NZ237417A - Synthetic hiv epitopic peptide (12 aa) and its use - Google Patents
Synthetic hiv epitopic peptide (12 aa) and its useInfo
- Publication number
- NZ237417A NZ237417A NZ237417A NZ23741791A NZ237417A NZ 237417 A NZ237417 A NZ 237417A NZ 237417 A NZ237417 A NZ 237417A NZ 23741791 A NZ23741791 A NZ 23741791A NZ 237417 A NZ237417 A NZ 237417A
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- NZ
- New Zealand
- Prior art keywords
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Description
New Zealand Paient Spedficaiion for Paient Number £37417
237 4 1 7
AMENDED under Section n m
Patents Act ! "53 from
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ASSISTANT COMMISSIONER OF PATENTS
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Patents Form No. 5
4r fh cfvuq-e. (
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CcM
NEW ZEALAND1
PATENTS ACT 19
COMPLETE SPECIFICS
SYNTHETIC POLYPEPTIDES
P'jT*LL
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(^LCl-V. TXUU«^.-<L<1 U\yMS 11/
1 -■/X^£C'L<!sfdd (JtwkJ Kt'XjsLuw
ME, PROTEUS MOLECULAR DESIGN LIMITED formerly
Proteus Biotechnoloty Ltd., a British company of Proteus House, 48 Stockport Road,
Marple, Cheshire, SK6 6AB, UNITED KINGDOM
hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:
(followed by page la)
■217 4
- la_-
55114/001.311
Synthetic Polypeptides
The present invention relates to synthetic 5 polypeptides. It particularly relates to synthetic polypeptides which emulate the three-dimensional structures and/or electrostatic surfaces and/or other physical, chemical and structural properties of specific regions of viral envelope proteins. It is of particular 10 interest to the design of vaccines, immunologically active therapeutic agents, diagnostics and other medical or scientific agents m relation to the Human Immunodeficiency Virus (HIV) known to be the causative agent of Acquired Immune Deficiency Syndrome (AIDS). 15 Within the last decade AIDS has emerged as an important medical problem throughout the world and there is currently an urgent need for agents for the study, diagnosis, treatment and/or prevention of infection by the HIV, the causative agent of the disease. With the 20 availability of the amino acid sequences of proteins produced by the HIV I and HIV II viruses (see, for example, Ratner, L. et al., Nature 313, 277 (1985); Meusmg, M.A. et al. , Nature 313. 450 (1985); Wain-Hobson, S. et al., Cell 40, 9 (1985)), it has been 25 possible to devise synthetic polypeptides which emulate the antigenic properties of the viral envelope proteins.
An object of the present invention is the development of synthetic polypeptides which can elicit the production of antibodies to the HIV virus, and most 30 preferably neutralising antibodies, that is, antibodies which prevent infection by and/or limit the spread of the HIV virus by passive or active immunisation.
Passive immunisation with such antibodies may constitute an effective means of treatment of AIDS patients thus 35 controlling the spread of the virus within and between individuals and hence slow or halt the progress of the disease.
Our invention provides a synthetic polypeptide
(followed by page 2)
— 2 —
&OW AMENDED
having at least one antigenic property of the envei/spe protein of at least one strain of Human Immunodeficiency Virus (HIV) , said polypeptide consisting substai}tially of an amino acid sequence of formula (I):-
X-R1 -R2-R3-R4-R5-R6-R7-Trp-Gly-Cys-Rs-R<?-R10-R11 -R12~Cys-Y
therein R is Asp or Glu;
R, is an amino acid res/due selected from
/ / Gly, Ala, Pro, Ser/ Thr, Asn or Gl'n,
R, is an amino acid^residue selectees from Gly, Ala, Pro, Ser, Thr, ^'sp, Glu,
3 7 ¥17
/
/
Asn, Lys, His/Gin or Arg;
/
Rs and Rn a»e each independently any amino acid/residue, /
R, and R0
ar/ i each independently an amino acid residue selected from Gly, Ala,
Pro, Ser, Thr, or Asnj
/
is aor amino acid residue selected from
/
Ala, Val, Leu.', lie, Ser, Thr, Asn,
Phe, Tyr, Trp, Cys, Met or Pro,
/
R12 are eac^/ independently an ammo acid residue selected from Gly, Ala, Leu, 1\&, Val, Met, Cys, Phe, Tyr or Trp,
is an amino acid residue selected from an ami1 , Hi/<
or Arg; and X and Y jpay each independently be absent or independently be one or more additional amino acid res/dues, with the proviso that when present, they dp not provide or form part of an epitope of the
/envelope protein of at least one strain of human immunodeficiency virus which is contiguous with the sequence to which X and Y are attached, according to formula I above without X and Y being
$
present will of course be useful, for example, m the production of antibodies to the HIV. However, when W or Y are present they may be any length but preferably less than 20 amino acids, more preferably less than lO^eg. 3 to 6. It will of course be
— — t
27ilLl313 j
^ 1 -
23 7 4 17
having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an ammo acid sequence of formula (I)"-
X-R1 -R2-R3-R4-R5-R6-R7-Trp-Gly-Cys—Rg-R,—R10-R„-R12—Cys-Y
(I)
rfnerem R1 is Asp or Glu;
R2 is an ammo acid residue selected from
Gly, Ala, Pro, Ser, Thr, Asn or Gin,
R3 is an ammo acid residue selected from Gly, Ala, Pro, Ser, Thr, Asp, Glu, 15 Asn, Lys, His, Gin or Arg;
R4, R5 and are each independently any amino acid residue,
R6 and Rs are each independently an ammo acid residue selected from Gly, Ala, 20 Pro, Ser, Thr, or Asn;
R7 is an ammo acid residue selected from Gly, Ala, Val, Leu, lie, Ser, Thr, Asn,
Gin, Phe, Tyr, Trp, Cys, Met or Pro,
Rg and R12 are each independently an 25 amino acid residue selected from Gly,
Ala, Leu, lie, Val, Met, Cys, Phe, Tyr or Trp;
R10 is an ammo acid residue selected from Lys, His or Arg; and 3 0 X and Y may each independently be absent or independently be one or more additional ammo acic residues, with the proviso that when present, the} do not provide or form part of an epitope of the envelope protein of at least one strain of human 3 5 immunodeficiency virus which is contiguous with the sequence to which X and Y are attached.
