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WO2000063245A9 - Vaccin - Google Patents

Vaccin

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Publication number
WO2000063245A9
WO2000063245A9 PCT/GB2000/001558 GB0001558W WO0063245A9 WO 2000063245 A9 WO2000063245 A9 WO 2000063245A9 GB 0001558 W GB0001558 W GB 0001558W WO 0063245 A9 WO0063245 A9 WO 0063245A9
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WO
WIPO (PCT)
Prior art keywords
name
resid
msp
assign
plasmodium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2000/001558
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English (en)
Other versions
WO2000063245A2 (fr
WO2000063245A3 (fr
Inventor
Anthony Holder
Berry Birdsall
James Feeney
William Morgan
Shabih Syed
Chairat Uthaipibull
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Medical Research Council
Original Assignee
Medical Research Council
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Filing date
Publication date
Priority claimed from GBGB9909072.2A external-priority patent/GB9909072D0/en
Priority to AU41330/00A priority Critical patent/AU779662B2/en
Priority to BR0009823-0A priority patent/BR0009823A/pt
Priority to MXPA01010701A priority patent/MXPA01010701A/es
Priority to HK02104693.0A priority patent/HK1043135A1/zh
Priority to JP2000612331A priority patent/JP2002543774A/ja
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to EP00920918A priority patent/EP1180120A2/fr
Publication of WO2000063245A2 publication Critical patent/WO2000063245A2/fr
Publication of WO2000063245A3 publication Critical patent/WO2000063245A3/fr
Priority to US09/978,756 priority patent/US7078043B2/en
Anticipated expiration legal-status Critical
Publication of WO2000063245A9 publication Critical patent/WO2000063245A9/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to modified Plasmodium MSP-1 protein variants and their use in producing a vaccine against malaria. It also relates to a method for the rational design of suitable variants.
  • Malaria is a devastating disease that causes widespread morbidity and mortality in areas where it is transmitted by anopheline mosquitoes. In areas of high transmission young children and non-immune visitors are most at risk from this disease, which is caused by protozoa of the genus Plasmodium. In areas of lower or unstable transmission, epidemics of the disease can result and afflict individuals of all ages.
  • the most dangerous form of malaria responsible for much of the morbidity and most of the mortality, is caused by the species Plasmodium falciparum. It has been estimated that 2 billion people are at risk from malaria, with 200-300 million clinical cases and 1-2 million deaths each year.
  • the parasite has a complex life cycle in its human and mosquito hosts.
  • the stage of the life cycle which is responsible for the clinical symptoms of the disease occurs in the bloodstream.
  • the parasite is largely hidden within host red blood cells.
  • the parasite grows and multiplies.
  • each P. falciparum parasite divides several times to produce approximately 20 new ones during a 48 hour cycle.
  • the red blood cell is burst open and the parasites (called merozoites at this stage) are released into the bloodstream.
  • the merozoites must enter new red blood cells in order to survive and for the cycle of replication in the blood to continue. If the parasites do not manage to enter red blood cells they cannot survive for very long and are rapidly destroyed.
  • Vaccines against microorganisms can be very cost effective and efficient ways to protect populations against infectious diseases.
  • the pre-erythrocytic stages are the sporozoites that are injected by an infected mosquito when it takes a blood meal and the initial development of the parasite in the liver.
  • the asexual blood stage is the infection and release of merozoites from red blood cells that occurs in a cyclic manner, and the stage responsible for the manifestation of the clinical symptoms.
  • the sexual stage takes place in the mosquito's gut after it has ingested gametocytes in a blood meal and this initiates the infection of the insect to complete the cycle; a vaccine against the sexual stages would not protect the individual but could reduce transmission and therefore the incidence of malaria in a given human population.
  • MSP-1 is a large protein that varies in size and amino acid sequence in different parasite lines. It is synthesised as a precursor molecule of -200 kDa by the intracellular parasite and located on the parasite's surface. During release of merozoites from red blood cells and the re-invasion of new erythrocytes the protein undergoes at least two proteolytic modifications. In the first modification as a result of a process called primary processing, the precursor is cleaved to four fragments of -83, 30, 38 and 42 kDa that remain together as a complex on the merozoite surface. This complex also contains two other proteins of 22 kDa and 36 kDa derived from different genes.
  • the complex is maintained by non-covalent interactions between the different subunits and is held on the merozoite surface by a glycosyl phosphatidyl inositol anchor, attached to the C-terminus of the 42 kDa fragment and inserted into the plasma membrane of the merozoite.
  • a glycosyl phosphatidyl inositol anchor attached to the C-terminus of the 42 kDa fragment and inserted into the plasma membrane of the merozoite.
  • the C-terminal 42 kDa fragment is cleaved by a second proteolytic cleavage in a process called secondary processing.
  • the result of secondary processing is that the entire complex is shed from the surface of the merozoite except for a C-terminal sub-fragment that consists of just under one hundred amino acids and which is carried into the newly invaded erythrocyte on the surface of the merozoite.
  • MSP-1 19 the structure of this small C-terminal fragment (called MSP-1 19 ) was suggested to consist of two epidermal growth factor (EGF)-like domains (see sequence in Figure 1) (Blackman et al, 1991).
  • EGF epidermal growth factor
  • An EGF-like motif consists of a 45-50 amino acid sequence with a characteristic disulphide bonding pattern and such domains occur frequently in extracellular modular proteins of animals.
  • each of the motifs contains six Cys residues proposed to form three disulphide bonds and each motif has a partial match to the EGF consensus (see Figure 1).
  • MSP-1 C-terminal fragment As comprised of EGF- like structures, the designation of the MSP-1 C-terminal fragment as comprised of EGF- like structures has been regarded as tentative. Other relatively divergent potential EGF- like sequences occur in Plasmodium proteins, but previous structure determinations have been confined to those from metazoan organisms (Campbell et al, 1998). A number of studies have implicated MSP-1 as the target of a protective immune response. Although the goal of this work is to develop a malaria vaccine for use in humans, out of necessity most of this experimental work has been done either in model animal systems or in vitro.
  • MSP-1 and in particular polypeptides based on the C-terminal sequence that forms the 42 kDa or the MSP-1 19 region should be very good candidates for malaria vaccine development.
  • epitopes or binding sites for antibodies on MSP-1 19 require a correct polypeptide tertiary structure, and that this is destroyed by treatments that reduce the disulphide bonds that are postulated to be present between the cysteine residues present in MSP-1 19 .
  • This limitation appears to have been overcome by the expression of recombinant protein in ways that allow antibodies that recognise the native parasite MSP- 1 to bind.
  • MSP-1 C-terminal fragment is currently the lead candidate for development of a vaccine against the blood stages of the malaria parasite (Diggs et al, 1993; Stoute et ⁇ Z.,1998).
  • P. falciparum merozoites are released from the infected erythrocyte to re-invade new red blood cells and during this time they are exposed to the host's immune system. Therefore, the question arises as to how the parasite has evolved to avoid the potentially lethal effects of, for example, neutralising antibodies.
  • antigenic variation and antigenic diversity are two mechanisms that involve presenting the immune system with "a moving target" such that even though an immune response to one variant of the micro- organism may kill that variant, new variants are produced that are at least partially or fully resistant to the immune response.
  • blocking antibodies In the case of malaria merozoites and in particular MSP-1 an alternative mechanism has been proposed whereby the binding of some antibodies (“blocking antibodies”) can prevent the binding of neutralising antibodies and thereby allow the parasite to successfully invade a red blood cell even in the presence of neutralising antibodies (Guevara Patino et al, 1997).
  • These blocking antibodies may be of two types, those against epitopes that are formed from amino acids that are distant in the linear primary sequence from the epitopes that are the target of neutralising antibodies, and those that are against epitopes that overlap with the epitopes of the neutralising antibodies. This represents a novel mechanism by which a parasite can evade an effective immune response, and unlike mechanisms based on antigenic polymorphism or diversity, it is not dependent upon amino acid sequence diversity.
  • mAbs monoclonal antibodies that bind to MSP-1 19 inhibit the proteolytic cleavage and erythrocyte invasion, suggesting that cleavage is a prerequisite for invasion.
  • inhibitory antibodies are ineffective and invasion proceeds.
  • the balance between inhibitory and blocking antibodies induced by immunisation may be a critical factor in determining whether or not the immune response is effective in preventing invasion (Guevara Patino et al, 1997). Summary of the Invention
  • An object of the present invention is therefore to provide an effective vaccine against the malaria parasite based on variants of the Plasmodium MSP-1 protein.
  • the following criteria should be met:
  • the amino acid sequence of the polypeptide to be used in the vaccine should contain epitopes that are the targets of, and can induce, neutralising antibodies.
  • the polypeptide should ideally not include amino acid sequences that only form epitopes for blocking antibodies.
  • polypeptide contains epitopes for both neutralising and blocking antibodies then it should be modified to remove the blocking antibody epitopes without affecting the neutralising epitopes.
  • modified MSP-1 1 structures either alone or coupled to other carriers, which may or may not contain other parts of MSP-1 to enhance the immunogenicity (for example a combination of the remainder of the MSP-1 2 kDa fragment with the modified MSP-1 1 ) and provide additional T cell epitopes, would be more effective vaccines than equivalent structures that have not been modified in this way.
  • the present invention provides a non-naturally occurring variant of a C-terminal fragment of a Plasmodium merozoite surface protein- 1 (MSP-1) wherein said variant has (i) a reduced affinity, compared with a naturally occurring Plasmodium MSP-1 19 , for at least one first antibody capable of blocking the binding of a second antibody, which second antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 2 and (ii) substantially the same affinity for said second antibody compared with said naturally occurring Plasmodium MSP-11 9 .
