Patent Application
20150366958
The present invention relates to polypeptides from Plasmodium and polynucleotides encoding the polypeptides. The invention further relates to compositions comprising the polypeptides and their use in the treatment and prevention of malaria.
Human malaria is caused by infection with protozoan parasites of the genus Plasmodium. Four species are known to cause human disease: Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax. However, Plasmodium falciparum is responsible for the majority of severe disease and death. Recent estimates of the annual number of clinical malaria cases worldwide range from 214 to 397 million (The world health report 2002: reducing risks, promoting healthy life. Geneva: World Health Organization; Breman et al., 2004), although a higher estimate of 515 million (range 300 to 660 million) clinical cases of Plasmodium falciparum in 2002 has been proposed (Snow et al., 2004). Annual mortality (nearly all from Plasmodium falciparum malaria) is thought to be around 1.1 million (Breman et al., 2004).
Malaria also significantly increases the risk of childhood death from other causes (Snow et al., 2004). Almost half of the world's population lives in areas where they are exposed to risk of malaria (Hay et al., 2004), and the increasing numbers of visitors to endemic areas are also at risk. Despite continued efforts to control malaria, it remains a major health problem in many regions of the world, and new ways to prevent and/or treat the disease are urgently needed.
Early optimism for vaccines based on malarial proteins (so called subunit vaccines) has been tempered over the last two decades as the problems caused by allelic polymorphism and antigenic variation, original antigenic sin, and the difficulty of generating high levels of durable immunity emerged, and with the notable failures of many promising subunit vaccines (such as SPf66) have led to calls for a change in approach towards a malaria vaccine. Consequently, this growing sense of frustration has lead to the pursuit of different approaches that focus on attenuated strains of malaria parasite or irradiated Plasmodium falciparum sporozoites (Hoffmann et al., 2002). Similarly, both the limited success achieved to date with protein-based vaccines and the recognition that cell mediated immunity may be critical to protection against hepatic and perhaps blood stages of the parasite has led to a push for DNA and vectored vaccines, which generate relatively strong cell mediated immunity. Unfortunately, DNA vaccines have demonstrated poor efficacy in humans with respect to antibody induction (Wang et al., 2001). Thus, there remains a need for methods of treating and preventing malaria.
The present inventors have identified novel recombinant fragments of Plasmodium polypeptides that can be used in vaccine compositions to generate antibodies that inhibit merozoite binding and invasion of erythrocytes.
Accordingly, in one aspect, the present invention provides a recombinant combination vaccine composition comprising an isolated and/or recombinant first and a second polypeptide, wherein the first polypeptide consists of SEQ ID NO: 60 or consists of SEQ ID NO: 60 having one or more point mutations selected from the group consisting of:
L at amino acid position 70 replaced with V,
A at amino acid position 152 replaced with G,
Y at amino acid position 382 replaced with N, and
I at amino acid position 436 replaced with M;
and wherein the second polypeptide consists of SEQ ID NO: 64 or consists of SEQ ID NO: 64 having one or more point mutations selected from the group consisting of:
S at amino acid position 8 replaced with N,
E at amino acid position 163 replaced with K,
K at amino acid position 172 replaced with E,
E at amino acid position 298 replaced with V, and
G at amino acid position 340 replaced with D;
or wherein the second polypeptide consists of SEQ ID NO: 36 or consists of SEQ ID NO:
36 having one or more point mutations selected from the group consisting of:
S at amino acid position 9 replaced with N,
E at amino acid position 164 replaced with K,
K at amino acid position 173 replaced with E,
E at amino acid position 299 replaced with V, and
G at amino acid position 341 replaced with D;
and an immunologically effective amount of an adjuvant.
In a particular embodiment, the first polypeptide consists of SEQ ID NO: 60 and the second polypeptide consists of SEQ ID NO: 64.
In another embodiment, the recombinant combination vaccine composition further comprises a Rh polypeptide, wherein the Rh polypeptide is an Rh5 polypeptide selected from the group consisting of: i) an amino acid sequence selected from any one of SEQ ID NOs:17 to 28, or ii) an amino acid sequence which is at least 70% identical to any one of SEQ ID NOs:17 to 28, or iii) or an amino acid sequence of SEQ ID NO: 18 comprising one or more point mutations selected from the group consisting of:
E at amino acid position 25 replaced with K,
Y at amino acid position 124 replaced with H,
H at amino acid position 125 replaced with N,
S at amino acid position 174 replaced with Y,
C at amino acid position 180 replaced with Y,
I at amino acid position 181 replaced with K or R,
N at amino acid position 324 replaced with Y or D,
Y at amino acid position 335 replaced with F,
E at amino acid position 339 replaced with D,
V at amino acid position 348 replaced with I,
I at amino acid position 384 replaced with V,
I at amino acid position 387 replaced with M, and
K at amino acid position 406 replaced with N.
In a more particular embodiment, the Rh polypeptide comprises an amino acid sequence of SEQ ID NO: 18.
Also encompassed herein is a recombinant combination vaccine composition comprising an isolated and/or recombinant first and a second polypeptide, wherein the first polypeptide consists of SEQ ID NO: 64 or consists of SEQ ID NO: 64 having one or more point mutations selected from the group consisting of:
S at amino acid position 8 replaced with N,
E at amino acid position 163 replaced with K,
K at amino acid position 172 replaced with E,
E at amino acid position 298 replaced with V, and
G at amino acid position 340 replaced with D; and
wherein the second polypeptide consists of SEQ ID NO: 36 or consists of SEQ ID NO: 36 having one or more point mutations selected from the group consisting of:
S at amino acid position 9 replaced with N,
E at amino acid position 164 replaced with K,
K at amino acid position 173 replaced with E,
E at amino acid position 299 replaced with V, and
G at amino acid position 341 replaced with D;
and an immunologically effective amount of an adjuvant.
In a particular embodiment thereof, the first polypeptide consists of SEQ ID NO: 64 and the second polypeptide consists of SEQ ID NO: 36.
In a further embodiment thereof, the recombinant combination vaccine composition further comprises a Rh polypeptide, wherein the Rh polypeptide is an Rh5 polypeptide selected from the group consisting of: i) an amino acid sequence selected from any one of SEQ ID NOs:17 to 28, or ii) an amino acid sequence which is at least 70% identical to any one of SEQ ID NOs:17 to 28, or iii) or an amino acid sequence of SEQ ID NO: 18 comprising one or more point mutations selected from the group consisting of:
E at amino acid position 25 replaced with K,
Y at amino acid position 124 replaced with H,
H at amino acid position 125 replaced with N,
S at amino acid position 174 replaced with Y,
C at amino acid position 180 replaced with Y,
I at amino acid position 181 replaced with K or R,
N at amino acid position 324 replaced with Y or D,
Y at amino acid position 335 replaced with F,
E at amino acid position 339 replaced with D,
V at amino acid position 348 replaced with I,
I at amino acid position 384 replaced with V,
I at amino acid position 387 replaced with M, and
K at amino acid position 406 replaced with N.
In a more particular embodiment, the Rh polypeptide comprises an amino acid sequence of SEQ ID NO: 18.
In one embodiment, at least one of the polypeptides in a composition of the invention is a fusion protein comprising at least one other polypeptide sequence. The at least one other polypeptide may be, for example, a polypeptide that enhances the stability of a polypeptide of the present invention, or a polypeptide that assists in the purification or detection of the fusion protein, or a polypeptide capable of eliciting an immune response in an animal, especially a human.
In one embodiment, the fusion protein comprises a polypeptide at least 90% identical to MSP-1 (SEQ ID NO:43) or a fragment of at least about 50 amino acids thereof. In a preferred embodiment, the MSP-1 fragment is MSP-1(42) (SEQ ID NO:44) or MSP-1(19) (SEQ ID NO:45).
In another particular embodiment, the fusion protein comprises a Histidine (His) tag.
In a particular embodiment, the composition is an immunogenic composition. In one particular embodiment, the composition is a vaccine.
In a more particular embodiment, the composition comprises an adjuvant and/or pharmaceutically acceptable carrier.
In yet another embodiment, a recombinant polypeptide fragment of the invention is immunogenic.
In another aspect, the present invention provides an isolated and/or exogenous polynucleotide comprising or consisting of:
i) a sequence of nucleotides as set forth in SEQ ID NO:58,
ii) a sequence of nucleotides encoding a recombinant polypeptide fragment of the invention,
iii) a sequence of nucleotides which is at least 70% identical to SEQ ID NO:58, and/or
iv) a sequence which hybridises with any one or more of i) to iii) under at least moderately stringent conditions.
In one embodiment, the isolated and/or exogenous polynucleotide comprises or consists of:
i) a sequence of nucleotides encoding a recombinant polypeptide fragment comprising or consisting of the amino acid sequence set forth in SEQ ID NO:60 and
ii) a sequence of nucleotides encoding a recombinant polypeptide fragment comprising or consisting of the amino acid sequence set forth in SEQ ID NO:64.
