Patent Grant
10940190
The present invention is a national stage application which claims priority from PCT Application No. PCT/AU2018/050155 filed Feb. 23, 2018, and AU Application No. AU2017900648 filed Feb. 27, 2017. Applicants claim the benefits of 35 U.S.C. § 120 as to the said PCT application, and priority under 35 U.S.C. § 119 as to the said AU application, and the entire disclosures of all applications are incorporated herein by reference in their entireties.
The present invention is a national stage application which claims priority from PCT Application No. PCT/AU2018/050155 filed Feb. 23, 2018, and AU Application No. AU2017900648 filed Feb. 27, 2017. Applicants claim the benefits of 35 U.S.C. § 120 as to the said PCT application, and priority under 35 U.S.C. § 119 as to the said AU application, and the entire disclosures of all applications are incorporated herein by reference in their entireties.
The present description relates to malaria vaccines comprising Plasmodium falciparum (Pf) polypeptides and methods of producing the same.
Bibliographic details of references in the subject specification are also listed at the end of the specification.
Reference to any prior art in this specification is not, and should not be taken as, acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Malaria in man is a tropical disease resulting from infection by one celled protozoan parasites of the genus Plasmodium which are deposited in to the blood stream when an infected mosquito takes a blood meal. Plasmodium falciparum and Plasmodium vivax are responsible for the most common and severe forms of malaria and despite recent significant reductions in the frequency of infection and deaths, last year it is estimated that over 200 million people were affected with approximately 400,000 deaths. Children in sub-Saharan Africa are particularly vulnerable to P. falciparum and account for most deaths from malaria. The parasite undergo a complex life cycle in man with rounds of infection and replication in erythrocytes or reticulocytes causing many of the early clinical symptoms of malaria, i.e., fever, shaking chills, headache, malaise, myalgia and fatigue. Current guidelines suggest that all new subjects with P. falciparum malaria should be admitted to hospital initially because of the speed with which patients can worse.
Subunit malaria vaccines are still in development 20 years after they were widely heralded as the most promising way forward for protecting vulnerable populations from Malaria. Much research has been undertaken during this period and knowledge concerning the molecular immunobiology of the host parasite relationship has accumulated. There are currently no available vaccines for malaria. The most advanced vaccine currently undergoing development is RTS,S which targets the liver stages of the parasite life cycle.
Another proposed vaccine approach is to target blood stage (merozoite) parasites. A large number of proteins are produced by the merozoite stage of the parasite that congregate at least in part at the apical end or complex of the merozoite prior to or at the time of erythrocyte engagement. Plasmodium merozoite proteins also form erythrocyte cell surface proteins that act as receptors for the invading merozoite. Illustrative merozoite proteins include MSP1, MSP2, MSP4, MSP5, MSP10, Pf12, Pf38, Pf92, Pf113, ASP, RAMA, EBA140, EBA175, EBA181, EBL1, AMA1, MTRAP, MSP5, MSP6, H101, H103, MSP7, Pf41, RAP1, RAP2, RAPS, RHopH1, RhopH3, Rh1, Rh4, Rh2b, Rh5, SPATR, PTRAMP, TLP, Pf34, PF14_0344, PF10_0323, PFF0335c, AARP, MSP3.4, MSP3.8, MSRP1, MSRP2, MSRP3, RON1, RON2, 4, 5 and 8) RON3, RON6, Pf12p, MSP5, GAMA, and PF11_0373. The merozoite surface proteins (MSPs) are thought to function early in merozoite invasion (a process which takes place in less than two minutes) and are highly polymorphic. The erythrocyte binding antigens (PfEBAs) and reticulocyte binding protein (PfRHs) are related to P. virax duffy binding proteins/reticulocyte binding proteins respectively and they orchestrate a variety of interations that result in host cell invasion by poorly defined pathways that display strain specific permutations. RH5 is more conserved PfRH merozoite secreted protein that binds the Ok blood group antigen, basagin. Improtantly antibodies raised against RH5 are able to prevent parasite growth in vitro and the blocking effect is not strain specific. In this case, the ability to produce a immunogenic RH5 able to engender inhibitory antibodies has been a problem for vaccine developers. Others have looked at the ability of antibodies against other merozoite proteins to enhance invasion blocking and found, for example that antibodies against PfRH4 and PfRH5 work synergistically to enhance invasion blocking effects.
These molecules are proposed as vaccine candidates based on their surface location as extracellular proteins and/or proposed role in host cell invasion and development within the host cell. One of the problems with this approach generally is the antigenic variability or allele polymorphism of the blood stage antigens which limits the ability of one allele of the protein to protect against different alleles present in the population of Plasmodium alleles. Another problem is redundancy in the host invasion pathway which means that blocking one pathway may not affect merozoite invasion levels.
Another set of problems faced by malaria vaccine developers in the field is the production of adequate amount of protein in a suitable form. For example, Plasmodium polynucleotides have a very high A:T content which leads to low codon usage compatibility in heterologous expression systems. The large size of some of these proteins and the presence of long stretches of repetitive amino acid sequences also hampers their expression in heterologous expression systems. Endogenous Plasmodium proteins are not modified by N-linked glycans thus expression in most available eukaryotic expression systems may lead to inappropriate or non-native glycosylations or disulfide bonding which foreshadow unpredictable effects on the immunogenicity of vaccine candidates. One approach with membrane proteins has been to express hydrophilic ectodomains without the more hydrophobic domain, or to express only about 200 bp fragments of the ectodomain recombinantly.
Accordingly, it remains challenging to develop and produce Plasmodium derived antigens, including modified or truncated forms that are able to engender effective immunity against blood stage forms of the parasite.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
As used herein the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes a single composition, as well as two or more compositions; reference to “an agent” includes one agent, as well as two or more agents; reference to “the disclosure” includes single and multiple aspects of the disclosure and so forth.
Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of sequence identifiers is provided after the brief description of the drawings. A sequence listing is provided after the claims.
Plasmodium falciparum (Pf) Ripr polypeptide is a peripheral membrane protein of over 1000 amino acids comprising 10 EGF domains mostly at the C-terminal portion of the polypeptide. EGF domains comprise multiple conserved cysteine residues which can lead to complex aggregate or multimer formation upon expression in vitro. Such polypeptides are typically difficult to express in prokaryotic expression systems and when expressed in E. coli, the present inventors identified that antibodies produced against the polypeptide were not inhibitory.
In one embodiment, the specification provides a modified arthropoda cell, modified to express a Plasmodium falciparum (Pf) Ripr polypeptide from a heterologous polynucleotide encoding a PfRipr polypeptide. In one embodiment, the cell is an insect cell. In one embodiment, the heterologous polypeptide comprises at least initially (prior to cleavage) a suitable signal sequence. In one embodiment, the polynucleotide is operably linked to an expression control sequence which induces PfRipr polypeptide expression in the cell. As determined herein, PfRipr polypeptide expressed in the insect cell induces parasite invasion inhibitory antibodies when administered to a subject.
In one embodiment, the specification provides a modified Drosophila derived cell modified to over express a Plasmodium falciparum (Pf) Ripr polypeptide (antigen) from a heterologous polynucleotide encoding a PfRipr polypeptide. In one embodiment, the heterologous polypeptide comprises at least initially (prior to cleavage) a suitable signal sequence. In one embodiment, the signal sequences is an insect signal sequence to direct the polypeptide through the host cell secretory pathway. In one embodiment, the polynucleotide is operably linked to an expression control sequence which induces PfRipr polypeptide expression in the cell. In one embodiment, insect promoter sequences are routinely employed. As determined herein, PfRipr polypeptide expressed in the Drosophila cell induces parasite and specifically merozoite invasion inhibitory antibodies when administered to a subject.
An illustrative Drosophila derived cell is a Drosophila melanogaster derived cell, such as a Scheider 2 cell line (S2), S3 cells or a derivative of the Kc cell line used for recombinant protein production. A wide variety of vectors in general allow for stable expression of recombinant proteins in insect cells. However, the production of such a large full length PfRipr polypeptide in Drosophila derived S2 cells was not a routine matter.
Other insect host cell expression systems are known in the art and include Spodoptera frugiperda derived cells, such as Sf9, Sf21 cell lines. Many types of viruses can infect insect cells and Baculovirus expression systems are widely employed to express recombinant proteins transiently or stably in insect cells.
Illustrative signal sequences include the insect BiP leader sequence, the honeybee melittin, signal peptide. As known to those of skill in the art, co-expression of BiP/Grp78 with a protein disulfide isomerase can enhance secreted expression in insect cells. Putative optimal signal sequences are tested to determine the optimal performance.
In one embodiment, the Pf polypeptides are expressed as soluble non-aggregated forms.
Expression may be transient or stable. Based upon the present disclosure, a wide range of selection procedures are known in the art to identify stable cell lines expressing high levels of antigenic Pf polypeptide, including a PfRipr polypeptide, and a PfRH5 polypeptide and a PfCyrPa polypeptide.
