CA2118033A1 - Synthetic polypeptides derived from the hiv envelope gylcoprotein - Google Patents

Abstract

2118033 9321218 PCTABScor01 A synthetic polypeptide has at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consists substantially of an amino acid sequence of the formula (I):
X-R1-Leu-R2-Leu-Thr-Val-Trp-Gly-R3-Lys-Y, wherein R1 is selected from Gln-Gln-R4-R5, Gln-R4-R5, R4-R5, R5 or R1 is absent; R2 is an amino acid residue selected from Gln, Lys, Glu or Arg; R3 is an amino acid residue selected from Ile, Thr or Ala; R4 is His or Glu; R5 is Leu or Met; and X and Y may each independently be absent or independently be one or more amino acid residues.
Preferred peptides are (Ia):
X-Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y; (Ib):
X-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y; (IIa) X-Gln-Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y; and (IIb):
X-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y and desirably X is absent and Y is Gly-Cys or Gly-Cys-Ala. The peptides are similar to certain epitopes of HIV
envelope proteins.

Description

,^- WO93/21218 h 1~ ~ D 3 3 PCT/GBg3/00808 SYNTHETIC POLYPEPTIDES DERIVED FROM THE HIV ENVELOPE GLYCOPROTEIN.

The present invention relates to synthetic polypeptides. It particuiarly 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 initerest to the desi~n of vaccines, immunologically active therapeutic agents, diagnostics and other medical or scientific agents in relation to the Human Immunodeficiency Virus (HIV) known to be the ca~sati~e 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 khe study, i;
: ~iagnosis, treatment and/or prevention of infection by ~: the HI~, the causative agent of the disease. With the 20 availabi1ity of the amino acid sequences of proteins ~.
~: ~ : produced by the HIV l and HIV 2 viruses (see~ for ~example, ~atner, L. et al., Nature 313r 277 (1985); .
Meusing, M.A. et al., Nature 313, 450 (19~5); Wain- .:
~obson, S. et al., Cell 40, 9 ~l985)), it has been .~`
:: 25 po~sible to devise synthetic polypeptides which emulatei the~antigenic properties of the viral envelope proteins~
: An object of the present invention is the :~
development of synthetic poly~eptides which can ~licit the production of antibodies to the HIV virus, and most preferably neutralising antibodies, that is, antibodies which pr~vent infection by and/or limit thei~pread of ! ii the ~IY YirUS by passive or active immunisation.
Passive immuni~ation with such antibodies may constitute an effective means of treatment of AIDS patients thus controlling the spread of the virus within and between individuals and hence slow or halt the ~ro~ress of the disease.
:: . o~r invention provides a synthetic polypeptide '.

W~93/21218 PCT/GB93/00808 f~,

2~ ~Q3~
-- 2 ~
havin~ at least one antigenic property of the en~elope protein of at least one strain of Human Immunadeficiency Virus (HIV), said polypeptide consisting substantially o~ an amino acid sequence of formula (I):-, ~ ;.
X~R1-Leu-R2-Leu-Thr-Val-Trp-Gly-R3-Lys-Y

wherein ~1 is selected from Gln-Gln R4-R5, Gln-F~-R~ R4~Rs~ Rs or R1 is absent;
R2 is an amino acid residue ~elected from Gln, Lys, Glu or Arg;
: R3 is an amino acid re~idue selecte~ from Ile, Thr or Ala; , ~4 iS His or Glu;
~ R5 is Leu or Met; and ~ ~
: : : X and Y may each independently be absent ~.
~' ~ or independently be one o~ moré~amino acid res~idues~
. No~mally,~hen R1 is Gln-Gln-R4-R5 and X and Y are ~-2~:~ present, X~and Y are not homologous with the natural enYelope protein:sequenre. More particularly/ when X
and/or~Y~are present and R1 is as defined above X and Y
do::~not pro~ide or ~orm part of an antigenic property of the~envelope~p~otein of at lea:st one strain o~Human 25 ~:Immunod~ficien:cy~irus. : ~ ;
Peptides accordin~to formula I above wi~hout X and Y being~present will of course be use~ul,: for èxam~le, in~the~production of antibodies to the HIV. Such : peptides will be:especially effectivs when coniugated to ~; ;:30 a ~arrier~im~lecu~l.e.~ ~, Howeve~, when !x~ or Y: are:~resent ~
hey may be an~ length but preferably less than~20:iamino .
acids~,~more~preferably less than 10, eg. 3 to 6O It will~of course ba ,appreciat2d that the s~u~nce~ -: :according to formula I may constitute a pr~tein~with~X
35~ and ~ béing major portions of the protein with the ntigenic sequence being, for exa~ple, part of an : exposed loop on a globular protein.
: . ' . ' ' ' : ~ ".

~,;. WO93/2121B 21 ~. 8 0 3 ~ PCT/CB93/00~08 ' ':':' Preferably, if R1 is present, R5 is either Leu or Met, more preferably Leu, and R~ if present, is His or Glu, more preferably His. R2 is preferably Gln, Lys or Glu, more preferably Gln and R3 is preferably Ile. ~
Preferably in a sequence according to formula (I), ';:
R1 is Gln-Gln-R4-R5 or is absent and when present, R4 is preferably His and Rs is Leu. Also it is preferable that R2 is Leu and R3 is Gln.
Preferred forms of polypeptide of formula (I) ,:
according to the invention consist of the sequences:' '' ' Seq. X.D. No: l X~Gln-Gln-~is-Leu-Leu-Gln-Leu-Th~-Val-Trp-~ly-Ile-Lys-Y ~:
(Ia); and :
:
Seq. I.D. No: 2 X-Leu-Gln-Leu-Thr-Val-Trp-Gly Ile-Lys-Y (Ib) ' ' "
~: wherein X and Y are as defined above aIthough it is :~
; 20 preferred that if X or Y are present they are relatively : ;:
short se~uences. Pre~erably, X is absent and Y is 2 or : 3 residues long, e.g. Gly-Cys or Gly-Cys-Ala. With .
regard to se~uences Ia and Ib above, it is preferred that X
is~absent and Y is Gly-Cys in sequence Ia and i5 Gly-Cys or .25 Gly-Cys-Ala in seguence Ib; such C-terminal extensions ; provide alternate sites for coupling:to a Garrier~
Polypep~ides acoording to formula I resem~le certain 2pitopic portions of HIV l en~Jelope protein. ::
A~other preferred form of polypeptide accordi~g to 30 the invention- consists substantially of an amino acid - ;
segu~nce:of formulal~tII~

X R1-Leu-Arg-Leu-Thr-Val-Trp-G1y-R3-Lys Y (~I3 ' . ~
:
: 35 wherein R1 is selected from Gln-Gln-Glu-R~, Gln-. Glu-~, Glu~ or ~1 i5 absent;
R3 is Thr ~r Alai R5 is Met or Leu; and : X and Y are as defined above. --CANCELLEO / ~NNUL~

