SYNTHETIC PEPTIDES CORRESPONDING TO PORTIONS OF HIV-2 VIRUS AND METHODS OF USING IN AN IMPROVED ASSAY

Abstract: The present invention relates to synthetic peptides of HIV-2 and method to use the synthetic peptides in an improved immunoassay for antibodies to HIV-2. The antigenic peptide of formula (Sequece Id. No. 1 or 2), the improvement involving adding Lysine substantially adjacent to the N or C terminus of the peptide and adding a glycine between Lysine and Aspartic acid if Lysine is added to the N-terminus of the antigenic peptide.

Description: SYNTHETIC PEPTIDES CORRESPONDING TO PORTIONS OF HIV-2 VIRUS AND MErøODS OF USING IN AN IMPROVED ASSAY $ BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to synthetic peptides 10 and method to use these synthetic peptides in an improved im unoassay for antibodies to HIV-2. 2. Description of the Prior Art HIV-2 is a virus related to HIV-1. Guyader et al.. Nature 326: 662-669 (1987). The complete nucleotide 15 sequence of HIV-2 shows a genetic sequence ho ology with HIV.-l of 42% I . However, studies have showed that patients with HIV-2 infection were not identified by serologic test which detect HIV-1. Certain HIV-2 antigens have been indentified as being recognized by 20 antibodies in the sera of patients infected by HIV-2 infection. These antigens may be produced either recombinantly or synthetically. See e.g. Vahlne et al., U.S. Patent No. 4,812,556. In the '556 patent, Vahlne disclosed the a ino acid sequence of certain 25 antigenic peptides. These antigenic peptide sequences (Seq. Id. No:l) (Seq. Id. No:2) can have at its N-terminus either a H of the amino terminal NH 2 group of the peptide or an additional amino acid bonded to the amino terminal NH 2 group of the peptide, the 30 additional amino acid being selected to facilitate coupling of the peptide to a carrier protein and at C-terminus either a OH or NH 2 . Although a peptide may by its self be antigenic, in coupling the peptide to various solid phases the antigenicity may be effected. This change in antigenicity may result in a solid phase that it not useful for conducting an immunoassay. In conducting an immunoassay the ability of the solid phase immunoreactant to detect HIV-2 antibodies in low concentrations is required, as these antibodies may exist in low concentration in the bodily fluid. SUMMARY OF THE PRESENT INVENTION The present invention relates to certain synthetic peptides corresponding to a portion of the glyco-protein gp-41 encoded by HIV-2 env gene, having the basic sequence disclosed by Vahlne et al. The improvement discovered by the inventors involves adding a Lysine substantially adjacent to either terminus of the peptide for coupling on to the solid phase and by adding a Glycine between the added Lysine and -Aspartic acid of the Vahlne et al. peptide, if Lysine is added to the N-terminus of the peptide. Surprisingly, this peptide modified for handling convenience showed improved sensitivity in an immunoassay. In particular this invention relates to an antigenic'peptide of the formula: (Sequence Id. No:l) wherein the improvement involves adding a Lysine substantially adjacent to either terminus of said peptide and adding a Glycine between the added Lysine and Aspartic acid, if Lysine is added to the N-terminus. Additionally, this invention relates to an antigenic peptide of the formula: (Sequence Id. No:2) wherein the improvement involves adding a Lysine substantially adjacent to either terminus of said peptide and adding a Glycine between the added Lysine and Aspartic acid if Lysine is added to the N-terminus. More specifically, the invention can be further defined as peptides having: a sequence of 26 amino acids (Sequence Id. No:3) 27 amino acids having (Sequence Id. No:4) as described in the sequence listing; 25 amino acids (Sequence Id. No:5) ; 26 amino acids (Sequence Id. No:6). These peptides can be used for detecting antibodies to HIV-2 in a sample. The method involves contacting the sample with the peptide under conditions such that an immunological complex will form between the peptide and antibodies to HIV-2 present in the sample, if such antibodies are present in the sample, and measuring the formation, if any of the•immunological complex to determine the presence of antibodies to HIV-2 in the sample.. DETAILED DESCRIPTION OF THE INVENTION The present invention provides improved peptides corresponding to a region of the transmembrane glyco-protein gp-41 of HIV-2 which has been synthesized and tested for immunoreactivity to HIV-2 positive serum samples as shown by Vahlne et al., (U.S. Patent No. 4,812,556 incorporated by reference). The Vahlne et al. peptide has the following amino acid