys Cysteine 
D Asp Aspartic acid 
E Glu Glutamic acid 
F Phe Phenylalanine 
G Gly Glycine 
H His Histidine 
I Ile Isoleucine 
K Lys Lysine 
L Leu Leucine 
M Met Methionine 
N Asn Asparagine 
P Pro Proline 
Q Gln Glutamine 
R Arg Arginine 
S Ser Serine 
T Thr Threonine 
V Val Valine 
W Trp Tryptophan 
Y Tyr Tyrosine 
______________________________________ 
In order to illustrate specific preferred embodiments of the invention in 
further detail, the following exemplary laboratory preparative work was 
carried out although it will be understood that the invention is not 
limited to these specific examples. 
EXAMPLE 1 
This example illustrates the isolation of the E. histolytica specific cDNA 
clone, designated cl, and the characterization and properties of the 
SREHP. 
MATERIALS AND METHODS 
E. histolytica isolates and culture conditions. The E. histolytica strain 
HM1:IMSS is virulent in vivo and in vitro [Li et al, Infect. Immun. 57, 
8-12 (1989); Mattern and Keister, Am. J. Trop. Med. Hyg. 27, 882-887 
(1977)]. The E. histolytica -like Laredo strain was isolated from a 
patient with diarrhea [Diamond, J. Parasitol. 54, 1047-1056 (1968)], but 
is avirulent in in vitro cytoxicity assays and in animal models. Both 
strains were grown axenically in TYI-S-33 media by conventional procedure 
as described previously by Diamond et al., Trans. R. Soc. Trop. Med. Hyg. 
4, 431-432 (1978). 
Construction and screening of a HM1:IMSS cDNA library. Total cellular RNA 
was isolated from exponentially growing HM1:IMSS and Laredo trophozoites 
using the method of Chirgwin et al., Biochemistry 18, 5294-5299 (1979). 
Poly-(A).sup.+ -RNA Was purified by chromatography on oligo(dT)-cellulose 
[Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring 
Harbor Lab., Cold Spring Harbor, N.Y. (1982)], and used to prepare double 
stranded cDNA [Gubler and Hoffman, Gene 25, 263-269, (1983)]. The double 
stranded cDNA was C-tailed using the enzyme terminal deoxynucleotidyl 
transferase, and annealed with G-tailed PstI digested pUC13 
[Villa-Komaroff et al., Proc. Nat. Acad. Sci. USA 75, 3727-3731 (1978)]. 
The chimeric plasmid was used to transform DH-5 E. coli [Hanahan, J. mol. 
Biol. 166, 557-580 (1983)], generating a cDNA library containing 50,000 
recombinants. Replica filters of 10,000 recombinants were probed with 
radiolabeled single stranded cDNA from HM1:IMSS or the non-pathogenic, E. 
histolytica -like, Laredo strain, respectively. 
Northern blot analysis. Northern blots were prepared after 20 .mu.g samples 
of total cellular RNA were subjected to electrophoresis through 1.5% 
agarose gels containing formaldehyde [Thomas, Proc. Nat. Acad. Sci. USA 
77, 5201-5205 (1980)]The Northern blots were probed with cDNA labeled with 
[.alpha.-.sup.32 P]ATP to a specific activity of 1000 cpm/pg by the random 
primer method of Feinberg and Vogelstein, Anal. Bicochem. 132, 6-13 
(1983). The hybridization and washing conditions were identical to those 
itemized by Li et al., Proc. Nat. Acad. Sci. USA. 83, 5779-5783 (1986), 
except the last three washes were done at 60.degree. C. Autoradiographs 
were exposed for 24 hours. 
Nucleotide sequence analysis. The cDNA clones were sequenced using the 
technique of Maxam and Gilbert, Methods Enzymol, 65, 499-560 (1980). The 
cDNA sequence of clone cl was completed using primer extension as 
described by Heuckeroth et al., J. Biol. Chem. 262, 9709-9717 (1987), 
utilizing the oligonucleotide TTCAGGACTAGCTTCGTTCTT derived from the 
sequence of cl. 
