JP2002513381A - Env-glycoprotein vaccine for preventing HTLV-I and HTLV-II infections - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel protein antigen derived from an HTLV envelope protein that can be used as a vaccine for preventing and treating HTLV-I and HTLV-II infections, and a novel method for producing the antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION   Env-glycoprotein vaccine to prevent HTLV-I and HTLV-II infections   This application is partly pending from co-pending application Ser. No. 08 / 681,054, filed Jul. 22, 1996. Request, the entire text of which is incorporated herein by reference. 1.Introduction   The present invention relates to human T-cell leukemia virus-I (HTLV-I) infection and HTLV-II infection. Novel protein antigens for use in therapeutic and prophylactic vaccines for And a novel method for efficiently producing such an antigen. The present invention further A nucleotide sequence encoding the novel antigen, and expressing the novel antigen And expression systems (both eukaryotic and prokaryotic) You. More specifically, the present invention provides useful amounts of insect and mammalian cell lines. Expressing the recombinant HTLV envelope (env) glycoprotein of The present invention relates to a method for producing a glycoprotein. 2.Inventionbackground   Human T cell leukemia virus type I (HTLV-I) and type 2 (HTLV-II) are genetic And members of the serologically related retrovirus group, which They have the common tropism for lymphocytes and are associated with rare lymphotropic diseases. (Hall et al., 1994, Seminars in Virology 5: 165-178). HTLV-I It is unique to several confirmed areas and when infected, adult T-cell leukemia Disease (ATL), malignant transformation of mature T lymphocytes, and HTLV-I associated Known as spinal cord disorder (myelopathy) and tropical spastic paraparesis (HAM / TSP) Chronic cerebral spinal cord disorders develop.   HTLV-I infection has been reported in Southwest Japan, West Indies, South America and Africa. It is unique to that area. HTLV-II infection is now a major source of American Indians It has been found to be peculiar to the population and is given intravenously in North America and Europe. Infected drug abusers (IVDA) have been shown to be infected at high rates (Hall et al., 1994, Seminars in Virology 5: 165-178).   The majority of infected individuals remain asymptomatic carriers and further spread of the virus It becomes a propagation source. The mode of transmission of HTLV-II is not as well understood as HTLV-I. But not from all the evidence available to date, It is suggested that they are similar. Transmission of HTLV-I is achieved by three main pathways. Happen. That is, the vertical route from mother to child (this is mainly Transmission between heterosexuals and homosexuals; and contaminants From blood products that contain drug abuse after transfusion or by intravenous injection (Hollsberg et al., 1993, New England J. Medicine 32). 8: 1173).   Over the past decade, HTLV-II infection has been associated with a range of neurological (possibly uncommon) There has been accumulating evidence that it can be associated with lymphoproliferative disorders. at present If HTLV-II is less pathogenic than HTLV-I, or a clinical disorder is observed Lack of infection simply means that the infection has been identified and evaluated clinically. It is unclear whether this reflects the small number of people.   The size and structural organization of the HTLV-II provirus is very similar to HTLV-I. (Shimotono et al., 1985 Proc. Natl. Acad. Sci. USA 82: 310. 1-3105). Most primary amino acid sequences are also identical, so HTLV-I and HTLV-I Antigen cross-reactivity with LV-II would be suggested. The genome is an RNA polymer Long terminal repeats containing a binding site for the enzyme and sequences that regulate viral transcription. It is sandwiched between columns (LTR). Four major genes have been identified and these are It occupies the position LTR-gag-pol-env-pX-LTR in the gnome (Selki et al., 1983).  Proc. Natl. Acad. Sci. 80: 3618-3622).   The gag gene is a processin that produces three internal viral structural proteins. Encodes the polyprotein that is being labeled. The pol gene is reverse transcriptase RNaseH And integrase, all of which encode provirus synthesis and pro Involved in the integration of the virus into the host genome. Putative viral protease Open reading frame is located at the 3 'end of the gag gene. this Extends to the pol region and is involved in the frameshift of ribosome from mRNA. (Sh1motono et al., 1985 Proc. Natl. Aca d. Sc1. USA 82: 3101-3105).   The env gene is located upstream of the 3 'end of the pol gene and partially overlaps with the pol gene. Duplicate. The env gene encodes the precursor protein p63. p63 is a protein It undergoes cleavage by degradation and is subsequently glycosylated to form two glycoproteins, gp46 and And gp2l. gp46 protein is found on natural virions by electron microscopy It constitutes the observed surface protrusion and is considered to have receptor binding activity, Contains domains responsible for the production of neutralizing antibodies. gp2l protein is a transmembrane glycoprotein And may be involved in cell fusion activity like HIV. The env protein is currently Interacts with an unidentified cell receptor to mediate viral entry .   From its nucleotide sequence, the env protein has a hydrophobic signal at the amino terminus. 5 possibilities for N-glycosylation with sequence and centrally attached carbohydrate With a hydrophobic receptor and a hydrophobic amino acid in the putative transmembrane domain. Presumed to have a second cluster (Seiki et al., 1983 Proc. Natl. Acad. Sc. l. 80: 3618-3622). Due to its sequence characteristics, env protein is a cell membrane glycoprotein It is suggested to have a typical structure of   Extensive research has been done on vaccine development against HTLV-I and HTLV-II I have. The env protein was the target of such vaccines, but was Efforts to develop a full-length gp63 for use have failed. Useful level Previous attempts to express the HTLV env protein have been successful Was. Thus, instead, several groups have identified antigens against HTLV-I. (US Pat. No. 5,378,805, US Pat. No. 5,066,579), and HTLV-II As antigens (US Pat. No. 5,378,805, US Pat. No. 5,359,029), env Synthetic peptides derived from protein sequences have been developed. The main of these peptides Another use is for disease diagnosis and vaccine development. On the protein surface Synthetic peptides to map antigenic determinants and as potential vaccines Pide is being used increasingly. These chemically synthesized peptides are responsible for HTLV-I infection. Sensitive to develop a re-vaccine to differentiate from HTLV-II infection Used in assays (US Pat. No. 5,476,765).   A viral vector capable of expressing the recombinant env protein (eg, HTLV-I Living adenovirus recombinant virus expressing the envelope protein) Has been proposed as a vaccine against HTLV-I (deThe et al., 1994, Ciba Founda tion Symposium 187: 47-60). HTLV-I env protein expressed in vaccinia virus Parkin has also been formulated into vaccine formulations (Seikl et al., 1990, Virus Gen. es 3: 235-249; Shida et al., 1987, EMBO J. 6: 3379-3384). Another group, HTLV Live recombinant Poxou expressing full-length envelope protein of -I Developed a vaccine consisting of irs (Franchini et al., 1995, AIDS Research and Human Retroviruses 11: 307-313). However, this vaccine does not contain the gp63 protein. Combination of two additional boosts of cytoplasmic subunit Gp63 protein subunit administration. It was suggested that chin abolished the prophylactic efficacy.   Purification of full-length glycosylated gp63 may be dependent on the presence of anti-HTLV antibodies in biological samples. It is described for use in an assay to check for nullity. The bulletin states that Although it has been suggested that cosylated gp63 could be used in vaccine formulations, this type of vaccine The efficacy of chin is not described (US Pat. No. 4,743,678, US Pat. No. 5,045,448). issue).   Therefore, effective full-length HTLV env antigens for use in vaccine formulations, and There remains a need for efficient means for producing such antigens. 3.Summary of the Invention   The present invention is used as a vaccine for preventing and treating HTLV-I and HTLV-II infections. Novel protein antigen derived from HTLV env protein which can be used, and said antigen A novel method for producing. The present invention relates to a nucleic acid encoding a novel antigenic protein. It relates to the nucleotide sequence, mutants and derivatives thereof. The present invention further provides expression Expression of novel antigens, including vectors and cell lines (both eukaryotic and prokaryotic) About the method. The invention further provides for the prevention and / or prevention of HTLV-I and HTLV-II infections. Or how to use this novel antigen as an immunogen in therapeutic vaccine formulations. You.   The present invention provides that the polypeptide, when expressed recombinantly, is anchored to the host cell membrane. Not all or one of its membrane spanning domains HTLV env protein with truncation. In a preferred embodiment of the present invention, The soluble HTLV env protein lacks all or part of its amino terminus. ing. The present invention further provides an amino-terminal that is soluble and accumulates in the cytoplasm of the host cell. For truncated HTLV env proteins. As a result, the HTLV env protein dissolves It is easily purified from live host cells.   More specifically, the present invention encodes amino-truncated HTLV env proteins. Nucleotide sequence, expression of recombinant HTLV env protein in host cell system Of recombinant HTLV env protein in vaccine formulations for the prevention of HTLV infection About.   