AU2001286336A1

AU2001286336A1 - HIV peptides from Tat, Rev and Wef conserved and their application as E.G. vaccine components

Info

Publication number: AU2001286336A1
Application number: AU2001286336A
Authority: AU
Inventors: Birger Sorensen
Original assignee: Bionor Immuno AS
Current assignee: Bionor Immuno AS
Priority date: 2000-09-04
Filing date: 2001-09-03
Publication date: 2002-06-13
Anticipated expiration: 2021-09-03

Description

Title : HIV regulatory and auxiliary peptides, antigens, vaccine compositions, immunoassay kit and a method of detecting antibodies induced by HIV."

The present invention relates to novel peptides based on conserved regions of the regulatory and auxiliary HIV proteins, antigens in free or carrier-bound form comprising at least one of the said peptides, vaccine compositions containing at least one of the antigens, immunoassay kits and a method of detecting antibodies, induced by human immunodeficiency virus (HIV) or HIV-specific peptides, using such antigens.

BACKGROUND

Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS) continues to present a formidable challenge to health in developing countries. In the Western world, therapeutic strategies that target HIV-1 replication and maturation have had a prominent impact on disease progression. The high cost of current treatment, high toxicity of the drugs and lack of cure , however, means that the development of safe and effective vaccines remains paramount for control of the AIDS pandemic.

HIV-1 is a complex retrovirus encoding six regulatory and auxiliary genes not found in the simple retroviruses, namely, tat, rev, nef, vif, vprand vpu (Table 1). In eukaryotic cells, only completely spliced mRNAs are exported to the cytoplasm for translation. Unspliced or partially spliced RNAs are retained and eventually degraded in the nucleus. In this way, proteins encoded by the tat, rev and nef genes (designated Tat, Rev and Nef) derived from multiply spliced RNA species, are expressed first and constitute early HIV-1 gene expression. In order to express singly spliced RNAs and also to transport the full length unspliced RNA genome into the cytoplasm for packaging, HIV-1 has developed means to overcome the restrictions on RNA transport. The regulatory proteins, Tat and Rev, are essential for HIV-1 replication since mutations in these proteins eliminate HIV-1 production (Dayton A.I., et.al. (1986) Cell, 44:941-947, and Fisher, A.G., et.al. (1986) Nature, 320:367-371.). The auxiliary genes are derived from exons positioned entirely upstream of the HIV-1 envelope gene e.g. vif or exons upstream of as well as within env but in different reading frames e.g. tat, rev. The efficiency of splicing in part regulates the levels of gene expression of the different auxiliary proteins.

Following integration of HIV-1 proviral DNA, predominantly truncated forms of mRNA are synthesised by the cellular RNA polymerase II which interacts with sites on the 5' long terminal repeat (LTR) of the proviral DNA. tat is one of the first genes to be expressed and carries a nuclear localisation signal. It is a potent transcriptional activator that enhances LTR-directed transcription up to a thousand fold. Continuous expression of Tat ensures a positive feed back loop for continued high level gene expression. Unlike conventional transcriptional activators that interact with DNA sequences, Tat binds directly to the 5' ends of all HIV-1 RNAs at a specific stem-loop secondary structure, TAR (transactivating response element). The structure of the loop is highly conserved and essential for Tat function. The structure of the Tat/TAR interaction has been analysed using nuclear magnetic resonance (NMR) (Puglisi et al. (1992) Science, 257:76-80). Tat binds TAR in association with the cellular protein, cyclin T, which in turn binds CDK9 that phosphorylates the RNA polymerase II C- terminal domain, thereby promoting the elongation of RNA transcripts. These Tat cellular cofactors are only present in activated cells, their absence represses transcription of proviral DNA resulting in a 'quasi latency' in T lymphocytes. Tat is expressed from two exons, both the nuclear localisation signal and the TAR binding region are located in the first exon. Tat is secreted from infected cells and can exert heterologous effects on neighbouring cells. These include cellular activation (Hofman et al. (1993) Blood, 82:2774-2780.), induction of cellular apoptosis (Macho et al. (1999) Oncogene, 18:7543-7551.1999) functioning as a secretable growth factor (Trinh, DP. et.al. (1999) Biochem. Biophys. Res. Commun., 256:299-306.) and modulating host cell protein synthesis in favour of viral protein synthesis (Xiao et al. (1998) Biochem. Biophys. Res. Commun., 244:384-389.1998). Therapeutic strategies targeting Tat will therefore have a strong impact on HIV-1 infection. The small multiply spliced mRNAs encoding Tat, Rev and Nef predominate during early phase after infection. When a threshold level of Rev is produced, unspliced and singly spliced RNAs accumulate in the cytoplasm for translation, allowing productive infection to proceed. Failure to generate this threshold level of Rev may contribute to HIV quasi latency. Rev can only bind to RNAs carrying an RNA structure, RRE (Rev responsive element) which is located in the env coding region of the genome. Rev interacts with the RRE as a multimer through a basic arginine rich region present in the amino terminal half of the protein. A consequence of this interaction is the transport of partially spliced RNAs that will provide gene products such as Env, Vif, Vpu and Vpr as well as unspliced RNAs that will serve as new genomes for incorporation into assembling particles. Structural analysis of the Rev/RRE interaction has also been carried out using NMR (Battiste et al. (1996) Science, 273:1547-1551.1996). Rev carries a leucine rich export signal that allows it to shuttle between the nucleus and the cytoplasm for continued transport of newly synthesised RNAs (Kalland et al. (1994). J. Virol., 68:1475-1485; Meyer & Malim, (1994) Genes Dev., 8:1538-1547.). In this way, Rev ensures that the structural genes are expressed late following the regulatory genes. Therapeutic strategies directed against Rev will interrupt the viral life cycle early in infection.

Although originally described as a negative factor, Nef has later been shown to have positive effects on virus replication and is expressed in larger quantities than that of Tat and Rev, both early and throughout infection. Nef is myristylated at the N-terminus and is associated with the inner side of the plasma membrane. Nef is partly responsible for down regulation and degradation of surface CD4 by endocytosis (Piguet et al. (1998) EMBO J., 17:2472-2481.). Removing CD4 from the cell surface prevents superinfection with other HIV-1 strains, or reinfection with newly released virus. Nef is also responsible for the down regulation of MHC class I thereby protecting infected cells from destruction by cytotoxic T-lymphocytes (Le Gall et al. (1997). Res. Virol., 148:43-47.). Nef is not an essential viral protein since it is not required for in vitro infection of peripheral blood lymphocytes or T-cell lines. Nef deletion mutants, however, are less pathogenic over long periods of time. Nef also has complex effects on signal transduction pathways in the cell and contains a proline rich region that can interact with the SH3 domain of kinases involved in T-cell activation, a feature necessary for efficient HIV replication (Moarefi et al. (1997). Nature, 385:650-653.). Nef containing viruses are capable of more viral DNA synthesis than viruses deleted in the Nef gene which suggests that Nef directly or indirectly activates the viral reverse transcriptase. The low level of Nef associated with virions may be responsible for this phenomenon. Low levels of Nef are also released from infected cells although the potential effect on neighbouring cells is unclear. Since Nef is expressed early in infection and has significant effects on CD4 and MHC class I expression as well as disease progression, it represents an important target for future therapeutic strategies. Tat and Nef are secreted and can be taken up by macrophages and expressed in association with MHC class II molecules. This improves their suitability as targets for peptide based therapies which also would be expressed in the context of MHC class II. It is clear that targeting early gene products that are essential for HIV-1 replication, such as Tat and Rev should be given priority in addition to Nef which is also expressed early and influences disease progression. Table 1 HIV-1 Regulatory and auxiliary proteins.

Naturally occurring HIV sequences in vaccine candidates are not capable of stimulating a stable longterm immune response due to HIVs inherent ability to hide by changing the appearance of the epitopes presented for the immune system. To overcome this variable presentation of epitopes, certain amino acid substitutions and amino acid combinations will support the immune system to present and recognize these foreign virus antigens in a reliable manner and thus to a greater extent.

Based on the above background, we decided to investigate the possibility of designing novel synthetic peptides which can mimic the epitopes from the regulatory and auxiliary HIV proteins in such a way that they can be exposed for both the humoral as well as the cellular part of the immune system, to meet the need for an effective therapeutic and/or prophylactic vaccine.

