EP1349571A2 - Enterotoxine nsp4 de rotavirus et ses methodes d'utilisation - Google Patents

Enterotoxine nsp4 de rotavirus et ses methodes d'utilisation

Info

Publication number
EP1349571A2
EP1349571A2 EP01992588A EP01992588A EP1349571A2 EP 1349571 A2 EP1349571 A2 EP 1349571A2 EP 01992588 A EP01992588 A EP 01992588A EP 01992588 A EP01992588 A EP 01992588A EP 1349571 A2 EP1349571 A2 EP 1349571A2
Authority
EP
European Patent Office
Prior art keywords
nsp4
peptide
nucleic acid
protein
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01992588A
Other languages
German (de)
English (en)
Other versions
EP1349571A4 (fr
Inventor
Mary K. Estes
Judith M. Ball
Peng Tian
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baylor College of Medicine
Original Assignee
Baylor College of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/705,621 external-priority patent/US6673355B1/en
Application filed by Baylor College of Medicine filed Critical Baylor College of Medicine
Publication of EP1349571A2 publication Critical patent/EP1349571A2/fr
Publication of EP1349571A4 publication Critical patent/EP1349571A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to the viral enterotoxin NSP4 and to methods for using it, or antibodies/antisera thereto, as diagnostic agents, vaccines and therapeutic agents for the detection, prevention and/or treatment of rotaviral disease, for the prevention of stunted growth in animals and children caused by rotaviral infection and for the treatment of cystic fibrosis.
  • This invention also relates to methods and animal models for 1) the screening for viral enterotoxins, 2) the detection of viral enterotoxins and 3) the identification of viral enterotoxins.
  • Rotaviruses are the leading cause of severe, life-threatening viral gastroenteritis in infants and animals (Kapikian et al, 1996) and are associated with sporadic outbreaks of diarrhea in elderly (Halvorsrud 1980) and immunoco promised patients (Holzel et al, 1980). These viruses have a limited tissue tropism, with infection primarily being restricted to cells of the small intestine (Estes et al, 1994). Rotavirus infections also cause morbidity and mortality in many animal species.
  • symptomatic infection i.e., diarrhea
  • mice the outcome of infection is age-related; although rotaviruses may infect individuals and animals of all ages, symptomatic infection (i.e., diarrhea) generally occurs in the young (6 months - 2 years in children, and up to 14 days in mice), and the elderly.
  • Age-related host factors which may influence the outcome of infection have been proposed to include 1) differences in the presence/quantity of virus-binding receptors on mature villus epithelial cells, 2) virus strains with a specific spike protein (VP4), 3) passive immunity acquired by maternal antibody or in colostrum, and 4) reduced levels of proteases in the young.
  • mice Disease resulting from rotavirus infection in mice has been studied more extensively than in any other species and an age restriction of disease has been reported by several investigators (Ramig 1988; Wolf et al. 1981; Riepenhoff-Talty et al, 1982). Only mice less than 14 days of age develop diarrhea following oral inoculation of murine rotavirus, and the peak age at which animals are most likely to develop diarrhea (6-11 days) corresponds to the age when rotavirus can bind to mouse enterocytes (Riepenhoff-Talty et al, 1982).
  • rotavirus infections in mice show minimal histologic alterations. That is, villus blunting is limited and transient, and crypt cell hyperplasia is not present. In addition, the loss of villus tip epithelial cells is more limited in mice than in other animals. Instead, vacuolization of enterocytes on the villus tips is a predominant feature in symptomatic rotavirus infection in mice and virus replication may be abortive (Greenberg et al, 1981). The lack of extensive pathologic alterations in the mouse intestine during symptomatic infections has remained a puzzle; one interpretation of this phenomenon is that a previously unrecognized mechanism of diarrhea induction may be active in symptomatic rotavirus infection in mice.
  • NSP4 rotavirus nonstructural ER glycoprotein
  • NSP4 disrupts ER membranes and may play an important role in the removal ofthe transient envelope from budding particles during viral morphogenesis.
  • NSP4 114-135, a 22 aa peptide of NSP4 protein, has been shown to be capable of mimicking properties associated with NSP4 including being able to (i) mobilize intracellular calcium levels in insect cells when expressed endogenously or added to cells exogenously (Tian et al, 1994; Tian et al, 1995), and (ii) destabilize liposomes.
  • NSP4 thapsigargin-sensitive store
  • the present invention discloses herein a method of immunization against rotavirus infection or disease comprising administering to a subject a peptide NSP4 112-175 or NSP4 112-150 or a toxoid thereof. Further, the present invention discloses a method of immunization against rotavirus infection or disease comprising administering to a subject a non-glycosylated NSP4 protein or a toxoid of NSP4.
  • the immunizations may result in both homotypic and heterotypic immunity.
  • a method of passive immunization against rotavirus infection comprising administering to an expectant mother a peptide NSP4 112-175, NSP4 112-150 or a toxoid thereof.
  • the present invention discloses a method of passive immunization against rotavirus infection comprising administering to an expectant mother a non-glycosylated NSP4 protein or a toxoid of NSP4.
  • the immunizations may result in both homotypic and heterotypic immunity.
  • NSP4 peptide e.g., NSP4
  • NSP4 112-175 or NSP4 112-150) or toxoid is produced by a synthetic method.
  • the NSP4 peptide or toxoid is produced by an expression vector.
