Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-09-30
2001-08-07
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C424S208100, C424S160100, C530S412000, C530S806000
Reexamination Certificate
active
06270959
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of immunology and is particularly concerned with Human T-cell Lymphotropic Virus type I envelope proteins and human monoclonal antibodies specific therefor.
BACKGROUND TO THE INVENTION
Human T-cell Lymphotropic Virus type I (HTLV-I) was the first human retrovirus to be associated with disease, Adult T-cell Leukemia/Lymphoma (ATL; ref. 1, 2—various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure) and later with HTLV-I Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP; 3). Recently this virus has been associated with arthropathy (ref. 4), uveitis (ref. 5) and infective dermatitis (ref. 6). HTLV-I has been found in almost every region of the world and it is estimated that approximately 10 to 20 million people are infected (ref. 7).
The envelope protein of HTLV-I is composed of an external surface glycoprotein, gp46 and a noncovalently associated transmembrane anchor protein, gp21; both of these are derived from a common precursor, gp63 (ref. 8). The gp46 HTLV-I envelope protein is one of the smallest retroviral envelope proteins known and exhibits little sequence variability (ref. 9, 10, 11). This genetic stability may be a reflection of the limited coding sequence and a need for structural conservation in order to preserve its functionality. While a number of studies have characterized the HTLV-I gp46 protein (refs. 12, 13), it has been difficult to heterologously express recombinant envelope protein in large amounts for use in biochemical and immunological studies. (refs. 40, 41, 42, 43, 44, 45) We have recently described the expression of the entire HTLV-I envrelope protein, gp63, in a baculovirus expression system (ref. LA). Although the recombinant protein was expressed in the amounts, it was insoluble and the majority of protein was not completely post-translationally processed. Following successful solubilization of this protein, the soluble and insoluble forms of gp63 have been used to generate human T-cells lines in vitro (ref. 15) and high anti-envelope antibody titres in rabbits (ref. 16). Unfortunately, only non-neutralizing antibodies were induced by the recombinant gp63 protein as either insoluble inclusion bodies (ref. 14) or in its soluble form. This baculovirus-expressed envelope protein thus cannot be in the natural conformation that it is present in the virus and thus is not optimal for vaccine or diagnostic purposes.
Our previous study demonstrated that a recombinant vaccinia virus (RVV E3) containing the HTLV-I coding region for gp46 alone, produced the conformationally correct envelope surface protein, induced neutralizing antibodies in mice (refs. 16, 17) and expressed envelope protein at much higher levels that it did when gp21was concomitantly expressed in another construct RVV E1 (ref. 17). In this previous work, however, there was no provision of an isolated and purified envelope protein of Human T-cell Lymphotrophic Virus Type I (HTLV-I) devoid of non-envelope proteins of HTLV-I having substantially the same conformation as the envelope protein in native HTLV-I, especially the Tox and p12
I
proteins which have demonstrated oncogenic potential.
It would be advantageous to provide a recombinantly-producted, isolated and purified envelope protein of HTLV-I which is devoid of other HTLV-I proteins and having substantially the same conformation as the native protein in high yields and methods of purification of such proteins. Such proteins have use as antigens, immmunogenic prepared including vaccines, as components of diagnose assays and for the generation of diagnostic reagents. It would also be advantageous to provide human monoclonal antibodies which are HTLV-I envelope protein specific and substantially non-binding to HTLV-I envelope protein in a denatured form.
SUMMARY OF INVENTION
In accordance with one aspect of the present invention, there is provided an isolated and purified envelope protein of Human T-cell Lymphotrophic Virus Type I (HTLV-I) devoid of non-envelope proteins of HTLV-I having substantially the same conformation as the envelope protein in native HTLV-I. This envelope protein is sometimes referred to herein as the “gp46 envelope protein”. The envelope protein is provided devoid of non-envelope protein of HTLV-I by production by recombinant means as described in more detail below.
The isolated and purified envelope protein generally is provided in a glycosylated form with an apparent molecular weight of about 47 to about 49 kDa, as determined by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The protein provided herein may be in the form of a mixture of two envelope proteins of HTLV-I having an apparent molecular weight of about 47 kDa and about 49 kda respectively.
The isolated and purified envelope protein provided herein generally binds to a HTLV-I envelope protein-specific human monoclonal antibodies which do not bind to denatured envelope protein of HTLV-I, particularly monoclonal antibodies which recognize conformational epitopes of the envelope protein of HTLV-I.
The recombinant procedure described herein produces a mixture of proteins of varying molecular weights and degrees of glycosylation. Accordingly, in another aspect of the invention, there is provided a mixture of at least two isolated and purified envelope proteins of HTLV-I devoid of non-envelope proteins of HTLV-I having an apparent molecular weight which is selected from about 39 kDa, about 43 kDa, about 45 kDa, about 47 kDa and about 49 kDa, as determined by SDS-PAGE, which may include a mixture of all such envelope proteins.
The envelope proteins provided herein are also recognized by antibodies specific for HTLV-II envelope proteins. Accordingly, in another aspect of the present invention, there is provided an isolated and purified envelope protein of HTLV-I devoid of non-envelope proteins of HTLV-I which is recognized by antibodies specific for the envelope protein of Human T-cell Lymphotrophic Virus Type II (HTLV-II).
The present invention also provides an immunogenic composition comprising an immunoeffective amount of an active component, which may be the novel envelope protein provided herein, which may be formulated along with a pharmaceutically acceptable carrier therefor. The immunogenic composition may be formulated as a vaccine for in vivo administration to a host.
The immunogenic composition may be formulated as a microparticle, capsule, ISCOM or liposome preparation. The immunogenic composition may be used in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces. Some targeting molecules include strain B12 and fragments of bacterial toxins, as described in WO 92/17167 (Biotech Australia Pty. Ltd.), and monoclonal antibodies, as described in U.S. Pat. No. 5,194,254 (Barber et al). The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant.
Suitable adjuvants for use in the present invention include, (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of anmino acid, a muramyl dipeptide polyphosphazare, ISCOPRP, DC-chol, DDBA and a lipoprotein and other adjuvants to induce a Th1 response. Advantageous combinations of adjuvants are described in copending U.S. patent application Ser. No. 08/261,194 filed Jun. 16, 1994, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference.
In a further aspect of the invention, there is provided a method of generating an immune respon
Arp Jacqueline
Dekaban Gregory A.
Foung Steven Kok Hing
Park Hankyel T.
Shoemaker and Mattare LTD
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