AU662534B2 - Peptides for use in induction of T cell activation against HIV-1 - Google Patents

Description

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OPI DATE 08/01/93 ,AO0.1P DATE 251/02/93 APPLN. ID 19065/92 PCT NUMBER PCT/SE92/00373 AU92 19065

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(51) International Patent Classification 5 (11) International Publication Number: WO 92/21377 A61IK 39/21, C07K 7/10, 13/00 Al (43) International Publication Date: 10 December 1992 (10.12.92) (21) International Application Number: PCT/SE92/00373 (74) Agents: ROTH, Michel et al.; G~teborgs Patentbyrfi AB, Box 5005, S-402 21 Gdteborg (SE).

(22) International Filing Date: 3 June 1992 (03.06.92) (81) Designated States: AT (European patent), AU, BB, BE Priority data: (European patent), BF (OAPI patent), BG, BJ (OAPI 709,709 3 June 1991 (03.06.9 1) us patent), BR, CA, CF (QAPI patent), CG (OAPI patent), CH (European patent), CI (QAPI patent), CM (QAPI patent), CS, DE (European patent), DK (European pa- -(7+)Applicant. S'.IR4TELLO VAEG-C144-EEIE-1GPNENT-A-B- tent), ES (European patent), Fl, FR (European patent), SE,'SEI, Gttdhi-gta 1 1, S 1116-Grtebrg--&E). GA (QAPI patent), GB (European patent), GN (OAPI patent), GR (European patent), HU, IT (European pa- (72) Inventors: VAHLNE, Anders ;Igelkottsstigen 14 A, S-430 tent), JP, KP, KR, LK, LU (European patent), MC (Eu- Hovds WVENNERHOLM, Bo ;Jakobsdalsga- ropean patent), MG, ML (OAPI patent), MN, MR (OAtan 48, S-4 12 68 Gbteborg RYMO, Lars Hiillekul- P1 patent,, MW, NL (European patent), NO, PL, RO, lavdgen 17, S-430 80 Hovhs JEANSSON, Stig RU, SD, SE (European patent), SN (OAPI patent), TD F6reningsgatan 33, S-411 27 Gtiteborg HORAL, (OAPI patent), TG (OAPI patent).

Peter ;Orangerigatan 21 B, S-4 12 66 Goteborg (SE).

CZtERKINSKY, Cecil Sveagatan 16-18, S-413 14 Goteborg HOLMGREN, Jan Korvettgatan I D, S-421 Published 74 Vdstra Frt~lunda With international search report.

Before the expiration of the rimte limit for amending the 71 Claimns and to be republished in the event of the receipt of 4A75/-1(7/ej r0?e,-ce (54)Title: PEPTIDES FOR USE IN INDUCTION OF T CELL ACTIVATION AGAINST HIV-1 (57) Abstract Peptides corresponding to regions of the human immunodeficiency virus protein gp-120 are provided for eliciting T-cell activation.

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I WO 92/21377 PCT/SE92/00373 -1- Description Peptides for Use in Induction of T Cell Activation Against HIV-1 pAnding TU. -pa rntappcti ril 07/57l,00, Background of the Invention AIDS and AIDS-related disorders (ARC) are caused by a retrovirus, the human immunodeficiency virus (HIV). Barr6-Sinoussi et al., "Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for Acquired Immune Deficiency Syndrome (AIDS), Science, 220:868 (1983); and Gallo et al., "Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) From Patients with AIDS and at Risk for AIDS", Science, 224:500 (1984).

Like most viruses, HIV often elicits the production of neutralizing antibodies. Unlike many other viruses and other infectious agents for which infection leads to protective immunity, however, HIV specific antibodies are insufficient to halt the progression of the disease. Therefore, in the case of HIV, a vaccine that elicits the immunity of natural infection could prove to be ineffective. In fact, vaccines prepared from the HIV protein gpl60 appear to provide little immunity to HIV infection although they elicit neutralizing antibodies. The failure to produce an effective anti-HIV vaccine has led to the prediction that an effective vaccine will not be available until the end of the 1990's.

The HIV genome has been well characterized. Its approximately 10Kb encodes sequences that contain i /y IT 0 WO 92/21377 PCT/SE92/00373 S-2regulatory segments for HIV replication as well as the gaq, pol and env genes coding for the core proteins, the reverse transcriptase-protease-endonuclease, and the internal and external envelope glycoproteins respectively.

The HIV env gene encodes the intracellular glycoprotein, gpl60, which is normally processed by proteolytic cleavage to form gpl20, the external viral glycoprotein, and gp41, the viral transmembrane glycoprotein. The gpl20 protein remains associated with HIV virions by virtue of noncovalent interactions with gp41. These noncovalent interactions are weak, consequently most of the gpl20 is released from cells and virions in a soluble form.

Previous studies have shown that the proteins encoded by the gag and especially the env regions of the HIV-1 genome are immunogenic since antibodies to the products of the gag and env genes are found in the sera of HIV infected, AIDS and ARC patients.

It has previously been shown that some antibodies obtained from sera of AIDS and ARC patients, as well as asymptomatic individuals infected with the virus are specific to gpl20 and gpl60. Occasionally these antibodies are neutralizing. The envelope glycoproteins are the HIV-1 antigen most consistently recognized by antibodies in AIDS and ARC patient sera. Allan et al., "Major Glycoprotein Antigens that Induce Antibodies in AIDS Patients are Encoded by HTLV-III", Science, 228:1091-1094 (1985); and Barin et al., "Virus Envelope Protein of HTLV-III Represents Major Target Antigen for Antibodies in AIDS Patients", Science, 228:1094-1096 (1985). In addition, antibodies in patient sera also recognize epitopes of the.viral core proteins encoded by the gag gene.

Immunologically important HIV-1 antigens for use in diagnosis and as potential vaccine compositions have i WO92/21377 PCT/SE92/00373 -3been prepared by cloning portions of the HIV-1 genome in various expression systems such as bacteria, yeast or vaccinia. Cabradilla et al., "Serodiagnosis of Antibodies to the Human AIDS Retrovirus With a Bacterially Synthesized env Polypeptide", Biotechnology, 4:128-133 (1986); and Chang et al., "Detection of Antibodies to Human f-Cell Lymphotropic Virus-III (HTLV-III) With an Immunoassay Employing a Recombinant Escherichia coli Derived Viral Antigenic Peptide", Biotechnology, 3:905-909 (1985). HIV-1 antigens produced by recombinant DNA methods, however, must still be exhaustively purified to avoid adverse reactions upon vaccination and false positive reactions in ELISA assays due to any antibody reactivity to antigens of the expression system which may contaminate the HIV-1 antigen preparation. Also, denaturation of HIV-1 antigens during purification may destroy important antigen activity. Preparation of proteins from intact viruses can also result in contamination by the virus.

Several publications have presented data showing immunologic reactivity of selected synthetic peptides corresponding to portions of the antigenic proteins of HIV-1. In one study, a peptide having the amino acid sequence Tyr-Asp-Arg-Pro-Glu-Gly-Ile-Glu-Glu-Gluf Gly-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Gly-Cys which corresponds to amino acid residues 735-752 of HIV-1 was Ssynthesized. Kennedy et al., "Antiserum to a Synthetic Peptide Recognizes the HTLV-III Envelope Glycoprotein", Science, 231:1556-1559 (1986). This peptide, derived from a portion of gp41, was used to immunize rabbits in an attempt to elicit a neutralizing antibody response to HIV-1. Furthermore, several sera from AIDS patients known to contain anti-gp41 antibodies were weakly reactive with this peptide, thus indicating that this peptide contains at least one epitope recognized, to i WO 92/21377 PCT/SE92/00373 -4some extent, by antibodies to native gpl60/gp41.

However, this peptide has not been shown to elicit neutralizing antibodies in mammals other than rabbits nor has it been suggested for use as a human vaccine.

Longitudinal studies conducted on cohorts of HIV-infected individuals have indicated that a stable clinical condition is associated with presence of high titers of neutralizing antibodies against the envelope glycoprotein gpl20 of HIV and especially against a specific segment of eight amino acids. Ranki et al., "Neutralizing Antibodies in HIV (HTLV-III) Infection: Correlation with Clinical Outcome and Antibody Response Against Different Viral Proteins", Clin. Exp. Immunol., 69:231 (1987); and Marx (1989).

Achieving protective immunity against HIV is likely to lie on the induction of gpl20 specific neutralizing antibodies. Marx, "New Hope on the AIDS Vaccine Front", Science, 244:1254 (1989). Potent T cell help might also be critical to promote the generation and the expansion of virus-specific cytotoxic T cells. Reinherz and Schlossman, "The Characterization and Function of Human Immunoregulatory T Lymphocyte Subsets", Immunol. Today, 2:69 (1981); Burns et al, "Thymus Dependence of Viral Antigens", Nature, 256:654 (1975); and Askonas et al., "Cytotoxic T-memory Cells in Virus Infection and the Specificity of Helper T Cells", Immunology, 45:79 (1982). To be durable and broad, protective immunity should rely on induction of immunologic memory to structurally conserved antigenic moieties comprising epitopes displaying limited MHC restriction for T helper cell recognition. Askonas et al. (1982).

Since production of antibodies, including neutralizing antibodies, by B cells is critically dependent on cognate T cell help, and antigenic determinants recognized by T cells are often distinct from the ones dt on c e T c WO 92/21377 PCT/SE92/00373 ilii recognized by B cells, identification of antigenic moieties recognized by T cells (so-called "T cell Sepitopes"), is important when considering vaccination strategies based on appropriate combinations of T and B cell epitopes.

It would therefore be useful in the treatment and prevention of AIDS and ARC to have an HIV vaccine capable of producing neutralizing antibodies and concomitantly eliciting T cell help.

Most antigenic determinants recognized by T cells are composed of continuous stretches of peptides. Streitcher et al., "Antigen Conformation Determines Processing Requirements for T-cell Activation", Proc. Natl. Acad. Sci. 79:4723 (1982); DeLisi and Berzofsky, "T Cell Antigen Sites Tend to be Amphipathic Structures", Proc. Natl. Acad.

Sci. 82:7048 (1985); and Margalit et al., "Prediction of Immunodominant Helper T Cell Antigenic Sites From Primary Sequence", J. Immunol., 138:2213 (1987). B and T cell recognition sites are often located in different regions of a complex antigen.

Milich et al., "Nonoverlapping T and B Cell Determinants on an Hepatitis B Antigen pre-S(2) Region Synthetic Peptide", J. Exp. Med., 164:532 (1986).

Within the functional T cell repertoire, T helper cells, T cytotoxic cells, and T suppressor cells, appear to recognize structurally distinct determinants.

Krzych et al., "Induction of Helper and Suppressor T Cells by Nonoverlapping Determinants on the Large Protein Antigen, 0-galactosidase", FASEB 2:141 (1988). This functional separation may have important bearing on the development of vaccines, since particular determinants recognized by T suppressor cells may be ablated resulting in important benefits for immunogenicity.

L. WO92/21377 PCT/SE92/00373 -6- AIDS and ARC are associated with progressive impairment of CD4 T cells, and increased susceptibility to opportunistic infections. In this respect, HIV-infected persons show decreased T helper cell activity for polyclonal B cell differentiation and decreased T cell proliferative responses to antigens and mitogens associated with an early loss of CD29 memory T cells. Terpstra et al., "Longitudinal Study of Leukocyte Functions in Homosexual Men Seroconverted for HIV: Rapid and Persistent Loss of B Cell Function After HIV Infection", Eur. J. Immunol., 19:667 (1989); Fahey et al., "Quantitative Changes in T Helper or Suppressor/Cytotoxic Lymphocyte Subsets that Distinguish Acquired Immune Deficiency Syndrome From Other Immune Subsets Disorders", JAMA, 76:95 (1984); Shearer et al., "Functional T Lymphocyte Immune Deficiency in a Population of Homosexual Men Who do not Exhibit Symptoms of Acquired Immune Deficiency Syndrome", J.