  • the expression vector is selected from the group consisting of mammalian, yeast, bacterial or insect.
  • Another embodiment ofthe present invention is a fusion protein comprising a protein that forms a virus-like particle linked to a NSP4 peptide.
  • the fusion protein further comprises a linker sequence.
  • An exemplary linker sequence includes, but is not limited to, three alanine residues and an alanine and serine residue. It is also contemplated that three glycine residues may be substituted for the three alanine residues.
  • One of skill in the art is cognizant that the scope of the invention is not limited to a five residue linker.
  • linkers may be used, for example, but not limited to, three alanine residues or 3 glycine residues.
  • the NSP4 peptide is NSP4 112-175 or a toxoid thereof or NSP4 112-150 or a toxoid thereof.
  • the protein that forms a virus-like particle is a viral protein or peptide isolated from the viral families Caliciviridae or Reoviridae.
  • the viral protein isolated from Caliciviridae is a Norwalk virus protein or peptide.
  • the Norwalk virus protein is ORF2 or ORF2 plus ORF3 or a fragment or toxoid of ORF2 or ORF2 plus ORF3.
  • ORF2 comprises amino acids 21-530 and ORF3 comprises 1-212.
  • Reoviridae is a rotavirus protein or peptide. More particularly, the rotavirus peptide is selected from the group of rotavirus proteins consisting of NP2, NP4, NP6 and NP7. In specific embodiments, the rotavirus peptide is NP2 or a NP2 fragment. Specifically NP2 comprises amino acids 94-881.
  • Another embodiment of the present invention discloses an expression vector comprising a nucleic acid sequence encoding a fusion protein operatively linked to a first promoter sequence, and a nucleic acid sequence encoding a viral peptide that is part of a virus-like particle operatively linked to a second promoter sequence.
  • the fusion protein comprises a viral peptide that is part of a virus-like particle linked to a ⁇ SP4 peptide or more than one NSP4 peptide.
  • the viral peptide linked to the NSP4 peptide is VP2 and forms the inner shell ofthe virus-like particle.
  • the viral peptide that is not linked to the fusion protein is rotavirus VP6.
  • the first promoter sequence is a polyhedrin promoter sequence and the second promoter sequence is a plO promoter sequence.
  • the expression vector is selected from the group consisting of insect, mammalian, viral and bacterial
  • the expression vector comprises a fusion protein that comprises a nucleic acid sequence encoding rotavirus NP2 amino acids 94-881 linked to a nucleic acid sequence encoding ⁇ SP4 amino acids 112-175.
  • the expression vector comprises a fusion protein that comprises a nucleic acid sequence encoding rotavirus VP2 amino acids 94-881 linked to a nucleic acid sequence encoding NSP4 amino acids 112-150.
  • Another embodiment of the present invention comprises an expression vector comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a viral peptide that is part of a virus-like particle linked to a NSP4 peptide.
  • the nucleic acid sequence is operatively linked to a promoter sequence.
  • the expression vector comprises the fusion protein that comprises a nucleic acid sequence encoding Norwalk virus ORF2 linked to a nucleic acid sequence encoding NSP4 amino acids 112-175.
  • the fusion protein comprises a nucleic acid sequence encoding Norwalk virus ORF2 linked to a nucleic acid sequence encoding NSP4 amino acids 112-150. It is also contemplated that the fusion protein may be a fragment of Norwalk virus ORF2 linked to NSP4 amino acids 112-175 or a fragment of Norwalk virus ORF2 linked to NSP4 amino acids 112-150.
  • the expression vector comprises the fusion protein that comprises a nucleic acid sequence encoding a nucleic acid sequence encoding Norwalk virus ORF2 and ORF3 linked to NSP4 amino acids 112-175.
  • the fusion protein comprises a nucleic acid sequence encoding Norwalk virus ORF2 and ORF3 linked to a nucleic acid sequence encoding NSP4 amino acids 112-150. It is also contemplated that the fusion protein may be a fragment of Norwalk virus ORF2 and ORF3 linked to NSP4 amino acids 112-175 or a fragment of Norwalk virus ORF2 and ORF3 linked to NSP4 amino acids 112-150.
  • a specific embodiment ofthe present invention also comprises a vaccine for inducing the formation of protective antibodies against rotavirus infection comprising administering a chimeric virus-like particle.
  • the chimeric virus-like particle comprises a peptide NSP4 112- 175, a first viral protein that is part of a virus-like particle, and a second viral protein that is part of a virus-like particle.
  • the first viral protein is rotavirus VP2, which forms an inner shell
  • said second viral protein is rotavirus NP6, which forms an outer shell surrounding the NP2 shell.
  • a method of immunization against rotavirus infection or disease comprising the step of administering to a subject a compound comprising a chimeric virus-like particle.
  • the chimeric virus-like particle comprises a peptide ⁇ SP4 112-175, a first viral protein that is part of a virus-like particle, and a second viral protein that is part of a virus-like particle. More particularly, the first viral protein is rotavirus NP2 and said second viral protein is rotavirus NP6.
  • the compound is administered orally, parenterally or intranasally. Another aspect comprises that the compound is administered with an adjuvant.
  • the compound is simultaneously or consecutively administered by at least two different routes of administration.
  • routes of administration include, but are not limited to, oral, parenteral or intranasal.