  • MSP-1 Plasmodium merozoite surface protein- 1
  • the Plasmodium MSP-1 1 9 and MSP-1 2 are Plasmodium falciparum MSP-1 19 and MSP-1 42 .
  • the first antibody is preferably selected from mAbs IE1, 2.2, 7.5, 9C8 and 111.4.
  • the second antibody is preferably selected from mAbs 12.8, 12.10 and 5B1.
  • the present invention further provides a non-naturally occurring variant of a C-terminal fragment of a Plasmodium merozoite surface protein- 1 (MSP-1) comprising an amino acid modification at any one of amino acid residues 14, 15, 27, 31, 34 , 43 48 and 53 of the Plasmodium falciparum MSP-11 9 amino acid sequence shown as SEQ LD. No. 1 or their equivalent positions in other Plasmodium MSP-1 19 polypeptides.
  • MSP-1 Plasmodium merozoite surface protein- 1
  • modifications are substitutions selected from Glnl4— A ⁇ g, Glnl4— >Gly, Asnl 5 ⁇ Arg, Glu27 ⁇ Tyr, Leu31 ⁇ Arg, Tyr34 ⁇ Ser, Tyr34 ⁇ Ile, Glu43 ⁇ Leu, Thr48- Lys and Asn53— >Arg and their equivalents in other Plasmodium MSP-1 19 polypeptides.
  • substitutions are combinations of substitutions selected from [Glu27-»Tyr, Leu31 ⁇ Arg and Glu43-»Leu], [Glu27 ⁇ Tyr, Leu31 ⁇ Arg, Tyr34 ⁇ Ser and Glu43-»Leu], [Asnl5 ⁇ Arg, Glu27 ⁇ Tyr, Leu31 ⁇ Arg and Glu43— »Leu] and their equivalents in other Plasmodium MSP-1 1 polypeptides.
  • a variant MSP-1 polypeptide of the invention further comprises a mutation at Cys 12 and/or Cys28 of the Plasmodium falciparum MSP-1 19 amino acid sequence shown as SEQ I.D. No. 1.
  • modifications are substitutions selected from Cysl2 ⁇ Ile and Cys28 ⁇ Trp, and Cys 12— ⁇ Ala and Cys28 ⁇ Phe.
  • substitutions are combinations selected from [Cysl2 ⁇ Ile, Asn 15 ⁇ Arg, Glu27 ⁇ Tyr, Cys28 ⁇ Trp, Leu31 ⁇ Arg, Glu43 ⁇ Leu], [Cysl2 ⁇ Ile, Asn 15 ⁇ Arg, Glu27 ⁇ Tyr, Cys28 ⁇ Trp, Leu31 ⁇ Arg, Glu43 ⁇ Leu, Asn53 ⁇ Arg], [Cysl2 ⁇ Ile, Asn 15 ⁇ Arg, Glu27 ⁇ Tyr, Cys28 ⁇ Trp, Leu31 ⁇ Arg, Tyr34 ⁇ Ser, Glu43 ⁇ Leu, Asn53 ⁇ Arg] and their equivalents in other PlasmodiumMS?- ⁇ ⁇ 9 polypeptides.
  • the present invention also provides a method for producing a Plasmodium MSP-1 variant for use in preparing a vaccine composition which method comprises modifying one or more amino acid residues of a Plasmodium MSP-1 C-terminal fragment such that the resulting derivative has (i) a reduced affinity, compared with a naturally occurring
  • Plasmodium MSP-1 19 for at least one first antibody capable of blocking the binding of a second antibody, which second antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 42 and (ii) substantially the same affinity for said second antibody compared with said naturally occurring Plasmodium MSP-1 19 .
  • the method of the invention preferably comprises as a preliminary step, selecting a candidate amino acid residue by reference to a three dimensional NMR model structure, preferably as set out in Table 2.
  • the 3D model structure is used to select a surface exposed amino acid residue.
  • a further step is included of computer modelling the three dimensional structure of the variant to exclude polypeptides that do not fold correctly.
  • the present invention also provides a non-naturally occurring Plasmodium MSP-1 variant obtained by the method of the invention.
  • the present invention provides a polynucleotide encoding a variant of the invention operably linked to a regulatory sequence capable of directing the expression of said nucleotide in a host cell.
  • the polynucleotide may comprise a sequence which has been optimised for expression in the host cell.
  • the host cell may be a Pic ia pastor is cell.
  • a nucleic acid vector comprising a polynucleotide of the invention, including viral vectors, and a host cell comprising a nucleotide or vector of the invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a variant of the invention, a polynucleotide of the invention or a vector of the invention together with a pharmaceutically acceptable carrier or diluent.
  • the composition further comprises an immunogenic Plasmodium polypeptide or fragment or derivative thereof such as MSP-1 33 or a fragment or derivative thereof which may be covalently attached to the non-naturally occuring MSP-1 19 . It is preferred not to use wild-type MSP-1 1 sequences.
  • the further immunogenic peptide may itself be derivatised in an analogous manner as described above for MSP-1 19 . Thus, epitopes present in the peptide may be identified and modified to prevent binding of blocking antibodies, without affecting the binding of neutralising antibodies. These epitopes may be capable of binding to antibodies which have similar properties to the first antibody described above, for example, in binding affinity.
  • the further immunogenic peptide may comprise several such modifications in its amino acid sequence.
  • the present invention also provides a method for producing anti-MSP-1 antibodies which method comprises administering a polypeptide variant of the invention, or a polynucleotide of the invention or a vector of the invention to a mammal, typically a non- human mammal.
  • the present invention provides a method for producing polyclonal anti-MSP-1 antibodies which method comprises administering a polypeptide variant of the invention, or a polynucleotide of the invention or a vector of the invention to a mammal, typically a non-human mammal, and extracting the serum from said mammal. Also provided is an antibody produced by the said methods.
  • the polypeptides, nucleotides and vectors of the present invention may be used in methods of treating and/or preventing malaria caused by Plasmodium species, in particular Plasmodium falciparum. Accordingly, the present invention provides a method of inducing immunity against malaria induced by Plasmodium falciparum which comprises administering to a person in need of such immunity an effective amount of a variant, a polynucleotide or a vector of the invention.
  • Also provided is a method of immunizing a mammal comprising administering an effective amount of a variant, a polynucleotide or a vector of the invention.
  • said mammal is immunized against malaria.
  • the mammal is a human.
  • the present invention also provides a method of treating a malaria infection in a human patient which comprises administering to the patient an effective amount of the pharmaceutical composition of the invention.
  • a nucleic acid encoding a Plasmodium MSP-1 polypeptide, in which the nucleic acid is optimised for expression in a heterologous host cell.
  • the heterologous host is a Pischia pastoris cell.
  • the MSP-1 polypeptide may be selected from the group comprising an MSP-1 42 polypeptide comprising a sequence shown in Figures 2C and 2E, an MSP-1 19 polypeptide comprising a sequence shown in Figure 2C, and an MSP-1 33 polypeptide comprising a sequence shown in Figure 2E.
  • the optimised nucleic acid may comprise a sequence selected from the sequences of Figure 2 A, Figure 2B and Figure 2D.
  • a vector comprising such a nucleic acid
  • a host cell comprising such a vector
  • a pharmaceutical composition comprising such a nucleic acid or a vector, together with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition may further comprise an immunogenic Plasmodium polypeptide or fragment or derivative thereof.
  • MSP-1 polypeptides of the present invention will be described with reference to Plasmodium falciparum MSP-1 amino acid sequences. However, it should be appreciated that except where otherwise stated, all references to MSP-1 polypeptides include homologues of MSP-1 found in other Plasmodium species, such as P. vivax, E. mala ae and E. ovale which all infect humans and E. yoelii which infects mice.
  • the variant MSP-1 polypeptides of the present invention are based on C-terminal fragments of the Plasmodium falciparum MSP- 1 2 polypeptide shown as S ⁇ Q I.D. Nos. 2 or 3.
  • Such polypeptides will comprise some or all of the MSP-11 region (S ⁇ Q I.D. No. 1), preferably at least substantially all of the domain 1 and/or domain 2 ⁇ GF-like sequences found in MSP-1 19 (approximately amino acids 1-47 and amino acids 48-96, respectively, of S ⁇ Q I.D. No. 1). It is particularly preferred to use regions that are conserved in most, more preferably all parasites of a single species to increase the effectiveness of the variant as a vaccine against a wide range of strains.
  • Variant MSP-1 polypeptides of the present invention comprise modifications to their primary amino acid sequence that reduce the ability of blocking antibodies to bind to the MSP-1 polypeptides.
  • any modifications made should maintain epitopes recognised by neutralising antibodies such that the affinity of the neutralising antibodies for the MSP-1 variant is substantially the same as for naturally-occurring MSP-1 polypeptides (such as an MSP-1 2 polypeptide having the sequence shown in S ⁇ Q I.D. Nos. 2 or 3).
  • Some reduction in the binding of some neutralising antibodies may be tolerated since the primary objective is to inhibit the binding of blocking antibodies and it is likely that an effective reduction in the binding of blocking antibodies will compensate in terms of overall vaccine efficacy for a small reduction in neutralising antibody binding.
  • Neutralising antibodies in the context of the present invention are antibodies that inhibit malaria parasite replication.
  • a variety of neutralising antibodies, polyclonal and monoclonal, are known in the art, including mAbs 12.8, 12.10 and 5B1 referred to in the Examples.
  • the activity of neutralising antibodies can be determined in a variety of ways that have been described in the art. For example, a convenient assay method described in Blackman et al, 1994 involves using preparations of merozoites (Blackman et al, 1993; Mrema et al, 1982) to measure cleavage of MSP-1 42 into MSP-1 33 and MSP-1 19 .
