# Patent

## Title
Multivalent antihelminthic vaccine

## Abstract

A multivalent anthelmintic vaccine targets both hookworm and schistosomiasis. The vaccine includes, at a minimum, a recombinant third-stage larval hookworm antigen, a recombinant adult stage hookworm antigen, and a recombinant schistosome antigen. Preferably, the hookworm antigens are Necator americanus antigens, although antigens from other hookworm species (e.g. Ancylostoma duodenale) may also be employed. The schistosome antigen is preferably a Schistosoma mansoni or a Schistosoma haematobium antigen although antigens from other schistosome species (e.g. Schistosoma japonicum) may also be employed. In some cases full or partial sequences of schistosome antigens may be fused with full or partial sequences of hookworm {Necator americanus) to produce recombinant chimeric antigens.

## Background
RELATED APPLICATION
This application claims benefit of U.S. Provisional Application No. 61/077,256, filed Jul. 1, 2008.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to a multivalent anthelminthic vaccine that targets both hookworm and schistosomiasis. In particular, the invention provides a vaccine that includes at least two hookworm antigens, of which one is a third-stage larval stage antigen and one is an adult stage antigen, and at least one schistosome antigen. In some cases, full or partial sequences of schistosome antigens may be fused with full or partial sequences of hookworm (Necator americanus) to produce recombinant chimeric antigens.
2. Background of the Invention
Hookworm infection and schistosomiasis are the two most important parasitic helminth infections of humans. Together these two helminthiases are responsible for an estimated 345,000 deaths and 26.6 million disability adjusted life years (DALYs) annually making them second only to malaria as the most important causes of human parasitic infection (Table 1).







TABLE 1







Disease burden of hookworm and schistosomiasis and comparison with malaria
















DALYs





Major

(Disability
Major clinical



Global
Geographic

adjusted life
features and


Disease
Prevalence
Regions
Deaths
years)
disabilities















Hookworm Infection
576 million
Sub-Saharan
65,000
22.1 million
Anemia, physical &


(Major species:

Africa, SE


cognitive retardation


Necator americanus;

Asia, Brazil


Minor species:


Ancylostoma duodenale)


Schistosomiasis
207 million
Sub-Saharan
280,000
 4.5 million
Anemia, liver disease,


(Major species:

Africa, Brazil


urogenital disease and


Schistosoma mansonia and




physical & cognitive


Schistosoma haematobium;




retardation


Minor species:


Schistosoma japonicum)


Hookworm and

Sub-Saharan
345,000
26.6 million


Schistosomiasis

Africa, Brazil


Malaria
300-500
Sub-Saharan
1,200,000
46.5 million
Anemia, cerebral



million
Africa


malaria, Acute







Respiratory Distress







Syndrome (ARDS)









