WO2017011380A1 - Vaccin et méthodes de dépistage et de prévention de la filariose - Google Patents

Vaccin et méthodes de dépistage et de prévention de la filariose Download PDF

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Publication number
WO2017011380A1
WO2017011380A1 PCT/US2016/041716 US2016041716W WO2017011380A1 WO 2017011380 A1 WO2017011380 A1 WO 2017011380A1 US 2016041716 W US2016041716 W US 2016041716W WO 2017011380 A1 WO2017011380 A1 WO 2017011380A1
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protein
seq
vaccine
proteins
antibodies
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PCT/US2016/041716
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English (en)
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Ramaswamy Kalyanasundaram
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The Board Of Trustees Of The University Of Illinois
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Priority claimed from US14/798,945 external-priority patent/US10072054B2/en
Application filed by The Board Of Trustees Of The University Of Illinois filed Critical The Board Of Trustees Of The University Of Illinois
Publication of WO2017011380A1 publication Critical patent/WO2017011380A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Vaccination is one strategy for controlling this infection and several subunit candidate vaccine antigens have been tested in laboratory animals with variable results (Bottazzi, et al. (2006) Expert Rev. Vaccines 5 (2) : 189-98; Chenthamarakshan, et al . (1995) Parasite Immunol. 17 (6) : 277-85; Dissanayake, et al. (1995) Am. J. Trop. Med. Hyg. 53 (3) : 289-94 ; Li, et al . (1993) J. Immunol. 150 (5) : 1881-5; Maizels, et al. (2001) Int. J. Parasitol.
  • Lymphatic filariasis is a multicellular organism with complex life cycle and produce large array of host modulatory molecules. Thus, fighting against this infection with a single antigen vaccine can be difficult.
  • a phage display cDNA expression library of the B. malayi parasite with sera from immune individuals, several potential vaccine candidates were identified (Gnanasekar, et al . (2004) Infect. Immun. 72 ( 8 ) : 4707-15 ) .
  • a varying degree of protection was achieved with each of the candidate vaccine antigens when given as a DNA, protein or prime boost vaccine (Veerapathran, et al . (2009) supra) .
  • the present invention is a fusion protein composed of (a) Brugia malayi Abundant Larval Transcript; and (b) Brugia malayi Small heat shock protein 12.6, Brugia malayi Tetraspanin, Brugia malayi Thioredoxin Peroxidase 2, or a combination thereof.
  • the Abundant Larval Transcript comprises or consists of SEQ ID NO: 37 or SEQ ID NO: 39;
  • the Small heat shock protein 12.6 comprises or consists of SEQ ID NO: 49 or SEQ ID NO: 64;
  • the Tetraspanin comprises or consists of SEQ ID NO: 45, SEQ ID NO: 63 or SEQ ID NO: 77;
  • the Thioredoxin Peroxidase 2 comprises or consists of SEQ ID NO: 71.
  • the fusion protein comprises or consists of SEQ ID NO: 70; SEQ ID NO: 73 or SEQ ID NO: 74.
  • a recombinant vector encoding the fusion protein, a host cell containing said vector and a vaccine containing the fusion protein are also provided, as is a method for immunizing an animal against filariasis.
  • the invention also provides a vaccine containing (a) an Abundant Larval Transcript of SEQ ID NO: 37 or SEQ ID NO: 39; and (b) a Small heat shock protein 12.6 of SEQ ID NO: 49 or SEQ ID NO: 64; a Tetraspanin of SEQ ID NO: 45, SEQ ID NO: 63 or SEQ ID NO: 77; a Thioredoxin Peroxidase 2 of SEQ ID NO: 71; or a combination thereof.
  • the proteins of (a) and (b) are expressed as a fusion protein.
  • the vaccine further includes an adjuvant.
  • a method for using the vaccine to immunize an animal against filariasis is also provided.
  • This invention further provides an assay and kit for detecting a filarial nematode.
  • the method of the invention includes the steps of contacting a biological sample, in vitro, with one or more binding agents against filarial nematode proteins selected from the group of ALT2, TSP, VAL-1, TPX2 and HSP or fragments thereof; and detecting binding between the binding agents and the filarial nematode proteins, wherein the presence of binding between the binding agents and the filarial nematode proteins indicates the presence of a filarial nematode.
  • the biological sample is from a human subject and the method further includes the step of treating the subject for filariasis.
  • the kit of the invention includes a device with a solid surface.
  • This invention also provides an assay and kit for determining the determining the presence of antibodies to filarial nematode proteins.
  • the method of the invention includes the steps of contacting one or more filarial nematode proteins selected from the group of ALT2, TSP, VAL-l, TPX2 and HSP, or fragments thereof, with a biological sample suspected of containing antibodies to one or more of the filarial nematode proteins; and detecting binding between the one or more filarial nematode proteins and antibodies in the biological sample, wherein the presence of antibodies to the one or more filarial nematode proteins is indicative of efficacy of a vaccine to the filarial nematode, prior exposure to filarial proteins, or an existing infection with a filarial nematode.
  • the one or more of the filarial nematode proteins are present on one or more solid surfaces or particles.
  • the kit of the invention includes a device with one or more solid surfaces.
  • Figures 2A-2B show the number of IL-4 ( Figure 2A) and IFN- ⁇ ( Figure 2B) secreting cells in the spleen of mice vaccinated with monovalent (BmHSP or BmALT2) or multivalent vaccine.
  • An ELISPOT assay was performed after stimulating the cells with rBmHSP or rBmALT (1 ⁇ g/ml) . Single cell preparations of spleen cells were stimulated with respective antigens for 48 hours and spot forming cells were counted. Results show that both monovalent and multivalent vaccine promoted IL-4 secreting cells. Multivalent vaccination induced the higher number of IL-4 producing cells than controls. IFN- ⁇ producing cells were comparatively low.
  • FIG. 3 shows the degree of protection conferred by a multivalent vaccine in a mouse model.
  • Balb/c strain of mice were immunized with HAT (HSP/ALT2/TSP) hybrid DNA, with recombinant HAT protein or a combination of both using a prime boost approach.
  • HAT hybrid DNA was used for priming.
  • mice Two weeks following the priming, mice were boosted with HAT hybrid protein.
  • Another group of mice were immunized with HAT hybrid DNA or with HAT hybrid protein.
  • Control groups of mice received only blank vector or alum adjuvant.
  • mice were challenged with 20 infective larvae of Brugia malayi by placing them in a micropore chamber in the peritoneal cavity of the immunized mice. After 48 hours, larval death was measured to determine the success of vaccination.
  • Figure 4 shows a western blot of recombinant BmTSP (lane 1) and two different fractions of isolated, recombinant 0. volvulus TSP (lanes 2 and 3) probed with sera from cHAT-vaccinated mice.
  • Figure 5 shows that the tetravalent vaccine formulation, BraHAXT, gave better protection than a trivalent vaccine formulation (BmRAT) against a challenge infection with Brugia malayi in a mouse model. ⁇ Statistically significant compared to AL019 control (P ⁇ 0.01) and BmHAT+AL019 (P ⁇ 0.05).
  • ALT2 Abundant Larval Transcript
  • TSP Tetraspanin
  • HSP Small heat shock protein
  • VAL-1 Vespid Venom Allergen homologue- Like protein
  • GST Glutathione S-Transferase
  • TPX-2 Thioredoxin Peroxidase 2
  • the present invention features fusion protein-based and DNA- based vaccines composed of filarial nematode antigens or nucleic acids encoding the same and use of the vaccines to prevent or control filariasis in humans and animals.
  • the present invention also provides assays and kits for detecting the presence of a filarial nematode.
  • a multivalent or polyvalent vaccine refers to a vaccine prepared from several antigens.
  • the antigen is a nucleic acid molecule, which is referred to herein as a "DNA-based” antigen.
  • the antigen is a protein or polypeptide, which is referred to herein as "protein-based” antigen.
  • a multivalent vaccine of the invention can be composed of two, three, four, five, six or up to ten antigens or their fragments in various permutation combinations.
  • the multivalent vaccine is composed of two, three or four antigens.
  • the multivalent vaccine is composed of solely of protein antigens.
  • the multivalent vaccine is composed solely of DNA-based antigens.
  • the multivalent vaccine is composed of a mixture of protein- and DNA-based antigens.
  • Antigens of this invention can be provided or expressed from a single nucleic acid molecule containing, e.g., internal ribosome entry sites between the antigens.
  • the antigens of the multivalent vaccine of this invention can be covalently attached to form a hybrid or chimeric molecule or fusion protein, wherein the antigens are immediately adjacent to one another ⁇ e.g., an in-frame fusion with or without a short spacer) .
  • antigens of the instant invention can be provided as a mixture of individual antigens.
  • the instant vaccine can be composed of a hybrid molecule containing, e.g., two antigens, in admixture with a third non-covalently attached antigen.
  • a multivalent vaccine of the invention can be composed of a chimeric TSP-HSP protein in admixture with a nucleic acid molecule encoding ALT2.
  • the antigens of the multivalent vaccine are different proteins from one species of filarial nematode.
  • the multivalent vaccine is composed of AL 2 , HSP, and TSP and/or TPX2 or GST antigens isolated from one or more strains of B. malayi.
  • the antigens are the same, but from different species of filarial nematodes.
  • the multivalent vaccine is composed of the ALT2 antigen isolated from W. bancrofti, B. malayi, B. timori, 0. volvulus and L. loa.
  • the multivalent vaccine is composed of a combination of different antigens from different species of filarial nematodes.
  • the multivalent vaccine can be composed of the ALT2 antigen isolated from W. bancrofti, O. volvulus and L. loa and the HSP antigen isolated from B. malayi and B. timori.
  • the DNA sequence of the gene of interest (also used interchangeably as DNA molecule) need not contain the full length of DNA encoding the corresponding protein.
  • the protein sequence need not contain the full length protein.
  • a fragment of the protein or gene which encodes an epitope region is sufficient for immunization.
  • the DNA/protein sequence of an epitope region can be found by sequencing the corresponding part of the gene from various strains or species and comparing them.
  • the major antigenic determinants are likely to be those showing the greatest heterology. Also, these regions are likely to lie accessibly in the conformational structure of the proteins.
  • One or more such fragments of proteins or genes encoding the antigenic determinants can be prepared by chemical synthesis or by recombinant DNA technology. These fragments of proteins or genes, if desired, can be linked together or linked to other proteins or DNA molecules, respectively.
  • the instant vaccine (e.g., fusion protein) includes the ALT2, TSP, VAL-1, TPX2, GST and/or HSP protein antigens and/or nucleic acid molecules encoding the ALT2, TSP, VAL- 1, TPX2, GST and/or HSP protein, or fragments thereof. Protein and nucleic acid sequences for these antigens are available in the art under the GENBANK accession numbers listed in Table 1.
  • nucleotide sequence encoding 0. volvulus TSP can be found under GENBANK Accession No. JN861043.
  • the protein antigens and nucleic acid molecules of the invention can be used as full length molecules or less than full length molecules.
  • the present invention further includes the use of fragments of the above-referenced protein antigens and nucleic acid molecules.
  • Fragments are defined herein as 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residue portions of full-length protein antigens (e.g., those listed in Table 1) or 60, 90, 120, 150, 180, 210, 240, 270, 300, 350, or 600 nucleotide portion of full-length nucleic acid molecules (e.g., those listed in Table 1).
  • Exemplary protein fragments include the large extracellular loop (LEL) domain of TSP (see, e.g., the LEL domain of B. malayi TSP of SEQ ID NO: 63 or SEQ ID NO: 77) and N-terminal deletion of HSP 12.6 (cHSP; see, e.g., the B .
  • fusion proteins of this invention include but are not limited to, fusion of HSP, ALT2 and TPX2; fusion of HSP, AL 2 and TSP; and fusion of HSP, ALT2 , TPX2 and TSP.
  • An exemplary fusion protein containing ALT2, HSP and TSP protein sequences is set forth in SEQ ID NO: 70.
