PLASMODIUM

PRIOR RELATED APPLICATION

[0001] The present application claims the benefit of priority of U.S. Provisional Application No. 62/463,011 filed February 24, 2017, incorporated herein by reference in its entirety.

FIELD

[0002] The products and processes described in this document relate to the fields of immunology, immunochemisiry, immunoassays, malaria studies, vaccines, malaria vaccines, transmission blocking vaccines, parasitology, protein expression and purification and other related fields.

BACKGROUND

[0003] Malaria, which is caused by protozoan parasites of the genus Plasmodium and is transmitted by Anopheles mosquitoes, is a global disease. In 2015, there were 214 million new cases and an estimated 438,000 malaria deaths worldwide. Currently, effective antimalarial vaccines are not available, while Plasmodium parasites throughout the world exhibit growing resistance to the existing anti -malarial drugs. Mosquitoes become infected when they ingest blood from an infected human, and the parasites undergo a complex developmental cycle in the mosquito in order to be transmitted to another person. Disease transmission from mosquitoes to humans is highly effective in malaria-endemic areas. In spite of this, malaria was eliminated in some of the areas by controlling the mosquito populations and reducing the rate of disease transmission. Thus, reduction in the transmission rates is one of the key steps required for malaria control and eradication. Compositions and methods reducing or blocking Plasmodium transmission would be highly useful in malaria-elimination efforts.

SUMMARY

[0004] This document describes compositions and methods that are useful, among other tilings, for blocking transmission of malaria-causing parasites, such as, but not limited to, Plasmodium, falciparum or Plasmodium vivax. The compositions and methods described in this document can reduce transmission of Plasmodium parasites from the vertebrate hosts to anopheline mosquitoes, thereby reducing Plasmodium infection transmission and the incidence of malaria. The terms ^" 'invention," "the invention," "tins invention" and "the present invention," as used in this document, are intended to refer broadly to all of the subject matter of this patent application and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein and not to limit the meaning or scope of the patent claims below. Covered embodiments of the invention are defined by the claims, not by this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the "Detailed Description" section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. Tire subject matter should be understood by reference to appropriate portions of the entire specification, any or all drawings and each claim. Some non-limiting exemplary embodiments of the present invention are summarized below. It is to be understood that various combinations the exemplary embodiments are envisioned and also fall within the scope of the present invention.

[0005] An exemplary embodiment of the present invention is an isolated nucleic acid encoding an isolated polypeptide corresponding to at least a portion of D2 domain of a Plasmodium P47 protein. The Plasmodium P47 protein can be Plasmodium falciparum P47 protein (Pfs47) or Plasmodium vivax P47 protein (Pvs47). The D2 domain can have at least 80%, at least 90% or at least 95% amino acid sequence similarity to SEQ ID NO:5. One or both cysteines in SEQ ID NO: 5, which together form a disulfide bond, can be substituted with same or different amino acids that do not lead to formation of the disulfide bond. For example, one or both cysteines in SEQ ID NO: 5 can be substituted with alanine. In some exemplary embodiments, the isolated polypeptide encoded by the nucleic acid does not contain any disulfide bonds. The isolated polypeptide, when administered to or expressed in a vertebrate animal in an immunologically effective amount, can induce production of immunoglobulins capable of specifically binding to D2 domain of the Plasmodium P47 protein and blocking transmission of a Plasmodium parasite in Anopheles mosquitoes. The isolated nucleic acid can be suitable for expressing the isolated polypeptide in a heterologous expression system. For example, the isolated nucleic acid can be codon-optimized for the heterologous expression system. The heterologous expression system can be a bacterial expression system, a yeast expression system, an insect cell expression system or a vertebrate cell expression system. For example, the heterologous expression system can be an E. coli expression system. The isolated polypeptide encoded by the isolated nucleic acid can contain a C-terminal hexahistidine tag. The isolated polypeptide encoded by the nucleic acid can have at least 80%, at least 90% or at least 95% sequence similarity to SEQ ID NOs 30, 3 , 40 or 41. In some embodiments, the nucleic acid encodes the isolated polypeptide, in which the portion of D2 domain has an N-terminal deletion corresponding to a deletion of 3-30 N- terminal residues of SEQ ID NO:5, a C-terminal deletion corresponding to deletion of 3-40 residues of SEQ ID NO:5, or the N-terminal deletion and the C-terminal deletions. Included among the embodiments of the present invention are compositions, expression vectors and viruses comprising the above isolated nucleic acids. Also envisions are the kits comprising the above nucleic acids, the compositions, the expression vectors, the viruses and one or more oilier reagents for heterologous expression of the isolated polypeptide. Genetically modified ceils, tissues or non-human organisms comprising the nucleic acids according to the embodiments of the present invention are also envisioned and included among the embodiments of the present invention. Methods of using the above isolated nucleic acids are also envisioned and included among the embodiments of the present invention. An exemplar} ⁷ embodiment is a method of heterologous!}' expressing the isolated polypeptide corresponding to at least the portion of D2 domain of a Plasmodium P47 protein, comprising introducing the isolated nucleic acid, the composition, the expression vector or the virus according to the embodiments of the present invention into a non-Plasmodium ceil; and, culturing the cell under conditions leading to the heterologous expression of the isolated polypeptide.

[0006] An exemplary embodiment of the present invention is an isolated polypeptide expressed in a heterologous expression system and corresponding to at least a portion of D2 domain of a Plasmodium P47 protein . The D2 domain can have at least 80%, at least 90% or at least 95% sequence similarity to SEQ ID NO:5. . In some embodiments, the isolated polypeptide does not contain any disulfide bonds. One or both cysteines in SEQ ID NO: 5, which together form, a disulfide bond, can be substituted with same or different amino acids that do not lead to formation of the disulfide bond. In one example, one or both cysteines in SEQ ID NO: 5 are substituted with alanine. The Plasmodium P47 protein can be Plasmodium falciparum P47 protein (Pfs47) or Plasmodium vivax P47 protein (Pvs47). The isolated polypeptide, when administered to or expressed in a vertebrate animal in an immunologically effective amount, can induce production of immunoglobulins capable of specifically binding to D2 domain of the Plasmodium P47 protein. The immunoglobulins can be capable of blocking or block transmission of a Plasmodium parasite in Anopheles mosquitoes. The isolated polypeptide can be expressed in the heterologous expression system that is a bacterial expression system, an insect cell expression system or a vertebrate cell expression system. In one example, the heterologous expression system is an E. coli expression system. The isolated polypeptide can be a fusion protein. The isolated polypeptide can contain a C-terminal hexahistidine tag. The isolated polypeptide can have at least 80%, at least 90% or at least 95% to SEQ ID NOs 30, 31, 40 or 41. In some embodiments, the isolated polypeptide corresponds to the portion of D2 domain containing an N-terminal deletion corresponding to deletion of 3-30 N-terminal residues of SEQ ID NO:5, a C-terminal deletion corresponding to deletion of 3-40 residues of SEQ ID NO:5, or both the N-terminal and the C-terminal deletions.

[0007] Among the exemplary embodiments of the present invention are immunogenic compositions and kits comprising the isolated polypeptide according to the embodiments of the present invention or one or more immunogenic fragment thereof and a pharmacologically acceptable carrier, an adjuvant or both. The immunogenic composition or the kit can also contain another immunogen capable of eliciting an immune response reducing Plasmodium infection in a vertebrate host, when administered to the vertebrate host. The immunogenic composition or the kit can also contain an immunogen capable of eliciting an immune response in a vertebrate host to one or more pathogens other than Plasmodium. Some other exemplary embodiments of the present invention are a screening agent or a screening kit comprising the isolated polypeptide according to the embodiments of the present invention and a carrier, a tag or a label. Methods of using such screening agents or kits are exemplified by (but are not limited to) a method of determining a presence, an absence or an amount of an antibody specifically binding to D2 domain of the Plasmodium P47 protein in a sample, comprising the steps of: contacting the sample with the screening reagent; and, detecting a specific binding of the screening reagent to the antibody in the sample, wherein presence of the detected specific binding is indicative of the presence or the amount of the antibody specifically binding to D2 domain of the Plasmodium P47 protein in the sample.

[0008] Included among the embodiments of the present invention are methods of blocking transmission of a Plasmodium parasite in an arthropod vector. Exemplary methods comprise the steps of: administering to a vertebrate animal capable of hosting the Plasmodium parasite the isolated polypeptide according to the embodiments of the present invention, in the immunologically effective amount, or of a nucleic acid encoding the isolated polypeptide, in an amount resulting in the expression of the immunologically effective amount of the isolated polypeptide; and, following the administering step and upon production of immunoglobulins specifically binding to D2 domain of the Plasmodium P47 protein in the vertebrate animal, allowing the arthropod vector to feed on the vertebrate animal. The arthropod vector can be Anopheles mosquito. The vertebrate animal can be a non-human primate or a human. Also included among the embodiments of the present invention are transgenic and paratransgenic animals. One example is a transgenic mosquito capable of expressing one or more monoclonal antibodies that specifically bind to D2 domain of a Plasmodium P47 protein and block transmission of a Plasmodium parasite in Anopheles mosquitoes, wherein the monoclonal antibodies are secreted into midgut lumen of the transgenic mosquito. Another example is a paratransgenic mosquito comprising gut microbes expressing one or more monoclonal antibodies that specifically bind to D2 domain of a Plasmodium P47 protein and block transmission of a Plasmodium parasite in Anopheles mosquitoes. The monoclonal antibodies can be single-chain monoclonal antibodies. The transgenic mosquito or the paratransgenic mosquito can be an arthropod vector of Plasmodium.

[0009] One more exemplary embodiment of the present invention is a method of inducing production of the antibodies specifically binding to D2 domain of the Plasmodium P47 protein in the mammal and blocking transmission of a Plasmodium parasite in Anopheles mosquitoes, comprising the step of administering to the mammal the immunogenic composition according to the embodiments of the present invention, in the immunologically effective amount. One more exemplary embodiment of the present invention is a method of producing a monoclonal antibody specifically binding to D2 domain of the Plasmodium P47 protein, comprising the steps of: administering to the mammal an immunogenic composition comprising the immunogenic polypeptide according to the embodiments of the present invention, or an immunogenic fragment thereof, in an amount effective to generate multiple antibody producing cells in the mammal: isolating an antibody producing cell from the multiple antibody producing cells; culturing the isolated antibody producing cell under conditions leading to production of the monoclonal antibody; and, testing the monoclonal antibody for specific binding to 1)2 domain of the Plasmodium P47 protein. One more exemplary embodiment of the present invention is a method of producing a monoclonal antibody specifically binding to D2 domain of the Plasmodium. P47 protein and blocking transmission of the Plasmodium parasite in Anopheles mosquitoes, comprising the steps of: administering to the mammal an immunogenic composition comprising the immunogenic polypeptide according to the embodiments of the present invention, or an immunogenic fragment thereof, in an amount effective to generate multiple antibody producing cells in the mammal; isolating an antibody producing cell from the multiple antibody producing cells; culturing the isolated antibody producing cell under conditions leading to production of the monoclonal antibody; and, testing the monoclonal antibody for blocking transmission of the Plasmodium, parasite in Anopheles mosquitoes. One more embodiment of the present invention is a method of producing a recombinant monoclonal antibody, by producing a monoclonal antibody according to one of the above methods, then determining one or more nucleic acid sequences encoding an antibody binding site of the monoclonal antibody, and then using the one or more nucleic acid sequences to produce the recombinant monoclonal antibody.

[0010] Also included among the exemplary embodiments of the present invention are compositions or kits comprising one or more antibodies specifically binding to D2 domain of a Plasmodium P47 protein. The one or more antibodies included in such compositions or kits can be capable of blocking transmission of a Plasmodium parasite in Anopheles mosquitoes. The one or more antibodies included in such compositions or kits can be polyclonal antibodies. The one or more antibodies included in such composition or kits can be one more monoclonal antibodies. A monoclonal antibody capable of specifically binding to D2 domain of a Plasmodium P47 protein is also included among the exemplary embodiments of the present invention. The monoclonal antibody can be capable of blocking transmission of a Plasmodium parasite in Anopheles mosquitoes. In the composition, the kit or the monoclonal antibody according to the above-described embodiments, the Plasmodium P47 protein can be Plasmodium falciparum P47 protein (Pfs47) or Plasmodium vivax P47 protein (Pvs47). The methods of using the above-described antibodies, compositions or kits are also included among the exemplar}' embodiments of the present invention. One such exemplary embodiment is a method of detecting a presence or absence of a polypeptide corresponding to D2 domain of the Plasmodium. P47 protein in a sample, comprising the steps of contacting the sample with the composition of or the monoclonal antibody according to the above- described embodiments, under conditions under which specific binding of the one or more antibodies or the monoclonal antibody capable of specifically binding to D2 domain of the Plasmodium P47 protein may occur; and, detecting the specific binding, wherein the detected specific binding is indicative of the presence of the polypeptide corresponding to D2 domain of the Plasmodium P47 protein in the sample. The above method can also comprise a step of determining an amount of the polypeptide corresponding to D2 domain of the Plasmodium P47 protein present in the sample. The sample in the above methods can be a cell sample, a tissue sample, an aqueous sample, a solution, a suspension, a blot or an electrophoresis gel. The specific binding can be detected in an ELISA, a Western Blot assay or in an immunofluorescence assay.

[0011] It is understood that the nucleic acids, the polypeptides, the vectors, the viruses, the cells and the tissues related to the embodiments of the present invention can be found in vivo, in vitro or ex vivo, depending on the context. It is also understood that the methods according to the embodiments of the present invention can be performed in vivo, in vitro or ex vivo, depending on the method and the context. When describing some of the embodiments of the present invention, the term "comprising," used interchangeably with the term "including," can be employed, meaning that the undescribed or unrecited elements of the embodiment are not excluded. It is envisioned that, when suitable, the description of the embodiments of the present invention can be modified to replace the term "comprising" with the terms "consisting of," which excludes the elements not explicitly recited or described, or "consisting essentially of," which limits the scope of the embodiment to the specified elements and to those elements that do not materially affect the characteristics of the embodiment.

BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure 1 shows cDNA (SEQ ID NO: 1 ) and amino acid (SEQ ID NO:2) sequences of P. falciparum Pfs47 (pf248-14 NIH Ghana3 Ghana isolate, also called GB4 isolate) GenBank: Locus ALQ44015.1, accession KT892054.1. Amino acid sequences of Dl and D3 are highlighted in gray in SEQ ID NO: l . Dl, D2 and D3 amino acid sequences are shown, respectively, as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Signal peptide and putative GPI anchor domain amino acid sequences, which were not included in Pfs47 thioredoxm fusion protein, are shown as SEQ ID NO:3 and SEQ ID NO: 7, respectively.

[0013] Figure 2 shows amino acid sequence of thioredoxin-Pfs47 fusion protein (SEQ ID NO:8).

[0014] Figure 3 is a photograph of a Coomassie blue-stained SDS-PAGE gel illustrating the steps of the expression and purification of Pfs47 thioredoxm fusion protein: lane 1 - induced cells' total lysate; lane 2 - induced cells' soluble fraction; lane 3 - first wash of the inclusion bodies (1% triton solution): lane 4 - second wash of the inclusion bodies; lane 5 - inclusion bodies solubilized with 8M urea; lane 6 - purified soluble protein after in-column refolding and nickel affinity purification. The unlabeled lanes show molecular weight standards.

[0015] Figure 4 is a dot plot illustrating the results of transmission blocking activity (TBA) assays conducted with four of the fourteen anti-Pfs47 mAbs obtained using Pfs47 thioredoxm fusion protein as an immunogen. Each dot represents the number of Plasmodium oocysts counted in a single mosquito midgut. Mouse IgG (mlgG) was used as a negative control; mAb against Pf25 was included as a positive control for TBA (mAb anti Pf25 ^" 'Gold standard").

[0016] Figure 5 is a dot plot illustrating the results of transmission blocking activity (TBA) assay conducted with a pool of four mAbs selected from fourteen anti-Pfs47 mAbs that were obtained using Pfs47 thioredoxin fusion protein as an immunogen. Each dot represents the number of Plasmodium oocysts counted in a single mosquito midgut. Mouse IgG (mlgG) was used as a negative control .

[0017] Figure 6 is a bar graph illustrating the results of Pfs47 domain epitope mapping.

[0018] Figure 7 is a bar graph illustrating the results of Pfs47 domain epitope mapping.

[0019] Figure 8 shows nucleotide sequence of Pfs47 D2 codon-optimized for E. coli expression (SEQ ID NO:9) and amino acid sequence of modified recombinant Pfs47 D2 polypeptide "Pfs47-D2-118" (SEQ ID NO: 10).

[0020] Figure 9 is a dot plot illustrating the results of transmission blocking activity (TBA) assays of anti-Pfs47 D2 polyclonal antibodies. Each dot represents the number of Plasmodium oocysts counted in a single mosquito midgut. Mouse IgG (mlgG) was used as a negative control; mAb against Pf25 was included as a positive control for TBA (mAb anti Pf25 "Gold standard").

[0021] Figure 10 is a dot plot illustrating the results of transmission blocking activity (TBA) assays of anti- Pfs47 D2 mAbs. Each dot represents the number of Plasmodium oocysts counted in a single mosquito midgut. Mouse IgG (mlgG) was used as a negative control; mAb against Pf25 was included as a positive control for transmission blocking (mAb anti Pf25 "Gold standard").

[0022] Figure 11 shows nucleotide sequence of P. vivax Pvs47 D2 codon-optimized for expression in E. coli (SEQ ID NO: 11) and amino acid sequence of modified Pvs47 D2 polypeptide (SEQ ID NO: 12). [0023] Figure 12 shows nucleotide (SEQ ID NO: 13) and ammo acid (SEQ ID NO: 14) sequences of P. falciparum P47 protein 7G8 strain.

[0024] Figure 13 shows amino acid (SEQ ID NO: 15) and nucleotide (SEQ ID NO: 16) sequences of P. falciparum P47 protein GB4 strain ,

[0025] Figure 14 shows amino acid (SEQ ID NO: 17) and nucleotide (SEQ ID NO: 18) sequences of P. vivax Pvs47 protein.

[0026] Figure 15 shows amino acid (SEQ ID NO: 19) and nucleotide (SEQ ID NO: 20 and SEQ ID NO: 21) sequences of P. reichenowi Prs47 protein.

[0027] Figure 16 shows amino acid (SEQ ID NO:22) and nucleotide (SEQ ID NO:23) sequences of P. cynomolgi B P47 protein.

[0028] Figure 17 shows ammo acid (SEQ ID NO:24) and nucleotide (SEQ ID NO:25) sequences of P. oelii 17NLX P47 protein.

[0029] Figure 18 shows amino acid (SEQ ID NO:26) and nucleotide (SEQ ID NO:27) sequences of P. herghei ANKA strain P47 protein.

[0030] Figure 19 is a dot plot illustrating the results of TBA assays conducted with polyclonal antisera ("Poli D2") obtained from the mice successively immunized with Pfs47- D2-118 polypeptide. Each dot represents the number of Plasmodium oocysts counted in a single mosquito midgut.

[0031] Figure 20 shows amino acid sequences of Pfs47 D2 immunogenic polypeptides: modified Pfs47 D2 amino acid sequence (SEQ ID NO:51), which corresponds to SEQ ID NO: 10 without the N-terminal methionine and C-terminal hexahistidine tag; D2 C-terminal deletion amino acid sequence - polypeptide Del 1 (SEQ ID NO:28); D2 C -terminal deletion amino acid sequence - polypeptide Del 2 (SEQ ID NO:29), modified D2 N-Terminal deletion amino acid sequence - polypeptide Del 3 (SEQ ID NO:30) and D2 N-terminal and C-terminal deletion amino acid sequence (SEQ ID NO:3 I).

[0032] Figure 21 is a dot plot illustrating TBA testing of stable hybridoma cultures (freeze/thawmg) corresponding to 200 ,ug/ml of mAbs IB2 BM2 and JH11. The data points shown represent the number of oocysts in individual mosquitoes, while the lines show the median values. The median values were compared using the Mann-Whitney test and labelled as follows: no label, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****p < 0.0001.

[0033] Figure 22 is a bar graph illustrating the results of ELISA testing of immunoreactivity of mAbs JH1 1 , IB2 and BM2 (0.1 jig/ml) against Pfs47 D2 domain deletion polypeptides (1 μπ/ηιΐ). The bars and the error bars represent, respectively, mean absorbance at 405 nm ± standard deviation of three replicate assays. Below the graph is a schematic representation of the polypeptide motifs presented in Pfs47 D2 domain: an enhanced transmission region (E), a transmission blocking region (TB), a cysteine loop (LOOP), with the location of the cysteines in the native sequence marked with asterisks .

[0034] Figure 23 is a line plot showing the immunoreactivity of mouse IgG obtained after a series of immunizations of the experimental mice with Pfs47 Del 2 and Del3 polypeptides. The immunoreactivity was measured by ELISA using a colorimetric assay, in which the absorbance was measured at 405 nm.

[0035] Figure 24 is a dot plot illustrating the results of TBA assays conducted with polyclonal antisera obtained from the mice immunized with Del 3 polypeptide (single boost). Each dot represents the number of Plasmodium oocysts counted in a single mosquito midgut. Mouse IgG (mlgG) was used as a negative control. mAb against Pf25 was included as a positive control for transmission blocking (mAb anti Pf25 "Gold standard").

[0036] Figure 25 is a dot plot illustrating the results of TBA assays conducted with polyclonal antisera obtained from the mice after multiple immunizations with Pfs47-D2-118 ("Pfs47D2" and Del 3 polypeptide ("Pfs47D2-Del3"). Each dot represents the number of Plasmodium oocysts counted in a single mosquito midgut. Mouse IgG (mlgG) was used as a negative control. mAb against Pf25 was included as a positive control for transmission blocking (mAb anti Pf25 "Gold standard").

[0037] Figure 26 is a dot plot illustrating the results of TBA testing of polyclonal sera raised in mice against Pfs47 Del 3 (top panel) and Del 4 (bottom panel) polypeptides after 1 or 3 boosts. Data points in represent the number of oocysts in individual mosquitoes, and the lines show the median values. The median values were compared using the Mann-Whitney test and labelled as follows: no label, no significant difference; *P < 0.05; ****p < 0.0001.

[0038] Figure 27 shows the alignment of Pfs47 D2 immunogenic region amino acid sequences (SEQ ID NOs 32-38) and amino acid sequence of "universal" Pfs47 immunogenic fusion protein (SEQ ID NO:39).

[0039] Figure 28 shows amino acid sequences of Pvs47 D2 deletion polypeptides (N- terminal deletion - SEQ ID NO:40; N- and C-terminal deletion - SEQ ID NO:41), alignment of Pvs47 D2 immunogenic region sequences (SEQ ID NOs 42-48) and P. falciparum - P. vivax immunogenic D2 fusion proteins (SEQ ID NOs 49 and 50). [0040] Figures 29 A-E are dot plots (Figures 29 A-D) and a bar graph (Figure 29 E) illustrating the experimental results of the inv estigation of whether human complement or the mosquito complement-like systems were required for the TBA observed for anti-Pfs47 Del 3 polyclonal mouse sera, TBA of polyclonal sera against Pfs47-D2 Del l after three boosts (300 μ§Λη1) in the presence (+) or absence (-) of Human Complement in A. gambiae (Figure 29A) or A. stephensi (Figure 29B). Figure 29C illustrates the effect of disrupting mosquito complement-like system by silencing ΤΈΡ1 by RNAi of the TBA of anti-Pfs47 Del 3 polyclonal sera after three boosts (300 }ig/ml). Figure 29D illustrates the effect of anti Pfs47 Del 3 polyclonal sera on ookinete formation 24 hours post-infection. The data points in Figures 29 A-D represent the number of oocysts in individual mosquitoes; the lines show the median values. The median values were compared using the Mann-Whitney test and labelled as follows: no label, no significant difference; **P < 0.01; ***P < 0.001; ****p <: 0.0001. Figure 29E is a bar graph illustrating the effect on the reduction of expression of ookinetes specific genes after Standard Membrane Feeding Assay (SMFA) in the presence of anti-Pfs47 Del 3 polyclonal sera 24 hours post-infection . CHTl = chitinase 1 , PIMMS2 = Plasmodium invasion of mosquito midgut screen candidate 2, SOAP= secreted ookinete adhesive protein and WARP= von Willebrand factor A domain-related protein. P<0.0001, ANOVA.

DETAILED DESCRIPTION

[0041] Compositions and methods for reducing or blocking Plasmodium transmission would be highly useful in malaria-elimination efforts, because reduction in the transmission rates is one of the key steps required for malaria control and eradication. As currently understood, Plasmodium P47 proteins mediate evasion of the mosquito innate immune response by suppressing midgut nitration responses that are critical for activation of the mosquito complement system. The inventors discovered that polypeptides based on specific and unexpected regions found in domain 2 (D2) of Plasmodium P47 protein, when administered to or expressed in a vertebrate animal in an immunologically effective amount, induce production of immunoglobulins specifically binding to D2 domain of the Plasmodium P47 protein and capable of blocking the propagation of Plasmodium parasites in Anopheles mosquitoes after the mosquitoes ingest Plasmodium parasites with the blood of Plasmodiu vertebrate hosts. Thus, the blocking or reduction of Plasmodium transmission in the mosquitoes is achieved. As an initial matter, the inventors used a creative strategy to achieve heterologous expression of polypeptides corresponding to P47 D2 region in E. coli, by creating a polypeptide not containing any disulfide bonds. The cysteines in P47 D2 amino acid sequences that together form a disulfide bond were advantageously substituted with amino acids that did not form a disulfide bond. Thus, the inventors addressed a previously- unresolved problem of heterologous expression of P47 D2 polypeptides, as P47 D2 polypeptides with the disulfide bond were toxic to E. coli.

[0042] Upon achieving efficient heterologous expression of full-length P47 D2 polypeptide, the inventors advantageously used such heterologously expressed polypeptide to generate polyclonal and monoclonal antibodies specifically binding to P47 D2 domain and possessing transmission-blocking properties. Thus, the inventors resolved a problem of producing antibodies blocking Plasmodium transmission in in Anopheles mosquitoes. The inventors then unexpectedly discovered that the transmission-blocking activity of polyclonal antibodies in laboratory animals decreased with successive immunizations of the animals with full-length P47 D2 polypeptide. Subsequent experimental investigation surprisingly revealed that, although the N-terminal region of P47 D2 polypeptide was "immunodominant," meaning that the immune responses in the experimental animals were predominantly mounted against the N-terminal region, the antibodies specifically binding to the N-terminal region of P47 D2 polypeptide did not have Plasmodium transmission blocking activity. Instead, transmission-blocking antibodies were specifically binding to the central region of P47 D2 polypeptide, which was less immunogenic than the N-terminal region. The discovery is surprising because the available genetic data, indicates that the C~terminai cysteine loop region of P47 D2 is very important for Pfs47 function of ensuring Plasmodium evasion from the mosquito's immune system and the parasite's survival and propagation in its mosquito host. Unexpectedly, none of the monoclonal antibodies with the transmission- blocking activity bound to the cysteine-loop region, located at the C-terminal end of the D2 domain. Instead, the transmission-blocking antibodies bound to the central region of the D2 domain, which precedes the cysteine loop region. It was also surprising found that several of the antibodies with the transmission-blocking activity did not bind to the full length P47 protein in its native conformation, which expressed in the insect cells using Baculovinis expression system. This finding indicated that, unexpectedly, at least under in vitro conditions, the D2 domain was buried, while the D 1 and D3 domains were on the surface of P47 protein. The fact that D2~specifie monoclonal antibodies nevertheless had transmission blocking activity suggested that P47 underwent a conformational change in vivo exposing the central region of the D2 domain. The inventors' findings led them to envision the invention related to various compositions and methods that may be useful, among other tilings, for blocking transmission of malaria-causing parasites, such as, but not limited to, Plasmodium, falciparum or Plasmodium vivax. For example, some of the compositions and methods described in this document can reduce transmission of Plasmodium parasites from the vertebrate hosts to anopheline mosquitoes, thereby reducing Plasmodium infection transmission and the incidence of malaria. Various embodiments of the present invention are described throughout this document.