Peptides according to formula I above without X and Y beinc present will of course be useful, for example, m the production of antibodies to the HIV. However, when X or Y are present they may be any len^h"^u"tr^^Sf^ifc[Bly less than 20 amino acids, more preferably l&£"S"~tlraTrr
24 SEP 1933
ttr-
to 6. It will of course be
237 4
appreciated that the sequence according to formula I may-constitute a protein with X and Y being major portions of the protein with the antigenic sequence being for example, part of an exposed loop on a globular protein 5 Preferably, R2 is selected from Gin or Thr, R3 is selected from Ser, Asn, Gin, Arg or Ala, R4 is selected from Leu, lie, Gin or Arg, R5 is either Leu or Lys, R6 is either Gly or Asn, R7 is selected from Gly, Ala, Leu, lie, Val, Met, Cys, Phe, Tyr, Trp or Ser, Rs is either 10 Ser or Ala, R9 is either Gly or Phe, R10 is either Arg or Lys, Rn is selected from Leu, His, lie, Gin and R12 is selected from Ala, lie or Val. The Cys residues at positions 10 and 16 may optionally be linked by an mtra-molecular disulphide bridge.
One preferred form of polypeptide according to the invention consists substantially of an amino acid sequence of formula (II):-
X-Asp-Gln-R3-Leu-R5-Gly-R7-Trp-Gly-Cys-Ser-Gly-Lys-R^-R^-Cys-Y
(II)
wherein R3, R5, Rn, R12, X and Y are as defined above; and
R7 is an amino acid residue selected from
Gly, Ala, Leu, lie, Val, Met, Cys, Phe, Tyr or Trp.
Preferably in a sequence according to formula (II), R3 is selected from Ser, Asn, Gin and Arg, R^ is 30 selected from Leu, His or lie and R12 is lie or Ala.
Advantageously, R7 is selected from lie, Phe, Met, Val or Leu. More preferably R3 is either Ser or Asn and R5 is Lys.
A preferred form of polypeptide of formula (II) 3 5 according to the invention consists of the sequence
X-Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y
237 4
wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an mtra-molecular disulphide bridge.
Another preferred form of polypeptide of Formula 5 (II) comprises R3 selected from Gin or Arg, R5 is Leu, R? is selected from lie, Phe and Met, R^ is selected from Leu, His or lie, and R12 is Ala. A preferred sequence has the formula:-
X-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-
Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an mtra-molecular disulphide bridge.
Polypeptides according to formula II resemble certain epitopic portions of HIV I envelope protein.
Another preferred form of polypeptide according to the invention consists substantially of an ammo acid sequence of formula (III):-
X-R1-R2-R3-R4-R5-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y (III)
wherein R, is either Glu or Asp,
R2 is Thr or Gin;
2 5 R3 is an ammo acid selected from Ser, Asn
Arg, Gin or Ala;
R4 is an amino acid selected from Leu, lie,
Arg or Gin;
R5 is Lys or Leu; and
wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an mtra-molecular disulphide bridge.
A preferred form of a polypeptide according to
Formula III consists substantially of an ammo acid sequence of formula (Ilia):-
237 4 1 7
X-Glu-Thr-R3-R4-Lys-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y
(Ilia)
wherein R3, is an ammo acid residue selected from
Ser, Asn, Arg, Gin or Ala, R4 is an ammo acid residue selected from Leu, lie, Arg or Gin, X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an mtra-molecular 10 disulphide bridge. It is preferred that R3 is Ser when R4 is lie and when R3 is Ala R4 is either Arg or Gin but preferably Arg.
Polypeptides according to formula III are similar to certain epitopes of HIV II envelope proteins. 15 Preferred polypeptide sequences according to the invention were chosen on the basis of their topographical similarity to more than one antigenic determinant of the HIV envelope proteins. For example, an antigenic determinant to which a given polypeptide 2 0 was originally designed to be an analogue may also show topographical similarity to one or more other regions of the HIV envelope proteins possibly due to duplication of ancestral genes, or because the polypeptide is an analogue of a discontinuous determinant, or because the
2 5 polypeptides have been designed to be polyvalent. A
discontinuous epitope may be viewed as being composed of closely opposed sequential epitopes which may be of antigenic significance in their own right and a polyvalent polypeptide may contain two or more 30 (continuous or discontinuous) determinant analogues m a single polypeptide chain, thus providing a means to simultaneously elicit the production of a range of antibodies which will recognise two or more determinants on the HIV envelope proteins
3 5 Peptides according to the invention may be synthesised for example using either standard 9-fluorenyl-methoxycarbonyl (F-Moc) chemistry (see, for example, Atherton, E. and Sheppard, R. C. (1985) J.
23 7 4
Chem. Soc. Chem. Comm. 165) or standard butyloxycarbonate (T-Boc) chemistry. The correctness of the structure and the level of purity, which will normally be excess of 85%, should be carefully checked, 5 and particular attention be given to the correctness of internal disulphide bridging arrangements when present. Various chromatographic analyses, including high performance liquid chromatography, and spectrographic analyses, including Raman spectroscopy, may for example 10 be employed for this purpose.
All the sequences herein are stated using the standard I.U.P.A.C. three-letter-code abbreviations for ammo acid residues defined as follows: Gly-Glycme, Ala-Alanme, Val-Valme, Leu-Leucme, Ile-Isoleucme, 15 Ser-Serine, Thr-Threonme, Asp-Aspartic acid, Glu-Glutamic acid, Asn-Asparagine, Gln-Glutamme, Lys-Lysme, His-Histidine, Arg-Argmme, Phe-Phenylalanme, Tyr-Tyrosme, Trp-Tryptophan, Cys-Cysteme, Met-Methionme and Pro-Prolme.
2 0 Polypeptides according to the invention or antibodies thereto may be administered on their own or with other agents such as 3 ' -azido-3 1 -deoxythymidme (AZT) (zidovudine), which acts at a different level by interfering with the replication of the genetic material
2 5 of the virus, and/or HIV protease inhibitors, which block the action of an enzyme essential to the development of the virus.
