CA2111681A1

CA2111681A1 - Mimic peptides of gp120

Info

Publication number: CA2111681A1
Application number: CA002111681A
Authority: CA
Inventors: Malcolm L. Gefter; Kenneth L. Melmon
Original assignee: Individual
Current assignee: Immulogic Pharmaceutical Corp
Priority date: 1991-06-19
Filing date: 1992-06-19
Publication date: 1992-12-23
Also published as: EP0590057A1; WO1992022579A1

Abstract

Disclosed are peptides having a primary amino acid sequence which is unrelated to gp120. The peptides are characterized by the ability to stimulate the production of anti-gp120, particularly anti-V3-loop, antibodies when administered to a mammal. The peptides are also characterized by the ability to interfere with viral function. The peptides can be formulated as a vaccine composition and administered as a method for stimulating an HIV-1 neutralizing immune response.

Description

W0~2/22579 1? ~ 81 PCI/US~210~972 MIMI~ PEPTIDES OF qPl~0 BackarQund of ~he InventiQn Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome and the HIV
05 external envelope glycoprotein~ gpl20, is associated with v1ral infectivity and cytopathology including cell fusion. gpl20 binds the cellular receptor of the virus, CD4, and cells e~cpressing the envelope :fuse with CD4-positive cells in culture. Fusion of infected and noninfected cells and infection by cell-free virions are important routes of HIY
infection.
It has been reported by Robey et al. ~çs~
~`~ Acad. S~i~ USA ~:7023-7027 (1986~) that gpl20 15 purified from ~irus-infected cells elicits antibodies that neutralize the infectivity of HIV. Recombinant~
gpl20 or gpl60 expressed in a~variety of cell types have also been shown to elicit~neutralizing antibodies (see e.g., Lasky et al.~ ~S~ 2~3:209-233 :(1986~; :
20 Rusche et al., Proc. ~a1~_A~ad S~i. USA~84:1-5 (1987);~and Steimer et al., Vaccines 87:236-241 (1987)). The amino a~id sequence:of the envelope is ~known to vary between different HIV~isolates and ~the -~
;. neutrali~ing antibod~ies: elicited by the envelope of:
25 one HIV isolate are usually:eff:ective against only a : sub~et~of he~erologous isolates. This observation suggests that such neutralizing antibodies are :
: directed to a variable region of the envelope. This idea is supported by the f act that observation suggests that such neutralizing antibodies are directed to a variable:region of the envelope;. This~

:

WO9~t/~2579 ~ 6~ ~ P~T/US92/04972

-2- ..

idea is supported by the fact that neutralizing antibodies are elicited b~ an E. oli-produced recombinant fragment;from the carboxyl-terminal region of gpl20, PBl, thattcontains 37% of gpl20 and is from :~
05 a variable region of the envelope. This fragment :~.
- contains the dominant neutralizing epitopes (epitope being the basic element or smallest unit of ~
recognition by a receptor such as an antibody), and a ~:
more complete understanding of the number, the 10 location, and the potential role of antibodies to ;
these epîtopes in preventing viral infection may ;
facilitate development of a subunit vaccine able to ~:
induce immunity to diverse HIV isolates. : ;
Matsushita et al. (J~_YirQl~ ~2107 2114 (1988)) 15 reported the identlfication of a monoclonal antib~dy ~.
which is characterized by the ability to neutralize infection by cell-free virus, and the ability to prevent fusion of virus-infected cells. The~ antibody binding site was mapped to a 25-amino acid segment of 20 gpl20, referred to herein as the V3~100p. This region ~: is known to contain major neutra~lizing epitopes.
Peptides which stimulate the production of antibodies .
which~are reactive with these immunodominant epitopes yet which are not found in the~primary sequence o~ the : 25 gpl20 protein would be useful~for immunization, for :
~: e~ample. Such peptides are referred to herein as mimic peptides.

W092/~2s79 -3- 2 1 ~ 1 6 ~ 1 PCT/~Sg2/U4972 Summary of th~ InventiQn This invention relates to peptides having a primary amino acid sequence which is unrelated to the primary gpl20 amino acid sequence and are, by 05 themselves, or conjugated to an immuno~enic carrier molecule, able to stimulate the production of anti-gpl20 antibodies. The anti-gpl20 antibodies produced in response to peptides of the pres~nt invention are directed toward a region of the gpl20 ~:~
lO molecule known as the V3-loop, which is known to contain important epitopes.
The peptides can be form~lated in a : - :
physiologically acceptable carrier for use aæ a vaccine composition. This composition can optionally 15 include an adjuvant. In addition, the peptide can be conjugated to an im~unogenic carrier mol~cule to increase immunogenicity. In this conte~t:the peptide serve as an immunogen.
~ The invention also pertains to peptides which -: ~ 20 serve as antiviral agen s and a method for interfering ::
:~ with viral propagation în an individual. The method ::~
: involves administering an effectiv~ amount of a therapeutic composition~comp;rising a peptide in a physiological carrier. The peptide has a primary :: 25 amino acid sequence which is unrelated to the primary amino acid sequence of an essential protein of a virus Se.g., gpl20) and the pepti~e has the ability to:~
- i~terfere with a component involved in the function of ~:~
: the virus t In this conte~t, the peptide serves as an 30 antiviral agent.
In another aspec~, the invention pertains to a method for ~creening an epitope librar~ with a non-isolate specific antibody. Such antibodies typically do not bind antigen with higA affinity.