  • This method is a particularly preferred method for assessing the efficacy of neutralising antibodies in the presence of antibodies believed to act as blocking antibodies.
  • candidate competing blocking antibodies are to be tested, the merozoite sample is preincubated with a blocking antibody for 15 mins on ice prior to incubation with a neutralising antibody at 37°C for 1 hour as described above.
  • blocking antibodies can readily be identified and/or characterised using such an assay method.
  • blocking antibodies are defined in the context of the present invention as antibodies that inhibit the binding of neutralising antibodies to MSP-1 but which do not themselves inhibit invasion of red blood cells by malaria parasites. Thus they "block” the neutralising function of the neutralising antibodies.
  • a variety of blocking antibodies have been characterised in the art, including mAbs IE1, 2.2, 7.5 and 1 11.4 referred to in the Examples. As discussed above, blocking antibodies can conveniently be identified and/or characterised using assays that test their effect on neutralising antibody function.
  • Modifications that may be made to produce MSP-1 variants of the invention include substitutions, deletions and insertions. It is particularly preferred to use substitutions to minimise disruption of the secondary/tertiary structure of the polypeptide. Furthermore, particularly preferred substitutions are those that replace one class of amino acid with another class, such as an aliphatic non-polar residue with a charged polar residue.
  • the twenty naturally occurring amino acids may be divided into four main groups (aliphatic non-polar [G, A, P, I, L and V], polar un-charged [C, S, T, M, N and Q], polar charged [D, E, K and R] and aromatic [H, F, W and Y]) and it is preferred to replace an amino acid from one group with an amino acid from another group.
  • Particularly preferred modifications are an amino acid modification at any one of amino acid residues 14, 15, 27, 31, 34 , 43, 48 and 53 of the Plasmodium falciparum MSP-1 19 amino acid sequence shown as SEQ I.D. No. 1 or their equivalent positions in other Plasmodium MSP-1 19 polypeptides. These residues are all almost within the EGF-like domain 1. It is known that the epitopes of some antibodies contain amino acid sequences that are within EGF-like domain 2, therefore equivalent modifications may also be made in EGF-like domain 2.
  • modifications include the following substitutions GlnH ⁇ Arg, Glnl4 ⁇ Gly, Asnl5 ⁇ Arg, Glu27- Tyr, Leu31- Arg, Tyr34 ⁇ Ser, Tyr34 ⁇ Ile, Glu43 ⁇ Leu, Thr48 ⁇ Lys and/or Asn53 ⁇ Arg and their equivalents in other Plasmodium MSP-1 19 polypeptides. It is especially preferred to carry out more than one modification, i.e. to use combinations of modifications, such as two or more or three or more.
  • an MSP-1 variant of the invention comprises a combination of amino acid substitutions selected from [Glu27 ⁇ Tyr, Leu31 ⁇ Arg and Glu43 ⁇ Leu], [Glu27 ⁇ Tyr, Leu31 ⁇ Arg, Tyr34 ⁇ Ser and Glu43 ⁇ Leu], [Asnl5 ⁇ Arg, Glu27 ⁇ Tyr, Leu31 ⁇ Arg and Glu43-»Leu] and their equivalents in other Plasmodium MSP-1 19 polypeptides.
  • a particularly preferred combination further comprises a modification to Cys 12 and/or Cys28 (and/or their equivalent residues in EGF-like domain 2) to disrupt the disulphide bond.
  • modifications are substitutions selected from Cysl2 ⁇ Ile and Cys28 ⁇ Trp, and Cysl2 ⁇ Ala and Cys28 ⁇ Phe.
  • substitutions are combinations selected from [Cys 12— Ile, Asn 15 ⁇ Arg, Glu27 ⁇ Tyr, Cys28 ⁇ Trp, Leu31 ⁇ Arg, Glu43 ⁇ Leu], [Cysl2 ⁇ Ile, Asn 15 ⁇ Arg, Glu27 ⁇ Tyr, Cys28 ⁇ Trp, Leu31- ⁇ Arg, Glu43 ⁇ Leu, Asn53 ⁇ Arg], [Cysl2 ⁇ Ile, Asn 15- ⁇ Arg, Glu27 ⁇ Tyr, Cys28 ⁇ Trp, Leu31 ⁇ Arg, Tyr34 ⁇ Ser, Glu43 ⁇ Leu, Asn53 ⁇ Arg] and their equivalents in other Plasmodium MSP-11 9 polypeptides.
  • substitutions are not confined to using naturally occurring amino acids - non-naturally occurring amino acid analogues may also be used, in particular where solid phase synthesis is to be used to chemically synthesise the variant, as opposed to recombinant technology.
  • Modifications to MSP-1 amino acid sequences may be carried out using standard techniques such as site-directed mutagenesis using the polymerase chain reaction. Alternatively, variants may be obtained by solid phase synthetic techniques.
  • the affinity of at least one neutralising antibody and at least one blocking antibody for the variant polypeptide compared with the naturally occurring MSP-1 sequence may be tested. Ideally more than one of each type of antibody should be used, for example two or three.
  • the ability of antibodies to bind to the variant and wild-type polypeptides may be determined using any one of a variety of methods available in the art for determining antibody-epitope binding.
  • One such method, described in the Examples, involves the use of MSP-1 sequences expressed as fusion proteins with a protein tag such as glutathione-S- transferase (GST). These GST-fusion proteins are typically immobilised to a solid phase such as glutathione sepharose beads or a BIAcore sensor chip. Binding of antibodies, such as monoclonal antibodies, to the fusion proteins may be determined using standard techniques such as Western blotting and/or by labelling the antibodies with a radioactive label such as 125 I. The use of BIAcore technology allows easy quantitation of the results.
  • the reduction in binding of at least one of the blocking antibodies tested is at least 50%) compared to wild-type MSP-1, more preferably at least 75, 80 or 90%>, typically as assessed using recombinantly expressed MSP-1 immobilised to a BIAcore sensor chip.
  • the binding of at least one, for example at least two or three, of the neutralising antibodies tested, more preferably at least half of the neutralising antibodies tested, more preferably substantially all of the neutralising antibodies tested is reduced by less than 50%>, more preferably less than 25%>.
  • the number of neutralising antibodies that need be tested to confirm compliance with the test criteria will not typically exceed from three to five different antibodies (three antibodies are used in the Examples).
  • the binding of at least one neutralising antibody is increased by at least 10%>.
  • the 3D NMR structure will enable the skilled person to carry out preliminary computer modelling studies of MSP-1 19 variants with specific modifications so that, for example variants that cannot fold properly may be discarded. This will assist in minimising the number of candidate MSP-11 9 variants that need be tested.
  • the present invention also provides a computer readable medium having stored thereon a model of the MSP-1 19 NMR structure.
  • said model is built from all or some of the NMR data shown in Tables A and B.
  • Variants of the present invention may optionally include additional MSP-1 sequences, in particular regions of the MSP-1 region of MSP-1 42 to confer additional immunogenicity to the variant.
  • additional sequences known to contain and promote T cell responses are advantageously included (i.e. T cell epitopes).
  • Other modifications may also be made that increase immunogenicity such as modifications that alter the pathway of antigen processing and presentation.
  • Polypeptide variants of the invention are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the immunogenicity of the MSP-1 variant.
  • Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended pu ⁇ ose of the polypeptide and still be regarded as substantially isolated.
  • a polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%., e.g. 95%., 98%) or 99% of the polypeptide in the preparation is a polypeptide of the invention.
  • the variants of the present invention may be produced recombinantly using standard techniques.
  • the present invention also provides a polynucleotide encoding a polypeptide MSP-1 variant of the invention.
  • Polynucleotides of the invention may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • Polynucleotides of the invention comprise can be inco ⁇ orated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
  • the host cell may be a methylotrophic yeast such as Pichiapastoris.
  • the coding sequence of natural or variant MSP polypeptides may be modified for optimal expression in a host cell. For example, secondary modification such as N-glycosylation may be prevented by removal of sequences necessary for such modification.
  • the sequence of the polypeptide may alternatively or in addition be modified with respect to codon usage for optimal expression in the host cell. Methods of mutagenising a sequence are known in the art; alternatively, the modified coding sequence may be generated by means of PCR gene assembly using overlapping synthetic oligonucleotides (Stemmer et al., 1995; Withers-Martinez et al., 1999).
  • a polynucleotide of the invention in a vector is operably linked to a regulatory sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • Such vectors may be transformed or transfected into a suitable host cell using standard techniques above to provide for expression of a polypeptide of the invention.
  • This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and optionally recovering the expressed polypeptides.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • the vector may also be adapted to be used in vivo, for example in a method of gene therapy.
  • Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.
  • prokaryotic promoters may be used, in particular those suitable for use in E. coli strains (such as E. coli HB 101 or DH5 ⁇ ).
  • mammalian promoters When expression of the polypeptides of the invention in carried out in mammalian cells, either in vitro or in vivo, mammalian promoters may be used. Tissue-specific promoters may also be used. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, he ⁇ es simplex virus promoters or adenovirus promoters. All these promoters are readily available in the art.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV promoter rous sarcoma virus
  • CMV human cytomegalovirus
  • variant MSP-1 polypeptides of the present invention and nucleic acid molecules may be used to treat or prevent malaria in animals, specifically humans.
  • the polypeptides of the invention may be administered by direct injection.
  • the polypeptides are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • each polypeptide is administered at a dose of from 0.01 to 30 ⁇ g/kg body weight, preferably from 0.1 to 10 ⁇ g/kg, more preferably from 0.1 to 1 ⁇ g/kg body weight.
