CA1341066C

CA1341066C - Small peptides which inhibit binding to t-4 receptors and act as immunogens

Info

Publication number: CA1341066C
Application number: CA000538202A
Authority: CA
Inventors: Candace B. Pert; William L. Farrar; Michael R. Ruff
Original assignee: US Department of Commerce
Current assignee: US Department of Commerce
Priority date: 1986-06-03
Filing date: 1987-05-27
Publication date: 2000-08-01
Anticipated expiration: 2017-08-01
Also published as: PT84992A; MX172337B; IL82719A0; IE61725B1; WO1987007613A1; IE871388L; FI885630A0; FI94352B; KR880701247A; FI885630A; FI94352C; DE3787927T2; HUT48907A; KR930008448B1; DK53288D0; NO176022C; AU7540887A; DK173667B1; NO880479D0; NO176022B

Abstract

Short peptide of the formula:
R a-Ser-Thr-Thr-Thr-Asn-Tyr-R b ~~(I) where R a represents an amino terminal residue Ala- or D-Ala and R b represents a carboxy terminal residue -Thr or -Thr amide or a derivative thereof with an additional Cys- residue at one or both of the amino and carboxy terminals, or a peptide of formula (II):
R1-R2-R3-R4-R5 (II) where R1 is an amino terminal residue Thr-, Ser-, Asn-,Leu-,Ile-,Arg- or Glu-R2 is Thr, Ser or Asp R3 is Thr, Ser, Asn, Arg, Gln, Lys or Trp R4 is Tyr and R5 is a carboxy terminal amnio group or a derivative thereof with a corresponding D- amino acid as the amino terminal residue, and/or a corresponding amide derivative at the carboxy terminal residue and/or additionally a Cys- residue at one or both of the amino and carboxy terminals, or a physiologically acceptable salt thereof.
Such peptides bind to T4 receptors are useful in preventing viral infectivity by viruses which bind to the T4 receptors. These peptides are believed to act as completitive blocking agents.

Description

BRIEF DESCRIPTION OF THE INVENTION
This invention relates to synthetically produced short peptide sequences which inhibit NTLV-III/LAf (hereiinafter referred to as HIY) binding to human cells by blocking receptor' cites on the cell surface, and thus preventing viral infectivity of human T celll. The peptides, while preventing infectivity, al~~o induce antibody production against the envelope protein of the HIV
virus.
He!~ce, these peptides also have use as vaccines to prevent development of Acquired Immune Disease Syndrome (AIDS). Monoclonal antibodies to the peptides could also be used as diaC~nostic agents to identify the HIV virus. Hence, pe~~tides and antibodies to the peptides would have use in preparing test kiss for identification of HIV carrier's or persons suffering from IDS.

Z
B,~C;<GROUND OF THE .INVENTION
~~ ~ 06 6 The complete nucleotide sequence of the AIOS .(HIV) virus has been reported by several investigators. (See lee Ratner et al., Nature 313 _ - . p. 277, January 1985; Muesing et al., Nature 313, p. 450, February 1985; and Main-Habson et al., Cell 40, pp. 9-17, January 1985.) The envelope gene has been_associated particularly with antigenicity and infectivity. However, the envelope portion ~is also known to have regions which are highly divergent. The HIV virus envelope glycoprotein has been shown to affix covalently to the brain membranes t of humans, rats, and monkeys and to cells of the immune system.
The realization th~it viruses may exert cell and tissue tropism by attachment at highly specific sites on ce'Il-~~r~embrane receptors has encouraged investigators to seek agents which would bind at the viral receptor sites of cell membranes and thus prevent binding of a specific virus to these cells. A demonstration of speci f i c receptor-medi af:ed vacc:i ni a vi rus i nfecti vi ty bei ng bl ocked by syntheti c ,peptides has been previously demonstrated (Epstein et al., Nature 318: 663-667).
The HI'J virus has i~een shown to bind to a surface molecule known as the COa or T.~ regicn, whici °s rrzsent on various cells susceatable to HI'J infection, s including T ly",phocyt~s and macrophages. (See Shaw et al., Science 226, pp.

