WO2014028852A1 - Use of p47 from plasmodium falciparum (pfs47) or plasmodium vivax (pvs47) as a vaccine or drug screening targets for the inhibition of human malaria transmission - Google Patents
Use of p47 from plasmodium falciparum (pfs47) or plasmodium vivax (pvs47) as a vaccine or drug screening targets for the inhibition of human malaria transmission Download PDFInfo
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- WO2014028852A1 WO2014028852A1 PCT/US2013/055372 US2013055372W WO2014028852A1 WO 2014028852 A1 WO2014028852 A1 WO 2014028852A1 US 2013055372 W US2013055372 W US 2013055372W WO 2014028852 A1 WO2014028852 A1 WO 2014028852A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/002—Protozoa antigens
- A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
- C07K16/205—Plasmodium
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- PLASMODIUM FALCIPARUM PFS47
- PLASMODIUM VIVAX PVS47
- the present invention comprises methods and compositions for delivering a
- Plasmodium P47 vaccine or an antibody to P47 to prevent Plasmodium falciparum or Plasmodium vivax malaria.
- the mosquito Anopheles gambiae is the main vector of Plasmodium falciparum malaria in large areas of sub-Saharan Africa. Mosquitoes become infected when they ingest blood from an infected human, and the parasites need to undergo a complex developmental cycle in the mosquito to be transmitted to another person. Work from animal models indicates that mosquitoes can defend themselves by mounting immune responses that can greatly limit Plasmodium survival. Thus, it is not clear why disease transmission is so effective in highly endemic areas. In all regions of the world where malaria has been eliminated, this has been achieved by controlling the mosquito vector populations, and reducing the rate of disease transmission is considered one of the key steps to eliminate malaria from endemic areas.
- Pfs230 is identified as the largest protein of a Plasmodium 10-member family characterized by cysteine-rich double domains with 1-3 di-S bridges in each half. Pfs230, Pfs 48/45, Pfs47 and others are mentioned as potential transmission-blocking vaccines due to their surface location on gametes.
- Plasmodium parasites evade the mosquito immune system. It has been reported that mosquitoes refractory to infection by some lines of Plasmodium falciparum can melanize other lines. Specifically, Anopheles gambiae L3-5 refractory line melanizes the Brazilian Plasmodium falciparum 7G8 line, but not the African Plasmodium falciparum 3D7, NF54 and GB4 strains. Investigation of this difference in parasite ability to infect the same refractory line suggested a role for Thioester containing protein 1 (TEP1) in the mosquito's capacity to inhibit Plasmodium transmission (Molina-Cruz (2012) PNAS 109(28): E1957- E1962).
- TEP1 Thioester containing protein 1
- Plasmodium falciparum has several strains that have been isolated including those found in Africa, America and Asia. Each of these strains expresses a slightly different version of the Pfs47 protein.
- the subject invention comprises the recognition that Pfs47 allows the parasite to suppress or evade the immune system, thereby ensuring the parasite's survival.
- the evolution of Pfs47 provides a very powerful mechanism by which the effective transmittal of Plasmodium falciparum is permitted in the field.
- the subject invention comprises the recognition that Pfs47 is, in fact, critical to the transmission of P. falciparum in the field.
- the novel use of Pfs47 and related compounds as transmission-blocking targets involves active participation of the mosquito immune system. Specifically, contact of a vaccine comprising Pfs47 or an antibody thereto, or a pharmaceutical agent that inhibits Pfs47, with the mosquito can prevent Pfs47 from interacting with and manipulating the mosquito immune system. By inhibiting or inactivating Pfs47 via such vaccine or agent, the mosquito immune system is able to "see" the parasite and destroy or substantially reduce it.
- the subject invention further comprises the recognition that P47 proteins, their antibodies and pharmaceutical agents found to be inhibitory to P47, can be effective as vaccines or transmission-blocking agents of malaria transmission.
- P47 proteins include, without limitation, Pfs47 produced by Plasmodium falciparum, and Pvs47 produced by Plasmodium vivax.
- one embodiment of the subject invention comprises vaccines and their administration, wherein the vaccines are pharmaceutical compositions comprising P47 protein (or immunological fragments or variants thereof); or pharmaceutical compositions comprising antibodies (or fragments thereof) to P47 protein (or its immunological fragments or variants).
- the vaccines are pharmaceutical compositions comprising P47 protein (or immunological fragments or variants thereof); or pharmaceutical compositions comprising antibodies (or fragments thereof) to P47 protein (or its immunological fragments or variants).
- the critical role of P47 is exploited to develop new pharmaceutical agents that can be used to disrupt P47 function in the field.
- the assay for identification of new agents comprises the screening of candidate compounds in Plasmodium falciparum infected mosquitoes.
- the assay involves screening of candidate compounds in a cell in which the JNK signaling pathway has been inactivated to identify those agents that can restore JNK signaling.
- the invention includes transgenic and paratransgenic mosquitoes capable of expressing antibodies or other pharmaceutical agents that can disrupt P47 function.
- the gene encoding the antibody or other pharmaceutical agent has been inserted into the genome of bacteria resident in the gut microbiota and expressed and exported, whereby the agent can interact with the P47 in the mosquito gut or other organs.
- Figure 1 shows Survival of the parental and progeny Plasmodium falciparum lines in refractory (R) mosquitoes and quantitative trait locus (QTL) mapping of the melanization phenotype.
- R refractory
- QTL quantitative trait locus mapping of the melanization phenotype.
- A P. falciparum GB4 and 7G8 parasites in the midgut of R mosquitoes.
- B Melanization phenotype of the parental and progeny lines of the GB4 x 7G8 genetic cross in R mosquitoes.
- Figure 2 shows linkage group selection, mRNA expression, and coding region sequence analysis.
- A Genotype frequency of homozygous African (AA; horizontal line), Brazilian (BB; vertical line), or heterozygous (AB, diagonal dashed line) markers along Chr. 13 in individual oocysts dissected from S or R mosquitoes, (black arrow, region with BB under extreme negative selection in R strain).
- B Relative mRNA expression of candidate genes in GB4/7G8 Plasmodium falciparum ookinete stage. Magenta dots, genes with non- synonymous single nucleotide polymorphisms (SNPs) between GB4 and 7G8; Arrows, SNPs shared between GB4-3D7 and 7G8-SL strains.