WO~3/2121~ 2 1 18 ~ 3 3 PC~/GBg3/00808 ~

Preferably, RS, if present, is Met and R3 is preferably Thr. ~lso, it is preferable that R1 is Gln-Gln-Glu-Me~ or absent.
Preferred forms of polypeptide according to formula II consist of the sequences~

Seq. I.D. No: 3 ~.`
X-~ln-Gln-Glu-Met-Leu~Arg-Leu-Thr-Val-Trp-Gly~Thr-Lys-Y
(IIa); and Sec~. I . D:. No: 4 .
X-Leu-Arg-Leu-Thr-V~l-Trp-Gly-Thr-Lys-Y (IIb). .-..
Preferably, X is absent and Y is 2 or 3 residues long, e.g. Çly-Cys~ o~ Gly-Cys-Ala.
Polypeptides according to formula II are similar to ertain epi~op~s o~ HIV 2 envelope proteins.
Preferred polypeptide seyuences according to the ~:~
:20~ invention~were chosen on the basis~of their topographical similarity to one or more antigenic .:
determinants of the ~HIV envelope proteins. For example,~
a~n:ant~igenic determinant to which a given polypeptide was;:;o}iginally designed to be an analogue may also:~how~
2~ topo~xaphical 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 : ,~
ana1~gue~of a dis~onti~uous dete ~ inant, or because the polypeptides~have been designed to be polyvalent. ~A ~;: 30 ~discontinuous epitope may be vie~ed as ~eing composed of clbsely opposed sequential epitopes ~hich m~,y be ~f antigenic signi~icanc~ in their own right and a polyva1ent polypeptide may con~ain two or more::~
continuous or discontinuous) determinan~ analogu s in a 35 single polypeptide chain, thus providing a means ~o ~ :
simultaneously elicit the production of a range of a ffl ibodies which will recognise two or more dete~minants on the HIV en~elope proteins.
Peptides according to the invention may be ~ :

WO93~21218 21 1~ IJ 3 ~ PCT/GB93/00808 synthesised for example using either standard 9-fluorenyl methoxycar~onyl (F-Moc~ chemistry (see, for example, Atherton, E. and Sheppard, R. C. (1985) J.
Chem. Soc. Chem. Comm. 165) or standard butyloxycarbonate tT-Boc) chemistry. The correctness of the structure and the level of purity, which will :
normally be in excess of 85%, should be carefully checked.
Various chromatographic analyses, including high per~ormance liquid chromatography, and spectrographic analyses, may for example be employed for this purpose.
All the sequences herein are stated using the standard I.U.P.A.C. three-letter-code ab~reviations for amino acid residues d fined as follows: Gly-Glycine, Ala-Alanine, Val-Valine, Leu-Leucine, Ile-Isoleucine, Ser-Serine, Thr-Threonine, Asp-Aspartic acid, Glu-Glutamic acid, Asn-Asparagine, Gln-Glutamine, Lys-: : Lyslne, ~is-Histidine t Arg-Arginine, Phe-Phenylalanine t ::~ Tyr-Tyrosine, Trp-Tryptophan, Cys-Cysteine, ~et-Methionine and Pro-Proline.
For therapeutic applications, polypeptides ; according to the i~vention or antibodies thereto may be administered on their own or with other agents such as

3'-azido-3l-deo~ythymidine (AZT) ~zi~ovudine), which acts at a dif$erent level by inter~ering with the 25 ~rep1ication of the genetic material of the virus, and/or ;;~
. ~ HIV~-pro~ease inhibitors, which block:the action of an ~ .
:: ~ : enzyme~ essential to~ the development of the virus.
Polypeptides according to the invention ~ay be used ~: to raise antibodies which will cross-react with envelope ~proteins produced by a wide range of ~IV l and/or HIV 2 s~rains. Our ana~yses have shown t~at, since the : conformational/topographic/electrostatic properti~s of polypeptides according to ~he in~ention are such that they are highly likely to Plicit the;produc~ion of antibodies which will cross-react with HIV envelope proteins from several or many strains, fur~her advantages may arise from combining se~eral variant polypeptides in a larger polypeptide. Such a ~--:

WO93/Z1218 2 1 1 ~ ~ 3 3 6 - PCT/GB93/00808 f~-polypeptide may have the general formula (III):

[La~F}m~[~~G]n~Lc (III) wherein F and G may each independently be a polypeptide according to any one of For~ul~e I to IIb, L is a linking sequence, a, b and c are each independently O or l and m and n are each positive numbers e.g. ~etween l and lO inclusive. ~ is preferably a short, conformationally flexible section of polypeptide chain such as, for example and without limit (Seq. I.D. No: 5 Gly-Gly-Gly-Gly-Gly, (Seq. I.D. No: 6) Gly-Pro-Gly-Pro-Gly-Pro or (Seq. I.D. No: 7) Gly-Ser-Ala-Gly-Ser-Gly- ~
Ala. It shouId be clear that each repeat ma~ optionally ~:
have a different variant of a polypeptide according to the invention.
~ : Polyvalent dete~minant analogues as defined by Formula III are referred to as pseudohomopolyvalent, wherein~variants~ o essentially the same determinant ;~
~analogue~are repeated in a single polypeptide chain. In ~ addition, simple homopolyvalent polypeptide immunogens, ~
: which:contain multiple copies of the same variant of one : of:th~ det~.rmi~ant~analogue according to any one of ormulae I to IIb~, would also be expected ta be~
25~ effe~tive, and are a1so included within the~scope of the present inven~ion. ;~
Pseudohomopolyva~lent immunogenic polypeptides are :;; expected to be particularly valuable as vaccines, where they should elicit the production of a range of :~
(neutralisin~) antibodies with a similar ~ut non-identical unde~lying spec`ificity, which ~etween them ..
would cross-react with envelope protein from a wider range of HIV strains, and would~thus be more e~fective at conferring protective immunity. There would also be 35 :: ad~antages in constructing heteropolyYalent polypeptides : which contain one or more copies, in any order, of one ~ o~ the polypeptides according to the present invention -~ and one or more other polypeptide analogues of determinant analogues. Such polypeptides, which are :, :; , "

~ WO93/21218 ~ 1 3 ~.3~ ~ 3 PCT/G893/0080~
. ., :

provided for in the present invention, have the general formula (I~):
"