Construction of trpE hybrid gene fusions. The pATH2 and pATH3 plasmids 
[described by Hardy and Strauss, J. Virology 62, 998-1007 (1988)] were 
used to express both open reading frames of the cl cDNA clone in E. coli, 
as trpE fusion proteins. The HindIII-SmaI fragment of cl (containing 
nucleotides 128-722 and a portion of the PUC13 polylinker region) was 
ligated into the HindIII site and the ClaI site (which had been made 
blunt-ended with Klenow DNA polymerase) of pATH3 to construct pORF1. The 
HindIII-SmaI fragment was ligated into the HindIII and blunt ended ClaI 
sites of pATH2 to construct pORF2. 
Expression and partial purification of the trpE fusion proteins. The trpE 
fusion proteins wre expressed as described by Hardy and Strauss, supra, 
except the bacteria were harvested after incubation with 
.beta.-indoleacrylic acid for 24h. An insoluble protein fraction was 
prepared from pelleted cells as previously described by Hardy and Strauss, 
supra, to partially purify the trpE proteins. The yield of fusion protein 
as determined by discontinuous SDS-PAGE using molecular mass standards for 
comparison was 15 to 30 .mu.g of protein per ml of culture. 
Immunizations. Polyclonal rabbit serum directed against HM1:IMSS 
trophozoites was obtained by immunizing New Zealand White Rabbits 
subcutaneously with a preparation of 2.times.10.sup.6 HM1:IMSS 
trophozoites suspended in complete Freunds Adjuvant. Booster immunizations 
were performed using the same preparation in incomplete Freunds Adjuvant. 
Serum was first collected two weeks after the initial booster injection, 
and subsequently two weeks after each consecutive booster. 
Gel purification of the fusion proteins, and immunization of female New 
Zealand White Rabbits with fusion proteins, was performed exactly as 
described by Hardy and Strauss, supra, using 75 to 100 .mu.g of protein 
per injection. Serum was collected from each rabbit before immunization to 
serve as a control, then 6 weeks after the initial immunization (two weeks 
after the first booster), and two weeks after each consecutive booster. 
Western blots. For Western blotting of the fusion proteins with E. 
histolytica immune human and rabbit serum, the insoluble protein fraction 
of bacterial cells from 0.3 ml cultures was separated by 10% SDS-PAGE 
under reducing conditions, then transferred to nitrocellulose. Blots were 
reacted with immune or pre-immune serum diluted 1/500, and immunoglobulin 
binding detected using .sup.125 I labeled staphlococcal protein-A. Blots 
were autoradiographed for 12 hr. 
For Western blotting of amebic lysates With antiserum against recombinant 
proteins, 5.times.10.sup.6 trophozites from 72 hr cultures were washed 
2.times. in phosphate buffered saline (PBS), then suspended in 2 ml of PBS 
containing 5 mM EDTA/2 mM leupeptin/5 mM N-ethylmaleimide/2 mM 
phenylmethlsulfonyl fluoride/2 mM benzamidine/5 mM 
trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (E-64). The 
trophozoites were lysed by sonication, and homogenates of the HM1:IMSS 
trophozoites were centrifuged at 100,000.times.g for 1 hr. Aliquots of the 
100,000.times.g supernatant and pellet fraction corresponding to 10.sup.5 
trophozoites were denatured, separated by 10% SDS-PAGE under reducing 
conditions, then transferred to nitrocellulose. Blots were reacted with a 
1/1000 dilution of rabbit antiserum to the fusion protein; subsequent 
steps were performed as described above. 
Adherence of .sup.3 H-thymidine labeled E. histolytica trophozoites to 
monolayers of 1021 Chinese Hamster Ovary Cells. These tests were performed 
by conventional procedures as described previously by Li et al., J. Exp. 
Med. 167, 1725-1730 (1988). 0.25 ml of media with or without the antisera 
to be tested were added to the monolayers immediately before addition of 
0.25 ml of trophozoites suspended at 8.times.10.sup.5 cells/ml. A 1:10 
dilution of each of the antisera tested was used. Duplicate wells were 
used for each assay. Data are presented as the % of trophozoite adherence 
seen in the media control group .+-. standard deviation. 
RESULTS 
Isolation of a cDNA clone from the pathogenic E. histolytica HM1:IMSS 
strain. 10,000 recombinants from the pUC13 cDNA library derived from 
HM1:IMSS mRNA were screened with radiolabeled single stranded cDNA 
transcribed from poly A.sup.+ RNA from HM1:IMSS or the non-pathogenic, E. 
histolytica -like Laredo strain, respectively. Four unique clones that 
hybridized to HM1:IMSS cDNA, but not to Laredo cDNA were isolated. The 
characterization of one of the clones, cl, is described in detail herein. 
