WO1998030237A9 - Polyoxime-based anti-malarial vaccines - Google Patents

Abstract

Vaccine compositions containing malaria parasite-derived T and/or B epitopes incorporated into a polyoxime are taught for vaccination.

Description

POLYOXIME-BASED ANTI-MALARIAL VACCINES

Field of the Invention

This invention relates to vaccines effective in eliciting protective immunity against malaria, in particular vaccines comprising polyoximes that elicit anti-malarial responses in individuals of differing genetic backgrounds.

Background of the Invention

The public health problems caused by malaria, which currently infects 400-500 million individuals world-wide, have been exacerbated by the emergence of multi-drug resistant parasite strains and insecticide-resistant mosquito vectors. These developments have led to increased efforts to provide an effective vaccine to prevent the mortality and morbidity due to malaria, in particular ³, fa/ciparum, the most virulent of the Plasmodial species.

In a mammalian host, malaria infection is initiated by the motile sporozoite stage of the organism, which is injected into the circulation by the bite of infected mosquitoes. The sporozoite is targeted to the host's liver cells through interaction of a major component of the sporozoite surface membrane, the circumsporozoite (CS) protein, with specific receptors on the hepatocyte surface. Following intracellular multiplication and release from ruptured hepatocytes, the parasites invade red blood cells and initiate the malaria erythrocytic cycle; this phase of infection is responsible for clinical disease and, in the case of P. falciparum, may be lethal.

A major focus of malaria vaccine development has been the CS protein, which is present in both sporozoite and liver stages of the parasite. Polyclonal and monoclonal antibodies specific for an immunodominant B-cell epitope within the repeat region of the CS protein, the (NANP)₃ peptide, neutralize the infectivity of sporozoites of rodent, primate and human malaria species (Nardin et al., J. Exp. Med. 156:20, 1 982) . Use of the (NANP)₃ peptide in a vaccine, however, resulted in only a limited immune response, most probably due to low epitope density and/or lack of a suitable T-cell epitope (Herrington et al., Nature 328:257, 1 987) .

The present inventors have defined parasite-derived T-cell epitopes using CD4 + T-cell clones derived from four human volunteers immunized by repeated exposure to the bites of irradiated P. falciparum malaria infected mosquitoes. When three of these volunteers were challenged with infective P. falciparum sporozoites, they were protected against malaria, as shown by the total absence of blood stage infection (Herrington et a\., Am.J. Trop.Hyg. 45:535, 1 991 ).

Using CD4 + T-cell clones derived from these sporozoite immunized volunteers, two T-cell epitopes have been identified, one located in the repeat region and one in the COOH-terminus of the P. fa/ciparum CS protein. The T-cell epitope contained in the NH₂-terminal repeat region, termed T1 , consists of alternating NVDPNANP repeats (Nardin et al., Science 246: 1 603, 1 989) . The T1 epitope is contiguous to, but antigenically distinct from, the COOH-terminal repeat region which contains the (NANP)₃ B cell epitope. The human CD4 + T-cell clones that specifically recognize peptides derived from various combinations of the NH₂- terminal repeat region and that contain the sequence NVDPNANP do not respond to the (NANP)₃ repeat peptide. The T1 repeat epitope is conserved in all P. fa/ciparum isolates sequenced thus far and therefore its inclusion in a vaccine is expected to induce immune responses reactive with parasites of diverse geographical regions.

The second T-cell epitope identified by sporozoite-specific human CD4 + T-cell clones is contained in a peptide spanning amino acid residues numbered 326-345, EYLNKIQNSLSTEWSPCSVT, of the P. falciparum NF54 strain CS protein (Moreno et al., Int. Immunol. 3:997, 1 991 ; Moreno et al., J. Immunol. 151 :489, 1 993). This epitope was shown to be recognized by cytotoxic and non- cytotoxic class ll-restricted human CD4 + T-cell clones and class l-restricted CD8 + CTL clones. The 326-345 sequence is unique in that it overlaps both a polymorphic, as well as a conserved region, RM (Dame et al., Science 225:593, 1 984), of the CS protein. The conserved Rll-plus contains a parasite ligand that interacts with hepatocyte receptors to initiate the intracellular stage of the malaria life cycle. The peptide-specific human CD4 + T-cells recognize a series of epitopes within the 326-345 peptide, all of which overlap the conserved RII found in the CS protein of all Plasmodium species.

The fact that these two CS epitopes were defined by CD4 + T-cells derived from human volunteers immunized by multiple exposures to the bites of malaria-infected mosquitoes suggests that these peptide sequences are efficiently processed for presentation by HLA class II molecules following exposure to the native CS protein on the sporozoite. It is contemplated that vaccines containing such parasite-derived T-cell epitopes can elicit anamnestic responses in naturally- infected individuals and can provide for vaccine-induced immunity to be maintained by continued exposure to the parasite under natural conditions. Class II restricted CD4 + T-cells play a central role in the induction of both cellular and humoral immunity to the pre-erythrocytic stages of the malaria parasite (Nardin et al., Ann. Rev. Immunol. 11:687, 1 993) . If the T-cell epitopes contained within a synthetic malaria vaccine bind to only a limited range of class II molecules, the vaccine may fail to elicit immune responses in individuals of diverse genetic backgrounds. Earlier studies have shown that the (NANP)₃ repeats of the P. falciparum CS protein induced low or undetectable T-cell responses in naturally-infected individuals living in malaria endemic areas (Herrington et al., Nature 328:257, 1 987; Etlinger et al., J. Immunol. 140:626, 1 988; Good et al., Proc.Natl.Acad.Sci. USA 85: 1 1 99, 1 988). Thus, there is a need in the art for immunogenic compositions and vaccines that provide protective immunity against malaria in individuals of diverse genetic backgrounds.

