Novel antigens for use in malaria vaccines

Abstract

The present invention provides a polypeptide useful as an antigen expressed in the pro-erythrocyte phase of a malaria parasite. The antigen can be used to induce an immune response and sterile protection against malaria in mammals by administering it as an antigen in a vaccine formulation or by expressing the antigen within a DNA or other recombinant protein expression system delivered to the vaccine formulation.

Description

Background Technology

[0001] Cross-reference of related applications

[0002] This application is based on U.S. Provisional Application No. 62 / 296,46, filed February 17, 2016, the entire contents of which are incorporated herein by reference, for the basis of a priority claim under 35 USC § 119(e).

[0003] Despite years of effort, there is no approved malaria vaccine. One of the obstacles facing the development of a malaria vaccine is the widespread heterogeneity of many malaria vaccine antigens. Potential vaccine antigens evaluated in humans have not so far induced a protective immune response.

[0004] Malaria kills approximately 863,000 people annually. Despite the existence of various antimalarial drugs, their cost can be prohibitively expensive in relatively poor regions of the world where malaria is endemic. The widespread use of the most commonly used drugs also leads to the proliferation of drug-resistant parasites, rendering many of these drugs ineffective. In the absence of inexpensive and highly potent drugs, vaccination is the most cost-effective way to complement traditional malaria interventions.

[0005] A successful malaria vaccine needs to protect humans from numerous antigenically diverse malaria parasites. Vaccines based on a single isolate of a single antigen cannot elicit a sufficiently broad immune response to protect individuals from these heterogeneous populations. One way to potentially improve the efficacy of antigen-based vaccines, or any other subunit malaria vaccine, may be to include additional malaria antigens in the vaccine, thereby expanding the immune response induced by the vaccine.

[0006] Malaria vaccine development efforts have been almost exclusively focused on a small number of well-characterized Plasmodium palsipharum ( P. falciparum Research has focused on antigens. Despite dedicated research by many researchers on other continents for over half a century, a successful malaria vaccine remains elusive. Sequencing of the Plasmodium falciferum genome has identified over 5,000 genes, but it has provided no indication as to which of these 5,000 genes are useful or how to identify potential vaccine targets.

[0007] Malaria is caused by mosquito-derived blood protozoan parasites belonging to the genus Plasmodium. Four species of Plasmodium protozoa (Plasmodium falciferum, Plasmodium bibox ( P. vivax ), Plasmodium Ovale ( P. ovale) and Plasmodium malariae ( P. malariae)) causes disease in humans. Plasmodium yoeli ( P. yoelii) and Plasmodium bergei ( P. berghei Others, such as ), also cause disease in animals. Plasmodium falciparum is the cause of most infections and deaths in humans. Malaria parasites have a life cycle consisting of four distinct stages. Each of these stages can induce a specific immune response against the parasite and the development of corresponding stage-specific antigens, but naturally induced malaria does not protect against reinfection.

[0008] Malaria parasites are transmitted to mammals by female Anopheles mosquitoes of various species. Infected mosquitoes deliver the malaria parasite in the form of sporozoites to the mammalian skin during a blood meal, which then invades the bloodstream. The sporozoites remain in the bloodstream for several minutes before attacking liver cells. During this stage, the parasite is located in the extracellular environment and is exposed to antibody attacks that primarily target circumsporozoite (CS) proteins, which are the main components of the sporozoite surface. When the sporozoites attack liver cells, the parasite differentiates and replicates to become schizonts. During this stage, the attacking parasite reproduces asexually, producing up to 20,000 merozoites per infected liver cell. During the aforementioned intracellular stage of the parasite, the host's immune response involves T lymphocytes, particularly CD8 + It contains T lymphocytes. Ten to 14 days after liver infection, thousands of newly formed cysts are released into the bloodstream to attack red blood cells (RBCs), becoming targets of antibody-mediated immune responses and T-cell secreted cytokines. After attacking red blood cells, the cysts replicate in several stages, transforming into trophozoites and saccharides, which rupture to produce a new generation of cysts that successively infect new red blood cells. The aforementioned stage of the parasite (the red blood cell stage) promotes a strong humoral response that can inhibit the cysts' attack on red blood cells and usually provides protection against the pathology associated with that stage. The red blood cell stage is associated with apparent clinical disease. A smaller number of trophozoites may become male or female gametocytes, which is the reproductive stage of the parasite. When a vulnerable mosquito ingests gametes, fertilization of these gametes triggers the formation of a zygote, followed by the development of a ookinete and oocyst, and finally a sporosome, which then migrates to the salivary gland to complete the cycle.

[0009] The two main branches of pathogen-specific immune responses that occur upon the entry of parasites into the body are cellular and humoral. One of these, the cellular response, involves CD8 cells participating in the immune response. + and CD4 + It is related to T cells. Cytotoxic T lymphocytes (CTLs) can specifically kill infected cells that express pathogen antigens on their surface. CD4+ T cells, or helper T cells, support the development of CTLs, produce various cytokines, and also assist B cells in dividing to produce antigen-specific antibodies. During the humoral response, B cells specific to a particular antigen are activated, replicate, and differentiate to produce antigen-specific antibodies.

[0010] The two branches of the immune response are related to protection against malaria infection. When infectious sporozoites migrate to the liver and penetrate hepatocytes, they become intracellular pathogens and spend almost no time outside the infected cell. At this stage, CD8 + T cells and CD4 + T cells are particularly important because these T cells and their cytokine products, such as interferon-γ (IFN-γ), contribute to the killing of hepatocytes in the infected host. The elimination of intracellular hepatic parasites in a mouse malaria model involved CD8 against peptides expressed by hepatic parasites. + It was found to be dependent on T cell response. CD8 + The decrease in T cells interferes with protection against sporosome provocation, and CD8 + Adoptive transfer of T cells to inexperienced animals confers protective ability.

[0011] When malaria infection reaches the erythropoietic phase, where cysts replicate in red blood cells, it appears that the cysts also circulate freely in the bloodstream for a short period until they attack new red blood cells. Since red blood cells do not express class I and II MHC molecules necessary for homologous interaction with T cells, antibody responses against the parasite are thought to be most associated with the hematopoietic phase of the parasite's life cycle. In conclusion, a possible malaria vaccine approach would be most useful if it could induce a strong cellular immune response as well as a strong humoral immune response to combat the different stages the parasite exhibits within the human body.

[0012] Current approaches to malaria vaccine development can be classified according to the different developmental stages of the parasite, as previously described. Three types of possible vaccines can be distinguished. The first is the pro-erythropoietic vaccine, which targets sporozoites and / or Malaria-infected hepatocytes. Historically, this approach has been most prominently represented by (CSP)-based strategies. Since there are no symptoms during the pro-erythropoietic stage of infection, the goal of the pro-erythropoietic vaccine is to confer sterile immunity mediated by humoral and cellular immune responses, thereby preventing latent malaria infection. This goal has not been met by any known treatments.

[0013] The second type of vaccine approach is an asexual blood-borne vaccine that targets infected red blood cells and the cysts themselves; it is designed to minimize clinical severity or prevent infection if antibodies block the cysts from attacking red blood cells. Attempts to manufacture these vaccines to date have not sufficiently reduced disease incidence and mortality rates or prevented the parasite from entering and / or developing into red blood cells. Transmission-blocking vaccines are designed to disrupt the development of the parasite in mosquito hosts. Attempts to manufacture this type of vaccine to date have not reduced malaria infection rates across the general population.

[0014] The final type of vaccine approach is a combination of malaria vaccines targeting multiple stages of the parasite's life cycle. This approach has attempted to develop multi-component and / or multi-stage vaccines. Attempts to manufacture such vaccines to date have not provided sufficient protection. As a result of these failures, there are currently no commercially available malaria vaccines.

[0015] Immunizing rodents, non-human primates, and humans with radioactive sporosome (RAS) is known to provide protection against successive provocations by live sporosome. However, the cost of producing irradiated sporosome, the lack of feasible large-scale culture systems, relatively short efficacy, the lack of cross-strain protection, and the necessity of intravenous delivery have been obstacles to the development of such vaccines.