  • Another embodiment of the present invention comprises a method of inducing an immune response comprising the step of administering to a mammal one expression vector, wherein said expression vector comprises a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises a first viral protein that is part of a virus-like particle linked to a ⁇ SP4 nucleic acid sequence, and a nucleic acid sequence encoding a second viral protein that is part of a virus-like particle.
  • the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the second viral protein are under separate transcriptional control and wherein the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the second viral protein are in tandem in the one expression vector.
  • a specific embodiment also provides a method of inducing an immune response comprising the steps of co-administering to a mammal or a cell two different expression vectors, wherein a first expression vector comprises a nucleic acid sequence encoding a first viral protein that is part of a virus-like particle and a second expression vector comprises a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises a second viral protein that is part of a virus-like particle linked to a NSP4 nucleic acid sequence.
  • FIG. 1 shows rotavirus NSP4 protein induces diarrhea in CD1 mice.
  • 0.1 to 5 nmols (2-100 ⁇ M) of purified NSP4 was administered by the IP or IL routes to 6-7, 8-9 and 17-18 day old CD1 pups.
  • Rotavirus protein NP6 was used as the control in 6-7 day old animals, the most sensitive.
  • the dose and route of the proteins, age of the animals, and mean diarrhea score (mean score) are indicated on the bottom of the graph.
  • the Y axis displays the % diarrhea. Above each column is the number of responders (mice with diarrheal disease) over the total number of animals tested.
  • FIG. 2 shows IP administration of NSP4 induces diarrhea in 6-7 day old CD-I pups.
  • FIG. 3 shows diarrheal response in CDl mice following IP administration of NSP4 114-135 peptide.
  • Young (6-7 day) mouse pups were injected with various doses of peptide (x axis) and monitored for disease. The number of responders over the total number animals tested is shown above each column.
  • 0.1 - 50 nmol (2 ⁇ M-lmM) of peptide elicited similar responses (30-40% diarrhea induction); and 100-400 nmols (2-8mM) of peptide elicited comparative responses with 60-70% of he animals sick.
  • FIG. 4 shows diarrheal response in Balb/C mice following IP administration of NSP4 114-135 peptide. Young (6-7 day) mouse pups were injected with various doses of peptide (x axis) and monitored for disease. The number of responders over the total number animals tested is shown above each column.
  • FIG. 5 shows IP delivery of NSP4 114-135 peptide induce an age-dependent diarrhea in CDl mice and Spraque-Dawley Rats.
  • IP inoculated
  • the age and species of the pups, dose of the synthetic peptide and indication of whether peptides were unlinked or cross- linked are indicated on the bottom of the graph.
  • the dose of the IP delivered peptide varied with the age of the animals, i.e., older animals received a higher dose to control for the differences in body weight.
  • the Y axis indicates the % diarrhea and above each column is indicated the number of responders over the total number of animals inoculated.
  • the peptide was diluted in sterile PBS and evaluated for sterility. A final volume of 50 ⁇ l per dose was used.
  • FIG. 6 shows IL delivery of NSP4 114-135 peptide induce an age-dependent diarrhea in CDl mice and Spraque-Dawley Rats.
  • Different age outbred mice and rats were inoculated (IL) with NSP4 114-135 peptide and evaluated for disease.
  • the age and species of the pups, dose of the synthetic peptide and indication of whether peptides were unlinked or cross- linked are indicated on the bottom of the graph.
  • the Y axis indicates the % diarrhea and above each column is indicated the number of responders over the total number of animals inoculated.
  • the peptide was diluted in sterile PBS and evaluated for sterility. A final volume of 50 ⁇ l per dose was used.
  • FIG. 7A and FIG. 7B shows the dose response for cross-linked NSP4 peptide delivered ip to 6-7 day old CDl and Balb/C Mice.
  • the NSP4 114-135 peptide was cross- linked to itself with glutaraldehyde and dialyzed against sterile PBS prior to IP delivery to CDl (FIG. 7A) and Balb/C (FIG. 7B) mice.
  • the number of responders over the total number of animals inoculated is shown above each column.
  • FIG. 8 illustrates the experimental designs to test the ability of NSP4 114-135 to induce protective immunity from infectious rotavirus challenge and the ability of NSP4- specific antibody to mitigate rotavirus diarrhea following infection.
  • the left hand side of the figure illustrates that mouse dams were immunized with NSP4 peptide or with control peptide and then bred. Pups born to the dams were orally challenged with virulent rotavirus at 6-7 days.
  • the right hand side of the figure illustrates how pups born to non-immunized dams were first orally challenged with virulent rotavirus, followed by oral gavage of NSP4- specific or control antisera. The results of these experiments are set forth in Tables 5 and 6.
  • FIG. 9 shows the results from an experiment to study the differences in weight and growth between normal animals and animals suffering from NSP4 114-135-induced diarrhea.
  • FIG. 10 shows the amino acid sequence comparison of NSP4 from OSU attenuated and OSU virulent virus.
  • the amino acid sequence ofthe NSP4 protein of OSU-a (a porcine rotavirus, tissue culture attenuated, avirulent strain, SEQ.ID.NO:7),,top line, is compared to the amino acid sequence of the NSP4 protein of OSU-v (a porcine rotavirus, virulent strain, SEQ.ID.NO:8), bottom line. Positions at which the two sequences are different are shown in bold.
