WO1990011778A1 - 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

"DENDRITIC POLYMER OF MULTIPLE ANTIGEN PEPTIDE SYSTEM USEF AS ANTI-MALARIAL VACCINE"

Vaccines often comprise an antigen on a natural carrie such as a protein, a carbohydrate, a lipid or a liposome. Suc vaccines are useful and have been employed for many years. There are however a number of art recognized problems wit them. Several of these problems are related to the carrier. Since the carriers are isolated from natural sources, they ar often not of uniform quality. Additionally, despite expensiv and arduous purification efforts, it is difficult, and ofte impossible, to provide products completely free of natura contaminants. Such contaminants may themselves be antigenic. They cause the undesirable side reactions often associated wit the use of vaccines, particularly fevers and tissue swelling Additionally, the concentration of antigen may vary from on batch to another because the amounts of antigen which reac with the carrier or are absorbed on its surface are no uniform. This problem has markedly increased the difficultie of preparing suitable vaccines for protection against malaria.

Malaria is a particularly important target for syn thetic vaccines, since it affects 200 million people worldwid and no immunoprophylaxis has yet been developed. It is know that protective immunity against rodent, simian and huma malaria sporozoites can be induced by immunization wit irradiated sporozoites. The major protein of the sporozoite i the circumsporozoite (CS) protein, and antibodies directe against the CS protein are known to neutralize the infectivit of parasites and inhibit their entry into the hepatocytes Thus, the CS protein has become an important target for the development of synthetic vaccines against the sporozoite stage of malaria. The im unodominant B-cell epitopes of the CS protein is contained within the repeating domain of the CS protein, a feature common to CS proteins of all malaria species. Mice immunized with a synthetic peptide, attached to tetanus toxoid as a protein carrier of this B-cell epitope, have been found to develop high antibody titers and resistance to challenge with 10^ sporozoites. However, vaccination attempts in humans, using a similar approach, have failed to induce good antibody titers.

Recently, several T-helper cell epitopes of the CS protein of P. berσhei (a rodent malaria) have also been identified (see Romero et al., Eur. J. Immunol. 1.8:1951, 1988). The identification of the B and T helper cell epitopes of the CS protein of P. berσhei has now made it possible to incor¬ porate these epitopes into one molecule in a specific and unambiguous manner using the MAP approach in which the epitopes are attached to a defined dendritic polymer, using the procedure developed by Tarn and his coworkers as described in J. Biol. Chem. 263, 1719 (1988). In addition, T-cell epitopes of other malarial species have been identified: See, e.g. , Sinigaglia, F. et al, Nature 336:778, 1988 (P. falciparum) ; Crisanti, A. et al., Science, 240:1324, 1988 (P. falciparum, blood stage); Kumar, S. et al.. Nature 334:258, 1988 (P. falciparum sporozoites) etc: Good, M.S. et al, Science 23_5:1059-1062, 1987; Good, M.S., et al, Proc. Nat'l. Acad. Sci. 85_.:1199-1203, 1988; Sinigaglia, F. , et al. , Eur. J. Immunol. 18:633-636, 1988; and Guttinger, M. , et al., EMBO J. 2:2555- 2557, 1988.

Dendritic polymers are a new class of polymers. They are characterized by higher concentrations of functional groups per unit of molecular volume than ordinary polymers. General¬ ly, they are based upon two or more identical branches origin- ating from a core molecule having at least two functional groups. Such polymers have been described by Denkewalter et al. in U.S. Patent No. 4,289,872 and by Tomalia et al. in several U.S. Patents including Nos. 4,599,400 and 4,507,466. Other polymers of the class have been described by Erickson in U.S. Patent 4,515,920. The polymers are often referred to as dendritic polymers because their structure may be symbolized as a tree with a core trunk and several branches. Unlike a tree, however, the branches in dendritic polymers are all substan¬ tially identical.

The products of this invention are based on such dendritic systems in which antigens are covalently bound to the branches which radiate from the core molecule. The system has been termed the multiple antigen peptide system and is some¬ times referred to herein as MAPS. As will be apparent from the discussion hereinafter, some of the carrier or core molecules used to form the products of the invention are of a molecular weight such that they might not usually be regarded as poly¬ mers. However, since their basic structure is similar to dendritic polymers, it is convenient to describe them as such. Therefore, the term "dendritic polymer" will be sometimes used herein to define the polymeric substrates of the products of the invention. The term includes carrier molecules which are sufficiently large to be regarded as polymers as well as those which may contain as few as three monomers.

It has now been discovered that dendritic polymers can function usefully as carriers for a wide variety of antigens. This invention will be better understood from a brief discussion of the structure of dendritic polymers.

Dendritic polymers are built upon a core molecule which is at lease difunctional. Each of the functional groups on the core molecule form at least two branches, the principal units of which are also at least difunctional. Each difunctional unit in a branch provides a base for added growth.

The system can be better visualized by reference t specific molecules. If, for example, lysine with two amin groups is joined in a peptide bond through its carboxyl grou to the amino group of alanine or glycine which may in turn be bound to a resin, the resulting molecule will have two fre amino groups. This dipeptide may be regarded as the firs generation. It may be joined to two additional lysine mole¬ cules by the formation of peptide bonds to produce a second generation molecule with four free amino groups. The process can be repeated to form third, fourth or even higher genera- tions of products. With each generation the number of free amino groups increases geometrically and can be represented by 2ⁿ, where n is the number of the generation.

Although none of these compounds are of particularly high molecular weight, it is convenient to refer to them as dendritic polymers.

Fig. 1 shows a three generation dendritic polymer core molecule based on lysine in which each of the eight available amino groups are joined to a peptide antigen through a glycine linker molecule. The same types of reactions can be carried out with aspartic or glutamic acid, both of which have two carboxyl groups and one amino group to produce polyaspartic or poly- glutamic acids with 2ⁿ free carboxyl groups.

