WO1991007664A1 - Proteines de diagnostic utilisees pour tester plus d'un anticorps - Google Patents

Proteines de diagnostic utilisees pour tester plus d'un anticorps Download PDF

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Publication number
WO1991007664A1
WO1991007664A1 PCT/US1990/006621 US9006621W WO9107664A1 WO 1991007664 A1 WO1991007664 A1 WO 1991007664A1 US 9006621 W US9006621 W US 9006621W WO 9107664 A1 WO9107664 A1 WO 9107664A1
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hiv
peptide fragment
htlv
virus
immunodiagnostic
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PCT/US1990/006621
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English (en)
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Elihu M. Young
James R. Storey
Gerald A. Beltz
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Cambridge Biotech Corporation
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Priority to JP91502660A priority Critical patent/JPH04502773A/ja
Publication of WO1991007664A1 publication Critical patent/WO1991007664A1/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention is directed to the discovery that certain envelope peptide fragments of the Human Immunodeficiency Virus 2 (HIV-2) are particularly immunoreactive against HIV-2 antibodies. These fragments can therefore be applied to immunodiagnostic tests for the detection of antibodies to HIV- 2.
  • This invention is also directed to certain chimeric peptide fragments that are made from immunogenic portions of the envelope gene of HIV-2, HIV-1, or HTLV-I that can be used to test for antibodies to HIV-2, HIV-1, HTLV-I, and HTLV-II.
  • HIVs Human Immunodeficiency Viruses
  • HIV-1 HIV-1
  • HIV-2 HIV-2
  • HIV-1 (originally HTLV-III) was isolated and identified by Gallo and Montagier et al., U.S. 4,520,113. Ratner et al., Nature 313:277-284 (1985); Muesing et al., Nature 313:450-458 (1985); Sanchez-Pescador et al., Science 227:484-492 (1985); and Wain-Hobson et al., Cell 40:9-17 (1985) disclose the complete DNA sequence of the HTLV-III virus. HIV-2 (originally LAV-2) was first isolated from West Africans with AIDS-like illness (Clavel, F. et al., Science 233:343-346 (1986); Clavel, F. et al., Nature 324:691-695 (1986)).
  • HIV-1 and HIV-2 are related to the human T-cell leukemia- lymphoma (HTLV) virus family of T4 tropic retroviruses.
  • HTLV- I is the etiological agent of T-cell leukemia and lymphoma.
  • HTLV-II originally isolated from a patient with hairy-cell leukemia, is associated with the development of malignant leukemia of mature T-lymphocytes. The complete nucleotide sequence of the provirus genome of HTLV-I is given in Seiki et al., Proc. Nat'l Acad. Sci. (USA) 80:3618-3622 (1983).
  • RNA dependent DNA polymerase RNA dependent DNA polymerase
  • the host cells transcribe the DNA of the viral gene and synthesize the proteins encoded by the virus, which are then assembled into new viruses.
  • the HIV viruses and HTLV viruses contain at least (i) a gag gene that encodes the internal structural (neurocapsid or core) proteins, (ii) a pol gene that encodes the reverse transcriptase, and (iii) an env gene that encodes the envelope glycoproteins of the virus.
  • Viral lysates of HIV-1 are currently widely used for the detection of antibodies to HIV-1 in human sera.
  • the viral lysate comes from HIV-1 that has been grown in tissue culture and partially purified as the antigen source.
  • the tests are quite sensitive, but suffer from a relatively high rate of false positives.
  • Many scientists have attributed these false positives to cellular proteins contaminating the virus preparations (Honter, J.B. et al., Lancet 1:1222-1223 (1985); Sayers, M.H., Transfusion 26:113-115 (1986)) and the presence of cross-reactive antibodies (Thiry, L. et al., Science 227:1484 (1985); Volsky, D.J., New Enol. J. Med. 315:457-458 (1986)).
  • the HTLV-I and HTLV-II virus can be readily transferred from the peripheral blood leukocytes of antibody-positive people to leukocytes of antibody-negative people when the two are cultivated together. Popovic et al., Science 219:856-859 (1983). Consequently, there is a risk of infection involved in whole blood transfusions when the transfused blood contains infected cells.
  • individuals infected with one retrovirus may also be harboring a second retrovirus infection.
  • Biological specimens from people with an HIV-1 infection often also give a positive test for antibodies to HTLV-I or HTLV-II.
  • Assays were conducted in the following studies for HTLV-I and HIV-1 antibodies in patient sera: "Rossi et al., Eur. J. Cancer Clin. Oncol 22:411-418 (1986); Aoki et al., Lancet. October
  • HTLV-I was detected by viral lysate (Rossi et al. and Aoki et al.) and with core antigen (Tedder et al. and Robert-Guroff et al.).
  • the Pasteur ELAVIA mixed assay has been reported to preferentially detect HIV-2 antibodies over HIV-1 antibodies. (Lelie, supra). However, this assay is based on the use of a HIV-2 viral lysate assay and thus requires the use of whole virus.
  • human blood In the United States, human blood must be screened for antibodies to HIV-1 and to HTLV-I and for hepatitis. It is expected that soon human blood sera will also need to be screened for antibodies to HIV-2. Thus, it would be desirable to have a reliable screening assay to test for the presence of antibodies to HIV-2 in human biological samples. It would also be desirable to have a single test that would be able to detect more than one antibody in human biological samples.
  • the invention comprises the amino acid sequences of immunoreactive env HIV-2 peptides that can be used for detecting antibodies to HIV-2.
  • the invention also comprises the amino acid sequences of immunodiagnostic chimeric env peptides which are able to detect more than one antibody. Specifically, these chimeric env peptides are able to detect antibodies to HIV-2, HIV-1, and HTLV-I and HTLV-II.
