WO2000040970A1 - Detecting and monitoring hiv viral infections - Google Patents
Detecting and monitoring hiv viral infections Download PDFInfo
- Publication number
- WO2000040970A1 WO2000040970A1 PCT/FR1999/003311 FR9903311W WO0040970A1 WO 2000040970 A1 WO2000040970 A1 WO 2000040970A1 FR 9903311 W FR9903311 W FR 9903311W WO 0040970 A1 WO0040970 A1 WO 0040970A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mixotope
- hiv
- reagent according
- antigenic peptide
- pol
- Prior art date
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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
-
- 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
- C12N2740/16011—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
- G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
- G01N2333/16—HIV-1, HIV-2
- G01N2333/161—HIV-1, HIV-2 gag-pol, e.g. p55, p24/25, p17/18, p.7, p6, p66/68, p51/52, p31/34, p32, p40
Definitions
- the present invention relates to a reagent for detecting and monitoring viral infections caused by the human immunodeficiency virus (HIV) and to its applications for the detection of human immunodeficiency.
- HIV human immunodeficiency virus
- the first HIV infection screening trials implemented in 1983 and 1985, used a viral lysate to capture anti-HIV antibodies. These tests had the major disadvantage of lacking both sensitivity and specificity.
- due to the use of a viral lysate there was a difficulty in reproducing viral antigens from batch to batch and the need to carry out virus cultures in large quantities, which presented a danger for the manipulator. .
- EP 181 150 and EP 387 914 describe the use of genetic engineering, for obtaining polypeptides (antigens), with a view to detecting anti-HIV1 antibodies.
- the immunoassays using such reagents generally have a low sensitivity, especially when there is a low affinity between the antibody to be tested and the selected antigen. This is particularly the case during a recent seroconversion and / or when a new HIV subtype appears.
- one or more amino acids selected from a mixture of amino acids selected from known variants of said epitopic domain or selected arbitrarily.
- compositions or mixtures generally contain between 2 and 2000 and up to 10 10 peptides which comprise different individual peptide sequences which are found as close as possible to a predefined statistical distribution; they can be used as immunological reagents.
- HIV1 virus is subject to frequent mutations; in this context, some of these viruses are little or not detected by these tests because of the mutations they carry, which results in the non-detection of subjects contaminated by these viruses (false negatives).
- the Applicant has set itself the goal of providing a new reagent for detecting HIV infections, capable of being used in immunoenzymatic tests, which is both specific and sensitive and which makes it possible to obtain a gain. sensitivity of at least 15 to 30% compared to the reagents of the prior art. Such a reagent better meets the needs of the practice than the reagents of the prior art, in the context of immunoenzymatic tests, in particular of the ELISA type.
- the subject of the present invention is a reagent for detecting an infection caused by a human immunodeficiency virus, characterized in that it comprises a mixture consisting of (1) an antigenic fragment or peptide encoded by the pol gene of HIV1 comprising at most 60 amino acids, preferably between 20 and 40 amino acids and (2) a mixture (called mixotope) of convergent combinatorial peptides, derived from said antigenic fragment.
- mixture of (1) and (2) means either the association of (1) and (2) in said mixture, or the sequential association of (1) and (2) on solid support.
- the term “mixotope” is intended to mean the mixture of all the combinatorial peptides obtained from the selected antigenic fragment by artificial or constructed degeneration; they are preferably obtained during a single synthesis and represent the peptide antigen and its variability, in its function of recognition of a population of antibodies; different mixotopes can be obtained from the same peptide; the factors which intervene in the constitution of a mixotope are:
- the percentage of degeneration of the selected native antigenic peptide (total or partial degeneration);
- the mode of selection of the substitution of amino acids of said native antigenic peptide for each position of the sequence of the native antigenic peptide chosen, the amino acid substitution is selected on the basis of the replacement matrix, established by HM GEYSEN et al. (J. Mol. Recog., 1988, 1, 32-41), or modified, as illustrated in FIG. 1, taking into account the tolerance of antibody recognition, depending on the substitution of amino acids in the linear epitopes: preferably, for a given position, the amino acids having the highest percentage of "replaceability" are chosen. However, it is better to take into account the conformation of natural epitopes, before degeneration.
- the mixotope within the meaning of the present invention consisting of convergent combinatorial peptides, derived from a native antigenic peptide, therefore represents an artificial and unnatural degeneration of the native structure by the systematic or partial replacement of each amino acid by another from the replaceable matrix of GEYSEN or of the matrix according to FIG. 1.