[0016] The CS protein is the only Plasmodium falcifarum antigen proven to prevent malaria infection when used as the basis for active immunization against mosquito-derived infections in humans. However, the level of protection provided by the antigen is not sufficiently high to support a successful treatment. Theoretically, for a treatment to be successful, the vaccine protection level must be at least 85%. If the protection level is lower than this, more lethal variants may escape from endemic areas. CS antigen-based vaccines have demonstrated only about 50% efficacy, and protective ability has not lasted for more than one year. Nevertheless, this was the best-known antigenic response prior to this specification.

[0017] The whole genome sequence of Plasmodium falciferum has been sequenced. See [Bowman et al., Nature, 400: 532-538 (1999); Gardner, et al., Nature, 419: 498-511 (2002)]. Another human malaria parasite, Plasmodium vivax, has also been sequenced. See [Carlton et al., Nature, 455: 757-763 (2008)]. The rodent malaria parasite, Plasmodium yoeli, has also been sequenced. See [Carlton et al., Nature, 419: 512-519 (2002)]. However, despite this, the development of effective anti-malaria vaccines has been severely hindered because promising antigens cannot be identified. Sequencing of the genomes of Plasmodium falciferum, Plasmodium vivax, and Plasmodium yoeli identified 5,369, 5,433, and 5,675 genes, respectively. However, knowing these sequences does not mean that new vaccine constructs can be created. Consequently, clinical trials are being conducted on only 0.2% of the Plasmodium falciferum proteome, and these trials have failed to induce a high level of protection in volunteers.

[0018] The present invention provides a polypeptide useful as an antigen expressed in both the pro-erythrocyte and erythrocyte phases of a malaria parasite. The antigen can be used to induce both cellular and humoral immune responses against malaria in mammals by administering the antigen within a vaccine formulation or by expressing the antigen within a DNA or other nucleic acid expression system delivered to the vaccine formulation. In a preferred embodiment, the mammal is a human.

[0019] In a preferred embodiment, the present invention is an immunogenic composition for protecting mammals from malaria infection, comprising one or more recombinant polypeptides of SEQ ID NO. 3 or SEQ ID NO. 6 or derivatives thereof in a pharmaceutically acceptable carrier The present invention provides an immunogenic composition comprising: generally, the derivative has at least 10 adjacent amino acids to the reference sequence and / or has 85% identity with the reference sequence. The immunogenic composition may be formed from an isolated or recombinant polypeptide or carrier virus expressing a recombinant antigen and may be paired with an acceptable adjuvant.

[0020] The antigens that are the subject of this specification are identified by different nomenclature in different contexts, as in the standards of this technical field. For convenience, the following table presents each antigen by its sequence, the various names and abbreviations used in the prior art and this specification:

[0021] Abbreviation PlasmoDB Identification Number (Identification) Sequence number Py E140 PY06306, PY17X_0210400, PYYM_0211900 1 (amino acid) 2 (nucleotide) Pf E140 PFA0205w, MAL1P1.31, PF3D7_0104100, XP_001350973 3 (amino acid) 4 (nucleotide) Pv E140 PVX_081555, PV081555, PVP01_0210600 6 (amino acids) 5 (nucleotides) Py falstatin PY17X_0816300, PY03424, PYYM_0816000 PyCSP PY03168, PYYM_0405600 Py E057 PY03396, PY17X_1006600, PYYM_1006600 Py E137 PY05693, PY17X_1006100, PYYM_1006100 Py UIS3 PY03011, PY17X_1402400 Pffalstatin, ICP PFI0580C or PF3D7_0911900 7 (amino acids) Pf CSP PFC0210C, MAL3P2.11, PF3D7_0304600 8 (amino acids) PF UIS3, ETRAMP13 PF13_0012, PF3D7_1302200 9 (amino acids)

[0022] The present invention may comprise a combination of two or more recombinant polypeptides in a pharmaceutically acceptable carrier, wherein one polypeptide is SEQ ID NO. 3, SEQ ID NO. 6, or a derivative thereof, and the other polypeptide is PyCSP, Py falstatin, Py UIS3, PY03396, PY05693, PY03424, and PY03011 falsiferum ( falciparum ) or Bbox ( vivax It is any of the speciative orthologs.

[0023] The present invention also comprises a method for inducing an immune response against malaria in mammals by administering an immunologically effective amount of a composition comprising one or more polypeptides encoded by SEQ ID NO. 3 or 6, or derivatives thereof. Alternatively, the method may comprise the step of administering one or more primary priming or enhancing immunizations against malaria, wherein the primary priming and enhancing immunizations comprise an immunologically effective amount of recombinant polypeptides as described. The method of administering the polypeptides may include the use of a suitable expression vector, such as a plasmid, a replicating viral vector, or a non-replicating viral vector. Suitable expression vectors may be DNA plasmids, baculovirus, rVSV, SpyVLP, alphavirus replicon, adenovirus, varicella virus, adeno-associated virus, cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus, RNA replicon, DNA replicon, alphavirus replicon particle, Venezuelan equine encephalitis virus, Semliki forest virus, or Sindbis virus.

[0024] The polypeptide useful as an antigen disclosed herein is a first Plasmodium ( Plasmodium) is a pro-erythrocyte antigen. These reactions are conveniently measured in mice as surrogates for human speciation homologs. Malaria infection, treatment, and immunization have been widely studied in both mice and humans, and mouse models are considered standard indicators of malaria vaccine efficacy in humans and other mammalian individuals. The PY06306 antigen disclosed herein alone protects 71% to 100% of CD1 mice against malaria and further induces an immune response capable of delaying the initiation of parasites in the blood of the remaining unprotected mice. Overall, 83% (384 / 461) of PY06306-immunized mice are protected from malaria infection. Such protection is reported in both crossbred (CD1) and inbreed (BABB / c) mice using efficacy evaluations as sterile protection and severe 300- and 100-spore-body provocations, respectively. The efficacy of the antigen disclosed herein causes a polypeptide to induce an immune response against malaria in mammals, considering the relationship between the malaria immune response and standard indicators of vaccine efficacy in rats, primates, and humans disclosed herein. Brief explanation of the drawing

