Patent Application
20140093530
The present invention relates to malaria vaccines including as an antigenic component a synthetic sequence representing a combination of fragments from the K1 type block 2 repeat region of Plasmodium falciparum merozite surface protein 1 (MSP1). The K1 type of the MSP1 block 2 region is highly polymorphic and the K1 type has been found to be the most common of the MSP1 block 2 region types in African populations. The present invention relates inter alia to harnessing the extensive sequence diversity of the K1 type block 2 region of MSP1 for vaccine use in a sequence referred to as the K1 type synthetic repeat (K1SR) sequence. This sequence is capable of inducing antibody responses having wide targeting for MSP1 K1 type block 2 region alleles. To improve breadth of effectiveness as an immunogen against P. falciparum, furthermore it can be incorporated into fusion constructs with other P. falciparum MSP1 epitopes.
Polymorphism in pathogen antigens presents a complex challenge for vaccine design, particularly when diversity is extensive. The N-terminal block 2 region of Plasmodium falciparum MSP1 exemplifies high diversity in a pathogen antigen. This region is known to be the target of naturally acquired human antibodies that are associated with a reduced risk of clinical malaria (Conway et al., (2000) Nature Med. 6, 689-692; Polley et al., (2003) Infect. & Immun. 71, 1833-1842; Cavanagh et al., (2004) Infect. & Immun. 72, 6492-6502). Hence, there has been interest in priming responses to the MSP1 block 2 region by vaccination, particularly in young children. However, there are three major allelic types of the MSP1 block 2 region (the K1 type, the MAD20 type and the R033 type) and much subtype polymorphism exists within two of these (K1 and MAD20).
Extensive sequence diversity is exhibited by both the K1 and MAD20 block 2 types within the central tri- and hexa-peptide repeat sequences of each type. The K1 and MAD20 types contain different tri- and hexa-peptide repeat sequences with serine at the first position and variations in the sequence and number of repeats produce subtype allelic differences within each of the types (Conway et al., (2000) Nature Med. 6, 689-692; Miller et al., (1993) Mol. Biochem. Parasitol. 59, 1-14). Despite this sequence diversity, most human sera from individuals in malaria endemic regions recognise multiple block 2 allelic variants within a type, and although some individuals have single allele-specific antibody responses to MSP1 block 2, this is less common (Cavanagh et al. (1998) J. Immunol. 161, 347-359; Cavanagh & McBride (1997) Mol. & Biochem. Parasitol. 85, 197-211). This can be explained in two ways. Firstly, there are shared type-specific flanking sequences associated with both the K1 and MAD20 block 2 types, which are the target of some antibody responses, and secondly the specific characteristics of the sequence diversity within the K1 and MAD20 types. Four different tripeptides are used in the K1 type and 5 (different) tripeptides are used in the MAD20 type. Thus, all K1 type block 2 alleles contain combinations of the tripeptides SAQ, SGA, SGP and SGT, flanked by common sequences unique to the K1 type of block 2. All MAD20 type block 2 alleles contain combinations of the tripeptides SGG and SVA (with minor variations at the N-terminal end of the repeats using the SVT, SKG and SSG tripeptides), flanked by MAD20 type common sequences. Variation within the K1 and MAD20 block 2 types appears to be restricted to these differences, plus some size constraint on the length of repetitive sequence allowed within the MSP1 molecule (Jiang et al. (2000) Acta Tropica 74, 51-61).
As already noted above, the K1 type is the most common of the types of the block 2 MSP1 region in African populations. For example, the inventors found in a Zambian population that 49 of 91 alleles of P. falciparum MSP1 block 2 region were of the K1 type and most of these had unique sequences due to variation in tri- and hexa-peptide repetitive motifs. There is therefore particular interest in priming immune responses which broadly target the allelic diversity associated with the repeats of MSP1 K1 type block 2 regions.