  • the polynucleotides of the invention may be administered directly as a naked nucleic acid construct.
  • the amount of nucleic acid administered is typically in the range of from 1 ⁇ g to 10 mg, preferably from 100 ⁇ g to 1 mg.
  • nucleic acid constructs Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • nucleic acid constructs are mixed with the transfection agent to produce a composition.
  • the polynucleotide may be administered as part of a nucleic acid vector, including a plasmid vector or viral vector, such as a vaccinia virus vector.
  • the amount of virus administered is in the range of from 10 to 10 pfu, preferably from 10 to 10 pfu, more preferably from 10 to 10 pfu.
  • typically 1-10 ⁇ l of virus in a pharmaceutically acceptable suitable carrier or diluent is administered.
  • the delivery vehicle i.e. naked nucleic acid construct or viral vector comprising the polynucleotide for example
  • a pharmaceutically acceptable carrier or diluent include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • Vaccines may be prepared from one or more polypeptides of the invention. They may also include one or more immunogenic Plasmodium polypeptides known in the art.
  • a vaccine of the invention may comprise one or more polypeptides of the invention and optionally, one or more polypeptides selected from, for example, the asexual blood stage proteins: apical merozoite antigen- 1, erythrocyte binding antigen 175, erythrocyte membrane protein- 1 ; the hepatic stage proteins: liver stage antigens 1 and 3; the sporozoite stage proteins: circumsporozoite protein , thrombospondin related adhesive protein; and the sexual stage proteins Pfs25 and Pfs28 polypeptides and immunogenic fragments thereof.
  • the other immunogenic Plasmodium polypeptides known in the art do not contain wild type MSP-11 9 sequences.
  • vaccines which contain an immunogenic polypeptide(s) as active ingredient(s), is known to one skilled in the art.
  • such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the protein encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanoi, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A,
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5%) to 10%>, preferably 1%> to 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%> to 95%> of active ingredient, preferably 25%> to 70%>.
  • the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
  • Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
  • the polypeptides of the invention may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanoi, histidine and procaine.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered which is generally in the range of 5 ⁇ g to 250 ⁇ g of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesise antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject.
  • the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgement of the practitioner.
  • the vaccine containing the immunogenic MSP-1 antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins.
  • the variant MSP-1 polypeptides prepared as described above can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing an MSP-1 epitope(s). Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an MSP-1 epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
  • Monoclonal antibodies directed against MSP-1 epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against MSP-1 epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
  • polypeptides of the invention can also be used to select for human monoclonal antibodies using the variable regions of immunoglobulin heavy and light chains cloned in the form of a phage display library, preferably from individuals who have been previously exposed to a natural malaria infection.
  • Antibodies both monoclonal and polyclonal, which are directed against MSP-1 epitopes are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired.
  • anti-idiotype antibodies may also be useful for treatment of Plasmodium infections, as well as for an elucidation of the immunogenic regions of MSP-1 antigens. It is also possible to use fragments of the antibodies described above, for example, F(ab') 2 , Fab, Facb and scFv fragments.
  • FIG. 1 MSP-1 sequences aligned according to the EGF-like motif consensus.
  • Top sequence P. falciparum (SWISS-PROT MSP1 PLAFW).
  • Second sequence P. vivax Belem strain (PIR A45604).
  • Third sequence human EGF (PDB legf).
  • Fourth sequence EGF-like domain consensus (Prosite EGFl).
  • Bottom sequence 14 residue EGF core region used for structure alignment in Figure 6. Black highlighting indicates conserved residues of the EGF-like domain. Dark shading shows hydrophobic residues at the EGF- module pair interface in the P. falciparum, and corresponding conserved residues in the E. vivax sequence.
  • FIG. 2 Sample of multidimensional heteronuclear NO ⁇ SY experiments showing planes containing NO ⁇ connections to the MSP-1 C-terminal fragment Lys35 NH proton.
  • Top 13 C (D4) and ⁇ (D3) plane from the 4D-[ 13 C]-HMQC-NO ⁇ SY-[ I 5 N]-HSQC experiment, taken at the chemical shift values of Lys35 NH in 15 N(D2) and ⁇ (D1).
  • Figure 3 Stereo drawing showing the backbone C, N, C a atoms of the 32 refined structures in the final ensemble.
  • the domain- 1 is on the left (red), with domain-2 on the right (green), and both the N- and C-termini are near the bottom.
  • Figure 4 MOLSCRIPT picture of the most representative model of the ensemble, showing the backbone C ⁇ trace, antiparallel ⁇ -sheet elements, and disulphide bridges (S ⁇ atoms in yellow). Domain- 1, red; Domain-2, green.
  • FIG 5 Alignment of typical EGF-like family members with the fitpdb program, using the 14 amino acid "reduced core” consensus (Bersch et al, 1998) (see Figure 1).
  • the aligned backbone segment in each structure is white.
  • the structures are aligned relative to the most representative structure of the group (factor Xa), with increasing divergence from left to right. Numbers indicate the rmsd value of the aligned C, N, C ⁇ atoms.
  • PDB identification codes factor Xa (crystal structure), lhcg; Complement Clr component, lapq (14 th model); human EGF, legf (11 th model); f ⁇ brillin-1, domains-32 and -33,_lemn (minimized average structure); transforming growth factor- ⁇ , 2tgf (minimized average structure); MSP-1 domains- 1 and -2, this study.
  • FIG 6 Backbone ribbon view of fibrillin-1 versus MSP-1 EGF module pair arrangements. Fibrillin-1 (lemn) cyan (domain-32) and magenta (domain-33) (Downing et al, 1996); MSP-1 domain- 1 (yellow) and domain-2 (green). Structures were aligned as in Figure 6 by the core consensus of the N-terminal domain of each pair. The bound Ca 2+ ions in the fibrillin-1 structure are shown as magenta spheres.
  • Figure 7 Two views, a and b, (rotated 180° about the y-axis) of the electrostatic potential surface of the MSP-1 EGF module pair, calculated with GRASP. Red indicates negative charge, blue indicates positive charge, and white is neutral. The orientation of the views is shown by the adjacent worm diagrams.
  • Figure 8 CPK model of the MSP-1 C-terminal fragment, showing the location of some mutations that affect binding of monoclonal antibodies. Domain- 1 is towards the top and right sides, and domain-2 towards the bottom left.
  • FIG 9 Examples of the binding of monoclonal antibodies to GST-MSP-1 19 detected by Western blotting. The binding of each monoclonal antibody to protein based on the wild type sequence and to proteins containing modified sequences is shown. The monoclonal antibodies are shown across the top.
  • the proteins WT, wild type sequence; 22, Leu22 to Arg; 26, Glu26 to He; 15, Asnl5 to Arg; 27, Glu27 to Tyr; 31, Leu31 to Arg; 43, Glu43 to Leu; 27+31+43, Glu27 to Tyr and Leu31 to Arg and Glu43 to Leu; 15+27+31+43, Asn 15 to Arg and Glu27 to Tyr and Leu31 to Arg and Glu43 to Leu.
  • FIG 10 The binding of monoclonal antibodies to GST-MSP-1 19 detected by BIAcore analysis.
  • the binding of each monoclonal antibody is normalised to 100%> binding to protein based on the wild type sequence and the binding of proteins containing modified sequences is expressed as a percentage of this.
  • WT wild type sequence; 15, Asn 15— > Arg; 26, Glu26 ⁇ Ile; 27, Glu27 ⁇ Tyr; 31, Leu31 ⁇ Arg; 34, Tyr34 ⁇ Ser; 43 Glu43 ⁇ Leu.
  • FIG 11 The binding of monoclonal antibodies to GST-MSP-1 19 containing multiple modifications detected by BIAcore analysis.
  • the binding of each monoclonal antibody is normalised to 100%) binding to protein based on the wild type sequence and the binding of proteins containing modified sequences is expressed as a percentage of this.
  • WT wild type sequence;
  • the combinations contain 3 mutations [27+31+43], or 4 mutations ([27+31+34+43] and [15+27+31+43]), at each site the changes are those identified in Figure 10.
  • Figure 12 Identification of blocking antibodies using a competitive binding assay and immobilised wild type GST-MSP-1 19
  • the ability of antibodies to compete with the binding of mAbs 12.8 and 12.10 to GST-MSP-1 19 was measured using BIAcore analysis. Individual antibodies (x-axis) were bound to the antigen and then the amount of either 12.8 or 12.10 (inhibitory mAb) that could subsequently bind was quantified. The amount of binding is presented as a percentage of the total amount of either 12.8 or 12.10 bound in the absence of pre-incubation with another antibody.
  • FIG. 13 Antibodies induced by immunisation with a modified recombinant MSP-1 19 assayed for their ability to inhibit secondary processing. Washed 3D7 merozoites were either analysed directly without incubation (0 h) or incubated for 1 hour at 37°C in the presence of no serum (no serum), 1 mM PMSF as a control for complete inhibition, normal rabbit sera (normal serum), or serum from a rabbit immunised with the 15+27+31+43 modified protein (immune serum), all at 1 :10 dilution in reaction buffer. The level of MSP-1 33 released into the supernatant as a results of secondary processing was measured using an ELISA method and is represented by Absorbance at 492nm.
  • FIG. 14 Pichia pastoris codon preference table used for input to the CODOP program.
  • FIG. 15 DNA and protein sequences for the optimized synthetic MSP-142 gene.
  • A Complete sequence designed for optimum codon usage and expression in P. pastoris.
