AU623097B2 - Oligopeptides and their use for diagnostic and vaccination purposes for aids and arc - Google Patents

Description

A uan' adppucaaon reterred to in paragraph 2 of this Declaration -s/are the first application made in a Convention country in respect of the invention the subject of the application.

ared at Lelystad this 3rd day of May 19 Stichting Centraal Di eserrdt-T rof. dr. C Decl OPI DATE 02/05/89 APPLN- B 24896 88 PCT wc AP T wE A 5/06/89 PCT NUMBER PCT/NL88/00040 INTERNATIONAL APPLICATIO.. -1L rk 7 i urncKA I r IuN I KA I Y (PC'T) (51) International Patent Classification 4 (11) International Publication Number: WO 89/ 03391 C07K 7/06, 7/08, G01N 38/68 A61K 37/02, 39/21, 39/395 1 (43) International Publication Date: 20 April 1989 (20.04.89) G01N 33/569 (21) International Application Number: PCT/NL88/00040 (74) Agent: SMULDERS, Th., Vereenigde Octrooibureaux, Nieuwe Parklaan 107, NL-2587 BP The (22) International Filing Date: 7 October 1988 (07.10.88) Hague (NL).

(31) Priority Application Number: 8702403 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (32) Priority Date: 9 October 1987 (09.10.87) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European pa- (33) Priority Country: NL tent), NL (European patent), SE (European patent),

US.

(71) Applicants (for all designated States except US): STICHTING CENTRAAL DIERGENEESKUN- Published DIG INSTITUUT [NL/NL]; Edelhertweg 15, NL- With international search report.

8219 PH Lelystad ACADEMISCH ZIEKEN- HUIS BIJ DE UNIVERSITEIT VAN AMSTER- DAM [NL/NL]; Meibergdreef 9, NL-1105 AZ Amsterdam (NL).

(72) Inventors; and Inventors/Applicants (for US only) GOUDSMIT, Jaap [NL/NL]; Oudezijds Voorburgwal 149, NL-1012 ES Amsterdam MELOEN, Robert, Hans [NL/NL]; Karveel 10-04, NL-8231 AP Lelystad (NL).

(54) Title: OLIGOPEPTIDES AND THEIR USE FOR DIAGNOSTIC AND VACCINATION PURPOSES FOR AIDS AND ARC (57) Abstract The invention relates to oligopeptides having utility in diagnosis of, or vaccination against AIDS and ARC, and to antibodies raised against such oligopeptides. The oligopeptides have a length of 8 to 17 amino acids. The amino acid sequence corresponds to a sequence occurring in the V 3 region of gpl20 and comprises the p-turn sequence GPG(R) at the positions 312-314 (315), or a variant p-turn sequence, and flanking sequences of at least one and preferably at least two amino acids on both sides of the p-turn sequence.

LL -i WO 89/03391 PCT/NL88/00040 Oligopeptides and their use for diagnostic and vaccination purposes for AIDS and ARC.

This invention relates to oligopeptides suitable for use in the diagnosis of, and vaccination against, AIDS (Acquired Immunodeficiency Syndrome) and ARC (AIDS-Related Complex) and, in addition to the new oligopeptides themselves, relates to their use for diagnostic purposes and vaccination compositions containing the new oligopeptides.

It is well known that AIDS and ARC are caused by a retrovirus, and that there are many variants of the virus.

Various names are used for the virus, such as HTLV-III (human T lymphotropic virus type III), HIV (human immunodeficiency virus), LAV (lymphadenopathy-associated virus) and ARV (AIDS-associated retrovirus). It is found that many patients with AIDS or ARC form specific antibodies against proteins of the virus, especially against antigenic determinants located on the envelope glycoprotein gpl20. However, the virus proves to be capable of avoiding the virus neutralizing effect of antibodies by changing certain parts of the gpl20. In the envelope protein 5 areas have been indicated which exhibit a strong variation of the amino acids sequence between different variants of the virus. As regards these variations, reference is made to "Human Retroviruses and AIDS, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences", eds.