Analysis of cl gene expression by RNA blot hybridization. The expression of 
the gene corresponding to the cl clone was examined by blot hybridization 
studies of RNA isolated from 4 E. histolytica strains, Laredo, Entamoeba 
invadens, and Entamoeba moshkovskii (FIG. 1). The cl clone hybridized with 
a 0.8 kb species in all four axenic strains of E. histolytica but did not 
hybridize with RNA from Laredo, or the two non-E. histolytica sp., E. 
invadens, and E. moshkovskii. When a Southern blot of EcoRl digested 
genomic DNA from the same ameba was probed with the cl clone, a 4.4 kb 
fragment was noted in all four E. histolytica strains, but not in Laredo, 
E. invadens, or E. moshkovskii. The results from RNA and DNA hybridization 
tests indicate that the gene corresponding to the cl clone is present and 
expressed only in the E. histolytica strains, and not in the other 
Entamoeba sp. surveyed. 
Nucleotide sequence of the cl cDNA clone. The cl cDNA sequence consisted of 
722 nucleotides (FIG. 2A), and contained two open reading frames, which 
were designated ORF1 and ORF2. ORF1 contained a continuous open reading 
frame from a putative initiator methionine beginning at nucleotide 4 to a 
TAA termination at nucleotide 703. Beginning at nucleotide 247 ORF1 
contained a stretch of 36 nucleotides encoding the dodecapeptide 
Ser-Ser-Ser-Asp-Lys-Pro-Asp-Asn-Lys-Pro-Glu-Ala. After a 24 nucleotide 
space encoding a similar octapeptide, the dodecapeptide was tandemly 
repeated 5 times, followed by 4 repeats of an octapeptide, 
Ser-Ser-Thr-Asn-Lys-Pro-Glu-Ala. The nucleotide sequence of the 
dodecapeptide repeats was highly conserved, with the only difference being 
the use of C or U in the third position of the codon for the first serine 
of each repeat. The repeated octapeptide represents a truncated version of 
the dodecapeptide, with a single nucleotide change substituting a 
threonine for the third serine, and nucleotides encoding both Asp 
residues, and one of the Lys and Pro residues absent. Serine constituted 
52 of the 233 amino acids, hence the derived amino acid sequence of ORF1 
is referred to as the serine rich E. histolytica protein (SREHP). Three 
contiguous serine residues were part of the dodecapeptide repeats, and 
were found in five other locations in the protein. The repeats were 
preceded by a highly charged region consisting of multiple lysine, 
glutamate, and aspartate residues. ORF1 terminated with 54 nucleotides 
encoding primarily hydrophobic amino acids, consistent with a possible 
membrane insertion or spanning region. The initial 13 amino acids of the 
NH.sub.2 -terminus possess some of the characteristics of a eukaryotic 
signal sequence as defined by the algorithm of von Heinje, Nuc. Acids Res. 
14, 4683-4690 (1986), with a possible cleavage site between the Ala at 
position 13 and Thr at position 14. The sequence differed from other 
signal sequences in the absence of one or more charged amino acids 
(n-domain) before the hydrophobic core (h-domain). 
The second ORF, ORF2, possessed 5 methionine codons at the 5' end, with the 
most 5' beginning at nucleotide 188 (FIG. 2A). ORF2 also encoded a 
tandemly repeated dodecapeptide, Gln-Val or 
Ala-Gln-Val-Ile-Asn-Gln-Ile-Ile-Asn-Gln-Lys, which began at nucleotide 
245, and had a pattern of repeats similar to ORF1, with 5 additional 
dodecapeptide repeats followd by 4 repeats of an octapeptide, 
Gln-Ala-Gln-Leu-Ile-Asn-Gln-Lys. ORF2 terminated with a relatively 
hydrophobic region. 