Clin. Invest., 74:496-506 (1984); Giorgi et al., "Early Effects of HIV on CD4 Lymphocytes in vivo", J.

Immunol., 138:3725 (1987); and van Noesels et al., "Functional and Phenotypic Evidence for a Selective Loss of Memory T Cells in Asymptomatic Human Immunodeficiency Virus-infected Men", 86:293 (1990).

The use of synthetic peptides as artificial T cell I: recognition sites in the composition of candidate subunit vaccines, offers attractive prospects. In this regard, the possibility to educate T helper cells with synthetic peptides for the development of subsequent antibody responses against overlapping and nonoverlapping B cell (antibody) recognition sites has been documented in several experimental systems.

Streitcher et al. (1982);.DeLisi and Berzovsky (1985); and Milich et al., "A Single 10-residue PreS(1) Peptide Can Prime T Cell Help for Antibody Production to Multiple Epitopes Within the pre-S(1), pre-S(2), and S l t SWO 92/21377 PCT/SE92/00373 -7regions of HBsAg", J. Immunol., 138:4457 (1987). It has now been found that peptides derived from two regions of the HIV genome elicit T cell activation.

These peptides are also capable of inducing the production of neutralizing antibodies to HIV-1.

Summary of the Invention In accordance with the present invention, novel peptides corresponding to epitopes of HIV-1 protein and analogues and homologs thereof are provided. These peptides can be utilized alone or in combination, uncoupled or coupled to other molecules or substrates. The peptides are useful in eliciting T cell activation, immunization against HIV infection, induction of a heightened immune response to HIV and in production of polyclonal and monoclonal antibodies.

Forty synthetic peptides corresponding to the entire primary sequence of the envelope gpl20 of the human immunodeficiency virus type 1 (HIV-1) were examined for their ability to induce antibody formation and/or T cell activation antibody formation was determined by measuring the amount of peptide-specific antibody formed. T cell activation was measured by the ability of the peptides to induce in vitro proliferative responses and/or IL-2 production when added to cultures of unfractionated, T cell enriched, Sand/or CD4 T cell enriched peripheral blood mononuclear cells (PBMC) from immune monkeys. Among four major areas of T cell recognition identified, two novel T cell activating regions were identified, both of which were also found to be capable of inducing, in Svivo, the production of neutralizing antibodies to HIV-1. One of these two novel areas corresponds to a highly conserved region of HIV-1 gp-120, the other area being located to a variable region of gp-120.

Recognition of the latter variable region does not i 1 WO 92/21377 PCT/SE92/00373 i appear to be restricted by MHC polymorphism, since all of six monkeys immunized with corresponding peptides were found to display in vitro proliferative responses to these peptides. The results of peptides thus have great utility for the development of synthetic subunit SAIDS vaccines.

Brief Description of the Drawings Fig. 1 is a graph depicting in vitro proliferative responses of monkey peripheral blood mononuclear cells (PBMC) to recall peptide after two and/or three peptide immunizations.

Fig. 2 is a graph depicting in vitro proliferative responses of monkey PBMC to half-overlapping peptides.

Detailed Description of the Invention A vaccine against AIDS, if an efficient one is to be found, is likely to contain components that are capable of inducing T helper cell activity to cognate B cells committed to the production of HIV neutralizing antibodies.

The present invention provides peptides, some of which have previously been found to elicit p oduction of HIV neutralizing antibodies by primate subjects and all of which have now been found to have the surprising property of eliciting T cell activation. The peptides correspond to regions of the gpl20 protein with amino acid coordinates as defined by Kennedy et al., "Antiserum to a Synthetic Peptide Recognizes the HTLV- III Envelope Glycoprotein", Science, 231:1556-1559 (1986). The peptides of the present invention are termed gpl20-11 (amino acid coordinates 141-164), gpl20-12 (amino acid coordinates 151-176), gpl20-13 (amino acid coordinates 164-192), gpl20-16 (amino acid coordinates 205-230) and gpl20-19 (amino acid coordinates 247-269), gpl20-29 (amino acid coordinates 366- SI I WO 92/21377 PCT/SE92/00373 -9- 389) and gpl20-30 (amino acid coordinates 377-400).

The peptides of the present invention have been described for use as immunogens in vaccine compositions and to elicit polyclonal or monoclonal antibody productions in United States patent application Serial Number 07/589,422 filed Sept. 27, 1990 which is incorporated herein by reference.

Four topographically related groups of peptides derived from gpl20 have now been identified which display T cell activating properties. Two of the regions found to elicit T cell activation are similar to previously identified T cell epitopes. Bolognesi, "HIV Antibodies and AIDS Design", AIDS 3:S111-S118 (1989). The present study indicates that recognition of the area defined by amino acid coordinates 295-343 by immune T cells may be submitted to strong MHCrestriction as one of the 2 monkeys injected with peptide gpl20-25 failed to respond to that particular peptide in vitro. The T cell antigenic determinants in this area seem to be more or less exclusively located within each of the immunizing peptides as none of the overlapping peptides gave rise to in vitro proliferative responses. However, PBMC isolated from monkeys immunized with peptide gpl20-24 secreted IL-2 when cultured in the presence of peptide gpl20-25, indicating the existence of a minor epitope shared by these two peptides.

The region of gpl20 corresponding to amino acid coordinates 295 to 343 (peptides gpl20-23, gpl20-24 and gpl20-25), is similar to a region (amino acid coordinates 301 to 338) which has previously been shown by other investigators to contain a major T cell recognition site (amino acid coordinates 303-337) whose sequence encompasses that of the neutralizing loop.

Bolognesi (1989); Javaherian et al., "Principal Neutralizing Domain of the Human Immunodeficiency Virus I L WO 92/21377 PCr/SE92/00373 Type 1 Envelope Protein", Proc. Natl. Acad. Sci. USA, 86:6768 (1989); and Rusche et al., "Antibodies That Inhibit Fusion of Human Immunodeficiency Virus infected Cells Bind a 24-amino Acid Sequence of the Viral Envelope, Proc. Natl. Acad. Sci. USA, 85:3198 (1988).

The area of gp-120 located between amino acid coordinates 409 and 466 (peptides gpl20-33, gpl20-34, gpl20-35 and gpl20-36), as described in the Examples presented below, was found to have potent T cell activating properties. The area between amino acid coordinates 409 och 466 has previously been shown to accommodate T cell activating domains. Bolognesi (1989). In this area, two T cell epitopes have been identified, one between amino acid coordinates 410 and 429, and one between amino acid coordinates 429 and 443. The latter area largely overlaps with the CD4-binding site (amino acid coordinates 420-463) of gpl20, the main site of virus attachment on permissive T cells.

Several other epitopes with T cell activating properties have now been identified in discrete areas of the molecule. Thus, peptide gpl20-4 (amino acid coordinates 53-74), gpl20-5 (amino acid coordinates 64-89), gpl20-8 (amino acid coordinates 100-126), gp 120-7 (amino acid coordinates 218-247) and peptide gpl20-21 (amino acid coordinates 269-295), and at least on of their overlapping peptides, were shown to be capable of inducing T cell responses in vitro. However, both of the monkeys immunized with peptide gpl20-21, when retested five months after the last booster dose, hade lost their in vitro T cell responsiveness to recall peptide. Thus, not every peptide capable of eliciting i1ir SUBSTITUTE SHEET 940901,,5 j WO92/21377 PCT/SE92/00373 -11- T cell activation is suitable for use in treatment and prevention of AIDS.

Surprisingly, peptide gp120-19, has now been shown to have T cell immunogenic properties as defined by in vitro proliferative responses of simian PBMC to cognate peptide. Additionally, PBMC from a monkey immunized with OVA-conjugated peptide gpl20-16 has now been found to secrete IL-2 after in vitro exposure to peptide gp120-16. Peptide gp120-16 therefore represents an additional novel T cell epitope.

Two novel areas with in vitro T cell activating properties have now been identified. Peptides 11, gp120-12 and gpl20-13, corresponding to amino acid coordinates 141 to 192, induce the most potent in vitro proliferative responses, with SI values exceeding sometimes 20 in cultures of T cells from monkeys immunized with corresponding OVA-conjugated peptides. The fact that six out of six monkeys from an outbred population, responded to peptides gpl20-11, gpl20-12 and gp120-13 strongly indicates that recognition of this bona fide T cell epitope area by simian T cells is not under strict MHC control. Accordingly, at least three distinct epitopes have now been recognized by immune monkey PBMC, one shared by peptides gpl20-11 and gp120-12, one shared by peptides gp120-12 and gp120-13 and one additional epitope within peptide gp120-13. In addition, in vitro proliferative responses to peptides gp120-12 and gp120-13 have now been demonstrated in cultures of CD2 T cells, and also CD4 T cells, initiated as late as five ionths after the second immunization indicating the presence of memory T helper (CD4 cell activating epitopes in that area.

Another novel area identified in this study, includes peptides gp120-29 and gpl20-30 (amino acid coordinates 366 to 400) which induced cell responses in 3 out of 4 monkeys. Recognition of this area SUBSTITUTE SHEET i MH cotro. Acordngl, atleat tree istnctepA reslaue roiiowed oy a hydroxy group; and a physiologically acceptable carrier therefor.

WO 92/21377 PCT/SE92/00373 -12by 4mmune T cells appears to be also under limited MHC restriction, or to involve epitope(>) associated with polymorphic MHC determinants. At least two epitopes would be expected within this area as the responses did not always overlap.

Importantly, among the novel T cell activating areas that have now been identified, three peptides were also found to be capable of inducing, in vivo, the production of neutralizing antibodies against HIV-1.

Thus, sera obtained from all monkeys immunized with peptide gpl20-12, peptide gpl20-16 and peptide gpl20-19 inhibited in vitro HIV induced p-24 antigen release and syncytia formation by human permissive T cell lines exposed to HIV-1 virions of the corresponding (BRU) isolate. Further, peptide gpl20-12 is derived from a partly conserved region of gp120 and is associated with a site recognized by neutralizing antibodies. Peptide gpl20-16 represents a highly conserved area of within all 14 different isolates investigated. The efficiency of the peptides, derived from a conserved region of HIV, at inducing the production of HIVneutralizing antibodies as well as at triggering a T cell response is noteworthy. Peptides gpl20-12 and gpl20-16 are thus the preferred embodiments of the present invention.

Less than 10% of HIV infected individuals produce J antibodies capable of recognizing peptides gpl20-12, gpl20-15, gpl20-16 and gpl20-19. Since antibodies are generated in response to immunization with these peptides it is possible to induce an increase in the repertoire of neutralizing antibody producing B cells in HIV positive individuals.

Proteins contain a number of antigenic determinants or epitopes which are the regions of the proteins comprising the recognition and binding sites for specific antibodies. An epitope contains a I II residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and a physiologically acceptable carrier therefor.

WO92/21377 PCT/SE92/00373 -13sequence of 6 to 8 amino acids. Epitopes can be either continuous wherein the sequence of 6-8 amino acids are linear or discontinuous in which case the amino acids are brought together by the three dimensional folding of the protein. Even though an epitope constitutes only a relatively few amino acids, its reactivity with an antibody may be influenced by the amino acids in the protein which surround the epitope.

Studies aimed at mapping antigenic sites or epitopes of proteins have been aided by the use of synthetic peptides corresponding to various regions of the proteins of interest. Lerner et al., in, The Biology of Immunological Disease: A Hospital Practice Book, (Dixon and Fisher, eds.) pp. 331-338 (1983); and Lerner, Adv. Immunol., 36:1 (1984). In addition to their usefulness in epitope mapping studies, synthetic peptides, if encompassing major antigenic determinants of a protein, have potential as vaccines and diagnostic reagents. Van Regenmortel, Ann. Inst. Pasteur Virol., 137E:497-528 (1986); and Van Regenmortel, Laboratory Techniques in Biochemistry and Molecular Biology, Buroden and Van Knippenburg eds. Vol. 19, Synthetic Peptides as Antigens, Elsevier ISBN 0-444-80974-0 (1988).