- SNPs single nucleotide polymorphisms
- FIG 3 shows phenotype of NF54 wild type (WT) or Pfs47 knockout (KO) on different mosquitoes.
- A Number of melanized (x-axis) and live (y-axis) KO parasites in R and S mosquitoes. Each dot represents an individual midgut. Medians are indicated by the horizontal lines.
- B Effect of TEP-1 silencing on KO infection in R mosquitoes.
- C Effect of silencing TEP1 on WT and KO infection in S mosquitoes.
- Figure 4 shows Effect of complementing Pfs47 knockout (KO) parasites with the Brazilian (7G8) and African (NF54) alleles of Pfs47. Infectivity of Pfs47 KO parasites complemented with the (A) NF54 or (B) 7G8 Pfs47 alleles in the Anopheles gambiae R strain.
- FIG. 5 shows Graphic representation of the markers that defined the chromosome 13 quantitative trait locus (QTL) associated with the Plasmodium falciparum (Pf) melanization phenotype.
- QTL quantitative trait locus
- MS Microsatellite
- Figure 6 shows genotype along chromosome 13 of individual oocysts dissected from the midgut of susceptible (S) G3 or refractory (R) L3-5 Anopheles gambiae mosquitoes infected with the un-cloned progeny of the cross between the GB4 African (A allele) and the 7G8 Brazilian (B allele) Plasmodium falciparum strains.
- the genotype of individual oocysts for each marker is indicated by different shading patterns: homozygous African (AA, in diagonal line), Brazilian (BB, in diagonal dashed line), and heterozygous (AB, in grid pattern). Frequency of genotypes for each marker is indicated at the bottom of each table.
- the chromosomal location and primers for the 26 markers along chromosome 13 are shown in Table 2.
- the QTL boundaries are indicated by the arrows.
- Figure 7 shows genotype of 50 additional individual oocysts dissected from refractory L3-5 Anopheles gambiae mosquitoes infected with the un-cloned progeny of the cross between the GB4 African and 7G8 Brazilian Plasmodium falciparum strains.
- Genotyping was done in the region of chromosome 13 under strong genetic selection.
- the genotype of individual oocysts for each marker is indicated by different shading patterns: homozygous African (AA, in diagonal line), Brazilian (BB, in grid pattern), and heterozygous (AB, in diagonal dashed line).
- the chromosomal location and primers for the markers along chromosome 13 are shown in Table 2.
- the QTL boundaries are indicated by the arrows.
- Figure 8 shows phenotype of Pfs48/45 knockout (KO) NF54 Plasmodium falciparum parasites in the An. gambiae refractory (R) strain. Number of melanized (x-axis) and live (y-axis) Pfs48/45 KO parasites per midgut in R mosquitoes. Medians are indicated by black lines (— ).
- Figure 9 shows phenotype of Pfs47 knockout (KO) NF54 Plasmodium falciparum parasites in Anopheles stephensi (Nijmegen Sda500 strain) mosquitoes. Number of melanized (x-axis) and live (y-axis) Pfs47 KO parasites per midgut in refractory mosquitoes. Medians are indicated by black lines (— ). This infection was done with the same gametocyte culture as that shown in Fig. 3A (left panel) with Anopheles gambiae susceptible strain mosquitoes. The intensity of infection in An.
- stephensi (Nijmegen) was significantly higher (median of 60 oocysts/midgut) than that in An. gambiae susceptible females (median of 1 oocyst/midgut; P ⁇ 0.0001).
- Figure 10 illustrates the pCBM-BSD plasmid with the Pfs47 gene used for complementation of Pfs47 KO parasites.
- the short arrows indicate the primers used to test the presence of the plasmid by PCR in the Pfs47 KO complemented (BSD 3' and 0248_b_F).
- Pfs47 sequence including ORF and contiguous regions are indicated in thick diagonal line and thin diagonal line respectively.
- Figure 11 shows PCR-based confirmation of the Pfs47 KO genetic
- the PCR products using primers BVS01 and L430 confirmed the Pfs47 KO background and the PCR products with primers BSD 3' and 0248_b_F confirmed the presence of the pCBM-BSD plasmid containing the corresponding Pfs47 alelles, 7G8 or NF54 (3D7 clone).
- the PCR reactions using NF54 (Wt) and Pfs47 KO lines genomic DNA templates and the no-template-control (NTC) were included as controls.
- Figure 12 shows confirmation of the Pfs47 KO background and the Pfs47 mRNA expression upon genetic complementation.
- Relative mRNA expression of Pfs47 was assessed by qPCR in stage IV- V gametocyte cultures. Detection of Pfs47 mRNA in the complemented lines confirms gene expression upon complementation of the Pfs47 KO line.
- Figure 13 shows confirmation of the Pfs47 KO background and the Pfs47 protein expression upon genetic complementation.
- Figure 14 shows effect of removing the complementation selection
- P47 protein refers generically to those proteins produced by the Plasmodium genus of parasites that can or may enable the parasites to manipulate, inhibit or other avoid the immunological repression by mosquitoes. It includes, without limitation, Pfs47 (SEQ ID NO: l), produced by Anopheles gambiae, and Pvs47 (SEQ ID NO:2), produced by Plasmodium vivax.
- the "Plasmodium” genus of parasites include, without limitation, Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium knowlesi (P.
- Plasmodium parasites include the Anopheles genus, which includes, without limitation, the species Anopheles gambiae (A. gambiae), Anopheles albimanus (A. albimanus), Anopheles darling (A. darlingi), Anopheles aquasalis (A. aquasalis), Anopheles freeborni (A. freeborni), Anopheles quadrimaculatus (A. quadrimaculatus) and Anopheles stephensi (A. stephensi).
- Anopheles gambiae A. gambiae
- Anopheles albimanus A. albimanus
- Anopheles darling A. darlingi
- Anopheles aquasalis A. aquasalis
- Anopheles freeborni A. freeborni
- Anopheles quadrimaculatus A. quadrimaculatus
- Anopheles stephensi A. stephensi
- An “epitope” is generally defined as a linear array of 3-10 amino acids aligned along the surface of a protein.
- a conformational epitope has residues that are not joined sequentially, but lie linearly along the surface due to the conformation (folding) of the protein. In either case, the epitope is immunoreactive.
- Immunoreactive means that the epitope or antigen in question will react specifically with antibodies of interest and, preferably, anti-P47 antibodies present, for example, in a biological sample from an individual having malaria.