[0025] Figure 1 shows the protective results for matrix experiments in which 14 CD1 xenobred mice per group were immunized with primary immuno-enhancing therapy using a combination of DNA expressing PY03396, PY05693, PY06306, PY00232, and PyCelTOS and a human adenovirus type 5 (Ad5) vector. Positive control mice were PyCS PImmunized using DNA and Ad5 vectors expressing [the antigen]. Negative control mice consist of mice immunized with 4X relative amounts of DNA and Ad5 vectors that do not express Plasmodium yoeli antigen, and naive mice. Gray and black bars represent antigen combination groups with and without PyCSP, respectively. Hatched and checkered bars represent the PyCSP and naive groups, respectively. Mice were provoked with 300 Plasmodium yoeli sporosome, and parasitemia was evaluated up to 14 days after provocation by performing Giemsa-stained blood smears. The numbers at the bottom indicate the number of sterile-protected mice per total provoked mouse in each group. Figure 2 shows the matrix deconvolution of experiments evaluating PY06306 and other antigens shown in Figure 1. Fourteen CD1 crossbred mice per group were immunized with a primary immuno-enhancing regimen containing DNA and adenovirus type 5 (Ad5) vectors expressing PY03396, PY05693, PY06306, PY03424, and PY03011. Positive control mice were immunized with DNA and Ad5 vectors expressing PyCSP. Negative control mice were immunized with 4X relative amounts of DNA and Ad5 vectors not expressing Plasmodium yoeli antigen. Gray and black bars represent antigen combination groups with and without PyCSP, respectively. Hatched and checkered bars represent PyCSP-immunized and unimmunized mice, respectively. Mice were provoked with 300 Plasmodium yoeli sporosome, and parasitemia was evaluated up to 17 days after provocation by performing Giemsa-stained blood smears. The numbers at the bottom indicate the number of sterile-protected mice per total provoked mice in each group. Figure 3 shows a Kaplan-Meier curve representing the percentage of mice protected against parasitemia after provocation as a function of time. Data were extracted and analyzed from Matrix Deconvolution Experiment 2.Closed circles represent CD1 mice immunized with the PY06306 antigen alone, and the symbols Xs, squares, and triangles represent PyCSP, 4X Null (unimmunized mice), and naive mice, respectively. Mice were provoked with 300 Plasmodium yoeli sporosome, and Giemsa-stained blood smears were performed to evaluate parasitemia up to 14 days (PyCSP, 4X Null, and naive) or 17 days (PY06306) after provocation. Figure 4 shows the antibody response to the matrix deconvolution experiment. Endpoint immunofluorescence assay (IFA) titers were measured for Plasmodium yoeli sporosome and hematological parasites. Serum collected one week after adeno-5 enhancement was gathered per group of antigen combinations, and reactivity to naturally dried parasites was analyzed. Gray bars and black bars represent sporosome and hematological reactivity, respectively. The positive control antibodies were NYS1 and NYLS3 monoclonal antibodies, respectively. Serum from 4X null and inexperienced animals served as negative controls. Figure 5 shows the antibody titers of protected and unprotected mice for the matrix deconvolution experiment illustrated in Figure 4. The endpoint immunofluorescence (IFA) titers were from a group of six PY06306 (E140)-containing mice. Plasmodium yoeli sporosome was measured in individual mice. One group from Matrix Experiment 2 (Mx2); E140, E137, E057 combination (closed circle) and five groups from Matrix Deconvolution Experiment 2 (MDx2); E140, E137, E057 combination (closed square), E140 alone (closed diamond), E140, E137 combination (closed star), E140, E057 combination (closed triangle), and E140, E137, E057, PY3424 combination (closed asterisk(*)). All protected mice were indicated by closed symbols, and all unprotected mice were indicated by X symbols. The Mann-Whitney non-parametric test indicated statistical significance; **, p<0.005, and ***, p=0.001. Figure 6 shows continuous protection at week 11 in the deconvolution study illustrated in Figure 2. Sterile-protected mice were provoked with 200 Plasmodium yoeli sporosome after resting for 11 weeks. Protection was measured up to 17 days after provocation by performing Giemsa-stained blood smears. Figure 7 shows Pf (human Plasmodium falciparum), Pv (human Plasmodium bibox), and Pc (rodent Plasmodium cavaudii ( P. Chabaudi )), Py (Rodent Plasmodium yoeli), Pb (Rodent Plasmodium bergei), Pk (Primate Plasmodium nouresi ( P. knowlesi )), Pr (Primate Plasmodium rhodiani ( P. rhodiani )), and Pg (primate Plasmodium gavonis ( P. gaboniFigure 8 shows the antigenic homology of PY06306 (Py E140) among Plasmodium species, including )). Figure 8 shows the amino acid conservation of Pf E140 (PFA0205w, MAL1P1.31, or PF3D7_0104100) among various Pf parasite species. These parasites were collected from various countries on different continents. The highest (99%) and lowest (92%) homology is highlighted. Figure 9 shows the results of an in vivo T-cell reduction experiment in mice. CD1 xeno-crossed mice were immunized with PY06306 DNA, enhanced with adeno-5 vaccine, and CD4 before and after provocation with 300 Plasmodium yoeli sporosome. + , CD8 + , CD4 + / CD8 +T cells were reduced (black bars). The rat Ig and non-reduced groups were used as positive controls. The unimmunized mouse group (gray bars) was also reduced in the same way and was used as a negative control. The PyCSP (diagonal bars) and inexperienced (striped bars) groups were the experimental positive and negative controls. Arrows indicate the type of reduction and the number of sterile-protected mice among the immunized mice. Parasitemia was evaluated up to 19 days after provocation by performing Giemsa-stained blood smears on provoked mice. Figures 10A and 10B show serotransfer studies in CD1 and BALB / c mice. In Figure 10A, a group of 14 BALB / c mice were immunized with DNA / adenovirus 5 encoding PY06306 (black solid line) and PyCSP (gray solid line). Serum from immunized and unprovoked mice was collected and delivered to inexperienced recipient mice (PY06306 (black dashed line) and PyCSP (gray dashed line)) 24 and 6 hours before provocation. After provocation with 300 Plasmodium yoeli sporosome, the mice were monitored for parasitemia for 17 days. In Figure 10B, a group of 14 CD1 mice was immunized with DNA / adenovirus 5 encoding PY06306 (black solid line) and PyCSP (gray solid line). Serum from immunized and unprovoked mice was collected and delivered to inexperienced recipient mice (PY06306 (black dashed line) and PyCSP (gray dashed line)) 24 and 6 hours before provocation. After provoking with 100 Plasmodium yoeli sporosome, mice were monitored for parasitic hemorrhage for 17 days. The percentage of sterile-protected mice for each group is shown in the legend box. Figure 11 shows the protection of PY06306 against hemorrhagic hemorrhage.Fourteen CD1 mice per group were immunized with a single dose of DNA and enhanced with adenovirus 5 expressing PY06306 (black bars), PY06306 + PyFalstatin (gray bars), and PyFalstatin alone. Unimmunized mice and naive mice were used as the negative control group for the mice. PyFalstatin is also known as PY03424. All mice were provoked with 10,000 infected Plasmodium yoeli-infected erythrocytes, and parasitemia was monitored for 17 days after provocation by Giemsa-stained thin film smears. Figure 12 shows protection using mammalian codon-optimized adenovirus 5 in a plot comparing the native (na) and codon-optimized (co) PY06306 with the immunization pathway. CD1 mice (14 per group) were primary immunized with co E140 DNA and augmented with either native PY06306 adeno-5 (black bars) or mammalian co PY06306 adeno-5 (gray bars). Both adeno-5 constructs were 10. ^ 10, 10 ^ 9, 10 ^ 8, and 10 ^It was administered intramuscularly (IM) at a reduced dose of 7 PU. Two additional mouse groups were augmented with Ad5 administered subcutaneously (SC) and intravenously (IV). Two additional mouse groups were not primary immunized with the DNA vaccine and were instead immunized with a single IM dose of na and co PY06306 Ad5 2 weeks prior to provocation. Unimmunized mice (striped bars) and naive (checkered bars) mice served as negative controls. All mice were provoked with 300 Plasmodium yoeli sporosome, and parasitemia was monitored for 18 days by Giemsa-stained thin-film blood smears. Figure 13 shows that Pf E140 (PFA0205w or MAL1P1.31 or PF3D7_0104100) is immunogenic in mice. IFA titers were induced by the PFA0205w vaccine. Both CD1 and BALB / c mice were immunized with the PFA0205w (PfE140) vaccine reagent (a full-length recombinant protein expressed as a GST and 6xHis fusion by the VR1020-DV plasmid DNA vaccine, adenovirus 5, and Malt System). The recombinant protein was emulsified in Montanide ISA 720 adjuvant and subcutaneously immunized at a dose of 5 μg / dose. Immunofluorescence (IFA) titers were measured for Plasmodium falciferum sporosome and various hematological mixtures. Figure 14 indicates that Plasmodium falciferum E140 (PFA0205w) is naturally immunogenic in humans. T cells respond to PFA0205w (PfE140 or PF3D7_0104100) by human individuals radioisotope (RAS)-immunized Plasmodium falsiferum. PBMCs were stimulated with the overlapping 15-mer peptide PFA0205w pool A for 21 hours using brefeldin A and stained for viability, phenotype (CD14, CD19, CD3, CD4, and CD8), and intracellular function markers (including IFN-γ and CD154).CD4 generating IFN-γ and intracellular CD154 with background excluded. + CD8 cells that produce T cells (A) and IFN-γ + The frequency of T cells (B) was plotted. In both experiments, positive reactions to PFA0205w pool A (filled symbols) were confirmed to exceed two standard deviations from the mean of the negative control (DMSO-stimulated) samples. Figure 15 indicates that PVX_081555 (PvE140) is relatively abundant in Plasmodium bibox sporozoites. 256 Plasmodium bibox sporozoite proteins sequenced using Multidimensional Protein Identification Technology (MudPIT) were graphed based on their relative abundance defined by their quantitative values. The locations of Plasmodium bibox sporozoite proteins (circumsporozoite proteins) and Plasmodium bibox E140 (PVX_081555) are indicated by black arrows in the graph. Specific details for implementing the invention

[0026] The inventors have identified that pro-erythrocyte proteins are of great importance in conferring protective immunity against malaria. Despite the relatively large number of malaria genes identified through the sequencing of the malaria parasite genome, the identification of vaccine candidates has been significantly limited by the relatively complex life cycle of the malaria parasite. Furthermore, many genes of the malaria parasite are not well defined, not only functionally but also antigenically.