  • B Sequence of the synthetic MSP-119 construct in the expression vector pPIC9K-HXa. Uppercase letters: vector sequences, including the His 6 tag and factor Xa cleavage site (IEGR). Lowercase letters: synthetic MSP-119 coding sequence. The cloned sequence in located at the SnaBI restriction site of the pPIC9K sequence.
  • C Expressed protein sequence of the synthetic MSP-119 construct. The sequence shown is produced as a fusion to the pPIC9K ⁇ -factor secretion signal, following the kex2/STE13 processing sites.
  • the synthetic MSP-119 is in bold-face type.
  • D Sequence of the MSP-133 construct. The cloned sequence is located at the Smal site of the pUCl l ⁇ vector.
  • E Predicted protein sequence of the synthetic MSP-133 construct translation product.
  • FIG. 16 Gene assembly PCR reactions for the MSP-133 and MSP-119 sequences. Reaction 1 : 10 ⁇ L aliquots of the assembly reactions. Reaction 2: 20 ⁇ L aliquots of the amplification reactions. The N-terminal and middle fragments were subsequently spliced together to form the MSP-133 synthetic construct. The C-terminal fragment synthesis reactions produced the optimized MSP-119 construct.
  • FIG. 17 Expression of the synthetic MSP-119 protein in P. pastoris.
  • Lanes 1-6 trichloroacetic acid precipitates of secreted recombinant protein from culture supernatants, without further purification (5 ⁇ L each). Samples from duplicate cultures of three independent transformants.
  • Lane 8,9 purified, deglycosylated MSP-1 19 produced from the original P. falciparum sequence.
  • Lane 7,10 NOVEX molecular weight markers.
  • Figure 18 A: ⁇ / 15 N ⁇ -HSQC spectrum of the protein (2.5 mM) expressed from the optimized synthetic MSP-119 gene.
  • B Control ⁇ / 15 N ⁇ -HSQC of deglycosylated protein (2.2 mM) expressed from the original P. falciparum sequence (Morgan et al., 1999).
  • the coding sequence of the MSP-1 C-terminal fragment was cloned by polymerase chain reaction with Vent polymerase (New England Biolabs) from a plasmid containing the Plasmodium falciparum strain T9/94 fragment (Blackman et al, 1991), using primers that included codons for a 6 residue N-terminal His tag (CACCATCATCATCATCAC), and inserted into the SnaBl restriction site of the pPIC9K vector (Invitrogen).
  • the sequence corresponds to residues 1526-1621 of the SWISS-PROT entry MSP1 PLAFW (accession number P04933).
  • a Mut + transformant was grown at 29.4 °C in a shaker-incubator in buffered minimal medium (100 mM potassium phosphate, pH 6.0, yeast nitrogen base (0.34 %> w/vol) (DIFCO: YNB without amino acids and without (NH 4 ) 2 SO 4 ), biotin (4xl0 "5 % w/vol), Sigma antifoam 289 (0.01% vol/vol), and carbon and nitrogen sources as described below. Unlabelled samples were initially grown in medium containing 1 %> w/vol (NH ) 2 S0 and 1 % w/vol glycerol, and induced by transfer to medium containing 0.5 %> CH 3 OH as the carbon source.
  • buffered minimal medium 100 mM potassium phosphate, pH 6.0, yeast nitrogen base (0.34 %> w/vol) (DIFCO: YNB without amino acids and without (NH 4 ) 2 SO 4 ), biotin (4xl0 "5 % w/vol
  • Labelled samples were initially grown in medium containing 0.2 % w/vol [ 15 N]-(NH 4 ) 2 SO 4 (Isotech), and 0.5 % w/vol glucose or [ 13 C 6 ]-glucose (Isotech), and induced by transfer to medium containing as carbon source 0.5 %> w/vol CH 3 OH or [ 13 C]- CH 3 OH (Isotech).
  • the initial cultures were grown in 150 ml to a density of -10 OD 60 o, then harvested and resuspended in methanol medium at 1 OD 600 in a volume of 1.5 L.
  • Methanol-induced cultures were grown for 4 d, with daily addition of 7.5 ml CH 3 OH or [ 1 C]-CH 3 OH, to a final density of -18 OD 6 o 0 .
  • This protocol produced a maximum yield of 24 mg/L of purified, 13 C/ 15 N uniformly labelled protein at the final stage (see below).
  • the YNB-based medium produced about 3 -fold higher yields than the FM22 medium (Laroche et al, 1994), for stable-isotope labeling of the MSP-1 C-terminal fragment.
  • the carbohydrate was completely removed (as shown by electrophoresis and mass spectrometry), with the Asn 1 residue presumably converted to Asp in the process.
  • the supernatant was clarified_by low- speed centrifugation, 5 M NaCl was added to a final concentration of 0.3 M, and the sample was applied to a 2 ml Ni-NTA affinity column (QIAGEN), washed, and eluted with 250 mM imidazole according to manufacturer's instructions.
  • the eluate was dialyzed against 50 mM sodium phosphate (pH 6.5), 50 mM NaCl, and then passed through a 1 ml Hi-Trap Q anion exchange resin (Pharmacia) to remove misfolded MSP-1 that bound to the column.
  • the MSP-1 fragment was characterized by Western blotting and electrospray mass spectrometry (data not shown). Two principal species of mass 11607 and 11807 Da were observed corresponding to the expected fragment, as well as a fragment with an additional N-terminal Glu - Ala dipeptide resulting from incomplete STE13 processing of the ⁇ -factor secretion signal.
  • Samples for NMR experiments were prepared in either 90 %> H 2 O/10 %> D 2 O with 0.01 %> w/vol NaN 3; or 100 % D 2 0, 50 mM sodium phosphate, 100 mM NaCl at pH 6.5, (pH uncorrected for deuterium isotope effects), at a concentration of 2.1 to 2.6 mM in 0.6 ml. Protein concentration was measured by UV absorbance at 280 nm, using a calculated molar extinction coefficient of 5220 liter mol " cm -1 . The protein was demonstrated to be monomeric by equilibrium ultracentrifugation of a 0.12 mM sample in the above buffer at 293 K.
  • NOE- and ROE-derived distance restraints between backbone and side chain amide protons were obtained primarily from the 3D 15 N-NOESY-HSQC, I5 N-ROESY-HSQC, and 4D 13 C-HMQC-NOESY- 15 N-HSQC experiments.
  • Aliphatic to aliphatic proton distance restraints were obtained from a 4D 13 C-HMQC-NOESY- 13 C- HSQC experiment.
  • a 3D 13 C-HMQC-NOESY experiment in D 2 O was used to identify aliphatic to aromatic proton NOEs and 2D NOESY experiments were used to measure aromatic to aromatic proton NOEs.
  • Crosspeaks were quantified by volume integration in Felix for 2D and 3D experiments and for the 4D 13 C-HMQC-NOESY- 15 N-HSQC experiment, and from peak height measurements in the 4D 13 C-HMQC-NOESY- 13 C- HMQC spectra. Crosspeaks were classified as strong, medium and weak and these were assigned to distance restraints of 0 - 2.8, 0 - 3.6, and 0 - 5.5 A. Restraints from backbone amide signals were initially treated in this manner, and then recalibrated more precisely using 3D- 1 :, N-ROESY-HSQC data into four classes involving maximum distances of 2.6, 3.1, 3.6, and 4.1 A.
  • Dihedral Angle Restraints % angles and stereospecific assignments of ⁇ -methylene protons were obtained using the grid-search program AngleSearch, with coupling constant and intraresidue ROE distance information (Polshakov et al, 1995).
  • the coupling constant information was provided by HNHB and HN(CO)HB spectral intensities for 3 J(HN-H ) and 3 J(CO-H ⁇ ), and intraresidue distances (HN-H , H ⁇ -H ) were obtained from 3D l5 N-ROESY-HSQC and 2D ROESY (D 2 O) experiments.
  • 3 J(HN-H ⁇ ) coupling constants were obtained from the HNHA experiment.
  • Residues with positive ⁇ angles were identified by large intraresidue H ⁇ crosspeak intensities in the HN(CO)HB experiment, and y angles near -60° degrees from strong H ⁇ ( j.i ) crosspeaks in the HNHB experiment. He and Leu ⁇ 2 angles and Leu ⁇ stereoassignments were derived from the LRCH experiment. Minimum ranges of 40 degrees ( ⁇ _ ⁇ 2 ) and 50 degrees ( ⁇ , ⁇ ) were used to account for errors and local dynamic effects on the coupling constants.
  • Disulphide Bonding Pattern An initial set of 20 structures was calculated by simulated annealing using approximately 550 unambiguous NOE-derived distance restraints and 36 ⁇ i and ⁇ dihedral angle restraints but with no hydrogen bonding or disulphide bond constraints. The Cys - Cys S ⁇ distances in these structures were examined in order to establish the probable bonding pattern. Prior to the calculations, the formation of disulphide bridges for 4 Cys residues (Cysl2 - Cys28, Cys78 - Cys92) was already established with high probability by the observation of H -H ⁇ NOEs between these pairs of Cys residues.
  • Non-exchanging amide groups involved in stable hydrogen bonds were identified in spectra of samples examined in 100 %> D 2 O.
  • the corresponding hydrogen bond acceptors were determined by examining the initial structural ensemble, using the Insight II and HBPlus (McDonald et al, 1994) programs, and hydrogen bond distance restraints were included in subsequent calculations. Further hydrogen bonds were identified in a similar manner in iterative calculations. Only 10 backbone hydrogen bonds in the antiparallel ⁇ sheets were used as restraints. Two distance restraints were used for each hydrogen bond, 1.7 - 2.3 A from proton to acceptor, and 3.0 - 3.6 A from donor nitrogen atom to acceptor. Structure Calculations
  • the final structure calculation and refinement used an expanded restraint list including hydrogen bonds, additional dihedral restraints, stereoassignments of ⁇ -methylene and Leu ⁇ signals, and more precisely calibrated ROE data (see Table 1).