- Said mixotope which can also be called convertope, differs from the mixotopes described in H. GRAS-MASSE et al. (Peptide Research, 1992, 5, 4, 211-216), which are divergent peptides obtained by natural degeneration taking into account natural antigenic variations or frequently observed during evolution and for vaccine purposes.
- the term constructed replaceability matrix means a matrix which does not reproduce the natural variations or frequently observed in evolution; the GEYSEN replaceability matrix or the matrix according to FIG. 1 are particularly suitable.
- the reagents according to the invention make it possible to obtain reliable, reproducible, very sensitive and very specific results, insofar as the combinations according to the invention have a synergistic effect in the detection of antibodies induced by viruses. ; in particular, a sensitivity gain of 15 to 30% is obtained.
- said antigenic peptide corresponds to an integrase epitope coded by the pol gene of HIV1, and preferably corresponds to the sequence KIQNFRNYYRDSRDPLWKGPAKLLWKGEGAVVIQDN (SEQ ID NO: 1) (HIV- POL); it has the advantage of having only few natural permutations within the groups M and O and of the subtypes of the class M.
- said selected antigenic peptide has been rigorously degenerated on the basis of the replacement matrix established by GEYSEN et al., Cited above or modified as illustrated in FIG. 1, which allows obtaining more than 10 10 peptides.
- an integrase epitope preferably an immunodominant fragment
- a mixotope derived from said epitope
- MIXO HIV-POL
- HIV-POL with its mixotope, MIXO (HIV-POL)
- MIXO MIXO
- the mixture of degenerate peptides, artificially produced, during the same synthesis is combined with the native peptide. Such a combination makes it possible, unexpectedly, to increase the reactivity of the mixture of antigens produced with respect to the antibodies naturally induced by the virus.
- the antigenic peptide (1) and the mixotope (2) are fixed on a solid support, preferably microtitration plates.
- said peptide (1) and said mixotope (2) are sequentially fixed on said support.
- the ratio of antigenic fragment: mixotope in the mixture is between 1: 10 and 1: 100.
- the present invention also relates to a method for diagnosing an HIV1 infection, by an immunoenzymatic method, characterized in that it uses a reagent according to the invention.
- a support such as a microtiter plate
- the present invention further relates to a kit or kit for diagnosing a viral HIV infection, characterized in that it comprises at least one reagent according to the invention.
- HIV 1 -POL determined 24 h after total acid hydrolysis (HAT) (black histograms), compared with the theoretical composition, calculated on the basis of an equi-molecular quantity of each amino acid, introduced in the degenerate positions (histograms white).
- the one letter code is used for amino acids.
- B represents Asn or Asp
- Z represents Glu and Gln.
- FIG. 3 represents the amino acid composition of the mixotop-MIXO (HIV-POL), determined 24 h after total acid hydrolysis (HAT) (black histograms), compared with the theoretical composition, calculated on the basis of an equimolecular quantity of each amino acid, introduced into the degenerate positions (white histograms).
- the one letter code is used for amino acids.
- B represents Asn or Asp
- Z represents Glu and Gln.
- FIG. 4 illustrates the IgG reactivity of 20 seropositive HIV1 sera (! and 26 seronegative HIV1 sera (O), characterized by immunofluorescence with the HIV-POL peptide fixed on a solid support at the rate of 0.1 ⁇ g / well ( Figure 4A) or at a rate of 1 ⁇ g / well ( Figure 4B).
- the horizontal line represents the threshold value, corresponding to the average obtained with seronegative sera + 3 standard deviations (SD).
- SD standard deviations
- FIG. 5 represents the effect of the MIXO mixotope (HIV-POL) on the reactivity of the IgGs of the seropositive HIV1 sera (!) and of the seronegative sera (O) at two different concentrations: 1 ⁇ g / well (FIG. 5 A) or at 10 ⁇ g / well (FIG. 5B), in an ELISA test.
- the horizontal line represents the threshold value corresponding to the mean of the control sera + 3 SD.
- FIG. 6 illustrates the effect of the HIV-POL peptide + MIXO mixotope (HIV-POL) combination on the IgG reactivity of seropositive (!) and seronegative (O) sera in ELISA tests.
- Each microtiter plate well is sequentially coated with 1 ⁇ g of HIV-POL peptide, then 10 ⁇ g of MIXO mixotope (HIV-POL) and brought into contact with the sera.