sequence: (Seq. Id. No:l) (Seq. Id. No:2) can have at its N-terminus either a H of the amino * terminal NH 2 group of the peptide or an additional amino acid bonded to the amino terminal NH 2 group of the peptide, the additional amino acid being selected to facilitate coupling of the peptide to a carrier protein and at C-terminus either a OH or NH 2 . The inventor's observed that the presence of Lysine in the peptide facilitates its covalent coupling to a solid surface. Consequently, the inventors extended "ttve Vahlne et al. peptide by addition of Lysine. Lysine can be incorporated at either end of the peptide for covalent coupling to the solid phase. The Lysine is positioned to be substantially adjacent to either terminus. The phrase substantially adjacent means within one or two amino acids of the terminus. In addition Glycine was added between Lysine and Aspartic acid of the Vahlne et al. peptide. Glycine was added to: 1) act as a spacer for better orientation of the peptide on the particles; and 2) to prevent the interaction between -COOH group of Asp and the NH group of Lysine. Thus, the modified peptides may have the amino acid sequences as shown in (Sequence Id. Nos:3-6) Peptides were synthesized in the amide form on a Milligen-Biosearch 9600 model peptide synthesizer using fluorenylmethoxy carbonyl (FMOC) amino protection scheme and 1-3 diisopropyl carbodiimide coupling chemistry. The amide form of the sequence was adopted because it could be expected to more closely mimic the biologically active analogue than the free acid form. Activated amino acids were coupled to a 2,4- dimethoxy benzhydrylamine resin. Peptide synthesis was monitored by ninhydrin analysis for all amino acids except proline for which an Isatin test was performed. The synthesized peptide was cleaved from the resin by Reagent R, which comprises trifluoroacetic acid, thioanisole, ethanedithiol and anisol in a volumetric ratio of 90:5:3:2. Peptides cleaved from resins were purified by high performance liquid chromatography (HPLC) , and characterized by Porton PI 20 90 E Integrated Micro Sequencing system to confirm the correct sequence. Purity was ascertained by HPLC on a reverse phase colu n using a linear gradient ((A) 0.1 trifluoroacetic acid in H J, (B)0.1% trifluoroacetic acid in CH-»CN) of 5% to 60% (B) in 45 minutes. Absorbance was followed at 230 nm. The peptides made by this process have the amino acid sequence as shown in (Sequence Id. No:3). These peptides were covalently coupled to carboxyl functionalized magnetic particles according to the following procedure: 1 ml of 2.5% weight/volume approximately 5 μm paramagnetic particles were separated on a magnetic separator in a 5 ml size disposable sterile cryogenic vial (corning, cat #25708). The supernatant was removed and.the particles resuspended with 1 ml of 70% ethanol for 10 minutes. The particles were then separated as before and supernatant was removed. The particles were resuspended in 1 ml of 0.1 M phosphate buffer, pH 5.5. The particles were separated as before and supernatant was removed. Further washing procedure with phosphate buffer was repeated twice as before and supernatant removed. To the slurry of particles was added 6 mg of Sulfo-N-Hydroxy Succinimide and 7 mg of i-Ethyl-3-(Dimethylaminopropyl) carbodiimide hydrochloride. The particles resuspended and left at room temperature for 5 minutes with occasional shaking. To the activated particles was added 125 ul solution of HIV-2 peptide (lmg/ml in 0.1 M phosphate buffer, pH 7.3) followed by the addition of 875 ul of 0.1 M phosphate buffer, pH 7.3. The particles were mixed thoroughly and then tumbled for 12-15 hours at room temperature. The covalently coupled peptide particles were then separated on a magnetic separator. Supernatant was removed and the particles were resuspended in isotonic buffered saline with 0.05% Tween 20 detergent. The particles were further separated and resuspended three times in isotonic buffered saline. The coated particles were then resuspended in isotonic buffered saline at final particles concentration of 0.25% weight to volume. It should be noted that the C-terminus can be comprised of either amide or acid groups depending on the desired end use. Peptide were also passively coated onto paramagnetic microparticles according to the following procedure: 1 ml of 2.5% of weight/volume approximately 5 μm paramagnetic particles were separated on a magnetic separator in a 5 ml size disposable sterile cryogenic vial (corning, cat #25708) . The supernatant was removed and the particles resuspended with 1 ml of 70% ethanol for 10 minutes. The particles were then separated as before and supernatant was removed. The