The current difficulty with mammalian recombinant gene expression is that many proteins Resistance to expression in a given system, thus predicting the likelihood of success It is difficult. Before trying to express high levels of HTLV env protein Attempts have not been successful. The present invention provides for truncation in baculovirus expression systems. This difficulty was overcome by expressing a truncated HTLV env gene. Amino terminal Transcription of the cDNA corresponding to the truncated HTLV env protein is unexpectedly abundant. Results in abundant transcribed proteins. Expression of immunologically useful HTLV-I env protein About 50-fold increase (compared to full-length gene expression in mammalian cells) Turned out to be.   The present invention further provides HTLV wherein the amino terminal leader or signal sequence has been deleted. -I or HTLV-II antigenic protein having the amino acid sequence of the env protein is H Protect recipients when challenged with TLV-I or HTLV-II Based on the applicant's finding that The immunogenicity of this protein is unexpected Powerful enough.   In a preferred embodiment of the invention, an amino-truncated HTLV env protein The nucleotide sequence encoding is antigenic or Produces an immunogenic polypeptide. The antigenic polypeptide binds to this antigen It can immunospecifically bind to an antibody against it. The immunogenic polypeptide An immune response to an antigen can be elicited, for example, by the polypeptide Immunization either produces antibodies that immunospecifically bind to this antigen, or Generates a cellular immune response to the original.   In another preferred embodiment of the invention, the antigenic protein of the invention is non-glycolytic. Expressed in a baculovirus system that produces silylated antigens, or It is expressed in producing stably transfected T cell lines. Further of the present invention In another preferred embodiment, the amino-truncated form of the HTLV env protein is Expressed as a fusion protein to facilitate purification of the protein.   In another aspect of the invention, methods of using these novel antigenic proteins are described. . Such methods may be merely prophylactic and / or if the recipient has HTLV-I After infection with either or both HTLV-II or HTLV-II, And the use of these novel antigenic proteins in pharmaceutical formulations. Also book The novel antigenic proteins of the invention are useful in diagnostic immunoassays, passive immunotherapy, and It is also useful in generating idiotype antibodies. 3.1.Definition   As used herein, the following terms have the following meanings:   The term “gp63” refers to the outer membrane proteins of the HTLV-I or HTLV-II virus. Refers to the 63 kilodalton precursor protein of the env protein. This term is gp63 Mutants, variants or fragments are also meant.   The term "gp46" refers to the region outside the 46 kilodalton of the HTLV-I or HTLV-II virus. Refers to membrane proteins, or env proteins. This term refers to gp46 mutant, mutation A type or fragment is also meant.   “Env protein” refers to HTLV-I and / or -II env proteins (full length and And truncated forms) and analogs thereof. . Preferred analogs are those that are substantially homologous to the corresponding natural amino acid sequence. , Most preferably at least one natural HTLV-I and -II env epitope, eg For example, a neutralizing epitope. More preferred types of HTLV-I and And -II env polypeptides are derived from the insect or mammalian cell expression hosts of the invention. Expression and / or secretion of HTLV-I and -II env proteins at the level Lacking a sufficient portion of the C-terminal transmembrane domain to promote is there.   An "effective amount" is one that elicits an immune response in a subject to whom the polypeptide is administered. Refers to the amount of HTLV-I and -II env polypeptide sufficient to Immune response is cellular And / or may include, but is not limited to, the induction of humoral immunity.   The term “treating or preventing HTLV infection” prevents the replication of the HTLV virus. Stopping, suppressing the transmission of HTLV, or allowing HTLV to Preventing establishment and showing signs of disease caused by HTLV infection It means to reduce or alleviate. Reduced viral load, mortality and / or Or, a reduction in morbidity is considered treatment.   The term "pharmaceutically acceptable carrier" refers to the effectiveness of the biological activity of the active ingredient. A carrier medium that does not interfere, is chemically inert, and is not toxic to the patient to whom it is administered Means   "Therapeutic agent" refers to the treatment of a viral infection or a disease caused by it. Any useful molecular compound, preferably an antiviral drug.                           4.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the nucleotide sequence and amino acid sequence of the HTLV-I env protein. The square part of this sequence is the amino-terminal truncated form of the HTLV-I env antigen of the invention. Correspond to those sequences deleted in one embodiment.   FIG. 2 shows the nucleotide sequence and amino acid sequence of the HTLV-II env protein . The square part of this sequence is the amino-terminal truncation of the HTLV-II env antigen of the present invention. It corresponds to those sequences that are deleted in one embodiment of the form.   Fig. 3 shows Western Blotty using HTLV-II infected human serum as a specific antibody. 63 kDa HTLV-II recombinant env glycoprotein expressed in H5 cells (rgp 63) (lane 1). This protein is not detected in uninfected insect cells. (Lane 2). HTLV-II negative human serum is HTLV-II e expressed in insect cells. No reaction with nv glycoprotein (lane 3).   FIG. 4 shows the detection of antibodies against env gp46 one week after immunization of R3 and R4 with rgp63. Out (lanes 2 and 3 respectively). The antigen used in this detection system was GSTLTLV-II gp4 Six fusion proteins. Pre-immune R3 serum against GST-gp46 fusion protein Did not react with any of the antibodies (lane 1).   FIG. 5 is a FACS analysis of an HTLV-II infected cell line using immunized rabbit R3. Sun Sex staining was detected in cell lines Vines and Mo-T. In contrast, CEM was negative. dotted line Is a control FITC-labeled anti-rabbit antibody. Solid line is anti-HTLV-II en used at 1:10 dilution v Protein rabbit serum. F1 is the fluorescence intensity.   FIG.6A is the antibody titer of the animal after HTLV-II-Vines inoculation as determined by the particle aggregation (PA) method. . The left figure (A) shows the antibody titer of a non-immunized rabbit. The right figure (B) shows the recombinant gp 3 shows the antibody response in rabbits immunized with 63. Black circle is R1, white circle is R2, white square is R3 , The black square is R4.   FIG. 6B shows detection of an antibody against GST-gp46 of R1 after inoculation of HTLV-II-Vines cells. You. Antibodies were first detected and appeared after two weeks. The antigen used in this detection system is It was a GST-gp46 fusion protein transcribed on the iron membrane.   FIG. 7 shows the results of Southern hybridization of nested PCR. II Detection of proviral DNA. The numbers indicate the number of weeks after inoculation. Only live rabbits (R1, R2 and R5) inoculated with HTLV-II-Vines cells after a long time Positive. In contrast, vaccinated animals (R3, R4, R8 and R9) or fever-free Rabbits (R6 and R7) injected with activated HTLV-II-Vines cells showed a negative reaction.                           5.Detailed description of the invention   The present invention relates to a novel antigenic protein derived from the HTLV env protein. this Vaccine formulation that helps prevent and treat HTLV-I and HTLV-II infections Can be used as an immunogen in. The present invention also relates to a method for producing such an antigen. I do.   The present invention relates to an HTLV env lipopepti that lacks all or part of a domain that spans a membrane. About This deletion allows the polypeptide to be expressed in the host when expressed recombinantly. It is not fixed in the cell membrane. In a preferred embodiment of the present invention, the soluble HTLVenv The protein lacks all or part of its amino terminus. The present invention further provides Soluble, amino-terminal truncated form of the HTLV env protein that accumulates in the cytoplasm of the main cell About the state. Because of this, the HTLV env protein is readily purified from lysed host cells. Made.   The present invention is directed, in part, to the deletion of the amino-terminal leader or signal sequence. Antigenic or having the amino acid sequence of HTLV-I or HTLV-II envelope protein Immunogenic protein is challenged by inoculation of HTLV-I or HTLV-II (antigen stimulation Applicants' finding that it is useful for protecting recipients when Is based on Further, in either eukaryotic or prokaryotic expression systems, Transcription of a cDNA transcript corresponding to the novel antigenic protein of the present invention is a transcription protein Led to unexpected enrichment.   The present invention relates to a nucleic acid encoding a novel antigenic protein, a variant and a derivative thereof. Reotide sequence. The present invention further relates to a method for expressing a novel antigen, Include expression vectors and eukaryotic and prokaryotic cell lines.   In a preferred embodiment of the present invention, the above-described nucleic acid is expressed upon expression in a suitable host cell. Otide sequences produce polypeptides that are antigenic or immunogenic. Antigenic port Re An antibody to the antigen can immunospecifically bind to the peptide. Immunogenicity A polypeptide may have an immune response against an antigen, for example, when immunized with the polypeptide. Can be elicited and induce the production of antibodies that immunospecifically bind to the antigen Or elicits a cellular immune response to the antigen.   In another preferred embodiment of the invention, the antigenic protein of the invention is baculovirus. Is expressed in the lus system to produce unglycosylated antigens, or the antigens are stable Is expressed in T cell lines transfected to produce glycosylated antigens . In yet another preferred embodiment of the invention, the amino terminus is truncated. The purified env protein is used as a fusion protein to facilitate purification of the protein. Expressed as quality.   