Patent 6,534,285

Diagnostic product and vaccine for Acquired Immuno-deficiency Syndrome (AIDS) and methods for making and using same, wherein viral polypeptide sequences from an AIDS associated retrovirus are expressed directly or as a fusion polypeptide in a prokaryotic or mammalian cell expression host to produce a diagnostic product which specifically binds complementary antibody produced by individuals afflicted with AIDS or a vaccine against AIDS which confers resistance to infection by AIDS associated retrovirus. The reverse transcriptase of an AIDS associated retrovirus is used separately or in a whole cell assay to identify compounds which selectively inhibit retroviral reverse transcriptase.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have demonstrated that viral protein from an AIDS associated retrovirus can be expressed directly or as a variant polypeptide in host cells and that such recombinant polypeptides are capable of specifically binding antibody to an AIDS associated retrovirus. Such variant polypeptides include viral polypeptides fused with one or more second polypeptide sequences as well as deletions, insertions, substitutions and derivatives of the viral polypeptide. In addition, directly expressed and certain variant polypeptides, each of which contain fragments of a predetermined polypeptide sequence of an AIDS associated retrovirus, also react with antibody to AIDS associated retrovirus. These results indicate that such recombinant polypeptides may be used as diagnostic agents to detect AIDS in individuals, donated blood and blood products.

Further, such polypeptides may be used as immunogens to induce the production of neutralizing antibodies which confer resistance to infection by an AIDS associated retrovirus.

Still further, the reverse transcriptase of an AIDS associated retrovirus may be used to identify compounds which may inhibit infection by AIDS associated retrovirus or the dissemination of such retrovirus in infected individuals.

The fusion polypeptides of the present invention comprise an AIDS associated polypeptide sequence and a second polypeptide sequence. These second polypeptide sequences may be used to: 1) promote secretion of the fusion polypeptide from a bacterial host into the extra-cellular environment or the periplasm of gram negative bacteria, 2) facilitate the functional association of the fusion polypeptide with the surface membrane of recombinant host cells or 3) provide a polypeptide sequence which may be used to purify the fusion polypeptide (e.g. purification of an HGH-AIDS fusion polypeptide by fractionation on an immunoadsorbent specific for HGH). Depending upon the particular applications, second polypeptide sequences used to form a fusion polypeptide with an AIDS associated retrovirus may be of prokaryotic or eukaryotic origin and may be positioned at the amino terminus, carboxy terminus, at both ends of the AIDS associated polypeptide sequence, or inserted within the AIDS associated polypeptide sequence.

Examples of second polypeptide sequences which may be used to promote secretion of the fusion polypeptide include (1) the signal sequence of Herpes Simplex Virus gD protein disclosed in copending U.S. patent application Ser. No. 527,917 filed Aug. 30, 1983; (2) the signal sequence of E. coli alkaline phosphatase or E. coli enterotoxin STII disclosed in copending U.S. application Ser. No. 658,342, filed Oct. 5, 1984, and references disclosed therein, and (3) pre-HGH disclosed in copending U.S. application Ser. No. 488,232 filed Apr. 25, 1984 or other higher eukaryotic signal sequences such as that of gamma interferon.

An example of a second polypeptide sequence which facilitates functional membrane association is the transmembrane sequence of Herpes Simplex Virus disclosed in U.S. application Ser. No. 527,917 filed Aug. 30, 1983.

Since many individuals at risk for AIDS also have antibodies to E. coli and other enterobacteria, the second polypeptide must be chosen to avoid false positive immunological reactivity with these antibodies. Polypeptide sequences from enterobacteria should therefore be used as a second polypeptide only if such sequences are removed during processing or otherwise prevented from reacting with the biologically derived samples to be assayed for the presence of antibody produced in response to infection by an AIDS associated virus, e.g. by recombinant expression such that the bacterial protein epitopes are modified so as to no longer be cross-reactive with the native protein (see the LE fusions described below).

When the fusion polypeptide of the present invention is used as a vaccine against AIDS infection, the second polypeptide sequence must be chosen to avoid the production of antibodies to polypeptides which are naturally occurring in the subject such vaccine is directed to. For example, in a vaccine for humans the second polypeptide sequence is preferably not HGH. Such vaccines, however, may contain prokaryotic polypeptide sequences or preferably eukaryotic polypeptide sequences other than those of yeast and primates.