  • FIG. 11A and FIG. 11B show purity of NSP4 112-175 and its interaction with an antiserum raised with synthetic SAl 1 NSP4 peptide 114-135.
  • FIG. 11A illustrates the silver stained SDS-15% polyacrylamide gel of purified NSP4 112-175.
  • FIG. 11B illustrates the Western blot of purified NSP4 112-175 tested by rabbit antiserum against synthetic SA11 NSP4 peptide 114-135 (1:300 dilution). Lane 1, NSP4 112-175 crude material. Lane 2, eluate from immune affinity column against baculoviral proteins. Lane 3, 0.5 ⁇ g of purified NSP4 112-175. Arrows indicate NSP4 112-175.
  • FIG. 12B show the serum antibody responses in dams and pups to parenterally administered NSP4 112-175. Mice were immunized three times with 15 ⁇ g of NSP4 aal 12-175 plus 20 ⁇ g of QS-21 or with 20 ⁇ g of QS-21 alone. Blood samples were collected at 0, 7, and 16 DPP.
  • FIG. 12A shows the serum antibody titers against NSP4 112- 175.
  • FIG. 12B shows the Serum antibody titers against full-length NSP4.
  • NSP4 dams immunized with NSP4 112-175 plus QS-21
  • NSP4- ⁇ ups delivered to an nursed by NSP4- dams
  • QS-21 -dams inoculated with QS-21 alone
  • QS-21 -pups delivered to and nursed by QS-21 -dams.
  • FIG. 13A and FIG. 13B show the serum antibody responses in pups during cross- nursing.
  • FIG. 13 A illustrates the serum antibody titers to NSP4 112-175 in pups.
  • FIG. 13B illustrates the serum antibody titers to full-length NSP4 in pups. Blood samples were s collected on 0, 7, and 16 DPP.
  • FIG. 14A, FIG. 14B and FIG. 14C show replication of simian rotavirus SA11 in seven-day-old mice.
  • SA11 at a dose of 20 DD 50 was orally gavaged in seven-day-old BALB/c mice born to NSP4 dams with QS-21 dams.
  • Titers of infectious virus in the combined small and large intestine homogenates were determined at various days postinfection by plaque assay. The limit of virus detection was about 50 PFU/ml. — • — , virus titers in intestinal homogenates from QS-21-pups. — o — virus titers in intestinal homogenates from NSP4-pups.
  • FIG 14A and FIG. 14B show the virus titers from 2 individual experiments and each experiment was composed of one inoculated and one control pup 1-7 DPI.
  • FIG. 14C shows the average virus titers given in FIG. 14 A and FIG. 14B.
  • FIG. 15 shows serum antibody responses against SAl 1 VP6 in dams and pups with or lacking antibody to NSP4 112-175 and challenged with a single dose of rotavirus SAI L Pups were challenged with a single dose of 20 DD 50 of SAl 1. Blood samples were collected on 0 DPI and 12 DPI. Bars: Bars: Bars: NSP4 dams, immunized with NSP4 112-175 plus QS-21; NSP4-pups: delivered to an nursed by NSP4-dams; QS-21-dams, inoculated with QS-21 alone; QS-21-pups: delivered to and nursed by QS-21-dams. n, number of mice per group. The antibody GMT between NSP4-pups and QS-21-pu ⁇ s were 3000 vs. 300.
  • FIG. 16 shows a Western blot analysis of NSP4-2/6-VLPs.
  • a fusion protein was constructed comprising NSP4 112-175 and aa 94-881 of VP2. The fusion was inserted into a baculovirus expression vector pBAC4x-l. Cells were transiently transfected with the expression vector. Virus-like particles were expressed, purified, and analyzed by Western blot.
  • FIG. 17 shows the effect of NSP4 on transepithelial resistance (TER) of MDCK-1 cells and neutralization by antibody. This is an in vitro assay that mimics the effect of antibody on induction of diarrhea in mice.
  • This invention stems from the discovery ofthe first known viral enterotoxin, rotavirus NSP4, previously called NS28, which encodes a viral toxin capable of inducing intestinal secretion through a heretofore unknown signal transduction pathway to cause diarrheal disease.
  • the present invention relates to the fortuitous discovery that the rotavirus nonstructural ER glycoprotein, NSP4, induces an age-dependent diarrhea in two rodent models. Induction of diarrhea following administration of this protein alone was completely unexpected because infection with rotavirus was not involved. Characterization of the parameters of these new models of rotavirus-induced diarrhea demonstrates that this enteric viral-encoded protein is an enterotoxin, similar to bacterial enterotoxins, which are well- known to induce diarrhea by stimulating signal transduction pathways following interaction with specific intestinal receptors. The ordinary practitioner will appreciate that these new findings on NSP4-induced diarrheal disease and the data presented herein support several novel therapeutic and preventive approaches to rotavirus-induced disease.
  • the present invention also demonstrates that a synthetic peptide corresponding to amino acids 114-135 of SAl 1 NSP4 also induces an age-dependent diarrhea in young mice comparable to NSP4 when administered by the IP and IL route. Since the NSP4 114-135 peptide was readily available in large amounts in pure form, the response to the peptide was studied in detail. The response to the peptide was specific as shown by 1) lack of response to control peptides, 2) blocking with peptide-specific antibody, and 3) a mutated peptide (differing by only a single residue) alone failed to induce the response. The concentration of peptide required for disease induction was considerably higher than that needed for a response to the protein.