- immunogenic means the ability of a substance to cause a cellular and/or humoral response. More specifically, immunogenic refers to the ability of a polypeptide to generate antibody that blocks malaria transmission.
- the substance may be linked to a carrier and may be admixed with an adjuvant.
- a "vaccine,” as used herein, means an immunogenic composition capable of eliciting partial or complete protection against malaria.
- a vaccine can be prophylactic for infection and/or therapeutic in an infected individual.
- a "variant" of an original polypeptide is one which has at least about 80% identity to the sequence of the original polypeptide or an immunogenic fragment of the original polypeptide, and which substantially retains the desired effect on the intended target of the original polypeptide (i.e., elicits an immunogenic response).
- the variant has at least about 85%, 90%, 95%, 97% or 99% identity to the sequence of the original polypeptide or an immunogenic fragment thereof.
- Antibody means a protein or immunoglobulin (Ig) produced by B cells of the humoral immune system in the body in response to the presence of an antigen.
- An antibody can also refer to polyclonal and monoclonal antibodies or to any active form of the antibody, including Fab and F(ab') 2 fragments and chimeric antibodies.
- Monoclonal antibodies can be obtained by methods known in the art (Kohler & Milstein (1975) Nature 256:495-497). Methods for production of antibodies in a variety of expression systems (plants, animals, and insects) are known in the art. Where an antibody is to be administered to a recipient species, it is preferred that they be compatible, so that the antibodies are not cleared before the parasite can be controlled. It is also preferred that the administered antibodies do not cause "serum sickness" in the individual.
- a "fragment" of an antibody refers to an antibody polypeptide fragment, e.g.,
- Fab and F(ab') 2 fragments capable of binding to the intended target and executing the desired effect, e.g., inhibition of Pfs47 function.
- Bio sample means a fluid or tissue of an individual that commonly contains antibodies produced by the individual, more particularly antibodies against malaria.
- the tissue or fluid can also contain P. falciparum antigen.
- Biological samples include, without limitation, blood, plasma, serum, white blood cells, myelomas, tears, saliva, milk, urine, spinal fluid, lymph fluid, respiratory secretions, and genitourinary or intestinal tract secretions.
- substantially reducing or “substantially blocking” are interchangeable, and can be used in reference to destruction, killing, or reduction in transmission of the Plasmodium parasite, e.g., P. falciparum or P. vivax, in mosquitoes, such as A. gambiae.
- the reduction in transmission is at least 10%, and, with increasing degrees of preference, is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and is most preferably 100%.
- Reduction in transmission of the parasite in mosquitoes can be determined by methods known in the art including without limitation, the Standard Membrane Feeding Assay (SMFA).
- the SMFA is a functional assay that measures the ability of antibodies to block transmission of parasites to mosquitoes (see
- Solid phase refers to a solid body to which the P. falciparum antigen or other compound or complex of interest is bound by covalent or non-covalent means such as by van der Waals, hydrophobic or ionic interaction.
- purified with respect to polypeptides (proteins) or polynucleotides means a composition in which the molecule of interest is, with increasing preference, at least 40% of the total matter in the composition, at least 50% of the total matter, at least 60% of the total matter, at least 70% of the total matter, at least 80% of the total matter, at least 90% of the total matter, or at least 95% of the total matter.
- An "essentially purified" polypeptide (protein) or polynucleotide is a polypeptide or polynucleotide that is substantially free from cellular matter that is not of interest and has been purified to homogeneity.
- an essentially purified molecule is at least 80% pure, at least 90% pure, at least 95% pure, at least 97% pure, at least 98% pure, at least 99% pure, or most preferably is 100% pure.
- polypeptide means a polymer of amino acids of unspecified length and can include proteins. It can include modified and unmodified polypeptides.
- a "recombinant polynucleotide or nucleic acid” refers to a polynucleotide or nucleic acid that is the result of splicing of two or more different sources, such as the splicing of genes from different organisms or of a gene with non-natural nucleic acid.
- a "vector,” as used in the context of cellular transfection or transformation, is an autonomous polynucleotide replication unit within a cell that comprises sequences for expression of a desired polynucleotide.
- the term "effective amount" for prophylactic or therapeutic treatment refers to an amount of epitope-bearing polypeptide (e.g., of Pfs47) sufficient to elicit an immunogenic response in the individual or an amount of antibody fragment sufficient to bind to and execute the desired effect on the intended target (e.g., Pfs47). It is believed that the effective amount(s) can be found within a relatively large, non-critical range. Routine experimentation can be used to determine appropriate effective amounts.
- a "pharmaceutically acceptable carrier” is a carrier of an antigen that does not itself induce production of antibodies harmful to the recipient individual.
- Carriers are slowly metabolized macromolecules including, without limitation, inactive virus particles, proteins, polysaccharides, polyglycolic acids, amino acid copolymers, and like carriers well known in the art.
- adjuvants are used to enhance efficacy of the composition and include, but are not limited to, aluminum hydroxide (alum), montanide, N-acetyl-normuramyl-L-alanyl- D-isoglutamine (the-MDP), N-acetyl-muramyl-L-threonyl-D-isoglutamine (nor-MDP), and the like adjuvants known in the art.
- “Pharmaceutically acceptable vehicle” refers to the water, saline, glycerol, ethanol, etc. used for dissolution, suspension, or mixing of components in the pharmaceutical composition.
- the term "manipulation" of the mosquito immune system by Pfs47 can mean, without wishing to be bound by theory, the suppression, evasion, or other avoidance of the mosquito's normal immune system mechanism that usually enables destruction or substantial reduction of the parasite.
- the present invention contemplates a pharmaceutical composition comprising
- P47 protein an immunogenic fragment thereof, a variant of either the protein or the immunogenic fragment, or a mixture thereof.
- the full length P. falciparum surface protein P47 (Pfs47) and P. vivax surface protein P47 (Pvs47) protein used in the present invention is set forth in SEQ ID NO: 1 and SEQ ID NO:2, respectively.
- the immunogenic fragment necessarily must not be missing any sequence essential to the formation or retention of an epitope.
- the variant sequence must retain sequences necessary for the desired function or effect on the intended target.
- the P47, the immunogenic fragment, or the variant can include other sequences that do not block or prevent the formation of the epitope of interest or other functional sequence.
