Schistosoma vaccine antigen modifications and uses thereof

Abstract

Provided herein are compositions, assays, kits, and methods for detecting sera that binds to, and inhibits the activity of, a recombinant calcium-activated neutral protease or calpain of Schistosoma mansoni, a recombinant calcium-activated neutral protease or calpain; such as a detectable substrate for a Schistosoma mansoni calcium-activated neutral protease or calpain; and instructions in the kit for combining sera from a subject suspected of having been exposed to Schistosoma mansoni, or a vaccine thereto, with the calcium-activated neutral protease or calpain, wherein the sera blocks an activity of the calcium-activated neutral protease or calpain against the detectable substrate and is a surrogate for effectiveness of the immunization to Schistosoma mansoni.

Description

SCHISTOSOMA VACCINE ANTIGEN MODIFICATIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 733,169, filed Dec. 12, 2024, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates in general to the field of immunizations against schistosomiasis, and more particularly, to a novel antigenic calpain (Sm-p80) subunit that is more stable and catalytically active.STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under AH79364-01 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] The present application includes a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on , 2025, is named > .xml and is , bytes in size.BACKGROUND

[0005] Without limiting the scope of the disclosure, its background is described in connection with vaccine formulations against Schistosoma mansoni.

[0006] Schistosoma mansoni is a flatworm parasite that inhabits the porto-mesenteric circulation of humans. Considerable morbidity and mortality results from the affliction of an estimated 207 million people worldwide by several species of schistosomes. An additional 779 million people are at risk of acquiring this infection. Disability-adjusted life years for schistosomiasis have been calculated to be somewhere between 1.7 million and 4.5 million years. Schistosomiasis is endemic in 74 developing countries.

[0007] The infective cycle of Schistosoma mansoni involves asexual reproduction within an intermediate snail host, followed by infection of a human host. Cercariae, the larval stage which exits from an intermediate snail host, infect humans by penetrating human skin. These juvenile schistosomes mature to schistosomula, undergo an intricate migration through the host’s lungs and liver, and develop into sexually mature egg-laying adults. Sexually mature male and female schistosomes begin the egg-laying phase of the life cycle within the intestinal venules. The constant production of large numbers of ova results in the excretion of some eggs with fecal matter, and in heavy infection, entrapment of eggs in visceral organs with ensuing host granulomatous immune responses directed against them. It is this egg-induced organ damagewhich results in complications such as hepatic fibrosis, portal hypertension, and esophageal varices, which lead to the death of chronically infected hosts.

[0008] The chronic nature of this debilitating disease results in cumulative damage to the liver, spleen, and colon due to the granulomatous reaction to accumulated embryonated eggs. Infection results in the production of circulating anti-schistosomal antibodies. The immune response is erratic, however, and does not lead to sterile immunity. Additionally, the adult parasites evade immune clearance by complex and multifactorial mechanisms.

[0009] Emphasis has been placed on chemotherapy as the preferred method for the treatment of schistosomiasis. Control programs based on chemotherapy are complicated, however, by the rapidity and frequency of re-infection and the difficulties and expense involved in maintaining these programs over a long term. The continuous drug treatment and re-infection cycle fails to reduce the overall egg output sufficiently to markedly reduce transmission of the disease in endemic areas. Additionally, concerns exist that the parasites may develop drug resistance. A critical need remains for the development of alternate approaches to control the disease.

[0010] No effective vaccine exists for schistosomiasis. Even though anti -parasitic drugs and other control measures, including public hygiene and snail control are available, the advent of an effective vaccine still remains the most potentially powerful means for the control of this disease. Vaccination of individuals at a young age would be the most efficient way of priming the immune system without the accompaniment of egg-induced pathology. A vaccine would also prevent severe infection and thus decrease transmission of eggs and help curb the cycle of S. mansoni infection. Boosting of immunity to schistosomes in vaccinated individuals would occur following subsequent exposure to infective cercariae.

[0011] Several adult S. mansoni proteins have been considered as potential vaccine candidates. Ideally, the most promising vaccine candidates may be those which are surface-exposed and are indispensable for the parasite’s survival within the human host.

[0012] A major problem that has hindered schistosomiasis vaccine research and development concerns the identification and selection of potential protective antigens encoded by the parasite. During the last two decades, many laboratories have attempted to identify schistosomal antigens that induce partially protective immune responses. More than 100 such antigens have been identified, about 25% of which confer protection of varying degrees. None of these candidate antigens, however, have induced levels of an immune response approaching immunity levels of about 80% that have been observed following vaccination with irradiated schistosome larvae. Independent examination of the six “priority antigens” (paramyosin, glutathione S-transferase,fatty acid binding 14 kDa protein, IrV-5, triose phosphate isomerase, and Sm23) via a standard comparative World Health Organization delineated procedure, resulted in none of the antigens providing the stated goal of 40% protection or better. Most schistosome vaccine candidates confer 30-50% protection in the mouse model system.

[0013] Thus, there is a great need to identify novel assays, antigens, adjuvant vehicles, and cocktail vaccine formulations to induce protection that ranges from 70% to 80%, as has been recorded with radiation-attenuated vaccines.SUMMARY

[0014] As embodied and broadly described herein, an aspect of the present disclosure relates to an assay for monitoring effectiveness of an immunization to Schistosoma mansoni comprising: providing a recombinant calcium-activated neutral protease or calpain; contacting a sera from a subject suspected of having Schistosoma mansoni or that was immunized with an immunization or vaccine against Schistosoma mansoni with the recombinant calcium-activated neutral protease or calpain; and adding a detectable substrate for a Schistosoma mansoni calcium -activated neutral protease or calpain; wherein the sera blocks an activity of the calcium-activated neutral protease or calpain against the detectable substrate and is a surrogate for monitoring the effectiveness of the immunization to Schistosoma mansoni. In one aspect, the assay further comprises one or more reagents or structures for performing an assay selected from the group consisting of a radioassay comprising one or more radioisotopes, a lateral flow assay comprising a test strip or dipstick, an ELISA-type assay comprising an enzyme adapted to convert a substrate into a detectable label, a nephelometric assay, a turbidimetric assay, a flow cytometry assay comprising one or more detectable particles, a fluorescent assay comprising one or more fluorescent labels, a chemiluminescent assay comprising one or more chemiluminescent labels, and a bead-type assay comprising detectably-labeled beads. In another aspect, the detectable substrate is a synthetic substrate selected from at least one of: Suc-LLVY-AMC (SEQ ID NO:2), Suc-LY-AMC, Edans- PLFAERK-Dab (SEQ ID NO:3), Edans-ALGIGTIPPK-Dab (SEQ ID NO:4), or Edans- ALGIGTIPPK-Dab (SEQ ID NO:5). In another aspect, the detectable substrate comprises a protein selected from at least one of: one or more cytoskeletal protein(s) (a-fodrin, spectrin, neurofilaments, desmin, talin, vimentin), one or more membrane protein(s) (ion channels, growth factor receptors, adhesion molecules), one or more nuclear proteins (lamin A / B), calpastatin, one or more kinases, or one or more phosphatases. In another aspect, the assay is adapted as a readout of, or a surrogate for, efficacy of a vaccine against Schistosoma mansoni. In another aspect, the recombinant calcium -activated neutral protease or calpain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NOS: 1 or 6.

