MXPA99006712A - Universal t-cell epitopes for anti-malarial vaccines - Google Patents

Abstract

The present invention provides methods and compositions for eliciting protective immunity against malaria. In particular, the invention relates to universal T-cell epitopes that elicit T-cell responses in individuals of differing genetic backgrounds. Immunogenic compositions and vaccines comprising malaria-specific universal T-cell epitopes are disclosed.

Description

EPITHOPES OF UNIVERSAL T CELLS FOR VACCINES AGAINST MALARIA Field of the Invention This invention relates to effective vaccines for producing protective immunity against malaria, in particular vaccines comprising universal T cell epitopes that produce T cell responses in individuals of different genetic backgrounds. Background of the Invention Public health problems caused by malaria, which currently infects 400 to 500 million individuals worldwide, have been increased by the emergence of strains of multi-drug resistant parasites and insecticide-resistant mosquito vectors. . This development has led to the increasing efforts to provide an effective vaccine in order to avoid mortality and morbidity due to malaria, in particular P. falciparum, the most virulent of the Plasmodia species. In a mammalian host, malaria infection is indicated by the mobile sporozoite stage of the organism, which is injected into the circulation by the bite of infected mosquitoes. The sporozoite targets host liver cells through the interaction of a major component of the sporozoite surface membrane, the protein of circunasporozoite (SC), with specific receptors on the surface of hepatocytes. After intracellular multiplication and the release of broken hepatocytes, the parasites invade the cells of red blood cells and initiate the erythrocytic cycle of malaria; This phase of infection is responsible for the clinical disease and, in the case of P. falciparum, it can be lethal. A major focus of the development of the malaria vaccine has been the SC protein that is present in the stages of both sporozoites and the liver of the parasite. The polyclonal and monoclonal antibodies specific for the immunodominant B-cell epitope within the repeat region of the SC protein, the peptide (NAN P) 3, neutralizes the effectiveness of sporozoites of the malaria species of rodents, primates and humans (Nardin et al., J. Exp. Med. 156: 20, 1982). However, the use of the peptide (NAN P) 3 in a vaccine resulted in only a limited immune response, most likely due to the low density of the epitope and / or the lack of a suitable T-cell epitope (Herrington et al. , Nature 328: 257, 1987). The inventors of the present invention have defined T cell epitopes derived from parasites 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 confronted with infectious P. falciparum sporozoites, they were protected against malaria, as shown by the total absence of infection in the blood stage (Herrington et al., Am.J. Trop.Hyg. 45: 535, 1991).

Using CD4 + T cell clones derived from these volunteers immunized with sporozoites, two T-cell epitopes have been identified, one located in the repeat region and one at the C-terminus of the P. falciparum SC protein. The T-cell epitope contained in the N-terminal repeat region H2, called T1, consists of alternating repeats of NVDPNANP (Nardin et al., Science 246: 1603, 1989). The epitope of T1 is contiguous to, but is antigenically different from, the repeat region of COOH that contains the B cell epitope of (NANP) 3. Human CD4 + T cell clones that specifically recognize peptides derived from various combinations of the N-terminal repeat region and containing NVDPNANP do not respond to the repeating peptide (NAN P) 3. The T1 repeat epitope is conserved in all P. falciparum isolates sequenced so far and, therefore, it is expected that its inclusion in a vaccine induces reactive immune responses with parasites from various geographic regions. The second epitopes of T cells identified by human CD4 + T cells specific for sporozoites are contained in expanded amino acid residues of peptides numbered 326-345, EYLNKIQNSLSTEWSPCSVT, of the SC protein of the NF54 strain of P. falciparum (Moreno et al. , Int. Immunol., 3: 997, 1991; Moreno et al., J. Immunol. 151: 489.1993). It was shown that this epitope is recognized by the clones of restricted human CD4 + T cells class I I cytotoxic and non-cytotoxic and restricted CD8 + CTL class I. The sequence of 326-345 amino acids is unique since it overlaps a polymorphic region as well as a conserved one, Rl l (Dame et al., Science 225: 593, 1984), of the SC protein. The most conserved Rll contains a parasite ligand that interacts with the hepatocyte receptors to initiate the intracellular stage of the malaria life cycle. Human CD4 + T cells specific for peptides recognize a series of epitopes within the peptide of 326-345 peptides, all of which overlap with the conserved Rll found in the SC protein of all Plasmodium species.

The fact that the T * epitope is defined by CD4 + T cells derived from human volunteers immunized by many exposures to mosquito bites infected by malaria suggests that this peptide sequence is efficiently processed for presentation by the class II molecules. LA after exposure to the native SC protein in the sporozoite. It is contemplated that vaccines containing this T cell epitope derived from parasites can produce anamnestic responses in naturally infected individuals and can provide vaccine-induced immunity to be maintained by continuous exposure to parasites under natural conditions. Restricted class I CD4 + T cells play a central role in the induction of cellular and humoral immunity for the pre-erythrocytic stages of the malaria parasite (Nardin et al., Ann.

