Note: Descriptions are shown in the official language in which they were submitted.
WO 95/00849 Z 16 ~ 2 0 2 PCT/US9~/06890
RECOMBIN~NT 170 KD SUBUr~llT LECTIN OF
ENTAMOEBA HlSTOlrYTlCA AND l\/IETHODS OF USE
This invention was made, in part, with support
supplied by the U.S. Government under Contracts AI 18841
and AI 26649 awarded by the National Institutes of
Health. The U.S. Government has certain rights in this
invention.
Field of the Invention
The invention concerns the use of epitope-
bearing regions of the 170 kD subunit of Entamoeba
histolytica Gal/GalNAc adherence lecl:in which are
produced recombinantly in procaryotic systems in
diagnosis and as vaccines. Thus, the invention relates
to the determination of the presence t absence or amount
o~ antibodies raised by a subject in response to
infection by E. histolytica using these peptides and to
vaccines incorporating them. This invention also
particularly relates to reagents specific for a novel
variant of the 170 kD subunit of E. ~listolytica
Gal/GalNAc adherence lectin and to the gene (hgl3 ) which
encodes this novel subunit form, whic:h represents the
third member of the multigene family encoding this 170 kD
subunit.
Backqround Art
Entamoeba histolytica infection is extremely
common and affects an estimated 480 million individuals
annually. However, only about 10~ of these persons
develop symptoms such as colitis or liver abscess. The
low incidence of symptom occurrence is putatively due to
the existence of both pathogenic and nonpathogenic forms
of the amoeba. As of 1988, it had been established that
2 ~ 2 pcTlus94lo689n
-- 2
the subjects who eventually exhibit symptoms harbor
pathogenic "zymodemes" whlch have been classi~ied as such
on the basis of their distinctive hexokinase and
phosphoglucomutase isoenzy~e~s. The pathogenic forms are
not conveniently disting~ishable from the nonpathogenic
counterparts using mor~hogenic criteria, but there is an
almost perfect correlation between infection with a
pathogenic zymodeme and development of symptoms and
between infection with a nonpathogenic zymodeme and
failure to develop these symptoms.
It is known that E. histolytica infection is
mediated at least in part by the "Gal/GalNAc" adherence
lectin which was isolated from a pathogenic strain and
purified 500 fold by Petri, W.A., et al., J Biol Chem
(1989) 264:3007-3012. The purified "Gal/GalNAc" lectin
was shown to have a nonreduced molecular weight of 260 kD
on SDS-PAGE; after reduction with beta-mercaptoethanol,
the lectin separated into two subunits of 170 and 35 kD
MW. Further studies showed that antibodies directed to
the 170 kD subunit were capable of blocking surface
adhesion to test cells (Petri, et al. J Biol Chem ~1989)
supra). Therefore, the 170 kD subunit is believed to be
of primary importance in meditating adhesion.
In addition, the 170 kD subunit is described as
constituting an effective vaccine to prevent E.
histolytica infection in U.S. Patent 5,004,608 issued 2
April 1991.
Studies of serological cross-reactivity among
patients having symptomology characteristic of
E. histolytica pathogenic infection, including liver
abscess and colitis, showed that the adherence lectin was
recognized by all sera tested (Petri, Jr., W.A., et al.,
Am J Med Sci (1989) 296:163-165). The lectin heavy
subunit is almost universally recognized by immune sera
3S and T-cells from patients with invasive amebiasis (Petri,
WO95/0084g 21~S202 PCT~S94/06890
-- 3
et al., Infect Immun (1987) 55-2327-233l; Schain, et al.,
Infect Immun (1992) 60:2143-2146).
DNA encoding both the heavy (170 kD) and light
(35 kD) subunits have been cloned. The heavy and light
subunits are encoded by distinct mRNAs (Mann, B., et al.,
Proc Natl Acad Sci USA (1991) 88:3248-3252) and these
subunits have different amino acid compositions and amino
terminal sequences. The sequence of the cDNA encoding
the 170 kD subunit suggests it to be an integral membrane
protein with a large cysteine-rich e:~tracellular domain
and a short cytoplasmic tail (Mann, 13., et al., Proc Natl
Acad Sci USA (1991) supra; Tannich, et al., Proc Natl
Acad Sci USA (1991) 88:1849-1853). The derived amino
acid sequence of the 170 kD lectin S]lOWS that the
extracellular domain can be divided :into three regions on
the basis of amino acid composition. The amino terminal
amino acids 1-187 are relatively rich in cysteine (3.2~)
and tryptophan (2.1~). Amino acid sequence at positions
188-378 does not contain cysteine, and the amino acid
sequence at positions 379-1209 contains 10.8~ cysteine
residues. The obtention of clones encoding the heavy
chain subunit is further described in U.S. Patent
5,260,429 issued 9 November 1993, the disclosure of which
is incorporated herein by reference. In that patent,
diagnostic methods for the presence of E. histolytica
based on the polymerase chain reaction and the use of DNA
probes is described.
The heavy subunit is considered to be encoded
by a multigene family (Mann, B., et al., Parasit Today
(1991) 1:173-176). Two different heavy subunit genes,
hgll and hgl2, have been sequenced by separate
laboratories. While hgl2 was isolated from an HM-l:IMSS
cDNA library in its entirety (Tannich, E. et al. Proc
Natl Acad Sci USA (1991) 88:1849-1853), hgll was isolated
in part from an H-302:NIH cDNA library and in part by PCR
amplification of the gene from the H~-l:IMSS genome
W095/00~9 216 ~ 2 0 2 PCT~S94/06890
- 4 - ~
(Mann, B.J. et al . Proc Natl A~d Sci USA (1991) 88:3248-
3252). As the amino acid sequence of these two genes is
87.6~ identical (Mann, ~ ; et al . Parasit Today (1991)
7:173-176), the differences could be explained by strain
variation alone. The presence of multiple bands
hybridizing to an hgl probe on Southern blots, however,
in consistent with the existence of a 170 kDa subunit
gene family (Tannich, E. et al. Proc Natl Acad Sci USA
(1991) 88:1849-1853).
Monoclonal antibodies specifically
immunoreactive with various epitope-bearing regions of
the 170 kD heavy chain subunit have also been disclosed
in U.S. Patent 5,272,058 issued 21 December 1993, the
disclosure of which is incorporated herein by reference
in its entirety. This application also describes use of
these antibodies to detect the 170 kD heavy chain and the
use of the 170 kD subunit to detect antibodies in serum
or other biological samples. The experimental work
described utilizes the native protein. Further
characterization of these antibodies is described in a
publication by Mann, B.J., et al., Infect Immun (1993)
61:1772-1778 also incorporated herein by reference.
Various ~mmllnoassay techniques have been used
to diagnose E. hi5tolytica infection. ELISA techniques
have been used to detect the presence or absence of
E. histolytica antigens both in stool specimens and in
sera, though these tests do not seem to distinguish
between the pathogenic and nonpathogenic strains. In a
semlnAl article, Root, et al., Arch Invest Med (Mex)
(1978) 9: Supplement 1:203, described the use of ELISA
techniques for the detection of amoebic antigen in stool
specimens using rabbit polyclonal antiserum, and various
forms of this procedure have been used, some in
conjunction with microscopic studies. Palacios et al.,
Arch Invest Med (Mex) (1978) 9: Supplement 1:203; ~n~
et al., Trans Rov Soc Tro~ Med Hyq (1984) 78:593; Grundy,
WOg5/00849 218~20~ PCT~S94/06890
Trans RoY Soc Trop Med Hyq (1982) 76:396; Ungar, Am J
Trop Med Hyq (1985) 34:465. These studies on stool
specimens and on other biological fluids are summarized
in Amebiasis: Human Infection bY Entamoeba HistolYtica,
J. Ravdin, ed. (1988) Wiley Medical Publishing, pp. 646-
64~.
Conversely, amebic serology is also a critical
component in the diagnosis of invasive amebiasis. One
approach utilizes conventional serologic tests, such as
the indirect hemagglutinin test. These tests are very
sensitive but seropositivity is persistent for years
(Krupp, I.M., Am J Trop Med Hyq (1970) 19:57-62; Lobel,
H.O. et al., Ann Rev Microbiol (1978) 32:379-347). Thus,
healthy subjects may give positive responses to the
asæay, creating an undesirable high l~ackground. Similar
problems with false positives are found in using
immunoassay tests involving a monoclonal antibody and
purified native 170 kD protein (Ravd:in, J.I., et al., J
Infect Dis (1990) 162:768-772.)