Synthetic peptides have several advantages with regard to specific antibody production and reactivity.

The exact sequence of the synthesized peptide can be selected from the amino acid sequence of the protein as determined by amino acid sequencing of the protein or T 30 the predicted amino acid sequence determined from the DNA sequence encoding the protein. The use of specific synthetic peptides eliminates the need for the fulllength protein in vaccination and the production of or assay for antibodies. Furthermore, the solid phase peptide synthetic techniques of Merrifield and coworkers allow for essentially unlimited quantities of j/ WO 92/21377 PCT/SE92/00373 -14the synthesized peptide of interest to be chemically produced. Erickson and Merrifield in The Proteins, 3rd Edit., Vol. 2, Academic Press, New York, Chapter 3 (1976). The availability of automated peptide synthesizers has further advanced such techniques.

Although a variety of criteria can be used to predict antigenic regions of proteins, peptides corresponding to such regions may not always be useful as vaccines. For example, antigenicity may be lost because the peptide is not in the proper spatial orientation to be recognized by antibodies which react with the protein. It has also been found that certain peptides derived from type C retroviruses and HIV act as immune-suppressive agents much as does HIV itself.

Cianciolo et al., J. Immunol., 124:2900-2905 (1980); and Cianciolo et al., Proc. Natl. Acad. Sci. USA, 230:453-455 (1985). Peptides such as these, which have a deleterious effect on the patient, would not be suitable for use as vaccines.

Furthermore, as is particularly evident with HIV-1 and HIV-2, there is significant genetic variability within each of these two virus groups leading to many serotypes, or isolates, of the viruses. This has put a significant constraint on choosing a region of a protein from which to derive a peptide for use in formu- S| lating immunogens. However, certain immunodominant portions of HIV-1 and HIV-2 proteins have been found to ~be relatively invariant. Synthetic peptides may also be key to viral vaccines in that they may induce an immune response against type common sequences not normally immunogenic in the native molecule. These otherwise silent epitopes may be of broad protective specificity. Stevard et al., Immunol. Today, 8:51-58 (1987). Several experimental vaccines have been formulated with the aim of preventing infection in those people who are likely to bi exposed to the virus.

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The HIV genome has been weli cnaracerimLtu. approximately 10Kb encodes sequences that contain

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WO92/21377 PCT/SE92/00373 Berman et al., "Protection of Chimpanzees from Infection by HIV-1 After Vaccination With Recombinant Glycoprotein gp120 but Not gpl60", Nature 345:622-625 (1990).

A number of neutralization epitopes on gpl20 have been found and defined by several investigators, for an overview see Bolognesi, AIDS (1989) 3(suppl 1): S111-s118. In his overview Bolognesi refers to four different virus neutralization epitopes with the following amino acid coordinates: 254-274, 303-337, 458-484 and 491-523. The peptide with amino acid coordinates 254-274 was used to immunize rabbits and the resulting antiserum was found to neutralize HIV-1 as described above. Ho et al., Science, 239:1021-1023 (1988).

The peptides encompassed by the invention comprise amino acid sequences each containing at least one continuous (linear) epitope that elicits production of activated T cells in the host in addition to eliciting the production of HIV specific antibodies.

The invention thus encompasses immunogenic peptides corresponding to regions of HIV gpl20 protein encoded by the envelope gene of HIV-1 HTLV III-B described by Muesing et al., "Nucleic Acid Structure and Expression of the Human AIDS/Lymphadenopathy Retrovirus", Nature, 313:450-458 (1985). The nucleotide sequence is given in Genbank Release 63 under the name HIVPV22. The invention further encompasses functionally equivalent variants of the peptides which do not significantly affect the immunogenic properties of the peptides. For instance, conservative substitution of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogues are within the scope of the invention.

g l• I inlyeuvln ainso h etdswihd i 0 ntsgiiatyafc h muoei rpriso Immunologically important HIV-1 antigens tor use in diagnosis and as potential vaccine compositions have WO 92/21377 PCT/SE92/00373 -16- Homologs are peptides which have conservatively substituted amino acid residues and peptides derived from corresponding regions of different HIV isolates.

Amino acids which can be conservatively substituted for one another include but are not limited to: glycine/ alanine; valine/isoleucine/leucine; asparagine/ glutamine; aspartic acid/glutamic acid; serine/ threonine; lysine/arginine; and phenylalanine/tyrosine.

Homologous peptides are considered to be within the scope of the invention if they are recognized by antibodies which recognize the peptides designated gpl20-12, gpl20-16 and gpl20-19 the sequences of which are shown below. Further, all homologous peptides corresponding to the peptides of the present invention but derived from different HIV isolates are also encompassed by th e of this invention.

So he invention also encompasses polymers of one or more of the peptides, and peptide analogues or homologs are within the scope of the invention. Also within the scope of this invention are peptides of fewer amino acid residues than the peptides but which encompass one or more immunogenic epitopes present in any one of the peptides and thus retain the immunogenic properties of the base peptide.

The invention further encompasses functionally equivalent variants of the peptides which do notm S significantly affect the antigenic or T cell activating properties of the peptides. For instance, various U analogues, or peptidomimetics are known in the art and can be used to replace one or more of the amino acids in the peptides. Analogues are defined as peptides which are functionally equivalent to the peptides of the present invention but which contain certain nonnaturally occurring or modified amino acid residues.

Additionally, polymers of one or more of the peptides are within the scope of the invention.

I- WO 92/21377 PCT/SE92/00373 -17- The use of peptide analogues can result in peptides with increased activity, that are less sensitive to enzymatic degradation, and which are more selective.

A suitable proline analogue is 2-aminocyclopentane carboxylic acid (AcSc) which has been shown to increase activity of a native peptide more than 20 times.

Mierke et al., "Morphiceptin Analogs Containing 2aminocyclopentane Carboxylic Acid as a Peptido.-imetic for Proline", Int. J. Peptide Protein Res., 35:35-45 (1990). See also Portoghese et al., "Design of Peptidomimetic S Opioid Receptor Antagonists Using the Message-Aerress Concept", J. Med. Chem., 33:1714-1720 (1990); and Goodman et al., "Peptidomimetics: Synthesis, Spectroscopy, and Computer Simulations", Biopolymers, 26:S25-S32 (1987).

The peptides were synthesized by known solid phase peptide synthesis techniques. Merrifield and Barany, The Peptides: Analysis, Synthesis, Biology, Vol. 1, Gross and Meinenhofer, eds., Academic Press, New York, Chap. 1 (1980). The synthesis also allows for one or more amino acids not corresponding to the original protein sequence to be added to the amino or carboxyl terminus of the peptide. Such extra amino acids are useful for coupling the peptides to another peptide, to a large carrier protein or to a solid support. Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof.

i Additional protein modification techniques may be used,

NH

2 -acetylation or COOH-terminal amidation, to provide additional means for coupling the peptides to another protein or peptide molecule 9r a support.

Procedures for coupling peptides to each other, carrier proteins and solid supports are well known in the art.

Peptides containing the above-mentioned extra amino acid residues either carboxy or amino terminally, a dent on cognate T cell help, and antigenic determinants recognized by T cells are often distinct from the ones WO 92/21377 PCT/SE92/00373 -18uncoupled or coupled to a carrier or solid support are consequently within the scope of the invention. Reference to the peptides of the present invention encompasses all of the embodiments discussed herein.

An alternative method of vaccine production is to use molecular biology techniques to produce a fusion protein containing one or more of the peptides of the present invention and a highly immunogenic protein.

For instance, fusion proteins containing the antigen of interest and the B subunit of cholera toxin have been shown to induce an immune response to the antigen of interest. Sanchez et al., "Recombinant System For Overexpression of Cholera Toxin B Subunit in Vibrio cholerae as a Basis for Vaccine Development", Proc.

Natl. Acad. Sci. USA, 86:481-485 (1989). It is thus implicit in the present invention that vaccine constructs based on appropriate constructions of B and T cell epitopes fused to a carrier protein like cholera toxin would represent important benefits in vaccination.

The novel peptide amino acid sequences are set forth below and in Table 2. The amino acid residues are derived from the nucleotide sequence previously described by Kennedy et al. (1986). The peptides may contain either an amido or carboxy group at their carboxy termini.

gpl20-11 i X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys-Gly-Glu-Ile- Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser-Thr-Ser-Y-Z gp120-12 X-Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser-Thr- Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala-Phe-Phe-Y-Z for immunogenicity.

WO 92237PCT/SE92/00373 -19- 4 gp 120-13 X-I le-Arg-Gly-Lys-Val-Glrl-Lys-Glu-Tyr-Ala-Phe-Phe-Tyr- Lys-Leu-Asp-Ile-Ile-Pro-Ile-Asp-Asn-Asp-Thr-Thr-Ser- Tyr-Thr-Y-Z gp12O-16 X-Pro-Lys-Val-Ser-Phe-Glu-Pro-Ile-Pro-Ile-His-Tyr-Cys- Ala-Pro-Ala-Gly-Phe-Ala-I le-Leu-Lys-Cys-Asn-Asn-Y-Z gpl2O-19 X-Thr-His-Gly-Ile-Arg-Pro-Val-Val-Ser-Thr-Gli-Leu- Leu-Leu-Asn-Gly-Ser-Leu-Ala-Glu-Glu-Glu-Y-Z gp 120-29 X-Gly-Asp-Pro-Glu-I le-Va l-Thr-His-Ser-Phe-Asn-Cys-Gly- Gly-Glu-Phe-Phe-Tyr-Cys-Asri-Ser-Thr-Gln-Y-Z gp 120-30 X-Cys-Gly-Gly-Glu--Phe-Phe-Tyr-Cys-Asn-Ser-Thr-Gln-Leu- Phe-Asn-Ser-Thr-Trp-Phe-Asn-Ser-Thr-Trp-Y- Z wherein X is either a hydrogen atom of the amino terminal NH 2 group of the peptide or an additional amino acid being selected to facilitate coupling of the peptide to a carrier; Y is absent or Cys; and Z is the carboxyl group of the carboxy terminal amino acid or an amido group. The amino acid abbreviations used are defined in Table 2.

In addition to eliciting T cell activation, several of the peptides are useful as vaccines to protect against future infection by HIV or to heighten the immune response to HIV in subjects already infected by HIV. Although any human subject could be vaccinated with the pqptides, the most suitable subjects are people at risk for HIV infection. Such subjects 4 include but are not limited to homosexuals, Can Prime T Cell Help for Antibody Production to Multiple Epitopes Within the pre-S(1), pre-S(2), and S -j WO 92/21377 PCT/SE92/00373 prostitutes, intravenous drug users, hemophiliacs and those in the medical professions who have contact with patients or biological samples. The invention also provides monoclonal and polyclonal antibodies which specifically recognize the peptides. The invention further provides antibodies produced in response to vaccination with the peptides which neutralize HIV.

In the preferred embodiment of the present invention, the peptides are formulated into compositions for use as immunogens. These immunogens can be used as vaccines in mammals including humans or to elicit T cell activation and/or production of polyclonal and monoclonal antibodies in animals. For formulation of such compositions, an amount sufficient to elicit T cell activation of at least one of the peptides (about 1-500 Pg) is admixed with a physiologically acceptable carrier suitable for administration to mammals including humans.

The peptides may be covalently attached to each other, to other peptides, to a protein carrier or to other carriers, incorporated into liposomes or other such vesicles, and/or mixed with an adjuvant or adsorbent as is known in the vaccine art. For instance, the peptide or peptides can be mixed with ii 25 immunostimulating complexes as described by Takahashi et al., "Induction of CD8+ Cytotoxic T Cells by Immunization With Purified HIV-1 Envelope Protein and ISCOMS", Nature, 344:873-875 (1990). Alternatively, the peptides are uncoupled and merely admixed with a physiologically acceptable carrier such as normal saline or a buffering compound suitable for administration to mammals including humans.

As with all immunogenic compositions for eliciting antibodies, the immunogenically effective amounts of the peptides of the invention must be determined empirically. Factors to be considered include the I uch esilesand/r mxed ith n ajuvat o being located to a variable region of gp-120.