  • a set of 100 structures was obtained using this list, and 38 structures with 0-2 NOE violations > 0.5 A and no dihedral angle violations > 5° were accepted.
  • These 38 structures were refined by the slow-cooling procedure described above, producing a final ensemble of 32 accepted structures with no NOE violations > 0.5 A and no dihedral angle violations > 5°.
  • These selection criteria produced an ensemble of structures that extend to the end of the continuum of total potential energies in order to include structures having large scale correlated motions (Abseher et al, 1998).
  • Statistics for the final ensemble are given in Table 1. Coordinates for the 32 refined structures have been deposited in the Brookhaven Protein Data Bank (coordinates ID code lcej; NMR restraints ID code rlcejmr).
  • Anti-MSP-1 19 monoclonal antibodies used in this study were : mouse IgG mAbs 1E1, 1E8, 2F10, 111.2, 111.4 2.2, 5.2, 7.5, 9C8, 12.8, 12.10, 12D11, 117.2, 8A12 (Holder et al, 1985; McBride & Heidrich, 1987; Blackman et al, 1987; Guevara Patino et al, 1997); and mouse IgM mAb 5B1 (Pirson & Perkins, 1985). Of these, mAbs 12.8, 12.10 and 5B1 are neutralising, inhibitory antibodies and 1E1, 2.2, 7.5, 9C8 and 111.4 are blocking antibodies. Some antibodies such as 111.2 are neither inhibitory nor blocking. Construction of modified MSP- I n? clones
  • MSP-1 1 domain of Plasmodium falciparum T9- 94/Wellcome strain
  • MSP-1 19 The DNA coding the wild type MSP-1 1 domain of Plasmodium falciparum (T9- 94/Wellcome strain) MSP-1 has been cloned in expression vector pGEX-3X to produce MSP-1 19 fused to the carboxy-terminus of the Schistosoma japonicum glutathione S- transferase (GST) in Escherichia coli (Burghaus & Holder, 1994).
  • GST Schistosoma japonicum glutathione S- transferase
  • the first method was a modification of the method of Perrin & Gilliland (1990) to carry out polymerase chain reaction (PCR)-mediated site specific mutagenesis.
  • DNA was amplified using the plasmid as a template together with one oligonucleotide to introduce the point mutation and a 5' primer from outside of the MSP-1 19 sequence.
  • the amplified product was purified after electrophoresis on an agarose gel and used in a second amplification step together with a 3' primer from outside of the other end of the MSP-1 19 sequence and the plasmid as template.
  • This second PCR product was digested with the restriction enzymes EcoRl and BamHX and the product consisting of the modified MSP- l ⁇ 9 coding sequence was inserted back into pG ⁇ X-3X and the products were used to transform DH5 ⁇ cells.
  • the second method used the QuikChangeTM Site-directed mutagenesis kit from Stratagene. Briefly, using the plasmid pGEX-MSP-l ⁇ 9 as a template, two complementary synthetic oligonucleotide primers containing the desired point mutation were designed and were extended on the template by temperature cycling with the enzyme Pfu DNA Polymerase. This inco ⁇ oration of the oligonucleotide primers results in the generation of a mutated plasmid containing staggered nicks in the DNA sequence. Following the temperature cycling, the product was treated with Dpnl endonulease which will digest the methylated parental DNA template and leaves the mutation-containing newly synthesised DNA intact. The DNA inco ⁇ orating the desired mutation was then transformed into E. coli strain DH5 ⁇ (Life technologies) competent cells where the nicks will be repaired.
  • Clones were screened by analysis of restriction enzyme digests and by PCR screening of the insert gene. The DNA sequence of the selected mutant clones was confirmed using a PerkinElmer Applied Biosystems ABI 377 automatic sequencer according to the manufacturer's instructions.
  • GST-MSP-1 19 was induced with 1 mM isopropyl- ⁇ -D- thiogalactopyranoside (IPTG; Melford Laboratories) for 1 hour in the E. coli strain TOPP 1 (Stratagene). The cells were then harvested by centrifugation and the cell pellet was resuspended in cell lysis buffer (50 mM Tris-HCl/1 mM EDTA pH 8.0 containing 0.2%. (v/v) Nonidet P40 (NP40; BDH). Phenylmethylsulphonyl fluoride (PMSF; Sigma) in isopropanol was added to a final concentration of 1 mM.
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • the cell suspension was sonicated, on ice, using VibraCell sonicator (Sonics & Materials) at 50%) duty cycle for 3 min (six 30 sec pulses with 30 sec in between).
  • the cell lysate was centrifuged at 65000 x g for 1 hour at 4°C.
  • Supernatant containing soluble GST-fusion protein was applied to a glutathione-agrose column (Sigma) and the GST-fusion protein was eluted with 5 mM reduced glutathione.
  • the eluted GST-fusion protein was dialysed extensively against phosphate buffered saline (PBS) at 4°C.
  • PBS phosphate buffered saline
  • Proteins were analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Samples were solubilised in SDS-PAGE buffer without reducing agents, then fractionated on a homogeneous 12.5%> polyacrylamide gel. The pre- stained low range molecular mass markers (24-102kDa) from Bio-Rad were used as markers. When required, SDS-PAGE-fractionated polypeptides were either stained with Coomassie Brilliant Blue R-250 (CBB; Sigma) or electrophoretically transferred to Optitran BA-S 83 reinforced nitrocellulose (Schleicher & Schull, 0.2 ⁇ m pore size) for analysis by western blotting.
  • CBB Coomassie Brilliant Blue R-250
  • Blots were blocked with 5%> BSA, 0.5%> Tween 20 in PBS (PBS-T) for 1 h at room temperature, then washed in PBS-T. Blots were probed with first antibodies for 2 h at room temperature, washed 3 times in PBS-T, and then incubated in 1/1000 dilution of horse radish peroxidase (HRP)-conjugated sheep anti-mouse IgG (H+L) (ICN Immunobiologicals) or Goat anti-mouse IgM ( ⁇ chain) (Sigma) for 1 h at room temperature. Blots were then washed 3 times in PBS-T and developed using Super Signal Substrate (Pierce) as HRP substrate for 1 min. Blots were then placed in plastic wrap and exposed to X-ray film (XB-200, X-ograph Imaging Systems). The films were processed with an Agfa Gevamatic ⁇ O film processor (Agfa).
  • HRP horse radish peroxid
  • GST-MSP-1 19 containing either the wild type or various modified sequences was used to coat a carboxymethyl dextran hydrogen sensor chip by the following methodology.
  • the binding of the GST-MSP-1 19 was via amino groups using EDC/NHS chemistry.
  • Immobilisation was done with the amine coupling kit (Pharmacia BIAcore).
  • the CM dextran surface was activated with 50 ⁇ l of 200 mM l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 5 mM N-hydroxysuccinamide (NHS) for 10 min.
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • NHS N-hydroxysuccinamide
  • GST-MSP-11 9 was then coupled to the BIAcore sensor surface using 50 ⁇ l of a solution at 100 ⁇ g ml "1 in coating buffer (0.0 IM sodium acetate buffer, pH 3.5) for 10 min. Unreacted carboxyl groups were blocked by adding 50 ⁇ l 1 M ethanolamine, pH 8.5 for 10 min.
  • the cells were washed with two pulses of 20 ⁇ l 10 mM glycine-HCl, pH 2.8 for 8 min in total to remove any non-covalently bound protein.
  • the immobilisation procedure was carried out at a flow rate of 5 ⁇ l min "1 . Measurements were performed on the BIAcore 2000 instrument.
  • the local backbone rmsd is highest at the N-terminus (up to Cysl2), in the loop Glu65 - Lys73, and following Cys92 at the C-terminus.
  • the Ramachandran plot quality is typical of that found for other EGF structures (Doreleijers et al, 1998).
  • the large loop at the end of the major b-sheet (Glu65 - Lys73) is relatively disordered, and high mobility for the segment Gly68 - Gly71 was confirmed by backbone amide L:> N ⁇ l H ⁇ heteronuclear NOE measurements (Barbato et al, 1992).
  • the heteronuclear NOE values are dramatically reduced for residues in this region.
  • the low NOE intensities correspond to increased mobility compared with the rest of the protein.
  • the interdomain linker region from Pro45 to Pro47 is distinct from other EGF-like module pairs.
  • Figure 5 shows the backbone C, N, C ⁇ atom alignments of the two MSP-1 C-terminal fragment domains made with typical examples of EGF-like domains from several proteins, using the fitpdb program. Pairwise alignments showed that the two domains from MSP-1 are more similar to the factor Xa structure and its close relative from Clr, than to each other or to the other structures tested.
  • the rmsd values for MSP-1 domains compared to factor Xa are comparable to those of the more distantly related structures fibrillin-1 and transforming growth factor- ⁇ .
  • each MSP-1 domain is thus similar to typical EGF family members, with the turns following the fifth Cys residue roughly equivalent, in spite of the divergence from the EGF consensus (C(5)xxG ⁇ ) where ⁇ is a Phe or Tyr -residue. Although some of the external loops are disordered, the scaffold is quite stable, as indicated by the non-exchangeable backbone amides (see above and in Protein Data Bank/BioMagResBank submission for details).
  • the MSP-1 C-terminal fragment lacks the conserved EGF Ca 2+ -binding sequence and there was no evidence of Ca 2+ binding to the MSP-1 C-terminal fragment.