Polypeptides according to the invention may be used to raise antibodies which will cross-react with envelope
3 0 proteins produced by a wide range of HIV I and/or HIV II
strains. Our analyses have shown that since the conformational/topographic/electrostatic properties of polypeptides according to the invention are such that they are highly likely to elicit the production of 3 5 antibodies which will cross-react with HIV envelope proteins from several or many strains, further advantages may arise from combining several variant polypeptides m a larger polypeptide. Such a
23 7 4 1 7
polypeptide may have the general formula (IV):
[La-^m-tLb-^n-Lc (IV)
wherein F and G may each independently be a polypeptide according to any one of Formulae I to Ilia, L is a linking sequence, a, b and c are each independently 0 or 1 and m and n are each positive numbers e.g. between 1 and 10 inclusive. L is preferably a short, 10 conformationally flexible section of polypeptide chain such as, for example and without limit Gly-Gly-Gly-Gly-Gly, Gly-Pro-Gly-Pro-Gly-Pro or Gly-Ser-Ala-Gly-Ser-Gly-Ala. It should be clear that each repeat may optionally have a different variant of a polypeptide according to 15 the invention.
Polyvalent determinant analogues as defined by Formula IV are referred to as pseudohomopolyvalent, wherein variants of essentially the same determinant analogue are repeated m a single polypeptide chain. In 2 0 addition, simple homopolyvalent polypeptide immunogens, which contain multiple copies of the same variant of one of the determinant analogues according to any one of formulae I to Ilia, would also be expected to be effective, and are also included within the scope of the
2 5 present invention.
Pseudohomopolyvalent immunogenic polypeptides are expected to be particularly valuable as vaccines, where they should elicit the production of a range of (neutralising) antibodies with a similar but 30 non-identical underlying specificity, which between them would cross-react with envelope protein from a wider range of HIV strains, and would thus be more effective at conferring protective immunity. There would also be advantages in constructing heteropolyvalent polypeptides
3 5 which contain one or more copies, in any order, of one of the polypeptides according to the present invention and one or more other polypeptide analogues of determinant analogues. Such polypeptides, which are
237 4 17
provided for in the present invention, have the general formula (V):
Ld-[G-L]m-F-[L"G]n-Le (V)
wherein F is a polypeptide according to any one of Formulae I to Ilia, G is a polypeptide according to any one of Formulae I to Ilia or other sequence, m and n are each positive numbers e.g. between 1 and 10 inclusive, and d and e are each independently 0 or 1. "L" is preferably a short, conformationally flexible section of polypeptide chain such as, for example and without limit Gly-Gly-Gly-Gly-Gly, Gly-Pro-Gly-Pro-Gly-Pro or Gly-Ser-Ala-Gly-Ser-Gly-Ala. In preferred forms of peptide V, G may comprise a polypeptide according to any one of Formula I to Ilia or the sequence:
X-Gln-Gln-Glu-Lys-Asn-Gly-Gly-Glu-Leu-Y
wherein each Gly may independently be replaced with any other amino acid and/or X and Y may each independently be absent or one or more e.g. three amino acid residues or G may comprise some other polypeptide sequence related to antigenic proteins from HIV.
It is to be understood that any antigemcally significant subfragments and/or antigemcally significant variants of the above-identifled polypeptide sequences which retain the general form and function of the parent polypeptide are included within the scope of this invention In particular, the substitution of any of the specific residues by residues having comparable conformational and/or physical properties, including substitution by rare (but naturally occurring, e.g. D-stereoisomers) or synthetic amino acid analogues, is included. For example, substitution of a residue by another m the same Set, as defined below, is included within the ambit of the invention; Set 1 - Ala, Val, Leu, lie, Phe, Tyr, Trp and Met; Set 2 - Ser, Thr, Asn
237 4 1 7
and Gin; Set 3 - Asp and Glu; Set 4 - Lys, His and Arg; Set 5 - Asn and Asp; Set 6 - Glu and Gin; Set 7 - Gly, Ala, Pro, Ser and Thr. D-stereoisomers of all ammo acid types, may be substituted, for example, D-Phe, D-Tyr and 5 D-Trp.
In preferred embodiments of the invention, X and Y if present may independently include one or more segments of protein sequence with the ability to act as a T-cell epitope. For example, segments of amino acid 10 sequence of the general formula 1-2-3-4, where 1 is Gly or a charged amino acid (e.g. Lys, His, Arg, Asp or Glu), 2 is a hydrophobic ammo acid (e.g. lie, Leu, Val, Met, Tyr, Phe, Trp, Ala), 3 is either a hydrophobic ammo acid (as defined above) or an uncharged polar 15 ammo acid (e.g. Asn, Ser, Thr, Pro, Gin, Gly), and 4 is a polar amino acid (e.g. Lys, Arg, His, Glu, Asp, Asn, Gin, Ser, Thr, Pro), appear to act as T-cell epitopes m at least some instances (Rothbard, J.B. & Taylor, W.R. (1988). A sequence pattern m common to T-cell 20 epitopes. The EMBO Journal 7(1): 93-100). Similarly segments can be of the sequence 11-21-3'-4'-51, wherein l1 is equivalent to 1 as defined earlier, 2' to 2, 3' and 4' to 3, and 5' to 4 (ibid). Both forms are included within the scope of the present invention and 25 one or more T-cell epitopes (preferably less than five) which may be of the type defined above or may be of other structure and which may be separated by spacer segments of any length or composition, preferably less than five ammo acid residues m length and comprising 30 for example residues selected from Gly, Ala, Pro, Asn, Thr, Ser or polyfunctional linkers such as non-a ammo acids. It is possible for a C- or N-terminal linker to represent a complete protein, thus obviating the possible need for conjugation to a carrier protein. 35 Also included within the scope of this invention are derivatives of the polypeptide according to formula I m which X or Y are or include a "retro-mverso" ammo acid, i.e. a bifunctional amine having a functional
23 7 4
group corresponding to an ammo acid. For example an analogue according to the invention and containing a retro-mverso ammo acid may have the formula:
R
I
A1-N-C-N-A2
I I I
H H H
where R is any functional group, e.g. a glycine side chain, and Al and A2 are preferably each a copy of one of the analogues defined herein (but not necessarily the same) attached by its N- or C-termmal end. T-cell epitopes may optionally be included as discussed
earlier.
Retro-mverso modification of peptides involves the reversal of one or more peptide bonds to create analogues more resistant than the original molecule to enzymatic degradation and offer one convenient route to
2 0 the generation of branched immunogens which contain a high concentration of epitope for a medium to large immunogen. The use of these compounds m large-scale solution synthesis of retro-mverso analogues of short-chain biologically active peptides has great potential.