In yet another embodiment, the isolated and/or exogenous polynucleotide further comprises:
i) a sequence of nucleotides encoding a recombinant polypeptide fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:36.
In another aspect, there is provided a vector comprising the isolated and/or exogenous polynucleotide of the invention. In a preferred embodiment, the polynucleotide is operably linked to a promoter.
In another aspect, the present invention provides a DNA vaccine comprising the isolated and/or exogenous polynucleotide of the invention and/or the vector of the invention.
In yet another aspect, the present invention provides a host cell comprising a recombinant polypeptide fragment of the invention, a polynucleotide of the invention, and/or a vector of the invention.
In another aspect, the present invention provides a method of making a recombinant polypeptide fragment of the invention, the method comprising:
(a) obtaining an expression vector comprising a polynucleotide sequence of the invention operably linked to a promoter; and
(b) introducing said expression vector into a cell or cell free expression system whereby said cell or cell free expression system produces the recombinant polypeptide fragment encoded by said polynucleotide sequence.
In one embodiment, the method further comprises isolating said recombinant polypeptide fragment.
In another aspect, the present invention provides a substantially purified antibody that specifically binds a recombinant polypeptide fragment of the invention.
In one embodiment, the antibody is detectably labelled.
In another aspect, there is provided a method of treating or preventing malaria in a subject, the method comprising administering to the subject a composition of the invention, a recombinant polypeptide fragment of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, and/or an antibody of the invention.
In yet another aspect, there is provided a method for raising an immune response in a subject, the method comprising administering to the subject a composition of the invention, a recombinant polypeptide fragment of the invention, a polynucleotide of the invention, a vector of the invention, and/or a host cell of the invention.
In another aspect, the present invention provides a composition of the invention, a recombinant polypeptide fragment of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, and/or an antibody of the invention for use in the treatment or prevention of malaria.
In another aspect, the present invention provides a non-human transgenic organism comprising an exogenous polynucleotide encoding a recombinant polypeptide fragment of the invention. In one embodiment, the non-human transgenic organism is a bacterium, for example, E. coli.
In another embodiment, the non-human transgenic organism is a plant. Preferably, the plant is selected from a fruit, vegetable or cereal.
In yet another aspect, the present invention provides a method of screening for an agonist or antagonist which modulates the activity of a recombinant polypeptide fragment of the invention, the method comprising contacting the recombinant polypeptide fragment with a candidate compound, and determining whether said compound binds the recombinant polypeptide fragment.
In one embodiment, the antagonist prevents a Rip recombinant polypeptide fragment from binding to an Rh5 polypeptide.
As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.
Some figures contain coloured representations or entities. Coloured versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.
SEQ ID NO:1—pfRip coding sequence
SEQ ID NO:2—pfRip amino acid sequence
SEQ ID NO:3—pfRip antigenic fragment 791-900
SEQ ID NO:4—pfRip antigenic fragment 238-368
SEQ ID NO:5—pfRip peptide 93-100
SEQ ID NO:6—pfRip peptide 101-114
SEQ ID NO:7—pfRip peptide 699-708
SEQ ID NO:8—pfRip peptide 760-769
SEQ ID NO:9—pfRip peptide 963-972
SEQ ID NO:10—pfRh1 amino acid sequence
SEQ ID NO:11—pfRh2a amino acid sequence
SEQ ID NO:12—pfRh2a/b 15 kDa antigenic fragment
SEQ ID NO:13—pfRh2a/b antigenic fragment 2030-2528
SEQ ID NO:14—pfRh2b amino acid sequence
SEQ ID NO:15—pfRh4 amino acid sequence
SEQ ID NO:16—pfRh4 antigenic fragment 28-766
SEQ ID NO:17—pfRh5 amino acid sequence
SEQ ID NO:18—pfRh5 antigenic fragment (minus leader sequence)
SEQ ID NO:19—pfRh5 antigenic fragment
SEQ ID NO:20—pfRh5 antigenic fragment
SEQ ID NO:21—pfRh5 antigenic fragment
SEQ ID NO:22—pfRh5 antigenic fragment
SEQ ID NO:23—pfRh5 antigenic fragment
SEQ ID NO:24—pfRh5 antigenic fragment
SEQ ID NO:25—pfRh5 antigenic fragment
SEQ ID NO:26—pfRh5 antigenic fragment
SEQ ID NO:27—pfRh5 antigenic fragment
SEQ ID NO:28—pfRh5 antigenic fragment
SEQ ID NO:29—pfRh5 peptide 187-197
SEQ ID NO:30—pfRh5 peptide 212-221
SEQ ID NO:31—pfRh5 peptide 237-247
SEQ ID NO:32—pfRh5 peptide 303-310
SEQ ID NO:33—pfRh5 peptide 358-366
SEQ ID NO:34—pfRh5 peptide 437-443
SEQ ID NO:35—pfEBA175 amino acid sequence
SEQ ID NO:36—pfEBA175 antigenic fragment 760-1271
SEQ ID NO:37—pfEBA181 amino acid sequence
SEQ ID NO:38—pfEBA140 amino acid sequence
SEQ ID NO:39—pfRip 238-368 codon optimised
SEQ ID NO:40—pfRip 791-900 forward primer
SEQ ID NO:41—pfRip 791-900 reverse primer
SEQ ID NO:42—pfRh2a/b 15 kDa DNA sequence
SEQ ID NO:43—MSP-1 amino acid sequence
SEQ ID NO:44—MSP-1(42) amino acid sequence
SEQ ID NO:45—MSP-1(19) amino acid sequence
SEQ ID NOs:46 to 57—Peptide linkers
SEQ ID NOs:58 and 59—nucleic acid sequence (coding and non-coding strands, respectively) encoding pfRip antigenic fragment 604-1086
SEQ ID NO:60—pfRip antigenic fragment 604-1086
SEQ ID NO: 61—N-terminal 6-Histidine (HIS)+FLAG tags, including a TEV cleavage site
SEQ ID NO:62—N-terminal 6-HIS tag (E. coli)
SEQ ID NO:63—C-terminal tag sequence (E. coli)
SEQ ID NO:64—pfEBA175 antigenic fragment 761-1298
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in immunology, protein chemistry, biochemistry, cell culture, microbiology, and molecular genetics).
Unless otherwise indicated, the immunological, microbiological and molecular genetic techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press (2001), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein, the term about, unless stated to the contrary, refers to +/−20%, more preferably +/−10%, of the designated value.
As used herein, the term “subject” refers to an animal, e.g., a mammal. In one embodiment, the subject is a human.
“Administering” as used herein is to be construed broadly and includes administering a composition or polypeptide as described herein to a subject as well as providing a composition or polypeptide as described herein to a cell.
As used herein the terms “treating”, “treat” or “treatment” include administering a therapeutically effective amount of a composition, polypeptide, polynucleotide, vector, cell and/or antibody the invention sufficient to reduce the severity of or eliminate at least one symptom of malaria in a subject such as prostration, impaired consciousness, respiratory distress (acidotic breathing), multiple convulsions, circulatory collapse, pulmonary oedema (radiological), abnormal bleeding, jaundice, and/or haemoglobinuria.
The term “preventing” refers to protecting a subject from developing at least one symptom of malaria, or delaying the onset of a symptom of malaria in a subject.
The terms “polypeptide” and “protein” as used herein are generally used interchangeably and refer to a polypeptide chain which may or may not be modified by addition of non-amino acid groups. Thus, the protein may be glycosylated, unglcosysolated, and/or may contain other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other polypeptides. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms “proteins” and “polypeptides” as used herein also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides described herein.
By “isolated polypeptide” we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other peptides, and other contaminating molecules with which it is associated in its native state. Preferably, the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.
The term “recombinant” in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state. In one embodiment the cell is a cell that does not naturally produce the polypeptide. However, the cell may be a cell which comprises a non-endogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced. A recombinant polypeptide of the invention includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. More preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. More preferably, the query sequence is at least 500 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 500 amino acids. More preferably, the two sequences are aligned over their entire length.
With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics, for example immunogenicity.
Mutant (altered) polypeptides can be prepared using any suitable technique known in the art. For example, a polynucleotide of the invention can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a “mutator” strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). These DNA shuffling techniques may include orthologous genes from closely related species. Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess desired characteristics.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. Amino acids are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of “exemplary substitutions”.
Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptides of the present invention. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Ca-methyl amino acids, Nα-methyl amino acids, and amino acid analogues in general.
Also included within the scope of the invention are polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or immunogenicity of the polypeptide of the invention.