Expression control sequences promote optimal transcription, processing, RNA export and translation of the subject Pf polypeptide/s. Transcription regulatory sequences, i.e., promoters/enhancers are selected for high levels of transcription. Putative optimal expression control sequences are tested to determine the optimal performance. Illustrative promoters include the actin 5 promoter, and PpIE promoters, OpIE (OpIE1/OpIE2 for example) promoters, and KanR promoters.
In one embodiment, the insect cell line grows in suspension, is tolerant to osmolarity changes, and does not require CO2 for cultivation. Protein is expressed into the culture media without requirement for cell lysis and thus purification steps are reduced.
Reference herein to a Plasmodium “polypeptide”, includes reference to an “antigen” or “one or more epitopes” and refers to proteins or parts or portions thereof that comprise naturally occurring amino acid sequences, alleles, recombinantly produced modified forms, processed products, or modified or truncated (biologically active fragments) forms thereof which induce antibodies in a subject that are effective in inhibiting the invasion of host cells by merozoites. In one embodiment the Pf polypeptide is a polypeptide that is secreted from the merozoite. Illustrative truncated forms include the ectodomain together with all or part of a transmembrane domain/GPI anchor. Illustrative modified forms include polypeptides with one or more amino acid deleted, substituted or modified. In one embodiment, modified or truncated forms essentially retain or improve on the antigenicity and/or stability of the unmodified or naturally occurring polypeptide sequence. Modified forms may be modified to ensure certain host cell post-translational modifications are avoided or reduced in the Pf polypeptide while others are enhanced to allow optimal folding and antigenicity. In one illustrative embodiment, modified forms may have amino acid substitutions, for example to Ala or Gly, to reduce host cell glycosylation events or to enhance stability for example in the case of unpaired cysteines.
In one embodiment, modified and truncated forms of Plasmodium polypeptides display at least one characteristic selected from the group comprising (i) reduced binding to non-invasion inhibitory antibodies, (ii) at least substantially the same binding to invasion inhibitory antibodies, (iii) elicits the production of lower titers of non-neutralizing antibodies than invasion inhibitory antibodies, (iv) elicits the production of invasion inhibitory antibodies, (v) elicits the production of broadly invasion inhibitory antibodies against more than one allele of the antigen/s, (vi) optionally elicits the production of higher titers of invasion inhibitory antibodies; and (vii) optionally elicits the production of high titers of broadly invasion inhibitory antibodies. Specifically, in one embodiment a modified or truncated polypeptide displays an enhanced ability relative to the unmodified form to elicit antibodies that facilitate invasion inhibition. Inhibition by an antibody includes complete inhibition of invasion and a significant reduction in invasion such as 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% or less than 30% invasion. By co-administering Plasmodium polypeptides, PfRipr, PfRh5, PfCyrPa individually or as a mixture or or as a complex, combinations of antibodies are generated against PfRipr, PfRh5, PfCyrPa that effectively inhibit growth of Plasmodium blood stages within the host. In another embodiment, modified and truncated forms may also be modified to comprise expression, detection, purification or anchorage tags to facilitate their isolation, purification, detection or administration etc. Tags may individually perform several such as enhancing folding and stability and facilitating purification. Several tags are known in the art and all such forms may be tested and employed. Modified and truncated forms may also comprise linker sequences to aid, for example, in their presentation to the immune system.
Illustrative purification tags include N-terminal hexa-His tag, biotin, Fc and Strepll (SAWSHPQFEK).
Reference to a Pf polypeptide/protein means one or more of PfRipr, PfRh5, PfCyrPa and PfEBA175 polypeptides or antigens, and includes any allele of the protein or modified or truncated forms thereof which induce inhibitory antibodies in a subject. In one embodiment, a Pf polypeptide may be expressed as a monomer. In one embodiment, a Pf polypeptide may be expressed as a homodimer. In one embodiment, a Pf polypeptide may be expressed as a homotrimer.
Reference to a Pf polypeptide/protein means one or more of PfRipr, PfRh5, PfCyrPa and PfEBA175 polypeptides or antigens, and includes any allele of the protein or modified or truncated forms thereof which induce inhibitory antibodies in a subject. In one embodiment, a Pf polypeptide may be expressed as a monomer. In one embodiment, a Pf polypeptide may be expressed as a homodimer. In one embodiment, a Pf polypeptide may be expressed as a homotrimer.
Reference to a Pf protein complex, or Pf protein mixture means substantially a complex or substantially a mixture comprising two or more of PfRipr, PfRh5, PfCyrPa and PfEBA175 polypeptides or antigens, and includes any allele of the protein or modified or truncated forms thereof which induce inhibitory antibodies in a subject.
In one embodiment, the Pf protein may be glycosylated.
In one embodiment, the PfRipr is essentially full length, lacking its signal sequence. The PfRipr signal sequence (MKLCILLAVVAFVGLSLG) is cleaved and removed during secretion. In an embodiment, PfRipr is expressed as a soluble monomer, a soluble homodimer or a combination thereof.
Illustrative strains or genotypes from which the polypeptides or their sequences may be derived include 7G8, 3D7 (reference strain), DD2, HB3, W2MEF, D10, GHANA1, T994, CSL-2, EBB, MCAMP, PREICH, RO-33, V1_S, SANTALUCIA, SENEGAL3404, K1, FCR-3, FCC-2, and D6.
“Derived from” includes directly or indirectly derived from.
In an embodiment, PfRipr comprises the amino acid sequence set forth in SEQ ID NO: 6 or a sequence or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical thereto, or a biologically or immunologically active fragment thereof.
In an embodiment, PfRh5 comprises the amino acid sequence set forth in SEQ ID NO: 8 or 9; or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical to SEQ ID NO: 8 or 9; or a biologically or immunological active fragment thereof.
In an embodiment, PfCyrPa comprises the amino acid sequence set forth in SEQ ID NO: 7 or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical thereto, or a biologically or immunologically active fragment thereof.
In an embodiment, PfEBA175 comprises the amino acid sequence set forth in SEQ ID NO: 10 or 27 or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical thereto, or a biologically or immunologically active fragment thereof.
In one embodiment, the heterologous polynucleotide encoding a PfRipr polypeptide and a suitable signal sequence operably linked to a promoter which induces PfRipr polypeptide expression in the cell comprises the sequence set forth in SEQ ID NO: 14.
In another embodiment, the cell further comprises a heterologous polynucleotide encoding PfRh5 polypeptide and a suitable signal sequence operably linked to a promoter which induces polypeptide expression the cell. In an embodiment, the polynucleotide comprises the sequence set forth in SEQ ID NO: 15. In one embodiment, co-expression of PfRipr and PfRh5 polypeptides in a Drosophila culture cell induces increased yields of both polypeptides compared to mono-expression of either polypeptide in the same cells.
In one embodiment, the cell further comprises a heterologous polynucleotide encoding a PfCyrPa polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in the cell. In an embodiment, the polynucleotide comprises the sequence set forth in SEQ ID NO: 16.
In one embodiment PfEBA175 is expressed together with PfRipr, PfRh5, PfCyrPa in the expression host cell.
Thus in one embodiment, the cell of the present invention may express a PfRipr, a PfRh5, or a PfCyrPa polypeptide, individually, a PfRipr and a PfRh5 polypeptide together, a PfRh5 and a PfCyrPa polypeptide together, a PfRipr and a PfCyrPa polypeptide together, or a PfRipr and a PfRh5 and a PfCyrPa polypeptide together. Reference to “together” includes substantially as a complex and/or substantially as an uncomplexed mixture of protein.
In another embodiment, the expression host cell expresses a PfRipr, a PfRh5, a PfCyrPa or a PfEBA175 polypeptide individually, a PfRipr, a PfRh5 and a PfEBA175 polypeptide together, or a PfRh5, a PfCyrPa and a PfEBA175 polypeptide together, or a PfRipr, a PfCyrPa and a PfEBA175, or a PfRipr and a PfRh5 and a PfCyrPa polypeptide and a PfEBA175 polypeptide together.
In one embodiment, the heterologous polynucleotide or vector expressing the subject polypeptides encodes one or two or three or four heterologous polypeptides.
In one embodiment, A Drosophila derived cell modified to express a Plasmodium falciparum (Pf) Ripr polypeptide, a PfRh5 polypeptide and a PfCyrPa polypeptide from a heterologous polynucleotide encoding a PfRipr polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfRipr polypeptide expression, a PfRh5 polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfRh5 polypeptide expression and a PfCyrPa polypeptide and a suitable signal sequence operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in a Drosophila derived cell wherein the PfRipr polypeptide, PfRh5 polypeptide and PfCyrPa polypeptide from said cell induces parasite invasion inhibitory antibodies when administered to a subject.
In one embodiment the cell further comprises a heterologous polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence operably linked to a promoter which induces EBA175 polypeptide expression in the cell. In one embodiment amino acids 760-1298 of EBA175 allele derived from 3D7 strain are expressed (SEQ ID NO: 17). In one embodiment, amino acids 761-1271 of EBA175 allele derived from W2MEF are expressed (SEQ ID NO: 27).
