Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
1999-12-23
2002-10-01
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S185100, C424S192100, C424S193100, C424S200100, C424S269100, C424S191100, C530S300000, C530S350000, C530S324000, C530S333000, C530S334000, C530S344000, C530S403000, C530S412000, C435S007100, C435S007220, C435S007400, C435S007920, C435S069100, C435S069300, C435S069700, C435S071100
Reexamination Certificate
active
06458359
ABSTRACT:
OBJECT OF THE INVENTION
The present specification relates to an application for an Invention Patent, regarding a chimeric gene formed of the DNA sequences that encode the antigenic determinants of four proteins of
L. infantum,
and to proteins encoded by said chimeric gene, useful for the prevention or treatment of leishmaniosis, in particular canine leishmaniosis. The obvious purpose of this lies in using the gene sequence or the protein obtained from the chimeric gene for providing pharmaceutical compositions for preventing or treating leishmaniosis, in particular canine leishmaniosis, that can be present in the body of a patient, for instance as a vaccine or a monoclonal antibody preparation. This patient does not have to be a dog but can also be a human being who suffers from diseases that involve immuno-depression. To achieve this, a chimeric gene will be produced that encodes a protein called PQ consisting of a chimeric product originating from an “in vitro” synthesis of a chimeric gene constructed “ad hoc”, which contains five of the antigenic determinants of four different proteins, The product is configured as highly sensitive and specific for—for instance—generating a protective immune responds against canine Leishmaniosis, or for preparing antibodies against canine Leishmaniosis.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is of utility within the industry dedicated to the manufacture of pharmaceutical products in general.
2. Description of the Related Art
The parasitic protozoa of the Leishmania genus are the aetiological agents that cause Leishmaniosis, a range of diseases that have a world-wide distribution and that are characterised in that they give rise to a wide variety of clinical symptoms.
The main forms of. Leishmaniosis are zoonotic in nature and humans are considered as secondary hosts.
The species denoted
L. Infantum,
widely distributed throughout many Mediterranean areas is the cause of visceral Leishmaniosis (LV) in humans and dogs,
In fact, dogs infected with
L. infantum
are the main animal reserve of this parasite, particularly during the long incubation period before the clinical symptoms can be observed.
The epidemiological data indicate that there is a direct correlation between the prevalence of canine Leishmaniosis and the transmission of the parasite to humans. For this reason, it is crucial to detect the disease or infection early on in campaigns undertaken to control the spread of the disease.
The parasite is transmitted to the host vertebrate as a flagellate promastigote, by means of a bite of a fly of the family “Phlebotominae”, and the parasite enter the cells of the mononuclear phages where they differentiate and reproduce as amastigotes, within the phago-lisosomal structure.
The infected cells gather in certain tissues, mainly spleen, liver and lymph nodes. It is estimated that around 15 million people are infected -with Leishmaniosis, and every year in the world 500,000 new clinical cases appear in the world, mainly in the underdeveloped and developing world.
In the south-western countries of Europe, Visceral Leishmaniosis (VL), is a zoonotic disease caused by the
L. Infantum
species, as was mentioned earlier. Recent data derived from epidemiological studies indicate that there is an alarming incidence of this infection.
In Italy the reported data for incidence of VL ranges from 14.4% to 37% according to the region.
In Portugal, more particularly in the area around Lisbon, seropositive rates of 8.4% have been found and in the region of the French Maritime Alps different centers of prevalence have been found that vary between 3.2% and 17.1%.
In Spain, the prevalence of Leishmaniosis depends on the zone being studied. In Catalonia an average incidence rate of 9.3% has been observed although in some hot-spots a prevalence of infected dogs of up to 18% has been found.
On the Island of Mallorca, the incidence rate is 14%, and other rates that have been found are: 2.4% in Murcia, 8.8% in Granada, from 10 to 15% in Salamanca, 5.25% in the province of Madrid, and 14% in Caceres.
Although the number of cases of VL in humans caused by
L. infantum
can be considered relatively low, the high percentage of patients with immuno-depression that become infected by Leishmania could be related to the high level of this illness in dogs.
In fact, in the South of Europe, 50% of adults that are infected by Leishmaniosis are also patients infected by the HIV virus. On the other hand, according to these data of Leishmania-HIV co-infection, it has been estimated that the level of infection (by parasites) can be one or two orders of magnitude higher than this figure due to the existence of a large number of undetected infections.
A common characteristic of the different types of Leishmania infection is that it induces a strong humoral response in the host. Therefore, diagnostic methods based on serological techniques are currently the most widely used.
It has been described that these antibodies are detected even during the asymptomatic phase of the disease in natural and experimental infections.
The sensitivity and specificity of these methods depends on the type, source and purity of the antigen used. In immunological processes that are currently commercialised, complete promastigotes and preparations more or less prepared from these are used as a source of antigen. This method normally leads to cross-reactions with serum from patients suffering from leprosy, tuberculosis, African tripanosomiasis, Chagas disease, malaria and other parasitosis.
The sensitivity and specificity of the serologic methods depend on the type, source and purity of the employed antigen. During the last years a great number of Leishmania antigens have been characterised, some of them can be considered as proteins specific to the parasite.
Among these proteins specific to the parasite, the surface protease GP63, the surface glycoprotein gp46 and the lipophosphoglicane associated KMP-11 protein deserve a mention.
An additional group of Leishmania antigens are formed of evolutionarily conserved proteins, such as kinesine, thermally induced proteins, actin and tubulin.
As part of a strategy to develop a specific serological diagnostic system for Leishmaniosis canine, a laboratory based project has been undertaken to identify the antigens of
L. infantum,
by means of a immuno-detection search of an expression library for genes of
L. infantum
using dog serum with active visceral Leishmaniosis.
It has been observed that most of the antigens isolated by this method belong to the family of proteins conserved during the course of evolution. The identification of the B epitopes of these antigens indicate, however, that in all cases the antigenic determinants were localised in regions that were not well conserved.
In particular, the acidic ribosomal proteins LiP2a and LiP2b are recognised by more than 80% of the VL sera.
It has been confirmed that these proteins contain disease specific antigenic determinants, and that the recombinant proteins LiP2a and LiP2b, from which a fragment had been removed, could be used as a specific instrument able to distinguish between VL and Chages disease.
It has also been shown that the PO ribosomal protein of
L. Infantum,
very highly conserved on the evolutionary scale, is recognised by a high percentage of VL dog sera. Furthermore, the antigenic determinants are found exclusively on the C-terminus of the protein, that is to say, in the region that has been poorly conserved during the course of evolution.
It has been observed that in 78% of the VL dog sera, antibodies against H2A protein are also present, and it has been confirmed that despite the sequence identity in all the H2A proteins among eukaryotic organisms, the humoral response to this protein in VL sera is particularly provoked by determinants specific to the Leishmania protein H2A.
The antigenic determinants recognised by the VL dog sera are found at both termini of the H2A protein.
The obvious solution to the problem currently encountered in this art would be to h
Alvarez Manuel Soto
Bedate Carlos Alonso
Requena Rolania Jose Maria
Baskar Padmavathi
Browdy and Neimark , P.L.L.C.
C.B.F. Leti S.A.
Smith Lynette R. F.
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