It should be noted that analogues incorporating retro-inverso amino acid derivatives cannot be made directly using a recombinant DNA system. However, the basic analogues can, and they can then be purified and chemically linked to the retro-mverso amino-acids using
3 0 standard peptide/organic chemistry. A practical and convenient novel procedure for the solid-phase synthesis on polyamide-type resin of retro-inverso peptides has been described recently [Gazerro, H., Pinori, M. & Verdini, A.S. (1990). A new general procedure for the
solid-phase synthesis of retro-mverso peptides. In
"Innovation and Perspectives m Solid Phase Synthesis" Ed. Roger Epton. SPCC (UK) Ltd, Birmingham, UK].
The polypeptides are optionally linked to a carrier
237 4
- n -
molecule, either through chemical groups within the polypeptides themselves or through additional ammo acids added at either the C- or N-termmus, and which may be separated from the polypeptides themselves or 5 surrounded by one or more additional ammo acids, m order to render them optimal for their immunological function. Many linkages are suitable and include for example use of the side chains of Tyr, Cys and Lys residues. Suitable carriers include, for example, 10 purified protein derivative of tuberculin (PPD), tetanus toxoid, cholera toxin and its B subunit, ovalbumin, bovine serum albumin, soybean trypsin inhibitor, muramyl dipeptide and analogues thereof, and Braun1s lipoprotein although other suitable carriers will be readily 15 apparent to the skilled person. When using PPD as a carrier for polypeptides according to the invention, a higher titre of antibodies is achieved if the recipient of the polypeptide-PPD conjugate is already tuberculin sensitive, e.g. by virtue of earlier BCG vaccination.
2 0 In the case of a human vaccine it is worth noting that in the UK and many other countries the population is routinely offered BCG vaccination and is therefore largely PPD-sensitive. Hence PPD is expected to be a preferred carrier for use in such countries. 25 The mode of coupling the polypeptide to the carrier will depend on the nature of the materials to be coupled. For example, a lysine residue m the carrier may be coupled to a C-termmal or other cysteine residue in a polypeptide by treatment with N-7
3 0 -maleimidobutyryloxy-succmimide (Kitagawa, T. & Ackawa,
T. (1976) J. Biochem. 79, 233). Other coupling reactions and reagents have been described m the literature
The polypeptides, either alone or linked to a 3 5 carrier molecule, may be administered by any route (eg parenteral, nasal, oral, rectal, intra-vagmal) , with or without the use of conventional adjuvants (such as aluminium hydroxide or Freund's complete or incomplete
23 7 4
adjuvants) and/or other immunopotentiating agents. The invention also includes formulation of polypeptides according to the invention in slow-release forms, such as a sub-dermal implant or depot comprising, for 5 example, liposomes (Allison, A.C. & Gregoriadis, G. (1974) Nature (London) 252, 252) or biodegradable microcapsules manufactured from co-polymers of lactic acid and glycolic acids (Gresser, J. D. and Sanderson, J. E. (1984) m "Biopolymer Controlled Release Systems" 10 pp 127-138, Ed. D L. Wise).
It is to be understood that the polypeptides according to the invention may be synthesised by any conventional method, either directly using manual or automated peptide synthesis techniques as mentioned 15 above, or indirectly by RNA or DNA synthesis and conventional techniques of molecular biology and genetic engineering. Such techniques may be used to produce hybrid proteins containing one or more of the polypeptides inserted into another polypeptide sequence.
2 0 Another aspect of the present invention therefore provides a DNA molecule coding for at least one synthetic polypeptide according to the invention, preferably incorporated into a suitable expression vector replicable m microorganisms or in mammalian 25 cells. The DNA may also be part of the DNA sequence for a longer product e.g. the polypeptides may be expressed as parts of other proteins into which they have been inserted by genetic engineering. One practical guide to such techniques is "Molecular 30 cloning: a laboratory manual" by Sambrook, J., Fritsch, E.F. and Maniatis, T. (2nd Edition, 1989).
Polypeptides according to the invention may be used either alone or linked to an appropriate carrier, as:
(a) Peptide vaccines, for use to prevent infection by
3 5 one or more strains of HIV;
(b) As ligands m assays of, for example, serum from HIV positive patients;
(c) As quality control agents m testing, for example,
23 7 4
binding levels of antibodies raised against the polypeptides;
(d) As antigenic agents for the generation of monoclonal or polyclonal antibodies by immunisation of 5 an appropriate animal, such antibodies being of use for (l) the scientific study of the HIV virus, (n) as diagnostic agents, e.g. as part of histochemical reagents, (m) for the passive immunisation of HIV patients, either as a treatment for AIDS m itself, or 10 m combination with other agents such as, for example
AZT and/or HIV protease inhibitors, and (iv) as a means of targeting other agents (e.g. AZT or HIV protease inhibitors) to HIV infected cells expressing HIV envelope proteins on their surfaces, such agents either 15 being linked covalently or otherwise associated, e.g. as in liposomes containing such agents and incorporating antibodies raised against any of the antigenic polypeptides. The invention further provides for genetically engineered forms or sub-components, 20 especially VH regions, of antibodies raised against the polypeptides, and of humanised forms of antibodies initially raised against the polypeptides m other animals, using techniques described m the literature; and
(e) The treatment of HIV infections, either by displacing the binding of HIV virus to human or animal cells or by disturbing the three-dimensional organisation of the virus m vivo; as well as aiding the scientific study of HIV viruses m vitro. 30 In respect of detection and diagnosis, of HIV or antibodies against HIV, the skilled person will be aware of a variety of immunoassay techniques known in the art, inter alia, sandwich assay, competitive and noncompetitive assays and the use of direct and indirect 35 labelling.
A further aspect of the invention provides a kit for detecting HIV or antibodies against HIV which comprises at least one synthetic polypeptide according
23 7 4
to the invention.