Thus the cell of the present invention may express a PfRipr polypeptide and one or more of a PfRh5 polypeptide, a PfCyrPa polypeptide, and a PfEBA175 polypeptide. In one embodiment, the cell may express a PfRipr polypeptide and a PfRh5 polypeptide, or a PfRipr polypeptide and a PfCyrPa polypeptide, or a PfRipr polypeptide and a PfEBA175 polypeptide. In one embodiment, the cell may express a PfRipr polypeptide, a PfRh5 polypeptide and a PfCyrPa polypeptide.
In one embodiment PfRipr is expressed in a Drosophila cell. In one embodiment PfRh5 and/or PfCyraPa and/or PfEBA175 are expressed in an expression system other than a Drosophila cell. A wide range of expression systems are known in the art and some are described herein. Protein expression systems suitable for clinical biomanufacture include baculovirus expression systems and mammalian expression systems and prokaryotic expression systems.
In another aspect the description provides an expression vector comprising, a polynucleotide encoding a PfRipr polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfRipr polypeptide expression in a Drosophila continuous culture cell, wherein the PfRipr polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.
In another aspect the description provides an expression vector comprising, a polynucleotide encoding a PfRh5 polypeptide including a truncated form and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfRh5 polypeptide expression in an expression host cell, wherein the PfRh5 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.
In on embodiment, the PfRh5 polypeptide is a truncated from of PfRh5 polypeptide comprising amino acids D126 through to Q526 of full length PfRh5. In one embodiment the truncated form of PfRh5 is formed in S2 cell culture.
In another aspect the description provides an expression vector comprising, a polynucleotide encoding PfCyrPa polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in an expression host cell, wherein the PfCyrPa polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.
In one embodiment, the description provides an expression vector comprising, a polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfEBA175 polypeptide expression in an expression host cell, wherein the PfEBA175 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.
In one embodiment, the expression host cell and expression vector are compliant with current good manufacturing practices.
In another aspect the description provides an expression vector comprising, a polynucleotide encoding a PfRh5 polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces high level of PfRh5 polypeptide expression in a Drosophila continuous culture cell, wherein the PfRh5 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject, and wherein the polypeptide is a truncated from of PfRh5 polypeptide comprising amino acids D126 through to Q526 of full length PfRh5.
In another aspect the description provides an expression vector comprising, a polynucleotide encoding PfCyrPa polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces high level of PfCyrPa polypeptide expression in a Drosophila continuous culture cell, wherein the PfCyrPa polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.
In one embodiment, the description provides an expression vector comprising, a polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence, said polynucleotide operably linked to a promoter which induces PfEBA175 polypeptide expression in a Drosophila continuous culture cell, wherein the PfEBA175 polypeptide from said cell induces parasite growth inhibitory antibodies when administered to a subject.
In one embodiment the polynucleotide encoding a PfEBA175 polypeptide and a suitable signal sequence operably linked to a promoter induces EBA175 polypeptide expression in a host cell. In an embodiment, the polynucleotide comprises the sequence set forth in SEQ ID NO: 17.
In one embodiment, amino acids 760-1298 of EBA175 allele derived from the 3D7 strain are encoded.
In one embodiment, amino acids 761-1271 of EBA175 allele derived from W2MEF are encoded.
In another aspect the description provides a Drosophila cell comprising one, two or three vectors selected from a vector as described herein. In an embodiment, the one, two or three vectors are stably transfected into the Drosophila cell. In an embodiment, the one, two or three vectors are transiently transfected into the Drosophila cell.
In another aspect the description provides a Pf polypeptide or Pf protein complex or Pf protein mixture purified from the cell as described herein.
In another aspect the description provides a purified Pf protein complex or mixture wherein the complex or mixture comprises PfRipr polypeptide, PfRh5 polypeptide comprising only amino acids D126 through to Q526 of full length PfRh5 (lacking amino acids 1 to 125), and PfCyrPa polypeptide, and wherein the purified Pf protein complex or mixture induces parasite invasion inhibitory antibodies when administered to a subject.
In another aspect the description provides an immunogenic composition comprising the purified Pf complex or mixture as described herein. Thus, in one embodiment, the immunogenic composition comprises a PfRipr polypeptide, a PfRh5 polypeptide, and a PfCyrPa polypeptide. In one embodiment, the PfRh5 polypeptide comprises only amino acids D126 through to Q526 of full length PfRh5 (lacking amino acids 1 to 125) or a modified form thereof. In one embodiment, the immunogenic composition further comprises a PfEBA175 polypeptide. In one embodiment, the EBA175 polypeptide comprises amino acids 760-1298 of EBA175 allele derived from the 3D7 strain. In one embodiment, the EBA175 polypeptide comprises amino acids 761-1271 of EBA175 allele derived from W2MEF.
In another aspect the description provides a Virus like particle (VLP) or virosome comprising the purified Pf protein or complex or mixture as described herein.
Thus in one embodiment, the description provides VLP or virosome comprising one or more of PfRipr polypeptide, a PfRh5 polypeptide, a PfCyrPa polypeptide, and a PfEBA175 polypeptide. In one embodiment, the VLP or virosome is assembled using a conjugation system to covalently attach the Pf protein to the surface of the VLP or virosome to form a medium or high density array of Pf polypeptide. VLP or virosome particles may be prepared using one, two, three or four Pf polypeptides, or complexes of two or three Pf polypeptides as described herein. Single Pf polypeptide VLPs or virosomes may then be combined to form substantially mixtures of Pf polypeptides as described herein.
Thus, in one embodiment, the VLP or virosome or mixture thereof comprises a PfRipr polypeptide, a PfRh5 polypeptide, and a PfCyrPa polypeptide. In one embodiment, the PfRh5 polypeptide comprises only amino acids D126 through to Q526 of full length PfRh5 (lacking amino acids 1 to 125) or a modified form thereof. In one embodiment, the VLP or virosome or substantially mixture thereof further comprises a PfEBA175 polypeptide. In one embodiment, the EBA175 polypeptide comprises amino acids 760-1298 of EBA175 allele derived from the 3D7 strain. In one embodiment, the EBA175 polypeptide comprises amino acids 761-1271 of EBA175 allele derived from W2MEF.
In one embodiment the description provides an immunogenic composition comprising the purified Pf protein or complex or mixture as described herein or the VLP or virosome as described herein, and a pharmaceutically acceptable carrier such as a diluent or excipient, or an adjuvant.
The term “carrier” applied to pharmaceutical compositions refers to a diluent, excipient, or vehicle with which the Pf protein mixture or complex is administered. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
In one embodiment the description provides a method of producing a Plasmodium falciparum (Pf) Ripr polypeptide comprising transfecting a Drosophila derived cell with a vector of as described herein and isolating PfRipr from said cells. In one embodiment, the Pf polypeptides comprise one or more tags to facilitate isolation and purification.
In one embodiment the description provides a method of producing a Plasmodium falciparum (Pf) polypeptide mixture or complex comprising transfecting a population of Drosophila derived cells with one, two, three or four vectors of as described herein. In one embodiment, the method comprises culturing the cells to identify a stable cell line expressing one or more Pf polypeptides. In one embodiment, the method comprises culturing a Drosophila derived cell line comprising one or more vectors as described herein and isolating the Pf polypeptides or complex comprising same from cell culture media. In one embodiment, the Pf polypeptide/s comprise/s one or more tags to facilitate isolation and purification.
In one embodiment the description provides a method of producing an immune response in a subject, the method comprising a administering to the subject an effective amount of the immunogenic composition as described herein.
In one embodiment the description provides a method of treating and/or preventing malaria caused by P. falciparum in a subject, the method comprising administering to the subject an effective amount of the immunogenic composition as described herein.
In one embodiment the description provides an immunogenic composition comprising the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein for use in the treatment and/or prophylaxis of P. falciparum malaria.
In one embodiment the description provides an immunogenic composition comprising the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein for use in the manufacture of a preparation for the treatment or prophylaxis of P. falciparum malaria.
In another embodiment, the immunogenic composition further comprises a pharmaceutically or physiologically acceptable carrier or diluent.
In a further embodiment, the immunogenic composition further comprises an adjuvant. In one illustrative embodiment, the adjuvant is a saponin based adjuvant.
In one illustrative embodiment, the adjuvant is a Virus-like particle (VLP).
In a further embodiment the description provides a kit for detecting if a subject (a) has or had a P. falciparum malaria infection or (b) has been vaccinated against P. falciparum malaria, the kit comprising:
(i) a Pf polypeptide or Pf protein complex or Pf protein mixture as described herein capable of binding an anti-P. falciparum malaria antibody and forming and antigen-antibody complex, and
(ii) one or more antigen-antibody complex detection reagents.