  • An exemplary fusion protein containing ALT2, HSP and TPX2 protein sequences is set forth in SEQ ID NO: 73.
  • An exemplary fusion protein containing ALT2, HSP, TSP and TPX2 protein sequences is set forth in SEQ ID NO: 74.
  • the protein or protein fragments of this invention have one or more antigenic sequences for eliciting an immune response in an animal.
  • the ALT2 protein of the invention is a B. malayi ALT2 protein or fragment comprising or consisting of the sequence VSESDEEFDDSAADDTDDSEAGGGSEGGDEYVT (SEQ ID NO:78) and/or EFVETDGKKKECSSHEACYDQREPQ (SEQ ID NO:79), which, based upon the Bepipred Linear Epitope Prediction -limethod (Larsen, et al . (2006) Immunome Res. 2:2), are predicted B-cell epitopes.
  • the HSP protein of the invention is a B. malayi HSP protein or fragment comprising or consisting of the sequence SAEQ DWPLQH (SEQ ID NO: 80) and/or KLPSDVDTKTL (SEQ ID NO:81), which are predicted B-cell epitopes.
  • the TSP protein of the invention is a B. malayi TSP protein or fragment comprising or consisting of the sequence KTGESEDEMQ (SEQ ID NO: 82), which is a predicted B-cell epitope.
  • the TPX2 protein of the invention is a B . malayi TPX2 protein or fragment comprising or consisting of the sequence FIGQPAPNFKT (SEQ ID NO: 83) and/or GEVCPANWHPGSETIKPGVKESKA
  • the multivalent vaccine of the invention includes other known antigens from W. bancrofti, B. malayi, 0. volvulus, L. loa and B. timori.
  • suitable antigens include, but are not limited to, glutathione peroxidase (see Cookson, et al . (1992) Proc. Natl. Acad. Sci. USA 89:5837-5841; Maizels, et al . (1983) Parasitology 87:249-263; Maizels, et al . (1983) Clin. Exp. Immunol.
  • the antigen is obtained from a filarial nematode selected from the group of W. bancrofti, B. malayi, 0. volvulus , L. loa and B. timori.
  • the antigens of the fusion protein and vaccine are isolated from a filarial nematode.
  • an isolated nucleic acid molecule or protein is a nucleic acid molecule or protein that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) .
  • isolated does not reflect the extent to which the nucleic acid molecule or protein has been purified.
  • the antigens are purified (e.g., purified to greater than 95% homogeneity) .
  • nucleic acid molecule or protein of the present invention can be obtained from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis.
  • Isolated nucleic acid molecules and proteins can also include, for example, natural allelic variants or isomers that induce an immune response in the host.
  • One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into a vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, that are nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule (s) are derived.
  • the vector can be either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating the nucleic acid molecules of the present invention.
  • the present invention also includes an expression vector, which includes a nucleic acid molecule of the present invention in a recombinant vector that is capable of expressing the nucleic acid molecule when transformed into a host cell.
  • 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, parasite, insect, other animal, and plant cells.
  • Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, helminth or other parasite, insect and mammalian cells.
  • 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 nucleic acid molecules of the present invention.
  • recombinant molecules 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.
  • transcription control sequences include those which function in bacterial, yeast, helminth or other endoparasite , or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p L and lambda p R and fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins ) .
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with parasitic helminths, such as B. malayi transcription control sequences.
  • Recombinant molecules of the present invention may also contain (a) secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed protein of the present invention to be secreted from the cell that produces the protein and/or (b) fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins.
  • suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention.
  • Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA) , interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments.
  • a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment.
  • Eukaryotic 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 recombinant host cell harboring one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid 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 nucleic acid 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 nucleic acid molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines).
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid 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 bacterial, fungal (including yeast) , parasite (including helminth, protozoa and ectoparasite), other insect, other animal and plant cells.
  • Preferred host cells include bacterial, mycobacterial, yeast, helminth, insect and mammalian cells. More preferred host cells include Salmonella , Escherichia , Bacillus, Listeria , Saccharomyces, Spodoptera , Mycobacteria r
  • Trichoplusia BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line) , CRFK cells (Crandell feline kidney cell line) , CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus) , COS (e.g., COS-7) cells, and Vero cells.
  • MDCK cells Merrell-Darby canine kidney cell line
  • CRFK cells Cell feline kidney cell line
  • CV-1 cells African monkey kidney cell line used, for example, to culture raccoon poxvirus
  • COS e.g., COS-7 cells
  • Vero cells particularly preferred host cells are Escherichia coli, including E.
  • coli K-12 derivatives Salmonella typhi; Salmonella typhimurium; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246) .
  • Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines) , myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK 31 cells and/or HeLa cells.
  • the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention and one or more transcription control sequences, examples of which are disclosed herein.
  • Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid 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 nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules 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 nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation.
  • transcription control signals e.g., promoters, operators, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
  • non-codon-optimized sequences may be used to express fusion proteins in host cells such as E. coli (see Table 1)
  • the nucleic acid molecule may be codon-optimized to facilitate expression in mammalian cells.
  • malayi Tetraspanin are set forth in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, respectively.
  • the protein sequence can be manipulated.
  • the insertion of a glycine residue after the N-terminal methionine residue of the B. malayi AL 2 protein was found to improve expression of this protein in E. coli.
  • Isolated protein-based antigens of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins.
  • an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein.
  • a preferred cell to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective, medium refers to any medium in which a cell is cultured to produce a protein of the present invention.
  • Such medium typically includes 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.
  • resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
  • Recovery of proteins of invention can include collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification.
  • Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • Proteins of the present invention are preferably retrieved in substantially pure form thereby allowing for the effective use of the protein as a therapeutic composition.
  • a therapeutic composition for animals for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
  • One embodiment of the present invention is an immunogenic composition or vaccine that, when administered to an animal in an effective manner, is capable of protecting that animal from filariasis caused by a filarial nematode.
  • Immunogenic compositions include two or more of the following protective molecules, an isolated antigenic protein of the present invention, an isolated nucleic acid molecule of the present invention, and hybrids and mixtures thereof.
  • the vaccine of the invention is protective in that, when administered to an animal in an effective manner, it is able to treat, ameliorate, and/or prevent disease caused by a filarial nematode including, but not limited to, W. bancrofti , B. malayi, 0. volvulus r L.
  • Vaccines of the present invention can be administered to any animal susceptible to such therapy, preferably to mammals, and more preferably to humans, pets such as cats, and economic food animals and/or zoo animals.
  • the preferred animals to protect against elephantiasis include humans.
  • a vaccine of the present invention when administered to the host can develop antibodies that can kill the parasites in the vector in which the filarial nematode develops, such as in a mosquito when they feed the host.
  • an immunogenic composition of the present invention is administered to the animal in an effective manner such that the composition is capable of protecting that animal from a disease caused by the filarial nematode.
  • Compositions of the present invention can be administered to animals prior to infection in order to prevent infection (i.e., as a preventative vaccine) and/or can be administered to animals after infection in order to treat disease caused by the filarial nematode (i.e., as a therapeutic vaccine).
  • compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
  • excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • buffers examples include phosphate buffer, bicarbonate buffer and Tris buffer
  • preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
  • Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection.
  • the excipient in a non-liquid formulation, can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration .
  • the vaccine can include an adjuvant.
  • Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF) , granulocyte colony stimulating factor (G-CSF) , macrophage colony stimulating factor (M-CSF) , colony stimulating factor (CSF) , erythropoietin (EPO) , interleukin 2 (IL-2), interleukin-3 (IL-3) , interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10) , interleukin 12 (IL-12), inter
  • toll-like receptor agonists e.g., endotoxins, in particular superantigens , exotoxins and cell wall components
  • toll-like receptor agonists e.g., endotoxins, in particular superantigens , exotoxins and cell wall components
  • TLR4 agonists examples include aluminum-based salts; calcium- based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants
  • Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the techniques described herein .
  • a vaccine can include a carrier.
  • Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
  • a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal.
  • a controlled release formulation includes a composition of the present invention in a controlled release vehicle.
  • Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles , bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
  • Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ.
  • Preferred controlled release formulations are biodegradable (i.e., bioerodible) .
  • a preferred controlled release formulation is capable of releasing a vaccine of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition to protect an animal from disease caused by a filarial nematode.
  • the vaccine is preferably released over a period of time ranging from about 1 to about 12 months.
  • a controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
  • Vaccines of the present invention can be administered to animals prior to infection in order to prevent infection and/or can be administered to animals after infection in order to treat disease caused by a filarial nematode.
  • proteins, nucleic acids and mixtures thereof can be used as immunotherapeutic agents.
  • Acceptable protocols to administer compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art.
  • a suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period.
  • a preferred single dose of a protein-based vaccine is from about 1 microgram (pg) to about 10 milligrams (mg) of protein-based vaccine per kilogram body weight of the animal.
  • Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease.
  • a preferred administration schedule is one in which from about 10 ⁇ g to about 1 mg of the vaccine per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months.
  • Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal and intramuscular routes.
  • the vaccine can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a protective protein in the animal.
  • Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, administering a naked (i.e., not packaged in a viral coat or cellular membrane) nucleic acid as a genetic vaccine (e.g., as naked DNA molecules, such as is taught, for example in Wolff, et al.
  • nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle
  • ⁇ genetic (i.e., naked nucleic acid) vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent.
  • a genetic vaccine of the present invention can include one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule.
  • Preferred genetic vaccines include at least a portion of a viral genome (i.e., a viral vector).
  • Preferred viral vectors include those based on alphaviruses , poxviruses, adenoviruses, herpesviruses, picornaviruses , and retroviruses, with those based on alphaviruses (such as Sindbis or Semliki forest virus) , species-specific herpesviruses and poxviruses being particularly preferred.
  • Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production.
  • Particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron-A) , Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a "strong" polyadenylation signal is also preferred.
  • Genetic vaccines of the present invention can be administered in a variety of ways, including intramuscular, subcutaneous, intradermal, transdermal, intranasal and oral routes of administration. Moreover, it is contemplated that the vaccine can be delivered by gene gun, skin patch, electroporation, or nano-based delivery. In this respect, DNA-based and protein-based vaccines can be administered at the same time.
  • a preferred single dose of a genetic vaccine ranges from about 1 nanogram (ng) to about 600 , depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically.
  • Genetic vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate-buffered saline) alone or in a carrier (e.g., lipid-based vehicles).
  • a recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration.
  • the recombinant molecule is packaging- or replication-deficient and/or encodes an attenuated virus.
  • a number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses , poxviruses, adenoviruses, herpesviruses, picornaviruses , and retroviruses.
  • Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species- specific herpesviruses and species-specific poxviruses. Examples of methods to produce and use alphavirus recombinant virus vaccines are disclosed in PCT Publication No. WO 94/17813.
  • a recombinant virus vaccine of the present invention When administered to an animal, infects cells within the immunized animal and directs the production of a protective protein that is capable of protecting the animal from filariasis caused by filarial nematodes.
  • a single dose of a recombinant virus vaccine of the present invention can be from about 1X10 4 to about 1X10 8 virus plaque forming units (pfu) per kilogram body weight of the animal.
  • Administration protocols are similar to those described herein for protein-based vaccines, with subcutaneous, intramuscular, intranasal and oral as routes of administration.
  • a recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express a protein of the present invention.
  • Preferred recombinant cells for this embodiment include Salmonella , E. coll, Listeria , Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae and Pichia pastoris) , BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, DCK and CRFK recombinant cells.
  • Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 10 8 to about 10 12 cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines.
  • Recombinant cell vaccines can include whole cells, cells stripped of cell walls or cell lysates .
