CA2031197A1 - Dendritic polymer of multiple antigen peptide system useful as anti-malarial vaccine - Google Patents

Abstract

Multiple antigen peptide systems are described in which a large number of each of T-cell and B-cell malarial antigens are bound to the functional groups of a dendritic core molecule providing a high concentration of antigen in a low molecular volume. The products elicit a very strong immunogenic response.

Description

k ~ ~ 7 W O 90/~1778 ~ ~ Pcr/US90/~ 9 . .' . . .

~I)ENDRITIC POLYMER OF MUL~IPLE ANTIGEN PEPT LDE SYSTEM USEF~L
AS ANTI--I~AhARIAL VP~CCINE n Vaccinei of~en cDmprise an antigen on a natural carrier such as a protein, a carbohydrate, a lipid or a liposome. Such vaccines are useful and bave been employed for many years.
There are ho-~ever a number of art r~cognized problems with them. Several of these problems are related to the carrier.
Since the carriers are isolatet from natural soùrces, they are of~en not of uniform quality. Additionally, despite expensive and arduous purifica~ion e~forts, it is dlff~.cult, and often impossible, to provide products completely ree of natural contaminants. Such contaminants ~ay themselves be antigenie.
They cause the undesirable side reac~ions often asso~iated wi~h the use of vaccines, particularly fevers and tis~ue swelling.
Additionally, the concentration of antigen ~ay vary from one bat-h to another becau~e the amounts of antigen ~hich react with the carrier or are ab~orbed on its ~ur~ace are not unifonm. This problem has markedly increased the difficulties o~ preparing suitabl~ vaccine3 for p~otection asainst ~alaria.
Malaria is a particularly i~portant targot for syn-thetic vaccines, since it ~ff~cts 200 ~illion people worldwide and no immunoprophylaxis has yet been developed. It i~ ~own that p~otective immunit~ against rodent, si~ian and human malaria porozoites can be induced by immunization with irradiated ~porozoites. The major protein of the sporozoite is the circumsporozoite ~CS) protein, and antibodies directed again3t the CS protein ~r~ known tv neutralize th~ infectivity of parasites and inhi~it their entry into the hepatocy~es.

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W0 90/11 ~ ~ ~ ~CT/~S~/32039 Thus, the CS protein has become ~an importaat target for the development of syn.hetic vaccines ~gainst the sporozoi~e st~ge of malaria. The immunodominant 8-cell epitopes of the CS
protein is ~ontained within the repeating ds~ain of the CS
protein, a feature co~on to CS protein~ of ~ alaria species. Mice im~unized with a synth~tic peptide, ~tDched to tetanus toxoid ~s a protein carrier of thi~ B-eell epitope, hav~ been found to develop hi~h antibody tite~ and resistance ~o çhAllenge w~th 103 ~porozoite~s. ~owev~r, v~ccination attempts in humans, using a simi'ar approach, have ~ailed to induce good antibody titers.
Recently, several T-helper cell epioopes of the CS
protein of P. ~-ergh-ei (a roten~ malaria) have al30 been identified Isee Romero et al., EY~- J- ~mmY~Q~ 1951~ 1988)-li ~he identification of the B and T helper cell epitopes of theCS protein of P. be~qh~i has now made it possiole to incor-porate th0s¢ epitopes into one ~olecule in a spe^ific and unambiguous manner ~sing the MAP approa^h-in which the epitopes ~re attached to a defined dendritic poly~er, using the procedure developet by Tam and his coworkers as de~cribed in ~ol. Chem. ~, 1719 11988). In addition, ~-cell ~pitope~ of o~her malarial species have been identified: ~ee, ~.q., Sinigaglia, F. et al, ~ature ~ 778, 1988 (~. ~alcipa u~);
Crisanti, ~. et al., ~ciençe, ~Q:1324, 1988 (~. f~lçi~arum, blood stage); Rumar, S. et al., NatuFe 334:258, 1988 ~P.
fa~ç~a~u~ sporozoites) ~~; G~od, M.S. et ~ çience 235:1059-1062, 19~7; Good, H.S., et al, Prçc- Nat'l. a3~ ~Si-85:1199-1203, 198a; Sinigaglia, F., et al., ~Y~
~Q:633-636, 1988; and Guttinger, M., et al., E~Q ~. 7:2555-255~, 1988.
Dendritic poly~ers are a new class of polycers. They are characterized by higher concentrations of Sunc~ional groups per unit of molecular volume than ordinary poly~ess. General-ly, they are ~ased upon two or more identical branches ori~in-ating from a core molecule having at leas~ ~wo func~ional groups. Such polyDer~ have been de~csibed by Den~ewal~er et al. in U.S. Patent No. 4,289,872 and by Somalia et ~1. in 3 tJ ~
! ., ~ , ~ 1~
,~ ~ WO 90/11778 ~ PCT/US~0/~20~9 everal U.S. Patents including Nos. 4,599,400 and 4,5~7,466.
Other polymers of the cla~s have been described by E~ickson in U.S. Patent 4,515,920. The po1ymers are often referred to as dendritic polymers because their structure ~ay be ~y~bolized a~
a tree with ~ core trunk and everal ~r~nche~. Unlik~ ~ tr~e, , ; however, the branches in dendritic poly~er are all substan-- tially identical.
The products of this invention are based on such dendritic 3ystems in which a~tigens ar~ cov~lently bound to the branche3 which radiate fro~ the core ~olecule. The system has be~n termed the ~ultip}e ~tigen peptide system and is some-tin,es re~erred to herein as ~APS. As will be apparent from the discussion hereinafter, some of the ca~rier or core molecules used to form the produ~ts of the inven~ion are of a mole~ular weight such that they might not usually be regarded as poly-mers. Howeve., since their basic structure is similar to de~dritio polymers, it is con~enient to describe them as such.
Therefore, the term ~dendrltic polymer~ will be sometimes used herein to define the polymeric substrates of the product3 of the invention. The term includes carrier molecules which are sufficiently large to be re~arded a~ polymers as well as those which ~ay contain ~s ~ew as three ~onomers.
It has now been discovered that tendritio polyme-s can function usefully as carriers ~or a wide variety of an~igens.
~his invention will be bet~er understood ~rom a brief discussion of the structure of dendritic polymers.
Dendriti~ polymers are built upon n core ~ole~ule which is dt lease difunctional. Each of the funotional groups on the core ~olecule for~ at l~ast two branches, the principal units of which are- also at lea~t tifunctio~al. Each difunc~ional unit in a branch provide5 ~ base for added 9rowth.
The system can be better visualized by reference to specifie molecules. If, for e~ample, lys,ne with two a~ino groups is joinet in a peptide ~ond through its e~rboxyl group to the amino group of alanine or glyçine whlch may in turn be bound to a resin, the resulting mol~cule will have two fr~e a~ino groups. This dipeptide ~ay ~e regarded as the irst , ; , .