[0027] Under these circumstances, the inventors decided to perform high-throughput screening of antigens encoded by many genes to identify potential protective responses. The inventors developed a novel strategy for identifying and testing potential malaria antigens that overcomes the difficulties presented in the prior art. This novel approach involved identifying specific characteristics that the inventors determined would serve as indicators for potential human vaccine candidates. Subsequently, the inventors compiled a list of 146 speciation homologs of Plasmodium yoeli of Plasmodium falciferum genes believed to possess these characteristics. The inventors then designed cloning primers and established a strategy for gene cloning and screening using transfection ELISpot. Transfection ELISpot included the steps of transfecting A20 cell lines with the VR1020 vaccine construct, expressing the antigen, and presenting the antigen on an ELISpot assay using these transfected cells. The use of such ELISpot was a novel strategy for screening antigens. Preferential antigens were identified from Plasmodium falciferum proteins in a large panel. Preferential antigens were evaluated based on several criteria determined by the inventors to be relevant to protection against malaria. One of these criteria was to select antigens expressed during the sporozoite and liver stages of the malaria parasite, namely, pro-erythrocyte phase antigens. Among the antigens selected based on the above criteria, specific antigens exhibited a protective response in mice, indicating that the speciational homologs of these genes code for human antigens useful as potential vaccine agents in humans. In particular, one gene, PY06306, was later identified as PY17X 0210400, which is the subject of this specification, exhibited a dramatic and consistent protective response suggesting, surprisingly, that its speciational homolog codes for an antigen useful as a major vaccine agent.

[0028] However, the sequence recorded for the PY06306 gene is only a partial one (479 aa) and is derived from early genome annotations. In order to perform the protection experiment disclosed herein using the full-length antigen (816 aa), the inventors had to re-clone the gene. A similar situation occurred with Plasmodium falsiferum (human homolog), which also had to be re-cloned from what is known in the art. The sequence disclosed in the list provided herein, used in all examples, and reflected in all data examples corresponds to the version of the gene modified by the inventors, rather than the version previously considered to be the relevant sequence in the art.

[0029] The present invention relates to DNA and amino acid sequences encoding recombinant proteins of Plasmodium falciferum and Plasmodium bibox. Specifically, the present invention relates to highly protective, pre-erythrocyte-phase Plasmodium yoeli and its speciative homologous antigens of Plasmodium falciferum and Plasmodium bibox for use in malaria vaccines. The related sequences may be used to express proteins encoded for use as subunit immunogenic antigens or to induce an immunogenic response. It can be incorporated into a vector suitable for in vivo host expression. The antigen can be combined with an immunogenic agent or used alone.

[0030] In one embodiment, the immunogenic composition is a DNA-based vaccine. DNA has been identified as a successful platform for delivering the immunogenic composition of this specification. The DNA-based vaccine may be delivered by a recombinant virus such as Modified Vaccinia Ankara (MVA) attenuated varicella virus, vesicular stomatitis virus (VSV), or GC46 (gorilla adenovirus). Other human adenovirus substitutes such as baculovirus may also be used.

[0031] In another embodiment, the composition comprises an immunogenic protein. In this embodiment, the protein may be prepared by a first insertion of DNA encoding the protein in a suitable expression system. This includes, for example, an adenovirus-based system, a varicella-zoster virus-based system, or a DNA plasmid system. The expressed and purified protein may then be administered to mammals, such as humans, in one or more doses. In this embodiment, the purified protein may be expressed individually, or DNA encoding a specific protein may be recombinantly linked to form a single immunogenic composition. This immunogenic composition may then be administered in one or more doses to induce an immunogenic response.

[0032] One embodiment of the present invention relates to a recombinant polypeptide expressed as a whole length or as a fragment by a heterologous expression system. Examples of such a system are: Escherichia coli ( Escherichia coli ), yeast (Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Or Pikia Pastoris ( Pichia pastorisThese are mammalian cells (HEK293 or CHO cells), baculovirus-infected insect cells, and Drosophila S2 stable cells. Recombinant proteins may be included in immunogenic agents to induce an immune response. In the present embodiment, polypeptides may be included individually or in combination. The immunogenic composition of the present invention may also include an adjuvant to improve or enhance the immune response induced by polypeptides. Suitable adjuvants include ALFQ, a non-toxic agent comprising a monophosphoryl lipid A-containing liposomal composition together with saponins.

[0033] Adjuvants have traditionally been classified into approximately two main types based on their compositional origin, physicochemical properties, or mechanisms of action: (i) immunostimulators, such as TLR ligands, cytokines, saponins, and bacterial exotoxins, which act directly on the immune system to increase the response to antigens, and (ii) vehicles, such as inorganic salts, emulsions, liposomes, virosomes, and biodegradable polymer microspheres, which present vaccine antigens and immunostimulators that are co-administered to the immune system in an optimal manner. Recently, it has become clear that many of these vehicles also exert direct effects on the immune system and can be considered as immunostimulators.

[0034] Examples of adjuvants acceptable for inclusion in malaria vaccines include Army Liposome Formulation (ALF) derivatives such as ALF, ALFA (and aluminum), and ALFQ (and QS21). Other options include lipid A derivatives within liposomal formulations such as QS21 and 3D-monophosphoryl lipid A (a non-toxic derivative of lipopolysaccharide), as well as saponins, LPS, MPL, or 3D-MPL, acylated monosaccharides, saponin derivatives (Quil-A, ISCOM, QS-21, AS02, and AS01), soluble triterpene glycosides, Toll-like receptor 4 (TLR4) agonists, montanide (ISA51, ISA720), immunostimulatory oligonucleotides, and structurally similar to imidazoquinoline. Includes other immunostimulators. The adjuvant can be manufactured within a cholesterol-containing liposome carrier.

[0035] As used herein, the term “polypeptide” refers to an amino acid polymer and does not refer to a specific length of the product. Proteins are included in the definition of polypeptide. The term “mer,” accompanied by a number such as 15-mer, refers to the length of a polypeptide in many amino acids.

[0036] As used herein, the protein may be prepared and then isolated to include one or more polypeptides in an effective amount as described herein in an immunogenic composition by first expressing a suitable gene fragment by a molecular method such as expression from another expression system, such as a plasmid or viral system. A further aspect of the invention is the ability of the protein to induce a humoral and / or T-cell immune response.

[0037] Embodiments of the present invention involve the integration of DNA encoding a polypeptide into a vector expression system, wherein the system enables the expression of one or more polypeptides within a mammalian host cell, such as a human, to induce an immune response. The expression system may be a DNA plasmid or a viral system. Methods for preparing and administering a DNA vaccine expressing a plasmodium protein are well known in the art.

[0038] In other embodiments, a derivative of the protein may be used in the immunogenic composition. In a variation of the present embodiment, the immunogenic derivative of the Plasmodium falsipalum and Plasmodium bibox proteins comprises a full-length polypeptide amino acid sequence comprising the amino acid sequence disclosed herein and at least 10 adjacent amino acids. The immunogenic derivative of the polypeptide may be prepared by the expression of a suitable gene fragment or by other methods such as peptide synthesis. Furthermore, the derivative may be a fusion polypeptide comprising an additional sequence encoding one or more epitopes of the Plasmodium falsipalum polypeptide disclosed herein. In these embodiments, the protein may be directly included in the immunogenic agent or may be expressed from a DNA plasmid or a viral expression system.

[0039] In some embodiments, Plasmodium falsiferum and Plasmodium bibox polypeptide comprise immunogenic derivatives having 80% or more amino acid sequence identity with the sequences disclosed herein. In this context, the term “identity” refers to two or more sequences or subsequences that are identical or have a specific proportion of identical amino acid residues when aligned to have maximum relevance. Where the sequences differ in conservative substitutions, i.e., substitutions of residues having the same characteristics, the percentage sequence identity may be adjusted upward to modify the conservative characteristics of the substitutions.

[0040] When a composition is prepared for administration, it is preferably combined with a pharmaceutically acceptable carrier, diluent, or excipient to form a pharmaceutical formulation or unit dosage form. A "pharmaceutically acceptable carrier" is a carrier, diluent, excipient, and / or salt that is compatible with other components of the formulation and is not harmful to its recipient. The active ingredient for administration may exist as a dry powder or granules; as a solution, suspension, or emulsion. The composition exists as a dry powder before being reconstituted in a liquid carrier.

[0041] Pharmaceutical formulations comprising the immunogenic composition of the present invention can be manufactured by procedures well known in the art using well-known and readily available ingredients. The therapeutic agent of the present invention can also be formulated as a solution suitable for parenteral administration, e.g., intramuscular, subcutaneous, or intravenous routes. The pharmaceutical formulation of the therapeutic agent of the present invention may also take the form of a water-soluble or anhydrous solution or dispersion, or an emulsion or suspension.