  • lymphatic filariasis As is known in the art, there are three groups of filarial nematodes, classified according to the niche within the body that they occupy: lymphatic filariasis, subcutaneous filariasis, and serous cavity filariasis. Lymphatic filariasis is caused by the worms W. bancrofit , B. malayi and B. timori. These worms occupy the lymphatic system, including the lymph nodes, and cause fever, lymphadenitis (swelling of the lymph nodes), lymphangitis
  • Subcutaneous filariasis is caused by Loa loa (the African eye worm) , Mansonella stretocerca , 0. volvulus and Dracunculus medinensis . These worms occupy the subcutaneous layer of the skin, in the fat layer, and present with skin rashes, urticarial papules, and arthritis, as well as hyper- and hypopigmentation macules. Onchocerca volvulus manifests itself in the eyes, causing "river blindness.” Serous cavity filariasis is caused by the worms M. perstans and M. ozzardi , which occupy the serous cavity of the abdomen. Serous cavity filariasis presents with symptoms similar to subcutaneous filariasis, in addition to abdominal pain, because these worms are also deep tissue dwellers.
  • the efficacy of a vaccine of the present invention to protect an animal from filariasis caused by filarial nematodes can be tested in a variety of ways including, but not limited to, detection of protective antibodies (using, for example, proteins of the present invention) , detection of cellular immunity within the treated animal, and/or challenge of the treated animal with the a filarial nematode to determine whether the treated animal is resistant to disease and fails to exhibit one or more signs of disease.
  • Challenge studies can include implantation of chambers including filarial nematode larvae into the treated animal and/or direct administration of larvae to the treated animal.
  • therapeutic compositions can be tested in animal models such as mice, jirds (Meriones unguiculatus) and/or mastomys (e.g., Mastomys natalensis) .
  • animal models such as mice, jirds (Meriones unguiculatus) and/or mastomys (e.g., Mastomys natalensis) .
  • mastomys e.g., Mastomys natalensis
  • this invention also provides a method and kit for efficacy evaluation, as well as for detecting prior exposure to filarial proteins and/or infection with a filarial nematode.
  • one or more antigenic proteins/epitopes is contacted with a biological sample from an animal and binding between the antigenic proteins/epitopes and antibodies in the biological sample is quantitively or qualitatively determined as described herein, wherein the presence and/or amount of antibodies to the antigenic proteins/epitopes is indicative of vaccine efficacy, as well as prior exposure to filarial proteins or an existing infection with a filarial nematode.
  • the method and kit use an array-based format in which serial dilutions of one or more antigens or epitopes are printed.
  • the one or more of the filarial nematode proteins are present on one or more solid surfaces or particles.
  • the one or more of the filarial nematode proteins are in an array so that the presence of multiple antibodies can be assessed in a single assay due to the multiplexing capability of an array-based approach.
  • the array can contain one or more of ALT2, TSP, VAL-1, TPX2, GST or HSP protein or an epitope thereof.
  • the array at least contains each of the proteins used in the vaccine. For example, to assay for protective or neutralizing antibodies . against a vaccine containing HSP, ALT2 and TSP, the array would contain HSP, ALT2 and TSP, or a fusion protein thereof.
  • this invention also provides a method and kit for detecting a filarial nematode.
  • the assay method generally includes the steps of contacting, in vitro, a biological sample with one or more binding agents against filarial nematode proteins selected from the group of ALT2, TSP, VAL-1, TPX2, GST and HSP or fragments thereof. The bound binding agents are then detected.
  • the bound binding agents can be detected using automated detection of binding such as an image reader of an ELISA assay, and if a bound binding agent is detected, the data indicating that a bound binding agent has been detected can be transferred, e.g., to a computer display or on a paper print out.
  • Detection of a filarial nematode protein indicates that the sample or subject from which the sample was obtained has filariasis. Therefore, detection allows selection of treatment options for the subject. Thus, in one embodiment, if one or more of ALT2, TSP, VAL-1, TPX2, GST and HSP is detected, the patient will be given a treatment suitable for filariasis, including but not limited to treatment with diethylcarbamazine, mebendazole, flubendazole, albendazole, ivermectin or a combination thereof.
  • a biological sample is any material to be tested for the presence or amount of a protein of interest (e.g., an antibody or antigen/epitope ) .
  • the sample can be a fluid sample, preferably a liquid sample.
  • liquid samples that may be tested in accordance with this invention include bodily fluids including blood, serum, plasma, saliva, urine, ocular fluid, semen, and spinal fluid. Viscous liquid, semi-solid, or solid specimens (e.g., human tissue, or mosquito or fly tissue) may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples.
  • the biological sample is undiluted. In other embodiments, the sample is diluted or concentrated depending on the detection application.
  • the recovered captured proteins can then be analyzed using any suitable method described herein.
  • the solid surface can be, e.g., beads, such as magnetic beads, polystyrene beads, or gold beads, or in an array or a microarray format using a glass, a plastic or a silicon chip.
  • Such protein capture can be also a part of a channel in a microfluidic device.
  • Binding agents of use in this invention include an antibody, an antibody fragment, or an antibody derivative (e.g., an aptamer) which specifically binds to a cognate filarial nematode protein.
  • Specific binding between two entities generally refers to an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 "1 . Affinities greater than 10 8 M _1 are desired to achieve specific binding.
  • the binding agent is an antibody
  • the antibody can be produced by natural ⁇ i.e., immunization) or partial or wholly synthetic means.
  • Antibodies can be monoclonal or polyclonal and include commercially available antibodies.
  • An antibody can be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Bispecific and chimeric antibodies are also encompassed within the scope of the present invention. Derivatives of the IgG class, however, are desirable.
  • an antibody can be of human, mouse, rat, goat, sheep, rabbit, chicken, camel, or donkey origin or other species which may be used to produce native or human antibodies (i.e., recombinant bacteria, baculovirus or plants ) .
  • monoclonal antibodies can be generated using classical cloning and cell fusion techniques or techniques wherein B-cells are captured and nucleic acids encoding a specific antibody are amplified (see, e.g., U.S. Patent Application No. 20060051348).
  • a collection of proteins or an individual protein e.g., a peptide or polypeptide
  • the antigen can be used for the initial immunization and in the context of antibody production.
  • the antigen of interest is typically administered (e.g., intraperitoneal injection) to wild-type or inbred mice (e.g., BALB/c) or rats, rabbits, chickens, sheep, goats, or other animal species which can produce native or human antibodies.
  • wild-type or inbred mice e.g., BALB/c
  • rats e.g., BALB/c
  • the antigen can be administered alone, or mixed with an adjuvant.
  • the spleen or large lymph node such as the popliteal in rat
  • the spleen or large lymph node is removed and splenocytes or lymphocytes are isolated and fused with myeloma cells using well-known processes, for example, see Kohler and Milstein ((1975) Nature 256:495-497) or Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York (1988)).
  • the resulting hybrid cells are then cloned in the conventional manner, e.g., using limiting dilution, and the resulting clones, which produce the desired monoclonal antibodies, are cultured (see Stewart, S. (2001) Monoclonal Antibody Production. In: Basic Methods in Antibody Production and Characterization, Howard and Bethell (eds.), CRC Press, Boca Raton, FL, pp.51-67).
  • antibodies can be derived by a phage display method.
  • Methods of producing phage display antibodies are known in the art, e.g., see Huse, et al.
  • An antibody fragment encompasses at least a significant portion of the full-length antibody's specific binding ability.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , scFv, Fv, dsFv, diabody, Fd fragments or microbodies.
  • An antibody fragment can contain multiple chains which are linked together, for instance, by disulfide linkages.
  • a fragment can also optionally be a multi-molecular complex.
  • a functional antibody fragment will typically include at least about 50 amino acid residues and more typically will include at least about 200 amino acid residues.
  • the antibody fragment can be produced by any means.
  • the antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or it can be recombinantly-produced from a gene encoding the partial antibody sequence.
  • the antibody fragment can be wholly or partially synthetically-produced.
  • Peptide aptamers which specifically bind to a protein are, in general, rationally designed or screened for in a library of aptamers (e.g., provided by Aptanomics SA, Lyon, France) .
  • peptide aptamers are synthetic recognition molecules whose design is based on the structure of antibodies.
  • Peptide aptamers are composed of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to that of an antibody (nanomolar range) .
  • binding agents of this invention can be achieved using conventional molecular biology techniques and commercially available expression systems. Furthermore, binding agents can be produced using solid-phase techniques (see, e.g., Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154; Seeberger (2003) Chem. Commun. (Camb) (10) : 1115-21) . Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Boston, MA) . Various fragments of a binding agent can be chemically-synthesized separately and combined using chemical methods to produce a full-length molecule.
  • the binding agents described herein can be labeled.
  • the binding agent is an antibody labeled by covalently linking the antibody to a direct or indirect label.
  • a direct label can be defined as an entity, which in its natural state, is visible either to the naked eye or with the aid of an optical filter and/or applied stimulation, e.g., ultraviolet light, to promote fluorescence.
  • Examples of colored labels which can be used include metallic sol particles, gold sol particles, dye sol particles, dyed latex particles or dyes encapsulated in liposomes.
  • Other direct labels include radionuclides and fluorescent or luminescent moieties.
  • Indirect labels such as enzymes can also be used according to the invention.
  • Various enzymes are known for use as labels such as, for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease.
  • alkaline phosphatase horseradish peroxidase
  • lysozyme glucose-6-phosphate dehydrogenase
  • lactate dehydrogenase lactate dehydrogenase
  • urease for a detailed discussion of enzymes in immunoassays see Engvall
  • the proteins described herein can be attached to a surface.
  • useful surfaces on which the protein can be attached for diagnostic purposes include nitrocellulose, PVDF, polystyrene, nylon or other suitable plastic.
  • the surface or support may also be a porous support (see, e.g., US 7, 939, 342) .
  • the proteins of the invention can be attached to a particle or bead.
  • antibodies to the filarial nematode proteins or the filarial nematode proteins themselves can be conjugated to superparamagnetic microparticles, e.g., as used in LUMINEX-based multiplex assays .
  • the filarial nematode proteins of this invention may be isolated and/or purified or produced synthetically or using recombinant nucleic acid technology.
  • the purification may be partial or substantial.
  • fragment refers to a protein having an amino acid sequence shorter than that of the proteins described herein. Preferably, such fragments are at least 5 consecutive amino acids long or up to 35 amino acids long.
  • the protein fragment includes at least one epitope.
  • an “epitope” is a feature of a molecule, such as primary, secondary and/or tertiary peptide structure, and/or charge, that forms a site recognized by an immunoglobulin, T cell receptor or HLA molecule.
  • an epitope can be defined as a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex ( HC) receptors.
  • the protein fragment of the invention is a fragment of B. malayi ALT2 comprising or consisting of the epitope of SEQ ID NO: 78. In other embodiments, the protein fragment of the invention is a fragment of B.
  • the protein fragment of the invention is a fragment of B. malayi, W. bancrofti, or L. loa HSP comprising or consisting of the epitope of SEQ ID NO: 80 or SEQ ID NO: 81.
  • the protein fragment of the invention is a fragment of B . malayi TSP comprising or consisting of the epitope of SEQ ID NO: 82.
  • the protein fragment of the invention is a fragment of B . malayi or W. bancrofti TPX2 comprising or consisting of the epitope of SEQ ID NO: 83.
  • the protein fragment of the invention is a fragment of B . malayi, W. bancrofti, or L . loa HSP comprising or consisting of the epitope of SEQ ID NO: 84.
  • the fragments of the invention can be isolated, purified or otherwise prepared/derived by human or non- human means.
  • epitopes can be prepared by isolating the filarial nematode protein fragment from a bacterial culture, or they can be synthesized in accordance with standard protocols in the art.
  • Synthetic epitopes can also be prepared from amino acid mimetics, such as D isomers of natural occurring L amino acids or non-natural amino acids such as cyclohexylalanine .
  • the filarial nematode protein or protein fragment is conjugated or fused to a high molecular weight protein carrier to facilitate antibody production.
  • the high molecular weight protein is bovine serum albumin, thyroglobulin, ovalbumin, fibrinogen, or keyhole limpet hemocyanin.