Recognition of the latter variable region does not WO 92/21377 PCT/SE92/00373 -21immunogenicity of the native peptide, whether or not the peptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and route of administration for the composition, i.e. intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be administered. Such factors are knovi in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.

The invention is further illustrated by the following specific examples which are not intended in any way to limit the scope of the invention. In order to determine T cell activation, PBMC from monkeys immunized with OVA-conjugated HIV gpl20 peptides were tested for their ability to produce IL-2 and/or to proliferate when exposed in vitro to recall (immunizing), overlapping, and non overlapping peptide(s).

Example 1 Animals Used in Subsequent Examples Cynomolgus monkeys (Macaca fascicularis) were given 3 intramuscular doses of ovalbumin (OVA)conjugated peptides (see below), three weeks apart, each dose consisting of 100 gg of ovalbumin-coupled peptide emulsified in Freund's complete (first dose) or incomplete (booster doses) adjuvant.

Example 2 Peptide Synthesis HIV-1 gpl20 peptides (Table with an additional carboxy-terminal cysteine residue, were synthesized on solid phase with an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, CA, USA) using the polymer p-methylbenzhydryl amine resin as solid phase (Peptides Int., Louisville, USA).

W092/21377 PC'T/SE92/00373 -22- All amino acids for use in synthesis contained t-butylcarbonyl groups (t-Boc) protecting the a-NH 2 group and were obtained from Novabiochem AG, Switzerland. Amino acids with reactive side chain groups contained additional protective groups to prevent unwanted and undesirable side chain reactions.

The individual protected amino acids used in synthesizing all of the peptides are set forth in Table 1.

TABLE 1 AMINO ACIDS USED IN PEPTIDES SYNTHESIS Boc-Ala-OH Boc-Arg (Tos)-OH Boc-Asn-OH Boc-Asp (Obzl)-OH Boc-Cys (Pmeobzl)-Oh Boc-Glu (Obzl)-OH Boc-Gln-OH Boc-Gly-OH Boc-His-(Tos)-OH Boc-Ile-OH^l/2

H

2 0 Boc-Leu-OH^H 2 0 Boc-Lys (2-CI-Z)-OH (cryst.) Boc-Met-OH Boc-Phe-OH Boc-Pro-OH Boc-Ser (Bzl)-OH^DCHA Boc-Thr (Bzl)-OH Boc-Trp (Formyl)-OH Boc-Tyr (2-Br-Z)-OH Boc-Val-OH Tos: Tosyl or p-Toluene sulfonic acid Obzl Benzyloxy Pmeobzl p-Methylbenzyloxy 2-CL-Z Carbobenzoxy chloride 2-Br-Z Carbobenzoxy bromide The peptides were synthesized using the t-Boc synthesis protocol as suggested by the manufacturer.

All solvents were from Applied Biosystems and the side chain protected amino acids used were from Nova Biochem

I

WO92/21377 PCT/SE92/00373 -23- (Switzerland) and Applied Biosystems. Following each amino acid coupling, a sample was taken and a quantitative ninhydrin assay was performed. Only if the coupling efficiency exceeded 99% for each amino acid coupled was the peptide accepted for further processing. Completed peptides were cleaved from the solid phase and amino acid side chains were deprotected by acidic hydrolysis using anisole and ethanedithiol (Merck, Germany) as scavengers.

After completion of a particular synthesis, the protecting groups were removed from the synthesized peptide and the peptide was cleaved from the solid support resin by treatment with trifluoromethane sulfonic acid (TFMSA) according to the method described by Bergot et al., "Utility of Trifluoromethane Sulfonic Acid as a Cleavage Reagent in Solid Phase Peptide Synthesis", Applied Biosystems User Bulletin, Peptide Synthesizer, Issue No. 16, Sept. 2, 1986. The following is the detailed protocol used.

1. For 1 gram peptide-resin, 3 ml Thio-Anisol 1,2-Ethane-Dithiol was added as scavenging agent and the mixture was incubated with continuous stirring for 10 min. at room temperature.

2. Trifluoracetic Acid (TFA), 10 ml, was added and stirred continuously for 10 min. at room temperature.

3. TFMSA, 1 ml, was added dropwise with forceful stirring and reacted for 25 min. at room temperature.

4. Following cleavage, the peptides were precipitated with and washed with anhydrous ether.

The precipitated and washed peptides were dissolved in a small volume of TFA.

6. The dissolved peptides were again precipitated and washed as above in step 4 and the precipitate was dried under a stream of N 2 (sV I WO 92/21377 PCT/SE92/00373 -24- Prior to use in specific assays, the peptides can be further purified, if desired, by reverse phase high performance liquid chromatography (HPLC). A particularly suitable column for such purification is the reverse-phase Vydak® C-18 column using a water (TFA) acetonitrile (TFA) gradient to elute the peptides.

Forty peptides were synthesized having the amino acid sequences shown in Table 2.

The amino acid sequences of the peptides, 17-29 amino acids long, half overlapping each other and entirely encompassing gp-120, were obtained from the HIV-1 BRU isolate. Muesing et al., "Nucleic Acid Structure and Expression of the Human AIDS/Lymphadenopathy Retrovirus", Nature, 313:450 (1985).

r

A

WO 92/21377 PCT/SE92/00373 TABLE 2 Peptid 1 Amino Acid AmnIcdSqine Petie _Coordinates** I Imn cd eune gp12O-1 1-28 MRVKEKYQHLWRWGTMiLGMLMIC gpl2O-2 22-46 GMLMICSATEKLWVTVYYGVPVW( gpl2O-3 4 0-64 GVPVWKEATTTLFCASDAKAYDTE gpl2O-4 53-74 CASDAKAYDTEVHNVWATHAC gpl2 0-5 64-8 9 VHNVWATHACVPTDPNPQEVVLVNV gp12O-6 74-100 VPTDPNPQEVVLVNVTENFNMWKNDM gp12O-7 89-116 TENFNMWKNDMVEQMHEDIISLWDQSL gpl2O-8 100-126 VEQMHEDIISLWDQSLKPCVKLTPLC gpl2O-9 116-141 KPCVKLTPLCVSLKCTDLKNDTNTN cqp120-10 126-151 VSLKCTDLKNDTNTNSSSGRMIMEK gp12O-11 141-164 SSSGRMIMEKGEIKNCSFNISTS gpl2O-12 151-176, GEIINCSFNISTSIRGKVQKEYAFF gp120-13 164-192 IRGKVQKEYAFFYKLDIIPIDNDTTSYT gp12O-14 176-205 YKLDIIPIDNDTTSYTLTSCNTSVITQAC gp12O-15 192-218 LTSCNTSVITQACPKVSFEPIPIHYC gpl2O-16 205-230 PKVSFEPIPIHYCAPAGFAILKCNN gpl2 0-17 218-247 APAGFAILKCNNKTFI4GTGPCTNVSTVQC gp120-18 230-257 KTFNGTGPCTNVSTVQCTHGIRPVVST gp12O-19 247-269 THGIRPVVSTQLLLNGSLAEEE gpl2O-20 257-282 QLLLNGSLAEEEVVIRSAIIFTDNARK gp120-21 269-295 VVIRSANFTDNAKTIIVQL7UQSVEIN gpl2O-22 282-306 TIIVQLNQSVEINCTRPNNNTRKS gpl2O-23 295-320 CTRPNNNTRKSIRIQRGPGRAFVTI gpl2O-24 306-326 IRIQRGPGRAFVTIGKIGNMRQAH gpl2O-25 320-343 GKIGNMRQAHKNISRAKWNNTLK gpl2O-26 326-353 KNISRAKWNNTLKQIDSKLREQF 9p12O-27 343-366 QIDSKLREQFGNNKTIIFKQSSG gpl2O-28 1' 353-377 IGNNKTIIFKQSSGGDPEIVTHSFN 51 proteins comprising the recognition and binding sites for specific antibodies. An epitope contains a L1~

A

I

f.

WO 92/21377 PC'/SE92/00373 -26'- TABLE 2 Peptid Amino Acid AioAi eune Peptide Coordinates** I mn cd eune gqpl2O-29 366-389 GDPEIVTHSFNCGGEFFYCNSTQ gpl2O-30 377-400 CGGEFFYCNSTQLFNSTWFNSTW gp12 0-31 389-409 LFNSTWFNSTWSTEGSNNTE gp120-32 400-417 STEGSNNTEGSDTITLP gp120-33 409-429 GSDTITLPCRIKQFIN4WQE gp12O-34 417-444 -CRIKQFINMWQEVGKAMYAPPISGQIR gp12O-35 429-453 VGKAMYAPPISGQIRCSSNITGLL gpl2O-3 6 444-466 CSSNITGLLLTRDGGNNNESE gpl2O-37 453-476 LTRDGGNNNNESEIFRPGGGDMR gpl2O-38 466-488 IFRPGGGDMRDNWRSELYKYKV gpl20--39 476-497 DNfWRSELYKYKVVKIEPLGVA gpl2O-40 488-511 VKIEPLGVAPTKARRRVVQREKR acid abbreviations] Alanine Ala A Leucine Leu L -Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamine Gin Q Serine Ser S -Glutamic acid Glu E Threonine Thr IT Glycine Gly G Tryptophan Trp W Histid,',e His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V t1 As previously described by Kennedy et al.(18) (1986) workers allow for essentially unlimited quantities of WO 92/2137 PCT/SE92/00373 -27- Example 3 Preparation of Peptides for Immunization Peptides according to the present invention were covalently coupled to ovalbumin grade V (Sigma, St.

Louis, MO, USA) at an approximate 10:1 (peptide: ovalbumin) molar ratio using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), (Pharmacia, Uppsala, Sweden) as bifunctional linker according to the manufacturer's instructions (Pharmacia) i.e.

briefly as follows: Ovalbumin was dissolved in coupling buffer (0.2 M NaH 2

PO

4 Ph The dissolved ovalbumin was then run through a Sephadex G-25M column (Pharmacia, Sweden), using the same buffer. Protein concentration was measured at 280 nm and the recovery was determined.

SPDP was dissolved in 99.5% ethanol to a final concentration of 40 mM. SPDP was then added dropwise to the ovalbumin solution under stirring. The SPDP-ovalbumin mixture was then left at room temperature for approximately 30 minutes. The ovalbumin- SPDP conjugate was separated from unconjugated SPDP by running the mixture through a Sephadex G-25M column, using water as eluent. The degree of substitution for the ovalbumin-SPDP conjugate was determined after diluting 50 Al conjugate in 2 ml of water, by measuring the diluted conjugate at 280 nm and the diluted conjugate plus 100 gl Dithiothreitol (DTT) (Sigma) at 343 nm, in order to determine the amount to be added to the peptide solution.

Finally, the synthetic peptide to be coupled to the ovalbumin-SPDP conjugate was dissolved in acetic acid to a final concentration of 1 mg/ml and a suitable amount of ovalbumin-SPDP conjugate (as determined by the substitution degree above) was added and allowed to stand overnight at room temperature.

mulated with the aim of preventing infection in those people who are likely to bo exposed to the virus.

WO 92/21377 PCT/SE92/00373 -28- Example 4 Immunization Protocols M. fascicularis were used to generate antibodies.

Prior to the initial peptide injection, a blood sample was drawn from the monkeys. This initial blood sample is termed "pre-immune" (Tables 5-8) and is used as an internal control and analyzed simultaneously with respective immuneserum.

The monkeys were injected with 100 gg peptide- SPDP-ovalbumin suspended in 0.5 ml phosphate buffered saline (PBS). The monkeys were immunized intramuscularly three times, three weeks apart. As adjuvant, 0.5 ml of Freund's complete adjuvant was used for all initial immunizations and Freund's incomplete adjuvant was used for booster shots. Two weeks after the final immunization the monkeys were bled by removing a 10 ml blood sample from the fossa and pre-immune and hyperimmune sera were subject to neutralization assays as described in Example 9.

