Identification and molecular characterization of proteins,...

Details Identification and molecular characterization of proteins,... Identification and molecular characterization of proteins,...

2001-07-19

2004-09-21

Nolan, Patrick J. (Department: 1644)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C424S184100, C435S975000

Reexamination Certificate

active

06794166

ABSTRACT:

FIELD OF THE INVENTION
The present invention is related to the molecular characterization of DNA sequences, which encode proteins expressed in the salivary glands of the
Ixodes ricinus
arthropod tick. These proteins are involved in the complex mechanism of interaction between this arthropod and its mammalian host. The invention relates to newly identified polynucleotides, polypeptides encoded by them and the use of such polynucleotides and polypeptides, and to their production.
BACKGROUND OF THE INVENTION
Ticks are hematophagous arthropods that feed on a wide diversity of hosts {Sauer, Annu. Rev. Entomol, 1995}. Unlike this group of arthropods, the Ixodid adult female ticks have the characteristics to ingest blood for an extended period of over 2 weeks.
Completion of the blood meal is dependent on the relationships of ticks with hosts species {Brossard Med. Vet. Entomol 1997}. Resistance to tick infestation implicates both innate and acquired immunity, and is characterized by reduced feeding, molting and mating capabilities that may lead to the death of the parasite. Acquired immunity of resistant hosts is mediated by a polarized Th1-type immune response, involving IFN-&ggr; production and delayed type hypersensitivity reaction {Allen J R, Int. J. Parasitol. 1973}{Ganapamo, Immunol. 1995}.
Some hosts are unable to counteract the tick infestation {Ganapamo et al, 1995}. Indeed, during their blood meal, ticks circumvent host defences via pharmacologically active components secreted in their saliva. These factors can modulate both the innate and the acquired immunity of the host. In this way, the leukocyte responsiveness is modified during tick feeding {Ribeiro, Exp. Parasitol. 1987}{Kubes, Immunol. 1994}. For example, cytokines production is modulated, inducing a polarised Th2 immune response {Ganapamo, Immunol. 1996} {Kopecky, Parasite Immunol. 1998}.
Therefore, the complex tick-host molecular interaction can be considered as a balance between host defences raised against the parasite and the tick evasion strategies, facilitating feeding for an extended period. Although, there is extensive information about the effects of tick bioactive factors on host immune defences, little is known about the mechanisms of their actions. However, it has been observed that a wide range of new proteins is expressed during the blood meal {Wang, Parasitol. 1994}. Several of them might be essential for the completion of the tick feeding process.
SUMMARY OF THE INVENTION
The present invention is related to a new isolated and purified polynucleotide obtained from tick salivary gland and presenting more than 75% identity with at least one nucleotide sequence selected from the group consisting of SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, SEQ.ID.NO.16, SEQ.ID.NO.17, SEQ.ID.NO.19, SEQ.ID.NO.20, SEQ.ID.NO.21, SEQ.ID.NO.22, SEQ.ID.NO.23, SEQ.ID.NO.24, SEQ.ID.NO.25, SEQ.ID.NO.26, SEQ.ID.NO.28, SEQ.ID.NO.29, SEQ.ID.NO.30, SEQ.ID.NO.31, SEQ.ID.NO.33 or a sequence complementary thereto, or a fragment thereof, as defined hereafter.
Preferably, the polynucleotide of claim
1
, which presents at least 80% identity with at least one of said nucleotide sequences, more preferably at least 90% identity, more preferably with at least 95% identity, and even at least about 98 to 99% identity.
Preferably, the polynucleotide of claim
1
, which presents at least 99% identity with at least one of said nucleotide sequences.
The present invention is also related to a polypeptide encoded by the polynucleotide of the present invention or a biologically active fragment or portion thereof.
Said polypeptide may be modified by or linked to at least one substitution group, preferably selected from the group consisting of amide, acetyl, phosphoryl, and/or glycosyl groups.
Moreover, said polypeptide may take the form of a “mature” protein.
It may also be part of a larger protein or part of a fusion protein.
Preferably, the polypeptide of the present invention further includes at least one additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which help in purification such as multiple histidine residues, or additional sequences for stability during production of recombinant molecules.
Another object of the present invention concerns a variant of the polynucleotide or the polypeptide of the present invention, a precise definition of this term being given hereafter.
Preferably, said variant varies from the referent by conservative amino acid substitutions.
Preferably, at least one residue is substituted in said variant with another residue of similar characteristics.
Advantageously, the substitutions in said variant are among Ala, Val, Leu and Ile; among Ser and Thr, among the acidic residues Asp and Glu; among Asn and Gln; among the basic residues Lys and Arg; or among aromatic residues Phe and Tyr.
Preferably, in the variant of the present invention, several amino acids are substituted, deleted or added in any combination.
Preferably, 5-10, more preferably 1-5, more preferably 1-2 amino acids are substituted, deleted or added in any combination, in said variant.
Said variant may be a naturally occurring allelic variant of an
Ixodes ricinus
salivary gland polypeptide present in
Ixodes ricinus
salivary glands.
The present invention is also related to a recombinant vector comprising at least one element selected from the polynucleotide, the polypeptide, and the variant of the present invention or fragments thereof.
Another object of the present invention concerns a cell transfected by or comprising the recombinant vector according to the invention.
The present invention further includes an inhibitor directed against said polynucleotide, polypeptide, or variant.
Said inhibitor is preferably an antibody or an hypervariable portion thereof.
The present invention is also related to an hybridoma cell line expressing said inhibitor.
Another object of the present invention concerns a pharmaceutical composition comprising an adequate pharmaceutical carrier and an element selected from the group consisting of said polynucleotide, polypeptide, variant, vector, cell, inhibitor or a mixture thereof.
Preferably, said pharmaceutical composition presents anti-coagulant properties and advantageously contains at least one polynucleotide selected from the group consisting of SEQ.ID.NO.7, SEQ.ID.NO.17, and SEQ.ID.NO.26, and fragments thereof or contains at least one polypeptide encoded by said polynucleotides or fragments thereof.
Preferably, the pharmaceutical composition presents immunomodulatory properties, and contains at least one polynucleotide selected from the group consisting of SEQ.ID.NO.12, SEQ.ID.NO.21, SEQ.ID.NO.26, and SEQ.ID.NO.31, and fragments thereof, or contains at least one polypeptide encoded by said polynucleotides or fragments thereof.
Another object of the invention is an immunological composition or vaccine for inducing an immunological response in a mammalian host to a tick salivary gland polypeptide which comprises at least one element of the group consisting of
a) a polynucleotide of tick salivary glands according to the invention;
b) a polypeptide of tick salivary glands according to the invention;
c) a variant according to the invention;
d) epitope-bearing fragments, analogs, outer-membrane vesicles or cells (attenuated or otherwise) of components a) or b) or c);
e) possibly a carrier.
The present invention is also related to a method for treating or preventing a disease affecting a mammal, said method comprising the step of administrating to said mammal a sufficient amount of the pharmaceutical composition or the immunological composition or vaccine according to the invention, in order to prevent or cure either the transmission of pathogenic agents by tick, especially by
Ixodes ricinus
, or the symptoms of diseases induced by t

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