CA2122263A1 - Induction of protection against viral infection by synergy between virus envelope glycoprotein and peptides corresponding to neutralization epitopes of the glycoprotein - Google Patents

Abstract

The invention comprises a method of enhancing the immunogenicity of an envelope virus glycoprotein in a host organism. The method comprises administering to the host a composition comprising the virus envelope glycoprotein and at least one oligopeptide derived from the amino acid sequence of the envelope glycoprotein, wherein the oligopeptide contains or corresponds to virus-neutralization epitopes. The method and compositions are useful for vaccinating against viruses, such as HIV, SIV, HTLV-I, HTLV-II, or any retrovirus capable of inducing AIDS in its natural host.

Description

`` 2122263 De~criDtion INWCTION OF PROTECTION AGAlNST VIRAL INFECTION BY SYNERGY BETWEEN VIRAL
PROTEINS AND VIRAL PEPTIDES.

Teehnie~l Field Thi~ invention relates to ~ vaeein~tion process, whieh involves the simultaneous or eonseeutive use of a priming ~ntigen, in this ea~e the glyeoprotein from a viru~, sueh a8 HIV, SIV or any lentivirus cap~ble of inducing AIDS in its n~tural host, or fro-w ~n HTLV-I or HTLV-II type retrovirus, and an amplifying eomposition eomprised of synthetie oligopeptides, whieh are $ree or bound to a e~rrier moleeule . and in whieh the oligopeptidos eorrespond to the noutralization ep~topes for this s~me glyeoprotein. This ~nvention al80 relates to a eom~osition for use in the proee~s.
An effeetive vaeeine eowpo~ition ag~inJt viruoe~ mu~t produee rapid neutraliz~tion of the viru~s in order to prevent the viru~es fro-w po~sibly proteeting themselves in a latent proviru~ form w~thin the ehromoso~e~ of resting eell or from find~ng refuge in the cellul~r or ti~ue eompartwents where they would be beyond the reach of ~he immune sy~tsm.
Back~round Ar~
From pr~v~ou~ experiment~ conducted with ~oth chimpanzees in the case of ~rv and m~c~ques in the c~ of SIV, it i8 cle~r that inoculation of viru~ envelope glycoprotein ~lone does not make it po~ible to obtain a fully protectiYe immune response. In particular, the viru~
envelope gly~oprotein does not produce a sufficient le~el neutr~lizing ant~bodies in order to provide protection against infection.

SUE~STITUTE SHEET

Accordingly, there exists a need in the ~rt for o method of inducing ~ sufficient level of neutralizing ~ntibodies against virus infection in a host susceptible to the infection by the virus. In addition, there exists a need in the art for a pharmaceutical compo6ition for u~e in the method.
,Disclo~ure of the Invention This invention aid6 in fulfilling these needs in the ~rt. An ob~ect of thi6 invention i8 to reinforce the immunogenicity of at le~st one envelope glycoprotein of a viru~ by comb$ning the glycoprotein with at lea~t one peptide, ~nd preferably ~t different ti~es a group of peptides~ derived frnm the sequence of the envelope glycoprotein and corresponding to virus-neutralization epitopes, i.e. correspondinq to ~mino acid seguences involved in the production of neutr~lizing antibodies in the host to which they ~ro admini~tered.
Accordlngly, thi~ invention provides a method of enh~ncing the immunogenicity of an envelope glycoprotein of a v$ru~ in a ho~t and a compo~ition for u~e in this method.
The method compri~es ~*m~nistering to the host at least one nvelope glycoprot~n of the v$ru~ ~nd at le~st one peptide der$ved from the ~m$no acid seguence of the ~nvelope glycoprote$n. The pept$de compri~es at le~st one viru~-neutrall~tlon pitope. The envelope glycoprotein and the peptide ose adm$ni~tered in an ~mount ~ufficient to induce neutr~liz~ng ~ntibodies in the host.
The in~ention pro~ides a composition for enhancing the ~mmunogenicity of ~n envelope glycoprotein of o deter~ined viru6, wherein the compo6ition comprises 08 ~ combined prep~ration for simult~neous, seporate, or sequential use:
(A) ~t leost one envelope glycoprotein of the v~ru6 or o fr~gment of ~t least 50 amino acids of the glycoprotein ond, (B) at leost one peptide derived from the amino acid sequence of the envelope glycoprotein, ~nd wherein the peptide comprise6 ~t le~6t one virus-neutralization epitope `:

',.
~,:
~ ~ SUBSTITUTE SHEET

W0 93/08836 2 ~ 2 2 ~ 6 3 PCT/EP92/02459 ~nd wherein the envelope glycoprotein and the peptide are administered in ~n amount suffieient to induçe neutralizing ~nt~bodies in the host.
For the purpose of the invention, the word composition i~ intended to eompri~e eombined prep~ration in whieh the eomponents - in this ca~e the envelope glyeoprotein and the peptide or peptides derived from the envelope glycoprotein -e~n be pre~ented in a mixture or ean be presented side-by-side and therefore be applied simultaneou~ly, separ~tely or ~t intervals, to the ho~t. For instance, the peptide(~) present in the eomposition ean be m~intained separated from other eomponents in order to be a~m~nistered sequenti~lly to booster the immunogenie reaction whieh is primed with the envelope glyeoprotein.
In a preferred e~bodiment, the invention provides a eompo~ition whieh eomprises the ~bove envelope glyeoprotein and peptide providing the envelope glyeoprotein is present in an umount suffiei~nt for priming the induetion of neutralizing antibodies in a host to which it is ~d~ini~tered, and the ~t least one peptide is in an amount sufficient to enh~nee the induetion of persistent neutr~lizing antibodies in the ho~t to whieh it is ~dministered.
Aeeordingly, the invention eoneerns the use of at least one of the ~bove deseribed peptide6 for enhaneing the immunogenieity of ~n envelope glyeoprotein of a vir~, when thi~ glyeoprotein ifi administered to a host to induce neutralizing antibodies.
~ he eompo~ition of the invention ean be used for the preparation of an immunotherapeutic drug. In this caRe the CompoBition i8 administered to seropQsitive people in order to inerease the level of neutralizing ~ntibodie6 and aeeordingly to en~ble a control of the viru~.
Methods are deocribed ~y J. Salk in N 40 Colloque des Cent Garde6 - Retroviruses of human AIDS and related snim~l -dise~ses - ~d. M. Girard, L. V~lette - Found~tion Merieux 1 gso p. 273-278~ and in ~Nature 1989, vol. 327, p. 473-476 .

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4 ``~

This invention Also provides a composition fox vaeeinating A ho~t ag~inst infeetion by a vi~u~. The eomposition eomprise6 at least one envelope glyeoprotein of the virus in an amount suffieient for priming vaccination in A host to whieh the envelope glyeoprotein is _dmini~tered.
The eompo~ition al~o eontains at least one peptide derived from the ~ino aeid ~equence of the envelope glyeoprotein.
The peptide eompri~es At lea~t one virus-neutr~liz~tion epitope of the glyeoprotein. The eompo~ition contAins the peptide an amount ~uffieient to enh~nce the induction of persistent neutralizing antibodies in the host.
The deseription of the invention in eonneetion with the use as vaeeine of the defined eomposition ean al~o be applied to the use a6 immunotherApeutic drug of this eomposition, provided th_t the deseribed means enable6 the enhaneement of the produetion of neutralizing antibodies.
Peptide~ and ~nvelope glyeoproteins ean be ~ombined under condition~ allowing them to interaet by non-eovalent phy~ieal eombination or by eovalent ehemieal bonding.
Alternatively, ~nd in a preferred e~bodiment of the inv~ntion, a priming vaeein_tion (priming) i8 achieved by $n~eet$on~ of ~nvelope glyeoprotein, with proteetive immunity being subsequently onhaneed by the in~ection of immunogenic peptide~ corre~ponding to the neutralisation epitope6.
T~o of three ~mn~ized ehimp~nzee~ were ~ueees6fully proteeted agaln~t viru6 infeetion and virus W~8 ~uppressed in a third ~nimal for a long period usin~ the eomposition6 and methods of this invention. The~e results demon~trAte that thi~ invention makes it po6sible to eli~t protection again~t HIV-1 through immunization.
Brief De6cri~tion of the Drawinq~
This invention will be more fully de6eribed by reference to the following Figures in which:
Fig. 1 depiets anti-HIV antibody level mea6ured by ELISA
(Genetic Sy6tems ~it) in ehimpanzee FUNFACE (C-339) and a control (C-519). The result6 are ~hown a~ ~erum ELISA titre (1:dilution giving po6itive respon~e) versu6 time. Time zero SUBSTITUTE SHEET

21222~3 in the Figure corresponds to the dAy of the first boo6ter with inactivated HIV. The animal wa6 challenged at 70 weeks (arrow).
Fig. 2 depiets neutralizing antibody level in eh~mp~nzees FUNFACE (d~rk circles) and ROB~RT ~open circle6) in ro~pon~e to the in~ection of a R$H-~RU peptide eon~ugate (~rrows). The ani~als were inoeul~ted at 0, 3, and 19 weeks (arrows) and ehallenged at 24 weeks.
Fig. 3 depicts ~nti-HIV antibody level~ mea~ured by ELISA in ehimpanz~e ROBERT (C-433). The results are shown-as serum ELISA titre (lsdilution giving positive response) v r~u~ time. Time ~ero eorre~pond~ to the day of the first ~ntigen in~eetion (gpl60env, p27nef, p23vif, and pl8g~g).
The animal W~B ehallenged at 84 weeks (arrow).
Fig. 4 depiets neutr~liz~tion of HrV-l BRU as a function of the ~erum dilution in ehi~p~nzee~ JOJOTOO (499), IRA
(151), ~nd HENRY II (531) at tim~ t0 (t.l) and at 2 weeks (~]) and S week~ (- ) after a third inoeulation of~free peptid ~.
Fig. 5 depiets neutralization of ~IV-l BRU (dotted ~ ) and HrV-l ARV-2 (~olid eurves) as a function of the erum dilution in ehi~pan~ee JOJOTOO (C-499) at time t0 ([.~) and after the third inoeulation of froe peptide~ (~ ). ;
Fig. 6 shows total ~rV-l-~peeific antibody titer~ for ehi pan~-o~ C-339 (A), C-433 (B), and C-499 (C). At the indlc~t~d ti~e~, chimp~n~ees were inoculated with v~rioù~
i~munogen~ (~ee T~ble 1) or challonged with HrV-l. Titers ~re def~ned ~8 the reciprocal of the highes~ dilution of serum th~t W~8 positive using ~n ~IV-l ERA kit (G~netic ~;
Sy~tems).
Fig. 7 depicts neutralizinq ~ntibody titers in ~erum from C-339, C-433 ~nd C-499 during immuniz~tion with HIV-l ~ntigen~. Titer~ ~re the reciproc~l of the highest dilution of serum th~t g~ve 90% reduction in number of syncytia formed by CEM-SS cells (N~r~, P.L., H~tch, W.C., Dunlop, N.M., Robey, W.G., Arthur, L.O., Gonda, M.A. & Fischinger, P.J, (198~) AIDS Res. Hum~n Retroviru~es 3, 283-302.) when SUBSTITUTE SHEET

W093/08836 PCT/EPg2/02459 2,,22263 :~
compared to that obt~ined with control serum from a n~ive chimpanzee Fig 8 how~ PCR analy~is of DNA from PBMC ~nd lymph node ti~sue obt~ined 6 months ~fter ch~llenge of chimp~nzees C-339 and C-433 with HIV-l (A) ~thidium bromide-st~ined gel of amplifiod HIV
~equence~ following two rounds of PCR with ne~ted sets of pri~er~ The size of the HTV-~pecific amplifled fr~gment i~
141 ba-e p~irs Lane 1, O 5 ~g of OS174 DNA cle~ved with N~cIII ~8 lecular weight m~rker~
L4nes 2-7, positive controls for sen~itivity, e~ch containing tenfold fewer molecules of pHXB2 cleaved with Xb~I
than the previous o~mple, ~t~rting with 3000 ~olecules in lane 2 Each ~mple wa~ ~mplified in the presence of 1 ~g DNA (the ~ount of DNA in 1 5 x 105 cello) from an uninfected control ch~panzee, C-519 One neg~tive control sample (lane 14) w~- identified and u-ed as a ~ource of uninfected chl~p~nz~e collul~r DNA; all other s~mples were teoted bl~ndly C-487 w~ an ~rV-l infected ch~mpanzee, used a~ a po-itive control Lane- 8-11, DNA from P~MC of C-339, C-487, C-43 and C-519, re~pectively L~ne- 12-15, DNA from lymph node ti~-ue of C-487, C-433, C-519 ~nd C-339, re-pectively (B) Ethidium bromide-stained gel of an ~mplified port~on of the beta-globin gene (Scharf, S J , ~orn, G T &
Erlieh, H A (1986) Sc~Qnce 233, 1076-1078), _~ an internal eontrol (C) Oligonueleotide hybridization of PCR-amplified ~equence~ PCR reactlon produets shown in (A~ were denatured and annealed with l P~-labeled primer SR102, which anneals entirely within the amplified s~quence; the produet~ were examined following polyaerylamide gel eleetrophore~is and authoradiography aeeording to Rwok and Rellogg (~wok, S 6 Rellogg, D E (1990) in PCR Protocols: A Guido to ~othods ~nd Applic~tions: ed8. Innis, M A , Gelfand, D ~ , Sninsky SUBSTITUTE SHFFT

721222~3 J.J. ~ White ~.J. (Ac~demic Press, Inc., San D$ego, CA) pp.
337-347).
Fig. 9 depicts immunoblot an~ly~i~ of antibodies to ~pecific Hrv-l proteins following ~mmunization ~nd ch~llenge of ch~mpanz~e~ C-433, C-339 ~nd C-499. Serum ~mples were diluted ls200 and tested with ~ commercial kit (Di~gno~tics Pa~teur). For the ~mples shown, ~era w~re collected one month prior to challenge (marked by arrow) and then at 4 week interv~ls. Molecular weight~ of HrV-l proteinJ are ~hown for po~itive control serum.
Fig. 10 ~how~ anti-gpl60 ELISA titer~ in Rhesus monkey~
treated according to the invention.
Fig. 11 ~hows ~nti-v3 BRU antibody tlters in Rhe~us monkeys t-~ated according to the invention.
Fig. 12 shows the serum neutralization of cell-to-cell tr~u~ sion.
Fig. 13 ~hows the ~ntibody titer in ch~mpanzees, after cell-free EUrV challenge.
Fig. 14 Schedule of immunization. Purified recombinant gp160 and }~nD oligopeptide (V3) were infected at the t~e~ -indicated in the presence of either alu~, lFA or SAF-l.
Fig. 15 Time course of antibody re~ponse ~n the 3 group~ of monkeys as mea~ured by ~-~SA. Panel A: Anti-gp160 respo N e. P~nel B: anti-V3 respon~e. GsomQtric mean ant~body tit~rs in groups A (0), B t~]) and C (~) were computed from the data in tables 1 and 2 and plotted a~ a ~unction of time of ~mmunization.
Fig. 16 Time course of neutralizing antibody response in animals 57, 59 (group B), 61 and 64 (group C). Titers have been expressed as the reciprocal of the dilution of serum giving 50% reduction of syncitium formation.
Figs. 17 and 18 Correlation between ~nti-PND ELISA
titers and HIV-l neutralizing antibody titers. Panel A:
titers at 5 ~onths; panel B: titers at 7 ~onths. 0: group A;
t]: group B; ~: group C.

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Best ~ode or Ç~rxving_Q~t the_I~vention Previou~ ~ttQ~pts to protect chimpanzees again~t HI~
infsction bq vaccin~t$on h~ve f~iled, de~pite the use of -.
~evesal different types of v~ccines: ~yntheti~ peptides, ~-live recombinant vaccini~ ~irus ~W ) expre~s1ng HTV antigens, native or recombinant gpl20 or gp160 envelope antigen~, and ~:-inactivated whole viru~. The failure to protect a chimpanzee ~.
~gainst an infectious HTV challenge by prior ~accination with recombinant W followed ~y for~alin- and bet~propiolactone-inActivatQd whole ~rv wa~ previou~ly reported.
Thi% fAilure led to two considerations on which the pre~ent appro~h i~ ba~ed:
1 - Protection again~t lnfection with cell-free ~TV
probably r ~ ~e~ hi~h leYel~ of neutr~l~zing antibodies ~Ab). Should the ~iruR escape ar~dication ~y neutralizing AB
or ~ntibody dependent cellular ~ytoxic~ty (ADCC), the ~iru~
could e~ily sema~n ~heltered fr~m th~ immune ~y te~, either 8~ an int~gr~ted pso~iruR ~nd/or ~y lnfeetion of ~ell~ in the ~one m~sscw or central n~rvous ~y~tem. R~plic~tion of the ~iru~, e~n if li~itsd, could le~d to the e~rly amergen~e of n~utralization e~c~pe mutants. Ther~fore, rApid neutrnliz~tion of the ch~llen~e VirUB m~y ~e a ~ey to ~ucoe~ul ~accinat on.

21~2263 ;

2 - Up to 1990, induction of n~utr~lizing Ab by 811 the vaccines te~ted in chimpanzee~ ha~ been at best ~ediocre.
Thi~ may explain their failure to protect the animal6 against infection. To be efficacious, a vaccine, therefore, should induce higher neutr~lizing Ab titer~ th~n those obtained ~o far.
- It wa8, therefore, 60ught to elicit the highest possible neutralizing Ab titers in chimp~nzees through succes~ive immunization protocol~ using a variety of immunogen6.
One chimpanzee, C-339, wa8 immunized initially with four in~ections (~t 0, 1, 2 and 6 months) of 250 ~g of formalin ~nd betapropiolactone-inactivated whole HIV mixed with SAF-l usin~ a concentration of 1 mg threonyl NDP. The anim~l developed high HIV ELISA titers (1:200,000, using the ELAVIA
kit from Diagno~tic Pasteur with a cutoff of 0.1) and 6howed strong reactivity by Western blot to gpl60, gpl20, ~nd gp41 env, and to p55, p40, p25, and pl~ac. Its neutralizing Ab titer~ r~ached ls400 and 1:64, re~pe~tively, using two diferent neutr~liz~tion a6~ay~; the first a~s~y scored for 50% in~ibition of immunofluorescent foci formation on MT4 cell~, ~nd the second one for 90% ~nhibition of ~yncytia formation on CEM-SS cell~. Using a more stringen~ ~ssay (100~ inhibition of reverse transcripta~e production in fresh human PB~), the maximum titer of neutralizing Ab W~8 1:160, obtained $m~ediately ~fter the booster in~ection. These titor8, however, did not persist, but quickly declined to lower level~.
In ~n ~ttempt to inerease the neutralizing Ab titers of ch~mpanzee C-339, the animal w~s boo8tgd repeatedly with recombinan~ ~oluble gp160env ~urified from the ~upernatant of BHR-21 cell cultures infected with W -1163, a W -env recombinant expre~sing A gpl60 molecule containing a deletion of the tr~n~mambrane domain and a modification by site-d~rected mutagenesis of the gpl20/gp41 cleavage site to prevent cleavage. Vaccinia virus W -1163 can be made using the procedures deRcribed by gieny et al., Protein Engineering 2:219-226 (1988). The antigen was purified by ~equential S U B STITIIT F ~ ~ ~ ~T

WO 93/08836 PCT~EP92/02459 1 0 , lectin and cation-exchange chromatography, then was in~ected I.D. at multiple sites of the chest (125-150 pg per in~ection) with a human dose of BCG. This w~ followed by 3 succe~sive I.M. in~ections of the antigen formulated with SAF. ELISA and neutralizing Ab titer~ were followed on routinely; however, both remained unchanged during ~nd after this course of immunizations.
Failure of the gp160env to enhance antibody respon~es was not due to lack of immunogenicity, a8 ~hown by immunizing in parallel a naive chimpanzee, C-519, which previously had not been expos~d to HIV antigens. Using the same immunization protocol as for C-339, C-519 readily developed strong anti-gpl60 Ab respon~e, and its ELISA titer reached 200,00 after two in~ections. Therefore, failure of C-339 to re~pond to the in~ection~ of gpl60env was not due to lack of potency of the immunogen, but most likely to some unidentified, immunological block in the animal. It was reasoned that ~uch an impairment might be by-passed by in~ecting the animal with only those epitope~ of the gpl20 molecule that were r~quired for induction of neutralizing Ab.
~ t ha~ be~n shown that HIV neutr~lizing Ab are primarily directed against the type-~pecific, hyp4rvariable loop from the V3 region of gpl20. Therefore, using bi~-diazobenzidine, a 25-mer ollgopeptide with the sequence of that loop 4-(YNTRRSIRIQRGPG~AFVTIG~IGN) from the ~IV-I BRU (IIIb) strain was cross-linkQd to KLH. C-339 was in~ected with the peptide-carr~er con~u~ate in the pre~ence of SAF (3~0 ~g of peptid~) at 0, 3, And 19 wesks. ~o incre~e in ELISA titer was ob~erved, but ~ust~ined neutralizing Ab titerE w~re obtained following the ~econd in~ction. The animal was chall~nged on ~ ek 26 (see below), together with snother chimp~nzee, R08ER~, C-433, that had undergone parallel, albeit di6tinct, course of immunization.
Chimpanzee C-433 had been primed with W -1139, a W
recombinant expres~ing the same uncleaved ver ion of gpl60env a~ W -1163, but containing the transmembrane domain.
Vaccinia virus W -1139 can be made using the procedures SUBSTITUTE SHEET