In a further embodiment the present description provides a method for producing a purified antibody against one or more of the herein described proteins produced in a Drosophila derived cell, comprising administering to a subject an immunologically effective amount of the protein, mixture or complex, and isolating and purifying the antibody produced.
In an embodiment the description provides an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein.
In one embodiment, antibodies generated include those that at least partially neutralize an important part of the malaria life cycle such as host cell invasion or schizogony. In some embodiments, antibodies generated against the present Pf polypeptide mixture or complex, inhibit the formation of a complex between PfRipr, PfRh5, a PfCyrPa and thereby
The present description also provides for use of the herein described proteins, mixtures or complex in, or in the manufacture of a diagnostic agent (such as an antibody or protein mixture) for, the diagnosis or monitoring of infection by Plasmodium falciparum or monitoring of an anti-malaria treatment protocol.
In other aspects, screening methods are provided employing the instant proteins, mixtures and complexes to identify binding molecules such as antibodies, antigen-binding fragments or molecules, ligands, peptides, organic or inorganic molecules.
In vitro expression of proteins PfRipr, PfRh5, a PfCyrPa which form a complex in vivo facilitates screening assays to identify and test binding agents or other molecules able to prevent the complex formation or subsequent erythrocyte cell invasion.
In another aspect, the present description provides a kit or a solid or semi-solid substrate comprising one or more of the herein described proteins, mixtures or complex. The kits or substrates of the present invention are contemplated for use in diagnostic, prognostic, therapeutic or prophylactic applications as well as for use in designing and/or screening Plasmodium binding molecules or Plasmodium receptor molecules. The kits and substrates are also useful in monitoring the efficacy of a treatment protocol against Plasmodium falciparum. In an embodiment the present description provides a kit for detecting if a subject has a P. falciparum infection, the kit comprising:
(i) an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein capable of binding a P. falciparum malaria antigen, and
(ii) one or more antigen-antibody complex detection reagents.
In an embodiment the present description provides a kit for monitoring treatment of a subject having a P. falciparum infection, the kit comprising:
(i) an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein capable of binding a P. falciparum malaria antigen, and
(ii) one or more antigen-antibody complex detection reagents.
In an embodiment the present description provides a kit as described herein, wherein the kit is for use in an immunoassay. In an embodiment the kit is an Enzyme Linked Immunosorbent Assay (ELISA)-type kit. In an embodiment the kit is an indirect ELISA type kit.
ELISA—type assays and kits are widely used in the art. In one illustrative embodiment kit comprise one or more detection reagents include/s a binding reagent which binds the antigen-antibody complex. In an embodiment the binding reagent is selected from: (i) protein G or a recombinant version thereof; (ii) protein A or a recombinant version thereof; (iii) isolated and/or recombinant protein A/G; (iv) protein L or a recombinant version thereof; or (v) an antibody or fragment thereof. In an embodiment the binding reagent is detectably labeled. In an embodiment the binding reagent is linked to an enzyme. In an embodiment the one or more detection reagents includes an enzyme substrate.
In an embodiment the present description provides a kit as described herein, wherein the Pf polypeptide, Pf protein complex, Pf protein mixture or isolated antibody raised against the Pf polypeptide, Pf protein complex or Pf protein mixture is conjugated to a solid support. In an embodiment the solid support is a microtitre well or part of a point of care device such as a microfluidic or immunographic device.
In an embodiment, the steps, features, integers, compositions and/or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps or features.
Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.
The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present disclosure.
Many modifications will be apparent to those skilled in the art without departing from the scope of the present disclosure.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
SEQ ID NO: 1: PfRipr Full length (FL) nucleotide sequence;
SEQ ID NO: 2: PfCyrPa nucleotide sequence;
SEQ ID NO: 3: PfRh5 version 1 nucleotide sequence;
SEQ ID NO: 4: PfRh5 version 2 nucleotide sequence;
SEQ ID NO: 5: PfEBA175 nucleotide sequence;
SEQ ID NO: 6: PfRiprFL amino acid sequence;
SEQ ID NO: 7: PfCyrPa amino acid sequence;
SEQ ID NO: 8: PfRh5 Full length (FL) amino acid sequence;
SEQ ID NO: 9: PfRh5 version 1 amino acid sequence;
SEQ ID NO: 10: PfEBA175 amino acid sequence;
SEQ ID NO: 11: pExpreS2-1 vector nucleotide sequence;
SEQ ID NO: 12: pExpreS2-2 vector nucleotide sequence;
SEQ ID NO: 13: pExpreS2-PAC vector nucleotide sequence;
SEQ ID NO: 14: pExpreS2-1 vector comprising PfRiprFL;
SEQ ID NO: 15: pExpreS2-2 vector comprising PfRh5 version 1;
SEQ ID NO: 16: pExpreS2-PAC vector comprising PfCyrPa;
SEQ ID NO: 17: pExpreS2-1 vector comprising PfEBA175;
SEQ ID NO: 18: PfRh5 version 2 amino acid sequence;
SEQ ID NO: 19: His-tag nucleotide sequence;
SEQ ID NO: 20: StrepII purification tag nucleotide sequence;
SEQ ID NO: 21: N-terminal secretion signal nucleotide sequence;
SEQ ID NO: 22: Flag-tag nucleotide sequence;
SEQ ID NO: 23: His-tag amino acid sequence;
SEQ ID NO: 24: StrepII purification tag amino acid sequence;
SEQ ID NO: 25: N-terminal secretion signal amino acid sequence;
SEQ ID NO: 26: Flag-tag amino acid sequence;
SEQ ID NO: 27: PfEBA175 amino acid sequence (760-1271 W2MEF).
Table 1: Exemplary substitutions.
Table 2: Constructs present in each cell line.
Table 3: Lane order of gel from
The subject disclosure is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present disclosure. Practitioners are particularly directed to and Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Remington's Pharmaceutical Sciences (18th ed., Mack Easton, Pa. (1990)), for definitions and terms of the art and other methods known to the person skilled in the art.
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein “subjects” contemplated in the present description are humans or animals including laboratory or art-accepted test or vehicle animals. In an embodiment, the subject is a mammal. Preferably, the subject is a human, however the present description extends to treatment and/or prophylaxis of other mammalian patients including primates and laboratory test animals (e.g. mice, rabbits, rats, guinea pigs).
The terms “protein” and “polypeptide” are generally used interchangeably herein. A polypeptide may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. The % identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970; GCG program) with parameters of a gap creation penalty=5, and a gap extension penalty=0.3. In an embodiment, the query sequence is at least 50 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. In an embodiment, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. In another embodiment, the query sequence is at least 150 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 150 amino acids. Preferably, the GAP analysis aligns two sequences over their entire length. Preferably, the polypeptide has an antigenic activity of at least 10% of the activity of the reference polypeptide.
With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided herein will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide/protein comprises an amino acid sequence which is at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
As used herein, the term “Pf polypeptide” or “Pf protein” refers to a protein from Plasmodium falciparum, a protozoan parasite of the genus Plasmodium that causes malaria in humans.
As used herein, the term “Ripr” refers to the Rh5 interacting protein and “PfRipr” refers to the Rh5 interacting protein from Plasmodium falciparum. In an embodiment, the PfRipr protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof. In an embodiment, PfRipr has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 or a biological active fragment thereof.
As used herein, the term “a Rh5 polypeptide” refers to reticulocyte-binding protein homolog 5 and “a PfRh5 polypeptide” refers to reticulocyte-binding protein homolog from Plasmodium falciparum. In an embodiment, the PfRh5 polypeptide is encoded by nucleotide sequence set forth in SEQ ID NO: 3 or 4 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 3 or 4 or a biologically active fragment thereof. In an embodiment, a PfRh5 polypeptide has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8, 9 or 18 or a biological active fragment thereof. In an embodiment, the PfRh5 polypeptide comprising amino acids D126 through to Q526 of full length PfRh5 (SEQ ID NO: 18).
As used herein, the term “a CyrPa polypeptide” refers to the Plasmodium cysteine-rich protective antigen and “a PfCyrPa polypeptide” refers to the Plasmodium cysteine-rich protective antigen from Plasmodium falciparum. In an embodiment, the PfCyrPa polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO: 2 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 2 or a biologically active fragment thereof. In an embodiment, PfCyrPa has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical to the sequence set forth in SEQ ID NO: 7 or a biological active fragment thereof.
As used herein, the term “a EBA175 polypeptide” refers to the Erythrocyte binding antigen-175 and PfEBA175″ refers to the Plasmodium Erythrocyte binding antigen-175 from Plasmodium falciparum. In an embodiment, the PfEB175 protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 5 or a nucleotide sequence that is at least 50%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 5 or a biologically active fragment thereof. In an embodiment, PfEB175 has an amino acid sequence that is at least 45%, or at least 50%, or at least 54%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identical the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO:11 or a biological active fragment thereof.
The biologically active fragment may have the same or similar activity as, or an enhanced activity/immunogenicity relative to a reference polypeptide.
