Patent Application
20130216569
The present invention relates to a polypeptide or nucleic acid useful as a vaccine, or a component of a vaccine, against an apicomplexan parasite.
The protozoan phylum Apicomplexa contains pathogens of medical and veterinary importance including Toxoplasma gondii and the Eimeria, Plasmodium and Theileria species. Despite several decades of effort, vaccines protective against these and other related parasites are scarce. Empiric approaches to identify genuinely immunoprotective antigens as vaccine candidates have achieved mixed results and problems differentiating immunogenicity from real immune protection persist.
There is thus a need to develop improved vaccines against protozoan parasites.
As food security gains in importance, pathogens which impact upon economic poultry production are re-emerging as serious threats to global food supply and human poverty. Eimeria species parasites have a globally enzootic distribution and can cause severe enteric disease in all livestock, most notably poultry, where the annual cost is estimated to exceed £2 billion worldwide.
The total cost of coccidial infections in the UK poultry industry (circa 780 million broilers) has been estimated to be at least £42 m per annum, of which 74% is due to sub-clinical effects on weight gain and feed conversion and 24% is the cost of prophylaxis and therapy of commercial birds (Williams, 1999; A compartmentalized model for the estimation of the cost of coccidiosis to the world's poultry production industry. International Journal for Parasitology, 1209-1229).
Infections with Eimeria spp. in intensively reared poultry have to date been controlled primarily by prophylactic in-feed medication (especially for broiler production). Indeed, more than a third of all chemotherapeutics used in UK animal production are applied to control Eimeria (http://www.vmd.gov.uk/), but resistance is rife and alternatives, such as vaccination with attenuated parasites, are limited by the need to include live versions of multiple Eimeria spp., production issues and cost.
There is thus a need to develop new cost-effective control strategies for Eimeria species parasites.
FIG. 1—Putative apicomplexan Immune Mapped Protein-1 (IMP-1) homologues. (a) IMP-I intron/exon structure from Eimeria maxima, Eimeria tenella, T. gondii (XM—002370108) and Neospora caninum (NCLIV—000430; GeneDB). (b) Full length IMP-1 alignment using sequenced translated coding sequences.
FIG. 2—Eimeria maxima strain-specific traits. The influence of drug (robenidine) and W strain-specific immunity on subsequent H and W strain parasite replication and the persistence of strain-specific AFLP markers (SS) in H/W hybrid populations under selection.
FIG. 3—Fine mapping locus 1 correlated with susceptibility to strain-specific immune killing. (a) The distribution of strain-specific AFLP markers lost under immune selection across BAC EmaxBAC8fl 8. Solid markers remained absent after six generations of backcrossing under selection. (b) Recovery of strain-specific markers by immune selected backcross generations 1-6, the number represents the generation at which the marker first reappeared, −=did not reappear. (c) Transcribed sequences identified within locus 1 (ST=putative sulphate transporter, AMA-1=apical membrane antigen 1 homologue, TEF=putative transcription elongation factor). (4) Percentage reduction in oocyst output induced by immunisation with individual genes identified within locus 1 compared to mock immunised controls, candidates presented as an E. maxima H vectored transgene compared to no transgene (T), recombinant protein compared to thioredoxin alone (P) or DNA compared to vector only (D) vaccination. nd=not done. Bars marked with different letters were significantly different (p<0.05).
FIG. 4—Fine mapping locus 5 correlated with susceptibility to strain-specific immune killing. (a) The distribution of strain-specific AFLP markers lost under immune selection across BAC EmaxBAC2k08. (b) Recovery of strain-specific markers by immune selected backcross generations revealed that the entire BAC encoded-region remained absent in all generations (−). (c) Regions 1-8 selected for disruption by BAC recombineering. (d) The influence of targeted disruption on the ability of W strain-derived EmaxBAC2k08 to confer cross-protective immunity when delivered by the heterologous E. maxima H strain following transient transfection: the protective capacity retained by each recombineered BAC compared to the unmodified parent BAC. (e-f) Relative fluorescence (rf) illustrating cDNA hybridisation to a BAC-tiling NimbleGen array covering ˜22.4 Kb of locus 5 corresponding to the smaller Not I/Sfi I BAC digest product. (e) cDNA derived from sporozoite (blue) and first generation merozoite (red) lifecycle stages. (f) Profiles calculated from rf using cDNA derived from uninfected chicken intestinal tissue subtracted from chicken intestinal tissue 6 (blue) and 16 (red) hours post infection. (g) Transcribed sequences identified within locus 5 (repeats =a repetitive region, no evidence of transcription by targeted lifecycle stages, CRIP =putative cyclophilin-RNA interacting protein, RP=related protein. (h) Average total E. maxima W strain oocyst output per bird following challenge of birds immunised with E. maxima H vectored W strain transgenes presented as genomic LD-PCR amplicons. C1 =control, no transgene; C2 =control, no immunisation. Bars marked with different letters were significantly different (p<0.05).
FIG. 5—In vivo intracellular E. maxima W strain replication in naïve (red, solid line) and previously infected (homologous infection; blue, broken line) chickens. Total parasite genome numbers determined for an 8 cm intestinal section covering Meckel's diverticulum. Region A highlights the first significant difference in parasite replication between the immunised and unimmunised host. Importantly, whilst qPCR-detected parasite genome numbers are not equivalent to viable parasites, the residual parasite genomes detected after 20 hpi in the immune host were not seen to replicate and were unlikely to represent live parasites. Thus, regions A and B represent the period when immune killing must have occurred. *p<0.01, **p<0.005, ANOVA +Tukey's post hoc.