~ i ~ J
W O 90~1177Y ~ ~ PCT/US~/02039 generaticn. It ma~ be joined to two additional lysine mole-cule~ b~ the ~c.-a~ion cf pept~de bonds to produce d second ge~eration molecule with four free amino groups. The process can be repeated to for~ third, fourth or even higher genera-tionC of produets. With each generation the number Df free ' ~ amino groups iner~ase~ geometrically and can be represented ~y , wh~re n is the number of the qeneration.
Although none of these compo~nds ~re of particularly hiqh molecular weight, it i5 convenient to refer to them as dendritic polymers.
Fig. 1 shows a three generation dendritie polymer core molecule based on lysine in which each o~ the eight available amino groups are joined to a peptide antigen through a glycine linker molecule.
I; The same ty~es of reactions can be carried o~t with aspartic or glutamic acid, both of which have two carboxyl groups and one ~uno ~roup to produce polyaapartic or poly-glutamic acids with 2n free carboxyl groups.
The necessary ch~m stry for per~orming these types of syn~hesis i5 known and avsilableO With amino acids the ch?mist~y for blocking ~unctional groups which should not react and them removing the blocking groups when it is desired that the functional groups sh~uld react has been described in detail in nu~erous patents and articles in the teehnical literature.
The dendri:ic polymers can be produced on a resin as in the well-known Merrifield sy~thesis and the~ removed ~ro~ the poly~er.
~omali~ utili ed ammoni~ or ethylenedi~mine as the core molecule. I~ thi~ procodure, th~ core molecule is re~ted wit~
an acrylate ester by ~ichael addition and ~he ester groups removed by hydrolysis. The resulti~g first genera~ion ~ole-cules contain three free ~ar~oxyl groups in the case of ammonia and four free carboxyl groups when ethylenedia~ine i5 employed.
Tommalia extends the deDdritic polymer with ethylenedia~ine followed by another a~rylic ester ~onomer, and repeats the sequenee until the desired mole~ular weight is attained. It will, however, be re~tily apparent to one skilled in the art, h ) ;3 m ~ ~3;
W O 90/11778 ~ ~ PCT/U590/02039 tha~ e~cn branch of the dendritic polymer can be len~thened by a~y of a nu er of selected proceclures. ~or example, each branch can be e~tended by ~ultiple react.ons with lysin~
molecul~s.
S Erick~on utilized the clas~ic Herrifield technique in which a polyp~ptide of ~ubstantially any desired ~olecular weight is grown fro~ a ~olid resin ~upport. As the technigue i~ utilized for the preparation of dendriti~ polymers, the linking ~olecule which joins the polymer to the resin ~upport is trifunctional. One of the functional groups is involved in th~ lir,ka~e to the resin, t~e other tWO functional groups serve as ~he startins pcint for the grcwth of the po}ymer. The polymer is removed frcm the resin when the deslred molecular weight has been oDtained. Gne standard cleav~ge procedure is treatme~t with liquid hydrogen fluorite at 0-C for one hour.
Another, ahd mora satisfactory proceture, is tO utilize complex ~f hydrogen fluoride and dimethylsulfide ~F:DMF) as described by Tam et al. in '. Am. Soc. (1983) 105: 6442. ~his procedure greatly minimizes side reactions and lo s of peptide.
Denkewalter, in one example of his process, utilizes lysine as the eore mol~cule. The ~mino groups of the core moiecule are blocked by conversion eo ~rethane groups. The carboxyi group is blocked by reaction with benzhydrylamine.
Bydrolysis o~ the urethane groups qenerates a be~zhydrylamide of lysina with two free amino groups which serve as the starting points for th~ growth of the dendritic polymer.
This brief outline of three of the available procedures for procu~ing dendritic polymers should be ~dequate to teach those skilled in the art the basis principles of the current technology. They will also teach the ~killed artisan the salient ~e~tures of the polymers, one of the most important of ~hich is that the poly~er~ provide a large num~er of a~ail~le functional groups in a small ~olecular volume. ~he result i5 th~t ~ high concentration of antigens in a ~mall ~lume ean be achieved by joiniDg the antigen lo those availa~le fu~ctional ~roups~ Moreover, the resulting molecul~r pro~uet ~ontain~
high pr~portion vf antigen on a relatively s~all carrier. ~bis s~
PCIrU59:/0209 is in contrast to conventional products u~ed a~ a basis for vaccines. These convention~l products often a.-e compoced of a - small amount of antigen on a large amount of carrier.
Other important features o~ the! ~endritic poly~er aC an antigen carrier are that the exact ~tructure is known; there are no conta~inAnt3 which may be themselves antigenic, produce tissue irritation or other undesirable reactions; the exact concen~ration of the antigen i~ Xnown; tbe antigen is ~ym~etri-cally distributed on the carrier; and the carrier can be utilized as a bas~ for more than one ~ntigén so that multi-valent vaccines can be prcducei. The principal advantage of the MAPS te~hnique as the basis for malarial va~cines of this inventicn is that unlike previous systems using natural carrie-s such as ke}hole limpet hemoc~anin, tetanus toxoid and bovine serum albumin, the _arriers of this invention are fully defined cnemicai entities on which the antigens are dispersed in known concent:ations. Additionally the antigen comprises a large p~rt of the molecule no' a relatively small and undefined proportion of the molecule as in the case of natural carriers.
For the vaccines of this inve~tion, it i5 preferred that the core ~ole-hle be a naturally occurring amino acid such as lysine 50 that it can be dealt with by the body following the usual metabolic pathways. ~owever, as ~ill be explained more fully hereinafter, amino acids which ~re no~ na~urally occurrin~, even those which ~re not alpha-amino acids can be employe~. The acids, or any other asymmetric ~olecules used in building the core molecule can be in either the D or L for~.
Although the dendritic polymers have been principally described hereinabove as polyamlde polymers, i~ ~ill be xeadily apparent th~t the carrier~ of this invention are not limited to dendritic polyamides. Any of a wide variety of molecules having at lea t two avail~ble functional groups can serve ~s core mole~ules. Propylene glycol, for ex~pl~, can serve as the ba~is for a polyester dendritic poly~er. Succinic acid wi~h ~elec~ed glycols or amines can ~erve as a c~re molecule ~o generate polyes~ers or poly~mides. Diisocyanates can be used to generate polyurethanes. The important point is tha~ the S~ ~
~"' ~
W090/~778 0 ~CI/US911/020-9 core ~ole~ule has at least two av~ilable functional group~ fro~
which identical branches can be gener~ted by fiequential scaffolding-type reactions with additional ~olecules also .. having at le~st two ~vailable function~l or ~nchoring group~ on s each branch. In the mo~t si~ple ca3e in which the ~ore molecule has two available fun~tional group~ and each ~ucceed-ing gene~ation has two available functi~nal groups, the nu~ber of anchoring site~ to which ~alarial-origin T-eell and ~-cell antigens employed in this invention c~n be anchored is ex-presset by (2)n where n is the nunber o~ the qeneration, For a more complete discussion of the che~istry o~
dendritic polymers attention is directed to Tamalia et al., Polymer Journal 17 (1),- lli (1905), Akaroni et al, Mar-comolecules 15, 1093 (1982), and the following United States 15 Patents:
4,289,~72 4,'5~,12G
~,376,861 4,56~,737 4,507,466 4,587,329 4,515,920 4,59~,400 4,517,122 4,600,535 All cited patents, patent ~pplications and re~eren~es are incorporated by reference in tbeir entirety.
~HE IN~TENTION
This invention in its presently preferred e~bodiments provides a multiple antigen peptide system comprising a dendritic polymer b~se with a plurality of anch~ring sites covalently ~ound to antigenic T-ccll and ~-cell epitopes of ~alarial proteins such as the CS protein such that the result-ing construct be~rs both T and ~ epitopic peptides. The polymers comprise a central core molecule having ~t least two functisnal groups to which molecular branches having termir.al functional groups are covalently boundO ~he terminal func~ion-al groYpS on the branches are covalently b~nded to the epitopi~
peptides. The antigenic molecules are principally described herein ~s p~ptide antiqens, but they are not limited to peptide antigens or ~ven to antigens. Thus, peptides ~hat are no~
antigenic by-themselves ~ay be rendered antigenic when bound to f ~ , ~ .. _ _ J