  • this invention comprises chimeric env peptides comprising immunodiagnostic env peptide fragments of HIV-2-HIV-1; HIV- 2-HIV-1-HTLV-I; HIV-2-HTLV-I; and HIV-1-HTLV-I.
  • Figure 1 shows the construction of plasmid pK1 from plasmids K3D, containing the HIV-1 clone K3D, and pCBC1.
  • Figure 2 is an electrophoretic gel showing an induced 34 kd protein, called K1.
  • Figure 3 is a Western blot showing the reaction of the induced protein, K1, with human sera from HIV-2 infected individuals.
  • Figure 4 gives the complete DNA sequence of the coding region of the K1 and the derived amino acid sequence.
  • Figure 5 shows the construction of plasmid pK3 and a 1038 base pair fragment from position 7101 to position 8139 and the ligation of K3D into the expression vector pCBC2.
  • Figure 6 is an electrophoretic gel showing an induced 40 kd protein, called K3.
  • Figure 7 is a Western blot showing the reaction of the induced protein, K3, with human sera from HIV-2 infected individuals.
  • Figure 8 gives the complete DNA sequence of the coding region of the K3 and the derived amino acid sequence.
  • Figure 9 shows the construction of the plasmid pKlDG71 from plasmids pLCBCODG71A and pK1.
  • Figure 10 is an electrophoretic gel showing an induced 55 kd chimeric protein, called K1DG71.
  • Figure 11A is a Western blot showing the reaction of the induced protein, K1DG71, with human sera from HIV-1 infected individuals.
  • Figure 11B is a Western blot showing the reaction of the induced protein, K1DG71, with human sera from HIV-2 infected individuals.
  • Figure 12 gives the complete DNA sequence of the coding region of the pK1DG71 and the derived amino acid sequence.
  • Figure 13 shows the construction of the plasmid pDG71353.
  • Figure 14 shows the construction of the plasmid PK1DG71353.
  • Figure 15 gives the complete DNA sequence of the coding region of the pKlDG71353 and the derived amino acid sequence.
  • Figure 16 is an electrophoretic gel showing an induced 68 kd chimeric protein, K1DG71353.
  • Figure 17 is a Western blot showing the reaction of the induced protein, K1DG71353, with human sera from HIV-1 positive individuals.
  • Figure 18 is a Western blot showing the reaction of the induced protein, K1DG71353, with human sera from HIV-2 positive individuals.
  • Figure 19 is a Western blot showing the reaction of the induced protein, K1DG71353, with human sera from HTLV-I positive individuals.
  • Figure 20 shows that no specific reactivity was seen with negative sera in a Western blot of the induced protein K1DG71353.
  • Figure 21 gives the complete DNA sequence of the coding region of the K1DG71353 protein and the derived amino acid sequence.
  • Promoter A DNA sequence located proximal to the start codon at the 5' end of the transcribed sequence. At the promoter region, transcription of an adjacent operably linked gene(s) is initiated.
  • Gene A DNA sequence that contains information for transcription of a mRNA which codes for a polypeptide or protein. Typically, the nucleotide of the first transcribed codon is numbered +1, and the nucleotides are numbered consecutively with positive integers through the transcribed regions of the gene. Nucleotide +1 may or may not also code for the first translated amino acid.
  • a gene may have regions at the 5' end and 3' end which are transcribed but which are not translated. Similarly, the gene may or may not contain intron information which must be spliced out prior to translation of the mRNA. The mRNA may or may not have regions at the 5' end and/or the 3' end which are not translated.
  • the numbering of nucleotides in the promoter and transcriptional regulatory region 5' to the transcribed region proceeds consecutively with negative integers with the 5' nucleotide next to the first transcribed codon being numbered -1.
  • a "complementary DNA” or "cDNA” gene includes recombinant genes synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed.
  • Operably linked means that two elements are physically arranged such that factors which influence one element also influence the other. For example, factors which induce a specific promoter to function also induce the transcription of a gene operably linked to that promoter.
  • Expression is the process by which the information encoded within a gene is revealed. If the gene encodes a protein, expression involves both transcription of the DNA into mRNA and translation of the mRNA into protein.
  • Cloning vehicle A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning.
  • the cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance. The word "vector” is sometimes used for "cloning vehicle.”
  • Expression vehicle A vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
  • the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • Host Any organism that is the recipient of a replicable expression vehicle.
  • peptide fragment is meant to include any amino acid sequence which represents a segment of HIV-2, HIV-1, and/or HTLV-I, which is capable of immunologically reacting with the respective antibody and includes naturally-occurring peptide sequences; synthetic, chemically-synthesized peptide sequences; and expressed, genetically engineered peptide sequences.
  • the invention comprises the amino acid sequences of immunoreactive env HIV-2 peptides that can be used for detecting antibodies to HIV-2.
  • the invention also comprises genetic sequences coding for the immunoreactive env peptides, expression vehicles containing the genetic sequences, and hosts transformed therewith.
  • the invention also comprises the amino acid sequences of immunoreactive chimeric env peptides which are able to detect more than one antibody. Specifically, these chimeric env peptides are able to detect antibodies to HIV-2, HIV-1, and HTLV-I and HTLV-II.
  • this invention comprises chimeric env peptides comprising immunodiagnostic env peptide fragments of HIV-2-HIV-1; HIV-2-HIV-1-HTLV-I; HIV-2-HTLV-I; and HIV-1- HTLV-I.
  • a chimeric env peptide is meant a peptide wherein said peptide contains an amino acid sequence which encodes domains of different viral peptides.
  • Two peptides are said to be different peptides if they are not covalently joined in their mature form in the native virus.
  • the inventors have discovered that by combining domains of peptides from different retroviruses there is created a chimeric peptide that is able to detect antibodies to HIV-2, HIV-1, and HTLV-I and HTLV-II, depending on the particular construction of the chimeric peptide.