- the pharmaceutical composition can additionally include other human pathogen antigens that are useful in eliciting an immune response, including influenza, measles, mumps, diphtheria, tetanus, pertussis, poliovirus, hepatitis B virus, varicella, N. meningitides, and rubella.
- human pathogen antigens that are useful in eliciting an immune response, including influenza, measles, mumps, diphtheria, tetanus, pertussis, poliovirus, hepatitis B virus, varicella, N. meningitides, and rubella.
- the "immunogenic fragment” has a length of at least five amino acids. With increasing preference, the length of the immunogenic fragment is at least 7, 9, 11, 13, 15, 17 and 19 amino acids. The fragment can generate antibodies that block malaria transmission.
- a "variant" of P47 or its immunogenic fragment is a polypeptide having at least about 80% identity to either P47 protein (Pfs47 or Pvs47) or an immunogenic fragment thereof, and which retains the function of being immunogenic.
- the variant has at least 85%, 90%, 95%, 98%, or 99% identity to the P47 protein or an immunogenic fragment thereof.
- the invention also comprises a recombinant polynucleotide comprising a nucleotide sequence encoding an immunogenic fragment of the P47 protein, a variant of the protein or the immunogenic fragment.
- the P47 fragment or variant can be, e.g., Pfs47 or Pvs47.
- the immunogenic fragment is at least about five amino acids long.
- the variant encoded by the nucleotide sequence has at least about 80% identity to the immunogenic fragment.
- the invention encompasses a vector comprising the polynucleotide that comprises a nucleotide sequence encoding a P47 immunogenic fragment or a variant of the P47 immunogenic fragment.
- Appropriate vectors including plasmid and viral vectors are known in the art.
- the vector can further be used to transfect a host cell using methods known in the art.
- An expression vector, such as the VR1020 plasmid vector can be used to immunize animals or to express P47 in vertebrate cells, such as human kidney cells and obtain recombinant protein.
- the P47 protein, its immunogenic fragment, or variant can be made by any method that provides the desired epitope or functional sequence. A preferred method is recombinant expression in E. coli to provide non-glycosylated antigens in native
- P47 can be expressed using the baculovirus system and glycosylation can be chemically removed from the recombinant protein.
- the P47 protein, its immunogenic fragment, or its variant can be modified as appropriate to enhance properties such as in vitro stability and the like, or in vivo properties including its pharmacokinetics.
- compositions of the subject invention can be prepared by known methods of combination of compounds in admixture with a pharmaceutically acceptable carrier. Suitable carriers and their formulation with proteins are described in Remington's Pharmaceutical Sciences (16 th ed. Osol, E. ed., Mack Easton Pa. (1980)).
- the subject invention also comprises a method of substantially inhibiting or substantially reducing Plasmodium parasite transmission by administration to vertebrates of a pharmaceutical composition comprising P47 protein, its immunogenic fragment, or a variant thereof.
- the composition can also include a pharmaceutically acceptable carrier, an adjuvant, and/or a pharmaceutically acceptable vehicle.
- the pharmaceutical composition comprising P47, its immunogenic fragment, or its variant can be used as a vaccine to block Plasmodium transmission by administration to humans and higher primates.
- the pharmaceutical composition can additionally include other human pathogen antigens that are useful in eliciting an immune response, including influenza, measles, mumps, diphtheria, tetanus, pertussis, poliovirus, hepatitis B virus, varicella, N. meningitides, and rubella.
- Methods of administration of a pharmaceutical composition of the subject invention to a human or higher primate can be carried out by any suitable means, including intravenously, intramuscularly, intranasally, and subcutaneously.
- the subject invention also encompasses a pharmaceutical composition for substantially blocking or substantially reducing transmission of a Plasmodium parasite in vertebrates, wherein the composition comprises an antibody or a fragment thereof, which is specifically reactive to P47 or an immunogenic fragment or variant thereof, and a
- the antibody can be a monoclonal antibody or a polyclonal antibody.
- the parasite can be, e.g., P. falciparum or P. vivax.
- the invention further comprises a method of substantially blocking or substantially reducing transmission of a Plasmodium parasite in a population of humans or higher primates comprising administering, to at least one human or higher primate, a pharmaceutical composition comprising antibodies or fragments thereof, which are specifically reactive to P47, or immunogenic fragments or variants thereof.
- the composition can further include a pharmaceutically acceptable carrier.
- composition used in the foregoing method can further include one or more antigens of a human pathogen, including, without limitation, influenza, measles, mumps, diphtheria, tetanus, pertussis, poliovirus, hepatitis B virus, varicella, N. meningitides, and rubella.
- the composition can further include an adjuvant.
- the method of administering the pharmaceutical composition comprising the antibody or related compounds includes methods known in the art, including, without limitation, intravenous, intramuscular, intranasal, and subcutaneous.
- the invention comprises a method of providing one or more antibodies or fragments or variants thereof, specifically reactive to one or more antigens, to a mosquito, comprising administering to a human a composition comprising the one or more antigens.
- the antigen can be P47 protein or an immunogenic fragment or variant thereof.
- the P47 can be, e.g., Pfs47 derived from P. falciparum or Pvs47 derived from P. vivax.
- the invention comprises a recombinant plasmid comprising a polynucleotide that encodes P47 protein (or an immunogenic fragment or variant thereof), or an antibody (or antibody fragment) specific to P47 (or its immunogenic fragment or variant).
- the translation of the polynucleotide can be under the control of a viral promoter such as the Rous Sarcoma Virus or Cytomegalovirus.
- the plasmid can also include a polyadenylation signal such as the bovine growth hormone or rabbit beta-globulin polyadenylation sequences.
- the plasmid can be used in a method of treating mammals comprising administering a pharmaceutically effective amount of the recombinant plasmid to an individual in need thereof.
- the invention further comprises a method for identifying pharmaceutical agents or drugs that can interfere with the function of P47.
- this method involves producing an infected mosquito population by contacting or feeding the mosquito population with a blood meal comprising Plasmodium gametocytes using, e.g., a MFA.
- the candidate pharmaceutical agent can be added to the gametocyte culture to determine whether the candidate molecule affects the capacity of the mosquitoes to substantially reduce the Plasmodium parasite transmission.
