WO2009114202A2 - Vaccine and immunization method using plasmodium antigen 2 - Google Patents

Abstract

A vaccine comprising an immunogenic preparation containing a Plasmodium antigen or a fragment thereof, wherein said antigen is selected from a group consisting of PyAg2, PfAg2, PvAg2, PkAg2, PoAg2, PmAg2, PbAg2, PcAg2 and a combination thereof. A vaccination method comprising administering a priming immunization preparation containing a Plasmodium antigen or fragment thereof, wherein said antigen is selected from a group consisting of PyAg2, PfAg2, PvAg2, PkAg2, PoAg2, PmAg2, PbAg2, PcAg2 and a combination thereof, and administrating a boosting immunization preparation containing said Plasmodium antigen or fragment, wherein said antigen is selected from a group consisting of PyAg2, PfAg2, PvAg2, PkAg2, PoAg2, PmAg2, PbAg2, PcAg2 and a combination thereof.

Description

VACCINE AND IMMUNIZATION METHOD USING PLASMODIUM ANTIGEN 2

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application number 61/036,666 filed March 14, 2008.

SEQUENCE LISTING

[0002] Incorporated by reference in its entirety herein is a paper copy of the nucleotide/amino acid sequence listing submitted concurrently herewith.

BACKGROUND OF THE INVENTION

[0003] ^' Malaria is one of the most devastating parasitic diseases affecting humans. Indeed, 41% of the world's population lives in areas where malaria is transmitted (e.g., parts of Africa, Asia, the Middle East, Central and South America, Hispaniola, and Oceania). The World Health Organization (WHO) and the Centers for Disease Control (CDC) estimate that malaria infects 300-500 million people and kills 700,000-3 million people annually, with the majority of deaths occurring in children in sub-Saharan Africa. Malaria also is a major health concern to U.S. military personnel deployed to tropical regions of the world. For example, in August 2003, 28% of the 26^th Marine Expeditionary Unit and Joint Task Force briefly deployed to Monrovia, Liberia, were infected with the malaria parasite Plasmodium falciparum. In addition, one 157-man Marine Expeditionary Unit sustained a 44% malaria casualty rate over a 12-day period while stationed at Robert International Airport in Monrovia. In all conflicts during the past century conducted in malaria endemic areas, malaria has been the leading cause of casualties, exceeding enemy-inflicted casualties in its impact on "person-days" lost from duty. [0004] To combat malaria during U.S. military operations, preventive drugs, insect repellants, and barriers have been used with some success, but developing drug resistance by the malaria parasite and insecticide resistance by mosquito vectors has limited the efficacy of these agents. Moreover, the logistical burden and side effects associated with the use of these agents often is associated with high non-compliance rates. Vaccines are the most cost effective and efficient therapeutic interventions for infectious diseases. In this regard, vaccination has the advantage of administration prior to military deployment and likely reduction in non-compliance risks. However, decades of research and development directed to a malaria vaccine have not proven successful. Recent efforts have focused on developing vaccines against several specific malaria genes and delivery vector systems including adenovirus, poxvirus, and plasmids. The current status of malaria vaccine development and clinical trials is reviewed in, for example, Graves and Gelband, Cochrane Database Syst. Rev., 1: CDOOO 129 (2003), Moore et al., Lancet Infect. Dis., 2: 737-743 (2002), Carvalho et al., Scand. J. Immunol., 56: 327-343 (2002), Moorthy and Hill, Br. Med. Bull, 62: 59-72 (2002), Greenwood and Alonso, Chem. Immunol, 80: 366-395 (2002), and Richie and Saul, Nature, 415: 694-701 (2002).

[0005] Over the past 15-20 years, a series of Phase 1/2 vaccine trials have been reported using synthetic peptides or recombinant proteins based on malarial antigens or gene-based approaches. Approximately 40 trials were reported as of 1998 (see Engers and Godal, Parisitology Today, 14: 56-64 (1998)). Most of these trials have been directed against the sporozoite stage or liver stage of the Plasmodium life cycle, where the use of experimental mosquito challenges allows rapid progress through Phase 1 to Phase 2a preliminary efficacy studies. Anti-sporozoite vaccines tested include completely synthetic peptides, conjugates of synthetic peptide with proteins such as tetanus toxoid (to provide T cell help), recombinant malaria proteins, particle-forming recombinant chimeric constructs, recombinant viruses, and bacteria and DNA vaccines. Several trials of asexual blood stage vaccines have used either synthetic peptide conjugates or recombinant proteins. There also have been trials of transmission blocking vaccines (e.g., recombinant P/s25). A recurring problem identified in all of these vaccination strategies is the difficulty in obtaining a sufficiently strong and long lasting immune response in humans, despite the strong immunogenic response in animal models.

[0006] The malarial parasite infects both mammalian and mosquito hosts during its complex life cycle. When the parasite changes hosts, it develops motile stages that can invade the target organ in the new host, heading for the site where it can proliferate to the next motile stage. In this migration, the parasites encounters host cell barriers that hamper its advance. To circumvent these barriers and penetrate the organ, the parasite invades and traverses host cells. Cell-traversal ability is thus essential for the parasite to establish an infection in a new host.