The initital work was based on the native Tat amino acid sequences published by Korber B., et al., Human Retroviruses and AIDS 1997 Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM. The first Tat epitope is located between amino acid 1 and amino acid 24 of the tat protein:

Table 2 Tat epitope

AA no Naturally occurring AAs

1 M S

2 E D V

3 S Q P L V A

4 V I

5 D N

6 P H A

7 R N S K E D

8 L I Q R V M

9 E D P

10 P S

11 W

12 K N E H L

13 H R Q

14 P

15 G P

16 S N A

17 Q K T

18 P H

19 K T S A R P Q

20 T A I

21 A P D V

22 C S

23 T N S

24 N K R Q A P T

The one letter as well as the three letter codes defining the amino acids in the sequences given throughout this specification are in accordance with International standards and given in textbooks, for instance Lehninger A.L., «Principles of

Biochemistry», Worth Publishers Inc., New York, 1982. The amino acids given to the right of the left column, represent the natural variation of the sequence. Our analyses resulted in a sequence containing this modified epitope: C S W V N P R L E P W L H P G S Q P N1 T A C T N

Wherein Nl indicates 2-aminohexanoic acid (Norleucine, abbreviated Nle and Nl in the three letter and one letter code, respectively) and the cysteine residues are in an oxidized state, i.e. are forming an intrachain disulphide bridge. Since the Cystein residue to the C-terminal part (in position 22) of the peptide is part of an intramolecular disulfide bond outside this selected epitope, a similar intrapeptide disulphide bond is formed by placing a Cystein in the N-terminal part of the selected epitope. Another alternative is to form an intermolecular disulphide bond by dimerization of the sequences :

WVNPRLEPWLHPGSQPNITACTN

A further alternative is dimerization with another epitope selected from Tat. The second epitope on Tat is located between amino acid 35 and 57 in C-terminal direction, separated from the first epitope by 10 amino acids containing 5 Cys residues in addition to the Cys residue in each of the epitopes. The relatively high number of Cys residues . offers a variety of inter- and intramolecular crosslinking possibilities. It is likely that this Cys-rich domain will dominate the immunological exposure of this protein and hence cause a "hiding" of the two relevant epitopes. Selection and modification of the two adjacent epitopes can expose an essential part of the Tat protein in a more optimal way.

In order to reduce the probability for development of escape mutants, the number of epitopes is further increased and two additional peptide sequences were selected. These sequences are located on Rev (residues 58-78) and Nef (residues 65-85). The native sequences have been published in Human Retroviruses and AIDS 1999; A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos : Table 3 Tat epitope

a. no. Naturally occurring A

35 Q P I L T Y V

36 V A C L

37 C

38 F

39 I L Q M T

40 T N K R

41 K Q

42 G A

43 L

44 G S

45 I

46 S ' F Y

47 Y N

48 G

49 R K S

50 K

51 K

52 R

53 R K S G

54 Q R P

55 R

56 R

57 R G S

Table 4 Rev epitope:

AA no. Naturally occurring AAs

58 R W Q

59 I V L F

60 L I P

61 G S N D C V T A R

62 T A D N S

63 Y C F R H V

64 L V

65 G H

66 R G

67 S P F L

68 A T E Q V

69 E K Q D N

■ 70 P S A N

71 V N G P

72 P Q H S L R T D

73 L F V

74 Q L P H D

75 L

76 P

77 P L E

78 L I V P

and the Nef epitope, Table 5 :

AA no. Naturally occurring AAs

65 E G D S

66 N G D E

67 E V

68 A G

69 L F

70 P

71 I V

72 T R K A M

73 P

74 Q H

75 V L l

76 P

77 L V T

78 R

79 P

80 M V I

81 T D

82 Y F R

83 K R

84 A G S E Q

85 A S V

Several modified peptides have been synthesized in order to determine the uniqueness of the sequences as well as their properties for stimulation of the immune system in combination with their specificity and sensitivity as HIV-1 antigens.

DESCRIPTION OF THE INVENTION

The peptides according to the invention are originating from the four different conserved areas of the HIV-1 Tat, Rev and Nef proteins which are described above, having the properties of maintaining the uniqueness (immunogenicity, sensitivity and specificity) of the HIV-1 epitopes. Further the new peptides according to the invention possess no recognized cytotoxic T lymphocyte (CTL) antagonistic effect and shall have at least one potential CTL epitope.

The peptides, according to the invention, which have met the above criteria are selected from the following groups;

Xaa-i Xaa₂Xaa₃Xaa Xaa5 Xaaβ Xaa Leu Glu Pro Trp Xaaι₂ His ProXaa-is Xaa-|₆ Xaa-|₇ Xaa-isXaaig Xaa₂o Xaa₂ι Xaa₂₂ Xaa₂₃ Xaa₂ (SEQ ID NO : 1) wherein the amino acids of the chain could have the following meanings

Xaa n position 1 of the peptide derivative is Met, Ser, Cys or none, Xaa n position 2 is Glu, Asp, Val, Ser or none Xaa n position 3 is Ser, Gin, Pro, Leu, Val, Ala, Trp, Tyr or Phe, Xaa n position 4 is Val or lie, Xaa n position 5 is Asp, Asn or lie, Xaa n position 6 is Pro, His or Ala, Xaa n position 7 is Arg, Asn, Ser, Lys, Glu or Asp Xaa n position 12 is Leu, lie or Nle Xaa n position 15 is Gly or Pro Xaa n position 16 is Ser, Asn or Ala, Xaa n position 17 is Gin, Lys or Thr Xaa n position 18 is Pro or His, Xaa n Position 19 is Lys, Thr, Ser, Ala, Arg, Pro, Glu, Leu, lie or Nle Xaa n position 20 is Thr, Ala or lie Xaa n position 21 is Ala, Pro, Asp or Val Xaa n position 22 is Cys or Ser Xaa n position 23 is Thr, Asn or Ser Xaa n position 24 is Asn, Lys, Arg, Gin, Ala Pro or Thr

the peptide comprises at least six consecutive amino acids of the sequence of SEQ ID NO : 1 , furthermore two or more of the Cys residues may form part of an intrachain- or interchain disulphide binding, a -S-(CH₂)_P-S- or a - (CH₂)_p-bridge wherein p = 1-8, optionally intervened by one or more hetero atoms such as O, N or S,

Xaai Xaa₂ Xaa₃ Phe Xaa₅ Xaa₆Xaa Xaa₈Xaa₉XaaιoXaaιι Xaaι₂ -Z-Tyr Xaai Gly Xaais Lys Lys Arg Xaa-ιgXaa₂₀ Xaa₂ι Xaa₂₂Xaa₂₃ (SEQ ID NO : 4)

wherein the amino acids of the chain have the following meaning;

Xaa in position 1 is Pro, lie, Leu, Thr, Tyr or Val

Xaa in position 2 is Val, Ala Cys, Leu,

Xaa in position 3 is Cys, lie, Leu, Val or Nle

Xaa in position 5 is lie, Leu, Gin, Met or Thr Xaa in position 6 is Thr, Asn, Lys or Arα Xaa in position 7 is Lys, Arg or Gin

Xaa in position 8 is Gly or Ala

Xaa in position 9 is Leu or lie

Xaa in position 10 is Gly, Ser or Ala Xaa in position 11 is lie or Gly

Xaa in position 12 is Ser, Phe or Tyr

Xaai inserted before position 14 is Leu, lie, Nle

Xaa in position 15 is Arg, Lys, Ser or Citrulline (Cit)

Xaa in position 19 is Arg, Lys, Ser, Gly or Cit Xaa in position 20 is Gin, Arg or Pro

Xaa in position 21 is lie or leu

Xaa in position 22 is Gly, Leu, lie, Cys or none

Xaa in position 23 is Gly or none wherein the sequence of SEQ ID NO : 4 comprises at least six consecutive amino acids, -Z- is an optional linker and have the meaning PEG, modified PEG and/or [Gly]_n wherein n = 1 , 2 or 3, furthermore two or more of the Cys residues may form part of an intrachain- or interchain disulphide binding, a -S-(CH₂)_P-S- or a - (CH₂)_p-bridge wherein p = 1-8, optionally intervened by one or more heteroatoms such as O, N or S,

Xaai lie Leu Xaa₄Xaa₅ Xaaβ Leu Gly Arg Xaa-ioXaan -Z- Xaa-ι₂ Leu Xaai Xaai Xaaι Xaa-i5 Xaa-1₆ Xaa-|₇ Xaa-|₈ Xaa-|₉ Leu Pro Pro Leu (SEQ ID NO : 7)

wherein Xaa in position 1 is Phe, Tyr, Trp or Arg Xaa in position 4 is Gly, Ser, Asn, Asp, Cys, Val, Thr, Ala, or Arg