  • the 2D ⁇ -NOESY spectra were virtually identical in the absence or presence of 20 mM CaCl 2 , indicating that any binding that might occur has, at most, only a small affect the overall structure.
  • the most striking feature of the MSP-1 C-terminal fragment structure is the interface between the domains, which consists of several nonpolar amino acids (Phe 19, Leu31, Leu32, Leu86, Phe87, Ile90 and Phe91) involved in hydrophobic interactions. These residues join the base of the major ⁇ -sheet and the tight turn in domain-1 with the final bend from residue 86 to 91 in domain-2.
  • the domain interactions result in the domains forming a U-shaped structure which contrasts with structures observed for other pairs of EGF domains (Downing et al, 1996; Brandstetter et al, 1995).
  • the proximity of the C- and N-terminal positions may be significant, since it suggests that the proteolytic processing site that produces the C- terminal 96 amino acid fragment may be very close to the GPI membrane attachment site at or near residue 96. This proximity is consistent with the idea that a membrane-bound Plasmodium proteinase is responsible for secondary processing.
  • the electrostatic potential surface of the MSP-1 C-terminal fragment is shown in two views in Figure 7.
  • the surface in Figure 7a is highly charged, especially in the protruding loop regions 23 - 27, 35 - 40 and 64 - 66.
  • the surface in Figure 7b contains more neutral hydrophilic residues as well as a small hydrophobic patch from Pro85 - Phe87 near the center of the surface. In the future, such information could assist in understanding how these different surfaces may be involved in interactions with the rest of the MSP-1 precursor, the processing proteinase, other proteins on the merozoite surface, or unknown targets on the erythrocyte or parasite vacuolar membrane surfaces.
  • Glu65 to Lys73 also appears to be the most variable region among different Plasmodium species (Daly et al, 1992; Holder et al, 1992).
  • the fifth site has a substitution between hydrophobic residues (Leu86/Phe86). This partially-exposed side- chain is located at the hydrophobic domain interface, and the conservative substitution is consistent with a role in this interaction.
  • the Glu26 mutation shown in cyan, is closest to the N-terminal proteolytic processing site (magenta) at Asnl, and is the only one of this group of mutations that affects binding of a processing-inhibitory antibody, i.e. one that is capable of preventing both proteolytic processing of the MSP- 1 precursor and erythrocyte invasion in vitro.
  • the other three mutations abolish binding of blocking antibodies that bind to the native C-terminal fragment and interfere with the binding of processing-inhibitory antibodies.
  • modified recombinant proteins will also be used to affinity select antibodies from pooled serum from individuals exposed to malaria. We hypothesise that the modified proteins will select less blocking antibody than the wild type protein and that therefore these selected antibodies will be more effective in inhibiting parasite invasion in vitro and secondary processing.
  • cysteines 2 and 4 are not present in the first EGF-like domain of MSP-1 from the rodent, primate and P. vivax malaria parasites.
  • cysteines 2 and 4 are not present in the first EGF-like domain of MSP-1 from the rodent, primate and P. vivax malaria parasites.
  • This does not have appear to have any effect on the binding of any of the inhibitory antibodies, but does abolish the binding of the blocking antibody mAb 2.2.
  • T cell recognition is more effective or that processing by antigen processing cells proceeds by a different degradation pathway that drives the fine specificity of the antibody response in a more productive direction (see for example Egan et al, 1997).
  • Removal of the cysteine pair may improve the immunogenicity of the modified protein and this will be assessed by measuring the level of antibodies induced by the P. falciparum protein without the two cysteines with the level of antibodies induced by the wild type protein.
  • Table 2 The location of amino acid sequence changes and their effect on the binding of monoclonal antibodies
  • MSP-1 merozoite surface protein- 1 Plasmodium falciparum (C-terminal fragment)
  • Aromatic atoms on opposite sides of the ring# e.g. Tyr HE1 and HE2 protons
  • Intraresidue ambiguities e.g. Lys HG and HD protons
  • MSP-1 merozoite surface prote ⁇ n-1
  • Example 3 Identification of blocking antibodies using a competitive binding assay and immobilised wild type GST-MSP-119.
  • a recombinant fusion protein comprising wild type MSP-11 9 fused to GST was coupled to the sensor chip and competitor antibody was first allowed to bind to the antigen. Then a solution of either mAb 12.8 or 12.10 was passed over the chip and the amount of binding of this second antibody was quantified. If the first antibody interferes with the binding of the second antibody then this is reflected in a reduction in the amount of second antibody bound.
  • the wild type GST-MSP-1 1 was coupled to a CM5 sensor chip.
  • the binding assays were performed with a constant flow rate of 5 ⁇ l min "1 at 25°C.
  • purified mAbs 1E1, 8A12 and 2F10 at 100 ⁇ g ml "1 in HBS-EP buffer (lOmM HEPES pH7.4 containing 150mM NaCl, 3mM EDTA and 0.005%v/v polysorbate 20); mAbs 1E8, 9C8, 12D11, 1 11.2 and 111.4 in cell culture medium supernatant; mAbs 2.2, 7.5 and 89.1 at 1 :10 dilution of ascitic fluid in HBS-EP buffer; and mouse ⁇ -GST antibody at 1 : 10 .dilution serum in HBS-EP buffer were allowed to interact with immobilised wild type GST-MSP- l ⁇ for 10 min.
  • Example 4 Immunization of small animals with modified GST-MSP-lic) and analysis of the antibodies induced
  • the rabbits were immunised subcutaneously with MSP-1 19 protein in Freund's complete adjuvant and then boosted on three occasions with 200 ⁇ g of the protein in Freund's incomplete adjuvant 21, 42 and 63 days later, and serum samples were collected.
  • the presence and level of antibodies binding to the native MSP-1 protein in the parasite was assessed by indirect immuno fluorescence using acetone fixed smears of parasite- infected erythrocytes.
  • the sera were diluted serially in phosphate buffered saline (PBS) and incubated on the slide for 30 min at room temperature. After washing, the slides were incubated with FITC conjugated goat anti-rabbit or anti-mouse IgG, washed, and then examined by fluorescence microscopy.
  • PBS phosphate buffered saline
  • the sera were also analysed in an MSP-1 secondary processing assay. Analysis and quantitation of secondary processing of MSP-1 in merozoite preparations was by a modification of an assay described previously (Blackman et al, 1994). Washed P. falciparum 3D7 merozoites were resuspended in ice-cold 50 mM Tris-HCl pH 7.5 containing 10 mM CaCl 2 and 2 mM MgCl 2 (reaction buffer). Aliquots of about 1 x IO 9 merozoites were dispensed into 1.5 ml centrifuge tubes on ice, and the parasites pelleted in a microfuge at 13,000 x g for 2 minutes at 4°C.
  • the processing was assayed using the western blot-based method and by a modified processing assay.
  • Supernatants from the assays were obtained after centrifugation for 30 min at 4°C, 13,000 x g to remove the insoluble material.
  • the amount of MSP-1 3 in the supernatants was measured using an ELISA method. Fifty microlitres of diluted sample supernatants were added to the wells of an ELISA plate (NUNC F96 Cert. Maxisorp) that had been coated with 100 ⁇ l/well of 4 ⁇ g ml "1 human mAb X509 in PBS. Plates were incubated for 4 hours at 37°C and then washed 3 times with 0.01% PBS-Tween (PBS-T).
  • Bound MSP-1 33 protein was detected by addition of 100 ⁇ l of 1 :4000 dilution of mouse mAb G13 for 1 hour at 37°C, followed by washing and the addition of 100 ⁇ l of 1 : 1000 dilution of sheep anti-mouse IgG (H+L) HRP-conjugated antibody. After incubation for 1 h at 37°C, the plates were washed again and HRP was detected by the addition of 100 ⁇ l of freshly prepared substrate solution (400mg l "1 o-phenylenediamine dihydrochloride in 0.05 M phosphate buffer, 0.024 M citric acid and 0.012% H O 2 ) at room temperature for 20 min. The reaction was stopped by adding 10 ⁇ l of 1 M sulphuric acid and the absorbance of each sample was measured at 492 nm.
  • substrate solution 400mg l "1 o-phenylenediamine dihydrochloride in 0.05 M phosphate buffer, 0.024 M citric acid and
  • the results are shown in Figure 13.
  • the two modified proteins produced antibodies that reacted with MSP-1 in the parasite-infected erythrocyte, with a serum titre of 1 : 10,000, which was an identical titre to that of a serum produced in the same way by immunisation with a recombinant protein containing the wildtype MSP-1 sequence.
  • the antibodies induced by immunization were able to partially inhibit processing at the concentration used in a preliminary experiment, whereas in the control serum no antibodies that inhibited processing were present.
  • the coding sequence of the Plasmodium falciparum merozoite surface protein-1 (MSP1) 41.1 kDa processed fragment (MSP-142) has been redesigned for optimal heterologous expression in the yeast Pichia pastoris.
  • the optimized DNA sequence was synthesized by PCR gene assembly, in the form of two fragments, MSP-133 and MSP-1 19.
  • E. pastoris was transformed with an expression vector containing the optimized MSP-119 construct. Recombinant strains were shown to express high levels of non-glycosylated, properly folded MSP-119 protein.
  • Proteins encoded by the AT-rich genome of the human malaria parasite Plasmodium falciparum are generally poorly expressed in heterologous systems (Withers-Martinez et al., 1999).
  • the methylotrophic yeast Pichia ( Komagataella) pastoris is an appropriate system for expression of disulphide-bridged proteins such as the C-terminal fragment of the P. falciparum merozoite surface protein-1 (MSP1) (White et al., 1994; Morgan et al.,
  • the optimized MSP-142 sequence was synthesized by gene assembly polymerase chain reaction (Stemmer et al., 1995, Withers-Martinez et al., 1999), in the form of separate MSP-133 and MSP-119 fragments.