As an approach to addressing the above-noted need, a minimal length novel synthetic polypeptide sequence of 78 amino acid residues has been designed which contains a large proportion of all antibody epitopes within the repeat sequences of the MSP1 K1 type block 2 region (the K1 type synthetic repeat sequence referred to as the K1SR and presented as SEQ. ID. No. 1). This was achieved using sera from malaria endemic regions and 23 synthetic peptides representing all the 12 mer amino acid sequences (with the serine of tri-peptides at position 1) derived from a collection of MSP1 K1 type block 2 repeat sequences present in the GenBank database. The resulting synthetic sequence has been expressed as a recombinant fusion protein having at the N-terminus a glutathionc-S-transferase (GST) sequence and in this form has been shown to exhibit broader type specific reactivity with human sera than known individual alleles of the MSP1 K1 type block 2 region.
In one aspect, the present invention thus provides a polypeptide comprising the following amino acid sequence as an antigenic sequence (SEQ ID no. 1; see also
The invention also extends to antigenic polypeptides comprising a functional analogue of the K1SR sequence which is of the same length and which retains substantially the same immunogenicity as the K1SR sequence when in the form of a GST-fusion protein, i.e. joined at the N-terminus to a GST sequence. Such a sequence may be any conventional GST sequence as commonly employed for expression and purification of recombinant proteins as fusion proteins, e.g. a GST sequence as encoded by a pGEX expression vector (Smith et al., Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase (1988) Gene 67, 31-40). It has been found that MSP1 block 2 repeat regions are poorly immunogenic unless conjugated to a carrier protein.
“Substantially the same immunogenicity” may be judged using a panel of monoclonal antibodies which bind to epitope-presenting fragments spanning the whole K1SR sequence. Such a panel may be obtained by animal immunisation with appropriate peptides. A variant with “substantially the same immunogencity” will desirably bind with all of the same antibodies or at least about 90 to 99% thereof, preferably at least about 99% thereof. Such a functional analogue may have one or more substitutions, e.g. one or more conservative substitutions, which retain the desired immunogenicity.
A functional analogue as discussed above can be expected to share with the sequence of SEQ. ID. NO. 1 ability to bind with monoclonal antibodies to each of the following sequences: (i) SGASAQSG (SEQ ID NO 3) (ii) SAQSGTSGTS (SEQ ID NO 4) and (iii) SAQSGTSGT (SEQ ID NO 5). Such monoclonal antibodies are exemplified by the monoclonal antibodies Mab 12.2, Mab 123D3 and Mab CE2 respectively as referred to in the exemplification below. Such monoclonal antibodies may be used in initial screening for functional analogues of SEQ. ID. No. 1.
Preferred polypeptides of the invention are fusion proteins wherein a synthetic repeat sequence selected from the K1SR and functional analogues thereof is joined at the N-terminus and/or the C-terminus to an additional amino acid sequence. For example, as indicated above, a K1 type synthetic repeat sequence as described above may be preferably expressed as a fusion protein in which it is joined at the N-terminus to a GST sequence or another amino acid sequence which may be used to aid purification, e.g. an N-terminal sequence to provide a His-tagged protein. Such fusion constructs exhibit broader type-specific reactivity with sera from P. falciparum infected individuals than known MSP1 K1 type block 2 alleles, more particularly, for example, the alleles designated 3D7 and Palo Alto.