Both hookworm and schistosomiasis are co-endemic in many regions of the world. In both sub-Saharan Africa and Brazil these two helminth infections not only exhibit a high degree of geographic overlap, but there is evidence that co-infections with hookworm and schistosomes are extremely common, and there is evidence that hookworm promotes susceptibility to schistosomiasis (Fleming et al, 2006; Raso et al, 2007). The co-morbid effects of hookworm and schistosomiasis are profound and include severe anemia and physical and intellectual growth retardation in children, as well as adverse pregnancy outcome (Hotez and Ferris, 2006; Hotez et al, 2006). There is additional evidence that both hookworm and schistosomiasis increase susceptibility and worsen the severity of malaria and HIV/AIDS (Hotez et al, 2006; 2007).
SUMMARY OF THE INVENTION
It is an object of this invention to provide multivalent anthelminthic vaccines that target both hookworm and schistosomiasis. The multivalent anthelminthic vaccine includes one or more recombinant hookworm antigens and one or more recombinant schistosome antigens. In some embodiments, the vaccine contains 1) at least one recombinant hookworm third-stage larval antigen; 2) at least one recombinant hookworm adult stage antigen; and 3) at least one recombinant schistosome antigen. By “antigen” it should be understood that full length sequences or fragments with antigenic sequences, i.e. capable of causing the body to raise antibody titers for protection against hookworm and schistosome infection, can be used in the practice of the invention. In some embodiments, the one or more recombinant hookworm antigens are Necator americanus hookworm antigens such as, for example Na-ASP-2, Na-SAA-2, Na-APR-1 and Na-GST-1. In other embodiments, Necator americanus hookworm antigens such as, Na-APR-2, Na-CP-2, Na-CP-3, Na-CP-4 and Na-CP-5 may be employed. In yet other embodiments, full or partial sequences of one or more different Necator americanus antigens may be linked to produce recombinant chimeric antigens. An example of this is a chimeric protein comprised of Na-GST-1 linked to a fragment of Na-APR-1 (e.g. fragment 5, described below).
In other embodiments, the adult stage and third-stage larval hookworm antigens are Ancylostoma duodenale hookworm antigens.
In some embodiments of the invention, the one or more recombinant schistosome antigens are Schistosoma mansoni antigens, for example, Sm-TSP-2, Sm-TSP-3, Sm-TSP-4 and Sm-29. In other embodiments, the one or more recombinant schistosome antigens are Schistosoma haematobium antigens, for example, Sh-TSP-2. In yet other embodiments, the one or more recombinant schistosome antigens are Schistosoma japonicum antigens. In other embodiments full or partial sequences of schistosome antigens may be fused with full or partial sequences of hookworm (e.g. Necator americanus) to produce recombinant chimeric antigens. Examples of these chimeric proteins include but are not limited to the second extracellular domain (EC-2) of Sm-TSP-2 linked to Na-GST-1, or the EC-2 of Sm-TSP-2 linked to a fragment of Na-APR-1.
In addition, the multivalent anthelminthic vaccine may also include one or more adjuvants. Examples of suitable adjuvants include but are not limited to an aluminum-based adjuvant, CpG and Synthetic Lipid A.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and B. Na-ASP-2: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 2A and B. Na-SAA-2: A, nucleic acid sequence; B, amino acid sequence.
FIG. 3A-D. Na-APR-1: A, nucleic acid sequence encoding Na-APR-1, Shanghai strain; B, amino acid sequence of Na-APR-1, Shanghai strain; C, nucleic acid sequence encoding Na-APR-1, Australian strain; D, amino acid sequence of Na-APR-1, Australian strain.
FIGS. 4A and B. Na-CP-2: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 5A and B. Na-CP-3: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 6A and B. Na-CP-4: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 7A and B. Na-CP-5: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 8A and B. Na-GST-1; A, nucleic acid sequence; B, amino acid sequence.
FIGS. 9A and B. Sm-TSP-2: A, nucleic acid sequence; B, amino acid sequence. Residues corresponding to extracellular loop-2 (EC-2) are underlined.
FIGS. 10A and B. Sm-29: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 11A and B. Sh-TSP-2: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 12A and B. Sm-TSP-3: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 13A and B. Sm-TSP-4: A, nucleic acid sequence; B, amino acid sequence.
FIGS. 14A and B. Na-APR-2: A, nucleic acid sequence; B, amino acid sequence.
