WO2010036293A1 - Malaria vaccine - Google Patents
Malaria vaccine Download PDFInfo
- Publication number
- WO2010036293A1 WO2010036293A1 PCT/US2009/004243 US2009004243W WO2010036293A1 WO 2010036293 A1 WO2010036293 A1 WO 2010036293A1 US 2009004243 W US2009004243 W US 2009004243W WO 2010036293 A1 WO2010036293 A1 WO 2010036293A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fertilization
- subject
- post
- antigens
- plasmodium
- Prior art date
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- 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
- Malaria is one of most dangerous infectious diseases in tropical and subtropical countries, afflicting about 300 million people.
- the pathogen of the disease is a protozoan parasite, Plasmodium sp. which is transmitted by Anopheles mosquitoes.
- Four species of malaria parasites can infect humans under natural conditions: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae.
- P. falciparum and P. vivax cause the most infections worldwide.
- P. falciparum is the agent of severe, potentially fatal malaria. Malaria caused by P. falciparum is responsible for nearly 1 million deaths annually. Based on recent estimates from the WHO, worldwide, there were an estimated 247 million malaria cases among 3.3 billion people at risk living in 109 countries [I].
- a transmission blocking vaccine [8] specifically targeting the sexual development of the parasite in the mosquito vector may elicit immunity which can effectively block transmission of the parasite from invertebrate mosquito vector to vertebrate host.
- Transmission of malaria depends upon the presence of infectious male and female gametocytes in the peripheral blood of infected persons and successful ingestion of these gametocytes by Anopheles mosquitoes. Soon after ingestion, exflagellation occurs within the mosquito midgut, and emergent male gametes fertilize female gametes, resulting in the formation of zygotes.
- the zygotes undergo post-fertilization transformation into motile ookinetes which traverse the midgut epithelium and develop into oocysts resulting in the production of infective sporozoites. Finally the sporozoites are released into the hemocoel, invade the salivary glands and are transmitted to vertebrate hosts during subsequent blood feeding [9].
- the targets of transmission blocking antibodies include pre-fertilization antigens (Pfs230 and Pfs48/45) expressed in the circulating gametocytes and post- fertilization antigens (Pfs25 and Pfs28) expressed during mosquito stage ookinete development [10].
- pre-fertilization antigens are also targets of the natural immune response and thus immunity induced by a vaccine based on any of these antigens will have the added benefit of natural boosting of immunity.
- Pfs25 and Pvs25 P. vivax homolog of Pfs25
- a vaccine targeting sexual stages of the parasite will not only reduce malaria transmission by female Anopheles mosquitoes, but also reduce the spread of parasites able to evade immunity elicited by vaccines targeting pre-erythrocytic and erythrocytic asexual stages.
- the present invention features immunogenic compositions based on pre-fertilization antigens expressed in the circulating gametocytes in the peripheral blood of infected persons.
- the present invention makes use of an approach that harmonizes codon usage frequency of the target gene with those of the expression host for heterologous expression of protein. Taking these concepts into account, an algorithm termed "codon harmonization" [19] was developed where synonymous codons from E. coli were selected that closely resemble the codon usage of a native pre-fertilization gene, for example the native Pfs48/45 gene, including regions coding 'link/end' segments of proteins in P. falciparum.
- Also featured in the invention are antibodies, and methods to prevent the transmission of malaria using the immunogenic compositions of the invention.
- the invention features a method of blocking transmission of Plasmodium falciparum or Plasmodium vivax in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization antigens, thereby blocking transmission of Plasmodium falciparum or Plasmodium vivax in the subject.
- the invention features a method of immunizing a subject against Plasmodium falciparum or Plasmodium vivax comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization antigens, thereby immunizing the subject against Plasmodium falciparum or Plasmodium vivax.
- the invention features a method for treating or preventing malaria in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization antigens, thereby preventing malaria in the subject.
- the invention features a method of blocking transmission of Plasmodium falciparum or Plasmodium vivax in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax post-fertilization antigens, thereby blocking transmission of Plasmodium falciparum or Plasmodium vivax in the subject.
- the invention features a method of immunizing a subject against Plasmodium falciparum or Plasmodium vivax comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax post- fertilization antigens, thereby immunizing the subject against Plasmodium falciparum or Plasmodium vivax.
- the invention features a method for treating or preventing malaria in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax post-fertilization antigens, thereby preventing malaria in the subject.
- the one or more pre- fertilization or post-fertilization antigens are selected from the group consisting of Pfs48/45, Pfs 230, Pfs25, Pvs48/45, Pvs 230 and Pvs25.
- the pre-fertilization antigen is Pfs48/45.
- the post-fertilization antigen is Pfs25.
- the one or more pre- fertilization antigens or post-fertilization antigens is derived from a codon harmonized gene.
- the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 1.
- the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 3.
- the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 5.
- the present invention features a method of blocking transmission of Plasmodium falciparum or Plasmodium vivax in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax surface antigens, thereby blocking transmission of Plasmodium falciparum or Plasmodium vivax in the subject.
- the invention features a method of immunizing a subject against Plasmodium falciparum or Plasmodium vivax comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax surface antigens, thereby immunizing the subject against Plasmodium falciparum or Plasmodium vivax.
- the invention features a method for treating or preventing malaria in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax surface antigens, thereby preventing malaria in the subject.
- the surface antigens are gametocyte or gamete surface antigens.
- the gametocyte or gamete surface antigens are selected from the group consisting of: Pfs48/45, Pfs230, Pvs48/45 and Pvs230.
- the surface antigens are midgut parasite surface antigens.
- the midgut parasite surface antigens are selected from the group consisting of: Pfs25, Pfs28, Pvs25 and Pvs28.
- the one or more surface antigens is derived from a codon harmonized gene.
- the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 1. In another embodiment, the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 3. In another further embodiments, the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 5.
- blocking transmission is measured by the reduction of mosquito oocysts by sera or plasma from a subject treated with the immunogenic composition compared to a control subject.
- pre-immune sera from the treated subject and the control subject are used as a measure of 100% transmission for the corresponding test sera.
- the method further comprises administering an adjuvant.
- the adjuvant is selected from water-in-oil emulsion or Aluminum hydroxide.