Of particular interest for vaccine design are fusion constructs in which a K1 type synthetic repeat sequence as described above exhibits immunogenicity, i.e. is capable of raising an immune response to a number of alleles of the MSP1 K1 type block 2 region, and is joined to one or more additional epitope-containing sequences capable of raising an antibody response or a cellular immune response in humans to a P. falciparum antigen. Of especial interest are fusion constructs in which a K1 type synthetic repeat sequence of the invention exhibits desirable immunogenicity, e.g. as when joined to an N-terminal GST sequence, and is joined to one or more additional epitope-containing sequences selected from other MSP1-derived sequences, preferably human T cell epitope-containing sequences capable of inducing a cellular immune response to P. falciparum and MSP1 block 2 regions of the types MAD20 and RO33. Preferably, such a hybrid fusion construct may, for example, incorporate, or consist of, for example, all of the following sequences joined consecutively in the N-terminal to C-terminal direction: (i) an N-terminal sequence, for example, to assist with purification such as a GST sequence or a His tag sequence; (ii) a sequence comprising one or more human T cell epitope-presenting sequences derived from an MSP1 K1 type allele such as the 3D7 allele and capable of activating a cellular immune response and (iii) a K1 type synthetic repeat sequence of the invention. Component (ii) may, for example, be a complete K1 type block 1-block 2 allele or a portion or portions thereof. It may comprise an artificial sequence providing a combination of T cell epitopes, e.g. the T cell epitope 1 (T1) and the T cell epitope 2 (T2) derived from the 3D7 block 1-block 2 allele. Such a hybrid sequence may be desirably further extended at the C-terminus by joining to both an MSP1 block 2 region of the MAD20 type and an MSP1 RO33 type block 2 region.
In another aspect, the invention provides a composition for inducing an immune response to P. falciparum including as an antigenic element a K1 type synthetic repeat sequence in the form of a polypeptide or fusion protein as discussed above.
The above-noted aspects of the invention are discussed in more detail below with reference especially to the following listed sequences and figures.
SEQ. ID no 1: the amino acid sequence (78 amino acid residues) of the synthetic K1 type super repeat antigen designated K1SR;
SEQ. ID no. 2: DNA insert as shown above for cloning of an optimised coding sequence for the K1SR in pPCR-Script Amp and transfer of the coding sequence to the expression vector pGEX-2T for expression of the K1SR as a GST fusion protein in E. coli.
SEQ. ID. No. 36: DNA insert as shown in
SEQ. ID no. 37: the hybrid multi-allelic protein expressed using the DNA insert of SEQ. ID no. 36. The T cell epitope 1 (T1) sequence and T cell epitope 2 (T2) sequence are underlined.
The following figures are referred to below in further describing design and construction of the K1 type synthetic repeat sequence noted above and longer fusion constructs containing that synthetic repeat sequence:
Final product: [Plus K1 Bk1/T]-K1SR-RO33-Wellcome [3RW] 22.8 kDa
A construction scheme for the final hybrid protein is given in Example 3.
As indicated above, the present invention provides a polypeptide comprising as an antigenic sequence a synthetic sequence designed to contain multiple epitopes that occur amongst different serological subtypes of the highly polymorphic K1 type block 2 region of the P. falciparum protein MSP1. More particularly, there is provided such a polypeptide wherein said antigenic sequence is a synthetic repeat sequence selected from the sequence of SEQ. ID. No. 1 (the K1SR; see also
The KISR of the invention and functional analogues thereof are thus proposed as highly advantageous sequences for provision in compositions for inducing immune responses to P. falciparum, particularly in humans. As indicated above, there is particular interest in such compositions, for example, for priming immune responses to P. falciparum in young children in regions where there is high prospective risk of clinical malaria. Such a composition will desirably contain additional epitope-presenting sequences which are capable of inducing immune responses to a P. falciparum antigen. As indicated above, one or more such sequences may be provided in a fusion construct incorporating the K1SR or a functional analogue thereof. However, such sequences may also be provided as one or more separate polypeptides as further discussed below.
As indicated above, a K1 type synthetic repeat sequence of the invention is typically expressed as a recombinant fusion protein. For example, to ease purification a K1 type synthetic repeat sequence may be expressed joined at the N-terminus to a GST sequence or His residue tag sequence. Moreover, as indicated above such a fusion protein may be used directly as an immunogen to induce antibody responses to P. falciparum.