The present invention specifically discloses the cloning and expression of certain HTLV-III-encoded polypeptides. However, the present invention also contemplates the cloning and expression of other HTLV-III polypeptides. HTLV-III polypeptides which possess antigenic determinants to antibodies for AIDS and pre-AIDS patents include gp-160, gp-120, gp-65, gp-41, p-60/p-55. The gp-160 polypeptide appears to be a precursor polypeptide for gp-120 and gp-41. These particular HTLV-III polypeptides are illustrative and are not intended to limit the scope of the invention.

In addition, the present invention contemplates the generation of a library of products, each containing different antigenic determinants that may be used to determine which antigenic determinants are best suited for detection of AIDS or pre-AIDS. Such a library, for example, may be used to determine which antigenic determinants are immunologically reactive to serum derived from healthy individuals who are serologically positive for AIDS. Those antigenic determinants which test positive to such serum but negative to serum from AIDS patients may be prime candidates for a vaccine to induce the production of neutralizing antibodies. Further, diagnostic products containing such antigenic determinants may be used to identify individuals with neutralizing antibodies who are unlikely to develop the severe clinical manifestations associated with AIDS.

Although the present invention is based on studies of HTLV-III it is to be understood that HTLV-III may be similar or identical to LAV or ARV. As so related, polypeptide products derived from those retroviruses are within the scope of the present invention. Accordingly, the designation AIDS associated retrovirus refers to HTLV-III, LAV, ARV, and/or other retrovirus that may cause AIDS or ARC (AIDS-associated complex).

As used herein, a polypeptide sequence of an AIDS associated retrovirus is the full length native polypeptide sequence or the predetermined sequence derived from genomic sequencing.

A naturally occurring (native) polypeptide sequence is the polypeptide formed in virus infected cells or found in the culture fluid of such cells.

Variant polypeptide sequences of an AIDS associated retrovirus include: (1) fusions of viral polypeptide or fragments thereof with second polypeptide sequences including N and C terminal fusions and insertions; (2) deletions of the N-terminal, C-terminal, or an internal region of the polypeptide sequence of viral polypeptide to produce a fragment of a polypeptide sequence of an AIDS associated retrovirus; (3) substitutions of one or more amino acids in a polypeptide sequence of an AIDS associated retrovirus and (4) derivatives such as labelled or bound viral polypeptide sequences which may be labelled by well known techniques or bound to a solid phase such as that disclosed in U.S. Pat. No. 3,720,760 incorporated herein by reference.

"Second polypeptides" are sequences which are fused with a polypeptide sequence of an AIDS associated retrovirus or fragment thereof to form the fusion polypeptide sequences of the present invention. These second polypeptides may be full length or partial protein sequences of eukaryotic, non-AIDS viral or prokaryotic origin and may be used to promote secretion of the fusion polypeptide, facilitate association of the fusion polypeptide with the surface membrane of an expression host or aid in the purification of the fusion polypeptide. When used as a vaccine, the second polypeptide of a fusion polypeptide is a sequence which is not normally capable of inducing antibodies which are cross-reactive with naturally occurring polypeptides, in the subject such vaccine is directed to.

"Complementary antibody" refers to antibody raised against a corresponding naturally occurring viral epitope or epitope encoded by AIDS-associated retrovirus.

A DNA sequence of an AIDS associated retrovirus encodes the polypeptide and variant polypeptide sequences of the present invention described above.

"Biologically derived sample" includes any biological fluid or tissue sample taken from a human or animal subject which may be assayed to detect the presence of complementary antibody produced in response to exposure to or infection by an AIDS associated retrovirus. Such samples typically comprise blood, urine, semen, and saliva but may include any biological material in which such complementary antibody or AIDS associated retrovirus may be found.

Prokaryotes are preferred for cloning and expressing DNA sequences to produce the diagnostic product and vaccine of the present invention. For example, E. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains which may be used include E. coli strains such as E. coli B, and E. coli X1776 (ATCC No. 31537), and E. coli c600 and c600hfl, E. coli W3110 (F^-, .lambda.^-, prototrophic, ATTC No. 27325), bacilli such as Bacillus subtilus, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various pseudomonas species. When expressed in prokaryotes the polypeptides of the present invention typically contain an N-terminal methionine or a formyl methionine, and are not glcosylated. These examples are, of course, intended to be illustrative rather than limiting.