Example Isolation and Fractionation of Lymphocytes from Immunized M. fascicularis Heparinized venous blood was collected from the S1 femoral vein, at least two weeks after the second and/or the third injections. Peripheral blood mononuclear cells (PBMC) were obtained by gelatin sedimentation followed by density gradient centrifugation by the following method. A solution of 3% (weight/volume) gelatin (gelatin L936, PB Gelatins UK LTD, GB) in Hank's Balanced Salt Solution was mixed with the blood at a 1:3 ratio and erythrocytes were allowed to sediment for one hour at 37 0 C. The erythrocyte-free supernatant was layered onto a Ficoll-Hypaque cushion (Pharmacia, Sweden) and centrifuged fbr 15 minutes at 930 x g, at 20 0

C.

SWO 92/21377 PCT/SE92/00373 -29- Interface PBMC were washed twice by centrifugation (500 xg, 20 0 C, 5 min) with isotonic phosphate-buffered saline (PBS, 0.01 M phosphate buffer in 0.15 M NaC1, pH 7.4).

In some Examples represented below, T cells were enriched by rosetting with AET-treated sheep red blood cells as described by Kaplan and Clare, "Improved Rosetting Assay for Detection of Human T Lymphocytes", J. Immunol. Met., 6:131 (1974) followed by density centrifugation on Ficoll-Hypaque. The rosetted cells (nominal T cells) were collected from the pellets and resuspended for 20 seconds with distilled water to lyse sheep red blood cells. Further enrichment into CD4 T cells was obtained by paramagnetic depletion of CD8 T cells using microspheres coated with monoclonal anti-CD8 antibodies (Dynal AS, Norway), according to the manufacturer's instructions.

Example 6 Lymphocyte Proliferation Assays 20 Unfractionated PBMC were resuspended in complete :medium (see below) and dispersed in round-bottomed 96 micro-well plates (Nunc, Denmark) at three different I cell densities (2x10 5 Ix10 5 and 5x10 4 cells per well) in I Iscove's medium supplemented with 10% fetal calf serum 25 (FCS, Biological Industries, Israel), 3 gg/ml 7 L-Glutamine (Gibco, UK) and 0.1 mg/ml Gentamycinsulfate (Essex Lakemedel AB, Sweden). Fractionated T cells (2x10 5 or 1.2x10 5 nominal T cells, or 4x10 4 CD4 T cells) were dispersed in separate sets of wells together with 4x10 4 or 2x10 4 autologous T cell-depleted irradiated (2500 rad), PBMC as a source of accessory cells.

Synthetic peptides were dissolved in dimethylsulfoxide Ag/mnl) and further diluted in culture medium.

Uncoupled peptides were added at different Additionally, polymers of one or more of the peptides are within the scope of the invention.

WO 92/21377 PCT/SE92/00373 concentrations (10, 1 and 0.1 pg/ml) to the culture wells.

Concanavalin A (Sigma) (10 pg/ml) was added to separate cultures as a positive control. Cells, in a final volume of 0.2 ml, were incubated for five days at 37°C in a humid atmosphere with 7.5% CO 2 After four days, 25 ul of culture supernatant were collected from each well and frozen at until assayed for IL-2 activity according to the method described in example 5. 16 hours prior to the completion of the culture period, 20 pl culture medium containing 1 pCi of 3 H]thymidine (Amersham, UK) were added to each well. The harvesting and subsequent measurement of incorporated radioactivity was performed on an automated filter cell harvester coupled to an argon activated B-scintillation counter (Inotech, Switzerland). Data were expressed as arithmetic mean stimulating indexes the latter SI being defined as the mean ratio of [H]-thymidine incorporated in peptide stimulated cultures (mean from three cultures divided by corresponding triplicate of control cultures (unstimulated). A mean SI of at least 2.4 is considered positive. SI values equal to at least 2.4 twice the Ssum of the mean plus 3.3 times the SD of the replicate cultures exposed to irrelevant peptides (confidence interval, p 0.001, Student's test)] were considered as significantly increased.

I IAs seen i Figure 1, a substantial number (18/40) of peptides that were injected into monkeys in an OVA-substituted form, induced in vitro proliferation of PBMC from corresponding immune animals. In Figure 1, results are expressed as mean SI SD of all triplicates tested SD (if tested on two monkeys). Black bars indicate a positive result. The frequency of responding monkey(s) is indicated. Four major areas corresponding to the additive sequence of 2 to 3 overlapping peptides were found to accommodate this activity. Peptides gpl20-11, gpl20-12 and gpl20-13 SUBSTITUTE SHEET acid residues either carboxy or amino terminally, 2 I 1 .WO 92/21377 PCT/SE92/00373 31 (amino acid ccordinates 141-192) correspond to one such area. Five out of six monkeys immunized with one of these three peptides responded to recall peptide. Another major area comprises peptides gpl20-23, gpl20-24 and gp120-25 (amino acid coordinates 295-343) which induced proliferative responses of PBMC from at least one out of 2 monkeys immunized with the corresponding peptide. A third area, comprising peptides gpl20-29 and gpl20-30, accommodates a site(2) of proliferation inducing activity on PBMC from monkeys immunized with the corresponding OVA-conjugated paptides. The fourth area consists of peptides gpl20-33, gpl20-34, 35 and gpl20-36 (amino acid coordinates 409-466) where each peptide could induce profilerationof PBMC from at least one of the immunized monkeys.

Apart from these major areas, five peptides, i.e.

peptides gpl20-4 (amino acid coordinates 53-74), gp 120-5 (amino acid coordinates 64-89), gpl20-17 (amino acid coordinates 218-247) and gpl20-21 (amino acid coordinates 269-295), were shown to induce in vitro profilerative responses when added to PBMC from monkeys immunized with the corresponding OVA-conjugated peptide.

Peptides found to be capable of inducing a proliferative response of PBMC from monkeys immunized with the corresponding OVA-coupled peptide were reassayed on PBMC from at least three other monkeys immunized with a non-cognate OVA- Scoupled peptide. Peptides gpl20-4, gp120-13 and gp120-34 induced proliferation of PBMC from 1 out of 3 monkeys and peptide gpl20-30 in 1 monkey out of 7 (SI ranging between and 2.5) while the other peptides failed to induce any significant proliferative responses.

Peptides capable of inducing a proliferative response in one or two immunized monkeys were retested after the third immunization. On this occastion, the in vitro proliferative responses of PBMC from immune SUBSTITUTE

SHEET

i ~i~u~ WO 92/21377 PCT/SE92/00373 32 monkeys to each of 2 peptides containing a sequence half overlapping with the immunizing peptide were also evaluated.

As seen in Figure 2, PBMC from both monkeys immunized with OVA-conjugated peptide gpl20-11 responded also in vitro to peptide gp120-12, but none of the peptide gpl20-12-immunized monkeys responded to peptide gpl20-11. Cells were obtained two weeks after the third immunization. The peptides tested were selected on the basis of in vitro responsiveness to the immunizing peptide after two immunizations. In Figure 2, results are expressed as mean SI of all triplicates tested SD (if tested on two monkeys). Black bars indicate a positive result.

Similarly, monkeys immunized with peptide gp120-12 responded to peptide gp120-13 but none of the peptide 13 immunized monkeys responded to peptide gp120-12. In the next area of in vitro profilerative activiti., i.e. peptides gpl20-23, gp120-24 and gp120-25, none the overlapping peptides induced in vitro profiler .i of PBMC from any of the monkeys immunized with OVA-conjugated peptides. The same holds true for PBMC from monkeys immunized with peptides gpl20-29 and gp120-30 (OVA-conjugated) as no response to overlapping peptides is achieved. In the area consisting of peptides gp120-33, gp120-34, and gp120-35, none of the overlapping peptides induced in vitro proliferation of PBMC from any of the monkeys immunized with OVA-conjugated peptides. PBMC from the other monkeys were also negative in this respect. Of the other epitopes identified, only PBMC from the peptide gpl20-4 immunized monkey responded in vitro to an overlapping peptide, i.e. to peptide gp120-5.

SThe profilerative responses of different cell fractions from two monkeys (immunized with peptide gpl20-12 or peptide gpl20-13) were examined. As seen SUBSTITUTE

SHEET

1 n people but Lb'are not limited to homosxu include but are not limited to homosexuals, VVO 92/21377 PCT/SE92/00373 -33in Table 3, enrichment of CD2 T cells increased the proliferative response of PBMC obtained from both monkeys when cultured in the presence of immunized (but uncoupled) peptide. Also, existing responses to overlapping peptides remained relatively constant.

After a further depletion of CD8 T cells, the CD4 T cell enriched fractions (containing 9 to 18% of the original CD2 T cell fractions) still proliferated in response to incubation with immunized peptide.

However, the CD4 T cell enriched fraction from the peptide gpl20-12 immunized monkey did not proliferate in response to any of the overlapping peptides.

TABLE 3 IN VITRO PROLIFERATIVE RESPONSES TO RECALL PEPTIDES OF TOTAL PBMC AS WELL AS CD2 AND CD4 ENRICHED FRACTIONS OF CELLS OBTAINED FROM MONKEYS 5 MONTHS AFTER IMMUNIZATION WITH OVA-CONJUGATED PEPTIDES Stimulation Index (SI) of CD2 J CD4 Immunized In vitro Total Enriched Enriched Peptide Peptide PBMC Fraction Fraction gp120-12 gpl20-11 2.7 2.8 1.1 gp120-12 2.5 8.6 gpl20-13 3.9 3.6 1.4 gpl20-13 gp120-12 1.0 0.4 0.8 gpl20-13 2.5 2.9 5.6 qpl20-14 L 2.1 2.4 2.4 In Table 3, the various columns were obtained as follows. Immunized peptide: 100 Ag of OVA-conjugated peptide was immunized at three occasions in Freund's WO 92/21377 PCT/SE92/00373 -34complete (1st dose) or incomplete (boosting doses) adjuvant; In vitro peptide: unconjugated peptide; Total PBMC: mean SI of four triplicates of different cell densities and peptide concentrations; CD2 enriched fraction: 2x10 5 SRBC-rosetted PBMC incubated with 4x10 4 irradiated, non-rosetted cells together with 10g/ml of peptide(s): CD4 enriched fraction: 1.25x10 5 (peptide gpl20-12 immunized monkey) or 4x10 4 (peptide gpl20-13 immunized monkey) SRBC-rosetted PBMC further enriched in CD4 T cells by incubating with anti-CD8+ coated beads were incubated with 2x10 4 irradiated, non-rosetted cells together with 10 Ag/ml of peptide(s).

Example 7 IL-2 assay The IL-2 content of individual cell microcultures was determined by the bioassay performed as described by Gillis et al., "T Cell Growth Factor: Parameters of Production and a Quantitative Microassay for Activity", J. Immunol., 120:2027 (1978). Briefly, supernatants were added at a final dilution of 1:4 to 104 CTLL-2 cells. Cells were incubated for 24 hours at 37 0 C in flat-bottomed 96 microwell plates (Nunc, Denmark) in Iscove's medium supplemented with 10% FCS, 3 gg/ml L-Glutamine, 0.1 mg/ml Gentamycinsulfate and 5x10 5

M

0-Mercaptoethanol. Six hours prior to completion of the culture period, 1 gCi of [3H]-thymidine was added.

The cells were harvested and 3 H]-thymidine incorporation was determined as described in Example 6.

IL-2 content in the supernatants was determined by extrapolation from a standard dose-response curve generated by culturing CTLL-2 cells in the presence of i resin as SOiJat pna rjL.u.. J.II.. ukuV=vr= -tui ~1 WO 92/21377 PCT/SE92/00373 known amounts of recombinant human IL-2 (Genzyme, Boston, MA).

As seen in Table 4, several cell culture supernatants contained detectable amounts of IL-2 in cultures of PBMC from monkeys immunized with OVA-conjugated peptides gpl20-11, gpl20-12, gpl20-13, gpl20-16, gpl20-21, gpl20-25, gpl20-30 and gpl20-34, secreted IL-2 could be detected after in vitro challenge with the corresponding, unconjugated, peptide. The ratio of secreted IL-2 found after 4 days of in vitro culturing ranged from 0.2 to 1.0 U/ml.