[0042] Accordingly, the immunogenic composition may be formulated for parenteral administration (e.g., by injection, e.g., single injection or continuous infusion) and may be present in unit doses within ampoules, pre-filled syringes, small-volume infusion containers, or multi-dose containers with added preservatives. The composition may be suitable for intravenous, subcutaneous, or intramuscular administration. The active ingredient may appear in a form such as a suspension, solution, or emulsion in an oily or aqueous carrier and may include formulation materials such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from a solution, to be composed with a suitable carrier, e.g., sterile, pyrogen-free water, before use.

[0043] Furthermore, the immunogenic composition may include formulation materials that do not naturally occur in the cellular environment where the peptide is expressed. Although such formulation materials do not naturally occur in the cellular environment where the peptide is expressed, they nevertheless occur before, after, or during administration to mammals. It includes any surfactant, diluent, solubilizer, emulsifier, buffer, thickener, preservative, surfactant, adjuvant, excipient, and antimicrobial agent provided to artificially enhance biocompatibility, efficacy, delivery, storage, administration, absorption, stability, safety, or function within the immunogenic composition of the peptide.

[0044] Alternatively, the immunogenic composition may be provided as a dry powder. The dry powder composition may be prepared by freeze-drying, spray-drying, and freeze-spray-drying a solution or suspension containing the polypeptide described herein, and may optionally include the step of grinding or freeze-drying together with grinding. The dry powder may be suitable for direct administration to a patient, for example, by inhalation or capsule ingestion, or may be suitable for suspension or reconstitution in a fluid carrier. The dry powder formulation may include physiologically acceptable carrier powders such as excipients, dispersants, stabilizers, wetting agents, anti-caking agents, or other additives.

[0045] The immunogenic compositions of the present invention in dry powder and fluid embodiments may comprise, as optional components, pharmaceutically acceptable carriers, diluents, solubilizers, or emulsifiers, and salts of forms well known in the art. Specific, non-limiting examples of carriers and / or diluents useful for formulations of the compositions of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate-buffered saline (pH 7.0-8.0). The compositions of this specification may also comprise combinations of other substances such as diluents, water, salts, glycerol or other suitable alcohols, wetting or emulsifiers; buffers; thickeners, such as cellulose or cellulose derivatives, for example; preservatives; surfactants; antimicrobial agents, etc.

[0046] When an immunogenic composition is used as a vaccine, the composition comprises an immunologically effective amount of peptide as described herein. The "immunologically effective amount" of an antigen is an amount effective for the treatment or prevention of malaria infection when administered to an individual in a single dose or a series of doses. Such amount varies depending on the health and physical condition of the individual to be treated and the antigen. Determining the effective amount of an immunogenic or vaccine composition for administration to an organism is within the ability of a person skilled in the art.

[0047] The composition according to the present invention may be administered orally, systemically, parenterally, topically, mucosally, intramuscularly, intravenously, intraperitoneally, intradermally, subcutaneously, intranasally, vaginally, rectalally, transdermally, sublingually, by inhalation, or by nebulization. The composition may be prepared to be administered as a single dose or as part of a multiple dosing schedule. Multiple doses may be administered as primary immunization leading to one or more booster injections. Primary immunization may comprise a single formulation, such as a virus (GC46) or DNA vaccine, followed by one or more booster injections using a single or multiple formulation, such as other viruses (e.g., MVA) or recombinant proteins. The appropriate timing between primary immunization and booster injections may be determined by custom. The composition according to this specification may be used separately or in combination with one or more other immunogenic or vaccine compositions and / or with one or more other therapeutic regimens.

[0048] Accordingly, the present specification provides a method for protecting a human or non-human mammal from the effects of malaria infection, comprising the step of administering a composition described herein to a human or non-human mammal. The composition may be a vaccine. The present specification further provides a method for increasing an immune response in a human or non-human mammal, comprising the step of administering a pharmaceutical composition described herein to a human or non-human mammal. The immune response is preferably protective. The method may increase an enhancing response in a patient who has already been primary immunized. The immune response may be prophylactic or therapeutic.

[0049] Examples

[0050] Example 1: Verification of E140

[0051] Novel, highly protective pre-erythrocyte (PE) plasmodium yoeli ( Plasmodiumyoelii (Py) antigen, a human speciative homolog identified for use in human malaria vaccines. The said antigen is identified according to the nomenclature used as PlasmoDB ID 10: PY06306, or PY17X_0210400, PYYM_0211900, or ID: 2121.m00052. The said antigen is also referred to as E140 or Py E140 as an abbreviation in the experimental tests disclosed herein. The novel antigen is highly expressed in the sporozoite, liver stage, and blood stage of the parasite and in CD8 in mice immunized with Plasmodium yoelii radiotoxin sporozoites (RAS). + It induces a T cell response. This elicits a strong antibody and cellular response during antigen-specific vaccine immunization and provides 71% to 100% sterile protection against infectious Plasmodium yoeli sporosome and hemoglobin provocation, both alone and in combination with other mouse antigens. First, to identify antigens for vaccine development, the responsiveness of T cells from RAS-immunized mice to Plasmodium yoeli pro-erythroid antigens was screened as a platform. The process included the identification, cloning, and generation of the DNA plasmid (VR1020), screening of Py antigens, and evaluation of mouse protective ability. It is well known that mouse models allow for the assessment of the success of human speciation homologs. The gene encoding the PY06306 antigen was identified as a pro-erythroid target for vaccine development, and a portion of the gene was cloned. Subsequent experiments revealed that the aforementioned protein can remember the cytokine (IFN-γ) response from splenocytes observed in mice immunized with Plasmodium yoeli RAS. These results provide strong evidence that the PY06306 antigen is involved in the RAS immune response and protection, thereby demonstrating the importance of pro-erythrocyte vaccination in humans.

[0052] Example 2: E140 Protection Verification

[0053] For protection studies in mice, two vaccine reagents expressing the PY06306 antigen were prepared. These reagents were prepared using the full-length gene: the VR1020 plasmid (PY06306-E140) and the DNA vaccine within adenovirus serotype 5 (AdE1(t.PY06306)E3(10X)E4(TIS1)). Evidence for the potential of the PY06306 antigen as a vaccine was demonstrated in two independent animal matrix studies intended to evaluate the antigen's ability to induce an immune response capable of sterile protection in mice from infectious Py sporosome provocation. Sterile protection was measured by the absence of parasites in the mice's blood up to 14 or 17 days after sporosome provocation. Xenobred CD1 mice were first immunized with the DNA vaccine (100 μg, IM) and then, 6 weeks later, with the adenovirus serotype 5 construct (10 10 Immunization was performed using a regimen consisting of boosting with (PU, IM). A 3-antigen combination strategy (called the matrix) was employed to test PY06306 antigens and other novel Py pro-erythrocyte antigens with and without Plasmodium yoeli sporosome protein (PyCSP).

[0054] The first matrix animal study shown in Figure 1 illustrates two PY06306-containing antigen combinations (groups) exhibiting significant protection. The first combination induced sterile protection of 64% and 86% when used alone and with PyCSP, respectively. The antigen components of the first combination were E140 (PY06306), E137 (PY05693), and E057 (PY03396). The 86% protection provided by the 3-antigen mixture combined with PyCSP was twice as high as that of the PyCSP-alone group (43%), indicating a significant improvement in the efficacy of the vaccine of the optimal standard. The second 3-antigen combination exhibited sterile protection of 14% and 71% when used alone and with PyCSP, respectively. The second combination above consisted of E140 (PY06306) combined with two additional antigens having vaccine potential, Py325 (PY00232) and PyCelTOS (PY17X_1434600). As shown in the corresponding figure, any or all of these five antigens (PY06306, PY05693, PY03396, PY325, and PyCelTOS) contribute to protective ability, but PY06306 was the only antigen common to all three antigen combinations, and thus a second experiment for deconvolution of these antigen combinations is required.