1171 for a discussion of tropism of HTLV-III.) In addition to symptoms arising from immunodeficiency, patients with LIDS show neuropsychological defects. The central nervous and imnune syster~s <.~hare a large number of specific cell-surface recognition molecules, serving <<s receptors for neu ropeptide-mediated intercellular communication. The neuropeptides and their receptors show profound evolutionary stability, being highly conserved in largely unaltered form in unicellular organisms as well as higher animals. Furthermore, the central nervous and immune systems show common,DC4 (T4) cell-surface recognition molecules which serve as receptors for the binding of HIV envelope glycoprotein (gp 120). Since the same highly . 1 341 06 6 conserved neuropeptide informational substances integrate immune and brain function through receptors remarkably similar to those of HIY, we postulated a very similar amino acid sequence between the HIV glycoprotein gp 120 and a short peptide previously identified in another context from the envelope region of the Epstein Barr-Virus might indicate the core peptide essential for viral receptor 6indi~ng. It was postulated that such a peptide would be useful in preventing infection of cells with the HIV by binding with receptor cells and blocking the binding ~of HIV gp 120, that such peptides binding to the receptor cites would give rise to production of antibodies directed to the peptide sequence, and that 'these peptides might be used to provide immunological basis for prevention of AIDS.

PURPOSE
It was the object o~F the present invention to provide peptides that would act to alleviate symptoms of AIDS by preventing binding of HIV (AIDS
virus) to receptor sites of cel'Is of brain membranes and the immune system.
It was also an objeca of the present invention to provide peptides for use as vaccines to be usf~d to giive rise to antibodies that would protect against development of AIDS in persons who might become exposed to~the HIV
(~4IDS virus).
It was a further object of the present invention to provide diagnostic means of identifying presence of antibodies to HIV or HIV envelope protein.

/f DETAILED DESCRIPTION OF,THE INVENTION ' 3 ~~~66 An octapeptide in the HIV envelope glycoprotein {gp 120) was identified by computer-assisted anal,ysis_ This peptide, termed "peptide T" because of the high threonine content, has been shown to inhibit binding of gp 120 to the brain membranes. The peptide has the sequence Ala-Ser-Thr-Thr-Thr-Asn-T3~r-Thr. Later analysis disclosed a class of related pentapeptides having similar binding properties.
According to a first aspect of the present invention there is provided a peptide of formula (I):-Ra-Ser-Thr-Thr-Thr-Asn-Ty r-Rb (I) where Ra represents an amino terminal residue Ala- or D-Ala and Rb represents a carboxy terminal residue -Thr or -Thr amide or a derivative thereof with an additional Cys- residue at one or both of the amino and carboxy terminals, or a peptide of formula (II):-Rl-R2_R3-R4-R5 (II) w~~ere R1 is an amino terminal residue Thr-, Ser-, Asn-, Glu-, Arg-, Ile- or Leu-, R2 is Thr, Ser or Asp, R3 is Thr, Ser, Asn, Arg, Gin, Lys or Trp R4 is Tyr -and R5 is preferably a carboxy terminal residue -Thr, -Arg or -Gly or a derivative thereof with a corresponding D- amino acid as the amino terminal residue, and/or a corresponding amide derivative at the carboxy terminal residue and/or additionally a Cys- residue at one or both of the amino and carboxy terminals. While the preferred amino acids at R5 have been designated, it is known the amino acid at this position may vary widely. In fact, it is possible to terminate the peptide with R4 (Tyrosine) as the carboxy terminal amino acid wherein R5 is .absent. Such peptides retain the binding properties of the group taught herein. Serine and threonine appear to be interchangeable ' 6 far purposes of biologi c~7 properti es taught herei n. The ac~i ve compounds ~I
of the i nventi on ~~ay exi s t as .phys i ologi ca i 1y acceptable salts of the pepti des.
This class of peptides has been found to bind tn the T~ viral receptors.
~!ost prefers ed peptides, as well as peptide T above, arz the following octapeptides 4. for",ula (I):-D-.~Ia-Ser-~i hr-T hr-Thr-;sn-Tyr-T hr and D-;ia-Ser-Thr-Thr-Thr-.=,sn-Tyr-Thr-amide and t5e following pentapeptidea of for~~~ tar (II):-Thr~-asp-nsn-Tyr-Thr T hr-T hr-Ser-Tyr-Thr Thr-Thr-~sn= yr-Thr and t~~cei r analogues wi th D-T hr as the ami no termi na1 resi due and/or an _ ami de derivate at the carboxy, terminal.
A peptide the selected from group consisting of peptides having the general formula I:
Ra-Ser-Thr-Thr-'T'hr-A sn-Tyr-R5, C I ) peptides having the c;ene'ral formula II:
R~ _Rz_R3_TYr_R5. C I I ) peptides having the general formula I or II with an additional c.ys residue at the amino terminus, peptides having the general forrula I or II with an additional Cys at the carboxy t?rrtinua, peptides having the gE=_neral formula I or II with an additional c.ys residue at the amino terminus and at the carboxy terminus, peptides having the general formula II, and physiologically acceptable salts of any of: the preceding peptides, ~,rherein 6a Ra is Ala or D--Ala;
R~ is an amino acid residue selected from the group consisting of Thr and Thr amide;
R' is an amino acid residue selected from the group consisting of Thr, Ser, Asn, Lau, Ile, Arg, Glu, D-Thr, D-Ser, D-Asn, D-Leu, D-Ile, D-Arg, and D-Glu;
R2 is an amino acid residue selected from the group consisting of Thr~ Ser, and Asp ;
R is an amino acid residue selected from the group consisting of Thr, Ser, Asn, A:rg, Gln, Lys, and Trp; and R' is an amino acid selected from the group consisting of Thr, Arg, Gly, Thr-amide, Arg-amide, Gly-amide, D-Thr, D-Arg, and D-Gly.
The compounds of th~~ invention may be beneficially modified by methods known to enhance passage of mo lecules across the blood-Srain barrier.
Acetylation has proven to be e~pecialiy useful for enhancing binding activity of the peptide.
The terminal amino and carboxy sites are particularly preferred sites far ~r~odi fi cati on. -The peptides of this may also be modified in a constraining conformation to provide improved stability an d oral availability.
The following abbreviations are used hereinafter:-Amino Acid . Three Letter Code One Letter Code arginine - arg asparagine asn N

