WO1987002989A1

WO1987002989A1 - Aids virus gene expression

Info

Publication number: WO1987002989A1
Application number: PCT/US1986/002374
Authority: WO
Inventors: Anna Aldovinni; Christine Marie Debouck; Martin Rosenberg; Flossie Wong-Staal
Original assignee: United States Of America, Represented By The Unite; Smithkline Beckman Corporation
Priority date: 1985-11-06
Filing date: 1986-11-05
Publication date: 1987-05-21
Also published as: FI872960A0; JPS63502000A; AU6595986A; EP0245456A1; PT83682A; PT83682B; FI872960A; EP0245456A4

Abstract

The tat-3 gene of HTLV-III is expressed at high levels in E. coli and is reactive with antibodies induced in response to infection by HTLV-III and can induce production of antibodies which are reactive with HTLV-III.

Description

TITLE

AIDS Virus Gene Expression

FIELD OF THE INVENTION

This invention relates to the field of molecular biology and more particularly to expression of a gene from the HTLV-III Virus in E. coli and uses thereof.

BACKGROUND OF THE INVENTION Human T - lymphotropic virus type -III (HTLV-III) , also known as Lymphadenopathy virus (LAV) or AIDS-associated retro-virus (ARV) , is the etiologiσal agent of the acquired immuno-deficiency syndrome (AIDS) and related disorders. While HTLV-III is evolutionar ily more closely related to the ungulate lenti-retroviruses, it shares many common features with the previously isolated human T-lympho tropic viruses, types I and II (HTLV-I and HTLV-II), particularly those biological and pathogenic properties that are consequences of their capacity to infect helper T-lymphocytes and impair immune function. Furthermore, one unusual property unites HTLV types I, II and III, related animal retroviruses, such as bovine leukemia virus and simian T-lymphotropic viruses, type I, and the ungulate lenti-retroviruses, namely, the presence of a viral encoded protein which mediates activation of transcription initiated in the viral long terminal repeat (LTR). It has been speculated that this trans criptional activator (tat) plays a critical role in the biological activities (transformation or cytopathic effects) of this group of viruses.

The severity of AIDS makes early and accurate diagnosis of infection by HTLV-III and detection and elimination of HTLV-III-contaminated samples from blood banks extremely important. Gallo et al., U.S. Patent 4,520,113, disclose use of antigens derived from HTLV-III to detect presence of anti- HTLV-III antibodies in serum. Montagnier et al., EP-A-138,667, disclose use of a specified HTLV-III antigen to detect infection by the virus. Papas et al., united States Patent Application Serial No. 6-664,972 (Derwent Accession No. 85-110268/18) , disclose expression of a HTLV-I envelope protein coding sequence in E. coli and use of the protein expressed thereby to detect infection by HTLV-I. Crowl et al., Cell 41; 979(1985), report expression in E. coli of portions of the HTLV-III envelope protein gene, env, and use of such proteins for detection of infection by HTLV-III. Casey et al., j. Virol. 55: 417 (1985), report purification of the gag gene product, an internal structural protein of HTLV-III referred to as ρ24, and use of the protein to detect infection by HTLV-III.

Seiki et al., Proc. Nat'l. Acad. Sci USA 80: 2618 (1983), and Haseltine et al.. Science 225: 419 (1984), report identification of proteins which mediate activation of transcription of the LTR in HTLV-I and HTLV-II, referred to as the tat-1 and tat-2 proteins, respectively.

Sodroski et al., Science 227: 171 (1985), report in trans activation of gene expression from the LTR in HTLV-III.

Arya et al., Science 229:69(1985), and Sodroski et al., Science 229: 74 (1985), report a tat protein encoded by HTLV-III and identification and cloning in E. coli of a cDNA coding for said tat protein, referred to as tat- 3. SUMMARY OF THE INVENTION

The invention is, in one aspect, an E. coli expression vector which comprises a DNA coding sequence operatively linked to a regulatory element wherein the DNA coding sequence codes for the tat-3 protein of HTLV-III or for a derivative thereof, which derivative is a polypeptide which is reactive with antisera to tat-3 induced in response to infection in an animal by HTLV-III.

In another aspect, the invention is a method for detecting infection in an animal by HTLV-III which comprises contacting a sample of serum from the animal with tat-3, or a derivative thereof which derivative is a polypeptide which is reactive with antisera to tat-3 induced in response to infection in an animal by HTLV-III, and assaying for reactivity of the sample with the tat-3 or the tat-3 derivative.