In another aspect of the invention, a method of using the above novel antigenic proteins is described. Will be posted. These methods may be used alone in vaccine formulations in a prophylactic manner, and / or Or, if the recipient is already on HTLV-I or HTLV-II or both The use of the above antigenic proteins for therapeutic treatment after infection with s. The novel antigenic proteins of the present invention also provide diagnostic immunoassays, active immunotherapy, and And anti-idiotype antibodies.                        5.1New ENV glycoprotein antigen   The present invention addresses, in part, the difficulty of expressing useful amounts of HTLV-env protein. Amino-cleaved form of HTLV env protein in insect cells or lung T lymphocytes Based on the applicant's unexpected finding that Have been. By transcription of the cDNA corresponding to the amino-cleaved form of the HTLV env protein, Unexpected enrichment of the transcribed protein results. Immunologically useful HTLV env protein An approximately 50-fold increase in the expression of was achieved.   The present invention further provides that the antigenicity of this protein is surprisingly strong. Based on human knowledge. HTLV env protein lacking the amino-terminal leader sequence The antigenic protein having the amino acid sequence can be used as an anti-HTLV-II Induced body production and challenged with HTLV-I or HTLV-II inoculation It was useful in some cases to protect the recipient. HTLV-I and HTLV-II High level of amino acid sequence identity between amino acid sequences of regionally different strains of In addition, the HTLV-I env antigen of the present invention is useful for protection against many regional isolates of HTLV-I. HTLV-II env antigens of the present invention may be used in many regional isolates of HTLV-II. Would be useful for protection against   In a preferred embodiment of the present invention, the antigen protein of the present invention comprises baculoyl To produce a non-glycosylated antigen or to express the antigen protein The quality is expressed in a stably transfected T cell line to Generate. 5.2.Nucleotide sequence encoding the antigen   The present invention relates to HTLV env proteins and fragments, truncations and variants thereof. The nucleotide sequence to be encoded. In a preferred embodiment of the present invention, Includes the nucleotide sequence encoding the truncated HTLV env protein. According to the invention In a more preferred embodiment, the amino terminal 33 amino acid truncation of the HTLV-I env protein is Encompasses the encoding nucleotide sequence. The present invention further provides an amino terminal 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, or 6 Nucleotide sequence encoding HTLV-I env protein with 5 to 70 amino acids cleaved Includes columns. The present invention further provides amino-truncated HTLV-I env proteins. The recombinantly expressed polypeptide Although not anchored to the main cell membrane, it has the same antigenic activity as the full-length HTLV-I env protein. Encompasses the nucleotide sequence as it remains. Further, the present invention Intermediate deletions, which involve recombinantly expressed polypeptides in host cells. Does not adhere to the vesicle membrane but retains the same antigenic activity as the full-length HTLV-I env protein Includes deleting portions of the signal sequence domain sufficient to remain . HTLV-I env nucleotide sequences of the present invention include the following DNA sequences: : (1) HTLV-I env protein immunologically reactive with anti-HTLV-I env antibody (2) HTLV-I en containing the amino acid sequence shown in FIG. All the DNA sequences encoding the v protein; (3) the complementary strand of the DNA sequence shown in FIG. Hybridized under highly stringent conditions (e.g., 0.5 M NaHPO_Four , 7% sodium dodecyl sulfate (SDS), filter binding in 1 mM EDTA at 65 ° C Hybridization to DNA and washing in 0.1 × SSC / 0.1% SDS at 68 ° C. Pure (Ausubel F.M. et al., Eds., 1989, Current Protocols in Molecular Biology, Vol. . I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New  York, at p. 2.10.3)), all nucleic acids encoding functionally equivalent gene products Otide sequence; (4) moderately stringent to the complement of the DNA sequence shown in FIG. Hybridize under less stringent conditions such as mild conditions (eg, Washing at 42 ° C. in 0.2 × SSC / 0.1% SDS (Ausubel FM et al., 1989, supra)), still Function as All nucleotide sequences encoding the HTLV env gene product which are equivalently equivalent; and (5) The complementary strands of the DNA sequence shown in FIG. Under stringent conditions (eg, 0.2 × SSC / 0.1% SDS). Medium, washing at 37 ° C.), still encoding all functionally equivalent gene products Nucleotide sequence of Functionally equivalent gene products are produced at high levels and exempt Gene products that are epidemiologically reactive with the anti-HTLV-I env antibody are included.   In another preferred embodiment of the invention, the amino terminal 17 amino acids of the HTLV-II env protein Includes nucleotide sequences encoding amino acid truncations. The present invention further provides 1 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 ~ 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 Encodes an HTLV-II env protein truncated at ~ 65 or 65-70 amino acids Nucleotide sequences. The present invention further provides amino-truncated HTLV-II en v a nucleotide sequence encoding a protein, expressed by recombinant methods Polypeptide is not anchored to the host cell membrane, but is not the full-length HTLV-II env protein. Said nucleotide sequence that retains the same antigenic activity as the quality You. In addition, the invention encompasses intermediate deletions, which are expressed by recombinant methods. Does not anchor the polypeptide to the host cell membrane, but is a full-length HTLV-II env protein A portion of the signal sequence domain sufficient to retain the same antigenic activity as To delete. In addition, the present invention encompasses intermediate deletions, Signal sequence domain sufficient to prevent anchoring of the expressed polypeptide In part is deleted. The HTLV-II env nucleotide sequence of the present invention includes the following: (1) Immunologically reactive with anti-HTLV-II env antibody All DNA sequences encoding the HTLV-II env protein; (2) the amino acid sequence shown in FIG. All DNA sequences encoding the HTLV-II env protein containing the acid sequence; (3) FIG. 2 under highly stringent conditions. (For example, 0.5M NaHPO_Four, 7% sodium dodecyl sulfate (SDS) in 1 mM EDTA Hybridization to filter-bound DNA at 65 ° C. and 0.1 × SSC / Washing at 68 ° C. in 0.1% SDS (Ausubel F.M. et al., Ed., 1989, Current Protocols  in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and Jo hn Wiley & Sons, Inc., New York, at p. 2103)), encoding a functionally equivalent gene product. (4) moderately stringent to the complementary strand of the DNA sequence shown in FIG. Hybridizes under less stringent conditions, such as under stringent conditions (For example, washing at 42 ° C. in 0.2 × SSC / 0.1% SDS (Ausubel F.M. et al., 1989, supra) )), Still all functionally equivalent HTLV-II env gene product encoding (5) the nucleotide sequence shown in FIG. Hybridizes under less stringent conditions, such as under stringent conditions (Eg, washing in 0.2 × SSC / 0.1% SDS at 37 ° C.) and still functionally equivalent All nucleotide sequences encoding various gene products. Functionally equivalent gene products Has high levels of production and is immunologically reactive with anti-HTLV-II env antibody Gene products are included.   The present invention also provides a nucleic acid encoding an immunologically equivalent fragment of the HTLV env protein. Also encompasses expression of the reotide sequence. Such an immunologically equivalent fragment of HTLV env Is an analog of the nucleotide sequence encoding the protein, and And / or 3'-terminal truncations and / or 1 or more intermediate deletions Making an analog, expressing the analog nucleotide sequence, and obtaining the resulting fragment Whether it interacts immunologically with HTLV antibodies, or such antibodies, especially neutralizing Identify by determining whether to induce antibody production in vivo Can be. For example, in a preferred embodiment of the present invention, the amino-terminal signal sequence domain HTLV en which lacks an intermediate region that may promote secretion of Includes expression of the nucleotide sequence encoding the v protein.   The present invention also provides for operable regulatory elements that govern the expression of the above coding sequences. A DNA expression vector containing any of the linked coding sequences, and a host cell. Operably linked to regulatory elements that govern the expression of the coding sequence in the cell Encompassing the genetically engineered host cell containing any of the coding sequences. Include. As used herein, regulatory elements include inducible and non-inducible promoters. As enhancers, enhancers, operators, and those that manipulate and regulate expression Other elements known to those skilled in the art include, but are not limited to.   The env glycoprotein gene product or a peptide fragment thereof can be obtained by techniques known in the art. It can be produced by recombinant DNA technology using techniques. Therefore, the present invention v Glycoprotein gene polypeptides and peptides are converted to env glycoprotein gene sequences. The method of preparing by expressing a nucleic acid containing is described herein. ing. Using methods known to those skilled in the art, the env glycoprotein gene product coding sequence And constructing an expression vector containing appropriate transcription and translation control signals Can be. Such methods include, for example, in vitro recombinant DNA technology, synthetic Technology, and in vivo genetic recombination. For example, see Sambrook et al. (1989) above. And the techniques described in Ausubel et al. (1989). Or env sugar An RNA capable of encoding a protein gene product sequence, for example, a synthesizer And may be chemically synthesized. For example, "Oligonucl (Oligonucl eotide Synthesis) ”(Gait, MJ, 1984, IRL Press, Oxford) (Incorporated herein by reference in its entirety).   