JP2002513381A - env-glycoprotein vaccines for preventing HTLV-I and HTLV-II infections - Patent Application 20070233334 - Google Patents

Abstract

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

Description

[DETAILED DESCRIPTION OF THE PRESENT APPLICATION] env-Glycoprotein Vaccine for Preventing HTLV-I and HTLV-II Infection This application is a continuation-in-part of co-pending application Ser. No. 08/681,054 (filed Jul. 22, 1996), the entire contents of which are incorporated herein by reference. 1.IntroductionThe present invention relates to novel protein antigens for use in vaccines for the treatment and prevention of human T-cell leukemia virus-I (HTLV-I) and HTLV-II infections, and to novel methods for the efficient production of such antigens. The invention further relates to nucleotide sequences encoding the novel antigens, and vectors and expression systems (both eukaryotic and prokaryotic) for expressing the novel antigens. More specifically, the invention relates to methods for the production of recombinant HTLV envelope (env) glycoproteins using insect and mammalian cell systems to express useful amounts of the envelope glycoproteins. 2.The inventionContext Human T-cell leukemia viruses types I (HTLV-I) and II (HTLV-II) are members of a group of genetically and serologically related retroviruses that share a common tropism for T-lymphocytes and an uncommon lymphotropic disease (Hall et al., 1994, Seminars in Virology 5:165-178). HTLV-I is endemic to several well-established geographic regions and causes adult T-cell leukemia (ATL), malignant transformation of mature T-lymphocytes, and chronic cerebrospinal disorders known as HTLV-I-associated myelopathy and tropical spastic paraparesis (HAM/TSP). HTLV-I infection is endemic to southwestern Japan, the West Indies, South America, and some areas of Africa. HTLV-II infection is now known to be endemic in many Native American populations, and a high proportion of intravenous drug abusers (IVDA) have been documented in North America and Europe (Hall et al., 1994, Seminars in Virology 5:165-178). The majority of infected individuals remain asymptomatic carriers, providing a source of further transmission of the virus. The mode of transmission of HTLV-II is less well understood than that of HTLV-I, but all available evidence to date suggests that they are similar, if not identical. Transmission of HTLV-I occurs by three major routes: There are three ways HTLV-II infection can be transmitted: vertically from mother to child (which occurs primarily through breast-feeding); through heterosexual and homosexual transmission; and through contaminated blood products (which can occur after transfusion or through intravenous drug abuse) (Hollsberg et al., 1993, New England J. Medicine 32 8: 1173). Over the past decade, evidence has accumulated that HTLV-II infection can be associated with a range of neurologic (and possibly unusual lymphoproliferative) disorders. At present, it is unclear whether HTLV-II is less pathogenic than HTLV-I or whether the lack of observed clinical disorders simply reflects the small number of infected individuals that have been identified and clinically evaluated to date. The size and structural organization of the HTLV-II provirus have been found to be very similar to that of HTLV-I (Shimotono et al., 1985 Proc. Natl. Acad. Sci. USA 82:310 1-3105). Most of the primary amino acid sequence is also identical, suggesting antigenic cross-reactivity between HTLV-I and HTLV-II. The genome is flanked by long terminal repeats (LTRs) that contain binding sites for RNA polymerase and sequences that regulate viral transcription. Four major genes have been identified, which occupy the following position in the genome: LTR-gag-pol-env-pX-LTR (Selki et al., 1983 Proc. Natl. Acad. Sci. 80:3618-3622). The gag gene encodes a polyprotein that is processed to produce the three internal viral structural proteins. The pol gene encodes the reverse transcriptase RNaseH and integrase, all of which are involved in the synthesis of the provirus and its integration into the host genome. An open reading frame for a putative viral protease is located at the 3' end of the gag gene. It extends into the pol region and is thought to be expressed from the mRNA by a mechanism involving ribosomal frameshifting (Sh1motono et al., 1985 Proc. Natl. Acad. Sc1. USA 82:3101-3105). The env gene is located upstream of the 3' end of the pol gene and partially overlaps with it. The env gene encodes the precursor protein p63, which undergoes proteolytic cleavage and subsequent glycosylation to produce two glycoproteins, gp46 and gp2l. The gp46 protein constitutes the surface protrusions observed on native virus particles by electron microscopy, is thought to have receptor binding activity, and contains the domain responsible for the production of neutralizing antibodies. The gp21 protein is a transmembrane glycoprotein that may be involved in cell fusion activity, similar to HIV. The env protein interacts with a currently unidentified cellular receptor to mediate viral entry. The nucleotide sequence of the env protein predicts that it has a hydrophobic signal sequence at the amino terminus, five potential receptor sites for carbohydrate-linked N-glycosylation in the central region, and a second cluster of hydrophobic amino acids in the putative transmembrane domain (Seiki et al., 1983 Proc. Natl. Acad. Scl. 80:3618-3622). The sequence features suggest that the env protein has a typical structure for cell membrane glycoproteins. Extensive research is being conducted toward the development of vaccines against HTLV-I and HTLV-II. The env protein has been the target of such a vaccine, but efforts to develop full-length gp63 for use as an effective antigen have failed. Previous attempts to express useful levels of the HTLV env protein have been unsuccessful. Instead, several groups have developed synthetic peptides derived from the env protein sequence as antigens for HTLV-I (U.S. Pat. No. 5,378,805; U.S. Pat. No. 5,066,579) and for HTLV-II (U.S. Pat. No. 5,378,805; U.S. Pat. No. 5,359,029). The primary use of these peptides is for disease diagnosis and vaccine development. Synthetic peptides have been increasingly used to map antigenic determinants on protein surfaces and as potential vaccines. These chemically synthesized peptides have been used in sensitive assays to develop vaccines that distinguish between HTLV-I and HTLV-II infections (U.S. Patent No. 5,476,765). Viral vectors capable of expressing recombinant env proteins (e.g., live adenovirus recombinants expressing HTLV-I envelope proteins) have been proposed as vaccines against HTLV-I (deThe et al., 1994, Ciba Foundation Symposium 187:47-60). HTLV-I env proteins expressed in vaccinia virus have also been formulated into vaccine preparations (Seikl et al., 1990, Virus Genes 3:235-249; Shida et al., 1987, EMBO J. 6:3379-3384). Another group developed a vaccine consisting of a live recombinant poxvirus expressing the full-length envelope protein of HTLV-I (Franchini et al., 1995, AIDS Research and Human Retroviruses 11:307-313). However, combining this vaccine with two additional boosts of the gp63 protein subunit failed to provide protection, suggesting that administration of the gp63 protein subunit abolished the protective efficacy of the vaccine. Purification of full-length glycosylated gp63 has been described for use in assays to determine the presence of anti-HTLV antibodies in biological samples. The same publication suggests that full-length glycosylated gp63 could be used in vaccine formulations, but does not describe the efficacy of this type of vaccine (U.S. Patent No. 4,743,678; U.S. Patent No. 5,045,448). Thus, there remains a need for effective full-length HTLV env antigens for use in vaccine formulations, and efficient means for producing such antigens. 