The present application is directed to stabilized envelope glycoprotein trimers. The trimers are stabilized by introducing disulfide bonds at certain sites in the gp41 ectodomain. DNA molecules encoding such trimers can be used to generate an immunogenic reaction.

DETAILED DESCRIPTION OF THE INVENTION

We have now discovered an improved immunogenic gp120-gp 41oligomer, sometimes referred to as gp 160 and DNA sequences encoding them. This oligomer is stabilized by the creation of cysteine-SH-cysteine bonds. Moreover, by appropriate placement of the cysteine residue in the gp 41 portion, the resulting oligomer forms spikes similar to that seen in the native wild type virus. Consequently, antibodies generated by these polypeptides are more likely to recognize and interact with native virus.

The gp160 glycoprotein is the precursor for gp120 and gp 41. Following oligomerization of the precursor the gp 160 glycoprotein is transported to the Golgi apparatus where cleavage by a cellular protease generates the gp120 and gp41 glycoproteins, which remain associated through non-covalent interactions (Earl, P. L., et al., J Virol 1991, Kowalsid, M., et al., Science 1987). In mammalian host cells, addition of complex sugars to selected, preferably surface-exposed, carbohydrate side chains of the envelope glycoproteins occurs in the Golgi apparatus (Leonard, C. K., et al, J Biol Chem 1990).

The mature envelope glycoprotein complex is incorporated into virions, where it mediates virus entry into the host cell. The gp120 exterior envelope glycoprotein binds the CD4 glycoprotein, which serves as a receptor for the virus (Klatzmann, D., et al., Nature 1984, Dalgleish, A. G., et al, Nature 1984). Because gp120 is external as discussed above it was proposed as a natural target for trying to develop an immune response to prevent viral entry. However, in part due to the numerous variable regions which can mutate rapidly, the wild type gp 120 has not proven to be a successful target. An approach to using a modified gp 120 polypeptide wherein at least portions of the variable region have been removed, while the overall 3-dimensional conformation is retained [Sodroski, et al, 39813 which is incorporated herein by reference] has avoided some of these problems.

The importance of the envelope glycoprotein has been underscored by recent discoveries. The binding of gp 120 to CD4 is followed by interaction of the gp120-CD4 complex with one of the chemokine receptors, which are seven-transmembrane G protein-coupled receptors (Feng, Y., et al., Science 1996; Choe, H., et al., Cell 1996; Doranze, et al., Cell 1996; Dragic, et al., Nature 1996; Alkhatib, G., et al., Science 1996). The chemokine receptor interaction is believed to bring the viral envelope glycoprotein complex nearer to the target cell membrane and to trigger additional conformational changes in the envelope glycoproteins (Wu, L., et al., Nature 1996; Trkola, A., et al., Nature 1996). These changes are proposed to result in the interaction of the gp41 glycoprotein with the target cell membrane, culminating in fusion of this membrane with the viral membrane. Such a model is consistent with mutagenic analysis. Amino acid changes in the hydrophobic gp41 amino terminus (the "fusion peptide"), in the amino-terminal half of the ectodomain, or in the transmembrane region all result in fusion-defective envelope glycoproteins (Kowalski, M., Science 1987; Freed, E. O., Proc Natl Acad Sci 1990; Cao, J., J Virol 1993). All these factors confirm the importance of the envelope glycoprotein. However, in nature an oligomeric form is seen. Thus, being able to prepare a stable oligomer containing the gp 120 portion is extremely important. Yet, the stable oligomer must approximate the conformation of the oligomer formed naturally. This has proven difficult. First, the HIV-1 envelope glycoprotein oligomer is naturally labile, disassociating into individual subunits readily. Second, the introduction of cysteine residues in inappropriate positions can result in non-native structures. Since these molecules are folded differently than the native HIV-1 envelope glycoproteins, their utility in raising antibodies that recognize and neutralize the viral envelope spike is limited. We have discovered that there are only a limited number of positions in the gp 41 portion that can be used to create a stable oligomer that approximates the native conformation.