W092/22579 PCTlUSg2/04972 ~ 6~ -4-Brief Descri~tion of the Drawinqs Fi~ure 1 is a schematic representation of results from a competition assay for V3-loop binding.
Figure 2 is a schematic representation of rgsults 05 from a competition assay fbr recombinant gpl20 binding. ~`
Figure 3 is a schematic repr~sentation of results -;
from a direct binding assay with serum from mice immun~zed with M306 G3.
~igure 4 is a ~chematic repr~sentation of results from a direct binding assay with serum from mice i~munized with M306 G3 conjugated to an immunogenic carrier molecule. ~ :
Figure 5 is a schematic representation of results 15 from a competition esperiment in which Y3-loop was attached to an ELISA plate and reacted with serum from j~
mic~ immunized with ~306 G3 conjugated ~o an immunogenic carrier molecule~mi~ed with~variou~
: peptides. ;
Figure 6 is a schematic represent.ation of res~lts from a comp~tition e~periment in which M306 ~3 conjugated to bovine serum al~umin was fised to an ELISA plate and:reacted with se~um from mice immunized:
with M306 G3 and conjugated to an immunogenic ~arrier 25 molecule~mi~ed with various peptides.
Figure 7 is a schematic reprssentation of results from a competition experiment in which V3-loop was fixed to an ELISA plate and the antibody was an affinity purified rat monsclonal known to b~ HIV ~ ;
30 neutralizing. ~ :

. ~
W092/2~79 -5- 2 1 ~ 1 6 8 1 P~T/US92~972 Figure 8 is a schematic representation of results from a competition experiment in which recombinant ::
gpl20 was fixed to an ELISA plate and the antibody was an affinity purified rat monoclonal known to be HIV
05 neutralizing.
..
Detailed De~criPtion of ~he ~n~ntiQ~ - .
One embodiment of subject invention is~based on Appl~cants':discovery of pepti~des~, unrelated in : primary amino acid sequence to the primary amino acid 10 sequence of gpl20, which stimulate the prodùction of anti-gpl20 antibodies when administered ia:vivo to a ~ :
mammal. The primary amino acid sequence:refers to the linear order of amino acid residues in a peptide or protein. - ~-: 15 Peptides of the invention~were identifie~
initially by screening an epitope library with a rat :~
monoclo~al a~tibody known to be HIV neutra~l:izi:ng in an n:vitro assay. Such a method is des ribed, for example, by Scott and smith (Sçi~nÇ~ 249:3~86-390 20 (~ggo)). Briefly, an epitope~;~library is a vast misture Qf filamentous phage clones,:each displaying one :peptide sequence on the v~irion surface.~ The survey is ~ ~ -:~ accomplished using a:binding protein (e.g., a monoclonal antibody) to affinity-purify~phage that ~ :
25 display tightly-binding peptides~ and~propagating:the:
purified pha~e in~E~ ~Qli. The~amino acid ~equence of:
: the peptides displayed on the phage are then determined by sequencing the corresponding coding region in the viral DNAs. Peptides identified in this W092/~2S79 PCT/US92/04972 6~ -6-manner that are also capable of competing for antibody binding with the natural epitope are referred to herein as mimic peptides because they mimic the natural epitope, but in most instanc~s are unrelated 05 in primary sequence to;the natural epitope.
More specifically, the steps invol~ed in the screening of an epitope 1ibrary include providing an epitope library containing bacterial cells infected with fusion phage. The fusion phage are then 10 contacted with a biotinylated antibody under condition~ appropriate ~or the binding of the antibody to peptides displayed on the surface of the bacteria1 cells infected by the fusion pha~e. The mi~ture is then contacted with a streptavidin-coated ~ubstrate 15 under conditi~ns appropriate for the binding of biotin to streptavidin. ~on-~pecifical1y bound phage are removed with a wash step. Boun~ phage are eluted and :
used to infect E ~oli cells and peptide s~quences binding to the rat monoc1Ona1 antibody were 20 identified. Although these peptid~ sequences are ~: fourteen amino acid residues in 1ength~ it is be1ieved that some or all of the he~apeptide amino acid residu~s 5 through 10 are essential for the binding of the peptides to the rat monoclonal antibody since ;~
25 amino acid residues 1 through 4 and 11 through 14 were : identical for all the peptides in the epitope 1ibrary. However, it is possib1e that-some or a11 of the amino acid residues 1 through 4 and 11 through 14 ar~ critical since amino acid residues 1 throuqh 4 and 30 I1 through 14 were part of the peptide that actually demonstrated function in the E~emplification.