Rev. immunol. 1 X687, 1993). If the T cell epitopes contained within a synthetic malaria vaccine bind only on a limited scale of class II molecules, the vaccine may not produce immune responses in individuals of diverse genetic backgrounds. Previous studies have shown that repeats of (NAN P) 3 of the P. falciparum SC protein induced low or undetectable responses of T cells in naturally infected individuals living in endemic areas of malaria (Herrington et al., Nature 328 : 257, 1987; Etlinger et al., J. Immunol., 140: 626. 1988; Good et al., Proc. Nati, Acad. Sci. USA 85: 1 199, 1988). Therefore, there is a need in the art for T cell epitopes derived from parasites that bind to most, if not all, of class II molecules for inclusion in immunogenic compositions and vaccines, to provide protective immunity. against malaria in individuals of diverse genetic backgrounds. Brief Description of the Drawings Figure 1A is a fluorescence histogram obtained by incubation of EBV-B 9008 cells with biotinylated peptides. Figure 1 B is a hystogram of the fluorescence obtained by incubating 9065 cells of EBV-B with biotinylated peptides. Figure 2A is a graphic illustration of peptide proficiency ELISA using class I molecules DR4 (DRB1 * 0401). The variable concentrations of competing peptides 326-345, T1 or NAN P3 were tested for their ability to inhibit the binding of a biotinylated reporter peptide of GFK (A) 7 to soluble DR molecules. The peptide / MHC complexes were captured on ELISA plates coated with anti-DR Mab and revealed by incubation with HRP-avidin and peroxidase substrates. Figure 2B is a graphic illustration of peptide proficiency ELISA using class II molecules of DR 13 (DRB 1 * 1301), carried out as described for Figure 2A. Figure 3A is a graphic illustration of a peptide proficiency analysis using soluble class I I molecules of DQ 9 (DQ A1 * 0201 / DQ B1 * 0303) which are transported as described for Figure 2A. Figure 3B is a graphic illustration of a peptide proficiency analysis using soluble class I molecules of DQ 7 (DQ A1 * 0501 / DQ B 1 * 0301) which are transported as described for Figure 2A. Figure 4A is a graphic illustration of anti-PAM ELISA titers measured in mice that had been immunized intraperitoneally with 50 μg (T *) PAM. Figure 4C is a graphic illustration of anti-PAM ELISA titers measured in mice that had been immunized intraperitoneally with 50 μg (T * T1) 4 PAM. SUMMARY OF THE INVENTION The present invention encompasses immunogenic compositions that produce protective immunity against malaria. The compositions comprise a first malaria-derived peptide comprising a "universal" T-cell epitope, which produces a T-cell response against malaria in mammals of various genetic backgrounds. As used in this, mammals of "diverse genetic background" include without limitation mammals expressing a multiplicity of class I I haplotypes of MHC. In one embodiment, the universal T cell epitope comprises the sequence EYLNKIQNSLSTEWSPCSVT. Preferably, the compositions of the invention further comprise at least one second malaria peptide comprising a B cell epitope, which stimulates the production of anti-malarial antibodies in mammals. The compositions may also comprise additional T cell epitopes. The compositions are preferably formulated into vaccines, which also comprise a pharmaceutically acceptable carrier or diluent and, optionally, an adjuvant. In another aspect, the invention provides methods for inhibiting the spread of malaria organisms in a susceptible animal, preferably by producing a protective immunity against malaria in the mammal. The methods were carried out by administering to mammals an immunogenically effective amount 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 incorporated herein by reference in their entirety. In the case of inconsistencies, the present description, including definitions, will be the one that controls. Definitions: 1. An "immunogenic composition" is a composition that produces 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 yields 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 form that is specific for the peptide derived from parasites 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 form that activates the T cell function in a manner restricted to class II or class I. The activated T cells can be auxiliary cells (CD4 +) and / or cytotoxic cells (CD4 + restricted to class II and / or CD8 + restricted to class I). In one embodiment, the universal T cell epitope comprises the sequence EYLNKIQNSLSTEWSPCSVT. In another embodiment, the universal T cell epitope consists essentially of the sequence EYLNKIQNSLSTEWSPCSVT. As used herein, an epitope "consisting essentially of" is a sequence of peptides encompassing peptides in which one or more amino acids can be deleted or substituted while retaining the ability of the peptide to bind to a multiplicity of molecules of the peptide. MHC class II and / or to activate T cell functions as cells carrying said molecules. It will be understood that the deletion or substitution of one or more amino acids may alter the ability of the peptide to bind to one or more MHC Class I molecules but will still allow binding to a multiplicity of other MHC Class II molecules. An epitope of universal T cells specific for malaria or specific for parasites, has the potential to expand, or induce, T cells specific for parasites in naturally infected individuals without affectation, 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 is encoded by the organism's genome. It will be understood that changes can be made in the sequence of a peptide relative to the polypeptide from which it is derived, which does not negate the ability of the altered peptide, when used as part of an immunogenic composition, to give an immune response that is specific for the polypeptide from which the peptide is derived.

. "Multiple Antigen Peptide" (PAM) refers to the peptide multimer formed from a polylysine core and containing a branched table on which the peptides are conjugated (Tam, J. Immunol. Meth. 196: 17, 1996; Nardin et al., Adv. Immunol. 60: 1 05, 1995). The present invention provides immunogenic compositions and methods for producing protective immunity against malaria, in particular against P. falciparum. The compositions comprise one or more of the following components: (i) at least one malaria-derived peptide comprising a universal T-cell epitope capable of producing a T-cell response against malaria in vaccines of various genetic backgrounds; and (ii) at least one malaria-derived peptide comprising a B-cell epitope capable of stimulating the production of anti-malaria (ie, neutralizing) antibodies directed against the sporozoite stage of the malaria organisms. Preferably, the immunogenic compositions of the present invention comprise at least one B cell epitope and at least one T cell epitope, more preferably a universal T cell epitope. B cell epitopes preferably give the production of antibodies that specifically recognize and bind to the malarial circumsporozoite (SC) protein. The compositions may comprise epitopes of B cells and / or T cells derived from, and reactive with, other malarial components, such as, for example, the sporozoite surface protein of P. falciparum designated Trombospondin-related Accession Protein (Anonymous) (TRAP), also called Esporozoite Surface Protein 2 (SSP2, for its acronym in English); LSA I, hsp 70; SAUCE; STARP, Hep17; MSA; RAP-1; and RAP-2. In one embodiment, the B cell epitope and the universal T cell epitope components are incorporated into multiple antigen peptides (PAMs), which form a synthetic macromolecular polypeptide containing a high epitope density. Methods for the synthesis of MAP are described in (Tam, Proc Nati, Acad Sci USA 85: 5409, 1988, Tam, Meth, Enzymol, 168: 7, 1989). The present invention encompasses epitopes of B cells and T cells derived from plasmodial species including without limitation P. falciparum, P. vivax, P. malariae, P. ovale, P. reichenowi, P. knowlesi, P. cianomolgi, P. brasilianum, P. yoelii, P. berghei and P. chabaudi. The epitopes usually comprise at least 5 amino acid residues, preferably at least 7 residues and more preferably at least 10 residues, derived from a plasmodial protein. B cell epitopes can be identified by methods well known in the art, such as, for example, (i) preparing synthetic peptides whose sequences are derived from the SC protein of a plasmodial species; and (ii) testing the ability of synthetic peptides to give anti-malarial antibodies in a model system. The B cell and T cell epitopes specific for malaria are described in Nardin et al., Ann. Rev. Immunol. 1J_: 687, 1993.