- the method further comprises administering the immunogenic composition in one or more booster administrations.
- the booster immunization is administered at 4 weeks. In another particular embodiment, the booster immunization is administered at 12 weeks.
- the method further comprises treatment with an additional agent.
- the additional agent is used to treat or prevent malaria.
- composition is administered by one or more routes selected from the group consisting of: subcutaneous, intradermal, intramuscular, intravenous and transdermal delivery.
- the composition is administered in a concentration between 1 - 100 ⁇ g.
- the invention features an immunogenic composition comprising one or more pre-fertilization or post-fertilization antigens from P.falcipa ⁇ um or P.vivax.
- the one or more pre-fertilization or post-fertilization antigens are selected from the group consisting of Pfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.
- the pre-fertilization antigen is Pfs48/45.
- the post-fertilization antigen is Pfs25.
- the one or more pre-fertilization or post- fertilization antigens is derived from a codon harmonized gene.
- the codon harmonized gene encodes a protein represented by the amino acid sequence corresponding to SEQ ID NO: 1.
- the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 3.
- the codon harmonized gene is encoded by the amino acid sequence corresponding to SEQ ID NO: 5.
- composition is designed for expression in E. coli.
- the invention features a vector comprising a codon harmonized Pfs48/45 sequence suitable for expression in a cell.
- the invention features a vector comprising a codon harmonized Pvs48/45 sequence suitable for expression in a cell.
- the invention features a vector comprising a codon harmonized Pfs25 sequence suitable for expression in a cell.
- the codon harmonized Pfs48/45 sequence corresponds to the nucleic acid sequence comprising SEQ ID NO: 2.
- the codon harmonized Pfs25 sequence corresponds to the nucleic acid sequence comprising SEQ ID NO: 4.
- the codon harmonized Pvs48/45 sequence corresponds to the nucleic acid sequence comprising SEQ ID NO: 6.
- the invention features a cell expressing the vector of any one of the aspects described.
- the cell is an E. coli cell or an E.coli derivative cell.
- the invention features a kit comprising an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization or post-fertilization antigens, and instructions for use in reducing transmission of Plasmodium falciparum or Plasmodium vivax.
- the invention features a kit comprising an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization or post-fertilization antigens, and instructions for use in preventing malaria.
- the one or more pre-fertilization or post-fertilization antigens are selected from the group consisting of Pfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.
- the one or more pre-fertilization or post- fertilization antigens is derived from a codon harmonized gene.
- the invention features a method for preparing a codon harmonized pre-fertilization or post-fertilization antigen sequence encoded by a P. falciparum or P. vivax pre-fertilization or post-fertilization gene comprising determining the frequency of codon usage of the pre-fertilization or post-fertilization gene coding sequence; and substituting codons in the coding sequence with codons of similar frequency from a host cell which code for the same pre-fertilization or post- fertilization antigen, thereby preparing a codon harmonized pre-fertilization or post- fertilization antigen sequence.
- the one or more pre-fertilization or post-fertilization genes are selected from the group consisting of Pfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.
- the invention features a method for preparing a codon harmonized Pfs48/45 antigen sequence encoded by a pre-fertilization or post- fertilization gene comprising determining the frequency of codon usage of the pre- fertilization or post-fertilization gene coding sequence, wherein the Pfs48/45 sequence corresponding to the nucleic acid sequence represented by SEQ ID NO: 7, and substituting codons in the coding sequence of SEQ ID NO: 7 with codons of similar frequency from a host cell which code for the Pfs48/45 antigen, thereby preparing a codon harmonized Pfs48/45 antigen sequence.
- the codon harmonized gene is expressed in a host cell.
- the host cell is E.coli or an E.coli derivative.
- the invention features a codon harmonized nucleotide sequence prepared according to the methods described herein.
- Figure 1 shows the purification and characterization of CH-rPfs48/45.
- Lysates of uninduced and induced cells either non-reduced (left panel), or reduced (treated with 10 mM 2-mercaptoethanol, right panel) were run on SDS-polyacrylamide gel and stained with Coomassie stain.
- Lane 1 Protein molecular weight marker; lane 2, uninduced cell lysate; lane 3, induced cell lysate.
- the induced protein band in both non-reduced and reduced gels are encircled for easy recognition,
- (c) Flow diagram of various major steps including differential detergent extractions used for protein purification,
- Lane 1 Western blot analysis of purified CH-rPfs48/45 using anti-His mAb. Lane 1 , eluate from Ni-NTA column; lane 2, dialyzed CH-rPfs48/45. (f) Recognition of CH-rPfs48/45 by conformation specific mAb IIC5B10. Lane 1, non-reduced CH-rPfs48/45; lane 2, reduced CH-rPfs48/45.
- Figure 2 (a & b) is two panels that show (a) ELISA analysis of CH-rPfs48/45 immunized individual mouse sera in three different adjuvant formulations: Complete Freund's adjuvant (top panel), Montanide ISA-51 (middle panel), and Alum (bottom panel). All the results are representative of three independent experiments. Pooled pre-immune sera + 3SD are shown by broken lines.
- mice 1 filled triangle
- mouse 2 open square
- mouse 3 filled square
- mouse 4 open circle
- mouse 5 filled circle
- IgGl filled columns
- IgG2a hatched columns
- IgG2b shadeled columns
- IgG3 bladed columns
- Figure 3 shows recognition of native Pfs48/45 in P. falciparum gametocyte extract
- Stage V gametocyte extract was run either in non-reduced or reduced (10 mM 2-mercaptothanol) form in SDS-PAGE and transferred to nitrocellulose membrane. Mice sera were allowed to react at 1 :1000 dilution for 1 h at 22 0 C.
- Figure 4 shows analysis of anti-Pfs48/45 antibody production by Olive baboons (Papio anubis).
- CH-rPfs48/45 50 ⁇ g in 0.25 ml endotoxin free PBS
- Montanide ISA-51 0.25 ml
- mice were immunized with CH-rPfs48/45 (50 ⁇ g in 0.25 ml endotoxin free PBS) formulated in Montanide ISA-51 (0.25 ml) in baboons, administered intra muscularly (quadriceps, two sites).
- Schedules for immunization and bleeds are indicated and sera were stored at -2O 0 C until shipped frozen from Kenya to Baltimore for ELISA and MFA.