[0015] As embodied and broadly described herein, an aspect of the present disclosure relates to a kit comprising: a recombinant calcium -activated neutral protease or calpain; a detectable substrate for a Schistosoma mansoni calcium-activated neutral protease or calpain; and instructions in the kit for combining sera from a subject suspected of having been exposed to Schistosoma mansoni, or a vaccine thereto, with the calcium-activated neutral protease or calpain, wherein the sera blocks an activity of the calcium-activated neutral protease or calpain against the detectable substrate and is a surrogate for effectiveness of the immunization to Schistosoma mansoni. In one aspect, the kit further comprises one or more reagents or structures for performing an assay selected from the group consisting of a radioassay comprising one or more radioisotopes, a lateral flow assay comprising a test strip or dipstick, an ELISA-type assay comprising an enzyme adapted to convert a substrate into a detectable label, a nephelometric assay, a turbidimetric assay, a flow cytometry assay comprising one or more detectable particles, a fluorescent assay comprising one or more fluorescent labels, a chemiluminescent assay comprising one or more chemiluminescent labels, and a bead-type assay comprising detectably-labeled beads. In another aspect, the detectable substrate is a synthetic substrate selected from at least one of: Suc-LLVY-AMC (SEQ ID NO:2), Suc-LY-AMC, Edans-PLFAERK-Dab (SEQ ID NO:3), Edans-ALGIGTIPPK-Dab (SEQ ID NO:4), or Edans-ALGIGTIPPK-Dab (SEQ ID NO:5). In another aspect, the detectable substrate comprises a protein selected from at least one of: one or more cytoskeletal protein(s) (a-fodrin, spectrin, neurofilaments, desmin, talin, vimentin), one or more membrane protein(s) (ion channels, growth factor receptors, adhesion molecules), one or more nuclear proteins (lamin A / B), calpastatin, one or more kinases, or one or more phosphatases.

[0016] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for preventing schistosomiasis comprising the steps of: administering a vaccine comprising an antigenic protein having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or 6, wherein a dosage amount is an amount sufficient to provide worm reduction in the host, antifecundity effect, or protection against acute schistosomiasis. In one aspect, at least one of: the antigenic protein is defined further as a fusion protein comprising a peptide tag for at least one of identification, isolation, or purification; or the peptide tag is selected from a His-tag, a myc-tag, an S-peptide tag, a MBP tag, a GST tag, a FLAG tag, a thioredoxin tag, a GFP tag, a BCCP, a calmodulin tag, a streptavidin tag, an HSV-epitope tag, a V5-epitope tag and a CBP tag. In another aspect, the vaccine is administered with a primary immunization at week 0, a first boost at week 4, and a second boost at week 8; a primary immunization at week 0, a first boost at week 4 comprising the antigenic protein in the presence of an adjuvant, and a second boost at week 8 also comprising the antigenic protein in the presenceof the adjuvant; or both; and optionally comprises an adjuvant. In another aspect, the antigenic protein is enzymatically active, is soluble when expressed in a bacteria, or both.

[0017] As embodied and broadly described herein, an aspect of the present disclosure relates to a vaccine comprising a recombinant antigenic protein having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or 6, wherein a dosage amount is an amount sufficient to provide worm reduction in the host, antifecundity effect, or protection against acute schistosomiasis, wherein the antigenic protein is enzymatically active, is soluble when expressed in a bacteria, or both, and optionally comprises an adjuvant, and optionally the adjuvant is Glucopyranosyl Lipid Adjuvant (GLA) or GLA squalene emulsion (GLA-SE).

[0018] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for preventing schistosomiasis, said method comprising the steps of: administering a vaccine comprising a cDNA expressing an antigenic protein having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or 6; or cloned into an expression vector, wherein the dosage amount is an amount sufficient to provide worm reduction in the host, antifecundity effect, or protection against acute schistosomiasis. In one aspect, the vaccine is administered with a primary immunization at week 0, a first boost at week 4, and a second boost at week 8. In another aspect, the vaccine is administered with a primary immunization at week 0, a first boost at week 4 comprising the antigenic protein in the presence of an adjuvant, and a second boost at week 8 also comprising the antigenic protein in the presence of the adjuvant. In another aspect, the method further comprising adding an adjuvant and optionally the adjuvant is Glucopyranosyl Lipid Adjuvant (GLA) or GLA squalene emulsion (GLA-SE). In another aspect, the protein further comprises a peptide tag for identification, isolation, or both, of the presence of the subunit.

[0019] As embodied and broadly described herein, an aspect of the present disclosure relates to a polynucleotide encoding a subunit of S. mansoni calpain (Sm-p80) cloned into a vector having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO:6. In one aspect, the polynucleotide further comprises a peptide tag for identification, isolation, or both, of the presence of the subunit. In another aspect, the vector is in a host cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] For a more complete understanding of the features and advantages of the present disclosure, reference is now made to the detailed description of the disclosure along with the accompanying figures and in which:

[0021] FIGS. 1 A to IF show the kinetics and inhibition of B7, , which is a soluble deletion variant of the Sm-p80 antigen. Inhibition of B7-mediated activity was tested using pools of sera obtained from murine and baboon models as well as from human volunteers vaccinated with the schistosomiasis vaccine, SchistoShield® (Sm-p80 + GLA-SE). Enzymatic reactions were performed in a final volume of 100 pL [20mM Tris (pH 7.1), 50mMNaCl, 0.12 pg / pL B7 enzyme, 10 mM CaCE, 0.1 mM Suc-LLVY-AMC (SEQ ID NO:2)]. Enzyme-like kinetic behavior of the B7 protein was observed in a concentration and time-dependent manner that was inhibited by leupeptin, shown in logio scale (FIG. 1 A). Important to note is the inhibition of B7 activity in the presence of human sera obtained from individuals vaccinated with SchistoShield® in first in human trial in USA (FIG. IB) and in Africa (FIG. 1C). High IgG titer pools of mouse antisera to Sm-p80 also inhibited the activity (FIG. ID). The B7-dependent activity was significantly inhibited by baboon anti-Sm-p80 sera (FIG. IE). Purified IgG from high titer anti-Sm-p80 sera obtained from vaccinated baboons exhibited inhibition in a dose dependent manner, lower concentrations of IgG provided higher inhibition (FIG. IF). NS / NHS refers to normal sera and VHS / VS refers to sera from the vaccinated group.

[0022] FIG. 2 is a graph that shows the kinetics of B7 enzymatic activity. Shown are the B7 enzyme activity (in relative fluorescence units) in presence or absence of leupeptin a well known protease inhibitor (200 pM).

[0023] FIG. 3A to 3E show the biomarker / endpoint readout: calpain (B7)-inhibition in humans. FIGS. 3 A and 3B are artists renditions of the binding site on the B7 catalytic domain interacting with the fluorogenic substrate Suc-LLVY-AMC (SEQ ID NO:2), in FIG. 3C the substrate is cleaved by the enzyme and the product can be detected by using a fluorescent readout. In 3D the presence of antibodies from subjects vaccinated with the schistosomiasis vaccine SCHISTOSHIELD® sera inhibit the fluorescent assay readout leading to less signal. Inhibition of B7-mediated activity was tested using pools of sera obtained from murine and baboon models as well as from human volunteers vaccinated with the schistosomiasis vaccine SCHISTOSHIELD® demonstrating that the B7-based assay is a useful readout to monitor the vaccine performance in human clinical trials in Africa. FIGS. 3C to 3E show the B7 inhibition by antibodies generated in humans following vaccination with Sm-p80-based (NIH trial, Seattle, WA, USA) for a NIH trial high dose-unadjuvanted (FIG. 3C), NIH trial mid dose-unadjuvanted (FIG. 3D), and Madagascar trial low dose-unadjuvanted (FIG. 3E).