In a further embodiment, the present invention thus provides a fusion construct in which a K1 type synthetic sequence of the invention is joined at the N-terminus and for C-terminus to an additional amino acid sequence. The synthetic sequence may, for example, be joined directly or indirectly at the N-terminus to a GST sequence or His tag sequence, e.g. a conventional His6-containing tag sequence. In such a fusion construct, or in fusion construct of the invention with an N-terminal GST or His tag sequence, the K1 type synthetic repeat sequence may be provided as part of a longer hybrid construct containing additional epitope-presenting sequences.
For improvement of breadth of targeting, as indicated above, a K1 type synthetic repeat sequence of the invention may be provided in a fusion protein in the form of a longer antigenic sequence in which it is joined to one or more additional epitope-containing sequences capable of raising an antibody response or a cellular immune response in humans to a P. falciparum antigen. Such additional epitope-containing sequences will desirably be P. falciparum MSP1-derived sequences selected from (i) human T cell epitope-containing sequences capable of inducing a cellular immune response to P. falciparum (ii) a sequence providing an MSP1 block 2 repeat region of the MAD 20 type and (iii) a sequence providing an MSP1 block 2 repeat region of the RO33 type. Of especial interest for vaccine design are fusion proteins including all of (i) to (iii) and a K1 type synthetic repeat sequence as described above.
Evidence from previously published work (Parra et al. (2000) Infect. Immun. 68, 2685-2691; Quakyi et al. (1994) J. Immunol. 153, 2082-2092), and from use of computer-based predictive algorithms, has indicated the presence of human T cell epitopes within the flanking sequences of the MSP1 block 2 region, i.e. in the type specific regions preceding and following the block 2 repeats, in the junction region between MSP1 block 2 and MSP1 block 1 and in MSP1 block 1 itself. Thus, component (i) in a hybrid construct as described above may be a complete MSP1 K1 type block 1-block 2 allele, such as the MSP13D7 block 1-block 2 allele (amino acid residues 20 to 133 of Genbank accession number NP—704838.1; derived from P. falciparum 3D7 sequence) or a portion or portions thereof providing one or more human T cell epitopes. As indicated above, it may be an artificial sequence providing a combination of T cell epitope 1 and T cell epitope 2 derived from the same block 1-block 2 allele as illustrated by
Component (ii) may be one or more MSP1 MAD 20 type block 2 alleles, e.g. the known Wellcome allele (amino acid residues 54-118 of Genbank accession no. CAA33163).
Component (iii) may desirably be a complete RO33 type MSP1 block 2 sequence (e.g. amino acid residues 54-106 of GenBank accession no. AB116601).
Relevant MSP-1 block 2 allele sequences (3D7, Palo Alto, MAD 20, Wellcome and RO33) can also be found in FIG. 2 of Cavanagh and McBride (1997) Mol. & Biochem. Parasitol. 85, 197-211: Antigenicity of recombinant proteins derived from Plasmodium falciparum merozoite surface protein 1. The same figure also provides the sequence of the MSP1 block 1 alleles Palo Alto and MAD 20 which may be used in a hybrid construct as described above.
In a preferred embodiment, a fusion construct is thus provided in which a single K1 type block 1-block 2 allele, e.g. the MSP1 3D7 allele, is joined to the N-terminus of the K1 synthetic repeat sequence. Such a hybrid construct may be desirably further extended, typically at the C-terminus, by addition of an MSP1 block 2 region of the RO33 type and an MSP1 block 2 region of the MAD 20 type. Such a fusion protein with an N-terminal sequence to aid purification (a His tag sequence) and including both an MSP1 RO33 type block 2 allele and an MSP1 MAD 20 type block 2 allele (the Wellcome allele) is illustrated by
A particularly preferred type of fusion construct of the invention is a fusion construct in which the K1SR is joined at its N-terminus to an artificial sequence providing one or more T cell epitopes derived from an MSP1 K1 type allele, e.g. the T cell epitope 1 and T cell epitope 2 derived from the 3D7 block 1-block 2 allele and is joined at its C-terminus to a sequence providing both an MSP1 block 2 region of the RO33 type and an MSP1 block 2 region of the MAD 20 type, e.g. a complete RO33 type block 2 allele and a complete MAD 20 type allele such as the Welcome allele as illustrated by
In another aspect, the present invention provides polynucleotides, which may be DNAs or RNAs, comprising a coding sequence for a polypeptide or protein of the invention, including fusion constructs as discussed above. As particularly preferred embodiments of DNA according to the invention, there are provided DNAs comprising the K1SR coding sequence shown in SEQ. ID no. 2 (nucleotide position 28 to nucleotide position 261). This sequence is codon optimised for expression in E. coli cells.