In a preferred embodiment, the immunogenic composition of the invention comprises a peptide comprising the B cell epitope (NAN P) 3 and a peptide comprising the universal T cell epitope represented by amino acid residues numbered 326-345, EYLN KIQNSLSTEWSPCSVT, of the CF protein of the NF54 strain of P. falciparum, or immunogenic variants derived therefrom. In another preferred embodiment, the immunogenic composition of the invention encompasses (NAN P) 3 EYLN KIQNSLSTEWSPCSVT and the epitope T1. Related sequences in other isolates and in other malarial species share an identical pattern of aliphatic and aromatic residues at positions 327, 328, 331, 335 and 339. These residues are thought to represent critical supports for peptide attachment within the cleft of peptide binding of class II or class I molecules. Consequently, the sequences related to EYLN KIQNSLSTEWSPCSVT that share these structural aspects and / or efficiently bind to different class I I or class I molecules can be used in the invention. Other universal T cell epitopes can be identified for use in the present invention using experimental methods described below for EYLNKIQNSLSTEWSPCSVT. Identification of Malarial Epitopes of Universal T Cells In the practice of the present invention, the universal T cell epitopes specific for malaria were identified using one or more of the following methods: (i) experimentally measuring the interaction of different malaria-derived peptides with Class II polypeptides isolated in vitro; and (ii) analyzing by computer different peptide sequences to identify specific motifs for high affinity class I I alleles. 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 (MAP) peptides containing these T cell epitopes. Similarly, a TRAP / SS derived peptide from P. falciparum was previously understood to comprise a universal T cell epitope, it has been experimentally shown to bind to multiple class II molecules in vitro. These methods for the identification of universal T cell receptors are described in more detail below. I. In vitro analysis: MATERIALS AND METHODS Peptides: The synthesis of multiple antigen peptides (PAM) was carried out as originally described (Tam, Proc Nati, Acad Sci USA 85: 5409. 1988). The solid-phase step synthesis based on the Boc peptide chemistry was used to synthesize the T cell epitopes in a tetra-branched matrix constructed using the alpha amino and epsilon groups of lysine. Two mono-epitope MAPs containing one of the epitopes T1 (DPNANPNV) 2, abbreviated (T1), or only the T cell epitope 326-345 of the SC protein of the NF54 strain of P. falciparum, EYLNKIQNSLSTEWSPCSVT, abbreviated were constructed. (T *) 4. A PAM of two tetra-branched epitopes containing both the T * epitope and the T1 epitope [T * T1] 4, synthesized as the 36-mer sequence with the distal T * epitope of the lysine matrix, was also concluded . Peptides T1, 326-345 and (NANP) 3 biotinylated with terminal NH2 were purchased from AnaSpect (Anaheim, CA). The purity of more than 90% of the peptides by HPLC and biotinylation of the peptides was confirmed by mass spectrometry. Mice: Four strains of offspring of 6-8 week old mice were obtained from Jackson Laboratories, Bar Harbor, ME: groups of 5-10 mice of strains A / J (H-2a), C57B1 / 10 (H -2), BALB / c (H-2d) and C3H (H-2k) were immunized by three intraperitoneal injections of 50 μg of mono PAM or di-epitopes emulsified in Freund's adjuvant. Sera were recovered 14-20 days after immunization for serological analyzes. Serological ELISA assays: Enzyme linked immunosorbent assay (ELISA) was carried out using mono PAM or di-epitopes as antigens (Munesingh et al., Eur. J. Immunol. 12: 3015, 1991). ELISA wells coated with blocked PAM were incubated twice with dilutions of serum in PBS / 0.05% Tween / 2.5% BSA. After washing, the bound antibody was detected using peroxidase-labeled anti-mouse IgG (specific for chain?) (Kirkegaard and Perry Gaithersburg, MD) and ABTS (2,2'-Asino-di- (3-ethylbenzthiazoline sulfate) / H2O2 as a substrate The geometric mean titrations (TMG) were determined for each group using the final dilution of serum that has OD higher than the average +3 SD of pre-immune serum as the end point .AIF: indirect immunofluorescence (AI) F) was carried out using P. falciparum sporozoites fixed with glutaraldehyde and FITC-labeled anti-mouse IgG to detect the bound antibody.The sporozoites were excised from the salivary glands of Anopheles mosquitoes infected by feeding gametocytes of P. falciparum (strain N F54) derived from in vitro blood-phase cultures Peptide Binding Analysis Peptide binding of cells expressing class II molecules finidas: The binding of biotinylated peptides to EBV-B cells of defined haplotypes, or L cells transfected with DR molecules, were evaluated by flow cytometry (Busch et al., J. Immunol., Meth. 134: 1, 1990). The lines of EBV-B cells 9065 and 9008, which present peptides to clones of CD4 + T cells more specific to T1 were tested for the ability to bind to biotinylated T1, (NAN P) 3 or peptides 326-345. For flow cytometry, EBV-B or L cells (2x105 cells) were incubated with an equal volume of (100 μl) of biotinylated peptide (200 μg / ml) in each well of a 96-well plate with background of U shape. After a 4 hour incubation on ice with moderate agitation, the unbound peptides were removed by washing. To increase the sensitivity of the fluorescent signal, two layers of FITC-Avidin were used to label the cells by first incubation with FITC-Avidin D, followed by biotinylated anti-Avidin D and again with FITC-Avidin DCS (Vector, Brulingame CA ). Propidium iodide (2.8 ug / ml) was added before the FACS analysis to allow regulation in viable cells. ELISA binding peptide: Peptide interactions with soluble DR or DQ molecules were measured using an ELISA binding peptide (Hammer et al., J. Exp. Med. 180: 2353. 1994). Class II molecules were obtained from approximately 109 EBV-B cells by lysis and extraction using 1% NP-40 (v / v) and a cocktail of protease inhibitors. The class II molecules in the cell extracts were purified by immunoaffinity on a Sepharose-Protein A anti-class II Mab column constructed using Mab specific for DR (ATCC HB-55) or DQ (ATCC 144 or SPV-) molecules. YOU). The homozygous EBV-B lines were used as the source of the class II molecules for each of the DR peptide proficiency analyzes: DR 1 HOM-2 molecules (DRB1 * 0101), DR 3 - WT49 (DRB1 * 0301), DR4-BSM or PREISS (DRB1 * 0401), DR 7 - EKR (DRB 1 * 0701), DR 8 - BM9 (DRB 1 *? 0801); DR 1 1 -SWEIG (DRB1 * 1 101) and DR 13 -HHKB (DRB1 301). DR 2a (DRB5 * 0101) were isolated from L cells transfected to L416-3. The DQ peptide proficiency analysis used soluble DQ 7 molecules (DQA1 * 0501 / DQB 1 * 0301) derived from SWEIG EBV-B cells. DQ 9 aβ dimers (DQA 1 * 0201 / DQB 1 * 0303) were produced in insect cells using the baculovirus expression system. In the peptide-binding assay, an optimal concentration of purified DR or DQ molecules was added to each well of a 96-well plate along with the biotinylated reporter peptide in citrate-phosphate buffer containing 2% n-octyl -glucoside, PMSF, EDTA and protease inhibitors. A binding buffer at pH 7 was used for all DQ and DR analyzes, with the exception of the DRB1 * 0701 buffer that was pH 5. After incubation overnight at room temperature (RT) or at 37 ° C, the peptide / class II complexes were transferred to the wells coated with L234 antibody from Mab anti-DR (15 μg / ml) or HB 144 Mab anti-DQ (3.5 μg / ml). After a two-hour incubation, the wells were washed with PBS + 1% Tween, and the capture of biotinylated peptide / class II molecule complexes was revealed by the addition of alkaline phosphatase labeled streptavidin and substrate, p nitrophenyl phosphate (Kierkegaard and Perry, Gaithersburg, MD). The optical densities were determined in a Titertek MC Multiscan ELISA vector (Flow Labs) using a 405 nm filter.