The present invention also provides nucleotides encoding peptide fragments of the HTLV env gene product. The sequence. In a preferred embodiment, the invention relates to HT from raised host cells. Soluble and stored in the host cell cytoplasm to facilitate purification of the LV env protein. Polypeptide or peptide corresponding to the amino-terminal truncated HTLV env protein to be deposited About Chid. For example, a polypeptide corresponding to the extracellular domain of the HTLV env protein Peptides or peptides are useful as "soluble" proteins that promote secretion . The HTLV env protein gene product or its peptide fragment can be identified using a commercially available antibody. Known heterologous epitopes, which are also included in the present invention. You. Durable fusion proteins can also be designed. This means that HTLV e It has a cleavage site located between the nv sequence and the heterologous protein sequence, A fusion protein that can be excised from a part. For example, collagenase The cleavage recognition consensus sequence is combined with the HTLV env protein or peptide It can be designed between tides or proteins. The HTLV env domain is It can be released from this fusion protein by lagenase treatment. The present invention In a preferred embodiment, glutathione-S-transferase and HTLV env46kd Protein fusion proteins can be designed. 5.3.HTLV ENV Antigenic proteins and polypeptides   The present invention relates to the ability of a polypeptide to be expressed in a host cell membrane when the polypeptide is genetically expressed. Lacks all or part of the signal sequence or transmembrane domain so that it is not anchored to HTLV env polypeptide. In a preferred embodiment of the invention, this soluble The sex HTLV env protein lacks all or part of its amino terminus. Of the present invention A preferred embodiment is a HTLV-I env polypeptide lacking the amino terminal 33 amino acids And HTLV-II env polypeptides lacking the 17 amino acids at the amino and amino termini.   HTLV env proteins, polypeptides and peptides, variants, truncated, or Is a truncated form of the HTLV env protein that is useful for treating and preventing HTLV-I and -II infections. It can be prepared for cutin preparations and as a medicament.   This env protein has a hydrophobic sequence at the amino terminus due to its nucleotide sequence. Signal, five access points that may be linked to the N-glycoside sugar chain at the center. A second cluster of hydrophobic amino acids in the putter site and in the putative transmembrane domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. 80: 3618-3). 622). The properties of the sequence indicate that it has the typical structure of a cell membrane glycoprotein. And suggests.   This env gene undergoes proteolysis and is catalyzed by subsequent glycosylation. Encoding the precursor protein gp63, which produces the glycoproteins gp46 and gp21. You. gp46 protein is observed by electron microscopy on native virus particles Surface protrusions, believed to have receptor binding activity, and Contains domains responsible for the production of sum antibodies. gp21 protein is a transmembrane glycoprotein Yes, and may be involved in cell fusion activity.   Furthermore, the present invention also relates to features (including, but not limited to, those recognized by HTLV env antibodies). As determined by the number of (capabilities), the nucleotide sequence described in Section 5.2. Includes proteins functionally equivalent to the loaded HTLV env protein. like this The equivalent HTLV env gene product is the gene encoded by the HTLV env gene sequence described above. Amino acid deletions, additions, and substitutions within the amino acid sequence may be That produce a functionally equivalent HTLV env gene product. You. Amino acid substitutions may include the polarity, charge, solubility, hydrophobicity, hydrophilicity, And / or similarities in the amphipathic nature. For example, non-polar (Hydrophobic) amino acids include alanine, leucine, isoleucine, valine, Phosphorus, phenylalanine, tryptophan and methionine; polar neutral Amino acids include glycine, serine, threonine, cysteine, tyrosine, Paragine and glutamine; positively charged (basic) amino acids include Arginine, lysine and histidine; and negatively charged amino acids Examples include aspartic acid and glutamic acid. For use in this specification The term “functionally equivalent” refers to a protein that can be recognized by the HTLV env antibody. This elicits an immunological response substantially similar to the endogenous HTLV env gene product described above. It is a protein that can be emitted.   On the other hand, random mutations (using random mutation techniques well known to those of skill in the art), HTLV env Can be performed on the nucleotide sequence and the resulting HTLV env protein Activity is tested. In addition, site-specific mutations in the sequence encoding HTLV env (Using site-specific mutagenesis techniques that are well known to humans) and have improved function (e.g., HTLV env protein (which leads to altered expression or antigenicity).   The HTLV env proteins of the invention for use in vaccine preparations are substantially Pure or homogeneous. At least 60-70% of the protein is single Are considered to be substantially pure or homogeneous. Substantive Pure protein is preferably 60-90%, more preferably about 95% and most Preferably it contains 99% of the protein sample. Methods known to those skilled in the art (e.g., The sample is stained after polyacrylamide gel electrophoresis and the single polypeptide Visualizing the bands) is used to determine protein purity or homogeneity. You can be. More detailed analysis (higher resolution) is known by HPLC or in the art. May be performed using other similar methods.   The present invention expresses recombinant nucleotide sequences encoding these proteins. Polypeptides that are typically purified from different host cells. Such a protein Purification can be achieved by various methods well known in the art. In a preferred embodiment The HTLV env protein of the present invention is a glutathione-S-transferase Is expressed as a fusion protein. The resulting recombinant fusion protein is HTLV env domain is purified by affinity chromatography To yield a substantially pure HTLV env protein sample. Other people The method can also be used. For example, "Methods In Enzymology", 1990, Academic Press, I nc., San Diego, "Protein Purification: Principles and practice", 1982, Sp. See ringer-Verlag, NewYork. 5.4.Expression system   The present invention includes expression systems wherein both eukaryotic and prokaryotic expression vectors are used. Can be used to express truncated and full-length HTLV env proteins You.   In a preferred embodiment of the present invention, the nucleotide sequence of FIG. Region has been deleted and encodes a truncated HTLV-I env protein) Expression in either a prokaryotic or prokaryotic expression vector.   A preferred embodiment of the present invention provides for full-length and truncated HTLV env gene products. , Including expression in baculoviruses, to produce non-glycosylated antigens.   Another embodiment of the present invention relates to the safety of full-length and truncated HTLV env gene products. Non-glycosylated antigens, including expression in routinely transfected T cell lines Is produced.   Various host-expression vector systems provide env glycoproteins encoding the sequences of the present invention. Can be used to express. Such a host-expression system may include a coding sequence of interest. Vehicles that can be produced and that can be substantially purified, In situ when transformed or transfected with a leotide coding sequence In this context, the term also refers to a cell that can exhibit the env glycoprotein gene product of the present invention. these Include, but are not limited to, the following: Recombinant bacteriophage DNA, including protein gene product coding sequences, plus Bacterium transformed with mid DNA or cosmid DNA (eg, E. coli, B. subtilis). B. subtilis); recombinant containing the env glycoprotein gene product coding sequence Yeast transformed with a yeast expression vector (e.g., Saccharomyces (Saccharomy ces), Pichla); recombinant containing env glycoprotein gene product coding sequence Transformed with a viral expression vector (e.g., baculovirus) Insect cell line; recombinant virus containing env glycoprotein gene product coding sequence Expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic) Virus, TMV) or a plant cell line infected with a recombinant plasmid expression vector (e.g., Plant cell lines transformed with Ti plasmid); or mammalian cell genome Promoters of origin (e.g., metallothionein promoter) or mammalian viruses Promoters (eg, adenovirus late promoter; vaccinia) A mammalian cell line (e.g., Cos, CHO, BHK, 2 93, 3T3).   