Xaa in position 5 is Thr, Ala, Asp, Asn or Ser

Xaa in position 6 is Tyr, Cys, Phe, Arg, His, Ser, Val or Leu

Xaa in position 10 is Ser, Pro, Phe, Leu or lie

Xaa in position 11 is Ala, Thr, Glu, Gin, Val Pro or Ser Xaa in position 12 is Glu, Lys, Gin, Asp, Asn, Tyr, Trp or Phe

Xaai inserted after position 13 is Ser, Pro, Phe, Leu or lie

Xaai inserted before position 14 is Ala, Thr, Glu, Gin, Val, Pro, or Ser

Xaa in position 14 is Glu, Lys, Gin, Asp or Asn

Xaa in position 15 is Pro, Ser, Ala or Asn Xaa in position 16 is Val, Asn, Gly or Pr Xaa in position 17 is Pro, Gin, His, Ser, Leu, Arg, Thr, Asp or lie

Xaa in position 18 is Leu Phe or Val

Xaa in position 19 is Gin, Leu, Pro, His, Asp or Glu

wherein the sequence of SEQ ID NO : 7 consists of at least six consecutive amino acids, the linker -Z- is optional and have the meaning PEG, modified PEG and/or [Gly]_n wherein n = 1 , 2 or 3,

Xaai Leu Val Gly Xaa₅ Pro Xaa₇Xaas Pro Xaa₁₀ Xaa-n Pro -Z-[Arg]_m Xaaj Xaa-ι₃Xaaι₄ Pro Xaa-₁₆ Xaaι XaaisXaa-ig Xaa₂oXaa₂ι (SEQ ID NO : 10)

wherein the Xaa in position 1 is Lys or Arg Xaa in position 5 is Phe or Leu Xaa in position 7 is lie or Val Xaa in position 8 is Thr, Arg, Lys, Ala or Met Xaa in position 10 is Gin or His Xaa in position 11 is Val, Leu or lie Xaai inserted before position 13 is Leu

Xaa in position 13 s Leu, Val or Thr Xaa in position 14 s Arg or Citrulline (Cit) Xaa in position 16 s Met, Val, lie or Nle, Leu Xaa in position 17 s Thr or Asp Xaa in position 18 s Tyr, Phe or Arg Xaa in position 19 s Lys or Arg Xaa in position 20 s Ala, Gly, Ser, Glu or Gin Xaa in position 21 s Ala, Ser or Val

wherein the sequence of SEQ ID NO : 10 consists of at least six consecutive amino acids, the linker -Z- is optional and have the meaning PEG, modified PEG and/or [Gly]_n wherein n = 1 , 2 or 3 and independently from n, m in [Arg]_m is = 0, 1 , 2 or 3, the terminal ends of the sequences may be free carboxyl- or amino groups, amides, acyls, acetyls or salts thereof, and/or the said peptide sequences are immobilized to a solid support. The new peptide sequences have the potential to serve as a good antigen wherein the antigen comprises at least one peptide selected from the group of sequences of SEQ ID NO : 1 , SEQ ID NO : 4, SEQ ID NO : 7 or SEQ ID NO : 10 . The antigenicity may be adapted through adjusting the ratio or concentration of different peptides or size of the peptides by for instance dimerization or polymerization and/or immobilization to a solid phase. The antigen comprises one or more polypeptide sequences, according to the invention, which could be either linked by a bridge for instance a disulphide bridge between the Cys residues of the chains or bridges like Ci-C₃ alkylen possibly intervened by one or more heteroatoms like O, S, or N or preferably they are unlinked. The chains may be immobilized to a solid phase in monomeric, dimeric or oligomeric forms. Further amino acids may be added to the ends in order to achieve an «arm» to facilitate immobilization.

PEG is polyethylene glycol (HO(CH₂CH₂O)_a H and can be part of the linker -Z-, optionally PEG is modified by a dicarboxylic acid (HO(CH₂CH₂O)_a CO(CH₂) COOH) or a terminal carboxylic group where a = 1-10 and b = 2-6, prior to linking.

The linker -Z- can either consist of PEG, modified PEG, or a combination thereof and/or one or more Gly residues combined. Alternatively the linker -Z- can consist of a Gly-bridge [Gly]_n where n = 1 , 2 or 3.

All amino acids in the peptides of the invention can be in both D- or L-form, although the naturally occurring L- form is preferred.

The C- and N-terminal ends of the peptide sequences could deviate from the natural sequences by modification of the terminal NH₂-group and/or COOH-group, they may for instance be acylated, acetylated, amidated or modified to provide a binding site for a carrier or another molecule.

The peptides according to the invention are consisting of 6 to 50 amino acids, preferably between 10 and 30 amino acids. They are covering all natural variation of amino acids in the identified positions. The polypeptide antigen according to the invention is either in a free or in a carrier- bound form. The carrier or solid phase to which the peptide is optionally bound can be selected from a vide variety of known carriers. It should be selected with regard to the intended use of the immobilized polypeptide as a diagnostic antigen or as an immunizing component in a vaccine.

Examples of carriers that can be used for e.g. diagnostic purposes are magnetic beads or latex of co-polymers such as styrene-divinyl benzene, hydroxylated styrene-divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy-activated porous magnetic glass, gelatine or polysaccharide particles or other protein particles, red blood cells, mono- or polyclonal antibodies or fab fragments of such antibodies.

According to a further embodiment of the present invention, the antigens may form part of a vaccine possibly combined with carriers, adjuvants or combined with other immunostimulating elements such as canarypox virus carrying the env gene. Examples of carriers and/or adjuvants for vaccine purposes are other proteins such as human or bovine serum albumin and keyhole limpet haemocyanin and fatty acids. Immunostimulatory materials may be divided into three groups; adjuvants, carriers for antigens and vehicles. Examples of adjuvants include aluminum hydroxyd, aluminum salts, saponin, muramyl di and tripeptides, monophosphoryl lipid A, palmitic acid, B. ertussis and various cytokines including the Th1 cytokine IL-12 and IL-1. A number of protein toxins can be used to carry passenger proteins across cellular membranes into the cytosol, which are useful in developing CTL vaccines. Carriers include bacterial toxoids such as inactivated tetanus and cholera toxins, genetically detoxified bacterial toxins such as heat labile enterotoxin from E.coli, fatty acids, live vectors such as polio chimeras and hybrid proteins that form particulates for example yeast retrotransposon hybrid TY particles and HBcAg particles. Vehicles which are frequently occurring components in modern vaccines are consisting of mineral oil emulsion, Freunds complete and incomplete adjuvant, vegetable oil emulsions, nonionic block co-polymer surfactants, squalene or squalane, lipopeptides, liposomes and biodegradable microspheres. Two novel adjuvants which possess significant potential for the development of new vaccines include an oil-in-water microemulsion (MF59) and polymeric microparticles. Any substance that can enhance the immunogenicity of the antigen may be used and several further alternatives of carriers or adjuvants are given in the US or European Pharmacopoeia.

A suitable formulation of the antigen for immunostimulatory uses may also comprise interferons such as INF-γ, antiviral chemokines or haematopoietic growth factors such as granulocyte macrophage growth factor.

Another approach in order to enhance the stimulation and absorption in for instance the intestine is to administer the peptides of the invention, with small peptides such as di, tri or tetrapeptides. These peptides can be administered in addition to or in combination with the peptides of the invention. Preferably the peptides are administered together with the tripeptide YGG, consisting of amino acids in the D- or L-forms, preferably in the D-form. Recent approaches to non-parenteral delivery of vaccines, for instance via mucosa include; gene fusion technology to create non-toxic derivatives of mucosal adjuvants, genetically inactivated antigens with a deletion in an essential gene, coexpression of an antigen and a specific cytokine that is important in the modulation and control of a mucosal immune response, and genetic material itself that would allow DNA or RNA uptake and its endogenous expression in the hosf s cells.

One approach for developing durable responses where cell-mediated immunity is required, is to vaccinate with plasmid DNA encoding one or more specific antigen(s).

In order to protect against HIV infection, vaccines should induce both mucosal and systemic immune responses and could be administered by any convenient route, parenterally or non-parenterally, such as subcutanously, intracutanously, intravenously, intramuscularly, perorally, mucosally or intranasally for example.

In a preferred embodiment of the vaccine according to the present invention it comprises antigens containing peptides selected from at least one of the groups of the SEQ ID NO: 1 , 4, 7 and 10, more preferred the different peptides occur in equal amounts.

In a further preferred embodiment the vaccine composition contains the antigens FVIHRLEPWLHPGSQHNITASTN -NH₂ (SEQ ID NO :14)

and

RLVGFPVKPQVPGLLRPLTYKAA-NH₂ (SEQ ID NO :15).