The necessary chemistry for performing these types of synthesis "is known and available. With amino acids the chemistry for blocking functional groups which should not react and them removing the blocking groups when it is desired that the functional groups should react has been described in detail in numerous patents and articles in the technical literature. The dendritic polymers can be produced on a resin as in the well-known Merrifield synthesis and then removed from the polymer.

Tomalia utilized ammonia or ethylenediamine as the core molecule. In this procedure, the core molecule is reacted with an aerylate ester by Michael addition and the ester groups removed by hydrolysis. The resulting first generation mole¬ cules contain three free carboxyl groups in the case of ammonia and four free carboxyl groups when ethylenediamine is employed. Tommalia extends the dendritic polymer with ethylenediamine followed by another acrylic ester monomer, and repeats the sequence until the desired molecular weight is attained. It will, however, be readily apparent to one skilled in the art, that each branch of the dendritic polymer can be lengthened by any of a number of selected procedures. For example, each branch can be extended by multiple reactions with lysine molecules. Erickson utilized the classic Merrifield technique in which a polypeptide of substantially any desired molecula weight is grown from a solid resin support. As the technique is utilized for the preparation of dendritic polymers, the linking molecule which joins the polymer to the resin support is trifunctional. One of the functional groups is involved i the linkage to the resin, the other two functional groups serv as the starting point for the growth of the polymer. Th polymer is removed from the resin when the desired molecula weight has been obtained. One standard cleavage procedure i treatment with liquid hydrogen fluoride at 0°C for one hour.

Another, and more satisfactory procedure, is to utilize complex of hydrogen fluoride and dimethylsulfide (HF:DMF) a described by Tarn et al. in J. Am. Soc. (1983) 105: 6442. Thi procedure greatly minimizes side reactions and loss of peptide. Denkewalter, in one example of his process, utilize lysine as the core molecule. The amino groups of the cor molecule are blocked by conversion to urethane groups. Th carboxyl group is blocked by reaction with benzhydrylamine

Hydrolysis of the urethane groups generates a benzhydrylamid of lysine with two free amino groups which serve as th starting points for the growth of the dendritic polymer.

This brief outline of three of the available procedure for producing dendritic polymers should be adequate to teac those skilled in the art the basis principles of the curren technology. They will also teach the skilled artisan th salient features of the polymers, one of the most important o which is that the polymers provide a large number of availabl functional groups in a small molecular volume. The result i that a high concentration of antigens in a small volume can b achieved by joining the antigen to those available functiona groups. Moreover, the resulting molecular product contains high proportion of antigen on a relatively small carrier. Thi is in contrast to conventional products used as a basis for vaccines. These conventional products often are composed of a small amount of antigen on a large amount of carrier.

Other important features of the dendritic polymer as an antigen carrier are that the exact structure is known; there are no contaminants which may be themselves antigenic, produce tissue irritation or other undesirable reactions; the exact concentration of the antigen is known; the antigen is symmetri¬ cally distributed on the carrier; and the carrier can be utilized as a base for more than one antigen so that ulti- valent vaccines can be produced. The principal advantage of the MAPS technique as the basis for malarial vaccines of this invention is that unlike previous systems using natural carriers such as keyhole limpet hemocyanin, tetanus toxoid and bovine serum albumin, the carriers of this invention are fully defined chemical entities on which the antigens are dispersed in known concentrations. Additionally the antigen comprises a large part of the molecule not a relatively small and undefined proportion of the molecule as in the case of natural carriers. For the vaccines of this invention, it is preferred that the core molecule be a naturally occurring amino acid such as lysine so that it can be dealt with by the body following the usual metabolic pathways. However, as will be explained more fully hereinafter, amino acids which are not naturally occurring, ^'"even those which are not alpha-amino acids can be employed. The acids, or any other asymmetric molecules used in building the core molecule can be in either the D or L form.

Although the dendritic polymers have been principally described hereinabove as polyamide polymers, it will be readily apparent that the carriers of this invention are not limited to dendritic polyamides. Any of a wide variety of molecules having at least two available functional groups can serve as core molecules. Propylene glycol, for example, can serve as the basis for a polyester dendritic polymer. Succinic acid with selected glycols or amines can serve as a core molecule to generate polyesters or polyamides. Diisocyanates can be used to generate polyurethanes. The important point is that the core molecule has at least two available functional groups fro which identical branches can be generated by sequentia scaffolding-type reactions with additional molecules als having at least two available functional or anchoring groups o each branch. In the most simple case in which the cor molecule has two available functional groups and each succeed ing generation has two available functional groups, the numbe of anchoring sites to which malarial-origin T-cell and B-cel antigens employed in this invention can be anchored is ex pressed by (2)ⁿ where n is the number of the generation.

For a more complete discussion of the chemistry o dendritic polymers attention is directed to Ta alia et al.

Polymer Journal 17 (1), 117 (1985), Akaroni et al, Mar comolecules 15, 1093 (1982), and the following United State Patents:

4,289,872 4,558,120

4,376,861 4,568,737

4,507,466 4,587,329

4,515,920 4,599,400 4,517,122 4,600,535

All cited patents, patent applications and reference are incorporated by reference in their entirety.

THE INVENTION This invention in its presently preferred embodiment provides a multiple antigen peptide system comprising dendritic polymer base with a plurality of anchoring site covalently bound to antigenic T-cell and B-cell epitopes o malarial proteins such as the CS protein such that the result ing construct bears both T and B epitopic peptides. T polymers comprise a central core molecule having at least t functional groups to which molecular branches having termin functional groups are covalently bound. The terminal functio al groups on the branches are covalently bonded to the epitop peptides. The antigenic molecules are principally describ herein as peptide antigens, but they are not limited to pepti antigens or even to antigens. Thus, peptides that are n antigenic by themselves may be rendered antigenic when bound the core molecule.