To increase the sensitivity, the known biotinylated reporter peptides to optimally bind to the different DR alleles were used in the peptide proficiency analysis. The polyalanine designer peptides containing specific parallel binding motifs were used as reporter peptides since these peptides allowed the detection of competitors with increments of 100 fold decreases in binding affinity. GIy-Phe-Lys- (Ala) 7 biotinylated designated G FK (A) 7 was used as a reporter peptide in the DR 1, 4, 7 and 13 analyzes and in the DQ analyzes. The DR 3 analysis used biotinylated IAYD (A) S and the DR 8 assays used a biotinylated GYR (A) 6L indicator peptide. Competition analyzes of DR 4 were also carried out using biotinylated peptide U D4, YPKFVKQNTLKAA, designed for optimal binding to all DR 4 allotypes. Binding to DR 2 molecules (DRB5 * 0101) was measured using biotinylated peptide of MBP of myelin basic protein. For peptide proficiency analysis, an optimal concentration of the biotinylated reporter peptide (0.1 μM - 5 μM) was incubated with ten-fold dilutions (0.01 μM - 100 μM) of the unlabeled competing peptides, T1, aa 326-345 or (NANP) 3. In each competition analysis, an unlabeled peptide of defined class I binding specificity was included as a positive control and allowed for relative affinity determination. The ability of the unlabeled competitor peptide to compete with the biotinylated reporter peptide to bind to the class II molecule was revealed by measuring the optimum density (D.O.). The inhibition was calculated as a percentage using the formula: 100 X 1 - (? D.O. in the presence of the competing peptide /? D. O. in the absence of competitor). The concentration of the competing peptide required to inhibit 50% binding of the biotinylated reporter peptide (ICso) was determined and the ICSo 100 μM was taken as an indication of the binding of the peptide to the class II molecule. RESULTS Binding of T cell epitopes of SC to class II molecules associated with cells: Human CD4 + T cell clones derived from volunteers immunized with sporozoites recognized the T cell epitopes on the P. falciparum SC protein in the context of class II molecules of DR or DQ. The clones specific for the T cell epitope 326-345 (T *) of the P. falciparum SC protein were restricted by multiple DR alleles, including DR 1, DR 4, DR 7 or DR 9. Genetic restriction of the epitope of T1, located in the repeated region of the P. falciparum SC protein, was recently defined. Monoclonal antibodies specific for monomorphic determinants of DQ, but not for DR molecules, significantly inhibited the proliferative response of T cell clones specific for T1 peptides. When the EBV-B cells expressing the DR / DQ haplotype of the T-cell donor immunized with sporozoites (DRB 1 * 1502 / * 1301, DQB1 * 0602/0603) was used as APC, only the cells expressing DQB 1 * 0603 could present e! T1 peptide to T cell clones. However, the number of T cells specific for the peptides available for the study of genetic constraints, has been limited by a small number of volunteers immunized by sporozoites. To obtain additional information about the scale of class II molecules that could potentially function in the presentation of T1 and 326-345 T cell epitopes, in vitro binding analyzes were carried out using defined haplotype or transfectant cell lines. DR. to. Binding analysis using EBV-B cells of defined class ll haplotypes. To determine if EBV-B of known haplotypes can be used to screen molecules capable of binding to the SC epitopes, cell lines were tested to bind the peptides of T1 and 326-345. The biotinylated peptide (NANP) 3, known to be poorly recognized by human T cells, was also tested. It is known that two EBV-B cell lines, one expressing DR 4 (BSM) and one expressing DR 7 (EKR), function as APC for the presentation of peptide 326-345 to clones of restricted T cells of DR 4 and DR 7. As measured by flow cytometry , the biotinylated peptide 326-345 to the lines bound to the BSM and EKR cell lines with medium fluorescent channels (CFM) of 251 and 142, respectively. However, no detectable binding of the T1 epitope or the biotinylated peptide (NAN P) 3 to these cells was obtained (CFM <; 35). In the inverse analyzes, the EBV-B cell lines known to function as APC for the T cell clones specific for the T1 peptide were tested for their ability to bind to detectable levels of biotinylated SCs. Peptide binding T1 biotinylated to cell lines of EBV-B 9008 and 9065, which express DRB1 * 1501 / DQB 1 * 0602/0603 and haplotypes DRB1 * 1301 / DQB1 * 0603, could not be detected (Figures 1A and 1 B). In contrast, peptide 326-345 bound to both of these cells EBV-B (9008 or 9065) with a CFM of 403 and 758, respectively. b. Peptide binding to transfected L cells: Because EBV-B cells express multiple class II isotypes, the positive fluorescence obtained with peptide 326-345 could reflect the binding of DR and / or DQ molecules, or other molecules of HLA The class II specificity of the peptide binding was determined by measuring the interaction of the biotinylated SC peptides with L cells transfected with DR. The level of expression of DR on the surface of the different transfectants was compared with that observed in EBV-B cells, varying CFM from 443 to 964 following the tinsion with monoclonal anti-DR antibodies (L243) (Table 1).