Ld-[G-L]m F-~L-G]n-Le (IV) ;
wherein F is a polypeptide according to any one of Formulae I to IIb, G is a polypeptide according to any on~ of Formulae I to IIb or other sequence, m and n are each positive numbers e.g. between l and lO inclu~ive, and d and e are each independently 0 or l. I'L" is preferably a short, conformationally flexible section vf polypeptide chain:such as, for example and without limit (Seq, X~D. No: 5) Gly-Gly-Gly-Gly-Gly, (Seq. I.D.
No: 6) Gly-Pro~Gly-Pro-Gly-Pro or (Seq. I,D. No: 7) Gly- ;
15 Ser-Ala~Gly Ser-Gly-Ala. .:
It is to be understood that any antigenically ; significant subfragmen~s and/or antigenically significant variants of the above-identified polypeptide :~
: sequences which rétain the general form and function of ` 20 the parent polypeptide are included within the scope of this invention. In particular, the substitution of any of:thè:specific residues:by residues having comparable : ` conformational and/or physical properties, including . substitution by rare amino acids ~e.g. D-stexeoisomers~
:25 ` or s~nthetic amino acid analogues, is included:O For :example~, substituti~n of a residue by another in ~he same~Set, as defin d below, is included within the ambit of the in~entionl Set 1 - Ala, Val, Leuf Ile, Phe, ~yr, Trp and Met; Set 2 - Ser, Thr, Asn and Gln; Set~3 - Asp : 30 and 1u; Set 4 - Lys, His and Arg; 5et 5 - ~sn and Asp;
~: - Set 6 - ~lu and ~In; Set 7 - Gly, Ala, Pro, ~erland Thr.
D-stereoisomers of a1l amino acid types, may be : :subs~itutéd, for example, D-Phe, D-Tyr and D-T~p.
In preferred embodim~nts of the inven~ion, X and Y
~if present may independently include one or~more se~ments of protein sequence with the ability to act as a T-cell epitope. For example, segments of amino acid : 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 , fJ 1 ,,~
WO93/21218 PCT/GB93/00808 ~ ;

Glu), 2 is a hydrophobic amino acid (e.g. Ile, Leu, Val, Met, Tyr, Phe, Trp, Ala), 3 is either a hydrophobic amino acid (as defined above) or an uncharged polar amino acid (e.g. Asnj Ser, Thr, Pro, Gln, Gly), and 4 is 5 a polar amino acid (e.g~ Lys, ~rg, His, ~lu, Asp, Asn, ~:
Gln, Ser, Thr, Pro), appear to act as T-cell epitopes in ~ ~
at least some instances (Rothbard, J.B. & Taylor, W.~. :
(1988). A sequence pattern in common to T-cell epitopes. The EMBO Journal 7(l): 93-lO0). Similarly segments can be of the sequence 1'-2'-3'-41_51, wherein 1' is equivalent to 1 as defined earlier, 2' to 2, 3' and 4' to 3, and 5' to 4 (ibid). Both forms are included wi~hin the scope of the present invention and one or more T-cell epitopes (preferably less than five) may be incorporated into a polypeptide according to any one of formulae I to IIb. Therefore each epitope may be of the type defined above ox may be of other structure and may be separated by spacer segments of any length or composition (preferably less than five amino acid 20 residues in lengthj and comprise for example residues `~
selected from Gly, Ala, Pro, Asn, Thr, Ser or polyfun~tional linkers such as non-~ amino acids. It is : possible for a C- or N-terminal linker to represent a :complete protein, thus obviating the possible need for ~conjuga~ion to a carrier protein~
:; : Also included within the scope of this invention ~: ~ are derivati~es of the polypeptide according to formula .`
; I in which X sr ~ are or include a "r~tro inverso" amino acid, i.e. a bifunctional amine ha~ing a functional ~;
group corresponding to an amino acid. For example an analogùe according ~o the in~entioniand containing a :
retro i~verso amino acid may have the formula:
R ~

Al - N - C - N - A2 H H H

:

.~- WO93/21218 21 ~ 0 3 3 PCT/GB93/00B08 g _ , where R is any funotional 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) a~tached by its N- or C-terminal end~ T-cell epitopes may optionally be included as discussed earlier.
Re-tro-in~erso 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 the generation of branched immunogens which contain a ~;
high concentration of epitope for a medium to large immunogen. The use of these compounds in large-scale ;~
solution synthesis of retro-inverso analogues of short- ~:
chain biologically ac~ive peptides has great potential.
It should be noted that analogues incorporating retro in~erso amino acid derivatives cannot be made directly using a recombinant DNA system. However, the basic analogues can, and they can then be purified an~
chemically linked to the retro-inverso amino-acids using 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. &
;~ ~ 25 Verdini, A.S. (199Oj. A new general procedure for~the ~.
- solid-phase synthesis:of retro-inverso peptidPs. In 'Innovation and Perspecti~es in Solid Phase Synthesis~
Ed. Roger Epton.::~ SPCC (UK~ Ltd, Birmingham, UK].
The polypeptides are opkionally linked to a carrier molecule, either ~hrough chemical groups within the polypeptidés the~selves'~or through additional amino l i :
`acids added at eikher the C- or N-terminus, and which :~
may ba separated ~rom the polypeptides themselves or surrounded by one or more addikional amino acids, in 3S order to render them optimal ~or their immunological function. ~ Many linkages are suitable and include fo~
xample use of the side chains of Tyr, Cys and Lys residues~ Suitable carriers include, for example, WO93/21218 2 11. ~ O ~ ~ PCT/GB93/00808 r~

- 10 -- .
purified protein derivative of tuberculin (PPD), tetanus toxoid, cholera toxin and i~s B subunit, ovalbumin, bo~ine serum albumin, soybean trypsin inhibitor, muramyl dipeptide and analogues thereoff and Braun's lipoprotein although other suitable carriers will be readily apparent to the skilled person. For example, multiple , ankigen peptides may be used such as those comprising a polylysyl core, e.g. heptalysyl, bearing reactive amin~
termini. Polypeptide antigens according to the in~ention may be reacted wi~h, or synthesised on, the amino termini and different polypeptide antigens may be reacted with the same core or carrier.
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 vlr~ue of earlier BCG vaccination. In the case of a human vaccine it is worth noting that in the UK and many :other Gountries the population is routinely offered BCG
:: 20 vaccination and is therefore largely PPD-sensitive.
;: Hence PPD is e~pected to be a preferred carrier for use in such countries.
The mode of coup~ing the polypeptide to th oarrier ~.
will depend on the nature of the materials to be : 2S coup1ed~ For example, a lysine residue in the carrier may be coupled to a C-terminal or other cysteine residue in a polypeptide by treatment with N-~~maleimidobutyryloxy-succinim~de (Kitagawal T. & Ackawa, : ~ T. ~1976) 3. Biochem. 79, 233). Other coupling reactions and reagents have been described in the iiterature. ; ~
The polypeptides, either alone or linked to a carrier moleculP, may be administered by any route (eg parenteral, nasal, oralg rectal, intra-vaginal), with or 3S without the use of conventional adjuvants (such as aluminium hydroxide or, when not ~or administration to :: humans, FreundCs complete or incsmplete adjuvants~ :
- and/or other i~munopotentiating agents. The invention .