- Those candidate molecules determined to improve the mosquitoes' capacity to reduce the transmission of Plasmodium are identified as
- the Plasmodium parasites can be provided in P. falciparum gametocyte culture or as blood from an infected donor. Further, contacting of P. falciparum with mosquitoes can be managed in a MFA. The candidate molecules can also be contacted with the infected mosquito population using the MFA.
- the invention comprises further methods for identifying pharmaceutical agents that can be useful as vaccines.
- the pharmaceutical agents can restore JNK signaling in a system in which inactivation of the JNK signaling pathway has been established.
- the system is contacted with a candidate agent, and the effect of the candidate agent on the restoration of JNK signaling is determined.
- the system comprises Drosophila S2 cells or other insect cell lines in which the intact JNK signaling pathway has been inactivated by exposing the cells to P47.
- the process by which the Drosophila S2 cells or other insect cells can have their intact JNK signaling pathway inactivated may be by, e.g.: a) exposing the surface of said cells to P47 by adding
- Drosophila S2 or other insect cell line is accomplished by measuring JNK phosphorylation or by using a reporter gene, such as green fluorescent protein (GFP).
- a reporter gene such as green fluorescent protein (GFP).
- the method for identification of pharmaceutical agents that can be useful as vaccines uses, as an assay system, mosquitoes that have been infected with Plasmodium parasites that express P47, resulting in the JNK signaling pathway of the mosquitoes being inhibited. This, in turn, results in a lack of activation of the mosquito complement-like system.
- a candidate agent that restores JNK signaling can be evident by the mosquitoes' capacity to substantially block parasite infection.
- the mosquitoes are A. gambiae or other anopheline mosquito
- the Plasmodium parasites are P. falciparum or P. vivax.
- the subject invention also comprises a transgenic mosquito that comprises cells that express an inhibitory factor that interferes with the function of P47.
- This inhibitory factor can be an exogenous polynucleotide sequence that encodes an antibody specifically directed to P47 and that interferes with the immuno-suppressive activity of P47.
- Antibodies, polyclonal or monoclonal, can be obtained using methods known in the art.
- the transgenic mosquito is paratransgenic, and bacteria native to the mosquito gut microbiota are transfected with a polynucleotide encoding the inhibitory factor.
- the symbiont bacteria of the gut microbiota can be readily transfected with, e.g., plasmids containing the exogenous polynucleotide, are grown easily in vitro, and can export the exogenous polypeptide.
- the engineered bacteria remain stable and are easily delivered to the gut of the mosquito, where they continue to export exogenous polypeptide.
- the exogenous polypeptide cannot be toxic to the symbiont bacteria or the mosquitoes. By this mechanism, the paratransgenic mosquitoes can disrupt the inhibitory function of
- Plasmodium Pfs47 permitting the mosquitoes' immune system to recognize the Plasmodium parasites and substantially reduce their transmission.
- suitable symbionts for mosquitoes include acetic acid bacteria (AAB), especially members of Acetobacter and Gluconacetobacter genera, and Pantoea agglomerans (P. agglomerans).
- AAB acetic acid bacteria
- P. agglomerans Pantoea agglomerans
- AAB bacterium Asaia spp. has been found to be "a dominant bacterium within the insect microbial community," including Anopheles (An.) stephensi, An. maculipennis, An. gambiae, and Aedes aegypti.
- the predominant habitat of the AAB in mosquitoes has been found to be the gastrointestinal tract, which ensures access to diet-derived sugars.
- the gastrointestinal tract is acidic, aerobic, and provides a sugar diet, thereby permitting growth and reproduction of the AAB.
- the AAB are spread naturally through the host mosquito population by vertical and horizontal transmission routes.
- the inhibitory factor employed in the paratransgenic mosquito can be an exogenous polynucleotide sequence that can, for example, encode an antibody specifically directed to P47 that interferes with the immuno-suppressive activity of P47.
- the antibody to P47, polyclonal or monoclonal can be obtained by methods known in the art.
- the invention includes a method of detection or determination of malaria antibodies in a biological sample.
- the method uses P47 (or its immunogenic fragment or variant), preferably purified or essentially purified, and is typically performed in vitro.
- the detection or determination of malaria antibodies in the biological sample of an individual can be for purposes of diagnosis, or for monitoring of response to malaria treatment and prognosis for patients previously diagnosed.
- the method includes the steps of providing or obtaining a biological sample from an individual who may have malaria and antibodies to P47; contacting the sample with a P47 protein or an immunogenic fragment under conditions which allow the formation of an immune complex; and detecting the presence of the complexes of P47 protein or fragment and the antibody.
- the assay methods use conditions that allow the P47 (or its fragment) to bind to antibody in the biological sample. These conditions include physiologic pH, temperature, and ionic strength with an excess of P47 (or fragment), followed by incubation.
- the contacting step of the method can be carried out in solution.
- the P47 or its fragment can be linked to a detectable label.
- the label can be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Labeled complex can be detected by known methods including fluorimetry, chemiluminescence, radiometry, or colorimetry. In another aspect, during incubation, the complex of antibody and antigen may immunoprecipitate .
- the contacting step of the method can be carried out with the
- the P47 immobilized to a solid support.
- the P47 may be labeled, e.g., with a fluorescent label.
- solid supports include, without limitation, nitrocellulose (in membrane or microtiter well form), polyvinylidine fluoride, polyvinyl chloride, polystyrene latex, activated beads, and the like.
- This method may further comprise a step of removing unbound components after complex formation on the support.
- the detection of antibody complexed to the P47 can be accomplished using methods known in the art, including, without limitation, fluorimetry.
- the invention further comprises a kit for detecting or determining the presence of malaria antibodies in a biological sample.
- the kit includes a P47 protein, its immunogenic fragment, or a variant thereof; a buffer for enabling immune complex formation between the P47 protein, fragment, or variant, and antibodies against malaria present in a biological sample.
- the P47, fragment, or variant is immobilized on a solid support (e.g., ELISA plate).
- a wash solution to remove uncomplexed components.
- Pfs47 is a key factor for survival of P. falciparum parasites in the mosquito A. gambiae, a major natural vector of human malaria in Africa. Pfs47 allows the parasite to infect the mosquito without activating the mosquito immune system.
- the genomic region responsible for the immune evasion by using classic QTL analysis with the progeny of a cross between Brazilian 7G8 X African GB4 parasites was mapped. The genomic location of the immune evasion gene was confirmed by linkage group selection analysis of individual oocysts from the recombinant population from the cross. These experiments defined a 171 kb region that codes for 41 genes.