FIG. 6—Anti-coccidial protection induced by vaccination with recombinant EmIMP-1 compared with thioredoxin, PBS and no immunisations (protein, immunisation and environmental controls respectively). Bars marked with different letters were significantly different (p<0.01, ANOVA +Tukey's post hoc).
FIG. 7—PCR confirmation of targeted EmaxBAC2k08 disruption by recombineering for eight selected regions (1-8). PCR assays confirming the targeted insertion of the recombineering cassette (knockout: k/o) and the absence of unmodified BAC copies (wild type: wt). +=recombineered candidate BAC, −=unmodified original BAC.
FIG. 8—PFGE resolution of Not I/Sfi I digested unmodified EmaxBAC2k08 (lane 1) and EmaxBAC2k08 recombineered to disrupt regions 1-8 (lanes 2-9).
The present inventors have identified two genuinely protective anti-coccidial vaccine candidates. The candidates, identified in Eimeria, have readily identifiable homologues encoded within the genomes of related apicomplexan parasites.
Thus, in a first aspect, the present invention provides a polypeptide comprising the amino acid sequence as shown as SEQ ID No. 1 or a homologue (such as those shown in
In a second aspect, the present invention provides a nucleic acid capable of encoding a polypeptide according to the first aspect of the invention. The nucleic acid sequence may comprise the nucleotide sequence shown as SEQ ID No. 2, 6, 7 or 8.
In a third aspect, the present invention provides a vector comprising a nucleic acid according to the second aspect of the invention.
In a fourth aspect, there is provided a polypeptide, nucleic acid or vector according to any of the first three aspects of the invention, for use in the treatment and/or prevention of a disease associated with an apicomplexan parasite.
In a fifth aspect, the present invention provides a vaccine for the treatment and/or prevention of a disease associated with an apicomplexan parasite, which comprises:
The apicomplexan parasite may be selected from, but is not limited to, the following group of species: Eimeria, Neospora, Plasmodium, Theileria and Toxoplasma.
The disease may be selected from, but is not limited to, the following: babesiosis, coccidiosis cryptosporidiosis, cyclosporiasis, isosporiasis, malaria and toxoplasmosis.
The vaccine may comprise a plurality of amino acid/nucleic acid sequences corresponding to SEQ ID No. 1 homologues from different species of apicomplexan parasite.
For example, a vaccine against Eimeria infection in chickens may comprise a plurality of amino acid/nucleic acid sequences corresponding to SEQ ID No. 1 homologues from two or more of the following species: Eimeria acervulina; Eimeria Brunetti; Eimeria maxima; Eimeria mitis; Eimeria necatrix; Eimeria praecox and Eimeria tenella.
The vaccine may also comprise:
(i) an amino acid sequence as shown as SEQ ID No. 3 or a homologue or fragment thereof; and/or
(ii) a nucleic acid sequence capable of encoding an amino acid sequence as shown as SEQ ID No. 3 or a homologue or fragment thereof.
The disease may be coccidiosis.
The vaccine may comprise a plurality of amino acid/nucleic acid sequences corresponding to SEQ ID No. 3 homologues from different Eimeria species and strains.
In a sixth aspect, the invention provides a method for treating and/or preventing a disease in a subject by administration of an effective amount of a polypeptide according to the first aspect of the invention, nucleic acid according to the second aspect of the invention, any vector according to the third aspect of the invention or a vaccine according to the fifth aspect of the invention, to the subject.
The subject may be a vertebrate subject. The subject may, for example, be a mammalian or avian subject. The subject may be a poultry subject, such as a bantam, chicken, duck, goose, guinea fowl, partridge, pheasant or turkey.
The term “polypeptide” is used in the normal sense to mean a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The term is synonymous with “protein”. The polypeptide may be isolated from its natural environment, for example, extracted from or produced outside of an apicomplexan parasite.
The first aspect of the invention relates to a polypeptide comprising the amino acid sequence of Immune Mapped Protein-1 (IMP-1) shown as SEQ ID No. 1 below, or a homologue or fragment thereof.
The polypeptide shown as SEQ ID No. 1 is derivable from the Eimeria maxima Weybridge (W) strain. Immunisation of chickens with this protein has been shown to induce protection against challenge by the W strain (see Examples).
The corresponding sequence from the Eimeria maxima Houghton (H) strain is shown as SEQ ID No. 5. Comparison between the H and W coding sequences uncovered five nucleotide polymorphisms, two of which yield non-synonymous changes.
It is readily possible to identify homologues of this protein within the genome of other apicomplexan parasites. The amino acid sequences for homologues from E. tenella, T. gondii and N. caninum are shown in
Homologues may, for example, be identified by tBLASTx sequence analysis using the genomic or cDNA sequences, followed by RT-PCR, cloning and sequencing. Other methods include searching based upon structural features of all or part of the molecule.
The vaccine of the present invention may also comprise a polypeptide having the amino acid sequence shown as SEQ ID No. 3, or a homologue or fragment thereof.
The polypeptide shown as SEQ ID No. 3, is the Eimeria maxima W strain apical membrane antigen-1 (AMA-1). AMA-1 homologues from other apicomplexan parasites have also been identified elsewhere (i.e. Richie and Saul, 2002, Progress and challenges for malaria vaccines, Nature 415: 694-701; Zhang et al., 2010, Neospora caninum: Application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice, Exp Parasitol 125: 130436).
The polypeptide is or comprises at least part of the wild-type protein (e.g. the protein having the sequence shown as SEQ ID No. 5) or a mutant, variant, homologue or fragment thereof.
The term “wild type” is used to mean a polypeptide having a primary amino acid sequence which is homologous with the native protein (i.e., the protein from the apicomplexan parasite).