h 11~ 0~ ~
. WO93/21218 PCT/GB93/00808 -- 11 - . .
also includes formulation of polypeptides according to the inven~ion in slow-release formsl such as a sub-dermal implant or depot comprising, for example, liposomes (Allison, A.C. & Gregoriadis, G. (1974) Nature (London) 25~, 252) or biodegradable microcapsules manufactured ~rom co-polymers of lactic acid and glycolic acids (Gresser, J. D. and Sanderson, J. E.
(1984) in l'Biopolymer Controlled Release Systems" 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 us.ing manual or automated peptide synthesis techniques as mentioned above, or indirectly by ~NA or DNA synthesis and conventional techn.i~ues of molecular biology and genetic engineering. Such techniques may be used to produce hybrid:proteins containing one or more of the polypeptides inser~ed into another polypeptide se~uence.
Ano~her aspect of the presen~ invention therefore ~20 provides a DNA molecule coding for at least one synthetic~polypeptide according to the invention, pre:ferabIy incorporated into a suitable expression vector~replicable in microorganisms or in mammalian, ins~ct, p~lant, fungal or other cells. The DNA may also 25 ~ be;part:;:of the DNA:se~uence for a longer product:e.g.
. ~ the polypeptldes~ may be expressed as parts of other prote~in into which they have been inserted by geneti~
engineering. On2 practical guide to ~ch techniques i5 'Molecular cloning: a }aboratory manuali' by Sambrook, ~:
J., Fritsch, E.F. and Maniatis, T. (2nd ~dition, 1989j.
:i Polypeptides according to the`ihven~ion may be usedi ~:
either alone or linked to an appropriate carrier, as.
(a) ~Peptide vacGines~ for use to prevent infec ion ~y one or more strain of HIV; ~
lb) ~s ligands in assays of, for example, s~ra from HIV
: posi~ive patients; : -:; ~(c) As quality control agents in testing, for example, binding levels of antibodies raised against the ~'.
:

WO93/21218 2 ~ 1 8 0 ~ 3 PCT/CB93/00808 polypeptides;
~d) As immunogenic agents for the generation of monoclonal or polyclonal antibodies by immunisation of an appropriate animal, such antibodies being of use for 5 (i) the scientific study of the HIV virus, (ii) as ~:
diagnostic agents, e.g. as part of histochemical . ~ :
reagents, (iii) for the passive immunisation of ~IV
patlents, either as a treakment for AIDS in itself, or in 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 .
being Iinked covalently or otherwise associated, e.g. a~
in liposomes containing such agents and incorporating antibodie~ raised against any of the antigenic : ~ :polypeptides~ The invention further provides for gene~ically en~ineered forms or sub-components, espec:ially VH rPgions ~ of antibodies raised against the ~:20 polypeptides, and of~humanised forms of antibodies : :initially raised against the polypeptides in other ~.
animals, using techni~ues described in the literature;
and~
e~The:treatment of HIU infections,~e~ither by ~displ:acing the binding of HIV virus:to human or animal cells::or by disturbing the three-dimensional organisation of the virus in vivo;~as well~as aiding the scientif ic study of HIV viruses in vi~ro. ::
In respect of~detection and diagnosis,~of HI~ or 30 antibodies against HIV, the skilled person will be aware `~
of a~varie~y of~imm~unoassay techniques known i~ ~he a~t j ;
inte ~ ~ a, sandwich assay, ~ompetitive and non-competitive assays and the~use of direct and indirect labelling. - ~;
35 ~ A further aspect of the inYention provides a kit~ ~ ;
for detecting HIV or antibodies against HIV which comprises at least one synthetic polypeptide according to the invention.

~ .

~: ' A ~ WO 93t21218 ~ 3 t PCT/GB93/008U8 The preparation of polyclonal or monoclonal antibodies, humanised forms of such antibodies (see, for example, Thompson K. M. et al (1986) Immunology 58, 157-160), single domain antibodies (see, for example, Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. and Winter, G. (l~9) Nature 341, 544-546), and antibodies .
which might cross the blood-brain barrier, which bind specifically to a synthetic polypeptide according to the present invention, may be carried out by conventional means and such antibodies are considered to form part o~
this invention. Antibodies according to the invention are, inter alia, of use in a method of diagnosing ;
mammalian HIV infection which comprises incubating a .. . .
sample of tissue or body fluid of mammal with an ~;
15 effective amount of antibody as described herein and :-determining whether, and if desired the extent to which and/or rate at which, cross-r~action between said sample and said antibody occurs. Diagnostic kits which contain ~:~
~: at lea~t one of said antibodies also form part of this : ;, O invention.
A ~urther aspect of the invention pro~ides synthetic polypeptides ~or use in therapy or:prophy1axis -:
of mammalian HIV infection and/or stimulating the mammalian immune: system and/or blocking the:cellular 25~ receptors for t:e HIV virus and for the preparation of m~dicaments suitab~le for such uses. Also included are pharm~ceutical compositions containing, as active~
ingredient, at least ~one poly~eptide ~r polypeptide-carrier conjugate as described herein:in 30 ~association with one or more pharmaceutically acceptabl~ ~;
a;d~uvant~lcarrier~and/or1excipient. iThe:compo~it~lons may be formulated for oral, rectal, nasal or especially -:
parenteral administration (including intra-C~S
administration).
The invention further provides a method ~f therapy : : or prophylaxis of mammalian HIY infection and/or of stimulating the mammalian immune system and/or of ~; blocking the cellular receptors for the HIV virus, which : :

WO93/21218 h 1 1 3 0 ~ 3 PCT/GB93/00808 ~c~

comprises administering an effective amount of a polypeptide as hereinbefore defined, either in isolation or in combination with other agents for the treatment of AIDS such as AZT and/or inhibitors of the HIV protease.
The following examples are intended to illustrate the invention and are not limiting in any way. ~ :

ExamPl e ~
A C-terminally extended form of peptide Ib of lO sequence (Seq. I.D. No. 8) ~:;

Leu-Gln-Leu~Thr-Val-Trp-Gly-Ile-Lys-Gly-Cys-Ala :~

: was synthesised using standard solid-phase F~moc ~;
methodologies (Atherton, E. and Sheppard, RoC~ ~ 1985;
J.Chem. Soc~ Comm. 165-166). The C-terminal Gly-Cys-Ala ~
was introduced to provide an alternative coupling site ::
to the carrier. The peptide was cleaved from the resin in the presence of tri~luoroacetic acid and subsequent purification of peptide wa5 achieved by gel filtration, ion exchange chromatography and reverse-phase high : : per~ormance li~uid chromatoyraphy. The purity of the resultant peptide was in excess of 85%. The C-terminal ;.
aIanin was included to assist the conjugation.~ The peptid was di5sol~ed in phosphate-buffered saline ~PBS;
5mg~ml:) and mixed with an equal ~olume of ovalbumin~ -(5mg/'mI~ prior to the:addition of glutaraldehyde to a final con entration of 0.1%(w/v)~ The conjugate mixture ::
5 allowed to s~and for 30 minutes prior to em~lsification with Freund's adjuvant. Each sheep (5 animals'in éach group) was;immunised~with the eguivatentl ;
of 250 ~g of peptide in Freund's Complete Adjuvant (F~ ach animal was given a boo5ter injection at 2 weeks and then again at 5-6 weeks with a similar amount 3S ~ of peptide in Freund's Incomplete AdjuYant (F A). Blood ~: : samples were taken 7-lO days after the final booster injecti~n and assayed for binding to HIV gpl60 en~elope ~ protein.
; ~wo HIV-l strains were used in the study. One of .