The sequences can activate the cellular immune system, and contribute with CTL- epitopes. The amino acid changes implemented within the frame of the CTL-epitopes are designed to achieve enhanced binding. Other amino acid changes have been conducted in order to facilitate the synthesis of the peptide and/or increase the solubility of the peptide.

A method for detecting antibodies, induced by HIV-1 or HIV-1 specific peptides or proteins, in a sample of body fluid using the present antigens is a further embodiment of the invention. Also immunoassay kit designed for this detection and antibodies capable of selectively reacting with the said antigens are encompassed by the present invention.

DESCRIPTION OF THE PREPARATION OF THE PEPTIDES

The peptides of the invention can be produced by any known method of producing a linear amino acid sequence, such as recombinant DNA techniques. A nucleic acid sequence that encodes one or more peptides of the invention or a multimer of the said peptides, is introduced into an expression vector. Suitable expression vectors are for instance plasmids, cosmids, viruses and YAC (yeast artifical chromosome) which comprise necessary control regions for replication and expression. The expression vector may be stimulated to expression in a host cell. Suitable host cells are for example bacteria, yeast cells and mammal cells. Such techniques are well known in the art and described for instance by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. Other well- known techniques are degradation or synthesis by coupling of one amino acid residue to the next one in liquid phase or preferably on a solid phase (resin) for instance by the so-called Merrifield synthesis. See for instance Barany and Merrifield in the Peptides, Analysis, Synthesis, Biology, Vol.2, E. Gross and Meinhofer, Ed. (Acad. Press, N.Y., 1980), Kneib-Coronier and Mullen Int. J. Peptide Protein Res., 30, p.705-739 (1987) and Fields and Noble Int.J. Peptide Protein Res., 35, p.161-214 (1990).

In case a linked or cyclic peptide is desired, the amino acid sequence is subjected to a chemical oxidation step in order to cyclize or link the two cysteine residues within one or between two peptide sequences, when the appropriate linear amino acid sequences are synthesized, see Akaji et al., Tetrahedron Letter, 33, 8, p.1073-1076, 1992.

GENERAL DESCRIPTION OF SYNTHESIS All peptide derivatives prepared in the Examples given below were synthesized on a Milligen 9050 Peptide Synthesizer using a standard program. The resin used was Tenta Gel P RAM with a theoretical loading of 0,20 meq/g (RAPP POLYMERE GmbH, Tubingen). The final product of the synthesis was dried in vacuuo overnight. The peptide was then cleaved from the resin by treatment with 90% trifluoroacetic acid in the presence of ethandithiol (5%) and water (5%) as scavengers (1 ,5 hours at RT). Then the resin was filtered and washed on filter with additional trifluoro acetic acid (100%) (2 x 20 ml). The combined filtrates were evaporated in vacuuo (water bath at RT) and the residue was triturated with ethyl ether (200 ml) and the precipitated product filtered off. The solid was promptly dissolved on filter with glacial acetic acid (100 ml) and added to 1 ,5 I of 20% acetic acid in methanol and treated with 0,1 M solution of iodine in methanol until a faint brown colour remained. Then Dowex 1 x 8 ion exchange in acetate form (15g) (Bio-Rad, Richmond, CA) was added and the mixture filtered. The filtrate was evaporated and the residue freeze-dried from acetic acid. The product was then purified by reversed phase liquid chromatography on a column filled with Kromasil® 100 - 5 C8 (EKA Nobel, Surte, Sweden) in a suitable system containing acetonitrile in 0,1 % trifluoro acetic acid water solution. The samples collected from the column were analyzed by analytical high performance liquid chromatography (HPLC) (Beckman System Gold, USA) equipped with a Kromasil® 100 - 5 C8 Column (EKA Nobel, Surte, Sweden). Fractions containing pure substance were pooled, the solvent was evaporated and the product freeze-dried from acetic acid. The final HPLC analysis was performed on final product, and the structure of the peptide was confirmed by amino acid analysis and mass spectrometry (LDI-MS). All amino acids used during the synthesis were L-amino acids and they were protected with a fluorenyl methoxy-carbonyl group at the α-amino function. The side chains were protected as follows:

Cys (Trt), Gln(Trt), Glu(OtBu), Thr(tBu).

The abbreviations, within the brackets are : Trt = triphenyl methyl t-Bu = tert. Butyl OtBu = tert. Butyl ester

The amino acid derivatives were supplied by Bachem AG, Switzerland.

EXAMPLE 1

Preparation ofCSWVNPRLEPWNIHPGSQHNITACTN-NH₂ (SEQ ID NO : 2). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The peptide was then cyclizated by oxidation with l₂. The peptide was dissolved in acetic acid/methanol (1:4) and 0,1 M l₂ in methanol was added. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI- MS).

Purity (HPLC) : more than 97% (single impurity less than 1%) Molecular weight (free base) : 2746.2

EXAMPLE 2 Preparation ofFVIPRLEPWNIHPGSQPNITACTN-NH₂ (SEQ ID NO : 3). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS). Purity (HPLC) : more than 97% (single impurity less than 1%) Molecular weight (free base) : 2538,0 EXAMPLE 3

Preparation ofYLLFLTKGLGIS GGGYNIGCitKKRCitQILG-NH₂ (SEQ ID NO : 5). The peptide is synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity is determined by HPLC analysis and the structure is confirmed by amino acid analysis and mass spectrometry (LDI-MS).

EXAMPLE 4

Preparation of Y L Nl F L T R G L G I S G G G Y Nl G Cit K K R Cit Q I C G - NH₂ (SEQ ID NO : 6). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 97% (single impurity less than 1%) Molecular weight (free base) : 3097,7

EXAMPLE 5

Preparation of RILSTYLGRISGGGWLSAEPVPLQLPPL-NH₂ (SEQ ID NO : 8). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 97% (single impurity less than 1%) Molecular weight (free base) : 2990,6

EXAMPLE 6

Preparation of RILSTYLGRISGGGYLSAEPVPLQLPPL-NH₂ (SEQ ID NO : 9). The peptide is synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity is determined by HPLC analysis and the structure is confirmed by amino acid analysis and mass spectrometry (LDI-MS). EXAMPLE 7

Preparation of KLVGFPVKPQVPGGGRLLCitPNITYKAA-NH₂ (SEQ ID NO : 11). The peptide is synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity is determined by HPLC analysis and the structure is confirmed by amino acid analysis and mass spectrometry (LDI-MS).

EXAMPLE 8

Preparation of RLVGFPVKPQVPGGGRLLRPLTYKAA-NH₂ (SEQ ID NO : 12). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 97% (single impurity less than 1%) Molecular weight (free base) : 2790,4

Molecular formula : Cι₃oH₂₇O₃₉N₂₉

EXAMPLE 9

Dimerisation via disulphide bridge. The peptide sequences of the Example 2 are linked via an oxidation step to form a dipeptide wherein the cysteine residues form a disulphide bridge. The bridge is formed in either ways;

A) Oxidation with l₂.The peptides (equal amounts if different) are dissolved in acetic acid/methanol (1:4) and 0,1 M l₂ in methanol is added yielding a mixture of the homodimer, or

B) Oxidation via [Cys(Spy)²²]-SEQ ID NO : 3.2,3mM of the peptide of SEQ ID NO : 3 dissolved in 2 M AcOH (aq) and 2-propanol (1:1) is treated with 2,2 dithiodipyridin (3 eqv) to yield [Cys(Spy)²]-SEQ ID NO : 3. [Cys(Spy)²²]-SEQ ID NO : 3 is dissolved in mM NH₄Oac (aq pH=6, 5) and methanol (5:2) to yield the dimer of SEQ ID NO : 13.

The purity of the resulting dimer is determined by HPLC analysis and the structure is confirmed by amino acid analysis and mass spectrometry (LDI-MS). EXAMPLE 10

Preparation of FVIHRLEPWLHPGSQHNITASTN -NH₂ (SEQ ID NO :14). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 98% (single impurity less than 1%) Molecular weight (free base) : 2540,2

EXAMPLE 11

Preparation ofRLVGFPVKPQVPGLLRPLTYKAA-NH₂ (SEQ ID NO : 15). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 99% (single impurity less than 1%) Molecular weight (free base) : 2520,3

EXAMPLE 12 - REFERENCE EXAMPLE Preparation of a nativ tatl sequence ; MESVDPRLEPWKHPGSQPKTAC T N -NH₂ (SEQ ID NO :16). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS). Purity (HPLC) : more than 97% (sing le impurity less than 1%) Molecular weight (free base) : 2708,1

EXAMPLE 13 - REFERENCE EXAMPLE

Preparation of a nativ tat2 sequence ;QVCFITKGLGISYGRKKRRQRRR -NH₂ (SEQ ID NO :17). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 94% Molecular weight (free base) : 2806,4 EXAMPLE 14- REFERENCE EXAMPLE

Preparation of a nativ Nef sequence ; E E V G F P V R P Q V P L R P M T Y K A A - NH₂ (SEQ ID NO :18). The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS). Purity (HPLC) : more than 95% Molecular weight (free base) : 2384,8

EXAMPLE 15

A vaccine comprising the peptides of the SEQ ID NO : 3 and 12 is prepared. The freeze-dried peptides are dissolved in sterile water at a final concentration of 4 mg/ml. A preparation of a granulocyte-macrophage-colony stimulating factor (GM-CSF) is also prepared, according to the manufacturers directions for use, to a final concentration of 0,3 mg/ml. The two solutions are administered intracutaneously. A typical injection dose is 100 μl.