particles were resuspended in 1 ml of 0.1 M CHES buffer (2-(N-Cyclohexylamino) ethansulfonic acid) at pH 9.3. The particles were separated as before and supernatant was removed. Further, washing procedure with CHES buffer was repeated twice as before and supernatant removed. To the slurry of particles was added 875 ul of 0.1M CHES buffer and 125 ul of peptide solution (1 g/ml in 0.1M CHES buffer) . The particles were resuspended and then tumbled for 18 hours at room temperature. The passively adsorbed peptide particles were then separated on a magnetic separator, supernatant removed and particles resuspended in isotonic buffered saline with 0.05% Tween 20 detergent. The particle were further separated and resuspended three times in isotonic buffered saline. The coated particles are then resuspended in isotonic buffered saline at final particle concentration of 0.25% weight to volume. EXAMPLE 1 A paramagnetic particle assay using particles coated with peptide prepared as previously described was performed as follows: Human serum or plasma was diluted 1:100 in well buffer (20% Neonate Calf Serum, 1.06 M Sodium Chloride, 0.03 M Tris-HCL, pH 7.4, 0.018 ° M Phosphate buffer, pH 7.4 + or - 0.3, 0.09% Sodium Azide, and 1.01% NP-40) . 50 μl of the diluted sample was added to each well of a Pandex black microtiter plate. Samples were tested in replicates of at least 2. Paramagnetic 5 particles, coated with peptides as describe in example 1 or 2, were added to each well (20 μl) . The plate was then placed at 42°C for 30 minutes. Upon completion of the incubation, the particles in the wells were washed with 100 μl phosphate buffered 0 saline and Tween-20 (2.06 g sodium phosphate dibasic, 0.318 g sodium phosphate monobasic 0.5 ml Tween-20, 8.76 g sodium chloride, and 1.0 g sodium azide per liter; pH 7.4). During the wash steps, the paramagnetic particles were held in the microtiter 5 plate well via a magnetic field applied to the bottom of the plate. Particles were washed in this manner six times. Particles in each well were resuspended in 30 μl of particles resuspension buffer (4.346 g sodium 0 phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, and 1 g sodium azide per liter; pH 7.4) 20 μl of goat antihuman IgG (H+L) conjugated ,9-Galactosidase (conjugate) and diluted 1:2000 in conjugate dilution buffer (0.1 M Tris-HCL pH 7.5, 0.5 M sodium chloride, 5% glycerol, 5.25 mM magnesium chloride, 0.1% sodium azide and 20% neonate calf sera pH 7.5 + or - 0.3) was then added to the wells. After incubation with conjugate for 15 minutes at 42°C the particles in the wells were washed six times with phosphate buffer saline and Tween-20 as described above to remove essentially all of the unbound conjugate. The Tween-20 in the wash solution enhanced the washing process and removed non specifically bound conjugate. Finally, 50 μl of a substrate solution of 4-methyl-umbelliferyl-3-D-galactoside (MUG) was added to each well (0.178 MUG, 3.58 g tricine, 5.1 ml dimethyl sulfoxide, 30 ml methyl alcohol, 0.2 g sodium azide, 0.5 ml Tween-20, per liter, pH 8.5). The presence of ,9-galactosidase (ie: conjugate) in the wells triggered the cleavage of MUG to generate a fluorescent cou arin product. This reagent and conjugate were used as a sensitive detection system. Fluorescence (excitation wavelength 400 nm/emmision wavelength 450 nm) was measured at two time intervals (i.e. 2 and 14 minutes) post MUG addition. The difference between the two values was a kinetic measurement of fluorescent product generation and is a direct measurement of conjugate and human IgG/IgM bound to the particles. Fluorescent values were converted to nM coumarin values using various concentrations of coumarin itself and its resultant fluorescence to establish a standard curve. EVALUATION OF TWO HIV-2 PEPTIDES COVALENTLY COUPLED TO PARTICLES IN HIV ASSAY* TEST SAMPLES HIV-2 POS. HIV-2 POS. HIV-1 POS. 1:100 85 65 NEG.CONTROL. 1:100 90 83 SAMPLE DILUENT 12 16 * Assay conditions were 0.05% particles suspension, Goat anti Human IgG-B Galactosidase conjugate. ** Assay signal is in fluorescent units. EVALUATION OF TWO HIV-2 PEPTIDES PASSIVELY ADSORBED ON PARTICLES IN HIV ASSAY* TEST SAMPLES HIV-2 POS. HIV-2 POS. NEG.CONTROL. 