In bacterial systems, the intended use for expression of the env glycoprotein gene product is Depending on the application, a suitable number of expression vectors may be selected. For example, env sugartan For the preparation of a pharmaceutical composition of protein or raise antibodies against the env glycoprotein Therefore, when producing such proteins in large quantities, high expression levels That direct the expression of a fusion protein product that can be easily purified. May be desired. Such vectors include, but are not limited to, E. coli. Expression vector pUR278 (Ryther et al., 1983, EMBO. 2: 1791) (in this expression vector The env glycoprotein gene product coding sequence is PIN vector, which can be individually ligated into the vector in frame with the lacZ coding region). (Inoue and Inoue, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke and Sc huster, 1989, J. Biol. Chem. 264: 5503-5509). PGEX vector momo As a fusion protein with glutathione-S-transferase (GST), It can be used to produce proteins. Generally, such a fusion protein Soluble and free glutathione after adsorption on glutathione-agarose beads Can be readily purified from lysed cells by elution in the presence of pGEX vector Thrombin so that the cloned target gene product can be released from the GST moiety. Alternatively, it is designed to include the Xa protease cleavage site.   In insect systems, Autographa california ) Nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. Used. The virus is Spodoptera frugipa erda) Proliferate in cells. The env glycoprotein coding sequence is individually non-essential for the virus. Region (e.g., the polyhedrin gene) and It is placed under the control of a motor (eg, a polyhedrin promoter). env sugartan Successful insertion of the protein gene results in inactivation of the polyhedrin gene, Non-occluded recombinant virus (i.e., encoded by the polyhedrin gene) Virus that lacks a protein coat). These recombinant viruses are then: The insect Spodoptera frugiperda (Spodopterafru) in which the inserted virus is expressed giperda) used to infect cells. (E.g., Smith et al., 1983, J. Virol. 46: 584 See Smith, US Patent No. 4,215,051).   Many mammalian host-based expression systems are used in mammalian host cells. Can be If adenovirus is used as an expression vector, The coding sequence of the env glycoprotein gene, which is the subject of Translation control complex (eg, late promoter and tripartite leader sequence). Can be. Next, the chimeric gene is added to the adeno by in vitro or in vivo recombination. Virus genome. Non-essential regions of the viral genome (eg, When inserted into the region E1 or E3), it is viable in the infected host and A recombinant virus capable of expressing the protein gene product will be obtained (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 3655-3659). specific The initiation signal is also sufficient for the inserted env glycoprotein gene product coding sequence. You may request to translate. These signals are the ATG start codon and adjacent Contains columns. env glycoprotein gene (with its own initiation codon and adjacent sequences If included in an appropriate expression vector, additional translation control signals May not be required. However, the env glycoprotein gene coding sequence If only a portion is inserted, an exogenous translation possibly containing the ATG start codon A translation control signal must be provided. In addition, the start codon may be Must be aligned with the reading frame of the sequence to ensure translation of the entire insert. I have to. These exogenous translational control signals and initiation codons are native and synthetic. Both can be of various origins. The effectiveness of expression is determined by the appropriate transcriptional enhancer. -Elements, transcription terminators, etc. (Bittner  et al., 1987, Methods in Enzymol. 153: 516-544).     5.5 cell line   The invention includes the expression of HTLV env glycoprotein in animal and insect cell lines . In a preferred embodiment of the invention, the env glycoprotein is baculo in an insect cell line. Expression in a viral vector produces an unglycosylated antigen. Others of the present invention In a preferred embodiment, the stably transfected T lymphocyte cell line Express the v-glycoprotein and generate glycosylated antigen.   The host cell system may be one that alters the expression of the inserted sequence, or in a particular desired manner. Those that modify and process the gene product can be selected. Protein Such modifications (eg, glycosylation) and processing (eg, cleavage) ) May be important for the function of the protein. Different host cells are Characteristic and specific mechanisms for post-translational processing and modification of quality and gene products Have a structure. Select an appropriate cell line or host system to express the foreign protein expressed. Ensure correct modification of the quality of the material. For this purpose, the appropriate processing of the primary transcript Retains cellular machinery for singing, glycosylation, and phosphorylation of gene products Eukaryotic host cells can be used. Such mammalian host cells are CHO, V Including but not limited to ERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38 cell lines It is not something to be done.   High-yield production and stable expression of recombinant proteins over long periods of time are preferred. An example For example, it is possible to manipulate cell lines that stably express the env glycoprotein gene product. Wear. Rather than using an expression vector containing the viral origin of replication, the host cell Appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators) And polyadenylation sites) and DNA controlled by a selectable marker. Can be exchanged. Following the introduction of the foreign DNA, the engineered cells are placed in enriched media for 1-2 Can be grown daily, and subsequently replaced with a selective medium. Selection in recombinant plasmid A selectable marker confers resistance to selection and allows cells to add The stable incorporation of the mide allows the growth to form foci, which are then cloned and Allows to become vesicular. This method expresses the env glycoprotein gene product It can be used to advantage to manipulate cell lines. Such a cell line , A compound that affects the endogenous activity of the env glycoprotein gene product It will be particularly useful for marking and evaluation.   Many selection systems can be used. Herpes simplex virus thymidine Kinase (Wigler, et al., 1977, Cell 11: 223), hypoxanthine-guanine phospho Ribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy, et al., (1980, Cell 22: 817)^-, Hgprt^-Or aprt^-Recruit into cells Including but not limited to: Also antimetabolites object Quality resistance can be used as a basis for selecting for the following genes: Meso Dhfr (Wigler, et al., 1980, Natl, Acad. Sci. USA 77: 3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); Mykov Gpt (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo (Colberre) conferring resistance to aminoglycoside G-418 -Garapin, et al., 1981, J. Mol. Biol. 159: 1); and resistance to hygromycin Hygro (Santerre, et al., 1984, Gene 30: 147).   Alternatively, use antibodies specific for the expressed fusion protein. Thus, any fusion protein can be easily purified. For example, Ja The system described by nknecht et al. Enables rapid purification of sex fusion proteins (Janknecht, et al., 1991, Proc. .Acad.Sci.USA 88: 8972-8976). In this system, the genetics of interest Offspring into a vaccinia recombination plasmid and Translation into the amino-terminal tag consisting of six histidine residues Try to fuse. Extract from cells infected with recombinant vaccinia virus The Ni²⁺Place on a nitriloacetic acid-agarose column and buffer with imidazole-containing buffer. Selectively elute stidine-added proteins.   5.6 Vaccine preparation and administration method   Since the HTLV env glycoprotein antigen of the present invention can be produced in large quantities, The antigens produced and purified have use for vaccine preparations. HTLVenv The protein may also be present, for example, in a body fluid sample from a vaccinated subject. Detect or measure the presence of antibodies to the antigen, thus diagnosing the infection, and / or Is used in immunoassays to monitor the immune response of subjects after vaccination. Has the usefulness to   The preparation of a vaccine containing an immunogenic polypeptide as an active ingredient is known to those skilled in the art. Have been.   5.6.1. Determining vaccine efficacy   To monitor the immune response in test animals after immunization with the HTLV env antigen Or using any immunoassay known to those skilled in the art. Can determine the immunopotency of the HTLV env antigen. Humoral (antibody) response and Production of cell-mediated immunity can be taken as an indicator of an immune response. Trial Experimental animals include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc. And finally it can include human subjects.   Methods for taking the vaccine are oral, cerebral, intradermal, intramuscular, intraperitoneal, intravenous , Subcutaneous, intranasal or any other standard route of immunization. Of the tested subject The immune response can be analyzed by various approaches. For example, By known techniques such as Luvent assay (ELISA), immunoblot, radioimmunoprecipitation, etc. The reactivity of the immune serum obtained against the HTLV env antigen, as assayed If the HTLV env antigen displays antigenicity or immunogenicity, Protecting the immunized host and / or preventing HTLV infection in the immunized host The reduction in symptoms can be analyzed.   As an example of a suitable animal test of the HTLV env vaccine, the vaccine of the invention is  Rabbits can be tested for their ability to induce an antibody response to the env antigen. Wear. Young adult New Zealand white rabbits free of male-specific pathogen-free (SPF) Can be used. Each test group receives a fixed concentration of vaccine. Control group Receive an injection of 1 mM Tris-HCl pH 9.0 without HTLV env antigen.   Blood samples can be taken from rabbits every week or two and HTLV env The serum can be analyzed for antibodies to the protein. Specific for antigen The presence of such antibodies can be assayed using, for example, an ELISA.   