3.Summary of the InventionThe present invention relates to novel protein antigens derived from HTLV env protein that can be used as vaccines for the prevention and treatment of HTLV-I and HTLV-II infections, as well as novel methods for producing said antigens. The present invention relates to nucleotide sequences encoding the novel antigenic proteins, mutants and derivatives thereof. The present invention further relates to methods for the expression of the novel antigens, including expression vectors and cell systems (both eukaryotic and prokaryotic). The present invention further relates to the use of the novel antigens as immunogens in vaccine formulations for the prevention and/or treatment of HTLV-I and HTLV-II infections. The present invention relates to HTLV env proteins that lack all or part of their membrane spanning domain, such that the polypeptide is not anchored to the membrane of a host cell when recombinantly expressed. In a preferred embodiment of the present invention, the soluble HTLV env protein lacks all or part of its amino terminus. The present invention further relates to amino-terminally truncated HTLV env proteins that are soluble and accumulate in the cytoplasm of a host cell. As a result, the HTLV env protein is easily purified from lysed host cells. More specifically, the present invention relates to nucleotide sequences encoding amino-terminal truncated HTLV env proteins, the expression of recombinant HTLV env proteins in host cell systems, and the use of recombinant HTLV env proteins in vaccine formulations for preventing HTLV infection. A current difficulty with mammalian recombinant gene expression is that many proteins are resistant to expression in many systems, making it difficult to predict the likelihood of success. Previous attempts to express HTLV env proteins at high levels have been unsuccessful. The present invention overcomes this difficulty by expressing truncated HTLV env genes in a baculovirus expression system. Transcription of cDNAs corresponding to amino-terminal truncated HTLV env proteins results in unexpectedly abundant transcribed proteins. It has been found that an approximately 50-fold increase in expression of immunologically useful HTLV-I env protein (relative to expression of the full-length gene in mammalian cells) can be achieved. The present invention is further based on the applicant's discovery that an antigenic protein having the amino acid sequence of an HTLV-I or HTLV-II env protein with a deleted amino-terminal leader or signal sequence can protect a recipient when challenged with HTLV-I or HTLV-II vaccination. The immunogenicity of the protein is unexpectedly strong. In a preferred embodiment of the present invention, a nucleotide sequence encoding an amino-terminal truncated HTLV env protein produces an antigenic or immunogenic polypeptide when expressed in a suitable host cell. An antigenic polypeptide is capable of immunospecifically binding to an antibody against the antigen. An immunogenic polypeptide is capable of eliciting an immune response against the antigen, e.g., immunization with the polypeptide produces antibodies that immunospecifically bind to the antigen or generates a cellular immune response against the antigen. In another preferred embodiment of the invention, the antigenic proteins of the invention are expressed in a baculovirus system producing non-glycosylated antigens or in a stably transfected T cell line producing glycosylated antigens. In yet another preferred embodiment of the invention, an amino-terminal truncated version 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 include using these novel antigenic proteins in vaccine formulations, either solely in a prophylactic manner and/or in a therapeutic manner after the recipient has already been infected with either HTLV-I or HTLV-II or both. The novel antigenic proteins of the invention are also useful in diagnostic immunoassays, passive immunotherapy, and for the generation of anti-idiotypic antibodies. 3.1.DefinitionAs used herein, the following terms have the following meanings: The term "gp63" refers to the 63 kilodalton precursor protein of the envelope protein or env protein of the HTLV-I or HTLV-II virus. This term also refers to mutants, variants, or fragments of gp63. The term "gp46" refers to the 46 kilodalton envelope protein or env protein of the HTLV-I or HTLV-II virus. This term also refers to mutants, variants, or fragments of gp46. "env protein" refers to a polypeptide that includes the native sequence of the HTLV-I and/or -II env protein (full-length and truncated), as well as analogs thereof. Preferred analogs are those that are substantially homologous to the corresponding native amino acid sequence, and most preferably encode at least one native HTLV-I and -II env epitope, e.g., a neutralizing epitope. A more preferred type of HTLV-I and -II env polypeptide lacks a sufficient portion of the C-terminal transmembrane domain to facilitate efficient expression and/or secretion of HTLV-I and -II env proteins at high levels from the insect or mammalian cell expression hosts of the invention. An "effective amount" refers to an amount of HTLV-I and -II env polypeptide sufficient to elicit an immune response in a subject to which the polypeptide is administered. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity. The term "treating or preventing HTLV infection" refers to inhibiting HTLV viral replication, inhibiting the spread of HTLV, or preventing HTLV from establishing itself in the host, and reducing or alleviating the symptoms of disease caused by HTLV infection. A reduction in viral load, mortality and/or morbidity is considered treatment. The term "pharmaceutical acceptable carrier" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert, and is not toxic to the patient to whom it is administered. "Therapeutic agent" refers to any molecular compound, preferably an antiviral agent, useful in treating a viral infection or a disease caused thereby. 4.BRIEF DESCRIPTION OF THE DRAWINGS1 shows the nucleotide and amino acid sequences of the HTLV-I env protein. The boxed portions of this sequence correspond to those sequences deleted in one embodiment of the amino-terminal truncated form of the HTLV-I env antigen of the present invention. 2 shows the nucleotide and amino acid sequences of the HTLV-II env protein. The boxed portions of this sequence correspond to those sequences deleted in one embodiment of the amino-terminal truncated form of the HTLV-II env antigen of the present invention. 3 shows the detection of the 63 kDa HTLV-II recombinant env glycoprotein (rgp63) expressed in H5 cells by Western blotting using HTLV-II-infected human serum as a specific antibody (lane 1). This protein was not detected in uninfected insect cells (lane 2). HTLV-II-negative human serum did not react with the HTLV-II env glycoprotein expressed in insect cells (lane 3). Figure 4 shows the detection of antibodies against env gp46 one week after immunization of R3 and R4 with rgp63 (lanes 2 and 3, respectively). The antigen used in this detection system was GSTHTLV-II gp46 fusion protein. Pre-immunization serum from R3 did not react with antibodies against the GST-gp46 fusion protein (lane 1). Figure 5 shows the FACS analysis of HTLV-II infected cell lines using immunized rabbit R3. Positive staining was detected in cell lines Vines and Mo-T. In contrast, CEM was negative. The dotted line is the control FITC-labeled anti-rabbit antibody. The solid line is the anti-HTLV-II env protein rabbit serum used at a 1:10 dilution. F1 is the fluorescence intensity. Figure 6A shows the antibody titers of animals after HTLV-II-Vines vaccination, as determined by the particle agglutination (PA) method. The left panel (A) shows the antibody titers of non-immunized rabbits. The right panel (B) shows the antibody response in rabbits previously immunized with recombinant gp63. The black circle is R1, the white circle is R2, the white square is R3, and the black square is R4. Figure 6B shows the detection of antibodies against GST-gp46 in R1 after inoculation with HTLV-II-Vines cells. The antibodies were first detected and appeared after 2 weeks. The antigen used in this detection system was a GST-gp46 fusion protein transferred to a nylon membrane. Figure 7 shows the detection of HTLV-II proviral DNA by Southern hybridization of nested PCR. The numbers indicate the number of weeks after inoculation. Only live rabbits inoculated with HTLV-II-Vines cells (R1, R2, and R5) showed positivity after a long time. In contrast, vaccinated animals (R3, R4, R8, and R9) or rabbits injected with heat-inactivated HTLV-II-Vines cells (R6 and R7) showed negative responses. 5.Detailed Description of the InventionThe present invention relates to novel antigenic proteins derived from the HTLV env protein. The proteins can be used as immunogens in vaccine formulations to aid in the prevention and treatment of HTLV-I and HTLV-II infections. The present invention also relates to methods for producing such antigens. The present invention relates to HTLV env polypeptides lacking all or part of the membrane-spanning domain. This deletion prevents the polypeptide from anchoring in the membrane of a host cell when recombinantly expressed. In a preferred embodiment of the present invention, the soluble HTLV env protein lacks all or part of its amino terminus. The present invention further relates to soluble, amino-terminally truncated forms of the HTLV env protein that accumulate in the cytoplasm of host cells. This allows the HTLV env protein to be easily purified from lysed host cells. The present invention is based, in part, on the applicant's discovery that antigenic or immunogenic proteins having the amino acid sequence of HTLV-I or HTLV-II envelope proteins with the amino-terminal leader or signal sequence deleted are useful for protecting recipients when challenged by inoculation with HTLV-I or HTLV-II. Furthermore, transcription of cDNA transcripts corresponding to the novel antigenic proteins of the present invention in either eukaryotic or prokaryotic expression systems led to an unexpected enrichment of the transcribed proteins. The present invention relates to nucleotide sequences encoding the novel antigenic proteins, variants and derivatives thereof. The present invention further relates to methods of expressing the novel antigens, including