  • peptides from this protein which have the same effect are also included in the present invention.
  • These peptides include, but are not limited to, NSP4, non-glycosylated NSP4, NSP4 114-135, 120-147, 112-175 or 112-150.
  • toxoids ofthe above listed NSP4 proteins or peptides are also contemplated in the present invention.
  • proteins and or peptides according to the present invention may contain the entire amino acid sequence of NSP4 protein or any other fragment of NSP4 as set forth herein.
  • the following amino acid sequences are sequences corresponding to NSP4 proteins and are within the scope of the invention and some are referenced with the corresponding GenBank Accession Numbers (http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html): SA11 (SEQ.ID.NO:ll, AAC61867); SA11 clone 3 (SEQ.ID.NO:12); Murine EC (SEQ.LD.NO:13, AAB58700); Porcine OSU (SEQ.ID.NO:7, BAA13728); Gott-v (SEQ.ID.NO:14) and Gott-a (SEQ.ID.NO:15).
  • the present invention illustrates an analogous age dependence in the induction of diarrhea with purified NSP4 protein and NSP4 114-135 peptide.
  • Mice were most sensitive to the effects of the protein or peptide at 6-7 days of age.
  • Diarrhea induction by NSP4 or NSP4 114-135 decreased as the age of the animal increased, regardless of the route of administration.
  • the observed diarrhea in this study mimics the properties of symptomatic infection observed in experimental and natural rotavirus infection.
  • the present invention also shows that the inoculation of NSP4 114-135 peptide-specific antiserum prior to IP delivery of peptide results in a dramatic reduction of disease. (90% reduction in disease).
  • the present invention shows that diarrheal disease in pups born to dams immunized with the NSP4 114-135 and NSP4 112-175 peptide is significantly reduced in severity, duration, and in the number of pups with diarrhea. Further, the present invention demonstrates that immunization of dams with highly pure NSP4 112-175 induces passive protection of suckling mice against diarrhea induced by challenge viruses homotypic or heterotypic to the NSP4 112-175 immunogen.
  • NSP4-specif ⁇ c antibody significantly reduces diarrheal disease.
  • NSP4 protein or NSP4 114-135 and NSP4 112- 175 -specific antibodies' are sufficient to block the induction or severity of diarrhea
  • NSP4, NSP4 114-135 or NSP4 112-175 and/or antibodies specific thereto will be useful as vaccines and as therapeutic agents.
  • new drugs can now be developed to block or minimize the effects of interaction of NSP4 with its receptor or the effects ofthe disruption ofthe calcium homeostasis in affected cells.
  • viral enterotoxins associated with rotavirus and other gastroenteritis viruses such as caliciviruses, astroviruses, enteric adenoviruses, coronaviruses, and parvoviruses
  • human volunteers shall be considered to be within the scope of "animals.” It is a further embodiment of the present invention to provide methods for the screening and identification of new viral enterotoxins including administration of expressed proteins or peptides or synthetic peptides to CDl mice, Balb/C mice and/or Sprague-Dawley rats and monitoring for diarrhea.
  • diarrhea-genic viral protein may be construed to differ from the meaning ofthe term viral enterotoxin, if the term “diarrhea-genic viral protein” is substituted for the term viral enterotoxin, the subject matter of this and all following embodiments and all claims is also considered to be within the scope of the invention, and fully described for all purposes.
  • examples include human immunodeficiency virus (HIV) and cytomegalovirus (CMN).
  • HIV human immunodeficiency virus
  • CMV cytomegalovirus
  • This method includes administering expressed proteins or peptides or synthetic peptides of a selected virus to animals and monitoring the animals for diarrhea.
  • the present invention provides antibodies that bind with high specificity to ⁇ SP4 or fragments thereof (i.e., NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150) provided herein.
  • ⁇ SP4 or fragments thereof i.e., NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150
  • antibodies may also be generated in response to smaller constructs comprising epitopic core regions, including wild-type and mutant epitopes.
  • antibodies shall mean polyclonal and monoclonal antibodies unless otherwise indicated. Methods for the preparation of polyclonal and monoclonal antibodies to any protein or peptide are well known to the practitioner having ordinary skill in the art.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single domain antibodies
  • scFv single chain Fv
  • the techniques for preparing and using various antibody-based constructs and fragments are well known in the art.
  • Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting
  • IgA functions as the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts.
  • Humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
  • Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the NSP4 protein, peptide or fragments thereof of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds ofthe present invention.
  • fragments of NSP4 protein include, but are not limited to NSP4 114-135, NSP4 120-174, NSP4 112-175 or NSP4 112-150.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a NSP4 polypeptide or fragment thereof (i.e., NSP4, NSP4 114-135, NSP4 120-174, NSP4 112-175 or NSP4 112-150) and collecting antisera from that immunized animal.
  • an immunogen comprising a NSP4 polypeptide or fragment thereof (i.e., NSP4, NSP4 114-135, NSP4 120-174, NSP4 112-175 or NSP4 112-150) and collecting antisera from that immunized animal.
  • an animal used for production of anti-antisera is a non-human animal including rabbits, chickens, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • NSP4 protein to NSP4 114-135 peptide, to NSP4 120-147 peptide, to NSP4 112-175 peptide, to NSP4 112-175 and to other peptides of NSP4.
  • Monoclonal antibodies are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred.