The invention also extends to vectors encoding a polypeptide or protein of the invention. The invention additionally provides expression constructs, e.g. expression vectors, in which a DNA coding sequence for a polypeptide or protein of the invention is operably-linked to a promoter sequence and optionally other control sequences. Such expression constructs which are capable of expressing a polypeptide or protein of the invention in human cells may be administered to humans for the purpose of promoting an immune response to P. falciparum as further discussed below.
In another aspect of the invention, there is provided a method of producing a polypeptide or protein of the invention which comprises culturing host cells containing an expression vector of the invention under conditions whereby said polypeptide or protein is expressed and isolating said polypeptide or protein. The host cells may be any cells conventionally employed for expression of recombinant proteins including in addition to E. coli cells, other bacterial cells, yeast cells and mammalian cells. Such in vitro host cells containing an expression vector for use in an expression method as described above constitute a still further aspect of the invention.
For administration to induce an immune response to P. falciparum, a polypeptide or protein of the invention may be administered to an individual, e.g. a human, together with a pharmaceutically acceptable diluent or adjuvant. Such a composition may take the form of a conventional vaccine composition. It is envisaged that relevant polypeptides or proteins of the invention may, for example, be delivered adsorbed to Alum (Alhydrogel or Adju-Phos, superfos Biosector, Kvistgørd, Denmark) or emulsified in the adjuvants Montanide ISA51 or Montanide ISA720 (Seppic, France). Other known adjuvants for human use might be employed. Such adjuvants include AS02 (GSK, Belgium) or MF59 (Chiron, Siena, Italy). However, as indicated above it may be preferred to administer a DNA composition for expression of a polypeptide or protein of the invention in vivo. Thus, the invention also provides a composition for inducing a response to P. falciparum which comprises an expression construct as described above together with a carrier. Such a composition may, for example, comprise DNA deposited on gold beads in conventional manner for formulation of a DNA vaccine.
A composition of the invention comprising, or capable of expressing in human cells, a K1 type synthetic repeat sequence as described above, may comprise a fusion construct which provides in the cells not only the synthetic repeat sequence but also other epitope-containing sequences as discussed above. However, it will be appreciated that such additional epitope-containing sequences may alternatively be provided by one or more further polypeptides or polynucleotides in the same composition or one or more different compositions.
In a further aspect, the present invention provides use of a polypeptide or protein of the invention or use of a polynucleotide of the invention capable of expressing such a polypeptide or protein in the manufacture of a composition for use in inducing an immune response to P. falciparum.
In a still further aspect, there is provided a method of inducing an immune response to P. falciparum in an individual, e.g. a human, which comprises administering to said individual a polypeptide or protein of the invention. As will be appreciated from the discussion above, such administration may be direct administration of the polypeptide or protein or indirect administration by expression in vivo from a DNA encoding the polypeptide or protein.
Polypeptides presenting as an antigenic element a K1 type synthetic repeat sequence of the invention and sera raised to such sequences may also be used to type the MSP1 block 2 region in sera from individuals infected with P. falciparum and in cultured P. falciparum schizonts.