  • the chimeric env peptides may contain domains from any HIV-2, HIV-1, and HTLV-I env peptide as long as those domains are capable of being immunoreactive against their respective antibodies or of being combined with another domain in such a way as to maintain, enhance or induce antibody recognition.
  • the preferred peptide fragments are those described in Examples 1, below, and shown in Figure 4, which gives the complete DNA sequence of the coding region of the K1 peptide fragment and the derived amino acid sequence and in Example 2, below, and shown in Figure 8, which gives the complete DNA sequence of the coding region of the K3 peptide fragment and the derived amino acid sequence.
  • the preferred env peptides of HIV-1 are those described in Beltz et al., U.S. 4,753,873, particularly the clone G peptide fragment and those peptide fragments derived therefrom.
  • the most preferred env peptide fragment is CBre3, derived from the gp120 and gp41 regions of the HIV-1 env gene.
  • CBre3, also identified as delta G71A is described in Thorn et al., "An enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus," J. CIin. Microbiol. 25:1207-1212 (1987) and in Beltz et al., U.S. 4,753,873.
  • a cell line expressing the recombinant antigen is also on deposit at the American Type Culture Collection (ATCC), accession number 53455.
  • ATCC American Type Culture Collection
  • Other recombinant HIV-1 antigens may be used in this invention, provided that the antigens exhibit immunoreactivity to HIV-1 antibodies. Examples of such recombinant HIV-1 antigens are described in Chang, et al., Bio/Technology. 3:905-909 (1985); Cabradilla et al., Bio/Technology, 4:128-133 (1985); and U.S. 4,629,783, all incorporated herein by reference.
  • HTLV-II HTLV-I envelope gene from base pairs 6101-6118 and 6170-6499 based on the published sequence of Seiki et al. (Proc. Natl. Acad. Sci. USA 80:3618 (1983)) incorporated herein by reference.
  • Other preferred peptide fragments are those described in Samuel et al., Science 126:1094-1097 (1984), incorporated herein by reference, which gives a restriction map of the env gene of HTLV-I.
  • HTLV-II antibodies can also be detected with use of HTLV-I antigens.
  • peptide fragments there may also be some variation in the peptide fragments, provided however, that these peptides retain immunoreactivity to antibodies to HIV-2, HIV-1, or HTLV-I, respectively.
  • the ranges in the length of the peptide fragments need not be precisely fixed.
  • Amino acids of the peptide fragments may be deleted or added without loss of immunoreactivity.
  • amino acids could be exchanged, e.g. a neutral amino acids such as a valine could be exchanged with another neutral amino acid such as leucine.
  • the changes in amino acids may be either conservative or non-conservative.
  • HIV-2 env peptide fragments are used for illustration.
  • the HIV-1 and HTLV-I env peptides used in the chimeric env diagnostic peptides may also be used in these described methods.
  • the native peptide fragments of the HIV-2 virus may be used as a source of the peptide domains and may be obtained directly from the infected host cell.
  • the peptide fragment would then be obtained by fragmenting the naturally-occurring virus using suitable enzymes or chemical methods.
  • a preferred method of obtaining the peptide fragment is by cloning a polynucleotide fragment which codes for the desired peptide, using genetic engineering techniques.
  • the advantages of using genetic engineering and recombinant clones are twofold: the first advantage is that it is difficult and time-consuming to obtain large amounts of the viral peptides either by direct isolation from the virus or by chemical synthesis; the second advantage is that recombinant peptides are devoid of human antigens that may reduce the reliability of a diagnostic test.
  • the genetic constructs and the methods for using them can be utilized for expression of the peptide fragments in hosts, including prokaryotic and eukaryotic hosts.
  • the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in a prokaryotic host.
  • a plasmid or viral vector capable of autonomous replication in a prokaryotic host. Any of a wide variety of vectors may be employed for this purpose, as outlined below.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • the procaryotic hosts may include bacteria such as E. coli. S. typhimurium, Serratia marcescens or Bacillus subtilis.
  • the peptides of the invention are expressed in prokaryotes and especially in E. coli.
  • the preferred bacterial host for expression is an E. coli strain that contains a temperature sensitive bacteriophage lambda CI857 gene, such as MZ1, described in Lautenberger et al., Gene Anal. Tech. 1:63-66 (1984).
  • Suitable vector systems for expression in E. coli are pCBCl, described by Beltz, U.S. 4,753,873, and pJL6 Lautenberger, et al., Gene Anal. Tech.
  • Eukaryotic hosts may also be used and include yeast, filamentous fungi, insect cells and mammalian cells (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and especially mammalian cells which have been immortalized and may be maintained in cell culture.
  • Preferred eukaryotic plasmids include those derived from the bovine papilloma virus, vaccinia virus, SV40, and, in yeast, plasmids containing the 2-micron circle, etc., or their derivatives.
  • Such plasmids are well known in the art (Bot- stein, D., et al., Miami Wntr. Svmp. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomvces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.
  • mammalian expression vector systems which utilize the MSV-LTR promoter to drive expression of the cloned gene, and in which it is possible to cotransfect with a helper virus to amplify plasmid copy number, and, integrate the plasmid into the chromosomes of host cells have been described (Perkins, A.S. et al., Mol. Cell Biol. 3:1123 (1983); Clontech, Palo Alto, California).
  • the DNA sequence of the peptide fragments can also be inserted into the genome of viruses which is used to infect a cell; for example, vaccinia virus and bacculovirus may be used.
  • viruses which is used to infect a cell
  • vaccinia virus and bacculovirus may be used.
  • the recombinant vaccinia virus replicates in any mammalian cell and the fragment of interest appears on the envelope or in internal viral proteins.
  • the DNA sequence may be chemically constructed if it is not desired to utilize the HIV-2 genome as the source of the genetic information.