5.6.2. Vaccine preparation   Suitable preparations of such vaccines are either liquid lysates or suspensions. Suitable for dissolving or suspending in a liquid prior to injection Solid dosage forms can also be prepared. The preparation can be emulsified or The polypeptide may be encapsulated in liposomes. The active immunogenic component is the active ingredient Can often be mixed with pharmaceutically acceptable and compatible excipients. Wear. Suitable excipients include, for example, saline, dextrose, glycerol, ethanol And combinations thereof. In addition, if desired, the vaccine preparation Are adjuvants that increase the effectiveness of wetting agents, emulsifiers, pH buffers, and / or vaccines. Small amounts of auxiliary substances such as paints.   Examples of adjuvants that may be useful are aluminum hydroxide, N-acetyl-mura Mil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L- Alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutami Nyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl Loxy) -ethylamine.   The effectiveness of the adjuvant is determined by the immunogenic potential of the HTLV env polypeptide epitope. It can be determined by measuring the induction of antibodies to the repeptide. So The antibody may also be a polypeptide as described above in a vaccine that also contains various adjuvants. Is obtained.   The polypeptide may be formulated as a vaccine in a neutral or salt form. it can. Pharmaceutically acceptable salts include: The acid addition salts formed, inorganic acids (eg, hydrochloric acid, phosphoric acid, etc.) or organic acids (eg, Acid addition salts formed with acetic acid, oxalic acid, tartaric acid, maleic acid, etc.) It is. Salts formed by free carboxyl groups also include inorganic bases (eg, Sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, Ferric hydroxide, etc.) or an organic base (eg, isopropylamine, trimethylamido) , 2-ethylaminoethanol, histidine, procaine, etc.) Can be.   The vaccine of the present invention may be either polyvalent or monovalent. Multivalent vaccine Can be made from a recombinant virus that can express more than one antigen .   The vaccine formulation of the present invention can be introduced by a number of methods. This Such methods include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, etc. Path, or a path due to random cutting (for example, using a bifurcated needle to scratch the upper layer of skin) But not limited thereto.   The patient to which the vaccine of the present invention is administered is preferably a mammal, most preferably Or humans, but non-human animals (eg, cows, horses, sheep, pigs, birds). (Eg, chickens), goats, cats, dogs, hamsters, mice, rats, and the like. However, the present invention is not limited thereto.   The vaccine formulation of the present invention provides an effective immunizing amount of HTLV env protein and a pharmaceutically acceptable dose. It contains a carrier or excipient that can be contained. Vaccine preparations contain an effective immunizing dose of one or more It contains the original and pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable Carriers are well known to those skilled in the art and include, for example, saline, buffered saline, dext Loose, water, glycerol, sterile isotonic aqueous buffer, or a combination thereof But not limited thereto. Such an acceptable burden One example of a body is one or more stabilizers (eg, stabilized hydrolyzed protein). , Lactose, etc.). Such a carrier is Preferably, it is sterilized. The formulation should suit the mode of administration.   The compositions optionally contain small amounts of wetting, emulsifying, or pH buffering agents. You may. The composition can be used as a solution, suspension, emulsion, tablet, pill, capsule, Preparations or powders. Oral formulations include standard carriers (eg, Pharmaceutical products such as mannitol, lactose, starch, magnesium stearate, Sodium saccharine, cellulose, magnesium carbonate, etc. ).   In general, the above ingredients, separately or mixed together, Lyophilized powder or anhydrous concentrate in a closed container such as pull or sachette Supply. When administering the above composition by injection, the above components are administered before administration. It can be supplied as an ampoule of a sterile diluent so that it can be mixed.   In a specific embodiment, the lyophilized HTLV env polypeptide of the invention is Feed to the first container and 50% glycerin, 0.25% phenol, and And a diluent consisting of preservatives (eg 0.005% brilliant green) .   The exact dose of the vaccine formulation to be formulated will depend on the route of administration and the characteristics of the patient. Depending on the judgment of the field physician and the condition of each patient according to standard clinical techniques. Should be specified. The effective immunizing amount is based on the antigen in the host to which the vaccine preparation is administered. An amount sufficient to cause an immune response.   Use of purified antigen as a vaccine should be performed by standard methods Can be. For example, a purified protein is prepared at an appropriate concentration, and an appropriate vaccine is prepared. It can be formulated with an adjuvant, packaged and used. With an appropriate adjuvant Then, for example, a mineral gel (for example, aluminum hydroxide), a surfactant ( For example, lysolecithin, pluronic polyols), polya Nions, peptides, oil emulsions, alum, and MDP However, the present invention is not limited to these. Also, the immunogen can be incorporated into liposomes Conjugated to polysaccharides and / or other polymers that can be used in vaccine formulation You may let it. For example, if the recombinant antigen is a hapten (ie, selectively Anti It is antigenic in that it can respond, but immunogenic in that it cannot elicit an immune response. The hapten is shared by the carrier or immunogenic molecule. They may be combined. For example, large proteins such as serum albumin bind to it Hapten can be rendered immunogenic. The hapten-carrier is formulated It may be used as a vaccine.   The effective dose (immune dose) of the vaccine of the present invention is obtained from an animal model test system. It can be estimated based on the dose-response curve obtained.   The invention includes one or more containers containing one or more of the components of the vaccine formulation of the invention. Formulation packs or kits are also provided. Such containers contain pharmaceutical or biological products. A notice in a form prescribed by a government body that regulates the manufacture, use or sale of goods. It can be affixed to the manufacturer, use or sale for human administration. This represents the permission of the government agency.   Thus, the present invention provides a method of immunizing an animal, or various diseases or diseases in an animal. Or methods for treating or preventing afflictions, these methods being effective Administering the vaccine of the present invention to an animal. 5.6.3Use of antibodies produced by the vaccine of the present invention   Immunization with the HTLV env protein of the invention raised against antigen Antibodies include diagnostic immunoassays, passive immunotherapy, and anti-idiotypic antibodies It is also useful in the production of   The resulting antibodies can be obtained using standard techniques known to those skilled in the art (eg, immunoaffinity Chromatography, centrifugation, precipitation, etc.) Can be used for immunoassays. Further, the antibody may be used for treatment and / or disease. It can also be used to monitor the progress of qi. Public to those skilled in the art as described above. Any known immunoassay system may be used for this purpose. Less but an example As shown, radioimmunoassay, ELISA (enzyme-linked immunosorbentass ay), "sandwich" immunoassay, sedimentation reaction, gel diffusion sedimentation reaction, immunology Diffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent assays Technologies such as munoassay, protein A immunoassay and immunoelectrophoresis assay Include, but are not limited to, competitive and non-competitive assay systems It is not done.   Production of antibodies for use in passive immunotherapy using vaccine formulations of the invention In passive immunotherapy, it can be pre-formed against a heterologous organism. By administering the antibody, the host can be protected for a short period of time.   The antibody prepared by the vaccine preparation of the present invention is used for preparing an anti-idiotype antibody. It can also be used for Anti-idiotype antibodies are also used to immunize To generate antibodies that bind to the early antigens of the pathogenic microorganism (Jerne, 1974, Ann. Immunol. (Paris) 125c: 373; Jerne et al., 1982, EMB). O J. 1: 234).   The amount of immunogen to be used in the immunization method and the immunity schedule are determined by those skilled in the art. The doctor in the field can make the decision and refer to the immune response and the antibody titer of the subject. Can be given. 5.6.4Packaging   The composition, if necessary, contains one or more single-dose preparations containing the active ingredient. Pack or dispenser device. Pack is like For example, a metal or plastic foil such as a blister pack may be included. Pa The pack or dispenser device may be accompanied by instructions for administration. Also Preparing a composition comprising a compound of the invention formulated in a compatible pharmaceutical carrier, Placed in a clean container and labeled to be processed under the indicated conditions. 6.Example: Expression of HTLV-II envelope protein   In the examples provided herein, amino-truncated HTLV envelope proteins are used. Demonstrate quality expression. Furthermore, the resulting recombinant HTLV envelope protein is immune Prove that it is reactive.6.1 . Materials and methods Construction of baculovirus transformation vector and GST fusion vector   Nucleotide sequence of the HTLV-II envelope gene used in these constructs Is shown in FIG. Diagram showing the nucleotide sequence deleted from the HTLV-II envelope gene This is shown in the boxed area 2. Used to amplify the truncated version of the HTLV-II envelope gene. The primers used are underlined in FIG.   