  • a particularly preferred carrier is keyhole limpet hemocyanin.
  • Any suitable immunoassay method may be used, including those which are commercially available, to determine the level of at least one of the specific filarial nematode proteins, protein fragments or protective/neutralizing antibodies according to the invention. Extensive discussion of the known immunoassay techniques is not required here since these are known to those of skill in the art.
  • Typical suitable immunoassay techniques include sandwich enzyme-linked immunoassays (ELISA) , radioimmunoassays (RIA) , competitive binding assays, homogeneous assays, heterogeneous assays, etc.
  • ELISA sandwich enzyme-linked immunoassays
  • RIA radioimmunoassays
  • competitive binding assays e.g., homogeneous assays, heterogeneous assays, etc.
  • Various of the known immunoassay methods are reviewed, e.g., in Methods in Enzymology (1980) 70:30-70 and 166-198.
  • the immunoassay method or assay includes a double antibody technique for measuring the level of the filarial nematode proteins or protein fragments in the biological sample.
  • one of the antibodies is a "capture” antibody and the other is a “detector” antibody.
  • the capture antibody is immobilized on a solid support which may be any of various types which are known in the art such as, for example, microtiter plate wells, beads, tubes and porous materials such as nylon, glass fibers and other polymeric materials.
  • a solid support e.g., microtiter plate wells, coated with a capture antibody, preferably monoclonal, raised against the particular protein of interest, constitutes the solid phase.
  • the biological sample which may be diluted or not, typically at least 1, 2, 3, 4, 5, 10, or more standards and controls are added to separate solid supports and incubated.
  • the protein of interest is present in the sample it is captured by the immobilized antibody which is specific for the protein in question.
  • a detector antibody e.g., a polyclonal rabbit anti-marker protein antibody
  • the detector antibody binds to the protein bound to the capture antibody to form a sandwich structure.
  • an anti-IgG antibody e.g., a polyclonal goat anti-rabbit IgG antibody, labeled with an enzyme such as horseradish peroxidase (HRP) is added to the solid support.
  • HRP horseradish peroxidase
  • the degree of enzymatic activity of immobilized enzyme is determined by measuring the optical density of the oxidized enzymatic product on the solid support at the appropriate wavelength, e.g., 450 nm for HRP.
  • the absorbance at the wavelength is proportional to the amount of protein of interest in the sample.
  • a set of marker protein standards is used to prepare a standard curve of absorbance vs. filarial nematode protein concentration. This method is useful because test results can be provided in 45 to 50 minutes and the method is both sensitive over the concentration range of interest for each filarial nematode protein and is highly specific.
  • the standards may be positive samples containing various concentrations of the protein to be detected to ensure that the reagents and conditions work properly for each assay.
  • the standards also typically include a negative control, e.g., for detection of contaminants.
  • the positive controls may be titrated to different concentrations, including non-detectable amounts and clearly detectable amounts, and in some aspects, also including a sample that shows a signal at the threshold level of detection in the biological sample.
  • the method of the invention can be carried out in various assay device formats including those described in US 6,426,050, US 5,910,287, US 6,229,603, and US 6,232,114 to Aurora Biosciences Corporation.
  • the assay devices used according to the invention can be arranged to provide a quantitative or a qualitative (present/not present) result.
  • the method includes the use of a microtiter plate or a microfluidic device format.
  • the assays may also be carried out in automated immunoassay analyzers which are known in the art and which can carry out assays on a number of different samples. These automated analyzers include continuous/random access types.
  • Point-of-care systems, assays and devices have been well described for other purposes, such as pregnancy detection (see, e.g., US 7,569,397 and US 7,959,875). Accordingly, the invention also provides devices, such as point-of-care test strips and microfluidic devices to perform the in vitro assays of the present invention.
  • an assay device of the latter type is one which can provide a semi-quantitative result for the filarial nematode protein or antibodies measured according to the invention, i.e., one or more of ALT2, TSP, VAL-1, TPX2, GST and HSP, or antibodies thereto.
  • the assays or immunoassays of the invention include beads coated with a binding agent against a filarial nematode protein or a fragment thereof, or antibody. Commonly used are polystyrene beads that can be labeled to establish a unique identity. Detection is performed by flow cytometry. Other types of bead-based immunoassays are known in the art, e.g., laser bead immunoassays and related magnetic bead assays (see, e.g., Fritzler, et al. (2009) Expert Opinion on Medical Diagnostics 3:81-89).
  • the methods of the invention can be automated using robotics and computer directed systems.
  • the biological sample can be injected into a system, such as a microfluidic devise entirely run by a robotic station from sample input to output of the result.
  • the step of displaying the result can also be automated and connected to the same system or in a remote system.
  • the sample analysis can be performed in one location and the result analysis in another location, the only connection being, e.g., an internet connection, wherein the analysis is subsequently displayed in a format suitable for either reading by a health professional or by a patient.
  • the presence of any one or any combination of protective/neutralizing antibodies described herein identifies a subject as having been immunized with a vaccine against a filarial nematode.
  • the subject may or may not receive additional booster vaccinations.
  • the presence of any one or any combination of the filarial nematode proteins described herein identifies a subject as having a filarial nematode infection.
  • the subject is diagnosed as having filariasis and, in certain embodiments of this invention, treated with diethylcarbamazine, mebendazole, flubendazole , albendazole, ivermectin or a combination thereof.
  • the diagnosis can be made if the presence of any one of the filarial nematode proteins is detected in the subject's sample.
  • treatment is prescribed or administered if at least two of the filarial nematode proteins are identified positively in the biological sample.
  • Kits provided according to this invention include one or more binding agents, e.g., antibodies or antibody fragments, or filarial nematode proteins, and optionally a device with a solid surface.
  • the solid surface is a bead, slide, assay plate ⁇ e.g., a multiwell plate) or a lateral flow device, to which the binding agents/proteins are bound.
  • the kit further includes one or more standards or controls.
  • each well can contain a single antibody against at least one of the listed filarial nematode proteins. In other embodiments, each well contains an array of antibodies against at least two or more of the listed filarial nematode proteins. In certain embodiments, each well of the plate includes an antibody to two, three, four, or five of the following proteins: ALT2, TSP, VAL-1, TPX2, GST and HSP. In particular embodiments, each well of the plate includes an antibody to each of AL 2 , TSP, VAL-1, TPX2, GST and HSP.
  • each well contains an array of at least two or more of the filarial nematode proteins of this invention.
  • each well of the plate includes two, three, four, or five of the following proteins: ALT2, TSP, VAL-1, TPX2 , GST and HSP.
  • each well of the plate includes each of AL 2 , TSP, VAL-1, TPX2, GST and HSP.
  • the invention provides simple to use point-of-care diagnostic test strips akin to pregnancy detection strips, wherein the strip includes at least one antibody against at least one of the listed filarial nematode proteins.
  • the invention provides simple to use point-of-care diagnostic test strips, wherein the strip includes at least one of the instant filarial nematode proteins.
  • the test strip may include a positive and negative control to show the user that the reagents work properly and/or that the sample has been added to the strip properly.
  • the strips may be provided with or without a casing and with or without additional reagents. Diagnostic test strips for lateral flow assays, such as the test strip assay described herein, may be constructed as described in the art, see, e.g., US 2010/0196200; US 2010/0129935; US 2009/0253119; and US 2009/0111171.
  • Suitable materials for test strips include, but are not limited to, materials derived from cellulose, such as filter paper, chromatographic paper, nitrocellulose, and cellulose acetate, as well as materials made of glass fibers, nylon, dacron, PVC, polyacrylamide, cross-linked dextran, agarose, polyacrylate, ceramic materials, and the like.
  • the material or materials of the test strip may optionally be treated to modify their capillary flow characteristics or the characteristics of the applied sample.
  • the sample application region of the test strip may be treated with buffers to correct the pH or specific gravity of an applied sample, to ensure optimal test conditions.
  • a three-dimensional model of BmHSP protein was constructed by homology modeling. BLAST sequence homology searches were performed to identify template proteins in the PDB database. Human alpha-crystallin A, a recently crystallized protein, showed significant sequence identity and was therefore chosen as the template for modeling BmHSP. Model building was performed using MODELLER 9v6 (Sali & Blundell (1993) J. Mol. Biol. 234:779-815). The 3-D structure obtained was subsequently validated using PROCHECK program (Laskowski, et al . (1993) J. Appl . Cryst . 26:283-29). The best model predicted by PROCHECK had a score of -0.46 and was chosen for further modeling and for generating the 3-D structure using Rasmol program.
  • BmHSP Analysis of the Structure of BmHSP.
  • the secondary structure and protein-protein interaction site of BmHSP was predicted at PDBsum and the Predict Protein E-mail server at the European Molecular Biology Laboratory, Heidelberg (Roos, et al. (1995) Parasitol. Today 11:148-150). Motif scanning was carried out via PROSITE pattern analysis to identify the functional motifs in BmHSP.
  • B-cell, T-cell and CTL epitopes in BmHSP sequences were predicted using Immune Epitope Database and Analysis Resource (1EDB) .
  • Chaperone Assay One of the typical characteristics of chaperone is that they can bind to and protect cellular proteins from heat damage. When proteins are exposed to heat damage, they aggregate (thermal aggregation) . Chaperones prevent this aggregation. To determine whether BmHSP could prevent thermal aggregation, a citruline synthase (CS) (Sigma, St. Louis, MO) thermal aggregation assay was used. CS was selected because this protein is highly sensitive to heat denaturation . An established method was used (Gnanasekar, et al . (2009) Biochem. Biophys. Res. Commun. 386:333-337).
  • CS citruline synthase
  • CS CS was exposed to 45°C in the presence or absence of BmHSP (2 ⁇ ) suspended in 50 mM of sodium phosphate pH 7.4 buffer containing 100 mM NaCl .
  • BSA was used as a control.
  • CS was incubated with BmHSP at a molar ratio of (1:2) for various time intervals from 0 to 40 minutes. Thermal denaturation (aggregation) was monitored spectrophotometrically at 360 nm.
  • Anti-BmHSP Antibody Levels in Human Sera A total of 20 sera samples belonging to different clinical groups such as Mf, CP, EN and NEN were analyzed for the presence and titer of anti-BmHSP IgG antibodies using an indirect ELISA (Cheirmaraj, et al . (1992) J. Trop. Med. Hyg. 95:47-51). Briefly, wells of a 96-well microtiter plate were coated with rBmHSP (1 g/ml) in carbonate buffer, pH 9.6, overnight at 4°C and blocked with 3% BSA for 1 hour at 37 °C. Sera samples were added to the wells and the plates were incubated overnight at 4°C.
  • HRP-labeled mouse anti-human IgG was added (1:5000) and incubated further for 1 hour at 37 °C. Color was developed using ABTS substrate. Absorbance was measured at 405 nm in a microplate reader (BIO-RAD, Hercules, CA) . The isotype of anti-BmHSP IgG antibodies in the sera of subjects was also determined using an isotype-specific ELISA. Biotinylated mouse monoclonal antihuman IgGl, IgG2, IgG3 and IgG4 were used as the secondary antibodies and color was developed with avidin-HRP conjugate (Sigma, St. Louis, MO) as the secondary antibodies.
  • Codon-optimized BmHSP into pVAX Vector for DNA Vaccine.
  • Codon-optimized Bmhsp genes were cloned into eukaryotic expression vector pVAX (Invitrogen) using insert-specific primers (forward primer, 5 ' -CGC GGA TCC ATG GAA GAG AAG GTG GTG-3 ' (SEQ ID NO:l) containing BamEI site and reverse primer, 5 ' -CCG GAA TTC TCA CTT GTC GTT GGT G-3 ' (SEQ ID NO: 2) containing EcoRI site).