  • Methods of chemically synthesizing DNA are well known in the art (Oligonucleotide Synthesis, A Practical Approach, M. J. Gait, ed., IRS Press, Washington, D.C., 1984; Synthesis and Applications of DNA and RNA. S.A. Narang, ed., Academic Press, San Diego, CA, 1987). Because the genetic code is degenerate, more than one codon may be used to construct the DNA sequence encoding a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357).
  • HIV-2 peptides of the invention transcriptional and translational signals recognizable by an appropriate host are necessary.
  • the cloned HIV-2 peptide encoding sequences obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a host cell, either prokaryote or eukaryote, to produce the recombinant HIV-2 peptides of the invention or functional derivatives thereof.
  • HIV-2 peptides in different hosts may result in different post-translational modifications which may alter the properties of the peptides. It is necessary to express the peptides in a host wherein the ability of the peptides to be immunologically recognized by HIV-2 antibodies is not hindered.
  • expression vectors containing transcriptional regulatory sequences such as promoter sequences, which facilitate the efficient transcription of the inserted gene fragment, and which are derived from species compatible with the host cells, are used in connection with these hosts.
  • the expression vector typically contains discrete elements such as, for example, a) either an origin of replication which allows for autonomous replication of the vector or elements which promote insertion of the vector into the host's chromosome, b) a suitable transcriptional promoter to which the sequence of interest can be operably linked, c) a transcriptional terminator sequence if necessary, and d) specific genes which are capable of providing phenotypic selection in transformed cells.
  • the precise nature of the regulatory regions needed for gene expression will vary between species or cell types and there are many appropriate expression vector systems that are commercially available (For example, through Pharmacia, Boehringer Mannheim or Clontech).
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are “operably linked” to the nucleotide sequence which encodes the polypeptide.
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • Two DNA sequences (such as HIV-2 peptide encoding sequence and a promoter region sequence linked to the 5' end of the encoding sequence) are said to be operably linked if induction of promoter function results in the transcription of the peptide encoding mRNA and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the HIV-2 peptide, or (3) interfere with the ability of the HIV-2 peptide template to be transcribed by the RNA polymerase.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • HIV-2 peptides in eukaryotic hosts requires the use of regulatory regions functional in such hosts, and preferably eukaryotic regulatory systems.
  • a wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the eukaryotic host.
  • the transcriptional and translational regulatory signals can also be derived from the genomic sequences of viruses which infect eukaryotic cells, such as HIV, SIV, adenovirus, bovine papilloma virus, Simian virus, herpes virus, or the like.
  • these regulatory signals are associated with a particular gene which is capable of a high level of expression in the host cell.
  • a fusion product of the HIV-2 peptides may be constructed.
  • sequence coding for the HIV-2 peptides may be linked to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular host.
  • signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable to subsequent removal.
  • Transcriptional initiation regulatory signals can be selected which allow for repression or activation, so that expression of the operably linked genes can be modulated.
  • regulatory signals which are temperature- sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.
  • sequences functional in the host cell may be substituted.
  • the vectors of the invention may further comprise other operably linked regulatory elements such as enhancer sequences, or DNA elements which confer species, tissue or cell-type specific expression on an operably linked gene.
  • the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, for example by transformation of bacterial cells. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the HIV-2 peptide or in the production of a fragment of this peptide. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner.
  • the HIV-2 peptide DNA encoding sequence and an operably linked promoter is introduced into a recipient host cell as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replication, the expression of the HIV-2 peptide may occur through the transient expression of the introduced sequence.
  • a non-replicating DNA (or RNA) molecule which may either be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replication
  • Genetically stable transformants may be constructed with vector systems, or transformation systems, whereby the HIV-2 peptide DNA is integrated into the host chromosome. Such integration may occur de novo within the cell or be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with retroviral vectors, transposons or other DNA el ements which promote integration of DNA sequences in chromosomes.
  • a vector is employed which is capable of integrating the desired gene sequences into a mammalian host cell chromosome.
  • Cells which have been transformed with the HIV-2 peptide- containing DNA vectors of the invention are selected by also introducing one or more markers which allow for selection of host cells which contain the vector, for example, the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the transformed host cell can be fermented (if prokaryotic) or cultured (if eukaryotic) according to means known in the art to achieve optimal cell growth, and also to achieve optimal expression of the cloned HIV peptide sequence fragments.
  • high level of HIV-2 peptide expression for the cloned sequences coding for peptide fragments can be achieved according to a preferred procedure of this invention.
  • the fragments will typically be recovered and purified according to means known in the art.
  • high-level expression of the clones usually results in the formation of insoluble inclusion bodies or aggregates. To purify the expressed proteins, the insoluble inclusion bodies must be made soluble.
  • the expressed peptide fragments are purified in a process using N-acylation of amino groups, for example, by citraconylation (Marciani, D.J. et al., Protein Purification: Micro to Macro, Alan. R. Liss, Inc., 1987 pp. 443-458.
  • An alternative to recombinant genetic engineering techniques for producing peptide fragment antigen includes enzyme polymerase directed in vitro transcription and translation systems.
  • An amplification system for producing peptide fragments is described, for example, in U.S. 4,683,202.
  • the purified immunogenic and diagnostic peptide fragments according to this invention are specifically recognized by antibodies produced in response to the HIV-2 virus.
  • the HIV-2 antibodies in blood or tissue samples can be detected using the peptide fragments in immunoassays wherein the peptides can be utilized in liquid phase or bound to a solid phase carrier.
  • the peptide fragments can be detectably labeled in various ways for use in immunoassays for virus.
  • the preferred immunoassays for detecting HIV-2 antibodies using the peptide fragments of this invention include radioimmuno- assays, enzyme-linked immunosorbent assays (ELISA), or other assays known in the art, such as immunofluorescent assays, chemiluminescent assays, or bioluminescent assays.