[0024] FIGS. 4A and 4B show the vaccine efficacy of B7 in murine model using the B7 RNA and a SARS-CoV2 RNA as a control, for total worm and female worm reduction (FIG. 4A) and reduction in eggs (FIG. 4B).DETAILED DESCRIPTION

[0025] While the making and using of various aspects of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific aspects discussed herein are merely illustrative of specific ways to make and use the disclosure and do not delimit the scope of the disclosure.

[0026] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific aspects of the disclosure, but their usage does not delimit the disclosure, except as outlined in the claims.

[0027] A next-generation Sm-p80 schistosome vaccine represents a novel approach to combating schistosomiasis, a parasitic disease caused by schistosome flatworms. The core of this invention are modifications to the sequence of the Sm-p80 antigen, which has been shown to be effective in stimulating immune responses that confer protection against schistosomiasis. Building on the success of the earlier version, this vaccine utilizes key modifications to enhance immunogenicity, solubility, activity and expression of the antigen. The modifications to Smp-80 include introducing structural stability enhancements. By altering specific amino acid sequences, the new Sm-p80 is designed to overcome expression-, solubility- as well as aggregation-issues. Additionally, this invention incorporates embodiments based on RNA delivery technology, an approach that has been validated as safe and highly effective for other infectious diseases. The mRNA-based embodiment of the Sm-p80 vaccine encodes the modified Sm-p80 antigen and is formulated within a nanoparticle delivery system. This delivery mechanism ensures that the RNA is efficiently transported into host cells, allowing for in situ protein expression that mimics natural infection and elicits a strong cellular and humoral immune responses. The RNA version of the vaccine is engineered for rapid synthesis, scalability, and adaptability, allowing for modifications to the antigen if necessary. Finally, the new constructs are catalytically active providing a basis for functional studies that could demonstrate inhibition of catalytic activity and its impact on protective efficacy.

[0028] As used herein, the terms an “immunogenic composition” and “vaccine” refer to a composition that comprises an antigenic calpain (Sm-p80) subunit that is more stable and catalytically active, or a nucleic acid that expresses the antigenic calpain (Sm-p80) subunit, where administration of the immunogenic composition or vaccine to a subject results in the developmentin the subj ect of a humoral and / or a cellular immune response to the antigenic molecule of interest, and by extension, to the virus.

[0029] As used herein, the term “substantially purified” refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample a substantially purified component comprises 70%, 75%, 80%-85%, 90-95%, or 95%, 96%, 97%, 98%, 99%, or 100% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0030] As used herein, the term a “coding sequence” or a sequence which “encodes” a antigenic calpain (Sm-p80) subunit, refers to a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences (or “control elements”) and in vitro or in vivo. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3’ to the coding sequence.

[0031] As used herein, the term “control elements”, includes, but is not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3’ to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences, and / or sequence elements controlling an open chromatin structure.

[0032] As used herein, the term “nucleic acid” includes, but is not limited to, DNA or RNA that encodes the antigenic calpain (Sm-p80) subunit of the present disclosure, whether expressed or optimized for prokaryotic or eukaryotic expression. The term also captures sequences that include any of the known base analogs of DNA and RNA.

[0033] As used herein, the term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when active. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0034] As used herein, the term “recombinant” refers to a polynucleotide that encodes antigenic calpain (Sm-p80) subunit whether from the viral genome, cDNA, semi synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and / or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting prokaryotic microorganisms or eukaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.

[0035] Techniques for determining amino acid sequence “similarity” are well known in the art. In general, “similarity” means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and / or physical properties such as charge or hydrophobicity. A so-termed “percent similarity” then can be determined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby and comparing this to a second amino acid sequence. In general, “percent identity” refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. For use with the present invention, the antigenic calpain (Sm-p80) subunit can have 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 at the nucleic acid or amino acid level.

[0036] As used herein the phrase “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0037] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A “comparison window” includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full-length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995)).

[0038] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always< 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0039] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0040] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0041] The term “antigenic calpain (Sm-p80) subunit” as provided herein includes any of the recombinant or naturally-occurring forms of antigenic calpain (Sm-p80) subunit (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared calpain (Sm-p80) full-length protein. In aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring antigenic calpain (Sm-p80) subunit. In embodiments, antigenic calpain (Sm-p80) subunit is theprotein as identified by the UniProt reference number P27730, or a variant, homolog or functional fragment thereof.

[0042] As used herein, the term a “vector” refers to a nucleic acid capable of transferring gene sequences to target cells (e.g., bacterial plasmid vectors, viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of one or more sequences of interest in a host cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors. The term is used interchangeable with the terms “nucleic acid expression vector” and “expression cassette.”

[0043] Many suitable expression systems are commercially available, including, for example, the following: baculovirus expression (Reilly, P. R., et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992); Beames, et al., Biotechniques 11 :378 (1991); Pharmingen; Clontech, Palo Alto, Calif.)), vaccinia expression systems (Earl, P. L., et al., “Expression of proteins in mammalian cells using vaccinia” In Current Protocols in Molecular Biology (F. M. Ausubel, et al. Eds.), Greene Publishing Associates & Wiley Interscience, New York (1991); Moss, B., et al., U.S. Pat. No. 5,135,855, issued Aug. 4, 1992), expression in bacteria (Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media Pa.; Clontech), expression in yeast (Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued, Mar. 17, 1998, herein incorporated by reference; Shuster, J. R., U.S. Pat. No. 5,629,203, issued May 13, 1997, herein incorporated by reference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(l-2):79-93 (1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D. V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink, Methods in Enzymology 194 (1991)), expression in mammalian cells (Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary (CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11 :687-706 (1983); 1983, Lau, Y. F., et al., Mol. Cell. Biol. 4: 1469-1475 (1984); Kaufman, R. J., “Selection and coamplification of heterologous genes in mammalian cells,” in Methods in Enzymology, vol. 185, pp 537-566. Academic Press, Inc., San Diego Calif. (1991)), and expression in plant cells (plant cloning vectors, Clontech Laboratories, Inc., Palo- Alto, Calif., and Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.; Hood, E., et al., J. Bacteriol. 168: 1291- 1301 (1986); Nagel, R., et al., FEMS Microbiol. Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in Plant Molecular Biology Manual A3: 1-19 (1988); Miki, B. L. A., et al., pp. 249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al., eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology: Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley, 1997; Miglani, Gurbachan Dictionary of Plant Genetics and Molecular Biology, New York, Food Products Press, 1998; Henry, R. J., Practical Applications of Plant Molecular Biology,New York, Chapman & Hall, 1997), relevant portions of any of the above are incorporated herein by reference.