[0043] Some embodiments of the present invention provide immunogenic compositions, including polypeptide and nucleic acid -containing compositions based on the sequences of P47 D2, which are capable of inducing production of immunoglobulins specifically binding to D2 domain of Plasmodium P47 protein and capable of blocking transmission of Plasmodium parasites in Anopheles mosquitoes. Thus, embodiments of the present invention include P47 D2 polypeptides, products, compositions and kits containing such polypeptides and methods (processes) of making and using such polypeptides and related compositions and kits in analytical, research, diagnostic and human and animal health applications. Embodiments of the present invention also include nucleotide sequences encoding P47 D2 polypeptides, compositions and kits containing such nucleotide sequences and methods (processes) of making and using such nucleotide sequences and related compositions and kits in analytical, research, diagnostic and human and animal health applications. In some contexts, the above compositions and methods may be used to reduce transmission of Plasmodium parasites. In some other contexts, the above compositions and methods may be used to produce antibodies having advantageous properties, such as blocking of transmission of Plasmodium in the invertebrate host, after the invertebrate host feeds on the vertebrate host's blood containing the parasite. Some other embodiments of the present invention provide anti-P47 D2 antibodies, including polyclonal and monoclonal antibodies having advantageous properties, such as Plasmodium transmission-blocking activity, products, compositions and kits containing such antibodies, methods (processes) of making the monoclonal antibodies and related compositions, as well as methods of using the monoclonal antibodies in analytical, research, diagnostic and human and animal health applications. In some other embodiments, the present invention includes cells, including bacterial and animal cells, tissues and transgenic and paratransgenic animals capable of expressing immunogenic polypeptides and antibodies according to the embodiments of the present invention, as well as the methods (processes) of making and using such ceils, tissues and animals.

Definitions

[0044] To facilitate the understanding of the invention, a number of terms are defined below. These terms may be further defined and understood based on the accepted con ventions in the fields of the present invention and the description provided throughout the present document. Some other terms can be explicitly or implicitly defined in other sections of tliis document, and may be used and understood based on the accepted conventions in the fields of the present invention and the description provided throughout the present document . The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present invention and interpreted in the context of the present document.

[0045] The term "sample" may be used in reference to any organism, tissue or cell sample or extract originating from organisms, cells or tissues, and includes samples of human or animal (including vertebrate, invertebrate and protozoan animals) organisms, cells or tissues as well as organisms or cells of non-human or non-animal origin, including bacterial samples. A sample can be directly obtained from an organism, propagated or cultured. Samples can be subjected to various treatments, storage or processing procedures. Generally, the terms "sample ^" ' or "samples" are not intended to be limited by their source, origin, manner of procurement, treatment, processing, storage or analysis, or any type of modification. Samples encompass samples of healthy or pathological organisms, cells or tissues. Samples also encompass samples of naturally occurring organisms, cells or tissues, samples derived from naturally occurring organisms, cells or tissues, and modified and artificially generated organisms, cells, tissues and other related structures. Samples can contain or be predominantly composed of cells or tissues, or can be prepared from cells or tissues. Some examples of the samples are organisms, cells, tissues, organs, cultures, solutions, suspensions, supernatant., precipitates (cell precipitates), pellets, extracts, such as cell extracts (for examples, cell lysates), biological fluid samples, such as hemolymph, blood or plasma samples, tissue sections, microscopy slides, including fixed slides (for example, formalin- fixed or paraffin-embedded), frozen and desiccated organisms, cells or tissues, flow cytometry samples and fixed cell samples. Samples, such as cells and tissues, may be mixed in a slurry of an inert support with or without use of optimal cutting temperature or other compounds before freezing. Electrophoresis gels and blots (such as those used in Western blotting) are also included within the scope of the term "sample.' ¹

[0046] As used herein, the term "subject" and the related terms are used to refer to animal organisms, meaning multicellular, eukaryotic organisms of the kingdom Animalia, including vertebrate and invertebrate animals. For example, a subject may be a mammal, such as a primate, including a human. The term "subject" also includes non-primate and non-human animals, for example, domesticated animals, such as cats, dogs, etc., livestock (such as cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (such as mice, rabbits, rats, guinea pigs, etc.). In another example, a subject may be an animal belonging to phylum Arthropoda, including the animals of subphylum Insecta, including, in turn, the animals of order Diptera. in some contexts, the term "subject" can be used to refer to invertebrate Plasmodium hosts, such as mosquitoes (family Culicidae), including anopheline mosquitoes (subfamily Anophelinae), such as mosquitoes of genus Anopheles. In some other contexts, the terms "subject," "individual," and "patient" can be used interchangeably to refer to an organism, such as but not limited to a human or a primate, capable of being a vertebrate host for Plasmodium, hosting Plasmodium parasites, or having or suspected of having malaria. In these contexts, the terms do not imply any kind of relationship to a medical professional, such as a physician or a veterinarian.

Malaria

[0047] Malaria is a mosquito-borne infectious disease affecting humans and other animals. Malaria is caused by parasitic protozoans of genus Plasmodium. The disease is most commonly transmitted by an infected female Anopheles mosquito. In the affected humans, malaria causes symptoms that typically include fever, fatigue, vomiting, and headaches. In severe cases it can cause yellow skin, seizures, coma or death. Symptoms usually begin ten to fifteen days after being bitten by an infected mosquito. If not properly treated, infected subjects may have recurrences of the disease months later.

[0048] Plasmodium life cycle is complex. The parasite always has two hosts in its life cycle: a Dipteran insect host and a vertebrate host. Sexual reproduction always occurs in the insect. Infection in the vertebrate host begins when young malarial parasites or "sporozoites" are injected into the bloodstream of the vertebrate host by a mosquito. After injection, the parasite localizes in liver cells. Approximately one week after injection, the parasites or "merozoites" are released into the bloodstream to begin the "erythrocytic" phase. Each parasite enters a red blood ceil in order to grow and deveiop. When the merozoste matures in the red blood cell, it is known as a trophozoite and, when fully developed, as a schizont. A schizont is the stage when nuclear division occurs to form individual merozoites, which are released to invade other red cells. After several schizogonic cycles, some parasites, instead of becoming schizonts through asexual reproduction, develop into large uninucleate parasites. These parasites undergo sexual development.

[0049] Sexual development of the malaria parasites involves the female or "macrogametocyte" and the male parasite or "microgametocyte " These gametocytes do not undergo any further development in the vertebrate host. Upon ingestion of the gametocytes by the mosquito, the complicated sexual cycle begins in the mosquito's midgut. The red blood cells disintegrate in the midgut of the mosquito after 10 to 20 minutes. The microgametocyte continues to deveiop through exflageliation and releases eight highly flagellated microgametes. Fertilization occurs with the fusion of the microgamete and a macrogamete. The fertilized parasite, which is known as a zygote, then develops into an "ookinete." The ookinete penetrates the midgut wall of the mosquito and develops into an oocyst, within which many small sporozoites form. When the oocyst ruptures, the sporozoites migrate to the salivary gland of the mosquito via the hemolymph. Once in the saliva of the mosquito, the parasite can be injected into the vertebrate host, repeating the life cycle.

[0050] Plasmodium genus of parasites includes, but is not limited to, Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium knowlesi (P. knowlesi), Plasmodium ovale (P. ovale), Plasmodium malariae (P. malariae), Plasmodium berghei (P. herghei), Plasmodium chabaudi (P. chabauai), Plasmodium gallinaceurn (P. gallinaceurn), Plasmodium reichenowi (P. reichenowi) and Plasmodiu yoelii (P. yoelii). The species most common for human malaria transmission are P. falciparum, P. vivax, P. knowlesi, P. ovale and . malariae. Mosquitoes that are commonly susceptible or vulnerable to infection by Plasmodium parasites include the Anopheles genus, which, in turn, includes, but is not limited to, the species Anopheles gambiae (A. gambiae), Anopheles albimanus (A. albimanus), Anopheles darlingi (A. darlingi), Anopheles aquasalis (A. aquasalis). Anopheles freeborni (A. freeborni), Anopheles quadn macula tus (A. quadri macula ius) and Anopheles stephensi (A. siephensi).

[0051] The term, "transmission" may be used herein in reference to transmission of Plasmodium parasites among their hosts. Some of the compositions and methods described in this document may be used to reduce or block malaria transmission in a Dipteran insect host. As such, the compositions and methods described in this document may be described as blocking transmission, transmission blocking, having "transmission-blocking activity" (TBA) etc., even when complete blocking of transmission is not achieved. In this context, reduction or blocking of malaria transmission may be modified by the terms "substantially" or "substantial," and with or without such term may mean at least 10%, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% reduction of transmission of the parasite in mosquitoes, as determined by standard methods, such as (but not limited to) the Standard Membrane Feeding Assay (SMFA). The SMFA is a functional assay that measures the ability of antibodies to block transmission of parasites in mosquitoes and is described, for example, in van der Kolk et al. (2005) Parasitology 130(Pt. 1): 13-22,

Nucleic Acids and Polypeptides, Sequence Similarity

[0052] Embodiments of the present invention encompass nucleic acids and polypeptides, which can be characterized by their respective sequences. Embodiments of the present invention encompass homologues, variants, isoforms, fragments, mutants, modified forms and other vari ations of the amino acid and nucleic aci d sequences described in this document. The term "homologous," "homologues" and other related terms used in this document in reference to various amino acid and nucleic acid sequences are intended to describe a degree of sequence similarity among protein sequences or among nucleic acid sequences, calculated according to an accepted procedure. Homologous sequences may be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% similar to reference sequences. As used herein, "% similarity" of two amino acid sequences or of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul, which is incorporated into the NBLAST and XBLAST programs, available for public use through the website of the National Institutes of Health (U.S.A.). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. The term "% similarity" and the related terms may be used in this document to describe fragments, variants or isoforms of amino acids and nucleic acid sequences, but other ways of describing fragments, variants or isoforms may be employed alternatively to or in conjunction with calculated "% similarity." [0053] For example, another indication that nucleotide sequences are homologous is when two polynucleotide molecules hybridize to each other, or to a third nucleic acid, under stringent conditions. Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH), at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 1 molar at pH 7 and the temperature is at least about 60° C. In one example, RNA polynucleotides representing the embodiments of the present invention can be identified in Northern blots under stringent conditions using nucleotide sequence disclosed in this document or their fragments of, for example, at least about 100 nucleotides. For the purposes of this document, stringent conditions for such RN A-DNA hybridizations are those which include at least one wash in 6 < SSC for 20 minutes at a temperature of at least about 50°C, usually about 55°C. to about 60°C, or equivalent conditions. Thus, nucleic acid molecules according to some embodiments of the present invention include nucleic acid molecules that hybridize, under defined stringent conditions, with the nucleic acid molecules encoding P47 D2 polypeptides.

[0054] In another example, an indication that protein amino acid sequences are homologous may be that one protein is immunologically reactive with antibodies raised against the other protein. Thus, poly peptides and proteins according to some embodiments of the present invention include proteins immunologically reactive with antibodies raised against P47 polypeptides, such as P47 D2 polypeptides.

[0055] Fragments of a polypeptide or an amino acid sequence can include any portion of a polypeptide or an amino acid sequence of at least 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids. Variants of a polypeptide may result from sequence variations, such as amino acid substitutions, deletions, and insertions, as well as from post-translational modifications and their variations. Variations in post-translational modifications can include variations in the type or amount of carbohydrate moieties of the protein core or any fragment or derivative thereof. Variations in amino acid sequence may arise naturally as allelic variations (such as due to genetic polymorphism) or may be produced by human intervention (such as by mutagenesis of cloned DNA sequences), the examples being induced point, deletion, insertion and substitution mutants. Variations in a nucleic acid sequence may result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Variants of a polypeptide may also be conformational variations, with or without the changes the ammo acid sequence and/or post-translational modifications.

[0056] The term '"mutation" or "mutated sequence," when used in reference to nucleotide or amino acid or nucleotide sequence can be used interchangeably and/or in conjunction with the terms "variant," "allelic variant," "variance," or "polymorphism." Ammo acid sequence modifications include substitutions, insertions or deletions. Insertions include amino and/or carboxyl terminal fusions as well as mtrasequence insertions of single or multiple ammo acid residues. Insertions ordinarily will be smaller insertions than those of ammo or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Amino acid substitutions are typically of single residues but may include multiple substitutions at different positions; insertions usually will be on the order of about from 1 to 10 amino acid residues but can be more: and deletions will range about from 1 to 30 residues, but can be more. Ammo acid substitutions may be characterized as "conservative," meaning substitution for an amino acid with similar properties. Some examples of conservative substitutions are shown in Table 1, below. Conservative amino acid substitutions in monoclonal antibodies may have substantially no effect on antigen binding or other immunoglobulin functions. See, for example, Harlow & Lane, "Antibodies, A Laboratory Manual, Cold Spring Harbor Publications," New York (1988). A variant or an isoform can contain one or more of substitutions (including, for example, conservative amino acid substitutions, such as 1 -5, 1 - 10, 1 -20, 1 -50 or more conservative amino acid substitutions), deletions or insertions.

[0057] An isoform or a variant can be a result of post-translational modifications, derivatizations or lack thereof. For example, variants may arise as a result of differences in glycosylation, such as N- and O-glycosylation. Glutaminyl and asparaginyi residues are frequently post-transiationaily deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (acetylation of the N -terminal amine and, in some instances, amidation of the C-terminal carboxyl. Modifications can also include modifications in glycosylation. Table 1. Conservative amino acid substitutions

Vectors, cells and organisms

[0058] As discussed throughout this document, the embodiments of the present invention include various polypeptides, for example, P47 polypeptides, such as P47 D2 immunogenic polypeptides, and anti-P47 D2 monoclonal antibodies, as well as nucleic acids encoding such polypeptides. The polypeptides described in this document can be produced using the nucleic acids encoding them with the aid of recombinant technologies. Accordingly, the embodiments of the present invention include expression vectors containing one or more nucleic acids encoding one or more of the polypeptides described in this documents. In the expression vectors, the encoding nucleic acid is typically operabiy linked to one or more regulatory sequences. Such useful regulator}' sequences include, for example, the early or late promoters, such as promoter sequences of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3 -phosphogly cerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (for example, Pho5), the AOX 1 promoter of methylotrophic yeast, the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses.