Recombinant E. histolytica proteins other than
the 170 kD subunit have been use,d as the basis for
serological tests. Western blotting using a recombinant
form of the "52 kD serine-rich prote:in" was highly
specific for invasive disease and had a higher predictive
value (92 vs. 65~) than an agar gel diffusion test for
diagnosis of acute amebiasis (Stanley, Jr., S.L., et al.,
Proc Natl Acad Sci U.S.A. (1990) 87:4976-4980; Stanley,
Jr., S.L., et al., JAMA (1991) 266:1984-1986). However,
the overall sensitivity was lower than for the
conventional agar gel test (82~ vs. 90-100~).
Thus, there r~m~; n~ a need for serological
tests which will provide optimum sensitivity while
minimizing the number of false positives retained. The
present invention provides such a test by utilizing, as
antigen, epitope-bearing portions of the 170 kD subunit
W095/00849 5202 i 6 - PCT~S94/06890
of the adherence lectin produced recombinantly in
procaryotic systems.
It is particularly advantageous to use
recombinantly produced, nonglycosylated peptides or
5 proteins in this a~say since these peptides are easily
and efficiently obtained and are easily standardized.
Furthermore, since selected portions of the lectin heavy
chain subunit can be produced, epitopes characteristic of
the pathogenic or nonpathogenic forms of E. histolytica
can be produced and used to distinguish these forms in
the assays. Subsequent to the invention herein, a report
of ;mmllnoreactivity of recombinant 170 kd lectin with
immune sera was published by Zhang, Y, et al. J. Clin
Micro-immunol (1992) 2788-2792. Applicants incorporate
15 by reference their own publication: Mann, B.J et al.
Infect and Immun (1993) 61: 1772-1778.
Similarly, although it is known that the 170 kD
subunit may be used as a vaccine as described in the
above-referenced U.S. Patent 5,004,608, recombinantly
produced forms of the 170 kD subunit, specifically those
obtained from procaryotic cells that lack glycosylation
may offer advantages in reproducibility of product and in
ease of preparation of subunit vaccines. The present
invention is directed to this desirable result.
Disclosure of the Invention
The invention provides diagnostic tests which
permit the assessment of patients for invasive E.
hi~tolytica infection and vaccines for prevention of
infection. The invention also provides a novel third
variant of the 170 kD subunit of the Gal/GalNAc adherence
lectin and a gene (hgl3 ) which encodes this novel
protein. Accordingly, the diagnostic tests of the
invention are based on the genetic sequences of all three
35 variants of the 170 kD subunit of the Gal/GalNAc
W095/00~9 216S2~ PCT~S94/06890
, . . .
-- 7
adherence lectin which are encoded by three different
genes in a multigene family.
Pathogenic and nonpathogenic strains can be
distinguished by use of the invention diagnostic method,
if desired. The tests use, as antigen, an epitope-
bearing portion of the 170 kD subunit of the Gal/GalNAc
adherence lectin recombinantly produced in procaryotic
systems. Despite the absence of glycosylation from such
portions and despite the lack of post-translational
modifications characteristic of the native protein or
peptide, the recombinantly produced proteins are
effective antigens in these assays.
Thus, in one aspect, the invention is directed
to a method to detect the presence or absence of
antibodies immunoreactive with pathogenic and/or
nonpathogenic E. histolytica in a biological sample which
method comprises contacting the fluid with an epitope-
bearing portion of the 170 kD heavy chain of the
Gal/GalNAc adherence lectin wherein the lectin is
nonglycosylated and in a form obtainable from procaryotic
cells. If distinction between antibodies to the
pathogenic and nonpathogenic forms i~ desired, the
portion may be chosen so as to be characteristic of the
pathogenic or nonpathogenic form. Alternatively, the
assay may be conducted as a competition assay using MAbs
with such characteristics. The contacting is conducted
under conditions where the epitope-bearing portion forms
complexes with any antibodies present in the biological
fluid which are lmmllnoreactive with an epitope on the
portion. The presence, absence or amount of such
complexes is then assessed, either directly or in a
competition format, as a measure of the antibody
contained in the biological sample. The invention is
also directed to materials and kits suitable for
performing the methods of the inventi.on.
W095/00849 PCT~S94/06890
6 ~ 20`~ - 8 -
In a second aspect, the invention is directed
to methods to prevent E. histolytica infection using
vaccines containing, as active ingredient, epitope-
bearing portions of the 170 kD subunit produced
recombinantly in procaryotic syste~s, as described above.
The invention is also directed;~o vaccines containing
this active ingredient. ~
In other aspects, t~e invention is directed to
epitope-bearing portions of the 170 kD subunit produced
recombinantly in procaryotic systems and thus in a form
characteristic of such production. One characteristic is
lack of glycosylation; in addition, secondary structure
of proteins produced by procaryotic hosts differs from
that of proteins produced by the natural source.
In yet another aspect, the invention is
directed to a DNA in purified and isolated form which
consists essentially of a DNA encoding the 170 kd heavy
chain subunit of pathogenic E. histolytica Gal/GalNAc
adherence lectin, which subunit is encoded by the hgl3
20 gene for which the nucleotide sequence and deduced amino
acid sequence are shown in Figure 4. In further aspects,
the invention is directed to both nucleic acid and
immunological reagents which are enabled by the discovery
of the hgl3 gene, reagents which are specific for each of
25 the hgll, hgl2 or hgl3 genes, as well as reagents which
detect common regions of all three hgl genes or their
nucleic acid or protein products. For example,
oligonucleotide probes specific for any one of these
three genes or for a sequence common to all three genes
may be identified by one of ordinary skill in the art,
using conventional nucleic acid probe design principles,
by comparisons of the three DNA sequences for these
genes. See Example 6.
In still further aspects, the invention is directed
3 5 to a method to detect the presence, absence, or amount of
a pathogenic or nonpathogenic form of Entamoeba
WO95/00~9 PCT~S94/06890
~ ?l~t~
g
histolytica, where E. histolytica has both pathogenic and
nonpathogenic forms, in a biological sample, which method
comprises contacting the sample with a monoclonal
antibody immunospecific for an epitope of the 170 kd
subunit of Gal/GalNAc lectin unique to the pathogenic or
to the nonpathogenic form, or shared by the pathogenic
and nonpathogenic forms of E. histolytica, to form an
immunocomplex when the pathogenic and/or nonpathogenic
form is present, and detecting the presence, absence or
amount of the immunocomplex. In this method, the epitope
is selected to be specific for one of 170 kD subunits
encoded by the hgll, hgl2 or hgl3 genes, or for a common
region of the subunits from all three hgl genes.
In another aspect, the invention is directed to a method
to determine the presence, absence or amount of
antibodies specifically immunoreactive with the
Gal/GalNAc lectin derived from E. histolytica, which
method comprises contacting a biological sample with the
Gal/GalNAc lectin or the 170 kd subunit thereof in
purified and isolated form, under conditions wherein
antibodies ~mml~nospecific for said lectin or subunit will
forma complex, and detecting the presence, absence or
amount of the complex, wherein the purified and isolated
Gal/GalNAc lectin or subunit is derived from either a
pathogenic or nonpathogenic form of ~. histolytica, and
is a 170 kD subunit encoded by one of the hgll, hgl2 or
hgl3 genes. Detailed descriptions of these and related
methods for detecting pathogenic or nonpathogenic forms
of E. histolytica and antibodies specifically
immunoreactive with the Gal/GalNAc lectin derived from E.
histolytica, as well as reagent kits suitable for the
conduct of such methods, are disclosed in U.S. Patent
5,272,058, the entire disclosure of which is incorporated
herein by reference.
W O 9S/00849 PCT~US94/06890
al6~2~ ~
-- 10
Brief DescriPtion of the Drawinqs
Figure lA shows t~e DNA and amino acid sequence
deduced from the nucl~o~lde sequence corresponding to the
170 kD heavy chain o` t:~`e adherence lectin from
5 pathogenic strain HMI-IMSS, designated hgll .
Figure lB shows the deduced amino acid sequence
of hgll with the amino-terminal amino acid of the mature
protein designated as amino acid number 1.
Figure 2A is a diagram of the construction of
expression vectors for recombinant production of
specified portions of the 170 kD subunit; Figure 2B shows
the pattern of deletion mutants.
Figure 3 is a diagram of the location of human
B cell epitopes and pathogenic-specific epitopes on the
15 170 kD heavy chain.
Figure 4A shows the DNA and amino acid sequence
deduced from the nucleotide sequence corresponding to the
170 kD heavy chain of the adherence lectin from
pathogenic strain HMl:IMSS, designated hgl3.