- Pfs47 and Pfs48/45 are expressed in gametocyte stages of Plasmodium and have been investigated as candidates for transmission-blocking vaccines.
- Knockout (KO) lines for these two genes were generated by Drs. Sauerwein and Eling from the Radboud University Nijmegen Medical Center and collaborators in the African NF54 P. falciparum Strain, and their effect on Plasmodium infectivity to mosquitoes has been published (van Dijk et al., 2001; van Schaijk et al., 2006). It was confirmed that Pfs48/45 KO parasites infect mosquitoes at low levels due to deficient fertility.
- TEP1 -mediated killing of Pfs47 KO parasites was also observed in the A gambiae G3 strain, which is highly susceptible to infection with wild type NF54 P.
- Pfs47 is a key factor for survival of P. falciparum parasites in the mosquito A. gambiae, a major natural vector of human malaria in Africa.
- a combination of genetic mapping, linkage group selection, and functional genomics was used to identify Pfs47 as a P. falciparum gene that allows the parasite to infect A. gambiae without activating the mosquito immune system.
- Disruption of Pfs47 greatly reduced parasite survival in the mosquito and this phenotype could be reverted by genetic complementation of the parasite or by disruption of the mosquito complement-like system.
- Pfs47 suppresses midgut nitration responses that are critical to activate the complement-like system.
- A. gambiae L3-5 strain was selected to be refractory (R) to Plasmodium cynomolgi (simian malaria), but also eliminates most other Plasmodium species including P. falciparum strains from the New World, and forms a melanotic capsule ⁇ i.e., deposition of melanin, a black insoluble pigment) around the dead parasites.
- this strain is highly susceptible to infection with some African P. falciparum strains, such as NF54, 3D7, and GB4. Some parasite lines from malaria-endemic areas where A. gambiae is the natural vector are able to evade the mosquito immune system. Co-infection experiments reveal that the immune response (or lack thereof) to a P. falciparum strain did not affect the fate of other parasites present in the same mosquito midgut;
- Linkage group selection analysis a method that allows de novo location of loci encoding selectable phenotypes of malaria parasites, was used to obtain independent confirmation of the locus in Chrl3.
- the un-cloned recombinant progeny from the original genetic cross was used to generate gametocytes and infect either the R strain or a permissive susceptible (S) A. gambiae G3 line in which both parental parasite lines survive.
- Individual oocysts were isolated, subjected to whole genome DNA amplification, and genotyped for multiple markers along Chrl3.
- Oocysts derive from the diploid ookinete stage and can be homozygous for the African GB4 (AA) or Brazilian 7G8 alleles (BB), or heterozygous (AB).
- BB genotype is highly abundant in the central region of Chrl3, reaching a frequency of > 90% (Fig. 2A; Fig. 6, Table 2), which was already observed in the progeny clones from the genetic cross and is not due to selection by the mosquito, because both parental strains survive in the S strain.
- Fig. 2A Fig. 6, Table 2
- a well-defined region was identified, indicated by the dotted line, in which the BB genotype is under strong negative selection and is totally absent (0%) (Fig. 2A).
- Fig. 2B black arrows; Table 4
- the GB4 SNP alleles are shared with the NF54 and 3D7 strains that also survive, and the 7G8 SNP alleles with the SL strain that is melanized by the R strain.
- Five genes were selected as top candidates for detailed genetic analysis based on large differences in gene expression and/or on polymorphisms that correlates with survival in other strains (Table 5).
- Pfs47 and Pfs48/45 code for members of the 6- cysteine protein family that are expressed on the gametocyte surface. Previous gene disruption experiments in the NF54 line revealed that Pfs48/45 is critical for gamete fertility. Pfs47 is expressed in female gametocytes but is not essential for P. falciparum fertilization, although its homolog in Plasmodium berghei is required for female gamete fertility.
- the infection level in the S strain (median of 1 oocyst/midgut) is much lower than that in An. stephensi mosquitoes (60 oocysts/midgut median) (Fig. 9). Notably, this An. stephensi strain has been selected to be highly permissive to P. falciparum infection.
- A. gambiae complement- like system was disrupted by silencing TEP1. Reducing TEP1 expression completely reversed melanization of Pfs47 KO parasites in the R strain (Fig. 3B). In the A. gambiae S strain (G3), neither NF54 WT nor Pfs47 KO parasites were melanized (Fig. 3C); however, while TEP1 silencing had no significant effect on infection with NF54 WT parasites (Fig. 3C), it dramatically increased both the intensity (P ⁇ 0.0001 Mann- Whitney test) and prevalence of infection (P ⁇ 0.001; ⁇ test) of Pfs47 KO parasites (Fig. 3C). This indicates that Pfs47 is necessary for P. falciparum parasites to evade two well- characterized immune responses mediated by TEP1 in A. gambiae: killing followed by melanization in the R strain and parasite lysis without melanization in the S strain.
- Pfs47 protein is present on the surface of WT NF54 ookinetes, the stage that invades the midgut, but is absent in Pfs47 KO parasites (Fig. 3D).
- the expression of HPX2 and NOX5, two enzymes that mediate midgut nitration in response to P. berghei infection and promote TEP1 activation (2) was evaluated in S mosquitoes.
- HPX2 and NOX5 were not induced by NF54 WT parasites and nitration levels were lower than in uninfected controls (Fig. 3E).
- Pfs47 KO parasites induced expression of HPX2 and NOX5, and a robust nitration response, indicating that Pfs47 may prevent TEP1 -mediated lysis by suppressing midgut epithelial nitration responses (Fig. 3E).
- Pfs47 was identified as an essential survival factor for P. falciparum that allows the parasite to evade the immune system of A. gambiae, a major mosquito vector in Africa.
- other parasite genes may also be involved in this process.
- Pfs47 is a highly polymorphic gene with a marked population structure in field isolates and exhibits extreme fixation in non- African regions of the world.
- Our findings suggest that the population structure of Pfs47 may be due to adaptation of P. falciparum to the different Anopheles vector species present outside of Africa.
- Pfs47 evolved a function in P. falciparum that increases parasite survival in A. gambiae mosquitoes and may be responsible, at least in part, for the very high rates of malaria transmission in hyperendemic regions in Africa. Disruption of the immunomodulatory activity of Pfs47 may prove to be an effective strategy to reduce malaria transmission to humans.