[0059] An expression vector according to the embodiments of the present invention can be designed to produce anti-P47 monoclonal antibodies, such as anti-P47 D2 monoclonal antibodies, or P47 immunogenic polypeptides, such as P47 D2 immunogenic polypeptides, described in tins document. An expression vector can be suitable for expression in eukaryotic or prokaryotic cells and thus include DNA molecules capable of integration into a prokaryotic or eukaryotic chromosome and subsequent expression . The inserted genes in viral and retroviral vectors usually contain promoters and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. Specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types. For example, the glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin. Expression vectors used in eukaryotic host cells (for example, yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary' for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct. Some examples of the vectors are PUC vectors, pcDNA3 vectors, pEE series vectors, pGL3 vectors or pEGFP vectors. pFUSE-CLlg and pFUSE-CHIg plasmid vectors can be employed, which are designed to change a monoclonal antibody from one isotype to another, thus pennitting the generation of a variety of antibodies with the same antigen affinity. [0060] The vectors according to the embodiments of the present invention include viral vectors that transport the nucleic acids encoding polypeptides described in this document into cells without degradation and include a promoter yielding expression of the nucleic acids in the cells into which it is delivered. Viral vectors are derived from viruses, including retroviruses, such as Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus. Polio virus, AIDS virus, neuronal trophic vims, Sindbis virus and other RNA viruses. Also preferred are any viral families that share the properties of these viruses that make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Some other examples of viral vectors are simian virus 40 (SV40) and Baculovirus vectors. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promoter cassette is inserted into the viral genome in place of the removed viral DNA. The necessary functions of the removed early genes are typically supplied by cell lines that have been engineered to express the gene products of the early genes in trans.

[0061 ] Cells containing the expression vectors are also included among the embodiments of the present invention. Such cells can thus produce the polypeptides, for example, anti-P47 monoclonal antibodies or P47 immunogenic polypeptides, described in this document. A cell can be either a eukaryotie or prokaryotic cell. Some examples of the cells are bacterial cells, for example, cells of E. coli, Pseudomonas, Bacillus or Streptomyces, fungal cells, such as yeast cells (for example, cells of Saccharomyces, and methylotrophic yeast such as Pichia, Candida, Hansenula, and Torulopsis); animal cells, such as CHO, Rl . 1, B-W and LM cells, African Green Monkey kidney cells (for example, COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (for example, Sf9 cells), human cells (such as human embryonic kidney ceils, for instance, HEK293) and plant cells. The ceils according to the embodiments of the present invention can be found in cell or tissue culture.

[0062] When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, eiectroporation, insertion of a plasmid encased in liposomes, or virus vectors, may be used. Eukaryotie cells can also be co-transformed with polynucleotide sequences encoding the antibody, labeled antibody, or antigen binding fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian vims 40 (SV40) or bovme papilloma vims, to transiently infect or transform eukaryotic cells and express the protein. Expression systems, such as plasmids and vectors, can be employed to produce proteins in cells, including higher eukaryotic cells, such as the COS, CHO, HeLa and myeloma cell imes.

[0063] Multicellular organisms and unicellular organisms modified to produce P47 polypeptides or anti-P47 antibodies are also included among the embodiments of the present invention. Among the exemplary embodiments are transgenic and paratransgenic mosquitoes capable of expressing anti-P47 D2 antibodies having transmission-blocking activity. In paratransgenic mosquitoes, the nucleic acid sequence encoding the antibody is produced by bacteria residing in the mosquito gut, which were modified with nucleic acid sequences encoding the antibody . For example, symbiont bacteria of the anopheline mosquitoes can be transfected with the vectors containing the nucleic acid sequences, cultured, and delivered to the gut of the mosquito, where they continue to express the antibody. Suitable symbionts for mosquitoes, such as A. gambiae, include acetic acid bacteria, such as Acelobacter or Ghiccmacetobacter, and Pantoea agglomerans (P. agglomerans). The antibodies thus expressed in transgenic or paratra sgenic mosquitoes can disrupt the function of P47, permitting the mosquitoes' immune system to recognize the Plasmodium parasites, thus substantially reducing their transmission.

P47 and P47 1)2 nucleic acids and polypeptides and related compositions, products and kits

[0064] The term ^" 'Ρ47" and the related terms refer to a genus of Plasmodium female gametocyte surface proteins P47, polypeptides and nucleic acids encoding such proteins, as well as their fragments and variants. As currently understood, Plasmodium P47 proteins mediate evasion of the mosquito innate immune response by suppressing midgut nitration responses that are critical for activation of the mosquito complement system. However, the mechanism of action is not intended to limit the scope of P47-related embodiments of the present invention, unless explicitly stated. One example of a Plasmodium P47 protein is P. falciparum Pfs47 protein, which is described, for example, in van Schaijk B.C, et al. ^" 'Pfs47, paralog of the male fertility factor Pfs48/45, is a female specific surface protein in Plasmodium falciparum." Mol. Biochem. Parasitol. 149:216-222 (2006), Molina-Cruz et al., "The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system. Science 340:984-987 (2013) and in International Patent Publication WO2014028852. Another example of P47 protein is Pvs47; produced by P. vivax, Amino acid and nucleotide sequences of Pfs47 and Pvs47 are illustrated, respectively, in Figures 1, 8 11-14, 20, 24 and 25. Amino acid and nucleotide sequences of other exemplary Plasmodium P47 proteins are illustrated in Figures 15-18.

[0065] P47 proteins are members of the 6-cysteine protein family. The 6-cysteine (6-cys) domain is found in Plasmodium proteins that are expressed in all stages of the parasite life cycle in both the vertebrate and mosquito hosts. 6-cys domain is of roughly 120 amino acids and contains six positionally conserved cysteines. P47 contains three domains, Dl, D2 and D3. Two domains of P47, Dl and D3, have the classic 6-cysteine fold, while the central one (D2) has only two cysteines. The terms "Dl ," "D2" and "D3" are used in this document to refer to domains 1, 2 and 3 of P47 protein, including P47 amino acid sequences of the domains, polypeptides including such sequences and nucleotide sequences encoding the domains. For example, the term "P47 D2" may be used to refer to any or all of the following: full or partial amino acid sequences of D2 domain of P47 protein, an isolated polypeptide containing full or partial amino acid sequence of D2, a stretch of nucleic acid sequence encoding full or partial D2 or a nucleic acid sequence or molecule encoding full or partial D2. The term "P47" as used in this document, used separately or in conjunction with the term specifying the domains, such as Dl, D2 and D3, includes orthologs from different Plasmodium species, variants and isoforms, including postransiationally modified (for example, glycosylated or lipidated) variants and isoforms, mutants, homologs and fragments. Thus, the term "P47 D2" may also refer to fragments and/or variants of P47 sequences or polypeptides. For example, the term "P47 D2 polypeptide" may be used to refer to various polypeptides and ammo acid sequences derived from D2, such as native full-length D2 polypeptides or amino acid sequences (for example, SEQ ID NO:5), modified D2 polypeptides or amino acid sequences (for example, SEQ ID NOs 10, 12, 30, 40, 41 and 51), or D2 fragments and variants (for example, SEQ ID NOs 28-38 and 40-48).

[0066] In the context of recombinant nucleic acid sequence encoding P47 or its domains, such as expression cassettes or vector-expressed sequences or transgenes, P47 or P47 D2 nucleic acid sequence need not be identical to the corresponding natural sequences, and may be variants of the naturally occurring nucleic acid sequences from which they were derived. For example, due to codon degeneracy a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically included among the nucleotide sequences according to the embodiments of the present invention. Some of the nucleotide sequences according to the embodiments of the present invention may be modified to improve heterologous expression. Heterologous expression generally refers to the expression of a nucleic acid in a cell or organism, which does not naturally have this nucleic acid. In the context of P47 or P47 D2 nucleotide sequences, ' ^" heterologous expression" means expression in a noa.-Plasmod.ium cell or organism.. A modification of the nucleic acid sequence to improve heterologous expression may be referred to as "codon optimization," and the modified nucleotide sequences as "codon-optimized." These terms are used to reflect the process and the result, in which the codons used for coding particular amino acids that are changed without changing the amino acid sequence of the protein itself. P47 or P47 D2 nucleotide sequences according to the embodiments of the present invention specifically include the sequences encoding poly peptides capable of inducing Plasmodium transmission- blocking immune response.

[0067] Nucleic acid sequences encoding P47 polypeptides, including P47 D2 polypeptides, such as those described above and elsewhere in this document, are included among the embodiments of the present invention. Such sequences may be referred to as "isolated," meaning that they are not found in Plasmodium in their natural state. Such isolated nucleic acid sequences may be used in various ways. For example, they can be incorporated in other nucleic acid sequences (such as vector sequences) for manipulation and/or expression of P47 D2 polypeptides. Amino acid sequences corresponding to P47 polypeptides, including P47 D2 polypeptides, such as those described above and elsewhere in this document, are also included among the embodiments of the present invention. Such amino acid sequences may be referred to as "polypeptides" or "isolated polypeptides" (singular - "polypeptide" or "isolated polypeptide"). In this context, the term "isolated" means that the amino acid sequence or polypeptide is not found in Plasmodium in their natural state. Such sequences may be referred to as "isolated," meaning that they are not found in Plasmodium in its natural state. The isolated amino acid sequence or isolated polypeptide can be modified, incorporated into or joined with other amino acid sequence (or sequences) or polypeptide (or polypeptides), for example, to produce modified polypeptides or proteins or fusion polypeptides or proteins, as illustrated, for instance, in Figures 2, 8, 11, 20, 24 and 25. [0068] The nucleic acid sequences used to express P47 D2 polypeptides and to transfect the host cells can be synthetically produced and/or modified according to standard techniques to encode P47 D2 polypeptides and their variants or fragments with a variety of desired properties. For example, P47 D2 nucleic acid sequences can be modified to manipulate or enhance the properties of the resulting polypeptides, such as in vitro stability, solubility, or in vivo toxicity or pharmacokinetics. For instance, P47 D2 nucleic acid sequences can be modified in a site-specific manner to change or delete some amino acid residues in the resulting polypeptide, such as cysteine residues. Modified P47 D2 nucleic acid sequences are included among the embodiments of the present invention. Site-specific mutations can be introduced into P47 D2-eneoding nucleic acids by a variety of techniques used in the field of molecular biology. The modifications may be evaluated by routine screening in a suitable assay for the desired characteristic. For instance, the effect of various modifications on the ability of the poly peptide to elicit transmission blocking can be easily determined using the assays described elsewhere in this document or otherwise known in the field of malaria studies. Changes in the immunological character of the polypeptide can be detected by an appropriate competitive binding assay or other immunochemical assays. Modifications of other properties such as redox or thermal stability, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques. In some embodiments of the present invention, P47 D2 nucleic acids can be used in recombinant expression methods and systems to produce immunogenic polypeptide. One exemplary method is recombinant expression in E, colt Other exemplary expression methods are expression in Baculoviriis expression system, expression in Pichia pastoris or expression in mammalian cell lines, such as Human Embryonic Kidney cells. Nucleic acid sequences encoding P47 D2 polypeptides can be used as immunogens. For example, the nucleic acid sequences can be incorporated into expression cassettes or vectors and administered as "naked" DNA to a subject. In another variation, cationic liposome-entrapped DNA may be used.

[0069] P47 and P47 D2 polypeptides and nucleic acids may be described by using amino acid or nucleotide sequences, respectively, of Pfs47 or Pvs47 proteins or their fragments and homologous sequences, such as the respective amino acid or nucleotide sequences with an extent of homology (as defined elsewhere in this document) of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%. Homologues and variants of P47 including P47 D2, include variants of the native proteins constructed by in vitro techniques, and proteins from parasites related to P. vivax and P. falciparum. For certain uses, it is advantageous to produce a polypeptide sequence that is lacking a structural characteristic; one may remove a transmembrane domain to obtain a polypeptide that is more soluble in aqueous solution. Embodiments of the present invention include P47 lacking one or more domains and P47 D2 polypeptide sequences containing deletions. For example, P47 D2 polypeptide sequences containing N-terminal and/or C-terminal deletions are among the embodiments of the present invention. P47 and P47 D2 variants include posttranslational modification variants, such lipidated or delipi dated, glycosy lated or degiycosyiated forms of the polypeptides, structural variants, such as variants with or without one or more (at least some to all) disulfide bridges, folding (conformational) variants, and the nucleic acids which encode these variations.

[0070] Hie inventors discovered that, for unexpected improvements expression of P47 D2 polypeptides in some heterologous organisms, such as E. coli, it is desirable to replace at least one of the two cysteine residues in the native amino acid sequence of P47 D2, which form a disulfide bridge, with amino acids, such as alanine, that do not result in the formation of disulfide bridge in D2 polypeptide. Thus, some other exemplary embodiments reflecting the inventors' discovery are isolated or recombinantly produced polypeptides having at least 80%, at least 90% or at least 95% sequence similarity to a naturally found P47 D2 sequence, such as SEQ ID NO:5, in which at least one of the two cysteine residues are substituted with amino acids that prevent formation of disulfide bridges, such as alanine (different ammo acids may be substituted in different positions). Such polypeptides may also contain additional residues. For example, a methionine may be added at the N terminus, and/or histidine residues can be added at the C-terminus to form a polyhistidine (such as hexahistidine) tag.