The preparation of polyclonal or monoclonal antibodies, humanised forms of such antibodies (see, for example, Thompson K. M. et al (1986) Immunology 58., 157-5 160), single domain antibodies (see, for example, Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. and Winter, G. (1989) Nature 341. 544-546), and antibodies which might cross the blood-brain barrier, which bind specifically to a synthetic polypeptide according to the 10 present invention, may be carried out by conventional means and such antibodies are considered to form part of this invention. Antibodies according to the invention are, inter alia, of use in a method of diagnosing mammalian HIV infection which comprises incubating a 15 sample of tissue or body fluid of mammal with an effective amount of antibody as described herein and determining whether, and if desired the extent to which and/or rate at which, cross-reaction between said sample and said antibody occurs. Diagnostic kits which contain 20 at least one of said antibodies also form part of this invention.
A further aspect of the invention provides synthetic polypeptides for use in therapy or prophylaxis of mammalian HIV infection and/or stimulating the 25 mammalian immune system and/or blocking the cellular receptors for the HIV virus and for the preparation of medicaments suitable for such uses. Also included are pharmaceutical compositions containing, as active ingredient, at least one polypeptide or 30 polypeptide-carrier conjugate as described herein in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients. The compositions may be formulated for oral, rectal, nasal or especially parenteral administration (including intra-CNS 35 administration).
The invention further provides a method of therapy or prophylaxis of mammalian HIV infection and/or of stimulating the mammalian immune system and/or of
237 4 17
blocking the cellular receptors for the HIV virus, which comprises administering an effective amount of a polypeptide as hereinbefore defined, either in isolation or m combination with other agents for the treatment of 5 AIDS such as AZT and/or inhibitors of the HIV protease.
The following examples are intended to illustrate the invention and are not limiting m any way.
Example 1
A C-termmally extended form of basic peptide (a)
with sequence
Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys
was synthesised using standard solid-phase F-moc methodologies and an intra-molecular disulphide bridge was formed between the two Cys residues. The peptide was cleaved from the resin in the presence of 20 trifluoroacetic acid and subsequent purification of peptide was achieved by gel filtration, ion exchange chromatography and reverse phase high performance liquid chromatography. The purity of the resultant peptide was in excess of 85%. The C-terminal alanine was included to 25 assist the conjugation. The peptide was dissolved in phosphate-buffered saline (PBS; 5mg/ml) and mixed with an equal volume of ovalbumin (5mg/ml) prior to the addition of glutaraldehyde to a final concentration of 0.1%(w/v). The conjugate mixture was allowed to stand 30 for 30 minutes prior to emulsification with Freund's adjuvant. Each sheep (5 animals/group) was immunised with 250 fig of peptide m Freund's complete adjuvant (FCA) and subsequently (14 days) challenged with a further similar quantity m Freund's incomplete adjuvant 3 5 (FIA). Further challenges were performed after a period of 3-4 weeks in FIA. Blood samples were taken 7-10 days post-challenge and assayed for binding to HIV envelope protein.
237 4 17
The determination of anti-synthetic peptide antibodies which recognise HIV envelope protein was undertaken by employing a recombinant vaccinia virus (see, for example, Macket, M. and Smith, G. L. (1986) J.
gen. Virol. 67, 2067, for general methodology) which has been constructed to express the HIV envelope proteins on the surface of infected cells. This method of assay is preferred as binding to the antigen is measured on the surface of virus infected cells and hence may be more 10 representative than binding to solution phase antigen
(e.g. because certain potential epitopes on the envelope proteins may be masked m vivo through interaction with membrane phospholipids). CV1 monkey kidney cells, grown m monolayer cultures m 96-well microtitre plates, were 15 infected with the recombinant vaccinia virus. The virus construct contained the gene encoding the HIV envelope glycoprotein and this is known to be processed and expressed on the surface of the infected cells.
Antisera from sheep were assayed m duplicate. A 20 quantity of 50 nl of antiserum at various dilutions was added to the wells and incubated for 4 hours prior to washing (twice) and adding a second peroxidase-labelled anti-sheep antiserum. Positive wells were determined by reading the optical density m a microplate reader.
2 5 The anti-peptide antisera were found to cross-react with high affinity with HIV envelope protein expressed on the surface of infected cells (see Tables 1 and 1A).
These results confirm that these anti-peptide antibodies would be valuable as research tools and as diagnostic
3 0 agents
237 A 17
Table 1 Cross-reaction between serum from sheep immunised with conjugate of peptide (a) and recombinant HIV envelope protein expressed on the surface of monkey 5 kidney cells
Cross reaction
Control monkey
Monkey kidney
kidney cell line cell line
expressing HIV
envelope protein
Serum from
immunised
+++
sheep
Control serum
-
—
Table 1A
Typical titres of positive antisera from sheep immunised with a conjugate of peptide (a) when reacted 2 0 with recombinant HIV envelope protein expressed on the surface of monkey kidney cells
Mean optical density value
(serum dilution)
Serum sample (titre)
1
50
1 250
1
1250
1
31250
1
156250
Control sheep
0. 344
0. 288
0.197
0. 012
0. 016
serum
Peptide (a)
0.736
0. 636
0.432
0.140
0. 052
treated sheep
serum diluted
1/5000
The above table shows that even with significant dilution of the serum from test sheep (relative to the control) there is a greatly increased titre.
Example 2
40 One role of neutralising antibodies is to inhibit
237 4
the transmission of virus from infected to non-infected cells. Preventing or reducing the rate of transmission of virus from T-cells to macrophages is crucial since this may extend the life of AIDS patients considerably 5 Anti-peptide antisera were assayed for their ability to inhibit syncitia formation m vitro. Syncitia are multinucleated giant cells which result from "bridge"-formation between HIV-infected cells.
The C-termmally extended form of basic peptide (a)
with sequence
Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys
and containing an intra-molecular disulphide bridge between the two Cys residues was synthesised, purified, conjugated and used to immunise groups of sheep as described in Example 1. The anti-peptide antisera were used m an m vitro HIV neutralisation assay. The
2 0 anti-peptide antibodies were introduced into an in vitro culture of human T-lymphocytes infected with a highly virulent strain of HIV, and uninfected human macrophages. The anti-peptide antisera were found to inhibit syncitia formation amongst this mixed
population of T-cells and macrophages exposed to a virulent strain of HIV I, i.e. they inhibit spread of HIV infection from T-cell to macrophage.
The antibodies were found to prevent infection of the macrophages with HIV, and are therefore neutralising
3 0 m this m vitro assay, whereas the macrophages became infected m a control culture, as shown m Table 2.