The term “mutant” is used to mean a polypeptide having a primary amino acid sequence which differs from the wild type sequence by one or more amino acid additions, substitutions or deletions. A mutant may arise naturally, or may be created artificially (for example by site-directed mutagenesis). The mutant may have at least 80% sequence identity with the wild type sequence and/or up to and including 20 amino acid substitutions, deletions or insertions.
The term “variant” is used to mean a naturally occurring polypeptide which differs from a wild-type sequence. A variant may be found within the same parasitic strain (i.e. if there is more than one isoform of the protein) or may be found within different strains. The variant may have at least 80% sequence identity with the wild type sequence and/or up to and including 20 amino acid substitutions, deletions or insertions.
Here, the term “homologue” means a polypeptide derivable from another strain or species, which performs the same function as the original protein, and/or can be predicted to share a common evolutionary history in terms of their sequence.
A homologous protein (or fragment thereof) may be identified by sequence homology (including the location of critical residues), structural, biophysical biochemical or functional homology.
A homologous sequence may be identified by a conserved putative structure or similarity between amino acid sequences such that alignment using BLASTp comparison yields an E value of 1e-20 or less across a significant portion of the full sequence.
A homologous sequence may be identified by similarity between amino acid sequences or a conserved putative structure. For example, functionally validated homologues to SEQ ID No. 3 specific to Plasmodium falciparum and T. gondii share only 29% amino acid homology, but retain the same number of cysteine residues in a similar sequence position and consequentially can be predicted to have a conserved protein structure. Typically, the homologues will include some critical sites (e.g. active sites) or structural conservation
Identity or similarity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs, such as: the GCG Wisconsin Bestfit package; the BLAST and FASTA packages; and the GENEWORKS suite of comparison tools, can calculate % identity between two or more sequences.
The term “fragment” indicates that the polypeptide comprises a fraction of the wild-type amino acid sequence. It may comprise one or more large contiguous sections of sequence or a plurality of small sections. The polypeptide may also comprise other elements of sequence, for example, it may be a fusion protein with another protein. The fragment may comprise at least 50%, more preferably at least 65%, most preferably at least 80% of the wild-type sequence. The fragment may be or comprise a sub-unit of the subject sequence.
With respect to function, the mutant, variant, homologue or fragment should be capable of inducing a protective immune response in a subject, against subsequent challenge with the relevant apicomplexan parasite.
In a second aspect, the present invention provides a nucleic acid capable of encoding a polypeptide comprising the amino acid sequence shown as SEQ ID No. 1.
The nucleic acid may be any suitable type of nucleotide sequence, such as a synthetic RNA/DNA sequence, a recombinant RNA/DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence. The nucleic acid may be isolated from its natural environment, for example, extracted from, or produced outside of, an apicomplexan parasite.
The nucleic acid may comprise the coding part of nucleotide sequence shown as SEQ ID No. 2.
The start codon is underlined, and the stop codon is shown in bold.
The nucleic acid shown as SEQ ID No. 2 codes for IMP-1 and is derivable from the Eimeria maxima Weybridge (W) strain.
The corresponding sequence from Eimeria Houghton (H) strain is shown as SEQ ID No. 6. Comparison between the H and W coding sequences uncovered five nucleotide polymorphisms, two of which yield non-synonymous changes.
The corresponding sequence from Toxoplasma gondii is shown as SEQ ID No. 7.
The corresponding sequence from Neospora canium is shown as SEQ ID No. 8.
The vaccine of the present invention may also comprise a nucleic acid sequence capable of encoding a polypeptide having the amino acid sequence shown as SEQ ID No. 3, or a homologue or fragment thereof.
The polypeptide shown as SEQ ID No. 3, is the Eimeria maxima W strain apical membrane antigen-1 (AMA-1).
The nucleic acid may comprise the coding part of the nucleotide sequence given as SEQ ID No. 4.
The start codon is underlined, and the stop codon is shown in bold.
The nucleic acid may comprise the coding sequence part of the sequences shown as SEQ ID No. 4, or a mutant, variant, homologue or fragment thereof
The term “mutant” is used to mean a nucleotide sequence which differs from the subject sequence by one or more base additions, substitutions or deletions. A mutant may arise naturally, or may be created artificially (for example by site-directed mutagenesis). The mutant may have at least 80% sequence identity with the subject sequence or up to and including 20 mutations. The mutation(s) may be “in frame” so there is not a frameshift over a significant proportion of the encoded sequence.
The term “variant” is used to mean a naturally occurring nucleic acid sequence which differs from a subject sequence. A variant may be found within the same parasitic strain (e.g. a splice variant) or may be found within different strains. The variant may have at least 80% sequence identity with the subject sequence or up to and including 20 mutations.
Here, the term “homologue” means a nucleic acid derivable from another genus, strain or species, which encodes a protein that performs a similar function as the subject protein, and/or can be predicted to share a common evolutionary history in terms of its sequence.
In the present context, a homologous sequence is taken to include a nucleotide sequence which may be identified by direct comparison, similarity between amino acid sequences when translated or a conserved putative protein structure. For example, functionally validated homologues to SEQ ID No. 3 specific to Plasmodium falciparum and T. gondii share only 29% amino acid homology, but retain the same number of cysteine residues in a similar sequence position and consequentially can be predicted to have a conserved protein structure. Typically, the homologues will include some critical sites (e.g. active sites) or structural conservation as the subject amino acid sequence.
The present invention also provides a vector comprising a nucleic acid of the present invention.
As used herein, a “vector” may be any agent capable of delivering or maintaining nucleic acid in a host cell, and includes viral, bacterial and eukaryotic vectors, plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules and nucleic acids immobilised onto solid phase particles.
Nucleic acids in accordance with the present invention can be delivered by viral or non-viral techniques.
Non-infectious delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-infectious vector to deliver nucleic acid of the invention to a target cell.