[0071 ] P47 polypeptides, including P47 D2 polypeptides, also include fusion proteins. A ''fusion protein" refers to a composition containing at least one polypeptide or peptide domain which is associated with a second domain. The second domain can be a polypeptide, peptide, polysaccharide, or the like. The "fusion" can be an association generated by a peptide bond, a chemical linking, a charge interaction (for example, electrostatic attractions, such as salt bridges, H-bonding, etc.) or the like. If the polypeptides are recombinant, the ^" 'fusion protein" can be translated from a common nucleic acid. Alternatively, the compositions of the domains can be linked by any chemical or electrostatic means. P47 and P47 D2 fusion proteins can include non-malarial sequences, such as linkers, epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals, and the like. Some exemplary embodiments of P47 D2 polypeptides are conjugated to heterologous polypeptides, such as fusion proteins containing polypeptides intended to improve expression and/or purification, for example, histidine tagged fusion proteins, thioredoxin fusion proteins etc. For example, a P47 D2 polypeptide according to the embodiments of the present invention may contain a thioredoxin protein sequence (thioredoxin fusion protein) to improve expression and purification in E. con. In another example, a P47 D2 polypeptide according to the embodiments of the present invention may contain a C-terminal polyhistidine, such as hexahistidine sequence (tag) to improve purification. Some fusion proteins according to the embodiments of the present invention include more than one P47 D2 sequence. For example, such fusion proteins may include two, three, four, five, six, seven or more P47 D2 sequences originating from various strain or species of Plasmodium. One example of such fusion protein is shown in Figure 24 as SEQ ID NO:39 and includes several Pfs47 D2 immunogenic sequences. Two more examples are fusion proteins shown in Figure 25 as SEQ ID NOs 49 and 50, which include Pfs47 and Pvs47 D2 immunogenic sequences.

Immunogenic P47 D2 polypeptides, nucleic acids encoding such polypeptides, their uses and related compositions, products and kits

[0072] The terms "immunogen," "immunogens," '"immunogenic" and the related terms can be used in this document to refer to ammo acid sequences, molecules or compositions containing a peptide, polypeptide or protein capable of eliciting, augmenting or boosting a cellular and/or humoral immune response, either alone or in combination or linked or fused to another substance. For example, an immunogen can be a polypeptide or an amino acid sequence of at least about 5 ammo acids, of at least 10 amino acids in length, of at least 15 amino acids in length, at least 20 amino acids in length, at least 30 amino acids in length, at least 40 amino acids in length, at least 50 amino acids in length or greater. For example, an immunogen can be a polypeptide 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-20, 10-30, 10-40, 10- 50, 10-60, 20-30, 20-40, 20-50, 20-60, 30-40, 30-50, 30-60, 40-50, 40-60 or 50-60 amino acids in length. An immunogen can comprise a "carrier" polypeptide and a hapten or a fusion protein or a carrier polypeptide fused or linked (chemically or otherwise) to another composition. An immunogen can also be an antigenic determinant or epitope, which are typically 3 to 10 amino acids in length. For example, that some epitopes can contain only three amino acids. Furthermore, since epitopes (antigens) can involve residues that are not adjacent in peptide sequence, but are next to each other in three-dimensional structure, it is possible to achieve a special configuration of an epitope using smaller fragments of a polypeptide sequence included discontinuously within a longer sequence.

[0073] An immunogen can be encoded by a nucleotide sequence, which, in turn, can be incorporated in a vector for expression. Such a vector can be simply naked DNA containing the immunogen' s coding sequence operably linked to a promoter - a simple expression cassette. More complex vectors can also be used. Depending on the context, such nucleic acid sequences and molecules and sequences, molecules or structures incorporating them, such as vectors or artificial chromosomes, can also be referred to as "immunogen," "immunogens" or "immunogenic." Immunogens and immunogenic composition capable of partially or completely reducing (blocking) transmission of malaria can be referred to as "vaccines." Transmission-blocking vaccines according to the embodiments of the present invention are vaccines administered to Plasmodium vertebrate hosts, such as humans, which induce production of antibodies blocking the portion of Plasmodium life cycle that takes place in the mosquito or other arthropod vector after it ingests Plasmodium gametocytes.

[0074] Among the embodiments of the present invention are polypeptides of P47 D2 and nucleic acid sequences encoding such polypeptides, which can be useful, among other things, for production of anti P47 D2 antibodies, in which case they can be referred to as "immunogenic." Immunogenic polypeptides of P47 D2 and nucleic acid sequences encoding such polypeptides can be useful as transmission-blocking vaccines. The antibodies that arise from administration of P47 D2 immunogens to animals generate an immune response by blocking transmission of the parasite malaria by interfering with the portion of Plasmodium life cycle that occurs in the mosquito. In other words, when an immunogenic polypeptide based on P47 D2 sequence is administered to or expressed in a vertebrate animal in an immunologically effective amount, it induces production of immunoglobulins specifically binding to D2 domain of Plasmodium P47 protein and blocking transmission of a Plasmodium parasite in Anopheles mosquitoes. In some embodiments, the D2 domain has at least 80%, at least 90% or at least 95% amino acid sequence similarity to SEQ ID NO:5. It was unexpectedly discovered that, although the N-terminal region of P47 D2 polypeptide was immunodominant in vertebrates, the antibodies specifically binding to the N-terminal region of P47 D2 polypeptide did not have Plasmodium transmission-blocking activity. Transmission-blocking antibodies were specifically binding to the central region of P47 D2 polypeptide, which was less immunogenic in vertebrates than the N-terminal region. Accordingly, some embodiments of the present invention provide immunogenic P47 D2 polypeptides, in which the portion of D2 domain has an N-terminal deletion corresponding to a deletion of 3-30 N-terminal residues of SEQ ID NO:5; a portion of D2 domain can also have a C-terminal deletion corresponding to deletion of 3-40 residues of SEQ ID NO:5. Suitable exemplary amino acid sequences corresponding to the above polypeptides are illustrated in Figures 20, 24 and 25. Some exemplary embodiments of immunogenic polypeptides are isolated, synthetic or recombinantly produced polypeptides with amino acid sequences having at least 80%, at least 90% or at least 95% sequence similarity to SEQ ID NOs 30, 31, 40 oi- 41.

[0075] As also discussed elsewhere in this document, molecules, compositions, products, kits including the above immunogenic polypeptides and nucleic acids, as well as methods of making and using the above polypeptides and nucleic acids, for example, for producing, testing or screening antibodies, inducing immune response in the subjects, such as laboratory animals, cells or tissues are also included within the scope of the present invention. For instance, compositions or molecules containing above polypeptides or nucleic acids may be employed in the methods of producing monoclonal antibodies described in this document as probes in the screening assays. In another example, the compositions or molecules containing above polypeptides or nucleic acids may be administered to subjects, such as animals, cells or tissues to induce immune response, including production of antibodies. The compositions or molecules containing above poiypeptides or nucleic acids may also be used to reduce or block Plasmodium parasite transmission by administering them to vertebrate hosts, in immunologically active amounts, to induce production of malaria transmission- blocking antibodies, subsequently transferred to mosquitoes with a blood meal.

[0076] Immunogenic P47 D2 poiypeptides and nucleic acids of the present invention can be used for blocking transmission of malaria. In some embodiments of the present invention compositions containing P47 D2 polypeptides or nucleic acids are administered to a vertebrate host of Plasmodium, such as a primate or a human, giving rise to an anti-P47 D2 immune response in the mammal entailing the production of anti-P47 D2 immunoglobulins. The P47 D2 polypeptide-specific immunoglobulins are then transferred to the arthropod host, such as mosquito, with the blood meal and block either fertilization of the female gamete and/or evasion of Plasmodium from the arthropod's immune system, thus blocking transmission of the parasite from the vertebrate host to the arthropod vector. It should be noted that the embodiments of the present invention are not limited by the mechanism leading to the blocking of transmission. An amount of an immunogenic composition sufficient to result in a titer of antiserum which, upon ingestion by the arthropod host, such as mosquito, is capable of blocking transmission or is capable of decreasing ability of the oocyte to mature in the mosquito (resulting in fewer infective particles passed to the mosquitoes' next target blood meal), is defined to be an immunologically effective dose or amount, or simply as an effective dose or amount.

[0077] In some embodiments of the present invention, compositions comprising immunogenic P47 D2 polypeptides or nucleic acids of the present invention can further included immunogens (polypeptides or nucleic acids) useful for eliciting immune response to other pathogens. For example, pharmaceutical compositions for administration to human subjects can include immunogenic P47 D2 polypeptides eliciting Plasmodium transmission- blocking immune response and further include immunogenic polypeptides eliciting protective immune response to other human pathogens, such as, but not limited to, influenza, measles, mumps, diphtheria, tetanus, pertussis, poliovirus, hepatitis B virus, varicella, N, meningitides or rubella. In another example, pharmaceutical compositions for administration to human subjects can include nucleic acids encoding immunogenic P47 D2 polypeptides eliciting Plasmodium transmission-blocking immune response and further include nucleic acids encoding immunogenic polypeptides eliciting protective immune response to other human pathogens, such as, but not limited to, influenza, measles, mumps, diphtheria, tetanus, pertussis, poliovirus, hepatitis B virus, varicella, N, meningitides or rubella.

[0078] In some other embodiments, compositions comprising immunogenic P47 D2 polypeptides or nucleic acids of the present invention can further included immunogens (polypeptides or nucleic acids) useful for eliciting immune response to Plasmodium stages found in a vertebrate host. For example, pharmaceutical compositions for administration to human subjects can include immunogenic P47 D2 polypeptides eliciting Plasmodium transmission-blocking immune response and further include immunogens preventing or reducing development of Plasmodiu in the vertebrate hosts, for example, Plasmodium circumsporozoite protein. In another example, pharmaceutical compositions for administration to human subjects can include nucleic acids encoding immunogenic P47 D2 polypeptides eliciting Plasmodium transmission-blocking immune response and further include nucleic acids encoding immunogenic polypeptides preventing or reducing development of Plasmodium, in the vertebrate hosts, for example Plasmodium, circumsporozoite , The compositions comprising immunogenic P47 D2 polypeptides can also include other antigens that also elicit transmission-blocking such as the gametocyte surface proteins Pfs230, Pf48/45 or Pfs25.

[0079] The isolated nucleic acid sequences encoding P47 D2 polypeptides can be used in viruses to transfect host cells in the susceptible organism, particularly, a human. Live attenuated viruses, such as vaccinia or adenovirus, are convenient alternatives for vaccines because they are inexpensive to produce and are easily transported and administered. Suitable viruses include, but are not limited to, pox viruses, such as, canarypox and cowpox viruses, and vaccinia viruses, alpha viruses, adenoviruses, and other animal viruses. The recombinant viruses can be produced by methods well known in the art: for example, using homologous recombination or ligating two plasmids together. A recombinant canarypox or cowpox virus can be made, for example, by inserting the nucleic acid sequence encoding the P47 D2 into a plasmid so that it is flanked with viral sequences on both sides. The gene is then inserted into the virus genome through homologous recombination. A recombinant adenovirus virus can be produced, for example, by ligating two plasmids each containing 50% of the viral sequence and the DNA sequence encoding P47 D2 polypeptide. Recombinant RNA viruses such as the alpha virus can be made via a cDNA intermediate using methods known in the art. The recombinant viruses can be used to induce anti- P47 D2 polypeptide antibodies in mammals, such as primates or humans. In the case of vaccinia vims, the sequence encoding P47 D2 polypeptide can be inserted into the viral genome by a number of methods including homologous recombination using a transfer vector.

[0080] Immunogenic P47 D2 polypeptides or nucleic acids can be used in pharmaceutical compositions that are useful for administration to subjects in a single administrations or a series of administrations. When given as a series, inoculations subsequent to the initial administration are given to boost the immune response and are typically referred to as booster inoculations. The pharmaceutical compositions are intended for parenteral, topical, oral or local administration. In other words, pharmaceutical compositions are administered parenteral!}', intravenously, subcutaneously, intradermal!} ⁷ , or intramuscularly. Pharmaceutical compositions according to the embodiments of the present invention contain as an active ingredient an immunologically effective amount of the P47 D2 polypeptide, nucleic acid or recombinant virus in combination with a suitable carrier. A variety of aqueous carriers may be used, such as, phosphate buffered saline, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. Other ingredients, such as thyroglobu!in, albumins such as human seram albumin, tetanus toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid) etc., can also be included in the pharmaceutical compositions. The compositions can also contain an adjuvant, such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum. The compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The compositions can include, for example, anti-oxidants, buffers, and bacteriostatic agents, and may include suspending agents and thickening agents. For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%. For aerosol administration, the polypeptides or nucleic acids are preferably supplied in finely divided form along with a surfactant and propeiiant. The surfactant must, of course, be nontoxic, and preferably soluble in the propeiiant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic poiyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery. The compositions of the present invention can include nanospheres or microspheres, such as lipid nanospheres or microspheres, neutral, anionic or cationic polymeric nanoparticles or microparticles, site- specific emulsions, long-residence emulsions, sticky-emulsions, micro-emulsions, nano- emulsions, microspheres, nanospheres, nanoparticles etc. Microspheres, microparticles, nanospheres, nanoparticles etc. can be prepared from various natural or synthetic polymers, including anionic, neutral or cationic polysaccharides and anionic, neutral cationic polymers or copolymers. The active components of the compositions according to some embodiments of the present invention can be used with any one, or any combination of, carriers. [0081] As noted elsewhere in this documents, immunogenic P47 D2 polypeptides or nucleic acids polypeptides of this invention may also be used to make monoclonal antibodies. Such antibodies may be useful as potential research, diagnostic or therapeutic agents. P47 D2 polypeptides or nucleic acids polypeptides themselves may also find use as diagnostic reagents. For example, a polypeptide of the invention may be used to diagnose the presence of antibodies against Plasmodium in a patient. Alternatively, the polypeptides can be used to determine the susceptibility of a particular individual to a particular treatment regimen, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an infected individual.

Anti-P47 D2 antibodies; methods of making and using anti-P47 antibodies; related compositions, molecules, products and kits

[0082] Embodiments of the present invention include anti-P47 polyclonal and monoclonal antibodies described in this document, as well as their various modifications and variations. Anti-P47 monoclonal antibodies according to some embodiments of the present invention were obtained by carefully selecting potentially immunogenic amino acid sequences from P47 D2 amino acid sequences, using the nucleic acid sequences encoding the above immunogenic amino acid sequences to generate immunogenic peptides, then using the immunogenic peptides to generate polyclonal antibodies (polyclonal sera) as well as monoclonal antibodies via a hybridoma technology. The resulting monoclonal antibodies were screened for their Plasmodium transmission-blocking activity. The antibodies are useful in a wide range of analytical, diagnostic and public health applications, in which specific binding of a monoclonal antibody to a P47 protein is desired.