EXAMPLE 16

A vaccine comprising the peptides of the SEQ ID NOS : 14 and 15 is prepared. The freeze-dried peptides are dissolved in sterile water at a final concentration of 4 mg/ml. A preparation of a granulocyte-macrophage-colony stimulating factor (GM-CSF) is also prepared, according to the manufacturers directions for use, to a final concentration of 0,3 mg/ml. The two solutions are administered intracutaneously. A typical injection dose is 100 μl.

EXAMPLE 17

An antigen solution or suspension is mixed with equal parts of Freund^'s adjuvant of Behring, complete or incomplete, and is then finely emulsified by being drawn up into, and vigorously pressed out of, an injection syringe, or with a homogenator. The emulsion should remain stable for at least 30 minutes. The antigen-adjuvant emulsions is best injected subcutaneously as a depot. EXAMPLE 18

Immunoassay for detection of antibodies induced by HIV-1.

The magnetic particle reagents are to be prepared according to the manufacturers recommended protocol. Dynal AS, is the manufacturer of the Dynabeads, which are employed. The magnetic particles coated with ligand are called Reagent 1. A peptide according to the invention is covalently coupled to the pre-activated surface of the magnetic particles. It is also possible to physically absorb the peptide to the surface of the magnetic particles. The concentration of particles in Reagent 1 is within the range from 1 mg/ml to 15 mg/ml. The particle size varies between 0,2 μm to 15 μm. The concentration of peptides is within the range from 0,01 mg/mg particle to 1 mg/mg particle.

The anti human Ig Alkaline Phosphatase (AP) conjugated antibody reagent is prepared according to the recommended protocol of Dako AS. This protocol is a standard procedure in this field. This reagent is called Reagent 2. The substrate solution phenolphtaleine-monophosphate is to be prepared according to the recommended protocol of Fluka AG. This protocol is a standard procedure in this field. The substrate solution is called Reagent 3.

The washing and incubation buffer which is used is standard 0,05M tris-base buffer with the following additional compounds; Tween 20 (0,01% to 0,1%), glycerol (0,1% to 10%) and sodium chloride (0,2% to 0,1%).

The assay procedure comprises an incubation step wherein 1 drop of Reagent 1 is mixed with 2 drops of washing buffer in each well. After mixing, 30 μl of sample is added and the solution is incubated for 5 minutes. The magnetic particles can be trapped by a magnet and the liquid removed, before the magnet is separated. Then the wells are washed twice in 4 drops of washing solution, before incubation with Reagent 2. 1 drop of Reagent 2 is added with 2 drops of washing buffer and the solution is incubated for 5 minutes. The magnetic particles can be trapped by a magnet and the liquid removed, before the magnet is separated. Then the washing step is repeated before incubation with Reagent 3. 2 drops of Reagent 3 is added to each well and the solution is incubated for 3 minutes. The results can be read against a white background. Positive results are red (3+ = strong red) whereas negative results are clearly light yellow/brown solutions as obtained in the negative control. The immunoassay kit could be used in detection of antibodies, induced either by HIV virus or HIV-specific peptides or proteins, for instance the peptides of the present invention.

EXAMPLE 19

Therapeutic or prophylactic vaccine.

At least one of the polypeptides of the invention, selected from the group of sequences SEQ ID NO : 1 , SEQ ID NO : 4, SEQ ID NO : 7 or SEQ ID NO : 10 can form antigens and constitute the active principle of a prophylactic or therapeutic vaccine intended to provide protection against the human immunodeficiency virus type 1 (HIV-1). The vaccine may include compounds having beneficial effects in protecting or stimulating the hosts immune system (human being or vertebrate animal) for instance interleukins, interferons, granulocyte macrophage growth factors, haematopoietic growth factors or similar. Preferably the vaccine composition further contain an adjuvant or vehicle, more preferably the adjuvant or vehicle is Monophosphoryl Lipid A (MPL ®) possibly with alum, Freund^'s adjuvant (complete or incomplete) or aluminum hydroxyd. The optimal amount of adjuvant/vehicle will depend on the type(s) which is chosen.

The peptides of the invention might be modified by C-terminal addition of a single fatty acid such as a single palmitoyl chain to form a lipopeptide vaccine. Further the lipopeptides can be introduced into liposome membranes by the freeze-thaw method resulting in liposomes bearing the peptide ligands on their surface.

The peptide or vaccine formulation can be freeze-dried prior to storage. The freeze- dried peptides can be dissolved in sterile water to a final concentration of 0,1-100 mg/ml. The vaccine may be stored preferably at low temperature, in ampoules containing one or more dosage units, ready for use. A typical dosage unit of the peptide according to the invention is within the concentration range : 0,05 μg-1mg per kg bodyweight, preferably within 0, 15 μg-0.15 mg per kg body weight. Persons skilled in the art will appreciate that a suitable dose will depend on the body weight of the pasient, the type of disease, severity of condition, administration route and several other factors. When used as a therapeutic vaccine, the vaccine might be administered up to 12 times, through injections. Further boosters might follow and can in some cases take place throughout the patients life. In preparation of an injection solution the peptides are dissolved in sterile water at a final concentration of 1 mg/ml per peptide. Typically an injection volume is 100 μl to 200 μl (2 x 100 μl). The peptide is preferably co-administered with a suitable adjuvant and/or a granulocyte-macrophage growth factor for instance Leucomax® «Shering Plough» made within a concentration range of from 0,1 mg/ml to 1 mg/ml, or according to the manufacturers recommendations for use. Particularly preferred is a combination therapy where the present peptides are administered together with the peptides as described in the published International patent application no. PCT/NO00/00075 and/or the co-pending Norwegian patent application no. 2000 4413 . These peptides may be administered simultaneously or sequentially. Suitable administration may be intracutane, subcutane, intravenous, peroral, intramuscular, intranasal, mucosal or any other suitable route. Booster administrations may be required in order to maintain protection. For persons skilled in the art it will be understood that the vaccine compositions according to the invention are useful not only in prevention of infection, but also in treatment of infection.

No toxic effects of the peptides according to the invention, are observed when injected in mice in a dosage of 100 μg per kg body weight.

The above Examples are only meant as illustrating the invention. It must be understood that a person skilled in the art can modify the peptides, antigens and vaccines herein described without deviating from the concept and scope of this invention as set forth in the claims.

Claims

PATENT CLAIMS

1. Peptide c h a r a c t e r i z e d i n t h a t it comprises at least one amino acid sequence selected from the groups of amino acid sequences :

Xaai Xaa₂ Xaa₃ Xaa₄Xaa₅ Xaa₆ Xaa₇Leu Glu Pro Trp Xaaι₂ His ProXaa-|₅ Xaaι₆ Xaaι₇ Xaaι₈Xaa-i9 Xaa₂o Xaa₂ι Xaa₂₂ Xaa₂₃ Xaa₂₄ (SEQ ID NO : 1)

wherein the amino acids of the chain could have the following meanings ;

Xaa in position 1 of the peptide derivative is Met, Ser, Cys or none,

Xaa in position 2 is Glu, Asp, Val, Ser or none

Xaa in position 3 is Ser, Gin, Pro, Leu, Val, Ala, Trp, Tyr or Phe,

Xaa in position 4 is Val or lie,

Xaa in position 5 is Asp, Asn or lie,

Xaa in position 6 is Pro, His or Ala,

Xaa in position 7 is Arg, Asn, Ser, Lys, Glu or Asp

Xaa in position 12 is Leu, lie or Nle

Xaa in position 15 is Gly or Pro

Xaa in position 16 is Ser, Asn or Ala,

Xaa in position 17 is Gin, Lys or Thr

Xaa in position 18 is Pro or His,

Xaa in Position 19 is Lys, Thr, Ser, Ala, Arg, Pro, Glu, Leu, lie or Nle

Xaa in position 20 is Thr, Ala or lie

Xaa in position 21 is Ala, Pro, Asp or Val

Xaa in position 22 is Cys or Ser

Xaa in position 23 is Thr, Asn or Ser

Xaa in position 24 is Asn, Lys, Arg, Gin, Ala Pro or Thr the peptide comprises at least six consecutive amino acids of the sequence of SEQ ID