In general, plasmid vectors containing replication and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (18). Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying and selecting transformed cells. The pBR322 plasmid, or microbial plasmid must also contain, or be modified to contain, promoters which can be used by the microbial organism for an expression of its own proteins. Those promoters most commonly used in recombinant DNA construction include .beta.-lactamase (penicillinase) and lactose promoter systems (19-21) and tryptophan (trp) promoter system (22, 23). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning the their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (24). In the specific embodiments disclosed, a trp promoter (22, 23) was used to express the diagnostic product and vaccine of the present invention.

In addition to prokaryotes, eukaryotic cells may be used to express the AIDS associated virus polypeptides including particularly the reverse transcriptase of an AIDS associated retrovirus. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (25-27) is commonly used. The plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (28). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (29) or other glycolytic enzymes (30, 31), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate, decarboxylase, phosphofructokinase, glucose-6-phosphate, isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA termination. Other promoters, which have the additional advantage of the transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and the enzymes responsible for maltose and galactose utilization. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.

Cultures of cells derived from multicellular organisms also are employed for expression of AIDS associated retrovirus proteins. Mammalian or vertebrate cells are of particular interest, such as VERO and HELA cells, Chinese Hamster ovary (CHO) cell lines, and WI38, BHK, COS-7 and MDCK cell lines. Expression vectors for such cells ordinarily include an origin of replication, a promoter for controlling expression of the DNA encoding the AIDS associated retroviral polypeptide, along with a mammalian selection marker, RNA splice site, polyadenylation site and transcriptional terminator sequences as required.

Vectors capable of transforming mammalian host cells to expression of AIDS associated polypeptides are preferably introduced into host cells with a selection marker, e.g. the gene encoding DHFR (dihydrofolate reductase) in known fashion and then amplified by exposing the transformants to increasing concentrations of selection agent, e.g. methotrexate. For example see U.S. Pat. No. 4,399,216.

For use in mammalian cells, the transcriptional and translational control functions are conventionally obtained from viral sources. For example, commonly used promoters are derived from polyoma, Simian Virus 40 (SV40) and most particularly Adenovirus 2. The early and late promoters of SV40 virus are useful as is the major late promoter of adenovirus as described above. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from adenovirus or other viral (e.g. Polyoma, SV40, VSV, BPV, etc.) source, or may be provided by the host cell chromosomal replication mechanism, if the vector is integrated into the host cell chromosome.

For vectors of the invention which comprise DNA sequences encoding both AIDS associated polypeptide and a cotransformation, selection and amplification gene such as the DHFR enzyme, it is appropriate to select the host according to the type of DHFR protein employed. If wild type DHFR protein is employed, it is preferable to select a host cell which is deficient in DHFR, thus permitting the use of the DHFR coding sequence as a marker for successful transfection in selective medium which lacks hypoxanthine, glycine, and thymidine.

On the other hand, if DHFR protein with low binding affinity for MTX is used as the controlling sequence, it is not necessary to use DHFR resistant cells. Because the mutant DHFR is resistant to methotrexate, MTX containing media can be used as means of selection provided that host cells are themselves methotrexate sensitive.

Alternatively, a wild type DHFR gene may be employed as an amplification marker in a host cell which is not deficient in DHFR provided that a second drug selectable marker is employed, such as neomycin resistance.

An example, which is set forth hereinafter, contemplates the use of CHO cells as host cells and an expression vector which encodes the reverse transcriptase of an AIDS associated retrovirus.

As more fully set out below, the diagnostic product of the present invention is utilized in place of its counterpart derived from a live pathogen in analogous immunoassays. In that regard, a commercial diagnostic test kit would include the above diagnostic products with a variety of other immunological products, at least one of which is labeled, for detection of its complementary antibody or the antigen. The system has been described with respect to the molecular cloning and expression of specific proteins of HTLV-III which possess sufficient antigenic determinants to render them capable of specifically binding complementary antibody, namely antibody to HTLV-III. The specific techniques for cloning and expressing exemplary polypeptides are set forth in more detail in the examples that follow.