[0055] Example 3: Sporosome provocation

[0056] A second study (Matrix Deconvolution Experiment 2) was designed to evaluate various antigen combinations having PY06306 as the common denominator antigen. The experimental format and immunization followed the same regimen described for the first matrix experiment. Figures 1 and 2 show the significantly high efficacy of all antigen combinations containing the PY06306 (E140) antigen, with the range of protected mice ranging from 71% to 100%. Overall, 89% (137 / 154) of PY06306-immunized mice were protected from malaria infection. The PY06306 vaccine alone showed 71% protective efficacy, which was significantly higher compared to the PyCSP alone group (36%). Furthermore, as shown in Figure 3, there was a significant delay in the onset of parasitaemia in unprotected mice. Detailed analysis of blood smear data from the PY06306-immunized group revealed that 3 out of 4 unprotected mice tested positive for malaria 7, 10, and 12 days after sporosome provocation. This was significant when compared to the onset of parasitemia in the PyCSP, 4X Null, and naive groups, and all unprotected mice tested positive for malaria on day 5 after sporosome provocation.

[0057] Example 4: Antibody titer

[0058] The PY06306 antigen induced high antibody titers against the sporosome stage of Plasmodium yoeli and low antibody levels against the hematological stage, depending on the individual mouse. This evidence is shown in Figure 4 (PYQ6306 group), which lists the immunofluorescence (IFA) antibody titers against sporosome and hematological parasites measured in serum collected from mice in Matrix Deconvolution Experiment 2. In summary, anti-sporosome antibodies were detected in all groups immunized with PY06306, including combinations, which supports the immunogenicity of the PY06306 antigen. Titers ranged from 1:5,120 to 1:20,480. Antibodies induced by Plasmodium yoeli PY06306 immunization cross-react with Plasmodium bergayi sporosome. Detection of high antigen titers (1:5,120) in mice immunized with PY06306 alone demonstrates that the PY06306 antigen induces antibodies against sporosome.

[0059] Based on the review of the above data, two important findings are: (i) that no protection was observed (0%) for the antigen group lacking PY06306 (PY03396 and PY05693) in Figure 2, and no antibody response was observed. This confirms that PY06306 is the major component, if not the sole component in this combination that induces protection. The other is (ii) that an anti-sporosomal antibody response was specifically induced by the PY06306 antigen. Comparison of anti-sporosomal IFA titers for protected and unprotected mice strongly suggests that the antibodies detected in these mice are associated with the protective outcome. All protection studies were performed under animal protocols D02-09 and 14-IDD-13. The results of the protection studies demonstrate that the PY06306 speciational homolog plays a role as an important component in malaria vaccines.

[0060] Example 5: Spleen and Liver Analysis

[0061] Further studies showed that PY06306-immunized mice exhibited over 10% CD8+ T cells expressing IFNγ and fewer (less than 0.6%) CD4+ T cells in the spleen. In the liver, levels were observed in the range of 5% to 16.2%. High protective efficacy was maintained for 11 weeks following the second sporosome provocation. The decrease in T cells indicates that high levels of E140-specific T cells are not required for protection in mice. Furthermore, PY06306 immunization induces high levels of CD8+ T cells expressing IFNγ in the spleen and liver. Anti-PY06306 serotransmission to CD1 and BALB / c mice significantly delayed the onset of parasitemia. Additionally, mice receiving E140 sero showed significantly lower IFA titers compared to protected mice immunized with PY06306. PY06306 serum collected before sporosome provocation reacted with sporosome alone. However, after provocation, some of the protected mice developed antibodies positive for IFA in the blood group.

[0062] PY06306 provides sterile protection against blood-related provocation in up to 100% of CD1 and BALB / c mice (Fig. 11). Immunization with PY06306 prevents blood infection in 88% (30 / 34) of unprotected mice and delays the onset of detectable parasiticemia. Furthermore, delivery of anti-PY06306 antibodies to unprotected mice significantly delays infection. High levels of CD8+ T cells expressing IFNγ are found in the spleen and liver of PY06306-immunized mice. A decrease in these cells did not reduce sterile protection. PY06306-specific IFA antibody titers are associated with protection.

[0063] Example 6: In vivo T cell reduction

[0064] Figure 9 shows the results of the study on in vivo T-cell reduction. Several groups of crossbred CD1 mice were immunized. T-cell reduction was induced by injecting T-cell-specific monoclonal antibodies according to a standard protocol. Subsequently, the mice were provoked with 300 Plasmodium yoelie sporosome, and protective ability was evaluated by the absence of parasites in thin-film blood smears up to day 19 after provocation. All T-cell reduced PY06306-immunized mice were protected, confirming that neither CD4+ nor CD8+ T cells are required for PY06306 protection. On day 13 after sporosome provocation, malaria was detected in the blood of one unprotected mouse in the CD4 / CD8 group, whereas all other mice showed positive smear samples on day 5. A total of 68 protected mice out of 70 were immunized, demonstrating an overall efficacy of 97%. The above study demonstrates a surprising mechanism in which protection induced by pro-erythrocyte antigens in response to sporosome provocation does not rely on T cells.

[0065] Example 7: Serum Delivery Study

[0066] Figures 10A and 10B show serum delivery studies in CD1 and BALB / c mice. The study demonstrated the role of antibodies in protection induced by PY06306 (E140). The study design followed a standard serum delivery protocol, collecting serum from PY06306-immunized CD1 and BALB / c mice, delivering it to naive animals (at a 1:1 ratio), and then provoking them with Plasmodium yoeli sporosome. Serum delivery occurred for 2 days; 24 hours; and 6 hours prior to provocation with the sporosome. The results of protection are shown in Figure 10A for CD1 mice and Figure 10B for BALB / c mice. Figures 10A and 10B show that sterile protection was not transferred to serum from mice immunized with the PY06306 vaccine (7% of CD1 mice (1 in 14) and 0% of BALB / c mice (0 in 14)). Compared to other random groups in the same study, there was a statistically significant delay in the onset of parasitemia in all unprotected mice from PY06306 serum recipients (dotted line) (Mantel-Cox ***, p=0.0001). This demonstrates that the anti-PY06306 antibody plays a role in protection by effectively influencing the progression of parasites in the blood. The significantly lower antibody titers in recipient CD1 (1:2,560) and BALB / c (1:575) mice compared to donor CD1 (1:7,994) and BALB / c (1:18:549) mice explain why these mice were not protected from provocation.

[0067] Example 8: Detection of PY06306-specific CD8 T cells in the spleen and liver

[0068] PY06306-specific CD8 T cells are found in the spleen and liver of PY06306-immunized and naive mice. Due to the fact that PY06306 is a macromolecule, the 15-mer overlapping peptide is divided into two pools throughout the entire protein; containing peptides from the N-terminus Pool Aand from the C-terminus of PY06306 Pool B T cells were measured by a flow cytometry method specified for CD8+ cells expressing interferon gamma (IFNγ) and expressed as a proportion of the total T cell population. The data showed that only peptides from pool A were able to remember IFNγ CD8 cells, confirming that PY06306 T cell epitopes are prone to being confined to the N-terminus of the antigen. Very high levels of IFNγ-expressing CD8+ T cells were detected in the spleen (average 18%) and liver (average 11%) of PY06306-immunized mice. For intracellular cytokine staining, splenocytes and liver-present T cells were prepared from PY06306-immunized and unimmunized (Null) mice using a standard protocol and then stimulated for 6 hours with PY06306 (E140) peptide pools A and B at a final concentration of 2 μg / ml. Data were obtained using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo (Tree Star Inc.).

[0069] Example 9: PY06306 induces protection in BALB / c mice

[0070] The PY06306 antigen effectively protects BAB / c mouse lines against sporosome provocation. 14 BALB / c mice per group were immunized with a single dose of DNA and PY06306, PYQ6306 + PyCSP, and PyCS PIt was enhanced with adenovirus 5 encoding [variant]. Unimmunized mice and naive mice were used as mouse negative controls. All mice were provoked with 100 infectious Plasmodium yoeli sporosome, and parasitemia was monitored by Giemsa-stained thin film smears for 17 days after provocation. At provocation, all (100%) PY06306-immunized mice were sterile protected (PY06306 and PY06306+PyCSP), whereas 57% were protected with PyCSP. Therefore, PY06306 can protect inbred mouse strains, and mixing with PyCSP antigens does not inhibit the protection of PY06306.

[0071] Example 10: PY06306 induces protection against blood-related provocations.