237 4
Table 2 Neutralisation of virulent strain of HIV m in vitro culture of HIV-infected human T-lymphocytes and uninfected human 5 macrophages. "+++" indicates a high level of infection and indicates effective protection against infection.
Cell culture Level of infection of macrophages
HIV infected T-lymphocytes and uninfected +++ 15 macrophages only
HIV infected T-lymphocytes, uninfected 2 0 macrophages and anti-
peptide(a) antibodies
Also, positive antisera to peptide (a) were evaluated in a syncitium assay. This assay determines 25 the capability of antisera to prevent spread of live HIV from infected to uninfected cells and to prevent fusion between cells mediated through reactivity between the virus glycoprotein (gpl60) and the CD4 molecule. Measurement is carried out by syncitium detection and 3 0 enumeration.
The syncitium assay is performed as follows: a known concentration of an HIV producer (CD+4) cell line supporting active virus replication is washed three times and mixed with the antiserum under test and used 35 at specified dilutions. After incubation for 30 minutes at 37"C these cells are mixed, at specified proportions with an indicator CD4+ cell line, highly susceptible to HIV infection and syncitium induction. The cells are
237 4 17
observed daily for syncitium formation.
Antibodies to peptide(a) were found to inhibit syncitium formation as shown m Table 3.
Table 3 Syncitium inhibition by antisera between HIV-infected producer cells and an indicator cell line
Serum Sample Syncitium inhibition
Anti-peptide (a) antiserum + (3/5 animals)
Normal sheep serum
Example 3
A panel of sera from HIV-1 infected individuals m various stages of progression to acquired immunodeficiency syndrome disease (AIDS), ranging from asymptomatic to full-blow symptoms were obtained in order to produce a representative picture of the reactivity of sera from such patients with the synthetic peptides m ELISA.
The ELISA procedure determines the degree of reactivity of the synthetic peptides (analogous to regions of either gpl2 0 or the gp41 transmembrane protein of HIV-1) with antibody to the surface glycoprotein of HIV-1 in sera of HIV-1 positive individuals.
One hundred antisera from HIV positive individuals were reacted with synthetic peptide (a). Two of these sera (from asymptomatic individuals) contained HIV neutralising antibodies and cross-reacted with peptide (a) as shown m Table 4.
237 4
Table 4 Cross reactivity of peptide (a) with two HIV
positive antisera from asymptomatic individuals
Sample
OD (peptide fa))
Buffer
0. 056
Normal serum 1/100
0.476
1/1000
0. 140
HIV positive (1) 1/100
o cm a
1/1000
>2 . 0
HIV positive (2) 1/100
>2.0 )
>2.0 ) replicates
1.37 )
23 7 4 17
Claims (30)
1. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an amino acid sequence of formula (I): X-R^-I^-R^-R^-R^-Rg-R-y-Trp-Gly-Cys-R8-R9-R10-Rll-R12-Cys-Y (I) wherein R^ is Asp or Glu; Rj is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asn or Gin; R^ is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asp, Glu, Asn, Lys, His, Gin or Arg; R^, Rj. and R^ are each independently any amino acid residue; Rg and Rg are each independently an amino acid residue selected from Gly, Ala, Pro, Ser, Thr or Asn; R^ is an ammo acid residue selected from Gly, Ala, Val, Leu, lie, Ser, Thr, Asn, Gin, Phe, Tyr, Trp, Cys, Met or Pro; Rg and R12 are each independently an ammo acid residue selected from Gly, Ala, Leu, lie, Val, Met, Cys, Phe, Tyr or Trp; R10 is an amino acid residue selected from Lys, His or Arg; and X and Y may each independently be absent or independently be one or more additional amino acid residues, with the proviso that when present they do not provide or form part of an epitope of the envelope protein of at least one strain of human immunodeficiency virus which is contiguous with the sequence to which X and Y are attached.
2. A synthetic polypeptide as claimed in claim 1 where R2 is selected from Gin or Thr, R3 is selected from Ser, Asn, Gin, - 23 - 237 417 Arg or Ala, is selected from Leu, lie, Gin or Arg, is either Leu or Lys, Rg is either Gly or Asn, R^ is selected from Gly, Ala, Leu, lie, Val, Met, Cys, Phe, Tyr, Trp or Ser, Rg is either Ser or Ala, Rg is either Gly or Phe, R^q is either Arg or Lys, R^ is selected from Leu, His, lie, Gin and R^ is selected from Ala, lie or Val.
3. A synthetic polypeptide as claimed m claim 2 consisting substantially of an ammo acid sequence of formula (II): X-Asp-Gln-R^-Leu-Rg-Gly-R7-Trp-Gly-Cys-Ser-Gly-Lys-R^-R^-Cys-Y (II) wherein R^, R^, Rn' Ri2 are as defined in claim 2; Ry is an amino acid residue selected from Gly, Ala, Leu, lie, Val, Met, Cys, Phe, Tyr or Trp; and X and Y may each independently be absent or independently be one or more additional amino acid residues, with the proviso that when present they do not provide or form part of an epitope of the envelope protein of at least one strain of human immunodeficiency virus which is contiguous with the sequence to which X and Y are attached.
4. A synthetic polypeptide as claimed in claim 3 wherein R^ is selected from Ser, Asn, Gin and Arg, R^ is selected from Leu, His or lie and R^ is lie or Ala.
5. A synthetic polypeptide as claimed m claim 4 wherein R7 is selected from lie, Phe, Met, Val or Leu.
6. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an amino acid sequence of formula (II): - 24 - 23 7 4 1 7 X-Asp-Gln-Rg-Leu-Rjj -Gly-R^ -Trp-Gly-Cys-Ser-Gly-Lys-R-^-R.^-Cys-Y (II) wherein R^ is Ser or Asn; Rjj is Lys; R^ is selected from lie, Phe, Met, Val or Leu; R^ is selected from Leu, His or lie; R^2 1S He or Ala; and X and Y may each independently be absent or independently be one or more additional ammo acid residues.
7. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an amino acid sequence: X-Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y may each independently be absent or independently be one or more additional ammo acid residues.
8. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an ammo acid sequence of formula (II): X-Asp-Gln-R^-Leu-R<- -Gly-R^-Trp-Gly-Cys-Ser-Gly-Lys-R11~R12-Cys-Y (II) wherein R^ is Gin or Arg; Rg is Leu; R7 is selected from lie, Phe or Met; ! ,j 7 pateimt office i -- - 25 - WOW AMENDED is selected from Leu, His or lie; R^2 is Ala; and X and Y may each independently be abse be one or more additional amino acid residu 9. A synthetic polypeptide as claimed p.xi claim 8 consisting of the sequence: X-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Ly s -lieu-Ala-Cys -Y or independently wherein X and Y may each independently be Absent or independently be one or more additional amino Acid residues. 10. A synthetic polypeptides having at leajrc. one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Vi^rus (HIV), said^polypeptide consisting substantially of an aminp acid sequence/of formula (III): X-R1-R2-Rn-R2-Asn-Ser-Trc£Gly-Cys-Ala-Phe-Arg-Gln-V^^-Cys-Y (III) wherein RVis either Glu R2 is Thr or Gin; ammo acid se/ected •> / or Ala; R^ is an amino acid/selected Re; /is Lys or Leu; ^find independently be absent or Asp; from Ser, Asn, Arg, Gin from Leu, lie, Arg or Gin; 5/ wherein X and Y independently be one Sr more additional ammo acid residues. 11./ A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodef jrciency Virus (HIV), said polypeptide consisting substantially jbf an amino acid sequence of formula (Ilia): 2 7 JUL 1933 - 25 - } ~ 23 7 ^17 R^ is selected from Leu, His or lie; R^2 1S Ala; and X and Y may each independently be absent or independently be one or more additional amino acid residues.
9. A synthetic polypeptide as claimed in claim 8 consisting of the sequence: X-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y may each independently be absent or independently be one or more additional ammo acid residues.
10. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an amino acid sequence of formula (III): X-R^-R2-R3-R4-R5-Asn-Ser-Trp-Gly-Cys-Ala- Phe-Arg-Gln-Val-Cys-Y (III) wherein is either Glu or Asp; R2 is Thr or Gin; R^ is an amino acid selected from Ser, Asn, Arg, Gin or Ala; R^ is an amino acid selected from Leu, lie, Arg or Gin; R^ is Lys or Leu; and wherein X and Y may independently be absent or independently be one or more additional ammo acid residues.
11. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an ammo acid sequence of formula (Ilia): - 26 - 237417 X-Glu-Thr-R^-R^-Lys-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y (Ilia) wherein R^ is an amino acid residue selected from Ser, Asn, Arg, Gin or Ala; R^ is an amino acid residue selected from Leu, lie, Arg or Gin; wherein X and Y may each independently be absent or independently be one or more additional amino acid residues.
12. A synthetic polypeptide as claimed in claim 11 wherein R^ is Ser when is lie or is Ala when is Arg or Gin.
13. A synthetic polypeptide as claimed m any one of the preceding claims wherein Cys residues at positions 10 and 16 are linked by an intra-molecular disulphide bridge.
14. A synthetic polypeptide having the general formula (IV) : [L -F] -[L, -Gl -L 1 a Jm L d Jn c (IV) wherein F and G may each independently be a polypeptide according to any one of Formulae I to Ilia (as defined in claims 1, 3, 10 and 11), L is a linking sequence, each linking sequence L being the same or different, a, b and c are each independently 0 or 1 and m and n are each positive numbers.
15. A synthetic polypeptide having the general formula (V): L [G-L]-F-[L-Gl-L d L Jm L Jn e (V) - 27 - 237417 wherein F is a polypeptide according to any one of Formulae I to Ilia (as defined in claims 1, 3, 10 and 11)/ G is a polypeptide according to any one of Formulae I to Ilia or other sequence, m and n are each positive numbers and d and e are each independently 0 or 1.
16. A synthetic polypeptide as claimed in any one of the preceding claims including a retro-inverso amino acid.
17. A synthetic polypeptide which comprises an antigemcally significant subfragment or variant of a polypeptide as claimed m any one of claims 1 to 13.
18. A synthetic polypeptide as claimed m any one of the preceding claims additionally comprising at least one T-cell epitope.
19. A synthetic polypeptide as claimed in any one of claims 1 to 18 linked to a vaccine carrier.
20. A vaccine comprising at least one synthetic polypeptide as claimed in any one of claims 1 to 19 effective to promote immunity against at least one strain of HIV.
21. A kit for detecting HIV or antibodies against HIV which comprises at least one synthetic polypeptide as claimed in any one of claims 1 to 18.
22. A DNA molecule coding for at least one synthetic polypeptide as claimed in any one of claims 1 to 18.
23. A pharmaceutical composition containing, as active ingredient, at least one polypeptide as claimed in any one of claims 1 to 19 in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients. 28 23 7 4 1 7
24. Use of a synthetic polypeptide as claimed in any one of claims 1 to 19 for the preparation of a medicament for the therapeutic or prophylactic treatment of mammalian HIV infection and/or for stimulating the mammalian immune system and/or blocking the cellular receptors for the HIV virus.
25. An in vitro method of detecting HIV or antibodies against HIV or antigen binding fragments thereof, which comprises incubating a sample with at least one polypeptide as claimed in any one of claims 1 to 19.
26. An antibody or antigen binding fragment thereof which specifically binds to a synthetic polypeptide as claimed in any one of claims 1 to 19.
27. A diagnostic kit for detecting HIV or antibodies against HIV which contains at least one antibody as claimed in claim
28. A pharmaceutical composition containing, as active ingredient an antibody as claimed in claim 26 m association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients.
29. A pharmaceutical composition as claimed m claim 23 or m claim 28 further comprising AZT and/or HIV protease inhibitor.