WO9~/22579 2 1 1 1 ~ 1 PcT/US92/04972 ;

Peptides having the same sequences as the peptides identified by screening the epitope library, ~`~
e~cept for the addition of amino acid 1~ (alanine) were synthesized and assayed for the ability to 05 stimulate an anti-gpl20 or anti-V3-loop response when administered to mice n v vo, as described in the E~emplification below. Murine antisera was tested for reactivity in a direct binding assay as described in detail in the E~emplification section. Three 10 peptides were identified which stimulate a response which is cross-reactive with the natuxal HIV ~.
epitope. The three peptides are 15 amino acid residues in length and can be represented generically as follows~

15 Ala Asp Gly Ala AAl Gln AA2 Asn Arg AA3 Gly Ala Ala Gly Ala.
~.
In the generic formula above the symbols AAl, ~A2 and AA3 represent amino acid residues which are not i ~:: perfectly conserv~d in the three mimic peptides described herein. Thu~, the peptides of this 20 invention include peptides~of the generic formula set forth above wherein AAI, AA2:and AA3 represent any amino acid residue. In addition, the peptides~of the invention~include fragments of the 15 amino acid sequence which have the desired properties discussed 25 hereinO More preferably, AAl and AA3 are selected from the ~roup of amino acids consisting:of serine, threonine, methionine and cysteine and AA2 is selected from the group consisting of lysine, arginine, histidine, serine and threonine. Due t~
30 the critical nature of the he~apeptide, the peptides of this invention can also be represented generically including the following sequence:

W092/22579 PCT/US921~4972 `;
~ 6~ -8-;;,.

AAl ~ln AA2 Asn Arg AA3 where symbols AAl, AA2 and ~A3 are the same as above described~ the preferred peptides including the .~
following sequences being as follows: ;

05 Ser Gln Thr Asn Arg Met; ~;~
Met Gln Ser Asn Arg Ser; and Ser Gln Arg Asn Arg Ser.

Peptides of the generic formula set forth above can be routinely synthesized by standard chemi~al .10 techni~ues or by recombinant DNA methodology. Such ~
techni~ues are well known in the art. Peptidé ~:
synthesizers are available commercially and standard techniques have been described in numerous publications including, for example,~Merrifield;(~
15 ~hem._~Q~. 35:~2149-2154 (1963~9 and Hun~apill:ar et al. :(Na~ure~lQ:105~ 1984)). :It iæ well known to one skilled in the art that the immunogenicity of ~:
small non-, or weakly-immu~ogenic molecules (haptens) can be increased by c:onjugating th~ weakly- :
20 immuno~enic molecule to a carrier protein. Commonly used carrier:proteins include~ keyhol~ limpet : hemacyanin a~d bovine serum albumin. The peptides of his invsntion can be conjugated to a carrier mole~u~e, usin~ conventional metho~s, to enhance their 25 immunogenic:properties.
Another embodiment of the subject invention is based on Applicants' discovery of peptides, unrelated in primary amino acid seguence to the primary amino acid sequence of an essential protein of a virus (e.g., HIV gpl20), which have the ability to wo 92,2257g 2 1 1 1 6 ~ 1 P~TIUS92/04972 `~
_9 _ interfere with a component involved in the function of the virus. In a pre$erred embodiment, these peptides have the ability by themselves, or conjugated to an immunogeni~ carrier molecule, to 05 stimulate the production of antibodies specifically reactive with the essential viral protein when :
administered to a mammal such as the peptides set forth above which are mimic peptides for the V3 loop.
One skilled in the art wilI recogniz~ that the 10 amino acid seguences of th~ p2ptides set forth above represent only the essential portion required for .
immunogenic or anti-viral a~tivity. ~ peptide~which is useful as an immunogen or anti-vi~ral aqent is not~
necessarily limited to a mintmal sequence shown 15 above, but mu:st have at least a portion of an amino acid sequence shown above~which provides immunogenic or anti-viral activity. In addition, peptides which include modifiea amino acids~a~re included~within the scope of the invention provided that the ~ modifications (e.g., conservative substitutions) do not adversely affect the functional characteristics ~`~ of the psptides.

Uses ~Qr the m~m~ ~eptides t is w~ll known in~the art:~that antibodies 25 directed to the V3-loop can prevent viral infection - by both ~ree and cell-associated virus. The mimic peptides can be used, for example, as immunogens to stimulate an anti-gpl20 immune response in an individual. Such a response could be protective in 30 the sense that the individual to whom the peptides are administered may be immune to infection by the HIV virus.