FIG. 15A-C. Chimera including Na-GST-1 and fragment 5 of Na-APR-1. A, schematic of the construct; B, nucleic acid sequence C, amino acid sequence. Solid underline=Na-GST-1; dotted underline=fragment 5; no underline (between Na-GST-1 and fragment 5)=linker; no underline (after fragment 5)=histidine tag.
FIG. 16A-C. Chimera including Sm-TSP-2 EC-2 and fragment 5 of Na-APR-1. A, schematic of the construct; B. nucleic acid sequence; C, amino acid sequence. Solid underline=Sm-TSP-2-extracellular loop-2 (EC-2); dotted underline=fragment 5; no underline (between Sm-TSP-2-EC-2 and fragment 5)=linker; no underline (after fragment 5)=histidine tag.
FIG. 17A-C. Chimera including Na-GST-1 and Sm-TSP-2 EC-2. A, schematic of the construct; B. nucleic acid sequence; C, amino acid sequence. Solid underline=Na-GST-1; dotted underline=Sm-TSP-2 EC-2; no underline (between Na-GST-1 and Sm-TSP-2 EC-2)=linker; no underline (after Sm-TSP-2 EC-2)=histidine tag.
FIG. 18. Parasite gut immunolocalization of three Na-APR-1 monoclonal antibodies Each of these monoclonal antibodies exhibits the ability to inhibit Na-APR-1 enzymatic activity (see FIG. 19)
FIG. 19. Monoclonal antibody neutralization of enzymatic activity using mAB 11F3. Similar results with 2H12 and 9E10 antibodies (data not shown).
FIGS. 20A and B. Monoclonal antibody mapping to Na-APR-1 fragment 5. A, shows the relative placement of fragments that were tested compared to the mature protein; B, results of binding of antibodies 2H12, 9E10 and 11F3 to Na-APR-1 fragments 1-5.
FIG. 21. Amino acid sequence of Na-APR-1 fragment 5 (Necator americanus) and its orthologues in other parasitic helminths. Necator americanus, SEQ ID NO:37; Ancylostoma ceylanicum, SEQ ID NO:38; Ancylostoma duodenale, SEQ ID NO:39; Anclostoma canimun, SEQ ID NO:40; Schistosoma mansoni, SEQ ID NO:41; Opisthorchis viverrini, SEQ ID NO:42; Fasciola hepatica, SEQ ID NO:43; Brugia malayi, SEQ ID NO:44; Onchocerca volvulus, SEQ ID NO:45; Human pepsinogen, SEQ ID NO:46; Human cathepsin D, SEQ ID NO:47; Human rennin, SEQ ID NO:48.
FIG. 22. Antibody against either the Na-GST-1-Na-APR-1 fragment 5 chimeric protein or the Sm-TSP-2 EC2-Na-APR-1 fragment 5 chimeric protein neutralize Na-APR-1 enzymatic activity.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
The present invention is based on the development of a multivalent antithelminthic vaccine (MAHV) that targets both hookworm and schistosomiasis. The high frequency of polyparasitsm with hookworms and schistosomes and the co-morbidity and synergy with malaria and HIV/AIDS provided the impetus for the development of the vaccine. The MAHV of the invention comprises at least two recombinant hookworm antigens and at least one schistosome antigen. In some embodiments, one of the hookworm antigens is a third-stage larval antigen and another of the hookworm antigens is an adult stage antigen. Preferably, the antigens are from species of worms that infect humans, although this may not always be the case. For example, preferred hookworm antigens include N. americanus antigens and preferred schistosome antigens include S. mansoni antigens (which may be surface or adult stage antigens) and S. haematobium antigens (especially surface antigens). Preferably, the antigens are from major species as listed in Table 1, e.g. Necator americanus for hookworm and Schistosoma mansonia and Schistosoma haematobium for schistosomes. However, several orthologous proteins from other minor species may also be used, including but not limited to Ancylostoma duodenale for hookworm and Schistosoma japonicum for schistosomes.
To reduce the number of antigens in the MAHV, in some cases recombinant chimeric or fusion proteins are created. For example, the Na-GST-1 gene product may be expressed in a Na-GST-1-Sm-TSP-2 EC-2 (EC-2 is described in detail below) hookworm/schistosome recombinant fusion protein or a hookworm/schistosome fusion recombinant protein comprised of Sm-TSP-2 EC-2 and a partial length fragment of Na-APR-1 such as fragment 5 (which is described in detail below). A hookworm chimeric recombinant protein comprised of Na-GST-1 linked to a partial length fragment of Na-APR-1 (e.g. fragment 5) may also be produced.
The development of a MAHV for hookworm and schistosomiasis combined represents a major global public health breakthrough, and will facilitate meeting several goals such as sustainable poverty reduction, eradicating poverty and hunger, achieving universal primary education, reducing child mortality, improving maternal health, and combating HIV/AIDS, malaria, and other diseases.
Table 2 lists several exemplary candidate N. americanus, S. mansoni and S. haematobium antigens that may be used in the practice of the present invention, each of which exhibits strong preclinical evidence for protective immunity.