1:100 23 38 SAMPLE DILUENT 22 13 * Assay conditions were 0.05% particles suspension. Goat anti Human IgG-B Galactosidase conjugate. ** Assay signal is in fluorescent units. The modified peptide, passively as well as covalently coupled on magnetic particles, show significantly better sensitivity compared to unmodified peptide coated onto particles under identical coating and test conditions. This better sensitivity would allow the detection of HIV-2 antibody in lower concentrations in the patent sample. Although the invention has been described primarily in connection with special and preferred embodiments, it will be understood that it is capable of modification without departing from the scope of the invention. The following claims are intended to cover all variations, uses, or adaptations of the invention, following, in general, the principles thereof and including such departures from the present disclosure as come within known or customary practice in the field to which the invention pertains, or as are obvious to persons skilled in the field. SEQUENCE LISTING (1) GENERAL INFORMAΗON: (i) APPLICANT: Shah, Dinesh 0 Schneider, Andrew (ii) TITLE OF INVENTION: Synthetic Peptides Corresponding to a Portion of HIV-2 Virus and Method To Use The Same in an Improved Immunoassay (Hi) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Baxter Diagnostics Inc. (B) STREET: One Baxter Parkway, DF2-2E (C) CITY: Deerfield (D) STATE: Illinois (E) COUNTRY: usa (F) ZIP: 60015 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: (C) CLASSIHCAπON: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Barta, Kent (B) REGISTRATION NUMBER: 29,042 (C) REFERENCE DOCKET NUMBER: 91I83A (ix) TELECOMMUNICAΗON INFORMATION: (A) TELEPHONE: 708/948-3308 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPΗON: SEQ ID NO:l: Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gin Val Cys 1 5 10 15 His Thr Thr Val Pro Trp Val Asn 20 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPΗON: SEQ ID NO:2: Asp Gin Ala Arg Leu Asn Ser Tip Gly Cys Ala Phe Arg Gin Val Cys 1 5 10 15 His Thr Thr Val Pro Trp Val Asn Cys 20 25 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Lys Gly Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gly 1 5 10 15 Val Cys His Thr Thr Val Pro Trp Val Asn 20 25 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPΗON: SEQ ID NO:4: Lys Gly Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gly 1 5 10 15 Val Cys His Thr Thr Val Pro Trp Val Asn Cys 20 25 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPΗON: SEQ ID NO:5: Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gin Val Cys 1 5 10 15 His Thr Thr Val Pro Trp Val Asn Lys 20 25 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPΗON: SEQ ID NO:6: Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gin Val Cys 1 5 10 15 His Thr Thr Val Pro Trp Val Asn Cys Lys 20 25

Claims: We claim: 1. An antigenic peptide of the formula: (Sequence Id. No:l) the improvement comprising: a) adding a Lysine substantially adjacent to either terminus of said peptide; b) adding a Glycine between said added Lysine and Aspartic acid if Lysine is added to the N-terminus of said antigenic peptide. 2 A method for detecting antibodies to HIV-2 in a sample comprising: a) contacting-said sample with the peptide of claim 1 under conditions such that an immunological complex will form between the peptide and antibodies to HIV-2, if such antibodies are present in the sample and b) measuring the ' formation, if any of the immunological complex to determine the presence of antibodies to HIV-2 in said sample. 3. An antigenic peptide of the formula: (Sequence Id. No:2) the improvement comprising: a) adding a Lysine substantially adjacent to either terminus of said antigenic peptide; b) adding a Glycine between said added Lysine if Lysine is added to the N-terminus. 4. A method for detecting antibodies to HIV-2 in a sample comprising: a) contacting said sample with the peptide of claim 2 under conditions such that an immunological complex will form between the peptide and antibodies to HIV-2, if such antibodies are present in the sample and b) measuring the formation, if any of the immunological complex to determine the presence of antibodies to HIV-2 in said sample. 5. A synthetic peptide compound capable of binding to antibodies to HIV-2 consisting of a peptide selected from the group of peptides represented by the following N-terminal to C-terminal amino acid sequences: a. Sequence Id : No 3 b. Sequence Id : No 4 b. Sequence Id : No 5 c. Sequence Id : No 6 6. A method for detecting antibodies to HIV-2 in a sample comprising: a) contacting said sample with the peptide of claim 5 under conditions such that an immunological complex will form between the peptide and antibodies to HIV-2, if such antibodies are present in the sample and b) measuring the formation, if any of the immunological complex to determine the presence of antibodies to HIV-2 in said sample.

Inventors: SCHNEIDER ANDREW, SHAH DINESH O, SCHNEIDER, ANDREW, SHAH, DINESH, O.
Applicants: BAXTER DIAGNOSTICS INC, BAXTER DIAGNOSTICS INC.
Application year: 1992
Main classification: C12Q1
IPC codes: C07K14, G01N33