[0007] Malarial transmission to the mammalian host is accomplished by sporozoite infection of the hepatocyte, in which it forms a parasitophorous vacuole and develops into thousands of erythrocyte-invasive form. Sporozoites gathered in the mosquito salivary glands are injected into the mammalian host by a mosquito bite. The sporozoites enter the blood circulation and are transported to the liver sinusoid. There they leave the circulation by crossing the liver sinusoidal layer, which is the boundary between the circulation and hepatocytes. Because motile stages of malarial parasite have no locomotory organelles, such as flagella or cilia, their motility requires substrates and is achieved by highly developed cytoplasmic secretary organelles, called micronemes. A secretory microneme protein, named cell -traversal protein of Plasmodium ookinetes and sporozoites (CeITOS or antigen 2, Kariu et al 2006) was found to have crucial role in the parasite invasive motility. [0008] Targeted disruption of the CeITOS gene in Plasmodium berghei reduced parasite infectivity in the mosquito host approximately 200-fold. The disruption also reduced the sporozoite infectivity in the liver and almost abolished its cell-passage ability. Liver infectivity was restored in Kupffer cell-depleted rats, indicating that CeITOS is necessary for sporozoite passage from the circulatory system to hepatocytes through the liver sinusoidal cell layer. Electron microscopic analysis revealed that celtos-disrupted ookinetes invade the midgut epithelial cell by rupturing the cell membrane, but then fail to cross the cell, indicating that CeITOS is necessary for migration through the cytoplasm. These results suggest that conserved cell-passage mechanisms are used by both sporozoites and ookinetes to breach host cellular barriers.

[0009] In this study, antigen 2 (same as "CeITOS", hereafter "Antigen 2" or "Ag2") is first identified as one of malarial vaccine candidate from the genomic sequences of complex pathogens via an antigen identification strategy, which integrates bioinformaic predictions, HLA-supertype consideration, and in vitro cellular assays. Antigen 2 has also been shown to be highly expressed in the P. falciparum sporozoite/liver proteome as evidenced by MudPIT of P. falciparum sporozoites and P. falciparum sporozoite gene transcript profiles. In immune screening studies, Ag2 is recognized by volunteers either experimentally immunized with radiation-attenuated P. falciparum sporozoites or naturally exposed to malaria, and is preferentially recognized by irradiated sporozoite immunized volunteers who were protected against parasite challenge as compared to unprotected volunteers. Furthermore, animal data indicates that Ag2 is protective against P. yoelii challenge.

DETAILED DESCRIPTION OF THE FIGURES

[0010] FIG. IA. Antigen-specific reactivity in irradiated sporozoite-immunized volunteers - subject response rate. [0011] FIG. IB. Antigen-specific reactivity in irradiated sporozoite-immunized volunteers - test response rate.

[0012] FIG. 2. Antigen-specific reactivity in irradiated sporozoite-immunized volunteers protected or non-protected against sporozoite challenge.

[0013] FIG. 3. Alignment of amino acid sequences of P. falciparum, P. vivax, P. knowlesi and P. voelii Ag2 orthologues.

[0014] FIG. 4A. IFN-g ELIspot of PfAg2 DNA/DNA in immunized inbred and outbred mice.

[0015] FIG. 4B. Intracellular cytokine staining (ICS) of PfAg2 and PyAg2 DNA/DNA or

DNA/POX immunized inbred.

[0016] FIG. 5. Ag2 enzyme-linked immunosorbent assay (ELISA) against recombinant

Ag2 protein. (A) Pre-boost sera. (B) Post-boost/ Pre-challenge sera.

SUMMARY OF THE INVENTION

[0017] The inventive subject matter relates to a method for inducing immune response and protective immunity in humans and animals using antigen 2 or CeITOS or a fragment thereof comprising immunizing with a priming immunization preparation that selected from a group consisting of recombinant virus expression system (such as recombinant poxvirus or recombinant adenovius); a recombinant protein antigen or a recombinant polypeptide; a synthetic peptide; a DNA vector; a whole organism or extract thereof; and a combination thereof, and subsequently immunizing with a boosting immunization preparation that is selected from a group consisting of a recombinant virus expression system such as recombinant poxvirus and recombinant adenovius; a recombinant protein antigen or a recombinant polypeptide; a synthetic peptide; a DNA vector; a whole organism or extract thereof; and a combination thereof. Antigen 2 is selected from the group consisting of PfAg2 {P. falciparum), PyAg2 (P. yoelii), VvKgI (P. vivax), PkAg2 (P. knowlesi), PoAg2 (P. ovale), PmAg2 (P. malariae) PbAg2 (P. Berghei), PcAg2 (P. Chabaudi), and combination thereof.

DETAILED DESCRIPTION OF THE INVENTION Definition:

[0018] As used herein, the term "antigen" means an immunogenic peptide or protein which induces an immune response (see below) to a malarial pathogen capable of infecting a mammal.