  • Radioactive isotopes which are particularly useful in assays are 3 H , 125 I , 131 I , 32 P , 35 S , 14 C , 51 Cr, 36 Cl, 57 Co,
  • the peptide sequence or antibodies thereto may also be labeled using fluorescent labels, enzyme labels, free radical labels, avidin-biotin labels, or bacteriophage labels, using techniques known to the art (Chard, Laboratory Techniques in Biology, "An Introduction to Radioimmunoassay and Related Techniques," North Holland Publishing Company (1978).
  • Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.
  • Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and the oxalate esters.
  • Typical bioluminescent compounds include luciferin, luciferase, and aequorin.
  • Typical enzymes include alkaline phosphatase, ⁇ - galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, glucose oxidase, and peroxidase.
  • EMIT enzyme-linked immunosorbent assay
  • EMIT enzyme-multiplied immunoassay
  • the immunoassays within the scope of the present invention include latex agglutination assays, immunometric assays and competitive assays.
  • Latex agglutination assays have been described in Beltz, G.A. et al., in Molecular Probes: Techniques and Medical Applications, A. Albertini et al. , eds., Raven Press, New York, 1989, incorporated herein by reference.
  • a antigen-coated latex particles and serum to be tested are mixed and the results then read.
  • the latex particles With samples lacking HIV-2 antibodies, the latex particles remain in suspension and retain a smooth, milky appearance. However, if antibodies reactive with the recombinant antigen are present, the latex particles clump into visibly detectable aggregates.
  • the latex agglutination assay is especially suitable for small volume users, emergency situations, and areas lacking the sophisticated laboratory equipment and supplies needed for immunometric assays.
  • An agglutination assay can also be used to detect HIV-2 antibodies wherein the desired peptide fragment is immobilized on a suitable particle other than latex beads, for example, on gelatin, red blood cells, nylon, liposomes, gold particles, etc.
  • a suitable particle other than latex beads for example, on gelatin, red blood cells, nylon, liposomes, gold particles, etc.
  • the presence of antibodies in the same causes agglutination, similar to that of a precipitation reaction, which can then be detected by such techniques as nephelometry, turbidity, infrared spectrometry, visual inspection, colorimetry, and the like.
  • Immunometric assays include forward sandwich, reverse sandwich immunoassays and simultaneous assay. Each of these terms is well understood by those skilled in the art.
  • the immunometric assays will be described for the detection of antibodies to HIV-2.
  • the peptide fragment is bound to the solid-phase carrier and anti-IgG antibodies are detectably labeled.
  • a forward sandwich immunoassay a sample suspected of containing antibodies against HIV-2 is first incubated with a solid-phase immunoabsorbent containing the peptide fragment. Incubation is continued for a period of time sufficient to allow the antibodies in the sample to bind to the immobilized peptide fragment.
  • the solid-phase immunoabsorbent is separated from the incubation mixture and washed to remove interfering substances which also may be present in the sample.
  • Solid-phase immunoabsorbent- containing antibodies bound to the immobilized peptide fragments are subsequently incubated for a second time with soluble labeled antibody cross-reactive with a different domain on the antibody to be detected.
  • another wash is performed to remove unbound labeled antibody from the solid-phase immunoabsorbent and to remove non-specifically bound labeled antibody.
  • Labeled antibody bound to the solid-phase immunoabsorbent is then detected and the amount of labeled antibody detected serves as a direct measure of the amount of antibodies present in the original sample.
  • labeled antibody which is not associated with the immunoabsorbent complex can also be detected, in which case the measure is in inverse proportion to the amount of antigen present in the sample.
  • Forward sandwich assays are described, for example, in United States Patents 3,867,517; 4,012,294; and 4,376,110.
  • a reverse sandwich assay the sample suspected of containing test antibodies against HIV-2 is initially incubated with labeled anti-antibody, after which the solid- phase immunoabsorbent containing immobilized peptide fragment cross-reactive with a different domain on the test antibody is added thereto, and a second incubation is carried out.
  • the initial washing step required by a forward sandwich assay is not required, although a wash is performed after the second incubation.
  • Reverse sandwich assays have been described, for example, in U.S. Patent No. 4,098,876 and 4,376,110.
  • a simultaneous sandwich assay In a simultaneous sandwich assay, the sample, the immunoabsorbent having immobilized peptide fragment thereon and labeled soluble antibody specific to a different domain of the test antibody are incubated simultaneously in one incubation step.
  • the simultaneous assay requires only a single incubation and does not require washing steps.
  • the use of a simultaneous assay is a very helpful technique, providing ease of handling, homogeneity, reproducibility, linearity of the assays, and high precision.
  • a simultaneous sandwich assay is described, for example, in U.S. Patent No. 4,376,110.
  • delayed immunometric assays can also be utilized, as are, for example, described in Chu, U.S. Patent No. 4,289,747, and Wolters, U.S. Patent No. 4,343,896.
  • Another immunometric assay involves capturing the Fc capture technique .
  • Fc capture immunoassay total serum antibodies are captured through anti-human Fc antibodies, typically bound to a solid support. Thus, bound, the Fc region of the antibody to be detected does not participate in other protein-protein interactions.
  • the antibodies to be detected can then be screened with the HIV-2 peptide fragment(s) or with the appropriate chimeric peptide fragment(s) of this invention.
  • a preferred Fc capture immunoassay is described in U.S. Serial No. 07/203,730, filed June 8, 1988, incorporated herein by reference.
  • the sample-containing antibody, solid-phase immunoabsorbent with immobilized peptide fragment and labeled soluble antibody are incubated under conditions and for a period of time sufficient to allow the test antibodies to bind to the immobilized peptide fragments and to the soluble antibodies.