A search of the Genbank and NBRF data banks revealed no sequences with 
significant homology with either the nucleotide or derived amino acid 
sequences from cl. In addition, the derived amino acid sequences from cl 
differ from the partial sequence of the E. histolytica cDNA clone recently 
reported by Tannich et al , Proc. Natl. Acad. Sci. USA 86, 5118-5122 
(1989). Tandem repeats have been found in antigens from other parasites, 
but are most prominent amoung the antigens of malaria species [Kemp et 
al., Ann. Rev. Microbiol. 41, 181-208 (1987)]. The primary structural 
motif of SREHP is similar to the repetitive antigens of malarial species, 
most notably the circumsporozoite proteins [Kemp et al, supra, and Ozaki 
et al., Cell 34, 815-822 (1983)]. Circumsporozoite proteins cover the 
surface of the sporozoite stage of the malaria parasite, range in size 
from 40 to 60 kDa, and contain a species specific pattern multiple tandem 
repeats. The predicted structure of the P. knowlesi circumsporozoite 
protein consists of a hydrophobic NH.sub.2 terminal region followed by a 
series of tandemly repeated amino acids flanked by two domains containing 
predominantly charged amino acids, and concluding with a COOH-terminal 
region, consisting of a hydrophobic anchor region (FIG. 2B) [ Ozaki et 
al., supra]. The predicted structure of SREHP consists of a hydrophobic 
NH.sub.2 -terminal region, followed by a region containing primarily 
charged amino acids, followed by tandemly repeated amino acids, and ends 
with a COOH-terminal hydrophobic region (FIG. 2B). In the NH.sub.2 
-terminal charged area of the P. knowlesi circumsporozoite protein, 27 of 
the 48 amino acid residues are charged (primarily lysine and glutamate 
residues [Ozaki, supra.], whereas 21 of the 36 residues are charged 
(primarily lysine, glutamate, and aspartate) in the comparable NH.sub.2 
-terminal region of SREHP. Unlike circumsporozoite proteins SREHP does not 
possess a second COOH-terminal group of charged amino acids. An additional 
similarity between SREHP and circumsporozoite proteins lies in the amino 
acids utilized in the repeating units. Circumsporozoite protein repeats 
appear to be derived from a repertoire of 8 amino acids, Ala, Pro, Gly, 
Asn, Gln, Asp, Arg, and Glu [Galinski et al, Cell 48, 311-319 (1987)]. 
Five of these 8 amino acids, Ala, Pro, Asn, Asp, and Glu, are among the 7 
amino acids which make up the dodecapeptide repeats of SREHP. 
cl cDNA encodes a naturally immunogenic E. histolytica protein. Since it 
was not known which of the ORF of cl were translated in E. histolytica 
both open reading frames of cl in E. coli using the trpE containing 
vectors pATH2 and pATH3 were expressed. Constructs, pORF1 and pORF2, 
containing most of the sequence of the cl insert fused in the appropriate 
reading frame to the N-terminal two-thirds of the E. coli trpE gene were 
expressed. Coomassie blue staining of SDS-PAGE separated insoluble pellets 
from cells containing the pORF1 or pORF2 vector which had been induced to 
produce trpE by .beta.-indoleacrylic acid revealed fusion proteins with 
molecular weights of approximately 60 kDa (the predicted molecular weights 
for the fusion proteins encoded by ORF1 and ORF2 are 57 kDa and 59 kDa 
respectively). To determine whether either of the ORF of cl produced a 
naturally immunogenic E. histolytica protein, Western blotting was 
performed (FIG. 3). Immune serum from a patient with amebic liver abscess 
and serum from a rabbit immunized with HM1:IMSS trophozoites both bound to 
a series of bands, (highest MW approximately 60 kDa) in bacteria 
expressing the fusion protein (predicted MW 57 kDa) encoded by pORF1 (FIG. 
3B and D, lane 2). Immune serum did not bind to the fusion protein encoded 
by pORF2. Control human serum and preimmune rabbit serum did not bind 
either of the fusion proteins (FIG. 3A and C). In subsequent studies it 
was found that serum from 25 additional patients with invasive amebiasis 
also bind the fusion protein produced by pORF1. It has been confirmed that 
the recombinant protein recognized by immune serum contains the tandemly 
repeated peptides encoded by ORF1 by demonstrating that antiserum to a 
synthetic peptide containing the dodecapeptide repeat 
Ser-Ser-Ser-Asp-Lys-Pro-Asp-Asn-Lys-Pro-Glu-Ala binds the recombinant 
protein. 