h . ~ ;,t,~
,.-. . WO 93/21218 PCT/GBg3/00808 these was a fully~characterised, well-recognised and widely~used HIV-l strain named GB8 available from Dr G~
Farrarr, Centre for Applied Microbiology and Research .
(CAMR), Porton Down, Salisbury, UK and discussed in AIDS 1: 147-150 (1987). Th~ other was the RF strain of HIV-1 used in the syncytium inhibition assay. RF strain of HIV-1 is known from Science 224: 497~500 (1984).
Sequences from two or more HIV-1 isolates from a single individual have been obtained and the capabil.ities of the test materials to inhibit growth and replication of these ~irus strains wexe assessed comparatively. The isolates were obtained from Dr~ G. Farrarr under designations GB8A, GB8B and ~B8D.
Cel1 lines ~9, C8166 and JM are known to support replication or show syncytium formation when infected with HI~-1 'laboratory strains' of GB8 or RF.

) Assa~s of inhibition of live HIV-I antl-~eptide . antisera :
Three standard assay procedures have been used to assess :, the inhibition of live HIV-l by anti-peptide antisera. :~
; ~ : : , , i) Neut:ralisation assay : ~ ~
: Thi5 a6say measures the capability of~the anti-peptide anti6era ~o prevent attachment and infection of su6ce~tible ceIls by live, ce}l-free virus. The measurement of this is determined by syncytium counts.
: 30 Tèst~antisera wer~ inactiivated ~y heat at 56~C for 30 ;!
; :minutes, diluted appropriately in RPMI 1~40 and incubated with an equal volume of HIV-l conta~ning fluid .
of known titre for 30 minutes at room temperature ~RT) .
A CD4~ cell line (JM) at a known concentration and highly susceptible to infection with HIV-l GB8 strain : was exposed to the HIV-l antiserum mixture, left for 2 hours at 37C, washed, resuspended in cell culture medium, incu~ated and assessed by syncytium cvunts at ~; ~

WO93/21218 2 1 ~ 8 0 ~ ~3 PCT/GB93/00808 ,~
- 16 ~
specified times. Neukralising activity of the antisera ~;
was assessed by reduction in syncytium counts compared to controls.
,~
S Controls included with each batch of tests were known negatîve and positive sera replacing the test antiserum .
in an otherwi~e identical procedure.

(ii) Inhibition Assay , This assay de-termines the ability of the anti-peptide antisera to inhibit replication of HIV-l subsequent to cellular infection and measures levels of extracellular and intracellular HIV~l p24 antigen. In addition, 1~ syncytium detection and enumeration are perfo~med. ~;

A highly susceptible CD4+ cell line was exposed to HIV-l at a known multiplirity of infection and incubated ~or 2 hours at 37C. The ceIls were then washed 3 times with : 20 ~PMI 1640 and an equal volume of appropriately diluted, : heat-inactivated test antiserum was added. The infected cell serum suspension was incubated for l h~our at 37C, amL~ed microscopically and the culture medlum sampled : :at~specified time-points subsequently (at days 3 and 5~ ;
~or measurement of p24 antigen. Intracellular p24 ~ntigen lavels wers determined at similar time points ~ollowing lysis o~ the cells using appropr}ate lysis bu fer. Samples to be tested.for HIV l p24 antigen by ; ELISA were stored until the assays were performed.
Syncytium counts were measured on day 5.

~on~rols for each batch of tests included known negative ,, and po~itiVQ sera replacing the test antiserum in an otherwise identica1 procedur Syncytium assay This as ay determinPs the capability of anti-peptide antisera to prevent spread of live HIV-l from infected J~3 to uninfected cells and to prevent fusion between cells mediated through reactivity between the virus ~:
glycoprotein (gpl60) and the CD4 molecule. The measurement is carried out by syncytium detection and 5 enumeratio~O ~:

A known concentration of an HIV-l producer ~CD4~) cell line supporting active virus replication (H9 cells chronically infected with HIV-l RF strain) was washed 3 ~;
; 10 times and mixed with the antiserum under test used at specified dilutions. After incubation for 30 minutes at 37C these cells were mixed in specified pxoportions :~
~: with an indicator CD~+ cell line (C8166), highly susceptible to HIV-l infection and syncytium induction.
These cells were observed dail~ for syncy~ium formation~

Controls for each batch of tests included known positive ~.
and negative sera replacin~ the test serum in an ,.
otherwise identical procedure.
; 20 . RESULTS

Neutralisation:assay 25: ~A number of sera raised to peptide Ib~exhibited in vitrQ
ne~tralising activity against HIV-l GB8 strains~ Table : 1 indicates greater acti~ity of serum 17 ayains~ all GB8 sequential isolates. The assay demonstrates that the sera neutralise~cell-free virus.

: : :

`

:' ' ~ . ' ' ' ~::: ' ,~.

W093/~l2l8 21 1 ~ 0 3 3 PCT/GB93tO0808 ~

Table 1 : Anti-peptide sheep sera showing in v}tro neutralising activity against HIV-1 GB8 strains ~ ~ . __ .
Serum Serum Neutralising activity*
against HIV-l strain :.
~ _ Dilution _ _ _ _ _ ~ ~.
Lab No. GB8 GB8AGB8B GB~D ;
. Tested , - , _ ~ _ _ _ 17 1:5 4/72(g4) 0/98(100)3/80(96) 19/77(75) .~;
1:10 13/72(~2) 14/98(86)22/8~(72) 21/77(73) ~7 1:5 15/85(~2)4/48(9~) 5/60(92) :
1:10 49/85(-)27/48(-) 10/~(83) 17 ~:5 2/51(96) . 1:10 17/51(67) 21 1:5 10/68(85)18/59(69) ~/36(78) 1:10 . 28/68(59)18/59(69) 22/36(-) 37 1:5 ~7s~99) S/48(90) 23/59~61) 1:10 4~/7s( ) 15/~(69) 39/59(-) 37 : 1:5 15/58(74) 16/60(73) 1:10 32/58(-) 54/60(-) 37 : 1:5 29/7Z(60) 23/48(52) : 1:10 68/72(-) 25/48(-) ~ :
__ ~ ~
* - mean no. of synctia in test/mean no. of syncytia in control C% reducti:on) ) = reduction < 50%

:
: Replication inhibition assay Sera 17 and 37 show comparable activity in ~rms of reduction in syncytium counts at day 5 in the replication inhibition assay as demonstrated in Table 2, : Furth r, replication is preYented as indicated by p~4 antigen determination. It should be noted that the sera use~ in this assay were not diluted.

.