Typical transfection methods include electroporation, nucleic acid biolistics, lipid-mediated transfection, compacted nucleic acid-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs), multivalent cations such as spermine, cationic lipids or polylysine, 1,2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes and combinations thereof.
Non-viral delivery systems may also include, but are not limited to, bacterial delivery systems. Bacteria have previously been used as anticancer agents and as delivery agents for anticancer drugs.
Cell adhesion molecules are a large group of molecules involved in a variety of cell-to-cell and cell-to-extra-cellular matrix (ECM) interactions and are exploited by a number of pathogenic micro-organisms as receptors for cell entry. These molecules may be used for the targeting and uptake of both gene and drug delivery systems.
A gene gun delivery system may also be used for the delivery of DNA.
Viral delivery systems include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors or baculoviral vectors, venezuelan equine encephalitis virus (VEE), poxviruses such as: canarypox virus (Taylor et al 1995 Vaccine 13:539-549), entomopox virus (Li Y et al 1998 XIIth International Poxvirus Symposium p144. Abstract), penguine pox (Standard et al. J Gen Virol. 1998 79:1637-46) alphavirus, and alphavirus based DNA vectors.
Other examples of vectors include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation, DNA biolistics, lipid-mediated transfection and compacted DNA-mediated transfection.
The vector may be a plasmid DNA vector. As used herein, “plasmid” refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof.
The fourth aspect of the invention relates to a polypeptide, nucleic acid or vector according to any of the first three aspects of the invention, for use in the treatment and/or prevention of a disease associated with an apicomplexan parasite.
The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; as well as the Cryptosporidium spp., Eimeria spp., Theileria spp. and Toxoplasma gondii, parasites of considerable medical, zoonotic and/or agricultural importance.
The Apicomplexa are protozoa with a structure called the apical complex involved in penetrating a host's cell and most possess an organelle called the apicoplast.
Most members have a complex life-cycle, involving both asexual and sexual reproduction. Typically, a host tissue is infected via an active invasion by the zoite form of parasites, which then divide intracellularly. Eventually, the cells burst, releasing zoites which infect new cells. This may occur several times, until gamonts are produced, forming gametes that fuse to create new cysts. There are many variations on this basic pattern, however, and many Apicomplexa have more than one host.
The Apicomplexa are an extremely large and diverse group, with well over 5000 named species. There are seven genera which infect humans: Babesia, Cryptosporidium, Cyclospora, Isospora, Plasmodium, Sarcocystis and Toxoplasma.
Plasmodium, as the causative agent of malaria, has the greatest impact on human health. Babesia is a relatively rare zoonotic infection. The other five species are all classified as coccidia.
Several apicomplexan parasites are also important in terms of veterinary medicine and agriculture. Most notable are Babesia and Theileria in cattle and Eimeria in poultry, cattle and sheep.
The vaccine may be for treating and/or preventing a disease.
To “treat” means to administer the vaccine to a subject having an existing disease in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
To “prevent” means to administer the vaccine to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease (e.g. infection) or to reduce or prevent development of at least one symptom associated with the disease.
The vaccine may comprise a plurality of amino acid/nucleic acid sequences corresponding to SEQ ID No. 1 homologues and/or SEQ ID No. 3 homologues from different strains and/or species of apicomplexan parasites.
Such “multi-strain” vaccines may comprise, for example, homologues from between two and ten strains of an apicomplexan parasite. The strains may, for example, represent those most commonly associated with pathogenesis in a particular territory or region, or they may be based on the strains associated with a recent (e.g in the last year, or 5 years) outbreak of the disease.
Diseases caused by apicomplexan organisms include, but are not limited to Babesiosis (Babesia spp.) and Malaria (Plasmodium spp.), as well as the Coccidian diseases including: Coccidiosis (Eimeria spp.); Cryptosporidiosis (Cryptosporidium spp.); Cyclosporiasis (Cyclospora spp.); Isosporiasis (Isospora spp.); and Toxoplasmosis (Toxoplasma gondii).
The disease coccidiosis may be caused by Eimeria.
Coccidiosis is one of the economically most important diseases in modern livestock production. For chickens the disease is caused by the replication within the intestine of the asexual and/or sexual stages of seven species of Eimeria (usually several species occur concurrently) and although overt clinical disease in intensively reared poultry is now relatively uncommon, sub-clinical infections are the norm. Sub-clinical infection can have a massive economic impact on livestock production, incurring reduced growth rates, poor feed conversion and a requirement for prophylactic control.
Coccidiosis is a parasitic disease that can cause severe losses in poultry meat and egg production. The parasites multiply in the intestines and cause tissue damage, lowered feed intake, poor absorption of nutrients from the feed, dehydration, and blood loss. Birds are also more likely to get sick from secondary bacterial infections. Eimerian infection can also predispose towards clinical disease caused by other pathogens, for example Clostridium perfringens, a cause of necrotic enteritis in chickens.
Chicken coccidia species include: Eimeria acervulina; Eimeria brunetti; Eimeria maxima; Eimeria mitis; Eimeria necatrix; Eimeria praecox and Eimeria tenella.
Turkey coccidia species include: Eimeria adenoeides; Eimeria dispersa; Eimeria gallopavonis and Eimeria meleagrimitis.
Bovine coccidia species include: Eimeria alabamensis, Eimeria bovis and Eimeria zuernii.
The vaccine may be administered in a convenient manner such as by the oral, intravenous, intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (e.g. using slow release molecules).
For avian subjects, the vaccine may be administered individually to hatched chicks or chickens. Although accurate, these methods can be expensive e.g. for large broiler flocks. Alternative methods for treating the whole flock include in ovo vaccination, spray inoculation, provision of vaccine-containing gel blocks or administration to drinking water or feed.