[0072] Figure 11 shows the protection of PY06306 against hemoglobin provocation. The PY06306 antigen alone and in combination with PyFalstatin protect mice from an urgent provocation using 10,000 hemoglobin parasites. In this study, mice were immunized with PY06306 alone and in combination with PyFalstatin and provoked with Plasmodium yoeli-infected erythrocytes. Mice in both groups were 100% sterile protected (black and gray bars). The PyFalstatin antigen is also known as PY03424. Protection against hemoglobin provocation is an important characteristic of malaria vaccines, providing a second-stage defense induced by the PY06306 vaccine.

[0073] Example 11: Protection using a smaller single-dose codon-optimized PY06306 Ad5

[0074] Figure 12 shows protection using codon-optimized adenovirus 5. The study evaluated adenovirus 5 constructs prepared using the codon-optimized (co)PY06306 gene designed for expression in mammalian cells. Changes in the original PY06306 codon sequence do not alter the amino acid sequence expressed by the Ad5 virus. The study evaluated the in vitro ( in vitro ) expression was examined and compared. After probing with mouse polyclonal serum, coPY06306 Ad5 expressed much higher levels of PY06306 protein compared to the original construct. In mouse group 1, the enhancement dose was 10 per dose for the original (na) and codon-optimized (co) PY06306 Ad5. ^ 10, 10 ^ 9, 10 ^ 8 and 10 ^Titration was performed within a range of 7 PU. All mice in these eight groups were primary immunized with the same coPY06306 DNA vaccine dose (100 μg) and intramuscularly (IM) enhanced with various doses of naPY06306 (black bars) and coPY06306 (gray bars) Ad5 constructs. Overall efficacy showed that the coPY06306 Ad5 vaccine induced higher protection (100%, 100%, 86%, and 93%) compared to naPY06306, which exhibited lower protection (86%, 93%, 86%, and 71%) in CD1 mice at the same Ad5 dose. The study also compared the subcutaneous (SC) and intravenous (IV) routes for Ad5 administration. The SC route showed similar levels of protection for the na and coPY06306 vaccines (50% and 57%, respectively). The IV route for co PY06306 Ad5 resulted in 100% sterile protection, whereas na showed 79%. The IV route for na PY06306 Ad5 resulted in 79% sterile protection, while the subcutaneous route resulted in 50% protection. The group of mice immunized with a single dose of coPY06306 Ad5 induced 93% sterile protection, compared to 29% for the naPY06306 vaccine. These mice did not undergo primary immunization with the DNA vaccine. All protection studies were performed under animal protocols D02-09 and 14-IDD-13.

[0075] Example 12: Human Plasmodium falciparum is immunogenic.

[0076] Figure 13 shows that Plasmodium falsiferum PFA0205w (E140 speciative homolog) is immunogenic in mice. Four vaccine reagents were generated for PFA0205w (also known as PF3D7_0104100): VR1020 DNA vaccine construct, human adenovirus 5 construct, protein expression plasmid pEU-E01-GST, and pEU-E01-His. The DNA vaccine and Ad5 were prepared in large quantities for mouse immunization. Recombinant proteins were produced on a small scale using a malt cell-free system at the NMRC. CD1 and BALB / c mice were immunized with various primary immunoadjuvant therapies as shown in Figure 13. Ad5 primary immunity and recombinant protein enhancement is the most immunogenic regimen, inducing IFA titers of up to 1:4,000 against the parasite Plasmodium falciferum at the blood and sporosome stages. A single dose of PFA0205w adenovirus 5 induces antibodies against the parasite. This demonstrates the potential for success of PFA0205w as a vaccine formulation, either as a single dose of recombinant virus (adenovirus 5) or as a primary immunity-enhancing agent using Ad5 protein therapy.

[0077] Example 13: Plasmodium falsiparum E140 (PFA0205w) is immunogenic in humans

[0078] Figure 14 shows that Plasmodium falsiferum E140 (PFA0205w) is immunogenic in humans. T cells from individuals immunized with radioactive sporosome (RAS) were able to respond to stimulation by the PFA0205w peptide pool (A). The peptide mixture contained 15-mer overlapping peptides covering most of the N-terminal region of the PFA0205w protein. Due to the size of the protein, the peptides are divided into two pools: Pool A, covering the N-terminus of the PFA0205w protein, and Pool B, covering the C-terminus. Data from both graphs indicated that immunization with the radioactive sporosome vaccine induced both CD4 and CD8 T cells in humans. CD4+ and CD8+ T cells play a role in PFA0205w-induced protection against pro-erythrocyte parasites. High levels of Plasmodium yolei E140 response are observed in the spleen and liver of E140-immunized mice.

[0079] Example 14: PFA0205w is expressed in pygmy worms and is localized on the surface of sporosome and the cell matrix.

[0080] The PFA0205w antigen is expressed in both the sporosome and vegetative stages of Plasmodium falciferum. IFA reactivity was obtained using CD1 mouse serum produced by primary immunization with PFA0205w adenovirus 5 and enhancement with recombinant PFA0205w protein. The serum was positive for 36-hour Plasmodium falciferum erythropoietic vegetative stages and negative for the early vegetative stage and trophosome. Intracellular localization of the PFA0205w antigen in sporosome stages was confirmed by immunoelectron microscopy (EM). Analysis of electron microscope images revealed that the PFA0205w antigen was localized on both the surface and the cellular matrix of Plasmodium falciferum sporosome. Immunofluorescence and immunoelectron microscopy demonstrated the reactivity of serum from CD1 mice immunized with PFA0205w adenovirus 5 and enhanced with recombinant PFA0205w protein. Naturally dried IFA slides were prepared using the NF54 Plasmodium falciferum parasite for approximately 36 hours after attacking erythrocytes. IFA was diluted to a 1:500 serum solution and developed with FITC-labeled goat anti-mouse Ig. For immunoEM, salivary glands containing Plasmodium falciferum sporosome were isolated from infected mosquitoes. Fixed salivary glands were inserted and excised, placed on an electron microscope grid, and stained using the same serum and colloidal gold-labeled anti-mouse antibody. Microscopic images showed that the PFA0205w antigen was localized on both the surface and the cellular matrix of the Plasmodium falciferum sporosome.

[0081] Example 15: PVX_081555 (PvE140) is expressed in Plasmodium bibox sporosome.