aspartic acid _ 0 asp .

cystei ne cys C

c~lyci ne 91Y

Seri ne ser threanine ~ thr T -tyrosi ne tyr Y

Unless otherHise indicated the amino acids are, of course, in the natural form of L-stereoisc~r.~.
A comparison of amino acid sequnces of 12 pentapeptides is presented in Table 1. Although hi~s~=orically our initial computer search revealed peptide T
(contained in the AR'! isolate) to be the relevant moiety, as additicnal viral sequences became available it became clear that the relevant, bioactive sequence, mi ght be a shorter pentapepti de comori si ng~, nomi nal ly, pepti de T[4-8], or the sequence TT~YT. In the isolates we compared (Table 1) substantial homologies were discerned only in this, shorter, regien. The majority of changes are the i nterconversi ons of s~eri ne (S ) and threcni ne (T), t:vo cl osely ref ated amino acids. The tyrosine of position 7 of peptide T is an invariant feature of all these c;,ns~ruct~c indicating that it may be obligatory for bioactivity.
Subs titui:ions occurring at position 5 include T, G, R or S. Position 4 and 6 were first restricted (with one exception) to S, T and N, all amino acids containing uncharged polar groups with closely similar steric properties.
nn assessment of ce.~.er:~l s2qu~ence concordance among 5 vari ous AIDS vi rat isolates (9,10) reveal~~ that the region around and including the peptide T
sequence is a highly variable area. Such variability may indicate specialization through s trong selecti ve di ve:rsi fi cati on of the functi on(s ) whi ch may be defi nod at this locus. Like the opiat a peptides, these peptide T analogs seem to exist in multiple forms, reminiscent of met and leu enkephalin. These pentapeptide sequences represented in these various AIDS virus Isolates are biologically active and capable of interacting as agonists of the CD4 receptor - previously known largely as a surface "marker" of T helper cells.

f' 8 Table i. Comoarision of EhIV Seguence from Multiple AIDS Virus Isolates Isolate Sequence Ref- erence peptide T ASTTTNYT Pert, C.B. et al.
.
PNAS (in press ' t lARV (195-199) TTNYT Willey, R.L. et a1.
LAV TTSYT PNAS 83: 5038; 1~$6 B10(HTLV-III) TTSYT Starcich, B.R, et al.
WMJ-1 SSTYR Cell 45: 637, 1985 Sequential isolates ~ STNYR
WMJ-1 SSTYR Hahn, B.L. et al.
WMJ-2 SSRYR Science 232: 1548, TTSYS
lNumbers refer to relative positions of amino acids within the ARV env sequence (9).
The seven amino acid peptide CYS-THR-THR-AS~~-TYR-THR-CYS is also active.
~dditien of cys~eines to a core does not adversely affect activity.
The peptides veri~ custom synthesized by Peninsula laboratories under a confidentiality agreement between the inventors and the manufacturer. The Merrifield method of ~~olid phase peptide synthesis was used. (See U.S.
Patent No. 3,531,258) The synthesized peptides are especial~!y preferred. While peptide T and the pentapeptide which is a portion thereof could be isolated from the virus, the peptides prepared in accord with Merrifielc! are free of viral and cellular debris. Hence, untoward reactions to contaminam is does not occur when the synthesized peptides are used.
The peptides of t:he invEantion may be produced by conventional methods of peptide~synthesis. Both solid phase and liquid phase methods may be used.
We have found the solid phase method of Merrifield to be particularly convenient.