Brief Description of the Drawings Figure 1 A is an illustration of an electrospray ionization mass spectrography (EIS-MS) spectrum of a di-epitope (T1 B)₄ polyoxime construct, shown on a true mass scale. Figure 1 B is an illustration of an EIS-MS spectrum of a di-epitope (T1 B)₄-P₃C polyoxime construct, containing a lipopeptide adjuvant, showing the expected series of multi-charged forms of the product characteristic of this ionization mode. Figure 1 C is an illustration of a matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrum of a tri-epitope (T1 BT*)₄ polyoxime construct. Figure 1 D is an illustration of a MALDI-TOF mass spectrum of a tri-epitope (T1 BT*)₄-P₃C polyoxime construct, containing a lipopeptide adjuvant. Figure 2 is a graphic illustration of the Geometric Mean Titers (GMTs) measured in sera obtained 20 days after subcutaneous injections of a (T1 B)₄-P₃C polyoxime construct on days 0, 21 , and 42. The titers were measured by enzyme- linked immunoassay (ELISA) using (T1 B)₄ polyoxime-coated plates.

Figure 3 is a graphic illustration of the GMT of sera obtained from mice 20 days after a third injection of a (T1 B)₄-P₃C polyoxime construct, measured by ELISA using (T1 B)₄ as antigen (closed bars), or by indirect immunofluorescence (IFA) using glutaraldehyde-fixed P. falciparum sporozoites (hatched bars) .

Figure 4A is a graphic representation of the peak antibody response obtained following immunization of different murine strains with the (T1 BT*)₄ polyxoxime construct when conjugated to P₃C (closed bars); in PBS (open bars); or adsorbed to alum (hatched bars) . The antibody response was measured by ELISA, using (T1 B)₄ as antigen. Figure 4B is a graphic representation of the peak antibody response obtained following immunization of different murine strains with the (T1 BT*)₄ polyxoxime construct when conjugated to P₃C (closed bars); in PBS (open bars); or adsorbed to alum (hatched bars). The antibody response was measured by indirect immunofluorescence as described for Figure 3.

Figure 5A is a graphic illustration of the kinetics of anti-repeat antibody response in mice immunized with (T1 BT*)₄ in PBS. Figure 5B is a graphic illustration of the kinetics of anti-repeat antibody response in mice immunized with (T1 BT*)₄ adsorbed to alum. Figure 5C is a graphic illustration of the kinetics of anti-repeat antibody response in mice immunized with (T1 BT*)₄ constructed as a P₃C conjugate.

Summary of the Invention The present invention encompasses immunogenic compositions constructed as polyoximes that elicit protective immunity against malaria. The compositions comprise one or more malaria-derived peptides comprising T-cell epitopes, which elicit anti-malarial T-cell responses. Malaria-derived universal T-cell epitopes, which elicit T-cell responses in mammals of diverse genetic backgrounds, may also be included. Preferably, the compositions of the invention further comprise at least a second malaria-derived peptide comprising a B-cell epitope, which stimulates the production of anti-malarial antibodies in mammals. The polyoxime-based compositions are preferably formulated into vaccines, which may also comprise a pharmaceutically acceptable carrier or diluent and, optionally, an adjuvant. Adjuvants may also be covalently incorporated into the polyoxime constructs, including without limitation tri-palmitoyl-S-glyceryl cysteine.

In another aspect, the invention provides methods for inhibiting the propagation of malarial organisms in a susceptible mammal, preferably by eliciting protective immunity against malaria in the mammal. The methods are carried out by administering to mammals immunogenically effective amounts of the immunogenic compositions and vaccines described above.

Detailed Description of the Invention All patent applications, patents, and literature references cited in this specification are hereby incorporated by reference in their entirety. In the case of inconsistencies, the present description, including definitions, will control. Definitions:

1 . An "immunogenic composition" is a composition that elicits a humoral and/or cellular immune response in a host organism.

2. A "B-cell epitope" as used herein refers to a peptide or other immunogenic molecule, or a fragment thereof, that elicits the production of specific antibodies (i.e., antibodies that recognize the parasite as well as the immunogenic molecule) in a mammalian host. A "T-cell epitope" refers to a peptide or immunogenic molecule, or fragment thereof, that activates T-cells in a manner that is specific for the parasite-derived peptide as well as the immunogenic molecule.