All of these embodiments of the invention, as well as others described herein, are readily attainable and are considered as further aspects of the same invention.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that the tat-3 protein of HTLV-III can be expressed in E. coli in readily recoverable quantities and that the protein so expressed is reactive with sera from animals infected by the HTLV-III virus.

The E. coli expression vector of the invention is prepared by recombinant DNA techniques or by a combination of recombinant DNA and synthetic techniques. It comprises at least a coding sequence for the tat-3 protein of HTLV-III or for a derivative of the tat-3 protein which is immunologically equivalent to tat-3. By "immunologically equivalent" is meant that the derivative polypeptide is reactive with antibodies to authentic tat-3 induced in response to infection in an animal by HTLV-III or, conversely, is capable of inducing an immune response which is reactive with authentic tat-3. In the expression vector of the invention, said coding sequence is operatively linked to a regulatory element.

The coding sequence for authentic tat-3 can be prepared by known techniques from HTLV-III virus or from HTLV-III-infected cells by isolation of viral mRNA and preparing cDNA by reverse transcription. Such preparation of a tat-3 coding sequence is disclosed by Arya et al., Science 229: 69 (1985) and by Sodroski et al., Science 229: 74 (1985), both of which are incorporated by reference herein.

Derivatives of the coding sequence so obtained can be prepared by standard recombinant DNA and/or synthetic techniques. These include mutation techniques reviewed by Botstein, Science 229:1193 (1985). Such derivatives can comprise addition, substitution or deletion of one or more base pairs such that upon expression, the resulting fused, mutated or truncated polypeptide is immunologically equivalent to authentic tat-3. Typically, for use as a diagnostic, such derivative will comprise a truncated protein, for example a polypeptide of 5 to 10 amino acids which retains immunologic cross-reactivity with tat - 3 , such as a tat-3 protein in which N- or C-terminal amino acids have been deleted.

The coding sequence for the polypeptide can be inserted into any E. coli expression vector, many of which are known and available. By "regulatory element" is meant the expression control sequences, for example, a promoter and ribosome binding site, required for transcription and subsequent translation. Regulatable regulatory elements, that is, regulatory signals which are not constitutive but require induction or derepr ession, are preferred. Such vectors typically comprise, in addition to the regulatory element, a region which permits the vector to be stably maintained in a host cell population, that is, a replicon or origin of replication, and one or more selection markers, that is, genes which confer a selectable phenotype upon hosts carrying the vector. One exemplary expression vector of the invention is the plasmid pASl, described by Rosenberg et al., Meth. Enzym., 101: 123 (1983) and Shatzman et al., in Experimental Manipulation of Gene Expression, edit, by M. Inouye, Academic Press, New York, 1982. pASl carries the _PBR322 origin of replication, an ampicillin resistance marker and a series of fragments from bacter iophage lambda, which comprise the regulatory element including PL, N anti-termination function recognition sites (NutL and NutR), the rho-dependent transcription termination signal (tRl) and the cll ribosome binding site (rbs), including the cll translation initiation site, the G residue of which is followed immediately by a BamHI cleavage site as follows:

5' ...cll...CA:TATG*GATCC...3'

wherein the symbol, *, indicates the clevage site for BamHI and the symbol, ;, indicates the cleavage site for Ndel.

pASl can be derived from pKC30cII by deleting nucleotides between the BamHI site at the lambda-pBR322 junction of pKC30cII and the ell ATG and religating the molecule to regenerate the BamHI site immediately downstream of the ATG. pKC30cII is constructed by inserting a 1.3 kb Haelll fragment from lambda which carries the ell gene into the Hpal site of pKC30. See Shatzman et al., cited above, and Rosenberg et al., cited above. pKC30 is described by Shimatake et al., Nature, 292: 128 (1981). It is a pBR322 derivative having a 2.4 kb Hindlll-BamHI fragment of lambda inserted between the HindIII and BamHI sites in the tetR gene of ρBR322. Constructions similar to pASl are described by Courtney et al.. Nature, 313, 145 (1985) and Kotewicz et al., Gene 35: 249(1985). Derivatives of pASl, comprising the PL, NutL, NutR and ell rbs regulatory element, can be constructed by standard techniques. The coding sequence is operatively linked, that is, in correct orientation and in proper reading frame, to a regulatory element of an E. coli expression vector by standard techniques to construct an expression vector of the invention.