. 1 341 06 6 Ire this process the peptide is synthesized in a stepwise manner while the carboxy er~d of the chain is covalnntly attached to the insoluble support. During the intermediate synthetic st~~ges the peptide remains in the solid phase and therefore can be conveniently manipulated. The solid support is a chioromethylated styrene-divinylbenzene copolymer.
An N-protected form of the carboxy terminal amino acid, e.g. a t-butoxy carbonyl protected (Boc-) amino aciid, is reacted with the chloromethyl residue of the chloromethylated styrene divinylbenzene copolymer resin to produce a protected amino acyl derivative of the re~~in, where the amino acid is coupled to the resin as a benzyl ester. This is de~~rotected and reacted with a protected form of the next required amino acid thus producing a protected dipeptide attached to the resin.
The amino acid will genere~lly be used in activated form, e.g. by use of a ca~rbodiimide or active ester. This sequence is repeated and the peptide chat n grows one residue at a tine by condensation at the amino end with the required .1-protected amino acids until thEa required peptide has been assembled on the resin. The peptide-resin is then treated with anhydrous hydrofluoric acid to cleave the ester linking the <issembied peptide to the resin, in order to liberate the required peptide. .'side chain functional groups of amino acids which must be blocked during the synthetic procedure, using conventional methods, may also be simultaneausly removed. Synthesis of a peptide with an amide group on its carboXy terminal can be carried out in conventional manner, us i ng a 4-methylbenahydryl ami ne res i n.
The compounds of the inventiion were found to effectively block receptor sites of cells and to prevent celil infectivity with HIV (AIDS virus) in monkey, rat, and human brain membranes and cells of the immune system.
As an aspect of the invention, therefore, we provide a pharmaceutical composition comprising a peptide compound of the invention in association ro with a pharmaceutically acceptable carrier or excipient, adapted for use in human or veterinary medicine.. Such compositions may be presented for use in conventional manner in admixture with one or more physiologically acceptable carriers of excipients. The compositions may optionally further contain one or more other therapeutic: agents which may, if desired, be a different antiviral agent.
Thus, the peptides <rccordin g to the invention may be formulated for oral, buccal, parenteral" topic<~1 or rectal administration.
In particular, the_Ereptides according to the invention may be formulated for injection or for infusion and may be presented in unit dose form in ampoules or in multidose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formu'latory agents such as suspending, stabilizing and/
or dispersing agents. A~Iternatiively, the active ingredient may be in powder form for constitution with a :suitable vehicle, e.g. sterile, py rogen-free water, before use.
The pharmace~~tical compositions according to the invention may also contain other active ingredients such as antimicrobial agents, or preservatives.
The compositions ma~~ contain from 0.001-99x of the active material.
The invention further provides a process for preparing a pharmaceutical composition which compri<.~es bringing a peptide of the invention into association with a pharmaceutically acceptable excipient or carrier.
For administration by injection or infusion, the daily dosage as employed far treatment of an adult: human of approximately 70 kg body weight will range from 0.2 mg to 10 mg, preferably 0.5 to 5 mg, which may be administered in 1 to 4 doses,~for example, depending on the route of administration and the condition of the patient., ~ 341 06 6 It was postulated that the affinity constants are similar to those of morphine. On the basis of this affinity, dosage of .33-.0003 mg/kg per day w<<s suggested. This has ,proven to be effective. A blood concentration 10-6 to 10-11 molar blood concentration is suggested. In monkeys 3 mg/kg per day achieves a serum concentration of 150 x 10-g M. This concentration is 15 times greater than necessary to achieve a concentration of 10-$ M. Primates generally require 10 timea the dose used in humans.
A further aspect of this invention relates to vaccine preparations containing a peptide according to the invention, to provide protection against infection by AIDS virus. The vaccine will contain an effective immunogenic amount of peptide, e.g. l,tcg to 20 mg/kg of host, optionally conjugated to a protein such as human serum albumin, in a suitable vehicle, e.g. sterile water, saline or buffered saline. Adjuvants may be employed, such as aluminum bydroxide gel. Administration may be by injection, e.g. i~ntramuscularly, interperitoneally, subcutaneousl,y or intravenously. Administration may take place once or at a plurality of times, e.g. at 1-4 week intervals.
Antigenic sequences from crab as well as proteins from other invertebrates ca.n also be added to the peptides of the invention to promote antigenicity.
A yet further aspect of this invention relates to test kits for the detection of the AIDS virus and antibodies to the AIDS virus containing a peptide according to the invention as source of antigen, or a monoclonal antibody elicited by a peptide according to the invention. For example, a peptide according to the invention may be used in a test kit to detect AIDS
infection and to diagnose AIDS and pre-AIDS conditions by using it as the test reagent in an enzyme-linked immunosorbent assay (ELISA) or an enzyme irtrcrnrnodot assay. Such test kits may include an insoluble porous surface or solid substrate to which the antigenic peptide or monoclonal antibody has teen preabsorbed or covalently bound, such surface or substrate preferably in the form of microtiter plates or wells; test sera; heteroantisera which specifically bind to and saturate the antigen or antibody absorbed to the surface or support; various diluents and buffers; labelled conjugates for t;he detection of specifically bound antibodies and other signal-generating ~~eagents such as enzyme substrates, cofactors and chromogens.
The peptide according to the invention may be used as an immunogen to elicit monoclonal antibodies which specifically bind to the relevant portion c~f the envelope sequence of the AIDS virus, using conventional techniques;
such monoclonal antibodies form a further feature of the invention.