3. A "universal" T-cell epitope as used herein refers to a peptide or other immunogenic molecule, or a fragment thereof, that binds to a multiplicity of MHC class II molecules in a manner that elicits activated T-cell function. The activated T-cells may be helper cells (CD4 + ) and/or cytotoxic cells (class II- restricted CD4 + and/or class l-restricted CD8 + ). A malaria-specific or parasite- specific universal T-cell epitope has the potential to expand, or induce, parasite- specific T-cells in naturally-infected and naive individuals, respectively, in the general population. 4. A peptide epitope that is "derived from" a particular organism or from a particular polypeptide comprises an amino acid sequence found in whole or in part within the particular polypeptide and encoded by the genome of the organism, including polymorphisms. It will be understood that changes may be effected in the sequence of a peptide relative to the polypeptide from which it is derived that do not negate the ability of the altered peptide, when used as part of an immunogenic composition, to elicit an immune response that is specific for the polypeptide from which the peptide is derived.

5. "Multiple Antigen Peptide" (MAP) refers to peptide multimer formed from a polylysine core and containing a branched scaffolding onto which peptides are synthesized in a stepwise fashion (Tarn, J. Immunol. Meth. 196: 1 7, 1 996; Nardin et al., Adv. Immunol. 60: 105, 1 995) .

6. "Polyoxime" as used herein refers to a macromolecule of defined structure which comprises a first organic molecule, termed a baseplate, to which other organic molecules are attached via oxime linkages. Methods for synthesis of immunogenic polyoxime compositions are disclosed in International Patent Application WO 94/25071 .

The present invention provides immunogenic compositions and methods for eliciting protective immunity against malaria, in particular against P. falciparum. The compositions comprise immunogenic components that are formed into polyoximes. The polyoximes may contain one or more of the following components: (i) at least one malaria-derived peptide comprising a T-cell epitope capable of eliciting an anti-malarial T-cell response; and (ii) at least one malaria- derived peptide comprising a B-cell epitope capable of stimulating the production of anti-malarial (i.e., neutralizing) antibodies directed against the sporozoite stage of the malarial organism. Preferably, the immunogenic compositions of the present invention comprise at least one B-cell epitope and at least one T-cell epitope, most preferably a universal T-cell epitope. The B-cell epitopes preferably elicit the production of antibodies that specifically recognize and bind to the malarial circumsporozoite (CS) protein. The compositions may also comprise B-cell and/or T-cell epitopes derived from, and reactive with, other malarial components, such as, for example, the P. falciparum sporozoite surface protein designated Thrombospondin Related Adhesion (Anonymous) protein (TRAP), also called Sporozoite Surface Protein 2 (SSP2); LSA I; hsp70; SALSA; STARP, Hep1 7; MSA; RAP- 1 ; and RAP-2. The polyoximes of the present invention may comprise homo- polyoximes, in which a single type of organic molecule is conjugated via oxime linkages to a baseplate molecule, as well as hetero-polyoximes, in which a plurality of different organic molecules are conjugated via oxime linkages to a baseplate molecule. Preferably, the baseplate structure is a peptide having a backbone in which at least some of the amino acid residues have side-chain groups suitable for modification to oxime-forming reactive groups, such as, for example, lysine, ornithine, and cysteine. Suitable reactive groups include without limitation amino- oxy-acetyl (AOA) or aldehyde groups such as glyoxylyl (GXL) . Malaria-specific "complementary orthogonal specifically active molecules", or "COSMs", that are reacted with the baseplate molecule may comprise peptides or other immunogenic molecules having site-specifically placed oxime-forming reactive groups. The synthesis and analysis of malaria-specific polyoxime constructs are described in Example 1 below. The present invention encompasses B-cell and T-cell epitopes derived from plasmodial species, including without limitation P. falciparum, P. vivax, P. malariae, P. ovale, P. reichenowi, P. knowlesi, P. cynomo/gi, P. brasilianum, P. yoelii, P. berghei, and P. chabaudi. Epitopes typically comprise at least 5 amino acid residues, preferably at least 7 residues, and most preferably at least 10 residues, derived from a plasmodial protein. Overlapping epitopes may be contained within a single peptide. B-cell epitopes may be identified by methods well known in the art, such as, for example, by (i) preparing synthetic peptides whose sequences are derived from the CS protein of a plasmodial species; and (ii) testing the ability of the synthetic peptides to elicit anti-malarial antibodies in a model system. Malaria-specific B-cell and T-cell epitopes are disclosed in Nardin et al., Ann. Rev. Immunol. 1 1 :687, 1 993.

In a preferred embodiment, the immunogenic composition of the invention comprises (i) a peptide comprising the malarial B-cell epitope (NANP)₃; (ii) a peptide comprising the T1 malarial epitope (DPNANPNV)₂; and (iii) a peptide comprising the universal T-cell epitope represented by amino acid residues numbered 326-345, EYLNKIQNSLSTEWSPCSVT, of the P. falciparum NF54 strain CS protein, or immunogenic variants derived from any of the above.

Other universal T-cell epitopes for use in the present invention may be identified by one or more of the following methods: (i) experimentally measuring the interaction of different malaria-derived peptides with isolated class II polypeptides />7 vitro; and (ii) computationally analyzing different peptide sequences to identify high-affinity class II allele-specific motifs. The interactions that have been measured in vitro have been correlated with in vivo immunogenicity, as measured by the immune response of mice of different genetic backgrounds when immunized with multiple antigen peptides (MAP) containing these T-cell epitopes. Similarly, a peptide derived from P. falciparum TRAP/SS2 that was predicted to comprise a universal T-cell epitope has been shown experimentally to bind multiple class II molecules in vitro.