Polypeptides of the present invention can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. A preferred cell to culture is a host cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
The terms “antigen”, “antigenic”, “antigenic fragmant” and the like are well understood in the art and refer to the portion of a macromolecule, for example a polypeptide defined herein, which is specifically recognized by a component of the immune system, for example, an antibody or a T-cell antigen receptor. The term “antigen” therefore refers to a peptide, a polypeptide, or other macromolecule to which an immune response can be induced in a host. Thus, the invention includes an antigenic fragment of a polypeptide defined herein. Preferably, the antigenic fragment is capable of raising an immune response against a pathogen of the genus Plasmodium, for example Plasmodium falicparum, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, and/or Plasmodium knowlesi. In one embodiment, the antigenic fragment is 6 amino acids in length, more preferably 7 amino acids in length, more preferably 8 amino acids in length, more preferably 9 amino acids in length, more preferably at least 10 amino acids in length. Alternatively the antigenic fragment is at least 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids in length. In an embodiment, the antigenic fragment when administered to a subject is able to elicit an immune response against at least one polypeptide comprising an amino acid sequence as provided in any one of SEQ ID NOs:2 to 4, 10 to 28 or 35 to 38. Further examples of antigenic fragments useful for the invention are described in WO 2010/022452, US 2009/0175895 and US 2009/0202579, some of which are outlined in further detail below.
In a particularly preferred embodiment, a composition of the invention comprises a Rip polypeptide, or antigenic fragment thereof. An example of an P. falciparum Rip polypeptide is provided as SEQ ID NO:2. It is known to the skilled person that there are a large number of single nucleotide polymorphism in Rip and these and any other mutations are included within the scope of the invention. Particular polymorphisms include changes to amino acids N144 to K, V190 to A, H511 to R, L673 to V, A755 to G, Y985 to N, and/or I1039 to M.
In a particularly preferred embodiment, the Rip antigenic fragment comprises, more preferably consists of, EGF domains 5 and 6 of the group of 8 EGF domains (see
i) an amino acid sequence as set forth in SEQ ID NO:3,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:3, and/or
iii) an antigenic fragment of i) or ii).
Examples of other RIP antigenic fragments include those comprising or consisting of, one, preferably two or more, and up to all 8 (such as about amino acid 636 to about amino acid 979) of the EGF domains towards the C-terminal end of pfRip (see
Yet another exemplary Rip antigenic fragment comprises or consists of amino acids 604-1086 of pfRip (SEQ ID NO:2). The pfRip fragment spanning amino acids 604-1086 of pfRip is designated herein as SEQ ID NO:60.
In another particular embodiment, the Rip antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:60,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:60, and/or
iii) an antigenic fragment of i) or ii).
In an embodiment, a composition of the invention comprises or consists of an Rh1 polypeptide, or antigenic fragment thereof. An example of an P. falciparum Rh1 polypeptide is provided as SEQ ID NO:10. It is known to the skilled person that there are a large number of single nucleotide polymorphism in Rh1 and these and any other mutations are included within the scope of the invention.
In one embodiment, the antigenic fragment comprises or consists of the region between about amino acid residue 1 to the transmembrane domain of Rh1.
In an embodiment, a composition of the invention comprises or consists of an Rh2a polypeptide, or antigenic fragment thereof. An example of an P. falciparum Rh2a polypeptide is provided as SEQ ID NO:11. It is known to the skilled person that there are a large number of single nucleotide polymorphism in Rh2a and these and any other mutations are included within the scope of the invention. Examples of such mutations are whereby A at amino acid 2546 is replaced with D, E at amino acid 2613 is replaced with G, R at amino acid 2723 is replaced with K, or K at amino acid 2725 replaced with Q.
In one embodiment, the antigenic fragment of Rh2a comprises or consists of the region between about 31 amino acids N-terminal of the Prodom PD006364 homology region to about the transmembrane domain of Rh2a. The antigenic fragment may also comprise or consist of the region from about residue 2133 to about residue 3065, the region from about residue 2098 to about residue 2597, or the region from about residue 2616 to about residue 3115, of Rh2a.
In a particularly preferred embodiment, the Rh2a antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:12,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:12, and/or
iii) an antigenic fragment of i) or ii).
In another particularly preferred embodiment, the Rh2a antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:13,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:13, and/or
iii) an antigenic fragment of i) or ii).
In an embodiment, a composition of the invention comprises or consists of an Rh2b polypeptide, or antigenic fragment thereof. An example of an P. falciparum Rh2a polypeptide is provided as SEQ ID NO:14. It is known to the skilled person that there are a large number of single nucleotide polymorphism in Rh2b and these and any other mutations are included within the scope of the invention. Examples of such mutations are whereby D at amino acid 2546 is replaced with A, K at amino acid 2635 is replaced with E, K at amino acid 3165 is replaced with N, or N at amino acid 3191 replaced with T or Y.
In one embodiment, the antigenic fragment of Rh2b comprises or consists of the region between about 31 amino acids N-terminal of the Prodom PD006364 homology region to about the transmembrane domain of Rh2b. The antigenic fragment may comprise or consist of the region from about residue 2027 to about residue 3115, more particularly from about residue 2027 to about residue 2533, of Rh2b. In other examples, the antigenic fragment may comprise or consist of the region from about residue 2098 to about residue 2597, or the region from about 2616 to 3115, of Rh2b.
In a particularly preferred embodiment, the Rh2b antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:12,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:12, and/or
iii) an antigenic fragment of i) or ii).
In another particularly preferred embodiment, the Rh2b antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:13,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:13, and/or
iii) an antigenic fragment of i) or ii).
In an embodiment, a composition of the invention comprises or consists of an Rh4 polypeptide, or antigenic fragment thereof. An example of an P. falciparum Rh4 polypeptide is provided as SEQ ID NO:15. It is known to the skilled person that there are a large number of single nucleotide polymorphism in Rh4 and these and any other mutations are included within the scope of the invention. Examples of such mutations are whereby Y at amino acid 12 is replaced with A, L at amino acid 143 is replaced with I, N at amino acid 435 is replaced with K, Q at amino acid 438 is replaced with K, T at amino acid 506 replaced with K, N at amino acid 771 is replaced with S, N at amino acid 844 is replaced with I, K at amino acid 1482 is replaced with R, or N at amino acid 1498 is replaced with I.
In one embodiment, the antigenic fragment of Rh2b comprises or consists of the region from about the MTH1187/YkoF-like superfamily domain to about the transmembrane domain of Rh4. The antigenic fragment may comprise or consist of the region from about residue 1160 to about residue 1370 of Rh4.
In another particularly preferred embodiment, the Rh4 antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:16,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:16, and/or
iii) an antigenic fragment of i) or ii).
In an embodiment, a composition of the invention comprises or consists of an Rh5 polypeptide, or antigenic fragment thereof. An example of an P. falciparum Rh5 polypeptide is provided as SEQ ID NO:17. It is known to the skilled person that there are a large number of single nucleotide polymorphism in Rh5 and these and any other mutations are included within the scope of the invention. Examples of such mutations are whereby E at amino acid 48 is replaced with K, Y at amino acid 147 is replaced with H, H at amino acid 148 is replaced with N, S at amino acid 197 is replaced with Y, C at amino acid 203 is replaced with Y, I at amino acid 204 is replaced with K or R, N at amino acid 347 is replaced with Y or D, Y at amino acid 358 is replaced with F, E at amino acid 362 is replaced with D, V at amino acid 371 is replaced with I, I at amino acid 407 is replaced with V, I at amino acid 410 is replaced with M, or K at amino acid 429 is replaced with N.
In one embodiment, the antigenic fragment lacks the 23 amino acid N-terminal leader sequence (SEQ ID NO:18). In alternate embodiments, the antigenic fragment may comprise or consist of one of the amino acid sequences provided as SEQ ID NO:19 to SEQ ID NO:28, or variants thereof such as where one or more of the above-mentioned mutations of Rh5 are present. In further embodiments, the antigenic fragment may comprise or consist of residues from about residue 203 to about residue 224, 317, 329, 345, or 351; or residues from about residue 224 to about residue 317, 329, 345, or 351; or residues from about residue 329 to about residue 345 or 351, or residues from about residue 345 to about residue 351. In one embodiment, cysteines 203 (polymorphic in P. falciparum) and 329 (absent in P. reichenowi) pair in the molecule by way of disulfide bridge to form a loop. Accordingly, in one form of the invention the antigenic fragment may comprise or consist of amino acid residues from about residue 203 to about residue 329. It is further proposed that cysteines 224 and 317 pair with either cysteine 345 or cysteine 351, such that the antigenic fragment may comprise or consist of residues from about residue 224 to about residue 345 or 351; or from about residue 317 to about residue 345 or 351.