- Plasmodium falciparum strains used— GB4, 7G8, GB4x7G8 cross progeny (cloned and un-cloned), NF54, NF54-Pfs47KO, complemented NF54-Pfs47KO and NF54-Pfs48/45KO— were maintained in 0 + human erythrocytes using RPMI 1640 medium supplemented with 25 mM HEPES, 50 mg/1 hypoxanthine, 25 mM NaHC0 3 , and 10% (v/v) heat-inactivated type 0 + human serum (Interstate Blood Bank, Inc., Memphis, TN) at 37°C and with a gas mixture of 5% 0 2 , 5% C0 2 , and balance N 2 .
- Example 2.2 Artificial infection of mosquitoes with P. falciparum and QTL analysis
- Microsatellite (MS) genotyping of P. falciparum clonal progeny lines was carried out by PCR amplifying MS markers using DNA from asexual P. falciparum cultures and primers described in Table SI. PCR products were run in an ABI 31000 DNA Sequencer (ABI, Fullerton, CA) to determine their size. Individual P. falciparum oocysts were dissected from infected midguts under the microscope using fine needles, placed in 9 ⁇ TE buffer, and frozen at -70°C until used.
- qPCRs were performed under standard conditions using 0.5 ⁇ of each primer with an initial denaturation step of 15 min at 95°C and then 45 cycles of 10 sec at 94°C, 20 sec at 50°C, and 30 sec at 60°C, with a final extension of 5 min at 60°C.
- Gene expression of A. gambiae Hpx2 and Nox5 was assessed in a similar way using SYBR green qPCR and the A.
- qPCRs were performed under standard conditions using 0.5 ⁇ of each primer with an initial denaturation step of 15 min at 95°C and then 45 cycles of 10 sec at 94°C, 20 sec at 55°C, and 30 sec at 72°C, with a final extension of 5 min at 72°C. Sequencing of candidate genes in chromosome 13 QTL region in both GB4 and 7G8 strains was done on extracted DNA.
- Example 2.5 - dsRNA-mediated mosquito gene knockdown [00104] Individual female An. gambiae mosquitoes were injected 1-2 day post emergence as previously described in Molina-Cruz et al. (2008) J Biol Chem 283, 3217. Briefly, mosquitoes were injected with 69 ⁇ of a 3- ⁇ / ⁇ 1 dsRNA solution 3-4 days before receiving a Plasmodium- fected blood meal. dsRNA TEP1 and LacZ were produced using the MEGAscript ® RNAi Kit (Ambion, Austin, TX) using DNA templates obtained by PCR using An. gambiae cDNA and the primers previously described with T7 polymerase promoter sites added in the 5 '-end.
- TEP1 gene silencing was assessed in whole sugar-fed mosquitoes by quantitative real-time PCR using primers TEPl-qF (5 -GTTTCTCACCGCGTTCGT-3 ') (SEQ ID NO: 225), TEPl-qR (5 -AACCAATCCAATGCCTTCTC-3 ' ) (SEQ ID NO:
- L-lysine (0.01 ) coated glass slides and stored at 4°C until use. Dry smears were fixed in 4% paraformaldehyde in PBS for lhr at room temperature (RT) in a humidified chamber. Smears were blocked with 5% BSA in PBS for 30 min RT and rinsed with 0.01% Tween 20 for 10 min RT. Incubation with prinary antibody (rat monoclonal anti-Pfs47 antibodies 47.1, 47.2 and 47.3 or mouse monoclonal 4B7 anti Pfs25, diluted 1:500 in 1%BSA in PBS) was done overnight at 4°C. The slides were rinsed twice with 0.01% Tween 20 and once with PBS for 10 min RT.
- prinary antibody rat monoclonal anti-Pfs47 antibodies 47.1, 47.2 and 47.3 or mouse monoclonal 4B7 anti Pfs25, diluted 1:500 in 1%BSA in PBS
- Nitration assays were performed as previously described in Oliveira et al.
- Nucleic Acids Res 29, 850 150 ⁇ of leukocyte cleared red blood cells (RBC) (Sepacell R-500II, Fenwall) were washed once with incomplete cytomix (120 mM KC1, 0.15mM CaCl, 2 mM EGTA, 5 mM MgC12, 10 mM K2HP04/KH2P04, 25mM HEPES, pH 7.6 adjusted with KOH) and resuspended in 400 ⁇ 1 cytomix.
- RBC leukocyte cleared red blood cells
- the plasmid (100 ⁇ g at a concentration of 1 ⁇ g/ ⁇ l in cytomix) was added to RBC's in a chilled electroporation cuvette (Bio-Rad, 0.2 cm electrode) under sterile conditions, Electroporation was done in a Bio-Rad Gene Pulser II at 310V and 975 ⁇ capacitance. Electroporated RBC's were washed three times with 12ml complete culture media and mixed with P. falciparum NF54-Pfs47KO schizonts purified by Percoll-Sorbitol gradient.
- Plasmids containing Pfs47 alleles from 3D7 (a clonal line obtained from NF54) or 7G8 and their presumed endogenous 5' promoter region (l,030bp upstream of the start ATG) and 3' UTR (162 bp downstream of stop codon) were prepared by amplifying Pfs47 from P.
- P/s47_inf_F 5'- AGCTGGAGCTCCACCGCGGTTTATAAAAACATTCCTAACACATT-3'(SEQ ID NO: 227) and Pfs47_M_R 5'-CGGGGGATCCACTAGTATTTACCTTACATTTATCTCCA- 3' (SEQ ID NO: 228) (the sequence directed to Pfs47 is indicated in bold characters, the rest of the sequence is complementary to the plasmid used for In-Fusion cloning).
- the PCR product included noncoding regions upstream (1 kb) and downstream (0.16 kb) of the Pfs47 open reading frame (ORF).