Myers, Josephs, Rabson, Smith (1987).

After infection with the AIDS virus, human beings and also experimentally infected chimpanzees develop antibodies against the exterior envelope protein gpl20. These antibodies are demonstrable soon after infection, and continue to circulate irrespective of the clinical condition. Antibodies ;1 WO 89/03391 PCT/NL88/00040 -2neutralizing the prototype AIDS virus HTLV-III, measured by means of a CD4-dependent cell fusion inhibition test or HIV replication inhibition test, do not occur until later.

People developing these virus neutralizing antibodies appear to remain healthy for a longer period of time than do people doing this to a lesser extent. The recognition of parts of gp120 by immune globulines of Man is associated with the virus neutralizing capacity of such sera. In this connection it has been demonstrated that goat sera directed against a recombinant protein PBI (DNA sequence PvuII 2d BglII restriction site) neutralize the homologous virus (Putney et al., Science 234, (1986) 1392-1396) and the mouse sera against two synthetic peptides (amtino acid 458-484/503-532 and 298-314) located within this recombinant protein also have virus neutralizing activity (Ho et al. J. Virol 61 (1987) 2024-2028). However, no oligopeptides of gpl20 have been described which are immunogenic in the natural or experimental host (Man and chimpanzee) and may or may not be associated with the possible induction of virus neutralization. A knowledge of such short amino acid sequences is of interest to diagnostics and vaccine development.

By using the PEPSCAN method of Geysen et al. (Proc.

Natl. Acad. Sci. USA 81 (1984) 3998-4002; Proc. Natl. Acad.

Sci. USA 82 (1985) 178-182; "Synthetic peptides as antigens" 1986, 130-149, Wiley; W084/03506 and W084/03564) and polyclonal sera of HIV infected human beings and chimpanzees, we have now localized the sequence 305-321 (in the amino acids numbering of HTLV-IIIB (BH10)) on the gpl20 of the virus in the variable region V 3 It has been found that this sequence is an immunodominant epitope for neutralizing antibodies of infected human beings and chimpanzees, that is to say, the a natural hosts, and that antibodies generated by a given variant of the virus, such as the BH10 variant, do react with the BH10 sequence 305-321, but not with the corresponding sequence of other variants, such as the RF variant, and the LI WO 89/03391 PCT/NL8/00040 -3other way round.

The sequence referred to comprises the amino acids sequence GPG or GPGR, responsible for a S-turn in the protein structure. This S-turn sequence is necessary for the protein structure and therefore highly conserved, but not responsible for immunogeneity, in view of the fact that sera specific for, for example, BH10, do not react with, for example, RF, and the other way round; cross reactions do not occur. The specific immunogeneity is therefore evidently caused by the flanking amino acid sequences.

This understanding is used, according to the present invention, by providing oligopeptides composed of 8-17 amino acids in a sequence corresponding with a sequence occurring in the envelope protein gpl20 of an AIDS or ARC causing or related virus in the variable V 3 area, consisting of the 8-turn amino acids sequence GPG or GPGR at the positions 312-314 or 312-315 in the amino acids numbering of HTLV-III and flanking amino acids sequences at least 1, preferably at least 2 amino acids long, and variants thereof in which the GPG or GPGR sequence has been replaced by a different B-turn sequence, and variants in which the free amino group of the amino terminal amino acid and/or the free carboxyl group of the carboxy-terminal amino acid is blocked or otherwise modified.

These oligopeptides can be used for diagnostic purposes, in particular for determining, by means of a body fluid, such as serum of mammals, such as humans or chimpanzees, infection with an AIDS or ARC causing or related virus, and for determining the variant or variants of the virus with which the mammal has been infected, or which have formed during the infection, using known per se methods for detecting the binding of the antibodies present in the sample being investigated to the oligopeptides. Also, the new oligopeptides according to the new invention can be used for preparing vaccine preparations protecting against AIDS or

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WO 89/03391 PCT/NL88/0004 0 ARC causing or related viruses.