EXPERIMENTAL METHODS AND DATA 1 ~ 4 1 0 6 6 Radiolabelinv of gp 1120, Preparation of Brain Membranes, Bindiny and Crosslinking of gp120 to Receptor, and Immunoprecipitation of T4 Antiyen.
HTLY-IIIb isolate of HIV was prop aged in H9 cells, and the gp120 was isolated by irtmunoaffinity chromatography and preparative NaDodS04/PAGE. Purified gp120 was labeled with 12'~I by the chloramine-T method.
Fresh human, monkey, and rat hippocampus were quickly homogenized (Polytron, 8rinkmann Instruments) in 100 vo'i. of ice-cold 50 mM Hepes (pH 7.4). The membranes collected by centrifug<ition (15,000 x g) were washed in the original buffer volume and were used fresh or stored at -70°C. Before use, brain membranes and highly purified T cells (ref. 16; gift of Larry Wahl) were preincubated for 15-30 min in phosphate-buffered saline (PBS). Membranes deri ved from 2mg (i ni ti al wet wei ght ) of brai n (x100,ccg of p rotei n ) were incubated with 28,000 cpm of 125'.f-gp120 for 1 hr at 37°C in 200,c,~1 (final volume) of 50 rt~M Hepes containing 0.1% bovine serum albumin and the peptidase inhibitors bacitracin (0.00 5 ), aprotinin (0.005%), leupe~tin (0.001X), and chymostatin (0.001:). Incubations were rapidly vacuum-filtered and counted to determine the receptor-bound material.
Immunoprecipitation. Immunoprecipitates were prepared by incubation (overnight at 4°C) of 0.5.: Triton X-100/PBS-solubilized, lactoperoxidase/
glucose oxidase/125I-iodinated brain membranes or intact T cells with indicated mAbs at l0,ug per reaction. A solid-phase immunoabsorbant (immunobeads, "
Bio-Rad) was used to precipitate immune complexes prior to their resolution by NaDodS04/PAGE. Control incubations contained no primary mAb or a subclass control mAb (OKTB).
Chemical Neuroanatomy and Computer-Assisted Densitometry. Cryostat-cut ,, 25 ~m sectiflns of fresh-frozen human, monkey, and rat brain were thaw-mounted and dried onto gelatin-cocited slides, and receptors were visualized as described (18).

1 ~f 'Incubations, with or without antibodies (l0,ug/ml) against T4, T4A, T8, and '~11, were conducted overnight.a~t 0°C in RPMI medium, crosslinked onto their ~3ntigens, and visualized with 1251-labeled goat anti-mouse antibody.
Incubations of slide-mounted tissue sections to label the antigen-receptor with 1251-gp120 sere conducted in 5-mi slide carriers with (l,uM) or without unlabeled gp120 or rnAb OKT4A (l0,ug/ml ) (Ortho Diagnostics). ~-~-Separation of T-Lymoh~ ocyte Subsets. Subsets of T cells were obtained try treatment of Percoll density-purified peripheral blood T cells with specific monoclonal antibodies (T4 or T8) at l0,ug/ml. The treated cells were then panned (21) on a plastic Petri dish that was coated with goat [F(ab')2] anti-mouse immunoglobulin (Sero Lab, Eastbury, MA) for 30 min at 4°C. The nonadherent cells were then removed, washed, and analyzed for reactivity by flow cytometry.
The separated T4 and T8 cell populations have < 5X contamination of other T-cell subsets. Cells were then cultured with phytohemagglutinin (l,ug/ml) for 72 hr a.nd exposed to HIV as described below. Infected cells were.phenotypically characterized when cytotoxicity assays were performed.
Virus Infection. The HTLV-III virus used for infection was isolated from an interleukin 2 (I'L-2)-dependent cultured T-cell line established from fresh AIDS patient material and passaged into HuT 78, a permissive IL-2-independent cell line.