  • PCR parameters were as follows: 94°C of denaturation for 30 seconds, 50°C of primer annealing for 30 seconds, 72°C of primer extension for 30 seconds for 30 cycles; and a final extension of 5 minutes was performed at 72 °C. Insert DNA was sequenced to ensure authenticity of the cloned nucleotide sequence on both strands. Plasmids were maintained and propagated in E. coli TOP10F' cells. Subsequently, plasmids were purified using endotoxin-free plasmid extraction kit (Qiagen, Hilden, Germany) . DNA was analyzed by agarose gel electrophoresis and quantified by spectrophotometry (OD 260/280, ratio> 1.8).
  • mice Six-week-old male Balb/c mice purchased from Charles River Laboratories were used in these experiments. Humane use of animals in this study and the protocol was approved by the IACUC committee at the College of Medicine, University of Illinois Rockford. Each group was composed of five (5) mice and all mice were immunized intraperitoneally using three different immunization regimens. Group A mice were immunized using a prime-boost regimen. Mice were primed twice at two week intervals with 100 pg of endotoxin-free, codon-optimized pVAX Bmhsp DNA suspended in 50 ⁇ volume.
  • mice Following priming, all mice received two booster doses of 15 pg of rBmHSP protein (50 ⁇ each) suspended in alum at two weeks interval.
  • Group B mice were immunized with rBmHSP protein alone. These mice received four doses of 15 ⁇ ig of rBmHSP protein suspended in alum given at two week intervals.
  • Group C mice were immunized with DNA alone. These mice received four doses of 100 g of pVAX Bmhsp DNA given at two week intervals.
  • Group D animals received 100 yg of pVAX vector control and adjuvant at the same interval and remained as negative controls. Blood samples were collected from each mouse before immunization and one month after the last booster dose.
  • Anti-BmHSP Antibody Levels in the Sera of Mice Anti-BmHSP IgG antibody levels in the sera of immunized and control groups of mice were determined using an indirect ELISA (Veerapathran, et al . (2009) PLoS Negl . Trop. Dis. 3:e457). IgGl, IgG2a, IgG2b and IgG3 anti-BmHSP antibody levels were also determined using a mouse antibody isotyping ELISA kit (ThermoFisher Scientific, Rockford, IL) .
  • ABTS 2 , 2 ' -azinobis (3-ethyl benzothiazoline-6-sulfonic acid) chromogen substrate and the absorbance was measured at 405 nm in an ELISA reader (BIO-RAD) .
  • Anti-BmHSP antibodies were depleted from pooled sera of EN subjects and immunized mice by incubating the pooled sera with cobalt IMAC resin coupled with his-tagged rBmHSP according to established methods (Veerapathran, et al .
  • Anti-BmHSP IgGl, anti-BmHSP IgG2a, anti-BmHSP IgG2b, anti-BmHSP IgG3 and anti-BmHSP IgG4 antibodies from pooled sera of EN subjects and pooled sera of immunized mice were depleted using NHS (N-hydroxysuccinimidyl ) resin (Thermo fisher scientific) . Briefly, 1 g of respective monoclonal antibodies were coupled to NHS resin column. After washing the resin twice with PBS (pH.8), 100 ⁇ of sera were passed through the column. The flow through was collected as the antibody depleted sera.
  • NHS N-hydroxysuccinimidyl
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • ADCC assay was also performed with pooled human sera samples as described herein except that the human sera samples were incubated with 2 x 10 5 PBMCs collected from normal health subjects and 6-12 B. malayi L3 for 48 hours at 37 °C and 5% CO 2 . Larval viability and death was determined as described above.
  • Protection Studies in Mice Vaccine potential of BmHSP was evaluated in a mouse model of challenge infection. Mice were immunized as described above using prime-boost, DNA alone or protein alone approach. Vector and alum group served as negative controls. Immunized and control animals were challenged using a micropore chamber method as known in the art (Abraham, et al. (1986) Immunology 57:165-169). Briefly, micropore chambers were assembled using 14 x 2 mm PLEXIGLASS (acrylic) rings
  • Splenocyte Proliferation Assay Spleens were collected from all mice from the above experiment and single-cell suspension of spleen cells was prepared. Approximately 2 x 10 5 cells/well suspended in complete RPMI 1640 medium supplemented with 10% heat-inactivated FCS were incubated at 37 °C and 5% C0 2 for 72 hours with rBmHSP (1 g/ml) , ConA (1 ⁇ g/ml) or with medium alone. After incubation, cell proliferation was determined using cell counting kit (CCK-8) purchased from Dojindo Molecular Technologies, Inc. (Gaithersburg, MD) . Stimulation index of spleen cell proliferation was calculated using the formula: Absorbance of stimulated cells ⁇ Absorbance of unstimulated cells .
  • Cytokine Analysis Spleen cells from immunized and control mice were cultured at 37 °C and 5% C0 2 for 72 hours with rBmHSP (1 ⁇ g/ml) , ConA (1 ⁇ g/ml) or with medium alone as described above. After 72 hours, culture supernatants and cell pellets were collected separately for cytokines analysis. For measuring cytokine mRNA, cell pellets were suspended in TRIZOL (phenol, guanidinium and thiocyanate) reagent (GIBCO-BRL, Life technologies, Carlsbad, CA) and total RNA was extracted as per the manufacturer's instructions.
  • TRIZOL phenol, guanidinium and thiocyanate
  • RNA pellets were dissolved in RNAse-free water (Sigma) and treated with DNase I before determining total RNA concentration using a Beckman spectrophotometer at 260 nm. Reverse transcription of total RNA was performed using first strand cDNA synthesis kit (SABiosciences , Frederick, MD) as per manufacturer's recommendations. Relative quantification of the expression of genes of interest was measured in an Applied BioSystem 7300 real-time PCR machine (Applied BioSystems, Foster City, CA) . PCR amplifications were performed with the LIGHTCYCLER-DNA SYBR Green (cyanine dye) mix ( SAbiosciences ) .
  • the reaction was performed using the following PCR conditions: 15 minutes activation step at 95°C for one cycle, 15 seconds denaturation step at 95°C, annealing of primers for 20 seconds at 50 °C and elongation step for 15 seconds at 72°C.
  • DNA was amplified for 50 cycles.
  • the fluorescent DNA binding dye SYBR Green (cyanine dye) was monitored.
  • RT-PCR data array set was generated and analyzed using SABiosciences web-based data analysis system.
  • Culture supernatants were then collected from splenocyte cultures 72 hours after incubation with rBmHSP (1 ⁇ g/ml), ConA (1 ⁇ g/ml) or with medium alone.
  • Secreted levels of IL-2, IL-4, IFN- ⁇ and IL-10 protein in the culture supernatants were determined using a sandwich ELISA kit purchased from ThermoFisher Scientific. Concentration of each cytokine was determined from a standard curve plotted using recombinant mouse IL-2, IL-4, IFN- ⁇ or IL-10.
  • BmHSP BmHSPl2.6
  • BmHSP was cloned in pRSET A vector and was expressed as a histidine-tagged (his-tagged) fusion protein in E. coli BL21 (DE3)PLysS.
  • Recombinant BmHSP protein was subsequently purified using IMAC column. The molecular mass of the purified recombinant his-tag fusion protein was found to be approximately 18 kDa .
  • the column-purified recombinant protein appeared as a single band in SDS-PAGE.
  • BmHSP is a Chaperone. Most of the heat shock proteins reported to date have chaperone function. To determine whether BmHSP also has similar chaperone function, a thermal aggregation reaction was performed using a model substrate, Citrulline synthase (CS) . Incubation of CS at 42 °C resulted in unfolding of the protein and subsequent aggregation within 10 minutes. Addition of BmHSP to CS protein (at a molar ratio of 1:2), before the heat treatment, significantly (P ⁇ 0.01) inhibited the thermal aggregation of CS protein. A non-chaperone control protein, BSA, had no effect on the heat-induced aggregation of CS protein.
  • CS Citrulline synthase
  • chaperone proteins Another function of chaperone proteins is that they can specifically bind to denatured proteins.
  • rBmHSP was incubated with native and denatured CS or native and denatured luciferase substrates. These studies showed that rBmHSP preferentially bound to denatured protein substrates compared to native or control protein. These findings thus confirmed that BmHSP can act as a molecular chaperone potentially protecting the parasite cellular proteins from the damaging effects of the host.
  • Mf carriers had only significant levels of anti-BmHSP IgG2 antibodies in their sera.
  • CP individuals had only significant levels of anti-BmHSP IgG4 antibodies in their sera.
  • Anti-BmHSP IgGl and IgG3 levels were very low in the sera of these Mf and CP individuals.
  • Anti-BmHSP antibodies were not detectable in the sera of NEN subjects.
  • mice immunized with rBmHSP developed significant levels of anti-BmHSP IgG antibodies. More specifically, prime-boost vaccine regimen induced significantly higher titer of IgG antibodies compared to DNA vaccine alone group (p ⁇ 0.05) . However, rBmHSP protein vaccine induced the highest IgG antibody titer. Analysis of the isotype of anti-BmHSP IgG antibodies showed that predominantly IgGl, IgG2a and IgG2b anti-BmHSP antibodies were present in the sera of vaccinated animals. The ADCC assay was also performed with mouse sera. These studies showed that sera from BmHSP-vaccinated mice promoted adherence of peritoneal exudate cells to L3 and participated in ADCC function (83.02% larval killing) compared to control sera (13%) (p ⁇ 0.002) (Table 5).
  • Vaccine Potential of BmHSP in Mice was assessed in Balb/c mice using a micropore chamber method. Results showed that mice immunized using the prime-boost vaccination regimen and protein vaccine of BmHSP exhibited nearly 72% and 58% mortality, respectively, of L3s implanted into the peritoneal cavity of the immunized mice (Table 7). While chambers implanted in the control groups of animals showed only 7% mortality of the parasite, the difference between the protection of control group of mice and vaccinated mice was significant (P ⁇ 0.001). On the other hand, mice immunized by DNA vaccine alone induced only 31% protection. Thus, the prime-boost vaccination regimen appeared to be highly efficient in conferring vaccine-induced protection against a challenge infection compared to DNA alone or protein alone immunization protocols.
  • Spleen cells from the control group of animals failed to proliferate in response to rBmHSP (stimulation index of 0.98 ⁇ 0.013) and was similar to media alone controls. Since the spleen cells from vaccinated animals were proliferating significantly to recall response to rBmHSP, levels of cytokines in the culture supernatants were measured. These results showed that IFN- ⁇ was the predominant cytokine secreted by spleen cells from vaccinated animals at 72 hours after stimulation with rBmHSP. A real time-PCR cytokine gene array was performed on mRNA collected from the spleen cells stimulated with rBmHSP. These results showed that both Thl
  • cytokine genes were significantly increased in vaccinated animals .
  • Brugia malayi L3s were obtained from the NIAID/NIH Filariasis Research Reagent Resource Center (FR3) at the University of Georgia, Athens, GA.
  • Monovalent DNA vaccine was composed of Bmhsp or Bmalt2 in pVAXl vector.
  • codon optimized Bmhsp or BmALT2 genes were cloned into the eukaryotic expression vector pVAXl (Invitrogen, Carlsbad, CA) using insert-specific primers (Gnanasekar, et al . (2004) supra).
  • the multivalent vaccine was composed of Bmhsp and Bmalt2 genes in the same pVAXl vector.
  • Codon optimized Bmhsp gene was first cloned into pVAXl vector with no stop codon in the reverse primer (5'-CCG GAA TTC TCA CTT GTC GTT GGT G-3' ; SEQ ID NO:24) but contained a Pstl site. Codon optimized Bmalt2 gene was then inserted into this clone using gene specific primers (Gnanasekar, et al. (2004) supra). PCR parameters for all the three constructs were: 94 °C denaturation for 30 seconds, 50°C primer annealing for 30 seconds, 72 °C primer extension for 30 seconds for 30 cycles/ a final extension of 5 minutes was performed at 72 °C.