In this specification, the amino acids are designated by the one-letter code, that is to say that, for example, G stands for glycine, P stands for proline, R stands for arginine, etc.

As the 8-turn sequence GPG or GPGR is only of importance for the protein structure and not for the specific immunogeneity, the oligopeptides according to the invention may comprise a different 8-turn sequence instead of the natural sequence GPG or GPGR. Examples of other 8-turn sequences are GLGQ and GLGK, which occur in the gp 120 of various African HIV-1 strains. Such variants are therefore comprised by the invention.

In view of the contemplated uses, it may further be desirable that the terminal amino acids be modified.

Possibilities in this connection are blocking the terminal free amino and carboxyl groups, or chemically modifying these groups otherwise. By way of example, the free amino group of the amino terminal amino acid could be acetylated and the free carboxyl group of the carboxy terminal amino acid could be amidated. Such variants are therefore also comprised by the present invention.

The oligopeptides according to the invention have a length of 8-17 amino acids, preferably 9-15, and most preferably 10-13 amino acids. The 8-turn sequence is flanked by amino acid sequences having a length of at least 1 and preferably at least 2 amino acids each, preferably at least 3 and most preferably at least 4 amino acids.

Examples of oligopeptides according to the invention from 17 amino acids are:

KSIRIQRGPGRAFVTIG

KKIRIQRGPGRAFVTIG

VRKSITKGPGRVIYATG

ISQSIRIGPGKVFYAKG

VRRSLSIGPGRAFRTRE

RRRHIHIGPGRAFYTGE

ARRRIHIGPGRAFYTGK

VRKSIYIGPGRAFHTTG

RKSIIQRGPGRAFVTIG

1 i WO 89/03391 PCT/NL88/00040 Examples of shorter oligopeptides according to the invention are:

IRIQRGPGRAFVTIG

IQRGPGRAFVTIG

IQRGPGRAFVT

ITKGPGRVIYATG

ITKGPGRVIY

ITKGPGRVI

IQRGPGRAFV

IRIQRGPGRAF

TKGPGRVIYA

Methods of producing oligopeptides are generally known, so that their preparation will not be described in any detail herein.

The new oligopeptides according to the invention can De used for diagnostic purposes. In particular, one possibility is an examination of infected human of chimpanzee sera by means of a set of oligopeptides according to the invention to determine which variant of the virus has caused the infection and/or what new variants have formed during the infection. An insight into this is of diagnostic, prognostic and therapeutic interest. Techniques suitable for such examinations are known per se. By way of example we can mention here ELISA's, RIPA's, dot-blots, etc. According to a more concrete example, serum from an infected person can be contacted with a nitrocellulose strip on which different oligopeptides according to the invention have been arranged.

By washing, adding an anti-hlg-antibody coupled to a peroxydase, re-washing and adding peroxidase substrate, colour formation can be obtained in the site of the oligopeptide with which the antibodies present in the sample react.

The new oligopeptides according to the invention also open the way for vaccines against AIDS and ARC, in WO 89/03391 PCT/NL88/000 4 0 particular multivalent vaccines or vaccine cocktails directed against a plurality of variants of the virus. The vaccin compositions according to the invention:comprise one or (preferably) a plurality of oligopeptides according to the invention, in combination with one or a plurality of carriers and/or adjuvants suitable for vaccination purposes. Carriers and adjuvants suitable for vaccines are known per se. By way of example, we mention the possibility of coupling the oligopeptide by means of a suitable coupling agent to KLH (Keyhole Limpet Hemocyanin) or BSA (Bovine Serum Albumine).