WO 90/~ 2039 ~."~ r, 8 .,~. i - ~he core molecule.
The selected antigen may be sep~ately synthesized (by synthetic methods, including but not limited to rec~binant DNA
n' techniques, as i~ now well-known in the art) or otherwise obtained ~nd joined to the carrier. Preferably, the antigen may be ~ynthesized on the carrier by extending e~ch branch of : the poly~er utilizing ~nown peptide synthesi~ techniques.
Fig. 1 ~hows the structure of 2 dendritic polymer which may be employed in the p~acti_e of this invention. As will be ~0 seen, it is a three generAtion dendritic polylysin~ product.
I~ may be produced by a conventional s lid phase teehniques by gener~ting the polymes or. a Pam or a Pop resin. See Mitchell et al., J. Org. Chem. ~197a) 43, 2845 and T~m et al., J. A~.
Chem. Soc., ~19~0) l0? 6117. The polymer i5 then cleaved from lS the res~n usins, preferacly HF:DMS. The dendritic polylysine, as shcwn, was b~ilt from a glycine linker originally joined through a ben~yl linkar to the resin. Other linkers such as alanina can be employed. Of course, the linker can be omitted, or a plurality of linker molecules can be utilized.
Fig. 1 shows a dendritic poly~.er each molecule of which carries eight peptides some of w}ich rep_esent T-cell epitopic peptides and others B-cell epitopic peptides of a PlasmQdium species responsihle for ~alaria, e.s., Plasmodium b~er~
Plasmodium _alciP~ or Plasmodi~ viva~, P. Yo~ , P-malariae, P. o~ale, P. _ÇvnomO~s , P, kn wl~i; etc. joined directly to each of the ~ailable ~unctional groups on each terminal lysin~ moiety. $t i5 preferable that the ~- and T-epitopes on the pol~mer are of the same ~alarial species. ~he the pr~sent invention is Dot limited to poly~ers bearing only one T- and B-epitope co~bination from a single species. For example, MAPS bearinq simultaneously S- ant B-epitopes from p.viva~ CS protein and T- and B-epitnpes f~om P.falci~rum CS
protein are within ehe BCope of the in~en~ion. In addition, the ability of ~ peptide to function ~s a ~-helper epitope is not flece~sarily dependent upon the copse~ence of a ~ cell epitope from the same malarial speci~s. ~ence, cross-species combination3 of T-helper and 8-cell epitopic peptides are ~lso 3 ~
W091)/11778 ~ ~@ PCr/US9~/0~039 ~" 9 ccntemplated. When the 3ele~~e~' epitopic ~tructures are rela;ively short, e.g. 6 to 1~ resid~les, it has been observed that it is best tO extend the polylysine b~ a linker such .,s a sim~le tri- or tetr~peptide of glycine, ~lanine or beta-alanine. ~owever, for ~ntigenic peptides with ~ore than 14 residues, the linker i3 nor~ally unnece~sary, This invention has been described for convenience, principally a~ applied to prDducts built on ly~ine as the core molecule. In fact lysine .,nd lysine like molecules such as ornithine, nor-lysine and beta-amino alanine are preferre~
mciecules ~or building the products of this invention bPcause they are rel6tively easy to o~tain, they are easy ~o work with and they aSford good yields.
Such molecules can be r~resented by the seneral form~la:
( C~32 ) j~ H2 ~2N - ~CH2)x ~ C - ~C~21 C~OH
H
wherein x, y and z are in~egers ~rom 0 to 10, preferably 0 to 4 provided th~t at lea~t one of them is 1 and tha a~ino groups can~ot be atSached to the ~ame carbon atom. In the most preferred molecules the total of x, y and z is from 2 to 6 and the amino gro~ps are ~eparated by at least two ~ethylene g~oups .
O~her pre~erred core ~o~ecules include e~hylene diamine and like molecules with longer cha.ns such as propylene diamine and butylene diamine. Su~h molecules may ~e repre3ented by the general for~la:
~ C~2 1cH2)n ~ C~2 NH2 wherein n is an integer ~rom 0 to 10, preferably 0 to 3.
Of course, am~onia can dlso be e~ployed as the core molecule.
~he develop~ent of synthetic vaccines aqainst a lasge nu~ber of dis~ases ha~ recently been ~e~tly accelerated ~ecause of the recognition that a va2cine need not be based on a native protein, ~ut ~ay be ba~ed on a low molecular weight seg~ent of the native protein. These segm~n~s, nor~ally called '. . :, .

WO 90/11778 ~ /L _ oe~J9 ~ ~mmunogenic determinants o~ epito,D~s are capable of stm.. ~lating - ~he production of antibodies which will proteot against infection by sporo20ites bearing the native prote`n antigen and in turn in~rod~ced in the ~ammalian host by the bite of a S mo~quito vector.
~ his invention is concerned with ~alarial-origin T- and 9-cell epitopic peptides such as tho~e described by Romero, et al., L~ i~. whieh is incorporated herein by reference. ~y way of nonlimiting example, some of the ~. berghei ~-cell epitopi~ peptides are:
Desigr.a~ion YNRN~-~NRLLAD

NEKIERNNXLXQP N

NDDSYIPSAEK~ 3 2~9 260 KQIRDSITEEWS ~_4 GS~IRVRRhRGS~ 5 SSI~NIVSNS~G 6 317 32a NERIERNNX~XQPDPPPPNPNDPPPPNPN~ Ntl~.l KQIRDSITEEWSDPPPPNPNDPPPPNPND ~-4+17.1 The las~ t~o antigen N~17.1 and B-4~17.1 represent a combination of T-cell epitopes N or B-4 with a ~-cell epitope 1~.1. ~he epitope 17.1 and it5 preparation are desc:i~ed in Zavala ~t al., J. E~ ~ 6:1591, l9e7, which is incor-porate~ by reference. ~t should be noted that in the oase ofcir~u~5porozoite protein, the P-cell ~pitope (which happ~ns to be the immunodominant epitope) is repetitive in nature, e.g., IDPPPPNPN)X for ~ e~ghe-; tDRAAGQpAG~x or ~DRADGQPAG)X or oo~bina~ions of the two f or P . vivax; ( N~NP ) ~ f or P ~
ip~ , (QAQGDGANA~QP)X for P. k~owle$i, e~. ~herein x is ~t least 2 ~or at least some malarial specie Repeats o~ cycli~
permutations of these mini~um repeating units will also yield WO 90/11778 0 ~ PCT/US~/~2039 .`;-:; B-c~ll opltoplc p~ptld~ .g. ~PNAN)X.
So~e o~ ~h~ antiqenic p~ptide~ ~hlch ~re curr~ntly avallable ei~her co~rcially or by known ~ynthetic or isolation technlques ~re listed in ~ablo 1, belov. The table lists the S peptides Vhich are seg~ents o~ protein~ associated ~ith ~he di~ease or pathogen identi~ied in the ~econd column. Ths referance8 identl~y the publi~atlon~ which dQ w rlb~ the peptld~
. ~ ~nd ~ow to obtain ~hem. The conventional abbrevlation6 ~r~ u~ed '- ~or th~ a~ino acid6.