[0082] Fig. 15 shows that PVX_081555 (PvE140) is expressed in Plasmodium bibox sporosome. Anopheles dirus ( Anopheles dirusBlood from a malaria-infected patient was supplied to mosquitoes via a membrane supply device. Fourteen days after membrane supply, mosquito salivary glands were extracted from 100 mosquitoes. The salivary glands were crushed using a mortar and pestle in a microcentrifuge tube containing phosphate-buffered salts to release the plasmodium bibox sporosome. Subsequently, the salivary gland fragment-sporosome mixture was centrifuged to remove the mosquito gland fragments, and the plasmodium bibox sporosome from the supernatant was transferred to a new microcentrifuge tube. The extracted plasmodium bibox sporosome was counted at 1 x 10⁶ 6The sporosome was digested using 1 µg of molecular biology-grade trypsin at 37°C for 18 hours. After digestion, the trypsin-digested sporosome peptides were desalted using a C8 reverse-phase column and freeze-dried. To identify Plasmodium Bivax sporosome proteins that could serve as vaccine candidates, Multidimensional Protein Identification Technology (MudPIT) was applied to the freeze-dried trypsin-digested peptides. Tandem mass spectra generated from Plasmodium Bivax sporosome were investigated against the combined Anopheles-Plasmodium Bivax protein sequence database using the Sequest algorithm. The result files from the Sequest investigation were loaded into the Scaffold protein viewer. Sequences matched to the Anopheles proteome were excluded using the Scaffold program to highlight proteins that specifically matched the Plasmodium Bivax proteome. Scaffold software was used to compare the abundance of each Plasmodium bibox sporosome protein identified by MudPIT. Protein abundance was defined by a scaffold "quantitative value" that normalizes the abundance of the mass spectrum matching the specified protein to its molecular weight. Twenty-fivety high-reliability Plasmodium bibox proteins were identified in this MudPIT experiment. Plasmodium bibox E14Q (PVX_081555) was the 39th most abundant Plasmodium bibox sporosome protein and the 5th most abundant among sequenced membrane-associated proteins. By comparison, the CSP vaccine antigen, which is also associated with the parasite membrane, was the 5th most abundant protein overall and the most abundant membrane-associated protein within the sample. The above results demonstrate that Plasmodium bibox E140 is the most abundant membrane-associated protein within the parasite. Therefore, due to the membrane association and abundance of E140, E140 becomes a prominent target of humoral responses. [Effective Mode]<Embodiment 1> An immunogenic composition for protecting mammals from malaria, comprising: a recombinant polypeptide, wherein the recombinant polypeptide comprises one of the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, and their derivatives, said derivative having at least 10 adjacent amino acids of SEQ ID NO. 3 and SEQ ID NO. 6 or having 85% identity with one of SEQ ID NO. 3 and SEQ ID NO. 6; a pharmaceutically acceptable carrier; and an adjuvant. <Embodiment 2> An immunogenic composition for protecting mammals from malaria, comprising a combination of two or more recombinant polypeptides within a pharmaceutically acceptable carrier, said first of the two or more recombinant polypeptides comprising one of the amino acid sequences of SEQ ID NO. 3 and its derivatives, said derivative having at least 10 adjacent amino acids of SEQ ID NO. 3 or having 85% identity with SEQ ID NO. 3; a pharmaceutically acceptable carrier; An immunogenic composition comprising an adjuvant. <Embodiment 3> The immunogenic composition of Embodiment 2, wherein the second of the two or more recombinant polypeptides comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, and derivatives thereof, and the derivative has at least 10 adjacent amino acids of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, and / or has 85% identity with one of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9.<Embodiment 4> A method for inducing an immune response to malaria in a mammal, comprising the step of administering to the mammal an immunologically effective amount of a composition comprising a polypeptide encoded by one of the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, and a derivative thereof, wherein the derivative has at least 10 adjacent amino acids of SEQ ID NO. 3 and SEQ ID NO. 6 or has 85% identity with one of SEQ ID NO. 3 and SEQ ID NO. 6. <Embodiment 5> The method of Embodiment 4, wherein the mammal is a human. <Embodiment 6> The method of Embodiment 4, wherein the method further comprises the step of administering one or more primary immunity or booster prophylaxis injections against malaria to the mammal, wherein the primary immunity and booster prophylaxis injections comprise an immunologically effective amount of a recombinant polypeptide, wherein the recombinant polypeptide comprises one of the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, and a derivative thereof, and wherein the derivative comprises at least 10 of SEQ ID NO. 3 and SEQ ID NO. 6. A method having adjacent amino acids or having 85% identity with one of SEQ ID NO. 3 and SEQ ID NO. 6. <Embodiment 7> A method of administering an immunologically effective amount of the composition of Embodiment 1 to a mammal by introducing a suitable expression vector for the expression of a polypeptide into the mammal, wherein the suitable expression vector is selected from the group consisting of a plasmid, a replica virus vector, and a non-replication virus vector. <Embodiment 8> The method of Embodiment 7, wherein the mammal is a human. <Embodiment 9> The immunogenic composition of Embodiment 1, wherein the recombinant polypeptide is expressed by a suitable expression vector selected from the group consisting of a plasmid, a replica virus vector, and a non-replication virus vector.<Embodiment 10> An immunogenic composition in Embodiment 1, wherein the recombinant polypeptide is expressed by a suitable expression vector selected from the group consisting of DNA plasmid, baculovirus, VSV, MVA, GC46, alphavirus replicon, adenovirus, varicella virus, adeno-associated virus, cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus, RNA replicon, DNA replicon, alphavirus replicon particle, Venezuelan equine encephalitis virus, Semliki forest virus, and Sindbis virus. <Embodiment 11> The method of Embodiment 4, wherein the composition is administered through a suitable expression vector expressing a recombinant polypeptide, and the suitable expression vector is selected from the group consisting of DNA plasmid, baculovirus, VSV, MVA, GC46, SpyVLP, alphavirus replicon, adenovirus, varicella virus, adeno-associated virus, cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus, RNA replicon, DNA replicon, alphavirus replicon particle, Venezuelan equine encephalitis virus, Semliki forest virus, and Sindvis virus. <Embodiment 12> An immunogenic composition for protecting mammals from malaria, comprising a recombinant polypeptide, wherein the recombinant polypeptide comprises one of the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, and their derivatives, and the derivative comprises at least 10 adjacent amino acids of SEQ ID NO. 3 and SEQ ID NO. 6 An immunogenic composition having 85% identity with either SEQ ID NO. 3 or SEQ ID NO. 6, wherein the immunogenic composition is a dry powder. <Embodiment 13> The immunogenic composition of Embodiment 12, wherein the dry powder is suspended or reconstituted in a pharmaceutically acceptable carrier and is suitable for administration to mammals.

Claims

Claim 1 As an immunogenic composition for inducing an immune response in an organism, the immunogenic composition comprises (a) a recombinant plasmodium comprising the amino acid sequence of SEQ ID NO. 3 ( Plasmodium ) E140 polypeptide; (b) a pharmaceutically acceptable carrier; and (c) an adjuvant, wherein the recombinant plasmodium E140 polypeptide is plasmodium falciferum ( P. falciparum ) An immunogenic composition that is the E140 antigen. Claim 2 In claim 1, the recombinant plasmodium E140 polypeptide is an immunogenic composition produced using an expression vector for the expression of a prokaryotic or eukaryotic organism. Claim 3 An immunogenic composition according to paragraph 2, wherein the expression vector is a plasmid, a replicating virus vector, or a non-replication virus vector. Claim 4 An immunogenic composition according to claim 2, wherein the expression vector is a DNA plasmid, baculovirus, rVSV, SpyVLP, alphavirus replicon, adenovirus, varicella virus, adeno-associated virus, cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus, RNA replicon, DNA replicon, alphavirus replicon particle, Venezuelan equine encephalitis virus, Semliki forest virus, or Sindbis virus. Claim 5 The immunogenic composition according to claim 1, wherein the adjuvant is selected from the group consisting of Army Liposome Formulation (ALF) derivatives, lipid A derivatives, saponins in QS21, saponins in 3D-monophosphoryl lipid A, lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), 3-O-diacylated monophosphoryl lipid A (3DMPL), acylated monosaccharides, saponin derivatives, soluble triterpene glycosides, Toll-like receptor 4 (TLR4) agonists, montanide ISA51, montanide ISA720, immunostimulating oligonucleotides, and imidazoquinoline. Claim 6 In paragraph 5, the immunogenic composition wherein the ALF derivative is selected from the group consisting of ALF, ALF plus aluminum (ALF A), and ALF plus QS21 (ALFQ). Claim 7 In claim 1, the adjuvant is an immunogenic composition selected from the group consisting of Quil-A, Immunostimulating Complex (ISCOM), QS-21, AS02, and AS01. Claim 8 The immunogenic composition of claim 1, wherein the immunogenic composition is a solution, suspension, or emulsion. Claim 9 In paragraph 1, the individual is a human, an immunogenic composition. Claim 10 In claim 1, the immunogenic composition is administered orally, systemically, parenterally, topically, mucosally, intramuscularly, intravenously, intraperitoneally, intradermally, subcutaneously, nasally, vaginally, rectalally, transdermally, sublingually, via a spray route, or by inhalation. Claim 11 The immunogenic composition of claim 1, wherein the immunogenic composition is formulated into an ampoule, a pre-filled syringe, a small-volume infusion container, or a multi-dose container having an added preservative. Claim 12 In claim 11, the immunogenic composition is an immunogenic composition formulated in a pre-filled syringe. Claim 13 An immunogenic composition for inducing an immune response in an individual, wherein the immunogenic composition comprises: (a) a recombinant plasmodium E140 polypeptide having the amino acid sequence of SEQ ID NO. 3; (b) a pharmaceutically acceptable carrier; and (c) an adjuvant, wherein the immunogenic composition further comprises one or more additional recombinant polypeptides having amino acid sequences selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO.

9. Claim 14 In claim 13, the recombinant plasmodium E140 polypeptide comprising the amino acid sequence of SEQ ID NO. 3 is an immunogenic composition produced using an expression vector for the expression of a prokaryote or eukaryote. Claim 15 In claim 14, the expression vector is an immunogenic composition that is a plasmid, a replicating virus vector, or a non-replicating virus vector. Claim 16 An immunogenic composition according to claim 14, wherein the expression vector is a DNA plasmid, baculovirus, rVSV, SpyVLP, alphavirus replicon, adenovirus, varicella virus, adeno-associated virus, cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus, RNA replicon, DNA replicon, alphavirus replicon particle, Venezuelan equine encephalitis virus, Semliki forest virus, or Sindbis virus.

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