'' WO92/2257g PCTiUS92/04972 .:
6~ o- ~

To be used as a vaccine, the mimic peptides are formulated in a composition and administered according to conventional protocols. Such a comp~sition includes at least one mimic peptide, by 05 itself or conjugated to~n immunogenic carrier molecule, in an amount sufficient to stimulate an `:
immune response in a host. The mimic peptide is :
contained in a physiologically acceptable carrier. ;:
Such carrier~ include, ~or example, fillers, 10 non-tosic buffers, physiological saline solution, etc~ The composition can also include adjuvants, protease inhibitors, lymphokines, or compatible drugs-. where such a combination is:seen as d~sirabl:e oradvantageous. -Although the role of the V3-loop has not been elucidated, it appears that the V3-loop has a viral:
function which leads to viral propa~ation. ~ Other patho~enic viruses contain proteins which have a viral function which lead to viral propagation~
Peptides which are unrelated in primary amino acid sequence to the primary amino acid seque~ce of an essential protein of a virus, preferably mimic peptides, can therefore be used-in a therapeutic composition to interfers with viral repl~ication in an 25 individual. This can occur, for e~ample, by interference of the binding of a viral product with a cellular or viral component. The mimic peptides~ can be used as decoy molecules to inhibit the natural function of an essential viral protein (e.g.,~ the HIV
30 gpl20 protein). By introducing an effective amount of the mimic peptides to an ir~fected or non-in~ected individual, it is reasonable to predict that their W092/22579 2 11 1~ 1 PCT/US92/~4g72 presence will interfere with viral replication thereby slowing or preventing the spread of the infection. `-EXEMP~IFICATION

05 Direct bindin~_~saY
Direct bindin~ between test peptides and test antibody (affinity purified or:in serum~ was determined by~ELISA assay. Costar~ELA/RIA plates w~r~ coated by overnight incubation with 50 ~1 of a 10 solution containing the test peptides or controls.
Following the overnight incubation, the p:lates were ~
washed 3 times with 1 X Phosphate Buffered Saline ~`
(PBS). The plates were then blocked with 1% Bovine :~-Serum Albumin (BSA) essentia~lly globulin free (Sigma~
15 ~at. #7638~ at room temperature for absut l~hour or more.
: The blocker was removed and about 100:~1 of affinity purified 1 antibody ~or test serum~ was ; added`in~a dilution series. The dilutions~ were made 20 :in PBS-T~FCS :(l X PBS, 0.05% Tween-20, 2:.5% Fetal Calf~Serum (FCS).~ The serum~was incubated~on the plate for~:about 1 hour~at room:temperature.
~:~: Following~this incubation:, the plates:were washed 3 times wit~ PBS-T/FCS. ~ ~ :
2 antibody was then added (at about 100 ~1 per well) and incubated at room temperature for:about 1 hour. The 2 antibody i~ typically an anti-species~
antibody which is biotin labeled. This incubation is followed by 3 PBS-T/FCS washes.
A Streptavidin-HRP (horseradish peroxidase) conjugate (Southern Biotechnology Associates, Birmingham, AL) was added at:an appropriate dilution : ~
.

W O 92/22579 P ~ /U~;92/04972 ~typically 1/10,000) in PBS-T/FCS and incubated at ~-room temperature for about 1 hour. ThiS ~tep was ;:.
ollowed by 3 washes with PBS-T/FCS. The assay was developed using the TMB Microwell Substrate System 05 (Kirkegaard & Pexry ~abs, Gaithersburg, :r!lD). The optical density (OD) was read on an ELISA reader at 450 nanom~ters. ` ' C:ompetit on çxp~iments Competition e~cperiments:wexe carried out by ~'~
10 mising each test peptide and co~trol peptide in ~, solution with a test antibody a~ determininq the ~, effect (if any) of the peptide in solution on bindin~ ~' of t~e antibody to a peptide attached to the plate in an ELISA assay. The protocol is essentially as 15 described above in connection with the ELI5A assay except that peptides (or peptide c:onjugates) to be tested or ability to compete with;the fixed peptides ~
are mi:~ed with the antibody and incubated for about ~;
: ~ 30 minutes prior to the addition of the antibody to 20 the ELISA plate.

Idçn~ification of antibodie ~reactive With V3-loop Four rat monoclonal antibodies which react:
specifi~ally with HIV-IIIB were tested for V3-loop binding. Each of these antibodies e~chibits 25 anti-syncytia activity in an n vitro assay. The antibodies were designated 24D3, 20G3, lG7 and 28G12.
In a direct binding a~say, of the type de~cribed above, pol~styrene plates were coated with cyclized V3-loop (50 ,ug/ml), non-cyclized V3 loop (50 ~giml) 30 and recombinant gpl20 (2 llg/ml). Each of the 4 rat monoclonal antibodies was tested in the assay. In W092t22S7~ PC~`/US92/04972 2111~1 addition a murine control which was known to bind V3-loop was included as a positive control. The results from this bindin~ assay clearly showed that all four rat monoclonal antibodies react specifically 05 with the cyclized and non-cyclized form of V3-loop.
Antibody lG7 demonstrated the strongest reactivity.
Two of the antibodies were biotinylated (lG7 and 28Gl~) by conventional methods. Specifically, in separate 1.5 ml polypropylene tubes, 167 ~1 (500 ~g) 10 of 28G12 and 250 ~1 of lG7 (500 ~g) wer~ added. To each tube was added 12 ~1 o~ a biotin solution and the tubes were incubated at room temperature, with -~
agitation, for 2 houræ. The biotin solution was prepared by dissolving 1-3 mgs NHS-LC Biotin (Pierce 15 Cat. #21335; MW 556.59 daltons) in 456.6 ~1 distilled water~mg of reagen~s. The final concentration waæ