Soluble forms of HIV-1 envelope glycoprotein oligomers should have advantages over monomeric gp120 preparations as immunogens, since the former are more likely to mimic the native envelope glycoprotein spike on virions (Broder, C. C., et al., Proc Natl Acad Sci USA 1994). Unfortunately, due to the lability of HIV-1 envelope glycoprotein, the preparation of high-quality stable oligomers that maintain high-order states has been difficult. We have found that preparation of a DNA sequence encoding complex having selective introduction of cysteine residues in the gp41 ectodomain helices results in disulfide bonds, between the expressed monomers resulting in stable envelope glycoprotein oligomers having a conformation approximating the native as demonstrated by the binding of antibodies to native of the oligomer to these constructs. Present in an N-terminal gp41 alpha helix is a heptad repeat of hydrophobic residues at the first (`a`) and fourth position (`d`), which is the hallmark of a coiled coil (O'Shea, et al., Science 1991). Coiled coils are believed to play a central role in influenza virus entry mediated by the hemagglutinin molecule, where the extension of a trimeric coiled coil in the transmembrane HA.sub.2 subunit is thought to mark the transition to a fusogenic conformation of this protein (Carr, C. M., et al., Cell 1993; Bullogh, P. A., Nature 1994). Recently, a crystal structure of an HIV-1 gp41 ectodomain fragment has been obtained, confirming the existence of a trimeric coiled coil that is bound and stabilized by three monomers of a C-terminal helix (Chan, D. C., Cell 1997). It was not clear from this data if this is the form used by the complex of gp 120-gp41 because the HIV-1 gp41 glycoprotein is thought to undergo conformational changes from its conformation in the gp160 precursor. Consequently, whether the crystallographic structure obtained for the gp41 ectodomain fragment corresponds to that found in the gp160 envelope glycoprotein precursor or represents a fusion-competent conformation was uncertain. The results we have obtained demonstrate the relevance of the available gp41 structures to the complete HIV-1 envelope gp 160 (gp 20-gp41) and imply that at least some of the molecular contacts observed are present before the induction of a fusogenic conformation.

By using DNA sequences encoding gp160 and/or gp41-gp120 proteins and by selective introduction of cysteines at specific locations in the HIV-1 gp41 coiled coil we can stabilize dimeric and trimeric forms of a conformational gp160 polypeptide such as based upon a processing-defective gp160 glycoprotein. This glycoprotein was expressed efficiently on the cell surface and was precipitated by antibodies that recognize conformation-dependent gp120 epitopes (Moore, J. P., et al., J Virol 1996; Thali, M., et al., J Virol 1993) but was gp160 processing defective. Thus, the impaired processing not appear to result from inefficient folding or transport along the secretory pathway. Although not wishing to be bound by theory we believe the processing defect could reflect a subtle conformational alteration in the envelope glycoprotein region recognized by the cellular protease, or could suggest that a degree of flexibility at the gp 120/gp41 cleavage site is necessary for efficient processing and is not present in the LQA/CCG mutant.

Traditional approaches at generating antibodies to env have typically focused on the gp 120 polypeptide. However, we found that creating a fusion protein containing a gp120 portion, preferably a modified gp 120 portion, and a modified gp 41 portion permits the creation of stable oligomers.

As will be discussed in detail below the preferred modified gp 120 portion is a gp 120 protein that has been modified to have variable loops or portions thereof.

The HIV-1 envelope glycoprotein oligomer may be stabilized through intersubunit disulfide bonds. One preferred structure has cysteine residues introduced at residues adjacent to the d and e positions of the coiled coil helix in gp 41. See FIG. 1B for the amino acid and a nucleotide sequence of this region. These positions correspond to 576 and 577 of HIV-1. These residues are highly conserved among HIV-1 and HIV-2 strains, indicating that the approach is applicable to both HIV-1 and HIV-2. These positions correspond to 576 and 577 of the HXBc2 isolate of HIV-1. The numbering varies slightly for different HIV-1 isolates, although the sequence in this region of the gp41 coiled coil is largely conserved. Therefore, the equivalently positioned residues are easily identified in other HIV-1 and, in fact, in HIV-2 envelope glycoproteins as well.

Other sites along the gp41 coiled coil could also be used for the introduction of cysteines (See FIG. 6). These sites are numbered 555/556, 562/563, 569/570, and 583/584 in the HXBc2 HIV-1 sequence. Analogous to the glycine substitution at position 578, glycines could be introduced adjacent to the introduced cysteines, at positions 557, 564, 571 and 584, respectively.