- the native HIV-POL peptide (SEQ ID NO: 1) is synthesized in solid phase using the conventional strategy of the Boc-benzyl type (or Fmoc), in an automated peptide synthesizer (model 43 OA, Applied Biosystems Inc.).
- the side chain protection groups are: Asn (Trt), Gin (Trt), Asp (OChx), Glu (OChx), Ser (Bzl), Thr (Bzl), Arg (Tos), Cys (4- MeBzl), and His (Dnp) (see RC SHEPPARD, Peptide, Synthesis, Comp. Org. Chem., 1979, 5, 321-363).
- Amino acids are introduced using the HBTU / ⁇ OBt activation protocol with systematic double coupling on a Boc-Gln-Pam resin (Applied Biosystems). After thiolysis of the dinitrophenyl Dnp group, followed by a final deprotection and a cleavage with hydrofluoric acid, the cleaved and deprotected peptide is precipitated and washed with cold diethyl ether, then dissolved in 5% acetic acid and lyophilized. The peptide is more than 90% purified on a 5 mm x column
- the purity of the peptide, greater than 96% is determined by analytical reverse phase HPLC.
- sequence identity of the purified peptide is confirmed by determining the amino acid composition and by mass spectrometry recorded on a mass spectrometer (MALDI) (calculated 4258.9 [M + H] + , found 4260.0) .
- MALDI mass spectrometer
- a first coupling is carried out with 1 mmol (total amount) of Boc-amino acid (or of a mixture of Boc-amino acids).
- a second coupling, using 2 mmol (total amount), is then systematically carried out.
- the crude peptide is dissolved in TFA (30 ml) and precipitated by adding the said peptide in a solution of cold diethyl ether (300 ml).
- the precipitate is dissolved in water and lyophilized. After oxidation in air of the solution at neutral pH, the mixotope is purified by gel filtration on a TSK HW40S column (Merck, Darmstadt, FRG). An aliquot of each purified mixotope is subjected to total acid hydrolysis for 24 hours with an HCl 6N: phenol mixture (10: 1), for the determination of the amino acid composition.
- FIG. 1 The replacement of the amino acids of MIXO (HIV-POL) is specified in FIG. 1.
- TSK HW 40S gel filtration
- a sample of the fraction used in the ELISA tests is subjected to a HAT, the result of which is presented in FIG. 3.
- HAT of the native peptide HAT of the native peptide
- I isoleucine
- valine being replaced by isoleucine in the mixotope and being part of a highly hydrophobic sequence of the mixotope, and therefore difficult to hydrolyze, makes it possible to explain the reduced quantities of Val and Ile in the result of this HAT (FIG. 3 ).
- the sequence of the HIV-POL peptide and the mixotopes derived therefrom are shown in FIG. 1. These reagents are preferably fixed on a solid support (microplate) at a concentration of 0.1 ⁇ g / well, for the HIV-POL peptide and at a concentration of 10 ⁇ g / well for the mixotopes.
- EXAMPLE 2 Immuno-enzymatic test using a reagent according to the invention for the serodetection of HIV1.
- HIV-POL peptide SEQ ID ⁇ O: l
- MIXO HV-POL
- MIXO mixotope
- phosphate buffer comprising 1.8% NaCl, pH 7.4 (PBS) and the excess binding sites are blocked with albumin (addition of 0.3 ml of 2% BSA (bovine serum albumin) in PBS, at 37 ° C, for 60 minutes).
- BSA bovine serum albumin
- PBS-T buffer the human sera to be tested are diluted 1/50 in PBS-T comprising 2% bovine serum albumin (BSA) and are incubated in wells containing the reagent according to the invention as specified above. above, for 120 minutes at
- the peroxidase-goat antibody-anti-human IgG-AM conjugates (Pasteur diagnostic), diluted to 1 / 10,000 in PBS-T buffer comprising 2% BSA, are incubated for 60 minutes at 37 ° vs.
- the conjugated antibody which binds to the Ig fixed on the support, is revealed for its peroxidase activity, using as substrate o-phenylenediamine dihydrochloride and H 2 O 2 , in a 0.05 M citrate buffer. , pH 5.5, for 30 minutes in the dark and at room temperature.
- the reaction is blocked by the addition of H 2 SO 4 4N (50 ⁇ l).
- the absorbance is recorded against a blank at 492 nm A 492 ), with a multi-channel automatic reader (M R 5000, Dynatech).