Cell culture supernatants of PBMC derived from monkeys immunized with peptides gpl20-11, gpl20-12, gpl20-13, gpl20-30 and gpl20-34 also contained IL-2 after in vitro exposure to one or two of the overlapping peptides. Accordingly, PBMC from a monkey immunized with peptide gpl20-11 secreted detectable levels of IL-2 in the cell supernatant after 4 days of stimulation with peptide gpl20-12, and a monkey immunized with peptide gpl20-12 secreted detectable levels of IL-2 after stimulation with peptide gpl20-13.

Cell culture supernatants containing IL-2 were identified from both PBMC cultures containing overlapping peptides (peptides gp120-12 and gpl20-14) together with PBMC from a peptide gpl20-13 immunized monkey and the same holds true for peptides gpl20-33 and gpl20-35 when co-cultured with PBMC from a peptide gpl20-34 immunized monkey. Finally, PBMC obtained from a peptide gp120-30 immunized monkey secreted detectable amounts of IL-2 not only when cultured in the presence Sof peptide gpl20-30, but also when peptide gpl20-29 had been added to the cultures.

chain protected amino acids used were from Nova Biochem I II imiuulimi WO 92/21377 PCI'/SE92/00373.

-36- TABLE 4 IL-2 CONTENT IN SUPERNATANTS OF PBMC FROM MONKEYS IMMUNIZED WITH OVA-CONJUGATED PEPTIDES AFTER IN VITRO EXPOSURE TO UNCONJUGATED, RECALL PEPTIDE(S).

Immunized IIn vitro 1IL-2 Content in Culture Peptidea I Peptide b 1.Supernatants (U/ml)c gpl2O-11 gp120-l1 0.28 gpl2O-12 0.26 gpl2O-12 gpl2O-12 0.28 gpl2O-13 0.22 gpl2O-13 gpl2O-12 0.36 gpl2O-13 1.01 gpl2O-14 0.61 gpl2O-16 gpl2O-16 0.22 gpl2O-21 gpl2O-21 0.37 gp12O-22 gpl2O-3 0.20 gpl2O-24 gpl2O-25 0.21 gpl2O-25 gp120-25 0.2 gp12O-30 gpl2O-29 0.23 gpl2O-30 0.20 gp12Q-33 gp12O-34 0.53 gpl2O-34 gpl2O-33 0.20 gp120-34 10.27 gp120-35 0.25 OVA-conjugated peptides.

b unconjugated peptides C IL-2 content of the highest triplicate from peptide-stimulated PBMC in vitro.

I

arlea unaer a stream or N 2 I 1 1 1 .WO 92/21377 PCT/SE92/00373 -37- Example 8 Cells and Virus Stocks All neutralization tests were performed using H-9 cells and HTLV-111B virus (originating from R.C. Gallo and supplied by Dr. William Hall, North Shore Hospital, Manhasset, New York). H-9 cells (designated H9 NY) were maintained in RPMI Medium (Gibco) supplemented with 20% fetal calf serum (FCS), penicillin/streptomycin (PEN/STREP 50 Ag/ml each and without any fungicides). Cells were subcultured at a dilution of 1:3 every 4 days.

Cells were scraped from the plates and pelleted by centrifugation at 325 x g. Pelleted cells were resuspended in 1 ml of stock virus previously diluted 1/10 and allowed to adsorb for 60 min at 37 0 C with frequent stirring. After adsorption of the virus, the cells were recentrifuged and resuspended in 10 ml of RPMI with 20% FCS and polybrene (2 Ag/ml) (giving a final concentration of 5x10 5 cells/ml) and incubated at 37 0 C in 5% CO 2 Infected cells were shown to be detectable at days post-infection by monitoring syncytia formation, positive cells in immunofluorescence and p-24 production (assayed by the Abbott p-24 antigen test). The peak of HIV production was seen 10 days p.i. at which time virus was collected. After low speed centrifugation to remove debris, supernatants containing virus collected from infected cells were frozen in stocks at -90 0 C. One virus stock with endpoint titer of 40,000 50% tissue culture infective doses (TCID 50 was used throughout the studies (referred to as NT3-NT19).

i.

WO 92/21377 PCT/SE92/00373 -38- Example 9 HIV-1 Neutralization Assay Sera containing antibodies that neutralize HTLV 111-B infectivity were detected by their ability to prevent HIV-1 syncytium formation, p-24 antigen production and decreased number of infected cells as determined by immunofluorescence markers, compared to control infections lacking peptide specific antisera.

Stock virus, described in Example 8 was diluted to 100

TCID

50 and mixed with serial fourfold dilutions F 1/20, and 1/80) of complement-inactivated immunesera obtained from the monkeys immunized as described in Example 4. As a positive control, a guinea pig hyperimmune serum (referred to as MSV) with known HIV neutralizing titer of 1/40 1/160 was included in all experiments (kindly provided by Prof. B. Morein, Dept.

Veterinary Virology, BMC, Uppsala, Sweden). After incubation for 60 min at 37 0 C or 16 hours at 4 0 C, the serum-virus mixture was added to 1x1l 6 3 cells and incubated for another 60 min at 37 0 C. Following incubation, the cells were washed once and placed in 24 well multidish plates with 2 ml of growth medium (RPMI, FCS, 2 Lg polybrene/ml) per well.

Cells were examined under the microscope (magnification x200) for the presence of syncytia on days 12 p.i. Supernatants from infected cells were assayed for the presence of p-24 antigen according to the manufacturer's instructions (Abbott ag test HIVAG-1®, Enzyme Immunoassay for the Detection of Human Immunodeficiency Virus Type I (HIV-1) Antigen(s) in Human Serum or Plasma) in tenfold serial dilutions (1/10 1/1,000) at 10 days p.i. The results are presented as absorbance values at 454 nm with higher absorbance values indicating higher P-24 antigen concentration and hence HIV infection. Serial dilutions of the supernatants were made so as to detect -I 1L~I SWO 92/21377 PCT/SE92/00373 -39p-24 concentrations in the most accurate range absorbance units).

The number of infected cells were determined at the end of experiment (usually on day 15 by acetone- fixation of cells on slides adopted for immunofluorescence An indirect IF test was used according to standard procedures described in Jeansson et al., "Elimination of Mycoplasmas from Cell Cultures Utilizing Hyperimmune Sera", Ex. Cell Res., 161:181-188 (1985), with 1/400 dilution hyperimmune sera from HIV-infected individuals and a fluorescein isothiocyanate (FITC) labeled antihuman IgG antibody (Bio-Merieux France) diluted 1/100. Tables 5-8 show the results obtained from screening of hyperimmune sera from monkeys immunized with peptides 1-40.

In Tables 5(A-D)-8 the p24 antigen content of the supernatants was analyzed by ELISA, indirect IF and syncytia formation as described above. The relative amount of antigen positive cells is depicted as AG POS cells wherein the percentages are represented by: 3-10% and 11-20% where the percentage interval indicates the number of antigen positive cells.

Table 5A (HIVNT3P1.XLS) depicts the results obtained with sera derived from monkeys immunized with peptides gpl20-1 gpl20-10. The cells used were H9 NY and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol was (virus plus serum) incubation at 37 0 C for one hour.

Table 5B (HIVNT4P1.XLS) depicts the results Sobtained with sera derived from monkeys immunized with peptides gpl20-11 gpl20-20. The cells used were H9 NY and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol was (virus plus serum) incubation at 37 0 C for one hour.

-vs WO 92/21377 PCU/SE92/00373 Table 5C (HIVNT5P1.XLS) depicts the results obtained with sera derived from monkeys immunized with peptides gpl20-21 gpl20-30. The cells used were H9 NY, and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol used was virus plus serum incubated at 37 0 C for one hour.

Table 5D (HIVNT6Pl.XLS) depicts the results obtained with sera derived from monkeys immunized with peptides gpl20-31 gp120-40. The cells used were H9 NY and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol was (virus plus serum) incubation at 370C for one hour.

Table 6 (HIVTAB4.XLS) shows the results of the first retest of putative neutralizing antibodies as determined by the first test (Tables 5A-D). In each test the virus used was HTLV-IIIB, Batch 18 and the cells used were H9 NY. The first retest results in rows 1-19 are the results of neutralization test number The incubation protocol was incubation at 37 0 C for one hour. The first retest results in rows 20-32 are the results of neutralization test number 7. The incubation protocol was incubation of at 37 0 C for one hour.

Table 7 (HIVTAB5.XLS) shows second, third and fourth retest results of the positive peptides. In each test the virus used was HTLV-IIIB Batch 18 and the cells used were H9 NY. The second retest results in rows 1-4 are the results of neutralization test number 7. The incubation protocol was incubation at 37 0 C for one hour. The second retest results in rows 5-13 are the results of neutralization test number 12. The third retest results are shown rows 14-16 are the results of neutralization test number 12. Th.

incubation protocol was incubation at 37 0 C for one hour. The fourth retest results in rows 17-39 are the results of neutralization test number 16. The Imum WO 92/21377 PCT/SE92/00373 -41incubation protocol was at 4 0 C for 16 hours. The second retest results in rows 40-53 are the result of neutralization test 19. The incubation protocol was cells plus virus at 40 for 16 hours.

Table 8 (HIVKOMBP.XLS) shows the neutralization assay results with combined hyperimmune sera. Note that the incubation of virus and cells was at 4 0 C for 16 hours.

The results depicted in Tables 5(A-D)-8 indicate that peptides gpl20-12, gpl20-16, and gpl20-19 elicit the production of HIV neutralizing antibodies in primate subjects. The use of the peptides in vaccination of human subjects is therefore applicable to prevent infection by HIV or to induce heightened immune response in subjects already infected by HIV.

i i{ __-TABLE 5A ASSAYS OF ANTISERA TO PEPTIDES gpl120-1 gpl120-10O- I PEPTIDE serum P-4ATGN(uentn I)RELATIVE AMOUNT jDilution 1/100 ]F 1/1000 OF AG P05 CELLS 1. Pos control 2.0 1.176 0.158..

2. Pos control 2.0 1.194 0.177..

3. Pos control 2.0 2.0 0.464 4. Neg control 0.056 guinea pig 1/10 0.178 0.066 0.063 6. Pos control 1/40 0.71 0.118 0.06 7. Antiserum 1/160 2.0 0.742 0.11 8. 1/320 2.0 0.484 0.093 9. preimmune ND ND ND ND gpl2O-1 /5 0.715 0.108 0.054 11. 1/20 2.0 0.36 0.073 12. 1/80 2.0 0.57 0.093 13. preimmune 2.0 0.437 0.081 14. gp12O-2 1/5 2.0 0.86 0.138 1/20 2.0 0.486 0.093 +4+ 16. 1/80 2.0 0.257 0.083 17. preimmune 2.0 0.466 0.09 18. gpl2O-3 1/5 2.0 0.367 0.079 19 1/20 2.0 0.512 0.094 1/80 2.0 0.724 0.113 Ij;j> ____-TABLE 5A ASSAYS OF ANTISERA TO PEPTIDES gpl2O-1 gp12O-1O PEPID sru P-24 ANTIGEN (Supernatant DIL) REAIE1ON Dilution 1/10 j 1/100 J 1/1000 OF AG P05 CELLS 21. preiminune 2.0 0.536 0.094 22. gp12O-4 1/5 2.0 0.638 0.092 23. 1/20 2.0 0.448 0.082 24. 1/80 2.0 0.592 0.097 preimmune 2.0 0.43 0.082 26. gpl2O-5 1/5 2.0 0.638 0.098 27. 1/20 2.0 0.737 0.11 28. 1/80 2.0 0.786 0.119..

29. preiminune 2.0 0.822 0.125 gpl2O-6 1/5 2.0 0.716 0.131..