- the samples were shipped under an export permit CITES # 008101.
- Figure 5 is a graph that shows follow up of immune responses elicited by CH- rPfs48/45 in baboons. Analysis of anti-CH-rPfs48/45 IgG titers (open bars) and percent transmission blocking activity (closed bars) up to 7 months post second boost in baboons. Results show mean antibody titer and mean transmission blocking activity of 5 baboons + 95% CI.
- amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxyglutamate, and O- phosphoserine, phosphothreonine.
- codon harmonization is meant to refer to a process that harmonizes codon usage frequency of a target gene with those of the expression host for heterologous expression of protein.
- codon harmonization refers to an algorithm where synonymous codons from E. coli are selected that closely resemble the codon usage of a native pre-implantation gene, for example the Pfs48/45 gene, including regions coding 'link/end' segments of proteins in P. falciparum.
- fragment is meant a portion (e.g., at least 10, 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, or 500 amino acids or nucleic acids) of a protein or nucleic acid molecule that is substantially identical to a reference protein or nucleic acid and retains the biological activity of the reference. In some embodiments the portion retains at least 50%, 75%, or 80%, or more preferably 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.
- the term "host cell” is meant to refer to a cell into which a foreign gene is introduced.
- the host cell can be prokaryotic or eukaryotic.
- the host cell is E.coli or an E.coli derivative.
- immunogenic composition refers to one or more Plasmodium pre-fertilization or post- fertilization antigens that is capable of eliciting protection against malaria, whether partial or complete.
- An immunogenic composition may also be useful for treatment of an infected individual.
- isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
- levels of purity may be applied as needed according to this invention in the different methodologies set forth herein; the customary purity standards known in the art may be used if no standard is otherwise specified.
- isolated nucleic acid molecule is meant a nucleic acid (e.g., a DNA, RNA, or analog thereof) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
- the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
- the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
- nucleic acid is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof. This term includes oligomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced stability in the presence of nucleases.
- RNA sequences refer to contiguous DNA or RNA sequences which have compatible nucleotides (e.g., A/T, G/C) in corresponding positions, such that base pairing between the sequences occurs.
- compatible nucleotides e.g., A/T, G/C
- the sense and anti-sense strands of a double-stranded DNA helix are known in the art to be complimentary.
- pre- or post-fetilization antigen is meant to refer to a protein target expressed in Plasmodium that is necessary for Plasmodium transmission in a host.
- prefertilizaation antigens refer to antigens expressed in the intraerythrocytic gamtocytes and male and female gametes prior to fertilization process. Examples include, but are not limited to, proteins such as Pfs230 and Pfs48/45 in P. falciparum or Pvs230and Pvs48/45 in P. vivax.
- post fertilization antigens refer to antigens expressed on the surface of zygotes formed after fertilization between female and male gametes and ookinetes. Examples include, but are not limited to, proteins such as Pfs25 and Pfs28 in P. falciparum and Pvs25 and Pvs28 in P. vivax.
- protein is meant any chain of amino acids, or analogs thereof, regardless of length or post-translational modification.
- reference is meant a standard or control condition.
- telomere binding molecule e.g., peptide, polynucleotide
- a sample for example, a biological sample, which naturally includes a protein of the invention.
- treat refers to reducing or ameliorating a disorder and/or symptoms associated therewith, for example malaria. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
- the terms "prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition, for example malaria. Other definitions appear in context throughout the disclosure.
- the present invention features immunogenic compositions comprising one or more pre-fertilization or post-fertilization antigens from Plasmodium, preferably P. falciparum or P.vivax.
- Plasmodium preferably P. falciparum or P.vivax.
- the genome of Plasmodium falciparum has been completely sequenced. (Gardner et al., Nature, 419:498-511 (2002)).
- the one or more of pre-fertilization or post-fertilization antigens are selected from the group consisting of Pfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230 Pvs 28, and Pvs25.
- the antigen is Pfs 48/45 or Pfs25 of P. falciparum.
- the antigen is Pvs 48/45 or Pvs25 of P. vivax.
- Pfs230 and Pfs48/45 can be classified as gametocyte and gamete (male and female) surface antigens.
- Pfs25 and Pfs28 can be classified as mosquito midgut parasite stages (zygote and ookinete) surface antigens.
- Targeted gene disruption studies have shown that Pfs48/45 plays a critical role in male gamete fertility, an important aspect of the sexual reproduction success of the parasite [14].
- Analysis of immune human sera in endemic areas has also suggested a strong correlation between naturally present anti-Pfs48/45 antibodies and transmission reducing activity of those human sera; thus making it a key candidate for vaccine development [15].
- Pfs25 is a P. falciparum antigen expressed on the surface of the malaria parasite.
- Pfs25 consists of four epidermal growth factor (EGF)-like domains located between a secretory signal sequence at the N-terminus, and a short C-terminal hydrophobic domain, which seems involved in the transfer of the EGF-like domains to a glycosyl-phosphatidylinositol (GPI) lipid anchor.
- EGF epidermal growth factor
- cysteine residues present as disulfide bonds in the four EGF-like domains of Pfs25.
- the disulfide bonds between the cysteine residues are essential for maintaining the structural integrity of the antigen.
- antibodies to the antigen can completely block transmission of P. falciparum.
- the one or more pre- fertilization or post-fertilization antigens is derived from a codon harmonized gene.
- nucleotide sequence capable of enhanced expression in host cells can be obtained by harmonizing the frequency of codon usage in the foreign gene at each codon in the coding sequence to that used by the host cell.
- the invention features a nucleic acid sequence encoding a polypeptide to enhance expression and accumulation of the polypeptide in the host cell. Accordingly, the present invention provides novel nucleic acid sequences, encoding a polypeptide or protein that is foreign to a host cell, and that is expressed at greater levels and with greater biological activity than in the host cell as compared to the wild-type sequence if expressed in the same host cell.
- codon harmonization has been described, for example, in US Application Nos. 20060088547 and 20080076161 and Angov et al. (PLOS, 2008. Volume 3, issue 5), which are incorporated by reference in their entireties herein.
- the methods of the present invention while directed to codon harmonization of pre- or post-fertilization antigens, are not limited as such, and are applicable to any coding sequence encoding a protein foreign to a host cell in which the protein is expressed.