Toxoids and liposomes are also suitable carriers, and so are poly-L-lysine, poly-L-glutanic acid, muramyl dipeptide, murabutidine, etc. Suitable adjuvants are, for example, aluminium hydroxide and other known adjuvants. Diluents suitable for the administration of the vaccine, such as distilled water, phosphate-buffered saline, and buffer solutions (such as a citrate buffer) are also known per se.

The particular properties of the oligopeptides according to this invention, and thus their particular suitability for use in the above applications, have been confirmed by the results of a large number of experiments.

Thus, for example, in a study of monoclonal antibodies, which had been established to neutralize the HIV-1 strain HTLV-IIIB, it was found that all antibodies investigated bind to oligopeptides according to the invention. A monoclonal antibody with a good virus neutralizing capacity was found to bind to an octapeptide IQRGPGRA, and another monoclonal antibody with a pronounced virus neutralizing capacity was found to bind to the octapeptide QRGPGRAF.

In a study of polyclonal antibodies, produced in a goat in response to recombinant gpl60 of HTLV-IIIB and of HTLV-IIIRF, expressed in baculo virus, the antiserum against recombinant gpl60-IIIRF, for example, was found to bind most strongly to the nonapeptide ITKGPGRVI from the V 3 region of HTLV-IIIRF, whereas it did not bind to the corresponding nonapeptide IQRGPGRAF of HTLV-IIIB.

WO 89/03391 PCT/NL8/00040 -7- Similar results were obtained with polyclonal antibodies produced in a rabbit in response to the carboxyterminal portion of gpl20 expressed in E. coli. All polyclonal antibodies neutralizing the homologue strain were found to bind to an oligopeptide according to the invention.

All these studies taken together justify the conclusion that antibodies produced by the immunization of, for example, mice, rabbits and goats with all sorts of potential vaccine products derived from the HIV-1 envelope precursor gpl60, or the external envelope and exhibiting strain-specific HIV-1 neutralization in vitro, always bind around the B-turn sequence, i.e., to an oligopeptide according to this invention. Neutralizing sera of chimpanzees and humans also bind in the area in question, as has also been determined by experimentation.

The experiments were not only carried out with octa- and nonapeptides, but, for example, also with the 17 amino acids long HTLV-IIIB oligopeptide KSIRIQRGPGRAFVTIG, which bound human sera inhibiting cell fusion and neutralizing cellfree virus, and also bound goat antibodies against and against the carboxyterminal part of gpl20, capable of neutralization and cell-fusion inhibition. Rabbit antibodies against this oligopeptide bound the HTLV-IIIB gp-120 and its precursor gp-160, while they also neutralized HTLV-IIIB and exhibited cell-fusion inhibiting properties.

The experiments conducted covered not only oligopeptides of various lengths, but also oligopeptides with widely divergent amino acid sequences, such as nonapeptides having the following sequences: IQRGPGRAF (LAV-1) IYIGPGRAF (SF-2) ITKGPGRVI (RF) IAIGPGRTL VTLGPGRVW (CDC-4) IHFGPGQAL (MAL) IRIGPGKVF (Z3) TPIGLGQAL (Z6) TPIGLGQSL (ELI) wn RQ/I391 PCT/NL88/00040 -8- The codes in brackets indicate the virus strains from which the sequences have been derived. Human sera collected in Europe turned out to recognize especially nonapeptides derived from European strains, whereas peptides derived from African strains (ELI, MAL, Z3, Z6) and Haitian strains (RF) were recognized to a much lesser extent.

Sera collected in Africa, on the other hand, were better in recognizing the peptides derived from African strains.

Each individual tested turned out to have a different, individual pattern of oligopeptides recognition.

The experiments also confirmed the suitability of oligopeptides according to the invention as components of vaccine cocktails. In fact, virtually all sera investigated were found to recognize at least one oligopeptide according to the invention. By determining the amino acid sequence of or at least its relevant part in the V 3 region, of mutants of the virus, valuable new components of such vaccine cocktails can be designed. Although the length of the oligopeptides to be used can vary from 8 or 9 to 17 amino acids, a length of about 12 amino acids will usually be selected, because such a length appears to give an optimum specificity and virus neutralizing activity.