In order to maintain the overall conformation it is desirable to substitute an adjoining amino acid residue with one that provides flexibility in turning. Preferably, the residue is Gly. For example, substituting gly for ala at position f of the helix in the above example of 576/577 corresponds to position 578. These monomers are useful in producing stable trimers for structural or vaccine purposes, where the lability of these higher-order forms has been problematic. Disulfide crosslinking of the HIV-1 envelope glycoprotein trimer stabilizes otherwise labile neutralization epitopes specific for the oligomer and the form can mask biologically irrelevant epitopes that are exposed on the gp 120 or gp160 monomer but buried on the functional oligomer, and lengthen the half-life of the intact vaccine construct in the body. With the availability of a crystallographic model of the gp41 exterior domain, the disulfide crosslinking strategy described herein can be used with other elements of the gp 41 coiled coil based upon our teaching (See FIG. 6).

Dimers as well as trimers of the mutant may be stabilized by the formation of disulfide bonds. The dimer form of the mutant was less abundant than the trimer and was more sensitive to a disruption by boiling (data not shown). Stable dimers could represent intermediates in the assembly or disassembly of the trimer. Alternatively, the dimer could result from the formation of an alternative disulfide bond between the cysteines in the d positions, excluding the possibility of forming the three d-e disulfide bonds presumably present in the trimer. However, we believe the dimer is an artifact.

The oligomer complexes can be used to generate a range of antibodies to gp120 and gp41. For example, antibodies that affect the interaction with the binding site can be directly screened for example using a direct binding assay. For example, one can label, e.g. radioactive or fluorescent, a gp120 protein or derivative and add soluble CD4. There are various soluble CD4s known in the art including a two-domain (D1D2 sCD4) and a four-domain version. The labeled gp120, or derivative, e.g., a conformationally intact deletion mutant such as one lacking portions of the variable loops (e.g. V1/V2) and in some instances constant regions and soluble CD4 can be added to medium containing a cell line expressing a chemokine receptor that the antibody will block binding to. In this example, the derivative will blocking binding to CCR5. Alternatively, when using a derivative from a T cell tropic gp120 one would use a cell line that expresses CXCR4. Binding can then be directly measured. The antibody of interest can be added before or after the addition of the labeled gp120 or derivative and the effect of the antibody on binding can be determined by comparing the degree of binding in that situation against a base line standard with that gp120 or derivative, not in the presence of the antibody.

A preferred assay uses the labeled gp120, or derivative portion, for example a gp120 protein derived from an M-tropic strain such as JR-FL, iodinated using for instance solid phase lactoperoxidase (in one example having a specific activity of 20 .mu.Ci/.mu.g). The cell line containing the chemokine receptor in this example would be a CCR5 cell line, e.g. L1.2 or membranes thereof. Soluble CD4 would be present.

In one embodiment, the conformational gp 120 portion should contain a sufficient number of amino acid residues to define the binding site of the gp120 to the chemokine receptor (e.g. typically from the V3 loop) and a sufficient number of amino acids to maintain the conformation of the peptide in a conformation that approximates that of wild-type gp120 bound to soluble CD4 with respect to the chemokine receptor binding site. In other embodiments the V3 loop can be removed to remove masking amino acid residues. In order to maintain the conformation of the polypeptide one can insert linker residues that permit potential turns in the polypeptides structure. For example, amino acid residues such as Gly, Pro and Ala. Gly is preferred. Preferably, the linker residue is as small as necessary to maintain the overall configuration. It should typically be smaller than the number of amino acids in the variable region being deleted. Preferably, the linker is 8 amino acid residues or less, more preferably 7 amino acid residues or less. Even more preferably, the linker sequence is 4 amino acid residues or less. In one preferred embodiment the linker sequence is one residue. Preferably, the linker residue is Gly.