Table 1: Binding of biotinylated malaria peptides to L cells of murine transfected with DR Peptide TRANSFECTANTS OF DR Biotinylated (CFMa) DRBI * 0401 DRBI * 0701 DRB 1501 Biotinil-326-345 217.1 203.8 167.7 Biotinil-T1 18.9 35.7 12.7 Biotinyl- (NAN P) 3 12.9 22.8 12.7 Anti-DR Mab 91 1 .4 443.5 964.4 Control Mab 18.5 23.8 19.3 to. The binding of biotinylated SC peptides (100 μg / ml) to L cells of murine transfected with DRA 1 * 0101 and DRB1 * 0401, * 0701 or * 1501 genes was measured by FACS. The results are expressed as fluorescent media channel (CFM). b. The expression of class I I in each of the transfectants was demonstrated by staining with Mab specific for human Class I I molecules (Mab L234) or a negative Mab control (3D 1 1) (50 ug / ml).

No significant fluorescence was obtained when the biotinylated T1 peptide or the peptide (NAN P) 3) was incubated with the transfected DR cell lines. The biotinylated 326-345 peptide bound to cells transfected with DRB1 * 0401 and * 0701 with CFMs of 217 and 203, respectively, which are consistent with the specificity of alleles of CD4 + T cell clones restricted with DR4 and DR7 specific for the peptide. In addition, peptide 326-345 was also shown to bind to L cells transfected with DR B1 * 1501 (CFM 167), which is consistent with the positive binding observed with DRV 15 positive cell line EBV-B 9008 (Figure 1 A). Binding of TS cell epitopes to soluble class II molecules In order to measure the binding affinity of peptides and regulate non-specific interactions with non-MHC cell surface molecules expressed on human cell lines and In the murine case, competitive peptide binding assays could be carried out using soluble class II molecules. 1. DR Molecules To increase the sensitivity and specificity of the peptide binding assays, competition analyzes were carried out using a biotinylated reporter peptide GFK (A) 7, a polyalanine peptide that binds to the DR molecule with an affinity that allows competition for peptides with a 100-fold affinity scale. As shown by the dose response curve for various concentrations of cold competing peptide, peptide 326-345, but not peptide T1 or (NAN P) 3, one could effectively inhibit GFK (A) indicator peptide binding. to the soluble DR4 molecules (Figure 2A). Similar results were obtained when peptide 326-345 was tested in peptide proficiency analysis using soluble DR 13 molecules (Figure 2B). The concentration of peptide 326-345 required to inhibit 50% binding of the biotinylated GFK (A) peptide (IC50) was comparable in both competition analyzes of peptides DR 4 (ICso 0.2 μM) and DR 13 (IC500.33 μM). Neither the T1 peptide, nor the peptide (NANP) 3, gave detectable inhibition at the highest concentration tested (ICso 100μM). The results of a series of peptide binding competition analyzes, carried out using different biotinylated peptides selected for optimal binding to each DR allele, are summarized in Table 2.