[0083] As used herein, the term "antibody" encompasses whole immunoglobulin of any class, including natural, natural-based, modified and non-natural antibodies, as well as their fragments. Natural antibodies are very large, complex polypeptide molecules (molecular weight (MW) of about 150,000, which about 1320 amino acids) with intricate internal structure. Natural antibodies are usually heterotetrameric glycoproteins. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable ("V") region involved in binding the target antigen, and a constant (' ^" C") region that interacts with other components of the immune system. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: gA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-l, IgG- 2, IgG-3, and IgG-4; IgA-1 and IgA-2. The prevalence of the isotopes differs among the species. For example, rabbit has only one IgG subclass, while mouse has IgGl , IgG2a, IgG2b, IgG2c, IgG3 subclasses. Most of rabbit research antibodies are of IgG isotope. They possess a number of advantages, in comparison to other antibody types. Some of the advantages are more diverse epitope recognition, improved immune response to small -size epitopes, high specificity and affinity, and greatly improved response to mouse antigens.

[0084] The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The term "variable" may be used in reference to antibodies to describe certain portions of their variable regions thai differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. The variability is not usually evenly distributed through the variable domains of antibodies but typically concentrated in the segments called complementarity determining regions (CDRs) or hypervariable regions, both in the light chain and the heavy chain variable domains. The other, more highly conserved portions of the variable domains, are called the framework (FR). Within each light or heavy chain variable region, there are three CDRs averaging 10 amino acids in length. The "Kabat Numbering Scheme" is a scheme for the numbering of amino acid residues in antibodies based upon variable regions. The scheme employs the so-called "Kabat numbers' ¹ to denote amino acid residues and is useful when comparing these variable regions between antibodies. The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold together in 3-D space to form the actual antibody binding site, which locks onto the target epitope within the antigen.

[0085] The term "antibody binding site" and the related terms may be used herein to describe a polypeptide structure capable of specifically binding an "epitope" - the region of its antigen to which the antibody binding site binds. As used herein, the terms "specific binding," "selective binding" or related terms refer to a binding reaction in which, under designated conditions, a molecule or a composition containing an antibody binding site binds to its epitope and does not bind in a significant amount to other potential binding partners. The absence of binding in a significant amount is considered to be binding that is less than 1.5 times background (i.e., the level of non-specific binding or slightly above non-specific binding levels). For example, the anti-P47 D2 antibodies of the present invention specifically bind to their respective P47 epitopes. Antibody binding site may also be described in reference to amino acid sequences of the polypeptides within the binding site, for example, CDR amino acid sequences, or in terms of the nucleic acid sequences encoding the amino acid sequences.

[0086] The antibodies according to some of the embodiments of the present invention are monoclonal. The terms "monoclonal antibody," "monoclonal antibodies," "mAb," "mAbs" and other related terms may be used in this document to refer to a substantially homogenous population of antibodies or to an antibody obtained from a substantially homogeneous population of antibodies. The antigen binding sites of the individual antibodies containing the population is composed of polypeptide regions similar (although not necessarily identical) in sequence. The nature of the monoclonal antibodies is easier understood in comparison to polyclonal antibodies. In laboratory conditions, polyclonal antibodies are typically produced by injecting an animal (such as a rodent, rabbit or goat) with an immunizing agent (which may be referred to as immunogen or antigen), which elicits animal's immune system lymphocytes to produce antibodies to the antigen, and extracting the antibody-containing serum from the animal. The extracted serum contains a population of immunoglobulin molecules produced by different B-cell lineages. This population is typically referred to as "polyclonal antibodies." Polyclonal antibodies react against the same antigen, but may bind to different epitopes within the antigen. Polyclonal antibodies have binding sites with different sequences and structures, as well as varying properties, such as affinity or specificity. In contrast to polyclonal antibodies, a population of monoclonal antibodies binds to the same epitope on an antigen and are understood to have antigen binding sites with similar sequence and structure. Polyclonal antibodies against P47 D2 are also included among the embodiments of the present invention.

[0087] Monoclonal antibodies may be prepared in the laboratory conditions using hybridoma methods. First, as during the production of polyclonal antibodies, a host animal, such as a mouse or a rabbit, may be administered an antigen. After the host animal mounts an immune response to the antigen, spleen cells or lymph node cells are extracted from the animal. Alternatively, the lymphocytes may be immunized in vitro. The cells are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma ceil line. Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized ceils. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoraas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells. For hybridoma, immortalized cell lines are useful that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. One example are immortalized cell lines are murine myeloma cell lines. The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies by various assays, such as immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELBA). After the desired hybridoma cells are identified, their clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Duibecco's Modified Eagle Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures.

[0088] Monoclonal antibodies may also be produced by recombinant DNA methods. For example, phage display/yeast display libraries are used for rapid cloning of immunoglobulin segments to create libraries of antibodies, from which antibody binding sites with desired properties may be selected. In another example, DNA encoding the monoclonal antibodies, such as those generated by hybridoma technology, or parts of the monoclonal antibodies, such as their binding sites, is isolated, synthesized and sequenced using conventional procedures from hybridoma cells. DNA encoding the monoclonal antibodies can be isolated and amplified by polymerase chain reaction (PCR) using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains antibodies. Once isolated, the DNA encoding antibodies or their parts, may be placed into expression vectors, which are then transfected into host cells to synthesize recombinant monoclonal antibodies in the recombinant host cells. Various types of mammalian cell expression systems may be employed, such as, but not limited to, simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, plasmacytoma cells, or myeloma cells, as well as other types of cells that do not otherwise produce immunoglobulin protein. Non-mammalian host cells and/or expression systems can also be employed, one example being insect cells expression systems or avian expression systems. In vitro translation/expression systems may also be used to produce monoclonal antibodies according to the embodiments of the present invention. Some of the compositions and methods related to recombinant production of monoclonal antibodies are discussed in more detail below and included within the scope of the embodiments of the present invention.

[0089] Monoclonal antibodies according to the embodiment of the present invention can be derived from naturally occurring monoclonal antibodies or artificially produced ("engineered), for example, by recombinant techniques, and encompass fragments, variants and modification of immunoglobulin molecules. In the broadest sense, the term, "monoclonal antibody" is used in this document to denote any product, composition or molecule that contains at least one antibody binding cite. For example, monoclonal antibodies encompass chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab, hybrid fragment, single chain variable fragments (scFv) and "third generation ^" (3G) fragments. Monoclonal antibodies also encompass fusion proteins, single domain and "miniaturized" antibody molecules.

[0090] Fragments can be made by known techniques, for example, they can be recombinant!}' and/or enzymatically produced, and can be screened for specificity and activity according to known methods, such as radioimmunoassays, ELBA, Western blotting or immunofluorescence assays and techniques. Digestion of whole antibody molecules can be employed to produce fragments, particularly, Fab fragments. For example, papain digestion of whole antibody molecules typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. In another example, pepsin treatment yields a fragment, called the F(ab')2 fragment, that has two antigen binding sites. The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the heavy chain domain, including one or more cysteines from, the antibody hinge region. F(ab')2 fragments are bivalent fragments containing two Fab' fragments, which may linked by a disulfide bridge at the hinge region. Fab'-SH is the designation that may be used Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments may be produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments may also be employed.

[0091] Various modifications of monoclonal antibodies may be produced, for example, by recombinant DNA techniques. As used herein, a "recombinant" or "recombinant produced" monoclonal antibody is a product, composition or molecule containing an antibody binding site produced with the help of recombinant DNA techniques. As used herein, the terms "recombinant" or "recombinant produced" encompass antibodies for which the genes have been constructed and/or placed in an unnatural environment, for example for expression, with the help of recombinant DNA techniques. In one example, the DNA encoding a monoclonal antibody may be modified, for example, by joining to the all or part of immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide, which is included within the scope of the "monoclonal antibodies" of the present invention, can be substituted for the constant domains of a monoclonal antibody. In another example, a chimeric antibody may be produced. A chimeric antibody, which also included within the scope of the monoclonal antibodies of the present invention, is an antibody in which parts of antibody molecules of different origins are combined.

[0092] Monoclonal antibodies according to the embodiments of the present invention may contain a label. The term "label" encompasses any detectable tag that can be attached directly (for example, a fluorescent molecule integrated into a polypeptide) or indirectly, by way of binding to a primary antibody a secondasy antibody with an integrated label or tag. The term "label" or "tag" also encompasses an epitope recognized by another (secondary) antibody or protein that can be conjugated to a label or tag. Detectable label may be an enzymatic label (such as but not limited to horse radish peroxidase (HRP) or alkaline phosphatase (AP)), a radio-opaque substance, a radiolabel, a fluorescent label, a nano-particle label or a magnetic label, a hapten, or a oligonucleotide or polynucleotide label. Detectable label may be a gamma-emitter, beta-emitter, alpha-emitter, gamma-emitter, positron-emitter, X-ray-emitter or fluorescence-emitter. Suitable fluorescent compounds include fluorescein sodium, fluorescein isothiocyanate, phycoerythrin (PE), Allophycocyanin (APC), Alexa Fluor ^® family of dyes (such as, Alexa Fluor ^® 350; Alexa Fluor ^® 405, Alexa Fluor ^® 488, Alexa Fluor ^® 532, Alexa Fluor ^® 546, Alexa Fluor ^® 555, Alexa Fluor ^® 568, Alexa Fluor ^® 594, Alexa Fluor ^® 647, Alexa Fluor ^® 680 or Alexa Fluor ^® 750), Cy ^® family of dyes (such as Cy ^® 3 or Cy ^® 5), BODIPY ^® FL, Coumarin, Oregon Green ^® , Pacific Blue ^® , Pacific Green ^® , Pacific Orange ^® , tetramethylrhodamine (TRITC), Texas Red ^® , Q-dot ^® probes (such as Qdoi ^® 525. Qdot ^® 565. Qdot ^® 605. Qdot ^® 655, Qdoi ^: 705, or Qdoi ^® 800), Expressed fluorescent proteins (such as Cyan Fluorescent Protein (CFP), Green Fluorescent Protein (GFP). Or Red Fluorescent Protein (RFP), haptens (such as biotin or DIG). Monoclonal antibodies according to the embodiments of the present invention can also be conjugated or fused to bioactive substances, drugs and radioactive or toxic moieties, such as diphtheria or ncm toxin.

Θ093] One of the properties used to describe the monoclonal antibodies according to the embodiments of the present invention is their ability to bind defined polypeptide '"antigens." For example, the monoclonal antibodies according to the embodiments of the present invention can be described in reference to their binding properties - as capable of binding P47 protein, its fragments and/or variants, such as P47 D2 polypeptide or its variants, including at least some orthologs. As discussed elsewhere in tins documents, anti~P47 monoclonal antibodies of the present invention can be obtained by using immunogenic polypeptides having amino acid sequences of P47 D2 domain or variants of such sequences, such as polypeptides having partial amino acid sequences of Pfs47 D2 domain or variants of such sequences (exemplified by SEQ ID NOs 30 and 31) and Pvs47 D2 domain or variants of such sequences (exemplified by SEQ ID NOs 40 and 41). Accordingly, monoclonal antibodies according to some embodiments of the present invention are capable of specifically binding D2 domain polypeptides of P47 proteins and P47 proteins containing D2 amino acid sequences, or their variants or homologues, such as polypeptides having amino acid sequences of Pfs47 D2 domain or variants of such sequences and Pvs47 D2 domain or variants of such sequences. Said differently, under appropriate binding conditions, monoclonal antibodies specifically bind D2 domain polypeptides of P47 proteins and P47 proteins containing D2 ammo acid domain sequences, or their variants or homologues, such as polypeptides having amino acid sequences of Pfs47 D2 domain or variants of such sequences and Pvs47 D2 domain or variants of such sequences. Binding of the monoclonal antibodies according to the embodiments of the present invention to their polypeptides may be described in terms of binding affinity. For example, monoclonal antibodies of the present invention typically have a binding affinity (K _a ) for their respective immunogen polypeptides of at least 10 ⁷ M ^"1 . Another property that may be used to describe the monoclonal antibodies according to the embodiments of the present invention is their ability to block transmission of Plasmodium in mosquitoes. This ability may be measured by a standard assay, such as SMFA. The ability to block transmission of Plasmodium in mosquitoes may be described as transmission-blocking .

[0094] Methods of making anti-P47 antibodies are included within the scope of the embodiments of the present invention. Monoclonal antibodies can be produced by processes containing a step of administering an immunogenic composition containing at least one of immunogenic polypeptides (described elsewhere in tins document) to subjects, cells or tissues, to induce antibody production in the subjects, cells or tissues. Administration of the immunogenic composition induces production of antibodies in the subjects, cells or tissues. Thus, antibody-producing subjects, cells or tissues are provided. Methods of making antibodies according to the embodiments of the present invention can include one or more steps typically employed in hybridonia technology, such as isolating antibody-producing cells from the subject, using the antibody-producing cells to generate a hybridonia cell culture and isolating an individual antibody -producing cell from the hybridonia cell culture. Antibody- producing cells may be expanded in culture and used to generate monoclonal antibodies. Θ095] Anti-P47 antibody production processes according to the embodiments of the present invention may include one or more screening steps, methods or processes. Such screening steps, methods and processes are included within the scope of the present invention. "Screening," which may also be referred to as "analysis," "characterization," "testing" or by other related terms The binding properties of thus generated monoclonal antibodies may be characterized or tested during the screening, which may involve the steps of contacting monoclonal antibodies with a screening composition containing at least one of immunogenic polypeptides under conditions of an assay intended to detect binding of the antibody to the one or more immunogenic polypeptides. The assay, which can be referred to as "screening assay," may be, but is not limited to, an immunoprecipitation assay, a Western blot, an ELISA, a flow cytometry assay, an immunofluorescence assay (IFA), an immunohistochemistry (IHC) assay, a cytospin assay, a fluorescence resonance energy- transfer (FRET) assay, or a reverse phase array. Another example of a screening assay that may be used for screening anti-P47 monoclonal antibodies according to the embodiments of the present invention is an assay detecting transmission blocking activity of the antibodies in mosquitoes, such as a Standard Membrane Feeding Assay, direct mosquito feeding of immunized infected volunteers from an endemic area, or membrane feeding assay using blood from volunteers naturally infected with P. falciparum or P. vivax parasites. [0096] More than one screening assay may he employed. A plurality of monoclonal antibodies may be obtained and tested by the above processes. Based on the results of the screening monoclonal antibodies with desired properties, such as binding strength, specificity, transmission blocking activity or a combination of properties, may be selected for further processes and uses. For example, a screening may reveal that an antibody exceeds a certain predetermined threshold values characterizing transmission blocking activity, binding strength or specificity, or that the antibody's characteristics are superior in comparison to other screened antibodies, which case the antibody may be selected for further processes and uses. Screening may involve conducting various screening assays.