3.93 mM. The solution was made fresh each ~ime the e~perime~t was performed. : ~:
To prevent biotinylation of the carrier 20 ovalbumin, 500 ~1 of lM ethanalamine was added (pH 9 a-nd the mi~ture was incubated at room temperature for 2 hours. 20 ~1 of carrier ovalbumin (10 ~g/ml was also added) and the biotinylated antibodies were diluted against 1 X TBS overnight. The concentration : 25 of each was determined to be about 400-500 ~g/ml.
The material was concentrated in a CentriconR
30-kilodalton ultrafilter (Amicon~j~ and washed three ~-times with 2ml TBS to re~ove unconjugated biotin.
300 ~1 of TBSJ0.02~ NaN3 and 40 ~1 (7.44 X 1012 30 virions) W kil led blocking phage was added directly to the CentriconR. The mi~ture was vortexed to thoroughly mi~ the blocking phage with the antibody solution, then reconcentrated. The blockin~ phage WOg2/22579 PCT/US92/04972 -14- .
6~ ~ `

are meant to block anti~odies that cross-react with the filamentous phage in general~ apart from the foreign peptide they display. The retentate was collected in the conical cup by back-centrifugation, 05 as described in the Centricon~ instructîons, and stored at 4C.

Screeninq of the:~it~Pe librar~ ~:
Biotinylated antibodies, prepared as described above, were u~ed to screen an ~pitope library (Scott 10 and Smith, Scien~e 249:386-390 ~1990)). The epitope library was screened essentially as descri~ed by . Scott and Smith (E~æ~). In this way, peptides were:
discovered which specifically bind the HIV :~
neutralizing rat monoclonal anti~odies described ~:
15 a~ove. Four 15 amino acid~ length synthetic peptides were produced which contain the 14 amino acid peptide identified by 6creening the~epitope library. These : p~ptides are:referred to herein as M303 G7,:~M305 G14, M306 G3 and M307 G10 and the sequences~of these : 20 peptides are as follows:

M303 G7: Ala Asp Gly Ala Ser Gln Thr Asn Ar~
Met ~Gly Ala ~Ia Gly Ala : M305 G14: Ala Asp Gly Ala Met Gln~Ser Asn Arg , Ser Gly Ala Ala~ Gly Ala M306 G3: Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala Ala Gly Ala M307 ~10: Ala Asp Gly Ala Ser Gln Asn Asn Gly Ser Gly Ala Ala Gly Ala '.

2111~1 For purposes of reference, the V3-loop sequence is: ;
~ly Cys Thr ~rg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys Gly. The 05 cyclized ~orm of the V3-loop is generated by joining the amino terminus and the carboxy terminu~ of the linear molecule. ~:-Reactiv ~y_of ~0~ ~7.~ ql4 and ~30~ G3 wi~h rat ~:
monQçl~nal 2~I2 The M303 G7, M305 G14, M306 G3 and M307 G10 :~
peptides were syn~hesized by conve~tional methods and tested for direct binding with xat monoclonal antibody 28G12 by the ELISA assay described above.
Each of the four peptides was attached to separate 15 wells of an ELISA plate. Appropriate controls were includ~d such a~:an V3-loop positive control in an ELISA well and a BS~ negative control in a separate ~
well. ~-In th~ direct binding assa~, khe antibody showed 20 strong binding to the positive controls (V3-loop and recombinant gpl20) but showed binding to ~303 G7, M305 G14, M306 G3 and M307 G10 at near background levels or sli~htly eIe~ated. This may be an artifact .

