Purified Gal/GalNAc adherence lectin of Entamoeba histolytica is used for development of a vaccine to prevent human amebiasis.

This invention relates to the control and reduction of human disease due to 
the protozoan parasite Entamoeba histolytica, which are parasitic amoebas 
of vertebrates which cause breakdown of body tissue. Amoebas are a large 
genus of naked rhizopod protozoans. 
Currently, 10% of the world's population is infected with E. histolytica. 
Worldwide there are 50,000,000 cases of invasive amebiasis resulting in 50 
to 100 thousand deaths per annum. Amebiasis is a disease or infection 
caused by amoebas. 
At present, there is no vaccine available to prevent invasive amebiasis. 
The molecule responsible for adherence of the parasite to tissue was not 
previously described. 
The present invention provides isolated lectin for use as a vaccine for 
prevention of amebiasis and also for use in diagnosis of virulent 
amebiasis. 
Purified Gal/GalNAc adherence lectin of Entamoeba histolytica is used for 
development of a vaccine to prevent human amebiasis. 
We have isolated and described the galactose binding lectin of Entamoeba 
histolytica which mediates the adherence of the parasite to mammalian and 
human cells (Clin Res 34:222A, 1986 and 34:529A, 1986; Petri et al, J Clin 
Invest 80:1238-44, 1987, attached). Immunization with this newly purified 
lectin protein as a vaccine or for the development of a recombinant DNA 
vaccine for prevention of human invasive amebiasis is the present 
invention. 
Ravdin and co-workers have established that adherence of axenic E. 
histolytica to trophozoites of Chinese hamster ovary (CHO) cells, human 
erythrocytes and white blood cells, fixed rat and human colonic mucosa, 
rat colonic submucosa, purified rat and human colonic mucus, and rat 
colonic epithelial cells is mediated by a galactose inhibitable lectin on 
the surface of the parasite (J Clin Invest 68:1313, 1981; J Infect Dis 
151:804, 1985; Infect Immun 48:292, 1985; J Clin Invest 76:481, 1985; 
Infect Immun 53:1, 1986; Clin Res 34:438A, 1986, J Clin Invest 80:1245-54, 
1987). Bracha and Mirelman (1982, 1983) demonstrated that the parasite's 
galactose inhibitable lectin mediated its adherence to opsonized bacteria 
or bacteria containing galactose or GalNAc residues in its 
lipopolysaccharide. Inhibition of parasite adherence prevents lysis of the 
target cells (J Exper Med 152:377, 1980; J Clin Invest 68:1313, 1981). The 
in vivo and in vitro virulence of different strains of E. histolytica 
correlates directly with their amount of lectin activity per mg of amebic 
protein (J Infect Dis 151:804, 1985 ) and adherence dificient E. 
histolytica clones are avirulent (Orozco, Rodriquez, Salata, Murphy, 
Ravdin--Exp. Parasit. 63:157, 1987. Therefore, we have ample evidence to 
conclude that the parasite's galactose inhibitable adherence lectin is 
absolutely essential for virulence; inhibition of the function of this 
parasite protein by using a vaccine composed of the adherence lectin or 
portions of the lectin in a recombinant DNA vaccine should prevent disease 
due to E. histolytica. 
We have recently isolated and described this lectin (Petri et al., J Clin 
Invest 80:1238-44.) The adherence lectin was purified by 
galactose-affinity chromatography and with adherence-inhibitory monoclonal 
antibodies. Monoclonal antibodies which inhibited amebic adherence 
exclusively recognized the denatured or native 170K dalton amebic protein 
isolated by galactose affinity chromatography; seven additional anti-E. 
histolytica monoclonal antibodies which do not inhibit adherence did not 
recognize the lectin. Indirect immunofluorescence with monoclonal antibody 
designated F-14, which inhibits amebic adherence by 86% confirmed the 
surface location of the lectin. The lectin molecule is a glycoprotein as 
determined by metabolic labeling with (3H) glucosamine and by 
concanavaline A binding. (Petri & Ravdin, unpublished results.) The lectin 
is antigenic; it was recognized on Western blots by human immune sera 
obtained from patients treated for liver abscess (Petri et al Infect Immun 
55:2327-2331, 1987.) We have sequenced the first 15 amino-terminil amino 
acids of the adherence lectin. Search of the NBRF library revealed no 
homologous sequence in any other protein that has been sequenced to date 
(Petri & Ravdin, unpublished). 
In summary, we have the first to isolate the adherence lectin of Entamoeba 
histolytica. We have ample evidence that this amebic molecule is 
absolutely essential for parasite pathogenicity. Eliciting a host immune 
response by immunization with this amebic protein should provide immunity 
against invasive amebiasis. Immunization with all or a portion of the 
lectin molecule may be optimal. The specific anti-lectin mouse monoclonal 
or rabbit polyclonal antibodies produced by the purified lectin can be 
utilized to screen a cDNA expression library for the lectin protein. 
Insertion of the lectin encoding DNA into a DNA virus vector such as 
vaccinia virus (by the method of Moss et al Nature 311:67 (1984)), and 
immunization with this recombinant virus or oral immunization of 
attenuated Salmonella dublin bacteria constitutively producing recombinant 
amebic lectin are alternate methods for use of the purified lectin and the 
DNA encoding the lectin in development of a vaccine against amebiasis.

In one example, a lectin protein of E. histolytica is purified as above 
described. The purified lectin protein is injected in a host. An immune 
reaction is elicited in the host which produces antibodies against the 
particular lectin protein. When amoebas within an alimentary system of a 
host produce the particular lectin protein, the host's antibodies attach 
the lectin protein and reduce it or render the particular lectin protein 
inoperative, thus preventing attachment or maintenance of the amoebas 
within the host. 
In another example, a portion of the lectin protein molecule is separated 
from a remaining portion and one of the portions is injected as a vaccine 
to elicit production of antibodies by the host. 
In another example, the DNA gene encoding the amebic lectin is inserted 
into attenuated Salmonella dublin. The lectin is gene is fused to the LT-B 
toxin of E. coli on a plasmid, permitting constitutive synthesis of the 
lectin fusion protein in S. dublin, which is then administered as an oral 
vaccine (by the method of Brey et al, abstract #66, Program of the 27th 
ICAAC, N.Y., N.Y.). 
In another example, the DNA encoding the lectin protein is inserted into 
the DNA of vaccinia virus making a recombinant virus, which is injected in 
the host to produce the production of antibodies specific to the lectin 
protein (see Moss et al Nature 311:67). 
While the invention has been described with reference to specific 
embodiments, modifications and substitutions may be made without departing 
from the scope of the invention defined in the following claims.