  • the specific concentrations of labeled antibodies and immobilized fragments, the temperature and time of incubation, as well as other such assay conditions can be varied, depending upon various factors including the concentration of antibody in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • solid-phase immunoabsorbents which have been employed and which can be used in the present invention.
  • Well-known immunoabsorbents include beads formed from glass, polystyrene, paper, polypropylene, dextran, nylon, and other material; tubes formed from or coated with such materials, and the like.
  • the immobilized peptide fragments may be covalently or physically bound to the solid-phase immunoabsorbent, by techniques such as covalent bonding via an amide or ester linkage or by adsorption.
  • suitable carriers for binding peptide fragments or will be able to ascertain such, using routine experimentation.
  • the raised antibody is preferably generated with antigenic peptide fragments of the invention.
  • the soluble antibody may be labeled with any detectable label, such as a radiolabel, a fluorescent label, an enzyme label, a free radical label, or a bacteriophage label.
  • a detectable label such as a radiolabel, a fluorescent label, an enzyme label, a free radical label, or a bacteriophage label.
  • the label is a radiolabel or an enzyme label.
  • the HIV-2 immunogenic peptide fragments according to this invention may be used to stimulate the production of antibodies.
  • the peptide fragment may be coupled to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanin (KLH), utilizing techniques well-known and commonly used in the art.
  • the carrier protein is KLH, linked to the peptide fragment through a cysteine residue.
  • HIV-2 peptide fragments can be admixed with an immunologically inert or active carrier.
  • Carriers which promote or induce immune responses such as Freund's complete adjuvant, can be utilized.
  • the antigenic material the peptide fragment hapten-carrier protein conjugate
  • the detection of appropriate antibodies may be carried out by testing the antisera with appropriately labeled tracer-containing molecules. Fractions that bind tracer- containing molecules are then isolated and further purified if necessary.
  • Antibodies thus obtained may then be utilized in various immunoassays to identify and quantitate the HIV-2 virus or fragments thereof.
  • Both polyclonal antibodies and monoclonal antibodies, produced by well-known techniques as described in Catty, supra, raised in response to the peptide fragments of this invention can be utilized in immunoassays.
  • two separate and distinct antibodies are required.
  • One of these antibodies is bound to the solid-phase support while the other is detectably labeled.
  • the two different antibodies although specific for HIV-2 virus, are cross-reactive with different domains on viral protein.
  • the two different antibodies may be prepared by using two different peptide fragments according to this invention. The use of antibodies to different peptide fragments, one bound to a carrier and the other detectably labeled, is useful in various sandwich assays.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, test tubes, and the like.
  • container means such as vials, test tubes, and the like.
  • Each of said container means comprises one of the separate elements to be used in the method.
  • one of said container means may comprise an immunoabsorbent-bound peptide fragment. Such fragment may be bound to a separate solid-phase immunoabsorbent or directly to the inner walls of a container.
  • a second container may comprise detectably labeled anti-antibody in lyophilized form or in solution.
  • the carrier may also contain, in addition, a plurality of containers each of which comprises different, predetermined and known amounts of antibody. These latter containers can then be used to prepare a standard curve from which can be interpolated the results obtained from the sample containing the unknown amount of antibody.
  • the presence of the HIV-2 antibody or the virus itself or portions thereof may be detected in biological fluids and tissues.
  • Any sample containing the unknown amount of HIV-2 antibodies or HIV-2 can be used.
  • a sample is a liquid such as, for example, urine, saliva, tear drops, cerebrospinal fluid, blood, serum and the like, or a solid or semi-sol id such as, for example, tissues, feces, and the like.
  • the HIV-2 virus and antibodies to the virus are associated with the T-cell disorder, Acquired Immune Deficiency Syndrome (AIDS) and pre-AIDS conditions, such as AIDS-related complex (ARC).
  • AIDS Acquired Immune Deficiency Syndrome
  • ARC AIDS-related complex
  • antibodies to HIV-2 may be present in a human's or animal's biological fluids or tissue, without such human or animal suffering from AIDS or ARC.
  • the peptide fragments according to this invention may also be used as a vaccine against the HIV-2 virus.
  • the peptide fragment may be prepared and administered to an animal, as is generally known in the art, to stimulate the production of antibodies.
  • the vaccinia virus can be used according to known means for the preparation of HIV-2 vaccines.
  • the K3D clone is a subgenomic fragment of a complete HIV- 2 clone HIV-2 SL/ISY (Franchini et al., Proc. Natl. Acad. Sci. USA 86:2433-2437 (1989)), prepared by insertion of a KPNI fragment of HIV-2 SBL/ISY from position 5295 to position 9012 into a Bluescript vector (Stratagene, Inc.).
  • the HIV-2 clone K3D was provided by Genoveffa Franchini.
  • the strategy for construction of pK1, a plasmid for expression of a peptide fragment of the env region of HIV-2 in E. coli, is illustrated in Figure 1.
  • K3D DNA was digested with restriction enzyme EcoRI with cuts at position 7297. The EcoRI end was made blunt with Klenow polymerase and Bglll linkers were added. The DNA was then digested with Bglll and Sau3AI. A fragment of approximately 848 base pairs extending from the EcoRI site (converted to Bglll) at position 7297 to the Sau3AI site at position 8139 was purified and cloned into Bam HI restricted the expression vector pCBCl to generate the clone pKl.
  • Plasmid pCBCl is an E. coli expression vector that makes use of the bacteriophage lambda pL promoter.
  • pCBCl is similar to pJL6 (Lautenberger et al., Gene Anal. Tech. 1:63 (1984)) whose construction is described completely in Beltz et al., U.S. Patent No. 4,753,873, incorporated herein by reference.
  • K3D (described in Example 1) was digested with Sau3AI. This cutting resulted in a 1038 base pair fragment from position 7101 to position 8139 ( Figure 5). After purifying the fragment, it was ligated into the expression vector pCBC2.