,:

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W093/21218 ~ 3 c~ PCT/GB93/OOB08 Syncytium inhibition assay Table 3 gi~es data for serum 17 in the syncytium inhibition assay. A dilution of 1:100 reduced the inhibiting activity of the serum to a little less than 50%~ This test suggests how even diluting the serum may prevenk cell fusion and hence spread of live HIV.
, Table 3: Syncytium inhibiting activing o f anti-peptide : against HIV-l ~ strain H9-RF) in vitro _ ... .
Serum S~rum .
Syncytium-inhibiting act.ivity _ Dilutionagainst H9-RF
Lab No. Tested __ _ _ _ _ .__ _.
17 l.50 3/101 (97)*

1017 1:2S 12/51 (77) :So: ~ 16/51 (69) ~: : ~:100 27/51 (47) _ ~
: ~, 15 ~ *`= Mean number of syncytia in test/Mean number of ::
s~ncytia in control ~% reduction) ` ~ : ~ ' ;' ~ ~ ' :: ~

?.e W093/21218 2 ~ J 3 3 PCT/GB93/OOB08 .~;
: - 21 -Exam~le 2 ELISA reactlvi~ _f HIV-2 positive human se_a with pePtides accordinq to formulae IIa and IIb A C-terminally extended peptide of formula XIa having the sequence (Seq. I.D. No: 9) Gln Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Gly Cys (2~) :
and a C-terminally extended peptide of formula IIb having the sequence (Seq. I.D. No: lO) :, Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Gly-Cys (2B) were synthesised and:purified as described in Example l.
: The peptid~s were then used in an ELISA with ~IV
positive human:sera. Peptid~ "lOA" (disclosed in ;~
~Example l of our cop~nding application~published as W09l/OOgO3) is a known to be an immunodominan~ peptide related to the HIV envelope protein and is known to :.
recognise substantially all HIV l infected individuals.
This peptide was used to investigate cross-reacti~ity : 25~;and confir~l the suitability of the protocoI.

LISA Protocol ~ ~

100 ~1 of coating buffer, containing 20:~g/~1 of :~30 ~ peptide, was added to ach well of the ELISA plates ::~
yne~ech) and the plates incubated overnight at 4~Co 2. Plates were washed once in Trls bufferled sali~e and : : : dried thoroughly.~ :
3.~ 200 ~l of blocking buf~er was added to each well, the plat~s shaken~for 10 seconds and:incubated for l hour ~t 37C.
4. The:sera to be tested were diluted l/lO0 or 1~200 using dilution buffer. O.l ml was added to each well, : and the pla~es incubated-for l hour at 37~C. This was done in triplicate for each sample.

.

w~ g3/2.2l8 2 1 1 ~ ~ 3 3 P~/GB93/00808 f~
-- 2 2 ~

5. The plates were washed three times for 2 minutes with washing buffer.

6. 100 ~l of alkaline phosphatase conjugated goat anti-human IgG (l/2000 in dilution buffer) was added to each well and plates incubated for 1 hour at 37C.

7. The wells were washed as in (5).
~. 50 ~l of alkaline phosphatase buffer was added to each well followed by 50 ~l of substrate (10 mg/ml in water~ and the plates incubated for 15 mins at 37C.
9. The absoxbence was read immediately at 4~4 nm.

The human sera samples tested were as follows: .
20 known HIV-2 po~itive sera that had previously been shown to exhibit no cross-reactivity with HIV-l sera. All twenty patien~s came from the Ivory Coast.
5 know~ HIV-2 positiye sera that had previously ;
been shown to exhibit no cross-reactivity with HIV-l ,::
sera. ~ll fi~e patients came ~rom Africa but not the Ivory Coast.;
7 known H~V 1 positive sera that had been shown to exhibit no cross-reactivity with HIV-2 sera. .
lO~known HI~ negative sera samples from age and sex matched subjects.:
he control:contained no serum, but the total ; :~ 25 volume was~ maintalned~by addition of~0:.1 ml of dilution .~: :buffer.
It~is clear ~rom the results in Table 4 below ~hat :
No~cross reactivity between HIV-l and HIV~2 :peptides was observed, that is HIV-2 sera did not ~recognîse the HIV l peptide and HIV-l sera did not ; recognise the HIV-2 peptides;
' 2)~ No peptide specific activity was found in the HIV
~: : negati~e samples;
35:
3) There is a distinction in reacti~ities between sera : of patients who came from the Ivory Coast, and-patients : who ¢ame from other parts 9f the African continent; and 4) The HIV-l~ serum samples had detectable antibodies : :, .

,~ û 3 ...~ WO93/21218 ~ 3 PCT/GB93/00808 to the HIV-l (lOA) peptide.

Table 4 : Reactivity of HIV positive sera with peptides 2A, 2B and lOA.
_ . _ __ Reactivities Number of positive sera/sample size _ _ ... ..
5ample panel 2A 2B lOA (reference) ~ _ HIV-2 positive Ivo-y Coast 0/20 0/20 0/20 ~ _ ~ _ .
HIV-2 positive Mon-Ivory Coast 5/5 5/5 0/5 __ . __ _ HIV-l positive ~/7 0/7 6/7 _ HI~ negati~e 0/lO - 0/lO 0/lO
_._ . _ ._ _ ~s exp~cted, no posi tlve resul ts occurre d with the control.
,:

These results show that HIV-2+ sera from a number of patients contain antibodies which react specifically with the HIV-2 peptides according to the formulae IIa and IIb of the invention~

2~ Example 3 . ~ :
-~
Immunisation of mice with ~_Eep ide accordinq to ~ormula :.Ia_con~uqated to a carrier Protein ~ . : :
.
. .
; 25 ~A C-te~minally extended according to formula Ia ha~ing ~ ~the~se~uence (Seq. I.D~ No~

; ~ ~Gln-Gln-~is-Leu-Leu-Gln-Leu-Thr Val-Trp-: - : : Gly-Ile-Lys-Gly-Cys (lA) was synthesised and purified as des~ribed i~ Example l.

Con;u ation ~ ;

The peptide was conjugated to ovalbumin using elther glut~raldehyde or MBS (M-maleimidobenzoyl-N-hydroxy-~: succinimide ester) as the coupling agent.
:: :
Glutaraldehy~de : The peptide was dissolved in PB5 (5 ~ .
' WO93/2121~ 2 1 1 ~ ~ 3 3 PCT/GB93/0080X ~;

mg/ml~ and ovalbumi~ added to give a molar ratio of 50 amino acids of carrier per l mole of peptide. The total ~olume was adjusted ~o 2 ml with PBS. 2 ml o~ 0.2%
solution of glutaraldehyde in PBS was slowly added to the stirring mixture and the solution left stirring at room temperature for l hour. The mixture was dialysed against large volumes of PBS overnight and stored at -20C for not longer than 3 weeks.