[0044] In some embodiments, for easier purification of the recombinant protein the single-chain polypeptide may be a fusion protein that further includes any purification tag sequences known in the prior art. Examples of polypeptides that aid purification include, but are not limited to, a His- tag, a myc-tag, an S-peptide tag, a MBP tag, a GST tag, a FLAG tag, a thioredoxin tag, a GFP tag, a BCCP, a calmodulin tag, a streptavidin tag, an HSV-epitope tag, a V5-epitope tag and a CBP tag.

[0045] As used herein, the term “subject” refers to any member of the subphylum chordata, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The system described above is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.

[0046] As used herein, the terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any unacceptable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0047] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequentialadministration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0048] As used herein, the term “co-administer” refers to a compound or composition described herein that is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. The preparations may also be combined with inhaled mucolytics (e.g., rhDNase, as known in the art) or with inhaled bronchodilators (short- or long-acting beta agonists, short- or long-acting anticholinergics), inhaled corticosteroids, or inhaled antibiotics to improve the efficacy of these drugs by providing additive or synergistic effects. The compositions of the present invention can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, nanoparticles, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).In another embodiment, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989).

[0049] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0050] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0051] The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0052] The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0053] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0054] The term “vaccine” refers to a composition that can provide active acquired immunity to and / or therapeutic effect (e.g., treatment) of a particular disease or a pathogen. A vaccine typically contains one or more agents that can induce an immune response in a subject against a pathogen or disease, i.e., a target pathogen or disease. The immunogenic agent stimulates the body’s immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any of the pathogen on subsequent exposure. Vaccines can be prophylactic (e.g., preventing or ameliorating the effects of a future infection by any natural or pathogen, or of an anticipated occurrence of cancer in a predisposed subject) or therapeutic (e.g., treating cancer or infection in a subject who has been diagnosed with the cancer or infection). The administration of vaccines is referred to vaccination. In embodiments, a vaccine composition can provide nucleic acid, e.g., mRNA that encodes antigenic molecules (e.g., peptides) to a subject. The nucleic acid that is delivered via the vaccine composition in the subject can be expressed into antigenic molecules and allow the subject to acquire immunity against the antigenic molecules. In the context of the vaccination against infectious disease, the vaccine composition can provide mRNA encoding antigenic molecules that are associated with a certain pathogen, e.g., one or more peptides that are known to be expressed in the pathogen (e.g., pathogenic bacterium or virus).

[0055] Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose (TM), agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).

[0056] As used herein, the term “adjuvant” refers to a compound that when administered in conjunction with the compositions provided herein including embodiments thereof, augments the composition’s immune response. Generally, adjuvants are non-toxic, have high purity, are degradable, and are stable.

[0057] Adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. The adjuvant increases the titer of induced antibodies and / or the binding affinity of induced antibodies relative to the situation if the immunogen were used alone. A variety of adjuvants can be used in combination with the agents provided herein including embodiments thereof, to elicit an immune response. Preferred adjuvants augment the intrinsic response to an immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Preferred adjuvants include Glucopyranosyl Lipid Adjuvant (GLA) or GLA squalene emulsion (GLA-SE), aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of GSK Bio). Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Patent No. 5,057,540), (Aquila BioPharmaceuticals, Framingham, MA). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute etaL, N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killed mycobacteria. Another adjuvant is CpG (WO 98 / 40100). Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent.

[0058] Other adjuvants contemplated for the invention are saponin adjuvants, such as Stimulon™ (QS-21, Aquila, Framingham, MA) or particles generated therefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvants include RC-529, GM-CSF and Complete Freund’s Adjuvant (CFA) and Incomplete Freund’s Adjuvant (IF A). Other adjuvants include cytokines, such as interleukins (e.g., IL-1 a and 0 peptides, IL-2, IL-4, IL-6, IL- 12, IL-13, and IL-15), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), chemokines, such as MIPla and 0 and RANTES. Another class of adjuvants is glycolipid analogues including N- glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants (see US Pat. No. 4,855,283). Heat shock proteins, e.g., HSP70 and HSP90, may also be used as adjuvants.

[0059] As used herein, the term “detectable substrate” refers to a substrate of a calcium-activated neutral protease or calpain from Schistosoma mansoni. The substrates can be made detectable directly, e.g., by adding a label, enzyme, or other detectable agent) or indirectly, by having a secondary agent with a detectable label, enzyme, or other detectable agent, such an an antibody. Non-limiting examples of substrates for the calcium-activated neutral protease or calpain fromSchistosoma mansoni for use in the assay for detecting the presence of antibodies against calpain, can include one or more proteins selected: cytoskeletal protein(s) (e.g., a-fodrin, spectrin, neurofilaments, desmin, talin), membrane protein(s) (e.g., ion channels, growth factor receptors, adhesion molecules), nuclear protein(s) (lamin A / B), kinases, or phosphatases. A synthetic fluorescent substrate (Suc-LLVY-AMC) (SEQ ID NO:2) in the presence of Ca2+. Other examples of synthetic substrates includes at least one of: Suc-LY-AMC, Edans-PLFAERK-Dab (SEQ ID NO:3), Edans-ALGIGTIPPK-Dab (SEQ ID N0:4), or Edans-ALGIGTIPPK-Dab (SEQ IDN0:5). Sera from a patient that has been exposed to Schistosoma mansoni or a vaccine against the calcium-activated neutral protease or calpain of subunits thereof, such as Smp80 or the B7 protein, is introduced in the assay and binds to the Smp80 or the B7 protein and blocks the enzymatice activity of the calcium-activated neutral protease or calpain (such as Smp80 or the B7 protein) on the substrate. A reduction in the activity of the calcium-activated neutral protease or calpain of subunits thereof, such as Smp80 or the B7 protein serves as a surrogate marker for the effectiveness of the immunization against Schistosoma mansoni.

[0060] As used herein, the term “treatment” refers to any of (i) the prevention of infection or reinfection with Schistosoma mansoni, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question. Treatment can be effected prophylactically (prior to infection) or therapeutically (following infection).

[0061] As used herein, the term “effective dose” refers to that amount of the antigenic calpain (Sm-p80) subunit of the disclosure sufficient to induce immunity, to prevent and / or ameliorate an infection or to reduce at least one symptom of an infection and / or to enhance the efficacy of another dose of antigenic calpain (Sm-p80) subunit. An effective dose refers to the amount of antigenic calpain (Sm-p80) subunit sufficient to delay or minimize the onset of an infection. An effective dose also refers to the amount of antigenic calpain (Sm-p80) subunit that provides a therapeutic benefit in the treatment or management of an infection. Further, an effective dose is the amount with respect to antigenic calpain (Sm-p80) subunit of the disclosure alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an infection. An effective dose is any amount sufficient to enhance a subject’s (e.g., a human’s) own immune response against a subsequent exposure to an infectious agent. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and / or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an “effective dose” is one that prevents disease and / or reduces the severity of symptoms.

[0062] As used herein, the term “immune stimulator” refers to a compound that enhances an immune response via the body’s own chemical messengers (cytokines). These molecules comprise various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interferons, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immune stimulator molecules can be administered in the same formulation as the antigenic calpain (Sm-p80) subunit of the disclosure or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.