  • pCBC2 differs from pCBCl by a single base pair just 5' to the BamHI cloning site. pCBC2 was used in this case rather than pCBCl to allow for proper reading frame alignment. pCBC2 is also described in Beltz et al., U.S. Patent No. 4,753,873, incorporated herein by reference. The resulting plasmid, pK3, was transferred into the E. coli host MZ-1.
  • Example 3 Construction of an HIV-2-HIV-1 chimeric envelope expressing clone MZ-1/pKlDG71
  • FIG. 9 The strategy for the construction of the plasmid to express a chimeric envelope clone is illustrated in Figure 9.
  • the plasmid clone pLCBCODG71A has been described in Beltz et al., U.S. 4,753,873, incorporated herein by reference. This plasmid is used for expression of HIV-1 envelope polypeptide.
  • pLCBCODG71A was digested with Bglll and BamHI. Bglll cleaves at position 407 and BamHI cleaves at position 944. A 537 BP fragment was isolated.
  • pK1 plasmid DNA described in Example 1, was digested with BamHI which cleaves at position 890.
  • the 537 BP Bglll- BamHI fragment of pLCBCODG71A was ligated to BamHI digested pK1.
  • the resulting plasmid, pK1DG71 was transferred to E. coli host MZ-1.
  • Upon temperature shift of MZ-1/pKlDG71 culture from 32oC to 42oC Upon temperature shift of MZ-1/pKlDG71 culture from 32oC to 42oC, synthesis of a recombinant protein of approximately 55 kd was induced ( Figure 10).
  • Western blot analysis was done with sera from HIV-I infected individuals, HIV-2 infected individuals and negative controls.
  • pCB1353 is an expression plasmid containing regions of the HTLV-I envelope gene from base pairs 6101-6118 and 6170-6499 based on the published sequence of Seiki et al. (Proc. Natl. Acad. Sci. USA 80:3618 (1983)) cloned into expression plasmid pCBCl.
  • the plasmid pCBC1353 was digested with BamHI and Sail to generate a 370 base pair fragment.
  • pCBC2DG71 is an expression plasmid coding for a polypeptide of the HIV-1 envelope protein. This plasmid has been described by Beltz et al., U.S. 4,753,873, incorporated herein by reference.
  • the resulting plasmid, pDG71353, codes for a chimeric polypeptide of HIV-1-HTLV-I.
  • the sequence of the coding region of pDG71353 is shown in Figure 15.
  • Figure 14 shows the remaining steps for the HIV-2-HIV-1- HTLV-I construction.
  • pDG71353 was digested with Hindlll and Sail.
  • the insert which contains sequences coding for the HIV- 1-HTLV-I polypeptide was ligated into the vector pKlDG71 which had been digested with Hindlll and Sail.
  • pKlDG71 is described in Example 3.
  • the resulting plasmid is called pK1DG71353.
  • pK1DG71353 was transferred into the bacterial strain MZ-1.
  • the following purification procedure has been used to purify both HIV-2 env (K-1) and HIV-2-HIV-1 chimeric antigens.
  • E. coli cells were lysed by enzymatic digestion with lysozyme (1 mg/gram of cells) for 10 minutes in 50 mM TrisHCl, pH 7.5 (3 ml/gram of cells) containing 2 mM PMSF, aprotinin (0.1 mg/gram of cells) and DNase I (0.1 mg/gram of cells). The solution was brought up to 1% triton X-100 and stirred for 30 minutes at room temperature. Insoluble material was collected by centrifugation at 12,000 xg for 30 minutes and redigested as above with the exception that RNase I (0.05 mg/gram of cells) and 2.5 mM MgCl 2 were added. After digestion, the pellet was collected by centrifugation as above.
  • the recombinant antigens remained in the insoluble pellet.
  • the pellet from the 8M urea wash was then solubilized with 7M guanidine-HCl pH 11.0/0.5% ⁇ - mercaptoethanol.
  • Both HIV-2 env antigen and HIV-2-HIV-1 chimeric protein are very hydrophobic and tend to form aggregates upon removal of guanidine-HCl.
  • the solubilized antigens were subjected to the following chemical modifications designed to increase the solubility of the antigens in aqueous buffers.
  • the solubilized protein in 6M guanidine-HCl was first alkylated with 1.2 fold excess of iodoacetic acid over ⁇ - mercaptoethanol at pH 8.5. Any unreacted iodoacetic acid was quenched with ß-mercaptoethanol.
  • the alkylated sample was dialyzed 3 times against 200 fold 50 mM borate, pH 9.0. The protein became insoluble upon dialysis. The insoluble material was collected by centrifugation and redissolved in 8M Urea, 50 mM borate, pH 9.0 for acylation.
  • citraconylated protein was soluble in aqueous buffer and amenable to standard chromatographic technique.
  • the modified protein sample was then applied to a DEAE- TSK column, equilibrated in 50 mM borate, pH 9.0.
  • the column was developed with a linear gradient of 0.3-1M NaCl to elute the recombinant antigen.
  • Fractions containing HIV-2 env, K-1, antigen or HIV-2-HIV-1 chimeric protein, K1DG71, were pooled.
  • the immunoreactivity of purified K1 and the chimeric K1DG71 antigens were tested using an indirect immunoassay (EIA). Recombinant antigen was coated onto microtiter wells at concentrations of 0.5 ug and 1.0 ug for K-2 and the chimeric antigen respectively.
  • EIA indirect immunoassay

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Abstract

L'invention a trait à la découverte que certains fragments peptidiques d'enveloppe du virus-2 d'immunodéficience humaine (VIH-2) sont particulièrement immunoréactifs contre des anticorps du VIH-2. Par conséquent, on peut appliquer ces fragments à des tests d'immunodiagnostic permettant la détection d'anticorps contre le VIH-2. L'invention a également trait à certaines protéines chimériques obtenues à partir de parties immunogènes du gène d'enveloppe du VIH-2, VIH-1, ou le HTLV-I, que l'on peut utiliser dans des tests visant à déterminer la présence d'anticorps contre le VIH-2, le VIH-1, le HTLV-I et le HTLV-II.