PEPTIDE SEQUENCES SU~ABLE FOR DEVELOPN~NT
OF VACCINES USING MA~S
Peptide Pathogen/Disease tprotein) Ref A. H-(Asn-Ala-Asn-Pro)n-CH n~3 Malaria, CS protein of ~~
B. ~-(Gly-Asp-Ar~-Ala-Asp-Gly Malaria, CS protein of Gln~Pro-Ala)n-OH n~2 P. vivax 2 C. Glu-Gln-Asn-Val-Glu-His- ~alaria, Pf 155 o~
Asp-Ala P. falciparu~ 3 D. Asn-Ala-Glu-Asn-Lys-Glu-Glu- Nalaria, ~arozoite sur~ace Leu-Thr-Ser-Ser-Asp-Pro-Glu- protein o~
Gly-Gln-~le-~at E. Asn-Ala-A~n-Pro-Asn-Val- Malaria, S protein o~ 5 ~sp-Pro-Asn-Ala-Asn-Pro P. ~alciparu~ -1. Za~ala, et al, Science 228:1436, 19~5 2. McCutc~an, ~t al, Science 230:1381, 1985; Arnot, D.E., et ~1, Science, 230:815 (1985) 3. Udomsangpetch, et al, Science 231:57, 1986 4. ~a~etch, et ~1, Science 227:1593, 19~4 5. Nardin, E.~. et al, Science 246:1603, 1989 In addition, ~alarial ~-helper cell epitopic pep~ides can be identi~ied, as des~ribed above in the references o~
Sinigaglia et ~1 ~tc. Briefly, once the a~ino a~id sequence of a ~alari~l prot~in is known, peptides corresponding ~o ~ragments o~ t~e proteln can ~e ~ynthesized ~nd injected in ~a~mals. ~-cell~
SUBSTIT~IT5E SH!~