DE:SCRIPT1,ON OF THE, DRAWINGS ~ 3 4' ~ 6 C
Figure IA shows a crosslin~;ing of 125I_gp120 to brain membranes and T
cells (a) 125I_gp120 only; (b) monkey; (c) rat; (d) human brain; and (e) human T cells.
Figures 1B and 1C show innurnoprecipitation of 125I_labeled-monkey brain membranes and human T cells, respectively; (f,i) no primary antibody control;
(g~, j ) OKT4 Mab; (h,k) OKf8 Mab.
Figure 2A shows a displacement of specific 125I_gp120 binding to fresh rat hippocampal membranes. Each determination was performed in triplicate;
the results of one experiment, which was performed three times with similar results, is shown. Specific binding displaceable by l0,ug/ml of OKT4 and 4A
ranged between 27 and 85x; of total binding, which was 2,201 ~ 74 cpm in the experiment shown.
Figure 29 shows that. viral infectivity is blocked by peptide T and its synthetic analogs. Each determination was performed in duplicate. Results represent a single exaeriment which was repeated three times with similar results.
Example I. A singles radiollabeled crosslinking product of about 180 Kd ins obtained after specific binding of 125I_gp120 to membranes from either s~auirrel monkey, rat or human brain membranes which are indistinguishable from that of human T cells (Fig,. lA). This result indicates that gp120 can b.' coupled to an approximately fi0 Kd protein; unreacted 125I-gp120 runs a~i jacent to the no membrane cont:rol ( 1 ane a ) .
Immunoprecipitation of radiioiodinated human brain membranes with OKT4 an d OKT8 (10 ~cg/ml) (Fig. 1B) shows that brain membranes contain a T4 antigen of about 60 Kd, indistincpishablle from that identified on human T lymphocytes (Fig. 1C); ,by contrast, OKT8 immunoprecipitates a low (about 30 Kd) molecular wei ght protei n from T lyn~phocytf~s (Fi g. 1C ) whi ch i s absent i n brai n membranes 1 341 06 fi (Fig. 18) indicating that brain T4 is not derived from resident lymphocytes.
Similar results are observed with monkey and rat (not shown) hippocampal mt~mbranes. These results show that the T4 antigen serves as the viral receptor and is a highly conserved 60 Kd molecule shared by the immune and central ncarvous systems.
The realization that Epstein-Barr and HTLV-III/LAV share an almost identical octapeptide sequence caused the synthesis and study of "peptide T."
Figure 2 demonstrates the high (0.1 nM range) affinity and saturability (Fig.