The selected antigen may be separately synthesized (by synthetic methods, including but not limited to recombinant DNA techniques, as is now well-known in the art) or otherwise obtained and joined to the carrier. Preferably, the antigen may be synthesized on the carrier by extending each branch of the polymer utilizing known peptide synthesis techniques.

Fig. 1 shows the structure of a dendritic polymer which may be employed in the practice of this invention. As will be seen, it is a three generation dendritic polylysine product. It may be produced by a conventional solid phase techniques by generating the polymer on a Pair, or a Pop resin. See Mitchell et al., J. Org. Chem. (1978) ϋ, 2845 and Tarn et al., J. Am. Chem. Soc, (1980) 102 6117. The polymer is then cleaved from the resin using, preferably HF:DMS. The dendritic polylysine, as shown, was built from a glycine linker originally joined through a benzyl linker to the resin. Other linkers such as alanine can be employed. Of course, the linker can be omitted, or a plurality of linker molecules can be utilized. Fig. 1 shows a dendritic polymer each molecule of which carries eight peptides some of which represent T-cell epitopic peptides and others B-cell epitopic peptides of a Plasmodium species responsible for malaria, e.g., Plasmodium berqhei, Plasmodium falciparum or Plasmodium vivax. P. yoelii. P. malariae, P. ovale, P. cynomolgi, P. knowlesi: etc. joined directly to each of the available functional groups on each terminal lysine moiety. It is preferable that the B- and T- epitopes o& the polymer are of the same malarial species. The the present invention is not limited to polymers bearing only one T- and B-epitope combination from a single species. For example, MAPS bearing simultaneously T- and B-epitopes from P.vivax CS protein and T- and B-epitopes from P.falciparum CS protein are within the scope of the invention. In addition, the ability of a peptide to function as a T-helper epitope is not necessarily dependent upon the copresence of a B-cell epitope from the same malarial species. Hence, cross-species combinations of T-helper and B-cell epitopic peptides are also contemplated. When the selected epitopic structures ar relatively short, e.g. 6 to 14 residues, it has been observe that it is best to extend the polylysine by a linker such as simple tri- or tetrapeptide of glycine, alanine or beta alanine. However, for antigenic peptides with more than 1 residues, the linker is normally unnecessary.

This invention has been described for convenience principally as applied to products built on lysine as the cor molecule. In fact lysine and lysine like molecules such a ornithine, nor-lysine and beta-amino alanine are preferre molecules for building the products of this invention becaus they are relatively easy to obtain, they are easy to work wit and they afford good yields.

Such molecules can be represented by the genera formula:

(CH₂)_y NH₂ H₂N - (CH₂)_X - C - (CH₂)₂ COOH H wherein x, y and z are integers from 0 to 10, preferably 0 to provided that at least one of them is 1 and the amino group cannot be attached to the same carbon atom. In the mos preferred molecules the total of x, y and z is from 2 to 6 an the amino groups are separated by at least two methylen groups. Other preferred core molecules include ethylene diamin and like molecules with longer chains such as propylene diamin and butylene diamine. Such molecules may be represented by th general formula:

H N CH₂ (CH₂)_n - CH₂ NH₂ wherein n is an integer from 0 to 10, preferably 0 to 3.

Of course, ammonia can also be employed as the cor molecule.

The development of synthetic vaccines against a larg number of diseases has recently been greatly accelerate because of the recognition that a vaccine need not be based o a native protein, but may be based on a low molecular weigh segment of the native protein. These segments, normally calle immunogenic determinants or epitopes are capable of stimulating the production of antibodies which will protect against infection by sporozoites bearing the native protein antigen and in turn introduced in the mammalian host by the bite of a mosquito vector.

This invention is concerned with malarial-origin T- and

B-cell epitopic peptides such as those described by Romero, et al., Loc. cit. which is incorporated herein by reference. By way of nonlimiting example, some of the P. berghei T-cell epitopic peptides are:

Designation YNRNTVNRLLAD 1

59 69

NEKIERNNKLKQP N 80 92

NDDSYIPSAEKI 3

249 260

KQIRDSITEEWS B-4

265 ^' 276 GSGIRVRKRKGSNK 5

283 296

SSIFNIVSNSLG 6

317 328

NEKIERNNKLKQPDPPPPNPNDPPPPNPND N-t-17.1 KQIRDSITEEWSDPPPPNPNDPPPPNPND B-4+17.1

The last two antigens N+17.1 and B-4+17.1 represent a combination of T-cell epitopes N or B-4 with a B-cell epitope 17.1. The epitope 17.1 and its preparation are described in Zavala et al., J. Exp. Med. , 166:1591, 1987, which is incor- porated by reference. It should be noted that in the case of circumsporozoite protein, the B-cell epitope (which happens to be the imraunodominant epitope) is repetitive in nature, e.g., (DPPPPNPN)_χ for P. berghei: (DRAAGQPAG)_χ or (DRADGQPAG)_χ or combinations of the two for P. vivax: (NANP)_X for P. fal- ciparum, (QAQGDGANAGQP)_χ for P. knowlesi. etc. wherein x is at least 2 for at least some malarial species. Repeats of cyclic permutations of these minimum repeating units will also yield B-cell epitopic peptides, e.g. (PNAN) .

Some of the antigenic peptides which are currently available either commercially or by known synthetic or isolation techniques are listed in Table 1, below. The table lists the peptides which are segments of proteins associated with the disease or pathogen identified in the second column. The references identify the publications which describe the peptides and how to obtain them. The conventional abbreviations are used for the amino acids.