W 0 90J1~778 O ~ ? `

~' - can then be harvested from blood samples of the im~unized mammalsand incubate~ in ~itro in the pre~ence of the peptide used for immunization. Such peptid~o are considered T-helper cell epitopic peptides if the T-cells proliferate duri.ng ~uch ln~ubatlon in the presence of such a peptide. To demon~trate whe~her these ~-cell ,. peptide~ are T-helper peptides, they al-e te~ted for elicitation of antibodies to ~ B-cell epitope by covalently linking the T-cell and the B-cell epitopic peptide and usinq the thus for~ed conjugate ~or i~unization.
10In the foregoing description the lette:s have the same maaning as is employed by those skilled ir. the peptide arts.
'rhese are:
A-alanine M-methionine C-cystine N-asparagine 15 D aspartic ,cid P-proline E-glutamic acid Q glutamine F-phenylalanine R-arginine G-slycine S-serine H-histidine 'r-threonine 20 I-isoleucine V-valine R-lysine ~-tryptophan L-leucine Y-~yrosine A particular advantage of this invention is that the dendritic polymer can ~erve as a carrier for t~o or more dif-ferent malasial antigens. This is particularly useful forproducing ~ultivalent vaccines li.e. vaccines directed against ~ore than one ~alarial species) ~nd/or for producing vaccines agains~ different stages of th~ malaria parasite. Vaccines produced from anti~enic products of the inYention in which both T-cell antigens ~nd ~-cell antigens aasociated with ~alaria are joined to the dendritic polymer in any of the v~rious configura-tions illustrated in a non-limiting fashion in Figure 2 are especially useful because they are capable of qeneratlnq extreme-ly high antibody titers.
i5It has been discovered that when ~he ~- and 9-cell epitopes of this invention are covalently bound to MAP sub-~trates, the resulting products will elicit levels o~ an~ibody W O 9S/11778 ~ ' ' PCr/US~/0~3 responqe which are 10 ~o 10~ fold grea~:er than those obtained in the past with recombinant CS protein or irradia~ed sporozoites.
I~ hds bee~ further ob~erved th~t, in ~ice, the ~-T monomeric di-epitope not supported on a MAP ~ubstrate, or ~ mixture of B-epitope MAP and T-epitope HAP produced ~ery low ~ntibody response and no protection. The pre~ently ~osl: preferred embodi~ent of the pre3ent inv~ntion i~ one where both a T ~nd ~ ~ epitopio peptide are linked in tandem on the sa~e ~unctional group of the dendritic polymer substrate~
The specifically selected ~- and T-epitop~s of this inve~tion can be placed cn the MAP substrate in a varlety of different arrangements as shown in Fig. 2. The figure shows alternate arrangement for the,~-epito~e (open blocks~ and the T-epitope ~solid blocks) which for P.__~erqhei include 15 PPPPNPDP~PPNP~:D an~ KQIRDSI~EEwS, respectively.
In Figure 2, T-(4i 2nd ~-~4) are monomeric maps with four branche~ but only one epitope ~gain the immunodominant ~-epitope for the CS protein comprises at least t~o occurrences of the repetitlve unit). T-~8) ~nd B-(8) are similar, but with ~
branches. In T~8)B and ~8)-T, there are a ~ or ~ epitopes on the branches of the dendritic polymer and on~ ~-epitope or T-epitope on the root of the poly~er. ~T-t4), TB-(4), BT-~8) and TB-(8) illustrate presently preferred products of the invention in which ~he epitopes are ~:ranged in tandem.
Naturally, it will be apparer.t to those skilled in the art that many combinations ~nd nu~be:s of ~larial T a~d B-epitopes are contemplated herein and are fully within the scope Oc the present invention.
It i8 ~lgo possible to produce prod~cts of the inven-tion in which the B- and T epitope3 are arranged alter~atively on the branches, i.e., one branch has onl~ B-epitopes, the other oaly T-epitopes. ~or instance, in Fig. 2, T/B(B~ represents an eiqht branch dendritic polymer base with alter~atiDg ~ ~nd ~
malaria antigens, within the scope of the inv~ntion; ~/B(4) is si~ilar cxcept that the poly~er ~ase has only four branches.
Thi~ is acco~pli~hed utiliziAg the orthogoDal protec-tion method by employinq a dendritic poly~er ba~ed OD a di~uno ~, t.~ , tJ, WO N/1l778 ~ PCr/US~/~03g :
- compound such as lysine in which ~he amino groups are blockedwith dlffere~t amino blocking groups, one of which is stable to - acid hydrolysis, t~e other of which is stable to alkallne hyd~olysis. (See, for example, the ~chenatic repre~enta~iOn of Fig. 2, E and F).
Fluorenylmethyloxycarbonyl (Pmoc~ b~3e labile protecting group and is completely stable to acidic deprotection.
~he t-bu~oxycarbonyl blocking group (Boc) i3 stable under muldly acidic conditions such as 50~ trifluoroacetic acid. By choosing ~oc-lys tBoc)-OH, ~oc-lys tF~oc)-O~, Fh~oc-lys ~oc)~OH or Fmoc-lys (hmoc)-OH, il is possible to place o~e set cf antigens on the alpha amino sroup of lysine and another on the omega a~ino group.
Those skilled in the art o~ peptide synthssis can reaaily devise methods of achieving the sa~e types of products using diverse blocking groups and other dendritic polymers.
It will be apparent to thoqe skilled in the art that many variations of the structures shown and discu~sed herein are possible. Fcr exampl~, the dendri~ic pclymer may have a str~c-ture in wkich segr.ents a e joined through a tisulfide bridge.
Such structures can be readily rormed from dendritic polymers in which the root con~ains a protected cystine which is oxidized by a mild cr.idizing agent such as molecula- iodine.
As another example, referring to Fig. l, the glycine at the root of the dendritic polymer, i.~., the free ~lycine could be joined to, or replaced with, a T- or B-malarial peptide antigen which may be the ~ame or different from the other peptide antigens on the branche~ of the dendritic polymer molerule. The T- and B-pep:ide antigens themselves ~ay sesve as the residue to which other lysine or simular molocules may be ~ttached to provide additional hran~hes to which still additional peptide antigens, antlbiotics or non-peptide aDtigens may be attached.
~ he p~oducts of thi~ invention can be employed to produce vaccines useful to protect against malarial infections of ma~m~ls including humans using any of ~he procedures known ~o those ~killed in the ~rt. ~he produc~s can, for exampl~, be su~pended in a phar~aceutically acc~p~a~le medium or diluent, such as inert oil, suit~bly a vegetable oil ~uch as sesa~e, W O ~/1177~ ~ ~ PCT/US9)/Ln~9 pea.qut or olive oil. Alternatively, t~ey c~n b2 suspended in an aqueous i~otonic buffer solution at a p~ of ~bout 5.6 to 7.4.
. Typically, ~uch solutions will be ~lde isotsnic with sodium - chloride ~nd buffered with sodium citr~te-citric ~cid or with 5 phos2hate. The solutions may be thic~e~d ~ith a thickening ~ : - agent such as ~ethyl cellulo~e.
~. Yaccines ~ay also be prepared in e~ulsified form, ; either water in oil or oil in water. Any of ~ wide variety of pharmaceutically acceptable emulsifyiny agents may be e~ployed lncluding, for exa~ple, acacia powder or an alkaryl polyether alcohcl, sulfonate or sulfate such as a Triton.
Stabilizers such as sorbitol or hydrolyze~ gel~tin may also be ad~ed to any of the above described cQmpositions. It is not unusual to incorporate an antibiotic such as neomycin or other anti-infective agents to prevent in~ection.
Beca~se the pro~cts os this inver.tion provide such high antibod~ titer~, in many instances they w!ll be employed withGut carriers or adj;vants. However, if an adjuvant is employed it may be 3electad from any of those normally employed to stimulate the immunogeric syste~ of mammals. These include, for exampl~, Freund's adjuvant (complete or incomplete~, Adjuvant Icontainin~ peanut oil, mannide monooleat2 and aluminum mcnostearateJ, and miner~l gel~ such as aluminum phosphate or alum; killed ~orde~ell~, tetanus toxoid, diphtheria toxoid, mura~yl dipeptide, ~luminum hydroxide, saponin, etc., but as sta~ed above, such ~djuvant~ or carri~rs are not necs~Sary when the polymeric substrate of the present invention is u~ed.
Freunt's adjuvant is no longer us~d in vaccine formulations for humans or ior food animals because it contain~ nonmetabolizable mineral oil and is a potential c~rcinogen. It can be u~ed in vaccines for non-food animals~ Mineral gels are widely used in commercial veterinary vaccines.
She v~ccines of the invention ~ay be defined as comprising a pharmaceutically acceptable carrier, of ~he general nature described above, toge~her with an amoun~ of an an~ige~ic product of thz invention , i.e., a ~elected T- or 9-c~ll epi~ope which is sufficient to produce an immunological response, i.e., a s l ~
W O 90/11778 ~ ~ PCT/USg0;02039 f ~ 16 protective antibody response in a ma~mal An effecti~e amount may be very small. It will, as i5 known, vary with the antigen.
The quantity which constitutes ~n effective ~mount may vary :depending on whether the vaccine is intended a~ a firs~ ~eatment - 5 or as a boo~ter treatment.
,~. `The ~mount of MAP will Yary depending upon the ~pecific ,:immunogeD, the response it elicit~ in various subjec~s, ~nd the presence os absence of h~terologous carri~r or ~djuvant.
Generally, amounts within the range from about 1 ~o about 1,000 micrograms of MhP are contemplated. Optimal amoun~s can be ascertained by routine experim~ntation involving measurement of antibod~ titers and oth~r parameters of mammdlian immune res-ponse, as is weli-known in the a~t. Repeat i~munizations axe preferred.
It may be ccnvenient to provide the products of this ir.vention as lyophil.ized or free2e dried powders ready to be reconstitutet with a pharmaceutica;ly accept~ble carrier just prior to use.
Additional infor~ation on vaccine preparations and 2G protocols is well-known. ~~ for ~xample, European Applica~ion No. ~l 19l,~48 of Smith~line ~eckman published on August 29, 1986; European Patent Application No. Al 192,626 of SmithRline ~eckman et al. p~blished August 27, 1986; U.S. Patent Nos.
4,693,994; 4,707,35,; ~,735,799; and 4,767,622.
All cited patents, patent applications ant licerature are incorporated by r~ferenc~ in their entirety.
Thus, this invention al30 provides a method of provid-ing L~munity in a mammal against infection by a ma~arial organism which comprises administering to the mammal an i~munogenically effective amount of a compound or compo~ition comprising a mal~rial T- and B-peptide-bearing MAP, such an amount being ef f ective to inhibit parasit~mia in a mammalian host pursuant to infection by ~ malarial organism, preferably prior ~o exposure of the mammal to the malarial organi~m.
Al~o contdmplated are vaccines u~eful for inhibiting ~alarial infection by the sporozoite or other stages of ~alaria, co~pri ing ~n effectiYe ~mount of an i~unogenic compound comprising a malarial-origin T- and B-peptide-bearing MAP, and, optionally, a pharmaceutically acceptable carrier or diluent.
It will be apparent to those skilled in the art that the products of this invention, once the concept is understood can be prepared by procedures well known to the skilled artisan.
The Tam procedures described in Proc. Natl. Acad. Sci. USA, 85:5409, 1988, Prosnett et al, J. Biol. Chem., 263:1719, 1988;
and Chenag et al, Proc. Natl, Acad. Sci. USA 86:4929, 1988, all of which are incorporated by reference are illustrative.
A few general observations applicable to the synthesis of MAPS will be of assistance to those skilled in the art. These are:
1. The syntheses generally require a long coupling time (2-4 hours).
2. Dimethyl formamide is generally a more suitable solvent than methylene dichloride.
3. The peptide resin should not be dried at any stage of the synthesis since resolvaton is extremely difficult.
4. Coupling should be closely monitored for completion of the coupling by the quantitative ninhydrin method.
5. The MAPS is best cleaved from the resin by the improved acid deprotection method with either HF or RFMSA (Tam, et al., J.
Am. Chem. Sic., 105:6442, 1983; and J. Am. Chem. Soc., 108:5242, 1986) in deimethyl sulfide to avoid strong acid catalyzed side reactions.

6. MAPS tend to strongly aggregate after cleavage from the resing support. Purification is best effected by extensive dialysis under basic and strongly denaturing conditions in a dialysis medium whichis 8M in urea and mercaptoethanol to remove undesirable aromatic additives of the cleavage reactions such as p-creosol and thiocresol. Further purficiation, if desire, can be effected using high performance gel-permeation or ion exchange chromatography. In most cases the MAPS could be used directly without further purification.
Table I summarizes the results of several tests con-ducted to determine the efficacy of the products of this inven-tion for eliciting an immunogenic response in mice. It will be h i ~. ~ ` C; ~

WO 9~/ll778 ~ ~CT/US~/0~039 ob~erved th~t the MAP ba~ed product~l of thiq invention have uniformly high antibody titers co~pared to irradiated sporozoite, recomDinant CS protein or mono~er ~T peptide. It will be observed also that the response v~ries with the ~tructure of the BT immunogens.
Table I. Comparison of antibody titers induc~d by different immunogens of P~_ ~grghçi and ~ sayed with the recom-binant CS protein and sporo20ites.