W093/08836 2 ~ ~ 2 ~ 6 3 PCT/EP92/02459 described by ~ieny et al., Protein Engineering 2:219-226 (1988). Scarific~tion wa6 done with 2 x 1o8 PFV of the W
recombinant and was repeated at 4 and ~2 weeks. The animal wa8 then immunized with 125-150 ~g each of recombinant soluble gpl60, purified a~ described above, and recombinant pl8aag, p27nef and p23vif (purified from E. coli) mixed with SAP. In~ections were at O, 1, 2, and 6 month~, and resulted in an ELI5A Ab titer of 1:400,000. Again, howaver, neutr~lizing Ab titers remained low (1:400 and 1:128, by the immunofluore~cent focu~ and ~yncytia-forming a~says, re~pectively)~ C-433, therefore, wa~ in~ected with the same V3 peptide-RLH con~ugate, according to the same immunization protocol, a~ C-339. The neutralizing Ab titer of C-433 was immediately boosted several fold and the animal W85 ch~llenged in parallel with C-339.
The two chimpanzees were challenged u~ing a titrated ViN~ ~tock (III B stock, lot No. 40) from the National Cancer Institute (a kind gift of Larry Arthur, NCI, Frederick, ~D). The ~tock, which contained 104 TCID50~ml, w~s d~luted lslOO, and l ml of the dilution wa~ in~ected I.V.
into both of the immunized animalfi. To prevent unnecessary use of an animal, ~nd in view of t~e fact th~t the virus ~tock had been titrated twice in chimpanzee~ and its inf~ctivity for chimpanzees had been a~ses~ed regulsrly, no control naive chimp was used in thi~ experiment. The chLmp ID50 of thi~ virus stock was equivalent to 4 TCIDSO, and in ~wo experiments, in~ection of chi~pan~ees with 40 TCID50 rQsulted ln the Hpp4arænce of detectable viru~ in PBL a~
early a~ 2 weeks hfter in~ection and was followed by ~eroconversion at 4 weeks.
By contrast, the challenge of chLmpanzees C-339 and C-433 with 100 TCID50 was not follo~ed by detectable incre~es in antibody titers during the 24 weQks that have elap~ed since time of challenge. In addition, C-433 ha~ no~
developed anti-p25qaa Ab, C-339 has not developed anti-p27nef Ab, nor have the 2 animal6 developed anti-p66Dol Ab.

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~22263 PCR tests done at 6 weeks, 12 week6, ~nd 24 weeks ~fter ehallenge on PBL from both ehimpanzoe~ were negative, where~
the in~uffieiently i~munized ehimp~nzee (C-487) th~t wa~
eh~llenged ~nd bee~me infected ~ year ~go, W~8 positive by PCR. Finally, virus h~s not been recovered by cocultiv~tion of P~L from eithor C-339 or C-433 with hum~n PBL, ~8 ~udged by absence of RT ~ctivity after 6 weeks of eulture.
It iB understood th~t the expre~sion ~neutraliz~t~on epitopes~ is taken to me~n, in the c~e of HIV-l, the m~or virus-neutr~lization epitope, sueh ~8 described, ~mong other~, by Putney et al. in 1986 (Science 234:1392-1395) and by Ru~ehe et al. in 1988 (Proc. N~tl. Ac~d. Sei. USA 85:3198-3202), for wh$eh the ~equenee eorre~pond6 approxim~tely to ~mino aeid6 296 to 331 of the HIV-l envelope glycoprotein ~6 de6erib~d in the work of Myers et ~1. ( Hum~n Retroviruse6 and AIDS 1989, ~08 Al~mo6, Natl. ~ab). Also covered by the invention are peptide~ eorresponding to equiv~lent region6 of different variants of HIV-l, or ~nother retroviru6, Hn'-2, HTLV-I, or HTLV-II in humans, FIV, FeLV, or ~nother lent1vlrus in ani~als, and whieh eorrespond to the neutralization epitopes of the virus under eonsideration.
Also ineluded in the seope of the invontion ~re peptides eorre~ponding to tho~e known as minor neutralization epitopes, eharaet-rized by the faet that they belong to eon~erved reg~ons of the envelopa glyeoprotein, and that they ~nduee antibodio~ eap~ble of neutr~lizing, ~t relativ~ly low titer~, several different i~olates of the VirUB under eonsideration, for ex~mple sever~l d~fferent isolates of HIV-1, or even different isolates of XIV-l, and al80 of HIV-2.
An ex~mple of A minor epitope can be found in the work of Ch~nh et al. in 1986 (The EM~0 Journal, 5:3065-3071) and in that of Ev~ns et al. in 1989 (Nature 339s385-388), or Almond et al. in ~Retroviruses of human AIDS and r~lated animal disease,~ M. Girard and L. Valette, Foundation Marcel Merieux, Lyon, 1990, in press).
Immunogenic peptides of ma~or and minor neutrnlization epitopes nre preferably mixed with each other to ensure the ~,~

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2Jl32 2 2 ~ 3 -greatost possible protection. They c~n be ~dministered in the free ~tate, not eoupled to a c~rrier molecule. Thoy ean al~o be eo~bined with a sequenee of amino acids having one or prefer~bly ssver~l T-epitope~ from one or sever~l ~tructural or non-struetural proteins of the same retrovirus or ~
rotrovirus immunologieally ero~s-re~etive with the former, partieularly ueh a~ de~cribed in Freneh patent applieation of Glrard-Gluekman-~ahraoui, No. 89.11044 of August 18, 1989.
In one partieularly preferred embodimont of the invention, immunogenic peptides corresponding to neutralization epitopes ~re ehemically coupled to soguences of amino aeids eorresponding to T-epitope~. In another ease, the poptide~ are eoupled to a earr~er moleeule whieh bears the desired T-epitope~, by allowing them to re~ct, for e~mple, with a bifunctional re~gent or any other coupling agent desired.
A~ a e~rrier mol eule, any protein eoded for by the viral genome e~n be`u~ed (in the ea~e of HrV, the protein~
produeed by $~, r~v, vif, EQL, v~r, VDX, v~u, ~ae, en~, or net gonos), or other (protein-type) moleeule~, sueh ~8 HB~
~ntigen, HBc antigen, tet~nu~ toxoid, hemocyanin, hum~n albu~in, or polypeptides (for example polyly-~ne) or appropriate lipopeptides.
In a partleular embodiment of the ~n~ention in whieh the envolope glycoprot~in molecules and ma~or and minor neutr~l~zing peptides (either free or bound to carrier lecules) ~re combined in tha s~me vaccine preparation, the primLng effeet of the en~elope glycoproteins appear~ af~er the first one or few in~ections of vaccine, and the amplifi~ation effect due to peptides immediately ~fterward.
Thu~, an ob~ect of the invention i~ to use a first ~ntigen, in this case the 6everal envelope glycoproteins of e~ch of the retrovirus serotypes under consideration, which has the effect of priming the respon~e of the immune system;
~nd a second antigen, in thi~ case the synthetic peptide~
correspondinq to ma~or and also possibly minor neutr~liz~tion epitopes of the different serotypes of the ~iru6 under SUBSTITUTE SHEET

eonsideration, for v~ccination (preferably eonsecutively, but in a mixture, if neeessary) with the purpose of amplifying ~nd eonsolidating the initial response, particularly through induetion of long-lasting, high-titer neutralizing antibodies. Thi~ invention make6 it possible to induce immunity that persists as long a6 about six months and even as long as one year or more.
The glyeoproteins usQd to prime the response of the immune ~ystem are preferably whole molecules a8 obtained before possible elsavage. Thus, in the ease of HIV-l, gpl60 is preferable to gpl20, and the same is true for other retroviruses. This allow~ anti-gp41 antibodies in particular to be indueed, whieh is a favorable sign in virus c~rriers (~lasse et al., Proe. N~tl. Aead. Sci. USA, Q5:5225-5229).
The peptides consti~uting the ~mplifier can be free or physieally bound (especially by hydrophobic bonding) or ehemieally bound (espeeially by eovalent bonding) to earrier moleeules. They CUl also be assoeiated with other peptides eorr ~ponding to T-epitope6, or even to peptides, lipop ptides, glyeopeptides, al$phatie ehains, fatty aeids, or ~ny eombination of these eapable of stimulating the immune sy~tem and/or speeifieally targeting the ~ampl$fier~ peptides to antigen-pre~enting eells.
From this point of view, a partieularly advantageou6 presentation of peptides eorresponding to HIV neutralization opitope~ i~ to bind them, preferAbly by eov~lent chem~cal bonding, to an aliphatic sequence, particularly a~ de~cribed in 1989 by Deres et 81. (Nature 342:561-564). The amplifying peptide~ presented in this way ean induce not only a B-cell respon~e, but also a CTL CD8 response, restricted HLA Class I, as described by Takana~hi et al. in 1988 (Proc. Natl.
Aead. Sci. USA 85:3105-3109).
When the viru6 has a high degree of antigenic variability, as in the case of HIV-l and HIV-2, it is neeessary to use a8 priming antigen not ~ust one, but several envelope glycoprotein~ with different sequences, each ~equence corresponding to an isolate or group of isolates of SUBSTITUTE SHEET

1s 21~2263 the virus under con~ideration, ~o a~ to obtain ~s many priming phenomena a8 de~ired, since each iB specific for single isolate or group of isolates. In th$s ca~e, it is understood th~t the ~mplifying pept$des ~re composed of the mixture of neutralization peptide~ of each of the isolate6 under consider~tion, a8 indic~ted below.
A prep~ration of HIV-l ~mplifier peptide6 according the invent$on is ch~r~cterized by the fact that it contain at least one of the sequence~ or one part of the ~equence~
described below in one letter amino ~cid code:
C-TRPNNNTR~R IRIQRGPGRA FVTIGR-IGN M-RQAEI-C
C-TR~NNND~S IRIQRGPGRA FVTIGI~-IGN M-RQA}I-C
C-TRPNNN~R~ IRIQRGPGRA ~VTIGI~-IGN M-RQAH-C
C-TRPNNNTRGS IRIQRGPGRA FVTIGl~-IGN M-RQAH-C
C-TRPNNNTR}~S IYI--GPGRA F}ITTGRIIGD -IRRAH-C
C-TRPYNNVRRS LSI--GPGRA FRTRE-IIGI -IRQAH-C
C-TRPGI~I~RG IHF--GPGQA L~TTGIV-GD -IRRAY-C
C-ARPYQNTRQR TPI--GLGQS I-YTTRSR-SI -IGQAH-C
C-TRPNNN~ ITR--GPGRV IYATGQIIGD -IRRAH-C
C-TRPNNNTII~ ITM--GPGRV lrYTTGQIIGD -IRRAH-C
C-TRPGSDRRQS TPI--GI ~ A IlrTTRGRTRI -IGQAH-C
C-TRPG8D~IR QSIRIGPG~V FYARGG---I -TGQAH-C
C-TRPNNNT~RG IAI--GPGRT ~YARERIIGD -IRQAH-C
C-TRPNN~I~$R VTL--GPGRV NYTTGEILGN -IRQAH-C
:~ C-TRFGNNT~G SHF--GPGQA ~YTTGrVGDI -RRAY-C
C-TRPDNRITSRQ-TPI-GLGQA ~Y~TRIRGD~ -RQAY-C.
C-TRPN~NVRRR-HIHI-GPGRA FYTGEIRNI -R~AH-C
- C-TRPYRNTRQS-TPI--GLGQA LYTTRTKSI -GQAH-C
C-TRPNNNTTRS-IHI--GPGRA F~ATGDIIGTIRQAH-C
C-TRPXY~RXR-IHI--GPGRA PYTTRNIIGDIRQAH-C :
The production of the amplify~ng mole~ules of the :.
invention by using a ~equence cont~ining at lea~t one neutralization epitope and particularly one of those from the list above and one carrier seguence having at lea~t one T-epitope, may be achieved by binding the~e sequence~ or by phy-ical co 'oination in the same compo~ition.

. :.
,.
~, ; SUBSTITUTE SHEET

2122263 1 6 ";

To be fully effective, priming and amplifying antigens must be enhanced, for example and preferably by lipid ad~uvants, 6uch a~ derivatives of muromyl dlpeptide ~n lipid emulsions, or incomplete Freund~6 ad~uvant.
The priming and amplifying sntigen6 are preferably administered ~ntramu~cularly to a host, such a6 a pr;~te, and especially a human. Following are typical immunizstion schedules thAt can be employed for gpl60 and peptides of HIV
gpl60 PQptides Imonth6~ ~month 0, 1, (2), 6 12, 13 0, 1, 2, 12 13, 14 0, 1, 2, 12 1, 2, (12) It will be under~tood that these immunization 6chedules are merely representstive and that the schedule6 can be varied to obtsin the optimum response in the host.
Similarly, the ~mount~ of the priming and amplifying antigens can be varied. ror ex~mple, about 150 ~g of gpl60 in Syntex SAF-l ~d~uvant can be admini~tered a8 indicated, followed by adm~ni~tration of the peptides in amounts of typically 100 ~g of each peptide.
~ nally, the relative proportions of th~ peptide~
involved can vary according to the de~ired final proportion~
of each peptide in the final prep~ration. In particular, the~e proportion~ will be ad~u~ted a8 a function of the immunog nicity of each peptide ~nd the number of fun.ctional grOUp8 carried by each one, which are c~pable of ent~ring into the con~ugation rehction with complament~ry functional group6, ~t least when these peptides are coupled to a carrier molecule.
In a particulnr applic~tion of the invention, the in~sction of omplifying peptides i8 rspl~ced by the admini~tration of particle6, viru~, or bacteria, which are recombinants expressing the nHutralization epitope of the viru6 under consideration on their surface ~nd/or during their multiplication and in thi~ way are capable of inducing neutralizing antibodie6 against ~aid retrovirus: HBc antigen - SU~3STITUTE SHEET

212,~;~6~

partieles; HBs antigen particles; bacteria expre~sing the neutr~lization epitope in surfaee or eytoplasmic protein6, ~ueh a~, for ex~mple, the l~mB reeeptor; pieorna virus ehimeras, sueh as, for example, poliovirus-HrV ehimeras;
poxvirus reeombinants; adenovirus reeombinants or adenovirus ehimer~s, ete. Depending on the live veetor seleeted for the presentation of the neutralization epitope, thi6 ~dminlstration ean be earried out in the form of live vaeeine administered orally (for example, ehimeras eonstrueted from S~bin poliovirus strains or from human adenoviruse6, or from attenuated ~trains of Salmonella, ~igella, or other nterob~eteria, or from any organi~m, vlrus, yea~t, ~cteri~
e~pable of indueing an immune response after oral administration) or in the form of live vaecine administered by the parenteral route (for example, recombinant poxvirus) or even in the form of inactivated vaeeine by the parenteral : route (for x~mple, ehimera~ eonstrueted from the Mahoney .:
~train of polioviru~, or inert partiele~ of HBsAg or HBcAg).
In ~nother particular embodiment of the invention, the ~ntigen (envelope:glyeoprotein), whieh i8 in~eeted for the priming of the vaeeination, i.e.! the envelope glycoprotein of the virus, is presented under the form of parttele~ sueh ISCOM (Immune.Stimulating Complex, eompri~ing an as~ociation of ~n ~ntlgenie protein with a glyeo~lde Quil A) or lipo~omes.
- The pr~ng antigen and/or the peptide can be al80 assoel~ted w$th live reeombinant mieroorganisms, such as viru~es or baeteria (for instanee the poxvirus or BCG: Bacile de Calmette Gerin) or sny live vaceinQ modified to expres~
the envelope glycoprotein or the peptide derived therefrom.
The envelope glycoprotein and/or the peptide derived therefrom can al80 be presented by inaetivated particle~, for ~nstance viral particles, such a~ the HIV virus or a part of thi8 virus, or particles without genome. Such particles without genome have been described to produce vaccine by Haffar 0. et al., Journal of Virology, 64:2653-2659 (lg90) The~e particles can be called HIV-like particles in the case SUBSTITUTE SHEET

W093/08836 PCT/EP92/024~9 . .

of HrV viru6: for the purpos~ of the invention they do not cont~in the complete HIV genome, but they enable the exposition at their ~urface of the virus component~ of the composition of the invent~on.
In another embodiment of the invention, the envelope glycoprotein ~ntigen i8 combined in a mixture with other antigens. For ~nstance, when the priming antigen is the HIV
envelope glycoprotein, one or several antigens, ~uch a6 g~g net, vif, pol, GPG or GLG antigens, c~n be combined with it, a8 they can be combined with the peptides of the compo~ition The invention al80 comprise~ the compositions above described, wherein the env glycoprotein i8 repl~ced by or a~80ciated with a fragment thereof. This fragment has advantageously more than 50 ~mino acids ~nd is char~cterize in that it has the immunogenic properties of the glycoprotein in the context of the invention.
The invention al80 concerns monoclonal or polyclonal antibodie~, which recognize the glycoprotein and/or peptides of the composition. These antibodies can be ~ssociated in a mi~ture ~nd used, for instance, for serotherapeutic purpo~es.

~XANPLE 1: Immunization of ~ chimp~nzee with HrV-l ~RV
and the qlycoprotein of this isolate;
~mplification of the respon~e with a BRU env oligopeptide coupled to RLH.
Ch~mpanzee 339 (FUNrACE) was first immunized with three in~ect~on~ at one ~onth intervals of 250 pg of purified HIV-1 ~RU viru~, inactivated by tre~tment with 0.025 percent formalin for 48 hours ~t 30C ~nd 0.025 percent betapropiolactone for 30 mlnute~ at 37C, combined with Syntex ad~u~ant containing 1 mg~ml threonyl-~DP in an emulsion of S percent ~qualane and 2.5 percent pluronic polymer. The~e in~ections were followed by ~ first booster at 7 months and 8 second booster one year later.
The anim~l then received five in~ections of BRU virus envelope glycoprotein (gp160~ purified from ~upernatant of BHR-~l cell cultures infected with a vaccinia virus recombin~nt (strain W env 1163) having a g~nome for which SUBSTITUTE SHEET

WO 93/08836 1 9 PCT/EPg2/02459 21222~;33 genetic recombination techniques were u~ed to in~ert the sequences of HIV-l BRU coding for gpl60env modified through oligonucleotide site-directed mutagenQsi~ to eliminate the sequences involved in gpl20/gp41 cleavage and from which the tr~nsmembrane hydrophobic zone wa~ deleted, a8 described in Rieny et al. in 1988 (Prot. ~ngineering 2s219-226). The purified protein wac used in an ~mount of 125-150 ~g per intra~uscular in~ection in the presence of Syntex ad~uvant.
To prepare the glycoprotein, the culture medium of BH~ cells infected with W -1163 wa~ concentrated by precipitation with ammonium sulfate, then with trichloracetic acid, and the glycoprotein wa~ then purified by three ~ucces~ive runs of affinity chromatography over lentil lectin, ion exchange over cation-exchange resin, and high-performance liquid chromatography (~PLC). The recombinant gpl60 obtained in this way i8 95 percent pure. It is recognized ~y monoclonal antibodie6 specific of the gpl60 of HIV-l and particularly by neLtralizing ant$bodies 110-4 6pecific for t~e ma~or neutralization epitope of the ~RU isolate. Moreover, it shows a strong affinity for the CD4 receptor of T4 lymphocytes. -The level of antibodies induced in respon~e to in~ections of inactivated viru~ (ELISA determination:
1/200,000 with the Diagno6tics Pasteur ELAVIA kit;
neutralizing t$ter 1/400 by mQasuroment of 50% inhibition of the formation of immunofluorewence foci; 1/64 by measurQment of 90% inhibit~on of syncytia formation in CEM-SS cells)~ was not changed appreciably by the in~ection of gpl60.
The animal was given 300 ~g of preparation of ~ynthetic peptide having the ~equence Y N T R ~ S I R I Q R G P G R A F
V T I G ~ I G N corre8ponding to the neutralization epitope of the BRU isolate, the tyrosine residue ~Y) being coupled to hemocyanine (RIH) with bi~(diazobenzidine) and combined with Syntox ad~uvant. The in~ection was repeated once three weeks later, then a second time at 19 weeks.
These in~ections did not result in any increa~e in antibody titers measured by ELISA (Figure 1), but they did SUBSTITUTE SH~ET

2 0 "

re~ult in ~ marked incre~e,in neutralizing ~ntibodies, ~8 can be seen in Table 1 ~nd Figure 2, ~ me~ured by three different ~ntibody titr~tion method~.