HTL Expressable in Baculovirus Transformation Vectors and GST Fusion Vectors The nucleotide sequence of the VI envelope gene is shown in FIG. HTLV-I envelope The nucleotide sequence deleted from the gene is shown in the boxed region in FIG. HTLV-I En Primers used to amplify the truncated form of the envelope gene are underlined in FIG. Was pulled.   Taq polymerase (Cetus), 30 cycles of amplification (94 ° C, 30 seconds, 56 ° C, 30 seconds , 72 ° C, 1 minute), and the following oligonucleotide primers: 5'-AAGGATCC ATGGGTAATGTTTTCTTC-3 '(5180-5197) and 5'-AAGGATCCTTATAGCATGGTTTCTGG -3 '(6643-6626) (BamH1 site is underlined, and SEQ ID NO: HTLV-II-Mo (Shimot ono) (derived from the published sequence). HTLV-II envelope gene was amplified by PCR from Sumid Mo (Shimotono) . The PCR-amplification product is cut with BamH1 and, after electrophoresis, in a low melting point agarose gel. Purified. Cut out the DNA band and place B downstream of the polyhedron promoter. It was ligated to the baculovirus transformation vector pVL1392 at the amH1 site. This PVLI, the baculovirus vector used to create these constructs   392 is obtained from Invitrogen, San Diego, CA. This recombinant plasmid is Used to transfect vesicles.   Similarly, glutathione S-transferase (GST) and HTLV-II envelope The fusion protein of the cleavage protein gp46 was prepared using the primer 5′-AAGGATCCATGGGTAAT. GTTTTCTTC-3 '(5180-5197) and 5'-AAGAATTCACGGCGGCGTCTTGTCGCGCCAGG- It was prepared by amplifying plasmid Mo-T using 3 '(6103-6086). BamH1 and Eco RI cohesive ends are introduced with these primers and used to ligate the fragments together. Used. Using the expression plasmid pGEX2T (Pharmacia, Upsala, Sweden), GST -The gp46 protein was expressed and purified according to the manufacturer's instructions. Production of recombinant baculovirus   To create a recombinant baculovirus, insect cells 10⁶Single layer containing (High Five (H5), Invitrogen, San Diego, CA) using the calcium phosphate method. The transformation vector DNA having the above envelope cDNA, and the linearized Transformed with baculovirus DNA (Baculogold, PharMingen, San Diego, CA) I did it. A single plaque containing the recombinant virus was purified from the supernatant and Amplified in cells. Western immunoblot analysis   Human antiserum of envelope protein expressed in baculovirus-infected cells Western blots were used to assess reactivity with. Recombinant baculo Whole cell extracts from H5 cells infected with irs were purified with 10% SDS polyacrylamide gel. (SDS-PAGE) for electrophoresis and transfection to PVDF membrane (Immobilon, Bedford, M). I did it. Serum from HTLV-11 infected subjects (200-fold diluted filter) Probed. 1: 5000 dilution of horseradish peroxidase conjugate to filter Inoculation with anti-human antibody (DAKO A / S, Denmark) followed by chemiluminescence (ELC, Am erciam, Buckinghamshire, England) to detect bound antibodies. Issued. Cells and viruses   T-cell line CEM as a T cell negative control for HTLV-II For B, the B-cell line BJAB was used. HTLV-II-Vines is a human immunodeficiency window HTLV-II-bearing human isolated from a male intravenous drug abuser not infected with Lus Used to establish a lymphoid cell line (Hall). HTLV-II-Mo-T is hairy HTLV-II- from a Mo subject with a T-cell variant of hairy cell leukemia Infected lymphoblastoid T cell line (Saladoon). All cell lines were fetal bovine serum 1 RPMI 16 supplemented with 0%, glutamine 2% and gentamicin 50 μg / ml Maintained in 40 media, 5% CO_TwoAnd cultured at 37 ° C.   Insect cells High Five (H5, Invitrogen, San Diego, CA) contain 10% fetal bovine serum. And a TC100 medium (Gibco BRL, Gaithersbur) containing kanamycin and 50 μg / ml. g, MD).6.1 . result   Purified recombinant baculovirus containing HTLV-II envelope gene was transferred to H5 insect Used to infect a monolayer of cells. Collect infected insect cells 4 days after infection Then, the expression of the HTLV-II envelope polypeptide was examined. Poly from lysed cells Separation of proteins by acrylamide gel, transfer to PVDF membrane, It probed with the serum (Hall) of the HTLV-II infected subject (FIG. 2). Apparent molecular weight 6 The 3 kDa protein was immunoreactive.   The location of the recombinant protein gp63 (rgp63) in insect cells was determined by immunofluorescence. I checked. Similarly, infected insect cells were collected two days after infection and tested for HTLV-II infection. Was incubated with normal serum. The majority of infected insect cells bind to the antibody and It was suggested that the recombinant gp63 is located on the surface of infected insect cells (data shown Not).   These results indicate that the immunoreactive HTLV envelope antigen is baculoyl. Proved to be successfully expressed in infected insect cell lines. 7.Example: Immunization with HTLV-II envelope protein   Examining the effect of inoculating rabbits with recombinant gp63 envelope protein For this purpose, the following analysis was performed. In this assay, rabbits were transformed into gp63-expressing insect cells. And immunize rabbits for antibodies to HTLV envelope protein. Assayed. The presence of the anti-HTLV envelope antibody was determined by (1) expression in bacteria Ability to detect recombinant GST-gp46 fusion protein, and (2) H in FACS analysis. It was measured by its ability to recognize TLV-II infected human cells. 7.1.Materials and methods Cells and viruses   T-cell line CEM as a T cell negative control for HTLV-II For B, the B-cell line BJAB was used. HTLV-II-Vines is a human immunodeficiency window HTLV-II-bearing human isolated from a male intravenous drug abuser not infected with Lus Used to establish a lymphoid cell line (Hall). HTLV-II-Mo-T is hairy HTLV-II-infected lymphoblastoid line from a Mo subject with a T-cell variant of leukemic leukemia T cell line (Saladoon). All cell lines are 10% fetal bovine serum, glutamine Maintained in RPMI 1640 medium supplemented with 2% and gentamicin 50 μg / ml And 5% CO_TwoAnd cultured at 37 ° C.   Insect cells High Five (H5, Invitrogen, San Diego, CA) contain 10% fetal bovine serum. And a TC100 medium (Gibco BRL, Gaithersbur) containing kanamycin and 50 μg / ml. g, MD). FACS analysis   HTLV-II-Vines, Mo-T, and CEM cell lines were stained with immunized rabbit serum and After three washes with BS, FITC-conjugated goat anti-human serum (2%) in the presence of 2% fetal bovine serum. DAK0 A / S, Denmark). Use relative fluorescence intensity It was detected by itometry. Rabbit   A commercial New Zealand white rabbit (2.5 kg) free of specific pathogens Obtained from breeders (SCL, Shizuoka, Japan). As shown in Table 1, the rabbit group 5 × 10⁷HTLV-II infected cells or heat-inactivated cells (70 ° C for 20 minutes) Injected and inoculated. Fluorescent antibody assay using serum from subjects infected with HTLV-II As a result of the measurement, HTLV-II infected cells were 90% infected (FIG. 5). Syncytium inhibition assay   HTLV-II-Vines cells were added to RPMI medium at 10 / ml.⁶Suspend with cells, Aliquot (50 μl per well) with 100 μl of heat-inactivated diluted rabbit serum. Both are incubated in a 96-well plate at 37 ° C. for 15 minutes, then Cell suspension (10 per ml⁶50 μl of cells) was added to each well. 5% CO_TwoInn After incubation at 37 ° C. for 16 hours in a cuvette, each well was inverted. The syncytium (giant polygonal cells) was examined using a microscope. Neutralizing power of antibody sample The titer refers to the sample in which syncytium formation is completely (100%) inhibited in the microculture. Expressed as the reciprocal of the dilution. Rabbit immunization   Recombinant-baculovirus-infected H5 cells were pelleted by centrifugation, Wash once with PBS, 10⁷Resuspended in PBS at a concentration of cells / ml. Complete (Day 0 ) Or incomplete (days 14, 28 and 42) emulsified in Freund's adjuvant Sample (10⁷Cells) were injected intramuscularly into female New Zealand white rabbits (2.5 kg). Was. Rabbits were purchased from SLC in Shizuoka, Japan. Rabbit immune serum on day 56 Collected. 7.2.result   Rabbits were immunized with recombinant gp63-expressing insect cells and truncated forms expressed in bacteria Serum was tested for recombinant GST-fusion protein using the gp46 envelope protein. Assayed for exit. Immunization because insect cells have different antigenicity from bacteria Later rabbit antisera does not show cross-reactivity with this fusion protein. Immunization 1 After a week, sera of rabbits 3 and 4 showed reactivity to GST-gp46 (FIG. 4, no. (Rows 2 and 3), but the serum of R3 before immunization did not react (row 1).   Rabbits and sera immunized with recombinant gp63-expressing insect cells were analyzed by FACS analysis. Assayed for the detection of HTLV-II infected human cells. Vine by FACS analysis Positive staining was shown in the s and Mo-T cell lines (FIG. 5). In contrast, the feeling Unstained T cell line CEM shows a negative staining pattern and rabbit serum is HTLV It was suggested that the antibody contained an antibody against the -II envelope protein.8.Neutralizing activity of serum from animals after vaccination   HTLV-II infected cells undergo cell fusion after co-culture with B-cell line BJAB (Hall) Trigger. Therefore, cells from rabbits immunized with recombinant gp63 were It was assayed for its ability to block vesicle fusion. 