NO : 1 ,

Xaai Xaa₂ Xaa₃ Phe Xaas XaaβXaaT-XaasXaagXaaioXaan Xaaι₂ -Z-Tyr Xaai Gly Xaa-i5 Lys Lys Arg XaaιgXaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ (SEQ ID NO : 4)

wherein the amino acids of the chain have the following meaning; Xaa in position 1 is Pro, lie, Leu, Thr, T r or Val Xaa in position 2 is Val, Ala Cys, Leu,

Xaa in position 3 is Cys, lie, Leu, Val or Nle

Xaa in position 5 is lie, Leu, Gin, Met or Thr

Xaa in position 6 is Thr, Asn, Lys or Arg Xaa in position 7 is Lys, Arg or Gin

Xaa in position 8 is Gly or Ala

Xaa in position 9 is Leu or lie

Xaa in position 10 is Gly, Ser or Ala

Xaa in position 11 is lie or Gly Xaa in position 12 is Ser, Phe or Tyr

Xaai inserted before position 14 is Leu, lie, Nle

Xaa in position 15 is Arg, Lys, Ser or Citrulline (Cit)

Xaa in position 19 is Arg, Lys, Ser, Gly or Cit

Xaa in position 20 is Gin, Arg or Pro Xaa in position 21 is lie or leu

Xaa in position 22 is Gly, Leu, lie, Cys or none

Xaa in position 23 is Gly or none wherein the sequence of SEQ ID NO : 4 comprises at least six consecutive amino acids, -Z- is an optional linker and have the meaning PEG, modified PEG and/or [Gly]_n wherein n = 1 , 2 or 3,

Xaai lie Leu Xaa₄Xaa₅ Xaaβ Leu Gly Arg Xaa₁₀Xaa-n -Z- Xaaι₂ Leuι₃ Xaai Xaai Xaau Xaa-i_δ Xaa₁₆ Xaa-ι₇ Xaais Xaa-ig Leu Pro Pro Leu (SEQ ID NO : 7)

wherein Xaa in position 1 is Phe, Tyr, Trp or Arg

Xaa in position 4 is Gly, Ser, Asn, Asp, Cys, Val, Thr, Ala, or Arg

Xaa in position 5 is Thr, Ala, Asp, Asn or Ser

Xaa in position 6 is Tyr, Cys, Phe, Arg, His, Ser, Val or Leu

Xaa in position 10 is Ser, Pro, Phe, Leu or lie Xaa in position 11 is Ala, Thr, Glu, Gin, Val, Pro or Ser

Xaa in position 12 is Glu, Lys, Gin, Asp, Asn, Tyr, Trp or Phe

Xaaj inserted after position 13 is Ser, Pro, Phe, Leu or lie

Xaai inserted before position 14 is Ala, Thr, Glu, Gin, Val, Pro, or Ser

Xaa in position 14 is Glu, Lys, Gin, Asp or Asn Xaa in position 15 is Pro, Ser, Ala or A°n Xaa in position 16 is Val, Asn, Gly or Pro Xaa in position 17 is Pro, Gin, His, Ser, Leu, Arg, Thr, Asp or lie Xaa in position 18 is Leu Phe or Val Xaa in position 19 is Gin, Leu, Pro, His, Asp or Glu wherein the sequence of SEQ ID NO : 7 consists of at least six consecutive amino acids, the linker -Z- is optional and have the meaning PEG, modified PEG and/or [Gly]_n wherein n = 1 ,2 or 3,

Xaai Leu Val Gly Xaa₅ Pro Xaa₇Xaa₃ Pro Xaa Xaan Pro -Z- [Arg]_m Xaai Xaa-ι₃Xaa-ι₄ Pro Xaa-i₆ Xaaι Xaa-isXaa-ig Xaa₂₀Xaa₂₁ (SEQ ID NO : 10)

wherein the Xaa in position 1 is Lys or Arg

Xaa in position 5 is Phe or Leu

Xaa in position 7 is lie or Val Xaa in position 8 is Thr, Arg, Lys, Ala or Met

Xaa in position 10 is Gin or His

Xaa in position 11 is Val, Leu or lie

Xaaj inserted before position 13 is Leu

Xaa in position 14 is Leu, Val or Thr Xaa in position 15 is Arg or Citrulline (Cit)

Xaa in position 17 is Met, Val, lie or Nle, Leu

Xaa in position 18 is Thr or Asp

Xaa in position 19 is Tyr, Phe or Arg

Xaa in position 20 is Lys or Arg Xaa in position 21 is Ala, Gly, Ser, Glu or Gin

Xaa in position 22 is Ala, Ser or Val wherein the sequence of SEQ ID NO : 10 consists of at least six consecutive amino acids, the linker -Z- is optional and have the meaning PEG, modified PEG and/or [Gly]_n wherein n = 1 , 2 or 3 and independently from n, m in [Arg]_m is 0, 1 , 2 or 3 ,

the terminal ends of the sequences may be free carboxyl- or amino groups, amides, acyls, acetyls or salts thereof, two or more of the Cys residues may form part of an intrachain- or interchain disulphide binding, a -S-(CH₂)_P-S- or a - (CH₂)_p-bridge wherein p = 1-8 optionally intervened by one or more heteroatoms such as O, N and S and/or the said peptide sequences are immobilized to a solid support.

2. Peptide according to claim 1, characterized in that the amino acid sequence of SEQ ID NO : 1 is selected from the groups of SEQ ID NO

2. SEQ ID NO : 3 and SEQ ID NO : 14.

3. Peptide according to claim ^characterized in that the amino acid sequence of SEQ ID NO : 4 is selected from the groups of SEQ ID NO 5 and SEQ ID NO : 6.

4. Peptide according to claim ^characterized in that the amino acid sequence of SEQ ID NO : 7 is selected from the groups of SEQ ID NO 8 and SEQ ID NO : 9.

5. Peptide according to claim ^characterized in that the amino acid sequence of SEQ ID NO : 10 is selected from the groups of SEQ ID NO

: 11, SEQ ID NO : 12 and SEQ ID NO : 15.

6. Antigen, characterized in that it comprises at least one peptide according to claim 1.

7. Antigen according to claim 6, characterized in that it comprises at least one peptide selected from at least one of the groups SEQ ID NO : 1 , SEQ ID NO : 4, SEQ ID NO: 7 and SEQ ID NO: 10.

8. Vaccine composition, characterized in that it comprises an antigen according to claim 6 with a pharmaceutically acceptable diluent and optionally an adjuvant, carrier and/or vehicle and optionally additional immunostimulatory compound(s).

9. Vaccine composition according to claim 8, characterized in that it comprises at least one peptide selected from at least one of the groups of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 7 and SEQ ID NO : 10.

10. Vaccine composition according to claim 9, characterized in that it comprises the peptides of the SEQ ID NO : 14 and SEQ ID NO : 15.

11. Vaccine composition according to the claims 8-10, characterized in t h a t the peptides are dissolved in a sterile water solution and the optional immunostimulatory compound is a granulocyte macrophage colony stimulating factor.

12. Vaccine composition according to the claims 8-11, characterized in that the composition comprises an adjuvant selected from the group Monophosphoryl Lipid A (MPL®), Freund^'s complete or incomplete adjuvant or aluminum hydroxyd.

13. Vaccine composition, characterized in that an antigen according to claim 6 is formulated as a lipopeptide and/or a liposome formulation.

14. A method of detecting antibodies, induced by a HIV or HlV-specific peptides or proteins, in a sample of body fluid, characterized in that subjecting the said sample to an immunoassay, wherein the antigen(s) is/are selected from the peptides of the claims 1 , 2, 3 , 4 and 5.

15. An immunoassay kit for the detection of antibodies, induced by a HIV or HlV- specific peptides or proteins, in a sample of body fluid, characterized in that the diagnostic antigen is a peptide of any one of the previous claims 1 to 5.