In one preferred embodiment, the gp120 portion also contains a CD4 binding site (e.g. from the C3 region residues 368 and 370, and from the C4 region residues 427 and 457). The chemokine binding site is a discontinuous binding site that includes portions of the C2, C3, C4 and V3 regions. By deletion of non-essential portions of the gp 120 polypeptide--such as deletions of portions of non-essential variable regions (e.g. V1/V2) or portions in the constant regions (e.g. C1, C5) one can increase exposure of the CD4 binding site. Another embodiment is directed to a gp120 portion containing a chemokine binding site. Similarly, by deleting the non-essential portions of the protein one can increase exposure of the chemokine binding site. The increased exposure enhances the ability to generate an antibody to the CD4 receptor or chemokine receptor, thereby inhibiting viral entry. Removal of these regions is done while requiring the derivative to retain an overall conformation approximating that of the wild-type protein with respect to the native gp120 binding region, e.g. the chemokine binding region when complexed to CD4. In addition, one can remove glycosylation sites that are disposable for proper folding. Maintaining conformation can be accomplished by using the above-described linker residues that permit potential turns in the structure of the gp120 derivative to maintain the overall three-dimensional structure. Preferred amino acid residues that can be used as linker include Gly and Pro. Other amino acids can also be used as part of the linker, e.g. Ala. Examples on how to prepare such peptides are described more fully in Wyatt, R., et al. J. of Virol. 69:5723 5733 (1995); Thali, M., et al., J. of Virol. 67:3978 3988 (1993); and U.S. application Ser. No. 07/858,165 filed Mar. 26, 1992 which are incorporated herein by reference. See for example Wyatt which teaches how to prepare V1/V2 deletions that retain the stem portion of the loop.

In one embodiment the gp120 derivative is designed to be permanently attached at the CD4 binding site to sufficient domains of CD4 to create a conformation of the chemokine binding site approximating that of the native gp120 CD4 complex.

An alternative gp120 derivative is one wherein the linkers used result in a conformation for the derivative so that the discontinuous binding site with the chemokine receptor approximates the conformation of the discontinuous binding site for the chemokine receptor in the wild-type gp120/CD4 complex. These derivatives can readily be made by the person of ordinary skill in the art based upon the above described methodologies and screened in the assays shown herein to ensure that proper binding is obtained.

The gp120 polypeptide portion is bound to at least a portion of gp41 polypeptide, namely the coiled coil. Some of these derivatives will lack the gp41 transmembrane region and will therefore be made as secreted, soluble oligomers. For example, gp41 portions lacking the transmembrane region but retaining the cytoplasmic region, others truncated beginning with the transmembrane region, and therefore also lacking the cytoplasmic region. In an alternative embodiment, one can substitute amino acid residues in the transmembrane region which results in anchoring the protein with other amino acid residues. Preferably, those amino acids although being residues that do not bind to the membrane, would be selected to have minimal conformational effect on the polypeptides. These amino acids can readily be selected by the skilled artisan based upon known knowledge in view of the present disclosure. This can be done by standard means using known techniques such as sets directed mulogenesis. The gp41 polypeptide contains the indicated cysteine residues, which result in the formation of the SH bonds between the monomers thereby stabilizing the complex as a trimer having spikes similar to that found in the wild type. These immunogenic oligomers can be used to generate an immune reaction in a host by standard means. For example one can administer the trimeric protein in adjuvant. In another approach, a DNA sequence encoding the gp120-gp41 complex can be administered by standard techniques. The approach of administering the protein is presently preferred.

The protein is preferably administered with an adjuvant. Adjuvants are well known in the art and include aluminum hydroxide, Ribi adjuvant, etc. The administered protein is typically an isolated and purified protein. The protein is preferably purified to at least 95% purity, more preferably at least 98% pure, and still more preferably at least 99% pure. Methods of purification while retaining the conformation of the protein are known in the art. The purified protein is preferably present in a pharmaceutical composition with a pharmaceutically acceptable carrier or diluent present.

DNA sequences encoding these proteins can readily be made. For example, one can use the native gp 160 of any of a range of HIV-1 strains which are well known in the art and can be modified by known techniques such to deleted the undesired regions such as variable loops and to insert desired coding sequences such as cysteines and linker segments. In addition to DNA sequences based upon existing strains, the codons for the various amino acid residues are known and one can readily prepare alternative coding sequences by standard techniques.

DNA sequences can be used in a range of animals to express the monomer, which then forms into the trimer and generates an immune reaction.

DNA sequences can be administer to a host animal by numerous methods including vectors such as viral vectors, naked DNA, adjuvant assisted DNA catheters, gene gun, liposomes, etc. In one preferred embodiment the DNA sequence is administered to a human host as either a prophylactic or therapeutic treatment to stimulate an immune response, most preferably as a prophylactic. One can administer cocktails containing multiple DNA sequences encoding a range of HIV env strains.