Table 2: Peptide Binding Competition Analysis using soluble PR molecules DRe DRB1 * Peptide IC50 of Biotinylated Competitive Peptide HA307-319 326-345 T1 (NANP) 3 DR1 DRB1 * 0101 GFK (A) 7 0.10 20.0 > 100 > 100 DR2 DRB5 * 0101 MBP 0.03 80.0 > 100 > 100 DR3 DRB1 * 0301 IAYD (A) 5 10.00 70.0 > 100 > 100 DR4 DRB1 * 0401 UD4 1.00 0.7 > 100 > 100 DR7 DRB1 * 0701 GFK (A) 7 0.10 0.4 > 100 > 100 DR8 DRB1 * 0801 GYR (A) 6L 5.00 10.0 > 100 > 100 DR11 (5) DRB1 * 1101 T831-843 1.00 40.0 > 100 > 100 a. The results are expressed as ICso, the concentration (μM) of the unlabeled competitor peptide required to inhibit 50% of the binding of a biotinylated reporter peptide. Percent inhibition was calculated based on D.O. obtained in the presence of different concentrations of competing peptide (100 - 0.001 μM). An ICso < 100 μM indicates positive peptide binding. A known positive competitor peptide derived from influenza hemagglutinin, HA307-319 was included in each analysis in order to determine the relative binding affinity of the SC peptides to each DR allele. Based on these analyzes, peptide 326-345 could be shown to bind DRB1 * gene products that encode class II molecules of DR 1, DR 4, DR 7, DR 8, DR 11 and DR 13 (Figure 2, Table 2) . Peptide 326-345 was a competitor to bind DR 3 molecules (ICSO 70 μM) and DR 2a molecules, encoded by DR B5 * 0101 (ICSO 80 μM). Significant binding of the T1 peptide, or the peptide (NANP) 3, was not detected with any of the soluble DR molecules tested in the peptide binding assay (ICso 100μM). The binding affinity of peptide 326-345 was different for each allele of DR as determined by ICS0 and the relative affinity when compared to peptide HA307-319. In the case of DR alleles 4, 7, 8, the binding of the SC 326-345 peptide was comparable with the universal HA peptide, with ICso ratios of HA307-3? G / CS326-345 of 1.4, 0.25 and 0.5, respectively. Without. However, the relative binding affinity of peptide 326-345 to DR 1 and DR 11 was lower, with IC50 ratios of 0.005 and 0.025. 2. DQ Molecules The results of DR binding analysis indicated that peptide 326-345 could bind to multiple DR molecules, while peptide T1 and peptide (NANP) 3 did not bind with high affinity to any of the DR molecules tested. To determine if the epitope of T1 restricted by DQ 6 could bind to other DQ alleles, the peptide competencies using soluble DQ molecules were carried out. Peptide proficiency analyzes used soluble DQ 7 (DQA1 * 0501 / B1 * 0301) and DQ 9 molecules were established (DQA1 * 0201 / B1 * 0303). A known DQ binding peptide, CLIP83-10? Amino acid derivative 83-101 of the Invariant chain was included in each analysis to determine the relative binding affinity of the SC peptides to the soluble DQ molecules. The T1 peptide, which was known to bind DQ 6 molecules, did not bind either DQ 7 or DQ 9 molecules (Figure 3). Similarly, the (NANP) 3 peptide does not compete with the peptide CLIP83-? O? to join any of the DQ allele. In contrast, peptide 326-345 could compete with the CLIP peptide to bind to DQ molecules. In the competition analysis using soluble DQ molecules, peptide 326-345 gave an IC50 of 2 μM, a binding affinity on the scale obtained with the peptide CLIP83.?o? (IC50 0.5 μM) (Figure 3A). Peptide 326-345 binding was also detected with soluble DQ 7 molecules (IC5020 μM), although the affinity of the peptide / DQ interaction was somebody compared to the CLIP peptide (ICSo 0.5 uM) (Figure 3B).

Immunogenicity of synthetic peptide vaccines containing T * T1 epitopes: a. Immunization with T cell epitopes of SC containing PAM of a mono-epitope. The results of the peptide binding assays demonstrated that peptide 326-345 could be bound to a broad scale of class I molecules I, whereas the T1 peptide showed detectable binding only to the DQ 6 molecule in the T cell analyzes. In order to determine whether the genetic constraints of broad limited contra of peptides 326-345 and T1 were correlated with immunogenicity in vivo, the immune response of multiple antigen peptides (PAM) containing the epitope 326-345 or T1 was determined in different mouse strains. Preliminary studies have determined that the 326-345 epitope contained B cells, as well as the T cell epitopes and therefore the anti-PAM antibody response was used as an indicator of functional class II restricted T helper cells in immunized mice with PAM. Consistent with the binding of peptide 326-345 to multiple class II molecules in vitro, mono-epitope MAP containing only the sequence 326-345 (abbreviated T *) produced anti-peptide responses in the four strains of mice tested (Figure 4B). The magnitude of the response was genetically restricted, with high levels of anti-peptide antibody obtained in BALB / c (H-2d) and C51 B1 (H-2b) and intermediate levels in A / J mice (H-2a).

All mice in the high and intermediate response strains developed similar levels of anti-peptide antibody after immunization with 326-345 PAM (SE <10%). However, lower antibody responses were more variable, in C3H (H-2k) in which 2/5 mice immunized with PAM responded with detectable levels of antibodies. In contrast, for the PAM (T *) 4 response containing the 326-345 epitope, the PAM of a mono-epitope containing the T1 epitope produced anti-peptide antibody responses in only one strain of mice, H-2b ( Figure 4A), consistent with previously published results (36). The genetic restriction of the murine response to the N2 terminal repeating T1 epitope is therefore the same as that observed for the terminal COOH repeat (NAN P) 3 sequence, with T-cell epitopes recognized only by the C57B1 mice (H-2b). To determine whether anti-peptide antibodies produced by PAM containing the repeating T1, or the 326-345 COOH-terminal sequence, could recognize the sporozoite SC protein from P. falciparum, indirect immunofluorescence assays (AI F) they took place. It was previously found that immunization with PAM constructs containing terminal COOH sequences of the P. falciparum SC protein frequently produced high levels of anti-peptide antibodies that did not react with sporozoites. In agreement with these early findings, only the anti-PAM antibodies that recognized the repeat region of the SC protein were reactive with the sporozoites. Therefore, while BALB / c mice immunized with (T *) 4 developed the highest titers of anti-326-345 antibodies (ELISA TMG 163,840), no reactivity was detected with sporozoites of P. falciparum (AI F < 80). In contrast, the only strain of mice, C57B1, that responds to immunization with the PAM of (T1) 4 mono-epitope containing the N-terminal repeat T-cell epitope (Figure 3A), gave ELISA titrations of comparable anti-T1 peptides (TMG 327, 680) and titers of AI F with sporozoites of P. falciparum (163-840). b. Immunization with PAM of di-epitopes The results of the peptide-binding assays and the immunogenicity studies in different strains of mice demonstrate that the peptide 326-345 can be recognized by multiple class I I molecules of human and murine. To determine whether the inclusion of the T cell epitope 326-345 in a synthetic vaccine could overcome the genetic restriction of the immune response to the repeat region of the P. falciparum SC proteins, a PAM of (T * T1) was synthesized. 4 of di-epitopes containing the epitope 326-345 randomly with the T1 epitope. The anti-PAM antibody response in the mice immunized with PAM (T * T1) shows that, as was found with the PAM (T *) of mono-epitope, the four strains of mice responded to the immunization and produced high levels of anti-peptide antibodies (Figure 4C). The magnitude of anti-(T * T1) 4 antibody response in different strains demonstrated the same hierarchy as that obtained in mice immunized with monoamino-epitope PAM (T *) 4, i.e., BALB / c, C57B 1 > A / J > C3H. The kinetics of the anti-PAM antibody response was faster in the mice immunized with di-epitope (Figure 4C). Anti-PAM titers exceeding 105 could be detected after a single dose of (T * T1) 4 in C57B1 mice. The lowest antibody titers were obtained with C3H mice; however, in contrast to mice immunized with the PAM of mono-epitopes, all mice immunized with PAM (T * T1) 4 from di-epitopes developed anti-PAM antibodies. What is more impotent is that the analysis of the fine specificity of the antibody responses showed that all strains of mice immunized with PAM developed antibodies reactive with sporozoites of P. falciparum (Table 3). As observed with the construction of previous PAM that contains repetitions of the SC protein of P. falciparum, there was a positive correlation between the level of anti-repeat antibodies, as measured by PAM ELISA (T1) and the reactivity with sporozoites of P. falciparum in the serum of mice immunized with PAM of di-epitopes.