In a further embodiment, a composition of the invention comprises or consists of EBA175, or antigenic fragment thereof. An example of an P. falciparum EBA175 polypeptide is provided as SEQ ID NO:35. It is known to the skilled person that there are a large number of single nucleotide polymorphism in EBA175 and these and any other mutations are included within the scope of the invention. Examples of such mutations are whereby N at amino acid 157 replaced with S, E at amino acid 274 replaced with K, K at amino acid 279 replaced with E, K at amino acid 286 replaced with E, D at amino acid 336 replaced with Y, K at amino acid 388 replaced with N, P at amino acid 390 replaced with S, E at amino acid 403 replaced with K, K at amino acid 448 replaced with E, K at amino acid 478 replaced with N K at amino acid 481 replaced with I, N at amino acid 577 replaced with K, Q at amino acid 584 replaced with K, R at amino acid 664 replaced with S, S at amino acid 768 replaced with N, E at amino acid 923 replaced with K, K at amino acid 932 replaced with E, E at amino acid 1058 replaced with V, or G at amino acid 1100 replaced with D.
In one embodiment, the antigenic fragment is found in the region between the F2 domain and the transmembrane domain of the EBA175 protein.
In a particularly preferred embodiment, the EBA175 antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:36,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:36, and/or
iii) an antigenic fragment of i) or ii).
In another particular embodiment, the EBA175 antigenic fragment comprises, more preferably consists of,
i) an amino acid sequence as set forth in SEQ ID NO:64,
ii) an amino acid sequence which is at least 70% identical to SEQ ID NO:64, and/or
iii) an antigenic fragment of i) or ii).
In a further embodiment, a composition of the invention comprises or consists of
EBA181, or antigenic fragment thereof. An example of an P. falciparum EBA181 polypeptide is provided as SEQ ID NO:37. It is known to the skilled person that there are a large number of single nucleotide polymorphism in EBA181 and these and any other mutations are included within the scope of the invention. Examples of such mutations are whereby V at amino acid 64 replaced with L, Q at amino acid 364 replaced with H, V at amino acid 363 replaced with D, R at amino acid 358 replaced with K, N at amino acid 414 replaced with I, K at amino acid 443 replaced with Q, P at amino acid 878 replaced with Q, E at amino acid 884 replaced with Q, E at amino acid 1885 replaced with K, Q at amino acid 890 replaced with E, P at amino acid 1197 replaced with L, K at amino acid 1219 replaced with N, D at amino acid 1433 replaced with Y or N, or K at amino acid 1518 replaced with E.
In one embodiment, the antigenic fragment is found in the region between the F2 domain and the transmembrane domain of the EBA 181 protein. The antigenic fragment may comprise or consist of the region from about residue 755 to about residue 1339 of EBA181.
In a further embodiment, a composition of the invention comprises or consists of EBA140, or antigenic fragment thereof. An example of an P. falciparum EBA140 polypeptide is provided as SEQ ID NO:38. It is known to the skilled person that there are a large number of single nucleotide polymorphism in EBA140 and these and any other mutations are included within the scope of the invention. Examples of such mutations are whereby V at amino acid 19 replaced with I, L at amino acid 112 replaced with F, I at amino acid 185 replaced with V, N at amino acid 239 replaced with S, K at amino acid 261 replaced with T.
In one embodiment, the antigenic fragment is found in the region between the F2 domain and the transmembrane domain of the EBA 140 protein. The antigenic fragment may comprise or consist of the region from about residue 746 to about residue 1045 of EBA140.
In one embodiment, a composition of the invention comprises a polypeptide which is a fusion protein comprising at least one other polypeptide sequence. The at least one other polypeptide may be, for example, a polypeptide that enhances the stability of a polypeptide of the present invention, or a polypeptide that assists in the purification or detection of the fusion protein, or preferably a polypeptide capable of eliciting an immune response in an animal, especially a human. By way of non-limiting example, the at least one other polypeptide sequence may comprise one or more T cell epitopes for recruitment of T helper cells or activation of cytotoxic T cells, or one or more antigens, cytokines and/or chemokines.
In an embodiment, the at least one other polypeptide is a polypeptide from Plasmodium falciparum. Preferably, the at least one other polypeptide from Plasmodium falciparum comprises one or more T cell epitopes for recruitment of T helper cells, and/or one or more MHC class I or MHC class II motifs. Methods for the identification of T cell epitopes and MHC class I and MHC class II motifs are known in the art and described in, for example, Rammensee (1995), Ohta et al. (1998), and Singh et al. (2010).
In one particular embodiment, the at least one other polypeptide is merozoite surface protein-1 (MSP-1) or a fragment of at least 50 amino acids thereof. An example of MSP-1 is provided as SEQ ID NO:43 (GenBank Accession No. BAF62268.1 and related molecules). Examples of MSP-1 fragments include MSP-1(42) provided as SEQ ID NO:44 and MSP-1(19) provided as SEQ ID NO:45.
In addition, the fusion protein may comprise one or more linkers or spacers. A “linker” or “spacer” as used herein refers to a peptide, polypeptide or other molecule, for example a straight or branched-chain carbon linker or heterocyclic carbon linker, that may be included between two polypeptides in a fusion protein to enhance expression of the protein in a bacterial or eukaryotic cell or to decrease steric hindrance such that one or more of the polypeptides in the fusion protein may assume its desired tertiary structure and/or interact appropriately with its target molecule, such as, for example, a B cell receptor or T cell receptor. Thus, the fusion protein may comprise one or more spacers before, after, or between one or more polypeptide domains in the fusion polypeptide. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. For spacers and methods of identifying desirable spacers, see, for example, George, et al. (2003).
In one embodiment, the spacer comprises one or more amino acid sequences that are between 1-50 amino acid residues in length, or about 1-25 residues, or about 5-15 residues in length.
Non-limiting examples of peptide linkers include AAA, GGG, SGG, GGSGGS (SEQ ID NO:46), SAT, PYP, PSPSP (SEQ ID NO:47), ASA, ASASA (SEQ ID NO:48), PSPSP (SEQ ID NO:49), KKKK (SEQ ID NO:50), RRRR (SEQ ID NO:51), GGGG (SEQ ID NO:52), GGGGS (SEQ ID NO:53), GGGGS GGGGS (SEQ ID NO:54), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:55), GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:56), and GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:57).
As known in the art, various chemical groups may be incorporated in the spacer segment instead of amino acids. Examples are described in U.S. Pat. No. 5,910,300. In one embodiment the spacer is comprised of an aliphatic chain optimally interrupted by heteroatoms, for example a C2-C6 alkylene, or ═N—(CH2)2-6—N═. Alternatively, a spacer may be composed of alternating units, for example of hydrophobic, lipophilic, aliphatic and aryl-aliphatic sequences, optionally interrupted by heteroatoms such as O, N, or S. Such components of a spacer are preferably chosen from the following classes of compounds: sterols, alkyl alcohols, polyglycerides with varying alkyl functions, alkyl-phenols, alkyl-amines, amides, hydroxyphobic polyoxyalkylenes, and the like. Other examples are hydrophobic polyanhydrides, polyorthoesters, polyphosphazenes, polyhydroxy acids, polycaprolactones, polylactic, polyglycolic polyhydroxy-butyric acids. A spacer may also contain repeating short aliphatic chains, such as polypropylene, isopropylene, butylene, isobutylene, pentamethlyene, and the like, separated by oxygen atoms.
The term “antibody” as used in this invention includes polyclonal, monoclonal, chimeric and humanised antibodies, and includes intact molecules as well as molecules comprising or consisting of fragments thereof, such as Fab, F(ab′)2, and Fv which are capable of binding an epitopic determinant. Thus, antibodies may exist as intact immunoglobulins, or as modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light and heavy chain variable regions, or Fd fragments containing the heavy chain variable region and the CH1 domain. A scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term “antibody”. As outlined above, also encompassed are fragments of antibodies such as Fab, (Fab′)2 and FabFc2 fragments which contain the variable regions and parts of the constant regions. CDR-grafted antibody fragments and oligomers of antibody fragments are also encompassed. The heavy and light chain components of an Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region. The antibody may be of animal (for example mouse, rabbit, chicken or rat) or human origin or may be chimeric or humanized.
The antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (VH) chain. The light and heavy chains may be joined directly or through a linker. As used herein a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed. Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.
As used herein, the term “specifically binds” shall be taken to mean a protein of the invention reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or antigens or cell expressing same than it does with alternative antigens or cells. For example, a protein that specifically binds to an antigen binds that antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens. It is also understood by reading this definition that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen. As such, “specific binding” does not necessarily require exclusive binding or non-detectable binding of another antigen, this is meant by the term “selective binding”. Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.
The antibody may be detectably labelled, such as for example, labelled with a fluorescent label (e.g. FITC or Texas Red), radiolabel, or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or β-galactosidase.
A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with the polypeptides of the invention. For example, surface labelling and flow cytometric analysis or solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See Harlow & Lane (supra) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
By “isolated polynucleotide” we mean a polynucleotide which has generally been separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. Furthermore, the term “polynucleotide” is used interchangeably herein with the terms “nucleic acid molecule”, “gene” and “mRNA”.