The tat-3 expressed by E. coli, as shown in the Examples below, was reactive with 42 of 92 samples of sera (46%) from individuals exposed to HTLV-III, but was not reactive with sera from normal individuals. Thus, tat-3 and derivatives thereof can be used in detection of HTLV-III infection by standard assay techniques which permit detection of presence of tat-3 protein or an ti -tat-3 antibodies. The known host range for HTLV-III is limited to man and certain other higher primates, although presence in a larger animal pool at this time or in the future cannot be ruled out. Preferably, tat-3 is used in a battery of one or more other tests, such as immunoassays for presence of the env, sor, gag or 3'orf gene products in sera. Based on data gathered to date, a positive reaction with tat-3 is 100% diagnostic of HTLV-III infection. E. coli-derived tat-3 can also be used to screen samples of blood in blood banks. Techniques for employing tat-3 in such diagnostic immunoassays are well known. These include, for example, the technique disclosed by Casey et al., J. Virol. 55: 417 (1985) and by Crowl et al., Cell 41: 979 (1985). tat 3 can be employed in an enzyme linked immunosorbent assay (ELISA) or radioimmuno assay (RIA). Preferably, a western blotting assay is employed, as an ELISA assay has been found in preliminary experiments to result in a small percentage of false positives. Also, the tat-3 protein produced by E. coli can be used to stimulate, production of anti-sera which is reactive with HTLV-III. Thus the tat-3 protein can be used as an antigenic component of a vaccine against infection by HTLV-III, although the protein is not structural and appears to be localized in cell nuclei. The ability to raise polyclonal antibodies renders it possible also to produce monoclonal antibodies by the standard techniques originally described by Kohler and Milstein, Nature 256:495 (1975) or other techniques of cell fusion or transformation. Such polyclonal or monoclonal antibodies can also be useful in detecting presence of tat-3 gene product in sera or in a cell population such as a cell culture. Such antibodies are also useful in affinity purification of tat-3, in epitope mapping to localize functional domains within the protein, such as domains which function in DNA binding, and can be used as neutralizing agents in therapy for HTLV-III infection. The tat-3 gene product can also be used in regulation of LTR-con trolled gene expression units as disclosed by Arya et al. and Sodroski et al., et al, cited above. Because tat 3 is, by definition, functional in tr ans, an expression vector of the invention can be used to control expression for an integrated gene expression unit or a gene expression unit present in another plasmid.

The predicted amino acid sequence of the tat-3 gene product (see, Example 1, below) reveals a highly hydrophilic protein with at least three discernible domains in the first 57 amino acids: a proline-rich region (5 out of 16 residues from positions 2-18) , a cys teine-rich region (7 out of 16 residues from positions 22-37) and a lysine/arginine-rich region (8 out of 9 residues from positions 49-57). The availability of highly purified tat-3 protein in sufficient quantities will allow direct elucidation of its sites (s) and mechanism(s) of action, e.g.., its DNA binding properties. In addition, expression of various truncated and mutated forms of this protein will allow precise localization of its functional domains. Availability of E. coli-derived tat-3 will permit identification of effectors, especially inhibitors of tat-3 function which can be used in therapy for HTLV-III infection.

Although the tat-3 gene of HTLV-III may be functionally analogous to the tat-1 and tat-2 genes and all three are transcribed from three exons into an RNA of about 2 kilobases, tat-3 differs from tat-1 and - 2 including, for example, in its position in the genome, its size and its primary nuσleotide sequence.

The following Examples are illustrative, and not limiting, of the invention and of techniques for making and using the invention.