expression vectors and eukaryotic and prokaryotic cell systems. In a preferred embodiment of the present invention, the nucleotide sequences, upon expression in a suitable host cell, produce polypeptides that are antigenic or immunogenic. The antigenic polypeptides are capable of immunospecific binding by antibodies against the antigen. Immunogenic polypeptides are capable of eliciting an immune response against an antigen, for example, when immunized with the polypeptide, eliciting the production of antibodies that immunospecifically bind to the antigen, or eliciting a cellular immune response against the antigen. In another preferred embodiment of the invention, the antigenic proteins of the invention are expressed in a baculovirus system to produce a non-glycosylated antigen, or in a stably transfected T cell line to produce a glycosylated antigen. In yet another preferred embodiment of the invention, an amino-terminally truncated env protein is expressed as a fusion protein to facilitate purification of the protein. In another aspect of the invention, methods of using the novel antigenic proteins described above are described. These methods include the use of the antigenic proteins in vaccine formulations in a solely prophylactic manner and/or in therapeutic treatments after the recipient has already been infected with either HTLV-I or HTLV-II, or both. The novel antigenic proteins of the present invention are also used in diagnostic immunoassays, active immunotherapy, and for the generation of anti-idiotypic antibodies. 5.1Novel ENV glycoprotein antigensThe present invention is based, in part, on the applicant's surprising discovery that the difficulty of expressing useful amounts of HTLV-env protein could be overcome by expressing an amino-truncated form of the HTLV env protein in insect cells or pulmonary mammalian T lymphocytes. Transcription of a cDNA corresponding to the amino-truncated form of the HTLV env protein results in an unexpected enrichment of the transcribed protein. An approximately 50-fold increase in expression of immunologically useful HTLV env protein could be achieved. The present invention is further based on the applicant's discovery that the antigenicity of this protein was unexpectedly strong. An antigenic protein having the amino acid sequence of the HTLV env protein with the amino-terminal leader sequence deleted was useful for inducing the production of anti-HTLV-II antibodies in recipients and for protecting the recipients when challenged by vaccination with HTLV-I or HTLV-II. Due to the high level of amino acid sequence identity between the amino acid sequences of geographically distinct strains of HTLV-I and HTLV-II, the HTLV-I env antigens of the present invention will be useful for protection against many geographic isolates of HTLV-I, and the HTLV-II env antigens of the present invention will be useful for protection against many geographic isolates of HTLV-II. In a preferred embodiment of the present invention, the antigen proteins of the present invention are expressed in a baculovirus system to produce non-glycosylated antigens, or the antigen proteins are expressed in a stably transfected T cell line to produce glycosylated antigens. 5.2.Nucleotide sequence encoding the antigenThe present invention encompasses nucleotide sequences encoding HTLV env proteins and fragments, truncations, and variants thereof. In preferred embodiments of the present invention, nucleotide sequences encoding amino-terminal truncated HTLV env proteins are included. In more preferred embodiments of the present invention, nucleotide sequences encoding amino-terminal 33 amino acid truncations of HTLV-I env proteins are included. The present invention further encompasses nucleotide sequences encoding HTLV-I env proteins truncated by 1-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, or 65-70 amino acids from the amino terminus. The present invention further includes nucleotide sequences encoding amino-terminal truncated HTLV-I env proteins, where the recombinantly expressed polypeptide is not anchored to the host cell membrane but retains antigenic activity similar to the full-length HTLV-I env protein. The present invention further includes intermediate deletions, which include deleting a sufficient portion of the signal sequence domain to cause the recombinantly expressed polypeptide to not be anchored to the host cell membrane but retain antigenic activity similar to the full-length HTLV-I env protein. The HTLV-I env nucleotide sequences of the present invention include the following DNA sequences: (1) any DNA sequence encoding an HTLV-I env protein that is immunologically reactive with anti-HTLV-I env antibodies; (2) any DNA sequence encoding an HTLV-I env protein that contains the amino acid sequence shown in FIG. 1; (3) any DNA sequence that hybridizes to the complement of the DNA sequence shown in FIG. 1 under highly stringent conditions (e.g., 0.5M NaHPO₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washed in 0.1×SSC/0.1% SDS at 68° C. (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)) and still encode functionally equivalent gene products; (4) all nucleotide sequences that hybridize to the complement of the DNA sequence shown in FIG. 1 under less stringent conditions, such as moderately stringent conditions (e.g., washed in 0.2×SSC/0.1% SDS at 42° C. (Ausubel F.M. et al., 1989, supra)) and still encode functionally equivalent HTLV-binding proteins; All nucleotide sequences encoding the env gene product; and (5) all nucleotide sequences that hybridize to the complementary strand of the DNA sequence shown in FIG. 1 under less stringent conditions, such as low stringency conditions (e.g., washing in 0.2×SSC/0.1% SDS at 37° C.) and still encode a functionally equivalent gene product. Functionally equivalent gene products include gene products that are produced at high levels and are immunologically reactive with anti-HTLV-I env antibodies. Another preferred embodiment of the present invention includes a nucleotide sequence encoding an amino-terminal 17 amino acid truncation of the HTLV-II env protein. The invention further includes nucleotide sequences encoding HTLV-II env proteins truncated by 1-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, or 65-70 amino acids from the amino terminus. The invention further includes nucleotide sequences encoding amino-terminal truncated HTLV-II env proteins, such that the recombinantly expressed polypeptide is not anchored to the membrane of a host cell but retains antigenic activity similar to the full-length HTLV-II env protein. Additionally, the invention includes intermediate deletions, which include deleting a sufficient portion of the signal sequence domain to cause the recombinantly expressed polypeptide to not be anchored to the membrane of a host cell but retain antigenic activity similar to the full-length HTLV-II env protein. Additionally, the present invention encompasses intermediate deletions, which delete a sufficient portion of the signal sequence domain to render the recombinantly expressed polypeptide unanchored. The HTLV-II env nucleotide sequences of the present invention include the following DNA sequences: (1) any DNA sequence encoding an HTLV-II env protein that is immunologically reactive with anti-HTLV-II env antibodies; (2) any DNA sequence encoding an HTLV-II env protein that contains the amino acid sequence shown in Figure 2; (3) any DNA sequence that hybridizes to the complement of the DNA sequence shown in Figure 2 under highly stringent conditions (e.g., 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, hybridization to filter-bound DNA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (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. 