Figure 4B shows the deduced amino acid sequence
of hgl3 with the amino-terminal amino acid of the mature
protein designated as amino acid number 1. The putative
signal sequence and transmembrane domains are overlined
and underlined respectively. Conserved cysteine residues
25 (-) and potential sites of glycosylation (*) are
indicated.
Figure 5 shows in schematic form a comparison
of amino acid sequences of three heavy subunit genes.
The top diagram represents a schematic representation of
30 a heavy subunit gene. Starting at the amino terminus,
regions include the cysteine/tryptophan (C-W) rich
domain, the cysteine-free (C-free) domain, the cysteine-
rich (C-rich) domain, and the putative transmembrane (TM)
sequence and cytosolic domains (Mann, B . J. et al . Parasit
35 TodaY (1991) 7: 173 -176) . Amino acid sequence comparisons
of hgll, hgl2 and hgl3 are shown. Upright lines indicate
WOg5/00849 PCT~S94/06890
2~ 6 ~;2Q~
-- 11 -- .
nonconservative amino acid substitutions in the amino
acid sequence of the second gene as compared to the first
gene listed to the right. Downward arrowheads indicate a
deletion while upright arrowheads indicate an insertion.
The number of residues inserted or deleted are listed
below the arrowheads and the total percent amino acid
se~uence identity is listed at right.
Modes of Carryinq Out the Invention
The invention provides methods and materials
which are useful in assays to detect antibodies directed
to pathogenic and/or nonpathogenic forms of E.
histolytica and in vaccines. The diagnostic assays can
be conducted on biological samples derived from subjects
at risk for infection or suspected of being infected.
The assays can be designed to distinyuish pathogenic from
nonpathogenic forms of the amoeba if desired. The
vaccines are administered to subjects at risk for amebic
infections.
The assays of the invention. rely on the ability
of an epitope-bearing portion of the 170 kD subunit
produced recombinantly in procaryotic cultures to
immunoreact with antibodies contained in biological
samples obtained from individuals who have been infected
with E. hi~tolytica. Bven though the relevant peptide or
protein is produced in a procaryotic system, and is thus
not glycosylated or processed after translation in a
manner corresponding to the native protein, the epitope-
bearing portions thus prepared are useful antigens in
immunoassays performed on samples prepared from
biological fluids, cells, tissues or organs, or their
diluted or fractionated forms. Similarly, these peptides
are also immunogenic.
The use of recombinant forms of the antigen or
offers advantages of cost-effective, reliable production
of pure antigen, thus assuring the uniformity of the
W095t00849 PCT~S94/06890
~ fi5?~Q?~
j - 12 -
assay material`s. Recombinant production in bacteria is a
particularly efficient and useful method. It is
surprising that such procaryotic systems can produce
successful antigens and immunogens, since the peptides
produced are not processed in a manner analogous to the
reactive native forms.
Furthermore, recombinant production facilitates
the preparation of specific epitopes, thus providing a
means for detecting antibodies specifically
immunoreactive with pathogenic or nonpathogenic ~orms of
the amoeba, as well as offering the opportunity to
provide subunit vaccines.
Thus, the invention is directed to methods to
detect antibodies in biological samples and to immunize
subjects at risk using these recombinantly produced
epitope-bearing portions as antigens or immunogens as
well as to the recombinantly produced peptides
themselves and to materials useful in performing the
assays and in ~m; n; stering the vaccines.
Definitions
The diagnostic assays may be designed to
distinguish antibodies raised against nonpathogenic or
pathogenic forms of the amoeba. "Pathogenic forms" of E.
histolytica refers to those forms which are invasive and
which result in symptomology to infected subjects.
"Nonpathogenic forms" refers to those forms which may be
harbored asymptomatically by carriers.
The assays and vaccines of the invention
utilize an epitope-bearing portion of the 170 kD subunit
of the Gal/GalNAc lectin. "Gal/GalNAc lectin" refers to
glycoprotein found on the surface of E. histolytica which
mediates the adherence of the amoeba to target cells, and
which mediation is inhibited by galactose or N-
acetylgalactosamine. The Gal/GalNAc lectin refersspecifically to the lectin reported and isolated by
WOg5/00849' PCT~S94/06890
216l~2~21'
- 13 -
Petri, et al. (supra) from the pathogenic strain HMI-
IMSS, and to the corresponding lectin found in other
strains of E. histolytica. The "170 kD subunit" refers
to the large subunit, upon reduction of the Gal/GalNAc
lectin, such as that obtained by Petri, et al. and shown
in Figure 1 as well as to its corresponding counterparts
in other strains.
Diagnostic Assays
With respect to the diagnostic assays of the
invention, the complete 170 kD antigen or an epitope-
bearing portion thereof can be used in the assays. Such
epitope-bearing portions can be selec:ted as
characteristic of pathogens or nonpat:hogens or common to
both.
As shown hereinbelow, the portion of the 170 kD
protein which contains epitopes for all monoclonal
antibodies prepared against the lecti.n is found at amino
acid positions 596-1138. There appears to be an epitope
characteristic of pathogens between each of amino acid
positions 596-818, 1082-1138, and 1033-1082. Positions
895-998 contain epitopes which are shared by pathogens
and nonpathogens as well as epitopes characteristic of
pathogenic strains. Thus, to utilize fragments of the
recombinantly produced protein for detection of
antibodies, a peptide representing positions 596-818,
1033-1082 or 1082-1138 may be used to detect antibodies
raised against pathogens by hosts in general; however,
the epitope at positions 596-816 is not recognized by
human antisera. Mixtures of these peptides could also be
used. Alternatively, longer forms of the antigen can be
used by selecting the appropriate positions depending on
whether pathogenic and nonpathogenic amoebae are to be
distinguished.
As shown in Bxample 4, below, epitope-bearing
~ portions relevant for human testing include portions 2-
WO9~/00849 ~ PCT~S94/06890
~65 2~ ~ - 14 -
482, 1082-1138, 1032-1082 and 894-998. Only the portions
represented by 1082-1138 and 1032-1082 appears specific
for antibodies against pathogenic ameba. These epitope-
bearing portions may be used as sing~e peptides, as
uni~uely lectin-derived portions o chimeric proteins, as
mixtures of peptides or of such ~roteins, or as portions
of a single, multiple-epitope-~earing protein.
Procedures for preparing recombinant peptide proteins
containing only a single epitope-bearing portion
identified above, or multiples of such portions
(including tandem repeats) are well understood in the
art.
The assays are designed to detect antibodies in
biological samples which are ''immunospecific~l or
"immunoreactive" with respect to the epitope-bearing
portion -- i.e. with respect to at least one epitope
contained in this portion. As used herein,
"immunospecific" or "immunoreactive" with respect to a
specified target means that the antibody thus described
binds that target with significantly higher affinity than
that with which it binds to alternate haptens. The
degree of specificity required may vary with
circumstances, but typically an antibody immunospecific
for a designated target will bind to that target with an
affinity which is at least one or two, or preferably
several orders or magnitude greater than with which it
binds alternate haptens.
The assays can be performed in a wide variety
of protocols depending on the nature of the sample, the
circumstances of performing the assays, and the
particular design chosen by the clinician. The
biological sample is prepared in a m~nner standard for
the conduct of immunoassays; such preparation may involve
dilution if the sample is a biological fluid,
fractionation if the sample is derived from a tissue or
organ, or other standard preparation procedures which are
WO95/00849 2 ~ ff 5 2 ~} 2 PCT~S94/06890
~, ', .
- 15 -
known in the art. Thus, "biological sample" refers to
the sample actually used in the assay which is derived
from a fluid, cell, tissue or organ of a subject and
prepared for use in the assay using the standard
techniques. Normally, plasma or serum is the source of
biological sample in these assays.
The assays may be conductecl in a competition
format employing a specific binding partner for the
epitope-bearing portion. As used herein, "specific
binding partner" refers to a substance which is capable
of specific binding to a targeted substance, such as the
epitope-bearing portion of the 170 kD subunit. In
general, such a specific binding part:ner will be an
antibody, but any alterative substance capable of such
specific binding, such as a receptor, enzyme or
arbitrarily designed chemical compound might also be
used. In such contexts, "antibody" refers not only to
immunoglobulin per se, but also to fragments of
immunoglobulin which retain the immunospecificity of the
complete molecule. Examples of such fragments are well
known in the art, and include, for example, Fab, Fab',
and F(ab')2 fragments. The term "antibody" also includes
not only native forms of immunoglobulin, but forms of the
immunoglobulin which have been modified, as techniques
become available in the art, to confer desired properties
without altering the ;mmllnospecificity. For example, the
formation of chimeric antibodies derived from two species
is becoming more practical. In short, "antibodies"
referR to any component of or derived form of an
immuno~lobulin which retains the immunospecificity of the
immunoglobulin per se.