[0063] As used herein, the term “protective immune response” or “protective response” refers to an immune response mediated by antibodies against an infectious agent, which is exhibited by a vertebrate (e.g., a human), which prevents or ameliorates an infection or reduces at least one symptom thereof. The antigenic calpain (Sm-p80) subunit of the disclosure can stimulate the production of antibodies that, for example, neutralize infectious agents, blocks infectious agents from entering cells, blocks replication of said infectious agents, and / or protect host cells from infection and destruction. The term can also refer to an immune response that is mediated by T- lymphocytes and / or other white blood cells against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates flavivirus infection or reduces at least one symptom thereof.

[0064] As used herein, the term “antigenic formulation” or “antigenic composition” refers to a preparation which, when administered to a vertebrate, e.g., a mammal, will induce an immune response.

[0065] As used herein, the terms “immunization” or “vaccine” are used interchangeably to refer to a formulation which contains antigenic calpain (Sm-p80) subunit of the present disclosure, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and / or ameliorate an infection and / or to reduce at least one symptom of an infection and / or to enhance the efficacy of another dose of the antigenic calpain (Sm-p80) subunit. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present disclosure is suspended or dissolved. In this form, the composition of the present disclosure can be used conveniently to prevent, ameliorate, or otherwise treat an infection. Upon introduction into a host, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and / or cytokines and / or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and / or other cellular responses.

[0066] The practice of the present disclosure employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag); Fundamental Virology, Second Edition (Fields & Knipe eds., 1991, Raven Press, New York), relevant portion incorporated herein by reference.

[0067] Expression of full-length Sm-p80 in E. coli yields inclusion bodies and aggregates that are not catalytically active. Also, the full-length protein appears difficult to express off of mRNA and repRNA vaccine constructs. By modifying the protein, the inventors solved these problems and can express a functionally active, well-folded antigen. The new antigen is better behaved, functionally active, and can express from RNA since it folds efficiently.

[0068] Sm-p80 B7 sequenceMAHHHHHHMNVASKQYETLVKRLKTERTLWEDPDFPANDKAIGNLPDFRERIEWKRP HEINPNAKFFAGGASRFDIEQGALGDCWLLAVVASISGYPQLFDQVVPKDQELKGPEYV GVVRFRFWRFGHWVEVLIDDRLPVRQGRNTLVFMHSNDPTEFWSALLEKAYAKLNGC YAHLSGGSQSEAMEDLTGGICLSLELNQKERPSDLIDQLKIYAQRCCLMGCSIDSSVME QKMDNGLIGSHAYSLTGVYPVNYRGRTQWLMRLRNPWGDSHEWKGAWCDGSPQWR EISEQEKKNINLSFTADGEFWMSYEDFVTCFSRVEVCHLGLESLEYNQNFHGKRRLDEAI FSGQWQRNVNAGGCINNRTTYWTSPQFRITVED (SEQ ID NO:1)

[0069] Sm-p80 B7 M9A sequence

[0070] MAHHHHHHANVASKQYETLVKRLKTERTLWEDPDFPANDKAIGNLPDFRERIE WKRPHEINPNAKFFAGGASRFDIEQGALGDCWLLAVVASISGYPQLFDQVVPKDQELK GPEYVGVVRFRFWRFGHWVEVLIDDRLPVRQGRNTLVFMHSNDPTEFWS ALLEK AYA KLNGCYAHLSGGSQSEAMEDLTGGICLSLELNQKERPSDLIDQLKIYAQRCCLMGCSIDS SVMEQKMDNGLIGSHAYSLTGVYPVNYRGRTQWLMRLRNPWGDSHEWKGAWCDGS PQWREISEQEKKNINLSFTADGEFWMSYEDFVTCFSRVEVCHLGLESLEYNQNFHGKRR LDEAIFSGQWQRNVNAGGCINNRTTYWTSPQFRITVED (SEQ ID NO:6)

[0071] In Sm-p80 B7 M9A a methionine was deleted at the N-terminus just after the His tag and replaced with Alanine. There is significant activity in the non-His tag binding fraction. By way of explanation but not a limitation of the present invention, it is hypothesized that this methionine (M9) is close enough to the start methionine that a significant amount of transcription was starting here rather that before the His tag.

[0072] Disclosed herein is a calpain inhibition assay. In one example, the assay is performed by mixing fluorescent substrate and a calcium-ion containing solution of the B7 protein. The substrate - when intact - is designed to quench fluorescent signal; when cleaved by a calpain the fluorophore is released eliminating the quenching and resulting in a fluorescent signal that can be detected by a fluorometer. When an inhibitor is added (e.g., sera from an immunized subject that contains antibodies to calpain and that block its enzymatic activity), the release of the fluorescent signal is slowed due to the suppression of calpain activity. It is shown herein that when subjects are vaccinated with the SchistoShield® vaccine, the antibodies induced in a variety of species - including humans -inhibit calpain activity. As calpain is a key protein for parasite survival, immunizations that elicit antibodies that inhibit calpain activity can be used as a surrogate marker for vaccine effectiveness. The skilled artisan will understand that the assay can be modified to increase a signal, such as a fluorescent signal, or can use secondary methods for detection, such as the use of secondary antibodies that comprise a detectable label, enzyme, or other chromophore or chromogenic agent, and the like.

[0073] The present inventors found that enzymatic inhibition of the soluble deletion variant of Sm-p80 antigen - termed B7 (SEQ ID NO: 1) - by antibodies from mice, baboons, and human sources is summarized in FIGS. 1 A to IF. The reaction was carried out using the B7 protein and a synthetic fluorescent substrate (Suc-LLVY-AMC) (SEQ ID NO:2) in the presence of Ca2+. Other examples of synthetic substrates includes at least one of: Suc-LY-AMC, Edans-PLFAERK-Dab (SEQ ID NO:3), Edans-ALGIGTIPPK-Dab (SEQ ID NO:4), or Edans-ALGIGTIPPK-Dab (SEQ ID NO:5). The enzyme reaction followed Michaelis-Menten-like kinetic behavior, in a time dependent manner, reaching a Vmax and plateauing at 3 hours (FIG. 1 A). This B7-mediated activity was inhibited by the positive control protease inhibitor leupeptin with 85% inhibition in 3 hours (FIG. 1 A). The data obtained in FIG. 1 A is shown on a logio scale. The kinetic data used to plot the curve have been provided in FIG. 2. The B7-mediated activity was significantly inhibited by the sera obtained from rodent and non-human primate models and from the human volunteers vaccinated with SCHISTOSHIELD® (PAI life Sciences, Seattle, WA, USA). Using SCHISTOSHIELD® pre-clinical trial mouse and baboon sera, an 18% inhibition of the B7- activity was recorded for mice (FIG. ID) and 55% reduction for the baboon (FIG. IE). In addition,purified IgG from baboons that were vaccinated with SCHISTOSHIELD® in pre-clinical trials, produced up to a 54% enzyme activity inhibition in a dose dependent manner, lower concentrations of purified IgG provided the most significant inhibition (FIG. IF). Sera from the human volunteers vaccinated with SchistoShield® for both the US population and for the African population (Madagascar) exhibited an inhibition of 63% (FIGS. 1B-1C). In all cases, higher dilutions of serum provided elevated inhibition, therefore titration of serum / antibodies for each sample was necessary. Cumulatively, the data presented in FIGS. 1A to IF, highlights the effectiveness of the antigen in the B7-based assay, which is a direct readout to monitor the efficacy of the schistosomiasis vaccine in human clinical trials in Africa.