. ' 10 Antibody Response ImmunogenIFA titers RIA titers Sporozoite rCS protein sporo_oite~ 2,048 8,192 recombinant CS proteinb 2,048 2,048 monomer BT peptideC 800 1,024 ~T_~p(4)c 128,0C0 408,000 TB-MAP(4) 32,000 400,000 ~T-MAP(8) 2~,000 100,000 T~-MAP ( ~ ) 64, 000 4 00,000 a. Four mice of the ~-2a halotype ~lO,A strainl were injected intravenously two doses of lx105 irradiated p. berqhe~
sporozoites at two-week intervals. Sera were collected and pooled ten days after the last injection. Anti~ody titers expressed as the reciprocal of the highest posltive serum solution were obtained by UQiAg glutaraldehyde-fixed P. ~erghe~
sporozoites in ~n indirect im~unof1uorescense assay (IFA) or the reco~binan~ CS protein in a radioLmmunoa~say (RIA).
b. Four mice of the R-2a halotype ~A/J straiD) were injected i.p. with 25 ug of the recombinant CS ~rCYS) P. berghei protein, emulsified in CFA on day 0, and S.C. with 25 ug of the rCS protein in I~A on day 15. Sera wese collected ten days later.
c. Five mice of the ~-2a halotype l~/J strain~ were injected i.p. with each 50 ~icrograms of the peptide i~unogens consisting of two occurrences of the repeating unit of P- berqhel ~ WO 90/11778 ~ ~ PCT/USgOl02039 CS protein i~m~nodo~inant reqion and one w curreeee of a E
. berqh~i CS protein-derived T-cell epitop~ peptide. ~he immuniza-; ~ion schedule and the aqQay method~ wer~e ~imilar to thoqe for the : recombinant CS protein.
Upon chailenge o~ the thus i~unized ~ice with 2000 ~porozoites e~ch, the ~T-MAP~4) produced complete protection (i.e., prevented p~r~site~ia~ in ~O~ of the mic~; TB-MAP(4) . protected 60~ of the miee; BT MAP18) protected 50% of the mice;
and T~-MAP~8) protected 60~ of the ~ice.
MhPs according to the preRent invention may be syn-thesized as follows:
Some of the followins ~bbreviations are used in the -' synthetic examples, below:
~oc - t-butoxycarbonyl TFA - trifluoracetic acid DMF - dimethylformamide DCC - ticyclohexylcarbodiimide Tos - tosyl Bzl - benzyl Dnp - dinitrophenyl 2ClZ - 2-chlorocarbobenzoxy DIEA - diisopropoylethylamine TFMSA - trifluormethyl~ulfonic acid BSA - bovine ~eru~ alb~in ~PLC - high performance liquid ehromatography-~BR - tu~or bearing rabbit ATP - ~denosine triphosph~te Dnp - dinitrophenyl.
ClZ - chlorobenzyloxycarbonyl BrZ - ~romobenzyloxycarbonyl ELISA - enzyme linked immunoabsorbent assay ~amLL~
General Methods for the Synthesi~ of Multiple-Antigen Peptides The synthesis of an octabranched ~atrix core wi~h peptide ~igen ~as carried out manually be 3 s~epwise solid-phase procedure ~errifield, R.~. J. Am. Chem. Soc. ~1963) ~, S .

~ W0 90/11778 ~ ~ PCi/US90/02139 .... ,~.......................................................... .
2149j on Boc-bet~-Ala-OCH2-Pam resin wit~ a typical scale of O.S
g of resin (O.05 mmol and a resin substitution levei of 0.1 mmol/g for the present ~ynthesis but was 30mewhat lcwer when a higher branching of oore lysinlyl matrix was used). After the remov~l of the ~oc-group by S0~ TFA and neu~ralization of the , re-~ulting ~alt by DIEA, the ~ynt~esis of the fir t l.evel of the _ carri~r-core was achieved u3ing 4 ~o1ar exceq~ of preformed symmetrical anhydride of ~oc-Lys t~oc~ (O.2 ~ol) in DMF and was then recoupled ~ia DCC alone in C~2Cl2. The Qeoond a~d thi~d level ~ere ~ynthesized by the same protocol with 0.4 and 0.8 mmol r~spectively of preactiva~ed Boc-Lys (~oc) to give the oc-tabranching ~oc-Lys(Boc)-core ma rix. ~owever, all subsequen~
couplings of the peptide-a~tigen sequence require 1.6 m~ol of preactivated amino acids. The protecting groups for the sy~-thesis of the peptide antiqens were as follows: ~oc sroup ~orthe alpha-amino terminus and benzyl alcohol derivatives or most side chains of trifunct~onal amino acids i.e., Arg~Tos~, Asp(O~zl), Glu(0Uzl), ~is~Dnp), Lys(2ClZ), Ser(~21), Thr(Bzll, and Tyr(8rZ). Because of the geometric increase in weight gain and volume, a new volume ratiQ of 30 ml of solvent per g of resin was used. Deprotectio~ by TFA (20 min) was pre~eded by two TFA
prewa~hes for 2 ~in each. Neutralization by DIEA was in CH2Cl2 (5~ DIEA~ and there was an additional neutral~zation of DMF ~2%
DIEA~. ~or all residues ~xcept Arg, Asn, Gln, and Gly, the first coupling was done with the pr~formed symmetric anhydride in C92C12 and a secont coupling was perfor~ed ih DMF; each coupling was for 2 h. The coupling o~ Boc-Asn ~nd ~oc-Gly were mediated by the preformed l-hydroxybenzotriazole ester in DMF. Boc-Gly and ~oc-Arg were coupled with DC- alone to avoid the risk of formation of dipeptide ~nd lactam for~ation, respectively. ~ll couplings were monitored by a quantitative ninhydrin test ~Sarin, V.K., et al ~al. Biochem. 1193l) 117, 1471 after esch cycle, and if needed, ~ third ~oupling of symmetrical anhydride n DMF ~t 50- fcr 2 h was used tTam, J.P0 (1985) In ~Proo. Am. Pept.
35 Sympo., 9th~ ~C.M. Deber, R.D. Kopple a~d V.J.~. The syn~hesis ~as ter~inated with acetylation in ac~tic anhydride/DMF ~3 ~ol~
containing 0.3 ~mol of N,N-dimethylpyridine.