Table 3: Thin specificity of antibodies produced by immunization with PAM (T * T1) 4 of di-epitopes CEPA ELISA IT * T1) ELISA IT *) _. ELISA IT1) IFA BALB / C 1, 558, 718 48,710 1 15, 852 163,840 C57BL 702, 398 31, 042 100,855 133, 079 A / J 327,680 1, 810 40,960 27, 024 C3H 94, 101 452 1, 470 3,225 The results are shown as TMG for serum obtained +28 days after the third injection i. p. of MAP (T * T1) in Freund's adjuvant. The ELISA was carried out using a PAM of di-epitope or mono-epitope as antigen. AI F is based on sporozoites (N F54) of P. falciparum fixed with glutaraldehyde. The magnitude of the anti-repeat and anti-sporozoite antibodies produced in different murine strains reflected the genetic restriction pattern of the epitope 326-345. The high responders (C57B 1, BALB / c, A / J) and low (C3H) to the PAM of (T *) 4 mono-epitopes were also high and low responders in the production of anti-sporozoite antibodies after immunization with PAM of di-epitopes. Vaccines The compositions of the present invention can be used as immunogens to produce immunity, including protective immunity, in a susceptible host. Immunity may include the production of antibodies in the host (or in another host or in vitro, as passive immunization) that will recognize and bind to plasmodial cells. Immunity may also include activation of T cells specific for malaria. Therefore, immunogenic compositions comprising universal T cell epitopes can be used in vaccine preparations to confer prophylactic immunity or therapeutic immunity by preventing (totally or partially) the spread of the disease in the host, such as, for example, by inhibiting the development of the pre-erythrocyte stages of the organism. It should be noted that 100% inhibition of any stage in infection or spread of malaria by an immunogenic composition (or by a vaccine containing it, or by an antibody) is not necessary for these materials to be useful. Any substantial decrease in the degree of infection (measured, eg, by the degree of parasitemia) could substantially attenuate clinical symptoms and substantially increase the likelihood of survival and recovery of the host. There are many protocols for the preparation of known vaccines in the art. Normally, vaccines are prepared as injectables, either as solutions or liquid suspensions. Suitable solid forms can also be prepared to dissolve or suspend in liquid prior to injection. The preparation can also be emulsified, or the encapsulated protein, in liposomes. The active immunogenic ingredients can 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 regulating agents and / or adjuvants to increase the effectiveness of the vaccine. The immunogenic compositions could also be administered after incorporation into liposomes or other microcarriers. Repetitive immunizations may be necessary to allow the host to have an immune response. Both protocols of immunogenicity and immunization can be determined experimentally, as is well known in the art, using animal models (eg, primates) followed by clinical tests in humans. The information in vaccine and immunization compositions is described for example in the Patent of E. U.A. No. 4, 767,622 of Ristic (August 30, 1988); Patent of E. U.A. No. 4,735, 799 of Patarroyo (April 05, 1988) and Patarroyo, M. E., et al., Nature 332: 158. 1988; and the published European Application Ai 250,261 (published on December 23, 1987) of the Wellcome Foundation. Vaccines can be administered by subcutaneous routes, intramuscular, oral, intradermal or intranasal. Doses may vary from about 5 μg to about 5 mg per dose and a single or multiple dose regimen may be used. The amounts administered, number of administrations and program of administrations can be determined empirically, such as, for example, establishing a dose and frequency matrix and comparing a group of experimental units or subjects for each point in the matrix. The present invention also provides methods for inhibiting the spread of a malaria 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 a malaria-derived peptide containing the B cell epitope capable of stimulating the production of anti-malarial antibodies (ie, neutralization) against the sporozoite stage of the organism; and (ii) at least one malaria derived peptide comprising a universal T cell epitope capable of producing an anti-malarial T cell response in vaccines of various genetic backgrounds. An immunogenically effective amount is an amount effective to give protective immunity against the malaria organism determined as described above. In a further aspect, the composition can be administered to a mammal which was previously exposed to the malaria organism. In yet a further aspect, the polypeptide can be administered to a mammal prior to exposure of the mammal to the malaria organism. The following examples are intended to serve as a non-limiting illustration of the present invention.