[0074] FIG. 2 is a graph that shows the kinetics of B7 enzymatic activity. Shown are the B7 enzyme activity (in relative fluorescence units) in presence or absence of leupeptin (200 pM). The relative fluorescence unit (RFU) values were collected over a period of 3 hours, starting from the initial time point, TO. The B7 and B7+ Leupeptin curves are represented on a linear scale. Data represents average normalized RFUs. Values were normalized by subtracting the RFUs obtained from the substrate (Suc-LLVY-AMC) (SEQ ID NO:2) in reaction buffer without B7. Data was obtained from 9 independent experiments; each experiment performed in triplicate wells (n=27).

[0075] Sm-p80 B7 Cloning and Production.

[0076] A soluble deletion variant, termed B7, [44 kDa monomer; amino acid residues 79 to 450 of P27730 (UniProt)] has been generated from the full-length Sm-p80 antigen in which the hydrophobic regions and portions not containing the catalytic triad required for enzymatic activity have been removed. The B7 protein was tested for its enzymatic activity using the enzymatic assay described below.

[0077] The coding sequence for Sm-p80 B7 was obtained from the Seattle Structural Genomics Center for Infectious Disease (SSGCID, Seattle, WA, USA). Constructs were designed by comparing secondary structure predictions calculated by XTALPRED23with multiple sequence alignments (MSA) of the Sm-p80 peptide sequence. MSA were generated by BLASTP search of the Protein Data Bank24,25N and C-terminal boundaries were determined based on locations of the Sm-p80 sequence that had poor sequence conservation and no secondary structure predicted. Structure-based alignments included proteins with at least 35% sequence identity and >300 length matching residues. Gene fragments covering the coding sequence were produced (IDT, Coralville, IA, USA) and cloned into the pET29a expression vector using an E. coli Turbo competent bacterium (New England Biolabs, Ipswich, MA, USA). Plasmids obtained from the successfully transformed bacteria were verified via Sanger sequencing. pET29a containing Sm-p80 B7 wasthen transformed into an E. coli production strain, HMS174(DE3) (Millipore Sigma, Burlington, MA, USA).

[0078] Sm-p80 B7 protein was produced by induction of the recombinant HMS174(DE3) bacterial culture at an ODeoo of 0.5 with ImM isopropyl P-D-l -thiogalactopyranoside (IPTG) followed by further incubation at 25°C for 24 hours. The culture was then spun down at 14,000 x g, and the resulting pellet was resuspended for lysis in B-PER™ (Thermo-Fisher Scientific, Waltham, MA, USA) at a ratio of 10 mL per 1 g of cell pellet. The solution was then freeze- thawed 3 times to facilitate bacterial cell lysis, followed by centrifugation at 14, 000xg to isolate the protein-containing inclusion bodies (IB) pellet. The pellet was then resuspended in 8M Urea / 20mM Tris at 37°C until solubilization of the IBs occurred. A nickel-nitrilotriacetic acid (Ni- NTA) resin was then added to the supernatant to bind the His-tagged Sm-p80 B7 protein, and the mixture was left rotating overnight at 4°C. The resin-lysate was loaded into a gravity-flow column and the protein was then eluted off the column after a series of washes with gradually increasing concentrations of imidazole in 8M Urea / 20mM Tris. Purified B7 was refolded by dialysis three times each into 10 volumes of 20 mM Tris pH7.4. Following the third dialysis, the protein was recovered, tested in the calpain assay for enzymatic activity, and found to be active. Fractions were run on an SDS-PAGE gel to identify those containing Sm-p80 B7, and those fractions were pooled, concentrated and then buffer exchanged into 25mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol. The final product was analyzed by reducing SDS-PAGE gel and protein concentrations were calculated by comparing lane intensity to a standard load of bovine serum albumin (BSA) using ImageJ software (NIH, Bethesda, MD, USA). The B7 protein produced exhibited > 95% purity and < 500 EU / mg endotoxin levels. Endotoxin was measured using the FDA approved EndoSafe PTS reader from Charles River Laboratory (Wilmington, MA, USA).

[0079] Activity assay of the Sm-p80 protease core (clone B7).

[0080] Following screening of a number of synthetic (e.g., Suc-LLVY-AMC (SEQ ID NO:2), Suc-LY-AMC, Edans-PLFAERK-Dab (SEQ ID NO:3), Edans-ALGIGTIPPK-Dab (SEQ ID NO:4), Edans-ALGIGTIPPK-Dab (SEQ ID NO:5)) and protein substrates (e.g., casein, gelatin and fibronectin), a suitable fluorescent substrate (Suc-LLVY-AMC) (SEQ ID NO:2) for B7 was identified. Concentrations of 0.01 pg / pl, 0.02 pg / pl, 0.06 pg / pl and 0.12 pg / pl ofB7 were tested in the reaction mixture (20 mM Tris pH 7.1, lOmM CaCE). The 0.12 pg / pl concentration provided the most consistent reactivity. The Ca2+requirement for the assay reaction was titrated from 0.1 to 100 mM (mixtures at Ca2+>20 mM, precipitated) and 5 to 10 mM Ca2+was found to be the optimal for the reaction. B7 showed continued reaction progress up to 3 hours in the presence of 5 mM Ca2+and 0.1 mM Suc-LLVY-AMC (SEQ ID NO:2) and was thermostable at 37°C for the sameduration of time. B7 prefers neutral pH conditions, activity at pH 7.0-7.5 with Ca2+5-10 mM is defined as 100%. A number of inhibitors in different concentrations in the B7-based reaction were tested including EDTA, E64-c, Cast-dl, Ac-Cast, calpeptin, leupeptin, AEBSF and Bortezomib. Leupeptin, an established cysteine protease inhibitor was identified as the most consistent inhibitor of the reaction. Based on the above-mentioned parameter optimization steps, a consistent and optimally performing assay procedure was developed.

[0081] Optimized B7-based assay.

[0082] Enzymatic reactions contained the following: Reaction Buffer (20mM Tris, 50mM NaCl, pH 7.1); 12 pg B7 enzyme, 10 mM CaCl2, 0.1 mM Suc-LLVY-AMC (SEQ ID NO:2)(AnaSpec, Fremont, CA or Millipore-Sigma, Burlington, MA) in a final volume of 100 pL. Leupeptin 200 pM was used as a positive inhibitor control. Reaction mixture was set in a 96 well clear bottom plate. Fluorescence measurements were taken using a SpectraMax® iD3 multimode plate reader (Molecular Devices, San Jose, CA). Fluorescence was measured for 3 hours at 2-minute intervals using the following parameters: Wavelengths for extinction and emission, 345 and 445 nm, respectively, with integration times of 140 seconds, PMT setting low, shaking enabled, constant temperature at 37°C.

[0083] Inhibition of B7-based assay activity with rodent, nonhuman primate, and human sera vaccinated with Sm-p80 + GLA-SE vaccine.