of the small size ~f t~ese peptides (15 amino:acids 25 as compared to 38 for ~3-loop)~
Surprising results were obserYed when the test peptides were tested i~ the competition assay described above. Specifically, when three of the f our test peptid~s were incubated with the 28G12 30 ~ntibody in solution prior to incubation with plates coated with ~3-loop or recombinant gpl20 a significant decrease in antibody binding, as W092/22579~ PCT/VS92/04972 evidenced by a decrease on the OD4so determination, was observed. When tested in this manner, the peptide M307 G10 was the only test peptide that did not result in a significant decrease in antibody 05 binding. This M307 G10 peptide had glycine as the - ninth amino acid residue, whereas the other three test peptides, M303 G7, M305 G14 and M306 G3 all had arginine as the ninth amino acid residue. In the competition e~periment, the control peptides us~d 10 were lambda repressor amino acid residues 12-26, said peptide having the sequence Leu Glu Asp ~la Arg Arg Leu Lys Ala Ile Tyr Glu Lys Lys Lys (CO~T~OL in :
Figure~ 1-7) and a synthesized peptide, M304 P8, containing the sequence of a 14 amino acid peptide 15 that did not bind biotinylated antibody during the screening of the epitope library, said M304 P8 peptide having the sequence Ala Asp Gl~ Ala Ile Ser Asn Leu Ile Ser Gly Ala Ala Gly Ala. The resul'rs of this e~periment are shown in Figures 1 a~d 2. -20 Immunization Qf mice with ~m c ~Ptid~
BALB~C mice were immunized u~ing conventional methods with each of the three mimic peptid~s. The mice were primed on Day 0 with 50 ~9 per mouse of either M306 G3 in complete Freund's adjuvant ~CFA) or 25 M306 G3 conjugated to keyhole limpet hemacyanin (RLH~
în CFA. At 21 days post-immunization, thc mice were tail bled for a 1 response and the serum was collected. The mice were then boosted with M306 G3 or M306 G3 KLH in incomplete Freund's adjuvant 3~ (IFA)o Two weeks after the boost the mice were bled for a 2 response. Two weeks after the 2 bleedin~
the mice were bled for a late 2 response. The mice W092/22579 PCT~US92~04972 21115~1 were again boosted and bled 2 weeks later for a 3~ ~.
response. Two weeks after the 3 bleeding, the mice were bled for a late 3 response. The boost schedule was repeated until the late 5 r~sponse was 05 determined. All blood was collected in Becton-Dickinson serum collection tubes and the serum separated out and used in direct and competition binding experiments. ~:
The results of direct binding e~periments using 10 mouse immune serum are presented in Figures 3 and 4.
Figure 3 represents an avera~e value of assays performed after each bleed from 4 mice immunized with M306 G3. The figure legend lists the antigen which was bound to the plate. The control was bovine serum 15 albumin (BSA)-. As can be seen clearly in Figure 3, immunization with M306 G3 mimic peptide led to the production of murine antibodies specifically reactive with both V3-loop and the M306 G3 peptide. The immunized mice had never been esposed to KLH and ~`
20 the~refore the reactivity seen in Figure 3, middle bar, is due to specific reactivity with the mimic peptide. As can be seen~in the later bleeds, particularly the 4 bleed,~immunization with the mimic peptide results in the production of murine 25 antibodies which are specificaIly reactive with the V3-loop peptide.
Figure 4 shows the results of a similar experiment in which M306 G3 conjugated to KLH was ~;
used to immunize 4 mice. The data presented in 30 Figure 4 represents an average value for the 4 mice.
The antigen which was fixed to the plate is shown in the legend. KLH was not present on the plate and therefore the middle bar represents reactivi~y toward O ~/2257g PCI/US92/04972 ~ 18-the mimic peptide specif ically. Again the antiserawas reactive with V3-loop although a greater disparity was observed between ~he relative affinities of the antisera for V3-loop versus M306 G3 05 than was observed in the e~periment described in the preceding paragraph.
Competition experiments were carried out using the mimic peptides and the mouse immune serurn. As -shown in Figu~e 5, in a f irst set o escperiments, 10 serum from a mouse immunized wi~h Mi306 G3 KLH was ;-:
mi~ed with peptides prior to incubation:with V3-loop f i~ed to an ELISA plate. A significant dscrease in ~ ;`
the OD reading was o~served when t~e serum was ~ ~.
preincubated with either V3-loop or M306 G3 as 15 compared with the OD reading when the seru~ was preincubated~with PBS. This result indicates that mice immunizeid with the M306 G3 K~H conjugate produce ~::
antibodie& which cross-react with both M306 G3 and `~
~3-loop.
:Figure:6 shows the results of competition e2periments in which M306 G3 BSA was fixed~ to the ELISA plate and the serum was from a mouse immunized with M306 G3 KLH. As ca~ be seen in the figure~ the M306 G3 peptide was able to compete effecti~ely at 25 an~igen concentrations exceeding l0 ~g/ml. ::~
Competition ~periments were also carried out using the affinity purified 28G12 monoclonal antibody. Figure 7 shows the results~of a competition experiment with this antibody using ELISA
30 plates to whieh V3-loop had been attached. Again, M306 G3 mimic peptide showed:competitive behavior at concentrations e~ceeding 10 ~g/ml.

W092/22579 -19 2 1 ~ 1 6 8 1 PCT/US92/04972 . ~
Figure 8 shows the results of a 28G12 competition :~
experiment in which recombinant gpl20 was fixed to the ELISA plate. In this e~periment, the inclusion of the M306 G3 peptide resulted in a substantial 05 reduction in the OD determination indicating effective competition~

EquivalentS
Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, 10 many equivalents to the specific embodiments of the invention described herein. These and all other equivàlents are intended to be encompassed by the ~ following claims.