~able 1 Induction of neutr~lizing ~ntibodie~
in the chim~nzee PUNFACE ~C-339 Level of neutr~lizing antibodie~
D~te after me~sured bY ~ethod 1st in~ection (week~ A . ~ C

As 90% inhibition of syncyti~ in MT4 cells B: 90% inhibition of syncyti~ in CEM-SS cells ~ .
C: 75~ inhibition of immunofluorescence in H9 cells FUNFA~ w~ then challenged ~t 26 week~, by adm~ni~tering an intravenou~ in~ection of 1 ml of ~ lslOO
d~lutlon, or 100 TCID50 of an HrV-l stock titr~ting 104TCIDSO/~l, kindly provided by L~rry Arthur (NCI, Frederick). This stock 040 W~8 titered on two occ~ions in the chl3panzee, which allowed Arthur et al. ~o determine th~t its ID50 for the chimp~nzees w~s 4 TCIDSO. ~he in~ection of 40 TCID50 of this stock in un~munized chimpanzeas resulted in the app~arance of detectable virus in the ly~phocytes of the ~nimal st~rting two weeks after in~ection and wa~
followed by anti-HIV seroconversion within four weeks, as ob~erved in the two s~mples, and as published by Arthur et ~1. in 1989 (J. Virol.).
The chimpanzee FUNFACE demon~tr~ted ~pparently total protection against infection with 100 TCID50 of ~he stock 040 ~virU8~ because at up to six month~ after the challenge inlection, no virus w~6 detected in his lymphocytes (~

SUBSTITUTE SHEET

2~22263 messurod either by gene amplifieation with pol and g~g probos, or by coeulture with human lymphocytes and assay of r~verse tran~eripta~e in 100,000 x g pellets obtained from eulture supernatants) and at 6iX months, there wa6 no anti-HIV ~namnostie response as measured by ELISA or by Western blot (T~ble 2) and no anti-nef antibody deteetable by Western blot Table 2 Fate of anti-gpl60 and anti-ma~or BRU neutrslization e~ito~e antibodie6 after ehallonoe in1eetion of FUNFASE
ELISA titer on date lndieated day of Antigen challenge +1 month +2 month6 ~3 month~ +4 months gpl60 1?9,000 127,000 89,000 44,000 18,000 BRU
p~ptld- 6,000 3,000 2,500 1,000 1,000 ~ .
~XA~PLE 2s Immuniz~tion of a ehimp~nzee with rocombin~nt ~ntlgens onv, gag, n t, and ~if of HIV-l;
~mplifieation of th~ r ~pon~e by a BRU env oligopoptide eoupled to ~LH
Chimpanzeo 433 (ROBERT) w~s fir~t primed with three eon~eeutive ~earifieations of 2 x 108 PFU of a recombinAnt vaeeini~ virus (W onv 1139) expre~sing the gpl60env of HIV-l ~RU, then by the intr~venou~ administr~tion of his own lymphocyte6 whieh previously had been infeeted in vitro by the reeo~binant viru~ W env 1139 and fixed in form~ldehyde The ~nim~l then received three consecutive intr~muscul~r in~eetions at one month interv~ls, then three booster6 at 33, 38, and 40 weeks ~nd a la6t booster ~t 66 weeks consisting of mixture of 125-150 ~g of eaeh of the following ~ntigen6 combined with Syntex ad~uvant gpl60env, purified as de~eribed in ~x~mple 1 above, and the protein~ pl8qa~, p27nef, ~nd p23vif expressed in E coli and purified a6 d~serlbed in French patent applieation No 89 11044 of ~ SUBSTITUTE SHEET

WO 93/08836 PCT/EPg2/02459 i 21~263 August 18, 1989. Finally, ROBERT received the ~ame BRU
peptide coupled to RLH ~nd combined with Syntex ad~uvant on the ~me inocul~tion 8chedule as FUNFACE did in the previous ex~mple.
In~ections of the peptide-RLH con~ugate did not re~ult in any incre~e in antibody levels a8 measured by ELISA (Fig.
3), but did result in a marked incre~se in neutralizing antibodies, a~ can be ~een in Fig. 2 and in Table 3. The neutralizing antibodies were al80 measured using three different methods:

Table 3 Induction of neutralizing antibodies in the chimD~nzee ROBERT (C-433~
.

Level of neutralizing antibodie~
Date ~fter me~sured bv method l~t in~ection.
_ (week6) A B C

8 >800256-512 >1600 ~s 90% inhibition of syncytia in NT4 cells Bs 90% inhibltion of ~yncytia in CEM-SS cells Cs 75% inhlbition of immunofluore~cence in H9 cells Robert W~8 then challenged in p~rallel with FUNFACE, by the intr~venou~ inoculation of 100 TCID50 of the same stock 040 of HrV-l viru8 from NCI as in the previou~ example. Here ag~in, total protection against infection appear5 to have been obtained ~ ~udging from the absence of virus in the ~nimal's lymphocytes and the negativity of the PCR six months ; after ch~llenge and by the absence of anti-p25aaa and Anti-p27nef antibodies, as well as the absence of anamnestic anti-HIV response as measured by SLISA or by Western blot six months after challenge. Table 4 shows the s~me ab~ence of :~ `

SUBSTITUTE SHEET

WO 93/08836 2 ~12 2 2 ~ 3 PCT/EP92/024S9 anamnestic effect on the anti-gpl60 and anti-BRU
neutralization epitope.

Table 4 Fate of anti-gpl60 and anti-ma~or ~RV neutralization ,eDitope anti~dies after ch~llenge inlection of RO~ERT
ELISA titer on date indicated day of Antigen challenge ~1 month +2 months +3 months +4 month gpl60 545,000 421,000 200,000 95,000 32,000 BRU peptide 9,000 6,000 3,000 3,000 4,000 ~XaPPLE 3: Immunization of a chimp~nzee with gp160,env and pl8aaa of HIV-l antig~ns; ~mplif ication with HIV-l env peptides not coupled to ~ carrier molecule. ,-Three chimpansees were u-ed in this e~p~riment: the ch~mp~nzees JOJOTOO (49g), IRA (151) and HENRY II (531).
The first, JOJOTOO, received three in~ections, at one ~onth ~ntervals, of 120-lS0 ~g of gpl60env and pl8aaa, purified a8 described above, ~nd mixed with Syntex ad~u~ant.
This first series of in~ections was followed by three boosters of the same ~ntigen given at weeks 33, 3B, and 40, ~nd ~ f~nal ~008ter at 14 months. The~e in~ections resulted in the appaarance of a high antibody level detectable ~y Western blot and by ELISA starting immediately ~fter the first three in~ections, although the level of neutralizing antibodies was relatively low, ~s described ~elow.
,The ~econd chimpanzee, IRA, was immunized with 108 PFU
of each of the four recombinan~ vaccinia viru8 ~tocks expre~ing, re~pectively, gpl60env, p55g~, p27nef, and p23vif of ~IV-1 BRV. ThQse inoculations given ~y the intradermal route r did not lead to the appearance of any neutralizing antibody, but a barely significant level SUBSTITUTE SHEET

2 1 2 2 ! 3 2 4 < i ~

(<1:200) of antibody was detectable by We~tern blot or by ELISA. Chimp_nzee IRA wa8 then rested for two year~.
The fourth chimpanzee, HENRY I I, w~ naive in reg~rd to cont~ct with HIV or SIV antigens before the day of the experiment.
On thAt day the three anim~ls described above were in~ected intr~muscularly with a cocktail compo~ed of 21 ~ynthetic peptides, correspondin~ to the 21 seguence~ of the ma~or neutr~lization epitope (loop V3) of HIV-l published in Myers et al. (1989), in the amount of 50 ~g per peptide, in the presence of Syntex ad~uvAnt. Each of the peptides had a cy~teine at the N-terminal po~ition and anoth~r at the C-terminal, and thus represented the entire V-3 loop of a given isolate (amino acids 296-331 of the BRU isolate and corresponding amino acid6 according to the alignment of Myers et al. (1989)). The animals were rein~ected with the same mixture, re~pectively, 1 and 2 months after the fir~t in~ction. This immunization with the mixture of peptides (1.05 mg per in~ection) was followed in JOJOTOO with a ~ignif~cant anamne~tic re~pon~e dir~cted against the gpl60 of the BRU i801ate and against its ma~or n~utralization epitope, as ~ea~ured by ELISA and by u~ing purified gp160 BRU or BRU
peptide as antigen (TablQ~ 5 and 6).

- SUBSTITUTE SHEET

2 52122'263 Table 5 Induction of ~nti-gp160 BRU antibodies in re~ponse to the in~ection of a cocktail of free peptide~
corre~ponding to 21 ~eguence~ of the HIV-l neutralization e~ito~e rELISA titer: anti-oD160 BRU~ :

Time l~t 2nd 3rd 4th in~ection in~ection in~ect~on in~e~tion Chimp~n~ee ltime O~ rl month~ ~2 month~ ~3 month~
JOJO~OO (499) 300,000 450,000 2,500,000 700,000 IRA (151) Negative ND 13,000 7,000 HENRY II (531) Negative ND Negative Neg~tive ND: not determined Table 6 Induction of BRU ~nti-neutralization epitope ~nt~bodie~ in response to the in~ection of a . cocktail cont~inina 21 oe~tide~ IELISA ~nti-BRU titer~

Time l~t 2nd 3rd 4th in~ection in~ection in~ectlon in~ection Chimp~nzee (time Q) (1 month~ (2 months~ (3 month~
: JOJOTOO (499) 6,000 10,000 380,000 2pO,000 IRA (151)Ne~ative ND 4,000 2,000 HENRY II (531) Negative ND Neg~tive Negative ND: not determined The titers obtained in IRA remained vory low, and they -`
were completely negative in H~NRY II. The~e re~ults clearly illu6tr te the priming effect on the immune re~ponse resulting from pre-immunization with gp160.

. .

~ :~ SUBSTITUTE SHEET

W093/08836 2 6 pcr/Ep92/o245g - :
2 1 ~ 3 - ~

"~
The increa~e in the anti-peptide and anti-gpl60 titer in JOJOT00 was, however, not accomp~nied by ~ m~rked increase in the ~nti-HIV ELISA titer, ~s can be seen (Table 7) by using a commercial diagnostic kit (ELAVIA Diagnostic~ Pasteur) Table 7 Anti HIV ~ntibodv level ae measured bv ELAVIA

Date Ti~e 02 months 5 month~
Chimpanzee 1st in~ection2nd in~ection 3rd in~ection JOJ0~00 (499) 1,000,0001,600,000 400,000 IRA (151) Negati~e 800 100 H~NRY II (531) Negative 200 Negative ~ In contrast, the in~ection6 of the mixture of synthetic - peptidQs~ oorresponding to neutraliz~tion epitopes of the 21 :~
ola:te8 of HIV-l wera followed by a very clear incre~e in th l~vel of antibodies neutr~lizing the ~RU isol~te, ~s ;~;
~hown in Table 8 and Figure 4. It i8 rem~rkable that thi6 incre~se wa~ seen only in JOJOT00, but not in IRA nor in HENRY II, demonstrating the specificity of the priming effect :~ - of pre-im~unization with gpl60 (Figure 4). ;~-JOJOTOO~s neutralizing anti~ody r~spon~e i~, mo~eover, ~pecif~c for the ~RU i~olate, 8~ can be ~een in Figure S:
his ~erum doee not neutralize the S~2 isolate (ARV-2), but only neutr~lize~ the BRU isolate (HTLV-3~LAYl~.

~ ~ SUBSTITUTE SHEET
, . . . , ... . .. , .. .... .. ..... , .. . ~ .. . . ..... o ... ~ . . . . . . .

WO 93/0883~ PCT/EP92/02459 2~222~3 Table 8 Level of neutralizing nntibodies induced by three in~ections of a mixture o~ peptide~ corr~Eponding to the 21 known ~equences of the mR~or neutraliz~tion epitope of HIV-l: 75~ neutr~lizing titer measured on CEM-T4 cells (Method C in Table 1~.
. _ Time 1 month before +1 month af~er ~he the first iniection _ _ _third in~ect~on 250 2,500 * ~ ~ ..
Foll~w-~D Y~Deri~2ntal Result~
The mofit stringent te~t for effi~acy of experimental vaccines again t the humAn Lmmunodeficiency virus t~pe 1 (HIV-1) is protection of chLmpanze~s from infection following live virus challenge. In the ~tudy reported here, ustained high tit~rs of neutralizing antibodie~ were elicited in three chimp~nzees ~fter sequential in~ctions of different HIV-lBRU
antigen pr~parations that included whole inactivated viru~ or purifiod re~omb~n~nt proteins, followed by ~ynthe~ic peptide&
identical to ~he ~a~or ~IV-l neutralizing ~pitope, V3. ~he ~nim~ls w~ro ch~llenged intr~enou~ly with 40 ~himpanz~e infectiou~ do~es (aguivalent to 100 S0~-ti~sue culture infec~ious do~es UTCID~) of a stock of HIV-lHTLV IIIB. After 6 mo~ths of follow-up, all thr~e animals sppeared uninfected by ~rologic and virologic criterial including PRC analysis and f~ilure to i~ol~te virus fr~m peripheral blood lymphocytes, bone m~rrow and lymph node ti~ue. Of two chimjpanz~es moni~ored for 1 year, VirUB waC iEolated initially from one anLmal at 32 week~, bu~ ~h~ second and third chi~panzees were vlrus negative by all as~ay~ through 12 m~nth6. The third animal ha~ r~mainsd viru8 negative through 7 months of follow-up. These re~ult~ indicate that it i8 possible to elicit protection again~t, or significantly delay inf~ction of, HIV-1 by immunization, thus layîng the foundation for development of an HIV-l v~ccine.

C'TIT ~

Materials And Method~
Animals. Anim~ls used in thi6 study were adult male chimpanzee~ that h~d boen u~ed previously in hepatiti~ A, B
~nd non-A and non-B experiment~. The chimpanz~es were maintained ~t LEMSIP, New York University ~edical Center, in biosafety level 3 facilities. All experimental procedure6 were done ~ccording to institut$on~1 guideline6 for containment of infectiou6 diseas~s ~nd for humane care and h~ndling of primates (Moor-Jankowski, J. & Mahoney, C.J.
( 1989 ) J. ~0d. Prim~tol . 18, 1-26).
Immunogens. Sucrose gr~dient-purified whole HIV Wa6 in~ctivated by incub~tion with 0.025~ bet~-propiol~ctone, followad by 0.025~ formalin, ~nd wa8 ~hown not to contain infectious viru6 by failure to isolate virus from peripheral blood mononucle~r cells (PBMC) of immunized chimp~nzees (Gir~rd, M., ~ieny, M.P., Gluckm~n, J.C., B~rre-Sinous6i, F., Mont~gnier, L. & Fultz, P. (1990) in V~ccines for SexuAlly Tr~n~m~ tted DiJ0~ses ~ds . M~heus, A. & Spier, R. (Butterworth Co., Ltd., ~ondon), pp. 227-237). Recombinant gp160env W~6 purified-from the culture medium of BHg21 cells infected with W -1163, a recombin~nt v~ccini~ virus expressing the gpl60env gene modified by site-directed mut~genesis to de~troy the gpl20/41 cle8v~ge site ~nd to r~move the ~nchor domain of gp41 (Rieny, ~.P., L~the R., Riviere, Y., Dolt, X., Schmitt, D., Girard, M., Mont~gnier, L. & Lecocq. J.P. (1988) Prot .
Ehginear~ng 2, 219-226; ~nd Schmidt, D., Dezutter-Da~buy~nt, C., ~an~u, D., Schmitt, D.A., Rolbe, H.V.J., R~eny, ~P., Cazenave, J.P. & Thi~olet, J. (1989) Cvmpte~ Rondu~ Ac~d.
S~i. P~ris, 308(III), 269-275). Where indicated, the antigen was mixed with recombinant pl8g~g, p27nef and p23vif antigens that were purified from E. coli pTG21S3, pTG1166 and pTG1149, respe~tively, a6 de~cribed (Guy, B., Riviere, Y., Dott, ~.
Regnault, A. & Rieny, M.P. (1990) Virology 176, 413-425; and Rolbe, H.V., Jaeger, F., Lepage, P., Roitsch, C., Lacaud, G., ~ieny, M.P., Sabatie, J., Brown, S.W. & Lecocq, J.B. (1989 J.
Chromatography 476, 99-112). Before each immunization, inacti~ated whole HIV ~250 ~g vitsl protein) or the purified SUBSTITUTE SHEET

WOg3/08836 2 ~1 2 2 ~ ~ 3 PCT/EP92/02459 recombinant protein~ (125-150 ~g each per dose) were mixed with the ad~uv~nt SAF-l (Allison, A.C. & ~y~r~, N.E. (1986) J. Immunol. ~ethods 95, 157-168), and 2 ml of the mixture~
were in~ected intr~muscularly (IM).
An aliquot (19.8 mg) of ~ 25-amino ~cid peptide, with the sequence ~-NIRRSIRIQRGPGRAFVTIGRIGN (Putnay, S.D., N~tthew6, T.J., Robey, W.G., Lynn, D.L., Robert-Guroff, M., Mueller, W.T., Langlois, A.L., Ghr~yeb, J., Pettew~y, S.R., Weinhold, R.J., Fischinger, P.J., Wong-Staal, F., Gallo, R.C.
& Bolognesi, D.P. (1986) Science 234, 1392-1395; Rusche, J.R., ~vaherian, R., McDanal, C., Petro, J., ~ynn, D.L., Grim~ , R., Langlois, A., G~llo, R.C., Arthur, L.O., Fischinger, P.J., Bolognesi, D.P., Putney, S.D. & Matthews, T.J. (1988) Proc. N~tl. Acad. Sci. U.S.A. 85, 3198-3202; and LaRos~, G.J./ D~vide, J.P., Neinhold, K., Waterbury, J.A., Profy, A.T., Lewis, J.A., Langlois, A.J., Dressman, G.R.
Bo8well, R.N., Shadduck, P., Ho}ley, L.H., R~rplus, M., Bologne~i, D.P., Matthew~, T.J. Emini, E.A. & Putney, S.D.
(1990) Sci~nc~ 249 932-935) wa8 treated fir~t with citraconic acid and then wa8 coupled to 19.3 mg keyhole l~mpet hemocyanln (RLH) by N-terminal tyrosyl linkage using bi~-diazobenzld~ne (pH 9.0). After the block on a~no group6 wa~
removed, the peptide-RIH con~ugate was dialyzed for 24 hour~
~g~in~t PBS to remove exce~g free pept~de. After formulation w~th 5AF-l, lmmuniz~t~on6 with the V3 peptide-RIH con~ugate (300 ~g peptlde per do~e~ were done by the IM route.-Ch~llenge Virus. The ch~llenge inoculum was from a~tock of HrV-l ~tr~in HTLV-IIIB (obtained from L. Arthur), which had been titratffd in chimpanzeR~ and u~od in other HIV
~accine chnllenge studies (~rt~ur, L.O., Bes~, J.W., Waters, D.J., Pyle, S.W., ~elliher, J.C., N~ra, P.L., Rrohn, K., Robey, W.G., Langlois, A.J., Gallo, R.C. & Fi~chinger, P.J~
(1989) J. Virol. 63,5046-5053; and Berman, P.W., Gregory, T.J., Riddle, L., Nak~murs, G.R., Ch~mpe, N.A., Porter, J.P., Wurm, F.M., Hershberg, R.D., Cobb, E.R. ~ Eichberg, J.W.
(1990) N~ture ~rondonJ 345, 622-625). The infecti~ity titer of thi~ HIV-l stock i~ considered to be 104 TCID50 per ml and .-.
: ' ~:
~ ~ SUBSTITUTE SHEET ``

3 o ~ `

4 x 103 infectiou6 units per ml for chimpanzees. The chimpanzees were challenged rv with 1 ml of a lslOO dilution.
Aliquots of these same 1:100 dilution~ were titrated in quadruplicate by twofold 6erial dilution and infection of 1 x 105 H9 cQlls in 96-well microtiter plates. After incubation for 6 days, infection wa6 ~cored by immunofluorescence assay.
9y this method, the challenge inoculum had a titer of grester than 64 immunofluore6cent focus-forming units (end-point not reached) for the fir~t sliquot and 170 for the second.
Neutr~lization Assay. Neutralization activity in ~erum samples from immunized chimpanzees was determined by inhibition of syncytia formation in CEM-SS cell~, aB ;:
described (Nara, P.L., Hatch, W.C., Dunlop, N.M., Robey, W.G., Arthur, L.O., Gonda, M.A. L Fischinger, P.J. (1987) AIDS Re6. Human Retro~iruses 3, 283-302), or inhibition of immunofluorescent foci in H9 cell6.
V~rus Isolation. P9MC or bone marrow cells (obt~ined as aspirates) from i~ unlzed ar.d challenged chimpanzees were culturHd with normal human PBMC, as described ~Fultz, P.N., McClure, H.M., Swenson, R.B., ~cGrath, C.R., 9rodie, A., Getchell, J.P., Jensen, F.C., Anderson, D.C., Broderson, J.R.
L Francis, D.P. (1986) J. V~rol., 58, 116-124). In so~e e~periments, CD~4-Qnriched ly~phocytes were obtained from chimpanzee PBMC by separation with magnetic beads to which ~ere attached monoclonal antibodies specific for the CD8 cell-~urface antigen (Dynabead6, Robbin6 Scientific): The CD+4-enriched cells w~re ~timulated 2 days with concAnavalin A (10 ~g/ml) before b~ing cultured alone or cocultured with phytohemagglutinin (PHA)-stimulated normal human P~MC in ~P~I-1640 med~um with 10% fetal bovine serum, glut~mine, gentamicin nnd recombinant interleukin-2 (8 un~ts/ml;
90ehringer ~annhe~m). Lymph nods tiRsue obtained by biopsy wa8 minced with scissors and cultured with human P~MC. All cultures were maintained and monitored for reverse transcriptase act1vity for 6 weeks before being discarded.
Polymerase Ch~in Reaction (PCR). ~oth single- and double-round ~ested) PCR were performed periodically with .