A particularly useful form of specific binding
reagents useful in the assay methods of the invention is
as monoclonal antibodies. Three categories of monoclonal
antibodies have been prepared to the 170 kD subunit. One
~ category of antibody is immunospecific for epitopes
t "~ ~
W095/00849 ; PCT~S94/06890
2,16~2~ . ~
- 16 -
"unique" to pathogenic forms. These antibodies are
capable, therefore, of immunorea~tion to a significant
extent only with the pathogenic forms of the amoeba or to
the 170 kD subunit of lectin isolated from pathogenic
forms. A second set of monoclonal antibodies is
immunoreactive with epitopes which are "unique" to
nonpathogenic forms. Thus, these antibodies are
immunoreactive to a substantial degree only with the
nonpathogenic amoeba or their lectins and not to the
pathogenic forms. A third category of monoclonal
antibodies is immunoreactive with epitopes common to
pathogenic and nonpathogenic forms and these antibodies
are capable of immunoreaction with the subunit or with
the amoeba regardless of pathogenicity.
With respect to the monoclonal antibodies
described herein, those immunoreactive with epitopes 1
and 2 of the 170 kD subunit isolated from the pathogenic-
strain exemplified are capable of reacting, also, with
the corresponding epitopes on nonpathogens. On the other
hand, those immunoreactive with epitopes 3-6 are capable
of immunoreaction only with the 170 kD subunit of
pathogenic strains. By applying the techniques for
isolation of the pathogenic 170 kD subunit to amoeba
which are nonpathogenic, a 170 kD subunit can be obtained
for ;mmnn;zation protocols which permit the analogous
preparation of MAbs ;mml~noreactive with counterpart
epitopes 3-6 in the nonpathogenic forms.
Of course, with respect to antibodies found in
the biological sample, in general, these will be found in
the form of immunoglobulins. However, pretreatment of
the sample with an enzyme, for example, to remove the Fc
portions of the antibodies contained therein, does not
debilitate the sample with respect to its ability to
respond to the assay.
WO 95/00~9 2 I 6 ~ 2 0 2 PCT~S94/06890
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Assay Procedure
For the conduct of the assays of the invention,
in general, the biological sample is contacted with the
epitope-bearing portion used as an antigen in the
immunoassay. The presence, absence or amount of the
resulting complex formed between any antibody present in
the sample and the epitope-bearing portion is measured
directly or competitively.
As is well understood in the art, once the
biological sample is prepared, there is a multiplicity of
alternative protocols for conduct of the actual assay.
In one rather straightforward protocol, the epitope-
bearing portion provided as antigen may be coupled to a
solid support, either by adsorption or by covalent
linkage, and treated with the biological sample. The
ability of any antibodies in the sample to bind to
coupled antigen is then determined.
This ability may be determined in a "direct"
form of the assay in which the level of complex formation
by ~he antibody is measured directly. In one
particularly convenient format of this approach, the
antigen may be supplied as a band on a polyvinylidene
difluoride (PVDF) and contacted with the biological
sample; any resulting complexes ~ormed with antibody on
the PVDF membrane are then detected as described above
for Western blot procedure. This protocol is
substantially a Western Blot procedure. Alternatively,
microtiter plates or other suitable solid supports may be
used. The binding of antibody to the antigen coupled to
support can then be detected as described above for
Western blot procedure using conventional techniques
generally involving secondary labeling using, for
example, antibodies to the species from which the
biological sample is derived. Such labels may include
radioisotopes, fluorescent tags, enzyme labels and the
~ like, as is conventionally understood.
W095/00849 PCT~S94/06890
~6$~~ 18 -
The assay may also be formatted as a
competition assay wherein the an~igen coupled to solid
support is treated not only with the biological sample
but also with competing specific binding partner
immunospecific for at least one epitope contained in the
antigen. The competing bindi~g partner is preferably an
antibody. The competing antibody may be polyclonal or
monoclonal and may itself be labeled or may be capable of
being labeled in a secondary reaction. In a typical
conduct of such a competitive test, a competitive
specific binding partner for the antigen is generally
supplied in labeled form and the success of the
competition from the biological sample is measured as a
reduction in the amount of label bound in the resulting
complex or increased levels of label rem~;n;ng in the
supernatant. If monoclonal antibodies are used, the
assay can readily be made specific for pathogenic or
nonpathogenic reacting antibodies, if desired, by
choosing antibodies of the appropriate specificity.
Thus, if the assay is to be made specific for antibodies
raised against pathogenic forms of E. histolytica, the
competition will be provided by a monoclonal antibody
specific for an epitope characteristic of pathogenic
strains.
Another manner in which the assay may be made
specific for pathogenic or nonpathogenic forms is in the
choice of the epitope-bearing portion. If antibodies
specific to the pathogens are to be detected, an epitope-
bearing portion is chosen which bears only epitopes
characteristic of pathogenic strains. Conversely,
antibodies immunospecific for nonpathogens can be
conducted by utilizing as antigen only portions of the
subunit which contain epitopes characteristic of
nonpathogens. Where characterization as pathogen or
nonpathogen-specific antibodies is unnecessary, antigen
WOg5/00849 ~1652~ PCT~S94/06890
-- 19
containing both such epitopes or epitopes shared by both
forms may be used.
Additional ways to distinguish between
antibodies immunospecific for pathogens and for
nonpathogens employ competition assays with monoclonal
antibodies of such specificities, as described above.
Alternatively, the biological sample can be
coupled to solid support and the desi.red epitope-bearing
portion added under conditions where a complex can be
formed to the epitope-bearing portion, which is then used
to treat the support. Subsequent treatment of the
support with antibodies known to immunoreact with the
antigen can then be used to detect whether antigen has
been bound.
~hus, the biological sample to be tested is
contacted with the epitope-bearing portion, which is
derived either from a pathogenic or nonpathogenic from
one both of E. hi5tolytica so that a complex is formed.
The complex is then detected by suita.ble labeling, either
by supplying the antigen in labeled from, or by a
secondary labeling process which forms a ternary complex.
The reaction is preferably conducted using a solid phase
to detect the formation of the complex attached to solid
support, or the complex can be precipitated using
conventional precipitating agents such as polyethylene
glycol.
In a more complex form of the assay,
competitive assays, can be used wherein the biological
sample, preferably serum or plasma, provides the cold
antibody to compete with a specific binding partner, such
as a labeled monoclonal antibody preparation known to
bind specifically to an epitope unique to the Gal/GalNAc
lectin or its 170 kD subunit of a pathogenic or
nonpathogenic from. In this embodiment, the binding to
labeled specific monoclonal antibody is conducted in the
presence and absence of biological sample, and the
WO95/00849 . PCT~S94/06890
2 ~ 6S ~ 0 2 - 20 -
diminution of labeling of the resulting complex in the
presence of sample is used ;as an index to determine the
level of competing a~i~ody.
Kits suit~ble for the conduct of these methods
include the appropriate labeled antigen or antibody
reagents and instructions for conducting the test. The
kit may include the antigen coupled to solid support as
well as additional reagents.
Methods of Protection and Vaccines
The recombinant 170 kD subunit or an epitope-
bearing portion thereof may be used as active ingredient.
Preferred regions include positions 482-1138, 596-1138,
885-998, 1033-1082 and 1082-1138.
The 170 kD subunit or its epitope-bearing
regions may also be produced recombinantly in procaryotic
cells for the formulation of vaccines. The recombinantly
produced 170 kD protein or an epitope-bearing region
thereof can be used as an active ingredient in vaccines
for prevention of E. histolytica infection in subjects
who are risk for such condition. Sufficiently large
portions of the 170 kD protein can be used per se; if
only small regions of the molecules for example
containing 20 amino acids or less or to be used, it may
advantageous to couple the peptide to a neutral carrier
to enhance its ;~l1nogenicity. Such coupling techniques
are well known in the art, and include standard chemical
coupling techniques optionally effected through linker
moieties such as those available from Pierce Chemical
Company, Rockford, Illinois. Suitable carriers may
include, for example, keyhole limpid hemocyanin (KLH) E.
coli pilin protein k99, BSA, or the VP6 protein of
rotavirus. Another approach employs production of fusion
proteins which include the epitope-bearing regions fused
to additional amino acid sequence. In addition, because
of the ease with which recombinant materials can be
wo gs/oo~g ~ ~5 2~2 PCTN994/06890
21
manipulated, the epitope-bearing region may be included
in multiple copies in a single molecule, or several
epitope-bearing regions can be "mixed and matched" in a
single molecule.