[0084] To study antibody-mediated inhibition of B7 activity, a battery of sera from different studies were used: murine13and baboon sera6from animals vaccinated with Sm-p80 + GLA-SE in dilutions ranging from 1 : 1,000, 1 : 10,000 and 1 : 100,000. In addition, IgG purified from sera of baboons vaccinated with Sm-p80 + GLA-SE6was used at varying concentrations (0.032 to 1.6 ng / pL). Similarly, human serum samples from participants vaccinated with Sm-p80 with and without GLA-SE in the non-endemic population of the USA (ClinicalTrials.gov Identifier: NCT05292391) and schisto-endemic population of Madagascar, Africa (Ethical Approval, Ministere De La Sante Publique, CERBM: IORG00001212 No 122MSANP / SG / AMM / CERBM) were included to determine the inhibition of B7-mediated protease activity.

[0085] Data analysis on protease activity inhibition.

[0086] To quantify B7 enzyme inhibition, the assay from above was performed in the presence of a variety of serum samples. A minimum of three technical replicates were included for controls and experimental samples across all of the dilutions used. Raw data were collected and plotted as relative fluorescence units (RFUs) versus 3-hours reaction time (Y-axis = fluorescence; X-axis = time). The slope value of the plot indicated the B7 activity in RFU / min. Data are presented asmean ± SE. Significance of fluorescence intensity data differences was evaluated using Student’s / -test with GraphPad Prism™ Software version 10. P- values < 0.05 were considered statistically significant.

[0087] To normalize the RFU, the background RFU of each sample was obtained as the average RFU value of the wells containing buffer plus substrate but not B7 and subtracted from the RFU value of corresponding samples. The normalized relative RFU activity in each sample was obtained by dividing these with the B7 RFU values, and percent activity was then calculated. The relative inhibition in samples was calculated by subtracting the normalized activity from 100. A percent enzyme inhibition activity curve relative to antibody sera titer dilutions or IgG concentrations was plotted. Sm-p80-specific antibody titers were directly related to inhibition of B7-based activity; high titers and lower dilutions exhibited higher inhibition of the B7-based activity.

[0088] The B7 inhibition assay was successfully used to determine schistosomiasis vaccine- mediated effect in schistosome-naive population. The assay is robust in differentiating the inhibition in a dose-dependent manner. The B7 inhibition assay reiterates the importance of this readout as a powerful surrogate marker of protection, demonstrating the vaccine’s ability to neutralize the parasite at a molecular level. In a murine model of schistosomiasis, B7 as an antigen, in an RNA-based platform, provided significant efficacy with regards to worm reduction and egg- induced pathology.

[0089] B7 inhibition by antibodies generated in humans following vaccination with Sm-p80- based (NIH trial, Seattle, WA, USA) and kinetics and inhibition of soluble deletion variant of the Sm-p80 antigen (B7). FIG. 3A to 3E show the biomarker / endpoint readout. FIGS. 3A and 3B show the binding site on the B7 catalytic domain for the fluorogenic substrate Suc-LLVY-AMC (SEQ ID NO:2), and the presence of the schistosomiasis vaccine SCHISTO SHIELD® sera on the assay readout. Inhibition of B7-mediated activity was tested using pools of sera obtained from murine and baboon models as well as from human volunteers vaccinated with the schistosomiasis vaccine SCHISTOSHIELD® demonstrating that the B7-based assay is a useful readout to monitor the vaccine performance in human clinical trials in Africa. FIGS. 3C to 3E show the B7 inhibition by antibodies generated in humans following vaccination with Sm-p80-based (NIH trial, Seattle, WA, USA) for a NIH trial high dose-unadjuvanted (FIG. 3C), NIH trial mid dose-unadjuvanted (FIG. 3D), and Madagascar trial low dose-unadjuvanted (FIG. 3E).

[0090] Briefly, a multicenter, randomized, placebo-controlled, observer-blinded, dose-escalation study, assessed the safety, tolerability, and immunogenicity of a three-dose regimen, spaced four weeks apart, given intramuscularly in healthy adults (20-59 years old). Three different doseformulations of the study product with varying antigen contents were investigated. A total of 120 eligible participants were recruited in 3 sequential cohorts (A, B, and C) in Burkina Faso (N=60) and in Madagascar (N=60). Cohort A received a low-dose antigen formulation (10 pg) or placebo (a low dose as used herein is a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 pg), Cohort B a mediumdose antigen formulation (30 pg) or placebo (a medium dose as used herein is a dose of 25, 30, 40, 50, or 60, pg) and Cohort C a high-dose antigen formulation (100 pg) or placebo (as used herein a high dose is 70, 75, 80, 90, 100, 110, 115, 120, 125, 130, 140, or 150 pg); all antigens with 5 pg adjuvant (GLA-SE). In each cohort, volunteers are randomized in a blinded manner into one of two arms, candidate vaccine or placebo, by a 3: 1 ratio. A subset of five out of 20 subjects in each cohort are sampled by convenience to enable further characterization of the immune response using the peripheral blood mononuclear cells (PBMC).

[0091] To ensure that the study participants at enrollment do not have any active schistosomiasis or helminth infection and are schistosomiasis egg-negative, pre-screening activities including schistosomiasis treatment was carried out in potential study participants prior to enrollment. Potential study participants were identified in the catchment population and were offered antihelminth treatment using praziquantel (PZQ) and Albendazole (ABZ) as per local guidelines at study site. The pre-screening visit was conducted 6-8 weeks before the screening visit. The last dose of PZQ / ABZ is administered at least 5 weeks prior to the first dose of study product.

[0092] FIGS. 4 A and 4B show the vaccine efficacy of B7 in murine model using the B7 RNA and a SARS-CoV2 RNA as a control, for total worm and female worm reduction (FIG. 4A) and reduction in eggs (FIG. 4B).

[0093] Briefly, the rSm-p80 antigen was tested for its prophylactic and anti -pathology efficacy in murine challenge models. Briefly, groups of mice were immunized three times, four weeks apart. At week twelve, four weeks after the last boost, mice were challenged with 150 cercariae of S. mansoni via tail immersion. Serum samples were collected via tail bleed before every immunization and before challenge. The mice were sacrificed six to eight weeks after challenge. Protection (P) was calculated by comparing worm burdens from vaccinated (V) and control (C) animals by a standard formula: %P = [(CV) / C x 100)]. After sacrifice, liver and intestine samples were collected from each animal and digested in 4% KOH. The number of eggs present in the tissues and percent reduction in egg production was determined. Antibody responses to Sm-p80 in immunized mice were estimated by an enzyme-linked immunosorbent assay (ELISA). In sera collected from these mice distinct Sm-p80-specific antibody titers were obtained for total IgG.

[0094] Animals immunized with adjuvant alone did not exhibit any significant IgG titers. However, robust antigen-specific (Sm-p80) IgG titers were detected in the vaccine group. Thesetiters reached end-point titers of 1 : 1,638,400 at weeks 10 and 12. These results demonstrate that the Sm-p80 antigen produced is highly antigenic when given with GLA-SE.

[0095] It is contemplated that any aspects of the disclosure discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0096] It will be understood that particular aspects described herein are shown by way of illustration and not as limitations of the disclosure. The principal features of this disclosure can be employed in various aspects without departing from the scope of the disclosure. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims.