  • Plasmids were maintained and propagated in E. coli TOP10F' cells. Plasmids were purified using endotoxin- free plasmid extraction kit (Qiagen, Valencia, CA) . DNA was analyzed by agarose gel electrophoresis and quantified in a spectrophotometer (OD 260/280, ratio >1.8).
  • mice Six-weeks old male Balb/c mice purchased from Charles River Laboratories were used in these experiments. Humane use of animals in this study and the protocol was approved by the IACUC committee at the College of Medicine, University of Illinois Rockford. Mice were divided into four (4) groups of five (5) animals each. All mice were immunized subcutaneously using a DNA prime - protein boost vaccine regimen. All experimental groups of mice were primed with two injections of endotoxin-free codon optimized DNA given in 50 ⁇ volume and boosted with two doses of recombinant proteins suspended in alum (50 ⁇ each) given at two weeks interval.
  • mice were primed with 100 of pVAXBmhsp and boosted with 15 ⁇ g of rBmHSP;
  • Group B mice were primed with 100 pg of pVAX Bmalt2 and boosted with 15 ⁇ g of rBmALT2;
  • Group C mice were primed with 100 of pVAXBmhsp/Bmalt2 DNA and boosted with 15 ⁇ g of rBmHSP and 15 ⁇ g of rBmAL 2.
  • Group D mice received 100 ⁇ g of pVAXl vector plus 50 ⁇ of alum and served as controls. Blood samples were collected from each mouse before immunization and one month after the last booster dose. Sera were separated and stored at -80°C.
  • Absorbance was measured at 405 nm in a microplate reader (BIO-RAD, Hercules, CA) .
  • Vaccine potential of the monovalent and multivalent vaccine formulations were then evaluated in a mice model. Mice were immunized as described above using the prime boost approach. Vector plus alum group served as negative controls. Immunized and control animals were challenged using a micropore chamber method known in the art (Abraham, et al . (1989) Am. J. Trop. Med. Hyg. 40 ( 6) : 598-60 ) .
  • micropore chambers were assembled using 14 x 2 mm PLEXIGLASS (acrylic) rings (Millipore Corporations, Bedford, MA) and 5.0 ⁇ NUCLEOPORE polycarbonate membranes (Millipore Corporations) that were attached to the PLEXIGLASS (acrylic) rings with cyanoacrylic adhesive and dental cement.
  • the chambers were immersed overnight at 37 °C in sterile RPMI medium containing gentamycin and antimycotic solution.
  • 20 live infective L3s suspended in RPMI1640 medium supplemented with 15% heat inactivated fetal calf serum (FCS) were introduced into the micropore chambers and the opening was sealed with dental cement.
  • Micropore chamber containing the L3s were then surgically implanted into the peritoneal cavity of each mice under anaesthesia. Aseptic conditions were followed for the surgical procedures. After 48 hours of implantation, animals were sacrificed and the chambers were recovered from peritoneal cavity. Contents of each chamber were emptied and larvae were examined microscopically for adherence of cells and for larval death. Larval viability was determined microscopically at 100 x. The percentage of protection was expressed as the number of dead parasites ⁇ number of total parasites recovered x 100.
  • the multivalent vaccine also elicited significant IgG antibody titers. Following multivalent vaccine, the mice produced IgG antibodies against both BmHSP and BmALT2 equally, suggesting that the antigens do not interfere or compete for dominance. An interesting finding was that the multivalent vaccine elicited 1.5- to 1.75-fold higher (p ⁇ 0.005) titers of IgG antibodies compared to the monovalent vaccine ( Figure 1) . These finding indicated that the two antigens in the multivalent formulation can act synergistically by increasing the vaccine-induced antibody responses against each antigens in the vaccinated mice. The findings also indicated that combining these two antigens in the vaccine formulation has a great advantage.
  • Multivalent Vaccine Induces Significant Protection in Mice.
  • the results herein showed that significant IgG antibodies were elicited following vaccination with monovalent and multivalent vaccine preparations.
  • vaccinated animals were challenged with live third stage infective larvae (L3) of B. malayi. Since the parasites do not reach maturity in these animals, a better recovery of worms is obtained if the parasites are surgically implanted into the animals.
  • a standard micropore chamber challenge method (Abraham, et al . (1989) supra).
  • mice were immunized with various prime-boost combinations. As shown in Figure 3, 100% protection can be achieved in mice following immunization with HAT hybrid protein or after prime boost immunization with HAT hybrid DNA and HAT hybrid protein.
  • Sera Sera samples used in this study were from archived samples stored at the Mahatma Vogel Institute of Medical Sciences, Sevagram, India. These samples were collected as part of epidemiological surveys in and around Wardha, an area endemic for lymphatic filariasis.
  • circulating antigen was detected using an Og4C3 kit and a WbSXP-based enzyme-linked immunosorbent assay (ELISA) .
  • ELISA enzyme-linked immunosorbent assay
  • Subjects with no circulating antigen or microfilariae were classified as EN, whereas subjects with circulating microfilariae and/or circulating antigen, as detected by ELISA, were considered as MF.
  • Subjects showing lymphedema and other visible clinical symptoms of filariasis were grouped into CP. Control non- endemic normal (NEN) sera were collected at the University of Illinois Clinic at Rockford, IL.
  • Brugia malayi L3s were obtained from the NIAID/NIH Filariasis Research Reagent Resource Center (FR3) at the University of Georgia, Athens, GA.
  • PCR parameters for all the constructs were: 94°C denaturation for 30 seconds, 50°C primer annealing for 30 seconds, 72°C primer extension for 30 seconds for 30 cycles; and a final extension of 5 minutes was performed at 72 °C.
  • Insert DNA was sequenced to ensure authenticity of the cloned nucleotide sequence on both strands. Plasmids were maintained and propagated in E. coli TOP10F' cells. Plasmids were purified using endotoxin- free plasmid extraction kit (Qiagen, Valencia, CA) . DNA was analyzed by agarose gel electrophoresis and quantified in a spectrophotometer (OD 260/280, ratio > 1.8).
  • Recombinant BmVAL-1 and rBmALT2 were expressed in pRSET-A vector and purified using an immobilized cobalt metal affinity column chromatography according to published methods (Norimine, et al. (2004) Infect. Immun . 72:1096- 1106; Shinnick, et al. (1988) Infect. Immun. 56:4 6-451).
  • Endotoxin in the recombinant preparations were removed by passing the recombinant proteins through polymyxin B affinity columns (Thermo Fisher Scientific, Rockford, IL) and the levels of endotoxin in the final preparations were determined using an E-TOXATE kit (Sigma, St Louis, MO) as per manufacturer's instructions. Endotoxin levels in the final preparations (0.005 EU/ml) were below detection limits in these recombinant protein preparations.
  • Each experimental set had four groups (a) DNA prime plus DNA boost (homologous) , (b) protein prime plus protein boost (homologous), (c) DNA prime plus protein boost
  • mice were immunized subcutaneously with codon-optimized DNA (100 pg) in 50 ⁇ volume or with recombinant protein (150 pg) plus alum in 50 ⁇ volume.
  • Control group received 100 pg of pVAXl blank vector or 50 ⁇ of alum.
  • Blood samples were collected at frequent intervals, sera separated and stored at -80°C.
  • the protocol used for immunizing mice and jirds was as follows. Animals were prebled and given a first dose on day 0. A second dose was administered on day 14 and subsequently bled. Third and fourth doses were administered on days 28 and 42, respectively, and the animals were subsequently bled. Mice were challenged on day 56 and protection was determined on day 58. Jirds were challenged on day 60 and protection was determined on day 155.
  • Splenocyte Proliferation and Cytokine Assays Single-cell suspension of spleen cells (0.5 x 10 6 cells per well suspended in 200 ⁇ media) were prepared from each mouse and cultured in triplicate wells with either (1) 1 ⁇ g/ml rBmVAL-1, (2) 1 yg/ml rBmALT2, (3) 1 g/ml rBmVAL- l+BmALT2, (4) a nonspecific recombinant protein (1 g/ml of Schistosoma mansoni G-binding protein) or (5) were left unstimulated in the media. All cells were incubated for 3 days at 37 °C with 5% C0 2 .
  • SI stimulation index
  • BmVAL-1 and BmALT2 Specific IgG Antibodies in the Sera of Immunized Mice Titer of anti-BmVAL-1- and anti- BmALT2-specific antibodies was determined in the sera of immunized mice using an ELISA (Veerapathran, et al . (2009) supra; Gnanasekar, et al. (2004) Infect. Immun . 72:4707- 15) . Pre-immune sera served as controls. HRP-conj ugated goat anti-mouse IgG was used as the secondary antibody (Thermo Fisher Scientific) for mouse assays. OPD (Sigma) was used as the substrate and optical density (OD) was measured at 405 nm.
  • Anti-BmVAL-1- and anti-BmALT2-specific IgGl, IgG2a, IgG2b, IgG3 and IgG4 antibodies were determined in the sera of mouse using a mouse antibody isotyping kit purchased from Thermo Fisher Scientific. All ELISAs were performed as per the manufacturer's recommendation and absorbance was read at 405 nm. Respective HRP-labeled goat anti-IgG isotype antibody was used as the secondary antibodies and color was developed using OPD substrate.
  • Jirds were challenged with 100 B. malayi L3s and worm establishment was determined on day 95 after challenge according to established methods (Weil, et al . (1992) supra).
  • Jirds are permissive hosts for B. malayi and the worms mature into adult males and females in about 75 days. Presence of mature worms in the control group of jirds was confirmed by demonstrating microfilariae in their blood on day 80 after challenge. Percent reduction in the worm establishment was calculated using the formula: average number of worms recovered from control worms - average number of worms recovered from vaccinated animals / average number of worms recovered from control animals x 100.
  • Significant anti-BmVAL-1 and anti-BmALT2 IgG antibodies were present in the sera of EN subjects compared to MF subjects (p ⁇ 0.01) and CP subjects (p ⁇ 0.005).
  • NEN subjects did not carry IgG antibodies against either of the antigens.
  • IgG antibody subset analysis showed that BmVAL-1 vaccination elicited primarily IgGl and IgG2a isotype of antibodies, whereas, BmALT2 vaccination induced IgGl, IgG2a and IgG3 isotype of antigen-specific antibody responses. Antigen-specific IgG4 antibody responses were not evident. The prime boost approach significantly amplified the IgG isotype responses. Following multivalent vaccination regimen IgGl, IgG2a and IgG3 subset of antigen specific antibodies were present in the sera of mouse.
  • Spleen cells from immunized mice stimulated with either rBmVAL-1 or rBmALT2 proliferated significantly (SI 10.8 + 1.1 and SI 14.6 + 1.2, respectively) compared to the media control (SI 2.1 + 0.9) .
  • Spleen cells from mice immunized with the multivalent construct responded to both rBmVAL-1 (SI 18.9 ⁇ 2.6) and rBmALT2 (SI 23.5 ⁇ 3.1), indicating that a strong recall cellular response was generated to both BmVAL-1 and BmALT2 following vaccination with the multivalent construct.
  • Multivalent Vaccine Induces Significant Protection in Mice and Jirds The results herein indicated that significant IgG antibodies were elicited following vaccination with monovalent and multivalent vaccine preparations.
  • vaccinated animals were challenged with live, third stage infective larvae (L3) of B. malayi. Since the parasites do not reach to maturity in mice, a standard micropore chamber challenge method was used (Gnanasekar, et al . (2004) supra). These studies showed that 39% to 74% protection was achieved in mice following immunization with monovalent vaccine (Table 9) .
  • Brugia malayi L3s were obtained from the NIAID/NIH Filariasis Research Reagent Resource Center (FR3) at the University of Georgia, Athens, GA.
  • Codon-optimized Bmalt2 gene was amplified with the forward primer 5 ' -AAC TGC AGA TGG GTA ACA AGC TCC TCA TCG- 3' (SEQ ID NO: 27) and the reverse primer without the stop codon 5'-CGC GAA TTC GGC GCA CTG CCA ACC TGC-3' (SEQ ID NO:28). Underlined sequences indicate Pstl and EcoRI restriction sites in the forward and reverse primers, respectively.