Example 1 pOTS34 was derived from pASl by inserting a 189 bp fragment carrying a transcription terminator, the oop terminator, into the Nrul site downstream of the BamHI site in pASl and by inserting a synthetic linker (Xbal, Xhol, SacI) into the Sail site between the BamHI site and the added terminator. pOTS34 is identical to pOTS-5 or pOTSV (Devare et al., Cell 36: 43 (1984)) except that the linker is inserted in opposite orientation. A vector carrying a cDNA for tat-3, pCV-1, (Arya et al., cited above) was employed as a source of the tat-3 coding sequence. The nuσleotide and predicted amino acid sequence of the cDNA in pCV-1 are as follows:

The strategy used to express the complete tat-3 protein involved two stages. First, the tat-3 coding region lacking the first 12 base-pairs (bp) at its 5' end was obtained as a Mbol restriction endonuclease fragment from the cDNA clone. This fragment was inserted at the BamHI site of the pOTS34 vector. The resultant construct, pOTS-tatlllD, contains the tat-3 coding sequence deleted in codons 2 to 4 and positioned in-frame with the initiation codon provided by pOTS34. The second stage involved regeneration of the three missing codons at the amino-terminus. Since the 5' but not the 3' BamHI site was recreated in the pOTS-tatlllD plasmid, this vector was digested with BamHI, followed by Mung Bean exonuclease to create a blunt-end cloning site immediately adjacent to the initiation codon and the fifth codon of tat-3. A synthetic DNA linker reconstructing the missing codons was then inserted. The nucleotide sequence of the linker was slightly modified from the ta t-3 gene without altering the amino acid sequence such that the 2d, 3d and 4th codons were as follows: GAA CCG GTG. This construction resulted in a BamHI site between the 4th and 5th codons. The final construction, pOTS-tatlil, consists of the reconstructed full-length tat-3 coding sequence in frame with the ATG of ρOTS34. A sample of pOTS-tatlll has been deposited under the terms of the Budapest Treaty in the American Type Culture Collection, Rockville, Maryland, under Accession Number 53305. pOTS-tatlllD, pOTS-tatlll and a control vector without insert (pOTS34), were introduced into E. coli AR120, a cl lysogen inducible by nalidixic acid. See Mott et al., Proc. Nat'l. Acad. Sci. USA 82:88 (1985). Aliquots of the bacterial lysate at different times after induction were subjected to polyacrylamide gel electrophoresis. The AR120 bacterial cells containing POTS34, pOTS-tatlllD or pOTS-tatlll were grown to OD650 = 0.4-0.5 and induced by the addition of nalidixic acid to 60 μg/ml substantially as described by Mott et al., cited above Aliquots were taken 0, 3, and 5 hours after induction, spun down and resuspended in lysis buffer (60mM Tris-HCl (pH 7.0), 10% glycerol, 5% 2-merσaptoethanol, 2% SDS, 0.1% bromophenolblue). The proteins were resolved on a 15% SDS -polyacrylamide gel (acrylamide:bisacrylamide ratio of 30:0.8) and visualized by staining with Coomassie Brilliant Blue R-250.

A protein migrating with a 14 kd lysozyme marker was specifically induced in the cells transfectad with pOTS-tatlllD, and a slightly larger protein was detected in the cells transfected with pOTS-tatlll. Although the apparent molecular size of 14 kd is greater than that expected for tat-3, about 9.7 kd based on the amino acid sequence, the discrepancy is attributed to the high proline content of this protein which could have retarded its migration. This example demonstrates high level expression of the tat-3 coding sequence in E. coli.

Example 2 The production of highly expressed tat-3 protein in bacteria (2-5% of total cellular protein), as in Example 1, allowed preparation of specific polyclonal antibodies against it. For this purpose the E. coli-derived tat-3 protein, purified by electroelution following resolution on preparative polyacrylamide gels, was injected subscapularly into New Zealand white rabbits. Antisera from immunized rabbits reacted well against the 14 kd protein expressed in bacteria. Furthermore, a similar sized protein, 14 kd by polyacrylamide gel el.ectrophoresis, was detected at low levels in an infected T-lymphocyte cell line (H9/HTLV-III-B) by immunoprecipitation. This latter data was the first evidence that E. coli-derived tat-3, even after SDS gel electrophoresis, retained epi topes in common with native tat-3, thus demonstrating that bacterially-derived tat-3 can be used to produce antibodies which are reactive with authentic tat-3.

