AU657590B2 - Synthetic peptides corresponding to portions of HIV-2 virus and methods of using in an improved assay - Google Patents

Description

-1- The present invention relates to synthetic peptides and method to use these synthetic peptides in an improved immunoassay for antibodies to HIV-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 sequence of HIV-2 shows a genetic sequence homology with HIV-1 of 42% Id. However, studies have shown that patients with HIV-2 infection were not identified by serologic test whidh detect HIV-1. Certain HIV-2 antigens have been identified as being recognized by 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 15 the amino acid sequence of certain antigenic peptides. These antigenic peptide sequences (Seq. Id. No:1) (Seq.. Id. No:2) can have at their-N-terminus either an H of the amino 9* terminal NH 2 group of the peptide or an additional amino acid bonded to the amino terminal NH 2 group of the peptide, 20 the additional amino acid being selected to facilitate coupling of the peptide to a carrier protein and at Cterminus either an OH or NH 2 Although a peptide may by itself be antigenic, in coupling the peptide to various solid phases the antigenicity may be affected. This change in antigenicity may result in a solid phase that is not useful for conducting an immunoassay.

In conducting an immunoassay the ability of the solid phase immunoreactant to detect HIV-2 antibodies in low -2concentration is required, as these antibodies may exist in low concentration in the bodily fluid.

According to one aspect of the invention there is provided a synthetic peptide compound selected from the groupj of peptides represented by the following N-terminal toa'tJterminal amino acid sequences consisting of:a. Sequence Id No 3 b. Sequence Id No 4 C. Sequence Id No 5 and d. Seqtience Id No 6 wherein the peptides are immunologically teactive with :,::antibodies to HIV-2 and wherein the immunological reactivity of the peptides to said antibodies is substantially greter.\J than the immunological reactivity of' peptides having the amino acid sequence X-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly Cys-Ala-Phe-Arg-Gln-Val-CysHis-Thr-Thr- VlPoTpalAn OH, X-Asp -ln,-Ala-Arg-Leu-Asn-S er-TrpG ly-Cys -AI~i-Phe-Arg- G ln-al-Cys-His-Thr-hr-Val-Pr-Trp-Val-As-~2 X-Asp-Gln-s Ala-Arg-:Leu-Asn- Ser-Trp-G ly-Cys-Al a-Phe-Arg -lu ln-Val -His- 20 Thr-Thr-ValPro-Trp-Val-Asn-CysOH, or X-Asp-Gln-Ala-Ar-Leq- :..:Asn-Ser-Trp-Gly-CysAla-Phe-Arg-Gln-Val-CsH-Trh-Vl Pro-Tr-Val -Asn-Cys-NH2 wherein X is either H or Lys, 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 -3immunological 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., Patent No. 4,812,556 incorporated by reference). The Vahlne et al. peptide has the following amino acid sequence: (Seq. Id. No:1) (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 peptides, the

S*

additional amino acid being selected to facilitate coupling i ":15 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 i' the peptidt facilitates its covalent coupling to a solid surface. Consequently, the inventors extended WO 93/09252 PCT/US92/09376 -4the 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 Idded between Lysine and Aspartic acid of the Vahlne et al. peptide. Glycine was added to: 1) act as a spacer for better orientntion of the peptide on thz particles; and 2) to prevent the interaction between -COOH group of Asp and the NH2 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 P1 20 90 E Integrated Micro Sequencing system to confirm the correct sequence.

Purity was ascertained by HPLC on a reverse phase WO 93/092152 PCT/US92/09376 column using a linear gradient 0.1 trifluoroacetic acid in H20, trifluoroacetic acid in CH 3

CN)

of 5% to 60% 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 m 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 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 1-Ethyl-3-(Dimethylaminopropyl) carbodUimide hydrochloride. The particles resuspended and left at room temperature for 5 minutes with occasional shaking. To the ac;ivated particles was added 125 ul solution of HIV-2 peptide (img/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 WO 93/09252 PCT/US92/09376, -6buffered 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 gm 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 ethanol for 10 minutes. The particles were then separated as before and supernatant was removed. The particles were resuspended in I 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 mg/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.

WO 93/09252 PCT/US92/09376 -7- 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% Al 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 particles, coated with peptides as describe in example 1 or 2, were added to each well [20 Al). The plate was then placed at 420C for 30 minutes.

Upon completion of the incubation, the particles in the wells were washed with 100 M1 phosphate buffered saline and Tween-20 (2.06 g sodium phosphate dibasic, 0.318 g sodium phosphate monobaSic 0.5 ml 8.76 g sodium chloride, and 1.0 g sodium azide per liter; pH During the wash steps, the paramagnetic particles were held in the microtiter 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 il of particles resuspension buffer (4.346 g sodium phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, and 1 q sodium azide per liter; pH 7.4) 20 pl of goat antihuman IgG conjugated P-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 WO 93/09252 PCT/US92/09376 -8pH 7.5 or 0.3) was then added to the wells. After incubation with conjugate for 15 minutes at 420C 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 pl 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 The presence of P-galactosidase (ie: conjugate) in the wells triggered the cleavage of MUG to generate a fluorescent coumarin product. This reagent and conjugate were used as a sensitive detection system.