  • the amplified Bmalt2 DNA insert was then subcloned into pVAX Bmhsp* plasmid at the Pstl and EcoRI restriction sites, resulting in pVAX Bmhsp+Bmalt2* plasmid.
  • Bmalt2 forward primer, 5'-CCC TCG AG A TGA ATA AAC TTT TAA TAG CAT-3' (SEQ ID NO: 33) containing Xhol or 5 ' -AAC TGC AGA TGG GTA ACA AGC TCC TCA TCG-3 ' (SEQ ID NO: 27) and reverse primer, 5'-GGG TAC CCG CGC ATT GCC AAC CC-3' (SEQ ID NO: 34) containing Kpnl .
  • Bmtsp, forward primer, 5'-GGG GTA CCC CGG CAA GGA TCA ATT TAA AA-3' (SEQ ID NO: 35) containing Kpnl and reverse primer, 5' ⁇ CGG AAT TCT CAA TCT TTT TGA GAT GAA T-3' (SEQ ID NO: 36) containing EcoRI were used to amplify the Bmtsp fragment of SEQ ID NO: 77.
  • Primers were also designed to amplify a Tetraspanin Large Extracellular Loop (LEL) fragment of SEQ ID NO: 63.
  • Bivalent constructs (HA, HT and TA) were also cloned individually into a pRSETA vector.
  • mice Six-week-old Balb/C mice were immunized with 100 ⁇ g of DNA intradermally (i.d.) as DNA vaccine or with 15 pg of recombinant protein subcutaneously (s.c.) as protein vaccine or with two doses of DNA and two doses of protein as prime-boost vaccine. Mice were randomly divided into 15 groups with 5 mice per group. Animals from groups 1-3 were immunized with HSP+ALT2 (HA) . Groups 4-6 were immunized with HSP+TSP (HT) , and 7-9 were immunized with TSP+ALT2 (TA) .
  • HSP+ALT2 HSP+ALT2
  • HT HSP+TSP
  • TA TSP+ALT2
  • IgG antibody levels in the sera of immunized and control groups of animals against all the three proteins were determined using an indirect ELISA (Anandharaman, et al. (2009) supra) . Briefly, wells of a 96-well microtiter plate were coated with recombinant proteins (rHSP, rALT2 or rTSP; 1 ⁇ g/ml) in carbonate buffer, pH 9.6, overnight at 4°C and blocked with 3% BSA for 1 hour at 37°C. Sera samples were added to the wells and the plates were incubated overnight at 4°C.
  • rHSP recombinant proteins
  • HRP-labeled mouse anti-human IgG was added (1:5000) and incubated further for 1 hour at 37°C. The color was developed with OPD (o-phenylene diamine) substrate (Sigma Aldrich, USA) . Absorbance was measured at 450 nm in a microplate reader (BIO-RAD, Hercules, CA) .
  • ADCC Assay To evaluate the protection efficacy of the antigen combinations, in vitro ADCC was performed with the sera from the mice immunized with bivalent and trivalent vaccine constructs. The in vitro ADCC assay was performed according to known methods (Chandrasekhar, et al. (1990) supra) . Briefly, Peritoneal Exudates Cells (PEC) were collected from normal Balb/c mice by washing the peritoneal cavity with sterile RPMI 1640 media. The cells were washed and suspended in RPMI 1640 medium supplemented with 10% Fetal Calf Serum (FCS) . Ten L 3 of B .
  • PEC Peritoneal Exudates Cells
  • malayi were added to 2 x 10 5 peritoneal exudates cells (PEC) /well in 96- well culture plates (Thermo Fisher Scientifics, USA. ) , 50 ⁇ of immunized mice sera and 50 ⁇ of RP I 1640 media were added to the wells in triplicates and incubated for 48 hours in 5% C0 2 at 37 ° C. Larval viability was determined microscopically after 48 hours of incubation. Larvae that were limpid, damaged and with the clumps of cells adhered to it were counted as dead. ADCC was estimated as the percent larval death calculated using the formula: Number of Dead larvae ⁇ Total number of larvae x 100.
  • the parasite was considered dead if it was not motile and limpid, and had several adherent cells on the surface.
  • the percentage protection was calculated using the formula: number of dead parasites ⁇ number of recovered parasites x 100. This experiment was repeated twice with five animals in each group.
  • Splenocyte Proliferation Vaccinated and control mice were sacrificed on day 60 and the spleens were removed aseptically. Single-cell suspensions were prepared in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, passed through a NYLON (aliphatic polyamide) mesh (BD Biosciences, Bedford, USA) . After determining the viability of cells using trypan blue dye exclusion, approximately 2 x 10 6 cells per well in triplicates were plated in 96-well culture plates (ThermoFisher, USA) .
  • the splenocytes were stimulated with l g/100 l/well of recombinant proteins (rHSP, rALT2 or rTSP) or ConA or with medium alone (Unstimulated) for 72 hours at 37 ° C in the atmosphere of 5% C0 2 .
  • Cell proliferation was determined using cell counting kit (CCK-8) purchased from Dojindo Molecular Technologies, Inc. (Gaithersburg, MD) .
  • Stimulation index of spleen cell proliferation was calculated using the formula: Absorbance of stimulated cells ⁇ Absorbance of unstimulated cells. All cultures were taken in triplicates and the results expressed as mean S.I.1SEM.
  • RT-PCR Real Time-PCR
  • first-strand cDNA was synthesized by RT 2 First Strand Kit (SuperArray Bioscience Corporation, Frederick, MD) .
  • PCR array analysis was performed according to the manufacturer protocol with the RT 2 Real-Time TM SYBR Green (cyanine dye) PCR Master Mix. Aliquots from this mix were added to a 96-well plate, where each well contained predispensed gene-specific primer sets. Relative quantification of the genes of interest that expressed was measured in an Applied BioSystem 7300 real-time PCR machine (Applied BioSystems, Foster City, CA) .
  • RT-PCR data array set was generated and analyzed using SABiosciences web-based data analysis system. Results were expressed in terms of fold change of immunized mice compared to control mice by normalizing the expression of housekeeping genes.
  • Antibody-Dependent Cell-Mediated Cytotoxicity Antibody-mediated adherence and cytotoxicity of immune cells to B. malayi L3 larvae was observed after 48 hours of incubation of parasites, with the sera and normal immune cells. ADCC showed maximum cytotoxicity of approximately 90% (p ⁇ 0.001) in the sera of mice immunized with rBmHAT or rWbHA vaccine constructs (Table 11) . Bivalent vaccine constructs of rWbHT and rWbTA also gave better protection of 82% and 87%, respectively, which was significant compared to monovalent-vaccinated and control animals (p ⁇ 0.001). To evaluate the protection mediated by the antibodies generated against HSP, ALT and TSP antigens, IgG antibodies were depleted from the immunized sera and used in ADCC. Depleted antibodies showed only 6% protection against L3.
  • RT-PCR Array To determine the cellular immune responses to multivalent constructs in the vaccinated mice, spleen cells collected from vaccinated and control mice were cultured in the presence of respective recombinant proteins and their proliferative responses and cytokine profiles were evaluated. Since the spleen cells from vaccinated animals were proliferating significantly to recall response, levels of cytokine mRNA were measured. An RT-PCR cytokine gene array was performed on mRNA collected from the spleen cells stimulated with recombinant proteins. These results showed that both Thl (IFN- ⁇ , IL-2) and Th2 (IL-4) cytokine genes were significantly increased in vaccinated animals.
  • Thl IFN- ⁇ , IL-2
  • IL-4 Th2
  • IL-10 is an immunosuppressive agent
  • the IL-10 receptor binding sequences were deleted from HSP.
  • the truncated sequence was referred to as cHSP.
  • the cHSP was then used to replace the HSP gene and HSP protein in the multivalent HAT hybrid vaccine.
  • the resulting new vaccine was called cHAT .
  • Protection Studies Using cHAT-Fusion Protein Vaccine in Mice Mice were immunized with four doses of cHAT fusion protein at two week intervals.
  • O. volvulus tetraspanin was cloned from 0. volvulus L3 cDNA library and recombinant proteins were prepared. Sera sample from mice vaccinated with cHAT vaccine that gave the 81% protection in Table 13 was used to probe the recombinant O. volvulus tetraspanin after separating the protein in a 12% SDS-PAGE gel. B. malayl tetraspanin was used as a positive control. Results showed that the sera sample significantly reacted with 0. volvulus tetraspanin ( Figure 4) thereby indicating that the cHAT vaccine developed in Example 5 is of use as a vaccine against O. volvulus .
  • Multivalent Fusion Protein rBmHAT The multivalent fusion protein rBmHAT expressed in Escherichia coli BL21 (pLysS) , was purified and endotoxin removed by Pierce High Capacity Endotoxin removal resin column (Thermo Fisher Scientific, Rockford, IL) as described herein.
  • B. malayi L3 Challenge On day 84, one month after the final dose of vaccine, macaques were anesthetized with ketamine HC1 and challenged subcutaneously with 400-500 B. malayi L3. To facilitate the production of the relatively large number of L3 (500 L3/animal) required for challenging 10 immunized macaques, the animals were divided into 2 subgroups within each group. The subgroups were challenged one week apart. Before challenge, B. malayi L3 were counted and examined for viability under a microscope. Only viable parasites were used for challenge.
  • PBMC peripheral blood mononuclear cells
  • PBMC collected before the challenge was analyzed for T cell proliferation and IFN- ⁇ secretions.
  • PBMC collected after the challenge experiments were tested for T cell proliferation and ELISPOT assays.
  • Proliferation assay was performed with PBMC isolated on the same day of blood collection. PBMC suspended in RPMI media with 10% FBS were used for ADCC assay and for cytokines analysis.
  • CFSE Carboxyfluorescein diacetate succinimidyl ester
  • Cells were cultured and harvested after 5 days of stimulation. Following a washing step with PBS/0.2% FBS, cells were surface stained with an antibody cocktail of CD3-APC-Cy, CD4-PE and CD8-PerCP and incubated for 20 minutes at room temperature. After an additional washing step with PBS/0.2% FBS the cells were acquired on BD FACS CANTO II flow cytometer (BD, San Jose, CA) and analyzed on a BD FACS DIVA Software v6.1.2. At least 50,000 events within the live lymphocyte gate were acquired.
  • CBC Cell Counts, Serum Chemistry and Complete Blood Count (CBC) Analysis .
  • CBC serum chemistries and eosinophil counts were analyzed using commercial automated hematology and serum chemistry analyzers by IDEXX. Samples collected prior to the initiation of the study served as a normal reference baseline for each animal.
  • ELISPOT Assay An ELISPOT assay was performed to determine the antigen-specific IFN- ⁇ and IL-10 secreting cells in the PBMC of vaccinated and control macaques.
  • a monkey ELISPOT kit purchased from U-Cytech biosciences (Yalelaan, The Netherlands) was used to determine the spot forming units as per the manufacturer's instruction.
  • PBMC collected 20 weeks post challenge were plated in 96-well plates at 1*10 6 cells/ml and were stimulated with 100 ng/well of B. malayi adult soluble antigen (BmA) for 24 hours at 37 °C and 5% C0 2 .
  • ELISPOT plates were coated with 100 ⁇ /well of capture antibodies (anti-IL-10 or anti-IFN- ⁇ ) diluted in sterile coating buffer and incubated overnight at 4°C. Plates were washed 2 times with sterile coating buffer. After blocking the plates with 200 ⁇ /well of blocking buffer for 1 hour at room temperature, PBMC that were already stimulated with BmA antigens or only media (negative control) were added to the wells of the ELISPOT plates at 100 ⁇ /well and incubated for 24 hours at 37 °C and 5% C0 2 . All the cells were removed from the plates and the membrane was washed 3 times with sterile PBS.