TABLE 1

PEPTIDE SEQUENCES SUITABLE FOR DEVELOPMENT OF VACCINES USING MAPS

Peptide Pathogen/Disease (protein) Ref

A. H-(Asn-Ala-Asn-Pro) -CH n>3 Malaria, CS protein of P. falciparum 1

B. H-(Gly-Asp-Arg-Ala-Asp-Gly Malaria, CS protein of

Gin-Pro-Ala)n-OH n>2 P. vivax 2

C. Glu-Gln-Asn-Val-Glu-His- Malaria, Pf 155 of

Asp-Ala P. falciparum 3

D. Asn-Ala-Glu-Asn-Lys-Glu-Glu- Malaria, Marozoite surface

Leu-Thr-Ser-Ser-Asp-Pro-Glu- protein of P. falciparum 4 Gly-Gln-Ile-Mat

E. Asn-Ala-Asn-Pro-Asn-Val- Malaria, CS protein of 5 Asp-Pro-Asn-Ala-Asn-Pro P. falciparum

1. Zavala, et al. Science 228:1436, 1985

2. McCutchan, et al. Science 230:1381, 1985; Arnot, D.E., et al, Science, 230:815 (1985)

3. Udomsangpetch, et al, Science 231:57, 1986

4. Ravetch, et al, Science 227:1593, 1984 5. Nardin, E.H. et al, Science 246:1603, 1989

In addition, malarial T-helper cell epitopic peptides can be identified, as described above in the references of Sinigaglia et al etc. Briefly, once the amino acid sequence of a malarial protein is lcnown, peptides corresponding to fragments of the protein can be synthesized and injected in mammals. T- cells

SUBSTITUTESHEET can then be harvested from blood samples of the immunized mammals and incubated in vitro in the presence of the peptide used for immunization. Such peptide are considered T-helper cell epitopic peptides if the T-cells proliferate during such incubation in the presence of such a peptide. To demonstrate whether these T-cell peptides are T-helper peptides, they are tested for elicitation of antibodies to a B-cell epitope by covalently linking the T- cell and the B-cell epitopic peptide and using the thus formed conjugate for immunization. In the foregoing description the letters have the same meaning as is employed by those skilled in the peptide arts. These are:

A-alanine M-methionine

C-cystine N-asparagine D-aspartic acid P-proline

E-glutamic acid Q-glutamine

F-phenylalanine R-arginine

G-glycine S-serine

H-histidine T-threonine I-isoleucine V-valine

K-lysine W-tryptophan

L-leucine Y-tyrosine

A particular advantage of this invention is that the dendritic polymer can serve as a carrier for two or more dif- ferent malarial antigens. This is particularly useful for producing multivalent vaccines (i.e. vaccines directed against more than one malarial species) and/or for producing vaccines against different stages of the malaria parasite. Vaccines produced from antigenic products of the invention in which both T-cell antigens and B-cell antigens associated with malaria are joined to the dendritic polymer in any of the various configura¬ tions illustrated in a non-limiting fashion in Figure 2 are especially useful because they are capable of generating extreme¬ ly high antibody titers. It has been discovered that when the T- and B-cell epitopes of this invention are covalently bound to MAP sub¬ strates, the resulting products will elicit levels of antibody response which are 10 to 100 fold greater than those obtained in the past with recombinant CS protein or irradiated sporozoites. It has been further observed that, in mice, the B-T monomeric di- epitope not supported on a MAP substrate, or a mixture of B- epitope MAP and T-epitope MAP produced very low antibody response and no protection. The presently most preferred embodiment of the present invention is one where both a T and a B epitopic peptide are linked in tandem on the same functional group of the dendritic polymer substrate. The specifically selected B- and T-epitopes of this invention can be placed on the MAP substrate in a variety of different arrangements as shown in Fig. 2. The figure shows alternate arrangement for the B-epitope (open blocks) and the T- epitope (solid blocks) which for P. berghei include PPPPNPDPPPPNPND and KQIRDSITEEWS, respectively.

In Figure 2, T-(4) and B-(4) are monomeric maps wit four branches but only one epitope (again the immunodominant B- epitope for the CS protein comprises at least two occurrences of the repetitive unit). T-(8) and B-(8) are similar, but with 8 branches. In T(8)B and B(8)-T, there are 8 T or B epitopes o the branches of the dendritic polymer and one B-epitope or T epitope on the root of the polymer. BT-(4), TB-(4), BT-(8) an TB-(8) illustrate presently preferred products of the inventio in which the epitopes are arranged in tandem. Naturally, it will be apparent to those skilled in th art that many combinations and numbers of malarial T- and B epitopes are contemplated herein and are fully within the scop of the present invention.

It is also possible to produce products of the inven tion in which the B- and T-epitopes are arranged alternatively o the branches, i.e., one branch has only B-epitopes, the othe only T-epitopes. For instance, in Fig. 2, T/B(8) represents a eight branch dendritic polymer base with alternating T and malaria antigens, within the scope of the invention; T/B(4) i similar except that the polymer base has only four branches.

This is accomplished utilizing the orthogonal protec tion method by employing a dendritic polymer based on a diamin compound such as lysine in which the amino groups are blocked with different amino blocking groups, one of which is stable to acid hydrolysis, the other of which is stable to alkaline hydrolysis. (See, for example, the schematic representation of Fig. 2, E and F) .

Fluorenylmethyloxycarbonyl (Fmoc) is a base labile protecting group and is completely stable to acidic deprotection. The t-butoxycarbonyl blocking group (Boc) is stable under mildly acidic conditions such as 50% trifluoroacetic acid. By choosing Boc-l s (Boc)-OH, Boc-lys (Fmoc)-OH, Fmoc-lys Boc)-0H or Fmoc-lys (Fmoc)-OH, it is possible to place one set of antigens on the alpha amino group of lysine and another on the omega amino group. Those skilled in the art of peptide synthesis can readily devise methods of achieving the same types of products using diverse blocking groups and other dendritic polymers.

It will be apparent to those skilled in the art that many variations of the structures shown and discussed herein are possible. For example, the dendritic polymer may have a struc¬ ture in which segments are joined through a disulfide bridge. Such structures can be readily xormed from dendritic polymers in which the root contains a protected cystine which is oxidized by a mild oxidizing agent such as molecular iodine.