The active ingredient, or mixture of active
ingredients, in the vaccine is formulated using standard
formulation for administration of proteins or peptides
and the compositions may include an immunostimulant or
adjuvant such as complete Freund's adjuvant, aluminum
hydroxide, liposomes, ISCOMs, and the like. General
methods to prepare vaccines are described in Reminqtons's
Pharmaceutical Science; Mack Publish:ing Company Easton,
PA (latest edition). The compositions contain an
effective amount of the active ingredient peptide or
peptides together with a suitable amount of carrier
vehicle, including, if desired, preservatives, buffers,
and the like. Other descriptions of vaccine formulations
are found in "New Trends and Developn~ents in Vaccines",
Voller, A., et al., University Park Press, Baltimore,
Maryland (1978).
The vaccines are administered as is generally
understood in the art. Ordinarily, administration is
systemic through injection; however, other effective
means of administration are included. With suitable
formulation, for example, peptide vaccines may be
administered across the mucus membrane using penetrants
such as bile salts or fusidic acids in combination,
usually, with a surfactant. Transcutaneous means for
administering peptides are also known. Oral formulations
can also be used. Dosage levels depend on the mode of
administration, the nature of the suhject, and the nature
of carrier/adjuvant formulation. Typical amounts of
protein are in the range of .01 ~g-1 mg/kg. However,
this is an arbitrary range which is h.ighly dependent on
the factors cited above. In general, multiple
W095/00849 ~~ PCT~S94/06890
~6S ~ 22 -
administrations in standard immunization protocols are
preferred; such protocols are standard in the art.
A preferred epitope-bearing region of the 170
kD subunit is that represented by amino acids 482-1138
which includes the cysteine-rich domain. This region i8
encoded by nucleotides 1~92-3460 shown in Figure 1
herein. Preferred regions include those bearing epitopes
which are specific for antibodies against pathogenic
amoeba -- i.e., regions 1082-1138 and 1032-1082.
However, the epitope-bearing region at positions 894-998
may also be used. For regions of this length, production
of peptides with multiple copies of the epitope-bearing
regions is particularly advantageous.
Production of Recombinant E~ito~e-bearinq Portions
The epitope-bearing portions of the 170 kD
subunit can be conveniently prepared in a variety of
procaryotic systems using control sequences and hosts
ordinarily available in the art. The portions may be
provided as fusion proteins or as mature proteins and may
be produced intracellularly or secreted. Techniques for
constructing expression systems to effect all of these
outcomes is well understood in the art. If the epitope-
bearing portion is secreted, the medium can be used
directly in the assay to provide the antigen, or the
antigen can be recovered from the medium and further
purified if desired. If the protein is produced
intracellularly, lysates of cultured cells may be used
directly or the protein may be recovered and further
purified. In the Examples below, the epitope-bearing
portion is provided as a fusion protein using the
commercially available expression vector pGEX.
Alternative constructions and alternative hosts can also
be used as is understood in the art.
Reaqents and assaYs for a novel 170 kD lectin subunit
W095/00849~ ~ 2 PCT~594/06890
- 23 -
To determine the existence and complexity of
the 170 kDa subunit gene family, hgl, an amebic genomic
library in lambda phage was hybridized with DNA fragments
from the 5' or 3' ends of hgll. Termini from three
distinct heavy subunit genes were identified including
hgll, hgl2, and a third, ~nreported yene designated hgl3.
The open reading frame of hgl3 was sequenced in its
entirety (Figure 4A). Nonstringent hybridization of a
genomic Southern blot with heavy sub~mit specific DNA
labeled only those bands predicted by hgll-3. The amino
acid sequence of hgl3 (Figure 4B) was 95.2~ identical to
hgll and 89. 4~ identical to hgl2. A].l 97 cysteine
residues present in the heavy subunit were conserved in
hgll-3. Analysis of amebic RNA showed that all three
heavy subunit genes were expressed in the amebae and that
hgl message became less abundant as t:he amebae entered a
stationary growth phase.
Accordingly, the present invention provides
both nucleic acid and immunological reagents specific for
170 kDa subunits encoded by each of t:he hgll, hgl2 or
hgl3 genes, as well as reagents which detect common
regions of all three hgl genes and their nucleic acid or
protein products. For example, oligonucleotide probes
specific for any one of these three genes may be
identified by one of ordinary skill in the art, using
conventional nucleic acid probe design principles, by
comparisons of the three DNA sequences for these genesj
which sequences are disclosed in Figure lA and Figure 4A
for hgll and hgl3, respectively, and for hgl2, in
Tannich, E. et al. Proc Natl Acad Sci USA (1991) 88:1849-
1853, the entire disclosure of which is hereby
incorporated herein by reference. Example 6 illustrates
the use of oligonucleotide probes specific for each of
the three hgl genes, for determining the level of
expression of RNA from each gene using Northern blot
analyses. Other methods of using hg7.-specific nucleic
W095/00849 PCT~S94/06890
p ~6S ~ 24 -
acids for diagnostic purposes, for pathogenic and/or
nonpathogenic forms of E. histolytica, are described in
U.S. Patent 5,260,429, the entire disclosure of which is
incorporated herein by reference.
The following Examples are intended to
illustrate but not to limit t~e invention.
.~
Exa~nPle
Construction of Expression Vectors
The 170 kD subunit of the galactose lectin is
encoded by at least two genes. The DNA used for all of
the constructions described herein encodes the 170 kD
lectin designated hgll (Fig. lA) . The nucleotide
position designations refer to the numbering in Figure
lA.
The DNA sequence encoding hgll was expressed in
three portions:
fragment C (nucleotides 46-1833) included the
cysteine- and tryptophan-rich region, the cysteine-free
region, and 277 amino acids of the cysteine-rich domain,
i.e. amino acid residues 2-596;
fragment A (nucleotides 1492-3460) encoded the
majority of the cysteine-rich domain, i.e. amino acid
residues 482-1138;
fragment B (3461-3892) included 70 amino acids
of the cysteine-rich domain, the putative membrane-
spanning region, and the cytoplasmic tail, i.e. amino
acid residues 1139-1276.
See Fig. 2B.
Each of these three fragments was inserted in
frame by ligation into pGEX2T or pGEX3X to obtain these
proteins as GST fusions. A diagram of the vectors
constructed is shown in Figure 2A.
Fragment C was produced by PCR amplification.
Primers were designed so that a BamHI site was added to
the 5' end and an EcoRI site was added to the 3' end
wo 95/0084g 21 f~520~ PCT/USg4/06890
- 25 -
during the PCR process. The PCR product, fragment C, was
then digested with restriction enzymes BamHI and EcoRI,
purified, and ligated into similarly digested pGEX3X.
Fragments A and B were produced by digestion with EcoRI
from plasmid clones (Mann, BJ et al. Proc Natl Acad Sci
USA (1991) 88:3248-3252) and ligated into pGEX2T that had
been digested with EcoRI. In the pGEX expression system
a recombinant protein is expressed as a fusion protein
with glutathione S-transferase (GST) from Schistosoma
japonicum and is under the control of the tac promoter.
The tac promoter is inducible by IPTG-. The construction
of ~he vectors and subsequent expression is further
described in Mann, BJ et al. Infec and Immun (1993)
61:1772-1778, referenced above, and incorporated herein
by reference.
Expression in the correct reading frame was
verified for all constructs by sequencing and Western
immunoblot analysis by testing for reactivity with anti-
adhesion antisera (data not shown). Expression of the
hgll fusion proteins was shown to be inducible by IPTG.
The GST protein produced from the original pGEX2T did not
react with the anti-adhesion sera. The GST portion of
the fusion protein has a molecular mass of 27.5 kD.
Exam~le 2
Production of Recombinant Protein
The four vectors described above, as well as
the host vector were transfected into competent E. coli
hosts and expression of the genes encoding the fusion
proteins was effected by induction with IPTG. Production
of the fusion proteins was determined by Western blot
SDS-PAGE analysis of the lysates.