The Pf proteins as described herein may comprise one or more mutations e.g. an amino acid substitution, deletion or insertion or may comprise a chemical analog not present in the naturally occurring protein. Chemical analogs contemplated include modification of side chains, incorporation of unnatural amino acids and/or their derivatives during synthesis and the use of linkers or cross-linkers or other methods to inter alia impose conformational constraints.
Amino acid sequence mutants of the polypeptides defined herein can be prepared by introducing appropriate nucleotide changes into a polynucleotide defined herein, or by in vitro synthesis of the desired polypeptide. Such mutants include for example, deletions, insertions, or substitutions of residues within the amino acid sequence. A combination of deletions, insertions and substitutions can be made to arrive at the final protein, provided that the final polypeptide product possesses the desired characteristics.
Mutant (altered) polypeptides can be prepared using any technique known in the art, for example, using directed evolution or rational design strategies.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series for example, by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site. Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. Substitution mutants have at least one amino acid residue in the polypeptide removed and a different residue inserted in its place. Such conservative substitutions are shown in Table 1 under the heading of “exemplary substitutions”.
In one embodiment a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 1. Mutants with desired activity may be engineered using standard procedures in the art such as by performing random mutagenesis, targeted mutagenesis, or saturation mutagenesis on known genes of interest, or by subjecting different genes to DNA shuffling.
Modified Pf polypeptides are tested for their ability to bind to, engender or compete for binding with invasion inhibitory or non-inhibitory antibodies (binding agents) that bind to Pf merozoites in vitro or in vivo. Such assays are commonly employed in the art.
In an embodiment the mutation may comprise the insertion of a “linker”. The term “linker” is used herein to refer to a short, flexible, polypeptide sequence of one or more amino acid residues in length. In an embodiment, the mutation comprises removal of one or more amino acid residues in the polypeptide and replacement with one or more residues with a linker. In an embodiment, the linker permits disulfide bond linkages between cysteine residues leading to retention of the native or “wild-type” disulfide linkages, and in particular retention of the ability to bind to conformation dependent antibodies. Suitable linker sequences are discussed in review articles by George and Heringa, 2002, and Argos, 1990, and may consist of up to 20 amino acid residues such as Gly and Ser, and include, and comprise amino acids selected from the sequence group consisting of Gly, Ser, Ala, Thr and Arg, more particularly Gly- and Ser-Ser-Gly (GSSG). Suitable linkers include, by way of example, the sequences (Gly) 2-Ala-(Gly)2, (Gly)5 or (Gly)s (see Sabourin et al., 2007), (Gly)6, (Gly)7 or (Gly) lo, Gly-Ser-Gly-Ser-Gly (see Dipti et al., 2006), -21(Gly) 4 (see Gly-Ala-Gly, (Gly)2-Arg-(Gly) 2-Ser (see Bellamy-McIntyre et al., 2007), (Gly-Gly-Gly-Gly-Ser),=3˜4 (see Arai et al., 2006), and Ser-(Gly) 2-Ser-Gly (see Bahrami et al., 2007). One linker may have the sequence Gly-(Ser)2-Gly disclosed herein. It will be understood that selection of suitable linker is a matter of routine experimentation for a person skilled in this field, and the modified polypeptide contemplated herein is not limited to the particular linker sequences disclosed herein.
As used herein, the term “Pf protein complex” refers to a complex comprising a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide. The polypeptides co-localise microscopically and form a functional interacting complex during invasion in vivo and may be co-purified and migrate together in non-reducing SDS-PAGE.
In one embodiment, the complex or proteins are produced using the cells, expression vectors and methods as described herein. The Pf protein complex may comprise PfRipr and PfCyrPa. The Pf protein complex may comprise PfRipr and PfRh5. The Pf protein complex may comprise PfRipr, PfCyrPa and PfRh5. The Pf protein complex or mixture may comprise PfRipr, PfCyrPa and PfRh5. A mixture may include the three polypeptides in an uncomplexed arrangement or in a partially complexed arrangement.
Reference to “Co-expression” includes for example expression in a cell of two or more vectors per cell, and includes one vector expressing two or more Plasmodium polypeptides.
As used herein, the term “Pf protein mixture” refers to mixture comprising two or more Pf proteins wherein the two or more proteins are not present as a complex.
In one embodiment, the proteins are produced using the cells, expression vectors and methods as described herein. In one embodiment the Pf protein mixture or complex contains or consists essentially of PfRipr and PfCyrPa. In one embodiment, the Pf protein mixture or complex contains or consists essentially of PfRipr and PfRh5. In one embodiment the Pf protein mixture or complex contains or consists essentially of PfRipr, PfCyrPa and PfRh5.
The term “isolated” and “purified” isolated means material that is substantially or essentially free from other components that normally accompany it in its native state. For example, an “isolated nucleic acid molecule” refers to a nucleic acid or polynucleotide, isolated from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. In particular, an isolated Pf polypeptide, Pf protein complex or Pf protein mixture includes in vitro isolation and/or purification of a protein from its natural cellular environment, and from association with other components of the cell. Without limitation, an isolated nucleic acid, polynucleotide, peptide, or polypeptide can refer to a native sequence that is isolated by purification or to a sequence that is produced by recombinant or synthetic means. In an embodiment, the Pf polypeptides as described herein may be isolated or purified as a Pf protein complex. In an embodiment, the Pf polypeptides as described herein may be isolated individually or as a Pf protein mixture and subsequently folded into a Pf protein complex.
Polynucleotides
As used herein, the term “polynucleotide” or “nucleotide” refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A “heterologous polynucleotide” of the invention may be of genomic, cDNA, semisynthetic, or synthetic origin, double-stranded or single-stranded and by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature. The following are non-limiting examples of polynucleotides: messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, chimeric DNA of any sequence.
As used herein, an “isolated polynucleotide” refers to a polynucleotide which has been separated from the polynucleotide sequences with which it is associated or linked in its native state, or a non-naturally occurring polynucleotide. As used herein, an “exogenous polynucleotide” or “heterologous polynucleotide” refers to a polynucleotide produced or originating outside a particular host organism or cell.
The heterologous polynucleotide described herein encoding Pf polypeptides comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different to that found in nature. Such sequences may include a promoter, nucleotides encoding a signal sequences or sequences comprising or encoding a tag to aid expression and/or purification. In one embodiment, the heterologous polynucleotide comprises a promoter which allows for a high level of polypeptide expression when in a cell. The heterologous polynucleotide may comprise an N-terminal secretion signal sequence. In an embodiment the N-terminal secretion signal comprises the amino acid sequence “MKLCILLAVVAFVGLSLG” (SEQ ID NO: 25). The heterologous polynucleotide may comprise or encode a tag to aid purification of the expressed polypeptide. In one embodiment the tag is a His-tag comprising the amino acid sequence “HHHHHH” (SEQ ID NO: 23). In an embodiment, the tag is a StrepII purification tag comprising the amino acid sequence “SAWSHPQFEK”. In one embodiment the tag is a Flag-tag comprising the amino acid sequence “DYKDDDDK”. In another embodiment, the heterologous polynucleotide may comprise a sequence which aids the formation of virus like particles. In an embodiment, the tag facilitates controlled assembly of one or more Pf proteins as described herein in desirable (such as immune-protective) proportions in a VLP.
Polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Polynucleotides which have mutations relative to a reference sequence can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shuffling on the nucleic acid as described above).
In an embodiment, the polynucleotide may be codon optimised to increase expression of the isolated and/or recombinant protein or antigenic fragment thereof in a particular host cell. For example, the polynucleotide may by codon optimised for expression in Drosophila. The term “codon optimised” refers to modification of the codon encoding a particular amino acid to increase the expression of a protein in a given host cell. In one embodiment, endogenous glycosylation sites are retained.
Expression Systems
The selection of a suitable host organism for expression purposes is determined by various factors which are well known in the art. Factors to be considered include, for example, compatibility with the selected vector, toxicity of the expression product, expression characteristics, immunogenicity of the expressed product, necessary biological safety precautions and costs.
In some instances it may be desirable to insert the heterologous polynucleotide into an expression vector. In an embodiment, the expression vector may be transferred into a cell and the cell used to produce a Pf protein, Pf protein complex, or Pf protein mixture as described herein.