Vaccines

The compositions of the present invention may be used as immunogens to elicit immunity, including protective immunity, in a susceptible host. Immunity may include eliciting the production of antibodies in the host (or in another host or in vitro, as in passive immunization) that will recognize and bind to plasmodial cells. Immunity may also include the activation of malaria-specific T- cells. Thus, the immunogenic compositions comprising B-cell epitopes and/or universal T-cell epitopes may be used in vaccine preparations to confer prophylactic immunity against the blood stage of malaria or therapeutic immunity by preventing (totally or partially) propagation of the disease in the host through inhibition of the pre-erythrocytic stages.

It should be noted that 100% inhibition of any stage in malarial infection or propagation by an immunogenic composition (or by vaccine containing it, or by an antibody) is not necessary for these materials to be useful. Any substantial decrease in the extent of infection (as measured, e.g. by the extent of parasitemia) would substantially attenuate the clinical symptoms and substantially increase the probability for survival and recovery of the host.

There are many protocols for the preparation of vaccines known in the art. Typically, vaccines are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for dissolving or suspending in liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients may be mixed with excipients, such as, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants to enhance the effectiveness of the vaccine. When the immunogenic components of the vaccine are constructed as polyoximes, adjuvants may be covalently linked to the immunogenic components via oxime linkages. The immunogenic compositions could also be administered following incorporation into liposomes or other microcarriers.

Repeat immunizations may be necessary to enable the host to mount an immune response. Both amounts of immunogen and immunization protocols can be determined experimentally, as is well-known in the art, using animal (e.g. primate) models followed by clinical testing in humans. Information on vaccine compositions and immunization is described for example in U.S. Patent No. 4,767,622 of Ristic (August 30, 1 988); U.S. Patent No. 4,735,799 of Patarroyo (April 5, 1 988) and Patarroyo, M.E., et al.. Nature 332: 1 58, 1 988; and published European Application A, 250,261 (published December 23, 1 987) of the Wellcome Foundation. The vaccines may be administered by subcutaneous, intramuscular, oral, intradermal, or intranasal routes. Dosages may range from about 5 μg to about 5 mg per dose, and a single or multiple dosage regimen may be utilized. The amounts administered, number of administrations, and schedule of administrations can be determined empirically, such as, for example, by establishing a matrix of dosages and frequencies and comparing a group of experimental units or subjects to each point in the matrix.

The present invention also provides methods of inhibiting the propagation of a malarial organism in a susceptible mammal, which comprises administering to the mammal an immunogenically effective amount of an immunogenic composition comprising one or more of the following components: (i) at least one malaria-derived peptide containing a B-cell epitope capable of stimulating the production of anti-malarial (i.e., neutralizing) antibodies directed against the sporozoite stage of the organism; and (ii) at least one malaria-derived peptide that encompasses a T-cell epitope capable of eliciting an anti-malarial T-cell response in vaccinates. An immunogenically effect amount is an amount effective to elicit protective immunity against the malarial organism determined as described above. In a further aspect, the composition may be administered to a mammal which has been previously exposed to the malarial organism. In a still further aspect, the polypeptide may be administered to a mammal prior to exposure of the mammal to the malarial organism.

The following working examples are intended to serve as non-limiting illustrations of the present invention.

Example 1 : Construction and Testing of an Anti-Malarial Polyoxime

The following experiments were performed to compare the immunogenicity of di-epitope and tri-epitope malaria-derived polypeptides synthesized by MAP and polyoxime procedures. A. Construction: Peptides were synthesized on a model ABI 430A machine (Applied

Biosystems Inc.) modified according to Schnolzer et al., Int. J. Peptide Protein Res. 40: 1 80, 1 992. Boc chemistry was used with coupling times of 20 min. The side- chain Fmoc group was removed from Lys with 20% piperidine in DMF after assembly of the peptide on the resin, and the free amino group was acylated with Boc-Ser(Bzl)- or Boc-NHOCH2CO- in the normal way. After machine-assisted removal of the Boc group and neutralization with 10% diisopropylethylamine in DMF and extensive washing with dichloromethane/ methanol (1 : 1 ), the resin was dried in vacuo. Cleavage from the resin and side-chain deprotection were achieved with HF/para-cresol ( 1 9: 1 v/v, 1 h at 0°C). HF was removed under reduced pressure at 0°C and the crude peptide precipitated, then washed with cold diethyl ether. Crude peptide was dissolved in 50% aqueous acetonitrile containing 0.1 % TFA, filtered to remove resin, then lyophilized.

Peptides were purified by reversed-phase HPLC and characterized by mass spectrometry (Rose, J.Am. Chem.Soc. 1 16:30, 1 994). Baseplates (also referred to as templates) were synthesized according to published procedures (Zeng et al., J. Peptide Sci. 2:66, 1 996; Rose et al. Mol. Immunol. 32: 1031 , 1 995).