SREHP is a membrane protein. In order to determine whether SREHP is 
associated with a membrane fraction of E. histolytica HM1:IMSS 
trophozoites were lysed, then spun at 100,000.times.g and loaded onto 
SDS-PAGE as a supernatant and pellet fraction. As shown in the Western 
blot (FIG. 4), the anti-SREHP fusion protein antiserum specifically bound 
to two distinct bands at molecular weight 46 kDa and 52 kDa in the 
100,000.times.g pellet fraction. Little binding was detected in the 
100,000.times.g supernatant suggesting the native SREHP is primarily 
membrane bound. This finding is consistent with the primary structure 
data. The anti-SREHP fusion protein antiserum showed no binding to any 
species in whole Laredo lysates, suggesting SREHP is E. 
histolytica-specific. It is presently unclear why antiserum to the 
recombinant SREHP fusion protein detects two species with molecular 
weights approximately twice that predicted from the derived amino acid 
sequence of the SREHP cDNA (25 kDa) in the E. histolytica lysates. The 
results of the Northern blotting study (FIG. 1) demonstrated that the size 
of the cl clone (722 nucleotides) is close to the size of the SREHP 
transcript (approximately 800 nucleotides) suggesting that most, if not 
all, of the coding region should be contained in the cl clone. Hence it is 
unlikely that the discrepancy in size results from additional amino acids, 
and more likely it is secondary to post-translational modifications. 
Antiserum to the fusion protein encoded by pORF2 showed no binding to 
HM1:IMSS trophozoite lysates by Western blotting. Thus the results do not 
demonstrate the existence of a HM1:IMSS trophozoite protein encoded by cl 
ORF2. One cannot exclude the possibility that ORF2 might be translated in 
the cyst form of the parasite. 
Antiserum to the SREHP fusion protein inhibits E. histolytica HM1:IMSS 
adhesion to Chinese Hamster Ovary Cells. The adherence of E. histolytica 
HM1:IMSS trophozoites to a panel of Chinese Hamster Ovary cells was 
previously studied and it was shown that trophozoites adhere best to the 
1021 Chinese Hamster Ovary cell line, Li et al, J. Exp. Med. 167, 
1725-1730 (1988). The ability of antiserum to the SREHP fusion protein to 
inhibit the binding of radiolabeled E. histolytica HM1:IMSS trophozoites 
to 1021 cells was evaluated. Antiserum to the SREHP fusion protein with 
preimmune serum, antiserum to the fusion protein encoded by pORF2, and a 
media control were compared. Antiserum to the SREHP fusion protein reduced 
the binding of HM1:IMSS trophozoites to 1021 cells to 30.+-.2% of control 
levels (Table 1). Preimmune serum, and antiserum to the fusion protein 
encoded by pORF2, had no inhibitory effects. This finding suggests that 
SREHP is located on the trophozoite cell surface, and can play a role in 
E. histolytica adhesion. It is notable in this regard that a recent study 
by Rodriguez et al using a polyclonal antisera against total amebic 
proteins found that an E. histolytica protein of molecular weight 50 kDa 
was one of 7 E. histolytica proteins found on the surface of red blood 
cells incubated with trophozoites [Rodriguez et al, Mol. Biochem. 
Parisitol. 37, 87-100 (1989)]. 
______________________________________ 
% of Control Binding 
SERA to 1021 Cells 
______________________________________ 
Preimmune 94 .+-. 25 
Anti-SREHP fusion protein 
30 .+-. 2 
Anti-ORF2 fusion protein 
120 .+-. 25 
______________________________________ 
Results are the means from 3 tests and are presented as the % of 
trophozoite adherence seen in the media control group .+-. standard 
deviation. 
EXAMPLE 2 
This example illustrates the use of the SREHP as a target antigen in a 
serologic test for invasive amebiasis. 
INTRODUCTION 
The serologic diagnosis of invasive amebiasis is problematic. The indirect 
hemagglutinin test (IHA) is sensitive for the detection of invasive amebic 
infection, but seropositivity can persist for years after infection 
[Krupp, Am. J. Trop. Med. Hyg. 19, 57-62 (1970); Lobel and Kagen, Ann. 
Rev. Microb. 32, 329-47 (1978)]. Agar gel diffusion (AGD) and counter 
immune electrophoresis (CIE) offer improved detection of acute disease, 
but seropositivity may persist for six months or more after infection. 
[Lobel and Kagen supra; Krupp and Powell, Am. J. Trop. Med. Hyg. 20, 
421-24 (1971); Juniper et al., Ibid. 21, 157-68 (1972); Jackson et al., 
Trans. Roy. Soc. Trop. Med. Hyg. 78, 342-45 (1984)]. Given these findings 
it is not surprising that in regions endemic for invasive amebiasis 
between 6 and 20% of healthy subjects will have positive serology, 
probably secondary to prior infection with pathogenic E. histolytica. 
