Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1997-09-16
2001-02-13
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S265100, C424S269100, C530S300000, C530S350000
Reexamination Certificate
active
06187310
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention in the field of infectious diseases and immunology concerns vaccine and diagnostic compositions and methods. The compositions comprise peptides and proteins which include epitope-bearing regions of the 170 kDa subunit (or adhesin) of the
Entamoeba histolytica
Gal/GalNAc adherence lectin. The compositions are produced recombinantly in prokaryotic hosts. These peptides are used to measure subunit-specific antibodies in a subject infected by
E. histolytica
or responding to these vaccines. This invention includes the discovery of a novel variant of the 170 kDa subunit and the gene (hgl3) which encodes it. Hgl3 is the third member of a multigene family each member of which encodes a 170 kDa subunit of the lectin.
2. Description of the Background 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 symptoms is thought to be due to the existence of nonpathogenic as well as pathogenic strains of this ameba. As of 1988, it had been established that the subjects who eventually exhibit symptoms harbor pathogenic “zymodemes” classified on the basis of their distinctive hexokinase and phosphoglucomutase isoenzymes. The pathogenic forms are not conveniently distinguishable from the nonpathogenic counterparts using morphogenic criteria, but there is an almost perfect correlation between pathogenicity of the infecting zymodeme and development of 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. This nomenclature derives from the fact that adherence of the organism to target cells via this lectin is inhibited by the saccharides galactose and N-acetylgalactosamine. The purified lectin was shown to have a nonreduced molecular weight of 260 kDa on SDS-PAGE; reduction with &bgr;-mercaptoethanol yielded two subunits having molecular masses of 170 kDa and 35 kDa. The 170 kDa subunit is also referred to herein as the 170 kDa adhesin or the 170 kDa protein. Further studies showed that antibodies directed to the 170 kDa subunit were capable of blocking surface adhesion to test cells (Petri, et al., supra). Therefore, the 170 kDa subunit is believed to be of primary importance in mediating adhesion, hence is designation as the 170 kDa adhesin.
U.S. Pat. No. 5,004,608 (Apr. 2, 1991), describes the entire lectin as an effective vaccine to prevent
E. histolytica
infection.
Studies of serological cross-reactivity of sera from patients having symptoms, characteristic of pathogenic
E. histolytica
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 170 kDa subunit is recognized almost universally by immune sera and T-cells from patients with invasive amebiasis (Petri et al.,
Infect Immun
(1987) 55:2327-2331; Schain et al,
Infect Immun
(1992) 60:2143-2146).
DNA molecules encoding both the heavy (170 kDa) and light (35 kDa) 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 N-terminal sequences. The sequence of cDNA encoding the 170 kDa subunit suggests that it is an integral membrane protein with a large cysteine-rich extracellular domain and a short cytoplasmic tail (Mann, B. et al., supra; Tannich et al.,
Proc Natl Acad Sci USA
(1991) 88:1849-1853). The deduced amino acid sequence of the 170 kDa adhesin shows that the extracellular domain can be divided into three regions based on amino acid composition. The N-terminal amino acids 1-187 are relatively rich in cysteine (3.2%) and tryptophan (2.1%). The convention for amino acid numbering of the 170 kDa subunit is to start with the N-terminus of the mature (processed) protein as #1. The sequence from positions 188-378 lacks cysteine. In the stretch of residues from 379-1209, 10.8% are cysteine. Clones encoding the 170 kDa subunit are further described in U.S. Pat. No. 5,260,429 (Nov. 9, 1993), the disclosure of which is incorporated herein by reference. This patent describes methods for diagnosing the presence of
E. histolytica
using the polymerase chain reaction (PCR) and DNA probes.
The 170 kDa subunit is thought to be encoded by a multigene family (Mann, B. et al.,
Parasit Today
(1991) 1:173-176). Two different 170 kDa subunit genes, hgl1 and hgl2, have been sequenced by separate laboratories. While hgl2 was isolated in its entirety from an HM-1:IMSS cDNA library (Tannich, E. et al.
Proc Natl Acad Sci USA
(1991) 88:1849-1853), hgl1 was isolated in part from an H-302:NIH cDNA library and in part by PCR amplification of the gene from the HM-1 :IMSS genome (Mann et al., supra). As the amino acid sequences of these two gene products have 87.6% identity (Mann, B. J. 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, is consistent with the existence of a gene family (Tannich, E. et al.
Proc Natl Acad Sci USA
(1991) 88:1849-1853).
U.S. Pat. No. 5,272,058 (Dec. 21, 1993; incorporated herein by reference in its entirety) discloses monoclonal antibodies (mAbs) immunoreactive with various epitopes of the 170 kDa subunit. This document also describes use of these antibodies to detect the 170 kDa protein and use of the protein to detect antibodies in serum or other biological samples. It is noteworthy that all the experimental work described in this document was limited to the native protein. These mAbs were further characterized by the present inventors' group (Mann, B. J. et al.,
Infect Immun
(1993) 61:1772-1778; also incorporated by reference).
Various immunoassay techniques have been used to diagnose
E. histolytica
infection.
E. histolytica
antigens have been detected by ELISA of stool specimens and sera, though these tests do not seem to distinguish between the pathogenic and nonpathogenic strains. Root et al.,
Arch Invest Med
(Mex) (1978) 9:
Supplement
1:203, described ELISAs with rabbit polyclonal antiserum to detect amebic antigens in stool specimens, and various forms of this procedure have been adapted by others (Palacios et al.,
Arch Invest Med
(Mex) (1978) 9
Supplement
1:203; Randall et al.,
Trans Roy Soc Trop Med Hyg
(1984) 78:593; Grundy,
Trans Roy Soc Trop Med Hyg
(1982) 76:396; Ungar,
Am J Trop Med Hyg
(1985) 34:465). These studies on stool specimens and other biological fluids are summarized in
Amebiasis: Human Infection by Entamoeba histolytica
J. Ravdin, ed. (1988) Wiley Medical Publishing, pp. 646-648.
Serological analysis is also a critical tool in the diagnosis of invasive amebiasis. One approach utilizes conventional serologic tests, such as indirect hemagglutination. These tests are very sensitive, but seropositivity persists for years (Krupp, I. M.,
Am J Trop Med Hyg
(1970) 19:5762; Lobel, H. O. et al.,
Ann Rev Microbiol
(1978) 32:379-347). Thus, healthy subjects may give positive assay responses, creating an undesirably high background. Similar problems with false positives have been observed in immunoassays using a mAb and purified native 170 kDa protein (Ravdin, J. I. et al.,
J Infect Dis
(1990) 162:768-772.)
Recombinant
E. histolytica
proteins other than the 170 kDa subunit have been used as the basis for serological tests. Western blotting using a recombinant form of the “52 kDa serine-rich protein” 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 USA
(1990) 87:4976-4980; St
Dodson James M.
Mann Barbara J.
Petri, Jr. William A.
Gucker Stephen
Kunz Gary L.
Livnat Shmuel
Rader Fishman & Grauer
University of Virginia Patent Foundation
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