Oxidations and oximations were performed essentially as described (Rose, J.Am. Chem.Soc. 1 16:30, 1 994; Zeng et al., J. Peptide Sci. 2:66, 1 996; Rose et al. Mol. Immunol. 32: 1031 , 1 995), with minor modifications imposed by the solubility properties of the reaction partners. In the case of T1 BT*, 6 mg peptide ( 1 .1 /mol) was dissolved in 1 .25 ml acetonitrile plus 3.75 ml imidazole buffer (50 mM, pH 6.95, counter-ion chloride). To 1 ml of this solution (221 .5 nmol) was added 55 μ\ ( 1 1000 nmol) of methionine (200 mM in water). Oxidation was initiated by adding 8.8 //I (880 nmol) NalO₄ ( 1 00 mM in water) . After 5 min in the dark, the reaction was quenched by adding 1 7.6 /I ethylene glycol ( 1 00 mM in water). The mixture was acidified with 40 μ\ acetic acid and the oxidized peptide isolated by semi-preparative HPLC. This procedure was repeated several times to obtain sufficient oxidized peptide to synthesize oximes with and without P₃C on the template. In the case of P₃C, 6 mg oxidized T1 BT* was dissolved in 100 μ\ acetonitrile plus 400 //I acetate buffer (solid guanidine hydrochloride was dissolved in 0.5 M acetate buffer, counter-ion sodium, pH 4, and the pH readjusted to 4 with acetic acid). Then 35 μ\ template (possessing four aminooxyacetyl groups and one P₃C, at a concentration of 5 mM in acetonitrile/water 1 :2) was added. After acidification with 50 μ\ pure acetic acid, the reaction was allowed to stand for 24 h prior to product isolation by semi-preparative HPLC. B. Immunizations:

Six-to-eight week old mice were obtained from Jackson Labs (Bar Harbor ME) . C57BI/6, A/J (H-2d) and BALB/c (H-2d) mice were immunized with 50 μg of the tri- or di-epitope polyoxime or MAP constructs. Mice were injected subcutaneously at three week intervals, and sera were obtained at 10 and 20 days after each immunization. The final bleed was obtained at 85 days after the third and final immunization.

C. Results: I. Di-epitope (T1B)₄ peptides:

A. Characterization of di-epitope polyoxime and MAP synthetic constructs

Tetrabranched polypeptides were synthesized by different chemical methods to contain the (T1 B) sequence, (DPNANPNV)₂(NANP)₃ , representing the NH₂-terminal and COOH-terminal repeat region of the P. falciparum CS protein. Two (T1 B)₄ polyoximes were constructed by condensation of four copies of the (T1 B) sequence on a branched polypeptide template, while the (T1 B)₄ MAP was synthesized by standard step wise procedure.

Both polyoxime and the MAP constructs gave a single peak on HPLC. Mass spectra, however, could be obtained only with the polyoxime construct; the MAPconstruct yielded an uninterpretable heterogeneous pattern. ESI-MS of the (T1 B)₄ polyoxime provided an experimentally determined molecular weight of 1 2,923, in agreement with the calculated mass of 1 2,922.5 (Figure 1 A). Consistent with the MS results, silver stained SDS-PAGE gels and

Western blots of the di-epitope MAP yielded a broad smear, ranging in molecular mass from 60,000 - 20,000 daltons. In contrast, similar preparations of the (T1 B)₄ polyoxime yielded a limited number of bands in the range of 20,000 - 30,000 daltons. The apparent heterogeneity of the polyoxime construct by SDS-PAGE is believed to reflect decomposition during sample preparation for electrophoresis. (Heating polyoximes in the presence of the assay components required for SDS-PAGE, i.e., mercaptoethanol, dithiothreitol, glycerol (or other substances capable of reacting with aldehydes or with aminooxy groups), leads to partial cleavage of oxime bonds) . B. Immunogenicity of di-epitope (T1 B)₄ polyoximes:

The immunogenic potential of the (T1 B)₄ polyoxime, as compared to (T1 B)₄ MAP, was tested in inbred strains of mice representing different responder phenotypes. In the high responder C57BI strain, both MAP and polyoxime (T1 B)₄ constructs were highly immunogenic and could elicit antibody responses even in the absence of adjuvant (Table 1 ). Three subcutaneous injections of either construct in PBS elicited anti-(T1 B)₄ antibody responses greater than 10⁴ in the C57BI mice. No response were detected in the intermediate (A/J) or low responder (BALB/c) strains immunized with peptides in the absence of adjuvant.

Table 1: Immunogenicity of di-epitope (T1B)₄ synthetic peptides constructed as polyoximes or MAP

Strain³ Adjuvant (T1B)₄ MAP (T1B)₄ Polyoxime"

109 103

C57BI none 12,902 81,920 81,920

alum 163,840 163,840 n.d.