- the invention features a method for preparing a codon harmonized pre-fertilization or post-fertilization antigen sequence encoded by P. falciparum or P. vivax a pre-fertilization or post-fertilization gene comprising determining the frequency of codon usage of the pre-fertilization or post- fertilization gene coding sequence, and substituting codons in the coding sequence with codons of similar frequency from a host cell which code for the same pre- fertilization or post-fertilization antigen, thereby preparing a codon harmonized pre- fertilization or post-fertilization antigen sequence.
- the frequency of occurrence of each codon in the P. falciparum or P.vivax pre-fertilization or post-fertilization gene of interest can be calculated and replaced with an E. coli codon with a similar frequency for the same amino acid.
- the one or more pre-fertilization or post-fertilization genes are selected from the group consisting of Pfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.
- An existing DNA sequence can be used as the starting material and modified by standard mutagenesis methods that are known to those skilled in the art or a synthetic DNA sequence having the desired codons can be produced by known oligonucleotide synthesis, PCR amplification, and DNA ligation methods.
- the invention features a method for preparing a codon harmonized Pfs48/45 antigen sequence encoded by a pre-fertilization or post- fertilization gene comprising determining the frequency of codon usage of the pre- fertilization or post-fertilization gene coding sequence, wherein the Pfs48/45 sequence corresponding to the nucleic acid sequence represented by SEQ ID NO: 7, and substituting codons in the coding sequence of SEQ ID NO: 7 with codons of similar frequency from a host cell which code for the Pfs48/45 antigen, thereby preparing a codon harmonized Pfs48/45 antigen sequence.
- SEQ ID NO: 7 1 atgatgttat atatttctgc gaaaaaggct caagttgctt tcatcttata tatagtattg
- DNA sequences modified by the method of the present invention are expressed at a greater level in host cells than the corresponding non-modified DNA sequence.
- the host cell is E.coli or an E.coli derivative.
- the method can be applied to any DNA sequence for introduction into a host cell to provide protein product.
- the codon harmonized gene corresponds to the amino acid sequence corresponding to SEQ ID NO: 1, shown below, and the corresponding nucleic acid sequence, SEQ ID NO: 2.
- SEQ ID NO: 2 preferably includes a signal and anchor sequence.
- SEQ ID NO: 9 corresponds to the codon harmonized DNA sequence (SEQ ID NO:
- the codon harmonized gene corresponds to the amino acid sequence corresponding to SEQ ID NO: 3, shown below, and the corresponding nucleic acid sequence, SEQ ID NO: 4.
- SEQ ID NO:4 preferably includes a signal and anchor sequence.
- SEQ ID NO: 8 corresponds to the codon harmonized DNA sequence (SEQ ID NO:4, shown in italics) and restriction enzyme sites shown in bold, ⁇ xhistidine codons shown as underlined, as affinity tags, and linker sequences, preferably used to facilitate cloning.
- the codon harmonized gene corresponds to the amino acid sequence corresponding to SEQ ID NO: 5, shown below, and the corresponding nucleic acid sequence, SEQ ID NO: 6.
- SEQ ID NO: 5 shows the full length peptide sequence, where the signal peptide and the GPI anchor position are in bold.
- SEQ ID NO:6 corresponds to the codon harmonized Pvs48/45 DNA sequence consisting of 1274 bases, not including the signal sequence and minus GPI anchor sequence shown in SEQ ID NO:5.
- the codon harmonized gene corresponds to the nucleic acid sequence shown as SEQ ID NO: 10, where SEQ ID NO: 10 is a codon harmonized Pfs230 C-terminal modified.
- the bold, underlined sequence refers to restriction enzyme sites to facilitate cloning into an expression plasmid vector.
- the codon harmonized gene corresponds to the nucleic acid sequence shown as SEQ ID NO: 11, where SEQ ID NO: 11 corresponds to codon harmonized Pf230 A2B3.
- the bold, underlined sequence refers to restriction enzyme sites to facilitate cloning into an expression plasmid vector.
- the codon harmonized gene corresponds to the nucleic acid sequence shown as SEQ ID NO: 12, where SEQ ID NO: 12 corresponds to codon harmonized Pfs230 A4B5.
- the bold, underlined sequence refers to restriction enzyme sites to facilitate cloning into an expression plasmid vector.
- the codon harmonized gene corresponds to the nucleic acid sequence shown as SEQ ID NO: 13, where SEQ ID NO: 13 corresponds to codon harmonized Pfs230 A6B7.
- the bold, underlined sequence refers to restriction enzyme sites to facilitate cloning into an expression plasmid vector.
- compositions of the invention are designed for expression in a host.
- a host is E.coli or an E.coli derivative.
- the DNA encoding the desired recombinant protein can be introduced into a host cell in any suitable form including, the fragment alone, a linearized plasmid, a circular plasmid, a plasmid capable of replication, an episome, RNA, etc.
- the gene is contained in a plasmid.
- the plasmid is an expression vector.
- Individual expression vectors capable of expressing the genetic material can be produced using standard recombinant techniques. Please see e.g., Maniatis et al., 1985 Molecular Cloning: A Laboratory Manual or DNA Cloning, Vol. I and II (D. N.
- the present invention contemplates host cells transformed with a vector as described herein.
- the vector may comprise a codon harmonized Pfs48/45 sequence suitable for expression in a cell, a codon harmonized
- Pvs48/45 sequence suitable for expression in a cell a codon harmonized Pfs25 sequence suitable for expression in a cell.
- Plasmodium pre-fertilization or post-fertilization antigens for example Plasmodium falciparum or Plasmodium vivax pre-fertilization or post- fertilization antigens can be used singly, or in combinations.
- a combination may comprise antigens derived from different Plasmodium species that will be capable of blocking the infection and/or transmission of more than one
- an immunogenic composition comprising Pfs48/45 and Pvs48/45 will be capable of blocking the transmission of both P. falciparum and P. vivax.
- the present invention describes an approach that harmonizes codon usage frequency of the target gene with those of the expression host for heterologous expression of protein.
- Basic studies on regulation of protein expression have shown that synonymous codon substitutions from infrequent to frequent usage in regions where mRNA translation occurs relatively slowly can be detrimental to protein expression and stability [17].