- the PCR product was cloned using the In-Fusion HD cloning kit (Clontech) into the previously developed pCBM-BSD plasmid linearized by restriction enzyme digestion with Sacl and Spell (New England Biolabs) (Fig. 10). Plasmid purification was done using Plasmid Mega kit (Qiagen) and an extra ethanol precipitation wash to resuspend the plasmid (1 ⁇ g/ ⁇ l) in cytomix. The Pfs47 KO background of the complemented lines was confirmed by PCR using primers BVS01 and L430 as previously described in Van Schaijk et al. (2006) Mol Biochem Parasitol 149, 216 (Fig. 11). The presence of the pCBM- BSD plasmid with the Pfs47 insert in the complemented lines was confirmed by PCR using a primer directed to the Pfs47 coding sequence (0248_b_F 5'-
- FIG. 13 Expression of Pfs47 protein in gametocytes from different P. falciparum lines was detected by western blot (Fig. 13). Gametocytes were isolated by saponin treatment. Briefly, a volume of 30 ml of Gametocyte culture (14 day-old) was centrifuged and the pellet incubated in 5 ml PBS containing 0.08% saponin for 10 min at room temperature (RT). The isolated parasites were centrifuged, rinsed twice with PBS and frozen at -70°C until used.
- the frozen pellet was resuspended in 100 ul water and 5 ul of it was mixed with NuPage LDS Sample Buffer, heated at 70°C for 10 min , fractionated in a 4-12% NuPage Bis Tris gel (Novex), and transferred to nitrocellulose using the iBlot® dry blotting system (Invitrogen).
- the blot was blocked with 5% milk in Tris buffered saline with Tween 20 (0.05M Tris, 0.138M NaCl, 0.0027M KC1, pH 8; 0.05% Tween 20) overnight at 4°C, followed by incubation with a pool of anti-P/s47 rat monoclonal antibodies 47.1, 47.2, 47.3 (lmg/ml) diluted 1:200 in the milk solution for 2hr at RT. Subsequently the blot was incubated for 1 hr at RT with anti-rat IgG Alkaline Phosphatese conjugate (1 mg/ml Promega) diluted 1: 10,000 in milk solution. Antibody staining was detected with Western Blue stabilized substrate (Promega).
- a polynucleotide segment encoding a Pfs47 fragment that is immunogenic can be transfected into E. coli to produce recombinant Pfs47 polypeptide in large quantities.
- the recombinant immunogenic polypeptide is then purified.
- the purified immunogenic polypeptide or a DNA vaccine plasmid encoding the Pfs47 protein is used to immunize rabbits or mice repeatedly in order to generate polyclonal anti- serum. Serum is collected over a period of 4-6 weeks, and the quality of the antibodies is monitored by indirect ELISA.
- the polyclonal antibodies can be purified away from other serum proteins, if desired, using Protein A affinity chromatography.
- a polynucleotide encoding a Pfs47 immunogenic fragment is used to generate large quantities of Pfs47 immunogenic fragment.
- the purified immunogenic polypeptide is used to immunize mice.
- the spleen is isolated, and B cells from the spleen are screened to select those that produce antibodies to the Pfs47 immunogenic fragment.
- the selected B cells are then fused with a mouse tumor (immortal) cell line to form hybridomas.
- Hybridomas are screened for antibody production against Pfs47 immunogenic fragment.
- the selected hybridomas are then allowed to multiply in culture to produce desired monoclonal antibodies.
- Example 3 can be admixed with adjuvant and a pharmaceutically acceptable carrier to produce a pharmaceutical composition suitable for use as a vaccine in humans using current Good Manufacturing Practices.
- An adjuvant can be added to enhance the immune response to the P47or its fragment.
- Suitable adjuvants can include a traditional adjuvant such as alum or newer adjuvants such as the Glaxo Smith Kline (GSK) ASOl adjuvant or an experimental adjuvant such as the GLA-SE adjuvant developed by Steve Reed and Colleagues in
- stabilizers that can increase shelf life can be added. Suitable stabilizers can include monosodium glutamate and 2- phenoxyethanol. Further, preservatives can be added so as to prevent contamination with bacteria and permit multidose vials. Suitable preservatives can include phenoxyethanol and formaldehyde.
- NADP Isocitrate dehydrogenase
- SNPs Single nucleotide polymorphisms between the GB4 and 7G8 Plasmodium falciparum strains in the coding regions of the 41 candidate genes linked to the melanotic phenotype in the Anopheles gambiae L3-5 refractory strain. Also shown are polymorphisms present in strains 3D7 which survives in the R mosquito, and Santa Lucia (SL) which is melanized in the R mosquito.
- Table 5 Top candidate genes in the chromosome 13 quantitative trait locus (QTL) based on differences in expression in the ookinete stage and single nucleotide polymorphism (SNP) analysis.
- QTL quantitative trait locus
- SNP single nucleotide polymorphism
- Nucleic acid binding protein (MAL13_P1.233) 27.7 0 0
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EP13830027.2A EP2885410A4 (en) | 2012-08-17 | 2013-08-16 | Use of p47 from plasmodium falciparum (pfs47) or plasmodium vivax (pvs47) as a vaccine or drug screening targets for the inhibition of human malaria transmission |
CN201380054025.3A CN104736710A (en) | 2012-08-17 | 2013-08-16 | Use of P47 from plasmodium falciparum (PFS47) or plasmodium vivax (PVS47) as a vaccine or drug screening targets for the inhibition of human malaria transmission |
US14/421,964 US20150203547A1 (en) | 2012-08-17 | 2013-08-16 | Use of p47 from plasmodium falciparum (pfs47) or plasmodium vivax (pvs47) as a vaccine or drug screening targets for the inhibition of human malaria transmission |
BR112015003184A BR112015003184A2 (en) | 2012-08-17 | 2013-08-16 | use of plasmodium falciparum p47 (pfs47) or plasmodium vivax (pvs47) as a vaccine or drug screening targets for inhibiting human malaria transmission |
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DATABASE GENBANK [online] 27 May 2010 (2010-05-27), XP055187795, accession no. NCBI Database accession no. XP_001350182.1 * |
MOLINA-CRUZ, ALVARO ET AL.: "Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes", PNAS, vol. 109, no. 28, 23 May 2012 (2012-05-23), pages E1957 - 1962, XP055187794 * |
See also references of EP2885410A4 * |
THOMAS, ANA M. ET AL.: "P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions", THE EMBO JOURNAL, vol. 20, no. 15, 2001, pages 3975 - 3983, XP055187787 * |
VAN DIJK, MELISSA R. ET AL.: "Three members of the 6-cys protein family of Plasmodium play a role in gamete fertility", PLOS PATHOGENS, vol. 6, no. 4, April 2010 (2010-04-01), XP055187782 * |
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