PCT/US1990/006621 1989-11-13 1990-11-13 Proteines de diagnostic utilisees pour tester plus d'un anticorps WO1991007664A1 (fr)

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FR2676071A1 (fr) * 1991-05-02 1992-11-06 Transgene Sa Nouveau variant gp160 non-clivable, soluble, de forme hybride.
WO1994018322A1 (fr) * 1993-02-05 1994-08-18 Genelabs Technologies, Inc. Peptides derives de gp21 de htlv-i et htlv-ii utilises dans des diagnostics
WO1999009179A2 (fr) * 1997-08-15 1999-02-25 Abbott Laboratories Produits de recombinaison d'antigenes utiles dans la detection et la differenciation d'anticorps contre le vih
WO1999059615A1 (fr) * 1998-05-20 1999-11-25 Trimeris, Inc. Polypeptides hybrides dotes de proprietes pharmacocinetiques ameliorees
WO2001003723A1 (fr) * 1999-07-09 2001-01-18 Trimeris, Inc. Polypeptides hybrides possedant des proprietes pharmacocinetiques ameliorees
GB2396008A (en) * 2002-12-03 2004-06-09 Trinity Res Ltd A test for detection of antibodies to HIV
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Cited By (25)

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US5643714A (en) * 1986-12-31 1997-07-01 Genelabs Technologies, Inc. Method and assay for HTLV
FR2676071A1 (fr) * 1991-05-02 1992-11-06 Transgene Sa Nouveau variant gp160 non-clivable, soluble, de forme hybride.
WO1992019742A1 (fr) * 1991-05-02 1992-11-12 Transgene S.A. NOUVEAU VARIANT gp160 NON-CLIVABLE, SOLUBLE, DE FORME HYBRIDE
WO1994018322A1 (fr) * 1993-02-05 1994-08-18 Genelabs Technologies, Inc. Peptides derives de gp21 de htlv-i et htlv-ii utilises dans des diagnostics
US7803524B2 (en) 1994-02-23 2010-09-28 Siemens Healthcare Diagnostics Products Gmbh Antigenic HIV gp41 chimeric polypeptides comprising the MVP5180/91 epitope SKGKLIS
US7619061B2 (en) 1997-08-15 2009-11-17 Abbott Laboratories, Inc. Antigen constructs useful in the detection and differentiation of antibodies to HIV
US7615613B2 (en) 1997-08-15 2009-11-10 Abbott Laboratories, Inc. Antigen constructs useful in the detection and differentiation of antibodies to HIV
US7883845B2 (en) 1997-08-15 2011-02-08 Abbott Laboratories Antigen constructs useful in the detection and differentiation of antibodies to HIV
WO1999009179A2 (fr) * 1997-08-15 1999-02-25 Abbott Laboratories Produits de recombinaison d'antigenes utiles dans la detection et la differenciation d'anticorps contre le vih
WO1999009179A3 (fr) * 1997-08-15 2003-05-08 Abbott Lab Produits de recombinaison d'antigenes utiles dans la detection et la differenciation d'anticorps contre le vih
EP2305818A3 (fr) * 1997-08-15 2011-05-11 Abbott Laboratories Constructions d'antigène utiles dans la détection et la différentiation d'anticorps du VIH
US7615614B2 (en) 1997-08-15 2009-11-10 Abbott Laboratories, Inc. Antigen constructs useful in the detection and differentiation of antibodies to HIV
US8785596B2 (en) 1997-08-15 2014-07-22 Abbott Laboratories Antigen constructs useful in the detection and differentiation of antibodies to HIV
US6846905B2 (en) 1997-08-15 2005-01-25 Abbott Laboratories Antigen constructs useful in the detection and differentiation of antibodies to HIV
EP2055780B1 (fr) * 1997-08-15 2013-11-27 Abbott Laboratories Constructions d'antigène utiles dans la détection et la différentiation d'anticorps du VIH
US6562787B1 (en) 1998-05-20 2003-05-13 Trimeris, Inc. Hybrid polypeptides with enhanced pharmacokinetic properties
US7297784B2 (en) 1998-05-20 2007-11-20 Trimeris, Inc. DNA encoding T-1249 and other viral fusion inhibitor peptides useful for treating aids
KR100742789B1 (ko) * 1998-05-20 2007-07-25 트라이머리스 인코퍼레이티드 약물 동력학 성질이 강화된 하이브리드 폴리펩티드
US6656906B1 (en) 1998-05-20 2003-12-02 Trimeris, Inc. Hybrid polypeptides with enhanced pharmacokinetic properties
US6348568B1 (en) 1998-05-20 2002-02-19 Trimeris, Inc. Hybrid polypeptides with enhanced pharmacokinetic properties
US6258782B1 (en) * 1998-05-20 2001-07-10 Trimeris, Inc. Hybrid polypeptides with enhanced pharmacokinetic properties
WO1999059615A1 (fr) * 1998-05-20 1999-11-25 Trimeris, Inc. Polypeptides hybrides dotes de proprietes pharmacocinetiques ameliorees
WO2001003723A1 (fr) * 1999-07-09 2001-01-18 Trimeris, Inc. Polypeptides hybrides possedant des proprietes pharmacocinetiques ameliorees
GB2396008B (en) * 2002-12-03 2006-01-25 Trinity Res Ltd A test for detection of antibodies to HIV
GB2396008A (en) * 2002-12-03 2004-06-09 Trinity Res Ltd A test for detection of antibodies to HIV

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EP0453563A1 (fr) 1991-10-30

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