[0019] The term "immune response" or "immunization" refer to the development in a subject of a humoral and/or cellular immunological response to an antigen that has been administered to the subject by the methods of this invention. "Humoral" immune responses refer to the production of antibodies, and a "cellular" immune response refers to the activation of T-lymphocytes, particularly cytolytic T-cells ("CTLs") and helper T-cells. Specific T-cells involved in the cellular immune response include CD4+ and CD8+ T-cells. [0020] To stimulate the humoral arm of the immune system, i.e. the production of antigen-specific antibodies, an "immunogenic fragment" will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15- 25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, that define an epitope, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains immunogenic activity, as measured by an assay, such as the ones described herein. [0021] Regions of a given polypeptide that include an "epitope" can be identified using any number of epitope mapping techniques, well known in the art. (See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed., 1996, Humana Press, Totowa, N. J.). Generally, T-cell epitopes which are involved in stimulating the cellular arm of the subject's immune system, are short peptides of 8-25 amino acids, and these are not typically predicted by the above-described methods for identifying humoral epitopes. A common way to identify T-cell epitopes is to use overlapping synthetic peptides and analyze pools of these peptides, or the individual ones, that are recognized by T cells from animals that are immune to the antigen of interest, using an enzyme-linked immunospot assay (ELISPOT). These overlapping peptides can also be used in other assays such as the stimulation of cytokine release or secretion, or by the ability to interact with major histocompatibility (MHC) tetramers. Such immunogenic fragments can also be identified based on their ability to stimulate lymphocyte proliferation in response to stimulation by various fragments from the antigen of interest. The term "epitope" as used herein refers to a sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 1 ,000 amino acids (or any integer therebetween), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence or stimulates a cellular immune response. There is no critical upper limit to the length of the immunogenic fragment, which may comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes from a single or multiple malarial parasite proteins. An epitope for use in the subject invention is not limited to a polypeptide having the exact sequence of the portion of the parent protein from which it is derived. Indeed, there are many known species of Plasmodium and the parasite retains the ability to continue to adapt, and there are several variable domains in the parasite that exhibit relatively high degrees of variability between species. Thus the term "epitope" encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature). [0022] One component of the methods of the present invention is the use of a "priming" immunization, comprising the initial administration of one or more antigens to an animal, especially a human patient, in preparation for subsequent administration(s) of the same antigen. Specifically, the term "priming", as used herein, defines a first immunization using an antigen which induces an immune response to the desired antigen and recalls a higher level of immune response to the desired antigen upon subsequent re-immunization with the same antigen when administered in the context of the same or a different vaccine delivery system. Specifically as used in this application, a "priming immunization" refers to the administration of a composition comprising a preparation containing a malarial antigen. As used herein, a "priming immunogenic composition or preparation" refers to a preparation containing a malarial antigen or fragment thereof with the preparation being selected from the group consisting of: a recombinant virus expression system; a recombinant protein antigen or a recombinant polypeptide; a synthetic peptide; a polynucleotide vector; a whole organism or extract and combinations thereof.

[0023] Another component of the methods and compositions of the present invention is the use of a "boosting immunization", or a "boost", which means the administration of a composition delivering the same malarial antigen as encoded in the priming immunization, but often utilizing a preparation with different platform, such as those selected from the group consisting of: a recombinant virus expression system; a recombinant protein antigen or a recombinant polypeptide; a synthetic peptide; a polynucleotide vector; a whole organism or extract and combinations thereof. A boost is sometimes referred to as an anamnestic response, i.e. an immune response in a previously sensitized animal. A boosting immunization or a boosting immunogenic composition can comprise multiple doses, which may be the same or different amounts. As used in this application, a boosting immunization or a boosting immunogenic composition can comprise one, two, three or multiple doses.

Genomic and proteomic identifications of malaria vaccine candidate [0024] The ImmunoSense strategy (Doolan et al. 2003) was designed to identify target antigens and maps T cell epitopes from large and complex genomes, such as P. falciparum, by integrating genomic and proteomic data with bioinformatic predictions, HLA supertype considerations, high-throughput binding assays, and cellular assays. A central component of the strategy is the capacity of the antigen to be recognized by immune responses thought to contribute in protection, allowing for rational antigen selection and prioritization. [0025] T cells recognize a complex between a specific MHC type and a particular pathogen-derived linear T cell epitope, and a given epitope will thus elicit a response only in individuals who express an MHC molecule capable of binding that epitope. Human MHC molecules are extremely polymorphic, and different HLA types are expressed at dramatically different frequencies in different ethnicities. One means of circumventing the problem of genetic restriction relies on the selection of epitopes restricted by HLA types that can be grouped in broad families, called HLA supertypes, which are characterized by largely overlapping peptide repertoires and are expressed at high frequencies in all major ethnicities (Sette and Sidney 1999). By targeting HLA-Al, -A2, -A3/A11, -A24, -B7 and -B44 supertypes, coverage of virtually 100% of all populations irrespective of the ethnicity of the target population can be achieved.