31. 1/20 2.0 0.977 0.119 32. 1/80 2.0 0.861 0.124 33. preimmune 2.0 0.719 0.116 34. gpl2O-7 1/5 2.0 0.587 0.106 1/20 2.0 0.45 0.092 36. 1/80 2.0 0.756 0.117 37. prejinmune 2.0 0.507 0.096 38. gpl2O-8 1/5 2.0 0.555 0.098 3.1/20 2.0 0.59 0.103 TABLE 5A ASSAYS OF ANTISERA TO PEPTIDES gpl 20-1 gpl 20-10 PEPTID serumP-24 ANTIGEN (supernatant DIL) 1 EAIEAON Dilution 3110 1/100 J 1/1000 JOF AG P05 CELLS 1/80 2.0 0.308 0.081 41. preiminune 2.0 0.322 0.07q6 42. gp12O-9 1/5 2.0 0.358 0.09 43. 1/20 2.0 0.403 0.082 44. 1/80 2.0 0.612 0.102 preinunune 2.0 0.747 0.127 46. gp120-10 1/5 2.0 0.3 0.074 47. 1/20 2.0 0.426 0.092 48. 1/80 2.0 0.442 0.083 TABLE 5B ASSAYS OF ANTISERA TO PEPTIDES gpl2O-1 1 gpl2O-20 PEPTIDE Serum P-24 ANTIGEN (Supernatant DIL) IREAVEAON Dilution 1/10 TJ 1/100o 1/100 OF AG P05 CELLS 1. preimmuie 1/5 2.0 0.882 0.149 2. gPl2O-11 1/5 2.0 0.73 0.135 3. 1/20 2.0 1.73 0.299 4. 1/80 2.0 0.700 0.148 preimmune 1/5 2.0 1.07 0.151 6. gpl2O-12 1/5 0.157 0.07 0.076 7. 1/20 2.0 1.45 0.22 8. 1/80 2.0 1.37 0.221 9. preiminune 1/5 2.0 0.58 0.107 gpl2O-13 1/5 2.0 1.16 0.194 11. 1/20 1.816 0.37 0.095 12. 1/80 2.0 1.16 0.187 13. preimmune 1/5 2.0 2.0 0.281 14. gp120-14 1/5 2.0 0.81 0.142 1/20 2.0 1.39 0.219 16. 1/80 2.0 0.83 0.156 17. preimmune 1/5 2.0 1.13 0.192 18. gpl2O-15 1/5 2.0 1.43 0.243 w mio

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TABLE 5B ASSAYS OF ANTISERA TO PEPTIDES gp120-11 gp120-20 PEPTIDE Serum P-24 ANTIGEN (Supernatant DIL) RELATIVE AMOUNT Dilution 1/10 1/100 1/1000 OF AG POS CELLS 19. 1/20 0.069 0.05 0.05 1/80 2.0 0.57 0.104 21. preimmune 1/5 2.0 1.78 0.303 22. gp120-16 1/5 0.26 0.07 0.056 23. 1/20 0.067 0.06 0.054 24. 1/80 2.0 0.74 0.132 preimmune 1/5 2.0 1.13 0.171 26. gpl20-17 1/5 2.0 0.76 0.161 27. 1/20 2.0 1.56 0.285 28. 1/80 2.0 0.7 0.129 29. preimmune 1/5 2.0 1.41 0.177 gpl20-18 1/5 2.0 2.0 0.339 31. 1/20 2.0 1.36 0.218 32. 1/80 2.0 1.26 0.199 33. preimmune 1/5 2.0 0.39 0.097 34. qpl20-19 1/5 0.476 0.1 0.061 1/20 1.048 0.18 0.068 36. 1/80 2.0 1.62 0.303 37. preimmune 1/5 2.0 1.11 0.189 tABLE 5B ASSAYS OF ANTISERA TO PEPTIDES gpl20-11 gpl 20-20 PEPTID serumP-24 ANTIGEN (Supernatant DIL) REAIEAON Dilution 1/10 1/100 1/1000 JOF AG P08 CELLS 38. gpl2O-20 1/5 2.0 1.19 0.182..

39. 1/20 2.0 j 1.47 j 0.054 j++ 1/80 2.0 1.42 J 0.264 B, TABLE 5C ASSAY OF ANTISERA TO PEPTIDES 21-30 1 1 J J NO. OF PEsIeErium~ P-24 ANTIGEN (Supernatant DIL) RELATIVE SYNCYTIA/WELL_ /10 /100 OF AG Day S 1 Day7 I /0110 110 P00 CELLS1 41. 1pos control 2.0 0.65 0.09 12 72 42. pos control 1.85 0.24 0.061 6 27 43. neg control 0.4 0 0- 44. guinea pig 1/10 0.5 0.04 0.047 -0 0 pos control 1/40 0.05 0.04 0.04 -1 0 46. antiserum 1/160 0.04 0.05 0.043 1 3 47. 1/640 1.07 0.14 0.056 2 19 48. preimmune 1/5_ 2.0 1.57 0.275 12 49. gpl2O-21 1/5 2.0 0.4 0.075 3 28 1/20 1 0.17 0.059 5 21 51. 1/80 2.0 0.48 0.089 7 72 52. preimmufle 1/5 2.0 1.1 0.182 3 ND 53. gpl2O-22 1/5 2.0 1.48 0.221 2 54. 1/20 2.0 J 1.07 0.16 T0 0 tJ

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j 4 TABLE 5C ASSAY OF ANTISERA TO PEPTIDES 21-30 NO. OF T Serum P-24 AlTIGEN (Supernatant DIL) RELATIVE SYNCYTIA/WELL PEPTIDE Dilution AMOUNT 1/10 1/100 1/1000 OF AG Day 5 Day 7 POS CELLS 1/80 2.0 0.63 0.087 5 56. preimmune 1/5 2.0 0.4 0.083 4 52 57. gpl20-23 1/5 1.97 0.26 0.067 ND 0 58. 1/20 2.0 1.63 0.236 5 98 59. 1/80 2.0 0.35 0.084 5 >150 preimmune 1/5 2.0 2.0 0.355 2 49 61. gpl20-24 1/5 1.95 0.29 0.067 0 3 62. 1/20 2.0 0.37 0.081 5 34 63. 1/80 1.87 0.24 0.069 3 48 64. preimmune 1/5 2.0 0.83 0.145 0 91 gpl20-25 1/5 2.0 0.73 0.11 1 66. 1/20 1.63 0.23 0.062 0 67. 1/80 1.88 0.22 0.064 0 38 68. preimmune 1/5 2.0 0.48 0.089 0 79 69. gpl20-26 1/5 2.0 0.62 0.101 3 91 1/20 2.0 0.34 0.063 3 TABLE 5C ASSAY OF ANTISERA TO PEPTIDES 21-30 RELATIVENO.

OF

serum P-24 ANTIGEN (Supernatant DIL) REAIE SYNCYTIA/ WELL PEPTIDE Dilution I AMOUNT 1/10 1/100 1/1000 OF AG Day 5 Day 7 IF 71. gpl2O-26 1/80 1.27 0.19 0.061 0 21 72. preimlnune 1/5 2.0 0.66 0.11 2 52 73. gp120-27 1/5 2.0 0.58 0.098 1 26 74. 1/20 2.0 0.65 0.099 6 49 1/80 2.0 0.3 0.062 2 76. preimmune 1/5 2.0 2.0 0.317 7 31 77. gpl2O-28 1/5 2.0 0.39 0.078 2 22 78. 1/20 2.0 0.68 0.105 5 79. 1/80 0.99 0.15 0.05 3 >150 preimmune 1/5 2.0 1.29 0.187 5 97 81. gpl2O-29 1/5 2.0 0.55 0.096 3 112 82. 1/20 2.0 0.85 0.135 3 >150 83. 1/80 2.0 0.72 0.113 0 29 84. preimmune 1/5 2.0 2.0 0.326 10 130 gpl2O-30 1/5 2.0 0.27 0.073 3 38 86. 1/20 2.0 1.71 0.24 9 52 87. 1/80 2.0 0.44 0.082 6 ND I j2 TABLE 5D ASSAYS OF ANTISERA TO PEPTIDES 31-40 P-24ANTGEN Suprnatnt IL)NO.

OF

PPIEserum p-4ATGN(uentn I)RELATIVE SYNCYTIA/WELL PETIE Dilution AMOUNT 1/10 I 1/100 1/1000 OF AGDa 6 jP05 CELLSDa 6 r8. pos control 0.976 0.258 0.123 6 89. pos control 1.836 0.656 0.185 11 neg control 91. guinea pig 1/10 0.103 0.088 0.09 0 92. pos control 1/40 0.104 0.087 0.093 0 93. antiserum 1/160 0.749 0.29 0.1 4 94. 1/640 1.066 0.238 0.237 preimmune 1/5 0.824 96. gpl2O-31 1/5 1.769 0.675 0.186 471 97. 1/20 1 1.124 0.302 0.111 22 98. 1/80 C.978 0.258 ND 24 99. preimmune 1/5 0.883 100. gp120-32 1/5 1.163 0.258 ND 7 101. 1/20 1.482 0.311 ND 8 102. 1/80 0.996 0.263 ND- 0 103. lpreimmune 1/5 1.76 1 M- I TABLE 5D ASSAYS OF ANTISERA TO PEPTIDES 31-40 P-24ANTGEN Suprnatnt IL)NO.

OF

PPIE serum P2 iTGN SuenttDL)RELATIVE SYNCYTIA/ WELL PETIE Dilution

AMOUNT

1/10 1101/1000 OF AG Day 6 1 1,100 OB CELLS 104. gpl2O-33 1/5 0.84 0.239 0.156 105. 1/20 1.282 0.333 0.144 106. 1/80 0.76 0.207 ND 17 107. preimmune 1/5 108. gpl2O-34 1/5 0.293 0.134 0.12 18 109. 1/20 1.446 0.391 0.148 17 110. 1/80 0.42 0.15 ill. preimmune 1/5 ND 112. gpl2O-35 1/5 1.485 0.52 0.142 113. 1/20 1.778 0.873 0.194 26 114. 1/80 1,475 0.196 ND 115. preimmune 1/5 1.076 116. gp12O-36 1/5 0.957 0.26 0.149 28 117. 1/20 1.44 0.448 0.119 16 118. 1/80 1.148 0.486 ND 119. preimmune 1/5 1.563 120. Tgpl2O-37 1/5-T0.666 j 0.155 0.098 0 t'J In 0 0

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5D ASSAYS OF ANTISERA TO PEPTIDES 31-40 P-24ANTGEN suprnatnt IL)NO.