30. A method of diagnosing mammalian HIV infection which comprises incubating a sample of tissue or body fluid of a mammal with an effective amount of an antibody as claimed in claim 26 and determining whether, and if desired the extent to which and/or rate at which, cross-reaction between said sample and said antibody occurs. 26 PROTEUS MOLECULAR DESIGN LIMITED r By their attorneys BALDWIN, SON & CAREY
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB909005829A GB9005829D0 (en) | 1990-03-15 | 1990-03-15 | Synthetic polypeptides |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ237417A true NZ237417A (en) | 1993-10-26 |
Family
ID=10672665
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NZ237417A NZ237417A (en) | 1990-03-15 | 1991-03-13 | Synthetic hiv epitopic peptide (12 aa) and its use |
Country Status (17)
Country | Link |
---|---|
EP (1) | EP0519986A1 (en) |
JP (1) | JPH05505188A (en) |
CN (1) | CN1054772A (en) |
AP (1) | AP211A (en) |
AU (1) | AU636735B2 (en) |
BR (1) | BR9106159A (en) |
CA (1) | CA2078220A1 (en) |
CS (1) | CS68091A2 (en) |
FI (1) | FI923800A0 (en) |
GB (1) | GB9005829D0 (en) |
HU (1) | HUT63179A (en) |
IL (1) | IL97551A0 (en) |
MX (1) | MX24890A (en) |
NZ (1) | NZ237417A (en) |
OA (1) | OA09671A (en) |
WO (1) | WO1991013909A1 (en) |
ZA (1) | ZA911886B (en) |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0565704A4 (en) * | 1991-11-04 | 1995-10-25 | Baxter Diagnostics Inc | Synthetic peptides corresponding to portions of hiv-2 virus and methods of using in an improved assay |
US5395750A (en) * | 1992-02-28 | 1995-03-07 | Hoffmann-La Roche Inc. | Methods for producing proteins which bind to predetermined antigens |
BR9306058A (en) * | 1992-03-11 | 1997-11-18 | Narhex Ltd | Amine derivatives of oxo- and hydroxy-substituted hydrocarbons |
MXPA93002392A (en) | 1992-03-11 | 2005-02-04 | Narhex Ltd | Amine derivatives of oxo- and hydroxy-substitued hydrocarbons. |
US5888992A (en) * | 1992-03-11 | 1999-03-30 | Narhex Limited | Polar substituted hydrocarbons |
US6071895A (en) * | 1992-03-11 | 2000-06-06 | Narhex Limited | Polar-substituted hydrocarbons |
GB9208428D0 (en) * | 1992-04-16 | 1992-06-03 | Proteus Molecular Design | Synthetic polypeptides |
US6511845B1 (en) | 1992-08-07 | 2003-01-28 | Alan R. Davis | Methods for producing an immune response against HIV-1 |
HUT71860A (en) | 1992-08-27 | 1996-02-28 | Deakin Res Ltd | Retro-, inverso-, and retro-inverso synthetic peptide analogues |
DE4402756A1 (en) * | 1994-01-31 | 1995-08-03 | Boehringer Mannheim Gmbh | Specific binding substances for antibodies and their use for immunoassays or vaccines |
AUPM411994A0 (en) * | 1994-02-25 | 1994-03-24 | Deakin Research Limited | Epitopes |
US6764682B1 (en) | 1994-06-16 | 2004-07-20 | Aventis Pasteur Limited | Adjuvant compositions containing more than one adjuvant |
US6290971B1 (en) | 1995-06-15 | 2001-09-18 | Aventis Pasteur Limited | Adjuvant compositions comprising a mineral salt and another immunostimulating compound |
CN1472314A (en) * | 2002-07-29 | 2004-02-04 | 清华大学 | Immunity expression of AIDS virus O and its use |
CN100445296C (en) * | 2006-03-31 | 2008-12-24 | 浙江大学 | Polypeptide organic compounds and application in xenogeneic transplantation |
CA2743904A1 (en) | 2008-11-17 | 2010-05-20 | The Regents Of The University Of Michigan | Cancer vaccine compositions and methods of using the same |
GB201612108D0 (en) | 2016-07-12 | 2016-08-24 | Univ Strathclyde | Preperation of non-ionic surfactant vesicles and variants |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8714802D0 (en) * | 1987-06-24 | 1987-07-29 | Proteus Biotech Ltd | Synthetic polypeptides |
EP0316695B1 (en) * | 1987-11-16 | 1993-03-31 | F. Hoffmann-La Roche Ag | Recombinant hiv-2 polypeptides |
-
1990
- 1990-03-15 GB GB909005829A patent/GB9005829D0/en active Pending
-
1991
- 1991-03-13 NZ NZ237417A patent/NZ237417A/en unknown
- 1991-03-13 BR BR919106159A patent/BR9106159A/en not_active Application Discontinuation
- 1991-03-13 JP JP3505817A patent/JPH05505188A/en active Pending
- 1991-03-13 EP EP91906077A patent/EP0519986A1/en not_active Withdrawn
- 1991-03-13 MX MX2489091A patent/MX24890A/en unknown
- 1991-03-13 HU HU922939A patent/HUT63179A/en unknown
- 1991-03-13 CA CA002078220A patent/CA2078220A1/en not_active Abandoned
- 1991-03-13 WO PCT/GB1991/000392 patent/WO1991013909A1/en not_active Application Discontinuation
- 1991-03-13 AU AU74679/91A patent/AU636735B2/en not_active Ceased
- 1991-03-14 AP APAP/P/1991/000245A patent/AP211A/en active
- 1991-03-14 IL IL97551A patent/IL97551A0/en unknown
- 1991-03-14 ZA ZA911886A patent/ZA911886B/en unknown
- 1991-03-15 CS CS91680A patent/CS68091A2/en unknown
- 1991-03-15 CN CN91101549A patent/CN1054772A/en active Pending
-
1992
- 1992-08-24 FI FI923800A patent/FI923800A0/en not_active Application Discontinuation
- 1992-09-11 OA OA60275A patent/OA09671A/en unknown
Also Published As
Publication number | Publication date |
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FI923800A (en) | 1992-08-24 |
BR9106159A (en) | 1993-03-16 |
EP0519986A1 (en) | 1992-12-30 |
CN1054772A (en) | 1991-09-25 |
JPH05505188A (en) | 1993-08-05 |
CA2078220A1 (en) | 1991-09-16 |
HU9202939D0 (en) | 1992-12-28 |
ZA911886B (en) | 1991-12-24 |
FI923800A0 (en) | 1992-08-24 |
MX24890A (en) | 1993-12-01 |
IL97551A0 (en) | 1992-06-21 |
AP211A (en) | 1992-10-21 |
AU636735B2 (en) | 1993-05-06 |
AP9100245A0 (en) | 1991-04-30 |
WO1991013909A1 (en) | 1991-09-19 |
OA09671A (en) | 1993-05-15 |
HUT63179A (en) | 1993-07-28 |
CS68091A2 (en) | 1991-12-17 |
AU7467991A (en) | 1991-10-10 |
GB9005829D0 (en) | 1990-05-09 |
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