2103)) and encoding a functionally equivalent gene product; (4) all nucleotide sequences that hybridize to the complement of the DNA sequence shown in FIG. 2 under less stringent conditions, such as moderately stringent conditions (e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel F.M. et al., 1989, supra)) and still produce a functionally equivalent HTLV-II gene product; (5) all nucleotide sequences that hybridize to the complement of the nucleotide sequence shown in FIG. 2 under less stringent conditions, such as low stringency conditions (e.g., washing in 0.2×SSC/0.1% SDS at 37° C.) and still encode a functionally equivalent gene product. Functionally equivalent gene products include gene products that are produced at high levels and are immunologically reactive with anti-HTLV-II env antibodies. The present invention also encompasses the expression of nucleotide sequences that encode immunologically equivalent fragments of the HTLV env protein. Such immunologically equivalent fragments of HTLV env can be identified by making analogs of the nucleotide sequence encoding the protein, with truncations at the 5' and/or 3' ends of the sequence and/or one or more intermediate deletions, expressing the analog nucleotide sequence, and determining whether the resulting fragments immunologically interact with HTLV antibodies or induce the production of such antibodies, particularly neutralizing antibodies, in vivo. For example, preferred embodiments of the invention include expression of nucleotide sequences encoding HTLV env proteins that lack the amino-terminal signal sequence domain and an intermediate region that may facilitate secretion of the env protein. The invention also includes DNA expression vectors containing any of the coding sequences operably linked to regulatory elements that govern expression of the coding sequences, and genetically engineered host cells containing any of the coding sequences operably linked to regulatory elements that govern expression of the coding sequences in the host cells. As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators, and other elements known to those skilled in the art to drive and regulate expression. The env glycoprotein gene products or peptide fragments thereof can be produced by recombinant DNA techniques using techniques well known in the art. Thus, methods for preparing the env glycoprotein gene polypeptides and peptides of the invention by expressing nucleic acids containing env glycoprotein gene sequences are described herein. Methods known to those skilled in the art can be used to construct expression vectors containing the env glycoprotein gene product coding sequence and appropriate transcriptional and translational control signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1989) and Ausubel et al. (1989), supra. Alternatively, RNA capable of encoding the env glycoprotein gene product sequence can be chemically synthesized, for example, using a synthesizer. See, for example, the techniques described in "Oligonucleotide Synthesis" (Gait, M. J., ed., 1984, IRL Press, Oxford), which is incorporated herein by reference in its entirety. The present invention also encompasses nucleotide sequences encoding peptide fragments of the HTLV env gene product. In a preferred embodiment, the present invention relates to polypeptides or peptides corresponding to amino-terminally truncated HTLV env proteins that are soluble and accumulate in the cytoplasm of host cells, facilitating purification of the HTLV env protein from cultured host cells. For example, polypeptides or peptides corresponding to the extracellular domain of the HTLV env protein are useful as "soluble" proteins to facilitate secretion. The HTLV env protein gene product or peptide fragments thereof can be conjugated to heterologous epitopes recognized by commercially available antibodies, which are also included in the present invention. Durable fusion proteins can also be designed. This is a fusion protein that has a cleavage site located between the HTLV env sequence and the heterologous protein sequence, allowing the HTLV env to be excised from the heterologous moiety. For example, a collagenase cleavage recognition consensus sequence can be engineered between the HTLV env protein or peptide and the heterologous peptide or protein. The HTLV env domain can be released from this fusion protein by collagenase treatment. In a preferred embodiment of the present invention, a fusion protein of glutathione-S-transferase and the HTLV env 46 kd protein can be engineered. 5.3.HTLV ENV Antigenic Proteins and PolypeptidesThe present invention relates to HTLV env polypeptides lacking all or a portion of the signal sequence or transmembrane domain such that the polypeptide is not anchored in the membrane of a host cell when recombinantly expressed. In a preferred embodiment of the invention, the soluble HTLV env protein lacks all or a portion of its amino terminus. Preferred embodiments of the invention include HTLV-I env polypeptides lacking 33 amino acids at the amino terminus and HTLV-II env polypeptides lacking 17 amino acids at the amino terminus. HTLV env proteins, polypeptides, and peptides, mutant, truncated, or deleted forms of HTLV env proteins can be prepared for use in vaccine preparations and as pharmaceuticals useful for the treatment and prevention of HTLV-I and -II infections. The nucleotide sequence of the env protein predicts that it has a hydrophobic signal sequence at the amino terminus, five potential acceptor sites for N-glycosidic attachment in the central region, and a second cluster of hydrophobic amino acids in the putative transmembrane domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. 80:3618-3622). The sequence properties suggest that it has a typical structure of a cell membrane glycoprotein. The env gene encodes a precursor protein, gp63, which undergoes proteolysis and subsequent glycosylation to produce two glycoproteins, gp46 and gp21. The gp46 protein constitutes the surface protrusions observed by electron microscopy on native virus particles, is believed to have receptor binding activity, and contains a domain responsible for the production of neutralizing antibodies. The gp21 protein is a transmembrane glycoprotein and may be involved in cell fusion activity. Additionally, the present invention also includes proteins that are functionally equivalent to the HTLV env proteins encoded by the nucleotide sequences set forth in Section 5.2, as judged by a number of characteristics, including but not limited to the ability to be recognized by HTLV env antibodies. Such equivalent HTLV env gene products may contain deletions, additions, or substitutions of amino acids within the amino acid sequences encoded by the HTLV env gene sequences set forth above, which result in silent changes, thus producing functionally equivalent HTLV env gene products. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged amino acids include aspartic acid and glutamic acid. As used herein, "functionally equivalent" refers to a protein that can be recognized by HTLV env antibodies, which is a protein that can elicit an immunological response substantially similar to the endogenous HTLV env gene product described above. Alternatively, random mutations (using random mutation techniques well known to those skilled in the art) can be made into the HTLV env nucleotide sequence, and the resulting HTLV env protein tested for activity. Also, site-specific mutations of the HTLV env coding sequence can be made (using site-specific mutagenesis techniques well known to those of skill in the art) to produce mutant HTLV env proteins with improved function (e.g., leading to increased expression or antigenicity). HTLV env proteins of the invention for use in vaccine preparations are substantially pure or homogeneous. The protein is considered substantially pure or homogeneous when at least 60-70% of the sample exhibits a single polypeptide sequence. A substantially pure protein preferably comprises 60-90%, more preferably about 95% and most preferably 99% of the protein sample. Methods well known to those of skill in the art (e.g., polyacrylamide electrophoresis of the sample followed by staining to visualize a single polypeptide band on the gel) can be used to determine the purity or homogeneity of the protein. Higher resolution can be performed using HPLC or other similar methods well known in the art. The invention includes polypeptides that are typically purified from host cells expressing recombinant nucleotide sequences encoding these proteins. Such protein purification can be accomplished by a variety of methods well known in the art. In a preferred embodiment, the HTLV env protein of the present invention is expressed as a fusion protein with glutathione-S-transferase. The resulting recombinant fusion protein is purified by affinity chromatography to separate the HTLV env domain from the heterologous moiety, resulting in a substantially pure HTLV env protein sample. Other methods may also be used. See, e.g., "Methods In Enzymology," 1990, Academic Press, Inc., San Diego; "Protein Purification: Principles and practice," 1982, Springer-Verlag, New York. 5.4.Expression systemThe present invention includes expression systems, both eukaryotic and prokaryotic expression vectors, that can be used to express truncated and full-length HTLV env proteins. In a preferred embodiment of the invention, the nucleotide sequence of FIG. 1 (with the boxed region deleted and encoding a truncated HTLV-I env protein) is expressed in either a eukaryotic or prokaryotic expression vector. A preferred embodiment of the invention includes expression of full-length and truncated HTLV env gene products in baculovirus to produce non-glycosylated antigens. Another embodiment of the invention includes expression of full-length and truncated HTLV env gene products in stably transfected T cell lines to produce non-glycosylated antigens. A variety of host-expression vector systems can be utilized to express the env glycoprotein encoding sequences of the invention. Such host-expression systems refer to vehicles in which a coding sequence of interest may be produced and substantially purified, but also to cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of displaying the env glycoprotein gene product of the present invention in situ. These include, but are not limited to, bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA containing the env glycoprotein gene product coding sequence; yeast (e.g., Saccharomyces, Pichla) transformed with recombinant yeast expression vectors containing the env glycoprotein gene product coding sequence; and yeast (e.g., Saccharomyces, Pichla) transformed with recombinant viral expression vectors (e.g., baculovirus) containing the env glycoprotein gene product coding sequence. plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the env glycoprotein gene product coding sequence; or mammalian cell systems (e.g., Cos, CHO, BHK, 293, 3T3) carrying promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In bacterial systems, a number of suitable expression vectors may be selected depending on the intended use for the expression of the env glycoprotein gene product. For example, when producing large quantities of such protein for the preparation of pharmaceutical compositions of env glycoprotein or for raising antibodies against env glycoprotein, vectors directing the expression of fusion protein products having high expression levels and which can be easily purified may be desired. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ryther et al., 1983, EMBO. 2:1791), in which the env glycoprotein gene product coding sequence can be individually ligated into the vector in frame with the lacZ coding region to produce a fusion protein; pIN vectors (Inoue and Inoue, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 264:5503-5509), and the like. PGEX vectors can also be used to produce foreign proteins as fusion proteins with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa california nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. The env glycoprotein coding sequence is cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the env glycoprotein gene results in the inactivation of the polyhedrin gene and produces non-occluded recombinant viruses (i.e., viruses lacking the protein coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect insect Spodoptera frugiperda cells in which the inserted virus is expressed. (See, e.g., Smith et al., 1983, J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051). In mammalian host cells, a number of viral-based expression systems are available. When adenovirus is used as an expression vector, the coding sequence of the env glycoprotein gene of interest can be ligated to an adenovirus transcription/translation regulatory complex (e.g., the late promoter and tripartite leader sequence). This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable in an infected host and capable of expressing the env glycoprotein gene product (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of the inserted env glycoprotein gene product coding sequence. These signals include the ATG initiation codon and adjacent sequences. If the entire env glycoprotein gene (including its own initiation codon and adjacent sequences) is inserted into the appropriate expression vector, no additional translational control signals may be required. However, if only a portion of the env glycoprotein gene coding sequence is inserted, exogenous translational control signals, possibly including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. Efficiency of expression may be increased by the inclusion of appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544). 5.5 Cell Lines The present invention includes expression of HTLV env glycoprotein in animal and insect cell lines. In a preferred embodiment of the invention, the env glycoprotein is expressed in a baculovirus vector in an insect cell line to produce a non-glycosylated antigen. In another preferred embodiment of the invention, the env glycoprotein is expressed in a stably transfected T lymphocyte cell line to produce a glycosylated antigen. Host cell lines can be selected that alter expression of the inserted sequences or that modify and process the gene product in a particular desired manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system is selected to ensure correct modification of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38 cell lines. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the env glycoprotein gene product can be engineered. Rather than using an expression vector containing a viral origin of replication, a host cell can be transformed with DNA controlled by appropriate expression regulatory elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selection marker. Following introduction of the foreign DNA, the engineered cells can be grown in rich medium for 1-2 days, followed by switching to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that are then cloned into cell lines. This method can be advantageously used to engineer cell lines that express the env glycoprotein gene product. Such cell lines would be particularly useful for screening and evaluating compounds that affect the endogenous activity of the env glycoprotein gene product. Many selection systems can be used. The herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes were cloned into tk^-, hgprt^-or aprt^-These include, but are not limited to, the ability to employ genes in cells that are capable of inhibiting metabolic pathways, and antimetabolite resistance can be used as the basis for selection for genes that inhibit metabolic pathways, such as: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 159:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147). Alternatively, any fusion protein can be readily purified by utilizing an antibody specific for the expressed fusion protein. For example, a system described by Janknecht et al. allows for the rapid purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with the recombinant vaccinia virus are incubated with Ni²⁺The HTLV env glycoprotein antigen of the present invention can be produced in large quantities, and the antigen thus produced and purified has utility for vaccine preparations. The HTLV env protein also has utility in immunoassays to detect or measure the presence of antibodies against the antigen, for example in a body fluid sample from a vaccinated subject, thus diagnosing infection and/or monitoring the immune response of the subject after vaccination. The preparation of vaccines containing immunogenic polypeptides as active ingredients is known to those skilled in the art. 5.6.1. Determination of Vaccine Efficacy The immunopotency of the HTLV env antigen can be determined by monitoring the immune response in test animals after immunization with the HTLV env antigen, or by using any immunoassay known to those skilled in the art. The production of humoral (antibody) responses and/or cell-mediated immunity can be taken as indicators of immune response. Test animals can include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and finally human subjects. Methods of administering the vaccine can include oral, intracerebral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or any other standard route of immunization. The immune response of the tested subjects can be analyzed by various approaches, for example, by the reactivity of immune sera obtained against HTLV env antigens, as assayed by known techniques such as immunosorbent assay (ELISA), immunoblot, radioimmunoprecipitation, etc., or, if the HTLV env antigens display antigenicity or immunogenicity, by protection of the immunized host from infection with HTLV and/or by reduction of symptoms of HTLV infection in the immunized host. As an example of a suitable animal test for an HTLV env vaccine, the vaccine of the present invention can be tested in rabbits for its ability to induce an antibody response to HTLV env antigens. Male specific pathogen-free (SPF) young adult New Zealand white rabbits can be used. Each test group receives a fixed concentration of vaccine. A control group receives an injection of 1 mM Tris-HCl pH 9.0 without HTLV env antigens. Blood samples can be taken from the rabbits every week or two and the serum can be analyzed for antibodies to HTLV env proteins. The presence of antibodies specific to the antigen can be assayed, for example, using ELISA. 5.6.2. Vaccine Formulations Suitable preparations of such vaccines include injectables, either as liquid dissolutions or suspensions; solid dosage forms suitable for dissolution or suspension in liquid prior to injection can also be prepared. The preparations can also be emulsified or the polypeptides can be encapsulated in liposomes. The active immunogenic ingredient can often be mixed with excipients that are pharma- ceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vaccine preparation may contain small amounts of auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, and/or adjuvants that enhance the effectiveness of the vaccine. Examples of adjuvants that may be effective are aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine. The effectiveness of an adjuvant can be determined by measuring the induction of antibodies against an immunogenic polypeptide containing an HTLV env polypeptide epitope, which can be obtained by administering the polypeptide in a vaccine that also contains various adjuvants. The polypeptide can be formulated as a neutral or salt form to form a vaccine. Pharmaceutically acceptable salts include acid addition salts formed with free amino groups of the peptide, and acid addition salts formed with inorganic acids (e.g., hydrochloric acid, phosphoric acid, etc.) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, maleic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, etc.) or organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.). The vaccine of the present invention can be either multivalent or monovalent. Multivalent vaccines can be made from recombinant viruses capable of expressing more than one antigen. The vaccine formulation of the present invention can be introduced by many methods, including, but not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and scarification (e.g., scratching the upper layer of the skin with a bifurcated needle). The patient to whom the vaccine of the present invention is administered is preferably a mammal, most preferably a human, but may also be a non-human animal, including, but not limited to, cows, horses, sheep, pigs, poultry (e.g., chickens), goats, cats, dogs, hamsters, mice, and rats. The vaccine formulation of the present invention comprises an effective immunizing amount of HTLV env protein and a pharmaceutically acceptable carrier or excipient. The vaccine formulation comprises an effective immunizing amount of one or more antigens and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, or combinations thereof. One example of such an acceptable carrier is a physiologically balanced culture medium containing one or more stabilizers (e.g., stabilized hydrolyzed protein, lactose, etc.). Such carriers are preferably sterile. The formulation should be compatible with the mode of administration. The composition may contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, as required. The composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulations include standard carriers (e.g., pharmaceutical preparations such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.). Generally, the components are supplied separately or mixed together as lyophilized powders or anhydrous concentrates in a sealed container such as an ampule or sachette depending on the amount of active agent. If the composition is to be administered by injection, the components can be supplied in ampules of sterile dilution so that they can be mixed prior to administration. In a specific embodiment, a lyophilized HTLV env polypeptide of the invention is provided in a first container, and a second container contains a diluent consisting of 50% glycerin, 0.25% phenol, and a preservative (e.g., 0.005% brilliant green). The exact dosage of the vaccine formulation to be formulated will also depend on the route of administration and the characteristics of the patient, and should be determined according to the judgment of the practitioner and standard clinical techniques for each patient's condition. An effective immunizing amount is an amount sufficient to produce an immune response to the antigen in the host to which the vaccine formulation is administered. The use of purified antigens as vaccine formulations can be carried out by standard methods. For example, the purified protein can be prepared to an appropriate concentration, formulated with a suitable vaccine adjuvant, and packaged for use. Suitable adjuvants include, but are not limited to, mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols), polyanions, peptides, oil emulsions, alum, and MDP. The immunogen can also be incorporated into liposomes or conjugated to polysaccharides and/or other polymers that can be used in vaccine formulations. For example, if the recombinant antigen is a hapten (i.e., a molecule that is antigenic in that it can selectively react with cognate antibodies, but not immunogenic in that it cannot elicit an immune response), the hapten can be covalently linked to a carrier or immunogenic molecule. For example, large proteins such as serum albumin can confer immunogenicity to a hapten bound to it. The hapten-carrier may be formulated and used as a vaccine. An effective dose (immunizing dose) of the vaccine of the invention may also be estimated based on dose-response curves obtained from animal model test systems. The invention also provides a formulation pack or kit comprising one or more containers containing one or more of the components of the vaccine formulation of the invention. Such containers may bear a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, the notice indicating approval by the governmental agency for manufacture, use or sale for administration to humans. Thus, the invention provides a method of immunizing an animal or treating or preventing various diseases or disorders in an animal, which method comprises administering to the animal an effective immunizing dose of the vaccine of the invention. 5.6.3Uses of antibodies generated by the vaccines of the inventionAntibodies generated against antigens by immunization with the HTLV env protein of the present invention are also useful in diagnostic immunoassays, passive immunotherapy, and in the generation of anti-idiotypic antibodies. The antibodies generated can be isolated by standard techniques known to those skilled in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays. The antibodies can also be used to monitor the progress of treatment and/or disease. Any immunoassay system known to those skilled in the art as described above may be used for this purpose. Examples include, but are not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, enzyme-linked immunosorbent assays (ELISAs), "sandwich" immunoassays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoretic assays, to name a few. The vaccine formulations of the present invention can also be used to generate antibodies for use in passive immunotherapy, in which preformed antibodies against a foreign organism are administered to provide short-term protection to the host. The antibodies generated by the vaccine formulations of the present invention can also be used to generate anti-idiotypic antibodies. Anti-idiotypic antibodies can also be used for immunization, which generate a population of antibodies that bind to the initial antigens of pathogenic microorganisms (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 immunization schedule can be determined by a physician skilled in the art and can be administered with reference to the immune response and antibody titer of the subject. 5.6.4PackagingThe compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms of the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Alternatively, compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, placed in a suitable container, and labeled for processing under the indicated conditions. 6.Example: Expression of HTLV-II envelope proteinIn the examples presented herein, we demonstrate the expression of amino-terminally truncated forms of HTLV envelope proteins. We further demonstrate that the resulting recombinant HTLV envelope proteins are immunoreactive.6.1 Materials and Methods Construction of Baculovirus Transformation Vectors and GST Fusion Vectors The nucleotide sequences of the HTLV-II envelope genes used in these constructs are shown in Figure 2. The nucleotide sequences deleted from the HTLV-II envelope genes are shown in the boxed regions of Figure 2. The primers used to amplify the truncated forms of the HTLV-II envelope genes are underlined in Figure 2. The nucleotide sequences of the HTLV-I envelope genes that can be expressed in the baculovirus transformation vectors and GST fusion vectors are shown in Figure 1. The nucleotide sequences deleted from the HTLV-I envelope genes are shown in the boxed regions of Figure 1. The primers used to amplify the truncated forms of the HTLV-I envelope genes are underlined in Figure 1. Taq polymerase (Cetus), 30 cycles of amplification (94°C, 30 sec, 56°C, 30 sec, 72°C, 1 min), and the following oligonucleotide primers: 5'-AAGGATCCATGGGTAATGTTTTCTTC-3' (5180-5197) and 5'-AAGGATCCThe HTLV-II envelope gene was amplified by PCR from the 3' half genome-containing plasmid Mo (Shimotono) using the following sequence: TTATAGCATGGTTTCTGG-3'(6643-6626) (the BamH1 site is underlined, and the sequence number is derived from the published sequence of HTLV-II-Mo (Shimotono)). The PCR-amplified product was digested with BamH1 and purified in a low melting agarose gel after electrophoresis. The DNA band was excised and ligated into the baculovirus transformation vector pVL1 392 at the BamH1 site located downstream of the polyhedron promoter. The baculovirus vector used to generate these constructs, pVL1 392, is obtained from Invitrogen, San Diego, CA. This recombinant plasmid was used to transfect insect cells. Similarly, a fusion protein of glutathione S-transferase (GST) and the HTLV-II envelope cleavage protein gp46 was prepared by amplifying the plasmid Mo-T with the primers 5'-AAGGATCCATGGGTAATGTTTTCTTC-3' (5180-5197) and 5'-AAGAATTCACGGCGGCGTCTTGTCGCGCCAGG-3' (6103-6086). BamH1 and EcoRI sticky ends were introduced with these primers and used to ligate the fragments together. The expression plasmid pGEX2T (Pharmacia, Upsala, Sweden) was used to express the GST-gp46 protein and purified according to the manufacturer's instructions. Production of recombinant baculovirus To generate recombinant baculovirus, 100 ng of insect cells were cultured in 100 ng of ...