There are a number of known techniques for the determination of an unknown quantity of antigen or antibody in biological fluids such as serum, urine, or saliva or from skin samples or the like. In principle, the present invention utilizes such known techniques but substitutes certain molecularly cloned diagnostic reagents of a type set forth above in the otherwise known procedure.

Accordingly, the procedures themselves will be described only generally with reference being made to conventional immunology text for the details of the procedures. It would be well known to skilled workers in the field how to utilize the novel diagnostic products of the present invention in conventional immunological techniques.

For simplicity of description, the general term "diagnostic product" will be used in describing the antigen functional product of the present invention. The term "diagnostic product" is defined as a predetermined polypeptide sequence of an AIDS associated retrovirus with one or more antigenic determinants capable of specifically binding complementary antibody induced by a AIDS associated retrovirus. The diagnostic product is formed in a recombinant host cell capable of its production. The polypeptide sequence may be either functionally associated with a surface membrane of the recombinant cell or it may be recovered and used free of the host cell membrane. Further, the antigenic polypeptide sequence may be fused to a second polypeptide sequence.

The diagnostic methods used in assaying AIDS associated retrovirus, its constituent polypeptides and complementary antibodies are conventional. These include the competitive, sandwich and steric inhibition techniques. The first two methods employ a phase separation step as an integral part of the method while steric inhibition assays are conducted in a single reaction mixture. The methodology for assay of retrovirus or its polypeptides on the one hand and for substances that bind retrovirus or viral polypeptides on the other hand are essentially the same, although certain methods will be favored depending upon the size of the substance being assayed. Therefore the substance to be tested is referred to herein as an analyte, irrespective of its status otherwise as an antigen or antibody, and proteins which bind to the analyte are denominated binding partners, whether they be antibodies, cell surface receptors or antigens.

Analytical methods for AIDS associated retrovirus, its polypeptides, complementary antibody or cell surface receptors all use one or more of the following reagents: Labelled analyte analogue, immobilized analyte analogue, labelled binding partner, immobilized binding partner and steric conjugates. The labelled reagents also are known as "tracers".

The label used is any detectable functionality which does not interfere with the binding of analyte and it binding partner. Numerous labels are known for use in immuno assay, examples including enzymes such as horseradish peroxidase, radioisotopes such as ¹⁴ C and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein, spin labels and the like. Conventional methods are available to covalently bind these labels to proteins or polypeptides. Such bonding methods are suitable for use with AIDS associated retrovirus, viral polypeptides, complementary antibody and retrovirus receptors, all of which are proteinaceous.

Immobilization of reagents is required for certain assay methods. Immobilization entails separating the binding partner from any analyte which remains free in solution. This conventionally is accomplished by either insolubilizing the binding partner or analyte analogue before the assay procedure, such as by adsorption to a water insoluble matrix or surface (Bennich et al., U.S. Pat. No. 3,720,760) or by covalent coupling (for example using glutaraldehyde cross-linking), or by insolubilizing the partner or analogue afterward, e.g., by immunoprecipitation.

Steric conjugates are used in the steric hinderance method for homogeneous assay. These conjugates are synthesized by covalently linking a low molecular weight hapten to a small analyte so that antibody to hapten substantially is unable to bind the conjugate at the same time as anti-analyte. Under this assay procedure the analyte present in the test sample will bind anti-analyte, thereby allowing anti-hapten to bind the conjugate resulting in a change in fluorescence when the the hapten is a fluorophore.

Other assay methods, known as competitive or sandwich assays, are well established and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a labelled analogue (the "tracer") to compete with the test sample analyte for a limited number of binding sites on a common binding partner. The binding partner is generally insolubilized before or after the competition and then the tracer and analyte bound to the binding partner are separated from the unbound tracer and analyte. This separation is accomplished by decanting (where the binding partner was preinsolubilized) or by centrifuging (where the binding partner was precipitated after the competitive reaction). The amount of test sample analyte is inversely proportional to the amount of bound tracer as measured by the amount of marker substance. Dose-response curves with known amounts of analyte are prepared and compared with the test results in order to quantitatively determine the amount of AIDS associated retrovirus, viral polypeptide or complementary antibody present in the test sample. These heterologous assays are called ELISA systems when enzymes are used as the detectable markers.