Typically, a physician, veterinarian or producer will determine the actual dosage which will be most suitable for an individual subject or group of subjects and it may vary with the age, weight and response of the particular subject(s).
The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the delivery or immunogenicity of the vaccine.
For all vertebrate use, the vaccines may be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and oral formulations. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension.
Polypeptides 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 polypeptide) 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 ethanol, histidine and procaine.
The subject may be any organism susceptible to infection with an apicomplexan parasite. The subject may, for example, be a human.
The subject may be a livestock animal, such as a cow, sheep, goat or pig. The subject may be an avian subject, such as a bantam, chicken, duck, goose, guinea fowl, partridge, pheasant or turkey. The subject may be a farmed fish species.
Where the subject is an avian subject, the vaccine may be administered in ovo, to a chick (for example, a newly-hatched or one-day old chick) or to an adult bird.
The subject may be susceptible to apicomplexan infection.
Where the vaccine is used to treat an established infection, the subject may have been diagnosed as positive for the disease and/or show one or more symptom(s) associated with the infection.
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.
The E. maxima Houghton (H) and W strains are genetically and phenotypically distinct (
Comparison between the two strains revealed 1,122 markers to be polymorphic (34.7%; as shown in
Inheritance patterns of these strain-specific (SS) markers by a parasite mapping panel (Table 2), which included the uncloned progeny of eight independent H/W strain crosses before and after concurrent immune/robenidine selection, revealed the absolute correlation of 36 and 2 markers with the immune and drug barriers respectively (
Sequencing the 36 SS markers whose inheritance correlated with susceptibility to strain-specific immune selection identified 32 suitable for use as hybridisation probes. When radiolabeled with 32P and used to probe an E. maxima W strain BAC library representing ˜7.5-fold genome coverage a series of BACs was identified.
One of the identified BACs contained an AMA-1 homologue (BAC-1), a second BAC contained IMP-1 (BAC-2). Marker hybridisation to Southern blotted full PFGE-resolved karyotypes identified the chromosomal location of BAC-2 to be a band representing chromosomes 12, 13 or 14 (Table 3). These BACs were used in a protection trial (see figure legend for detail) and both conferred protection against challenge.
aSignificantly different (p < 0.01),
bsignificantly different (p < 0.005), using ANOVA + Tukey's post-hoc.
Confirmation of the potent immunogenicity of the early stages of the E. maxima lifecycle supported the development of an in vivo BAC-screening system to begin fine mapping for each locus (Table 4, trial 1). Utilising recent advances in transfection protocols for Eimeria, purified E. maxima H strain sporozoites were transiently transfected with each individual W strain BAC identified by hybridisation to an immune-correlated SS marker or a randomly selected control BAC. When Line C chickens were immunised by infection with 1×106 test-, control- or non-BAC transiently transfected H strain sporozoites, drug-cleared by exposure to 66 ppm dietary robenidine and challenged three weeks later with the W strain the capacity to induce cross-protective immunity was seen to have been conferred by a single BAC from each mapped locus (Table 4, trial 2 and Table 3). A reproducible hierarchy was observed where a single BAC from both loci 1 and 2 consistently induced high levels of immune protection (Table 3).
E. maxima H
E. maxima H
E. maxima H
E. maxima W
E. maxima H
E. maxima W
E. maxima H
E. maxima W
E. maxima W
E. maxima H
E. maxima W
E. maxima H
E. maxima W
The parasite mapping panel was supplemented by backcrossing and re-selecting one double barrier-selected population to the immune-targeted parental W strain on six successive occasions. Genotyping each selected backcross generation revealed the reappearance of 21/32 hybridisation SS markers, between one and three SS markers remaining absent for all mapped loci (e.g.
Examination of fine-mapped locus 1 identified three predicted coding regions, annotated as encoding (i) a sulphate transporter, (ii) an apical membrane antigen-1 (AMA-1) homologue and (iii) a transcription elongation factor (
Whole BAC transient transfection identified locus 2, contained within BAC EmaxBAC2k08, as capable of inducing the most protective immune response (Table 3,
Comparison of cross-protective immunity induced following infection by H strain sporozoites transiently transfected with the unaltered EmaxBAC2k08 or a deletion mutant daughter BAC revealed reduced protection across three disrupted regions, most notably region 7 (
Examination of the array-identified transcribed sequences revealed (i) a putative non-coding RNA, (ii) an unknown coding sequence temporarily termed IMP-1, (iii) a putative cyclophilin-RNA interacting protein and (iv) a SCY kinase-related protein. Infection by H strain sporozoites transiently transfected with LD PCR amplicons covering each predicted W strain coding sequence and flanking regions, including a section of repeats not found to be transcribed but identified as a cross-reactive feature by the array, induced a cross-protective immune phenotype only when using EmIMP-1 (
Further characterisation of EmIMP 1 has identified potential homologues within the genomes of other coccidial parasites including E. tenella (2e-87; NCBI BLASTp2seq), T. gondii (XM—002370108; 2e-36) and Neospora caninum (GeneDB NCLIV—000430; 4e-37), all of which share a common intron/exon structure (
The identification of new antigens as highly protective anti-coccidial vaccine candidates is a significant step towards a new generation of cost-effective vaccines. Such vaccines have the potential to revolutionise poultry production and welfare, as well as impacting on the lives of many of the poorest people in the world.
The E. maxima Houghton (H, sensitive to dietary robenidine) and Weybridge (W, resistant to 66 ppm dietary robenidine) strains were used as the parental parasites in these studies. Oocysts were propagated and genetic crosses were carried out in vivo as described previously (Blake et al. (2004) Mol Biochem Parasitol 138: 143-152).