⁶Monolayers containing baculovirus (High Five (H5), Invitrogen, San Diego, CA) were co-transfected with the above transformation vector DNA carrying the envelope cDNA and linearized baculovirus DNA (Baculogold, PharMingen, San Diego, CA) using the calcium phosphate method. Single plaques containing recombinant virus were purified from the supernatant and amplified in H5 monolayer cells. Western immunoblot analysis Western blots were used to assess the reactivity of envelope proteins expressed in baculovirus-infected cells with human antisera. Whole cell extracts from H5 cells infected with recombinant baculoviruses were electrophoresed on 10% SDS polyacrylamide gels (SDS-PAGE) and transferred to PVDF membranes (Immobilon, Bedford, MA). Filters were probed with 200-fold diluted serum from an HTLV-11-infected subject (Hall). Bound antibodies were detected by inoculating filters with a 1:5000 dilution of horseradish peroxidase-conjugated anti-human antibody (DAKO A/S, Denmark) followed by development with chemiluminescence (ELC, Amersham, Buckinghamshire, England). Cells and viruses The T-cell line CEM was used as a T-cell negative control for HTLV-II, and the B-cell line BJAB for the fusion assay. HTLV-II-Vines were isolated from a male intravenous drug abuser not infected with human immunodeficiency virus and used to establish an HTLV-II-carrying human lymphoid cell line (Hall). HTLV-II-Mo-T is an HTLV-II-infected lymphoblastoid T-cell line (Saladoon) derived from a Mo subject with the T-cell variant of hairy cell leukemia. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2% glutamine, and 50 μg/ml gentamicin and incubated at 4 °C for 3 h at 2 °C in 5% CO.₂The cells were cultured at 37°C in 100% CO. Insect High Five (H5, Invitrogen, San Diego, CA) cells were maintained in TC100 medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum and 50 μg/ml kanamycin.6.1 .resultPurified recombinant baculoviruses containing the HTLV-II envelope gene were used to infect monolayers of H5 insect cells. Infected insect cells were harvested 4 days after infection and examined for expression of HTLV-II envelope polypeptides. Proteins were separated from lysed cells on polyacrylamide gels, transferred to PVDF membranes, and probed with serum from an HTLV-II-infected subject (Hall) (Figure 2). A protein with an apparent molecular weight of 63 kDa was immunoreactive. The location of the recombinant protein gp63 (rgp63) in insect cells was examined by immunofluorescence. Similarly, infected insect cells were harvested 2 days after infection and incubated with serum from an HTLV-II-infected subject. The majority of infected insect cells bound the antibody, suggesting that recombinant gp63 was located on the surface of the infected insect cells (data not shown). These results demonstrate that immunoreactive HTLV envelope antigens were successfully expressed in baculovirus-infected insect cell systems. 7.Example: Immunization with HTLV-II envelope proteinTo investigate the effect of inoculating rabbits with recombinant gp63 envelope protein, the following assay was performed. In this assay, rabbits were immunized with gp63-expressing insect cells and their sera were assayed for antibodies against HTLV envelope protein. The presence of anti-HTLV envelope antibodies was measured by (1) their ability to detect recombinant GST-gp46 fusion protein expressed in bacteria and (2) their ability to recognize HTLV-II-infected human cells in FACS analysis. 7.1.Materials and MethodsCells and viruses The T-cell line CEM was used as a T-cell negative control for HTLV-II, and the B-cell line BJAB for the fusion assay. HTLV-II-Vines were isolated from a male intravenous drug abuser not infected with human immunodeficiency virus and used to establish an HTLV-II-carrying human lymphoid cell line (Hall). HTLV-II-Mo-T is an HTLV-II-infected lymphoblastoid T-cell line (Saladoon) derived from a Mo subject with the T-cell variant of hairy cell leukemia. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2% glutamine, and 50 μg/ml gentamicin and incubated at 4 °C for 3 h at 2 °C in 5% CO.₂The cells were cultured at 37°C in 10% fetal bovine serum (FACS analysis). High Five (H5, Invitrogen, San Diego, CA) insect cells were maintained in TC100 medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum and 50 μg/ml kanamycin. FACS analysis HTLV-II-Vines, Mo-T, and CEM cell lines were stained with immunized rabbit serum, washed three times with PBS, and then incubated with FITC-conjugated goat anti-human serum (DAK0 A/S, Denmark) at 1-50 in the presence of 2% fetal bovine serum. The relative fluorescence intensity was detected by flow cytometry. Rabbits Specific pathogen-free female New Zealand white rabbits (2.5 kg) were obtained from a commercial rabbit breeder (SCL, Shizuoka, Japan). Groups of rabbits were divided into 5 × 10⁷HTLV-II-infected cells or heat-inactivated cells (70°C for 20 min) were inoculated intravenously. HTLV-II-infected cells were 90% infected as determined by a fluorescent antibody assay using serum from a subject infected with HTLV-II (Figure 5). Syncytium inhibition assay HTLV-II-Vines cells were inoculated in RPMI medium at 10 per ml.⁶The cells were suspended in 100 μl of diluted rabbit serum and aliquots (50 μl per well) were incubated with 100 μl of heat-inactivated diluted rabbit serum in 96-well plates at 37° C. for 15 minutes, followed by addition of BJAB cell suspension (10⁶50 μl of cells was added to each well.₂After 16 hours of incubation at 37°C in an incubator, each well was examined for syncytia (giant polygonal cells) using an inverted microscope. The neutralizing titer of an antibody sample is expressed as the reciprocal of the sample dilution at which syncytia formation in microcultures is completely (100%) inhibited. Rabbit Immunization Recombinant baculovirus-infected H5 cells were pelleted by centrifugation, washed once with PBS, and diluted with 10⁷The samples were resuspended in PBS at a concentration of 1000 cells/ml. Samples (1000 cells/ml) were emulsified in complete (day 0) or incomplete (days 14, 28, and 42) Freund's adjuvant.⁷The cells were injected intramuscularly into female New Zealand White rabbits (2.5 kg). The rabbits were purchased from SLC, Shizuoka, Japan. The rabbit immune serum was collected on day 56. 7.2.resultRabbits were immunized with recombinant gp63-expressing insect cells, and sera were assayed for detection of recombinant GST-fusion proteins using a truncated form of the envelope protein gp46 expressed in bacteria. Because the antigenicity of insect cells is different from that of bacteria, rabbit antisera after immunization do not cross-react with this fusion protein. One week after immunization, sera from rabbits 3 and 4 were reactive to GST-gp46 (Figure 4, columns 2 and 3), whereas pre-immunization serum from R3 did not (column 1). Rabbits immunized with recombinant gp63-expressing insect cells and sera were assayed for detection of HTLV-II-infected human cells by FACS analysis. FACS analysis showed positive staining in Vines and Mo-T cell lines (Figure 5). In contrast, the uninfected T cell line CEM showed a negative staining pattern, suggesting that the rabbit serum contained antibodies against HTLV-II envelope proteins.8.Neutralizing activity of sera from vaccinated animalsHTLV-II-infected cells induce cell fusion after co-culture with the B-cell line BJAB (Hall). Therefore, sera from rabbits immunized with recombinant gp63 were assayed for their ability to block cell fusion. 8.1.material and method Rabbits Specific pathogen-free female New Zealand white rabbits (2.5 kg) were obtained from a commercial rabbit breeder (SCL, Shizuoka, Japan). Groups of rabbits were inoculated with 5 × 10⁷HTLV-II-infected cells or heat-inactivated cells (70°C for 20 min) were inoculated intravenously. HTLV-II-infected cells were 90% infected as measured by a fluorescent antibody assay using serum from a subject infected with HTLV-II (Figure 4). Syncytium inhibition assay HTLV-II-Vines cells were inoculated at 10 per ml in RPMI medium.⁶The cells were suspended and aliquots (50 μl per well) were incubated with 100 μl of heat-inactivated diluted rabbit serum in 96-well plates at 37° C. for 15 min, followed by incubation with BJAB cell suspension (10 per ml).⁶50 μl of cells was added to each well.₂After 16 hours of incubation at 37°C in an incubator, each well was examined for syncytia (large polynuclear cells) using an inverted microscope. The neutralization titer of an antibody sample was expressed as the reciprocal of the sample dilution at which syncytia formation in microcultures was completely (100%) inhibited. Immunization of rabbits Recombinant baculovirus-infected H5 cells were pelleted by centrifugation, washed once with PBS, and then diluted with 10⁷The samples were resuspended in PBS at a concentration of 1000 cells/ml. Samples (1000 cells/ml) emulsified in complete (day 0) or incomplete (days 14, 28, and 42) Freund's adjuvant were⁷The HTLV-II antigen titer was calculated using a particle agglutination kit (Fujirebio, Tokyo, Japan). Purified HTLV-I particle-attached beads were incubated with various dilutions of serum, and the maximum dilution at which agglutination occurred was recorded as the antibody titer. 