[0097] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0098] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0099] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In aspects of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character orfunction of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0100] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0101] As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0102] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the disclosure(s) set out in any claims that may issue from this disclosure. Specifically, and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any disclosure(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the disclosure(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that areprotected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure but should not be constrained by the headings set forth herein.

[0103] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0104] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0105] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.REFERENCES

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Claims

What is claimed is:

1. An assay for monitoring effectiveness of an immunization to Schistosoma mansoni comprising: providing a recombinant calcium-activated neutral protease or calpain; contacting a sera from a subject suspected of having Schistosoma mansoni or that was immunized with an immunization or vaccine against Schistosoma mansoni with the recombinant calcium-activated neutral protease or calpain; and adding a detectable substrate for a Schistosoma mansoni calcium-activated neutral protease or calpain; wherein the sera blocks an activity of the calcium-activated neutral protease or calpain against the detectable substrate and is a surrogate for monitoring the effectiveness of the immunization to Schistosoma mansoni.

2. The assay of claim 1, further comprising one or more reagents or structures for performing an assay selected from the group consisting of a radioassay comprising one or more radioisotopes, a lateral flow assay comprising a test strip or dipstick, an ELISA-type assay comprising an enzyme adapted to convert a substrate into a detectable label, a nephelometric assay, a turbidimetric assay, a flow cytometry assay comprising one or more detectable particles, a fluorescent assay comprising one or more fluorescent labels, a chemiluminescent assay comprising one or more chemiluminescent labels, and a bead-type assay comprising detectably- labeled beads.

3. The assay of claim 1, wherein the detectable substrate is a synthetic substrate selected from at least one of: Suc-LLVY-AMC (SEQ ID NO:2), Suc-LY-AMC, Edans-PLFAERK-Dab (SEQ ID NO:3), Edans-ALGIGTIPPK-Dab (SEQ ID NO:4), or Edans-ALGIGTIPPK-Dab (SEQ ID N0:5).

4. The assay of claim 1, wherein the detectable substrate comprises a protein selected from at least one of: one or more cytoskeletal protein(s) (a-fodrin, spectrin, neurofilaments, desmin, talin, vimentin), one or more membrane protein(s) (ion channels, growth factor receptors, adhesion molecules), one or more nuclear proteins (lamin A / B), calpastatin, one or more kinases, or one or more phosphatases.

5. The assay of claim 1, wherein the assay is adapted as a read-out of, or a surrogate for, efficacy of a vaccine against Schistosoma mansoni.

6. The assay of claim 1, wherein the recombinant calcium-activated neutral protease or calpain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NOS: 1 or 6.

7. A kit comprising: a recombinant calcium-activated neutral protease or calpain; a detectable substrate for a Schistosoma mansoni calcium-activated neutral protease or calpain; and instructions in the kit for combining sera from a subject suspected of having been exposed to Schistosoma mansoni, or a vaccine thereto, with the calcium-activated neutral protease or calpain, wherein the sera blocks an activity of the calcium-activated neutral protease or calpain against the detectable substrate and is a surrogate for effectiveness of the immunization to Schistosoma mansoni.

8. The kit of claim 7, further comprising one or more reagents or structures for performing an assay selected from the group consisting of a radioassay comprising one or more radioisotopes, a lateral flow assay comprising a test strip or dipstick, an ELISA-type assay comprising an enzyme adapted to convert a substrate into a detectable label, a nephelometric assay, a turbidimetric assay, a flow cytometry assay comprising one or more detectable particles, a fluorescent assay comprising one or more fluorescent labels, a chemiluminescent assay comprising one or more chemiluminescent labels, and a bead-type assay comprising detectably- labeled beads.

9. The kit of claim 7, wherein the detectable substrate is a synthetic substrate selected from at least one of: Suc-LLVY-AMC (SEQ ID NO:2), Suc-LY-AMC, Edans-PLFAERK-Dab (SEQ ID NO:3), Edans-ALGIGTIPPK-Dab (SEQ ID NO:4), or Edans-ALGIGTIPPK-Dab (SEQ ID NO:5).

10. The kit of claim 7, wherein the detectable substrate comprises a protein selected from at least one of: one or more cytoskeletal protein(s) (a-fodrin, spectrin, neurofilaments, desmin, talin, vimentin), one or more membrane protein(s) (ion channels, growth factor receptors, adhesion molecules), one or more nuclear proteins (lamin A / B), calpastatin, one or more kinases, or one or more phosphatases.

11. A method for preventing schistosomiasis comprising the steps of: administering a vaccine comprising an antigenic protein having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or 6, wherein a dosage amount is an amount sufficient to provide worm reduction in the host, antifecundity effect, or protection against acute schistosomiasis.

12. The method of claim 11, wherein at least one of: the antigenic protein is defined further as a fusion protein comprising a peptide tag for at least one of identification, isolation, or purification; or the peptide tag is selected from a His-tag, a myc-tag, an S-peptide tag, a MBPtag, a GST tag, a FLAG tag, a thioredoxin tag, a GFP tag, a BCCP, a calmodulin tag, a streptavidin tag, an HSV-epitope tag, a V5-epitope tag and a CBP tag.;13. The method of claim 11, wherein the vaccine is administered with a primary immunization at week 0, a first boost at week 4, and a second boost at week 8; a primary immunization at week 0, a first boost at week 4 comprising the antigenic protein in the presence of an adjuvant, and a second boost at week 8 also comprising the antigenic protein in the presence of the adjuvant; or both; and optionally comprises an adjuvant.

14. The method of claim 11, wherein the antigenic protein is enzymatically active, is soluble when expressed in a bacteria, or both.

15. A vaccine comprising a recombinant antigenic protein having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or 6, wherein a dosage amount is an amount sufficient to provide worm reduction in the host, antifecundity effect, or protection against acute schistosomiasis, wherein the antigenic protein is enzymatically active, is soluble when expressed in a bacteria, or both, and optionally comprises an adjuvant, and optionally the adjuvant is Glucopyranosyl Lipid Adjuvant (GLA) or GLA squalene emulsion (GLA-SE).

16. A method for preventing schistosomiasis, said method comprising the steps of: administering a vaccine comprising a cDNA expressing an antigenic protein having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or 6; or cloned into an expression vector, wherein the dosage amount is an amount sufficient to provide worm reduction in the host, antifecundity effect, or protection against acute schistosomiasis.

17. The method of claim 16, wherein the vaccine is administered with a primary immunization at week 0, a first boost at week 4, and a second boost at week 8; or the vaccine is administered with a primary immunization at week 0, a first boost at week 4 comprising the antigenic protein in the presence of an adjuvant, and a second boost at week 8 also comprising the antigenic protein in the presence of the adjuvant.

18. The method of claim 16, further comprising an adjuvant and optionally the adjuvant is Glucopyranosyl Lipid Adjuvant (GLA) or GLA squalene emulsion (GLA-SE).

19. The method of claim 16, further comprising a peptide tag for identification, isolation, or both, of the presence of the subunit.

20. A polynucleotide encoding a subunit of S. mansoni calpain (Sm-p80) cloned into a vector having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO:6.

21. The polynucleotide of claim 21, further comprising a peptide tag for identification, isolation, or both, of the presence of the subunit.

22. The polynucleotide of claim 21, wherein the vector is in a host cell.

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