- codon substitutions introducing rare codons in the regions containing high frequency codons can lead to erroneous protein conformation [18].
- an algorithm termed "codon harmonization" [19] was developed where synonymous codons from E. coli were selected that closely resemble the codon usage of native Pfs48/45 gene, including regions coding 'link/end' segments of proteins in P. falciparum.
- the present invention describes for the first time the efficient and successful expression of a pre-fertilization antigen, and in particular, full length TBV candidate Pfs48/45, in high yields and appropriate conformation.
- the recombinant Pfs48/45 elicits potent malaria transmission blocking antibodies in mice and non human primates (Olive baboons, Papio anubis) and indicates the development of a malaria TBV based on the CH-rPfs48/45 antigen.
- the pre-fertilization or post- fertilization antigens can be used to develop malaria transmission-blocking immunogenic compositions.
- the invention features methods of blocking transmission of Plasmodium falciparum or Plasmodium vivax in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization or post-fertilization antigens, thereby blocking transmission of Plasmodium falciparum or Plasmodium vivax in the subject.
- the invention also features methods of immunizing a subject against Plasmodium falciparum or Plasmodium vivax comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization or post-fertilization antigens, and thereby immunizing the subject against Plasmodium falciparum or Plasmodium vivax.
- the immunogenic compositions of the invention are also preferably used in methods for treating or preventing malaria in a subject comprising administering to a subject an immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization or post-fertilization antigens, thereby preventing malaria in the subject.
- the pre-fertilization or post-fertilization antigens can be classified as Plasmodium falciparum or Plasmodium vivax surface antigens.
- the surface antigens are gametocyte or gamete surface antigens. Exemplary gametocyte or gamete surface antigens are selected from the group consisting of Pfs48/45, Pfs230, Pvs48/45 and Pvs230.
- the surface antigens are midgut parasite surface antigens. Exemplary midgut parasite surface antigens are selected from the group consisting of Pfs25, Pfs28, Pvs25 and Pvs28.
- the one or more pre-fertilization or post-fertilization antigens are selected from the group consisting of Pfs48/45, Pfs 230, Pfs25, Pvs48/45, Pvs 230 and Pvs25.
- the pre-fertilization antigen is Pfs48/45.
- the post-fertilization antigen is Pfs25.
- codon harmonized genes are preferably employed in the methods of the invention, where, generally, a wild type nucleic acid sequence encoding a polypeptide is modified to enhance expression and accumulation of the polypeptide in the host cell by harmonizing synonymous codon usage frequency between the foreign DNA and the host cell DNA. Accordingly, in certain preferred embodiments, of the invention, one or more pre-fertilization antigens or post- fertilization antigens is derived from a codon harmonized gene.
- Codon harmonized genes are described herein, and in particular embodiments the modified (codon harmonized) nucleic acid sequence and/ or the encoded polypeptide are shown in SEQ ID NOs 1 - SEQ ID NO: 13.
- the immunogenic compositions of the present invention can be administered to a subject by different routes such as subcutaneous, intradermal, intramuscular, intravenous and transdermal delivery. Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the subject; the route of administration; the desired effect; and the particular composition. The course of the immunization may be followed by assays for activated T cells produced, skin-test reactivity, antibody formation or other indicators of an immune response to a malarial strain.
- Dosage form such as injectable preparations (solutions, suspensions, emulsions, solids to be dissolved when used, etc.), tablets, capsules, granules, powders, liquids, liposome inclusions, ointments, gels, external powders, sprays, inhalation powders, eye drops, eye ointments, and the like, can be used appropriately depending on the administration method.
- Pharmaceutical formulations are generally known in the art and are described, for example, in Chapter 25.2 of Comprehensive Medicinal Chemistry, Volume 5, Editor Hansen et al, Pergamon Press 1990.
- Pharmaceutically acceptable carriers which can be used in the present invention include, but are not limited to, an excipient, a stabilizer, a binder, a lubricant, a colorant, a disintegrant, a buffer, an isotonic agent, a preservative, an anesthetic, and the like which are commonly used in a medical field.
- Immunogenic compositions are administered in immunologically effective amounts.
- An immunologically effective amount is one that stimulates the immune system of the subject to establish a level of immunological response sufficient to reduce parasite density and disease burden caused by infection with the pathogen, and/or sufficient to block the transmission of the pathogen in a subject.
- a dose of the immunogenic composition may, in certain preferred embodiments, consist of the range of 1 ⁇ g to 1.0 mg total protein. In certain preferred embodiments, the composition is administered in a concentration between 1 - 100 ⁇ g. However, one may prefer to adjust dosage based on the amount of antigen delivered. In either case these ranges are guidelines. More precise dosages should be determined by assessing the immunogenicity of the composition so that an immunologically effective dose is delivered.
- the immunogenic composition can be used in multi-dose formats.
- booster doses may subsequently be administered to maintain antibody titers, e.g., the compositions of the present invention can be administered one time or serially over the course of a period of days, weeks, months and or years.
- An example of a dosing regime would be day 1 an additional booster doses at distant times as needed.
- the booster doses may be administered at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more weeks after the primary immunization.
- the booster doses are administered at 4 weeks.
- the booster doses are administered at 12 weeks.
- the immunogenic compositions are administered after, before or at the same time as treatment with an additional agent, such as an agent to treat or prevent malaria.
- the subject that would benefit from the immunogenic compositions described herein include any host that can benefit from protection against malarial infection.
- a subject can respond to inoculation with the immunogenic compositions of the present invention by generating an immune response.
- the immune response can be completely or partially protective against symptoms caused by infection with a pathogen such as Plasmodium falciparum, or can block transmission of the pathogen by Anopheles mosquitoes.
- the subject is a human.
- the subject is a non-human primate.
- the immunogenic compositions of the present invention can be used to immunize mammals including humans against infection and/or transmission of malaria parasite, or to treat humans post-infection, or to boost a pathogen-neutralizing immune response in a human afflicted with infection of malaria parasite.