TABLE 2







Candidate antigens for the multivalent antithelminthic vaccine









Major Reference












Necator americanus hookworm



antigens


Na-ASP-2
Asojo et al, 2005;



Goud et al, 2005


Na-SAA-2
Fujiwara et al, 2006


Na-GST-1
Asojo et al, 2007


Na-APR-1
Loukas et al, 2005;



Yang, Y. et al 2009


Na-APR-2
Williamson et al., 2003


Schistosoma mansoni antigens


Sm-TSP-2 and Sm-TSP-2 EC-2
Tran et al, 2006,



Loukas et al



unpublished data


Sm-TSP-3


Sm-TSP-4


Sm-29
Cardoso et al, 2006


Schistosoma haematobium antigens


Sh-TSP-2 (orthologue of Sm-TSP-2)


and Sh-TSP-2 EC2


Sh-TSP-3 (orthologue of Sm-TSP-3)


Sh-TSP-4 (orthologue of Sm-TSP-4)


Sh-29 (orthologue of Sm-29)


Recombinant fusion proteins


Na-GST-1 - Na-APR-1 fragment 5


Na-GST-1 - Sm-TSP-2 EC2


Sm-TSP-2 EC2 - Na-APR-1 fragment 5









The amino acid sequences of several suitable antigens and antigenic sequences, as well as the nucleic acid sequences which encode them, are provided in FIGS. 1-17. However, those of skill in the art will recognize that many variants of the sequences presented herein may exist or be constructed which would also function as antigens in the practice of the present invention. For example, with respect to amino acid sequences, variants may exist or be constructed which display: conservative amino acid substitutions; non-conservative amino acid substitutions; truncation by, for example, deletion of amino acids at the amino or carboxy terminus, or internally within the molecule; or by addition of amino acids at the amino or carboxy terminus, or internally within the molecule (e.g. the addition of a histidine tag for purposes of facilitating protein isolation, the substitution of residues to alter solubility properties, the replacement of residues which comprise protease cleavage sites to eliminate cleavage and increase stability, the addition or elimination of glycosylation sites, and the like, or for any other reason). Such variants may be naturally occurring (e.g. as a result of natural variations between species, strains, or individuals, such as Na-APR-1 Shanghai and Australia, FIGS. 3A-D); or they may be purposefully introduced (e.g. in a laboratory setting using genetic engineering techniques). All such variants of the sequences disclosed herein are intended to be encompassed by the teaching of the present invention, provided the variant antigen displays sufficient identity to the described sequences. Preferably, identity will be in the range of about 50 to 100%, or in the range of about 75 to 100%, or in the range of about 80 to 100%, or 85% to 100%, or 90% to 100%, or about 95, 96, 97, 98, 99 or 100% of the disclosed sequences. The identity is with reference to the portion of the amino acid sequence that corresponds to the original antigen sequence, i.e. not including additional elements that might be added, such as, for example, histidine tags, spacer or linker sequences between antigens in a chimeric construct that encodes multiple antigens, etc. Representative spacer sequences are depicted in FIGS. 15-17. Those of skill in the art will recognize that other suitable spacer sequences might be used in the constructs depicted in these figures, or in some cases, spacer sequences may not be necessary, i.e. they may be eliminated and thus not be present. The same is true for the His tag that is depicted. Other sequences suitable for tracking or purifying and isolating the proteins/polypeptides that contain the antigens may be used to either replace the His tag, or may, in some embodiments, be eliminated, i.e. may not be present.
The MAHV of the invention may be constructed in any of several ways. As stated above, the MAHV may comprise either peptide/protein antigens that are administered directly to a vaccine recipient or nucleic acid (e.g. DNA, stabilized RNA, etc.) that encodes the antigens. The ensuing discussion is intended to apply to both of these possibilities.
The particular design of the vaccine and the arrangement of the antigenic elements of which the vaccine is comprised may vary. For example, the antigens may be administered as a mixture of individual antigens, or as a mixture of chimeric constructs that contain two or more of the antigens (e.g. as depicted in FIGS. 15-17), or even as a single chimeric construct that contains all the antigens of the vaccine. Those of skill in the art will recognize that in a chimeric protein construct, individual antigenic sequences may be separated by linking or spacer sequences which may or may not be antigenic. Thus, in a DNA molecule that encodes a plurality of antigenic sequences, those sequences may be separated spatially by intervening sequences that are translated into linking or spacer sequences, or the intervening sequences may encode genetic elements (e.g. internal ribosomal entry sites, IRES) that result in the translation of separate polypeptides for some or all of the antigens. In addition, other beneficial sequences may also be included. For example: sequences which aid in isolation and purification of the protein (e.g. histidine tag, GST, and maltose binding protein); sequences which direct the protein to a particular intracellular location (e.g. yeast secretory protein); sequences that increase the antigenicity of the protein (e.g. KHL, haptens). Further, various non-coding sequences may be added to nucleic acid vaccine compositions, e.g. to enhance protein expression, promote stability, etc.