  • capture antibodies anti-IL-10 or anti-IFN- ⁇
  • biotinylated anti-monkey IgGl (1:2000), IgG2 (1:200), IgG3 (1:2000), IgA (1:2000) and IgE (1:1000) antibodies were used as secondary antibodies. After washing the plates, optimally diluted streptavidin conjugated horse radish peroxidase (HRP) was added and further incubated for 60 minutes at room temperature and the color was developed.
  • HRP horse radish peroxidase
  • PBMC peripheral blood mononuclear cells
  • B. malayi L3 serum from each animal (collected one month after the final dose of vaccine) in a 96-well round bottom tissue culture plate. Five replicates were performed for each serum sample. Control wells contained B. malayi L3 incubated in media, with sera alone or cells alone. The plates were incubated at 37 °C with 5% C0 2 for 48 hours. Following incubation, B.
  • malayi L3 were examined under a microscope at 24 and 48 hours to determine larval viability.
  • Dead L3 were defined as those having a limpid or straight appearance with no movements for an additional observation period of 8 hours at 37 °C.
  • Live larvae were active, coiled and motile.
  • the percentage larval death was expressed as the ratio of the number of dead L3 to that of the total number recovered within the experimental period multiplied by 100. Average larval death in 5 wells were calculated and expressed as percent protection in each animal.
  • PCR- based assays are more sensitive in detecting the presence of Mf in the blood samples (Mishra, et al. (2005) Acta Trop. 93:233-7; Tao, et al . (2006) J. Clin. Microbiol. 44:3887-93). Therefore, the PCR based assay was also used to confirm the presence of Mf in the blood samples of all macaques 20 weeks after challenge. Whole blood samples were centrifuged at 10,000 rpm for 5 minutes and the supernatant containing serum was stored at -20 °C.
  • DNA was isolated from the pellet using DNEASY Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. Primers were synthesized at Integrated DNA Technologies Inc., (Coralville, IA) for Hhal tandem repeats. Primer sequences for Hhal tandem repeats were: Forward 5 ' -GCG CAT AAA TTC ATC AGC-3' (SEQ ID NO: 75) and Reverse 5 ' -GCG CAA AAC TTA ATT ACA AAA GC-3 ' (SEQ ID NO:76).
  • PCR parameters were initial denaturation of 94 °C for 5 minutes, followed by 40 cycles of 1 minute at 94°C, 1 minute at 56°C, 1 minute at 72°C and a final extension of 10 minutes at 72°C. Following PCR reaction, 10 ⁇ of each PCR product was analyzed on a 1% agarose gel.
  • PBMC Proliferations Assay PBMC collected 10 weeks post-challenge were cultured in 96-well tissue culture plates at a concentration of lxlO 6 cells/well in RPMI 1640 supplemented with 10% FCS . Cells were stimulated either with rBmHAT antigen (1 mg/ml) or Concanavalin A (1 mg/ml) or with medium alone (unstimulated) in triplicate wells. PBMC were stimulated in triplicate wells and the plates were incubated at 37°C in 5% C0 2 . After 72 hours, cell proliferation was measured using cell counting kit (CCK-8) (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) . Stimulation index of PBMC proliferation was calculated using the formula: Absorbance of stimulated cells/Absorbance of unstimulated cells.
  • rBmHAT Vaccination Does Not Induce Any Adverse Reactions in Macaques.
  • the injection sites were monitored closely for signs of any adverse reactions (redness, swelling, etc.) for 7 days post-immunization. There were no adverse reactions in any of the vaccinated or control animals.
  • Clinical monitoring showed no dramatic loss of body weight (>10% of the original weight) , changes in eating habits or any other behavioral changes.
  • Temperature measurements obtained daily following immunizations did not show any significant variations. Temperature measurements were also performed at regular intervals using implanted transponders. There were no significant variations in the body temperature in vaccinated and control animals.
  • the lymph nodes in the left and right leg of all animals were monitored weekly starting approximately 2 weeks prior to challenge (to establish a baseline) and throughout the challenge period.
  • the lymph nodes were measured with a caliper and observed for edema.
  • the measurements showed an overall increase in the mean size of the inguinal lymph nodes in both legs during the 5-8 week post-challenge period in all groups.
  • the lymph node size in control animals were 22+1 mm and rBmHAT group were 26.211 mm. Following this period, the sizes of the lymph nodes decreased to near pre-challenge levels in all macaques.
  • Macaques were immunized with 200 g of rBmHAT with alum adjuvant.
  • Anti-rBmHAT antibodies against rBmHSPl2.6, rBmALT2, rBmTSP LEL or rBmHAT were evaluated.
  • Each animal differed in the antibody titer against each antigen.
  • Isotype analysis showed that nearly all of the antibodies were of IgGl isotype against all the four antigens tested (rBmHSP, rBmALT2, rBmTSP and rBmHAT) . Levels of IgG2, IgG3, IgA and IgE did not show any significant difference from the background values.
  • rBmHAT Responding Cells Were Present in the PBMC of Immunized Rhesus Macaques.
  • PBMC was collected four weeks after the final vaccination.
  • Cell proliferation was determined after stimulating CFSE labeled PBMC with rBmHAT proteins for 5 days and counting the labeled cells in a flow cytometer.
  • Anti-rBmHAT Antibodies in the Sera of Immunized Macaques can Participate in the Killing of B. malayi L3.
  • an in vitro ADCC assay was performed. Results showed that the PBMC from vaccinated macaque were able to participate in the killing of 35% of B. malayi L3 (Table 16) .
  • maximum killing potential in the ADCC was 45% in the sera of macaque #5258.
  • Sera from macaque #5242 and #5259 also showed significant killing potential with 38% and 35% killing respectively.
  • Sera from macaque #4996 and #5259 had the least ADCC property with 25% and 31% killing respectively. No larval death occurred when sera from control macaques were used in these assays.
  • rBmHAT Responding Cells were Present in the PBMC of Immunized Rhesus Macaques After Challenge .
  • PBMC of three animals #5242 (S.I. - 0.928+0.01), #5258 (S.I. - 1.09110.16) and #5256 (S.I. - 1.018110.13) from the vaccinated group that were negative for Mf showed significant proliferation upon rBmHAT stimulation.
  • two of the vaccinated animals #4996 (S.I. - 0.258+0.12) and #5254 S.I.
  • Eosinophil Numbers were High in Infected Macaques Showing Mf. Microfilaremic individuals show high eosinophil counts in their blood (Pearlman, et al. (1993) Exp. Parasitol. 76:200-8; Pearlman, et al. (1993) J. Immunol. 151:4857-64). A similar finding was observed in rhesus macaques as well. Absolute counts of eosinophils were determined on weeks 13, 9, and 5 prior to challenge, on the day of challenge and on weeks 1, 5, 10, and 14 post- challenge.
  • Results showed that the titer of IgG antibodies was significantly high in the three immunized macaques that did not develop the infection after the challenge. Similarly, PBMC from the same three macaques secreted higher levels of IFN- ⁇ when stimulated with the rBmHAT antigen. PBMC from the two immunized macaques that developed the infection after challenge were unable to secrete similar levels of IFN- ⁇ in response to rBmHAT stimulation. An ELISPOT assay was performed using PBMC from vaccinated and control macaques. Results showed that in all the infected macaques there was a significant increase in the number of antigen- specific IL-10 secreting cells compared to IFN- ⁇ secreting cells.
  • H HSP. A, ALT2. T, TSP. X, TPX. G, GST. cTrivalent vaccine.
  • **Mice and jirds were immunized with 15 g of rBmHAX plus 15 ⁇ g of alum with a total of four immunizations at 2 weeks interval. Blood was collected on day 0, 14, 28, 42, 49 and 70 to monitor the titer of antibodies against each of the component antigens. The following titers were observed on day 49 (ALT-2 1:60,000; HSP 1:40,000, TPX 1:40,000). All the animals were challenged on day 49 with 20 B. malayi L3 for mice and 100 B. malayi L3 for jirds. Worm establishment or worm death in immunized animals was observed at 48 hours after surgical implantation of L3 in mice or 90 days after infection in jirds. Percent protection was calculated as described herein.
  • Example 9 Protection Against Brugia malayi with a tetravalent vaccine
  • a construct encoding HSP, ALT2, TPX2 and TSP as a fusion protein was prepared by synthesizing the complete sequence of the multivalent genes.
  • the construct was packaged in the pUC57 vector and subsequently amplified using ATTTTCGGCCGTAGGC (SEQ ID NO: 87) and AGGTGGCCATTGACATGAT (SEQ ID NO: 88) primers with restriction sites of BamRI and EcoRI .
  • the amplified product was then double digested and ligated into expression vector pRSETA using Quick ligation kit (Thermo Fisher Scientific, Rockford, IL) as per the manufacturer's instructions.
  • the ligation was confirmed by PCR using specific primers for T7 promoter.
  • the ligated products were then transformed into E. coli DH5a competent cells and the transformants were selected on LB plates supplemented with 100 pg/ml ampicillin.
  • the colonies were cultured in 5 ml LB broth supplemented with 100 g/ml ampicillin, at 37 °C for 16 hours on a shaker incubator (200 rpm) .
  • the recombinant plasmids were isolated using plasmid extraction kit (Qiagen) and the sequence of the insert was confirmed. Positive clones were transformed into competent E. coli strain BL21 (DE3) and recombinant BfflHAXT proteins were expressed.
  • Endotoxin was removed using a column purchased from Thermo Fisher Scientific. The expression and purity of final recombinant protein was confirmed in a 12% SDS-PAGE gel. Groups of five mice each were immunized four times with 15 pg of the rBmHAXT and respective adjuvants s/c (for Alum and AL019) and s/c first dose and subsequent three doses given orally for MCA (Mannosylated chitosan) . Two weeks after the last immunization, all animals were challenged with 20 L3 of B. malayi using a micropore chamber method. Seventy-two hours after challenge, live and dead worms were counted to determine the percent larval death. Results presented in Figure 5 show that vaccination with BfflHAXT along with alum or AL019 gave 100% protection.

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Abstract

La présente invention concerne un vaccin multivalent servant à immuniser un animal contre la filariose. Dans certains modes de réalisation, les antigènes du vaccin multivalent sont à base de protéines, d'ADN ou d'une combinaison de ceux-ci. Une méthode et un kit permettant de détecter un nématode filarien et de déterminer l'efficacité d'un vaccin sont en outre décrits.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020176887A1 (fr) * 2019-02-28 2020-09-03 Bayer Animal Health Gmbh Anthelminthiques à base de lactone macrocyclique contre les nématodes
WO2022169835A1 (fr) * 2021-02-03 2022-08-11 The Board Of Trustees Of The University Of Illinois Vaccin et méthodes de prévention de la filariose et de la dirofilariose

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US20060177461A1 (en) * 1988-06-15 2006-08-10 Young Richard A Stress proteins and uses therefor
US20130236490A1 (en) * 2010-11-15 2013-09-12 The Board Of Trustees Of The University Of Illinois Multivalent Vaccine for Filariasis
US20150174237A1 (en) * 2012-07-26 2015-06-25 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Multimeric fusion protein vaccine and immunotherapeutic

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060177461A1 (en) * 1988-06-15 2006-08-10 Young Richard A Stress proteins and uses therefor
US20130236490A1 (en) * 2010-11-15 2013-09-12 The Board Of Trustees Of The University Of Illinois Multivalent Vaccine for Filariasis
US20150174237A1 (en) * 2012-07-26 2015-06-25 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Multimeric fusion protein vaccine and immunotherapeutic

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020176887A1 (fr) * 2019-02-28 2020-09-03 Bayer Animal Health Gmbh Anthelminthiques à base de lactone macrocyclique contre les nématodes
WO2022169835A1 (fr) * 2021-02-03 2022-08-11 The Board Of Trustees Of The University Of Illinois Vaccin et méthodes de prévention de la filariose et de la dirofilariose

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