~ j . s, _ __ L~, W O 9~f~77~ ~ ~ PCT/~90/02039 After completion of the MAPS, protected peptide-resin 0.39) was treat~d with 1 M thioph~nol in DMF for a h (3 ti~e~
and at 50-C if neces~ry to complet~ the reAction) to re~ove the Nim-dinitrophenyl protecting group of ai~ (~hen pre3ent), with 50~ ~FA/C~2C12 (10 mll for 5 min to r~ove the X -Boc group, and with the lo~/high-BF ~.ethod [T~m, J.P., ~eath, W.F. ~ Merri~ield, ~.~. J. Am. Chem. Soc. (1983) lQ~, 64421 or th~ low-high TFMSA
method lT~m, J.P. Heath, W.F. ~ Merrifi~ld, R.B. J. ~m. Chem.
Soc. (1986) lQ~, 5242~ of cleavage to give the cr~de MAPS. ~he crude peptide was then washed with cold ether ~ercaptoeth~nol (99 1, v/v, 30 ml) to remove p-thio~resol and p-cresol and extract~d into 100 ml of 8 M urea, 0.2M dithio~hreitol in 0.1 H
Tris buffer, pH 8Ø ~o remove all the remainin~ aromatic byproducts s~nerated in the cleavage step, the peptide in the dialysis tubing (Spectra Por 6,M.W. cutoff 1,000) was equi-librated in a deaerate~ and N2-purged solution containing 8 M
urea, 0.1 M NH4HC03 INH4)2C03, pH 8.0 with 0.1 M mercap~oethanol at O-C for 24 n. The dialysis was then co~ti~uet in 8M, and then in 2M urea, all in 0.1 M N~4Nc03-lNH4~co3 buffer, ph 8.0 ror 12 h and then sequentially in ~2 and 1 M HOAc to remove all ~he urea. Tha lyophili2ed ~APS was then purified ~atchwise by high performance gel-permeation or ion-exohange chro~atography. All of the purifled materi~l gave a satis~actory amino ~cid analysis.

Example ?
- Synthesis and Purification of (Asn-~la-Asn-Pro)g-MAP (NP-16 MAP), a Peptide Derived from the Sporozoite Stage of Pla ~odium ~21siparum.
~he peptide, tAsn-Ala-Asn-Pso)g-Lys~-Lys2-Lys-O~ w~s synthesized by the general proceduro described in ~xample 1.
The synthesis was initiated with Box-Lys(Boc)-~CH2-Pam-resin ta copoly(styre~e-1~-divinylben2ene resin) ~t a substitu tion of 0.11 Kmol/g of resiD. Tha substitution was found to be 0.88 mmol/g after the sequential addition of three levels of Boc-Lys~Boc) to give an octabranching 3tructure of IBos-Lys(~oc)4]
LyslBoc)2-LyclBoc)-OC~2-Pam resin. ~he syn~hesis continued with 2.5 g o~ resin in a modified Beck~ann 990 synthesizer IBeckman ' J ~
~-v ,s~ J :, W O g0~l77Y ~ ~ PCT/U590/0e~39 Instructions, Palo Alto, California). Synthesis w~s pesformed using a computer prosram th~t optimized all of the coupling st~ps. For example, the c~upling o~ aoc-Ala ~nd E~oc-~ro were mediated by the synunetric anhydride ~ethod in a ~olvent ratio of S C~2C12;dimethylfor~amide (1:3, v/v) tc1 ~iniDIi2e ~ggregation and incomplete couplinq. ~he coupling of ~ A~n wa~ by the per-f or~ed l-hydroxyben~otriazole ~ctive ~4ter in the sa~e solvent .
Each a~ino acid underwent a double coupling protocol to ~aximize the coupling yield ~nd essentially b~ing the reaction to >99 . 6 10 completion.
The protected pe?tide-rPsin was deprotected in por-tions . Tne initial deprotection ~as carried out with 1.5/ g of crie~ peptide-resin in a reaction vessel and underwent the following proced~lre to remove the Boc-?rotecting group and othe~
15 ~xtraneous Inaterials: CN2C12 (3 x 1 min wash); CF3C02H-CH2C12 ( 1:1, 3 x 2 min ) and CF3C02H t 3 x 2 min wash ) and then cleavage reaction cont~ining the following deprotecting reagents:
trif luoromethanesulfonic acid:trifluoroacetic aeid:tetrahydrothiophene: m-cresol t4:20:12:4, in ~1~ at 4C for 20 3 . 5 h. The peptide released by the acidolytis eleavage of ~he sulfide-assisted ~leavage proceduxs was collected and preci~itated by ethyl ether t230 Dll~ prechilled to -30-C. The precipitate was cer~trifuged to a pelle~ and the ethyl ether was removed .~Q. The peptide was then dissol~ed in O.OlM ~ c 25 and dialyzed in 12 liters of 0. 01M HOAc. The peptide was then lyophilized to dryness to obtain 60 mg of tAsn-Ala-A~n-Pro)~O~AP.
Hydrolysis of the resul~ing resin after cleavaqe showed ~hat abcut 90~ of the peptide had boen cleaved from the resin suppost.
~hc low yield w~s due to inoomplete preeipitation of the peptide by the ether. The sa~e peptide-re~in (l.Og) was al~o ~leaved by ~:ani~ole (9:1, vtv total 10~1) at 0-C for 1 h to give 220 ~9 of MAP after extensive extr~ction with 10 to 100% ~oacc ~nd a crude yield of 33~. The dialysis was carriet out with 10~ O~Ac.
The peptide after dialysi~ was then analyzed firs~ by a~ino cid ~nalysi~ (after hydrolysis by 6N HC1~. ~he molar ratio o~ the MAP ~ound was Asn:Ala;Pro;Lys: 1.97 ~2): 1.03 (1):1 t1~:0.26t0.22) which was i~ dgree~ent with th~se expected s;~ J - _ W0 90/11778 ~ ~CT/US9C/~2039 ;'theoretical value~ ~hown in p~renthe3i~.
.
, E ~t aJnp 1~3 : `General Methods ~or the Synthe~is of Di-epitope ; 5 Multiple Antigen Peptides Containing Malarial-Provena~ce T-cell ' ~ and B-ccll ~ntigens. ?
~. ;(~) ~ethod A. Linking Two epitope~ in T~ndem.
The 3ynthesis of di-epitop~ MAPS was ~cco~pli3hed :manually by a ~tepwi~e ~olid-pha~e procedure o~ ~oc-Al~-OCH2-Pa~
resin (O.l ~mol of Ala s pre~ent in 1 g of resin) similar to tho~e mono-epitope MAPS described in the previous examples.
After the removal of the Boc group by 50~ ~F~ and neutxalization of the resuiting salt b~ DIEA, the synthesis of the first level of the carrier core to form Boc-Lys(~oc)-Ala-ocH2-pam resin was ac~.ievet using a 4 mole excess of ~oc-Lys(3oc) via DCC alone in CH2Cl2. The second ~nd third level were synthesi2ed by the saMe protocol, to give the octabranching ~oc-Lys~oo1 core matrix.
From this point onwart, the syntbe~i~ of peptide ~ntigens or two epitcpes proceeded ~s those of the previous examples using the 2G tertbutox~carbonyl/benzyl protecting group strategy since they were arrangad in tandem and ~re treated as if they ~re one sntigen. Spacera such as tetra-peptide Gly-Pro-Pro-Gly ~re sometimes inserted ketween two peptide antigens to a~low fl~xibility. After comFletion of the ~ynthe3is, the MAP-re5in was treated with TFA to remove the N -~oc groups, then aoetylated with lO~ acetic anhydride/lO~ DIEA in Ch2Cl2, and finally cleaved wi~h the low-high ~F ~ethod to remove th~ MAP from the resin support. The ~rude peptide was then washed with cold ether/mer-captoethanol ~99:l vol/vol) to remo~e p-thiocresol and p-cresol, and extracted into 8 H urea in O.l M Tris.BCl huffer (pB 8.0).
To re~ove the re~aining ~romatic by-product~ qenerated in the cleavage tep, MAPs were dialyzed (Spectra ~or 6, ~olecular weight cut off l,000) in 8 M urea and th~n in O.l M acetic acid twice for 5-6 hours to remove the ure~. ~he HAPs were lyophil-i2ed fro~ ~ three ti~es to remov~ ac~tic acid.
(b) Method B. Linking ~wo or Mose Epi~ope~ by AlternatingBranching of the A~ino groups of Lysines ': :

.