The experiments also confirm the suitability of oligopeptides according to the invention for diagnostic purposes. Thus the invention could be used in the form of ELISA plates with a panel of oligopeptides according to the invention thereon, for tracing the spread of HIVstrains with variation in the neutralizing epitope by determining the reactivity of the serum of an infected individual. The invention could also be used in the form of a set of antisera against a panel of oligopeptides according to this invention, so that viruses can be typed by means of RIPA.

i PCT/NL88/00040 WO 89/03391 -9- Example I: Synthesis of acetyl-Ile-Gln-Arg-Gly-Pro-Gly-Arg- Ala-Phe-NH2 Solvents and reagents were pro-analyse grade and were purchased from Merck, Darmstadt, GFR. They were used without further purification; dichloromethane (DCM) was purified over a column of activated aluminum oxide. Boc-amino acids, 4-methylbenzhydrylamine (MBHA) resin and benzotriazole-1-yloxy-tris-(dimethylamino)-phosphonium-hexa fluorophosphate (BOP, Castro's reagent) were purchased from Novabiochem, Laufelfingen, Switzerland.. The side-chain of Arg was protected by the Tosyl group. The reactions were carried out in a glass vessel with a glass frit in a mechanically operated shaker.

Routine liquid volumes were 7 ml.

Amino acid analyses were carried out with the Waters Pico-tag amino acid analysis system, after hydrolysis of the peptide in 1 phenol in 6 N HC1 at 1500 for 1 hr. HPLC was carried out on an analytical reverse phase column (polygosil 60-10C18, 250 x 4 mm) using a Waters HPLC system consisting of a model 680 gradient controller and two model 510 pumps and a model WISP 712 automatic injector. Using a solvent system composed of eluent A, 20 methanol/water with 0.1 trifluoroacetic acid (TFA), and eluent B, 80 methanol/ water with 0.1 TFA, and at a flow rate of 1 ml/min the peptide was detected at 210 nm using a Spectroflow 757 spectrophotometer.

After 24 hr swelling in DCM, MBHA resin (1 DVB, meq/mg, 600 mg) was successively washed with DCM, 50 TFA/DCM DCM, 7 diisopropylethylamine (DIEA)/DCM and dimethylformamid (DMF).

Coupling procedure: To the MBHA resin, suspended in a minimal amount of DMF, was added 1 mmol of Boc-Phe in 1 ml of DMF. EOP (1 mmol in 1 ml of DMF) was added, directly followed by DIEA (3 mmol). After 1 hr the resin was successively washed with DMF (3 x 2 min), isopropanol (3 x 2 min), and DCM (3 x 2 min). The completeness of the coupling was examined I WO 89/03391 PCT/NL88/00040 by the Kaiser test (Anal. Biochem. 34, 1970, 594-598). If the reaction was not complete the coupling procedure was repeated. If the reaction was complete the Boc group was removed by reacting the resin with 50 TFA/DCM (5 min), 50 TFA/DCM (20 min), followed by washings with DCM (3 x 2 min), 7 DIEA/DCM (3 x 4 min), and DMF (3 x 2 min). Then the next Boc-amino acid was coupled. At the end of the synthesis the N-terminal amino group was acetylated by acetic anhydride (1 ml in 3 ml of DMF and 0.5 ml of triethylamine) for 30 min.

Yield: 908 ml. Deprotection: A part of the peptide resin (523 mg) was treated with 10 ml anhydrous HF containing 10 anisole for 1 hr at 0°C. After removal of HF in vacuo, the crude reaction product was dissolved in 5 ml of 10 acetic acid and the fluoride ions were exchanged on an ion-exchange resin (BioRad AG 2-X8; 3 x 1 cm, acetate form). The product was lyophilized from 1 acetic acid. Yield: 154 mg.