Exam~le 3
ReactivitY of Recombinant 170 kD Subunit
Fusion Proteins with MAbs
W095/00~9 , PCT~S94/06890
2 i6S 2~ 2 - 26 -
Induced cultures containing bacterial strains
expressing hgll fragment A, B, or C were harvested, lysed
in sample bu~fer, and applied to an SDS-polyacrylamide
gel. After electroph~esis, the proteins were
transferred to Immobilon and incubated with anti-170-kD
MAbs, specific for seven different epitopes.
Characteristics of the individual MAbs are shown in Table
1. It will be noted that all the known epitopes are in
the region of amino acids 596-1138.
TABLE 1. 'h~ t~ of mnnnr~ antibodies directed against
the galactose a&esion 170 kD subunit
Epitope # Decign~ti~n Isotypel Adherencel CytotoAicity2 C5b9 Rrcict:~nre3 P4 NP4 LocationS
3F4 IgG1 Tnrre~crc Decreases No effect + + 895-998
2 8A3 IgG1 Increases No effect Decreases + + 895-998
3 7F4 IgG2b No effect No effect Decreases + - 1082-1138
4 8C12 IgGl Inhibits Inhibits Decreases + - 895-998
20 5 lG7 IgG2b Inhibits Inhibits Decreases + - 596-818
6 H85 IgG2b Inhibits Inhibits Blocks + - 1033-1082
7 3D12 IgG1 No effect Not tested Blocks + 895-998
1Adherence was assayed by the1~nding of Chinese hamster ovary (CHO) cells to E. histolytica
2 5 ~lU~oZvi~ and by binding of I labeled purified colonic mucins to llu~hozvil~,i,. Petri, W.A.
Jr., et al., J Immunol (1990) 144:4803-4809.
25~he assay for ~iylOluAiCily was CHO cell killing by E. histolytica l~ù~hozvit~,s as Illed~.~,cd by
Cr release from labeled CHO cells. Saffer, L.D., et al. Infect Im~nun (1991) 59:4681-4683.
3o 3
CSb9 ~ l~ce was assayed by the addition of purified comp' co,.~ c to E. histolytica
llupho~ . The percent of amebic Iysis was d~ ,c ""I~ iclv~copicdlly. Braga, L.L., et al. J
Clin Invest (1992) 90:1131-1137.
P and NP refer to ca ilivily of the MAb wit_ p~hogenir (P) and n.."l. .ll")~ ir (NP) species of
E. histolytica as d~ rd in an Elisa assay. Petri, W.A. Jr., et al. Infect Immun (1991)
58: 1802-1806.
5Lvcation of antibody binding site by amino acid number. Results pl~scnL~l herein.
4o 6
Inhibits adh~ ncc to CHO cells but not human colonic mucin gly~ulJ-uteins. Petri, W.A. Jr., et
al., J Immunol (1990) 144:4803-4809.
W095/00849 2~ ~S~a~ PCT~S94/06890
- 27 -
Fusion proteins B and C failed to react with
any of the seven MAbs (data not shown). Fusion protein
A, representing positions 482-1132, reacted with all
seven MAbs representing all 7 epitopes and not a negative
control developed with an irrelevant MAb, MOPC21. The
MAbs were used at 10 ~g/ml and polyc]onal antibodies at
1:1000 dilution. These results indic~ated that these
seven epitopes were contained within the 542 amino acids
of the cysteine-rich extracellular domain of the 170 kD
subunit.
The generation of 3' deleti.ons by controlled
ExoIII digestion of fragment A of the 170 kD subunit is
outlined in Figure 2B. ~1 contains amino acid residues
482-1082; ~2 contains amino acid residues 482-1032; ~3
contains amino acid residues 482-998. The reactivities
of the fusion proteins that include fragment A or either
of two carboxy-terminal deletions (~3 and ~4) with the
seven distinct 170 kD-specific MAbs were determined.
Deletion 3 reacted with MAb against epitopes 1-2, 4-5,
and 7 but failed to react with MAbs recognizing epitopes
3 and 6; Deletion 4 which contains residues 498-894
reacted only with the MAb which recognizes epitope 5.
The five deletion derivatives of fusion protein
A shown in Figure 2B, ranging in estimated size from 35
to 68 kD, were tested for reactivity to each MAb, and the
reactivities of the deletions with each MAb are
summarized in Fig. 3. The endpoints of the various
deletions were determined by DNA sequencing with primers
specific for the r~;n;ng hgll sequence. MAbs
recognizing epitopes 1 and 2, which increase amebic
adherence to target cells, failed to react with
recombinant lectin fusion proteins lacking amino acids
895 to 998. Similarly, MAbs recognizing epitope 4, an
inhibitory epitope, and epitope 7, which has the effect
of abrogating amebic lysis by complement, failed to react
W095/00849 PCT~S94/06890
2 ~6S ~ 28 -
with deletion mutants lacking this region. The MAb
specific for epitope 6, which has inhibitory effects on
amebic adherence and abrogates amebic lysis by
complement, did not react with a r~combinant protein
missing amino acids 1033 to 1082. Recombinant proteins
lacking amino acids 1082 to 1138 did not react with a MAb
which is specific for the neutral epitope 3. Finally, a
construct containing amino aci~ds 482 to 818 was
recognized only by the adherence-inhibitory epitope 5
MAb. The thus predicted locations of the MAb epitopes
are listed in Table 1 above.
Exam~le 4
Reactivity of 170 kD Fusion Proteins
with Human Immune Sera
Since the galactose adhesion is a major target
of the humoral immune response in the majority of immune
individuals, the mapping of human B-cell epitopes of the
170 kD subunit was undertaken. The recombinant ~usion
proteins and ExoIII-generated deletion constructs of the
170 kD subunit were tested for reactivity with pooled
human immune sera in the same manner as described for MAb
reactivity. Nonimmune sera was used as a control.
Fusion proteins A and C reacted with immune sera, whereas
fusion protein B did not (data not shown). Human immune
sera also reacted with deletion constructs A1, ~2, and A3
but not with ~4 or A10. Reactivity of immune sera with
the different deletions localized major human B-cell
epitopes to be within the first 482 amino acids and
between amino acids 895 and 1138 (Fig. 3). This second
region is the same area which contains six of the MAb
epitopes. These results are consistent with a report by
Zhang et al. supra, who found that sera from immune
individuals reacted primarily with recombinant adhesion
constructs containing amino acids 1 to 373 and 649 to
1202.
W095/0084g PCT~S94/06890
- 29 _2~ ~5 ~02>
Thus, for use in assays to detect human
antisera against E. histolytica, the useful epitope-
bearing portions are as shown in Table 2.
Table 2
Positions Epitope # P/NP
2-482 ? ?
1082-1138 ` 3 p
1033-1082 6 P
895-998 1,2,4,7 both
The epitope-bearing portions indicated can be
used alone, as fragments or as portions of chimeric or
fusion proteins, or any combination of these epitope-
bearing portions can be used.
Exam~le 5
Immunization Usinq Recombinant Subunit Protein
A GST fusion protein with fragment A was
prepared in E. coli as described in Example 1 above.
This peptide contains an upstream GST derived peptide
se~uence followed by and fused to amino acids 432-1138
encoded by nucleotides 1492-34~0 in Figure 1 herein. The
protein is produced intracellularly; the cells were
harvested and lysed and the lysates subjected to standard
purification techni~ues to obtain the purified fusion
protein.
Gerbils were ;mmlln;zed by intraperitoneal
injection with 30 ~g of purified fusion protein in
complete Freund's adjuvant and then boosted at 2-4 weeks
with 30 ~g of the fusion protein in incomplete Freund's
adjuvant.
The gerbils were challenged at 6 weeks by
intrahepatic injection of 5xlOs amebic trophozoites and
sacrificed 8 weeks later. The presence and size of
amoebic liver abscesses was determined.
W095/00~9 PCT~S94/06890
~65~ 30 -
The results of the two experiments described
above are shown in the tables below. The administration
of the fusion protein reduced the size of abscesses in a
statistically significant manner.
In experiment 1, six animals were used as
controls and nine were administered the fusion protein;
in experiment 2, seven animals were used as controls and
seven were provided the fusion protein.
F~ F~ 2
Abscess% with Abscess % with
Wei~ht Abscess Wei~ht Abscess
Control 1.44:t1.64 71% 4.76~1.78 lOO~o
GST - (482-1138) 0.81iO.10100% 2.35il.99 100%
* P<0.03 ~Vlll~Ja.l~l to control.
+ P<0.24 cu~ d to control.