~ ~;

Claims

1. A peptide having a primary amino acid sequence which is unrelated to the primary amino acid sequence of HIV gp120, the peptide characterized by the ability to stimulate the production of anti-gp120 antibodies when administered to a mammal and consisting essentially of an amino acid sequence:
AA1 Gln AA2 Asn Arg AA3;
wherein AA1, AA2 and AA3 represent any amino acid residue.

2. A peptide of Claim 1 wherein AA1 and AA3 are selected from the group consisting of serine, threornine, methionine and cysteine and AA2 is selected from the group consisting of lysine, arginine, histidine, serine and threonine.

3. A peptide of Claim 1 comprising an amino acid sequence selected from the group consisting of:
a) Ala Asp Gly Ala Ser Gln Thr Asn Arg Met Gly Ala Ala Gly Ala;
b) Ala Asp Gly Ala Met Gln Ser Asn Arg Ser Gly Ala Ala Gly Ala; and c) Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala Ala Gly Ala, or fragments of (a), (b), or (c).

4. A fragment of Claim 3 consisting essentially of an amino acid sequence selected from the group consisting of:
a) Ser Gln Thr Asn Arg Met;
b) Met Gln Ser Asn Arg Ser; and c) Ser Gln Arg Asn Arg Ser.

5. A peptide having a primary amino acid sequence which is unrelated to the primary amino acid sequence of HIV gp120, the peptide characterized by the ability to stimulate the production of anti-gp120:
antibodies when administered to a mammal and consisting essentially of an amino acid sequence:
AA1 Gln AA2 Asn Arg AA3;
wherein AA1 and AA3 are selected from the group consisting of serine, threonine, methionine and cysteine and AA2 is selected from the group consisting of lysine, arginine, histidine, serine and threonine.

6. A peptide of Claim 5 comprising an amino acid sequence selected from the group consisting of:
a) Ala Asp Gly Ala Ser Gln Thr Asn Arg Met Gly Ala Ala Gly Ala;
b) Ala Asp Gly Ala Met Gln Ser Asn Arg Ser Gly Ala Ala Gly Ala; and c) Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala Ala Gly Ala, or fragments of (a), (b), or (c).

7. A fragment of Claim 6 consisting essentially of an amino acid sequence selected from the group consisting of:
a) Ser Gln Thr Asn Arg Met;
b) Met Gln Ser Asn Arg Ser; and c) Ser Gln Arg Asn Arg Ser.

8. A peptide having a primary amino acid sequence which is unrelated to the primary amino acid sequence of the HIV gp120 protein, the peptide characterized by the ability to stimulate the production of anti-gp120 antibodies when administered to a mammal and comprising an amino acid sequence:
Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala A1a Gly Ala.

9. A peptide of Claim 6 which is conjugated to an immunogenic carrier molecule.

10. A peptide of Claim 9 wherein the immunogenic carrier molecule is keyhole limpet hemacyanin.

11. A peptide conjugated to an immunogenic carrier molecule, the peptide having a primary amino acid sequence which is unrelated to the primary amino acid sequence of HIV gp120, the conjugate characterized by the ability to stimulate the production of anti-gp120 antibodies when administered to a mammal.

12. A peptide of Claim 11 consisting essentially of the amino acid sequence:
AA1 Gln AA2 Asn Arg AA3;
wherein AA1, AA2 and AA3 represent any amino acid residue.

13. A peptide of Claim 12 wherein AA1 and AA3 are selected from the group consisting of serine, threonine, methionine and cysteine and AA2 is selected from the group consisting of lysine, arginine, histidine, serine and threonine.

14. A peptide of Claim 13 comprising an amino acid sequence selected from the group consisting of:
a) Ala Asp Gly Ala Ser Gln Thr Asn Arg Met Gly Ala Ala Gly Ala;
b) Ala Asp Gly Ala Met Gln Ser Asn Arg Ser Gly Ala Ala Gly Ala; and c) Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala Ala Gly Ala, or fragments of (a), (b), or (c).

15. A fragment of Claim 14 consisting essentially of an amino acid sequence selected from the group consisting of:
a) Ser Gln Thr Asn Arg Met;
b) Met Gln Ser Asn Arg Ser; and c) Ser Gln Arg Asn Arg Ser.

16. A vaccine composition comprising a peptide and a physiologically acceptable carrier, the peptide having a primary amino acid sequence which is unrelated to the primary amino acid sequence of HIV
gp120 V3-loop, the peptide characterized by the ability to stimulate the production of anti-V3-loop antibodies when administered to a mammal.

17. A vaccine composition of Claim 16 further comprising an adjuvant.

18. A vaccine composition of Claim 16 wherein the peptide comprises an amino acid sequence selected from the group consisting of:
a) Ala Asp Gly Ala Ser Gln Thr Asn Arg Met Gly Ala Ala Gly Ala;
b) Ala Asp Gly Ala Met Gln Ser Asn Arg Ser Gly Ala Ala Gly Ala; and c) Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala Ala Gly Ala, or fragments of (a), (b) or (c).