[Lobel and Kagen, supra; Jackson et al., Lancet 1985; i:716-719.] This 
high background level of seropositivity in endemic areas can limit the 
usefulness of serology in the diagnosis of acute invasive disease. 
SREHP appears to be present only in E. histolytica, and has a structure 
consisting of multiple tandem repeats, resembling the circumsprozoite 
proteins of malaria. In Example 1, above, it was demonstrated that serum 
from a patient with invasive amebiasis bound to a recombinant fusion 
protein containing the multiple tandem repeats encoded by the SREHP gene. 
This Example 2 provides further analysis of the serologic response to 
SREHP. The objectives were to examine the prevalence of antibodies to 
SREHP among patients with amebic liver abscess in two different endemic 
regions, and to determine whether the recombinant SREHP fusion protein 
might be useful in a serologic test for the diagnosis of invasive 
amebiasis. This Example 2 describes the finding that the presence of 
antibodies to SREHP has a high correlation with the presence of acute 
invasive amebiasis. 
MATERIALS AND METHODS 
Production of SREHP Fusion Protein. The pATH3 plasmid vector was used to 
express nucleotides 128 to 722 of the SREHP cDNA (containing the multiple 
tandem repeats) in E. coli as a 60 kDa trpE fusion protein as described in 
Example 1. A second, smaller open reading frame (ORF2), of the SREHP cDNA 
which does not appear to be expressed in E. histolytica trophozoites, was 
expressed in pATH2, and served as a control fusion protein for Western 
blotting. The fusion proteins were partially purified from bacteria 
induced to produce trpE as described in Example 1. 
Western Blotting. For Western blotting the insoluble protein fractions from 
bacteria expressing the fusion proteins were separated by 10% SDS-PAGE 
under reducing conditions then transferred to nitrocellulose as described 
in Example 1. Blots were reacted with human serum samples to be tested at 
a 1:500 dilution, and immunoglobulin binding detected using .sup.125 I 
labeled staphylococcal protein-A. Blots were autoradiographed for 12 hr. 
Blots were read independently by four observers, (SLS, CKJ, AB, and EL). 
Criteria for a positive blot were binding to SREHP recombinant fusion 
protein, and minimal or no binding to the control ORF2 fusion protein. 
Study Population. The characteristics of the study population are outlined 
in Table 2, below. 33 sera samples from Mexico City were obtained from 33 
patients with the diagnosis of amebic liver abscess based on clinical 
presentation and positive serology. 14 samples were obtained from patients 
seen in San Diego, four with intestinal invasive amebic disease (3 
colitis, 1 ameboma) and 9 with the diagnosis of amebic liver abscess (one 
patient had two separate episodes of amebic liver abscess with a serum 
sample provided from each episode), all with positive serology. 36 sera 
were obtained from 36 patients in Durban; this sample group was 
specifically selected in Durban and contained sera from patients with 
amebic liver abscess (17), and sera from patients with other illnesses 
(19), some of whom had positive AGD serology. Samples were either sent 
encoded, or the blots were read by observers with no knowledge of the 
clinical history. 2 sera, an acute and 1 yr convalescent serum, were 
examined from one patient from St. Louis who contracted an amebic liver 
abscess in Mexico, five sera from healthy people in St. Louis were 
included as negative controls. 
RESULTS 
A total of 90 sera from 88 patients were examined for the presence of 
antibodies to SREHP. Of the 61 patients with the diagnosis of amebic liver 
abscess 48, or 79%, had antibodies to the SREHP fusion protein (Table 3). 
Seropositivity was higher in the samples from Mexico (30/33, 91%) than 
from Durban (11/17, 65%) or San Diego (7/10, 70%). A patient from San 
Diego with two distinct episodes of amebic liver abscess was seropositive 
in a sample taken after the second amebic liver abscess, but seronegative 
in a sample taken after the first episode. The one patient from St. Louis 
had anti-SREHP antibodies at the time of his presentation with amebic 
liver abscess, but no antibodies were present 1 year later. Only 4 
patients with intestinal amebiasis were examined in this study, all 4 had 
anti-SREHP antibodies. None of the healthy controls from St. Louis had 
anti-SREHP antibodies. 