[0026] In proof-of-concept studies (Doolan et al. 1993), starting with 27 ORFs defined by multidimensional protein identification technology (Florens et al. 2002), 16 novel proteins were identified and reproducibly recognized by at least two of the eight irradiated sporozoite immunized volunteers tested in two or more assays, but not by any of the four mock immunized controls. Nine antigens were highly antigenic (recognized by at least 50% of irradiated sporozoite volunteers in at least 25% of assays) three antigens were of intermediate reactivity (recognized by at least 25% of volunteers in at least 15% of assays), and four were of low reactivity (recognized by at least 10% volunteers in at least 5% of assays). Only one protein (Ag2) was recognized by all 8 volunteers. In comparison, CSP was recognized by 3 of 8 volunteers, SSP2 by 5 of 8, LSAl by 2 of 8, and Expl by 1 of 8 volunteers (Table 1).

Table 1: Summary of immune reactivities against the panel of 27 putative and four known P. falciparum antigens.

[0027] More specifically, in those studies, to identify for analysis a set of ORFs representing antigens potentially expressed in the hepatic stage of the parasite life cycle, MS/MS spectra of peptide sequences generated by multidimensional protein identification technology (hereafter "MudPIT", Washburn and Yates, 2000) of P. falciparum sporozoite preparations were scanned against the P. falciparum genomic sequence database. A total of 1049 proteins were identified, selecting those ORFs with the highest number of peptide hits. A panel of 27 ORPs representative of putative P. falciparum antigens with a range of expression levels, stage-specificity, and membrane-association were selected. Frequency of recognition in the P, falciparum proteome dataset ranged between 1-16 peptide hits from six different sporozoite runs. CSP, SSP2 and Expl were identified during the proteomic analysis, by between 2 (CSP) to 24 (SSP2) peptide hits associated with between 10% (CSP) to 73% (Expl) sequence coverage. LSAl was not identified since it is expressed only during the hepatic stage and MudPIT studies were not carried out with liver-stage preparations. When searched against the final published P. falciparum database (Gardner et al. 2002) using refined gene model predictions, and taking into consideration genomic sequence information from the Anopheles (vector) (Holt et al. 2002) and human (host) (Venter et al. 2001; Lander et al. 2001) databases, 19 of the 27 antigens were independently identified in either P. falciparum cDNA libraries (P Blair and J Aguiar, unpublished) or as orthologs in P. yoelii laser capture microdissection datasets (J Sacci and J Aguiar).

[0028] Amino acid sequences from the 27 ORFs and the four known P. falciparum antigens: circumsporozoite protein (CSP), sporozoite surface protein 2 (SSP2), liver-stage antigen (LSA) and exported proteins 1 (Exp 1) were scanned with HLA supertype specific algorithms (Al, A2, A3/A11, B7 and DR supertypes) (Sidney and Sette 1999), and peptide epitopes predicted to bind with the highest affinity to each of the five HLA supertypes were identified (top 10 scorers selected from each supertype/protein combination for a maximum of 50 peptides per protein).

[0029] Control peptides from PfCSP, SSP2, LSAl and Expl predicted according to the same criteria applied to the 27 ORFs were also synthesized. Analysis revealed that the predictive algorithms were highly effective in identifying the majority (approximately 81%) of previously validated epitopes from those antigens. Peptides representing predicted epitopes were synthesized and then tested by IFN-γ ELIspot for their capacity to induce recall IFN-γ immune responses using PBMC from volunteers immunized with irradiated P. falciparum sporozoites (n=4), or mock immunized control volunteers (n=4). Peptides were tested as pools, at 1 ug/ml each peptide, with each antigen represented by a separate pool, Positive and negative control epitopes from well characterized antigens (CMV, Influenza, EBV, HIV) were also included. A total of 16 of the 27 previously untested antigens were reproducibly recognized as antigens by at least two of eight irradiated sporozoite immunized volunteers tested in two or more assays, but not by any of the four mock immunized controls (Doolan et al. 2003) (Table 1, FIG. IA & IB).

[0030] Nine of the 27 antigens were recognized by at least 50% of irradiated sporozoite volunteers in at least 25% of assays and classified as highly antigenic. These antigens could be prioritized for vaccine development accordingly to their immune reactivity (frequency and magnitude of response). Three antigens were recognized by at least 25% of volunteers in at least 15% of assays and classified as intermediately antigenic. Four antigens were recognized by two volunteers and classified as weakly antigenic. Finally, 11 of the 27 untested antigens failed to induce reproducible IFN-γ responses of sufficient magnitude to meet our criteria of positivity. Positive control peptides derived from CMV, FLU and EBV were recognized by 87.5% of the volunteers tested. Pools of predicted epitopes from the known antigens, PfCSP, PfSSP2, PfLSAl and PfExpl, were recognized by about one third of the irradiated sporozoite volunteers.

[0031] The antigens identified as a result of these studies represent, to the best of our knowledge, the first antigens identified from P. falciparum genomic sequence data and shown to be recognized by sporozoite-induced T cell responses. Particularly noteworthy, the immune reactivity against several of the newly identified antigens greatly exceeded the reactivities observed against the well characterized antigens currently considered prime preerythrocytic stage vaccine candidate antigens, namely PfCSP, PfS SP2, PfLSAl and PfExpl. These data suggest that some of the novel antigens identified using the Immunosense screening strategy may represent better candidates for vaccine development. [0032] The most antigenic protein amongst those identified by the ImmunoSense approach was a single-exon 549-nucleotide gene (182 amino acid protein) located on P. falciparum chromosome 12 (PFL0800c), and was designated "Ag2". In immune T cell screening studies (Doolan et al. 2003), Ag2 was reproducibly recognized in the context of multiple genetic restrictions by 100% (8/8) volunteers experimentally immunized with radiation-attenuated P. falciparum sporozoites, but not by any mock-immunized control volunteers, in more than 50% of assays (Table 1).