8.2.resultThe results of incubation of HTLV-II-infected cells with various dilutions of antisera against gp62 showed that at most 1:50 dilutions of sera from samples R3, R4, R8, and R9 were sufficient to completely block fusion in the first bleeding. Fusion was blocked by incubation with antisera, but not with preimmune sera. This suggests that the epitope eliciting fusion activity is located in the gp63, env protein. Previous studies have shown that sera capable of blocking cell fusion always show neutralization of viral infection. Furthermore, since cell-free infection is not possible in HTLV-I and II infections, the inhibition of cell fusion induced by rabbit gp63-immunized sera shows inhibition of HTLV-II infection. Therefore, these results indicate that rabbit gp63-immunized sera has neutralizing activity against HTLV-II. 9.HTLV-II Protection of rabbits from infectionIn the Examples presented herein, the ability of the HTLV env antigen of the present invention to protect rabbits against HTLV-II infection was assayed. 9.1.material and methodHTLV-II challenge and detection of provirus by PCR HTLV-II-Vines cells were washed with PBS and either heat-inactivated or uninactivated intravenously injected into rabbits (5 × 10⁷Every week after infection, PBLs were isolated from heparinized blood samples using lympholyte-Rabbit (Cedar Lane Laboratories, Hornby, Ontario, Canada). DNA samples were prepared from PBMCs using DNAzol (Gibco BRL, Gaithersburg, MD), and 1 μg of DNA was used for PCR analysis. The primers used in the PCR to amplify the tax region were SK43 5'TGGATA CCC CGT CTA CGT GT3' (7248-7267) and SK44, 5'GAG CTG ACA ACG CGT CCA TCG3' (7406-7386), and the primer used in the second step of the PCR was SK43' 5'GCG ATT GTG TAC AGG CCG ATT GGT 3' (7271-7294), which is located immediately downstream of SK43 and acts as a nested primer with SK44 (SK43 and 44 were obtained from PCR protocols M.A. Innis, David H. Gelfand, J.J. Sninsky and T.J. White. Academic press). Southern hybridization 10 μl (from 50 μl of PCR sample) of nested amplified DNA was separated on a 1.5% agarose gel and blotted onto nylon membranes (Schlicher & Schuell). These membranes were hybridized in HybriQuick solution (Strategen, San Diego, CA) for 2 hours at 68°C. PCR fragments generated using S K43 and 44 primers were purified from the agarose gel and (α-³²P) It was randomly primed and labeled with dCTP and then used as a probe. 9.2.result 5x10 to rabbit⁷HTLV-II-Vines cells were inoculated intravenously. In the first experiment, non-immunized groups R1 and R2 seroconverted for HTLV-II 2 weeks after challenge and showed a maximal increase in antibody titers over the next 8 weeks (Figure 4). Western blot analysis of R1 demonstrated the presence of antibodies against recombinant cleaved env-fusion protein after challenge (Figure 6). In rabbits R3 and R4 immunized with insect cells expressing HTLV-II env, antibody titers remained at a plateau for the next 10 weeks after challenge (Figure 5). The presence of HTLV-II nucleotide sequences in peripheral blood lymphocytes (PBL) isolated from immunized rabbits was assayed by PCR analysis. In the first experiment, HTLV-II provirus was detected in DNA samples from PBL of non-immunized rabbits, but not in PBL from immunized rabbits over a 20-week period, as summarized in Table 1. Furthermore, HTLV-II provirus was detected only in the immunized rabbits after the second challenge. To confirm HTLV-II infection, further challenge with heat-inactivated HTLV-II-Vines did not detect provirus after 20 weeks as expected. To confirm the absence of provirus in the immunized rabbits and the presence of provirus in the non-immunized rabbits, HTLV tax gene PCR products were amplified after both challenges and subjected to Southern hybridization (Figure 6). The results of the second PCR analysis demonstrated that after HTLV-II challenge, the non-immunized rabbits contained HTLV provirus, but no HTLV-II provirus was detected in the immunized rabbits. These results indicate that the HTLV env antigen of the present invention conferred protection against HTLV-II viral challenge to the immunized rabbits. The present invention is not intended to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the claims. Various publications are cited throughout this specification, the disclosures of which are incorporated herein by reference in their entirety.

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Claims (1)

[Claims] 1. An antigenic or immunogenic polypeptide having an amino acid sequence corresponding to an HTLV-II envelope protein lacking a sufficient portion of the leader sequence domain such that the polypeptide is not anchored to the host cell membrane when expressed by a genetically engineered host cell, or an analog thereof, and a pharma- tically acceptable carrier. 2. An antigenic or immunogenic polypeptide having an amino acid sequence corresponding to an HTLV-I envelope protein lacking a sufficient portion of the leader sequence domain such that the polypeptide is not anchored to the host cell membrane when expressed by a genetically engineered host cell, or an analog thereof, and a pharma- tically acceptable carrier. 3. A method for treating or preventing a disease or disorder in a subject caused by HTLV-II infection, comprising administering to the subject a polypeptide according to claim 1 in an amount sufficient to protect the subject against challenge with the HTLV-II virus. 4. The method of claim 3, wherein the recombinant protein is produced by a baculovirus-insect cell expression system. 5. The method of claim 3, wherein a signal sequence at the amino terminus of the recombinant protein is truncated. 6. The method of claim 3, wherein the recombinant protein is expressed in a T cell line. 7. A method for treating or preventing a disease or disorder in a subject caused by HTLV-I infection, comprising administering to the subject a polypeptide of claim 2 in an amount sufficient to protect the subject against a challenge with an HTLV-I virus. 8. The method of claim 7, wherein the recombinant protein is produced by a baculovirus-insect cell expression system. 9. The method of claim 7, wherein a signal sequence at the terminal of the recombinant protein is truncated. 10. The method of claim 7, wherein the recombinant protein is expressed in a T cell line. 11. 11. An immunogenic polypeptide having the amino acid sequence of SEQ ID NO: 3, corresponding to a truncated gp63 subunit of an HTLV-II envelope protein lacking an amino-terminal leader sequence, or an analog thereof. 12. An immunogenic polypeptide having the amino acid sequence of SEQ ID NO: 1, corresponding to a truncated gp63 subunit of an HTLV-I envelope protein lacking an amino-terminal leader sequence, or an analog thereof. 13. A method for eliciting in a subject the production of an antibody that specifically binds to an HTLV-II envelope protein, comprising administering to the subject a polypeptide according to claim 1. 14. A method for eliciting in a subject the production of an antibody that specifically binds to an HTLV-I envelope protein, comprising administering to the subject a polypeptide according to claim 2. 15. An antigenic or immunogenic polypeptide having an amino acid sequence corresponding to an HTLV-II envelope protein having a deletion of 33 amino acids from the leader sequence domain. 16. An antigenic or immunogenic polypeptide having an amino acid sequence corresponding to an HTLV-I envelope protein having a deletion of 17 amino acids from the leader sequence domain. 17. A method for eliciting an immune response in a subject to treat or prevent a disease caused by HTLV-II infection, comprising administering to the subject an amount of the polypeptide of claim 15 sufficient to produce HTLV-II specific antibodies. 18. A method for treating or preventing a disease caused by HTLV-I infection, comprising administering to the subject an amount of the antigenic or immunogenic polypeptide of claim 16 sufficient to enhance an HTLV-I specific immune response. 19. The method of claim 17, wherein the signal sequence at the amino terminus of the recombinant protein is truncated. 20. The method of claim 17, wherein the recombinant protein is expressed in a T cell system. 21. The method of claim 17, wherein the recombinant protein is produced by a baculovirus-insect cell expression system. 22. The method of claim 18, wherein the amino-terminal signal sequence of the recombinant protein is truncated. 23. The method of claim 18, wherein the recombinant protein is expressed in a T cell line. 24. The method of claim 18, wherein the recombinant protein is produced by a baculovirus-insect cell expression system.