MBS : O.l ml of a solution o~ MBS (25 mg/ml in PBS) was added to a stirring solution of ovalbumin (lO mg/ml in .
P~S) and the reaction mixture stirred at room :
temperature for 30 mins. The activated ovalbumin was ~;
then separated ~rom free MBS by gel filtration on a , Sephadex G-25 column equilibrated with PBS. Peptide was dissolved in PBS and added to the activated ovalbumin so that a final concentration of l mole of peptide for each 50 amino acids of carrier resulted. The pH was adjusted to 7-7.5 and the reaction stirred for three hours at ..
:20 room temperature. The mixture was dialysed against large~volumes of PBS overnight and stored at -20C ~or :~not longer than 3 weeks.
To determine the peptide/carrier protein ratio a procedure d~scribed by Habeeb can be u~ed (Habeeb, A.F.S.A., Anal. Biochem., 14, 328 (l966)3. In this, the free amino acids are determined by the trinitro~enzene ;~ sulphonic acid (TNBS) method using borate buffer, pH
9Ø ~The absorbence of the solution is read at 335~ nm and the percentage of modified free amino acid groups i5 ~ calculated. The free thiol assay as described by Ellman :
can also be used to assess the ef~iciency of conjuga~ion (~nderson, W.L. and Wet1aufer, D.B., Anal. Biochem.~ 67.
493 (1975)~)-:
Immunisatisn C57 Black mice were pre-bled 2-3 days before the . primary inoculatiQn, the sera separated and stored at 20C. This was the negative control mouse sera. Each ~0 mouse was immunised with 40 ~g of peptide in FCA and rJ ~ rj r3 WO93/2121~ PCT/GB93~00808 subsequently with 20 ~g of peptide in FIA 2 to 3 weeks later. The inoculation volume was O.l ml in each case and all injec~ions were given subcutaneously. Animals were bled immediately prior to the booster injection, and then two weeks and four weeks later.

The ELISA protocol was as described in Example 2, except that sera were diluted l/50 and l/lO0 in dilution ~uffer, and the antibody used was alkaline pho~phatase conjugated goat anti-mouse IgG~
' _ sult~
l. 2/24 (8%) animals responded to the peptide according to formula Ia conjugated using glutaraldehyde when compared to controls after the third bleeding.
Thus:~it is possible to raise antibodies against this p~ptide when it has been non-specifically conjugated to : a~carrier~ : :

2. ~ 14~24 (58%) of the mice had high levels of ~.
an*ibadies to the peptide after only one immunisation 2~5`~ with~the peptide accor~ing to~formula Ia conjugated to :ova1bumin by the:specific linkage using MBS. This is a strong:and specif~ic response. : ~ -Tho results~show:that the peptide according to~
~ 30 ~formula Ia, when conjugated to ovalbumin using ~BS, is a :~ : powerful i~munogen inducing very high levels o~ antibody a~te~ a single immunisation and that antibodies c`an bel :~aised even if the peptide is non-specifical~ly bound~to ~ ovalbumin.

: .

8 2 ~ 3 3 - 26 - PCT/GB93/00808 SEQUENCE LISTING
Number of SequPnces ll (l) Information for Se~ I.D. No: l (i) Characteristation of sequence:
(A) Length: 13 Amino acids (B) Type: ~mino acid (D) Topology: Linear (ii) Type of molecule: Peptide (xi~ Description o~ sequence: Seq. I.~. No: l Gln Gln ~is Leu Leu Gln Leu Thr Val Trp Gly Ile Lys ~: ~ 1 S 1~ .

:~ ' : (2) Information for Seq. I.D. No: 2 :~
(;.) Characteristation of sequence: ;
: (A3 ;Length: 9 Amino acids (B) Typ~e: Amino acid (D); Topology: Linear (ii) Type of molecule: Peptide (xi) Description of sequence: Seq. I.D. Mo:
~ , ~
Leu~Gln Leu ~hr Val Trp Gly Ile Lys (3)~ Information;for:5eq. I.D. No: 3 i) ~Characterlstation of sequence~
(A) Length: 13 Amino acids (B) Type: Amino acid (Dj ~opolo~y:! Li~ear Type of molecule: Peptide (xi) ~Description of sequence: Seq. I.D. No: 3 :-Gln Gln G}u ~et Leu Arg Leu Thr Val Trp Gly Thr ~ys , ~ ~. :::

- , - -WO93121218 ~1 ~ Y 0 3 ~ PCT/GB93/00~08 ~4) Information for Seq. I.D. No: 4 (i) Characteristation of sequence:
(A~ Length: 9 Amino acids ':
~B) Type: Amino acid (D) Topology: Linear ,";", (ii) Type of molecule: Peptide .
~xi) Description of sequence: Seq. I.D. No: 4 Le~ Arg Leu Thr Val Trp Gly Thr Lys l 5 ~:

(5) In~ormatio~ for Se~. I.D. No: 5 , (i) Characteristation of sequence: ~ ~:
~A) Length: S Amino acids .
. (B) Type: A~ino acid ~: (D) Topology: Linear , ~ , .
Type of molecule: Peptide (xi~ Description~of~sequence: Seq.~ I.D. Mo: 5 Gly Gly Gly Gly Gly ;~

(6)~ Information~for Seq. I.~D. No::6; :
ij Characteristation o~ sequence~
::(A)~:Leng~h: 6 Am~no acids : ;:
f) Type: Amin~ acid :: (D~ ~Topolo~y: Linear ii) Type of ~molecule Peptide :
: (xi~ Description of sequence: Seq. IoD~ No: 6 Gly Pro~Gly Pro~Gly~Pro (7)~ Information-for:Seq. I.D. Nof 7 (i) Characteristation of sequencP
(A) Length: 7 Amino acids ~ ~
' (Bf) Type: Amino acid ~:

W093/21218 ~7t r ~ û 3 3 PCT/GB93/00~08 ~
~ 28 - ' ;
(D) Topology: Linear (ii) Type of molecule: Peptide (xi) Description of sequence: Seq. I.D. ~o: 7 Gly Ser Ala Gly Ser Gly Ala l 5 (8) Information for Seq. I.D. No: 8 (i) Characteristation of sequence:
(A) Length: 12 Amino acids : (B) Type: Amino acid (D) Topology: Linear (ii) Type of molec~le: Peptide .
(xi) Description of sequence: Se~ D. No: 8 ~ 1eu Gln Leu Thr Val Trp Gly Ile Lys Gly Cys Ala ,~ 1 5 1

(9) Information for Seq. I.D. No: 9 ~: ~ (i) Chara~teristation of sequence:
(Aj Length: 15 ~mino acids (B) Type: ~mino acid (D) Topology: Linear:

(ii) Type of molecu}e: Peptide (Xi) Description of sequence: Seq. I.D. No: 9 :
:: : ~ : :
Gln Gln G1u Met Leu;Arg Leu Thr Val Trp Gly Thr Lys Gly Cys l 5 l~ 15 ; ~ :
: : (lO) I~formation for Seq. I.D. No: lO ~ æ
: (i) Charactéristation of sequence:
~: (A) Length: ll ~mino acids (B) Type: ~mino acid ) Topology: Linear - (ii) Type of mol~cule: Peptide xi3 Description of sequence: Seq. I.D~ No: lO
~:
, WO93/21218 ~ 3 3 ~ PCr/~B93/00808 - 2~ -Leu Arg Leu Thr Val Trp Gly Thr Lys Gly Cys 5 lO ~;

(11) Information for Seq. I.D. No~
(i) Characteristation of sequence;
(A) Length: 15 ~mino acids ( B ~ Type: Amino ac id (D~ Topology: Linear (ii) Type o:E molecule: Peptide (xi) Description of sequence: Seq. ~.D. ~lo~

Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly I~e Lys Gly Cys ' `

":

~-;'"`, ,"~
- ~, :~ ',``

~: ~" `.'"' '~
', ~,, ~ , , , ~.
: - ~
~.