Example 1: Anti-Malaria Vaccines Comprising PAM Studies in mice of different genetic backgrounds have shown that vaccines based on peptides containing the T * epitope (see above) as immunogenic in the absence of adjuvant, ie, when administered in a single phosphate buffer. Improved antibody responses were obtained by the addition of adjuvants, such as alum (Rehydragel, Reheis NJ) or QS21 (Cambridge Biotech, Cambridge MA). A normal anti-malaria vaccine comprising PAM contains 1 mg of MAP (T * T1 B) mixed with 100 μg of QS21. The vaccine was administered by subcutaneous injection. Example 2: Production of Specific Antibodies for SC in Humans The following study was carried out to examine the effect of immunization with a vaccine containing universal T cell epitope on humans of various genetic backgrounds. Methods: a polioxime synthetic malaria vaccine was synthesized, called (T1 BT *) 4-P3C. The vaccine contains the universal T cell epitope (T *) described above in combination with a repeated sequence of 28 residues derived from P. falciparum SC repetitions (DPNAN PNV) 2 (NAN P) 3 (called T1 B). The vaccine also contained a synthetic adjuvant covalently linked tri-palmitoyl cysteine (Pam3Cys) bound to the lysine core. Methods for the synthesis of immunogenic polyoxime compositions are generally described in International Patent Application WO 94/25071. The methods for the synthesis of T * containing polyoximes are described in co-pending application series no. , based on the provisional application series no. 60 / 034,506, filed on December 24, 1996. The vaccine was administered subcutaneously without adjuvant or additional emulsifiers, to ten human volunteers who express a Class I I haplotype scale (Table 4). The vaccine was on day 0 and day 28. Sera were obtained before immunization, on day 14, and on day 42. Antibody titrations were determined using an enzyme-linked immunosorbent assay (ELISA) using plates coated with the polioxime immunogen of tri-epitopes (T1 BT *) 4 or a PAM of di-epitopes containing only the repeats of SC (T1 B) 4. The plates were incubated in serial dilutions of two-fold serum (starting with 1: 80 dilutions), after which the plates were washed and reacted with anti-human IgG labeled with peroxidase. The presence of bound antibody was revealed by the addition of a peroxidase substrate (ABTS) and the optical density (OD) was measured at 410 nm. The endpoint titrations represent the final dilution of the immune serum in which the D.O. was greater than the D.O. mean + 3 normal deviations obtained with serum from the ten volunteers before the vaccine. Results: As shown in Table 4, within 14 days after a single dose of vaccine, antibodies specific for the polioxime immunogen could be detected in 50% of the vaccines. The administration of a second dose of polioxime vaccine on day 28 increased the anti-peptide antibody responses and positive reactions were detected in the serum of the majority of vaccinated persons. In addition, antibodies that were specifically reacted with the SC repeats were detected, as demonstrated by ELISA carried out using the PAM of (T1 B) 4. The repeat region of the SC protein of P. falciparum is the target of protective antibodies that can neutralize the infectivity of sporozoites by blocking the invasion of host hepatocytes and preventing the beginning of the life cycle of malaria in the host of mammals. Finally, all individuals had positive IgM responses after the second dose of vaccine. to. Primary IgG antibody responses were measured in sera recovered in the 14 days after the subcutaneous injection of 1 mg of polyoxime vaccine. of (T1 BT *) 4. Secondary IgG antibody responses were measured in serum by recovering + 14 days after a second vaccine injection administered on day 28. These results indicate that a vaccine containing the universal T cell epitope is capable of producing anti-repeat antibodies of IgG or IgM specific for the P. falciparum SC protein in all vaccines. Therefore, the inclusion of this universal epitope overcomes the genetic restriction of the immune response of the SC repeats and provides a synthetic peptide vaccine that is immunogenic in individuals of diverse genetic backgrounds.

Claims (20)

1. REVIVAL NAME IS 1. An immunogenic composition comprising a first malaria derived peptide comprising a universal T cell epitope, wherein the composition produces an anti-malarial T cell response in mammals of diverse genetic backgrounds.
2. 2. An immunogenic composition as defined in claim 1, further comprising a second malaria derived peptide comprising a B cell epitope that stimulates the production of anti-malarial antibodies in mammals.
3. 3. An immunogenic composition as defined in claim 1, wherein the first peptide is incorporated into a peptide of multiple antigens.
4. 4. An immunogenic composition as defined in claim 2, wherein the first and second peptides are incorporated into a peptide of multiple antigens.
5. 5. An immunogenic composition as defined in claim 1, wherein the first peptide comprises the EYLN sequence KIQNSLSTEWSPCSVT.
6. 6. An immunogenic composition as defined in claim 1, wherein the first peptide consists essentially of the EYLN sequence KIQNSLSTEWSPCSVT.
7. 7. A vaccine comprising an immunogenic composition as defined in claim 1, and a pharmaceutically acceptable carrier or diluent.
8. 8. A vaccine as defined in claim 7, further comprising a pharmaceutically acceptable adjuvant.
9. 9. A method for inhibiting the spread of a malaria organism in a susceptible mammal, comprising administering to the mammal an immunogenically effective amount of a vaccine as defined in claim 7.
10. 10. A method for producing protective immunity against malaria in a mammal, which comprises administering to the mammal an immunogenically effective amount of a vaccine as defined in claim 7.
11. 11. An immunogenic composition comprising a first malaria-derived peptide comprising the sequence EYLNKIQNSLSTEWSPCSVT, wherein the composition gives a response of T cells anti-malaria in mammals of various genetic backgrounds.
12. 12. An immunogenic composition as defined in claim 11, further comprising a second malaria derived peptide comprising a B cell epitope that stimulates the production of anti-malarial antibodies in mammals.
13. 13. A vaccine comprising an immunogenic composition as defined in claim 11 and a pharmaceutically acceptable carrier or diluent.
14. 14. A vaccine as defined in claim 13, further comprising a pharmaceutically acceptable adjuvant.
15. 15. A method for inhibiting the spread of a malaria organism in a susceptible mammal, comprising administering to the mammal an immunogenically effective amount of a vaccine as defined in claim 13.
16. 16. A method for producing protective immunity against malaria in a mammal, which comprises administering to the mammal an immunogenically effective amount of a vaccine as defined in claim 13.
17. 17. A nimmunogenic composition comprising a first malaria derived peptide consisting essentially of the sequence EYLNKIQNSLSTEWSPCSVT, wherein said composition produces an anti-malarial T cell response in mammals of diverse genetic backgrounds.
18. 18. A vaccine comprising an immunogenic composition according to claim 17, and a pharmaceutically acceptable carrier or diluent.
19. 19. 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 18.
20. 20. A method for producing protective immunity against malaria in a mammal, which comprises administering to said mammal an immunogenically effective amount of a vaccine as defined in claim 18. RESU MEN The present invention provides methods and compositions for producing protective immunity against malaria. In particular, the invention relates to universal T cell epitopes that produce T cell responses in individuals of different genetic backgrounds. Immunogenic compositions and vaccines comprising universal T cell epitopes specific for malaria are described.