8.1.Materials and methods Rabbit   Rabbits targeting female New Zealand white rabbits (2.5 kg) without specific pathogens Obtained from a breeder (SCL, Shizuoka, Japan). As shown in Table 1, 5 × 10⁷HTLV-II infected cells or heat-inactivated cells (70 ° C for 20 minutes) were inoculated intravenously . Measured by fluorescent antibody assay using serum from subjects infected with HTLV-II As a result, HTLV-II infected cells were 90% infected (FIG. 4). Syncytium inhibition assay   HTLV-II-Vines cells in RPMI medium at 10⁶Suspend with cells, Aliquot (50 μl per well) with 100 μl of heat-inactivated diluted rabbit serum Incubate in a well plate at 37 ° C for 15 minutes, then add BJAB cell suspension (1 ml 10⁶Cells (50 μl) were added to each well. 5% CO_Two16 at 37 ° C in an incubator After incubation for a period of time, each well was washed using an inverted microscope with Multinucleated cells). Antibody sample neutralization titers were Expressed as the reciprocal of the sample dilution at which citium formation is completely (100%) inhibited . Rabbit immunization   Recombinant-baculovirus-infected H5 cells were pelleted by centrifugation and PB Wash once with S, 10⁷Resuspended in PBS at a concentration of cells / ml. Complete (Day 0) or Incomplete (Days 14, 28, and 42) sumps emulsified in Freund's adjuvant (10⁷Cells) were injected intramuscularly into female New Zealand white rabbits (2.5 kg). Husa Gi was purchased from SLC in Shizuoka, Japan. Rabbit immune serum was collected on day 56 . Particle agglutination method   The titer of rabbit serum against HTLV-II antigen was determined using the Particle Aggregation Kit (Fujire, Tokyo, Japan). bio). The beads to which the purified HTLV-I particles are attached Incubate with serum and determine the highest dilution that causes aggregation to be the antibody titer. Described. 8.2.result   Incubate HTLV-II infected cells with antisera at various dilutions against gp62. The results show that to completely prevent fusion in the first bleeding, At most a 1:50 dilution of serum from samples R3, R4, R8 and R9 should be sufficient. Was shown. Fusion blocked by incubation with antiserum Are blocked when incubated with preimmune sera Absent. This indicates that the epitope that elicits fusion activity is gp63, env protein Suggests that it is located inside. Conventional research blocks cell fusion Possible sera have always been shown to show neutralization of viral infection. In addition, HTLV In the case of -I and II infection, since cell-free infection is not possible, rabbit gp Inhibition of cell fusion induced by 63 immunized sera indicates inhibition of HTLV-II infection. Therefore, these results indicate that rabbit gp63-immunized serum has neutralizing activity against HTLV-II. To do so. 9.HTLV-II Rabbit protection from infection   In the examples provided herein, the ability of the HTLV env antigen of the invention to The rabbits were assayed for their ability to protect against infection. 9.1.Materials and methods Provirus detection by HTLV-II challenge and PCR   Wash HTLV-II-Vines cells with PBS and heat inactivate or not Rabbits were injected intravenously (5 × 10⁷cell). Heparinized weekly after infection From a blood sample, a density separation medium for rabbit lymphocytes, lympholyte-Rabbit (Cedar PBL was isolated by lane Laboratories, Hornby, Ontario, Canada).   DNA samples prepared from PBMC by DNAzol (Gibco BRL, Gaitherburg, MD) Then, 1 μg of the DNA sample was subjected to PCR analysis. PCR to amplify tax region Primers used in SK43 5'TGGATA CCC CGT CTA CGT GT3 '(7248-7267) and And SK44,5'GAG CTG ACA ACG CGT CCA TCG3 '(7406-7386) The primer used was located immediately downstream of SK43 and nested with SK44. SK43'5'GCG ATT GTG TAC AGG CCG ATT GGT 3 '(7271-7 (SK43 and 44 are PCR protocols M.A.Innis, David H. Gelfand, J.J.S. ninsky and T.J. White. Academic press). Southern hybridization   10 μl (from 50 μl PCR sample) of nested amplified DNA on 1.5% agarose gel Separated and blotted on a nylon membrane (Schlicher & Schuell). Hybri with these membranes Hybridized in Quick solution (Strategen, San Diego, CA) at 68 ° C for 2 hours. S The PCR fragment prepared using the K43 and 44 primers was purified from an agarose gel, (α-³²P) After random priming labeling with dCTP, it was used as a probe. 9.2.result   5x10 on rabbit⁷HTLV-II-Vines cells were inoculated intravenously. In the first experiment, Non-immunized groups R1 and R2 were seroconverged for HTLV-II two weeks after challenge The antibody titer was maximized in the next 8 weeks (FIG. 4). R1 Western Bro Analysis shows that antibodies to the recombinant cleavage env-fusion protein after challenge The presence was demonstrated (FIG. 6). Rabbit R immunized with insect cells expressing HTLV-II env In 3 and R4, antibody titers remain plateau in the next 10 weeks after challenge (FIG. 5).   HTLV-II nucleotide sequence in peripheral blood lymphocytes (PBL) isolated from immunized rabbits The presence was assayed by PCR analysis. In the first experiment, as summarized in Table 1, HTLV-II provirus was detected in DNA sample from PBL of immunized rabbit , Was not detected in PBL from immunized rabbits for 20 weeks. The second time HTLV-II provirus was detected only in immunized rabbits in the challenge. HTLV-II In order to confirm the infection, he further challenged with heat-inactivated HTLV-II-Vines. Of course, no provirus was detected after 20 weeks, as expected. On immunized rabbits The absence of provirus in mice and the presence of provirus in non-immunized rabbits To confirm, the HTLV tax gene PCR product was amplified after both challenges and It was subjected to hybridization (FIG. 6). From the results of the second PCR analysis, HTLV-II After challenge, non-immunized rabbits contained the HTLV provirus, but immunized rabbits Demonstrated that no HTLV-II provirus was detected. these The results show that the HTLV env antigen of the invention escapes protection from HTLV-II virus challenge. Plague Indicates that it was given to herons.   The invention is limited in scope by the specific embodiments described herein. Not something. Indeed, various modifications of the invention in addition to those described herein will now be described. And the accompanying drawings will be apparent to those skilled in the art. Such changes are also claimed Shall be included.   Various publications are cited in this specification, and the disclosures thereof are referred to as is. Well incorporated here for reference.

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Claims (1)

[Claims] 1. The following polypeptides when expressed by genetically engineered host cells   Enough of the leader sequence domain so that the   Has an amino acid sequence corresponding to the HTLV-II envelope protein lacking   Antigenic or immunogenic polypeptides, or analogs thereof, and pharmaceutically acceptable   Carrier. 2. The following polypeptides when expressed by genetically engineered host cells   Enough of the leader sequence domain so that the   Having an amino acid sequence corresponding to the HTLV-I envelope protein lacking   An immunogenic or immunogenic polypeptide, or analog thereof, and a pharmaceutically acceptable   Carrier. 3. Claims sufficient to protect the subject from HTLV-II virus challenge   HTLV-II infection, comprising administering to a subject the polypeptide of claim 1.   A method for treating or preventing a disease or disorder in a subject caused by the following. 4. A recombinant protein produced by a baculovirus insect cell expression system.   The method of claim 3. 5. 4. The amino-terminal signal sequence of the recombinant protein is truncated.   The method described in. 6. 4. The method of claim 3, wherein the recombinant protein is expressed in a T cell line. 7. Claim 2 in an amount sufficient to protect the subject from HTLV-I virus challenge.   Administering HTLV-I infection to a subject, comprising administering the polypeptide according to   A method for treating or preventing a disease or disorder in a subject caused by the disease. 8. A recombinant protein produced by a baculovirus insect cell expression system.   The method of claim 7. 9. 8. The method according to claim 7, wherein a signal sequence at a terminal of the recombinant protein is truncated.   the method of. Ten. 10. The method of claim 7, wherein the recombinant protein is expressed in a T cell line. 11． HTLV-II envelope protein lacking the amino-terminal leader sequence   Having the amino acid sequence of SEQ ID NO: 3 corresponding to the truncated gp63 subunit of   An immunogenic polypeptide, or an analog thereof. 12． HTLV-I envelope protein lacking the amino-terminal leader sequence   Having the amino acid sequence of SEQ ID NO: 1 corresponding to the truncated gp63 subunit   Epidemiological polypeptide, or analog thereof. 13. HTLV comprising administering a polypeptide according to claim 1 to a subject.   Production of antibodies that specifically bind to -II envelope protein in subjects   How to withdraw. 14. HTLV comprising administering a polypeptide according to claim 2 to a subject.   Induces the production of antibodies that specifically bind to the   How to get out. 15． HTLV-II envelope with a deletion of 33 amino acids from the leader sequence domain   Antigenic or immunogenic poly having an amino acid sequence corresponding to the rope protein   peptide. 16． HTLV-I envelope with a 17 amino acid deletion from the leader sequence domain   Or immunogenic polypeptide having an amino acid sequence corresponding to a loop protein   Puchido. 17． An amount of the polypeptide of claim 15 sufficient to produce an HTLV-II-specific antibody.   Caused by an HTLV-II infection, comprising administering to the subject   Eliciting an immune response in a subject that treats or prevents a disease. 18． An antigenic or pharmaceutically acceptable carrier according to claim 16 in an amount sufficient to enhance an HTLV-I specific immune response.   Administering immunized polypeptide to a subject, comprising administering to the subject an HTLV-I infection.   A method for treating or preventing a disease caused by. 19. The signal sequence at the amino terminus of the recombinant protein is truncated.   The method described in. 20. 18. The method of claim 17, wherein the recombinant protein is expressed in a T cell line. twenty one. A recombinant protein produced by a baculovirus insect cell expression system.   18. The method according to claim 17. twenty two. 19. The signal sequence at the amino terminus of the recombinant protein is truncated.   The method described in. twenty three. 19. The method of claim 18, wherein the recombinant protein is expressed in a T cell line. twenty four. A recombinant protein produced by a baculovirus insect cell expression system.   19. The method according to claim 18.