~ ~ ! t ~
WO S0/1~778 P~T/US90/02039 Because there are t~o ~mino groups in lysine and because these two a~ino groups could ~e protected selectively~
the core mat-ix could be synthesized in ~uch a way to produce that the N -NH2 group is protected with ~he acid-labile aOc group ~nd the N -NE~ group i~ prote~ted with the b~e-labile Fmoc (fluorenylmethoxy~arbonyl) group, or vi~ ver~a, i.e. N -N~2 group is protected by the F~oc group, ~nd the N -N~2 gxoup is pr~tected by the Boe group. To achie~e the ~ynthe~i~ of thi~
core ~atrix using this selectivity, ~ core ~atrix containing N--NH2-Boc and N -NH2-Fmoc ls ill~strated. The synthesis of the core matrix was similar to those describ~d in the previous examples using the Boc-Lys(~ocl for the br~nching ~o~ the first and second level. At tl:e third level, Fmoc-Lys~Boc) was used for the Lys branching o' the core to give for each Lys~Boc) and P~oc-Lys end groups. The s}~nthes,s oL Ihe firs~ epitope (or two epitopes in tandem) us~d the Boctbenzyl chemistry as described in the previous examples, but during this synthesis, neutraliz~tion time was reduced to 1 mir. to minimize the premature cleavage of the Fmoc group. ~he s~nthesis of the second epitope used ~he Fmoc/tertbutyl chemistry (i.e. the N ~ 2 group is protected wi~h Fmoc and the side chain is protected with tertbutyl aloohol derived protecting groups) and started after the completion of the firs~ epitope using the Boc-amino acid chain was assembled.
The F~oc-a~ino ~cids were used with the side chain protecting groups for the trifunctional a~ino acids as follows: GluloBut3~
Asp~O~ut), Lys(Boc) Thr(~ut), Ser(But), TyrlHut), Arg(Pmz), ~is~Trt), Trp~Forl, and Cys(~ut). Repetitive deprotection o~ N-Fmoc was by 20~ piperidine in dimethylformamide and was preceded by one piperidin~ prewash ~nd the coupling wa~ mediated with DCC:FOBut in DHF. After completion of synthesis, the MAP res.in was treated with low-high ~ to remo~e the peptide chains fro~
the resin. The workup and purification was essentially the same as those described in the pre~iou~ ~xamples. The proceture for asse~bli~g the peptide ehain usin~ the F~oc.tertbutyl ~hemistry was as follows: tl) 20 ~il DMF ~3 x 1 mlnl; t2) 20 ~1 piperidine/DMF ~1:1 vol~vol~)l ~in); (3) 20 ~1 piperidinetDMF
(1:1 vol/vol) (10 ~in); 14) 20 ml DMF l3 x 1 ~in); ~5) 20 ~1 f~, `~.. ~ '~ si W O 90/11778 ~ r~us90/0~-9 ~: CH2C12(3 x 1 min); (6) 20 ml DMF (2 x 1 min); ~7) amino acid (4 eguiv) in DI~F S ml (5 min), HOBt(4 equiv) in DMF, DCC(4 equiv) in CH2C12 were added for 2 h; (8) 20 ml DMF (4 x 2 ~un); (9) 20 ~1 CH2C12(2 x 2 ~in).
(c) Hetr.od r, Linking Two or Hore Epitope~ via Di~ulfide ~inkage of Two Pre~ormed Heterologou~ MAPS.
To link two or ~ore cpitopcDs together via di~ulfide linkage of two preformed MAPS, a dipeptide fiuch as Cy~(Acm)-Ala - is added ~t the carboxy terminus of the prefor~ed MAPS ~s desoribed in Example 3a or 3b. This ~ould be achieved con-venie~tly before the start of the synthesis of the core matrix by adding ~oc-Cys(Acm) to the Boc-Ala-GCH2-Pam-resin. Afte_ the formation of the dipeptide 9oc-Cys(Ac~)-Ala-OCh_Pam-resin, the synlhesis of the ~ore matrix, the incorporation of or.e or more peptide antigen~s~ us_ng the procedures deseribed above proceeded to give the preformed ~PS containing a Cys~Acm)-Ala dipeptide COOH-tail. The Cys~Acm) is stable to the HF deprotection method.
The preformed M~P5 cortaining the COO~ Cys~Acm)-Ala dipeptide tail were purified. The dimeri2ation of two heterologous preformed ~APs was achieved by oxidation with I2 to the disul-fide, ~nd whioh al~o concomutantly remoYe the Ac~-group from the cysteinyl residue. A detailed procedure Wa5 as follows. To 1 ~mol of MAP, the heterologous preormed di-epitope HAPs contain-ing Cys(Acmj ~as dissolved in a de-aersted and N2-purified 50 ace~ic acid solution at roo~ temperature, 50 ml of a ~olution of I2 in ~eO~ll M solu~ion) was Ddded batchwise ~or 1 hour at O-C.
The redction was quenched by adding 1 ~ aqueou3 sodium thiosul-fate ~or a3corbic aeidJ until the yellow color was remo~ed. MeOH
was re~ed by dialysis in 0.1 ~cetic acid and tbe desired MAPs were purified by gel permeation ch~omatography, ion-exchange chro~atogrAphy or se~erse-ph~se high pres~ure liquid chro~atog-raphy.

Claims (8)

WHAT IS CLAIMED IS:

1. An antigenic product comprising a dendritic polymer having functional groups to which a plurality of both T-cell and B-cell epitopic peptide molecules selected from the group consisting of malarial B-cell and T-cell epitopic peptides are attached.

2. The product of claim 1 wherein at least one T-cell and B-cell epitopic peptide are attached in tandem to the same functional group.

3. The product of claim 1 wherein said T-cell and B-cell epitopic peptides comprise T- and B-cell epitopic peptides derived from the circumsporozoite protein of at least one species of malaria selected from the group consisting of P. berghei, P.
knowlesi, P. yoeli, P. malariae, P. ovale, P. falciparum, and P.
vivax.

4. The product of claim 3 wherein said B-cell epitopic peptides comprise amino acid sequences selected from the group consisting of (a) (NANP)x (b) (DRAZGQPAG)x wherein Z is independently selected from A or D;
(c) (QAQGDGANAGQP)x (d) (DPPPPNPN)x (e) (YAAA(A)nGGG(G)mN)x wherein Y is D or G indepen-dently; and n = 0 or 1; and m = 0 or 1 independently;
(f) combinations of the foregoing;
(g) peptides consisting of cyclic permutations of each of the repeating units (a) through (e);

wherein x is an integer of at least 1; and the T-cell epitope is one or more T-cell epitopes derived from the CS protein of the same malarial species as the B-cell epitope.

5. The product of claim 4 wherein a T-cell epitopic peptide is appended directly to a functional group of the dendritic polymer and the B-cell epitopic peptide derived from the same malarial species is appended to the other end of the T-cell peptide, optionally via a linker.

6. The product of claim 4, wherein more than one T-cell epitopic peptide derived from the same malarial species is included along with at least one B-cell epitopic peptide derived from said species.

7. A vaccine against malaria comprising an immunogenic effective amount of the product of any one of claims 1-6.

8. A method for providing immunity against malaria in a mammal in need of such treatment comprising administering to said mammal an immunogenically effective amount of the product of anyone of claims 1-6.