Claims (29)

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):- (I) wherein R1 is selected from Gln-Gln-R4-R5, Gln-R4-R5, R4-R5, R5 or R1 is absent;
R2 is an amino acid residue selected from Gln, Lys, Glu or Arg;
R3 is an amino acid residue selected from Ile, Thr or Ala;
R4 is His or Glu;
R5 is Leu or Met; and X and Y may each independently be absent or independently be one or more amino acid residues with the proviso that when present, X and Y must not be such that the region of homology with the envelope protein of any strain of Human Immunodeficiency Virus (HIV) is extended beyond the sequence to which X and Y are attached into the adjacent amino acids in the X and Y groups.

2. A synthetic polypeptide as claimed in claim 1 wherein R2 is selected from Gln, Lys and Glu, and R3 is Ile.

3. A synthetic polypeptide as claimed in claim 1 or claim 2 consisting of the sequence (Seq. I.D. No 1) (Ia); or (Seq. I.D. No: 2) (Ib) wherein X and Y are as defined in claim 1.

4. A synthetic polypeptide as claimed in claim l consisting substantially of an amino acid sequence of formula (II):- (II) wherein R1 is selected from Gln-Gln-Glu-R5, Gln-Glu-R5, Glu-R5, R5 or R1 is absent;
R3 is Thr or Ala;
R5 is Met or Leu; and X and Y are as defined in claim l.

5. A synthetic polypeptide as claimed in claim 4 consisting of the sequence (Seq. I.D. No 3) (IIa); or (Seq. I.D. No: 4) (IIb).

6. A synthetic polypeptide as claimed in any one of the preceding claims wherein X is absent and Y is Gly-Cys or Gly-Cys-Ala.

7. A synthetic polypeptide of general formula (III) [La-F]m[Lb-Gn]n-Lc (III) wherein F and G may each independently be a polypeptide according to any one of Formulae I to IIb, 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.

8. A synthetic polypeptide of general formula (IV):
Ld-[G-L]m-F-[L-G]n-Le (IV) wherein F is a polypeptide according to any one of Formulae I to IIb, G is a polypeptide according to any one of Formulae I to IIb 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.

9. A synthetic polypeptide which comprises an antigenically significant subfragment and/or antigenically significant variant of any one of the polypeptide sequences as claimed in claims 1 to 8.

10. A synthetic polypeptide as claimed in any one of claims 1 to 5 or claims 7 to 9 additionally comprising a T-cell epitope.

11. A synthetic polypeptide as claimed in any one of claims 1 to 5 or claims 7 to 10 including a retro-inverso amino acid.

12. A synthetic polypeptide as claimed in any one of the preceding claims Linked to a carrier.

13. A DNA molecule coding for at least one synthetic polypeptide as claimed in any one of claims 1 to 10.

14. A vaccine comprising at least one synthetic polypeptide as claimed in any one of claims 1 to 12 effective to promote prophylaxis against HIV infection.

15. 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 12.

16. A pharmaceutical composition containing, as active ingredient, at least one synthetic polypeptide as claimed in any one of claims 1 to 12 in association with one or more pharmaceutically acceptable adjuvant, carrier and/or excepient.

17. A pharmaceutical composition as claimed in claim 16, further comprising AZT and/or a HIV protease inhibitor.

18. A 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 12.

19. An antibody or antigen binding fragment thereof which specifically binds to a synthetic polypeptide as claimed in any one of claims 1 to 12.

20.. A diagnostic kit for detecting HIV or antibodies against HIV which contains at least one antibody or binding fragment thereof as claimed in claim 19.

21. A method of detecting HIV or antibodies against HIV or antigen binding fragments thereof, which comprises incubating a sample with at least one one antibody or binding fragment thereof as claimed in claim 19.

22. A pharmaceutical composition containing, as active ingredient an antibody or antigen binding fragment thereof as claimed in claim 19, in association with one or more pharmaceutically acceptable adjuvant, carrier and/or excepient.

23. A pharmaceutical composition as claimed in claim 22, further comprising AZT and/or a HIV protease inhibitor.

24. A method of diagnosing HIV infection which comprises incubating a sample of tissue or body fluid of a mammal with an effective amount of an antibody or binding fragment thereof as claimed in claim 19 and determining whether, and if desired the extent to which and/or the rate at which cross-reaction between said sample and said antibody occurs.

25. An anti-idiotypic antibody raised against an antibody or antigen binding fragment, as claimed in claim 19.

26. Use of a synthetic polypeptide as claimed in any one of claims 1 to 12 in the preparation of a medicament for the therapeutic or prophylatic treatment of mammalian HIV infection and/or for stimulating the mammalian immune system and/or blocking the cellular receptors for the HIV
virus.

27. A method of therapy or prophylaxis of mammalian HIV infection and/or for stimulating the mammalian immune system and/or blocking the cellular receptors for the HIV
virus, which comprises administering an effective amount of a polypeptide as claimed in any one of claims 1 to 12.

28. A process for the manufacture of 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):- (I) wherein R1 is selected from Gln-Gln-R4-R5, Gln-R4-R5, R4-R5, R5 or R1 is absent;
R2 is an amino acid residue selected from Gln, Lys, Glu or Arg;
R3 is an amino acid residue selected from Ile, Thr or Ala;
R4 is His or Glu;
R5 is Leu or Met; and X and Y may each independently be absent or independently be one or more amino acid residues with the proviso that when present, X and Y must not be such that the region of homology with the envelope protein of any strain of Human Immunodeficiency Virus (HIV) is extended beyond the sequence to which X and Y are attached into the adjacent amino acids in the X and Y groups, the process comprising the steps of coupling the residues using chemical, biological or recombinant techniques known per se and isolating the polypeptide.

29. A process for the manufacture of an antibody which specifically binds to 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):- (I) wherein R1 is selected from Gln-Gln-R4-R5, Gln-R4-R5, R4-R5, R5 or R1 is absent;
R2 is an amino acid residue selected from Gln, Lys, Glu or Arg;
R3 is an amino acid residue selected from Ile, Thr or Ala;
R4 is His or Glu;
R5 is Leu or Met; and X and Y may each independently be absent or independently be one or more amino acid residues with the proviso that when present, X and Y must not be such that the region of homology with the envelope protein of any strain of Human Immunodeficiency Virus (HIV) is extended beyond the sequence to which X and Y are attached into the adjacent amino acids in the X and Y groups, the process comprising immunising a non-human mammal with said polypeptide and isolating the antibody.