A/J none < 80 < 80 < 80

alum 10,240 < 80 < 80

BALB/c none < 80 < 80 < 80

alum < 80 < 80 < 80

a. Mice were immunized s.c. with 50 ug of MAP or Polyoxime diluted in PBS (no adjuvant) or adsorbed to alum. Results shown as geometric mean titers (GMT) of sera collected

+ 20 days post third dose of peptide when assayed in an ELISA using (T1B)₄ MAP as antigen.

b. Two oxime isomers where tested: the oxime bonds in peptide 109 were formed through an aldehyde group on the peptide and an AOA group on the template; the 103 peptide contained the reverse polarity. Two isomers of the (T1 B)₄ Polyoxime, 109 and 103, were equally immunogenic and elicited anti-repeat antibody titers greater than 10⁴ in the C57BI mice when tested against the (T1 B)₄ MAP (Table 1 ) or the immunogen itself. The 109 isomer was constructed by ligation of an aldehyde-modified (T1 B)₄ peptide with branched template modified by four AoA groups, while the 103 isomer contained an oxime bond with the reverse polarity by condensation of an AoA-peptide with an aldehyde- modified template. The ability of the isomers to elicit the identical antibody responses indicates that the location of the aldehyde and AoA modifications on the peptide, or template, did not affect the immunogencity of the polyoximes.

The antibodies elicited by each of the polyoxime isomers cross- reacted equally well with the isomer of reciprocal polarity. The orientation of the oxime bonds also did not affect antigenicity, as both the 1 09 and the 1 03 polyoxime constructs were recognized with equal efficiency by the Mab 2A10, specific for the P. falciparum CS repeats region.

The (T1 B)₄ constructs were adsorbed to alum, the most common adjuvant used for human vaccine formulations, to determine if increased antibody responses could be obtained in the different strains of mice. Immunization with (T1 B)₄ MAP/ alum significantly increased the anti-repeat antibody titers of C57BI mice, while no significant increase was obtained with the polyoxime/alum formulation (Table 1 ) . The (T1 B)₄ MAP/alum also elicited antibodies in A/J mice. However, the alum-adjuvanted polyoxime was not immunogenic in these mice. The "non-responder" BALB/C mice also did not produce detectable antibody responses following immunization with either the (T1 B)₄ polyoxime or MAP adsorbed to alum.

The synthetic lipophilic adjuvant, tripalmitoyl-S-glyceryl cysteine (P₃C) is a potent adjuvant for synthetic peptide vaccines. Immunization with a (T1 B)₄-P₃C MAP construct has been shown to overcome genetic restriction and elicit high titers of antibodies in both responder and non-responder strains of mice. Four copies of an aldehyde modified (T1 B)₄ peptide were condensed on an AoA template containing the P₃C lipopeptide adjuvant. The

(T1 B)₄ -P₃C polyoxime yielded a single HPLC peak and could be characterized by ESI-MS (Figure 1 B). The experimentally determined MW of 14,549.8 was in close agreement with the calculated mass of 14,553.7.

The inclusion of the P₃C lipopeptide significantly enhanced immunogenicity of the (T1 B)₄ polyoxime and overcame genetic restriction of the antibody response (Figure 2). Following a single subcutaneous injection of the (T1 B)₄ polyoxime, positive anti-(T1 B)₄ ELISA titers were detected in all three strains of mice. Booster inoculations further increased the immune response and peak anti-peptide antibody titers of 8 X 1 0⁵ and 5 X 10⁴ were obtained in the high (C57BI) and intermediate (A/J) responder strains, respectively. Furthermore, significant anti-repeat antibody responses were induced in the "non-responder" BALB/c mice following immunization with the (T1 B)₄-P₃C polyoxime, although the peak antibody titers ( 1 .6 X 1 0⁴) were lower than those obtained in the high and intermediate responder strains.

The antibodies elicited by immunization with the (T1 B)₄-P₃C polyoxime recognized the CS protein on the P. falciparum sporozoite surface, as determined by indirect immunofluorescence (Figure 3) . Sera obtained from (T1 B)₄-P₃C-immunized C57BI mice gave IFA titers of over 1 X 1 0⁶ , comparable with peak antibody titers obtained with (T1 B)₄ MAP constructs using Freund's as adjuvant. In contrast to the good correlation with anti-repeat ELISA and IFA titers in the polyoxime-immunized C57BI mice, the sera of the A/J and BALB/c mice reacted at lower titers with sporozoites, with peak IFA titers of 1 ,280 and 320, respectively.

//. Tri-epitope (T1BT*) peptides:

A. Characterization of (T1 BT*)₄ polyoxime or MAP

While positive antibody responses were obtained in all three strains of mice following immunization with the di-epitope (T1 B)₄-P₃C polyoxime construct, the titers of anti-sporozoite antibodies were sub-optimal in the A/J and BALB/c mice. In order to increase antibody responses, tri-epitope polypeptides were synthesized to incorporate a parasite-derived universal T helper cell epitope that has been characterized in amino acids 326-345 of the P. falciparum CS protein.

The 326-345 epitope, EYLNKIQNSLSTEWSPCSVT (abbreviated T*), was combined with the (T1 B) epitope in a tri-epitope polypeptide containing the 48-mer sequence in each of four branches, (T1 BT*)₄. The tri- epitope (T1 BT*)₄ polyoxime constructs were prepared using ligation technology to condense four copies of the aldehydic 48-mer peptide with an AoA-template. A tri-epitope MAP was also synthesized using standard protocols in which each amino acid was added to the tetrabranched core in a step-wise fashion.

The step-wise synthesis of the tri-epitope MAP was difficult due to poor solubility and steric hindrance as the MAP increased in size and complexity. The final product did not yield an interpretable mass spectrum. In contrast, mass spectrometry of the (T1 BT*)₄ polyoxime provided the experimentally determined mass of 22,290 + /- 60, in agreement with the calculated mass of 22,340.7 (Figure 1 C).

