EP0245456A1 - Expresion du gene du virus du sida - Google Patents

Expresion du gene du virus du sida

Info

Publication number
EP0245456A1
EP0245456A1 EP86907113A EP86907113A EP0245456A1 EP 0245456 A1 EP0245456 A1 EP 0245456A1 EP 86907113 A EP86907113 A EP 86907113A EP 86907113 A EP86907113 A EP 86907113A EP 0245456 A1 EP0245456 A1 EP 0245456A1
Authority
EP
European Patent Office
Prior art keywords
tat
htlv
iii
derivative
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86907113A
Other languages
German (de)
English (en)
Other versions
EP0245456A4 (fr
Inventor
Anna Aldovinni
Christine Marie Debouck
Martin Rosenberg
Flossie Wong-Staal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Commerce
SmithKline Beecham Corp
Original Assignee
US Department of Commerce
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Commerce, SmithKline Beecham Corp filed Critical US Department of Commerce
Publication of EP0245456A1 publication Critical patent/EP0245456A1/fr
Publication of EP0245456A4 publication Critical patent/EP0245456A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • HTLV-III Human T - lymphotropic virus type -III
  • LAV Lymphadenopathy virus
  • ARV AIDS-associated retro-virus
  • 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.
  • 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.
  • LTR long terminal repeat
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 P BR322 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:
  • 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).
  • pBR322 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 ⁇ BR322. 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.
  • tat-3 expressed by E. coli 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.
  • 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.
  • 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).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmuno assay
  • 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.
  • the tat-3 protein produced by E. coli can be used to stimulate, production of anti-sera which is reactive with HTLV-III.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 nu ⁇ leotide sequence.
  • 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 nu ⁇ leotide 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.
  • 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.
  • 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.
  • pOTS-tatlil The final construction, pOTS-tatlil, consists of the reconstructed full-length tat-3 coding sequence in frame with the ATG of ⁇ OTS34.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 ele ⁇ trotransf 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,
  • HN healthy ser onega tive (no reactivity with gag or env proteins)
  • HN healthy ser onega tive (no reactivity with gag or env proteins)
  • HCV healthy HTLV-III carriers
  • HC healthy HTLV-III carriers
  • ARC AIDS -related complex
  • AIDS acquired immuno deficiency syndrome
  • 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.

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  • Health & Medical Sciences (AREA)
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Abstract

Le gène tat-3 du HTLV-III est exprimé en quantités élevées dans E. coli et réagit à des anticorps induits en réponse à l'infection par le HTLV-III et peut induire la production d'anticorps qui réagissent au HTLV-III.
EP19860907113 1985-11-06 1986-11-05 Expresion du gene du virus du sida. Withdrawn EP0245456A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US79555985A 1985-11-06 1985-11-06
US795559 1985-11-06

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EP0245456A1 true EP0245456A1 (fr) 1987-11-19
EP0245456A4 EP0245456A4 (fr) 1989-01-24

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EP19860907113 Withdrawn EP0245456A4 (fr) 1985-11-06 1986-11-05 Expresion du gene du virus du sida.

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EP (1) EP0245456A4 (fr)
JP (1) JPS63502000A (fr)
AU (1) AU6595986A (fr)
FI (1) FI872960A (fr)
PT (1) PT83682B (fr)
WO (1) WO1987002989A1 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5110802A (en) * 1987-07-14 1992-05-05 City Of Hope Oligonucleotide phosphonates and method of inhibiting a human immunodeficiency virus in vitro utilizing said oligonucleotide phosphonates
ATE208813T1 (de) * 1988-02-16 2001-11-15 Greatbatch Gen Aid Ltd Modifizierte zellen mit resistenz gegen retroviralinfektionen
AU3006789A (en) * 1988-02-24 1989-08-24 Smithkline Beckman Corporation Expression of hiv binding proteins
AU3756789A (en) * 1988-06-16 1990-01-12 St. Louis University Antagonists of viral transactivating proteins
GB9003010D0 (en) * 1990-02-09 1990-04-04 Glaxo Group Ltd Gene expression in yeast cells
EP0455424A3 (en) * 1990-05-02 1992-04-29 Merck & Co. Inc. Mammalian inducible promoter cascade system
WO1991018454A1 (fr) * 1990-05-18 1991-11-28 Centre National De La Recherche Scientifique Compositions capables de bloquer la cytotoxicite de proteines regulatrices de virus et les symptomes neurotoxiques associes aux infections par retrovirus
US5891994A (en) 1997-07-11 1999-04-06 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
IT1297090B1 (it) * 1997-12-01 1999-08-03 Barbara Ensoli Tat di hiv-1 o suoi derivati, da soli od in combinazione, a scopo vaccinale, profilattico e terapeutico, contro l'aids i tumori e le
US6399067B1 (en) 2000-04-28 2002-06-04 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
CA2597373A1 (fr) 2005-02-15 2006-08-24 Thymon, L.L.C. Methodes et compositions permettant d'alterer la multiplication de vih-1
GB0802224D0 (en) * 2008-02-06 2008-03-12 Inst Superiore Di Sanito Process for the production of vaccine components

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4520113A (en) * 1984-04-23 1985-05-28 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Serological detection of antibodies to HTLV-III in sera of patients with AIDS and pre-AIDS conditions

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL, vol. 36, 1984, pages 430-436, Cambridge, MA, US; S.G. DEVARE et al.: "Expression of the PDGF-related transforming protein of simian sarcoma virus in E. coli" *
SCIENCE, vol. 228, 5th April 1985, pages 93-96, Washington, US; N.T. CHANG et al.: "Expression in Escherichia coli of open reading frame gene segments of HTLV-III" *
SCIENCE, vol. 229, 5th July 1985, pages 69-73, Washington, US; S.K. ARYA et al.: "Trans-activator gene of human T-lymphotropic virus type III (HTLV-III)" *
See also references of WO8702989A1 *

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Publication number Publication date
WO1987002989A1 (fr) 1987-05-21
PT83682A (en) 1986-12-01
AU6595986A (en) 1987-06-02
FI872960A0 (fi) 1987-07-03
FI872960A (fi) 1987-07-03
EP0245456A4 (fr) 1989-01-24
PT83682B (en) 1988-12-05
JPS63502000A (ja) 1988-08-11

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