SUB`STITUTE SHEET

~!!122263 PBNC or lymph node eells from ehallenged chimpanzees.
Single-round PCR wa8 a~ deseribed (Laure, F., Rouzioux, C., Veber, F., Jaeomet C., Courgnaud, V., Blanehe, S., Burgard, M., Griscelli, C. & Breehot, C. (1988) L~ncet 2, 538-541).
Briefly, 2 ~g DNA were used with 2 unit~ Taq-l DNA
polymera~e for 40 cyeles at 94C, 55C, and 72C (1 min.
each). Two primer pairs were used: one corresponded to nucleotides 2393-2417 and 2675-2700, encoded by the pol gene, ~nd the other eorresponded to nucleotides 5367-5385 and 5694-5711, eneoded by the t~t gene. To ~how ~peeifieity of the PCR, ~mplified DNA fragments were hybridized wlth t P~-labeled internal pol and tat gene probe6. The positive eontrol eon~isted of DNA from the 8E5 eell line persistently infeeted with LAV-l. For ne~ted PCR, the primers for the first round of PCR, performed as deseribed (Mullis, R.B. &
Faloona, F.A. ( 1987) ~ethods ~n~ymol . 155, 335-350) were:
S'-GCTlCTAGATAATACAGTAGCAACCCTCTAT~G-3'r corr~sponding to a 3-b~- ela~p ~equenee, an Xbul restrletion site and nucleotldes 1025-1048 of the HXB2 genome, and:

5'-GTCGGCCT~AAAGGCCCIGGGGCTTGTTCCATCTATC-3'~ eorresponding to a 3-ba-e el~mp ~equenee, a Notl re~trietion ~ite and nuel otides 5573-5553 of thQ HXB2 genome. From the first round, 2.5 ~1 of the product w~s re~mpllfied with primers SR14S and SR150 (Rwok, S. & Rellogg, D.E. (1990) in PCR
P~otocol~s A Gu~de to ~lethod~ ~nd Applic~t~onss eds. Innis, M.A., Gelf~nd, D.~., Sninsky, J.J. & White T.J. (Ac~demic Press, Inc., S~n Diego, CA) pp. 337-347), o~er a re~ion from nucleotides 1366 to 1507 on the B B2 geno~e.

, ~

~:~ SUBSTITUTE SHEET

21222~3 Immunization Regimen6 (Table 9) TABLE 9. Immunization regimen6 of chimp~nzees with variou6 HIV-l antigens Recombin~nt Inactivated Recombinant antigens V3 ~nimalw -1139 HIV oD160aao net vifDe~tide C-433 ~ + + ~ + ~ +
C-339 - ~ ~ _ _ _ +
C-499 - _ + ~ _ _ +
_ ~or C-433 and C-339, times of immunization~ and virus challenge were cslculated from the time that C-433 received its fir~t immunizntion with W -1139, which is considered week 0. Chimpa~zee C-433 wa~ fir~t immunized with a recombinant vaccinia viru~, W -1139, that expre~e~ a non-cleavable ver~ion of the HlV-lBRU gpl60~nv antigen (Rieny, M.P., Lathe R., Riviere, Y., Dott, R., Schmitt, D., Girard, M., Nontagnier, L. ~ Lecocq, J.P. (1988) Prot. Eng~no~ring 2, 219-226). W -1139 wa~ admini~tered on we4ks 0, 8 and 21 by scarification on the upper b~ck with a two-pronged needle (2 x 108 PFU p~r inoculum). At week 27, PBMC from ~-433 were stimulated with PRA, cultured in medium cont~ining IL-2 and then infected with W -1139 at ~ mult~plicity of infection of 7. Following culture for ~n additional 16 hour~, the.~BNC
w~re f~xed with 0.8% paraformaldehyde and rein~ected into C-433 by the IV route (Z~gury, D., ~ern~rd, J., Cheynier, R., Desportes, I., L~onard, R., Foucha~d, M., Reveil, B., Ittele, F . D ., Lurh~m~ , Z ., Nbsyo , R ., Wane , J ., S~l~un , J . J ., Goussard, B., Dechazal, L., Burny, A., Nnra, P. & Gallo, R.C.
( 1988 ~ Nat~are (London) 32~, 728-731 ) . At weeks 48, 54, 58, 81, 86, 88, 114 and 124, C-433 W~!18 inoclllAted IN with mixture~ of purified gp160en~, pl8g~g, p27nef and p23~if (125-250 pg ~ach per do~e) formulated with SAF-l.
Chimpanzee C-339 was first immunized on week 33 by IM
in~ection of inactivated HIV (125 ~g viral protein) mixed SUBSTITUTE SHEET

with SAF-l (1 mg threonyl muramyl dipeptide), followed by booster inoeulations on week6 37, 41, 62 and 124. C-339 was then inoculated with purified gpl60env only (125 ~g per dose) on weeks 66, 74, 81, 85 and 87. The V3 peptide (300 ~g peptide per dose) was administered IM on weeks 105, 108 and 126.
C-339 and C-433 were challengQd on week 131 with lO0 HTLV-IIIB- C-449 was inoculated IM with m$xture of gpl60env, pl8g~ and SAF-l on weeks 0, 6, lO, 33, 38, 66 ~nd 76. (Note: week 0 for C-499 corre~ponds to week 48 for C-433 and C-339.) A mixture of 21 free V3 peptide~
(100 ~g eaeh per dose) wa6 administered IM w$th SAF-1 on weeks 79, 83, 87 and 102. C-499 and C-087, a naive control, were ch~llenged on week 106 ~nd 100 TCIDso f ~IVHTLV-IIIB-Results ~ mmunization of chimpanzee C-339 with formalin- ~nd beta-propiolacton~-inactivated whole HIV mixe~ with the ~d~uv~nt SAF-l re~ulted in high titers of antibodies to ~g-and env-encoded proteins, as moasured by en~yme immunoa~say (EIA), a low neutralizing antibody response, ~nd no detoct~ble eoll-mediated immune re-ponse. In ~n effort to enhanee im~une respon~e~, C-339 was immunized with purified rocombinant gpl60env. Following one intr~dermal inoeulation of gpl60~nr with BCG in multiple sites on the chest, C-339 w~s g$~en four ~ucce~ive IM in~ect~on~ of the same.~ntigen formul~ted with 5AF-l. Total ~RA ~ntibody and nautrallzing ~ntibody tit~rs were determined periodically; howa~er, during the course of im~unization, both rem~ined un~hanged and d~creased rapidly af~er the in~ections were discontinued ~Figure 6A).
In ~IV-infected p~rsons, the ma~ority of HIV- ~
neutralizing antibodie~ re directed again~t the third hypervari~ble region of the extern~l envelope glycoprotein, termed the V3 loop (Putney, S.D., N~tthews, T.J., Robey, W.G., Lynn, D.L., Robert-Guroff, N., Mueller, W.T., Langlois, A.L., Ghrayeb, J., Pettew~y, S.R., Weinhold, K.J., SUBSTITUTE SHEET

WOg3/08836 PCT/EP92/02459 Fischinger, P.J., Wong-Staal, F., Gallo, R.C. & Bologne~i, D.P. (1986) Sc~nc~ 234, 1392-1395; Ru~che, J.R., Xavaherian, ., McD~nal, C., Petro, J., Lynn, D.L., Gr~ail~, R., L~nglois, A., Gallo, R.C., Arthur, LØ, Fischinger, P.J., Bolognesi, D.P., Putney, S.D. & Matthew6, T.J. (1988) Proc.
N~tl. Ac~d. Sci. U.S.A. 85, 3198-3202; and L~Ro~a, G.J., Davide, J.P., Weinhold, K., Waterbury, J.A., Profy, A.T., Lewi~, J.A., Langlois, A.J., Dres8man, G.R., Boswell, R.N., Shadduck, P., Holley, L.H., R~rplus, M., Bolognesi, D.P., ~atthews, T.J., ~mini, E.A. & Putney, S.D. (1990) Science 249 932-935). Antibodies to epitopes within the loop abrogate virus i~f~ctivity, probably by preventing fusion of the viral en~elope to the tar~et cell membrane. Neutralizing antibodies to V3 epitopes csn, in fact, be added as long as 40 to 60 minutes ~fter virus binds to the cell and still prevent infection (Nara, P.L., (1989) in V~ccines 89, eds.
Lerner, R.A., Gin~berg, ~., Chanock, R.M. & Brown, F. (Cold Spring ~arbor L~boratory, Cold Spring Harbor, NY) pp. 137-144). Therefore, to determine whether immunization with the V3 loop would boost neutralizing antibody titers, C-339 was in~ected with an oligopeptide of 25 amino acids, having the V3 sequence of HrV-lBRU(IIIB), cro~J-linked to RLH and formulated with SAF-l. No chango in EIA titer was observed (Figure 6A), ~ut a significant increa~e in neutralizing ~ntibody t~ter~, which wore su~tained for several months, was obtain d following the ~econd immun~zation ~t week lp8 (Figure 7A).
Another chimpanz~e, C-433, that had been primed by vaccin~tion ~ith W -1139 (Rieny, M.P., Lathe R., Riviere, Y., Dott, ~, Schmitt, D., Girard, M., ~ontagnier, L. & Lecocq, J-P- tl988) Prot. Engineering 2, 219-226), wa8 immunized repeatedly with 125-250 ~g each of recombinant soluble gpl60~nv, pl8g~g, p27nef and p23rif (Table 1). The ~nti-HIV
_ntibody response induced by thi~ regimen w~ clearly tr~nsient, with titers rising shArply after each boo~ter in~ection ~nd then decreasing rapidly (Figure 6B). The neutr~lizing antibody ~nd EIA titers of C-433 fluctuAted in ~ SUBSTITUTE SHEET

WO 93/OX836 2 l 2 2 2 ~ 3 PCT/EP92102459 ~

parallel. Finally, C-433 was in~ected with the same V3 peptide-RLH con~ugate as C-339, ~ccording to the s~me immun$zation protocol. Noutralizing ~ntibody titers incre~sed signific~ntly ~fter the ~econd in~ection of the V3-peptide con~ug~te and remained high there~fter (Figure 7A); a third ~mmunization 4 month~ later (week 126) elicited no change in titers.
At the time C-433 fir6t received the purified recombin~nt protein~ (48 weeks), a third chimp~nzee, C-49g, received an IM in~ection of purified gpl60env and pl8g~
formulated with SAF-l. C-499 received BiX booster inocul~tion~ of the s~me antigens, followed by a serie6 of four in~ections of a mixture of 21 free (uncon~ugated) V3 peptides (Myer6, G. (1990) in Hum~n Retroviruse~ ~nd AIDS, ed~. Myers, G., Josephs, S.F., Wong-Staal, F., Rabson, A.B., Smith, T.F. & Berzofsky, J.A. (Los Alamo6 National Laboratory, Los Al~mo~, NM) in SAF-l. AS with C-339 and C-433, C-439'~ EIA titers declined rapidly after immunization with the purified H n antigens, ~nd there was no detect~ble effect of the V3 peptides on EIA titer. There wa~, however, a significant increase in neutr~lizing antibody titer6 (to >
2000) following the V3 peptide inoculations (Figure 7B).
Challengn ~th Infectious ~IV. Because sustained neutr~lizing antibody titer~ were ach~eved, chimp~nzee6 C-433, C-339 and C-499 were ch~llenged by IV inoculstion of 100 TCID50 (40 ch~mp~nzee infectious do~e~) of HIV-l.. At the time of challenge, 50% neutraliz~tion titers by an immunofluore~cence inhibition a8~ay were 1:2000, 1:280-350 and 1s2000, ~nd 90~ neutralization titer6 by a syncytin-inhibition ~ay (Nara, P.L., Hatch, W.C., Dunlop, N.M., Robey, W.G., Arthur, LØ, Gonda, M.A. & Fischinger, P.J.
( 1987 ) AIDS Re~. Wum~n R~trovirus~ 3, 283-302) were 1:512-1024, lsl2~ and 1:1024 for chimp~nzee~ C-433, C-399 and C-499, re~pectively- Bec~u~e immunization of C-499 W~8 initi~ted at a different time from the other two 8nimal8, challenge of C-499 occurred 6 month~ ~fter thnt of C-399 and C-433, but was done at the s~me time a8 that of a n~ive SUBSTITUTE SHEET

r~7t eontrol animal, C-087. Virus was isolated from C-087'~ PBMC
at 2 weeks post-inoeul~tion ~PI) a~ well a~ at all sub~equent times, ~how~ng that a lslOO d~lution of the HIV-l ~toek readily infeeted ehimpanzees under our eonditions.
Att~ pts to I~ol~te ~IV from I ~uni~ed and Challonged Ch~ F~n~ees. At various times after ehallenge with HIV-l, threo methods were u~ed to aJ~e~s the infeetion ~tatus of the immuni2ed anim~ls. Fir~t, attempt~ to deteet HIV ~equence~ in lymphoid eells by PCR were made periodieally (L~ure, F., Rouzioux, C., Veber, F., Jacomet, C., Courgnaud, V., Blanche, S., Burgard, M., Griseelli, C. 6 Breehot, C. (1988) L~ncet 2, 538-541; Nullis, ~.B. & F~loona, F.A. tl987) ~ethods Enzymol.
155, 335-350; and ~wok, S. & ~ellogg, D.E. (1990) in PCR
Protocols: A Guide to ~l~thods ~nd Applic~tionss eds. Inni~, .A., Gelfand, D.H., Sninsky, J.J. & White, T.J. (Academic Pre~, Ine., San Diego, CA) pp. 337-347). DNA 6amples obtainod from PBMC of the three ch~mpanzees ~t 3 week~ and 3 ~nd 6 months after eh~llenge were te~ted. ~nd6 with the expe ted eleetrophoretlc moblllty were deteeted in DNA from eontrol HIV-infeetod ehimpanzee, but not in PBNC from the v~eeinated and ehallenged animals or from a eontrol naive ~nimal (data not ~hown). At 6 month~ after ch~llenge, nested ~ets of pri~ers were u~ed to perform PCR analysis on DNA from both P~C ~nd lymph node tis~ue of the challenged and control chimpan~e~ (~ullis, R.B. & F~loona, F.A. (1987) ~ethods En~y~ol. 155, 335-350). Thi~ technique i8 more sensiti~e th~n ~t~ndard PCR, ~nd in the~e experiments (repeated at le~st seven times on ~ amples), ~pproxi~ely one molecule of vir~l DNA wa8 found to produce a strong signal when present in 1.~ x 105 cell-equivAlent~ of DNA. All P8MC and lymph node ~ample~ were con~istently neg~tive except those from a previougly infected chimpanzee, which were ~lways positive (~igure 8). Thu~, at 6 months after challenge, vir~l DNA was not present in PBMC and lymph node tissues at a frequency greater than one copy per 106 cell~.
Second, at weekc 2, 4, 6 and 8, and at monthly intervals thereafter, attempt~ were made to isolate v$rus from PBNC by ~ ~ SUBSTITUTE SHEET

37 2~%22~ ~

cocultivation of the chimpanzees~ PBMC with lymphocytes obt_ined from norm~l hum~ns (Fultz, P.N., McClure, H.M., Swenson, R.B~, McGr_th, C.R., Brodie, A., Getchell, J.P., Jensen, F.C., Anderson, D.C., Broderson, J.R. & Francis, D.P.
(1986) J. Virol ., 58, 116-124). Because CD8 cells h_ve been shown to suppress virus replic_tion not only in HIV-infected human~ (Walker, C.M., ~oody, D.J., Stite~, D.P. & Levy, J.A.
(1986) Science 234, 1563-1566; and Tsubota, H., Lord, C.I., Watkins, D.I., Morimoto, C. & Letvin, N.L. (1989) J. Exp.
Med. 169, 1421-1434) and chimpanzQes (P.N.F., unpubli~hed data), but also in SrV-infected macagues (Tsubota, H., Lord, C.I., Natkins, D.I., Norimoto, C. & Letvin, N.L. (1989) J. ~`
Exp. ~ed. 169, 1421-1434), in some experiments chimp_nzee PBMC were depleted of CD8 lymphocyt~s before cultures were e~tablished. In contrast to virus recovery from the control animal, C-087, virus was not recovered from either total PBMC
or CD4~-enriched cells from C-339, C-433, or C-499 at _ny time during the fir~t 6 month6 of follow-up. At 6 months PI, inguinal lymph node biopsies were perfonmed on all anim~l~ _s well a8 on uninfected _nd HIV-infected control chimp&nzees.
Upon cocultiv tion with normal human PBNC, viru8 was recovered from the lymph node of the infected control, but not from those of th~ immunized and challenged chimp_nzee~
(d_ta not shown). Despite the fact that all attempts to detect viru~ during the fir3t 6 month sfter chAllenge had fail~d, virus was i~olat~d from C-433 coculti~tion of PRMC
obt~ined at 32 w~eks ~nd thereafter of bone m~rrow obt~in~d 37 woeks after challenge.
Lastly, the challenged animals were moni~ored for possible ~eroconversion to HIV ~ntigen6 th~t were not included in their ~mmunization regimen~. Immunoblot An~ly6i6 (Diagno~tic Pa~teur) show~d that C-433 and C-499, which had been immunized with, among other antigen6, pl8~g but not p25~g, did not seroconvert to p25 dur~ng 7 ~onth6 follow-up;
however, at 32 weeks (7~ month6) PI, a faint p25 band wa6 observed on immunoblot6 for C-433, which increaEed in intensity with succeeding 6erum s~mple6 (Figure 9). For ~C-339, which h~d been immunized with whole inactivated HIV, Shere were no detectable increases in EIA antibody titer6 or in ~pparent level~ of antibodies to ~ny HIV--pecific proteins (Figure 9). Also, using purified ant~gens in immunoblot ~88~y~, no antibodies to the vif or nQf proteins were detected in serum from C-339 during 12 months follow-up.
The results pre~ented here, a8 well as those reported by Bsrman and colleague6 (~erman, P.W., Gregory, T.J., Riddle, L., N~kamura, G.R., Champe, M.A., Porter, J.P., Wurm, F.M., Hershberg, R.D., Cobb, E.R. ~ Eichberg, J.W. (1990) N~ture (London) 345, 622-625), clearly show that it i6 pos6ible to elicit a protective immune response in chimpanzee6 with ~rious HIV-l antigens. It has been shown that C-499 was protected against establishment of HrV infection, at least through 7 months follow-up, that C-339 was protected for 1 year, and that C-433 wa6 protected partially, a6 evidenced by the 7-month delay in appearance of virus. It i6 possible, howev-r, that C-433 al80 migh~. have been fully protected if the challenge do~e had been the ~me as that used by others (Berm~n, P.W., Gregory, T.J., Riddle, L., Nak~mura, G.R., Ch~pe, N.A., Porter, J.P., Wurm, F.M., H~r~hberg, R.D., Cobb, 8.g. & ~chberg, J.W. (1990) N~tur~ ~LcndonJ 345, 622- :-625), which was fourfold lower th~n the dose u-ed herein. ~-Protectlon wa8 demon~trated by: Il) failure to recover virus from p~u~ during ~onthly attempts ~nd from lymph node tissue ~t 6 ~onth~ Pls ~ii) negative hybridization ~ignals ~n PCR
~nalys~ of DNA fro~ PBMC at various intervals and from lymph node~ at 6 months PI, and (iii) the absen~e of antibody r~pon~es that norm~lly follow a primary XTV infection or that are characteristic of anamnestic respon-es in pre~iou61y v~ccinated a~d challenged anim~ls (Berman, P.w., Groopman, J.E., Gregory,`T., Clapham, P.R., Wei88, R.A., Ferriani, R.
Riddle, L., Shima~aki, C., Lucas, C., La~ky, L.A. & Eichberg, J.W. (1988) P~oc. N~tl . Acad . Sci . U. S.A. 85 5200-5204;
Arthur, L.O., Bes6, J.W., water6, D.J., Pyle, S.w., ~elliher, J.C., Nara, P.L., Krohn, ~., Robey, W.G., Langloi6, A.J., Gallo, R.C. & Fischinger, P.J. (1989) J. V~ol. 63, SUBSTITUTE SltEET