B. Immunogenicity of (T1 BT*) polyoxime The immune response to (T1 BT*)₄ polyoxime versus MAP constructs, administered in phosphate buffered saline (PBS) adsorbed to alum, was compared in mice of different genetic backgrounds. Biologically relevant anti-repeat antibodies were measured by ELISA, using (T1 B)₄ MAP, or (T1 B)₄ polyoximes, as antigen. The incorporation of the 326-345 universal T cell epitope into the tri-epitope polyoxime provided T cell help for antibody responses in all three strains of mice, even in the absence of adjuvant (Table 2). These anti-repeat antibodies efficiently recognized the native CS protein on P. falciparum sporozoites as shown by the positive correlation of ELISA and IFA titers. Adsorption of (T1 BT*)₄ polyoxime to alum increased antibody responses 3 - 6 fold in different strains of mice. The greatest differences were noted in the IFA responses, which were 5-6 fold higher in BALB/c and A/J mice immunized with (T1 BT*)₄/alum when compared to (T1 BT*)₄in PBS. Immunization with the tri- epitope polyoxime/alum reversed the usual hierarchy of responder phenotypes, with higher titers in the BALB/c and lower titers in the C57BI mice.

Table 2: Immunogenicity of tri-epitope (T1 BT*)₄ peptide constructed by chemoligation (Polyoximes) or by stepwise synthesis (MAP)

STRAIN Adjuvant Polyoxime MAP

ELISA IFA ELISA IFA

C57BI none 16,255 20,480 905 452

alum 81,920 65,020 12,902 20,480

A/J none 65,020 20,480 80 <80

alum 30,040 103,213 160 <80

BALB/c none 103,213 51,606 160 80

alum 327,680 327,680 905 320

(2+/3) (2+/3)

Results shown for sera collected +20 days after the third subcutaneous injection of 50 ug tri-epitope (T1 BT*) peptide, administered in PBS (no adjuvant) or adsorbed to alum. ELISA: geometric mean titers (GMT) using (T1 B)₄ MAP as antigen. IFA: GMT using glutaraldehyde- fixed P. falciparum sporozoites. Three to four mice were immunized in each group; the number in parentheses indicates that only 2/3 of the immunized mice developed antibody responses after immunization with the tri-epitope MAP. In contrast to the polyoximes, the tri-epitope MAP was poorly immunogenic in all the murine strains, whether administered with or without alum adjuvant. Maximal titers were observed in the C57BI mice immunized with the (T1 BT*)₄ MAP/alum formulation, although the peak titer was lower than that obtained with the di-epitope (T1 B)₄ MAP/alum (Table 1 ). While the presence of the universal T helper epitope in the tri-epitope MAP overcame the genetic restriction to the CS repeats, the antibody response in the BALB/c mice immunized with (T1 BT*)₄ MAP/alum was low ( < 10³ GMT), as measured by both ELISA and IFA. The response to MAP/alum immunization in the BALB/c was also variable, with only 2 out of 3 mice in each group developing detectable antibody levels.

Based on the enhanced immunogenicity observed with the di- epitope (T1 B)₄-P₃C constructs (Figure 2), the P₃C lipid moiety was explored as an adjuvant for a tri-epitope (T1 BT*)₄ . Due to the technical difficulties encountered in the synthesis of the tri-epitope MAP, the construction of a lipopeptide modified tri-epitope MAP by step-wise synthesis was not attempted. However, a (T1 BT*)₄-P₃C polyoxime lipopeptide was easily constructed by ligation technology by condensation of the 48mer peptide with an AoA-template containing P₃C. The tri-epitope lipopeptide polyoxime was shown by chemical analysis to be of high purity. Mass spectrometry gave an experimental MW of 23,936 + /- 60, in excellent agreement with the calculated mass of 23,973.1 (Figure 1 D). Despite the complexity of the (T1 BT*)₄-P₃C polyoxime lipopeptide construct, SDS-PAGE silver stained gels and Western blots revealed defined bands and limited heterogeneity. The (T1 BT*)₄-P₃C polyoxime was immunogenic in all three strains of mice and elicited similar levels of antibody specific for the immunogen in each strain (Table 3). In contrast to the increased immunogenicity noted with the P₃C modified di-epitope (T1 B)₄ polyoxime, the adjuvant effect of the lipopeptide in the context of the tri-epitope (T1 BT*)₄ polyoxime was less marked (Table 3). Antibody levels induced by (T1 BT*)₄-P₃C polyoxime were similar to those obtained following immunization with the (T1 BT*)₄ polyoxime/alum formulation, when antibodies to the immunogen, the (T1 B)₄ repeats, or IFA were measured.