Vectors include chemical conjugates such as described in WO 93/04701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses and HIV-based viruses. One preferred HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors are preferred. These vectors include herpes virus vectors such as a herpes simplex I virus (HSV) vector [Geller, A. I. et al. J. Neurochem 64: 487 (1995); Lim, F. et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci. U.S.A. 90: 7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87: 1149 (1990)], adenovirus vectors [LeGal LaSalle et al., Science 259: 988 (1993); Davidson, et al., Nat. Genet 3: 219 (1993); Yang, et al., J. Virol. 69: 2004 (1995)] and adeno-associated virus vectors [Kaplitt, M. G., et al., Nat. Genet. 8:148 (1994)].

The DNA sequence would be operably linked to a promoter that would permit expression in the host cell. Such promoters are well known in the art and can readily be selected. Stabilized forms of these complexes can readily be made, for example, by conjugates such as a poly(alkylene oxide) conjugate. The conjugate is preferably formed by covalently bonding the hydroxyl terminals of the poly(alkylene oxide) and a free amino group in the gp120 portion that will not affect the conformation of the discontinuous binding site. Other art recognized methods of conjugating these materials include amide or ester linkages. Covalent linkage as well as non-covalent conjugation such as lipophilic or hydrophilic interactions can be used.

The conjugate can be comprised of non-antigenic polymeric substances such as dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinyl alcohols, polyacryl amides or other similar substantially non-immunogenic polymers. Polyethylene glycol(PEG) is preferred. Other poly(alkylenes oxides) include monomethoxy-polyethylene glycol polypropylene glycol, block copolymers of polyethylene glycol, and polypropylene glycol and the like. The polymers can also be distally capped with C1 4 alkyls instead of monomethoxy groups. The poly(alkylene oxides) used must be soluble in liquid at room temperature. Thus, they preferably have a molecular weight from about 200 to about 20,000 daltons, more preferably about 2,000 to about 10,000 and still more preferably about 5,000.

One can administer these stabilized compounds to individuals by a variety of means. For example, these antibodies can be included in vaginal foams or gels that are used as preventives to avoid infection and applied before people have sexual contact.

The peptides or antibodies when used for administration are prepared under aseptic conditions with a pharmaceutically acceptable carrier or diluent.

Doses of the pharmaceutical compositions will vary depending upon the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 .mu.g/kg a day, more preferably 1 to 10,000 .mu.g/kg.

Routes of administration include oral, parenteral, rectal, intravaginal, topical, nasal, ophthalmic, direct injection, etc.

Changes in the viral envelope glycoproteins, in particular in the third variable (V3) region of the gp120 exterior envelope glycoprotein, determine tropism-related phenotypes (Cheng-Mayer et al., 1990; O'Brien et al., 1990; Hwang et al., Westervelt et al., 1992; Chesebro et al., 1992; Willey et al., 1994). Amino acid changes in the V3 region (Helseth et al., 1990; Freed et al., 1991; Ivanoff et al., 1991; Bergeron et al., 1992; Grimaila et al., 1992; Page et al., 1992; Travis et al., 1992) and the binding of antibodies to this domain (Putney et al., 1986; Goudsmit et al., 1988; Linsley et al., 1988; Rusche et al., 1988; Skinner et al., Javeherian et al., 1989) have been shown to disrupt a virus entry process other than CD4 binding. Accordingly, one can create derivatives and change the phenotype for a particular receptor by substituting V3 loops.

One can inhibit infection by directly blocking receptor binding. This can be accomplished by a range of different approaches. For example, antibodies. One preferred approach is the use of antibodies to the binding site for these chemokine receptors. Antibodies to these receptors can be prepared by standard means using the stable immunogenic oligomers. For example, one can use single chain antibodies to target these binding sites.

As used herein the inhibition of HIV infection means that as compared to a control situation infection is reduced, inhibited or prevented. Infection is preferably at least 20% less, more preferably at least 40% less, even more preferably at least 50% less, still more preferably at least 75% less, even more preferably at least 80% less, and yet more preferably at least 90% less than the control.

One preferred use of the antibodies is to minimize the risk of HIV transmission. These antibodies can be included in ointments, foams, creams that can be used during sex. For example, they can be administered preferably prior to or just after sexual contact such as intercourse. One preferred composition would be a vaginal foam containing one of the antibodies. Another use would be in systemic administration to block HIV-1 replication in the blood and tissues. The antibodies could also be administered in combination with other HIV treatments.