"Fluorescence (excitation wavelength 400 nm/.mmision wavelength 450 nm) was measured at two time intervals 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.

WO 93/09252 WO 9309252PCT/US92/09376 -9- EVALUATION OF TWO HIV-2 PEPITIDES COVALENTLY COUPLED TO PARTICLES IN HIV ASSAY* TEST SAMPLES fl~1=Q~i ASSAY-SIGNA UNMODIFIED MODIFIED HIV-2 POS.

HIV-2 POS.

HIV-1 POS.

NEG.CONTROL.

SAMPLE DILUENT 1:1600 1: 3200 1:6400 1:10,00 1: 3200 1: 6400 1:100 1:100 2187 1099 563 1857 880 408 5000 2799 1482 3009 1447 629 Assay conditions were 0.05% particles suspension, Goat anti Human IgG-B'Galactosidase conjugate, Assay signal is in fluorescent units.

WO 93/09252 PCT/US92/09376 EVALUATION OF TWO HIV-2 PEPTIDES PASSIVELY ADSORBED ON PARTICLES IN HIV ASSAY* TEST SAMPLES PILT ASSAY SIGNAL** UNMODIFIED MODIFIED HIV-2 POS. 1:1600 1210 2307 1:3200 570 1074 1:6400 242 485 HIV-2 POS. 1:3600 959 2580 1:3200 396 1242 1:6400 198 598 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 WO 93/09252 WO 9309252PCT/US92/09376 -1 J,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.

WO 93/09252 -12- PC/US92i09376 SEQUENCE LISTING GENERAL INFORMATION: 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 NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Baxter Diagnostics Inc.

STREET: One Baxter Parkway, DF2-2E CITY: Deerfield STATE: Illinois COUNTRY: usa ZIP: 60015 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Barta, Kent REGISTRATION NUMBER: 29,042 REFERENCE/DOCKET NUMBER: 91183A (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 708/948-3308 WO 93/09252 PCT/US92/09776 13 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gin Val Cys 1 5 10 His Thr Thr Val Pro Trp Val Asn INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino cid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Asp Gin Ala Arg Lu Asn Ser Trp Gly Cys Ala Phe Arg Gin Val Cys 1 5 10 His Thr Thr Val Pro Tip Val Asn Cys WO 93/09252 PCT/US9" 109376 14 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid STRANDEDNESS: unknown 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 Val Cys His Thr Thr Val Pro Trp Val Asn INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Lys Gly Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gly 1 5 10 Val Cys His Thr Thr Val Pro Trp Val Asn Cys WO 93/49252 PCr/US92/09376 15 INFORMATION FOR SEQ 1ED SEQUENCE CHARACTERSTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON: SEQ MD Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gin Val Cys 1 5 10 His Thr Thr Val Pro Trp Val Asn Lys INFORMATION FOR SiZ:Q MD NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ II) NO:6: Asp Gin Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg GM Val Cys 1 5 10 His Thr Thr Val Pro Trp Val Asn Cys Lys

Claims (3)

1. A synthetic peptide compound selected from the group of peptides represented by the following N-terminal to C- terminal amino acid sequences consisting of:- a. Sequence Id No 3 b. Sequence Id No 4 c. Sequence Id :No 5 and d. Sequence Id No 6 wherein the peptides are immunologically reactive with antibodies to HIV-2 and wherein the immunological reactivity of the peptides to said antibodies is substantially greater than the immunological reactivity of peptides having the amino acid sequence X-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly- Cys-Ala-Phe-Arg-Gln-Val-Cys-His-Thr-Thr-Val-Pro-Trp-Val-Asn- OH, X-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg- S G J.n-Val-Cys-His-Thr-Thti-Val-Pro-Trp-Val-Asn-Nh 2 X-Asp-Gln- Ala-Arg-Leu-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Oys-His- Thr-Thr-Val-Pro-Trp-Val-Asn-Cys-OH, or X-Asp-Gln-Ala-Arg-Leu- Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-His-Thr-Thr-Val- Pro-Trp-Val-As-Cs-NH2 Pro-Trp-Val-Asn-Cys-H 2 wherein X is either H or Lys.

2. A method for detecting antibodies to HIV-2 in a sample comprising: a) contacting said sample with the peptides of claim 1 under incubation conditions such that an immunological complex will form between the peptide and jLIAantibodies to HIV-2, if such antibodies are present in 71 d the sample and -17- b) measuring the formation, if any of the immunolog:cal complex to determine the presence of antibodies to HIV-2 in said sample.

3. A method for detecting antibodies to HIV-2 in a fluid sample comprising: a) providing a solid phase having the peptide of claim 1 immobilized thereon; b) contacting the sample with the solid phase; c) incubating the sample and solid phase to form an immunological complex between the peptide and antibodies to HIV-2; d) washing the solid phase to remove any unbound Smaterial; S* e) adding an indicator that binds with antibodies to HIV-2 to the solid phase; f) washing the solid phase to remove any unbound indicator; g) measuring the amount of indicator; and h) correlating the amount of indicator to the amnount of antibodies to HIV-2. DATED this 7th day of December, 1994. BAXTER DIAGNOSTICS INC. Patent Attorneys for the Applicant: A LiA PETER MAXWELL ASSOCIATES