Example 6
AnalYsis of the Gene Family Encodinq the 170 kD Subunit
of E. hiætolytica Gal/GalNAc Adherence Lectin
This Example shows that the adhesin 170 kDa
subunit of HM-l:IMSS strain E. histolytica is encoded by
a gene family that includes hgll, hgl2 and a previously
undescribed third gene herein designated hgl3. Since
hgll and hgl2 were originally sequenced, in part, from
different cDNA libraries, it was possible that they
represented strain differences o~ a single gene.
However, in this report both 5' and 3' termini of hgll,
hgl2, and hgl3 were isolated and sequenced from the same
lambda genomic library demonstrating unambiguously that
hgl is a gene family.
Comparison of the amino acid sequences of the
three heavy subunit genes showed that hgll and hgl2 are
89.2~ identical, hgll and hgl3 are 95. 2~ identical, and
hgl2 and hgl3 are 89.4~ identical. Sequence variation
within the gene family, however, appears to be
nonrandomly distributed within the coding sequence. The
WO9S/00~9 ~S ~ PCT~S94/06890
-- 31 --
majority of the nonconservative amino acid substitutions
as well as insertions and deletions occur in the amino
third of the molecule. Comparison o~ the amino acid
sequences of hgl2 and hgl3 reveal that ll of the 19
nonconservative amino acid substitutions and 11 of the 13
residues inserted or deleted reside within the first 400
amino acid residues. A similar pattern of variation is
present when hgll and hgl2 are compared. While hgll and
hgl3 contain only two nonconservative substitutions, both
are found within the first 400 residwes although the 57
conservative substitutions appear to be more randomly
distributed throughout the coding sequence. The high
degree of sequence conservation between hgl3 and hgll
suggest that they may have arisen from a recent gene
duplication event.
All 97 cysteine residues were maintained in the
three heavy subunit genes. The hgl2 gene was originally
reported lacking a single cysteine present in both hgll
and hgl3. However, this discrepancy has since been
recognized as a sequencing error (Dr. E. Tannich,
Bernhard Nocht Institute, Hamburg, Germany). The
cysteine residues are nonr~n~omly distributed throughout
the gene (Figure 4 ) with the highest concentration within
the cysteine-rich domain between amino acid residues 379-
1210. All seven identified epitopes recognized by murine
monoclonal antibodies map to this region (Mann, B.J. et
al. Infect Immun (1993) 61:1772-1778). As these
monoclonal antibodies can block target cell adhesion,
target cell lysis (Saffer, L.D. et al. Infect Immun
(1991) 59 :4681-4683), and/or resistance to host
complement-mediated lysis (Braga, L.L. et al . J Clin
Invest (1992) 90:1131-1137), the conservation of cysteine
residues may play an important role in maintaining the
conEormation of this important region of hgl.
3 5 A min;ml~m of three genes are shown to make up
the heavy subunit gene family. While it is not possible
W095/00~9 PCT~S94/06890
2~6s2o2 - 32 -
to rule out the existence of additional hgl genes, the
Southern blot and library screen data can be explained by
a gene family of three members. As the genomic library
was screened separateIy with a 5 ' and a 3' hgl specific
5 probe, additional heavy subunit genes would be isolated
even if they contained only partial identity with the
gene family at only one end or even if one termini of an
additional gene had been lost during library
amplification. The library screen looked at more than
3 . 2xlO~ bases of genomic DNA in an organism with an
estimated genome size of 10~5 bases (Gelderman, A.H. et
al. J Parasitol (1971) 57:906-911). Thus, a full genomic
equivalent was screened at low stringency for genes
containing identity at either end.
The Northern data indicated that all three genes
were expressed in the amebae. As the messages of hgll - 3
are predicted to comigrate at 4.0 kb, differential
hybridization was required to ascertain expression of
individual genes. Due to the high degree of identity
20 between hgll-3, relatively short oligonucleotides (17-21
bases) were synthesized specific for regions where the
three genes diverge. Each probe was compared by computer
analysis to the other hgl genes to be certain that they
were sufficiently divergent to prevent cross
25 hybridization. Hybridization and wash conditions were
highly stringent for such A/T rich probes and were done
at temperatures 5C or less below the predicted Tm based
upon nearest neighbor analysis. While it is impossible
to rule out cross hybridization with other hgl gene
3 0 members, these precautions make such an event less
likely.
The Northern blot also indicates that abundance of
mRNA for all three genes decreased as the amebae
progre~sed from log to stationary growth. This finding
3 5 correlates with data which indicate that late log and
stationary phase amebae have a decreased ability to
W095/00~ S~0 2 PCT~Sg4/06890
- 33 -
adhere to, lyse, and phagocytose target cells ~Orozco, E.
et al. (1988) "The role of phagocytosis in the pathogenic
mechanism of Entamoeba histolytica. In: Amebiasis:
Human infection by Entamoeba histol~ica (Ravdin J.I.,
ed), pp. 326-338. John Wiley & Sons, Inc., New York.
Details of the experimental methods and results of
the characterization of the hgl multigene family are
presented below.
Library Screen. A lambda Zap~ ]:I library containing
randomly sheared 4-5 kb fragments of genomic DNA from
HM-l:IMSS strain E. histolytica was k:indly provided by
Dr. J. Samuelson at Harvard University (Kumar, A. et al.
Proc Natl Acad Sci USA (1992) 89:10188-10192). Over
80,000 plaques from the library were screened on a lawn
of XL-1 Blue E. coli (Strategene, La Jolla, CA).
Duplicate plaque lifts, using Hybond-N membranes
(Amersham, Arlington Heights, IL), were placed in a
prehybridization solution consisting of 6x SSC (.89 M
sodium chloride and 90 mM sodium citrate), 5x Denhardts
solution, .5~ SDS, 50 mM NaPO4 (pH 6.7), and 100 ~g/ml
salmon sperm DNA for a m;n;ml~m of 4 hours at 55C. A 5'
and 3' DNA fragment of hgll (nucleotides 106-1946 and
3522-3940 respectively) were [~_32p] dCTP (Amersham)
labeled using the Random Primed DNA labeling Kit
according to the manufacturer' 8 instructions (Boehringer
Mannheim, Mannheim, Germany) and hybridized separately to
the membranes overnight at 55C in prehybridization
solution. Membranes were rinsed once and washed once for
15 minutes at room temperature in 2x SSC, .1~ SDS, then
washed once for 15 minutes at room temperature, and twice
at 55C for 20 minutes in .lx SSC, .l~ SDS. Plaques that
hybridized with the 5' or the 3 radiolabeled probe on
both duplicate filters were isolated and purified.
Northern blot and hybridization. Total RNA was
harvested from amebae using the guanidinium
isothiocyanate method (RNagen, Promega, Madison, WI).
-
W095/00849 PCT~S94/06890
.` -' " _
2 ~6S ~ ~ _ 34 _
Polyadenylated RNA was purified ~rom total RNA using
PolyATract System 1000 (Promega). RNA was
electrophoresed through a formald~hyde gel and
transferred to a nylon Zetabind membrane (Cuno) using 25
mM phosphate buffer (pH 7.5) as described (Sambrook, J.
et al. (1989) Molecular Cloni~: A laboratorY manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York). The membrane was incubated in
prehybridization solution and incubated at 37C for at
least two hours. Oligonucleotides (18-22 nucleotides
long) were end-labeled using polynucleotide kinase and
[~-P3Z]ATP (Sambrook, J. et al. (1989) Molecular Cloninq:
A laboratorY manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York), added to the hybridization
mixture and the membrane, and incubated at 37C
overnight. The membrane was then washed once at room
temperature for 10 minutes, once at 37C for 10 minutes;
and twice at 40-44C for 15 minutes each in 2x SSC, .1
SDS. The radiolabeled probes used were:
5'-TTTGTCACTATTTTCTAC-3', hgl l ; 5'-TATCTCCATTTGGTTGA-3',
hgl2; 5'-TTTGTCACTATTTTCTAC-3', hgl3; and
5'-CCCAAGCATATTTGAATG-3', EF-l~ (Plaimauer, B. et al . DNA
Cell Biol (1993) 12:89-96).
Characterization of the hql3 qene. The hgl3
open reading frame was 3876 bases and would result in a
predicted translation product o~ 1292 amino acids (Figure
4). The predicted translation products of hgll and hgl2
would be 1291 and 1285 amino acids respectively. A
putative signal sequence and a transmembrane domain were
identified in the amino acid sequence of hgl3 similar to
hgll and hgl2. The amino-terminal amino acid sequence of
the mature hgl3 protein, determined by Edman degradation
(Mann, B.J. et al. Proc Natl Acad Sci USA (1991) 88:3248-
3252), was assigned residue number 1. Previous analysis
of hgll and hgl2 identi~ied a large, conserved,
extracellular region which was 11~ cysteine, designated
W095/00849 ~ 6 S 2~2 PCT~S94/06890
- 35 -
the cysteine-rich domain (Mann, B. J. et al. Parasit Todav
(1991) 7:173-176) (Fig. 2). Sequence analysis of hgl3
revealed that all 97 cysteine residues present within
this region were also conserved in both of the previously
reported heavy subunit genes.