21~263 ~

5046-5053; Girard, M., Kieny, M.P., Gluckm~n, J.C., Barre-Sinoussi, F., Montagnier, L. & Fultz, P. (1990) in V~cc~ne~ fo~ S~xu~lly Tr~n6~tted D~ Je8 eds. Meheus, A. &
Spier, R. (Butterworth Co., Ltd., London), pp. 227-237).
That C-433 appeared to be protected for 7 month~, but actually wa~ infected from time of challenge, despite repeatedly negative re~ults for virus isolation and detection by PCR, is worri~ome and underscore~ the fact that HIV c~n be sequestered such that it defies detection by both virologic and ~erologic criteria. A similar occurrence was reported (Desrosier~, R.C., Wyand, M.S., Rod~ma, T., Ringler, D.J., Arthur, LØ, Sehgal, P.R., Let~in, N.L., Ring, N.W.
D~niel, M.D. (1989) Proc. N~tl. Ac~d. Sci. U.S.A. 86 86, 6353-6357) for ~ macaque immunized with inactivated whole virus ~nd then challenged with infectiou6 SIV. In that ~tudy, virus wa~ not recovered initially until 32 week~ and ~n an~mnestic response was not observed until 39 weeks ~fter challenge. The ob~rvation in n~tural HrV infections that per~on~ remained ~eronegative by conventional te~t~ for extended t~mes, but HIV was detected by PCR or virus isolation (R~nki, A., Valle, S.L., Rrohn, N., Antonen, J., All~in, J.P., Leuther, ~., Franchini, G. ~ ~rohn, R. (1987) L~nCQt 2, 589-593 and Jehuda-Cohen, T., Slade, B.A., Powell, J.D., Villinger, F., De, 8., Folks, T.M., ~cClure, ~.M., Sell, ~.W. L Ah~ed-An~ari, A. (1990) Proc. N~tl. Ac~d. Sci.
U. S.A. 87, 3972-3~76)/ suggests that h1gh-risk individual~, ~uch ~8 sexual partners of ~rV-infected per~ons, po~sibly could be infected despite negative serologic, virologic or PCR analyses.
In view of the co~plex regimen of immunization undergone by the three chimpanzee~, it is difficult to determine which of the m~ny antigens and/or antigen formulstion6 were instrumQntal in elic$ting partial protection. C-339 W~8 immunizQd succ~ssively with inactivated HIV, purified gpl60, and the V3 peptide-KLH conjugate. C-433 W~8 immunized fir~t with a vaccinia viru~-gpl60env recombinant, then with a mi~ture of purified env, pl8g~g, nef and vif antigen~, and SUBSTITUTE SHEET

"
21222~3 fin~lly with the V3 peptide-RLH con~ug~te. The simplest im~uni~tlon r~gimen w~ that of C-499; it con~i~ted of purlfl~d gpl60~nv and pl8g~g followed by uncon~ugat~d V3 peptides. The ~ntigens that were common to the three anlmals were gpl60~nv, pl8g~g ~nd the V3 peptide, but their relatlve import~nce rem~ins to be determined~ Adequ~te protection mlght require ~ultiple ~nt$genic determin~nts found on more th~n one vir~l protein, ~nd/or multiple presentations of the s~me ~ntigenic dete-m~n~nt.
It i8 of interest th~t previou~ly tested prototype vaccines (Berman, P.W., Groopman, J.E., Gregory, T., Claph~m, P.R., Nei~, R.A., Ferri~ni, R. Riddle, L., Shlmas~ki, C., Luc~s, C., La~ky, L.A. L Eichberg, J.W. (1988) ~roc. N~tl.
~c~d. Sc~. U.S.A. 85 5200-5204; Arthur, B.O., Be~s, J.W., ~-W~ters, D.J., Pyle, S.W., ~elllher, J.C., Nara, P.L., ~rohn, ., Robey, W.G., ~4nglois, A.J., G~llo, R.C. & Fischinger, P.J. (1989) J. V~rol. 63, S046-5053; Girard, M., ~ieny, M.P., Gluckm~n, J.C., Barre-Sinoussi, P., Montagnier, L. & Pultz, P. (19gO) ~n V~cc~n for Se~u~lly Tr~nsmlttod D~e~JQ~ eds.
Neheu~, a. ~ 8p~er, R. ~Butterworth Co., Ltd., London), pp.
227-237~ and Hu, S.L., Fultz, P.N., ~cClure, ~.~., Elchberg, J.W., Thomac, E.~., Zarling, J., S$ngh~1, M.C., Rosowski, 8.G., 8wen~on, R.B., Anderson, D., C. ~ Todaro, G. (1987) N~ture (LondonJ 328, 721-723) that did not ellcit significant titers of neutsali~ing antibodies in chimpanzees were not effective in preventlng exper~mental infection of the an~oal-. The observation that su~tained neutralizing antlbody titer~ were reached in C-339 and C-43~ after two in~eztions of the V3 peptide-XLH con~ugate and in C-499 after three in~ections of V3 paptides (Figure 7), suggests that V3 mlght be ~een differently by the chimpnnzee immune 8y8tem when presented as a peptide than when presented a~ part of the gpl60/120env molecule. We have found by immunoaffinity chromatography that ~irtually all HrV-neutralizing ~ctivity $n the ~erum of the protected chimp~nzees could be ad~orbed by the V3 peptlde (unpublished data of A.P.). The booster inoculatlon~ of the v3 peptide(~) might e~plain why -.

SUBST~TUTE ~:H~T

4 !2122~63 immunization with gpl60 resulted in protection of chimpanzees in the sub~ect e~per~ment~, but not in those reported by Berman et al. (Ber~an, P.W., Gregory, T.J., Riddle, L., N~k~mura, G.R., Champe, M.A., Porter, J.P., Wurm, F.M., Hershberg, R.D., Cobb, E.~. & Eichberg, J.W. (1990) N~ture (~ondonJ 345, 622-625). In thi6 latter study, two chimpanzees were protected after immunization with gpl20, and these animal6 had three- to four-fold higher titer6 to the principal neutralizing determinant (PND) found in the V3 loop than the two animals not protected from infection.
The question of whether the protection observed in the present experiment was due solely to neutralizing antibodie6 or whether other immune mechanisms were involved remains unanswered. At time of challenge, antibody-dependent cellular cytotoxic activity was detected in the ~erum of C-339, but not in that of the other two chimpanzees.
HIV-specific proliferative responses to the soluble proteins pl8g~g, gpl60~nv, and p27nef (Bahr~oui, E., Y~gello, M., B~llaud, J.N., Sabatier, J.M., Guy, B., Muchmore, E., Girard, . ~ Gluckman, J.C. ~1990) AIDS ~. Num~n RQtroviruses 6, 1087-1088; ~nd Van Eendenburg, J.P., Yagello, M., Girard, M., R~en~, M.P., Lecocq, J.P., Muchmore, E., Fults, P.N., ~iere, Y., ~ontagnier, L. & Gluckman, J.C. (1989) AIDS Res.
Hum~n RetroviruJ~ 5, 41-50) were d tected in PBMC from C-433 ; both before and ~fter viru~ challenge, but not in PBMC from C-339. Intere~tlngly, ~fter lmmunlz~tlon w~th the V3.-RLH
con~ug~te, C-433 displayed a ~ustained, strong T-helper cell reactivity to the V3 peptide, wh~le C-339 h~d only we~k response. The respon~e~ of C-449 ar~ currently under study.
~epeated attempts to detect cytotoxic ~ lymphocytes (CTL) in PBMC of the vaccinated chimp~nzee~ before, on the day of, and sfter challenge have failed. It appe~rs, therefore, th~t the observed protection did not correlate with the T-helper cell or CTL activity.
The results presented here indic~te that HIV vAccine6 c~n induce protection ~g~inst ~irus infection. The high neutralizing ~ntibody response induced by the V3 peptide was :
:' ~ SUBSTITUTE SHEET
:~ :

4 2 .`;~
2 12 ~ ~ ~3 `
type ~pecific; serum from the vaccinated animal6 at time of challenge neutralized the more diverse HIV-l isolates RF and MN only marginally (unpublished data). Therefore, it will be necessary to design a vaccine that will induce high titer6 of neutraliz~ng antibodie6 to the many HIV variant6, but the recent identification (LaRosa, G.J., Davide, J.P., Weinhold, ~., Waterbury, J.A., Profy, A.T., L~wis, J.A., Langlois, A.J., Dre~sm~n, G.R., Boswell, R.N., Shadduck, P., Holley, L.H., ~arplu~, M., Bologne6i, D.P., Matthews, T.J., ~m1ni~
E.A. ~ Putney, S.D. (1990) Science 249 932-935) of PND
sequences with which a ma~ority of ~era from HIV-infected persons re~ct may make thi6 le~s formidable than previously thought. The apparent success ~n protecting two chimpanzee6 and ~uppresslon of virus for an extended period in a third animal ~ustify further efforts to develop an HIV vaccine, with the expectation that it will provide long-lasting protective immunity in humans.

Further ~tudie~ were conducted to ascertain the validity of the dual immwnization procedure (priming with gpl60 followed by boosting with synthetic peptides with the sequence of the V3 loop of gpl20); to compare 3 ad~uvants: -Al(0~)3, the Syntex ad~uvant, SAF-l, and incomplete Freund ad~u~ant (IFA); and to test an accelerated ~chedule Qf ~mmunizat~ons gpl60 at 0 and 1 month, the V3 peptide at 3 and 4 months, and both gp160 and V3 as a l~st boo~t at 6 months.
The experiment wa6 carried out in ~hesus macaque6 (4 ~nlm~ls per lot) u6ing 100 ~g of gpl60 BRU for prim~ng and a mixture of 200 ~g each of V3-BRU ~gpl20 amino acid residues 302-335) and V3-MN (6ame residue6) for boo6ting. ~he animals were bled at mont~ly intervals and anti-V3 and anti-gp antibody (Ab) titers were determined by ELISA. Neutralizing Ab titers were determined by the inhibition of immunofluorescent foci formation assay.