The term “exogenous” in the context of a polynucleotide refers to the polynucleotide when present in a cell, or in a cell-free expression system, in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded polypeptide. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
“Polynucleotide” as used herein refers to a oligonucleotide, polynucleotide or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity defined herein.
The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. Even more preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. More preferably, the two sequences are aligned over their entire length.
With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shuffling on the nucleic acid as described above). It is thus apparent that polynucleotides of the invention can be either naturally occurring or recombinant.
Polynucleotides of the invention include those which hybridize under stringent conditions to a polynucleotide comprising a sequence of nucleotides which is at least 50% identical, preferably at least 70% identical, more preferably at least 90% identical, to SEQ ID NO:1, SEQ ID NO:39 or SEQ ID NO:42. The term “stringent hybridization conditions” and the like as used herein refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra). For example, “moderately stringent” hybridization conditions, as used herein, can refer to hybridization at 20° C. to 64° C. in 3.5×SSC, 0.1% w/v SDS, and “high stringency” conditions can refer to hybridization at 65° C. in 0.2×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 mM EDTA.
One embodiment of the present invention includes a recombinant vector, which comprises at least one isolated polynucleotide molecule of the present invention, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Such a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and may be a transposon, a virus or a plasmid.
“Operably linked” as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory element to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells.
In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention. In particular, expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed polypeptide of the present invention to be secreted from the cell that produces the polypeptide and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a polypeptide of the present invention. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
Another embodiment of the present invention includes a host cell comprising one or more recombinant molecules of the present invention. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides of the present invention or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include animal, plant, bacterial, fungal (including yeast), parasite, and arthropod cells. Preferably, the host cell is a bacterial cell, for example E. coli.
Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
The present invention provides compositions comprising the polypeptide, including antigenic fragments, defined herein. In one embodiment, the composition is an immunogenic composition. An “immunogenic composition” refers to a composition that comprises materials that elicit a desired immune response and includes a “vaccine”. The term “vaccine” covers any composition that induces an at least partially protective immune response against a targeted pathogen or which efficaciously protects against the pathogen; for instance, after administration or injection into the subject (for example, a mammal such as a human), elicits an at least partially protective immune response against the targeted pathogen or provides efficacious protection against the pathogen (e.g., Plasmodium falciparum). By inducing an “at least partially protective” immune response it is meant that a vaccine reduces infection and/or reduces at least one symptom caused by infection with a pathogen expressing at least one polypeptide as defined herein.
An immunogenic composition may select, activate or expand cells of the immune system including memory B and T cells to, for example, enable the elimination of infectious agents, such as pathogens expressing at least one polypeptide as defined herein.
In some embodiments, an immunogenic composition includes a suitable carrier, such as an adjuvant, which is an agent that acts in a non-specific manner to increase the immune response to a specific antigen, or to a group of antigens, enabling the reduction of the quantity of antigen in any given dose, or the reduction of the frequency of dosage required to generate the desired immune response. A desired immune response may include, for example, full or partial protection against infection by a Plasmodium species or full or partial protection from developing one or more symptoms of malaria. For example, a desired immune response may include any value from between 10% to 100%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, protection against infection by Plasmodium in a vaccinated subject when compared to a non-vaccinated subject.
Adjuvants are useful for improving the immune response and/or increasing the stability of vaccine preparations. Adjuvants are typically described as non-specific stimulators of the immune system, but also can be useful for targeting specific arms of the immune system. One or more compounds which have this activity may be added to the vaccine. Therefore, particular vaccines of the present invention further comprise an adjuvant. Examples of chemical compounds that can be used as adjuvants include, but are not limited to aluminum compounds (e.g., alum, aluminum hydroxide), metabolizable and non-metabolizable oils, mineral oils including mannide oleate derivatives in mineral oil solution (e.g., MONTANIDE ISA 70 from Seppic SA, France), and light mineral oils such as DRAKEOL 6VR, block polymers, ISCOM's (immune stimulating complexes), vitamins and minerals (including but not limited to: vitamin E, vitamin A, selenium, and vitamin B12), saponin-based adjuvants (for example as described in Sun et al. (2009)) and CARBOPOL®. Other suitable adjuvants, which sometimes have been referred to as immune stimulants, include, but are not limited to: cytokines, growth factors, chemokines, supernatants from cell cultures of lymphocytes, monocytes, cells from lymphoid organs, cell preparations and/or extracts from plants, bacteria or parasites (Staphylococcus aureus or lipopolysaccharide preparations) or mitogens. Specific adjuvants include MPL, adjuvants from GSK's Adjuvant Systems such as the AS range, eg. AS01, AS02, AS03, AS04, AS15, fractionss from Quillaja saponaria such as QH-B fraction, QS-7, QS-17, QS-18 and QS-21 fractions (Antigenics, New York, N.Y.). Further details regarding suitable adjuvants are provided in the following passages.
Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. (e.g. see chapters 8 & 9 of Powell & Newman (eds.) Vaccine Design (1995) Plenum), or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt (WO 00/23105).
A typical aluminium phosphate adjuvant is amorphous aluminium hydroxyphosphate with PO4/A1 molar ratio between 0.84 and 0.92, included at 0.6 mg A13+/ml. Adsorption with a low dose of aluminium phosphate may be used e.g. between 50 and 100 μg Al3+ per conjugate per dose. Where an aluminium phosphate it used and it is desired not to adsorb an antigen to the adjuvant, this is favoured by including free phosphate ions in solution (e.g. by the use of a phosphate buffer).
Oil emulsion compositions suitable for use as adjuvants in the invention include oil-in-water emulsions and water-in-oil emulsions.
A submicron oil-in-water emulsion may include squalene, Tween 80, and Span 85 e.g. with a composition by volume of about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85 (in weight terms, 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85), known as ‘MF595’ (57-59 chapter 10 of Powell & Newman (eds.) Vaccine Design (1995) Plenum; chapter 12 of O'Hagen (ed.) Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series)). The MF59 emulsion advantageously includes citrate ions e.g. 10 mM sodium citrate buffer.
An emulsion of squalene, a tocopherol, and Tween 80 can be used. The emulsion may include phosphate buffered saline. It may also include Span 85 (e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene tocopherol is preferably <1 as this provides a more stable emulsion. One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml squalene), then microfluidising the mixture. The resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250 nm, preferably about 180 nm.
An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100) can be used. An emulsion of squalane, polysorbate 80 and poloxamer 401 (“Pluronic™ L 121”) can be used. The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the “SAF-I” adjuvant, (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the “AF” adjuvant (Hariharan et al. (1995) Cancer Res 55:3486-9) (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used.
Saponin formulations may also be used as adjuvants in the invention (see for example Chapter 22 of Powell & Newman (eds.) Vaccine Design (1995) Plenum). Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs. QS21 is marketed as Stimulon™
Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS1 8, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. Saponin formulations may also comprise a sterol, such as cholesterol (WO 96/33739).
As discussed supra, combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexs (ISCOMs) (see for example Chapter 23 of Powell & Newman (eds.) Vaccine Design (1995) Plenum). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QHA and QHC. ISCOMs are further described in WO 96/33739, EP-A-0109942, WO 96/11711). Optionally, the ISCOMS may be devoid of additional detergent WO 00/07621.
Virosomes and virus-like particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi). VLPs are discussed further in WO03/024480 and WO03/024481.
Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostiinulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof
Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref 77. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 μm membrane (EP-A-0689454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosamine de phosphate derivatives e.g. RC-529. Lipid A derivatives include derivatives of lipid A from E. coli such as 0M-174. OM-174.
Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory. The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. WO02/26757 and WO99/62923 disclose possible analog substitutions e.g. replacement of guanosine with 2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in WO 98/40100, U.S. Pat. No. 6,207,646, U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199. The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. The CpG sequence may be specific for inducing a TH1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs. Preferably, the CpG oligonucleotide is constructed so that the 5′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3′ ends to form “immunomers” (see, for example, WO03/035836).
Other immunostimulatory oligonucleotides include a double-stranded RNA or an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.
Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (E. coli heat labile enterotoxin “LT”), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO 95/17211 and as parenteral adjuvants in WO 98/42375. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192.
Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-I5 IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-17, IL-18, IL-23, IL-27), interferons (e.g. interferon-γ), macrophage colony stimulating factor, tumor necrosis factor and macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta.
Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. S uitable bioadhesives include esterified hyaluronic acid microspheres or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention (WO 99/27960).
Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).
Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, EP-A-0626169.
Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters (WO 99/52549). Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO 01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152). Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
Phosphazene adjuvants include poly(di(carboxylatophenoxy)phosphazene) (“PCPP”).
Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
Imidazoquinoline adjuvants include Imiquimod (“R-837”) (U.S. Pat. No. 4,680,338 and U.S. Pat. No. 4,988,815), Resiquimod (“R-848”) (WO92/15582), and their analogs; and salts thereof (e.g. the hydrochloride salts). Further details about immunostimulatory imidazoquinolines can be found in U.S. Pat. Nos. 4,689,338, 4,929,624, 5,238,944, 5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937, 5,482,936, 5,494,916, 5,525,612, 6,083,505, 6,440,992, 6,627,640, 6,656,938, 6,660,735, 6,660,747, 6,664,260, 6,664,264, 6,664,265, 6,667,312, 6,670,372, 6,677,347, 6,677,348, 6,677,349, 6,683,088, 6,703,402, 6,743,920, 6,800,624, 6,809,203, 6,888,000 and 6,924,293.
Thiosemicarbazone adjuvants include those disclosed in WO 2004/060308. Methods of formulating, manufacturing, and screening for active compounds are also described in WO 2004/060308. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
Tryptanthrin adjuvants include those disclosed in WO 2004/064759. Methods of formulating, manufacturing, and screening for active compounds are also described in WO 2004/064759. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
Various nucleoside analogs can be used as adjuvants, such as (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine) and prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds disclosed in U.S. Pat. No. 6,924,271, US 2005/0070556 and U.S. Pat. No. 5,658,731, or (f) a pharmaceutically acceptable salt of any of (a) to (g), a tautomer of any of (a) to (g), or a pharmaceutically acceptable salt of the tautomer.
Small molecule immunopotentiators useful as adjuvants include N2-methyl-1-(2-methylpropyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2,N2-dimethyl-1-(2-methylpropyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2-ethyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2-methyl-1-(2-methylpropyl)-N2-propyl-1H-imidazo(4,5-c)quinoline-2,4-diamine; 1-(2-methylpropyl)-N2-propyl-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2-butyl-1-(2-methylpropyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2-butyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo(4,5-c)quinorme-2,4-diamine; N2-methyl-1-(2-methylpropyl)-N2-pentyl-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2-methyl-1-(2-methylpropyl)-N2-prop-2-enyl-1H-imidazo(4,5-c)quinoline-2,4-diamine; 1-(2-methylpropyl)-2-((phenylmethyl)thio)-1H-imidazo(4,5-c)quinolin-4-amine; 1-(2-methylpropyl)-2-(propylthio)-1H-imidazo(4,5-c)quinolin-4-amine; 2-((4-amino-1-(2-methylpropyl)-1H-imidazo(4,5-c)quinolin-2-yl)(methyl)amino)ethanol; 2-((4-amino-1-(2-methylpropyl)-1H-imidazo(455-c)quinolin-2-yl)(methyl)amino)ethyl acetate; 4-amino-1-(2-methylpropyl)-1,3-dihydro-2H-imidazo(4,5-c)quinolin-2-one; N2-butyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2-butyl-N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; N2,N2-dimethyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo(4,5-c)quinoline-2,4-diamine; 1-(4-amino-2-(methyl(propyl)amino)-1H-imidazo(4,5-c)quinolin-1-yl}-2-methylpropan-2-ol; 1-(4-amino-2-(propylamino)-1H-imidazo(4,5-c)quinolin-1-yl)-2-methylpropan-2-ol; N43N4-dibenzyl-1-(2-methoxy-2-methylpropyl)-N2propyl-1H-imidazo(4,5-c)quinoline-2,4-diamine
One potentially useful adjuvant is an outer membrane protein proteosome preparation prepared from a first Gram-negative bacterium in combination with a liposaccharide preparation derived from a second Gram-negative bacterium, wherein the outer membrane protein proteosome and liposaccharide preparations form a stable non-covalent adjuvant complex. Such complexes include “IVX-908”, a complex comprised of Neisseria meningitidis outer membrane and lipopolysaccharides. They have been used as adjuvants for influenza vaccines (WO 02/072012).
Other substances that act as immunostimulating agents are disclosed in Vaccine Design ((1995) eds. Powell & Newman. ISBN: 030644867X. Plenum) and Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series) (ISBN: 1-59259-083-7. Ed. O'Hagan). Further useful adjuvant substances include: Methyl inosine 5′-monophosphate (“MIMP”); a polyhydroxlated pyrrolizidine compound (WO 2004/064715), examples include, but are not limited to: casuarine, casuarine-6-α-D-glucopyranose, 3-epz-casuarine, 7-epz-casuarine, 3,7-diepz-casuarine, etc; a gamma inulin or derivative thereof, such as algammulin; compounds disclosed in PCT/US2005/022769; compounds disclosed in WO 2004/87153, including: Acylpiperazine compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ) compounds, Benzocyclodione compounds, Aminoazavinyl compounds, Aminobenzimidazole quinolinone (ABIQ) compounds (U.S. Pat. No. 6,606,617, WO 02/018383), Hydrapthalamide compounds, Benzophenone compounds, Isoxazole compounds, Sterol compounds, Quinazilinone compounds, Pyrrole compounds (WO 04/018455), Anthraquinone compounds, Quinoxaline compounds, Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole compounds (WO 03/082272); loxoribine (7-allyl-8-oxoguanosine) (U.S. Pat. No. 5,011,828); a formulation of a cationic lipid and a (usually neutral) co-lipid, such as aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine (“Vaxfectin™”) or aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine (“GAP-DLRIE:DOPE”). Formulations containing (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium salts are preferred (U.S. Pat. No. 6,586,409).
The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion (WO 99/11241); (2) a saponin (e.g. QS21)+a nontoxic LPS derivative (e.g. 3dMPL) (WO 94/00153); (3) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) (WO 98/57659); (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (EP 0835318, EP 0735898, EP 0761231); (6) Ribi™ adjuvant system (RAS), (Ribi Imrnunochern) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); and (7) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dMPL).
Generally, an adjuvant is administered at the same time as the antigen. However, adjuvants can also, or alternatively be administered within a two-week period prior to the vaccination, and/or for a period of time after vaccination, i.e., so long as the antigen persists in the tissues.
Vaccine may be administered in various ways known to the skilled person, for example in particulate form, such as on a microcarrier or a nanocarrier (Paolicelli et al., 2010). One particular system uses PRINTED technology by delivering the vaccine antigen on a dissolvable particle (Liquidia Technologies, NC, USA).
The immunogenic compositions and vaccines according to the invention may be further supplemented by the addition of other recombinant or purified antigens which may result in the production of antibodies of a variety of specificities when administered to a subject. Not all of these antibodies need to be protective against a disease. In a particular embodiment of this type, such antigens are also from Plasmodium, for example, from Plasmodium falciparum. Thus, a vaccine of the present invention may contain various other active or inactivated pathogenic factors, along with at least one polypeptide defined herein. Therefore, in accordance with the present invention, at least one polypeptide defined herein can be combined with other Plasmodium and non-Plasmodium antigens.
In one embodiment, the composition of the invention comprises an Rh polypeptide or antigenic fragment thereof. As would be known to the person skilled in the art, Rh polypeptides belong to the family of reticulocyte binding-like proteins in Plasmodium spp. that are important for invasion of erythrocytes by merozoites. In Plasmodium falciparum, the Rh polypeptide family includes pfRh1 (e.g., PlasmoDB accession PFD0110w (www.plasmodb.org); Genbank accession AF533700; AF411933; AF411930), pfRh2a (e.g., PlasmoDB accession PF13—0198; Genbank accession AY138497; AY138498; AY138499), pfRh2b (e.g., PlasmoDB accession MAL13P1.176; Genbank accession AY138500; AY138501; AY138502; AY138503), pfRh4 (e.g., PlasmoDB PFD1150c; Genbank accession AF432854; AF420309), and pfRh5 (e.g., PlasmoDB PFD1145c; Genbank accession XP—001351544). Further details of Rh polypeptides and antigenic fragments thereof are provided above.
In another embodiment, the composition of the invention comprises an EBA polypeptide or antigenic fragment thereof. As would be understood in the art, EBA polypeptides belong to the Plasmodium erythrocyte binding-like (ebl) protein family which have also been shown to be important in merozoite invasion of erythrocytes. In Plasmodium falciparum, the EBA polypeptide family includes EBA-175 (e.g., PlasmoDB accession MAL7P1.176; Genbank accession XP—001349207), EBA-181 (e.g., PlasmoDB accession PFA0125c; Genbank accession ACN62280), EBA-165 (e.g., PlasmoDB accession PFD1155w; Genbank accession XP—001351546), and EBA-140 (e.g., PlasmoDB accession MAL13P1.60; Genbank accession XP—001349859). Further details of EBA polypeptides and antigenic fragments thereof are provided above.
A composition of the invention typically comprises a pharmaceutically acceptable carrier. Such carriers include any excipient that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate-buffered physiologic saline is a typical carrier.
The pH of the composition is preferably between 6 and 8, preferably about 7. The pH may be maintained by the use of a buffer. A phosphate buffer is typical. The composition may be sterile and/or pyrogen-free. The composition may be isotonic with respect to humans. Compositions may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10+/−2 mg/ml NaCl is typical. Compositions may also comprise a detergent e.g. a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g. <0.01%.