Example 3 To evaluate whether the tat-3 protein could be of diagnostic or prognostic value, sera from diverse individuals were examined for reactivity against the partially purified protein by Western blotting. Specifically, following partial purification, resolution on SDS-polyacrylamide gel and eleσtrotransf er to a nitrocellulose membrane, the membrane was cut into strips such that each strip was estimated to contain lug of tat-3. The strips were incubated at room temperature for 1 hr in milk buffer (5% non-fat dry milk,

0.1% Antifoam A, 0.1% N_aN₃, 0.9% NaCl) and then were incubated at 4°C with a 1/100 dilution of patient sera. The strips were then washed for 20 min in phosphate buffered saline (PBS) and incubated for 1 hr at room temperature (20-25°C) in the milk buffer containing

125_I- labelled protein A. After three washes for 30 min each in PBS, the strips were dried and autoradiographed. A 14.3 kd band corresponded to tat-3. The results, as summarized in Table 1 below, indicate that while all healthy normal people not known to be exposed to HTLV-III lacked antibodies to tat-3, a substantial fraction of people who were seropositive for other viral structural proteins (envelope and core antigens) had detectable antibody level to tat-3.

One hundred seven serum samples were examined. They were divided into four categories, based on accepted Center for Disease Control definitions: (1) healthy ser onega tive (no reactivity with gag or env proteins) (HN) ; (2) healthy HTLV-III carriers (includes sera from individuals in high risk populations and positive for HTLV-III antibodies against env or gag protein, but free of clinical symptoms) (HC), (3) individuals with AIDS -related complex (ARC) ; and (4) individuals with acquired immuno deficiency syndrome (AIDS).

None of the serum samples from category (1) were reactive with the tat-3. Approximately similar percentages of samples (53%, 29% and 53%) in each of the other categories were reactive with the tat-3. Reactions ranged from strongly reactive to weakly reactive without correlation to the stage of progression of the disease.

The lower immunoreai ctivity of tat-3 as compared to env or gag proteins may be due to the lower level of the protein expressed in vivo, presence of fewer immunogenic epi topes and the presumed nuclear localization of tat-3.

This example demonstrates utility of the bacter ially-der ived tat-3 protein in diagnosing infection by HTLV-III in an animal.

The above description and Examples are illustrative of the invention and of preferred embodiments thereof.

The invention, however, is not limited to embodiments specifically disclosed herein but rather includes all modifications coming within the scope of the claims which follow.

Claims

Claims:

1. An E. coli expression vector which comprises a DNA coding sequence operatively linked to a regulatory element wherein the DNA coding sequence codes for the tat-3 protein of HTLV-III or for a derivative thereof, which derivative is a polypeptide which is reactive with antisera to tat-3 induced in response to infection in an animal by HTLV-III.

2. The vector of claim 1 wherein the DNA coding sequence codes for a polypeptide having the amino acid

1 and X is G or A.

4. The vector of claim 1, 2 or 3 wherein the regulatory element comprises the PL promoter of lambda, the Nut L and Nut R recognition sites and the cll ribosome binding site.

5. The vector of claim 1, 2 or 3 which is pASl, or a derivative thereof, into which the coding sequence for tat-3 has been inserted.

6. The vector of claim 5 which is pOTS-tatlllD or pOTS-tatlll.

7. A method for detecting infection in an animal by HTLV-III which comprises contacting a sample of serum from the animal with tat-3, or a derivative thereof which derivative is a polypeptide which is reactive with antisera to tat-3 induced in response to infection in an animal by HTLV-III, and assaying for reactivity of the sample with the tat-3 or tat-3 derivative.

8. The method of claim 7 wherein the tat-3 or tat-3 derivative is derived from E. coli transformed with an expression vector which comprises a DNA coding sequence operatively linked to a regulatory element wherein the DNA coding sequence codes for the tat-3 protein of HTLV-III or for a derivative thereof, which derivative is a polypeptide which is reactive with antisera to tat-3 induced in response to infection in an animal by HTLV-III.

9. The method of claim 8 wherein the tat-3 or tat-3 derivative has the following amino acid sequence: N-MET

10. The method of claim 9 wherein the DNA coding

wherein n is 0 or 1 and X is G or A.

11. The method of claim 8, 9 or 10 wherein, in the vector, the regulatory element comprises the PL promoter of lambda. Nut L and Nut R recognition sites and the ell ribosome binding site.

12. The method of claim 8, 9 or 10 wherein the vector is pASl, or a derivative thereof, into which the tat-3 coding sequence has been inverted.

13. The method of claim 12 in which the vector is pOTS-tatlllD or pOTS-tatlll.

14. The method of claim 7, 8, 9 or 10 which also comprises contacting the sample with one or more other

HTLV-III gene products.

15. The method of claim 7, 8, 9 or 10 in which the sample is contacted with the tat-3 in a western blotting assay.