SUBSTITUTE SHEET :

``` 43 21~2263 Anti-gpl60 Ab were me~sured by E~ISA using plaques coated with purified gpl60 aRU. A fa~t anti-gpl60 Ab respon~e was observed in the 3 groups of anim~ls (Fig. 10), but the response to the antigen in the group~ with IFA and SAF-l WaB from 5 to 10 fold higher than that in the group with alum. In~ection of V3 peptide6 had no effect on anti-gpl60 titers. Titer~ were boo~ted ~everal fold upon recall in~ection of gpl60 at 6 months, but again, the group with alum had a 2-8 fold lower respon~e than the other 2.
Anti-V3 Ab were measured by ELISA using plagues coated with the BRU peptide. The response to V3 W~8 clearly bipha:cic in all groups, with a strong boo~ter effect ~een upon in~ection of the V3 peptide at 3 month~ ~Fig. 11).
Thus, ~nti-V3 titers increased 10 fold between month~ 3 and 4 ~nd then plateaued, confirming the remarkable boostar effect of a V3 peptide in~ection in gpl60-primed animals. This was ob~erved irrespect$ve of the ad~uvant used in the experiment.
; The initial re~pon~e to V3, mea~ured at month 3, wa8, howev-r, 5-6 fold hlgher in the SAF-l and IFA group6 than in the group with alum. The final anti-V3 titer~ were ~ltogeth r about 10 fold higher ln the former 2 group~ than ~n the latter. A t~o-~tep immunization ~chedule can be defined as follow~ 5 primings gpl60 at 0 and 1 month boo~tings V3 peptides at 3 months ~econd boo~ting: gpl60 I V3 peptides at 6 month~. -The second boost can be placed at a later time, such a8 12 months, to in~re~se further the anamne~tic response.

: ~: SuBsTlTl ITF` ~I-I~ ~ T

W093/08836 PCT/EP92~024~9 ~4 2122263 "`

All pre-immune sera were negati~e for neutralizing Ab.
Titers of neutralizing Ab mea~ured at one month af~er the ~econd boost (month 7) were the following:

Ad~uvant Monk~ys Al(OH)3 SAF-l IFA

1 60 140 > 450 2 neg 135 340 3 123 > 450 292 4 neg > 4S0 440 _ Here again, there W~6 a definite advant~ge in using SAF-l or inco~plete Freund ~d~uvant over using alum, ~lthough t~e relative difference ~n titers wa~ ~o~ewh~t les~ pronounced between the various group~.
In ~onclu~ion, a fa~t 2-step anti-HIV i~muniz~tion schedule for primate~ i~ able to induce high anti-V3, high ~nti-gpl60, ~nd high neutr~lizing Ab re~pon~e~. Thi6 s~hedule ~nclude~s gp gp V3 v3 gp ~ ~3 ~C l I Q .C~TITI IT~ C` ~

WO 93/08836 PCT/EPg2/02459 4'52122263 . , .

An altern~tive to that schedule could be:

gp gp V3 gp ~ V3 There i8 ~n ~dv~nt~ge in using the Syntex ~d~uv~nt SAF-l or incomplete Freund ~d~uv~nt r~ther th~n Alum [Al(OH)3], ~g final Ab titers are from 5 to 15 fold higher with the former 2 ~d~uv~nt~ ~8 comp~red to the l~tter. ~:

* * * :

Further ~tudie~ on monkeys ~re reported in the following t~bl~

SUBSTITUTE SHEET

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SUBSTITUTE SHEET

VACCINE PROTECTION OF CHIMPANZEES ~GAINST CHALLENGE
WITH HIV-l-INFECTED PERIPRERAL BLOOD MONONUCLEPR CELLS
L ~ ~
Recent studies h~ve demonstrated that, irrespective of ~tage of infection or disea~e, blood of per~on~ infected with the human immunodeficiency virus (HrV) contains both viru6-infected cells (also c~lled cell-associated virus) and cell-free virus (). ~hese finding6 imply that transmission of HIV may occur with either or both forms of virus.
Although data regasding the quantity and primary form of HIV
in vaginal and seminal fluids are limited (), it probably can be assumed that both cell-free and cell-associated virus are al80 transmitted through sexual contact. Therefore, ~ny effective vaccine again6t HIV mu~t protect against both form~
of ~irus as well a8 from tran8mi88ion via mucosal surfaces (se~u~l) and intravenou61y (through exchange of blood).
Anim~l model sy6tem6 employing e~ther Hrv-l infection of chimpanzee6 or infection of variou~ macaque species with v-~ or the simi~n immunodeficiency virus (SIV) have been used to demon~trate that vaccination can elicit i~mune respon~es capable of protecting ag~inst infection with these viru~es (). However, in all cases, protection wa~ -demon~trated only against challenge with relatively low do6e~
- of ~nfectiou~ cell-free ~isus. In the present ~tudy we determined (i) whethes ~erum and/or peripheral ~lood mononuclear cell6 (PBNC) from ~IV-~mmunized chi~panzee6 could prevent tran~mission of cell-~ssociated 8rv-l in ~itro, and hether chimpanzees previously immunized with ~rious ~IV-l ~ntigen prepasations would be protected again~t intravenous challenge with PB~C from ~n ~IV-infected chimp~nz~e.
~ xe~orted previously (), chimp~nzee C-339 w~s immNnized with various HIV-l antigens and W~8 sub~equently chAllenged w~th ~n intrsvenou~ in~ection of 100 TCIDSo of cell-free ~IV-l. This animal had rem~ined ~isu6 negative ~y multiple criteria and did not develop ~n ~n~mn~tic antibody response to thé ~irus through 40 weeks ~fter challenge.
8ecause unrelated in ~i~o studies had indicated thAt immune stimulation induced incre~ses in HrV-l e~pression in long-term infected chimpanzees (), and to insure that C-339 had - SURSTl'rlJTE SHI~E~ `

wo 93/08836 2 1 2 2 2 6 3 PCT/EP92/02459 indeed been proteeted from infection, we attempted to reaetivate or induce deteetable expression of put~tive latent virus by stimulating the animal~s immune system. At week 40 after ehallenge, C-339 wa~ inoculated with the Syntex ad~uvant formulation, SAF-1, ~nd at week6 44 and 48, the animal wa~ in~eeted with a mixture of ~IV-l antigen~
(inaetiYated HIV-lLAV lS rocombin~nt antigen~ gpl60~nv, p25-and pl8-~; and peptides r~pre~ent$ng the V3 ~mmunodominant loop, all formulated with SAF-l). While none of these inoeulations re~ulted in detection of viru~ by cocultivation ~-of C-339~s P~MC with normal hum~n PEMC, the lnst two in~eetion~ of HIV-l antigen did ~erve a~ boo~ter i~munization~; inereases in total anti-HIV-l (Figure 1) and ;~
neutralizing (dat~ not shown) antib~dy titers were ob~erved.
To obt~in an indication aB to whether C-339~6 level of HrV-~peeific immun$ty might be suffieient to prevent infeetion by HIV-infeeted cells, ~n ~ltro assay~ for both humoral and eell-mediated inhibition of transmi~sion were performed. We fir~t tested whethQr ~erum from ehimpanzee C-339 eould prevent transmission of infeetious viru~ from PB~C from an HIV-l-infeeted ehimpanzee to PHA-~timulated normal human PBMC. AS a positlve eontrol, serum (from an HIV-l-infected chimpanzee) that completely inhibited cell-to- -cell tr~nsmi~sion (P.N.F., manuscript in preparation) wa~
$ncluded in each assay. Compared to serum obtained from C-339 prior to immunization, which had no inhibitory .
acti~ity, serum from weeks 0 (at time of challenge with cell-free ~iru8) and 52 inhiblted viru~ tran~mi~sion and production by 68% and 75~, re~pectively, whQreas ~erum from week 24 inhibited viru~ production by only 33% ~Figure 2A).
The week 24 vnlue i~ probably a reflection of gradual los~ of inhib~tory activity after the in~ti~l VirUB challenge, and that at week 52, of ~n increase in inhibitory activity due to the two HIV-l boo~ter in~ections given to C-339 at weeks 44 ~nd 48.
Second, we tested whether P9MC from C-339, when u~ed as indic~tor cell6, would prevent tr~nsmission and replication ~ SUBSTITUTE SHEET

- 5 0 ~ e 212~3 of viru8 when eoeultivated with PBMC from an Hrv-l-infected ehimpanzee (C-087). PBMC from C-339 w~re added at a fixed eoneentration (2-3 x 106 eells/well) to wells of 12-well ti6~ue culture plates. C-087~ PBMC were serially diluted ls4, and cell~ from each dilution were added to duplicate well~ containing PBMC from C-339 (or normal hum~n or . ~:
ehimpanzee PBMC, as eontrols), starting with a ratio of 1:1.
Culture supernatants were monitored periodieally for virus ::
production by rever~e tran~eriptase assay. Inhibitory .;.
aetivity wa~ eon~idered to be present in cell~ from the :
immunized animal6 if (i) a larger number of C-087'~ PBMC were -:
required to yield viru6-positive eultures w~thin 6 weeks of observation, and (ii) there was a del~y in time at which eulture6 bee~me viru6 positive, eompared with tho~e coeulture6 established with PBMC from HIV-l-nsive individuals. These assay6 indicated that C-339 had substantial inhibitory aetivity on week 40, whieh was before the two booster in~eetion6 of HIV-l antigen~ (Figure 2B).
Althoogh this inhibitory activity had declined by week 73, enrichment for CD8~ eell~ by magnet$c bead depletion of CD4+
e ll~ r-sulted in eomplete inhibition of virus recovery ~-(Figure 2C). The ~pparent enhancement of infection with the CD4~-enriehed population of C-339's PB~C probably i~ a funetion of the greatly increased number of c~lls capable of ~upporting replieation of HTV-l.
Bee~u-e the ~n r}tro a~say6 ~ndieated that both serum and PB~C from C-339 had at lea~t some ~bility to prevent eell-to-eell transmission of Hrv-l~ C-339 and a ne~ative control ehimp~nzee, C-435, were challeng~d intr~venously with HrV-l-infeeted PBMC. The challenge inoeulum eonsisted of eryopreserved P~NC th~t were obt~ined from hep~rinized blood of ~ ehimpanzee, C-087, that had been infected 14 weeks e~rlier with HIV-1HTLV IIIB (~8 a po8itive eontrol in ~nother vaeein~ study ~), A ehallenge inoculum consisting of PBMC
from ~n HrV-l-infeeted ehimpanzee wa~ believed to most neerly ~pproximate transmission that oecurs between, for ex~mple, intravenous drug users. Sinee the minimal infeetiou~ dose of ,:;.' `:'~'' SUBSTITUTE SHEET ``

5?~122~53 HIV-infected cells required for infection of chimp~nzees had not been determined, and bec~use of the limited number of ;~
avail~ble chimpan2ees, the dose of the challenge inoculum wa~
selected empirically. This selection wa~ based on the ~i results of in vitro titration~ of aliquot~ of the cryopreserved PBMC from chimpanzee C-087, using PHA-3timulated normal PBMC from both hum~ns ~nd chimpanzee6 as indicator cell~ (). From these assays, it wa8 determined that there w 8 an average of 382 infectious cells per 107 total PBMC in this cryopreserved stock. The two chimpanzees, C-339 and C-435, were inoculated intravenously with a volume of 1 ml, which contained 5.8 x 105 P8NC or 22 infectious PBMC. Thi~ number i~ a minimum estimate ~nd i~ based on the assumption that one infected cell is ~ufficient for a culture to bec e virus positive.
Following inoculation, the animals were observed daily, and blood sample6 were obtained every 2 weeks for 8 weeks and at nthly intervals thereafter. Viru6 isolation attempt6 were performed by cocultivation of PBNC from each animal with PHA-~timulated nor~al human PBMC in 25-cm tissue culture fla~ks. We also attempted to isolate virus from bone marrow biopsy sa~ple~ obtained at 3 and 9 months and from lymph node biopsie~ at 6 and 11 1/2 months after inoculation of infected PBNC. At 4 weeks after challenqe and at every time ther ~fter, viru~ wa6 isolated from P~MC, a~ well a6 bone ~arrow ~nd ly~ph node sample~, from the control anim~l, C-435. In contra~t, viru~ W~B not isolated at any time from P~MC, nor from bone marrow or lymph node biop~ie~, from the ~mmunized chimp~nzee, C-339. HIV-specific antibodie6 were detected in serum fro~ C-435 initially at 8 weeks after ch~llenge, and titers continued to ri~e through week 24 ~-(Figure 1). However, no anamnestic respons~ W~6 detected in ~erum from C-339, ~nd ~ntibody titer6 to HIV-l dimini6hed slightly, then re~ained stable.
~ hese results, therefore, indic~ted th~t it w~6 possible to prevent tr~nsmis~ion of infection by HIV-infected cells by prior immunization. A6 confirmation, two additional SUBSTITUTE SHEET

21222~
immunized chimpanzees were challenged with an equivalent number of infectious cell~ using an aliquot of the sa~e cryopreserved PBMC from chimpanzee C-087 (Table 1). One of the~e chimpanzee~, C-499, like C-339, had been immunized and challenged previously with cell-free ~IV-1 and had remnined virus negative for 1 year (). The ~econd chimpansee, C-447, had been immunized initially with purified recombinant gpl60en~, pl8g~g, v~f, and nef proteins in SAF-l, and then received booster immunizations with purified gpl60env and pl8g~g, followed by peptides representing the principal neutralizing determinant (V3 loop) of HIV-lHTLV IIIB and purified nef protein in SAF-l. Chimpanzee C-447 had not been e~po~ed previously to infectious HrV-l in any form.
Following challenge, with the same dose of approximately 22 infectious PBMC, these latter two chimp~nzees were ~onitored biweekly, then monthly, for change~ in HIV-specific antibody titQrs and for presence of viru~ in PEMC, bone marrow and lymph node. Antibod~ titers to HIV-1 in both ani~als re~ained ~table, and v$ru~ was not isolated from any of the blood or tissue sample6. At 7 month~ after challenge, C-499 was acrificed due to congestive heart failure.
Frag~ents of eight different tissues (including brain, ~pleen, various lymph node~, kidney, li~er and salivary gland) were minced with scissors; these tis~ue fragment6, a~
~ell a8 PB~C and hone ~arrow, were then cocultivated with PHA-~t~mulated normal human PBMC. All cultures were yiru~
negative throughout 6 week~ in culture, a~ monitored by re~erse tran~criptase as~ay. All PBMC, bone marrow and ly~ph node s~mples from the ~econd animal, C-447, h~e been negative for virus on all attempts through 9 months of follow-up. Thu~, three of three immunized chimpanzees were apparentIy protected from infection by HIV-l-infected cells.
Since peripheral blood cells contain monocyte~macrophages ns well a~ lymphocytes, the infected cell popul~tion was probably heterogeneous not only with respect to cell type but also according to levels of virus expression by individu~l cells. Although the inocula was prepared as PBNC suspended SUBSTITUTE SltEET

WO 93/08836 2 1 2 2 2 6 3 Pcr/EP92/o24s9 in 1 ml of medium, it i~ highly likely that ~ome of C-087's PBMC were aetively produeing HIV. It i~ pos~ible, therefore, that the ehimp~nzee inocula aetually eonsisted of a mi~ture of both cell-free and eell-~s~oci~ted HIV-l. These eon~ideration~ further enhance the importanee of our results.
At time of ehallenge with HIV-infeeted PBMC, C-447 and C-499 had fourfold lower HIV-l EIA antibody titer6 (1:6400 versu~ 1:25,600), but four- to eight-fold higher neutralizing antibody titer~ 256 and 1:512 versu 1:64), compared with those of C-33~. To asse~ further the potential of the in v~tro serum and PBNC inhibition ~say6 to prediet possible vaecine-induced proteetion against cell-associated virus ehallenge, serum samples from C-447 and C-499 were tested.
Serum obtained from C-447 and C-499 on day of ehallenge inhibited cell-to-eell tran~mis~ion of HIV-l by 25% and 52~, respeetively. Beeause these levels of inhibition were les~
than the 75~ inhibition of eell-to-eell tr~nsmis~ion observed with ~erum from C-339 on the day it was ehallenged, thi6 -~
a~-ay may not be a reliable predietor of proteetion against eell-a~-oeiated ehallenge. PBMC from the~e two chimpanzee~
on the day of eell-associated ehallenge were tested in parallel with P~MC from C-339 (~e Figure 2B, week 75). :
Results were equivalent to those obtained with C-339's PBMC
from week 75; that is, PBNC from both animals exhibited no app~rent inhib~tory aetivity against tranomi~on of viru~
from C-087'~ infocted cell~.
When C-339 had been proteeted from cell-associated HrV-l ehallenge for 1 year tweek 104 relative to ths initial eell-free virus challenge of C-339~, we again ehallenged th~
animal with an lnoculum of cell-free HIV-lHTLV IIIB that wa~
equivalent to th~t u~ed for the flrst challenge e~periment 2 years earlier. U~ing another cryopre~erv~d ~liquot of the same viru~ stock (obtained from LArry Arthur, NCI-FCRF), 100 TCID50 were in~ected intravenously in ~ total volume of 1 ml.
Hrv-l w~ initially detected in PBMC from C-339 (by cocultivation with normal human PBNC) th~t were obt~ined 4 week~ ~fter t~i~ third HIV-l challenge, and an increase in SUBSTIl UTE SHEET

21~2~3 54 -HIV-l EIA antibady titer w~s observed at 6 week6 after challenge (Figure 1, week 110). Becsuse C-339 had not received a booster i~muni~tion or been expo~ed to HIV-l for 1 ye~r prior to this second challenge with cell-free HIV~
the immune response elicited by vaccination did not per~ist at a level sufficient to protect against thi~ last expo~ure to virus. C-339 became infected despite the pre~ence of a stable HIV-l immune respon~e, and infection wa~ detected relatively ~oon after the third e~posure to virus. Thi~
finding show6 that C-339 w~s not inherently re~istant to HIV-1 infection, and furthermore, underscores the significance of the ob~erved protection against cell-as~ociated HIV-l challenge. The other ~urviving chimpanzee, C-447, will be challenged similarlr when it has remained virus neg~tive for 1 year.
The mechanism of protection of the three chimpanzees ~-aga~nst challenge with ~rV-infected cells is not known, but it i~ likely to be due to a combination of both humoral and eell-~d$ated immunity. In the ~n rftro assays with P~MC
obt~in d on the days of challenge, only cell~ from C-339, but -~
not from C-499 and C-447, exhibited significant inhibitory ~ctl~lty ~ga~nst recovery of HrV-l from C-087's PEHC. Thi~ -~
~ay h~ve resulted from the fact that C-339 wa8 boosted with multlple HTV-l ntigen~ 4 and 8 week~ prior to cell challenge, whereas C-499 had not been exposed to HTV-l antig ns for ~ore than 1 year. Also, C-447 had receivQd three booster immunizations with only V3 peptide~ and Nef protein during an interval 2 to 5 month~ earlier; these ~no~ulation~ had re~ulted in more than a tenfold increase in neutralizing antibody titers, but no detectable increa~e in ~-HrV-BPeCifiC EIA antibody titers. That PBMC from C-339 subsequently 10st the ability to prevent cell-to-cell tr~nsmission in vitro ~upport~ this po~sibility.
Irre~pective of this, it appe~r~ th~t neither of the in vi ~ro ~88ay8, ~8 performed with ~erum or PBMC, ~re predictive of protective iDlmUSlity .

~ SUBSTITUTE SHEET

wo 93/08836 2 1 2 2 2 6 3 PCT/EP92/02459 Because C-087 and the three chimpanzee~ that were challenged with HIV-infected PBNC from C-087 were not sibling~, the po~sibility that the four animals ~hared identical ma~or hi~tocompatibility complex (MHC) haplotype6 is extremely low. Thus, one would assume a priori that $nitial protection again~t C-087~ P~MC, ~ome of which had HIV antigen~ on their ~urface, WaB not mediated by classical MHC-re~tricted cytotoxic T-cell activity, even if pre~ent.
To date we have been unable to detect CTL activity directly in peripheral blood lymphocytes from immunized chimpanzee6 (). The most likely cell-mediated mechanism of protection would appear to be antibody-dependent cellular cytotoxicity (ADCC), an activity previously detected in serum from C-339 (). A~ indicated above, it is likely that both HIV-specific antibodie6 and cell-mediated activities synergized to effect protection.
Ideally, a vaccine against any pathogen should be one that elicit~ Iong-lasting immunity following a minimal number of i~munizations. While we ha~e ob~erved long-lasting, stable EIA and neutralizing antibody titers in our immunized chimpan~ees, the~e were achieved with a large number of immunization~ (no fewer than ?12?) o~er a minimum of 2 year~. ~
The~e regimen~, to ~ay the least, are not practical for u~e ~`
in Western n~tions, much less in developing countrie6. Based on studies to da~e in nonhuman primate model~, it appears as though immuni~ation again~t Hrv-l will require at loast three inoculations initially and booster inoculation~ at un~pecified interval~. If multiple inoculation~ are required, then they mu~t be easily administered (such as orally), and the vaccine preparation mu~t be stable under normal storage conditions. The~e latter two conditions are especi~lly import nt relative to HIV-l vaccine delivery to developing nations. Thus, although progre~s has been made to demonstrate that it is possible to elicit protection agninst . .

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WO 93/08836 PCT/EP92/024~9 21222~3 - 56 ~

intravenous infection with both cell-free snd aell-ass~ciated HIV-l, ma~or problam~ remain ~o b~ re~olved.

REFBRENCES AND NOTES

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.

~ , P .'.
~ ~ o ~ ~
~1 o ~ O ~ ~ o l ~ `
P U ~ ~
s ~ .' ~ ~ ~
~ o p ~ ~r U o 3 ~ o~ c ¦ e 3 e v v e ¦ ~e ~ ¦

~ ~ 6 ~ ~ U o ~r ~
o ~ o ~ o o ~ ~ C~. C ~
~1 ~ g~

_~ N U j ~ ~
3 ~ ~ P ~ ~

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212226~
Serum Neutralization of Cell-to-Cell Transmi~ion , Chimpanzee Serum date % Inhibi~ion -- , .
C-339 10/87 0 .
Roberta 3/88 98 ,~,'.

7/gO 49 1/91 . 52 1~91 25 6 X 105 PBMC from C-527, 4 mos. p.i.
Average of 3 experiment~

SUBSTITUTE SHZ~ET

WO 93~08836 PCT/EP92/024~9 5921222~3 `

The influenee of ad~uvant6 on the neutralizing ~ntibody response of rhesus macague~ to HIV-l gpl60 and env peptide.
INTRODUCTION
The envelope glycoprotein of the human immunodeficiency viru~ type 1 (HIV-l) is made of two moieties that ari~e by proteolytic eleav~ge of a large preeur~or, gpl60. The exterior surface glyeoprotein gpl20 eorresponds to the ~mino-términal region of gpl60, whereas the tran~membrane glyeoprotein gp41 i8 derived from its carboxyl-terminal region tl, 2). The principal neutralization determinant (PND) for the virus has been mapped to the third hyper-variable domain (V3) of gpl20, a conserved eysteine loop loeated at residue~ 303-338 for strain IIIB (3-5) (numbering aeeording to ref 6). In addition, both gpl20 and gp41 carry minor neutralization epitope6 (for a review ~ee ref 7). The neutralizat~on epitopes of the V3 loop are of a seguential nature (8, 9) but the 3-D conformation of the loop seem~ to be i~portant for reaot~vity (10, 11). PND~ from different HIV-l l-ol~te~ e~hibit extensive ~eguence divergence (4, 12, 13), which e~plain~ why antibodie~ to the PND neutralize v~rus infecti~ity in a type spoeific manner. These ~ntibodie~ al~o inhibit ~yncitia formation and virus spread from eoll-to-cell. PND-targeted ~ntibodio~ act at the l~vel of fu~ion betwsen the viru~ onvelope ~nd the mQmbrano of t~rg t oell~, or between the membranes of infected and non-infeeted t~rget cells t4- 14).