To compare the sensitivity and specificity of Western blotting using the 
SREHP fusion protein with AGD in a highly endemic area, sera from 36 
patients from Durban were analyzed separately (Table 4). Of the 17 
patients with amebic liver abscess and positive AGD, 11 or 65% had 
anti-SREHP antibodies. Nine patients with positive AGD did not have acute 
amebic disease. Only 1 of these patients had antibody to SREHP. 
Three finding emerge from this Example. First, is the discovery that 
antibodies to SREHP are found among most patients with the diagnosis of 
amebic liver abscess. This held true for patients from two endemic areas, 
Mexico City and Durban, which exhibit differences in zymodeme distribution 
[Gathiram and Jackson, Lancet 1985; i: 719-721]. This suggests that SREHP 
is probably found on most, if not all, pathogenic strains of E. 
histolytica, and is consistent with the limited data from Northern blot 
study of axenic strains in Example 1. The inability to detect anti-SREHP 
antibodies in 21% of patients with liver abscess may represent failure of 
those individuals to make antibodies to SREHP, sensitivity problems with 
the assay, or different timing of the development of a serologic response 
to SREHP. However one cannot exclude the possibility that there may be 
strains of E. histolytica that do not possess an antigenically similar 
molecule. 
A second finding is that a recombinant E. histolytica antigen can be 
utilized in a test for invasive amebiasis. The use of the recombinant 
fusion protein offers the advantages of testing with a defined antigen, 
rather than the crude preparations of amebic lysates used in most assays, 
and could facilitate the standardization of assays for amebiasis. In 
addition a recombinant fusion protein, produced in large quantities 
without the need for a source of E. histolytica trophozoites, may be a 
cost effective way of producing antigen for mass use. This is an important 
consideration in areas endemic for amebiasis where resources for health 
services are limited. 
The most noteworthy finding is the correlation between the presence of 
antibodies to SREHP and acute invasive amebiasis. While clearly less 
sensitive (65%) in the Durban population of patients with amebic liver 
abscess (all of whom had positive AGD), detection of antibodies to SREHP 
was a much more specific indicator of acute amebic disease. Among the 19 
ill patients who did not have acute invasive amebiasis, there was only 1 
"false positive" (5%) with the SREHP test, as opposed to 9 "false 
positive" (47%) (probably true positives, representing previous infection 
with pathogenic E. histolytica strains) with AGD. This difference may 
reflect the reduced sensitivity of a single antigen test with SREHP, as 
opposed to the multiple antigen AGD test. A recent study measuring the 
serologic response to a monoclonal antibody purified E. histolytica 
antigen among patients with positive IHA serology also demonstrated a 
decreased sensitivity with an increased specificity for the single antigen 
test [Torian et al, J. Infect. Dis. 159, 794-97 (1989)]. A longitudinal 
study of patients with amebic liver abscess will be needed to determine 
what proportion of patients develop SREHP seropositivity, and whether 
SREHP seropositivity is shorter lived than that detected with the AGD, 
CIE, or IHA tests. 
TABLE 2 
______________________________________ 
Characteristics of study population 
Mexico 
Diagnosis 
City San Diego Durban St. Louis 
TOTAL 
______________________________________ 
Amebic 33 10 17 1 61 
liver 
abscess 
Amebic 4 4 
colitis 
Non- 19 19 
amebic 
disease 
Healthy 6 6 
controls 90 
______________________________________ 
TABLE 3 
______________________________________ 
Serologic response to SREHP 
anti-SREHP 
Diagnosis antibodies % positive 
______________________________________ 
Amebic liver abscess 
49/61 79 
Amebic colitis 4/4 100 
Non-amebic disease 
1/19 5 
(endemic area) 
Non-endemic controls 
0/6 0 
______________________________________ 
TABLE 4 
______________________________________ 
Comparison of anti-SREHP Western blot vs. 
AGD in Durban samples 
anti-SREHP 
Diagnosis 
AGD % positive 
antibodies 
% positive 
______________________________________ 
All sera 26/36 72 12/36 33 
Amebic 17/17 100 11/17 65 
liver 
abscess 
Non-amebic 
9/19 45 1/19 5 
disease 
______________________________________ 
Various other examples will be apparent to the person skilled in the art 
after reading the present disclosure without departing from the spirit and 
scope of the invention. It is intended that all such other examples be 
included within the scope of the appended claims.