OF

PEPTIDE DiSerum P-4ATGN(uentn I)RELATIVE SYNCYTIA/ WELL Dlution AMOUNT 1/10 I 1/100 1/1000 OF AG Day 6 I P O S C E L L S 121. 1/20 1.143 0.33 0.129 12 122. 1/80 1.362 0.33 ND 123. preinunune 1/5 1.364 124. gpl2O-38 1/5 1.386 0.59 0.114 11 125. 1/20 0.576 0.214 0.106 17 126. 1/80 1.23 0.329 ND 127. prejimmune 1/5 1.854 128. gpl2O-39 1/5 1.376 0.495 0.182 129. 1/20 0.711 0.296 0.118 130. 1/80 0.929 0.237 131. preimmune, 1/5 ND 132. gpl2O-40 0.862_0.255 0.132 13 133. 1/20 0.989 0.273 0.143 134. 1/80 0.477 0.164

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TABLE 6 RETESTING OF HYPERIMMUNE SERA WITH THE CAPACITY TO NEUTRALIZE HIV NO. OF Serum P-24 ANTIGEN (DIL) RELATIVE SYNCYTIA/WELL PEPTIDE Dilution AMOUNT OF

AG

1/10 1/100 1/1000 POS CELLS Day 5 Day 7 |Frt Re First Retest 1. pos control 2.0 0.646 0.09 12 72 2. pos control ,1.853 0.244 0.061 6 27 3. neg control 0.039 0 0 4. guinea pig 1/10 0.051 0.04 0.047 0 0 pos control 1/40 0.052 0.042 0.04 1 0 6. antiserum 1/160 0.042 0.046 0.043 1 3 7. 1/640 1.067 0.144 0.056 2 19 8. preimmune 1/5 2 1.326 0.172 10 112 9. gpl20-12 1/5 1.083 0.153 0.06 1 24 1/20 2 1.487 0.171 7 175 11. 1/80 2 0.463 0.07 ND 12. preimmune 1/5 2 1.991 0.237 2 64 13. gpl20-16 1/5 2 0.355 0.07 0 13 14. 1/20 0.741 0.103 0.048 11 1/80 2 0.32 0.08 0

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ABLE 6 RETESTING OF HYPERIMMUNE SERA WITH THE CAPA!T Y TO NEUTRALIZE HIV NO. OF PPIE Serum P-24 ANTIGEN (DIL) RELATIVE BYNCYTIA/ WELL PETIE Dilution MOUNT 1/10 1 /10L 1/1000 OF AG Da 1 ay7 16. preimmxune 1/5 2.0 0.547 0.082 3 42 17. gpl2O-19 1/5 0.141 0.062 0.053 0 6 18. 1/20 1.134 0.164 0.054 0 26 19.- 1/80 2.0 0.455 0.081 1 1 First Retest 1/5 1/50 1/500 7 Day pos control 1.175 0.426 0.201 9 46 21. pos control 1.529 0.401 0.161 32 167 22. neg control 23. guinea pig 1/10 0.139 0.165 0.145 -0 0 24. pos control 1/40 0.211 0.159 0.168 -1 0 antiserum 1/160 0.961 0.299 0.163 9 26 26. 1/640 0.989 0.26 0.159 5 27. gpl2O-24 1/5 1.067 0.245 0.166 4 34 28. 1/20 0.795 0.204 0.167 5 41 29. 1/80 0.433 0.167 -15 s0 gpl2O-25 1/5 1.237 0.282 0.155 19 144 ""taw TABLE 6 RETESTING OF HYPERIMMUNE SERA WITH THE CAPACITY TO NEUTRALIZE HIV NO. or SETD erum P-24 ANTIGEN (DIL) RELATIVE BYNCYTIA/ WELL PPIE Dilution AMOUNT 1/10 1/100 1/1000 POF AGL Dy5 a 31.- 1/20 f 1.312 1 0.373 0.187 I42 116 32. 1/80 ND j ND J ND JND ND ~fl- ~TABLE 7 RETESTING OF HYPERIMMUNE SERA WITH CAPACITY TO NEUTRALIZE HTLV-111 P-24 ANITIGEN (Supernatant DIL) NO. OF PPIE SERUM *RELATIVE SYNCYTIA/ WELL PETIE DILUTION iAMOUNT I 1/50 1/500 OF AG Day 5 Dy7 1P08 CELLS SBecond Retest 1. Igpl2O-16 1/5 ND ND ND ND ND 2. 1/5 1.924 1.062 0.282 3. 1/20 0.365 0.172 0.145 -2 4. 1/80 0.163 0.133 -0 0 Second Retest 1/10 1/100 1/1,000 pos control 2.0 2.0 1.026 320 6. pos control 2.0 2.0 0.639 220 7. pos control 2.0 2.0 0.866 290 1 8. pos control 2.0 2.0 0.881 neg control 11. gpl2O-24 1/5 2.0 2.0 0.545 112 12. 1/20 2.0 2.0 0.819 138 13. 1/80 2.0 2.0 230 Third Retest j~j~ III I TABLE 7 RETESTING OF HYPERIMMUNE SERA WITH CAPACITY TO NEUTRALIZE HTLV-111 P-24 ANTIGEN (Supernatant DIL) NO. OF PETIE SERUM *RELATIVE BYNCYTIA/ WELL PPIE DILUTION _AMOUNT 1/50 1/500 OF AG Day 5i Day?7 P05 CELLS 14. Jgp120-16 1/5 0.122 0.1 j 0.115 0 j1/20 2.0 1.14 0.352 0 16. I1/80 2.0 2. 210 Fourth Retest__ 17. pos control 1.425 0.732 0.154 16 18. pos control 1.346 0.672 0.152 16 19. pos control 1.431 0.845 0.182 17 pos control 1.414 0.931 0.251 21. neg control 0.067 22. neg control 0.045 23. neg control 0.042 24. guinea pig 1/10 0.044 0.037 0.029 0 pos control 1/40 0.063 0.039 0.029 0 26. antiserum 1/160 0.036 0.035 0.055 0 27. 1/640 0.556 0.072 0.034 1 28. gp120-12 1/8 0.072 0.043 0.046 0 __-TABLE 7 RETESTING OF HYPERIMMUNE SERA WITH CAPACITY TO NEUTRALIZE HTLV-1I1 P-24 ANTIGEN (Supernatant DIL) NO. OF SERUM *RELATIVE BYNCYTIA/ WELL PEPTIDE DILUTION -AMOUNT 1/51 1/50 1/500 OF AG Day 5 Day 7 29. 1 0. 4 PO E CELLS 2.1/32 0.169 0.054 0 1/128 2.0 1.124 0.241 19 31. gpl2O-16 1/8 0.043 0.045 0.049 0 32. 1/32 0.052 0.043 0.048 0 3.1/128 1.54 0.903 0.014 4 34. gpl2O-19 1/8 0.105 0.043 0.042 0 1/32 0.358 0.08 0.045 36. 1/128 2.0 0.944 0.205 37. gpl2O-24 1/8 2.0 0.885 0.155 2 38. 1/32 2.0 1.174 0.293 15 39. 1/128 1.158 0.858 0.213 11 Second Retest 1/5 1/50 1/500 Day S Day 7 pos control 0.916 0.166 0.099 74 41. pos control 1.607 0.469 0.151 130 42. pos control 2.0 0.943 0.203 123 43. 1pos control 1.445 0.319 0.082 195 b, __-TABLE 7 RETESTING OF HYPERIMMUNE SERA WITH CAPACITY TO NEUTRALIZE HTLV-111 P-24 ANTIGEN (Supernatant DIL) NO. OF PEPTIDE SERUM *RELATIVE BYNCYTIA/ WELL DILUTION AM~OUNT 1/5 I 1/50 1/500 OF AG Day 5 Day 7 POS CELLS 44. neg control 0.145 neg control 0.328 46. guinea pig 1/10 0.09 0.111 0.075 0 147 pos control 1/140 0.096 0.082 0.078 48. antiserum 1/160 0.094 0.109 0.091 0 49. 1/640 0.996 0.212 0.104 1/5 2.0 0.444 0.162 0.155 0.094 0.111 ND 52. 1/20 J 0.152 0.109 0.158 53. 1/80 j 0.176 j 0.13 0.207 0 .w I i- TABLE 8 COMBINED NEUTRALIZATION EFFECTS OF SERA FROM MONKEYS P-24 ANTIGEN (Supernatant DIL) NO. OF Serum NT TITRE RELATIVE BYNCYTIA/WELL PEPTIDE Dilution OF SERUM AMOUNT 1/50 1/500 OF AG Day 6 POS CELLS i. Pos control 1.4 0.7 0.154 16 2. Pos control 1.3 0.7 0.152 16 3. Pos control. 1.4 0.8 0.182 17 4. Pos control 1.4 0.9 0.251 neg control 0.1 6. neg control 0 7. neg control 0 8. guinea pig 1/10 0 0 0.029 0 9. pos control 1/40 0.1 0 0.029 0 antiserum 1/160 0 0 0.055 160 0 11. 1/640 0.6 0.1 0.034 1 12. Group I 1/8 0 0 0.038 1 13. gpl20.mix 1/32 0 0 0.041 0 14. 12+16+19+24 1/128 0.2 0.1 0.043 128 0 •0 Group II 1/8 0.1 0 0.046 0 16. gpl2O.mix 1/32 0.1 0.1 0.046 0 TABLE 8 COMBINED NEUTRALIZATION EFFECTS OF SERA FROM MONKEYS P-24 ANTIGEN (Bupernatant:DIL) NO. oF Serum NT TITRE RELATIVE BYNCYTIA/ WELL PEPTIDE Dilution T-OF SERUM AMOUNT 11/50 I 1/500 OF AG Day 6 JPOB CELLB 17. 16+19 1/128 -0.2 0.043 128 -0 1. Group 111 1/8 0/ 0 0.051 19. gpl20.Inix 1/32 0.1 0.1 0.043 16+24 1/128 1 0.3 0.065 128 1 21. Group IV 1/8 0.2 0 0.044 22. gpl2O.xnix 1/32 0.1 0 0.045 23. 16+12 1/128 0.2 0.1 0.048 128 -0 24. gpl2O-12 1/8 0.1 0 0.046 1/32 0.2 0.1 0.047 32 0 26. 1/128 3 1.1 0.241 19 27. gp12O-16 1/8 0 0 0.049 0 28. 1/32 0.1 0 0.048 32 -0 29. 1/128 1.5 0.9 0.138 gpl2O-19 1/8 0.1 0 0.042 0 31. 1/32 0.4 0 1 0.045 32 32. 1/128 3 0.9 0.205 33. gp12O-24 1/8 3 0.9 0.155 neg 2 TABLE 8 COMBINED NEUTRALIZATION EFFECTS OF SERA FROM MONKEYS P-24 ANTIGEN (Superniatant DIL) NO. OF Serum NT TITRE RELATIVE BYNCYTIA/ WELL PEPTIDE Dilution OF SERUM AMOUNT 1/50 1 1/500 OF AGDa6 I/ 2 _0 2 P 08 CELLSDa 6 -34. 1/32 3 j 1.21 0.293 1.2 0.91 0.213__ 1

Claims (7)

1. A composition comprising an amount of a peptide sufficient to elicit T cell activation, said peptide having the amino acid sequence: X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys- Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser- Thr-Ser-Y, wherein X is either a hydrogen atom of the amino terminal NH 2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and a physiologically acceptable carrier therefor.

2. A composition comprising an amount of a peptide sufficient to elicit T cell activation, said peptide having the amino acid sequence: X-Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile- Ser-Thr-Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu- Tyr-Ala-Phe-Phe-Y, wherein X is either a hydrogen i atom of the amino terminal NH 2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group 950322, p:\oper\jmw, 195/92cL244,64 F- consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and a physiologically acceptable carrier therefor.

3. A composition comprising an amount of a peptide sufficient to elicit T cell activation, said peptide having the amino acid sequence: X-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala- Phe-Phe-Tyr-Lys-Leu-Asp-Ile-Ile-Pro-Ile-Asp- Asn-Asp-Thr-Thr-Ser-Tyr-Thr-Y, Swherein X is either a hydrogen atom of the amino terminal NH 2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine S'residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and a physiologically acceptable carrier therefor. 950322,p:\opcr\jmw, 19065/92cl. 2 44,65 r -66-

4. A peptide having the amino acid sequence X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys- Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser- Thr-Ser-Y wherein X is either a hydrogen atom of the amino terminal NH 2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.

A peptide having the amino acid sequence X-Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile- Ser-Thr-Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu- Tyr-Ala-Phe-Phe-Y wherein X is either a hydrogen atom of the amino terminal NH: group of said peptide or an additional amino acid selected to facilitat c.upling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.

6. A peptide having the amino acid sequence X-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala- Phe-Phe-Tyr-Lys-Leu-Asp-Ile-Ile-Pro-Ile-Asp- Asn-Asp-Thr-Thr-Ser-Tyr-Thr-Y wherein X is either a hydrogen atom of the amino terminal NH 2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a I 940901,p:\oper\jnm,19065/92cL2 44 ,66 g 7v i ^wfy a= 81 -67- Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.

7. A composition according to any one of claims 1 to 3 or a pe-tide according to any one of claims 4 to 6 substantially as hereinbefore described with reference to any one of the Examples. DATED this 22nd day of March, 1995 Syntello Vaccine Development AB by DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) 4 1 c I I 950323,p:\oper\]mw,19O65/92cL2 4 4, 6 7 i