[0097] The methods, processes and steps of producing monoclonal antibodies according to the embodiments of the present invention may include recombinant techniques. The nucleic acid sequences (for example, cDNA sequences and genomic DNA sequences) encoding antibody binding site or amino acid sequences of the selected monoclonal antibodies may be determined. Recombinantly produced monoclonal antibodies containing the antibody binding sites may be generated based on these sequences using appropriate vectors and expression systems, which are described in more detail elsewhere in this document. The methods, processes and steps of producing monoclonal antibodies according to the embodiments of the present invention may include various steps related to analysis and generation of nucleic acid and amino acid sequences and molecules, such as nucleic acid and polypeptide sequencing, amplification (for example, by polymerase chain reaction (PCR)), restriction, nucleic acid and polypeptide synthesis, mass spectroscopy, HPLC, and various other procedures. The methods, processes and steps of producing monoclonal antibodies according to the embodiments of the present invention may include isolation and purification steps, such as dialysis, precipitation, microfiltration, ultrafiltration, protein A or G affinity chromatography, size exclusion chromatography, anion exchange chromatography, hydroxylapatite (hydroxyapatite) chromatography, hydrophobic interactions chromatography, cation exchange chromatography, other forms of affinity chromatograph, gel electrophoresis, HPLC and other techniques and procedures. Compositions, methods and kits related to the antibody production and processes (such compositions, methods and kits for recombinant antibody production, are included within the scopes of the embodiments of the present invention).

[0098] In an illustrative example, anti-P47 monoclonal antibodies according to the embodiments of the present invention are recombinantly produced. In one example illustrating the process of using the above-described vectors and cells, a nucleic acid sequence encoding an anti-P47 monoclonal antibody is introduced into a plasmid or other vector, which is then used to transform, living cells. For instance, genes encoding light and heavy chain V regions are synthesized from overlapping oligonucleotides and inserted together with available C regions into expression vectors that provide the necessary- regulatory regions, such as promoters, enhancers, poly A sites and other sequences. Expression vectors may be employed, in which a cDNA containing the entire anti-P47 monoclonal antibody coding sequence, a fragment of the anti-P47 monoclonal antibody coding sequence, amino acid variations of the anti-P47 coding sequence, or fusion proteins of the aforementioned, is inserted in the correct orientation in an expression plasmid. In some cases, it may be desirable to express the coding sequence under the control of an inducible or tissue-specific promoter. The expression vectors may then be transfected using various methods, such as lipofection or electroporation, into cells of an appropriate mammalian cell line, thus generating cells expressing the monoclonal antibodies. The cells expressing the antibodies may be selected by appropriate antibiotic selection or other methods and cultured. Larger amounts of antibody may be produced by growing the cells in commercially available bioreactors. Once produced by the antibody-producing cells, anti-P47 monoclonal antibodies may be purified according to standard procedures, such as dialysis, filtration and chromatography. A step of lysing the cells to isolate the anti-P47 monoclonal antibody can be included. Thus, a method of making an anti-P47 monoclonal antibody may contain one or more steps of culturing a cell containing a vector under conditions permitting expression of the anti-P47 monoclonal antibody, harvesting the cells and/or harvesting the medium from the cultured cells, and isolating tlie anti-P47 monoclonal antibody from the cells and'or the culture medium. Compositions, methods and kits related to tlie antibody production and processes (such compositions, methods and kits for recombinant antibody production) are included within the scopes of the embodiments of the present invention.

[0099] The methods according to the embodiments of the present invention involve binding of the antibody to its epitope. Thus, the processes of using anti-P47 antibodies, including polyclonal and monoclonal antibodies according to some of the methods described in this document, include a step of contacting the anti-P47 monoclonal antibody (meaning one or more monoclonal antibody) with a sample under conditions that permit binding of the monoclonal antibody with its P47 epitope. The conditions under which the binding of anti- P47 monoclonal antibody to its epitope occurs depend on the context of the specific method. The terms "sample" or "samples" is not intended to be limiting and refers to any product, composition, cell, tissue or organism that may contain epitopes of the anti-P47 monoclonal antibodies described in this document.

[0100] The methods according to the embodiments of the present invention may include a step of detecting the binding of an anti-P47 antibody monoclonal antibody to its epitope. The detection may be accomplished by detecting epitope -antibody complexes using v arious types of labels. For example, for various assays, the monoclonal antibodies may be labeled with fluorescent molecules, metals, spin-labeled molecules, nano-particles, enzymes or radioisotopes. The labels are sometimes referred to as "reporter molecules." Anti-P47 monoclonal antibodies may be directly labeled, meaning that the labels can be directly attached (conjugated to the antibodies) or indirectly (non-covalently) labeled, meaning that antibody-binding molecules containing the labels may be employed. For example, labeled secondary antibodies or their fragments capable of binding anti-P47 monocional antibodies may be used. Some examples of antibody labels and procedures used in labeling are described, for example, in "Guide to Antibody Labeling and Detection," Innova Biosciences (2010), Cambridge (UK), Buchwalow & Bocker "Immunohistochemistry: Basics and Methods" Springer-Veriag Berlin Heidelberg (2.010) (see, for example, Chapter 2, "Antibody Labeling and the Choice of a Label," pages, 9-17). Detection of the labels, which includes qualitative and quantitative detection, is accomplished by various methods, depending on the label, the method in which it is used, and the result desired.

[0101] For example, in some of the embodiments of the methods described in this documents, the binding of anti-P47 monoclonal antibodies to their epitopes in cell and tissue samples may be visually detected, such as in die slides examined or imaged under the microscope, by using either a direct (covaiently attached) fluorescent label or fluorescentiy labeled secondary antibodies. In another example, fluorescence emitted by labeled anti-P47 monoclonal antibodies or by the secondary antibodies is quantitatively detected by registering light emitted by the sample at a particular wavelength. One or more monoclonal antibodies may be employed. In some exemplary methods, two monoclonal antibodies binding to different P47 epitopes (i.e., not competing for binding) may be simultaneously employed, for example, in a "sandwich" assays, such as ELBA. Anti-P47 monoclonal antibodies according to the embodiments of the present invention and may be provided in the form of kits with all the necessary reagents to perform the assay for P47 presence, absence or level. [0102] Monoclonal anti-P47 antibodies according to the embodiments of the present invention detect a range of P47 variants and isoforms, and are useful for various analytical procedures and protocols, including immunofluorescence assays, Western blotting, ELISA, flow cytometry and immunoprecipitation. A method can contain a step of detecting a level of P47 in a sample and comparing it to a control level. Control levels can be used to establish a threshold value. This threshold value can be determined empirically by comparing positive controls (for examples, samples with a certain level of P47 present) and negative controls (samples without P47), Such procedures and protocols employing anti-P47 monoclonal antibodies may be useful in a wide range of analytical and diagnostic applications, for example, in research and laboratory applications in which detection of P47 is desirable.

[0103] The following examples will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that resort may be had to various embodiments, modifications and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention,

EXAMPLE 1

Optimization of Pfs47 recombinant protein expression

[0104] Multiple attempts to express Plasmodium falciparum Pfs47 in heterologous expression systems were made, but recombinant expression of Pfs47 failed repeatedly due to Pfs47 structure. P. falciparum Pfs47 is a member of the 6-cysteine protein family and has three domains: two domains (Dl and D3) with the classic six-cysteine fold; and the central domain (D2) with two cysteine residues. This large number of cysteine residues in Pfs47 (a total of 14) has to form correct disulfide bonds in order to form a correctly folded recombinant protein.

[0105] Several different strategies to express Pfs47 in heterologous systems, either alone or as a fusion protein with a protein carrier intended to facilitate downstream purification and protein folding, were unsuccessful. In all the strategies, the nucleotide sequence of Pfs47 coding region was codon -optimized to suit the expression system used. The signal peptide and GPI anchor were removed, and a histidine tag was added. Expression of several different Pfs47-encoding constructs in mammalian cell lines (pVR2010, pBIO and pLAC -based plasmids in Human Embryonic Kidney cells) or insect cell lines (Sua5.1 from Anopheles gambiae and Aag2 from Aedes aegipty cells) were all unsuccessful, as summarized in Table 2. The expression in Sf insect cells using Baculovirus expression system resulted in very low yield of the recombinant protein (about 100 ,ug/L of harvested supernatant).

[0106] Expression of Pfs47 as a fusion protein with thioredoxin ("Pfs47 thioredoxin fusion protein") in a modified E. coli strain that enhances correct disulfide bond foiTnation (Rosetta-gami 2) resulted in insoluble protein that was mainly present in the form of inclusion bodies at the yield of about 1 mg/L. Pfs47 thioredoxin fusion protein included a sequence of 394 amino acids that encoded all three predicted domains of Pfs47, Dl, D2 and D3, as illustrated in Figure 1 . The N-terminal predicted signal peptide for secretion and the putative GP1 -anchoring C-terminal region peptide, also illustrated in Figure 1, were not included in Pfs47 thioredoxin fusion protein amino acid sequence, which is illustrated in Figure 2. P. falciparum Pfs47 coding region lacking the signal peptide and GPI anchor was codon optimized and cloned into plasmid pET32 generating an N-terminal thioredoxin (shown in dark grey in Figure 2) fusion protein with Pfs47 coding sequence (light grey in Figure 2) and a C-terminal histidine tag.

[0107] E. coli BL2I Rosetta-gami cells were transformed with the pET32-Pfs47 plasmid construct, and Pfs47 expression was induced in liquid culture by adding 100 μΜ IPTG. An "m column" HPLC nickel affinity purification/refolding protocol was developed and applied to die insoluble inclusion bodies, resulting in soluble purified Pfs47 thioredoxin fusion protein. The results of purification/refolding protocol are illustrated in Figure 3.

EXAMPLE 2

Production and testing of monoclonal antibodies to Pfs47 thioredoxin fusion protein [0108] Soluble purified Pfs47 thioredoxin fusion protein was used to immunize mice using the Magic Mouse® adjuvant system, Creative Diagnostics, Shirley, New York. The first and second immunogen boost were administered 15 days after the previous immunization .. The third (final) boost was administered 8 weeks after the second boost. Three days after the third boost, the mice were splenectomized to generate hybridoma fusions. These hybridoma fusions resulted in 14 independent cloned mAbs that bound to soluble purified Pfs47 thioredoxin fusion protein, but did not bound to thioredoxin alone. The transmission blocking activity (TBA) of the mAbs was tested using a Standard Membrane Feeding Assay (SMFA) Mtura et al. "Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays." PLoS ONE 2013;8:e57909. Briefly, in SFMA, mosquitoes are fed in v/fro-cultured P. falciparum, gametocytes. to which a particular monoclonal antibody has been added. Infected mosquitoes are kept in the insectary and fed a sugar solution. The infection intensity and prevalence of the mosquito are measured 7-11 days after the feeding by counting the number of parasite oocysts infecting each midgut. As controls, a negative control (mouse IgG from naive mice) and a positive control (4B7 mAb) that binds to the surface of the P25 parasite protein and is considered the "gold standard" for TBA were used in SFMA. Although all anti-Pfs47 mAbs recognized the soluble stable recombinant Pfs47 protein expressed in SF9 insect cells, none of the 14 tested m Abs showed robust and reproducible TBA. None of the 14 mAbs significantly reduced oocyst numbers in mosquito midgut compared to the mouse IgG (mlgG) negative control, while 4B7 mAb positive control exhibited robust TBA. The results obtained with four of these monoclonal antibodies are illustrated in Figure 4, showing a dot plot of TBA of mAbs generated against Pfs47 thioredoxin fusion protein, measured using SMFA with Plas odium falciparum NF54 in Anopheles gamhiae, and in Table 3. The dot plot of Figure 4 shows the number of oocyst/midgut after feeding Anopheles gambiae mosquitoes on a gametocyte culture containing 0.12 mg/ml of purified IgG from the different monoclonal antibodies. The level of inhibition was calculated relative to the mouse IgG negative control (mlgG). Positive control Anii-Pfs25 mAb, performed as expected (***** _; p<G.()001, Mann-Whitney test). Four mAbs out of 14 exhibited modest and inconsistent transmission blocking activity, as illustrated in Figure 4 (the corresponding results are summarized in Table 3). When these four mAbs ("low TBA mAbs") were combined in equal ratios and added to the gametocyte culture, consistent and significant (p<G.0 ! 8), but very modest transmission blocking activity was observed, as illustrated in Figure 5 and Table 4. The percentage of inhibition was calculated against the mouse IgG control (mlgG). Although the observed transmission blocking activity of the pooled mAbs was significant, the effect was modest (*, p<0.05, Mann-Whitney test).

Table 3. Summary of TBA testing of the rnAbs obtained against full-length Pfs47 thioredoxin fusion protein

Table 4. Summary of TBA testing of the pooled mAbs obtained against full-length Pfs47 thioredoxin fusion protein

EXAMPLE 3

Domain mapping of Pfs47

[0109] Epitope mapping was conducted in order to determine to which Pfs47 regions anti-Pfs47 mAbs described above were binding. The results of the epitope mapping are illustrated in Figure 6, which is a bar graph showing the immunoreactiviry of mAbs generated against full-length Pfs47 to recombinant full-length Pfs47 (dark bars) and to a Pfs47 protem lacking the D2 domain (Pfs47 D1-D3 fusion) (white bars) in ELISA assays. ELISA assays using both full-length Pfs47 protein and D1-D3 fusion Pfs47 protein lacking D2 revealed that all 14 mAbs recognized the Pfs47 D1-D3 fusion protein (see Figure 6), thus indicating that none of them recognized D2.