Pharmaceutic Compositions

An exemplary pharmaceutical composition is a therapeutically effective amount of a the oligomer, antibody etc. that for examples affects the ability of the receptor to facilitate HIV infection or for the DNA sequence or the oligomer that can induce an immune reaction, thereby acting as a prophylactic immunogen, optionally included in a pharmaceutically-acceptable and compatible carrier. The term "pharmaceutically-acceptable and compatible carrier" as used herein, and described more fully below, includes (i) one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to a human or other animal, and/or (ii) a system, such as a retroviral vector, capable of delivering the molecule to a target cell. In the present invention, the term "carrier" thus denotes an organic or inorganic ingredient, natural or synthetic, with which the molecules of the invention are combined to facilitate application. The term "therapeutically-effective amount" is that amount of the present pharmaceutical compositions which produces a desired result or exerts a desired influence on the particular condition being treated. For example, the amount necessary to raise an immune reaction to provide prophylactic protection. Typically when the composition is being used as a prophylactic immunogen at least one "boost" will be administered at a periodic internal after the initial administration. Various concentrations may be used in preparing compositions incorporating the same ingredient to provide for variations in the age of the patient to be treated, the severity of the condition, the duration of the treatment and the mode of administration.

The term "compatible", as used herein, means that the components of the pharmaceutical compositions are capable of being commingled with a small molecule, nucleic acid and/or polypeptides of the present invention, and with each other, in a manner such that does not substantially impair the desired pharmaceutical efficacy.

Dose of the pharmaceutical compositions of the invention will vary depending on the subject and upon particular route of administration used. Dosages can range from 0.1 to 100,000 .mu.g/kg per day, more preferably 1 to 10,000 .mu.g/kg. By way of an example only, an overall dose range of from about, for example, 1 microgram to about 300 micrograms might be used for human use. This dose can be delivered at periodic intervals based upon the composition. For example on at least two separate occasions, preferably spaced apart by about 4 weeks. Other compounds might be administered daily. Pharmaceutical compositions of the present invention can also be administered to a subject according to a variety of other, well-characterized protocols. For example, certain currently accepted immunization regimens can include the following: (i) administration times are a first dose at elected date; a second dose at 1 month after first dose; and a third dose at 5 months after second dose. See Product Information, Physician's Desk Reference, Merck Sharp & Dohme (1990), at 1442 43. (e.g., Hepatitis B Vaccine-type protocol); (ii) Recommended administration for children is first dose at elected date (at age 6 weeks old or older); a second dose at 4 8 weeks after first dose; a third dose at 4 8 weeks after second dose; a fourth dose at 6 12 months after third dose; a fifth dose at age 4 6 years old; and additional boosters every 10 years after last dose. See Product Information, Physician's Desk Reference, Merck Sharp & Dohme (1990), at 879 (e.g., Diptheria, Tetanus and Pertussis-type vaccine protocols). Desired time intervals for delivery of multiple doses of a particular composition can be determined by one of ordinary skill in the art employing no more than routine experimentation.

The antibodies, DNA sequences or oligomers of the invention may also be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of this invention. Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulphonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Thus, the present invention also provides pharmaceutical compositions, for medical use, which comprise nucleic acid and/or polypeptides of the invention together with one or more pharmaceutically acceptable carriers thereof and optionally any other therapeutic ingredients.

The compositions include those suitable for oral, rectal, intravaginal, topical, nasal, ophthalmic or parenteral administration, all of which may be used as routes of administration using the materials of the present invention. Other suitable routes of administration include intrathecal administration directly into spinal fluid (CSF), direct injection onto an arterial surface and intraparenchymal injection directly into targeted areas of an organ. Compositions suitable for parenteral administration are preferred. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing the active ingredients of the invention into association with a carrier which constitutes one or more accessory ingredients.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the nucleic acid and/or polypeptide of the invention in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.

Preferred compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the molecule of the invention which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectibles.

Antibodies The term "antibodies" is meant to include monoclonal antibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with polypeptides encoded by eukaryotic nucleotide sequences of the present invention. The term "selectively reactive" refers to those antibodies that react with one or more antigenic determinants on e.g. gp120 and do not react with other polypeptides. Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Antibodies can be used for diagnostic applications or for research purposes, as well as to block bindiner interactions.