Thus in a further aspect of the invention, there is provided a method of typing the MSP1 block 2 region in a serum from an individual infected with P. falciparum which comprises contacting the serum with a polypeptide comprising as an antigenic sequence a K1 type synthetic repeat sequence as discussed above and determining whether antibodies in said serum bind to the presented K1 type synthetic repeat sequence. Such a typing method may conveniently adopt a conventional ELISA format
In a still further aspect the invention provides a method of typing the MSP 1 block 2 region in cultured P. falciparum schizonts which comprises contacting the schizonts with a serum containing antibodies raised to a K1 synthetic repeat sequence of the invention and determining binding of said antibodies to said schizonts. Antibodies may be raised by immunisation using a fusion protein as discussed above, e.g. by mouse immunisation with the adjuvant Montanide ISA720. Such a typing method may conveniently take the form of an immunofluoresence assay employing secondary labelled antibodies labelled with a fluorescent label such as fluorescein isothiocynate.
The following examples illustrate the invention.
Sequencing of P. falciparum MSP1 Block 2 Region from Zambian Samples.
A portion of the msp1 gene spanning the block 2 region was amplified from genomic DNA isolated from peripheral blood samples of 91 individuals with P. falciparum infections in Northern Zambia. Polymerase chain reaction primers BK1F and BK3R that annealed to conserved sequences in block 1 and block 3 were used with amplification conditions as described previously (Conway et al., (1998) J. Immunol. 161, 347-359). Amplification products were run and visualised on 2% agarose gels. Allelic sizes of the gene fragment range from approximately 400-600 base pairs, and many isolates contain more than one genetic type of P. falciparum, so the predominant band was excised for each isolate. This was then purified and DNA sequencing of both strands was performed directly using the BK1F and BK3R primers, using BigDye v3.1 chemistry and electrophoresis on an ABI 377 sequencer (Applied Biosystems). Sequence data for each isolate was visually examined for the quality of every nucleotide using the Sequence Navigator program, with PCR and sequencing reactions being repeated in the case of any uncertainty. Data from the whole population sample were then compiled using the MEGALIGN programe (DNAStar Inc, Madison, Wis.).
Out of the 91 alleles of the MSP1 block 2 region which were sequenced, 49 (54%) were of the K1 type, 32 (35%) were of the R033 type and 10 (11%) were of the MAD20 type.
Although the variance in the overall repeat length was considerable (mean amino acid length of repeats=35.6, variance=146.1), it was much less than the sum of the variance in the length of each of the repeat sub-regions (SAQSGT region, mean=23.1, variance=196.6; SGPSGT region, mean=10.0, variance=43.6; SAQSGA region, mean=2.6, variance=36.0). This was due to significantly non-random negative correlations between the lengths of the different repeat sub-regions in each allele (SAQSGT region vs SAQSGA region, r=−0.50, p<0.001,
It was considered that negative correlations between the lengths of the different sub-regions of the repeats could be due to selection by immune responses specific to each, such that there would be strong selection against alleles with long sequences in more than one sub-region and thus constraint on overall length. To identify epitopes in these repeat sequences, antibodies were assayed against a panel of 23 synthetic peptides (see Table 1 below) representing all of the 12 mer amino acid sequences (with the serine of tri-peptides at position 1) in K1 type repeats of MSP1 block 2 sequences derived globally.
The above-noted twenty-three peptides were synthesised onto a cellulose solid support (Whatman) using methods as described in Frank, R. (1997) Immunology Methods Manual Vol. 2, ed. Lefkovits, I. (Academic Press, pp. 763-795). These peptides were designed to represent all of the deduced 12 mer amino acid sequences contained in the repeat region of all P. falciparum K1-like MSP1 block 2 alleles found in the GenBank database and in the above-noted study, starting with a serine at position one (rather than positions two or three) of each tri-peptide repeat (see Table 1). As controls for type-specific reactivity, 24 synthetic peptides of the MAD20-like allelic type (representing all of the known 12 mer repeat sequences starting with a serine) and 12 synthetic peptides of the RO33-like type (all contiguous peptides overlapping by 9 amino acids spanning this non-repeat allelic sequence), were also synthesised and tested with antibodies.