  • Monoclonal antibodies may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified NSP4 protein, polypeptide or peptide or cell expressing high levels of NSP4.
  • a selected immunogen composition e.g., a purified or partially purified NSP4 protein, polypeptide or peptide or cell expressing high levels of NSP4.
  • a selected immunogen composition e.g., a purified or partially purified NSP4 protein, polypeptide or peptide or cell expressing high levels of NSP4.
  • a selected immunogen composition e.g., a purified or partially purified NSP4 protein, polypeptide or
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, human, monkey, rabbit, chicken, chicken eggs or frog cells is also possible.
  • the use of rats may provide certain advantages (Goding 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to NSP4, NSP4 114-135, NSP4 120-174, NSP4 112-175 or NSP4 112-150 antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular NSP4 protein or polypeptide (i.e., NSP4 114- 135, NSP4 120-174, NSP4 112-175 or NSP4 112-150) of different species may be utilized in other useful applications.
  • NSP4 protein or polypeptide i.e., NSP4 114- 135, NSP4 120-174, NSP4 112-175 or NSP4 112-150
  • the present invention also contemplates the use of antibodies against NSP4 proteins, polypeptides or fragments thereof (i.e., NSP4 114-135, NSP4 120-174, NSP4 112-175 or NSP4 112-150), generally ofthe monoclonal type, that are linked to one or more other agents to form an antibody conjugate.
  • NSP4 proteins, polypeptides or fragments thereof i.e., NSP4 114-135, NSP4 120-174, NSP4 112-175 or NSP4 112-150
  • Any antibody of sufficient selectivity, specificity and affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art.
  • a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
  • Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides.
  • a reporter molecule is defined as any moiety which may be detected using an assay.
  • Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
  • antibody conjugates are those conjugates in which the antibody is linlced to a detectable label.
  • Detectable labels are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired.
  • Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, as may be termed "immunotoxins”.
  • Antibody conjugates are thus preferred for use as diagnostic agents.
  • Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging".
  • Antibody conjugates of the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
  • Preferred secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 ; each incorporated herein by reference.
  • fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODLPY-TMR, BODLPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
  • Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels.
  • hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction.
  • Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983).
  • 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al, 1985).
  • the 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al, 1989; King et al, 1989; and Dholakia et al, 1989) and may be used as antibody binding agents.
  • Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3 -6 ⁇ -diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).
  • DTP A diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid ethylenetriaminetetraacetic acid
  • N- chloro-p-toluenesulfonamide N- chloro-p-toluenesulfonamide
  • tetrachloro-3 -6 ⁇ -diphenylglycouril-3 attached to the antibody
  • Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
  • derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated.
  • Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).
  • Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shamiessy et al, 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
  • Another embodiment of the present invention is to provide a method of using NSP4 and the peptides thereof, including but not limited to NSP4 114-135, NSP4 120-147, NSP4 112-175 and NSP4 112-150 to measure the levels of antibodies to NSP4.
  • These antibody measurements may be a surrogate for measuring protective immunity.
  • NSP4 and peptides thereof may be used to measure binding of antibodies, such as in an ELISA, or to measure neutralizing antibodies, such as in a transepithehal resistance assay (TER).
  • TER transepithehal resistance assay
  • NSP4 120-147, NSP4 112-175 and NSP4 112-150 As rotavirus infection is transmitted rapidly, this method is considered to include the prevention or amelioration of disease following exposure to a known infected person, for example in day care centers and in hospitals.
  • the term "compound comprising amino acids in a sequence corresponding to NSP4 114-135" shall mean a compound which has within it a sequence of amino acids corresponding to the sequence of NSP4 114-135, including NSP4 114-135 and the NSP4 protein.
  • the term “compound comprising amino acids in a sequence corresponding to NSP4 120-147” shall mean a compound which has within it a sequence of amino acids corresponding to the sequence of NSP4 120-147, including NSP4 120-147 and the NSP4 protein.
  • the term "comprising amino acids in a sequence corresponding to NSP4 112-175” shall mean a compound which has within it a sequence of amino acids corresponding to the sequence of NSP4 112-175, including NSP4 112-175 and the NSP4 protein.
  • the term “compound comprising amino acids in a sequence corresponding to NSP4 112-150” shall mean a compound which has within it a sequence corresponding to the sequence of NSP4 112-150, including NSP4 112-150 and the NSP4 protein.
  • the term "derivative” shall mean any molecules which are within the skill of the ordinary practitioner to make and use, which are made by derivatizing the subject compound, and which do not destroy the activity of the derivatized compound. Compounds which meet the foregoing criteria which diminish, but do not destroy, the activity of the derivatized compound are considered to be within the scope of the term "derivative.”
  • a derivative of a compound comprising amino acids in a sequence corresponding to the sequence of NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150 need not comprise a sequence of amino acids that corresponds exactly to the sequence of NSP4 114-135, NSP4 120-147, NSP 112-175 or NSP4 112-175 so long as it retains a measurable amount of the activity of the NSP4 114-135, NSP4 120-147, NSP 112- 175 or NSP4 112-150 peptide.
  • Another aspect ofthe present invention is a method of immunization against rotavirus infection or disease comprising administering to a subject a peptide NSP4 112-175 or NSP4 112-150 or a toxoid thereof.
  • the immunization may result in homotypic or heterotypic immunity.