[0110] Two of the four low TBA mAbs (3B9 and 5D6 } bound Dl , while the other low TBA mAbs (FB7 and 5B7) recognized D3. See Figure 7, which is a bar graph showing the immunoreactivitv of mAbs generated against full-length Pfs47 to recombinant full-length Pfs47 protein (orange bars), and to Pfs47 Dl (red bars) and D3 (green bars) in ELISA assays. The above findings suggested that antibodies to D2 domain may be needed to block transmission.

EXAMPLE 4

Expression of Pfs47 D2 polypeptide and production of anti-D2 antibodies

[0111] Recombinant expression of Pfs47 D2 polypeptide as a thioredoxin fusion protein or alone in E. coli was tried, but the attempts to produce E. coli cells transformed with the corresponding expression piasmids were unsuccessful. Bacterial colonies failed to grow in the presence of D2 expression piasmids, suggesting that expression of the D2 domain polypeptide was toxic to E. coli cells. In an attempt to remove the toxicity by changing D2 polypeptide conformation, two cysteines in Pfs47 D2 amino acid sequence were replaced with two alanines. The amino acid substitution resulted in high yield expression of soluble Pfs47 D2 polypeptide in E. coli. The amino acid sequences of modified recombinant 118 aa D2 polypeptide (Pfs47-D2-118) is shown in Figure 8.

[0112] Pfs47 D2 polypeptide was purified from E. coli inclusion bodies using a similar procedure as with the full-length protein. Briefly, Pfs47-D2-118 was expressed in E. coli BL21 Rosetta-gami cells after inducing expression by adding 100 μΜ IPTG in liquid culture. Inclusion bodies were isolated, washed and purified soluble protein was obtained using nickel affinity purification, followed by dialysis. Purified Pfs47-D2-118 was used as an immunogen to generate anti~D2 polyclonal antibodies in mice and anti-D2 mAbs using hybridoma procedure.

EXAMPLE 5

Transmission-blocking activity of anti-D2 antibodies

[0113] Both the polyclonal sera obtained from immunized mice and several of the mAbs obtained against Pfs47-D2-118 were tested using SFMA and shown to have strong and reproducible TBA, as illustrated by Figures 9 and 10 and Tables 5 and 6. Figure 9 shows a dot plot showing the number of oocyst/midgut after feeding Anopheles gambiae mosquitoes on a gametoeyte culture containing 0.12 mg/ml of purified polyclonal mouse IgG obtained after immunization with full-length or D2-Pfs47. The percentage of inhibition was calculated relative to the mouse IgG negative control (mlgG). The corresponding results are summarized in Table 5. The dot plot showing the number of oocyst/midgut after feeding Anopheles gambiae mosquitoes on a gametoeyte culture containing 0.12 mg/ml of purified IgG from the different monoclonal antibodies generated against Pfs47 D2 polypeptide is shown in Figure 10, with the corresponding results summarized in Table 6. The percentage of inhibition was calculated relative to the mouse IgG negative control (mlgG) (***=p<0.001: *·* *= 0.0001. Mann-Whitney test).

Table 5. Summary of IgG TBA testing

Table 6. Summary of anti- Pfs47 D2 polypeptide mAb testing

[0114] Anti-D2 polyclonal serum reduced oocyst numbers by 77%, while the serum after immunization with the full-length protein had no detected TBA. Four anti-D2 mAbs reduced oocysts numbers/midgut by 78-88%, relative to the mouse IgG (mlgG) control, an three of them (marked with *** * in Table 3) were as effective as the anti-Pfs25 mAb gold standard. EXAMPLE 6

Mapping of Pfs47 D2 polypeptide

[0115] When mice were repeatedly immunized with purified Pfs47-D2-1 18 polypeptide, it was unexpectedly observed that the TBA of the resulting polyclonal sera decreased with successive immunizations.

[0116] Figure 19 illustrates the results of TBA assays conducted with the sera obtained from the mice successively immunized with Pfs47-D2-118 polypeptide. The high level of TBA exhibited after the first boost immunization (78%), decreased to 61% after the second boost immunization and was lost after the third boost immunization. The percentage of inhibition was calculated relative to the mouse IgG negative control (mlgG) (*=p<0.05; ****= p<0.0001 , Mann-Whitney test). The corresponding results are summarized in Table 7, These results indicated that Pfs47-D2-118 polypeptide contained a strongly immunogenic (immunodominant) region that induced production of immunoglobulins specifically binding to D2 domain of the Plasmodium P47 protein, but were not transmission-blocking.

Table 7. Summary of anti-Pfs47-D2-l 18 polypeptide polyclonal serum testing

[0117] To locate the immunodominant region, a series of deletions in D2 amino acid sequence was used to map the binding of five anti-D2 mAbs described in Example 2: one mAb exhibiting no TBA, and four mAbs exhibiting strong TB A. The amino acid sequences of the three D2 Pfs47 N- and/or C -terminal deletions (Del 1, Del 2, Del 3 and Del 4) used in the mapping are shown in Figure 20 as SEQ ID NOs 28-31. The recombinantly expressed polypeptides contained N-terminal methionine residue and a C-terminal 6-histidine tag, as shown in SEQ ID NOs 52-55. It was discovered that all the mAbs with strong TBA bound to a central region of 52 amino acid region of D2 (SEQ ID NO:31), shared by Del 1 and Del 3 polypeptides peptides, but did not bind to Del 2 polypeptide lacking the central region. Hie mAb with no TBA bound to Del 2 polypeptide containing the N-terminal region of D2, but did not bind to Del 3 polypeptide lacking the N-terminal region. These results showed that the 52 amino acid region in the center of the D2 domain of Pfs47 contained the epitope leading to production of transmission-blocking anti-D2 antibodies, while the N-terminal region of D2 was immunodominant but did not lead to production of transmission-blocking antibodies.

[0118] The mice immunized with D2 Pfs47 polypeptide were used in two further independent hybridoma fusions. Fourteen additional mAbs recognizing D2 domain of Pfs47 were obtained. Three hybridoma lines named 1B2-B7, BF1M2-H7 and JH11-H4 were stable, the corresponding mAbs IB2, BM2 and JHl l were re-tested to measure TBA, and selected for further epitope mapping. mAbs IB2 and BM2 exhibited strong TBA, which resulted in 99% and 81% reduction in mosquito infection, respectively; whereas mAb JHl l consistently enhanced infection, doubling the number of oocyst. 4B7 mAb was used as a positive control. The results of the TB A testing are illustrated in Figure 21 and Table 8 below. ELISA conducted with the three mABS and three deletion polypeptides (Del2, Del 3 and Del 4) revealed that JHl l mAb bound at the N-terminus of D2 domain of Pfs47 (E= enhanced transmission region), while IB2 and BM2 mAbs bound in the central regions (TB = transmission blocking region) upstream of the modified cysteine loop (LOOP; the asterisks represent the modified cysteines).

Summary of anti- Pfs47 D2 polypeptide mAb testing

B,

mlgG IB2 BM2 n (midguts) 34 38 23

Prevalence (%) 74 95 26

Inhibition (%) 0 82 [0119] To confirm the above conclusion, the immunoreactivity of IgG purified from mice immunized with the Pfs47-D2-118 polypeptide to different regions of the D2 domain was evaluated using ELISA, As expected, the antibodies recognized the full length D2 polypeptide (Pfs47~D2~l 18) with which the animals were immunized. The results of ELISA testing of anti-Del 2 and anti-Del 3 IgG are illustrated in Figure 23. Hie recognition of the Del 2 peptide containing the non-protective N-terminal domain, was stronger than the recognition of Del 3 peptide lacking the N-terminal region present in the Del 2 peptide since the first boost. Furthermore, the recognition of Del 2 polypeptide became stronger with subsequent immunizations, while the recognition of Del 3 peptide decreased. These data showed that the immunodominance of the non-protective N-terminal region of the D2 domain was exacerbated with each boost immunization.

[0120] When mice were immunized with Del 3 polypeptide lacking the N-terminal region, the serum of the mice exhibited significant TBA after the first boost, as shown in Figure 24. The assays conducted after the third boost immunization (illustrated in Figure 25; data summarized in Table 9) showed that Del 3-induced TBA did not decrease with successive immunizations, but actually increased from 65% to 85%.

Table 9. Summary of anti- Pfs47 D2 polypeptide polyclonal serum testing

[0121] Mice were immunized with Pfs47 Del 3 and Pfs47 Del 4 polypeptides and the resulting serum was TBA-tested. The results of the testing are illustrated in Figure 26 and summarized in Tables 10. These experiments demonstrated that the removal of the N- terminal region from Pfs47preserved and enhanced the TBA with subsequent immunizations. Table 10. Summaiy of anti- Pfs47 Del 3 polypeptide polyclonal serum testing

Summasy of anti- Pfs47 Del 3 polypeptide polyclonal serum testin

B. Summaiy of anti- Pfs47 Del 4 polypeptide polyclonal serum testing

EXAMPLE 7

Pfs47 immunogenic polypeptides for induction of transmission-blocking immune response [0122] The alignment of Pfs47 D2 central region sequences from 364 Plasmodium falciparum isolates from around the world (Africa = 232, America=36, Asia=73 and Papua New Guinea=23) showed only 7 variants of these sequences, 6 of which differing by a single amino acid from SEQ ID NO:31. One variant from Papua New Guinea (Hap 7) has two amino acid differences. The alignment of different variants is shown in Figure 24, with the amino acid differences highlighted. This degree of conservation supports the use of SEQ ID NO:31 for induction of the immune response in humans in order to block transmission of a variety of Plasmodium falciparum parasites in Anopheles mosquitoes. A fusion polypeptide containing several variants of Pfs47 D2 central region sequences, as illustrated by SEQ ID N():39, can be used for induction of an immune response blocking transmission of a variety of Plasmodium parasites in Anopheles mosquitoes.

EXAMPLE 8

Plasmodium vivax Pvs47 protein and Pvs47 immunogenic polypeptides for induction of transmission-blocking immune response

[0123] D2 polypeptide of Plasmodium vivax Pvs47 protein is recombinantly expressed in E. coli, purified and used as an immunogen to produce polyclonal antibodies and niAbs. The antibodies are tested and exhibit robust and consistent TBA. Hie sequences of Pvs47 D2 recombinant proteins used for antibody production are shown in Figure 11. In the recombinant protein, two cysteines in D2 domain are substituted by two alanines, a starting methionine is added at the N-terminus and a histidine tag is added at the C-terminus to facilitate the purification of the recombinant protein. Hie immunogenic region leading to production of transmission-blocking antibodies is mapped to the central portion of Pvs47 D2. When mice are immunized with Pvs47 D2 polypeptides lacking the N-terminal region of D2 (amino acid sequences are illustrated in Figure 25 - SEQ ID NOs 40 and 41, the polypeptides used for immunization can contain an N-terminal methionine and/or a C -terminal polyhistidine tag), the serum of the mice exhibits significant TBA after the first boost. The assays conducted after the second and the third immunizations show that TBA does not decrease with successive immunizations.

[0124] The alignment of Pvs47 D2 central region sequences from 141 Plasmodium vivax isolates from around the world (Africa = 1, America=114, Asia=24 and Papua New Guinea=2) showed full conservation of these region, as illustrated in Figure 24. The sequence conservation supports the use of SEQ ID NO:41 for induction of an immune response in humans to block transmission of a variety of Plasmodium vivax parasites in Anopheles mosquitoes. Fusion polypeptides containing Pfs47 and Pvs47 D2 central region sequences, as illustrated by SEQ ID NOs 49 and 50 , can be used for induction of an immune response of blocking transmission of a variety of P. vivax and P. falciparum in Anopheles mosquitoes.

EXAMPLE 9

Investigation of the transmission blocking activity of anti-Pfs47 Del 3 polyclonal sera

[0125] A set of experiments was conducted to investigate whether human complement or the mosquito complement-like system were required for the TBA observed for anti-Pfs47 Del 3 polyclonal sera. The experimental results are illustrated in Figures 29 A-E and briefly described in the corresponding description of the figures elsewhere in the present document. No significant difference in TBA in the presence (+ H. Comp.) or absence (- H. Comp) of human complement was observed in either A. gambiae (see Figure 29A and Table 11, Al and All) or A. stephensi (see Figure 29B and Table 11, BI and ΒΠ) mosquitoes. Disruption of the mosquito immune system by silencing the thioester-containing protein 1 (ΤΈΡΙ), a key effector of the complement-like system, also had no effect on the level of TBA observed. See Figure 29C and Table 11C. It is known that, in P. berghei, P47 is required for female fertility under in vitro culture conditions, and disruption of the gene also significantly impairs fertilization in vivo. Moreover, Pfs47 is localized on the surface of gametocytes and gametes, as well as of zygotes and ookinetes. It was investigated whether anti-Pfs47 Del 3 polyclonal sera disrupted Plasmodium fertilization and reduced ookinete formation. It was found that, indeed, anti-Pfs47 Del 3 polyclonal sera dramatically reduced the number of ookinetes present in the midgut lumen 24 hours post-feeding (P<0.0001, Mann- Whitney). See Figure 29 D and Table 11D. Furthermore, the expression level of four ookinete-specific genes (CHTi = ^: chitinase 1, PIMMS2 = Plasmodium invasion of mosquito midgut screen candidate 2, SOAP= secreted ookinete adhesive protein and WARP= von Willebrand factor A domain- related protein) was also greatly reduced (P<0.0001 , ANOVA). See Figure 29E.

Table 11. Summary of anti- Pfs47 Del 3 polypeptide polyclonal serum testing

AI: A. gambinae, + H. Comp.

BI: A. stephensi, + H. Comp.

BII: A. Stephens L -H. Comp.

c.

D.

[0126] All patents and non-patent publications and other information cited above are incorporated herein by reference in their entirety. Various embodiments of the invention have been described in fulfillment of the various objectives of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention as defined in the following claims.