Uncloned populations of hybrid parasites were derived from eight independent crosses between the W and H E. maxima strains in Line C White Leghorn chickens as described previously (Blake et al. (2004) as above; oocysts recovered from two-ten birds and pooled for each population). Hybrid sub-populations were derived from each cross following in vivo passage under a double selective barrier comprising W strain-specific immune (induced by previous infection with 100 oocysts of the pure W strain) and H strain-specific drug (66 ppm robenidine) selection (Blake et al. (2004) as above). All eight first generation selected parasite populations, together with four serially-selected populations (three to five rounds of selection, two-ten birds pooled per population per generation), were used to prepare the parasite mapping panel (Table 2).
One selected parasite population was backcrossed six times under double barrier selection, each backcross generation derived after in vivo phases of (i) cross fertilisation: in vivo passage using 100 sporulated hybrid oocysts with 400 sporulated W (immune-targeted) parental strain oocysts and (ii) selection: passage of 10,000 sporulated recovered parasites under double barrier selection. Parasites recovered from ten birds were pooled for each backcross stage.
Experiments to measure immune protection induced by (i) immunisation through previous parasite exposure, (ii) recombinant protein or (iii) DNA vaccination followed standardised protocols. All treatment groups comprised six individually caged specific pathogen free Line C White Leghorn chickens. Total daily oocyst excretion per bird was determined following daily faecal collection from days 6-7, 7-8 and 8-9 post infection by flotation in saturated salt solution using a modified McMaster protocol as described previously (Blake et al. (2004) as above). All experiments included a non-immunised control group.
(i) Previous parasite exposure. Three week old chickens were immunised by oral infection with 100 sporulated E. maxima oocysts (wild type) or 1.0×106 transfected E. maxima sporozoites (following oral gavage with 0.5 ml 5% w/v sodium bicarbonate solution; drug cleared three days post immunisation by inclusion of dietary robenidine at 66 ppm for three days). Six week old chickens were challenged with 250 sporulated E. maxima oocysts.
(ii) Recombinant protein. Two week old chickens were immunised subcutaneously using 100 μg PBS-dialysed recombinant protein in TiterMax Gold adjuvant (Sigma-Aldrich Ltd.) at two sites in the neck. Chickens were subsequently re-immunised at three and four weeks of age (as before but in Freund's Incomplete adjuvant; Sigma-Aldrich Ltd.) prior to challenge with 250 sporulated E. maxima oocysts when six weeks old. Unimmunised, PBS alone and PBS-dialysed thioredoxin were included as negative control groups.
(iii) DNA. Two week old chickens were immunised intramuscularly using 50 μg DNA vaccine plasmid in sterile endotoxin-free TE buffer (Qiagen) at two sites in the thigh. Chickens were subsequently re-immunised at three and four weeks of age (as before) in alternating legs prior to challenge with 250 sporulated E. maxima oocysts when six weeks old. TE alone and vector pcDNA3.1(+) (Invitrogen) were included as negative controls in addition to an unimmunised group. Tissue excised from the immunisation site post-mortem from one extra bird per group seven days post-immunisation was processed to extract total RNA using the Qiagen RNeasy mini kit (Qiagen) for cDNA preparation and PCR to confirm DNA vaccine transcription (data not shown).
Genomic DNA was extracted from oocysts as described previously using a physical smashing step followed by phenol/chloroform extraction (Blake et al (2003) Parasitology Research 90: 473-475) and from chicken intestinal tissue samples using a Qiagen DNeasy tissue kit as described by the manufacturer (Qiagen) followed by RNase A treatment (Blake et al (2006) Int J Parasitol 36: 97-105). Total RNA was purified from E. maxima sporozoite, merozoite (67 hours post infection), infected and uninfected chicken intestinal tissue using a Qiagen RNeasy kit as described by the manufacturer. A BAC library was constructed for the E. maxima W strain in the pBACe3.6 vector following the protocol of Osoegawa et al (Osoegawa et al. (1998) Genomics 52: 1-8) based upon chromosomal DNA prepared from E. maxima sporozoites as described elsewhere (Shirley et al (1990) Mol Biochem Parasitol 38: 169-173). BAC DNA was prepared using the Qiagen Large-Construct kit as described by the manufacturer (Qiagen).
Standard PCR amplification was completed using BIO-X-ACT Short DNA Polymerase (Bioline Ltd.). Each PCR reaction contained 5 ng template DNA, 20 pmol of relevant forward and reverse primers, 0.5 U Taq polymerase, 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl and 0.2 mM dNTPs. Standard cycle parameters were 1×(5 min at 94° C.), 30×(1 min at 94° C., 1 min at 54-58° C. and 1-5 min at 72° C.) and 1×(10 min at 72° C.). For LD PCR BIO-X-ACT Long DNA Polymerase (Bioline Ltd.) was used as recommended by the manufacturer. Where required cDNA was prepared using Invitrogen Superscript II reverse transcriptase and oligo dT as described by the manufacturer (Invitrogen Ltd.). PCR fragments were cloned using pGEM®-T Easy (Promega) in XL1-Blue Escherichia coli (Stratagene), miniprepped (Qiagen) and sequenced (Beckman CEQ 8000 genetic analysis system) as described by the respective manufacturers. Sequence assembly, annotation and interrogation were undertaken using VectorNTI v11.0 (Invitrogen) except where stated.