2~1 of 1251-gp120 binding to freshly prepared rat brain membranes. Specificity (fig. 2B) is demonstrated by blockade with OKT4 and OKT4A, but not OKT3 (0.1 pci/ml). Peptide T and two of its synthetic analogs (but not the irrelevant oca apeptide substance P [1-8]) significantly inhibited 1251-gp120 binding in this 0.1 nM range (Fig. 2C). Substitution of a D-threonine-amide in position 8 resulted in at least a 100-fold loss of receptor binding activity. The classical [0-Ala] substitution for [L-Ala] results in a consistently more patent, presumably more peptidase-resistant, analog than peptide T; amidation of the C terminal threonine also consistently produces somewhat greater potency (Figure 3).
When the synthetic peptides were tested for their ability to block viral infection of human T cells, experimentors were blind to binding assay results.
At. 10-~M the three peptides active in the binding assay are able to reduce detectable levels of reverse transcriptase activity by almost 9-fold. The less active binding displ,acer [D-Thre]-peptide T similarly showed greatly reduced blockade of viral infection, requiring concentrations 100-fold higher tc~ achieve significant inhibition. Thus, not only the rank order of potencies w of the fou n peptides (0-[~41a]1-peptide T-amide > D-[Ala]1-peptide T >
peptide T > 0-[Thre]g-peptide T-amide), but also their absolute concentrations in , ~34~ 066 .
inhibiting receptor binding and viral infectivity are closely correlated (Figure 3). , Example 2. An approximate 60-Kd protein, which is similar if not identical t.o human T cell T4 antigun, was present in apparently conserved molecular form a~n membranes prepared from human brain; furthermore, the radiolabeled HIV
envelope glycoprotein (1~'-5I-gp120) can be covalently crosslinked to a molecule present in three mammalian brains whose size and immunoprecipitation properties were indistinguishable from the T4 antigen. Using a method for visualizing antibody-bound receptors on brain slices, the neuroanatomical distribution pattern of brain T4, which is densest over cortical neuropil and analogously organized in all three m<rmmalian brains, was presented. Also, radiolabeled HIV
viral envelope glycoprotEain bound in an identical pattern on adjacent brain sections, once again suggesting that T4 was the HIV receptor.
Example 3. Chemical Neuroanatomy, Computer-Assisted Densitometry.
Cry ostat-cut 25 micron sections of fresh-frozen human, monkey, and rat brain , were thaw-mounted and dried onto gel-coated slides and receators visualized as described by Herkenharn and Pert, J. Neurosci., 2: 1129-1149 (1982).
Incubations, with or without antibodies (10 ~g/ml) against T4, T4A, T8 and T11, were conducted overnight at 0°C in RPMI, crosslinked onto their antigens and visualized with 125I..goat anti-mouse antibody. Incubations of slide-mounted tissue sections iin order to label antigen/receptor with 1251-gp120 were conducted in 5 ml slide carriers with (10-6M) or without unlabeled gpl 20 or Mab OKT4A (10 ,(~c~/ml ) (()rtho Di agnosti cs ) as descri bed above for membranes.
Computer-assisted transformation of autoradiographic film opacity into quantitative color image~~ was performed. Co-exposure of standards of known increments of radioactivity with the monkey brain sections generated a linear plot (4 = > ,g9) of log O.D, versus cpm from which the relative concentration of radioactivity can be meaningfully extrapolated. Cell staining of brain :~ections'with thionine was performed by classical methods and visualization of receptors overlying stained tissue.
Examo~le 4. Experiments have been conducted to determine the distribution of T4 antigen on a rostral to caudal series or coronal sections of squirrel monkey brain. These experiments show that there are detectable levels of T4 monoclonal antibody binding to cytoarchitectonically meaningful areas of the train stem (e. g., the substantia nigra), but the striking pattern of cortical enrichment is apparent at every level of the rieuroaxis. OKTB, a T-lymphocyte directed monoclonal anti~5ody from the same subclass as OKT4, exhibits no observable pattern. Generally, the more superficial layers within the cerebral cortex contain the densest concentrations of the T4 antigen; the frontal and perilimbic cortex overlying the amydala are particularly receptor-rich throughout the deep layers. The hippocamp~al formation has the densest concentration of receptors in the monkey, rat, and human brain. Dark field microscopy of squirrel monkey sections dipped in photographic ertxrlsion revealed that the band of densest receptor labelling is located within the molecular layers of the dentate gy rus and hippocampus proper (which contain very few neurons). Thus, receptors appear to be rightly distributed over l:he neuropil (the neuronal extensions of dendrites and axons) or may be loc~rlized i:o a specific subset of unstained astroglial cells.
Evidence of the specificity of the chemical neuroanatomy and results showing that T4 and the viral envelope recognition molecule are indistinguishable has been determined. Coronal sE~ctions of rat brain revealed a very similar cortex/hippocampus-rich pattern of receptor distribution whether OKT4 or -' 1251-gp120 was used for visuali~:ation. Furthermore, this pattern was not ap parent when incubation occurre d in the presence of unlabeled gp120 (l,uM), i9 ~ 341 Ofi 6 O~KT4A (10 ~g/ml ) or OKT4 (10 ~g/'ml ). Other mouse Mabs di rected against other human T cell surface antigens, including OKT8 and OKT11 gave no detectable pattern on rat brain when visualized by 1251-goat anti-mouse IgG secondary antibody gust as .there was no reproducible, detectable antigen/receptor with secondary antibody alone.

Claims

1. A peptide selected from the group consisting of peptides having the general formula I:
R a-Ser-Thr-Thr-Thr-Asn-Tyr-R b; ~(I) peptides having; the general formula II:
R1-R2-R3-R4-R5 ~~~(II) peptides having the general formula I or II with an additional Cys residue at the amino terminus;
peptides having the general formula I or II with an additional Cys at the carboxy terminus;
peptides having; the general formula I or II with an additional Cys reside at the amino terminus and at the carboxy terminus;
peptides having; the general formula II; and physiologically acceptable salts of any of the preceding peptides, wherein R a is Ala or D-Ala;
R b is an amino acid residue selected from the group consisting of Thr and Thr amide;
R1 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, Leu, Ile, Arg, Glu, D-Thr, D-Sar, D-Asn, D-Leu, D-Ile, D-Arg, and D-Glu;
R2 is an amino acid residue selected from the group consisting of Thr, Ser, and Asp;
R3 is an amino acid reside selected from the group consisting of Thr, Ser, Asn, Arg, Gln, Lys, and Trp;
R4 is Tyr; and R5 is an amino acid selected from the group consisting of Thr, Arg, Gly, Thr-amide, Arg-amide, Gly-amide, D-Thr, D-Arg, and D-Gly.