  • the .optimized MSP-1 19 fragment was subcloned into a novel modified Pichia expression vector, transformed into the P. pastoris host strain SMD1168, and several independent transformants were isolated. There transformants were shown to efficiently express non-glycosylated, properly folded MSP-119. Strong expression of the optimized gene was observed in low copy number transformants.
  • codon table should reflect usage in highly expressed genes, rather than average usage.
  • the random sequence that contained the minimum number of unfavourable codons (6) was selected, and these codons were changed manually to more preferred alternatives.
  • the sequence was then analysed with DNA- STAR to check for AT-rich sequences that may cause transcription termination, and for direct and inverted repeats.
  • a set of 50 overlapping oligonucleotides coding for the final sequence was then generated. This consisted of 49 oligonucleotides of length 42 nt, and one of length 48 nt. Each oligonucleotide had a 21 bp overlap with its neighbours, with no gaps.
  • T m s were in the range of 60°C to 77°C. Oligonucleotides were synthesised by Oswel (Southampton, UK) at 40 nmol scale, and supplied in deionised water without purification. Outside primers of various lengths for the amplification step were also synthesised, to give a T m of 62°C to 64°C, and contained a 5 '-terminal phosphate group for ligation following the amplification step. The reverse primers also included a translation termination codon (UAA in the complementary strand). All oligonucleotides were diluted to 10 ⁇ M in ddH 2 O before use.
  • PCR-mediated gene assembly and amplification were carried out as described (Stemmer et /., 1995; Withers-Martinez et al., 1999), using a Biometra cycler, in thin- walled 200 ⁇ L tubes, under the following conditions.
  • Oligonucleotide mix containing each oligonucleotide at 200 nM
  • Cycles 32 cycles (2 h 33 m) denaturation 94°C 30 s annealing 52°C 30 s extension 72°C 3 m
  • N-terminal fragment (bp 1-423) 21 oligonucleotides middle fragment (bp 337-786) 22 oligonucleotides C-terminal fragment (bp 787-1074) 14 oligonucleotides
  • the C-terminal fragment produces a 10.6 kDa fragment (MSP-119).
  • the N-terminal and middle fragments, which overlap between positions 337 and 423, were subsequently spliced together at the Bglll site (371-376) to give a 786 bp fragment that encodes the 30.5 kDa MSP-133 protein.
  • Cycles 32 cycles (2 h 55 m) denaturation 94°C 45 s annealing 52°C 45 s extension 72°C 3 m final extension 72°C 5 m
  • PCR products were then purified by filtration with Centricon-100 units (Amicon), and cloned directly into the vectors by blunt-end ligation overnight at 16°C with T4 DNA ligase.
  • the synthetic MSP-119 gene was cloned directly into a E. pastoris expression vector.
  • the HXa vector had been previously created by insertion of a 36 bp synthetic oligonucleotide, containing the His 6 tag, factor Xa cleavage site, and Pmll restriction site into the SnaBl site of the pPIC9K vector.
  • the N-terminal and middle fragment PCR products were cloned into the Smal site of the dephosphorylated pUC118 vector. Plasmid clones containing inserts were sequenced. Clones with the correct synthetic sequence were then digested and the two fragments were gel-purified. The N-terminal fragment clones were digested with EcoRI and Bglll, and the middle fragment clones were digested with Hindlll and Bglll. The recombinant fragments were purified on an agarose gel, and eluted with a QIAGEN extraction kit.
  • the purified N-terminal and middle fragments were then spliced together by ligation into a pUC1 18 vector that had been digested with Hindlll and EcoRI and treated with calf alkaline phosphatase. This created the complete synthetic MSP-133 coding sequence.
  • the N- terminal and middle fragment PCR products were cloned into the Smal site of the dephosphorylated pUC118 vector. Plasmid clones containing inserts were sequenced. Clones with the correct synthetic sequence were then digested and the two fragments were gel-purified. The N-terminal fragment clones were digested with EcoRI and Bglll, and the middle fragment clones were digested with Hindlll and Bglll.
  • the recombinant fragments were purified on an agarose gel, and eluted with a QIAGEN extraction kit.
  • the purified N-terminal and middle fragments were then spliced together by ligation into a pUC 118 vector that had been digested with Hindlll and EcoRI and treated with calf alkaline phosphatase. This created the complete synthetic MSP-133 coding sequence.
  • the methylotrophic yeast Pichia (Komagataella) pastoris strain SMD1 168 was transformed by electroporation as described previously (Morgan et al., 1999). In addition, some G418 -resistant clones were isolated using Hybond-N+ membranes (Fairlie et al., 1999).
  • Expression screening of transformants was performed by growing 10 ml cultures in buffered minimal glucose medium. Cells were harvested and resuspended in 10 ml buffered minimal methanol medium at 1.0 OD 6 Q O and grown overnight to a final OD 6 oo of 2.5 to 3.0. Cells were removed by centrifugation, and 1.2 ml of the supernatant medium was precipitated 30 min on ice with 15 % trichloroacetic acid. The samples were centrifuged for 30 min at 14000 rpm at 4 °C in a microfuge, and the protein pellets were washed twice with cold acetone.
  • MSP-119 Homogeneously purified MSP-119 was obtained as described previously (Morgan et al., 1999), except that enzymatic deglycosylation was omitted for the synthetic gene products.
  • PCR-gene assembly reactions for the MSP-133 (two sections) and MSP-119 synthetic fragments are shown on agarose gels in Figure 16. This demonstrated that a single, correct size major product was observed in each case.
  • the PCR products were subcloned, screened, and sequenced as described in the Methods section.
  • P. pastoris was transformed with the synthetic MSP-1 19 construct in the modified pPIC9K expression vector (pPIC9K-HXa; Figure 15).
  • Expression of the synthetic MSP- 119 product in three independent transformants is shown on a protein gel in Figure 17.
  • the protein samples were prepared by trichloroacetic acid precipitation from culture supernatants as described in the Methods section. This demonstrated that a single, major product was present in each sample, corresponding to the expected migration of the synthetic MSP-119 protein. This migrated slightly more slowly than the control .sample, which as described previously (Morgan et al., 1999) has a shorter N-terminal tag sequence.
  • MSP-119 is efficiently expressed by the transformed yeast.
  • the yield (measured by UV absorbance) of purified MSP-119 was 16 mg/L for low copy number transformants (resistant to 0.25 mg/ml G418), and increased to 24 mg/L for intermediate G418 resistance (resistant to 1.0 mg/ml G418). This can be compared with yields of 1-2 mg/L for low copy number transformants of P. pastoris with the original Plasmodium falciparum coding sequence, before isolation of a highly G418 -resistant strain (Morgan et al., 1999). This indicated that the synthetic MSP-119 construct is advantageous for recombinant protein expression, and that further improvement would result from isolation of higher copy number transformants.
  • MSP-1 Plasmodium falciparum merozoite surface protein-1
  • MSP-1 malaria merozoite surface protein
  • Plasmodium falciparum isolation and purification of spontaneously released merozoites by nylon sieve membranes. Exp. Parasitol. 54, 285.
  • MSP-1 Plasmodium falciparum merozoite surface protein 1

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Abstract

L'invention concerne une variante d'origine non naturelle d'un fragment C-terminal d'une protéine de surface de mérozoïte 1 (MSP-1) de Plasmodium. Cette variante présente (i) une affinité réduite, si l'on compare avec une MCP-119 de Plasmodium d'origine naturelle, pour au moins un premier anticorps pouvant bloquer la liaison d'un second anticorps inhibant le clivage protéolytique d'une MSP-142 de Plasmodium, et (ii) sensiblement la même affinité pour au moins un troisième anticorps, si l'on compare avec ladite MCP-119 de Plasmodium d'origine naturelle, ce troisième anticorps inhibant le clivage protéolytique de la MSP-142 de Plasmodium. Cette variante s'utilise dans un vaccin contre la malaria.
PCT/GB2000/001558 1999-04-20 2000-04-20 Vaccin Ceased WO2000063245A2 (fr)

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EP00920918A EP1180120A2 (fr) 1999-04-20 2000-04-20 Variante de la proteine de surface du merozoite (msp-1) de plasmodium et vaccin la comprenant
BR0009823-0A BR0009823A (pt) 1999-04-20 2000-04-20 Variante da proteìna de superfìcie de plasmodium merozoito (msp-1) e vacina compreendendo a variante
MXPA01010701A MXPA01010701A (es) 1999-04-20 2000-04-20 Vacuna.
HK02104693.0A HK1043135A1 (zh) 1999-04-20 2000-04-20 原蟲分裂殖子表面蛋白(msp-1)的異變物及含該異變物的疫苗
JP2000612331A JP2002543774A (ja) 1999-04-20 2000-04-20 ワクチン
AU41330/00A AU779662B2 (en) 1999-04-20 2000-04-20 Vaccine
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US6855322B2 (en) 2001-01-26 2005-02-15 The United States Of America As Represented By The Secretary Of The Army Isolation and purification of P. falciparum merozoite protein-142 vaccine
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US7256281B2 (en) 2002-04-01 2007-08-14 United States Of America As Represented By The Secretary Of The Army Recombinant P. falciparum merozoite protein-142 vaccine
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CN1372568A (zh) 2002-10-02
WO2000063245A2 (fr) 2000-10-26
MXPA01010701A (es) 2003-08-20
AU779662B2 (en) 2005-02-03
EP1180120A2 (fr) 2002-02-20
BR0009823A (pt) 2002-04-09
WO2000063245A3 (fr) 2001-05-03

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