Table 3: Fine specificity of antibodies induced by immunization with tri-epitope (T1BT*)₄ polyoxime administered with or without adjuvant

(T1 BT^»).P,C IIIBI4 LT!!,

Strain Adjuvant ELISA ELISA ELISA IFA

Results shown as GMT for sera collected 20 days after the third injection of tri-epitope polyoximes administered without adjuvant (none), adsorbed to alum, or containing a P₃C lipid moiety. Antibody to the tri-epitope polyoxime was measured using (T1 BT*)₄--P₃C. The (T1 B)₄ and (T*)₄ ELISA used MAP as antigen. IFA were carried out using fixed P. falciparum sporozoites. The fine specificity of the antibody response in the (T1 BT*)₄ polyoxime- immunized mice was similar regardless of the adjuvant formulation. In immunized mice of all strains, the predominant antibody response was specific for the CS repeats, as measured by ELISA using (T1 B)₄ MAP as antigen. Identical anti-repeat titers were obtained using (T1 B)₄ polyoximes as antigen in the ELISA. Reactivity with P. falciparum sporozoites correlated with the anti-repeat antibody titers of the polyoxime-immunized mice (Table 3).

Antibody responses to the universal T helper epitope were also detected following immunization with all formulations of the (T1 BT*)₄ polyoxime, as previously found with MAPs containing the universal T-cell epitope (T*). The anti-T* antibody titers in the (T1 BT*)₄ polyoxime immunized mice were relatively low (10³ GMT) and did not correlate with sporozoite reactivity. Previous studies have also noted the failure of anti-peptide antibodies specific for non-repeat sequences of the CS protein to react with P. falciparum sporozoites.

A comparison of peak anti-repeat and anti-sporozoite antibody titers obtained in the different strains of mice following immunization with different formulations of the tri-epitope (T1 BT*)₄ polyoximes is illustrated in Figure 4. A strong adjuvant effect provided by alum or P₃C was not apparent when compared to the no adjuvant (PBS) groups. The presence of the universal T helper epitope in the (T1 BT*)₄ polyoxime may stimulate the production of sufficient levels of cytokine/lymphokines to mimic the immunostimulatorγ effect of exogenous adjuvants.

The presence of adjuvant in the (T1 BT*)₄ polyoxime formulations can be seen to shift the kinetics of the antibody response to early time points, without altering the pattern or persistence of the antibody response (Figure 5). The addition of the Pam₃Cys lipid moiety elicited the most rapid response with positive antibody titers in all three strains of mice following a single subcutaneous injection of (T1 BT*)₄-P₃C polyoxime. Peak antibody titers were maintained in all of the (T1 BT*)₄ polyoxime immunized mice for over 85 days after the third and final dose of antigen, regardless of the presence or absence of adjuvant.

SUMMARY: The results described above indicate that a tri-epitope polyoxime vaccine that includes a universal T-celi epitope overcomes the genetic restriction, as well as adjuvant dependence, of the immune response to malarial B-cell epitopes and provides a synthetic malaria vaccine that is highly immunogenic.

Example 2: Anti-Malarial Vaccines Comprising Polyoximes Studies in mice of different genetic backgrounds have shown that peptide-based vaccines containing the T* epitope (see above) are immunogenic in the absence of adjuvant, i.e., when administered in phosphate buffer alone. Enhanced antibody responses were obtained by the addition of adjuvants, such as alum (Rehydragel, Reheis NJ) or QS21 (Cambridge Biotech, Cambridge MA), or the covalent coupling of a synthetic lipopeptide, tri-palmitoyl-S-glyceryl cγsteine, to the branched peptide core.

A typical anti-malarial vaccine comprising a polyoxime contains 1 mg (T1 BT*)4 polyoxime containing tripalmitoyl-S-glyceryl cysteine, which is administered by subcutaneous injection.

Claims

Claims: 1. An immunogenic composition which comprises a first malaria-derived peptide comprising a T-cell epitope, wherein said T-cell epitope is incorporated into a polyoxime.

2. An immunogenic composition as defined in claim 1 , further comprising a second malaria-derived peptide comprising a B-cell epitope which stimulates the production of anti-malarial antibodies in mammals, wherein said B-cell epitope is incorporated into a polyoxime.

3. An immunogenic composition as defined in claim 1 , wherein said T-cell epitope comprises a malaria-derived universal T-cell epitope.

4. An immunogenic composition as defined in claim 1 , wherein said T-cell epitope comprises a malaria-derived T1 epitope.

5. An immunogenic composition as defined in claim 2, wherein said T-cell epitope comprises a malaria-derived universal T-cell epitope.

6. An immunogenic composition which comprises: (i) a B-cell epitope which stimulates the production of anti- malarial antibodies in mammals; (ii) a malaria-derived universal T-cell epitope; and (iii) a malaria-derived T1 epitope, wherein said epitopes are incorporated into polyoximes.

7. An immunogenic composition as defined in claim 6, wherein said B-cell epitope comprises (NANP)3 and said T1 epitope comprises (DPNANPNV)₂.

8. A vaccine comprising an immunogenic composition as defined in claim 1 and a pharmaceutically acceptable carrier or diluent.

9. A vaccine as defined in claim 5, further comprising a pharmaceutically acceptable adjuvant.

10. A method for inhibiting the propagation of a malarial organism in a susceptible mammal, which comprises administering to said mammal an immunogenically effective amount of a vaccine as defined in claim 5.

1 1. A method for eliciting protective immunity against malaria in a mammal, which comprises administering to said mammal an immunogenically effective amount of a vaccine as defined in claim 5.