2. A peptide selected from the group consisting of peptides having the general formula II:
R1-R2-R3-Tyr-R5 ~~~(II) peptides having the general formula II with an additional Cys residue at the amino terminus, peptides having the general formula II with an additional Cys residue at the carboxy terminus peptides having the general formula II with an addition Cys residue at the amino terminus and at the carboxy terminus, and physiologically acceptable salts of any of the preceding peptides, wherein R1 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, D-Thr, D-Ser, and D-Asn;
R2 is an amino acid residue selected from the group consisting of Thr, Ser, and Asp;
R3 is an amino acid residue selected from the croup consisting of Thr, Ser, Asn, and Arg; and R5 is an amino acid selected from the group consisting of Thr, Arg, Gly, Thr-amide, Arg-amide, and Gly-amide.

3. A peptide selected from the group consisting of peptides having the formula:
R1-R2-R3-R4.
wherein R1 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, Glu, Arg, Ile, and Leu;
R2 is an amino acid residue selected from the group consisting of Thr, Ser, and Asp;
R3 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, Arg, Gln, Lys, and Trp; and R4 is Tyr.

4. A peptide of claim 1 selected from the group consisting of peptides having the formula:
Cys-R1-R2-R3-R4-R5-cys.

5. A peptide selected from the group consisting of Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, Thr-Thr-Asn-Tyr-Thr, Ser-Ser-Thr-Tyr-Arg, Asn-Thr-Ser-Tyr-Thr, Thr-Thr-Ser-Tyr-Thr, Asn-Thr-Ser-Tyr-Gly, Ser-Thr-Asn-Tyr-Arg, Ser-Ser-Arg-Tyr-Arg, Thr-Thr-Ser-Tyr-Ser, and Cys-Thr-Thr-Asn-Tyr-Thr-Cys.

6. A composition of matter containing as an active ingredient at least one peptide of claim 1 in association with a pharmaceutical carrier.

7. A composition of matter containing as an active ingredient at least one peptide of claim 2 in association with a pharmaceutical carrier.

8. A composition of matter containing as an active ingredient at least one peptide of claim 3 in association with a pharmaceutical carrier.

9. A composition of matter containing as an active ingredient at lease one peptide of claim 4 in association with a pharmaceutical carrier.

10. A composition of matter containing as an active ingredient at least one peptide of claim 5 in association with a pharmaceutical carrier.

11. The use of the composition of claim 6 for preventing binding of causative agent of AIDS to cells of mammals wherein an effective T4 receptor blocking amount of the composition is administered.

12. The use of the composition of claim 7 for preventing binding of HIV virus to cells of mammals wherein an effective T4 receptor blocking amount of the composition is administered.

13. The use of the composition of claim 8 for preventing binding of HIV virus to cells of mammals wherein an effective T4 receptor blocking amount of the composition is administered.

14. The use of the composition of claim 9 for preventing binding of HIV virus to cells of mammals wherein an effective T4 receptor blocking amount of the composition is administered.

15. The use of the composition of claim 10 for preventing binding of HIV virus to cells of mammals wherein an effective T4 receptor blocking amount of the composition is administered.

16. A composition containing a peptide of claim 1 conjugated to human serum albumin and a pharmaceutically acceptable carrier.

17. The use of the composition of claim 6 for preventing AIDS wherein an immunogenic effective amount of the composition is administered.

18. The use of a peptide of claim 1 for preventing AIDS wherein an immunogenic effective amount of the peptide is administered.

19. A test kit for detecting antibodies to causative agent of AIDS which bind to the T4 receptor containing a peptide of claim 1 bound to a porous surface or solid substrate.

20. The kit of claim 19 wherein the peptide is bound to wells of a microtiter plate.

21. The use of the composition of claim 6 for preventing illness arising from binding of an HIV virus to T4 receptor.

22. The use of the composition of claim 21 for preventing illness arising from HIV viruses which bind to T4 receptors wherein an immunogenic effective amount of the composition in series of 2-4 doses is given 1 to 4 weeks apart.