As used herein, a “vector” or “expression vector” is a DNA or RNA vector that is capable of transforming a host cell and effecting expression of one or more polynucleotides. In one embodiment, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic. Expression vectors include any vectors that function (i.e., direct gene expression) in host expression cells of the present invention. The vector may be, e.g., a plasmid, virus, artificial chromosome, or a bacteriophage. Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophages, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids. In an embodiment, the vector is suitable for expression of Pf protein, Pf protein complex, or Pf protein mixture in an Arthropoda host cell such as a Drosophila cell. In an embodiment, the vector is a DNA polynucleotide comprising a nucleotide sequence having promoter activity in a Drosophila derived cell. In an embodiment, the vector is a viral vector such as a baculovirus (such as for example, AcMNPV that infects Lepidopteran derived cells such as Sf- or Hi-5 cells) retrovirus, a lentivirus, an adenovirus, a herpes virus, a poxvirus or an adeno-associated viral vector. Viral infected cells have a finite life span due to lysis of infected cells. Non-lytic expression systems are also available where the expression vector is stably integrated into the insect genome. Here, expression and secretion does not require cell lysis, making purification straightforward. Expression may be induced or constitutive depending upon the expression control sequence employed. In an embodiment, the vector is pExpreS2-1, pExpreS2-2 pExpreS2-PAC (Expres2ion Biotecnologies, Denmark).
A person skilled in the art will appreciate that a Pf polypeptide, Pf protein complex or Pf protein mixture as described herein can be produced in cell culture by expression of a heterologous polynucleotide as described herein in a host cell. In an embodiment, the host cell is an Arthropoda cell. A person skilled in the art would appreciate that the Arthropoda cell can be any Arthropoda cell which can be cultured in vitro and in which Pf polypeptide, Pf protein complex or Pf protein mixture can be expressed. In one embodiment, the cells are from a “continuous cell line” or can be cultured in “continuous cell culture”. As used herein, the term “continuous cell culture” refers to a culture comprising of a single cell type that can be serially propagated in cell culture for a limited number of cell divisions or indefinitely. The cells as described herein can be cultured in any cell culture medium that allows the expansion of the cells in vitro.
In one embodiment the Arthropoda cell is a Drosophila cell. Methods of culturing Drosphila cells, including large scale production using a bioreactor, are known in the art and are described for example in Swiech et al (2008). In one example, the cells are cultured in at least a 500 mL, a 1 L, a 1.5 L, a 2 L, a 2.5 L or a 3 L volume.
“Cells,” “insect cells”, “Drosophila derived cell” “host expression cells,” “transformed host cells,” and the like are terms that not only refer to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A person skilled in the art will appreciate that the Pf protein, Pf protein complex, or Pf protein mixture as described herein (which can include purified protein or a subunit thereof or a viral particle or subunit thereof comprising the isolated and/or recombinant protein or an antigenic fragment thereof) can be purified by any method known to a person skilled in the art, including, for example one or more of the following steps: centrifugation, microfiltration, antibody purification, depth filtration ultrafiltration, diafiltration, precipitation, bead chromatography (for example size exclusion chromatography, ion exchange chromatography or affinity chromatography), membrane adsorber (for example ion exchange chromatography or affinity chromatography). The Pf proteins as described herein may comprise a tag, such as a His-tag or Flag-tag, which aids purification of the isolated and/or recombinant protein or antigenic fragment thereof.
The present description enables a composition comprising a Pf polypeptide complex or mixture of polypeptides wherein the Pf polypeptide complex or mixture is substantially free of Pf molecules other than a soluble PfRipr polypeptide, a soluble PfCyrPa polypeptide and a soluble PfRh5 polypeptide.
As used herein the phrases “substantially free” refers to a composition comprising less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1% aggregated Pf polypeptide (by weight).
In one embodiment, the proportion of Pf non-monomeric forms is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% by weight.
In a particular embodiment, the compositions are substantially free of aggregated Pf polypeptide having less than 1% or less than 0.1% aggregated Pf molecules.
In another embodiment, the present description enables a composition comprising a Pf polypeptide complex comprising or consisting of a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide wherein the Pf polypeptide complex is substantially free or depleted of uncomplexed Pf molecules.
In another embodiment, the present description enables a composition comprising a Pf polypeptide complex comprising a PfRipr polypeptide and a PfCyrPa polypeptide, or a PfRipr polypeptide and a PfRh5 polypeptide, or a PfCyrPa and a PfRipr polypeptide, a wherein the composition further comprises a PfRipr polypeptide, a PfCyrPa polypeptide or a PfRh5 polypeptide in uncomplexed form.
As used herein the phrases “substantially free or depleted” refers to a composition comprising less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1% uncomplexed Pf polypeptide (by weight).
In one embodiment, the proportion of uncomplexed Pf polypeptide is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% by weight.
In a particular embodiment, the immunogenic compositions are substantially free of uncomplexed Pf polypeptide having less than 1% or less than 0.1% uncomplexed Pf molecules.
In one embodiment reference to a “mixture of Pf polypeptides” includes a proportion of complex polypeptide such as a composition comprising from 0% to 80% complexed Pf polypeptide and more than 20% uncomplexed Pf polypeptide. In one embodiment reference to a “mixture of Pf polypeptides” includes a composition having more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (by weight) of uncomplexed Pf polypeptide.
In a related aspect, the immunogenic composition or VLP is enriched for or comprises a Pf polypeptide complex comprising two or three of a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide and includes preparations of the composition having more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (by weight) of the Pf complex. In an embodiment, the composition or VLP further comprises up to 30% uncomplexed Pf polypeptide selected from one or more of a PfRipr polypeptide, a PfCyrPa polypeptide, PfRh5 polypeptide and an EBA175 polypeptide.
In another embodiment, the present description enables a composition comprising a Pf polypeptide complex comprising a PfRipr polypeptide, a PfCyrPa polypeptide and a PfRh5 polypeptide, wherein the Pf polypeptide complex is conjugated at high density to a virus like particle or virosome. In another embodiment individual polypeptides of the complex are displayed separately on the VLP. Illustrative VLPs are known in the art and includes those using conjugation systems such as the SpyTag/SpyCatcher system and variations thereof. In one illustrative embodiment one or more of the Pf polypeptides is genetically modified to comprise a c-terminal Spycatcher tag which is used to display the complex via covalent interaction with a Spytag attached to Acinetobacter phage AP205 VLPs modified to display at least one tag per VLP subunit.
In some embodiment, the mixture or complex or composition further comprises EBA175 as described herein.
The term “immunogenic composition”, “vaccine” or “vaccine composition as used herein refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in a subject and possibly but not necessarily one or more additional components that enhance the immunological activity of said active component (for example an adjuvant).
As used herein, the term “pharmaceutically acceptable carrier” includes any and all solids or solvents (such as phosphate buffered saline buffers, water, saline) dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. The use of such media and agents for pharmaceutically active substances is well known in the art. Immunogenic compositions are described in a number of sources that are well known and readily available to those skilled in the art, for example, Remington's Pharmaceutical Sciences (Martin E. W., Easton Pa., Mack Publishing Company, 19.sup.th ed., 1995).
An immunogenic composition is formulated to be compatible with its intended route of administration, e.g., local or systemic. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, nasal, topical, transdermal, transmucosal, and rectal administration. Oral and nasal administration include administration via inhalation. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Immunogenic compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, non-aqueous solutions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminium monostearate or gelatine.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatine capsules. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatine; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays, drops, or suppositories. For transdermal administration, the active compound (e.g., polynucleotides of the description) are formulated into ointments, salves, gels, or creams, as generally known in the art.
The immunogenic compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the immunogenic composition further comprises an adjuvant. Subject responses to immunogens can be enhanced if administered as a mixture with one or more adjuvants. Immune adjuvants typically function in one or more of the following ways: (1) immunomodulation (2) enhanced presentation (3) CTL production (4) targeting; and/or (5) depot generation. Illustrative adjuvants include: particulate or non-particulate adjuvants, complete Freund's adjuvant (CFA), aluminum salts, emulsions, ISCOMS, LPS derivatives such as MPL and derivatives thereof such as 3D, mycobacterial derived proteins such as muramyl di- or tri-peptides, particular saponins from Quillaja saponaria, such as QS21 and ISCOPREP™ saponin, ISCOMATRIX™ adjuvant, and peptides, such as thymosin alpha 1 or a VLP. In some embodiments, a VLP includes proteins from one or more of the following: an influenza virus (e.g., a hemaglutinin (HA) or neuraminidase (NA) polyptide), Hepatitis B virus (e.g., a core or capsid polypeptide), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human papilloma virus, HIV, RNA-phages, Q-phage (e.g., a coat protein), GA-phage, fr-phage, AP205 phage, a Ty (e.g., retrotransposon Ty protein p1). See, e.g., WO03/024480, WO03/024481, WO08/061243, and WO07/098186. An extensive description of adjuvants can be found in Cox and Coulter, “Advances in Adjuvant Technology and Application”, in Animal Parasite Control Utilizing Biotechnology, Chapter 4, Ed. Young, W. K., CRC Press 1992, and in Cox and Coulter, Vaccine 15(3): 248-256, 1997.
The description includes a method for prophylactic or therapeutic treatment of Plasmodium infection in a patient, which comprises administration to the patient of an effective amount of a composition as described herein. Reference herein to “treatment” is to be understood in its broadest context. Accordingly, the term “prophylactic treatment” includes treatment to protect the patient against infection or to reduce the likelihood of infection. Similarly, the term “therapeutic treatment” of infection does not necessarily imply that the patient is treated until total recovery from infection, and includes amelioration of the symptoms of infection as well as reducing the severity of, or eliminating, the infection.