- M R 5000, Dynatech The mean A 492 + 3 standard deviations (SD) of the seronegative samples is used as a threshold value in the ELISA tests.
- the specificity of binding of positive sera to the different mixotopes in the solid phase is evaluated by the absorption of the antibodies by the native HIV-POL antigen in solution, using the method of B. FRIGUET et al. (J. Immunol. Methods, 1985, 77, 305-319).
- This method is based on the measurement of the concentration of free antibody by an indirect ELISA method, when the HIV-POL antigen and the antibodies are in equilibrium in solution.
- the HIV-POL antigen at different concentrations (10 "10 M to 2.10 “ 6 M) is first incubated in solution (PBS-T buffer + 2% BSA) with an seropositive serum, at constant concentration (1/50 ) until the equilibrium state is reached.
- MIXO HAV-POL
- NAHCO 3 50 mM, pH 9.6.
- the bound immunoglobulins are detected by the addition of goat anti-human IgG-A-M antibodies, coupled to peroxidase.
- the reactivity of human anti-HIV-POL IgG with respect to the HIV-POL peptide is analyzed by an ELISA test, on different sera: 20 sero-positive sera (confirmed in western blot) and 26 seronegative sera.
- the mixotopes were tested as antigens in solid phase, at two concentrations, namely 0.1 ⁇ g and 10 ⁇ g / well (FIG. 5).
- MIXO HV-POL
- FIGS. 5A and 5B The use of MIXO (HIV-POL) alone in the ELISA tests (FIGS. 5A and 5B) is not satisfactory since the sensitivity of the detection of IgG does not exceed 50% at the best coating concentration (FIG. 5B) .
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Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU30490/00A AU3049000A (en) | 1998-12-31 | 1999-12-29 | Detecting and monitoring hiv viral infections |
JP2000592638A JP2003515723A (en) | 1998-12-31 | 1999-12-29 | Detection and monitoring of HIV infection |
CA002358102A CA2358102A1 (en) | 1998-12-31 | 1999-12-29 | Detecting and monitoring hiv viral infections |
EP99964735A EP1141719A1 (en) | 1998-12-31 | 1999-12-29 | Detecting and monitoring hiv viral infections |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9816727A FR2788057B1 (en) | 1998-12-31 | 1998-12-31 | REAGENT FOR THE DETECTION AND MONITORING OF VIRAL HIV INFECTIONS AND ITS APPLICATIONS |
FR98/16727 | 1998-12-31 |
Publications (1)
Publication Number | Publication Date |
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WO2000040970A1 true WO2000040970A1 (en) | 2000-07-13 |
Family
ID=9534757
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/FR1999/003311 WO2000040970A1 (en) | 1998-12-31 | 1999-12-29 | Detecting and monitoring hiv viral infections |
Country Status (6)
Country | Link |
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EP (1) | EP1141719A1 (en) |
JP (1) | JP2003515723A (en) |
AU (1) | AU3049000A (en) |
CA (1) | CA2358102A1 (en) |
FR (1) | FR2788057B1 (en) |
WO (1) | WO2000040970A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1777522A1 (en) * | 2005-10-20 | 2007-04-25 | PENTAX Corporation | Plate, kit and method for the determination of a virus infection |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990010230A1 (en) * | 1989-02-23 | 1990-09-07 | University Of Ottawa | Polypeptide having immunological activity for use as diagnostic reagent and/or vaccine |
RU2085586C1 (en) * | 1992-02-28 | 1997-07-27 | Биотехнологическая компания "Биосервис" | Polypeptide 1106 designated for assay of antibodies to human immunodeficiency virus type-1, fragment of dna h 1106 encoding polypeptide 1106, recombinant plasmid dna ph 1106 encoding polypeptide 1106, strain of bacterium escherichia coli - a producer of polypeptide 1106 |