As another example, referring to Fig. 1, the glycine at the root of the dendritic polymer, i.e., the free glycine could be joined to, or replaced with, a T- or B-malarial peptide antigen which may be the same or different from the other peptide antigens on the branches of the dendritic polymer molecule. The T- and B-peptide antigens themselves may serve as the residue to which other lysine or similar molecules may be attached to provide additional branches to which still additional peptide antigens, antibiotics or non-peptide antigens may be attached.

The products of this invention can be employed to produce vaccines useful to protect against malarial infections of mammals including humans using any of the procedures known to those skilled in the art. The products can, for example, be suspended in a pharmaceutically acceptable medium or diluent, such as inert oil, suitably a vegetable oil such as sesame, peanut or olive oil. Alternatively, they can be suspended in an aqueous isotonic buffer solution at a pH of about 5.6 to 7.4. Typically, such solutions will be made isotonic with sodium chloride and buffered with sodium citrate-citric acid or with phosphate. The solutions may be thickened with a thickening agent such as methyl cellulose.

Vaccines may also be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder or an alkaryl polyether alcohol, sulfonate or sulfate such as a Triton.

Stabilizers such as sorbitol or hydrolyzed gelatin may also be adαed to any of the above described compositions. It is not unusual to incorporate an antibiotic such as neomycin or other anti-infective agents to prevent infection.

Because the products of this invention provide such high antibody titers, in many instances they will be employed without carriers or adjuvants. However, if an adjuvant is employed it may be selected from any of those normally employed to stimulate the immunogenic system of mammals. These include, for example, Freund's adjuvant (complete or incomplete). Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), and mineral gels such as aluminum phosphate or alum; killed Bordetella, tetanus toxoid, diphtheria toxoid, muramyl dipeptide, aluminum hydroxide, saponin, etc., but as stated above, such adjuvants or carriers are not necessary when the polymeric substrate of the present invention is used. Freund's adjuvant is no longer used in vaccine formulations for humans or for food animals because it contains nonmetabolizable mineral oil and is a potential carcinogen. It can be used in vaccines for non-food animals. Mineral gels are widely used in commercial veterinary vaccines.

The vaccines of the invention may be defined as comprising a pharmaceutically acceptable carrier, of the general nature described above, together with an amount of an antigenic product of the invention , i.e., a selected T- or B-cell epitope which is sufficient to produce an immunological response, i.e., a protective antibody response in a mammal. An effective amount may be very small. It will, as is known, vary with the antigen. The quantity which constitutes an effective amount may vary depending on whether the vaccine is intended as a first treatment or as a booster treatment.

The amount of MAP will vary depending upon the specific immunogen, the response it elicits in various subjects, and the presence or absence of heterologous carrier or adjuvant. Generally, amounts within the range from about 1 to about 1,000 micrograms of MAP are contemplated. Optimal amounts can be ascertained by routine experimentation involving measurement of antibody titers and other parameters of mammalian immune res¬ ponse, as is well-known in the art. Repeat immunizations are preferred. It may be convenient to provide the products of this invention as lyophilized or freeze dried powders ready to be reconstituted with a pharmaceutically acceptable carrier just prior to use.

Additional information on vaccine preparations and protocols is well-known. See, for example, European Application No. A 191,748 of SmithKline Beck an published on August 20, 1986; European Patent Application No. A_j 192,626 of SmithKline Beckman et al. published August 27, 1986; U.S. Patent Nos. 4,693,994; 4,707,357; 4,735,799; and 4,767,622. All cited patents, patent applications and literature are incorporated by reference in their entirety.

Thus, this invention also provides a method of provid¬ ing immunity in a mammal against infection by a malarial organism which comprises administering to the mammal an immunogenically effective amount of a compound or composition comprising a malarial T- and B-peptide-bearing MAP, such an amount being effective to inhibit parasitemia in a mammalian host pursuant to infection by a malarial organism, preferably prior to exposure of the mammal to the malarial organism. Also contemplated are vaccines useful for inhibiting malarial infection by the sporozoite or other stages of malaria, comprising an effective amount of an immunogenic 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 tha the products of this invention, once the concept is understoo can be prepared by procedures well known to the skilled artisan.

The Tarn procedures described in Proc. Natl. Acad. Sci. USA,

81:5409, 1988, Prosnett et al, _ _ Biol. Chem. , 26.3:1719, 1988; and Chenag et al, Proc. Natl. Acad. Sci. USA £56:4929, 1988, al of which are incorporated by reference are illustrative. A few general observations applicable to the synthesi of MAPS will be of assistance to those skilled in the art. Thes are:

1. The syntheses generally require a long coupling tim

(2-4 hours) . 2. Dimethyl formamide is generally a more suitable solven than methylene dichloride.

3. The peptide resin should not be dried at any stage o the synthesis since resolvation is extremely difficult.

4. Coupling should be closely monitored for completion o the coupling by the quantitative ninhydrin method.

5. The MAPS is best cleaved from the resin by the improve acid deprotection method with either HF or TFMSA (Tam, et al., J Am. Chem. Sic. , 105:6442, 1983; and J. Am. Chem. Soc. , 108:5242 1986) in dimethyl sulfide to avoid strong acid catalyzed sid reactions.

6. MAPS tend to strongly aggregate after cleavage from th resin support. Purification is best effected by extensiv dialysis under basic and strongly denaturing conditions in dialysis medium which is 8M in urea and mercaptoethanol to remov undesirable aromatic additives of the cleavage reactions such a p-cresol and thiocresol. Further purification, if desire, can b effected using high performance gel-permeation or ion exchang chromatography. In most cases the MAPS could be used directl 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 b observed that the MAP based products of this invention have uniformly high antibody titers compared to irradiated sporozoite, recombinant CS protein or monomer BT peptide. It will be observed also that the response varies with the structure of the BT immunogens.