[0033] Moreover, it appeared to be preferentially recognized by irradiated sporozoite- immunized voluteers who were protected against parasite challenge as compared to unprotected volunteers (FIG. 2). Interestingly, responses to the previously characterized antigens (CSP, etc) have been detected predominantly in nonprotected volunteers. In all in vitro studies to date, immune responses to Ag2 have been markedly better than responses to PfExpl, PfLSAl, PfCSP or PfSSP2, as evidenced by the frequency of responders (eg., 8/8 vs. 1/8, 2/8, 3/8 and 5l%; p = 0.001, 0.007, and 0.026), and magnitude of response (122.7 vs 41.3, 34.2, 41.6, 97.2) (Doolan et al. 2003) (Table 1). The relative immunodominance of Ag2 as compared to the historic antigens has been consistently demonstrated in subsequent studies. [0034] In independent studies, seven of the 27 ORFs evaluated in the ImmunoSense approach were assayed in a humoral immunoscreening assay designed to evaluate whether putative antigens could be recognized by malaria-immune sera. Of those, 4/4 antigens classified with high immune reactivity and 1/1 antigens classified with intermediate immune reactivity were shown to be recognized by malaria-immune sera but not by malaria-naϊve sera, in as many as 5/8 independent experiments (Regis et al. mns submitted). For Ag2, responses that met criteria of positivity were detected in 2 of 3 independent experiments. Expression analysis ofΛg2

[0035] Plasmodium falciparum antigen 2 (PfAg2) appears to be highly expressed specifically in Plasmodium sporozoites based on two large-scale expression analyses. Transcription profiling using an AFFYMETRIX GENE CHIP^® (LeRoch et al. 2003) demonstrated PfAg2 to have the 11^th highest expression level (5,585.2) overall in sporozoites and falling only behind PfCSP (28,684.1) and PfSSP2 (12,475.8) for sporozoite stage- specific genes. Secondly, MudPIT proteomic analysis found Ag2 only matching peptides present in sporozoite stages. Moreover, peptides identified in sporozoites were numerous (SeqCount = 17; SpecCount = 56) and achieved high sequence coverage (49.5%). These data relatively translate to PfAg2 being in the top four most abundant sporozoite specific antigens via MudPIT analysis. Only PfCSP, PfSSP2, and PFL0945w (hypothetical protein) possessed higher SpecCounts for sporozoite specific antigens.

Identification oforthologs ofPfAg2 in other Plasmodium spp

[0036] No ortholog of Pf Ag2 could be identified in the annotated P. yoelii genome (Carlton et al. 2002). However, upon scanning the corresponding region along chromosome 12 common stretch of sequence and gene homology was achieved between P. yoelii and P. falciparum. It was noticed that genes flanking Ag2 in the P. falciparum chromosome 12 genome all had orthologs present in P. yoelii. After obtaining the corresponding P. yoelii contig (chrPyl 00049) and scanning for ORFs a previously uncharacterized ORF was identified that shares size (single exon; predicted 185 amino acids for P. yoelii, 182 amino acids for P. falciparum) and sequence identify (44% identity at the amino acid level; 57% identify at the nucleotide level). Subsequently, PfAg2 orthologs were determined in corresponding contigs in P. vivax (185 amino acids; 42% identity at the amino acid level; 54% identify at the nucleotide level) and P. knowlesi (185 amino acids; 43% identity at the amino acid level; 56% identify at the nucleotide level), using BLAST searches against the incomplete sequence databases (Table 2). Table 2; Amino acid and nucleotide sequence identity

Sequence conservation ofAg2 relative to well characterized P. falciparum antigens [0037] The level of sequence identity between the identified Plasmodium spp orthologs of Ag2 is greater than that observed between PyHEP 17 and PfExp-1 (37% identity at the amino acid level), P. falciparum and P. yoelii circumsporozoite protein (26%) (Dame et al. 1984; LaI et al. 1987); P. falciparum and P. knowlesi circumsporozoite protein (25%); and P. falciparum and P. yoelii sporozoite surface protein (28%) (Robson et al. 1999, 1990; Rogers et al. 1992a, b); and similar to that between the P. falciparum and P. knowlesi sporozoite surface protein 2 (36%). There are no known P. knowlesi or P. vivax orthologs of PyHEP 17/PfExp-l, and no known P. yoelii, P. knowlesi or P. vivax orthologs of PfLSAl, so sequence identity between species cannot be assessed in those cases. The level of identity between Plasmodium orthologs of Ag2 is also greater than for leading blood-stage candidate antigens, merozoite surface protein- 1 (MAD20, 29%; Wellcome, 30%) and rhoptry antigen (overall identity 4%; best identity 11%). (FIG. 3) [0038] Analysis of the sequence of Ag2 from a number of different P. falciparum strains and isolates revealed a high degree of conservation between P. falciparum strains, and showed that the polymorphism was restricted to a limited number of amino acid variants.