Sera from seventy eight West African adults, thirty eight (ages 18 to 60) from Lagos in Nigeria and forty adults (ages 22 to 70) from the village of Brefet in The Gambia were employed in ELISA as described below and a subset were employed in synthetic peptide immunoassays. Twenty sera from adults living in the United Kingdom who had never had malaria were used as negative controls. All of these samples were obtained with informed consent under the approval of the relevant local and institutional ethical committees. Four murine monoclonal antibodies were also employed. Specificities of three of these (Mabs 12.2, 123D3 and CE2) for some allelic products of the K1-like type of MSP1 block 2 were known (Locher et al., (1996) Exp. Parasitol. 84, 74-83; Cavanagh & McBride (1997) Mol. Biochem. Parasitol. 85, 197-211) and a control (Mab 12.1) was known to react with MSP1 block 4.
(iii) ELISAs
50 ng/well of each recombinant antigen (expressed as a GST fusion protein) was coated overnight at 4° C. in 100 μl coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.3) onto Immulon 4HBX flat bottom microtitre plates (Dynex Technologies Inc). Plates were washed (in PBS with 0.05% Tween 20), blocked (1% skimmed milk in PBS with Tween) for five hours, and washed again. Monoclonal antibodies were diluted as indicated, sera were diluted 1/500, and duplicate 100 μl aliquots in blocking buffer were incubated overnight at 4° C. in antigen-coated wells. The wells were washed, and then incubated with 100 μl of HRP-conjugated rabbit anti-human IgG (at 1/5000) (Dako Ltd.) before detection with O-phenylenediamine/H2O2 (Sigma). The mean OD value of each serum-antigen reaction was calculated after correction for binding of the serum to GST alone (this background OD was generally <0.1). A serum was scored as positive if the corrected OD value was higher than the mean+3 SD of values for the 20 negative control sera from UK residents. Using selected positive sera, competition ELISAs between pairs of antigens were carried out to define the specificity of the antibodies. The protocol was identical to that for direct ELISA, except that sera diluted 1/500 were pre-incubated with soluble competing antigens at concentrations ranging from 0-10 μg ml−1 for 5 hours before incubation overnight with 50 ng of plate-bound antigen.
Replicate peptide arrays on cellulose membranes were incubated in blocking solution (TBS/tween-20, 5% sucrose, 2% BSA and 3% skimmed milk powder) at 4° C. overnight. Each membrane was drained on filter paper to remove excess blocking solution and then incubated with a 1/500 dilution of serum (or monoclonal antibodies diluted as specified) in fresh blocking solution and stored at 4° C. The following day, the membranes were washed twice in each of the following Tris-based solutions, TBS/T-20, TBS/T-20/NaCl, TBS/T-20/Triton X-100 and TBS/T-20. The membranes were blotted dry and then incubated with a 1/5000 dilution of HRP-conjugated goat anti-human IgG (DAKO) in blocking solution for 4 hours. The membranes were washed twice in each of the above solutions followed by two washes in TBS. The membranes were blotted dry of excess buffer and results visualised after development in stabilised TMB substrate (Promega) for 2 mins.
Each of the three Mabs 12.1, 123D3 and CE2 had a different profile of reactivity with the K1-like synthetic peptides (
Twelve sera from Nigerian adults and twelve from Gambian adults that had antibodies to MSP1 block 2 recombinant proteins as assayed by ELISA were then tested to see whether the specificities of human antibodies could be deduced with the synthetic peptide array. Each serum reacted with between three and nine different peptides. Reactivities of two of the sera are shown in
To express the composite of repeats as an antigen, a novel DNA sequence was constructed and cloned into a pGEX plasmid for expression as a GST fusion protein in E. coli.