- the immunogenic compositions of the present invention can be formulated according to methods known and used in the art. Guidelines for pharmaceutical administration in general are provided in, for example, Modern Vaccinology, Ed. Kurstak, Plenum Med. Co. 1994; Remington's Pharmaceutical Sciences 18th Edition, Ed. Gennaro, Mack Publishing, 1990; and Modern Pharmaceutics 2nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990. Immunogenic compositions of the present invention can be prepared as various salts. Pharmaceutically acceptable salts (in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases.
- salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thio
- Adjuvants are almost always required to enhance and/or properly direct the immune response to a given antigen.
- An ideal adjuvant should be safe, stable with long shelf life, biodegradable, inexpensive and promote an appropriate immune response while itself being immunologically inert.
- Adjuvants affect processes including antigen presentation, antigen uptake and selective targeting of antigens thus critically determining the magnitude and type of the immune responses [34 - 36]. While the mechanisms by which different adjuvants result in different outcomes remain a "black box", studies strive for developing a vaccine that can provide maximum efficacy with ease of delivery in as fewer doses as possible.
- an adjuvant is not the active component in a vaccine and immunization; outcomes can vary greatly from one adjuvant to another when used in combination with the same vaccine antigen. Any given adjuvant - vaccine combination has to be evaluated on a case-by-case basis for safety, reactogenicity and efficacy in pre clinical trials. Ultimately, safety considerations outweigh any anticipated benefit and need to be evaluated for the development of a plan leading to human clinical trial [37].
- the immunogenic compositions are formulated with an aluminum adjuvant.
- Aluminum based adjuvants are commonly used in the art and include aluminum phosphate, aluminum hydroxide, aluminum hydroxy-phosphate, and amorphous aluminum hydroxyphosphate sulfate. Trade names of aluminum adjuvants in common use include ADJUPHOS, ALHYD ROGEL, (both from Superfos Biosector a/s, DK-2950 Vedbaek, Denmark).
- Non-aluminum adjuvants can also be used.
- Non-aluminum adjuvants include, but are not limited to, QS21, Lipid-A, Iscomatrix., and derivatives or variants thereof, Freund's complete or incomplete adjuvant, neutral liposomes, liposomes containing vaccine and cytokines or chemokines.
- Emulsions of Montenide ISA 51 (a mineral oil adjuvant) and ISA 720 (oil- based non mineral oil) have been used in human clinical trials.
- a review of clinical trials (25 trials representing more than 4000 patients and 40,000 injections for Montanide ISA 51 and various trials representing 500 patients and 1500 injections for Montanide ISA 720) has revealed their general safety and strong adjuvant effect with mild to moderate local reactions.
- the method of the invention further comprises administering an adjuvant.
- the adjuvant is selected a water-in-oil emulsion.
- the adjuvant is Aluminum hydroxide.
- any adjuvant that is suitable for administration with the immunogenic composition in the methods of the present invention can be suitably used.
- kits suitable for providing compositions of the immunogenic compositions as described herein are kits suitable for providing compositions of the immunogenic compositions as described herein.
- one vial can comprise the immunogenic composition comprising one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization or post-fertilization antigens, and instructions for use in reducing transmission of Plasmodium falciparum or Plasmodium vivax.
- kits can preferably comprise one or more Plasmodium falciparum or Plasmodium vivax pre-fertilization or post-fertilization antigens, and instructions for use in preventing malaria.
- the one or more pre-fertilization or post-fertilization antigens are selected from the group consisting of Pfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.
- the one or more pre-fertilization or post-fertilization antigens is derived from a codon harmonized gene.
- the kit will contain instructions for using the composition. Such instructions can be in the form of printed, electronic, visual, and or audio instructions.
- Example 1 Expression and purification of correctly folded Pfs48/45 Generally speaking it is often difficult to express P. falciparum proteins in a heterologous expression system and such problems are largely due to high A/T content giving rise to changes in codon usage frequencies. It is now increasingly being realized that synonymous codon substitutions are not always silent and changes in the frequency of codon usage affects protein structure and function. Differences in synonymous codon usage between expression and natural hosts greatly affect expression and stability of proteins. These codon differences have even greater consequences if they occur infrequently and' within the domains that encode polypeptide regions of higher ordered structure. The presence of infrequently used codons is believed to cause ribosome pausing leading to incorrect folding and premature termination of protein during translation.
- the native sequence of Pfs48/45 is represented by NCBI accession number AF356146, corresponding to SEQ ID NO: 7.
- the native sequence of Pfs 48/45 as represented by SEQ ID NO: 7, lacking N-terminal signal sequence (amino acid residues 1-27) and C-terminal anchor (amino acid residues 428-448) was converted to 'codon harmonized' sequence designed for expression in E. coli.
- the amino acid sequence reported under AF356146 differs from that of Z22145 (NF54) by C32Y, K33N, T72N, K253N and N254K substitutions.
- the A/T contents of Pfs48/45 sequence before and after codon harmonization were 75% and 56%, respectively.
- the codon harmonized Pfs48/45 sequence containing a sequence tag coding for 6 x histidines at the 5 'end was synthesized and cloned into the pET (K-) expression vector [19] and expressed in BL21 (DE3) cells [Fig Ia]. Initial attempts to induce the cells with IPTG indicated that the expressed protein might negatively impact the growth of E. coli.
- the expression strategy was modified to slow down protein expression by growing the cells at 30°C in Luria-Bertani growth medium containing 1% glucose and induction with 0.1 mM IPTG for 3 h, which resulted in highly efficient induction of protein in the cell lysate at 50 kDa [Fig Ib, left panel, encircled].
- a similar observation was made with the native form of the protein on the gametocyte surface [22,23]. Western blot analysis of cell lysates either untreated or after treatment with
- 0.5% sarcosyl revealed that the recombinant protein was insoluble in the absence of sarcosyl detergent (data not shown), and hence the treatment with ionic detergent was required to facilitate extraction of the protein from the pellet.
- the lysate was first treated with non-ionic detergent Tween-80 to remove any soluble bacterial proteins followed by treatment with 0.5% sarcosyl in PBS [Fig Ic].
- the mAb recognized the non-reduced form of CH-rPfs48/45 yielding 3 immunoreactive bands at ⁇ 50 kDa of the gel [Fig If, lane I].
- purified CH-rPfs48/45 protein preparations consistently revealed the presence of a higher molecular weight band at ⁇ 98 kDa, presumed to be a dimer.