For example, cDNA clone encoding a gp120-gp41 complex of the present invention may be expressed in a host using standard techniques (see above; see Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.: 1989) such that 5 20% of the total protein that can be recovered from the host is the desired protein. Recovered proteins can be electrophoresed using PAGE and the appropriate protein band can be cut out of the gel. The desired protein sample can then be eluted from the gel slice and prepared for immunization. Preferably, one would design a stable cell could expressing high levels of the proteins which be selected and used to generate antibodies

For example, mice can be immunized twice intraperitoneally with approximately 50 micrograms of protein immunogen per mouse. Sera from such immunized mice can be tested for antibody activity by immunohistology or immunocytology on any host system expressing such polypeptide and by ELISA with the expressed polypeptide. For immunohistology, active antibodies of the present invention can be identified using a biotin-conjugated anti-mouse immunoglobulin followed by avidin-peroxidase and a chromogenic peroxidase substrate. Preparations of such reagents are commercially available; for example, from Zymad Corp., San Francisco, Calif. Mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production. Positive supernatants of such hybridomas can be identified using the assays described above and by, for example, Western blot analysis.

To further improve the likelihood of producing an antibody as provided by the invention, the amino acid sequence of polypeptides encoded by a eukaryotic nucleotide sequence of the present invention may be analyzed in order to identify desired portions of amino acid sequence which may be associated with receptor binding. For example, polypeptide sequences may be subjected to computer analysis to identify such sites.

For preparation of monoclonal antibodies directed toward polypeptides encoded by a eukaryotic nucleotide sequence of the invention, any technique that provides for the production of antibody molecules by continuous cell lines may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (Nature, 256: 495 497,1973), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies, and the like, are within the scope of the present invention. See, generally Larrick et al., U.S. Pat. No. 5,001,065 and references cited therein. Further, single-chain antibody (SCA) methods are also available to produce antibodies against polypeptides encoded by a eukaryotic nucleotide sequence of the invention (Ladner et al. U.S. Pat. Nos. 4,704,694 and 4,976,778).

The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. The present invention provides for antibody molecules as well as fragments of such antibody molecules.

Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.

Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom 1984, "Specific killing of lymphocytes that cause experimental Autoimmune Myasthenia Gravis by toxin-acetylcholine receptor conjugates." Jour. Immun. 133:1335 2549; Jansen, F. K., H. E. Blythman, D. Carriere, P. Casella, O. Gros, P. Gros, J. C. Laurent, F. Paolucci, B. Pau, P. Poncelet, G. Richer, H. Vidal, and G. A. Voisin. 1982. "Immunotoxins: Hybrid molecules combining high specificity and potent cytotoxicity". Immunological Reviews 62:185 216; and Vitetta et al., supra).

Preferred linkers are described in the literature. See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201 208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, Umemoto et al. U.S. Pat. No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbociimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing Linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

Antibodies of the present invention can be detected by appropriate assays, such as the direct binding assay discussed earlier and by other conventional types of immunoassays. For example, a sandwich assay can be performed in which the receptor or fragment thereof is affixed to a solid phase. Incubation is maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. After this first incubation, the solid phase is separated from the sample. The solid phase is washed to remove unbound materials and interfering substances such as non-specific proteins which may also be present in the sample. The solid phase containing the antibody of interest bound to the immobilized polypeptide of the present invention is subsequently incubated with labeled antibody or antibody bound to a coupling agent such as biotin or avidin. Labels for antibodies are well-known in the art and include radionuclides, enzymes (e.g. maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, and the like. The labeled antibodies are incubated with the solid and the label bound to the solid phase is measured, the amount of the label detected serving as a measure of the amount of anti-urea transporter antibody present in the sample. These and other immunoassays can be easily performed by those of ordinary skill in the art.
 

Claim 1 of 10 Claims

1. An isolated molecule containing a nucleotide sequence encoding an HIV-1 or HIV-2 envelope glycoprotein containing at least: i) a coiled coil portion of a gp41 transmembrane glycoprotein, wherein said coiled coil has a heptad repeat wherein each of said seven consecutive amino acid residues are designated a, b, c, d, e, f, and g corresponding to amino acid sequences selected from a group consisting of amino acids 555 561, 562 568, 569 575, 576 582 and 583 589 of SEQ ID NO: 11, wherein at least two amino acids in positions "a", "d" and "e" have been substituted by cysteine residues, and "f" is glycine; and ii) a gp120 glycoprotein or gp120 derivative, wherein the gp120 derivative contains multiple gp120 constant regions connected by variable regions and/or linker residues that permit potential turns in the polypeptide structure so that the derivative maintains a conformation approximating that of wild type gp120, wherein at least a portion of one variable region has been deleted.
 

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