16. Antibody, characterized in that it is capable of selectively reacting with the antigen of the claims 6 and 7.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAM E: Bionor Immuno AS

(B) STREET: Strømda]sjordet 4, P.O.Box 1893 Gulset (C) CITY: 3703 Skien

(E) CO UNTRY: Norway

(F) PO STAL CODE (ZIP): N-3705

(G) TELEPHONE: +47 35 50 57 50 (H) TELEFAX: + 47 35 50 57 01

(i) INVENTOR:

(A) NAME : Birger Sørensen

(B) STREET : Meierlia 3

(C) CITY : 3727 Skien

(D) COUNTRY : Norway

(ii) TITLE OF INVENTION: HIV regulatory and auxiliary peptides, antigens, vaccine compositions, immunoassay and a method of detecting antibodies induced by HIV. (iii) NUMBER OF SEQUENCES: 18

(iv) COMPUTER READABLE FORM:

(A) M EDIUM TYPE: Floppy disk

(B) CO MPUTER: IBM Compatible (C) O PERATING SYSTEM: Windows 2000

(D) SO FTWARE: Word 7.0

(v) CURRENT APPLICATION DATA: APPLICAT ION NUMBER:

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS: (A) LENG TH: 22-24 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No

(v) FRAGMENT TYPE : internal (ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 1

(D) OTHER INFORMATION: /note= " Xaa in position 1 is Met, Ser, Cys or none ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= " Xaa in position 2 is Glu, Asp, Val, Ser or none ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= " Xaa in position 3 is Ser, Gin, Pro, Leu, Val, Ala, Trp, Tyr or Phe ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 4 (D) OTHER INFORMATION: /note= " Xaa in position 4 is Val or lie ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 5 (D) OTHER INFORMATION: /note= " Xaa in position 5 is Asp, Asn or lie ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 6 (D) O THER INFORMATION: /note= " Xaa in position 6 is Pro, His or Ala ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 7 (D) OTHER INFORMATION: /note= " Xaa in position 7 is Arg , Asn, Ser, Lys, Glu or Asp ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 12 (D) OTHER INFORMATION: /note= " Xaa in position 12 is Leu, lie or Nle ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 15 (D) OTHER INFORMATION: /note= " Xaa in position 15 is Gly or Pro ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 16 (D) OTHER INFORMATION: /note= " Xaa in position 16 is Ser, Asn or Ala ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 17 (D) OTHER INFORMATION: /note= " Xaa in position 17 is Gin, Lys or Thr ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 18 (D) OTHER INFORMATION: /note= " Xaa in position 18 is Pro or His ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 19

(D) OTHER INFORMATION: /note= " Xaa in position 19 is Lys, Thr, Ser, Ala, Arg , Pro, Glu, Leu, lie or Nle ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 20 (D) OTHER INFORMATION: /note= " Xaa in position 20 is Thr, Ala, or lie ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 21 (D) OTHER INFORMATION: /note= " Xaa in position 21 is Ala, Pro, Asp or Val ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 22 (D) OTHER INFORMATION: /note= " Xaa in position 22 is Cys or Ser ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 23 (D) OTHER INFORMATION: /note= " Xaa in position 23 is Thr, Asn or Ser ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 24 (D) OTHER INFORMATION: /note= " Xaa in position 24 is Asn, Lys, Arg, Gin, Ala, Pro or

Thr ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 1

(D) OTHER INFORMATION: /note= " Cys in position 1 may form part of a disulphide bridge ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LO CATION: 22

(D) OTHER INFORMATION: /note= " Cys in position 22 may form part of a disulphide bridge

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

Xaa_! Xaa₂Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Leu Glu Pro Trp Xaa₁₂ His Pro Xaa-|₅ Xaa-|₆ Xaa-|₇ 1 5 10 15

Xaa-isXaa-ig Xaa₂oXaa₂-ι Xaa₂₂Xaa₂₃Xaa 20 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 1...22

(D) OTHER INFORMATION: /note= " Cys in position 1 and 22 may form part of an intrachain disulphide bridge

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :

Cys Ser Trp Val Asn Pro Arg Leu Glu Pro Trp Nle His Pro Gly Ser Gin His Nle Thr 1 5 10 15 20 Ala Cys Thr Asn

(2) INFORMATION FOR SEQ ID NO: 3 : (i) SEQ UENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 :

Phe Val lie Pro Arg Leu Glu Pro Trp Nle His Pro Gly Ser Gin Pro Nle Thr Ala Cys 1 5 10 15 20

Thr Asn

(2) INFORMATION FOR SEQ ID NO: 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24-27 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: single (D) TOPOLOGY: both (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 1

(D) OTHER INFORMATION: /note= " Xaa in position 1 is Pro, He, Leu, T hr, Tyr or Val ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 2

(D) OTHER INFORMATION: /note= " Xaa in position 2 is Val, Ala Cys, Leu,

ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 3

(D) OTHER INFORMATION: /note= " Xaa in position 3 is Cys, He, Leu, Val or Nle ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= " Xaa in position 5 is He, Leu, G in, Met or Thr

ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 6

(D) O THER INFORMATION: /note= " Xaa in position 6 is Thr, Asn, Lys or Arg ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= " Xaa in position 7 is Lys, Arg or Gin ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 8

(D) OTHER INFORMATION: /note= " Xaa in position 8 is Gly or Ala ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 9

(D) OTHER INFORMATION: /note= " Xaa in position 9 is Leu or lie ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 10

(D) OTHER INFORMATION: /note= " Xaa in position 10 is Gly, Ser or Ala ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: 11

(D) O THER INFORMATION: /note= " Xaa in position 11 is He or G ly ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LO CATION: 12

(D) OTHER INFORMATION: /note= " Xaa in position 12 is Ser, Phe or Tyr ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: Xaa, inserted before position 14

(D) OTHER INFORMATION: /note= " Xaa, inserted before the position 14 is Leu, lie, Nle

ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 15

(D) OTHER INFORMATION: /note= " Xaa in position 15 is Arg , Lys, Ser or Citrulline ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 19

(D) OTHER INFORMATION: /note= " Xaa in position 19 is Arg , Lys, Ser, Gly or Citrulline

ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 20

(D) OTHER INFORMATION: /note= " Xaa in position 20 is Gin, Arg or Pro ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LO CATION: 21 (D) OTHER INFORMATION: /note= " Xaa in position 21 is He or leu ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 22

(D) OTHER INFORMATION: /note= " Xaa in position 22 is Gly, Leu, lie, Cys or none ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 23 (D) O THER INFORMATION: /note= " Xaa in position 23 is G ly or none ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 12..13

(D) OTHER INFORMATION: /note= " optionally inserted linker -Z- ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 2, 3 and 22

(D) OTHER INFORMATION: /note= " Cys in the positions 2, 3 and 22 may form part of a disulphide bridge

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 :

Xaa-i Xaa₂ Xaa₃ Phe Xaas XaaβXaa_T-XaasXaagXaa_KjXaan Xaa₁₂ -Z-Tyr Xaai Gly 1 5 10 15

Xaa-₁5 Lys Lys Arg XaaιgXaa₂₀ Xaa₂₁ Xaa₂₂Xaa₂₃ 20 (2) INFORMATION FOR SEQ ID NO: 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 27 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 12..13

(D) OTHER INFORMATION: /note= " inserted G ly-bridge of 3 residues

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :

Tyr Leu Leu Phe Leu Thr Lys Gly Leu Gly He Ser Gly Gly Gly Tyr Nle Gly Cit Lys Lys 1 5 10 15 20

Arg Cit Gin He Leu Gly 25

(2) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERISTICS: (A) LENG TH: 28 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 12..13

(D) OTHER INFORMATION: /note= " inserted G ly-bridge of 3 residues ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 27

(D) OTHER INFORMATION: /note= " Cys in position 27 may form part of a disulphide bridge

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Tyr Leu Nle Phe Leu Thr Arg Gly Leu Gly He Ser Gly Gly Gly Tyr Nle Gly Cit Lys Lys 1 5 10 15 . 20

Arg Cit Gin He Cys Gly 25 (2) INFORMATION FOR SEQ ID NO: 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 25-28 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 1

(D) OTHER INFORMATION: /note= " Xaa in position 1 is Phe, Tyr, Trp or Arg ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 4

(D) OTHER INFORMATION: /note= " Xaa in position 4 is Gly, Ser, Asn, Asp, Cys, Val, Thr, Ala, or Arg ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LOCATION: 5

(D) O THER INFORMATION: /note= " Xaa in position 5 is Thr, Ala, Asp, Asn or Ser ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LO CATION: 6

(D) OTHER INFORMATION: /note= " Xaa in position 6 is Tyr, Cys, Phe, Arg, His, Ser, Val or Leu ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 10

(D) OTHER INFORMATION: /note= " Xaa in position 10 is Ser, Pro, Phe, Leu or He ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 11

(D) OTHER INFORMATION: /note= " Xaa in position 11 is Ala, Thr, Glu, Gin, Val, Pro or Ser ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 12