- the mAb rather unpredictably showed recognition of a single band around 65-68 kDa [Fig If, lane 2].
- mice immunized with CH-rPfs48/45 in the other two adjuvant formulations also showed high antibody titer, the range being ⁇ 70,000 to more than 100,000 in both Montanide ISA-51 and Alum formulations [Fig 2a, middle and bottom panels].
- Sera for IgG isotype distribution was also tested and all four subtypes (IgGl, IgG2a, IgG2b, IgG3) were more or less equally represented in sera from mice immunized with CFA formulation, [Fig 2b, top panel].
- IgGl and to a lesser extent IgG2 were the dominant subtype in Montanide ISA-51 formulation [Fig 2b, middle panel].
- the overwhelming presence of IgGl and negligible amount of IgG3 were noticed in the Alum formulation [Fig 2b, bottom panel].
- mice sera in the three adjuvant groups showed a strong (>98% reduction in the number of oocysts) transmission blocking activity at 1 :2 dilution [Table 1, shown below] as compared to corresponding pre-immune sera.
- Table 1 shows MFA with sera from mice immunized with CH-rPfs48/45 formulated in CFA, Montanide ISA-51 or Alum. Individual mouse sera were tested for transmission blocking activity with respect to corresponding pooled pre-immune sera. Data are represented as percent transmission blocking activity (reduction in the number of oocysts per mosquito midgut). Numbers within parenthesis represent total number of infected mosquitoes / total number of mosquitoes dissected for each feed.
- the decrease in oocyst number/midgut by each immune sera was significant (P ⁇ 0.02, Mann- Whitney test).
- the transmission blocking effect was dependent upon the antibody dose as revealed by a gradual decrease with increasing sera dilutions.
- Sera from mice immunized with CH-rPfs48/45 vaccine in CFA and Montanide ISA-51 formulations as compared to Alum appeared to be relatively more potent blockers as apparent from the stronger transmission blocking activities at 1 :8 dilution of sera in MFA.
- Example 3 Evaluation of functional immunogenicity of CH-rPfs48/45 in non human primates (Olive baboons) Backed by strong functional immunogenicity of CH-rPfs48/45 in three different adjuvant formulations in mice, the CH-rPfs48/45 vaccine was next evaluated in nonhuman primates (Papio anubis, Olive baboons). The vaccine trial in baboons was approved by the institutional and scientific review committee of the Institute of Primate Research with a protocol #19/10/2007. A group of 5 baboons (ranging 7.6 to 12.2 kg in body weight) were immunized with 50 ⁇ g of CH-rPfs48/45 in Montanide ISA-51, water-in-oil emulsion.
- Mosquito MFA showed strong transmission blocking activity by all animals for various bleeds obtained at different time points post immunization [Table 2, shown below].
- Table 2 shows transmission blocking activity of sera of different bleeds at various time points from baboons immunized with CH-rPfs48/45 in Montanide ISA- 51. MFA was done with sera collected on 12/10/2007 (1 mo post primary immunization), 01/10/2008 (1 mo post 1st boost, 02/21/2008 (15 d post 2nd boost), 03/06/2008 (1 mo post 2nd boost), and 05/05/2008 (3 mo post 2nd boost).
- the geometric mean of oocyst numbers / midgut in the presence of individual pre- immune sera were 22.24 (Pan 3104), 18.43 (Pan 3140), 22.1 (Pan 3163), 6.31 (Pan 3275), and 19.7 (Pan 3313), respectively. Numbers within parenthesis represent total number of infected mosquitoes / total number of mosquitoes dissected for each feed. Even after primary immunization, the sera (at 1 :2 dilution) were capable of spectacular transmission blocking with more than 93% average reduction in the number of oocysts in comparison to pre-immune sera of corresponding animal. Pre- immune sera from each animal were used as a measure of 100% transmission for the corresponding test sera.
- Pre-immune serum from one animal (Pan 3275) exhibited -30% reduced transmission when compared with the pre-immune sera of other four baboons. It is possible that natural infection by other Plasmodium-l ⁇ ke parasites, such as Enteropoides and Hepatocystis spp might elicit partly cross-reactive and inhibitory activity. The average transmission blocking activity increased to greater than 97% in all the animals after a booster dose. In order to titrate the blocking effectiveness of immune sera from these animals, sera obtained one month after the second boost were further tested at various dilutions (1 :4, 1 :8, 1 : 16) in MFA. While exhibiting strong blocking activity at 1 :4 and 1 :8 dilutions, the sera at 1 :16 dilution were still able to reduce transmission (ranging from 74% to 86%).
- the invention was performed with, but not limited to, the following methods and materials.
- the codon harmonized sequence of Pfs48/45 containing 6x Histidines at N- terminal end was synthesized by Retrogen Inc. and cloned into the expression vector pET(K-) in E. coli BL21 (DE3) competent cells (Invitrogen Corp.).
- the cells containing pET(K-)Pfs48/45 were grown overnight at 3O 0 C in LB media containing 1% glucose and 50 ⁇ g/ml Kanamycin.
- the overnight culture was diluted 100 fold into 1 1 culture in above mentioned media and grown at 3O 0 C with agitation until the OD600 of the culture reached 1.00.
- the cells were then induced with 0.1 mM of IPTG and grown for 3 h at 30 0 C.
- the cells were then harvested and centrifuged at 3800 x g at 4°C for 20 min. The pellet was kept at -8O 0 C for further processing.
- the frozen pellet was resuspended in Ix PBS (pH 7.4) at a pellet to buffer ration of 1 : 10 (w/v) and was lysed by micro fluidization (Model Ml 1OY, Microfluidics).
- the Iy sate was centrifuged at 24000 x g for 45 min at 4 0 C.
- the lysate pellet was resuspended in Ix PBS containing 1% Tween-80 (final v/v), extracted for 30 min at 22 0 C and centrifuged at 24000 x g for 30 min at 4°C.
- the pellet was resuspended in Ix PBS containing 0.5% Sodium Lauroyl Sarcosine (sarcosyl) (final v/v), extracted and centrifuged as before. The supernatant was then passed through Ni-NTA agarose column (QIAGEN) according to manufacturer's protocol. The protein was eluted as 1 ml fractions with 1 M imidazole in Ix PBS as elution buffer. The protein was dialyzed against Ix PBS (pH 7.4) containing 10% glycerol and 0.2% Tween-80. Finally, the protein content was estimated using BCATM Protein Assay kit (Pierce) and the endotoxin level in the protein was measured using QCL-1000 Endpoint chromogenic LAL assay kit (Lonza).