The immunogenic composition as described herein is administered in an effective amount. An “effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of the infection. The amount varies depending upon the health and physical condition of the individual to be treated, the racial background of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. If necessary, the administration of an effective amount may be repeated one or several times. The actual amount administered will be determined both by the nature of the infection which is being treated and by the rate at which the active immunogen or composition as described herein is being administered.
With regard to “prophylactic treatment” which can include vaccination a subject may be administered with one or more doses to achieve an effective immune response.
In accordance with the present description, the composition as described herein is administered to a patient by a parenteral or non-parenteral routes of administration. Parenteral administration includes any route of administration that is not through the alimentary canal (that is, not enteral), including administration by injection, infusion and the like. Administration by injection includes, by way of example, into a vein (intravenous), an artery (intraarterial), a muscle (intramuscular) and under the skin (subcutaneous). The composition as described herein may also be administered in a depot or slow release formulation, for example, subcutaneously, intradermally or intramuscularly, in a dosage which is sufficient to obtain the desired pharmacological effect.
In accordance with the methods and uses as described herein, a subject may receive a therapeutically effective amount of the composition as described herein in one or more doses. A person skilled in the art will understand that the composition as described herein, in particular a vaccine composition may be administered to a subject more than once and can be administered on any appropriate schedule, e.g., from one or more times per day to one or more times per week; including once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months, 6 months, or more, or any variation thereon. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat, prevent or ameliorate a condition in a subject, including but not limited to previous treatments, the general health and/or age of the subject, and other diseases present. The skilled person will further understand that the compositions as described herein may be administered with one or more other immunogens suitable for the methods and uses described herein.
Sustained-release preparations that may be prepared are particularly convenient for inducing immune responses. Examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. Liposomes may be used which are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30% cholesterol, the selected proportion being adjusted for the optimal therapy.
Stabilization of proteins may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. The in vivo half-life of proteins may be extended using techniques known in the art, including, for example, by the attachment of other elements such as polyethyleneglycol (PEG) groups.
Prime-boost immunization strategies as disclosed in the art are contemplated. See for example International Publication No. WO/2003/047617. Thus, compositions may be in the form of a vaccine, priming or boosting agent.
The present description enables antibodies or antigen binding portions raised against the herein described Pf polypeptide or Pf protein complex or Pf protein mixture, and their derivatives, by routine protocols.
Immunoassay
A person skilled in the art will appreciate that a Pf protein, Pf protein complex, or Pf protein mixture as described herein or an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein are suitable for use in an immunoassay. Exemplary immunoassay formats include immunoblot, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and enzyme immunoassay and assays suitable for point of care such as microfluidic assays and immunochromatographic device formats.
In an embodiment an immunoassay as described herein may comprise immobilizing an antigen, for example a Pf protein, Pf protein complex, or Pf protein mixture as described herein, to a solid support e.g. a membrane or microtitre well. A sample from a subject is then brought into physical relation with the Pf protein, Pf protein complex, or Pf protein mixture, and anti-P. falciparum malaria antibody/antibodies in the sample are bound or ‘captured’ by Pf protein, Pf protein complex, or Pf protein mixture. Binding of an anti-P. falciparum malaria antibody/antibodies to the Pf protein, Pf protein complex, or Pf protein mixture results in an antigen-antibody complex. The antigen-antibody complex is then detected by one or more detection reagents.
In an alternate embodiment an immunoassay as described herein may comprise immobilizing an antibody, for example an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein to a solid support. A sample from a subject is then brought into physical relation with the isolated antibodies and P. falciparum malaria antigens in the sample are bound or ‘captured’ by the isolated antibody forming an antigen-antibody complex. The antigen-antibody complex is then detected by one or more detection reagents.
In an embodiment, the immunoassay is an ELISA. A person skilled in the art will appreciate that the ELISA may be a colorimetric, chemiluminescent or fluorescent. Furthermore, a person skilled in the art will appreciate that the ELISA can be a direct or an indirect ELISA.
In a direct ELISA the antigen-antibody complex is bound (or detected) by a binding reagent that is detectably labelled. In an indirect ELISA the binding reagent may be conjugated to an enzyme which acts upon an enzyme substrate to produce a detectable signal. Alternatively, in an indirect ELISA the antigen-antibody complex is bound by a first binding reagent (e.g. a primary antibody), which is bound by a second binding reagent (e.g. a secondary antibody), wherein the second binding reagent is detectably labelled or is conjugated to an enzyme which acts upon an enzyme substrate to produce a detectable signal.
It will be appreciated by a person skilled in the art that detection reagents may include, but is not limited to, one or more of the following: a binding reagent, a binding reagent conjugated to a detectable label, a binding reagent conjugated to an enzyme, an enzyme substrate, a wash buffer, a blocking buffer, and a solution for stopping an enzymatic reaction.
As used herein the term “binding reagent” refers to any reagent that binds to the antigen-antibody complex as described herein. In an embodiment, the binding reagent is a native or isolated and/or recombinant protein of microbial origin that binds to mammalian immunoglobulin molecules. In an embodiment the binding reagent, may bind to an Fc region in the antigen-antibody complex. In an embodiment, the binding reagent may bind to a VL-kappa present in the antigen-antibody complex. In an embodiment, the binding reagent binds to IgG, IgM IgA, IgE and/or IgD immunoglobulins. In an embodiment, the binding reagent binds to IgG antibodies. In an embodiment, the binding reagent is protein G or a recombinant version thereof, protein A or a recombinant version thereof, isolated and/or recombinant protein A/G, protein L or a recombinant version thereof.
In an embodiment, the binding reagent is an antibody or an antigen binding fragment thereof. These include immunoglobulins, immunoglobulin fragments or non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. The term “antibody” as used herein includes monoclonal antibodies, bispecific antibodies, fusion diabodies, triabodies, heteroconjugate antibodies, chimeric antibodies including intact molecules as well as fragments thereof, and other antibody-like molecules. Antibodies include modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light (VL) and heavy chain (VH) variable regions which may be joined directly or through a linker, or Fd fragments containing the heavy chain variable region and the CH1 domain.
A scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody (Bird et al., 1988; Huston et al., 1988) and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term “antibody”. Also encompassed are fragments of antibodies such as Fab, (Fab′)2 and FabFc2 fragments which contain the variable regions and parts of the constant regions. Complementarity determining region (CDR)-grafted antibody fragments and oligomers of antibody fragments are also encompassed. The heavy and light chain components of an Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region. The antibody may be of animal (for example mouse, rabbit or rat) or may be chimeric (Morrison et al., 1984). The antibody may be produced by any method known in the art.
The antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (VH) chain in which the light and heavy chains may be joined directly or through a linker. As used herein a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed. Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.
In an embodiment, the binding reagent comprises a detectable label. In an embodiment, the binding reagent is conjugated to an enzyme. In an embodiment, the binding reagent is conjugated to a protein such as biotin or streptavidin so that it may interact with a detectable label or enzyme that is streptavidin or biotin conjugated.
In an embodiment, the detection reagent comprises an enzyme. In an embodiment, the enzyme, may be, but is not limited to being alkaline phosphatase (AP), a peroxidisae such as horseradish peroxidase enzyme (HRP), β-D-galactosidease. In an embodiment, the enzyme is HRP.
A person skilled in the art will appreciate that the enzyme substrate can be any enzyme substrate suitable for use with one of the aforementioned enzymes wherein when acted upon by an enzyme, a detectable signal is produced. A person skilled in the art will appreciate that the enzyme substrate may be, for example, a colorimetric substrate, chemiluminescent substrate or fluorescent substrate.
In an embodiment, the solid support may be, but is not limited to being a membrane or microtitre well. In an embodiment, the solid support may be polyethylene, polypropylene or glass. In an embodiment, the solid support is a microtitre well. In an embodiment, the microtitre well is the well of a 96-well or 384-well plate.
Kits
A person skilled in the art would appreciate that the a Pf protein, Pf protein complex, or Pf protein mixture as described herein may be packaged in a kit suitable for detecting the presence of a P. falciparum malaria infection or the presence or detecting vaccination against a P. falciparum malaria infection. A person skilled in the art would further appreciate that an isolated antibody raised against the Pf polypeptide or Pf protein complex or Pf protein mixture as described herein may be packaged in a kit suitable for detecting if a subject has a P. falciparum infection. Such kits may contain one or more immunoassays suitable for detecting a positive or negative marker.
In an embodiment, the kit is for use in an immunoassay. In an embodiment, the kit is an Enzyme Linked Immunosorbent Assay (ELISA) kit. In an embodiment, the kit is an indirect ELISA kit. In an embodiment, the one or more detection reagents of the kit at least includes a binding reagent which binds the antigen-antibody complex. In an embodiment, the kit as described herein is suitable for high-throughput screening. The term “high-throughput screening” refers to screening methods that can be used to test or assess more than one sample at a time and that can reduce the time for testing multiple samples.