Recognition ofΛg2 in the context of multiple genetic restrictions

[0039] Parasite polymorphism of identified targets of CD 8+ and CD4+ T cell mediated protective immunity poses a major obstacle to the development of an effective vaccine, since such polymorphism provides a means for the parasite to evade the host immune response (Doolan and Hoffman, 1997). Thus, an ideal vaccine candidate antigen would be highly conserved between Plasmodium strains and species. The high level of sequence identity noted between P. falciparum strains and between the P. falciparum, P. vivax, P. knowlesi, and P. yoelii orthologs of Ag2 (FIG. 3) support the potential of Ag2 as a good candidate antigen for vaccine development.

[0040] Another obstacle to vaccine development is genetic restriction of the host immune response to the CD8+ and CD4+ T cell epitopes that are the targets of T cell mediated protective immunity (Doolan and Hoffman, 1997). Thus, in addition to being conserved between species, an ideal vaccine candidate antigen would contain multiple T cell epitopes recognized in the context of defined panels of HLA supertypes to ensure that the antigen is capable of being recognized by vaccinees of diverse genetic background. Identification of peptide epitopes that bind with high affinity to multiple HLA supertypes is an integral component of the ImmunoSense strategy. PfAg2 contains multiple HLA degenerate HLA- Al, HLA- A2, HLA-A3/A11, HLA-A24 and HLA-B7 degenerate T cell epitopes, and deconvolution of HLA-supertypes used for the initial identification of Ag2 (Doolan et al. 2003) has established the capacity of these epitopes to bind to multiple HLA molecules as assayed by in vitro peptide binding assays, and to be recognized by T cells derived from volunteer immunized with irradiated-sporozoites as assayed by IFN-g ELIspot (Doolan et al. 2003, DL Doolan, unpublished).

[0041] Additionally, sera from 20/20 genetically diverse individuals resident in a hyperendemic region of Kenya recognized PfAg2 by western blot analysis of cells transfected with plasmid DNA encoding PfAg2 (data not presented), demonstrating the unrestricted nature of the antibody response to Ag2. Chicken embryo cells infected with recombinant PfAg2 poxvirus or 293 cells infected with recombinant PfAg2 adenovirus were also recognized by sera from malaria-exposed individuals (data not presented). [0042] Studies assessing the immunogenicity of PfAg2 and PyAg2 in vivo support the contention that Ag2 is recognized in the context of multiple genetic elements. In the studies, BALB/C (H-2d), C57BL/6 (H-2b), Bl 0.BR (H-2k), A/J (H-2a) and CDl (outbred) mice were immunized under various immunization designs. In study 1, mice were immunized with priming and boosting immunization preparations containing plasmid DNA encoding PfAg2 or PyAg2. In study 2, mice were primed with DNA encoding PfAg2 or PyAg2, and boosted with DNA containing PfAg2 or PyAg2, or boosted with a recombinant poxvirus expression system containing PfAg2 or PyAg2. In study 3, mice were primed with either priming preparation containing DNA encoding PfAg2, PyAg2 or PyCSP or a preparation containing recombinant adenovirus expression system containing PyAg 2, PfAg2 or PyCSP. The mice were boosted with preparation containing either a recombinant adenovirus expression system or a recombinant poxvirus expression system, containing PfAg2, PyAg2 or PyCSP. Splenocytes were harvested at 2 weeks post last immunization for assessment of T cell responses by IFN-g ELIspot (FIG. 4A) and multiparameter ICS (FIG. 4B). [0043] Sera was harvested at 2-4 weeks post each immunization for assessment of antibody responses by IFAT against P. yoelii or P. falciparum sporozoites. Table 3 shows antibody responses by IFAT against P. yoelii or P. falciparum sporozoites for study 1. Tables 4A & 4B shows antibody responses by IFAT against P. yoelii or P. falciparum sporozoites for study 2, in which sera post last immunization were pooled and assayed in triplicate. IFAT data for Study 3 are shown in Tables 5A & 5B. ELISA against recombinant PfAg2 protein are shown in FIG. 5 A & 5B, in which pooled sera post last immunization were assayed in quadruplicate. Other studies have established that PfAg2 antisera does not recognize blood-stage parasites (liver stage IFAT not done).

Table 3: Assessment of antibody responses by IFAT against P. yoelii or P. falciparum sporozoites. (Study 1: DNA/DNA).

CD-1 A/J C57BL/6 BALEVc B10.BR

1 40 40 80 40 0

2 160 80 40 80 0

3 320 320 80 40 0

4 160 80 20 40 0

5 40 40 80 160 0

6 320 80 320 80 0

7 640 80 1260 80 0

8 20 80 40 40 0

9 40 40 40 0 0

10 0 40 160 0 0

GeoMeaπ 109 70 92 62 0

Range 0-640 40-320 40-1280 0-160 0

Table 4 A: Assessment of antibody responses by IFAT against P. yoelii sporozoites. (Study 2: DNA/DNA and DN A/POX)

Table 4 B: Assessment of antibody responses by IFAT against P. falciparum sporozoites. (Study 2: DNA/DNA and DNA/POX).