- CH-rPfs48/45 was characterized by Western blot analysis. Briefly, samples from each purification step described above were run on SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked overnight with Ix PBS containing 5% non-fat dry milk and 0.1% Tween-20 (blocking buffer) at 4 0 C.
- the membrane was washed with Ix PBS containing 0.1% Tween-20 (wash buffer) and incubated with either 6xHis mAb (Clontech) at 1 : 1000 dilution [Fig 1 d, e] or IIC5B10 mAb (MR4) at 1 :5000 dilution [Fig IfJ for 1 h at 22 0 C.
- the membrane was washed 4x in wash buffer for 30 min at 22 0 C and incubated with HRP -conjugated anti-mouse IgG mAb (GE Healthcare) at 1 : 10000 dilution in blocking buffer for 1 h at 22 0 C. This was followed by washing 4 x with wash buffer and ECL Plus chemiluminescent substrate (GE Healthcare) was used as detection reagent.
- CFA Complete Freund's Adjuvant
- SEPPIC Montanide ISA-51
- Alhydrogel, Brenntag aluminum hydroxide
- mice immunized in CFA were boosted at 4 week intervals twice with the same quantity of protein in incomplete Freund's adjuvant.
- Mice in the other adjuvant groups were boosted at 4 week intervals thrice in Montanide ISA-51 or Alum, respectively.
- Groups of control mice were immunized with adjuvant formulations only. Blood was collected on day 0 (Pre-immune sera) and 4 weeks after primary immunization and 2 weeks post
- the vaccine trial in baboons was approved by the institutional and scientific review committee of the Institute of Primate Research with a protocol #10/10/2007. Because these animals were trapped from their wild habitats, they were quarantined for 3 months and screened for the presence of any worms and protozoan parasites and successfully treated appropriately, if found infected, prior to initiating vaccination. Moreover, animals were also screened by three intradermal tuberculin tests and found to be negative for mycobacterial infections. Detailed hematological tests were also administered on all five animals during their quarantine period, just prior to and at the termination of the vaccine trial, and were certified to be in excellent health at all time points with no observable trial-related effects.
- ELISA ELISA was done. Briefly, Immulon-2 plates were coated with 1.5 ⁇ g/ml CH-rPfs48/45 in carbonate-bicarbonate buffer (pH 9.6) overnight at 4 0 C, blocked with 5% milk in PBS and incubated with various dilutions of sera at 37 0 C for 1 h. The plates were washed 5 times in PBS- 0.05% Tween-20 (PBST) followed by further incubation with 1 : 10000 dilution of horseradish peroxidase conjugated anti-mouse IgG antibody for 1 hour at 37 0 C.
- PBST PBS- 0.05% Tween-20
- the ELISA with baboon sera was done following a similar protocol and endpoint titers were calculated using the same criteria.
- the secondary antibody was anti-human IgGl, IgG2, and IgG3 conjugated to peroxidase (The Binding Site, Birmingham, UK) and used at 1 : 5000 dilution.
- Various sera were tested at 1 :5000 dilution for IgG subclass analysis.
- Plasmodium falciparum NF54 parasites were maintained using normal red blood cells and normal human serum (O+ve blood group) as s described [32]. Stage V gametocytes were used in live IFA studies and membrane feeding assays. To extract gametocytes for Western blot analysis, the gametocyte culture was centrifuged at 1000 x g for 5 min at 22 0 C. The RBC was lysed with 0.15% saponin in PBS for 5 min at 22 0 C. The gametocytes were collected after centrifugation at 1800 x g for 5 min and washed thrice in Ix PBS. The gametocytes were resuspended in 25 mM Tris-Cl (pH 7.5) containing 150 mM NaCl and Ix protease inhibitor cocktail and kept frozen at -7O 0 C till use.
- Tris-Cl pH 7.5
- the gametocytes were harvested from 19 day culture and gametes were produced by incubating the gametocytes in exflagellation buffer and purified by discontinuous Nycodenz gradient centrifugation as described previously [33]. Extracellular gametes were incubated at 4 0 C with 1 : 100 to 1 :1000 dilution of immune murine sera for 60 min. Parasites were washed 3 times with 1% BSA in PBS followed by incubation with FITC-anti mouse antisera (Alexa fluor 488), 1 :500 dilution at 4 0 C for 60 minutes. After washing, cells were examined by upright fluorescent Nikon E800 microscope (Japan) at IOOX magnification.
- the murine and baboon immune sera were mixed with cultured P. falciparum (NF54) stage V gametocytes, normal red blood cells and normal human sera (donor blood group: O+) and fed to Anopheles gambiae (starved for 5-6 hours) mosquitoes through water jacketed glass membrane (stretched parafilm) feeders [10,33] Briefly, washed human red blood cells and cultures containing stage V gametocytes were resuspended to 66% hematocrit and 0.3% gametocytemia in normal human serum and maintained throughout at 37 0 C.
- P. falciparum NF54 stage V gametocytes
- normal red blood cells and normal human sera donor blood group: O+
- Anopheles gambiae starved for 5-6 hours
- mosquitoes through water jacketed glass membrane (stretched parafilm) feeders
- Midguts were dissected 9-10 days after blood meal for enumeration of oocysts after staining with 1% mercurochrome. Transmission blocking activity of individual sera was calculated as percentage of reduction in oocyst number per midgut with test sera in comparison with pooled pre-immune sera (pre-immune murine or baboon sera was taken as allowing 100% transmission).
- pre-immune murine or baboon sera was taken as allowing 100% transmission.
- Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites. Infect Immun 73: 287-297.
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WO2010036293A8 (en) | 2010-06-03 |
US8501926B2 (en) | 2013-08-06 |
AU2009297127A1 (en) | 2010-04-01 |
EP2331119A1 (en) | 2011-06-15 |
EP2331119A4 (en) | 2012-05-30 |
BRPI0919245A2 (en) | 2021-11-03 |
US20110171266A1 (en) | 2011-07-14 |
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