Amino acid analysis: Glu 0.96; Gly 2.00; Arg 2.23; Ala 0.96; Pro 0.95; Ile 0.83; Phe 0.91. Purity, according to HPLC, 70 j LL_ J WO 89/03391 PT/NL88/0040 -11- Example II: Detection of antibodies to Neu21 The peptide KSIRIQRGPGRAFVTIG (Neu21) composed of amino acids 305-321 of the envelope gpl20 of clone HTLV-IIIB was produced using the Merrifield solid phase synthesis.

The presence of antibodies was determined using a direct non-competitive solid phase immunoassay.

Wells of microtiter plates were incubated with 0.1 mol/l NaH 2

PO

4 (pH 5.0) and 0.2 (volume/volume) glutardialdehyde (Merck, Meppel, the Netherlands) at a volume of 100 pl per well and incubated for four hours at room temperature.

Subsequently, the plates were rinsed three times for minute, with 0.1 mol/l NaH 2

PO

4 (pH Then 10 ng peptide per well was incubated in 0.1 mol/l Na 2

HPO

4 (pH 100 pl/well, for sixteen hours at room temperature.

After coating, wells were washed ten times with PBS (0.1 volume/volume). Non-specific binding sites were blocked by incubating for one hour at 37 0

C

using 4 normal goat serum (NGS) in PBS-Tween-20. Sera to be tested were diluted 1:100 in PBS-Tween-20, 4 NGS and incubated for one hour at 37 0 C. After another washing step, horseradish peroxidase-labelled conjugates (goat antihuman IgG, KPL, Gaithersburg, Maryland, USA) were added, diluted 1:500 in PBS-Tween-20 with 4 NGS.

Bound antibodies were visualized using ophenylene diamine and the reaction was stopped with 1 N H 2

SO

4 Optical density was read at an absorbance of 450 nm.

Interassay variation was controlled by including a serial dilution of a positive human control serum on each plate. The mean optical density of 149 HIV-1 seronegative samples plus four times the standard deviation was used as cut-off value.

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Claims (3)

1. Oligopeptides composed of 8-17 amino acids in a sequence corresponding to a sequence occurring in the variable region V 3 in the envelope protein gpl20 of an AIDS or ARC causing or related virus, comprising the B-turn amino acids sequence GPG or GPGR at the positions