Table 5 A: Assessment of antibody responses by IFAT against P. yoelii and P. falciparum sporozoites pre-boost. (Study 3: DN A/POX and DNA/ ADENO)

P. yoelii Ag2 & P. falciparum Ag2 Pre-Boost

GROUP IMMUNOGENS GEOMEAN RANGE

1 PyAg2 DNATPOX 120 20-320

2 PyAg2 DNA/ADENO 23 20-40

3 PyAg2ADENO/POX 35 20-160

4 PfAg2 DNA/POX) 67 20-320

5 Pf Ag2 DNA/ADENO 48 20-160

6 Pf Ag2 ADENO/POX 31 20-40

7 PyCSP DNA/POX 80 20-320

8 PyCSP DNA/ADENO 36 20-160

_. 9 PyCSP ADENO/POX) 40 20-160

PyAg2 DNA prcboost 54 20-320

PfAg2 DNA preboost 57 20-320

PyCSP DNA preboost 53 20-320

Table 5 B: Assessment of antibody responses by IFAT against P. yoelii and P. falciparum sporozoites pre-boost. (Study 3: DNA/POX and DNA/ADENO)

P. yoelii Ag2 & P. falciparum Ag2 Pre-Challenge (Post-Boost)

GROUP IMMUNOGENS GEOMEAN RANGE

1 Py Ag2DNA/POX 214 80-320

2 Py Ag2DNA/ADENO 135 40-320

3 Py Ag2 ADENO/POX 254 160-320

4 PfAg2 DNA/POX 135 80-320

5 Pf Ag2 DNA/ADENO 127 80-640

6 PfAg2 ADENO/POX 285 80-640

7 PyCSP DNA/POX 269 20-20480

8 PyCSP DNA/ADENO 320 20-20480

9 PyCSP ADENO/POX 285 20-2560

[0044] Thus, we have established that plasmid DNA vaccine encoding Ag2 are immunogenic in mice, as demonstrated by the induction of antibodies which recognize P. falciparum sporozoites by IFAT after a single IM dose, and by the induction of T cells which recognize pools of PfAg2 synthetic peptides by IFN-g ELIspot after two or three IM immunizations. T cell responses after one immunization were not assessed. In other related studies in our laboratory, with plasmid DNA constructs encoding hypothetical P. falciparum antigens derived from the P. falciparum genome and generated using the Gate Way recombinatorial cloning system, mice were immunized via GeneGun particle mediated gene delivery (typically a better delivery method for induction of antibody responses than IM immunization), and the antisera has been analyzed for immunoreactivity against different stages of the parasite by IFAT (Aguiar et al., 2004). In those studies, a total of 22% (21/95) of Gate Way clones induced parasite-specific antibody responses, but only 3% (3/95) induced antibodies that recognized P. falciparum sporozoite stages (antisera tested after two immunizations). Those genes were not selected based on stage-specific expression and it is possible that only a small percentage may be expressed on the sporozoite. Nonetheless, the capacity of plasmid DNA encoding Ag2 to induce anti-sporozoite antibodies after a single IM immunization attests to the proposed immunogenicity of Ag2 in vivo, consistent with its demonstrated antigenicity in vitro.

Protective efficacy ofAg2

[0045] The capacity of Ag2 to protect against P. yoelii sporozoite challenge was determined in inbred and outbred mice. BALB/C (H-2d), C57BL/6 (H-2b), Bl O.BR (H-2k), A/J (H-2a) and CDl (outbred) mice were primed with plasmid DNA encoding PfAg2 or PyAg2, and boosted with recombinant Ag2 poxvirus (table 6A). Outbred mice were primed with either DNA encoding for PfAg2 or PyAg2 or preparation containing recombinant Ag2 adenovirus expression system and boosted with preparation containing recombinant Ag2 adenovirus expression system or recombinant Ag2 poxvirus expression system. Outbred mice immunized with P. yoelii CSP were evaluated in parallel, as a comparator group. Mice were challenged at 2 weeks post last immunization with infectious P. yoelii sporozoites (100 spz for inbred strains; 200 spz for outbred CDl) and monitored for development of blood- stage parasitemia by Giemsa stained blood smears on days 5-14. Mice immunized with Antigen 2 immunization have shown significant protection against P. yoelii sporozoite, which is comparable to PyCSP.

Table 6A: Protective efficacy of Ag2 against P. yoelii sporozoite challenge. Mice immunized with DNA and boosted with recombinant poxvirus.

P. yoelii Ag 2 DNA/POX P. falciparum Ag 2 DNA/POX

Strain H-2

#ιnfect/#challenge % protection #ιnfect/#challenge % protection

BALB/c H-2d 0/9 0 1/9 11.1

C57BL/6 H-2b 1/9 11.1 0/9 0

A/J H-2a 1/9 11.1 0/9 0

B10 BR H-2k 1/9 11.1 1/9 11.1

CD-1 outbred 4/9 44.4 5/9 55.6

Table 6 B: Protective effecacy of Ag2 against P. yoelii sporozoite challenge. Mice immunized with DNA or recombinant adenovirus and boosted with recombinant poxvirus or recombinant adenovirus.