19. A vaccine composition of Claim 16 wherein the peptide comprises an amino acid sequence selected from the group consisting of:
a) Ser Gln Thr Asn Arg Met;
b) Met Gln Ser Asn Arg Ser; and c) Ser Gln Arg Asn Arg Ser.

20. A method for stimulating the production of an anti-HIV
gp120 immune response in a mammal comprising administering an effective amount of a vaccine composition comprising a peptide and a physiologically acceptable carrier, the peptide having a primary amino acid sequence which is unrelated to the primary HIV V3-loop amino acid sequence, the peptide characterized by the ability to stimulate the production of anti-V3-loop antibodies when administered to a mammal.

21. A method of Claim 20 wherein the mammal is a human.

22. A method of Claim 21 wherein the vaccine composition further comprises an adjuvant.

23. A method of Claim 20 wherein the peptide comprises an amino acid sequence selected from the group consisting of:

a) Ala Asp Gly Ala Ser Gln Thr Asn Arg Met Gly Ala Ala Gly Ala;
b) Ala Asp Gly Ala Met Gln Ser Asn Arg Ser Gly Ala Ala Gly Ala; and c) Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala Ala Gly Ala, or fragments of (a), (b) or (c).

24. A method of Claim 20 wherein the peptide comprises an amino acid sequence selected from the group consisting of:
a) Ser Gln Thr Asn Arg Met;
b) Met Gln Ser Asn Arg Ser; and c) Ser Gln Arg Asn Arg Ser.

25. A method of Claim 23 wherein the peptide is conjugated to an immunogenic carrier molecule.

26. A method of Claim 25 wherein the immunogenic carrier molecule is keyhole linpet hemacyanin.

27. A peptide having:
a) a primary amino acid sequence which is unrelated to the primary amino acid sequence of an essential protein of a virus; and b) the ability to interfere with a component involved in the function of the HIV virus.

28. A peptide of Claim 27 wherein the essential protein of a virus is the HIV gp120 protein.

29. A peptide of Claim 27 further characterized by the ability to stimulate the production of antibodies specifically reactive with the essential protein of a virus when administered to a mammal.

30. A peptide of Claim 29 wherein the essential protein of a virus is the HIV gp120 protein.

31. A peptide of Claim 29 consisting essentially of the amino acid sequence:
AA1 Gln AA2 Asn Arg AA3;
wherein AA1, AA2 and AA3 represent any amino acid residue.

32. A peptide of Claim 29 comprising an amino acid sequence selected from the group consisting of:
a) Ala Asp Gly Ala Ser Gln Thr Asn Arg Met Gly Ala Ala Gly Ala;
b) Ala Asp Gly Ala Met Gln Ser Asn Arg Ser Gly Ala Ala Gly Ala; and c) Ala Asp Gly Ala Ser Gln Arg Asn Arg Ser Gly Ala Ala Gly Ala, or fragments of (a), (b), or (c).

33. A fragment of Claim 32 consisting essentially of an amino acid sequence selected from the group consisting of:
a) Ser Gln Thr Asn Arg Met;
b) Met Gln Ser Asn Arg Ser; and c) Ser Gln Arg Asn Arg Ser.

34. A method for interfering with viral propagation comprising administering to an individual an effective amount of a therapeutic composition comprising a peptide and a physiologically acceptable carrier, the peptide having:
a) a primary amino acid sequence which is unrelated to the primary amino acid sequence of an essential protein of a virus; and b) the ability to interfere with a component involved in the propagation of the HIV virus.

35. A method of Claim 34 wherein the essential protein of a virus is the HIV gp120 protein.

36. A method of Claim 34 wherein the peptide is further characterized by the ability to stimulate the production of antibodies specifically reactive with the essential protein of a virus when administered to a mammal.

37. A method of screening an epitope library with non-isolate specific antibody comprising the steps of:
a) providing an epitope library containing bacterial cells infected with fusion phage;
b) contacting the fusion phage with a biotinylated non-isolate specific antibody under conditions appropriate for binding of the non-isolate specific antibody to peptides displayed on the surface of the bacterial cells infected by the fusion phage;
c) contacting the mixture from step b) with a streptavidin-coated substrate under conditions appropriate for the binding of biotin to streptavidin;
d) removing non-specifically bound phage;
e) eluting specifically bound phage; and f) infecting E. coli cells with the eluted phage.

38. A peptide conjugated to an immunogenic carrier molecule, the peptide having a primary amino acid sequence which is unrelated to the primary amino acid sequence of an essential protein of a virus, the conjugate having the ability to interfere with a component involved in the function of the virus and the ability to stimulate the production of antibodies specifically reactive with the essential protein of the virus when administered to a mammal.