DE19727943A1 (en) * | 1997-03-10 | 1998-09-24 | Boehringer Mannheim Gmbh | Method for the simultaneous determination of HIV antigens and HIV antibodies |
-
1998
- 1998-12-31 FR FR9816727A patent/FR2788057B1/en not_active Expired - Fee Related
-
1999
- 1999-12-29 JP JP2000592638A patent/JP2003515723A/en not_active Withdrawn
- 1999-12-29 AU AU30490/00A patent/AU3049000A/en not_active Abandoned
- 1999-12-29 CA CA002358102A patent/CA2358102A1/en not_active Abandoned
- 1999-12-29 EP EP99964735A patent/EP1141719A1/en not_active Withdrawn
- 1999-12-29 WO PCT/FR1999/003311 patent/WO2000040970A1/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990010230A1 (en) * | 1989-02-23 | 1990-09-07 | University Of Ottawa | Polypeptide having immunological activity for use as diagnostic reagent and/or vaccine |
RU2085586C1 (en) * | 1992-02-28 | 1997-07-27 | Биотехнологическая компания "Биосервис" | Polypeptide 1106 designated for assay of antibodies to human immunodeficiency virus type-1, fragment of dna h 1106 encoding polypeptide 1106, recombinant plasmid dna ph 1106 encoding polypeptide 1106, strain of bacterium escherichia coli - a producer of polypeptide 1106 |
DE19727943A1 (en) * | 1997-03-10 | 1998-09-24 | Boehringer Mannheim Gmbh | Method for the simultaneous determination of HIV antigens and HIV antibodies |
Non-Patent Citations (7)
Title |
---|
DATABASE WPI DERWENT PUBLICATIONS LTD., LONDON, GB; 27 July 1997 (1997-07-27), ALATORTSEVA G I; GOLTSOV V A; SUKHANOVA L L: "New hybrid polypeptide 1106 - used for determination of antibodies to HIV-1, useful in diagnosis of AIDS", XP002117628 * |
GEYSEN HM, MASON TJ, RODDA SJ: "Cognitive features of continuous antigenic determinants", JOURNAL OF MOLECULAR RECOGNITION, vol. 1, no. 1, February 1998 (1998-02-01), pages 32 - 41, XP002117627 * |
GRAS-MASSE H, AMEISEN JC, BOUTILLON C, ROUAIX F, BOSSUS M, DEPREZ B, NEYRINCK JL, CAPRON A, TARTAR A: "Synthetic vaccines and HIV-1 hypervariability: a "mixotope" approach", PEPTIDE RESEARCH, vol. 5, no. 4, July 1992 (1992-07-01) - August 1992 (1992-08-01), pages 211 - 216, XP002117624 * |
HAAS, GABY; SAMRI, ASSIA; GOMARD, ELISABETH; HOSMALIN, ANNE; DUNTZE, JOERG; BOULEY, JEAN-MARC; IHLENFELDT, HANS-GEORG; KATLAMA ...: "Cytotoxic T-cell responses to HIV-1 reverse transcriptase, integrase and protease", AIDS (LONDON), vol. 12, no. 12, 1998, pages 1427 - 1436, XP002117622 * |
HASHIDA, SEIICHI; HASHINAKA, KAZUYA; ISHIKAWA, SETSUKO; ISHIKAWA, EIJI: "More reliable diagnosis of infection with human immunodeficiency virus type 1 (HIV-1) by detection of antibody IgGs to pol and gag proteins of HIV-1 and p24 antigen of HIV-1 in urine, saliva, and/or serum with highly sensitive and specific enzyme immunoassay (immune complex ...", JOURNAL OF CLINICAL LABORATORY ANALYSIS, vol. 11, no. 5, 1997, pages 267 - 286, XP002117623 * |
R VOLKMER-ENGERT, B EHRHARD, J HELLWIG, A KRAMER, W HOEHNE, J SCHNEIDER-MERGENER: "Preparation, analysis and antibody binding studies of simultaneously synthesized soluble and cellulose-bound HIV-1 p24 peptide epitope libraries", LETTERS IN PEPTIDE SCIENCE, vol. 1, no. 5, 1995, pages 243 - 253, XP002117625 * |
VINGA-MARTINS C; SCHNEIDER T; WERNO A; ROENSPECK W; PAULI G; MUELLER-LANTZSCH N: "Mapping of immunodominant epitopes of the HIV-1 and HIV-2 integrase proteins by recombinant proteins and synthetic peptides", AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 8, no. 7, July 1992 (1992-07-01), pages 1301 - 1310, XP002117626 * |
Also Published As
Publication number | Publication date |
---|---|
FR2788057A1 (en) | 2000-07-07 |
JP2003515723A (en) | 2003-05-07 |
CA2358102A1 (en) | 2000-07-13 |
EP1141719A1 (en) | 2001-10-10 |
FR2788057B1 (en) | 2002-01-04 |
AU3049000A (en) | 2000-07-24 |
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