kxperiments in the HIV-l chimp~nzee model have ~hown that neutralizing ~nt~bodies play a ma~or role in protection again~t experiment~l HIV infection (15, 16). Nost of tho8e ~n~mals that were not protected ag~in~t challenge infection had no neutr~lizing antibodie~ at the time of challenge or very low levels of neutralizing antibody only (17j. P~s~ive protection of chimpanzee6 again~t viru6 challenge could be achieved through administration of an ~nti-V3 domain s2ecific virus-neutralizing monoclonal antibody (Emini, personal communication). Similarly, pas~ive protection of cynomolgu~
monkeys ag~inst HIV-2 or SIV could be ~chieved by SUBSTITUTE SHEET

21222~3 admini~tration of high doses of neutralizing anti-HIV or anti-SrV serum, re~pectively (18). The induction of high t$tors of neutralizing antibodio~ i~ thoroforo an ~pp~rent ~:
requisite for the efficacy of HrV v~ccines, psrhap~ bec~u~e these ~ntibodies ~re required for the est~bli~hment ~nd maintenance of an anti-HrV-l ~terilizing immunity (19).
We previously observed th~t chimp~nzees th~t h~d been primed by hyperimmuniz~tion with ~ v~riety of Hrv-l ~ntigens, ~mong which gpl60, then boosted with either free or RLH-coupled PND peptides, developed high titers of PND-~pecific neutralizing antibodies and were protected ag~in~t subsequent HrV challenge (16). The 6implest lmmunization regimen ~ble to induce protective immunity consisted of gpl60env ~nd pl8a~a followed by uncon~ugated PND peptide6. There i~
little re~son to believe that pl8a~ could be involved in protection. Thu6, the minimal protective immunization regimen should consi6t of gpl60 followed by boosting with the corresponding PND peptide. The effic~cy of such a priming-booster immun$zation ~chedu}e i6 likely to depend on multiple par~meters ~uch a8 dose and physical status of the gpl60 ~ntigen, ~mount and sequence of the PND peptides, number ~nd -~-sp~cing of the in~ections, ~nd ~l-o on the n~ture of the ~d~uvant. In this study, the efficacy of ~lum, incomplete Freund ad~uv~nt (IFA), and Syntex ad~uv~nt for~ul~tion 1 ~SAF-l) (20) were comp~red in ~ simplified priming-booster ~unization regimon in rhesus monkeys.
A~ will be shown, the effic~cy of IFA ~nd SAF-l ~ppeared to b~ comp~r~ble, where~c alum W~B ~ ~uch 1eB~ potent ~d~uvant. The~e results could have important bearing on the design of future HIV v~ccines.
l~q~ERIALS .AND METHODS
Anti~ens Con~truction of W 1163, the recombin~nt v~ccinia virus used for the production of gp160 from the LAV-l (LAI) isolate of HIV-l (21) h~s been described previously (22). The gpl60 gene c~rried by W 1163 was mutagenized ~t the gp120-gp41 cle~v~ge site ~nd deleted of the tr~nsmembr~ne dom~in. The SUBSTI~UTE SHEET

61 2 t 2 2 2 63 antigen wa~ purified from the cell culture medium of W 1163-infected BHK-21 cells ~6 described (22-24). PND
peptide (LAI) wa~ prepared by solid phase ~ynthesi~ as a 34 amino acid re6idues peptide with the sequence C-RPNNNTRRSIRIQRGPGRAFVTIGRIGNMRQA ~re6idue~ 303-336 from the BH10 isolate) and resuspended in pho~phate buffered ~aline (Neosystem, Strasbourg).
Immunization of monkev6 Monkeys were immunized by the I.M. route with 2 in~ections of 100 ~g recombinant gpl60 at 1 month intervAl, followed by 2 in~ections of 200 ~g PND peptide at 3 and 4 months (see Fig. 1). All antigens were in fin~l volume of 1 ml. Four monkeys (group A) were immunized with the antigen6 adsorbed to 0.2% aluminium hydroxyde (Superfos), ~nother four ~nimals (group B) with the antigens emulsified in 1 ml IFA
(Difco) and the la~t four (group C) with the antigen~
emuls~fied in 1 ml SAF-l (20) containing 1 mg threonyl-MDP
per do~e. All animals were boo~ted at 6 ~onths by I.~.
- in~ection- of both 100 ~g gpl60 and 100 ~g PND peptide in the ~ame r~pective ad~uvant6. The animal~ were bled at regular intervals (10-15 mI). Serum wa~ stored frozen until ~ub~ected to the experiments de~crlbed below.
HIV seroloov Anti-PND and anti-gpl60 antibody tlters were determlned by ELISA u~ing m~crowell plaques (Nunc) coated with 0.10 ~g PND peptide or 0.15 ~g gp160 per well, respectively. .
Incubation with the appropriate dilutions of serum was for 1.5 hr at 37C, after which sera were replaced by horseradi6h peroxydase-labeled rabb~t ~nti-monkey immunoglobulin (Nordic) ~nd incubation was continued for another 1.5 hr at 37C.
Bound enzyme activity wa8 measured using orthophenylenediamine (Merck) with 0.03 ~ hydrogen-peroxide`
a~ ~ sub~trate. The reaction wa~ stopped after 30 min. with ~ulfuric acid and absorbAnce wa8 read at 490 nm in an automatic plate reader (Vmax ; Molecular Device corporation).
End point titers were calculated from a linearized st~ndard curve obt~ined with a selected pool of positive macaque sera SUBSTITUTE SHEET

WO 93/08836 PCT/EPg2/02459 21~22~3 62 used ~8 ~n internal 6tandard. Titers obtained correspond ~pprox~mately to the reciproc~l of the highest ~erum dilution -~
th~t re~ulted in ~n optic~l den~ity of ~t le~st 0.1.
~o me~sure ~pparent affinity const~nt (~A)~ serial -dilutions of the LAI PND peptide were m~de on peptide co~ted pl~tes ~nd ~ dilution of e~ch m~c~que serum th~t yielded ~n ~b~orb~nce (A0) between 1 and 1.5 in the titration EIA
described ~bo~e W~8 ~dded. EIA w~s then performed a~ alre~dy described. Dis~oci~tion con~t~nt~ (RD) were cAlculated ~6 described by Friguet et al (2s). RA (affinity con~t~nt) was c~lcul~ted ~B KA~ D.
Neutr~lizing ~ntibodie~ were mo~sured by inhibition of i~munofluore~cent foci formation on H9-cell~ or inhibition of synicit~ form~tion on CEM-SS cell6 (26) using a IIIB virus stock. Except where otherwise st~ted, neutralization titers were defined ~ the reciprocal of the seru~ dilution that reduced foci form~tion by 50% or synicita formation by 90% as compnred with control.
RESULTS --The str~tegy th~t wa~ devised ~nd followed for the immuniz~tion of rh~sus m~caques i8 outlined in Fig. 1.
Briefly, 3 groups of 4 monkey~ were ~munized with the same ~ntigen prep~rations mixed with either ~lum (group A), IFA
(group B) or SAF-l (group C). The ~nimal~ were pr~med with two in~ection~ of recombination HrV-l gpl60 given one month ~part, then boo~ted with two in~ection6 of PND peptide on the 3rd ~nd 4th months, followed by a la6t bo~ster in~ection of both gp160 ~nd PND peptide at 6 month~. Antibody rosponse~
to whole gpl60 and to the PND of the sa~e HIV-l i601ate (LAI
a6 ussd for immunization, as well a6 neutralizing antibody re~ponse6 to HIV-1 IIIB were monitored regul~rly during the 6 month immunization period and an addition~ 6 month follow-up.
Anti-gp160 antibody titer6 induced by the two initial in~ections of gp160 were approximntely ten time6 higher in the IFA ~nd SAF-l group~ than in the alum group (B, C, and A, re~pectively, table 1 And Fig. 2). These titers in~reased - SUBSTITUTE SHEET ~"

wo 93/08836 2 t ~ 2 ~ 6 ~ Pcr/EP92/o24s9 only ~lightly in response to the in~ection6 of PND peptide, and began decl$ning thereafter. The s~x month booster in~ection with gpl60 plus peptide led to a 4-10 fold anamnestic antibody response to all three groups, but, again, the gpl60 antibody titer in the alum group rem~ined about one order of magnitude lower than the IFA or SAF-l groups (Fig.
2A). After the peak at 7 months, titers progressively deelined but were still ~ignifieantly elevated at the 12th month bleed, except in the alum group (table 1).
Anti-PND antibody titers remained low after one in~eetion of gpl60 and became moderately high after two in~eetions (table 2). They were markedly (3 to more than 20 fold) enhanced by the first in~ection of PND peptide, eonfirming our previou6 observation in chimpanzees (16).
However, no further increase in titer wa8 observed after the seeond in~eetion of PND peptide (Fig. 2B). In several ani~al~, that in~ection wa~ actually followsd by a drop in ~nti-PND titer (table 2). Similarly, the effeet of the boo-t-r in~eetion at 6 months on the anti-PND titer was of ~-very limited amplitude, after which titers st~adily declined.
Anti-PND t~ters at 7 month~ were thus paradoxally lower than those at 4 month6. ~he differonce was sign~ficant at the 1%
l vel. Thi~ ~uggests that too many in~oetion~ of PND peptide could aetually lead to some sort of immu~e paralysis. Anti-PND t~ter~ in the alum group renained ~t all t~me8 about one order of m~gnitude lower than those in the IFA ~nd SA~-l group and reaehed low level value~ at 12 months.
N~u~raliz~ng antibody titers were dete d ned a~ 5 months (one month after the ~econd in~ection of ~ND peptide) and again at 7 months (one month after the 6 month booster). As shown in table 3, the highest neutralizing titers were generated in the IFA group (B) and the SAF-l group (C).
Neutralizing titers in the alum group (A) remained definitely lower ~nd some animals even scored nog~tive in that group when measured at 7 month6 by ~ynciti~m inhibition assay.
~ost neutralizing antibody titer~ were a8 high or even higher immediately after the 2 injections of PND peptide (at 5 .

m ~ ~ PCT/EP92/02459 ~<~
64 ` ;

months) than after the combined gpl60 and peptide booster in~ection (at 7 months), again suggesting that the repetition of in~ection~ of PND peptide might induce a certAin degree of immune paraly~is.
The full time-course of the neutralizing antibody response wa~ followed on two animals of group B and two animal~ of group C (Fig. 3). Neutralizing titers induced by the two in~ections of gpl60 were boosted about 50 fold following the in~ections of PND peptide. Thi6 is in agreement with our previou6 observation that high HIV
neutralizing antibody titers could be induced in chimpAnzee6 -through priming with gpl60 followed by boosting with PND
peptides (16). The subsequent decay of the neutralizing antibody response wa6 at the rate of approximately 50 % every ~-6 weeks (Fig. 3).
To study the possible correlation between the anti-PND
antlbody re~pon~e and the neutralizing antibody response in the immunized animal~, neutrslizing titers determined by the syncitium inhibition assay were plotted a~ a function of anti-PN~ peptide titer (Fig. 4). A clear correlation between both titers was ob~erved for all the animals. For the 5 month time point, the correlation coefficient co~puted for the 12 animal~ together W~6 r - 0.85 (P < 0.001), and for the 7 month t~me point, r - 0.89 (P ~ 0.001). The fact that -;~
correlation~ ~ere highly significant in spite of the limited numb~r of ~ni~als leads to ~uggest thAt the relationship between both paramet~rs wa~ very strong.
Finally, apparent affinity constants in ~olution of the different monkey sera for the PND peptide was determined by ELISA (25). No ma~or difference w~s observed between the 3 group6 of immunized ani~als (data not shown).
DISCUSSION
The ~im of the present ~tudy W~8 to compare the efficacy of three different ad~uvant formulAtion~ with re~pect to their capacity to induce HIv-l neutraliz$ng antibodie6 in rhesus macaques in a gp160 priming - PND peptide booster immunization ~chedule limited to 5 in~ections over the course ~-~ SUBSTlTUtE SHEE~T

WO 93~08836 2 12 2 2 g 3 PCT/EP92/02459 of a 6 month immunization period. The data pre~ented here ~how th~t high anti-gpl60, anti-PND, and neutralizing ~ntibody titers could be rai~ed in re~pon~e to ~uch a short regimen of immunization, provided the ad~uvant was suitably chosen. In agreement with reports from other~ (27-31) aluminium hydroxyde was un~ble to provide help for a strong ~nti-HrV-l antibody response. By contrast, IFA and the threonyl-~DP ba~e SAF-l were both found suitable to induce high-titered anti-HIV antibody re~ponses. A ~imilar low grading of alum in comparison to SAF-l and IFA was observed by H~rt et al (32) when studying the response of rhesu6 mac~ques to Tl-SP10, a synthetic peptide made of the HIV-l PND peptide and the envTl Th-cell epitope from gpl20. Alum wa6 also the poore6t of the three ~d~uvants with re~pect to the induction of antibodie6 to the HIV-l PND in rabbits (33).
In our study, the h$ghest HIV-neutralizing titers measured by either the $mmunofluore~cent foci or the syncitium inhibition ~s~ay6 wer~ ob~erved in monkey~ roceiving IFA.
Neutralization titers in ~onkeys receiving SAF-~ were sl~ghtly lower, but still highly significant. Titer~ in the ~nim~ls receiving the aluminium hydroxide-ad~uvanted antigen6 `;
were disappointingly low. In all three groups of ~n$mal~, the peak of neutr~lizing antibody titer WaB reached after the in~ect$on of PND peptide, confirming the ad~antage of the du~l gp160-PND pept$de immun$zat$on regimen previously de~cr~bed (16, 19). It should be emphasized that the~e wa~
no s~gnific~nt difference in the ~ean apparent half-life of the gp160- and PND-specific antibodie3 nor in the spparent ~ffinity constAnts of the PND-specific ~nti~odies between the 3 groups of immunized anim21~. The difference between alum ~nd the other two nd~uvants was, therefore, only in the actu~i level of antibodie~ elicited.
We have ob~erved in previous experiments that immunized chimpanzees with ~IV-l neutralizing nntibody titer6 of greater than 1:32 At time of challenge were protected against intravenous virus challenge (16, and unpublished observation6). ~king this tier as an indicative thre6hold ~::1 IQ~TITI ITF C ~F~T

2 1 2 2 2 ~ 3 ~r~

level, it is interesting to note that 3 of 4 rhesus monkeys in the SAF-l group and 4 of 4 in the IFA group, but none of the 4 animals in the alum group, had titers above that level ~-~s early a~ at the 4th month of immunization. Altogether, the 6 months immunization schedule followed in this study was able to elicit high neutralizing antibody titers in 7 of 8 animals in pooled groups B and C. We suggest that in future experiment~, the in~ection of PND peptide at 4 months ought to be omitted, in view of its possible induction of immune paraly~i~, thus reducing the total number of in~ections to 4 only. The possibillty of a further decrease of the number of in~ections and/or of the dose of gpl60 and PND peptides used for immunization awaits further experiments.
Immunizat~on of chimpanzees against HIV-l using aluminium hydro~ide as an ad~uvant has usually yielded di~appointing results. Thus, titers of neutralizing antibodies elicited in chimpanzees by alum-ad~uvanted gpl20 have most often r~mained low (27, 29, 31), which probably explains why no protection was ob~erved upon sub~equent viru~
challenge of the animals (28, 29). ~e recently immunized thr~ chimp~nzee~ with gpl60 in alum, then boosted them with PND peptides, al~o in alum, but in view of the low neutralizing antibody titer induced by this ~mmunization regimen, none of the animals was challenged (unpubli~hed ob~ervation). By contrast, ~tudies by Berman et al (15) have --~hown that chimp~nzees immunized with gpl20 in alum, but not w~th gp160, developed HIV-l neutr~lizing antibodies and were protected against challenge with a moderate dose of cell-free ~irus. Whether the difference between thi~ ob~ervation and the former ones has to do with the nature, the physical ~tate (fully native versus perhaps partly denatured) and/or the dose of the immunogen remains to be determined.
The reason why Alum was not able to elicit as strong an immune response as IFA or SAF-l in this snd other studies may have to do with the fact that aluminium hydroxide i6 a mineral carrier whereas both IFA and SAF-l contain oil. The importance of lipids for antigen presentation has been SUBSTITUTE SHEET

W093/08836 2 ~ ~ 2 2 ~ 3 PCT/EPg2/0245g previously recognized (20, 31, 34 35). Presentation of the HIV-l PND to the immuns ~y6tem could understAndably be made vory difforent by the presence or ~b~enco of o$1. The u~e of IFA in humans has already been described, with little adverse ..
reactions reported (34, 36). IFA could therefore represent a useful ad~uvant for future HIV vaccineR in humans.
Remarkably, the 12 animals u~ed in thi~ study showed a ~trong, significant correl_tion between anti-PND and neutralizing antibody titers at the 5 months and 7 months .
time point~, suggesting that the ma~ority of the neutralizing ~ntibodie~ were t~rgeted to the PND, independent of the ad~uvant used in the vAccine (Fig. 4). A ~imilar correl_tion was observod in chimpanzee~, although not as strict a~
observed here (unpublished observ_tions).
These results h_ve strong implications for HIV-l vaccino6. Fir~t, they show that ad~uvants such as IFA or :~
SAF-l should be pr~ferred to ~lum ln vlew of their greater ~-potoncy. Second, lt ha~ recently been shown that, in addition to the PND, gpl20 also contain~ conserved, conformation~l noutralization epitope~ thAt elicit broadly ::
cross-reactive neutr_l$zing antlbodies (37-41). Antlbodies to these conformational epitopes seem to neu.trali~e viru6 infectivity by interfering with the binding of the virus to ~ts CD4 receptor (38, 39, 42). The fact that the great ma~ority of the neutralizing antibodies elicited by gpl20 .
alone (15) or gpl60 followed by PND peptides (16, and thi6 study) is essentially directed to the PND alone, suggests that the conserved, conformational neutralization epitopes of gpl20 are not vi~ible to the immune system of primates under the~e conditions. It would be of ma pr importance for the future developm~nt of HIV ~accine~ to f ind A W~y to improve or modify the mode of the delivery of the antigen(s) and/or to m~nipulate the immune re~ponse of the host 80 _8 to elicit such a bro_dly-neutralizing antibody response, short of which HIV-l v~ccine~ will run the risk of remaining isol~te-specific _nd un~ble to cope with the wide antigenic v_riAtion of the virus in the field (6).

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2,~ 2~2~'3 6 8 ACRNOWLEDGNENTS
We th~nk J.-P. L~vy ~nd J.-P. Lecocq for continuou_ ~ncour~gRment ~nd support, J.-M Dupuy ~nd M.-P. R~eny for helpful sugge~tion_. A. Pinter for help with viru~ .
neutr~liz~tion, Is~belle Diaz for providing the gp160 and J.-P. S~uzet for eff~cient technic~l ~6sist~nc~. ~hi~ work w~ ~upported by P~teur ~erieux S~rums & V~ccins and by the French Nation~l Agency for AIDS Rese~rch ~ANRS).

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2gl2~2~3 REFERENCES
1. Allan JS, Loligan JE, Barin F. MeCane MF, Sodro~ki JG, Rosen CA, H~eltine WA, Lee T~, and ~ex Ms M~or glyeoprotein antigens th~t induce antibodies in AIDS p tients ~re eneoded by HT~V-III. Science 1985; 228slO91-1094.
2. Verone~e FD, Devieo AL, Copeland TD, Orozl~n S, Gallo RC, and S~rng~dh~r~n ~Gs Chsr~cteriz~tion of gp41 ~ the tran~membrane prote~n eoded by the HTLV-III/~AV envelop gene. Seienee 1985;229:1402-1405.
3. Palker TJ, Clark ME, Langlois AJ, Matthew~ TJ, Weinhold RJ, Randall RR, Bolognesi DP, and H~ynes BF: ~ype-specific neutraliz~tion of the human immunodefieieney virus with ~ntibodie~ to env-eneoded synthetic peptide~. Proc Natl Ae~d Sei (USA) 1988;85sl932-1936.
4. Ru~ehe JR, Javherian K, McDanal C, Petro J. Lynn DL, Grim~ila R, Langloi6 A, Gallo RC, Arthur LO, Fi~chinger PJ, Bologne~i DP, Putney SD, and Matthews TJ: Antibodies ~nd inhibit fu~ion of human immunodeficieney virus-infeeted cell6 --bi~nd-a 24-amino aeid equenee of the viral envelope, gp120.
Proe N~tl A~ad Sei (USA) 1988;85:3198-3203.
5. Jav~herian ~, Langloi6 AJ, MeDanal C, Ros6 RL, Eekler ~I, Jellis CL, Profy AT, Rusehe JR, Bologne~i DP, Putney SD, ~nd Matthews FJs Prineipsl neutralization domain of the human immunodefieieney ~iru~ type 1 envelope protein. Proe Natl Aead Sei (U&A) 1989;86s6768-6772.
6. My~rs G, ~orber B, Berzof6ki JA, S~i~h RF, and Pavlakis GNs Hum~n retroviru~e~ and AIDS, 8 compilation and snaly~is of nucleic acid ~nd amino acid sequences. ~08 Alamo8 National L~boratory, Lo6 Alzmo8, New ~exico 1991.
7. Robinson NE Jr, ~nd ~itchel WNs Neutralization and enhanc~ment of in vitro and in vivo HIV and 6imian immunodeficiencyivirus infection. AIDS 1990;4 (suppl l)sS151-S162.

8. Putney JR, M4tthews TJ, Robey WG, Lynn DL, Robert-Guroff N, ~euller WT, Langlois AJ, Ghr~yeb J, Petteway SRs HTL~-III/~AV-neutralizing antibodie~ to an E. coli produced frag ent of th vlru- envelopo. Scienco 1986;234:1392-1395.

, `:

~ ~ SUBSTITUTE SHEET ~`

9. La Rosa G, David JP, Weinhold R, Waterbury JA, Profy AT, Lewi~ JA, L~nglois AJ, Dre~sman GR, ~oswell RN, Shadduck P, Holley LH, Rarplus M, Bolognesi DP, M~tthew~ $J, Emini EA, and Putney SA: Conserved sequence ~nd structural element6 in the HrV-prineipal neutralizing determinant. Seience 1990;249s932-935.

10. Putney SD, and McKe~ting JA; Antigenic variation in HIV.
AIDS 1990; 4 (suppl l)sS129-S136.

11. Nara PL, Garrity RR, and Goudsmit J : Neutraliz~tion of HIV-l, a paradox of humor~l proportions. FASEB J
1991;5:2437-2455. -~

12. Matthews TJ, Langloi~ AJ, Robey WG, Chang NT, Gallo RC, Fisheinger PJ, and Bolognesi DP: Restricted neutralization of divergent hum~n T-lymphotropic virus type III isol~t~s by antibodie6 to the ma~or envelope glycoprotein. Proc N~tl Aead Sei (USA) 1986;83:9709-9713.

13. Nara PL, Robey WG, Pyle SW, Hatch WC, Dunlop NM, Bes~ JN
Jr, Relliher JC Arthur LO, and Fisehinger PJ: Purified envelope glyeoproteins fro~ ~um~n immunodeficieney viru6 type 1 variants induce individual type-sp~cifie neutralizing antibodies. J Virol 1988;62s2622-2628.