(D) OTHER INFORMATION: /note= " Xaa in position 12 is Glu, Lys, Gin, Asp, Asn, Tyr, Trp or Phe ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: Xaa, inserted after position 13 (D) OTHER INFORMATION: /note= " Xaa, inserted after position 13 is Ser, Pro, Phe, Leu or lie ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LOCATION: Xaai inserted before position 14

(D) OTHER INFORMATION: /note= " Xaai inserted before position 14 is Ala, Thr, Glu,

Gin, Val, Pro, or Ser

ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 14

(D) OTHER INFORMATION: /note= " Xaa in position 14 is Glu, Lys, Gin, Asp or Asn ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 15

(D) O THER INFORMATION: /note= " Xaa in position 15 is Pro, Ser, Ala or Asn ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 16

(D) OTHER INFORMATION: /note= " Xaa in position 16 is Val, Asn, G ly or Pro ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 17

(D) OTHER INFORMATION: /note= " Xaa in position 17 is Pro, Gin, His, Ser, Leu, Arg , Thr, Asp or He ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LO CATION: 18

(D) O THER INFORMATION: /note= " Xaa in position 18 is Leu Phe or Val ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LO CATION: 19 (D) OTHER INFORMATION: /note= " Xaa in position 19 is Gin, Leu, Pro, His, Asp or G lu ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LOCATION: 11..12 (D) OTHER INFORMATION: /note= " optionally inserted linker -Z- ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= " Cys in position 4 and 6 may form part of a disulphide bridge (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 :

Xaa_! He Leu Xaa₄Xaa₅ Xaa₆ Leu Gly Arg Xaa₁₀Xaan -Z- Xaa₁₂ Leu₁₃ Xaai Xaai Xaa₁₄ 1 5 10 15 Xaa-₁₅ Xaaι₆ Xaa₁₇ Xaa ₈ Xaa₁₉ Leu Pro Pro Leu 20 25

(2) INFORMATION FOR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 28 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 11..12

(D) OTHER INFORMATION: /note= " inserted [G ly]_n bridge, wherein n = 3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 :

Arg He Leu Ser Thr Tyr Leu Gly Arg He Ser Gly Gly Gly Trp Leu Ser Ala Glu Pro Val 1 5 10 15 20

Pro Leu Gin Leu Pro Pro Leu 25

INFORMATION FOR SEQ ID NO: 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 28 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 11..12

(D) OTHER INFORMATION: /note= " inserted [G ly]_n bridge, wherein n = 3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :

Arg He Leu Ser Thr Tyr Leu Gly Arg He Ser Gly Gly Gly Tyr Leu Ser Ala Glu Pro Val 1 5 10 15 20

Pro Leu Gin Leu Pro Pro Leu 25 (2) INFORMATION FOR SEQ ID NO: 10 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22-29 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 1

(D) OTHER INFORMATION: /note= " Xaa in position 1 is Lys or Arg ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 5

(D) OTHER INFORMATION: /note= " Xaa in position 5 is Phe or Leu ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 7

(D) OTHER INFORMATION: /note= " Xaa in position 7 is He or Val ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 8

(D) OTHER INFORMATION: /note= " Xaa in position 8 is Thr, Arg, Lys, Ala or Met

ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 10

(D) OTHER INFORMATION: /note= " Xaa in position 10 is Gin or His

ix) FEATURE:

(A) NA E/KEY: Modified-site (B) LO CATION: 11

(D) OTHER INFORMATION: /note= " Xaa in position 11 is Val, Leu or He ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: Xaa, inserted before position 13 (D) OTHER INFORMATION: /note= " Leu is inserted before position 13

ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: 13 (D) OTHER INFORMATION: /note= " Xaa in position 13 is Leu, Val or Thr

ix) FEATURE:

(A) NA E/KEY: Modified-site

(B) LO CATION: 14 (D) OTHER INFORMATION: /note= " Xaa in position 14 is Arg or Cit

ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 16

(D) OTHER INFORMATION: /note= " Xaa in position 16 is Met, Val, He, Leu or Nle ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 17

(D) OTHER INFORMATION: /note= " Xaa in position 17 is Thr or Asp

ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 18

(D) OTHER INFORMATION: /note= " Xaa in position 18 is Tyr, Phe or Arg

ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 19

(D) OTHER INFORMATION: /note= " Xaa in position 19 is Lys or Arg

ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LOCATION: 20

(D) OTHER INFORMATION: /note= " Xaa in position 20 is Ala, Gly, Ser, Glu or Gin

ix) FEATURE:

(A) NAM E/KEY: Modified-site (B) LO CATION: 21

(D) OTHER INFORMATION: /note= " Xaa in position 21 is Ala, Ser or Val

ix) FEATURE: (A) NAM E/KEY: Modified-site

(B) LO CATION: 12...(LeUjXaa₁₃)

(D) OTHER INFORMATION: /note= " optionally a linker -Z- and/or an [Arg]_m bridge is inserted between Xaa₁₂ and (LeUj Xaa₁₃), wherein m is 0,1 , 2 or 3.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 :

Xaa₁ Leu Val Gly Xaa₅ Pro Xaa₇Xaa₈ Pro Xaa-io Xaa^ Pro -Z- [Arg]_m LeUj Xaa₁₃ 1 5 10 Xaa-u Pro Xaa₁₆ Xaa₁₇ Xaa₁₈Xaa-ιg Xaa₂₀Xaa₂₁ 15 20

(2) INFORMATION FOR SEQ ID NO: 11 : (i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 26 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: both (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No ix) FEATURE: (A) NAM E/KEY: Modified-site

(C) LOCATION: 12...(LβU|Xaa₁₃....)

(D) OTHER INFORMATION: /note= " the linker -Z- is [Gly]_n wherein n is 3 and an [Arg]_m wherein m is 1.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11

Lys Leu Val Gly Phe Pro Val Lys Pro Gin Val Pro Gly Gly Gly Arg Leu Leu Cit Pro Nle 1 5 10 15 20 Thr Tyr Lys Ala Ala 25

(2) INFORMATION FOR SEQ ID NO: 12 : (i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 26 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site

(E) LOCATION: 12...(LeUjXaa₁₃)

(F) OTHER INFORMATION: /note= " the linker -Z- is [Gly]_n wherein n is 3 and an [Arg]_n wherein m is 1.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 :

Arg Leu Val Gly Phe Pro Val Lys Pro Gin Val Pro Gly Gly Gly Arg Leu Leu Arg Pro Leu 1 5 10 15 20

Thr Tyr Lys Ala Ala 25

(2) INFORMATION FOR SEQ ID NO: 13 :

(i) SEQ UENCE CHARACTERISTICS:

(A) LENG TH: 44 amino acids

(B) TYPE: amino acid (C) ST RANDEDNESS: double

(D) TOPOLOGY: both (ii) MOLECULE TYPE: dimeric peptide (iii) HYPOTHETICAL: No ix) FEATURE:

(A) NAM E/KEY: Modified-site

(B) LO CATION: disulphide-bond between position 20 in SEQ ID NO : 3 and position 20 in SEQ ID NO : 3

(2) INFORMATION FOR SEQ ID NO: 14 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: both (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 :

Phe Val He His Arg Leu Glu Pro Trp Leu His Pro Gly Ser Gin His Nle Thr Ala Ser Thr Asn 1 5 10 15 20

(2) INFORMATION FOR SEQ ID NO: 15

(i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 23 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: double (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 :

Arg Leu Val Gly Phe Pro Val Lys Pro Gin Val Pro Gly Leu Leu Arg Pro Leu Thr Tyr Lys Ala Ala 1 5 10 15 20

(2) INFORMATION FOR SEQ ID NO: 16 :

(i) SEQUENCE CHARACTERISTICS: (A) LENG TH: 24 amino acids (B) TYPE: amino acid

(C) ST RANDEDNESS: double

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No, tatl nativ sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 : Met Glu Ser Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gin Pro Lys Thr Ala Cys Thr 1 5 10 15 20

Asn

(2) INFORMATION FOR SEQ ID NO: 17 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 23 amino acids

(B) TYPE: amino acid (C) ST RANDEDNESS: double

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: No, tat2 nativ sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 :

Gin Val Cys Phe He Thr Lys Gly Leu Gly He Ser Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg 1 5 10 15 20

(2) INFORMATION FOR SEQ ID NO: 18 : (i) SEQUENCE CHARACTERISTICS:

(A) LENG TH: 21 amino acids

(B) TYPE: amino acid

(C) ST RANDEDNESS: double

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: No, nativ Nef sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 :

Glu Glu Val Gly Phe Pro Val Arg Pro Gin Val Pro Leu Arg Pro Met Thr Tyr Lys Ala Ala 1 5 10 21