The following methods are used in practising the present description.
Vectors.
The PfRiprFL (SEQ ID NO: 1) and PfEBA175 (SEQ ID NO: 5) nucleotide sequences were inserted into the pExpreS2-1 expression vector (ExpreS2ion Biotechnologies, Denmark; SEQ ID NO: 11) which has a marker allowing for Zeocin selection. The PfRh5 sequence (SEQ ID NO: 4) was inserted in to the pExpreS2-2 expression vector (ExpreS2ion Biotechnologies, Denmark; SEQ ID NO: 12) which has a marker allowing for G418 selection and the PfCyrPa sequence (SEQ ID NO: 2) was inserted into the pExpreS2-PAC expression vector (ExpreS2ion Biotechnologies, Denmark; SEQ ID NO: 13) which has a marker allowing for Puromycin selection.
Cell Transfection.
The above vectors comprising PfRiprFL, PfEBA175, PfRh5 or PfCyrPa were transfected into Drosophila melanogaster ExpreS2 S2 cells (ExpreS2ion Biotechnologies, Denmark). On the day before transfection ExpreS2 S2 cells were split by centrifugation and resuspended in Excell420 Serum-Free Medium for Insect Cells (Sigma Aldrich; 14420C) at a density of 8×106 cells/ml and incubated at 25° C. at 115 rpm. On the day of transfection the cells were split by centrifugation and resuspended in Excell420 medium at a concentration of 2×106 cells/mL. A volume of 5 mL of the cell suspension was transferred to a T25 T-flask (CELLSTAR; GR-690160) and 50 μl ExpreS2 Insect-TR 5× (ExpreS2ion Biotechnologies, Denmark) was added to the cell suspension followed by 12.5 μg DNA of the above vectors comprising PfRiprFL, EBA175, PfRh5 or PfCyrPa and mixed. The cells were then incubated for 3 hours at 25° C. before 1 mL of FBS was added. The day after transfection selection was added at the following concentrations: Zeocin 2000 μg/mL; G418 4000 μg/mL; and Puromycin 100 μg/mL.
For the next two weeks the T-flask was counted every 3-4 day and the cells were diluted to 1×106 cells/ml by adding the appropriate amount of fresh Excell420 medium comprising 10% FBS and selection to a final volume of 6 mL. After two weeks the cell suspension was transferred to a T75 flask (CELLSTAR; GR-658170) and 4 mL of Excell420 comprising 10% FBS and selection added. After 4 days, an additional 5 mL of media with selection was added. The cells were then transferred to a 125 mL shake flask (Sigma-Aldrich; CLS431143) after the cells had recovered and 10 ml of Excell420 added. After 4 days, the cells were split by centrifugation and resuspended in 50 ml Excell420 at a density of 8×106 cells/ml in a 250 ml shake flask (Signma-Aldrich; CLS431144). The cells were subsequently frozen in CryoStor® CS10 (Sigma; C2874-100ML) for later use.
The cell lines for co-expression was created by retransfecting a cell line already comprising a vector expressing one of the proteins PfRiprFL, PfEBA175, PfRh5 or PfCyrPa as described above with a second vector. The retransfection was the same as the initial transfection protocol. In this manor Rh5 was transfected into the cell line expressing PfRiprFL. A triple expressing cell line was created by transfecting PfCyrPa in the PfRiprFL, and PfRh5 expressing cell line.
Production of PfRipr in WAVE Bioreactor.
The production of PfRipr was performed by expanding the S2 cell line in shake flasks to a final volume of 1200 mL in Excell420 media (Sigma Aldrich; 14420C) in a 5 l shake flask (Thomson Instrument Company; 931116). The disposable WAVE Bioreactor system (GE Healthcare; WAVE Base 20/50) was used for the final production. The bag (GE Healthcare; CB0050L10-01) was inoculated to a density of 8×106 cells/mL with a volume of 5000 mL Excell420 at 25° C. The system parameters were set to 20 rpm, 8° angle, and 0.85 l/min of atmospheric air. After three days, an additional 20 mL of Excell420 was added to the bag and the parameters adjusted to 26 rpm, 9° angle, and 1.50 l/min of atmospheric air for 3 days. The contents of the bag were harvested by centrifugation and subsequently sterile filtered using 0.22 um filters.
Immunization Protocol for Raising Antibodies Against the PfRIPR/PfRh5/PfCyrPa complex.
Antigens were prepared for immunization by mixing the three individual proteins (PfRIPR/P/Rh5/PfCyrPa) expressed separately in equal molar ratio (1.1.1) based on quantitation of the individual proteins by UV spectroscopy. For each immunization the total antigen quantity of the combined proteins was 100 μg. Since the molecular weight of Ripr is roughly twice that of PfRh5 and PfCyrPa to ensure 100 μg of assembled complex 55 μg of each of PfRh5 and PfCyrPa was added to 125 μg of PfRipr. All proteins are in PBS pH 7.4. Previous analyses showed that complex formation occurs immediately and with high affinity (data not shown). Alternatively, a complex or mixture of proteins isolated from a cell expressing PfRIPR, PfRh5 and PfCyrPa may be used for immunizations. The protein mixture was then mixed with Freund's adjuvant (FA) before injection into a rabbit. A three dose schedule was followed with one immunization in complete FA followed at 28-day intervals with two immunization in incomplete FA.
Analysis of Antibodies Raised Against the PfRIPR/PfRh5/PfCyrPa Complex.
Serum was obtained from blood taken two weeks after the third immunization in the immunization schedule and IgG prepared by protein A sepharose chromatography.
The following non-limiting examples are provided.
Samples were taken from the three cell lines at day three of culture and analyzed by western blot to compare PfRipr expression as shown in
Vectors comprising PfRiprFL, PfRh5, PfCyrPa as outlined above in the Vectors section were transfected separately and in combination with each other in ExpreS2 S2 cells. Each established cell line with their respective expression constructs are listed in the Table 2. The theoretical size of the PfRiprFL, PfRh5, PfCyrPa proteins is 126.67 kDa, 53.80 kDa, and 40.18 kDa, respectively.
The samples from each cell line were prepared by mixing with buffer and reducing agent and incubation at 95° C. for five minutes. Reducing agent was omitted in the lanes noted as un-reduced. The samples were loaded in the order listed below in Table 3. The gel was run for 35 minutes at 165V and blotted with an iBlotter. The membrane was blotted with anti-His or anti-Strep and anti-mouse-HRP as secondary antibody. The blot was then detected by enhanced chemiluminescence and imaged. Results are shown in
Lane 1, 5, and 11 of
The triple-expression of PfRipr, PfRh5, and PfCyrPa (Lane 2, 3, 8, 9, 12, and 13) is also positive for all three constructs. The His-tag shows a positive band for both Rh5 and PfCyrPa in the reduced samples (Lane 2 and 8) while only, what is presumably PfCyrPa, one band is visible in the un-reduced samples (Lane 3 and 9). The band appears as a triplet and is slightly smaller compared to the reduced sample.
The Strep-tag shows a clear band in both the reduced and un-reduced samples. The un-reduced sample appears to be slightly smaller compared to the reduced sample.
Two additives were evaluated to determine if the expression level of PfRipr could be improved. No obvious difference in intensity is observed for the three samples (Lane 14 through 16).
All three constructs are expressed individually and confirmed by western blot and the observed size of each construct correspond to the theoretical size. The co-transfection of PfRipr and PfRh5 was successfully performed and stable cell line expressing both constructs was created.
The triple-expression of PfRipr, PfRh5, and PfCyrPa was successful and the cell line expresses all three constructs. The two additives tested did not have any obvious effect on the yield of PfRipr.
Anti-Ripr antibodies were raised by immunizing a rabbit three times with 100 ug purified Ripr_FL protein in Freund's complete/incomplete adjuvant at day 0, 28 and 56. Serum was obtained from blood taken two weeks after the third immunization and IgG prepared by protein A sepharose chromatography.
To assess if antibodies raised against full-length Ripr inhibit the growth of P. falciparum, P. falciparum 3D7 parasites were incubated with serially diluted purified IgG from either non-immune rabbit serum (NS) or anti-Ripr antiserum (R1682). The parasitaemia in triplicate wells was counted after 96 hours (2 invasion cycles). In the presence of anti-Ripr IgG, merozoite invasion of erythrocytes was inhibited by 55% compared with non-immune rabbit IgG (
The growth inhibition single cycle assay was performed according to methods described in Malkin et al. 2005.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017900648 | Feb 2017 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2018/050155 | 2/23/2018 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/152584 | 8/30/2018 | WO | A |
| Number | Name | Date | Kind |
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| 20150366958 | Chen et al. | Dec 2015 | A1 |
| Number | Date | Country |
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| 2016016651 | Feb 2016 | WO |
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| Number | Date | Country | |
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| 20200030427 A1 | Jan 2020 | US |