Compositions may comprise a sugar alcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose) e.g. at around 15-30 mg/ml (e.g. 25 mg/ml), particularly if they are to be lyophilised or if they include material which has been reconstituted from lyophilised material. The pH of a composition for lyophilisation may be adjusted to around 6.1 prior to lyophilisation.
The composition may further comprise an antimalarial that is useful for the treatment of Plasmodial infection. Preferred antimalarials for use in the compositions include the chloroquine phosphate, proguanil, primaquine, doxycycline, mefloquine, clindamycin, halofantrine, quinine sulphate, quinine dihydrochloride, gluconate, primaquine phosphate and sulfadoxine.
The compositions of the invention may also comprise one or more immunoregulatory agents. Preferably, one or more of the immunoregulatory agents include(s) an adjuvant. The adjuvant may be selected from one or more of the group consisting of a TH1 adjuvant and TH2 adjuvant, further discussed below.
The immunogenic compositions and vaccines of the present invention may be administered in any suitable form such as a liquid, emulsion, dried powder and/or in a mist through any parenteral route, intravenously, intraperitoneally, intradermally, by scarification, subcutaneously, intramuscularly, or inoculated by a mucosal route, e.g., orally.
The immunogenic compositions and vaccines of the present invention may be administered using a variety of vaccination regimes familiar to the skilled person. In one form of the invention, the vaccine composition may be administered post antimalarial treatment. For example, blood stage parasitaemia may be cleared with Fansidar (25 mg sulfadoxine/0.75 mg pyrimethamine per kg body weight) before each vaccination. In another form of the invention antimalarial (e.g. Fansidar) treatment is given 1 to 2 weeks before the doses (e.g. first and third doses). In another form of the invention antimalarial (e.g. Fansidar) treatment is given before the first dose.
In another form of the invention, 3 doses of vaccine composition (e.g. 0.5 mg adsorbed onto 0.312 g alum in 0.125 mL) is administered in 3 doses, 2 mg per dose to >5 year olds, 1 mg to under 5 year olds, at weeks 0, 4, and 25. In another form of the invention, 3 doses of vaccine composition (e.g. 1 mg per dose) are given subcutaneously at weeks 0, 4, and 26. In another form of the invention, 3 doses of vaccine composition is administered on days 0, 30, and 180 at different doses (e.g. 1 mg; 0.5 mg). In another form of the invention, 3 doses of vaccine composition is administered at 3 to 4 month intervals either intramuscularly or subcutaneously. In another form of the invention 3 doses of vaccine composition is administered subcutaneously on days 0, 30, and about day 180. In another form of the invention, the vaccine composition is administered in 2 doses at 4-week intervals (e.g. 0.55 mL per dose containing 4 ng or 15 ng or 13.3 ng of each antigen). In another form of the invention, 3 doses of the vaccine composition is administered (e.g. 25 ng in 250 nL AS02A adjuvant) intramuscularly in deltoid (in alternating arms) at 0, 1, and 2 months. In another form of the invention 4 doses of the vaccine composition is given (e.g. 50 ng per 0.5 mL dose) on days 0, 28, and 150; and dose 4 given in the following year. In another form of the invention, where the vaccine is a DNA vaccine, the vaccine composition is administered in two doses (e.g. 2 mg on days 0 and 21 (2 intramuscular injections each time, 1 into each deltoid muscle). In another form of the invention, where the vaccine composition comprises an immunogenic molecule covalently linked to another molecule (e.g. Pseudomonas aeruginosa toxin A) the composition is administered in 3 doses (e.g. at 1, 8, and 24 weeks).
The polypeptides of the invention may be employed in a screening process for compounds which activate (agonists) or inhibit (antagonists) the ability of the polypeptide to bind an erythrocyte receptor (receptor binding).
Examples of potential antagonists include antibodies, oligosaccharides and derivatives thereof. A potential antagonist includes a small molecule which binds to the polypeptide of the invention, making it inaccessible to a binding partner of the polypeptide. Examples of small molecules include, but are not limited to, small organic compounds, small peptides or peptide-like molecules. The small molecules may mimic the structure of a binding partner of the polypeptide according to the invention.
The invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit the biological activity (i.e., affect receptor binding activity) of a polypeptide of the invention. HTS assays permit screening of large numbers of compounds in an efficient manner. HTS assays are designed to identify “hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the “hit” or “lead compound” is often based on an identifiable structure/activity relationship between the “hit” and the polypeptide.
Processed 45 kDa pfRh5 from parasite culture supernatant was purified by ion-exchange chromatography. Analysis of pfRH5 by size exclusion chromatography on a Superdex 200 analytical column demonstrated that pfRh5 was eluted as an ˜150-200 kDa species (
To determine whether pfRh5 is in complex with other molecules or if it forms a homo-oligomer, the protein was incubated with pfRh5 antibody and analysed by size exclusion chromatography. A 300 μl pfRh5-containing fraction isolated from culture supernatant was loaded onto a Superdex 200 analytical column and eluted with PBS (
PfRh5 complex purified from culture supernatant of RhSHA parasite line by anti-HA affinity resin was subjected to trypsin digestion in solution and the resulting peptides analysed by mass spectrometry (LC-MS/MS) and identified by searching databases (Table 2). The results show that PfRh5 binds pfRip (SEQ ID NO:2).
A single Strep-tag and triple Haemaglutinin (HA) tag were added to the C-terminus of pfRip by 3′-single homologous cross-over recombination (
Culture supernatants from both wt 3D7 and 3D7-pfRipHA parasite lines were immunoprecipitated with anti-HA-Sepharose bead. Bound materials were separated by SDS-PAGE, transferred to nitrocellulose membrane to probe for pfRh5 using monoclonal anti-pfRh5 antibody (clone 2F1). Detection of pfRh5 in the bound material only from 3D7-pfRipHA line indicated that pfRh5 was specifically co-immunoprecipitated with pfRipHA (
Culture supernatants from both wt 3D7 and 3D7-pfRipHA parasite lines were immunoprecipitated with monoclonal anti-pfRh5 antibody coupled to Mini-bead, and culture supernatant of 3D7-pfRipHA parasites was incubated with just Mini-bead as additional control. Bound materials were separated by SDS-PAGE, transferred to nitrocellulose membrane to probe for pfRipHA using anti-HA antibody (
A 30 ml-dish of triple synchronized pfRipHA parasite culture (the third synchronization was done when the parasites were in the 8-12 hours ring stage) was distributed into six 5-ml dishes. One dish of culture was harvested for preparing saponin pellet immediately after the third synchronization. The second dish was harvested 16 hr later, the third dish another 8 hr later and subsequent dishes every 6 hr later until the end of schizogony. The saponin pellets prepared from the harvested parasites were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was firstly probed with monoclonal anti-HA antibody for pfRipHA and then stripped to probe for pfRh5 and pfhsp70. Both pfRh5 and pfRip were shown to be expressed at late life cycle stage of parasite development (
To generate recombinant fragment of pfRip-791-900 (amino acid 791 to 900), oligonucleotides
were used to PCR amplify the fragment from genomic DNA prepared from 3D7 parasite. The resulting PCR fragment was then cloned into pET14b vector with NdeI and BamHI site, transformed into BL21 RIL E. coli strain for expressing recombinant pfRip-791-900 as a hexa-His-tagged protein. The His-tagged protein was purified from soluble lysate of bacteria cells by Ni-resin affinity purification followed by gel-filtration chromatography on Superdex 75 column.
The construct for producing the recombinant fragment of pfRip-238-368 (amino acid 238 to 368) was made by synthesizing codon-optimized DNA sequence coding for pfRip amino acid sequence 238 to 368 and cloned into pET28a vector with NheI and BamHI sites. The construct was then transformed into BL21 RIL E. coli strain and produced hexa-His-tagged protein as inclusion body. The protein solubilised from the inclusion body was refolded, purified by Ni-resin affinity column.