Table I. Comparison of antibody titers induced by different Immunogens of P. berghei and assayed with the recom¬ binant CS protein and sporozoites.

Antibody Response

Immunogen ' IFA titers RIA titers

Sporozoite rCS protein

sporozoite^a 2,048 8 , 192 recombinant CS protein" 2,048 2 , 048 monomer BT peptide^c 800 1 , 024

BT-MAP(4)^C 128,000 408,000 TB-MAP(4 ) 32,000 400,000 BT-MAP(8) 24,000 100,000 TB-MAP(8) 64,000 400,000

a. Four mice of the H-2^a halotype (B10,A strain) were injected intravenously two doses of 1x10^s irradiated P. berghei sporozoites at two-week intervals. Sera were collected and pooled ten days after the last injection. Antibody titers expressed as the reciprocal of the highest positive serum solution were obtained by using glutaraldehyde-fixed P. berghei sporozoites in an indirect immunofluorescense assay (IFA) or the recombinant CS protein in a radioimmunoassay (RIA). b. Four mice of the H-2^a halotype (A/J strain) were injected i.p. with 25 ug of the recombinant CS (rCVS) P. berghei protein, emulsified in CFA on day 0, and S.C. with 25 ug of the rCS protein in IFA on day 15. Sera were collected ten days later. c. Five mice of the H-2^a halotype (A/J strain) were injected i.p. with each 50 micrograms of the peptide immunogens consisting of two occurrences of the repeating unit of P. berghei CS protein immunodo inant region and one occurrence of a P. berghei CS protein-derived T-cell epitope peptide. The immuniza¬ tion schedule and the assay methods were similar to those for the recombinant CS protein. Upon challenge of the thus immunized mice with 2000 sporozoites each, the BT-MAP(4) produced complete protection (i.e., prevented parasitemia) in 80% of the mice; TB-MAP(4) protected 60% of the mice; BT-MAP(8) protected 50% of the mice; and TB-MAP(8) protected 60% of the mice. MAPs according to the present invention may be syn¬ thesized as follows:

Some of the following abbreviations are used in the synthetic examples, below:

Boc - t-butoxycarbonyl TFA - trifluoracetic acid

DMF - dimethylformamide

DCC - dicyclohexylcarbodiimide

Tos - tosyl

Bzl - benzyl Dnp - dinitrophenyl

2C1Z - 2-chlorocarbobenzoxy

DIEA - diisopropoylethylamine

TFMSA - trifluormethylsulfonic acid

BSA - bovine serum albumin HPLC - high performance liquid chromatography

TBR - tumor bearing rabbit

ATP - adenosine triphosphate

Dnp - dinitrophenyl.

C1Z - chlorobenzyloxycarbonyl BrZ - bromobenzyloxycarbonyl

ELISA - enzyme linked immunoabsorbent assay

Example 1 General Methods for the Synthesis of Multiple-Antigen Peptides The synthesis of an octabranched matrix core wit peptide antigen was carried out manually be a stepwise solid phase procedure [Merrifield, R.B. J. Am. Chem. Soc. (1963) 85 2149] on Boc-beta-Ala-0CH -Pam resin with a typical scale of 0.5 g of resin (0.05 mmol and a resin substitution level of 0.1 mmol/g for the present synthesis but was somewhat lower when a higher branching of core lysinlyl matrix was used). After the removal of the Boc-group by 50% TFA and neutralization of the resulting salt by DIEA, the synthesis of the first level of the carrier-core was achieved using 4 molar excess of preformed symmetrical anhydride of Boc-Lys (Boc) (0.2 mmol) in DMF and was then recoupled via DCC alone in CH C1₂. The second and third level were synthesized by the same protocol with 0.4 and 0.8 mmol respectively of preactivated Boc-Lys (Boc) to give the oc- tabranching Boc-Lys(Boc)-core matrix. However, all subsequent couplings of the peptide-antigen sequence require 1.6 mmol of preactivated amino acids. The protecting groups for the syn- thesis of the peptide antigens were as follows: Boc group for the alpha-amino terminus and benzyl alcohol derivatives for most side chains of trifunctional amino acids i.e., Arg(Tos), Asp(OBzl), Glu(OBzl), His(Dnp), Lys(2ClZ), Ser(Bzl), Thr(Bzl), and Tyr(BrZ). Because of the geometric increase in weight gain and volume, a new volume ratio of 30 ml of solvent per g of resin was used. Deprotection by TFA (20 min) was preceded by two TFA prewashes for 2 min each. Neutralization by DIEA was in CH C1₂ (5% DIEA) and there was an additional neutralization of DMF (2% DIEA). For all residues except Arg, Asn, Gin, and Gly, the first coupling was done with the preformed symmetric anhydride in CH₂C1 and a second coupling was performed in DMF; each coupling was for 2 h. The coupling of Boc-Asn and Boc-Gly were mediated by the preformed 1-hydroxybenzotriazole ester in DMF. Boc-Gly and Boc-Arg were coupled with DCC alone to avoid the risk of formation of dipeptide and lactam formation, respectively. All couplings were monitored by a quantitative ninhydrin test [Sarin, V.K., et al Anal. Biochem. (1981) 117, 147] after each cycle, and if needed, a third coupling of symmetrical anhydride in DMF at 50" for 2 h was used [Tarn, J.P. (1985) In "Proc. Am. Pept. Sympo., 9th" (CM. Deber, K.D. Kopple and V.J.]. The synthesis was terminated with acetylation in acetic anhydride/DMF (3 mmol) containing 0.3 mmol of N,N-dimethylpyridine. After completion of the MAPS, protected peptide-resin (0.3g) was treated with 1 M thiophenol in DMF for 8 h (3 times and at 50^CC if necessary to complete the reaction) to remove the N^im-dinitrophenyl protecting group of His (when present), with 50% TFA/CH C1₂ (10 ml) for 5 min to remove the N -Boc group, and with the low/high-HF method [Tarn, J.P., Heath, W.F. & Merrifield, R.B. J. Am. Chem. Soc. (1983) 105, 6442] or the low-high TFMSA method [Tarn, J.P. Heath, W.F. & Merrifield, R.B. J. Am. Chem. Soc. (1986) lfl8, 5242] of cleavage to give the. crude MAPS. The crude peptide was then washed with cold ether mercaptoethanol (99:1, v/v, 30 ml) to remove p-thiocresol and p-cresol and extracted into 100 ml of 8 M urea, 0.2M dithiothreitol in 0.1 M Tris buffer, pH 8.0. To remove all the remaining aromatic byproducts generated in the cleavage step, the peptide in the dialysis tubing (Spectra Por 6,M.W. cutoff 1,000) was equi¬ librated in a deaerated and N₂-purged solution containing 8 M urea, 0.1 M NH4HCO3-(NH₄) C0₃, pH 8.0 with 0.1 M mercaptoethanol at 0°C for 24 n. The dialysis was then continued in 8M, and then in 2M urea, all in 0.1 M NH₄HC0₃-(NH₄)₂C0₃ buffer, ph 8.0 for 12 h and then sequentially in H 0 and 1 M HOAc to remove all the urea. The lyophilized MAPS was then purified batchwise by high performance gel-permeation or ion-exchange chromatography. All of the purified material gave a satisfactory amino acid analysis.