First, a synthetic gene sequence (SEQ. ID no. 2) encoding the designed super repeat construct was assembled from synthetic oligonucleotides for cloning into pPCR-Script Amp vector (GeneArt, Regensburg, Germany) KpnI and SacI restriction sites. This sequence, which provided a codon-optimised coding sequence for expression in E. coli and terminal Bam H1 and EcoRI sites, was subcloned from the pPCR-Script Amp vector into pGEX-2T (Amersham Pharmacia Biotech) for expression of the K1 type synthetic repeat sequence as a GST-fusion protein in BL21 (DE3) E. coli cells. Expression and purification followed the manufacturer's protocols as described previously for other MSP1 block 2 recombinant proteins (Polley et al., (2003) Infect. Immun. 71, 1833-1842; Cavanagh & McBride (1997) Mol. Biochem. Parasitol. 85, 197-211). The K1SR was expressed abundantly in soluble form and purified on a glutathione column (
Five MF1 outbred mice were immunised with the K1SR recombinant protein following a protocol used previously for immunisation with other MSP1 block 2 recombinant proteins (Polley et al., (2003) Infect, Immun. 71, 1833-1842; Cavanagh & McBride (1997) Mol. Biochem. Parasitol. 85, 197-211). All animals were given three 50 μg doses of purified protein in the adjuvant ImjectAlum (Pierce) at monthly intervals; serum was collected before immunisation and 12-14 days after each dose.
Results
Mice immunised with the K1SR recombinant protein produced antibodies to different primary sequence determinants as determined by peptide immunoassay (
Antibody reactivity was then tested against a panel of 5 P. falciparum cultured lines with different repeat sequences of the K 1-like MSP1 block 2 (
Results
All mice produced antibodies with endpoint titres of at least 1/6400 against schizonts of at least one of the cultured lines. The profile of reactivity to different parasites varied among the mice (
DNA was obtained from P. falciparum clone 3D7 and P. falciparum isolates RO33 and Wellcome maintained by the WHO Registry of Standard Strains of Malaria Parasites at the University of Edinburgh. A recombinant DNA with BamH1 and Sma I ends was inserted into the vector pQE30 so as to provide a sequence encoding an N-terminally His tagged tandem array fusion protein consisting of the following components from the N to C-terminus:
(a) a His tag of 6 His residues encoded by the vector;
(b) a MSP1 3D7 block 1-block 2 allele (Genbank accession no. NP—704838.1; amino acid positions 20-133; 3D7 MSP1 block 1, amino acid residues 20-53 and 3D7 MSP1 block 2 allele (including flanking sequences), amino acid residues 54-133.)
(c) the K1 type synthetic repeat sequence;
(d) the MSP1 RO33 block 2 allele (accession number AB 116601; amino acid residues 54-106; whole allele including flanking sequences) and
(e) the MSP1 Wellcome block 2 allele (accession number CAA33163; amino acid positions 54-118; MAD 20 type; whole allele including flanking sequences.)
Element (b) provided recognised human T cell epitopes (T cell epitope 1 (T1) at amino acid positions 20-29; T cell epitope [T2] at amino acid positions 44 to 63).
Corresponding partial constructs comprising combinations of single alleles and corresponding encoding DNA inserts are shown diagrammatically in
Two long oligonucleotides [5′flank hyb R1+5′ flank hyb F1] covering both desired T cell epitopes and the block 2 flanking region were fused together yielding a 163 bp product using a PCR protocol.
A K1SR forward primer was used in conjunction with a 3′ primer (3′ flank R1) which was designed with a K1SR complementary overhang to fuse seamlessly using PCR.
The purified products from Steps 1 and 2 can be combined in a PCR reaction and fused together using the primers 5′ flank hyb R1 and 3′ flank R1.
Each of the products generated in Steps 1-3 can be cloned into the pGEMT-easy vector and sequence verified, using both vector specific primers and insert specific primers (where appropriate).
Once each module has been sequence verified, the K1SR+flanking insert can be restriction digested and ligated into a his tag expression vector with the RO33 and Wellcome allele modules already in place.
The immunological characteristics of the final construct may be compared with those of the partial constructs shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 0510763.6 | May 2005 | GB | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11921118 | Nov 2008 | US |
| Child | 14049554 | US |