The diagram in
PfRipHA parasite (late schitzont stage)-infected red blood cells were hypotonically lysed with water, centrifuged and pellet fraction washed with PBS twice. The pellet fraction was then divided into four eppendorfs and incubated on ice for 2 hours with 10 mM Tris/pH 8.0; 100 mM sodium carbonate/pH 11.5; 2% Triton X100 and 2% CHAPS in 50 mM Tris/pH8.0, 1 mM EDTA and 100 mM sodium chloride respectively. The samples were centrifuged to separate soluble and insoluble fractions. The insoluble fraction was washed twice with PBS and analysed by western blot together with the soluble fraction (
Pre-incubation of purified merozoites with Protein-A purified rabbit polyclonal antibodies [R1155 (αpfRIP/1) and R1156 (αpfRTP/2) at 2 mg/ml] raised against recombinant pfRip for 2 minutes at 37° C. inhibited merozoites attachment to uninfected red blood cells by 40-55% (
The present inventors tested the anti-PfRIP/1 and anti-PfRIP/2 antibodies (αPfRIP/1 and 2 antibodies) for their ability to block parasite growth (growth inhibition assays, GIA) using the P. falciparum strains FCR3, W2mef, T994, CSL2, E8B, MCAMP, 7G8, D10, HB3, and 3D7 (
Among other P. falciparum strains tested αPfRIP/1 antibody exhibited significantly higher inhibitory activity for those that invade erythrocyte preferentially using sialic acid-dependent receptors (ie. glycophorins), which includes FCR3, W2mef, T994, CSL2 and E8B. The αPfRIP/1 antibody was titrated in GIAs in comparison with IgG from normal serum for both FCR3 and 3D7 parasite strains. Growth of FCR3, a parasite that invades preferentially by sialic acid-dependent pathways, was almost completely abolished at 3 mg/ml and significant inhibition still remained even at 1 mg/ml (40%). In comparison, the 3D7 parasite strain, which can efficiently use sialic acid-independent invasion pathways primarily by using the ligand PfRh4 and complement receptor 1, was inhibited at significantly lower levels with 40% at 3 mg/ml and this decreased to 25% at 1 mg/ml of antibody. This suggests that the PfRIP/PfRh5 complex may be more functionally important in P. falciparum strains that efficiently use sialic acid-dependent invasion pathways.
The region of PfRIP to which the anti-PfRIP antibodies were raised was from the 3D7 strain of P. falciparum; however, this domain does not show any polymorphisms in other strains that have been sequenced (http://plasmodb.org/). Also, the present inventors did not observe any cross-reactivity of the antibodies with other proteins that contain EGF-like domains such as MSP1. This was not surprising as the only conserved amino acids was the six cytseine residues that define each EGF-like domain (
To confirm the 85 kDa PfRh2a and b protein was directly responsible for binding to human erythrocytes recombinant proteins were made of different portions that covered this region. A protein of 15 kDa corresponding to amino acids 446 to 557 of the PfRh2a/b N-terminus (rRh215), expressed as an E. coli hexa-His tagged protein, bound to erythrocytes whereas the 2b1 protein from the C-terminal region of PfRh2b showed no detectable binding (
To show that binding of rRh215 to erythrocytes was specific it was determined if IgG antibodies raised to this domain block binding of both the 85 kDa fragment from parasite supernatants and the rRh215 fragment. The antibodies R1170 showed a dose-dependent inhibition of binding of the 85 kDa fragment in contrast to antibodies raised to a second recombinant protein of PfRH2a/b made from the N-terminus and IgG from normal rabbit serum (
To determine if antibodies to rRh215 (R1170) inhibit invasion they were tested in growth inhibition assays with normal and trypsin-treated erythrocytes. The anti-rRh215 antibodies showed approximately 18% inhibition into normal erythrocytes compared to no inhibition for antibodies to a second fusion protein close to the receptor binding site and this was increased for trypsin-treated cells to 38% (
To show that the inhibitory effect was specific and also to determine if it was acting on the function of both PfRh2a and PfRh2b the P. falciparum lines in which each gene had been specifically disrupted were used (Duraisingh et al., 2003). For normal erythrocytes anti-rRh215 antibodies inhibited growth at approximately the same level for 3D7Δ2a, which lacks expression of PfRh2a, and the 3D7 parent and this was enhanced for trypsin-treated erythrocytes. In contrast, the P. falciparum lines 3D7Δ2b (lacks expression of PfRh2b) and FCR3 (lacks expression of PfRh2a and PfRh2b) were not inhibited (
Antibodies against a combination of antigens were tested for their ability to inhibit invasion of P. falciparum into human red blood cells in vitro.
Rabbits were immunized with a total of 225 μg protein comprising 75 μg of each the following antigens: EBA175 R3-5 (amino acids 760-1271; SEQ ID NO:36), PfRh2a/b (15 kDa fragment; SEQ ID NO:12) and PfRIPr (791-900; SEQ ID NO:3).
Blood was taken and IgG fraction purified 34 days following a single immunization with the three antigens. Serial dilutions were made of the IgG with 2 mg/ml starting concentration. Antibodies were incubated together with P. falciparum parasites 3D7. Control Ab was non-immune rabbit IgG. Percentage invasion is calculated as 100×(mean invasion (triplicate wells) of control IgG/test IgG).
DNA encoding the PfRip fragment amino acids 604-1086 was synthesized by Life Technologies (Mulgrave, Victoria, Australia). Codon usage was adapted to the bias of E. coli resulting in a CAI (codon adaptation index) value of 0.96. See
For expression in Hi5 insect cells, the synthetic sequence was cloned into pTriEx-2 (Novagen) using the Kpn 1/Xho 1 restriction site.
The PfRip 604-1086 fragment (SEQ ID NO: 60) was expressed with N-terminal 6-HIS+FLAG tags in Hi5 insect cells in the vector pTriEx-2 (Novagen) modified to include a signal peptide before the His-tag as well as a flag-tag (italics). A TEV cleavage site was also included (underlined).
The predicted size of the fragment is 59456.9 Da.
PfRip (amino acids 604-1086) can be expressed in E. coli, for example in the vector pET-45 with a N-terminal HIS tag (underlined).
MAHHHHHHVGTGSNDDDDKSPDP (N-term tag in E. coli; SEQ ID NO: 62) PfRip (amino acids 604-1086) can also be expressed in E. coli, for example in the vector pET-303 with a C-terminal HIS tag protein. The fragment can also comprise an N-terminal Methionine residue. LEHHHHHH (C-term tag sequence; SEQ ID NO: 63)
The predicted sizes these latter PfRip fragments are 58 KD and 57 KD, respectively.
EBA175 amino acids 761-1298 from Pf strain 3D7 were cloned into the pET-45b (+) vector using Ban HI and Xho I restriction sites to produce a recombinant protein with an N-terminal hexa-His tag. The plasmid was transformed into BL21 E. coli and His-tagged recombinant protein was purified from soluble lysate.
Amino acid 1058 (Glu; E) shown as bold and underlined can alternatively be a Val (V) in some strains. Amino acid 1100 (Gly) shown as bold and underlined can alternatively be a Asp (D) in some strains. See
EBA175 fragment amino acids 761-1298 of SEQ ID NO: 35 (the fragment is designated herein SEQ ID NO: 64) may comprise an N-terminal hexa-His tag.
In order to select an optimal antigenic Pf Rip fragment for a vaccine composition, a new batch of antibodies was produced in rabbits. Thus, protein fragments corresponding to RIP/1 (amino acids 791-900 of PfRip), RIP/2 (amino acids 238-368 of PfRip) and amino acids 604-1086 of PfRip were used to immunize rabbits to raise antisera for testing. Because the vaccine is to be used in humans, a regimen optimized to be compatible with a human vaccination schedule was adopted. The vaccine schedule called for three immunizations: on day 1, day 28, and day 52, each followed by a final bleed for sera 14 days after the third immunization.
The growth inhibition single cycle assay was performed according to methods described in Malkin E M. et al. (2005, Infect Immun 73: 3677-3685), the entire content of which is incorporated herein by reference.
Results from the growth inhibition assay using anti-RIP/2 antiserum indicate that the antibodies raised using the “human compatible” immunization regimen were not effective in inhibiting parasite growth at any of the antibody concentrations tested (
Antibodies raised against merozoite antigens inhibit invasion of different parasite strains in a single cycle GIA assay. IgG raised against EBA-175 amino acids 761-1298 (3D7 strain) (
IgG against EBA175 amino acids 761-1298 shows a dose-dependent inhibition of parasite growth relative to parasites in non-immune IgG of up to 89% at the top IgG concentration of 2 mg/ml against 3D7 parasites, 76% inhibition of FCR3 and 67% inhibition of W2mef, while 3D7 parasites with a genetic deletion in eba-175 were not inhibited at all (
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
All publications discussed and/or referenced herein are incorporated herein in their entirety.
The present application claims priority from U.S. Ser. No. 61/411,598 and U.S. 61/435,602, the entire contents of which are incorporated herein by reference.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
The present application is a Continuation in Part application of co-pending U.S. application Ser. No. 13/884,604, filed Jul. 23, 2013, which claims priority from PCT Application No. PCT/AU2011/001442, filed Nov. 8, 2011, which in turn, claims priority from U.S. Provisional Application Ser. No. 61/411,598, filed Nov. 9, 2010, and U.S. Provisional Application Ser. No. 61/435,602, filed Jan. 24, 2011. Applicants claim the benefits of 35 U.S.C. §120 as to the U.S. application and the PCT application and priority under 35 U.S.C. §119 as to the U.S. provisional applications, and the entire disclosures of all applications are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 61411598 | Nov 2010 | US | |
| 61435602 | Jan 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13884604 | Jul 2013 | US |
| Child | 14813739 | US |