Example 2

Synthesis and Purification of (Asn-Ala-Asn-ProJs-MAP (NP-16 MAP), a Peptide Derived from the Sporozoite Stage of Plasmodium falciparum.

The peptide, (Asn-Ala-Asn-Pro)s-Lys₄-Lys -Lys-OH was synthesized by the general procedure described in Example 1.

The synthesis was initiated with Box-Lys(Boc)-0CH -Pam- resin (a copoly(styrene-l%-divinylbenzene resin) at a substitu¬ tion of 0.11 mmol/g of resin. The substitution was found to be 0.88 mmol/g after the sequential addition of three levels of Boc- Lys(Boc) to give an octabranching structure of [Boc-Lys(Boc) ] [Lys(Boc) -Lys(Boc)-OCH2~Pam resin. The synthesis continued with 2.5 g of resin in a modified Beckmann 990 synthesizer (Beckman Instructions, Palo Alto, California). Synthesis was performed using a computer program that optimized all of the coupling steps. For example, the coupling of Boc-Ala and Boc-Pro were mediated by the symmetric anhydride method in a solvent ratio of CH2Cl2;dimethylformamide (1:3, v/v) to minimize aggregation and incomplete coupling. The coupling of Boc-Asn was by the per¬ formed 1-hydroxybenzotriazole active ester in the same solvent. Each amino acid underwent a double coupling protocol to maximize the coupling yield and essentially bring the reaction to >99.6% completion.

The protected peptide-resin was deprotected in por¬ tions. The initial deprotection was carried out with 1.57 g of dried peptide-resin in a reaction vessel and underwent the following procedure to remove the Boc-protecting group and other extraneous materials: CH₂C1₂ (3 x 1 min wash); CF₃C0₂H- CH₂C1₂(1:1, 3 x 2 min) and CF₃C0₂H (3 x 2 min wash) and then a cleavage reaction containing the following deprotecting reagents: trifluoromethanesulfonic aciditrif luoroacetic acid:tetrahydrothiophene: m-cresol (4:20:12:4, in ml) at 4°C for 3.5 h. The peptide released by the acidolytic cleavage of the sulfide-assisted cleavage procedure was collected and precipitated by ethyl ether (230 ml) prechilled to -30^βC. The precipitate was centrifuged to a pellet and the ethyl ether was removed in vacuo. The peptide was then dissolved in 0.01M HOAc and dialyzed in 12 liters of 0.01M HOAc. The peptide was then lyophilized to dryness to obtain 60 mg of (Asn-Ala-Asn-Pro)gOMAP. Hydrolysis of the resulting resin after cleavage showed that about 90% of the peptide had been cleaved from the resin support. The low yield was due to incomplete precipitation of the peptide by the ether. The same peptide-resin (l.Og) was also cleaved by HF:anisole (9:1, v/v total 10ml) at 0^βC for 1 h to give 220 mg of MAP after extensive extraction with 10 to 100% HOACc and a crude yield of 33%. The dialysis was carried out with 10% OHAc.

The peptide after dialysis was then analyzed first by amino -acid analysis (after hydrolysis by 6N HC1). The molar ratio of the MAP found was Asn:Ala;Pro;Lys: 1.97 (2): 1.03 (1):1 (1) :0.26(0.22) which was in agreement with those expected theoretical values shown in parenthesis.

Example 3 General Methods for the Synthesis of Di-epitope Multiple Antigen Peptides Containing Malarial-Provenance T-cell and B-cell antigens.

(a) Method A. Linking Two epitopes in Tandem.