14. Nara PLs Neutraliz~tion of HrV-l s a binding /po6t-binding event medi~ted through the gpl20 immunodominant epitope. In: ~etroviruse6 of human and related animal disea~es. M, Gir~rd and L Valette (ed6) Fond~tion M~reel ~or~eux, Lyon 1988, pp 138-150.

15. ~erman PW, Gr~gory FJ, Riddle L, Nak~mura GR, Ch~mpe MA, Porter JP, Wurm FN, Her~ehberg RD, Cobb ER, ~nd Eiehberg JW:
Proteetion of chimpanzee~ from infeetion by HIV-1 ~fter v~ceinAtion with recombinant gpl20 but not gpl60. N~ture 1990;345:622-635.

16. Girard ~, Rieny MP, Pinter A, Barré-Sinous~i F, Nara P, ~olbe H, Kusumi R, Chaput A, Reinhart T, Muchmore E, Ronco J, Raezerk N, Gom~rd.E, Gluekman JC, and Fultz P: Immunization of ehimp~nzee~ eonfer~ protection ag~in~t ehallenge with human immunodefieiency ~irus. Proc Natl Acad Sci (USA) 1991;88:542-546.

, .
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SUBSTITUTE SHEET

?~122~63 17. Girard M, and Eiehberg JW:Progres~ in the development of HIV vaeeines; AIDS 1990;4 (~uppl l)sS143-S150.

18. Putkonen P, Thor~tens~on R, Ghavamzadeh L, Albert J, Hild ~, Biberfeld G, and Norrby Es Prevention of HIV-2 and SIV~m infeetion by passive immunization in eynomolgu6 monkeys. Nature 1991;3S2s436-438.

19. Girard M : The ehallQnge of HIV vaeeines. Vaecine l9gl;
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20. Allison AC, and ~yar~ NE : An ad~uvant formulation that seleetively elieit~ the formation of antibodie~ of protective isotype~ and of eell-mediated immunity. J.Immunol Methods 1986;95:157-168.

21. Wain-Hobson S, Vartanian JP, Henry M, Cheneiner N, Cheynier R, Delassus S, Pedroza Nartins L, Sala ~, Nugeyre NT, Guetard D, Barré-Sinou6si F, and Montagnier L: LAV
revis~ted: origins of early viral HIV-l isolates from Institut P~steur Seienee 1991;252:961-964.

22. Rieny NP, Lathe R, Riviere Y, Dott ~, Sehmitt D, G~rard M, Mont~gnior L, ~nd Leeoeq JP: Improved antigenieity of the HrV env protein by ele~vage site removal Protein Engineering 1988;2:219-225.

23. Sehmitt D, Dezutt~r-Dambuyant C, Hanau D, Sehmitt DA, Rolbe HV, ~ieny MP, Cazenave JP, and Thivolet J: Les proteines d'enveloppe du VIH 60nt fixee6 par les cellule6 de L~ngerh~ns épide-m~ques humaine6 sur un site de fixation qui diff~re du site porte p~r la moléeule CD4, et 6'internalisent p~r une ~endoeytose par r6cepteur6n. C R Aead Sci (Pari6) 1989;309(III):269-275.

24. Thoma~ DJ, Wall 36, Hainfield JF, ~aczorek M, Budy FP, Tru6 BL, Eiserling FA, and Ste~en AC: gp160, the envelope glyeoprotein of human immunodefieieney VirUB type 1 is a dimer of 125-kilodalton subunit6 stablilized through interaction~ between their gp41 domnin6. J Virol 1991;65s3797-3803.

25. Friguet B, Chaffotte AF, D~avadi-Ohaniance L, and Goldberg ME: Measurements of the true nffinity constant in SU8STITUTE SHEET `::
.. . . , . . .. . ...... . ... ....... . . ~ . ., .. . , ... ~ .... . . . . . . . .

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solution of antigen-ant~body complexes by enzyme-linked -~
immunosorbent assay. J Immun Method~ 1985;77:305-319. ~.

26. Nara PL, Hatch WC, Dunlop NM, Robey WG, ~rthur LO, Gonda ~:
~A, and Fishinger PJ: Simple, rapid, quantitative, syncitium forming microassay for the detection of human :~-i~munodeficiency virus neutralizing sntibody. AIDS Res Human Retroviruses 1987;3:283-302.

27. Arthur LO, Pyles SW, Nara PL Be~s JW Jr, Gonda MA, ~elliher JC, Gilden RV, Robey WG, Bolognesi DP, Gallo RC, and Fischinger PJ: Serological responses in chimpanzee~
inoculated with human immunodeficiency virus glycoprotein (gpl2o) subunit vaccine. Proc Natl Acad Sci (USA) 1987 84:8583. ~-28. Arthur LO, ~e8B JW Jr, Waters W, Pyle SW, Relliher JC, Nara PL, Krohn K, Robey WG, Langlois AJ, Gallo RC, and Fischinger PJ: Challenge of chimpanzee~ (Pan troalodvtes) immunized with human immunodeficiency viru~ en~elope glycoprotein gpl20. J Virol 1989;63:5046-5053.

29. Berman PW, Groopman JE, Gregory T, Claph~m PR, Weiss RA, Ferri`~ni R, Riddle L, Sh~mas~ki C, Lucas C, Lasky LA, and E~chberg, JWs ~uman i~mn~nodeficiency virus type 1 challenge of chi~p~n~sees immunizod with recombinant envelope glycoprotein gp120. Proc N~tl Acad Sci (USA) 1988;85:5200-5204.

30. Ander~on gP, Lucas C, Han~on CV, Londe ~F, Izu A, Grogory T, Amm~nn A, Berman PW, ~nd Eichberg JW: Effect of doco ~nd ~mmunization ~chedule on immune re~ponses of baboons to rocombin~nt glycoprotein 120 of HrV-1. J Infect Dis 1989;160:960-969. -31. Nann-Halter JW, Pum ~, Wolf HM, Rupcu Z, Barrett N, Dorner ~, ~der G, and Eibl MM: Immunization of chimpanzees with the HIV-l glycoprotein gp160 induces long-la~ting T-cell memory. AIDS Res Hum Retroviruse~ 1991;7s485-493.

32. Hart M~, Palker TJ, Matthew~ TJ, ~angloi~ AJ, Lerche NW, Martin ME, Scearce RM, McDan~l C, BolognQsi DP, and Hayne6 BF: Synthetic peptides containin~ T and B cell epitope~ from human immunodeficiency virus envelope gpl20 induce anti-HIV

SUBSTITUTE SHEET

prolif~rative re~ponse~ and high titers of neutralizing antibodie~ in Rhe~us monkeys. J Imnunol 1990;145:2677-2685.

33. Ronco J, Chev~lier ~, X~czorek M, De~landres A, B~rre-Sinou~si ~, and Girard M: Induction of HIV-l neutralizing antibodie~ by ~ynthetic peptides In: Vaccines 91, E ~erner, C
Chanock, F Brown, and H Gin~b~rg (eds) Cold Spring Harbor NY
1991 pp29-35.

34. Roof RW, Luescher IF, and Unanue E: Phospholipids enhance the bind$ng of peptide~ to class II ma~or histocompatibility molecules. Proc Natl Acad Sci (USA) 1990;87:1735-1739.

35. Salk J: Prospect~ for the control of AIDS by immunizing seropositive indi~iduals. Nature 1987;327:473-476.

36. Levine A, Henderson B, Groshen S, Munson ~, Allen J, Carlo D, Burnett K, Jensen F, ~erre F, Trauger R, Abrah~mson J, Gersten M, and Salk J: Immunization of HIV-infected ;-indi~iduals with inacti~ated HrV immunogen In: Retroviruse~
of human AIDS and related diseases. ~ Girard and L Valette (ed~) ~ondat$on MarceI ~erieux, Ly~n 1990 pp247-250.

37. Stei~er ~S, and Haigwood NL: Importance of conformation of the neutral$zing ant$body response to HIV-l gpl20.
Biotechnology Therap 1991; 2:63-89.

38.01~he~-~y U, Hel~eth E, Furman C, Li J, Haseltine W, and Sodrowski Js Identification of individu~l hu~an immunodeficiency ~iru- type 1 gpl20 ~mino acid~ important for CD4 receptor binding. J Virol 1990;64:5701-5707.

39. Ho D, Mc~eating JA, Li XL, Moudgil T, Daar ES, Sun N, and Robinson JFs Conformational epitope on gp120 i~portant in CD4 binding and human deficiency virus type 1 neutrslization ident~fied by a human ~onoclonsl antibody. J Virol 1991;65:489-493.

40. Posner MR, Hideshima T, Cannon ~, Mukher~ee M, Msyer RH, ~-and Byrn RA: An Igg human monoclonal antibody that reacts with HIV-l gpl20, inhibits viruQ binding to cells, and neutr~lizes infection. J Virol 1991;65:489-493.

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41. Rang CY, Nara P, Chamat S, Caralli V, Ry~ka~p T, H~igwood N, Newman R, and Xohler H: Evidence for non-V3-~pecific neutralizing antibodie~ that interfere with gpl20/CD4 binding in hu~n i~munodeficiency virus l-infected humans . ~roc Natl Acad Sci (USA) 1991; 88 s 6171-617S .

42. T~lley, SA, Honnen JW, ~cho M, Hilgartner M, and Pinter A: A human monoclonal antibody ag~in~t the CD4 binding site of HIV-l gp120 exhibits potent, broadly neutralizing -activity. Res Virol 1991; in pre~6.

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,~ :
~ ~ SUBSTITUTE SHEET

27~22263 11. Moor-Jankowski, J. & Mahoney, C.J. (1989) J. ~ed.
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(1988) Prot. Engineer~ng 2, 219-226.
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Thivolet, ~. (1989) CQmpt~s Rendus Ac~d. Sci. P~r~s, 308(III), 269-275.
14. Guy, B., Riviere, Y., Dott, R. Regnault, A. &
Xieny, M.P. (1990) Virology 176, 413-425.
15. ~olbe, H.V., Ja~ger, F., Lepage, P., Roit~ch, C., -~
Lacaud, G., Xi~ny, M.P., Sab~tie, J., ~rown, S.W. & Lecocg, J.P. (1989) J. Chrom~togr~p~y 476, 99-112.
16. Allison, A.C. & Byars, N.E. (1986) J. Immunol.
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17. Putn~y, S.D., ~atthew~, T.J., Robey, W.G., Lynn, D.L., Robert-Guroff, M., ~ueller, ~.T., Langloi6, A.L., Ghrayeb, J., Pett~way, S.R., Weinhold, R.J., Fischinger, P.J.~ ~ong-St~al, F., Gallo, R.C. ~ Bolognesi, D.P. (1986) Sc~enc~ 234, 1392-1395. -18. Rusche, J.R., Xavaherian, R., ~cDanal, C., Petro, J., Lynn, D.L., Grimail~, R., Langlois, A., Gallo, R.C., ---Arthur, L.O., Fischinger, P.J., Bologne~i, D.P., Putney, S.D.
L N~tthews, T.J. (1988) Proc. N~tl. Ac~d. Sci. V.S.A. 85, 3198-320~. .
19. LaRosa, G.J., Davide, J.P., Weinhold, R., ~-Waterbu~y, J.A., Profy, A.T., Lewi6, J.A., L~ngloi~, A.J., A.J., Dr~sman, G.R. Boswell, R.N., Shadduck, P., Holley, - ~-L.H., R~rplu6, M., Bologne~i, D.P., Matthew~, T.J. Emini, E.A. & Putney, S.D. (1990) Science 249 932-935.
20. N~ra, P.L., H~tch, W.C., Dunlop, N.M., Robey, N.G., Arthur, L.O., Gond~, M.A. & Fischinger, P.J. (1987) AIDS Res.
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21. Fultz, P.N., McClure, H.M., Swenson, R.B., McGr~th, C.R., Brodie, A., Getche11, J.P., Jen~en, F.C., Ander~on, ~' "'.

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23. Hullis, R.B. & Faloona, F.A. (1987) Methods , .:
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24. Rwok, S. & Xellogg, D.E. (1990) in PCR Protocol~: A ~-Guide to ~thods ~nd Application6~ eds. Inni6, ~.A., Gelfand, D.H., Sninsky, J.J. & White T.J. (Academic Press, Inc., San Diego, CA) pp. 337-347.
25. Zagury, D., Bernard, J., Cheynier, R., Desporte~, I., Leon~rd, R., Fouchard, M., Reveil, ~., Ittele, F.D., Lurh~ma, Z., Mbayo, R., Wane, J., Salaun , J.J., Goussard, B., Dech~z~l, L., Burny, A., N~ra, P. & Ghllo, R.C. (1988) N~ture ~ .
~London) 322, 728-731.
26. Nara, P.L., (1989) in V~cc~n~ ~9, eds. Lerner, ~.A., Ginsberg, H., Ch~noek, R.M. ~ Brown, F. (Cold Spring Harbor L~boratory, Cold Spring H~rbor, NY) pp. 137-144.
;27. Myers, G. (1990) in Humun Retroviru~e~ ~nd AIDS, eds. ~yers, G., Josephs, S.F., Wong-Staal, F., Rabson, A.B., Smith, T.F. ~ Berzofsky, J.A. (Lo6 Al~mos Nation~l L~bor~tory, Los AlamO8, NM).
28. Scharf, S.J., Horn, G.T. & ~rlich, H.A. (1986) Sc~Rnc~ 233, 1076-1078.
29. Walker, C.N., Noody, D.J., St~te6, D.P. & Levy, J.A. (1986) Scf~nce 234, 1563-1566.
30. Tsubota, H., Lord, C.I., Watkin~, D.I., Norimoto, C. & Letvin, N.L. (1989) J. Exp. ~d. 169, 1421-1434.
31. R~nki, A., Valle, S.L., Rrohn, M., Antonen, J., Allain, J.P., ~euther, M., Fr~nchini, G. & ~rohn, R. (1987) Lancet 2, 589-593.
32. Jehud~-Cohen, T., Sl~de, B.A., Powell, J.D., Villinger, P., De, B., Folks, T.M., McClure, H.M., Sell, ~.W.
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SUBSTITUTE SHEET

WOg3/08836 2 12 2 2 6 ~ PCT/EP~2/02459 33. Bahraoui, E., Yagello, M., Billaud, J.N., Sabatier, J.M., Guy, B., ~uchmore, E., Gtrard, M. & Gluckman, J.C.
(1990) A~DS Ae~. Human R~trov~ru~s 6, 1087-iO88.
34. Van Eendenburg, J.P., Yagello, M., G~rard, M., ~ieny, M.P., Lecocq, J.P., Muchmore, E., Fultz, P.N., ~:
Rlviere, Y., Montagnier, ~. & Gluckm~n, J.C. (1989) AIDS Res.
Hum~n Retroviruses 5, 41-50.

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Claims (51)

Claims

1. A method of enhancing the immunogenicity of an envelope glycoprotein of a virus in a host, wherein the method comprises administering to the host at least one envelope glycoprotein of the virus and at least one peptide derived from the amino acid sequence of the envelope glycoprotein, and wherein the peptide comprises at least one virus-neutralization epitope, and wherein the envelope glycoprotein and the peptide are administered in an amount sufficient to induce neutralizing antibodies in the host.

2. Method as claimed in claim 1, wherein the virus is selected from the group consisting of HIV, SIV, HTLV-1, HTLV-2, FIV, and FeLV.

3. A method of enhancing the immunogenicity of an envelope glycoprotein of a virus, wherein the method comprises administering to a host at least one envelope glycoprotein of virus in an amount sufficient for priming vaccination and at least one peptide derived from the amino acid sequence of said envelope glycoprotein, wherein the peptide comprises at least one virus-neutralization epitope of said glycoprotein of virus, and the peptide is administered to the host in an amount sufficient to enhance the induction of neutralizing antibodies in the host to confer to the host long-lasting immunity against the virus.

4. Method as claimed in claim 3, wherein said envelope glycoprotein and said peptide are simultaneously administered to said host.

5. Method as claimed in claim 3, wherein said envelope glycoprotein is administered to said host, and then said peptide is administered to said host.

6. Method as claimed in claim 3, wherein said envelope glycoprotein is gp120 or gp160 of HIV.

7. Method as claimed in claim 3, wherein said at least one peptide comprises a mixture of said peptides, which are administered to the host in a free state not coupled to a carrier molecule.

8. Method as claimed in claim 7, wherein the peptides are coupled to a carrier molecule.

9. Method as claimed in claim 7, wherein the carrier is a lipopeptide.

10. Method as claimed in claim 3, wherein the envelope glycoprotein comprises a mixture of HIV-1 and HIV-2 envelope glycoproteins, and said peptide comprises a mixture containing at least one peptide having an HIV-1 neutralization epitope and at least one peptide having an HIV-2 neutralization epitope.

11. Method as claimed in claim 3, wherein the envelope glycoprotein is orally administered to the host.

12. Method as claimed in claim 3, wherein the envelope glycoprotein is parenterally administered to the host.

13. Method as claimed in claim 3, wherein the peptide is orally administered to the host.

14. Method as claimed in claim 3, wherein the peptide is intradermally administered to the host.

15. Method as claimed in claim 3, wherein said envelope glycoprotein is a mixture of gp160 glycoproteins from different HIV isolates (serotypes) and said at least one peptide is a mixture of the corresponding neutralization epitopes.

16. Method as claimed in claim 3, wherein said envelope glycoprotein is a mixture of glycoproteins gp120 from different HIV isolates (serotypes) and said at least one peptide is a mixture of the corresponding neutralization epitopes.

17. Method as claimed in claim 3, wherein said envelope glycoprotein is gp120 of HIV.

18. Method as claimed in claim 3, wherein the envelope glycoprotein and the peptide are administered in combination with an adjuvant to the host.

19. Method as claimed in claim 18, wherein the adjuvant is muramyl dipeptide in a lipid medium or incomplete Freund's adjuvant.

20. Method as claimed in claim 19, wherein the peptide is selected from the group consisting of env, pol, gag, nef, vif, antigen, and mixtures of said antigens.

21. Method as claimed in claim 19, wherein said mixture of said antigens comprises p27nef and p23vif.

22. Method as claimed in claim 19, wherein the peptide is at least one peptide selected from the group consisting of:

23. Method as claimed in claim 19, wherein the peptide comprises the following amino acid sequence.
YNTRKSIRIQRGPGRAFVTIGKIGN.

24. Method as claimed in claim 19, wherein said at least one peptide is comprised of the major neutralization epitope (loop V3) of at least one HIV-1 isolate.

25. Method as claimed in claim 19, wherein said envelope glycoprotein is administered to said host, said at least one peptide is administered to said host after said envelope glycoprotein, and thereafter a mixture comprising at least one envelope glycoprotein of said virus and at least one peptide derived from the amino acid sequence of said envelope glycoprotein is administered to said host.

26. Method as claimed in claim 25, wherein said envelope glycoprotein is gp160 of HIV.

27. Method as claimed in claim 26, wherein said at least one peptide is comprised of the major neutralization epitope (loop V3) of at least one HIV-1 isolate.

28. A composition for vaccinating a host against infection by a virus, wherein the composition comprises (A) at least one envelope glycoprotein of the virus in an amount sufficient for priming vaccination in a host to which the envelope glycoprotein is administered; and (B) at least one peptide derived from the amino acid sequence of said envelope glycoprotein, wherein the peptide comprises at least one virus-neutralization epitope of said glycoprotein and said composition contains said peptide in an amount sufficient to enhance the induction of persistent neutralizing antibodies in the host.

29. Composition as claimed in claim 28, wherein said at least one peptide comprises a mixture of peptides of glycoprotein of HIV.

30. Composition as claimed in claim 28, wherein the peptides are bound to a carrier therefor.

31. Composition as claimed in claim 28, wherein the composition contains an adjuvant in an amount sufficient to enhance vaccination of the host.

32. Composition as claimed in claim 31, wherein the adjuvant is muramyl dipeptide or incomplete Freund's adjuvant.

33. Composition as claimed in claim 32, wherein the peptide is selected from the group consisting of env, pol, gag, nef, vif antigen, and mixtures of said antigens.

34. Composition as claimed in claim 33, wherein said mixture of said antigens comprises p27nef and p23vif.

35. Composition as claimed in claim 33, wherein the peptide is at least one peptide selected from the group consisting of:

36. Composition as claimed in claim 33, wherein the peptide comprises the following amino acid sequence:
YNTRKSIRIQRGPGRAFVTIGKIGN.

37. Composition as claimed in claim 33, wherein said at least one peptide is comprised of the major neutralization epitope (loop V3) of at least one HIV-1 isolate.

38. A composition for enhancing the immunogenicity of an envelope glycoprotein or a fragment of a virus, wherein the composition comprises as a combined preparation for simultaneous, separate, or sequential use:
(A) at least one envelope glycoprotein of the virus or a fragment of at least 50 amino acids of the glycoprotein; and (B) at least one peptide derived form the amino acid sequence of the envelope glycoprotein;
wherein the peptide comprises at least one virus-neutralization epitope;
and wherein the envelope glycoprotein and the peptide are administered in an amount sufficient to induce neutralizing antibodies in the host.

39. A composition for enhancing the immunogenicity of an envelope glycoprotein of a virus, wherein the composition comprises, as a combined preparation for simultaneous, separate, or sequential use:
(A) at least envelope glycoprotein of the virus or a fragment of the glycoprotein having its immunogenic properties in an amount sufficient for priming the induction of neutralizing antibodies in a host to which the envelope glycoprotein is administered; and (B) at least one peptide derived from the amino acid sequence of said envelope glycoprotein;
wherein the peptide comprises at least one virus-neutralization epitope of said glycoprotein and said composition contains said peptide in an amount sufficient to enhance the induction of persistent neutralizing antibodies in the host.

40. A composition as claimed in claim 39, wherein the envelope glycoprotein or a fragment is gp160 of HIV or gp120 of HIV.

41. Composition as claimed in claim 49, wherein said at least one peptide comprises a mixture of peptides of glycoprotein of HIV.

42. Composition as claimed in claim 41, wherein said at least one peptide is bound to a carrier molecule comprising an aliphatic sequence.

43. Composition as claimed in claim 42, wherein the peptide is selected from the group consisting of the env, gag and especially p18gag, nef, vif, pol, or GPG OR GLG antigens and mixtures of said antigens, particularly p27nef and p23vif.

44. Composition as claimed in claim 42, wherein the envelope glycoprotein of the virus is combined with at least one of the antigens selected from the group consisting of gag, pol, nef, vif, and particularly with a mixture of p27nef and p23vif.

45. Composition as claimed in claim 39, wherein the composition is suitable for oral, parenteral, or intradermal administration.

46. Composition as claimed in claim 39, wherein the envelope glycoprotein or a fragment thereof and the peptide(s) derived therefrom are presented side-by-side in order to be applied simultaneously, separately, or at intervals to the host.

47. Composition as claimed in claim 39, wherein the envelope glycoprotein is combined with a pharmaceutical vehicle for oral or parental administration.

48. Composition as claimed in claim 39, wherein the peptide is combined with a pharmaceutical vehicle for oral administration.

49. Composition as claimed in claim 39, wherein either said at least one envelope glycoprotein of the virus and/or said at least one peptide derived from the envelope glycoprotein are presented:
either under the form of particles, such as ISCOMs or liposomes, or by a live recombinant microorganism.

50. Composition as claimed in claim 49, wherein the microorganism is a live recombinant microorganism, such as viruses or bacteria, for instance a poxvirus or BCG, or any live vaccine modified to express the envelope glycoprotein or the peptide derived from the envelope glycoprotein.

51. Composition as claimed in claim 50, wherein the microorganism is derived from inactivated particles, for instance viral particles, such as the HIV virus, or particles without virus genome, especially without HIV genome.