Those of skill in the art will recognize that it may not be necessary to utilize the entire primary sequence of a protein or polypeptide in order to elicit an adequate antigenic response to the parasite from which the antigen originates. In some cases, a fragment of the protein is adequate to confer immunization. Thus, the present invention also encompasses antigenic fragments of the sequences disclosed herein, and their use in vaccine preparations. In general, such a fragment will be at least about 10-13 amino acids in length. Those of skill in the art will recognize that suitable sequences are often hydrophilic in nature, and are frequently surface accessible. For example, extracellular Loop 2 of S. mansoni Sm-TSP-2 (EC2) or an orthologous loop sequence from another schistosome species and/or genus may be utilized; as may “Fragment 5” from Na-APR-1 as described below, or corresponding, orthologous sequences from other hookworm species and/or genera (e.g. S. mansoni cathepsin D). A “corresponding” or “orthologous” sequence is a sequence from a related genus species that exhibits at least about 50, preferably at least about 75%, or more preferably 80, 85, 90, 95, or even 100% nucleic acid homology or encoded amino acid primary sequence identity. Alternatively, even if the level of homology or identity is not at least about 50% or greater, the secondary and/or tertiary structural elements of corresponding or orthologous sequences may be analogous and may perform the same function, and all such sequences are intended to be encompassed by the present invention.
Examples of using peptide fragments or chimeric proteins comprised of full-length peptides and peptide fragments include but are not limited to Na-GST-1 and/or Sm-TSP-2 EC2 fused with a fragment of Na-APR-1, e.g. “fragment 5” of Na-APR-1 (FIGS. 15A-C and 16A-C) or Na-GST-1 combined with Sm-TSP-2 EC2 (FIG. 17A-C). Through testing of monoclonal antibodies against Na-APR-1 a specific fragment known as “fragment 5” was identified as critical for the enzymatic activity of this molecule (see Example 2), and this fragment may be used alone or in combination with other antigens for production and use of vaccines as described herein.
With respect to the nucleic acid sequences disclosed herein, those of skill in the art will recognize that many variants of the sequences may exist or be constructed which would still function to provide the encoded antigens or desired portions thereof. For example, due to the redundancy of the genetic code, more than one codon may be used to code for an amino acid. Further, as described above, changes in the primary sequence of the antigen may be desired, and this would necessitate changes in the encoding nucleic acid sequences. In addition, those of skill in the art will recognize that many variations of the nucleic acid sequences may be constructed for purposes related to cloning strategy, (e.g. for ease of manipulation of a sequence for insertion into a vector, such as the introduction of restriction enzyme cleavage sites, etc.), for purposes of modifying transcription (e.g. the introduction of promoter or enhancer sequences, and the like), or for any other suitable purpose. In addition, Pichia pastoris or Escherichia coli optimized coding sequences may be utilized. In one embodiment, the sequences used in the vaccine are produced in Pichia. All such variants of the nucleic acid sequences disclosed herein are intended to be encompassed by the present invention, provided the sequences display about 50 to 100% identity to the original sequence and preferably, about 75 to 100% identity, and more preferably about 80 to 100% identity, and most preferably 95, 96, 97, 98, 99 or 100% identity. The identity is with reference to the portion of the nucleic acid sequence that corresponds to the original sequence, and is not intended to cover additional elements such as promoters, vector-derived sequences, restriction enzyme cleavage sites, etc. derived from other sources.
The vaccine of the invention may also include one or more adjuvants. In a preferred embodiment, the vaccine of the present invention includes an aluminum-based adjuvant such as the aluminum hydroxide adjuvant Alhydrogel® either alone or in combination with additional adjuvants such as 10104 CpGs and synthetic lipid A molecules. CpGs is a synthetic oligodeoxynucleotide adjuvant containing cytosine-guanine dinucleotides in particular base contexts or CpG motifs, (e.g. CpG ODN) and is an immunomodulatory molecule. In addition, various lipid A derivatives, including the synthetic lipid A known as gluopyrranosyl lipid A (GLA) may be used as adjuvants (Persing D H, Trends in Microbiology 2002 10 (10 Suppl) S32-7). Lipid A is the portion of lipopolysaccharide that is known to be the primary component with regard to adjuvanticity and toxicity. Derivatives of lipid A have been produced in an attempt to retain