Groups of four inbred SPF Line C White Leghorn chickens were either left naive or were immunised by infection with 100 E. maxima W strain oocysts at three weeks of age. All birds were subsequently challenged by infection with 1.0×106 E. maxima W strain oocysts at six weeks of age (homologous challenge). Unimmunised and immunised groups were culled at 0, 2, 4, 6, 8, 12, 16, 20, 24, 32, 40, 48 and 72 hours post challenge, when an 8 cm length of intestine centred on Meckel's diverticulum was recovered post-mortem from each test bird and homogenised in sterile TE using a Qiagen TissueRuptor (230 V, 50/60 Hz). Total genomic DNA was extracted from three 25 μl aliquots of each sample using a Qiagen AllPrep DNA/RNA Mini kit as described by the manufacturer (Qiagen). The total numbers of E. maxima genomes per host genome were determined from each sample using TaqMan quantitative PCR assays specific for the E. maxima microneme protein 1 (MIC1) and chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loci in duplex with the 7500 Fast Real-Time PCR System (Applied Biosystems) (Blake et al (2006) as above). TaqMan probes were 5′ labeled with FAM (MIC1) or Yakima Yellow (GAPDH) and 3′ quenched with Eclipse Dark Quencher (Eurogentec). TaqMan conditions and cycle parameters were modified from the standard Applied Biosystems Fast protocol (40 cycles: 1×95° C., 20 s; 40×95 ° C., 15 s and 60° C., 30 s). Quantitative calculations were facilitated and validated by comparison with known concentrations of the relevant genomic DNA template.
AFLP was used to generate the majority of the genetic markers used during these studies as described elsewhere (Vos et al. (1995) Nucleic Acids Research 23: 4407-4414). Approximately 50 ng total E. maxima genomic DNA was digested using one of five restriction enzyme combinations (Table 1; New England Biolabs) prior to ligation to adapters derived from those described by Vos et al, adapted for the respective restriction enzyme (Vos et al (1005) as above). Primer pairs (MWG Biotech (UK) Ltd.) were based on the adaptor sequences and provided 0 and 1 (primary amplification) or 1 and 2 (secondary amplification) selective bases, respectively. Markers of interest were gel excised, re-amplified, cloned and sequenced as described previously (Blake et al (2004) as above. Marker specific-primers were designed using Primer3 to amplify marker-associated DNA fragments.
Additional genetic markers were developed by sequencing 600-750 by sections of E. maxima H strain genomic DNA corresponding to targets distributed across each locus mapped in the W strain (Table 5 for the primers used). Strain-specific primer pairs were developed following sequence alignment and SNP identification (ClustalX; Table 6).
Chromosomal karyotypes were resolved by PFGE. Briefly, E. maxima W strain chromosomal DNA in ˜4 mm×3 mm×2 mm sections cut from agarose blocks were separated in 0.8% SeaKem HGT agarose gels (Lonza) prepared in 0.5× Tris-borate EDTA (TBE) buffer and subjected to PFGE in a 21 cm×14 cm gel using a CHEF DRII system (Bio-Rad) in 2 L of 0.5×TBE running buffer at 14° C. PFGE conditions were (i) 216 h at 1.3 volts/cm with a switch time of 3,000-3,500 s followed by (ii) 120 h at 1.3 volts/cm with a switch time of 3,300-3,600 s finishing with (iii) 48 h at 1.2 volts/cm with a switch time of 3,200-3,400 s. Gels were stained in a 0.5 μg/ml aqueous solution of ethidium bromide for 30 min, destained in water and then photographed. Hansenula wingei and Schizosaccharomyces pombe DNA plugs (Bio-Rad) were included as size markers.
Individual BAC clone insert size was determined by Not I (New England Biolabs) digestion followed by PFGE in 1% Bio-Rad pulsed field certified agarose prepared in 0.5×TBE as above. PFGE conditions were 18 h at 4 volts/cm with a switch time of 1-6 s. Low range PFG size markers (New England BioLabs) were included as size markers.
PFGE-resolved chromosomal DNA was transferred to Hybond-N+ membrane (Amersham Biosciences) as recommended by the manufacturers. 3072 BAC transformed E. coli DH10B were robotically gridded onto replicated filter arrays providing ˜7.5-fold coverage of the E. maxima W strain genome. PCR products derived from AFLP markers of interest were labelled with 32P using a Prime-It II random priming kit (Stratagene) and hybridised to filters for 16-24 h at 65° C. as described by Amersham Biosciences. Filters were washed three times at 65° C. in 0.1×SSC before exposure to X-ray film (Kodak BioMax MS) at −80° C. against intensifying screens.
BAC DNA was prepared from clones identified by hybridisation to AFLP markers of interest using the Qiagen Large-Construct kit as recommended by the manufacturer. Small insert libraries (2-4 Kb) were prepared by shearing the DNA by sonication, blunt ending and size selecting by agarose gel electrophoresis prior to sub-cloning into Sma I digested dephosphorylated pUC18 for use in a whole-BAC shotgun sequencing strategy. ABI PRISM BigDye Terminator (Applied Biosystems) forward and reverse plasmid end sequences generated using an ABI3730 capillary sequencer were assembled using the Staden-based PHRAP (P. Green, unpublished). Contig assembly was based upon LD PCR.
Preliminary annotation of each assembled BAC sequence was achieved using tBLASTx interrogation of all publically accessible sequences through the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/), supplemented by Eimeria species EST data from the Eimeria ORESTES and E. maxima EST sequencing projects (Gruber and Madeira, unpublished; Wan and Blake, unpublished, respectively).