The synthesis of di-epitope MAPS was accomplished manually by a stepwise solid-phase procedure on Boc-Ala-OCH₂-Pam resin (0.1 mmol of Ala is present in 1 g of resin) similar to those mono-epitope MAPS described in the previous examples. After the removal of the Boc group by 50% TFA and neutralization of the resulting salt by DIEA, the synthesis of the first level of the carrier core to form Boc-Lys(Boc)-Ala-OCH₂-Pam resin was achieved using a 4 mole excess of Boc-Lys(Boc) via DCC alone in CH C1₂. The second and third level were synthesized by the same protocol, to give the octabranching Boc-Lys(Boc) core matrix. From this point onward, the synthesis of peptide antigens or two epitopes proceeded as those of the previous examples using the tertbutoxycarbonyl/benzyl protecting group strategy since they were arranged in tandem and were treated as if they are one antigen. Spacers such as tetra-peptide Gly-Pro-Pro-Gly are sometimes inserted between two peptide antigens to allow flexibility. After completion of the synthesis, the MAP-resin was treated with TFA to remove the N -Boc groups, then acetylated with 10% acetic anhydride/10% DIEA in Ch₂Cl₂, and finally cleaved with the low-high HF method to remove the MAP from the resin support. The crude peptide was then washed with cold ether/mer¬ captoethanol (99:1 vol/vol) to remove p-thiocresol and p-cresol, and extracted into 8 M urea in 0.1 M Tris.HCl buffer (pH 8.0). To remove the remaining aromatic by-products generated in the cleavage step, MAPs were dialyzed (Spectra Por 6, molecula weight cut off 1,000) in 8 M urea and then in 0.1 M acetic aci twice for 5-6 hours to remove the urea. The MAPs were lyophil ized from H_Q three times to remove acetic acid.

(b) Method B. Linking Two or More Epitopes by Alternatin Branching of the Amino groups of Lysines Because there are two amino groups in lysine and because these two amino groups could be protected selectively, the core matrix could be synthesized in such a way to produce that the N -NH group is protected with the acid-labile Boc group and the N -NE₂ group is protected with the base-labile Fmoc (fluorenylmethoxycarbonyl) group, or vice versa, i.e. N -NH group is protected by the Fmoc group, and the N -NH group is protected by the Boc group. To achieve the synthesis of this core matrix using this selectivity, a core matrix containing N-~ ^NH2-Boc and N -NH₂-Fmoc is illustrated. The synthesis of the core matrix was similar to those described in the previous examples using the Boc-Lys(Boc) for the branching for the first and second level. At the third level, Fmoc-Lys(Boc) was used for the Lys branching of the core to give for each Lys(Boc) and F oc- Lys end groups. The synthesis of the first epitope (or two epitopes in tandem) used the Boc/benzyl chemistry as described in the previous examples, but during this synthesis, neutralization time was reduced to 1 min to minimize the premature cleavage of the Fmoc group. The synthesis of the second epitope used the Fmoc/tertbutyl chemistry (i.e. the N -NH₂ group is protected with Fmoc and the side chain is protected with tertbutyl alcohol derived protecting groups) and started after the completion of the first epitope using the Boc-aπ.ino acid chain was assembled. The F oc-amino acids were used with the side chain protecting groups for the trifunctional amino acids as follows: Glu(OBu^'*-), AsptOBu¹^), Lys(Boc) ThrfBu^, Ser(Bu^t), Tyr(Bu^t), Arg(P z), His(Trt), Trp(For), and Cys(Bu^t). Repetitive deprotection of N- Fmoc was by 20% piperidine in dimethylformamide and was preceded by one piperidine prewash and the coupling was mediated with DCC:HOBut in DMF. After completion of synthesis, the MAP resin was treated with low-high HF to remove the peptide chains from the resin. The workup and purification was essentially the same as those described in the previous examples. The procedure for assembling the peptide chain using the Fmoc.tertbutyl chemistry was as follows: (1) 20 mil DMF (3 1 min); (2) 20 ml piperidine/DMF (1:1 vol/vol()l min); (3) 20 ml piperidine/DMF (1:1 vol/vol) (10 min); (4) 20 ml DMF (3 x 1 min); (5) 20 ml CH₂C1₂(3 x 1 min); (6) 20 ml DMF (2 x 1 min); (7) amino acid (4 equiv) in DMF 5 ml (5 min), HOBt(4 equiv) in DMF, DCC(4 equiv) in

CH C1 were added for 2 h? (8) 20 ml DMF (4 x 2 min); (9) 20 ml

CH₂C1₂(2 x 2 min). (c) Metnod C. Linking Two or More Epitopes via Disulfide Linkage of Two Preformed Heterologous MAPS.

To link two or more epitopes together via disulfide linkage of two preformed MAPS, a dipeptide such as Cys(Acm)-Ala is added at the carboxy terminus of the preformed MAPS as described in Example 3a or 3b. This could be achieved con¬ veniently before the start of the synthesis of the core matrix by adding Boc-Cys(Acm) to the Boc-Ala-OCH -Pam-resin. After the formation of the dipeptide 3oc-Cys(Acm)-Ala-0Ch_Pam-resin, the synthesis of the core matrix, the incorporation of one or more peptide antigen(s) using the procedures described above proceeded to give the preformed MAPS containing a Cys(Acm)-Ala dipeptide COOH-tail. The Cys(Acm) is stable to the HF deprotection method. The preformed MAPS containing the COOH Cys(Acm)-Ala dipeptide tail were purified. The dimerization of two heterologous preformed MAPs was achieved by oxidation with I₂ to the disul¬ fide, and which also concomitantly remove the Acm-group from the cysteinyl residue. A detailed procedure was as follows. To 1 mmol of MAP, the heterologous preformed di-epitope MAPs contain¬ ing Cys(Acm) was dissolved in a de-aerated and N₂-purified 50% acetic acid solution at room temperature, 50 ml of a solution of I₂ in MeOH(l M solution) was added batchwise for 1 hour at 0°C. The reaction was quenched by adding 1 M aqueous sodium thiosul- fate (or ascorbic acid) until the yellow color was removed. MeOH was removed by dialysis in 0.1 acetic acid and the desired MAPs were purified by gel permeation chromatography, ion-exchange chromatography or reverse-phase high pressure liquid chromatog¬ raphy.

Claims

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)_χ (b) (DRAZGQFAG)_χ wherein Z is independently selected from A or D; (c) (QAQGDGANAGQP)_χ (d) (DPPPPNPN)_χ (e) (YAAA(A)_nGGG(G)_mN)_χ 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 immunogenically 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.

SUBSTITUTESHEET