A schematic comparison (Fi~ure 5) of heavy
subunit gene sequences revealed a high degree of amino
acid sequence identity. However, seven sites, ranging
~rom 3-24 nucleotides, were found where an insertion or
deletion had occurred in one subunit relative to another,
all of which maintained the open reading frame. Both
hgll and hgl3 contained a large number of nonconservative
amino acid substitutions when compared to hgl2, making
them 89.2~ and 89.4~ identical to hgl2 respectively.
While the comparison of hgll and hgl3 revealed only two
nonconservative substitutions, 57 conservative amino acid
substitutions and 3 single residue insertion/deletions
making them 95.2~ identical.
All 16 potential sites of glycosylation present
in hgll were conserved in hgl3. A sequence analysis of
hgl2 indicated that it contained only 9 such sites,
alt].lough all 9 were present in hgll and hgl3.
Glycosylation appears to account for approximately 6~ of
the heavy subunits' apparent molecular mass (Mann, B.J.
et al. Proc Natl Acad Sci USA (1991) 88:3248-3252).
All three heavY subunits are exressed. Since
hgl3 was isolated from a genomic library, it was unknown
if this gene was transcribed. Polyadenylated RNA was
harvested from amebae in both log and stationary phase
growth. Probes specific for hgll, hgl2, or hgl3 were
hybridized to a Northern blot and identified an RNA band
of the predicted size of 4.0 kb.
As the messages of hgll-3 are predicted to
comigrate at 4.0 kb, differential hybridization was
required to ascertain expression of individual genes
using Northern blots. Due to the high degree of identity
W095/00849 . i PCT~S94/06890
2~6Sa~ ~ -36 -
between hgll-3, relatively short oligonucleotides (17-21
bases) were synthesized specific fo;~ regions where the
three genes diverge. Each probe was compared by computer
analysis to the other hgl genes ~o be certain that they
were sufficiently divergent to prevent cross
hybridization. Hybridization and wash conditions were
highly stringent for such A/T rich probes and were done
at temperatures 5C or less below the predicted Tm based
upon nearest neighbor analysis. While it is impossible
to rule out cross hybridization with other hgl gene
members, these precautions make such an event less
likely.
The message abundance decreased significantly
as the amebic trophozoites passed from log phase growth
(lane A) to stationary phase growth (lane B~ while the
control gene, EF-1~, either remained constant or
increased slightly. This finding correlates with data
indicating that late log and stationary phase amebae have
a decreased ability to adhere to, lyse, and phagocytose
target cells (Orozco, E. et al. (1988) "The role of
phagocytosis in the pathogenic mechanism of Entamoeba
histolytica. In: Amebiasis: Human infection bY
Entamoeba histolYtica (Ravdin J.I., ed), pp. 326-338.
John Wiley & Sons, Inc., New York.
Estimation of the number of heavy subunit qenes.
The observations herein confirm that the adhesin 170 kDa
subunit of HM-l:IMSS strain E. histolytica is encoded by
a gene family that includes hgll, hgl2 and a previously
undescribed third gene which iB designated hgl3. Since
hgll and hgl2 were originally sequenced, in part, from
different cDNA libraries, it was possible that they
represented strain differences of a single gene.
However, in the present work both 5' and 3' termini of
hgll, hgl2, and hgl3 were isolated and sequenced from the
same lambda genomic library, demonstrating unambiguously
that hgl is a gene family.
WOg5/00849 21 6~202 PCI~S94/06890
- 37 -
Comparison of the amino acid sequences of the three
heavy subunit genes found that hgll and hgl2 are 89.2
identical, hgll and hgl3 are 95.2~ identical, and hgl2
and hgl3 are 89.4~ identical. Sequence variation within
the gene family, however, appears to be nonrandomly
distributed within the coding sequence. The majority of
the nonconservative amino acid substitutions as well as
insertions and deletions occur in the amino third of the
molecule. Comparison of the amino acid sequences of hgl2
and hgl3 reveal that 11 of the 19 nonconservative amino
acid substitutions and 11 of the 13 residues inserted or
deleted reside within the first 400 amino acid residues.
A similar pattern of variation is present when hgll and
hgl2 are compared. While hgll and hgl3 contain only two
nonconservative substitutions, both are found within the
first 400 residues although the 57 conservative
substitutions appear to be more randomly distributed
throughout the coding sequence. The high degree of
sequence conservation between hgl3 and hgll suggest that
they may have arisen from a recent gene duplication
event .
All 97 cysteine residues were maintained in the
three heavy subunit genes. The hgl2 gene was originally
reported lacking a single cysteine present in both hgll
and hgl3. However, this discrepancy has since been
recognized as a sequencing error (Dr. E. Tannich,
Bernhard Nocht Institute, Hamburg, Germany, personal
communication). The cysteine residues are nonrandomly
distributed throughout the gene (Fig. 1) with the highest
concentration within the cysteine-rich domain between
amino acid residues 379-1210. All seven identified
epitopes recognized by murine monoclonal antibodies map
to this region (Mann, B.J. et al. Infect Immun (1993)
61:1772-1778). As these monoclonal antibodies can block
target cell adhesion, target cell lysis (Saffer, L.D. et
al. Infect Immun (1991) 59:4681-4683), and/or resistance
WO95/00849 ~2 1 38 - PCT~S94/06890
to host complement-mediated lysis (Braga, L.L. et al. J
Clin Invest (1992) 90:1131-1137), the conservation of
cysteine residues may play an important role in
maintaining the conforma~tion of this important region of
hgl.
A mlnlm1~m of three genes have been shown to make up
the heavy subunit gene family, as described herein.
While it is not possible to rule out the existence of
additional hgl genes, Southern blot analyses and library
screen data can best be explained by a gene family of
three members. For Southern blots, two restriction
enzymes were identified, DdeI and HindIII, t~at cut
genomic DNA to completion and resulted in analyzable
restriction fragments. As the membrane was hybridized
with a fragment of hgll corresponding to nucleotides 1556
to 3522, two bands of ~976 and 1965 nucleotides should
have been present from hgl3. This central hgll
radioprobe would hybridize with three bands of 1158, 810
and ~1080 nucleotides from hgll and would hyribidze with
five bands of 819, 312, 55, 755, and >1080 nucleotides
from hgl2. The Southern blot showed 7 bands for genomic
DNA disgested with DdeI, at 4200, 3700, 2100, 1800, 1300,
840, and 760 nucleotides. As the 819 and 810 nucleotide
bands would be expected to comigrate, all the bands
observed with DdeI digestion are explained by the
restriction maps of hgll-3.
HindIII has no restriction sites in hgll-3
within the coding region and would result in each gene
being represented by a single band greater than 4.0 kb.
30 The Southern blot showed three bands at 17500, 5600, and
4200 nucleotides. Should an additional heavy subunit
gene exist, its DdeI and HindIII fragments would need to
comigrate with hgll-3 bands, be so divergent that they
failed to hybridize with the hgll probe under very low
35 stringency, or be too large to be resolved and
transferred.
W095l0084~ Zl ~S 2az PCT~S94/06890
-- 39 --
As to the genomic screening data, the genomic
library was screened separately with a 5' and a 3 ' hgl
specific probe, additional heavy subunit genes would be
isolated even if they contained only partial identity
with the gene family at only one end or even if one
termini of an additional gene had been lost during
library amplification. The library screen looked at more
than 3 .2x108 bases of genomic DNA in an organism with an
estimated genome size of 107-5 bases (Gelderman, A. H . et
al. J Parasitol (1971) 57:906-911). Thus, a full genomic
equivalent was screened at low stringency for genes
containing identity at either end. Of 7 clones
identified with the 5' heavy subunit-specific probe, 4
contained inserts that matched the reported sequence for
15 hgl l, 2 matched the sequence of hgl 2 1 and 1 clone
represented hgl 3 . Of eight clones obtained using the 3 '
radiolabeled fragment, 1 matched the sequence for hgl l, 5
matched the sequence of hgl 2, and 2 represented hgl 3 . No
termini were found that did not match the sequence of
2 0 hgl l, hgl 2 or hgl 3 .
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