The E. coli DY380 strain (kindly supplied by the National Cancer Institute, Frederick, USA) was initially transformed with E. maxima W strain BAC Emax2K8 as described elsewhere (Thomason et al. (2007) Curr Protoc Mol Biol Chapter 1: Unit 1 16). Subsequently, the DY380/EmaxBAC2K8 line was transformed with each of eight PCR products representing (i) unique EmaxBAC2K8 sequences for targeted homologous recombination and (ii) the f3-lactamase coding sequence amplified from pGEM Teasy (Promega; primers as shown in Table 7; PCR as above, purified using the Qiagen Gel Extraction kit as described by the manufacturer)[27]. The resulting bacterial strains were sub-cloned and tested for evidence of correctly targeted insertion in a pure clonal line and the absence of widespread BAC disruption by (i) positive PCR between insert and flanking BAC sequences (
E. maxima W strain genomic DNA, presented as whole, recombineered or partial BAC-encoded templates or LD PCR amplicons, was used to transiently transfect the E. maxima H strain. For whole and recombineered BAC transfection purified plasmid DNA was re-suspended at 50 μg/10 μl TE. BAC EmaxBAC2k08 was subdivided by Not I/Sfi I digestion (New England Biolabs) and subsequent PFGE (as above), yielding ˜63.7 and ˜22.4 Kb fragments of the insert as well as the vector. The 63.7 and 22.4 Kb fragments were gel excised following large-scale PFGE, electroeluted into dialysis bags in TE, precipitated and re-suspended at 20 μg/10 μl in TE. LD PCR amplicons representing three EmaxBAC8f18 candidate regions and four EmaxBAC2k08 regions were amplified in triplicate, (primers shown in. Table 9, designed to lie >1 Kb outside any hit with an E value of 1e-05 or below or to yield an amplicon >7 Kb in size across the predicted coding sequence, whichever was the greater). All triplicates were electrophoresed to check for purity and target size, identified by test secondary PCR (Table 9), pooled once validated, precipitated and re-suspended in 10 μl TE.
Transient transfection was accomplished following a protocol modified slightly from that described previously (Clark et al. (2008) Mol Biochem Parasitol 162: 77-86). Briefly, freshly excysted and purified E. maxima H strain sporozoites were washed in incomplete cytomix and re-suspended in AMARA Basic Parasite Nucleofector Solution 2 at 3.0×107/ml immediately prior to nucleofection. Subsequently, 100 μl sporozoite suspension was mixed with 10 μl DNA at room temperature, transferred to a cuvette and nucleofected as described by the manufacturer using a Nucleofector II with program U-033 (Lonza). Post-nucleofection all sporozoites were immediately re-suspended in 3 ml PBS+1% glucose (w/v) and left to rest for 20 mins at room temperature. Sporozoites were then either used as an oral dose to initiate in vivo infection or incubated overnight at 41° C. in a 5% CO2 incubator. For oral dosing the output from two nucleofections were pooled and dosed using 1.0×106 sporozoites (counted pre-nucleofection) per bird. Total RNA was extracted from incubated sporozoites using the Qiagen RNeasy mini kit for cDNA preparation and PCR of one or more transfected DNA-specific transgenes to confirm transfection (data not shown).
A custom-made NimbleGen genome tiling array containing 79,955 50-75 by probes (presented as forward and reverse strands in duplicate) was designed and produced using BAC sequences obtained during these studies (Roche). E. maxima W strain genomic DNA covered by BACs EmaxBAC8fl8 and EmaxBAC2k08 were represented by 9,798 and 7,387 probes respectively (EmaxBAC8fl8: 8,321 unique, 1,477 twice, 84.5% coverage; ErnaxBAC2k08: 7,315 unique, 72 twice, 95.5% coverage). Total RNA extracted from purified E. maxima W strain sporozoites, merozoites (harvested 67 hpi), uninfected chicken intestinal tissue sampled at Meckel's diverticulum and chicken intestinal tissue sampled 6 and 16 hpi by 1×106 E. maxima W strain oocysts were processed to produce labelled cDNA and hybridised to the array as described by the manufacturer (Roche). Arrays were scanned using a GenePix 4000B scanner and the images were visualised using GenePix Pro software (Axon). Array design was viewed using NimbleGen SignalMap v1.9 software. Arrays were read using NimbleGen NimbleScan v2.5 software with alignment and uniformity cut off points of 0.15 and 0.3 respectively.
cDNA sequences corresponding to the predicted AMA-1 ectodomain and IMP-1 proteins (loci 1 and 2 respectively: AMA-1 coding nucleotides 79-1,347, IMP-1 161-1,275) were amplified from E. maxima W strain sporozoite cDNA, cloned into the expression vector pET32b (Novagen) using Barn HI/Hind III and Nco I/Eco RI restriction sites respectively and sub-cloned in E. coli BL21(DE3)pLysS (Novagen). Recombinant proteins were expressed and purified using HisTrap FF purification columns (GE Healthcare) as described by the respective manufacturers, dialysed overnight against PBS and finally mixed with an equal volume of adjuvant shortly before use. Thioredoxin expressed in the same manner using the unmodified pET32b vector was purified and used as a negative control.
cDNA sequences corresponding to the predicted AMA-1 ectodomain and sulphate transporter proteins (locus 1: AMA-1 coding nucleotides 79-1,347, sulphate transporter 1-2,988) were amplified from E. maxima W strain cDNA, cloned into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen) using Hind IIIIBam HI restriction sites and sub-cloned in E. coli XL1-Blue MRF (Stratagene). Plasmid DNA was purified using the Qiagen Plasmid Maxi kit as recommended by the manufacturer (Qiagen), precipitated and re-suspended in TE at 250 μg/ml.
Arithmetic mean and associated standard error of the mean (SEM) for each sample or group were calculated using Excel (Microsoft Excel 2002, Microsoft Corporation, 2001). Statistical analyses were performed using the t-test, ANOVA, Chi2 or Kruskal-Wallis tests in Minitab (Minitab Release 14, Minitab Inc., 2003), complimented by post hoc analysis using the Tukey's test. Differences were deemed significant with a p value<0.05.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1013006.0 | Aug 2010 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2011/051456 | 8/2/2011 | WO | 00 | 5/10/2013 |