Another species of competitive assay is a homogenous assay which does not require a phase separation. Here, a conjugate of an enzyme with the analyte is prepared so that when anti-analyte binds to the analyte the presence of the anti-analyte modifies the enzyme activity. In this case, a polypeptide of an AIDS associated retrovirus or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates are selected for use with complementary antibody so that binding of the complementary antibody inhibits or potentiates enzyme activity. This method per se is widely practiced under the name EMIT.

Sandwich assays particularly are useful for the determination of polypeptides of an AIDS associated retrovirus, complementary antibody or retrovirus cell surface receptors, i.e., large molecules. In sequential sandwich assays an immobilized binding partner is used to adsorb test sample analyte, the test sample is removed by washing, the bound analyte is used to adsorb labelled binding partner and bound material then separated from residual tracer. The amount of bound tracer is directly proportional to test sample analyte. In a "simultaneous" sandwich assay, test sample is not separated before adding the labelled binding partner.

The foregoing are merely exemplary assays for AIDS associated retrovirus, polypeptides of an AIDS associated retrovirus, complementary antibody and retrovirus cell surface receptors. Other methods now or hereafter developed for the determination of these analytes are included within the scope hereof.

In order to simplify the examples certain frequently occurring and well-known methods employed in recombinant constructions will be referenced by shorthand phrases or designations.

Plasmids are generally designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids or sources of DNA herein are commercially available, are publicly available on a restricted basis, or can be constructed from available plasmids or polynucleotides in accord with published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan since the plasmids generally only function as replication vehicles for the preprotein and its control sequences, or for elements thereof in intermediate constructions.

"Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain locations in the DNA. Such enzymes are called restriction enzymes, and the sites for which each is specific is called a restriction site.

The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements as established by the enzyme suppliers were used. Restriction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters and then, generally, a number representing the microorganism from which each restriction enzyme originally was obtained. In general, about 1 ug or plasmid or DNA fragment is used with about 1 unit of enzyme in about 20 ul of the buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37^oC. are ordinarily used, but way vary in accordance with the supplier's instructions. After incubation, protein is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered with aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme infrequently is followed with bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to prevent the two restriction cleaved ends of a DNA fragment from "circularizing" or forming a closed loop upon ligation (described below) that would impede insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed by 5' terminal dephosphorylation. Procedures and reagents for dephosphorylation are conventional (32).

"Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the digest by polyacrylamide gel electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the DNA from the gel, generally by electroelution. This procedure is known generally.

A "Western Blot" is a method by which the presence of polypeptide is confirmed by reaction with labelled complementary antibody. The polypeptide is separated electrophoretically on a polyacrylamide gel and electrophoretically transferred to nitrocellulose. The nitrocellulose is incubated with labelled complementary antibody, unbound antibody removed and the location of residual label is identified.

"Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or chromosomal integrant. Unless otherwise provided, the method used herein is the CaCl₂ transformation method (33).

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic fragments (34). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar amounts of the DNA fragments to be ligated.

"Fill in" or "blunting" refers to the repair of sticky ended (overhanging) restriction enzyme fragments in order to create a blunt end that will ligate to other blunt terminal DNA. Generally 2-15 .mu.g of DNA are incubated in 50 mM NaCl, 10 mM Tris (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol with 250 .mu.M of each of four deoxynucleoside triphosphates and 8 units DNA polymerase Klenow fragment at 20^oC. for 30 minutes. The reaction is terminated by phenol and chloroform extraction and ethanol precipitation.

"Preparation" of DNA from transformants means isolating plasmid DNA from microbial culture. Unless otherwise provided, the alkaline/SDS method was used (34).

"Oligonucleotides" are short length single or double stranded polydeoxynucleotides which are made by chemically known methods and then purified on polyacrylamide gels (35).

Claim 1 of 47 Claims

What is claimed is:

1. A composition comprising a predetermined polypeptide sequence of an AIDS-associated retrovirus which composition is essentially free of other naturally occurring AIDS-associated polypeptide sequences or human proteins from cells for which the AIDS-associated retrovirus is naturally infective, said predetermined polypeptide sequence comprising at least one antigenic determinant that specifically binds complementary antibody.

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