312-314 312-315 in the amino acids numbering of HTLV-IIIB and flanking amino acids sequences having at least one amino acid, and variants in which the GPG or GPGR sequence has been replaced by a different B-turn sequence, and variants in which the free amino group of the 10 amino terminal amino acid and/or the free carboxyl group of the carboxy terminal amino acid has been blocked or modified otherwise. 2. Oligopeptides as claimed in claim 1 having flanking amino acids comprising at least two amino acids. 4* 3. Oligopeptides as claimed in claim 2 composed of 9-15 amino acids. 4. Oligopeptides as claimed in claim 2 composed of 10-13 amino acids. Oligopeptides as claimed in claim 1, comprising the B-turn sequence GPG or GPGR and flanking amino acids sequences having a length of at least 3 amino acids. 6. Oligopeptides as claimed in claim 1, comprising the B-turn sequence GPG or GPGR and flanking amino acids sequences having a length of at least 4 amino acids. 7. Antibodies (monoclonal or polyclonal) raised against oligopeptides l 922 19 mwspc#5432 rr 13 a a a. a as claimed in any of claims 8. The use of one or more oligopeptides as claimed in claim 1 or of one or more antibodies raised against oligopeptides as claimed in claim 1, for determining, by means of a body fluid, such as serum, or mammals, such as Man or chimpanzee, infection with an AIDS or ARC causing or related virus, and for determining the variant or variants of the virus with which the mammal is infected, or which have beenformed during the infection, using known per se methods for detecting the binding of antibodies present in the sample being investigated to the oligopeptides or the binding of gp120 present in the sample to the antibodies. 9. A vaccine composition for protecting mammals, such as Man and chimpanzee, against AIDS or ARC causing or related viruses, comprising one or more oligopeptides as claimed in claim 1 and one or more carriers and/or adjuvants suitable for vaccination purposes. A diagnostic aid for use in testing HIV contaminations and/or typing viruses causing AIDS or ARC, or related viruses, comprising one or more oligopeptides as claimed in claim 1 or one or more antibodies induced against oligopeptides as claimed in claim 1. 11. Oligopeptides according to any one of claims 1 to 6 substantially as hereinbefore described. 12. Antibodies according to claim 7 substantially as hereinbefore described. 92 2 19 "1. 7-VTO mwspe#5432 14- 13. The use of oligopeptides according to claim 8 substantially as hereinbefore described. 14. A vaccine composition according to claim 9 substantially as hereinbefore described. 15. A diagnostic aid according to claim 10 substantially as hereinbefore described. DATED this 19 February 1992 CARTER SMITH BEADLE Fellows Institute of Patent Attorneys of Australia a. 10 Patent Attorneys for the Applicant: STICHTING CENTRAAL DIERGENEESKUNDIG INSTITUUT ACADEMISCH DIEKENHUIS BIJ UV S" UNIVERSITEIT VAN AMERSDAM S9... *221 mwspe#5432 INTERNATIONAL SEARCH REPORT International Application No PCT/NL 88/00040 I. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC 4 C 07 K 7/06; 7/08; G 01 N 33/68, A 61 K 37/02; 39/21; IPC A 61 K 39/395; G 01 N 33/569 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System I Classification Symbolt IPC C 07 K 7/00; G 01 N 33/00; A 61 K 37/00; SA 61 K 39/00 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document, "I with Indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 3 X,Y WO, A, 87/02775 (SOUTHWEST FOUNDATION FOR 1-9 BIOLOGICAL RESEARCH) 7 May 1987, see the whole document; especially pages 6,7; pages 28-31; claims 1-30 X WO, A, 86/02383 (INST. PASTEUR) 24 April 1-9 1986, see page 55, claim 1; page 57, lines 1-3; page 59, lines 11,12; page claims 14,15 X Chemical Abstracts, vol. 106, 1987 (Columbus, Ohio, US) see page 469, abstract no. 16942b US, A, 779431 DEPT. OF HEALTH AND HUMAN SERVICES) 14 March 1986 Y Chemical Abstracts, vol. 106, 1987 1,6-9 S (Columbus, Ohio, US) S. Modrow et al.: "Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable Special categories of cited documents: o1 later document published after the international filing date domen defiin t st t art which is not or riority date and not in conflict with the alicaton but A document deining the nal state of the art cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but publlahed on or after the international document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be consiaered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance;' the claimed Invention citation or other special reason (as specified) cannot be considered to Involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means menta, such combination being obvious to a person skilled document published prior to the international filing date but In the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 18th November 1988 DEC 1988 International Searching Authority SIgna eI- thorized car EUROPEAN PATENT OFFICE Form PCT/ISA/210 (second sheet) (January 1985) internaional Application No, PCT/NL 8 00 0 40 -2 Ill. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, withl ,ndkcatIO4', where appropriatel, of line r.4vant passages Relevant to Claim No regions", see page 506, abstract 100556m, J. Virol. 1987, 61(2),

570-8 Y Chemical Abstracts, vol. 107, 1987 1-9 (Columbus, Ohio, US) V. KRCHNAK et al.: "Computer prediction of potential immunogenic determinants from protein amino acid sqec" see page 485, abstract no. 113939 j I Anal. Biochem. 1987, 165(l), 200-7 Formi PCT ISA:'210 (extra iiheet) (.January 1965)