  • a non-glycosylated peptide of NSP4 may be administered to a subject or to an expectant mother.
  • NSP4 is a glycosylated protein.
  • the amino terminus of NSP4 contains two N- linked high mannose glycosylation sites, which are located in the first of three hydrophobic domains. Glycosylation of NSP4 is required for removal of the transient envelope from the budding particles. It is also contemplated that the glycosylation sites may be mutated using standard mutagenesis techniques well known and used in the art e.g., site-directed mutagenesis.
  • N-linked glycosylation will be preformed utilizing standard procedures that are well known in the art.
  • Exemplary inhibitors of N-linked glycosylation include, but are not limited to tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin or swainsonine.
  • NSP4 protein or peptides thereof including but not limited to NSP4 114-135, NSP4 120-147, NSP4 112-175 and/or NSP4 112-150 as a treatment for or vaccine against rotavirus diarrhea.
  • animals given peptide twice showed a rapid onset of severe diarrhea followed by stunted growth. The weight of these animals was 20-30% ⁇ lower for three weeks after administration of peptide.
  • the present invention also illustrates that NSP4 114-135 promotes and augments cAMP-dependent CI " secretion in mouse intestinal mucosa and induces diarrhea in rodents in a time frame similar to STB (about 3 hrs).
  • the electrophysiological data show that NSP4 induces calcium increases in the intestines of mice in an age-dependent maimer and these increases in calcium result in chloride secretion as measured by short-circuit currents.
  • Direct addition of cross-linked NSP4 114-135 to mouse ileal mucosal sheets resulted in a rise in current, similar to that evoked by the calcium agonist, carbachol.
  • induction of chloride secretion from intestinal mucosal sheets was site- dependent. Zero to minimal responses were observed when mouse jejunum, duodenum or colon tissue was employed, and maximum responses were induced when the ileum was utilized.
  • the present invention also includes, NSP4 112-175, a cleavage product of the SA11 NSP4 C-terminus, which was detected in the extracellular medium of rotavirus-infected cells.
  • the cleavage product mobilizes calcium and has enterotoxin activity in mice, similar to full- length NSP4 (Zhang et al, 2000).
  • NSP4 is released from virus-infected cells and such extracellular NSP4 initiates a signaling pathway leading to diarrhea (Ball et al, 1996).
  • NSP4 stimulation of a Ca 2+ -dependent signal transduction pathway resulting in disruption of normal intestinal epithelial transport, is similar to that reported for guanylin and the heat-stable enterotoxins.
  • NSP4 can be considered a viral enterotoxin.
  • NSP4 induces diarrhea by activating an age-dependent, calcium-sensitive anion (chloride) permeability in the small and large intestine mucosa in both normal mice and mice with cystic fibrosis that lacks the cystic fibrosis transmembrane regulation.
  • chloride calcium-sensitive anion
  • NSP4-induced diarrhea may be altered.
  • the scope of the present invention is not limited to one specific model of NSP4-induced diarrhea, but includes and is not limited to alterations and variations of the model.
  • the data from the Cystic Fibrosis mice suggest that age-dependence may be downstream of Ca 2+ mobilization, thus a channel may be involved instead of a receptor for the regulation of age-dependent disease.
  • viral enterotoxins associated with rotavirus and other gastroenteritis viruses such as caliciviruses, astroviruses, enteric adenoviruses, coronoviruses and parvoviruses
  • ⁇ SP4 protein or peptides thereof including but not limited to NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-175 to identify and/or characterize a new intestinal receptor whose signaling induces secretion.
  • NSP4 protein or peptides thereof including but not limited to NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150, to enhance fluid secretion.
  • NSP4 protein or peptides thereof including but not limited to NSP4 114-135, NSP4 120- 147, NSP4 112-175 or NSP4 112-150.
  • NSP4 peptides or fragments thereof i.e., NSP4 114-135, NSP4 120-174, NSP4 112-175 or NSP4 112-150
  • other viral enterotoxins to prevent, ameliorate or stop diarrheal disease.
  • small molecule inliibitors shall mean any ligand that can bind with high affinity to a target molecule, thereby inhibiting the target molecule's activity.
  • Small molecule inhibitors include, but are not limited to, peptides, oligonucleotides, amino acids, derivatized amino acids, carbohydrates, and organic and inorganic chemicals. Libraries of small molecule inhibitors are available to the practitioner either according to known methods, or commercially.
  • this method includes identifying a viral enterotoxin, screening the purified enterotoxin against one or more random small molecule libraries, for example, a random peptide library, a random oligonucleotide library, or a pharmaceutical drug library, and identifying those small molecules that bind with high affinity to the viral enterotoxin.
  • random small molecule libraries for example, a random peptide library, a random oligonucleotide library, or a pharmaceutical drug library
  • the term “candidate substance” refers to any molecule that may potentially inhibit or enhance NSP4 or any fragment of NSP4 (i.e., NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150) activity.
  • the candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to NSP4 or any fragment of NSP4 (i.e., NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150).
  • Using lead compounds to help develop improved compounds is know as "rational drug design" and includes not only comparisons with know inhibitors and activators, but predictions relating to the structure of target molecules.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs, which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a target molecule, or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.
  • Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
  • Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inliibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators.
  • modulators include antisense molecules, ribozymes, and antibodies
  • an antisense molecule that bound to a translational or transcriptional start site, or splice junctions would be ideal candidate inhibitors.