0 wks 8 wks Sterile Pr otection

Group Strain Prime Boost # protect % protect

1 CD-1 P yoelii Ag2 DNA P yoelii Ag2 POX 6/14 43%

2 CD-1 P yoelii Ag2 DNA P yoelii Ag2 Adeno 7/14 50%

3 CD-1 P yoelii Ag2 adeno P yoelii Ag2 POX 9/14 64%

4 CD-1 P falciparum Ag2 DNA P falciparum Ag2 POX 5/14 36%

5 CD-1 P falciparum Ag2 DNA P falciparum Ag2 Adeno 8/14 57%

6 CD-1 P falciparum Ag2 adeno P falciparum Ag2 POX 10/14 71%

7 CD-1 P yoelii CSP DNA P yoelii CSP POX 7/14 50%

8 CD-1 P yoelii CSP DNA P yoelii CSP Adeno 8/14 57%

9 CD-1 P yoelii CSP adeno P yoelii CSP POX 8/14 57%

10 CD-1 Infectivity control - 200 spz - 0/14 0%

11 CD-1 Infectivity control - 50 spz) - 0/7 0%

12 CD-1 Infectivity control - 10 spz) - 1/7 14%

13 CD-1 Infectivity control - 2 spz) - 2/7 29%

14 CD-1 Infectivity control - 0 4 spz) - 3/7 43%

DNA dose = 100 ug route = IM

POX dose = 1E7, route = IM

Adeno dose = 1E8, route = IM

Spz chl = 200 spz Conclusion - The potential ofΛg2for vaccine development

[0046] In summary, Ag2 is a novel and promising next generation vaccine candidate antigen identified by integrating bioinformatics, genomics, and molecular immunology. It is a single-exon 549 nucleotide gene (182 amino acid protein) located on P. falciparum chromosome 12. It is highly expressed in the P. falciparum sporozoite/liver proteome as evidences by MudPIT of P. falciparum sporozoites and P. falciaprum sporozoite gene transcript profiles. In immune screening studies, Ag2 is recognized by volunteers either experimentally immunized with radiation-attenuated P. falciparum sporozoites or naturally exposed to malaria, and is preferentially recognized by irradiated sporozoite immunized volunteers who were protected against parasite challenge as compared to unprotected volunteers. Significantly, Ag2 has proved to be more immunogenic than all well- characterized P. falciparum antigens currently in clinical evaluation. Overall, an abundance of data implicates Ag2 as an extremely promising candidate antigen for malaria vaccine development. Table 7: Nucleotide and Amino Acid Sequences for Ag 2 Orthologs

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Claims

What is claimed is:

1. A method to induce protective immunity comprising: a. administrating a priming immunization preparation containing a Plasmodium antigen or a fragment thereof; and b. administrating a boosting immunization preparation containing said Plasmodium antigen or fragment thereof.

2. The method of claim 1, wherein said Plasmodium antigen is selected from a group consisting of PfAg2, PyAg2, PvAg2, PkAg2, PoAg2, PmAg2, PbAg2, PcAg2 and a combination thereof.

3. The method of claim 1, wherein said priming immunization preparation containing said antigen or fragment thereof, is selected from a group consisting of: a. a recombinant virus expression system; b. a recombinant protein antigen or a recombinant polypeptide; c. a synthetic peptide; d. a DNA vector; e. a whole organism or extract thereof; and f. a combination thereof.

4. The method of claim 1 , wherein said boosting immunization preparation containing said antigen or fragment thereof, is selected from a group consisting of a. a recombinant virus expression system; b. a recombinant protein antigen or a recombinant polypeptide; c. synthetic peptide; d. a DNA vector; e. a whole organism or extract thereof; and f. a combination thereof.

5. The method of Claim 3 or claim 4, wherein said recombinant virus expression system comprises Plasmodium antigen 2, fragment thereof, or epitope derived from said antigen, or combination thereof.

6. The method of claim 3 or claim 4, wherein said recombinant virus expression system is selected from the group consisting of poxvirus, adenovirus, adeno-associated virus, and retrovirus.

7. The method of Claim 5, wherein said poxvirus is selected from the group consisting of cowpox, canarypox, monkeypox, and fowlpox.

8. The method of claim 1, wherein said antigen is native or recombinant.

9. The method of Claim 1, wherein the number of doses of priming agent is 1-4 and the number of doses of boosting agent is 1-4.

10. The method of claim 1, wherein said immunization preparations are administered by routes selected from the group consisting of subcutaneous, intramuscular, intradermal, mucosal, oral, transcutaneous, and by injection devices, or combinations thereof.

1. A vaccine comprising an immunogenic preparation containing a Plasmodium antigen or fragment thereof, wherein said antigen is selected from a group consisting of PyAg2, PfAg2, PvAg2, PkAg2, PoAg2, PmAg2 and a combination thereof.