the immunostimulatory activity of Lipid A yet reduce the toxicity. One such derivative, monophosphoryl lipid A (MPL, available from Chiron), has been shown to exhibit strong Th1 adjuvant activity but with a considerably reduced toxicity compared to LPS. MPL has adjuvant activity whether used alone, or in combination with other immunostimulants, such as CpG oligodeoxynucleotides (ODN), or an aluminum hydroxide based adjuvant. Another synthetic lipid A derivative that is very similar to the lipopolysaccharide derivative lipid A monophosphoryl (MPL) by Chiron is available from the Infectious Disease Research Institute, Seattle, Wash. Seppic, Quil A is another suitable adjuvant. A publication by McCluskie and Weeratna (Infectious Disorders, 2001, 1, 263-271) gives examples of several different suitable adjuvant systems. Other possible adjuvants include but are not limited to AS04; AS03, a proprietary formulation manufactured by Glaxo Smith Kline that contains an oil-in-water emulsion; AS02A, a proprietary formulation manufactured by Glaxo Smith Kline that contains the same oil-in-water emulsion as in AS03, plus two immunostimulants “3D-MPL” and “QS-21”. AS03 and AS02A are described (under their original designations SBAS3 and SBAS2, respectively) in Stoute et al NEJM 1997 336:86-91. It is noted that, AS02A and AS03 are designed to be used with the aluminum based adjuvant AS04, also available from GlaxoSmithKline.
The present invention provides compositions for use in eliciting immune responses to both hookworm and schistosomes. By “eliciting an immune response” we mean that an antigen or antigenic region or epitope stimulates synthesis of specific antibodies at a titer of about >1 to about 1×106 or greater. Preferably, the titer is from about 10,000 to about 1×106 or more, as measured by enzyme Linked Immunosorbent Assay (ELISA) or greater than 1,000 antibody units as defined previously (Malkin et al., 2005a; 2005b). By “vaccine” we mean an antigen or antigen preparation that elicits an immune response that preferably results in a protective immune response. However, those of skill in the art will recognize that many benefits may accrue even if a total protective response does not occur. Thus, the vaccines of the invention may also serve to decrease symptoms of both hookworm and schistosome infection, and thereby provide much benefit to the vaccine recipient. For example, a decrease in hookworm burden of a least about 30% in an organism in relation to a non-vaccinated (e.g. adjuvant alone) control organism represents such an outcome. This worm burden reduction has been calculated to restore a child's daily iron requirements that would otherwise be lost from a moderate (i.e. infections with between 2,000 and 4,000 hookworm eggs per gram of feces) infection with hookworm. Preferably, however, the level of the decrease in hookworm burden would approach 50%, or more. With respect to schistosome burden, the level of decrease in parasite egg production and/or worm burden would exceed 40%, as per standards set previously by the World Health Organization.
The compositions of the invention include substantially purified and/or isolated recombinant hookworm and schistosome antigens or combinations of antigens as described herein, or nucleic acids encoding such antigens, and a pharmacologically suitable carrier. The preparation of such compositions for use as vaccines is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients and/or detergents which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, zwiterrionic detergents and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. In addition, the composition may contain other adjuvants. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of antigens in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%.
The present invention also provides methods of eliciting an immune response to hookworm and methods of vaccinating a mammal against both hookworm and schistosome infection. The methods generally involve identifying a suitable vaccine recipient, and administering to the recipient a composition comprising the antigens and adjuvants described herein in a pharmacologically acceptable carrier. The vaccine preparations of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, orally, intranasally, by ingestion of a food product containing the antigens, etc. In preferred embodiments, the mode of administration is subcutaneous or intramuscular. The vaccine recipient is typically a mammal, and is usually but not always a human.
Immunization of vaccine recipients may be carried our in conjunction with chemotherapy. For example, in one embodiment, preventive strategy that combines the MAHV with benzimidazoles and praziquantel chemotherapy is utilized. Chemotherapy may be provided before, together with, or after vaccination. However, in a preferred embodiment, chemotherapy is provided to the vaccine recipient prior to vaccine administration.
The invention may be more fully appreciated in light of the ensuing non-limiting Examples.
EXAMPLES
Example 1
Construction of MAHV
The following is a list of suitable exemplary antigen combinations for use in the vaccines of the invention:

    1) Na-ASP-2+Na-GST-1+Sm-TSP-2+Adjuvant;
    2) Na-ASP-2+Na-APR-1+Sm-TSP-2+Adjuvant;
    3) Na-SAA-2+Na-GST-1+Sm-TSP-2+Adjuvant;
    4) Na-SAA-2+Na-APR-1+Sm-TSP-2+Adjuvant;
    5) Na-ASP-2+Na-GST-1+Sm-29+Adjuvant;
    6) Na-ASP-2+Na-APR-1+Sm-29+Adjuvant;
    7) Na-SAA-2+Na-GST-1+Sm-29+Adjuvant;
    8) Na-SAA-2+Na-APR-1+Sm-29+Adjuvant;
    9) Na-APR-1+Na-GST-1+Sm-TSP-2+Adjuvant;
    10) Na-APR-1+Na-GST-1+Sm-29+Adjuvant;
    11) Chimera of Na-GST-1−Na-APR-1 fragment 5+Sm-TSP-2+Adjuvant;
    12) Chimera of Na-GST-1−Sm-TSP EC2+Adjuvant; and
    13) Chimera of Sm-TSP 2 EC2−Na-APR-1 fragment 5+Adjuvant;


Example 2
Identification of Na-APR-1 Fragment that is Critical for Enzyme Activity
Through testing of monoclonal antibodies against Na-APR-1 a specific fragment known as “fragment 5” was identified as critical for the enzymatic activity of this molecule. The evidence that fragment 5 is a critical Na-APR-1 peptide required for protection against either hookworm or schistosome challenge infections is as follows: As shown in FIG. 18, three monoclonal antibodies against Na-APR-1 known as 2H112, 9E10, and 11F3 bind to the hookworm alimentary canal at the immunolocalized site of Na-APR-1. FIG. 19 shows results obtained when monoclonal antibody (mAB) 11F3 was used to neutralize enzymatic activity of Na-APR-1. As can be seen, a concentration of 10 μg of mAb 11F3 was sufficient to completely eliminate activity. As shown in FIG. 20, these monoclonal antibodies map to a unique fragment of Na-APR-1 known as “fragment 5”, the amino acid sequence of which is shown in FIG. 21. Also shown in FIG. 21 are the sequences homologous to Na-APR-1 fragment 5 from several other species. Monoclonal antibodies against fragment 5 of Na-APR-1 immunologically recognize recombinant Na-GST-1−Na-APR-1 fragment 5 chimeric protein, and recombinant Sm-TSP-2 EC2-Na-APR-1 fragment 5 chimeric protein expressed in bacteria (Escherichia coli) (data not shown). Conversely, antibodies to these two fusion proteins neutralize the enzymatic activity of Na-APR-1 recombinant enzyme (FIG. 22).
Example 3
Vaccine Comprising the Sm-TSP-2 EC2-Na-APR-1 Fragment 5 Chimeric Protein Causes a Reduction of Worm Burden In Vivo
A vaccine composition comprising a chimeric recombinant protein that included schistosome antigen Sm-TSP-2 EC2 fused to hookworm antigen Na-APR-1 fragment 5 (“Sm-TSP-2 EC2-Na-APR-1 fragment 5”; see FIGS. 16A-C for sequence) was prepared and tested in vivo. Briefly, mice were inoculated with Schistosomes according to methods known in the art. A preparation which included the chimeric polypeptide and the adjuvants alum and CpG was administered to experimental mice (n=10). Control mice were inoculated with Escherichia coli maltose binding protein plus adjuvant.
After a suitable time period, the worm burdens of the animals were determined. The results showed that mice which received the Sm-TSP-2 EC2-Na-APR-1 fragment 5 chimeric protein showed a 59% reduction in Schistosome worm burden compared to control mice.
While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.
REFERENCES


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## Claims

1. A fusion protein including at least one amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36.

2. The fusion protein as recited in claim 1, having the protein sequences of SEQ ID NO: 32.

3. The fusion protein as recited in claim 1, having the protein sequences of SEQ ID NO: 34.

4. The fusion protein as recited in claim 1, having the protein sequences of SEQ ID NO: 36.