  • the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the structure of the modulators.
  • Such compounds which may include peptidomimetics of peptide modulators, may be used in the same manner as the initial modulators.
  • An inl ibitor according to the present invention may be one which exerts its inhibitory or activating effect upstream, downstream or directly on NSP4 or any fragment of NSP4 (i.e., NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150). Regardless ofthe type of inhibitor or activator identified by the present screening methods, the effect of the inhibition or activator by such a compound results in NSP4 or any fragment of NSP4 (i.e., NSP4 114- 135, NSP4 120-147, NSP4 112-175 or NSP4 112-150) as compared to that observed in the absence ofthe added candidate substance.
  • a quick, inexpensive and easy assay to run is an in vitro assay.
  • Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time.
  • a variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.
  • a cell free assay is a binding assay. While not directly addressing function, the ability of a modulator to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions.
  • the target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determination of binding. ' Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enliance binding.
  • Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.
  • a technique for high throughput screening of compounds is described in WO 84/03564.
  • Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods.
  • the present invention also contemplates the screening of compounds for their ability to modulate NSP4 or any fragment of NSP4 (i.e., NSP4 114-135, NSP4 120-147, NSP4 112- 175 or NSP4 112-150) in cells.
  • Various cell lines can be utilized for such screening assays, including cells specifically engineered for this purpose.
  • culture may be required.
  • the cell is examined using any of a number of different physiologic assays.
  • molecular analysis may be performed, for example, protein expression, rnRNA expression (including differential display of whole cell or polyA RNA) and others.
  • mice are a preferred embodiment, especially for transgenics.
  • other animals are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons).
  • Assays for modulators may be conducted using an animal model derived from any of these species.
  • one or more candidate substances are administered to an animal, and the ability ofthe candidate substance(s) to alter one or more characteristics, as compared to a similar animal not treated with the candidate substance(s), identifies a modulator.
  • the characteristics may be any of those discussed above with regard to the function of a particular compound or cell, or instead a broader indication such as behavior, anemia, immune response, etc.
  • Treatment of these animals with test compounds will involve the administration ofthe compound, in an appropriate form, to the animal.
  • Admimstration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, intranasal, buccal, or even topical.
  • administration may be by a parenteral route, e.g intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.
  • Another method for identifying small molecule inhibitors includes the steps of identifying viral enterotoxins, determining the high resolution structure of these proteins and/or peptides thereof, determining the active domain(s) and designing small molecule inhibitors which bind with high affinity to the active domain(s).
  • Another method includes identifying viral enterotoxins, identifying the intestinal receptor which binds the viral enterotoxin, and designing small molecule inliibitors which competitively bind the receptor, without inducing secretion.
  • this method includes identifying the molecules active in the signaling pathway and identifying compounds which inhibit their activity.
  • Such compounds will include but not be limited to small molecule inhibitors which block binding of NSP4 to its receptor, blocking of G protein mediated or other signal transduction secondary messengers and pathways which lead to chloride secretion or diarrhea.
  • NSP4 protein or peptides thereof including but not limited to NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150 to induce the formation of protective active or passive antibodies.
  • Fragments of NSP4 include, but are not limited to, NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150.
  • It is another embodiment of the present invention to provide a method for immunization against rotavirus infection comprising administering to a subject a vaccine including the NSP4 protein or peptides thereof, including, but not limiting to, NSP4 114-135, NSP4 120-147, NSP4 112-175 orNSP4 112-150 peptides.
  • an antigenic composition must induce an immune response to the antigen in a cell, tissue or animal (e.g., a human).
  • an "antigenic composition” may comprise an antigen (e.g., a NSP4 peptide or NSP4 polypeptide or NSP4 protein or toxoid thereof), a nucleic acid encoding an antigen (e.g., an antigen expression vector), or a cell expressing or presenting an antigen.
  • the antigenic composition comprises the nucleic acid sequence that encodes NSP4, or any fragments thereof, including but not limiting to NSP4 112-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150, or an imrnunologicaHy functional equivalent thereof.
  • the antigenic composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent.
  • Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant.
  • one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination.
  • the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.
  • an antigenic composition or immunologically functional equivalent may be used as an effective vaccine in inducing a humoral and/or cell-mediated immune response in an animal.
  • the present invention contemplates one or more antigenic compositions or vaccines for use in both active and passive immunization embodiments.
  • a vaccine of the present invention may vary in its composition of proteinaceous, nucleic acid and/or cellular components.
  • a nucleic encoding an antigen might also be formulated with a proteinaceous adjuvant.
  • various compositions described herein may further comprise additional components.
  • one or more vaccine components may be comprised in a lipid or liposome.
  • a vaccine may comprise one or more adjuvants.
  • a vaccine of the present invention, and its various components may be prepared and/or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light ofthe present disclosure.
  • an antigenic composition of the present invention may be made by a method that is well known in the art, including but not limited to chemical synthesis by solid phase synthesis and purification away from the other products ofthe chemical reactions by HPLC, or production by the expression of a nucleic acid sequence encoding a peptide or polypeptide comprising an antigen ofthe present invention in an in vitro translation system or in a living cell.
  • the antigenic composition isolated and extensively dialyzed to remove one or more undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.
  • additional amino acids, mutations, chemical modification and such like, if any, that are made in a vaccine component will preferably not substantially interfere with the antibody recognition of the epitopic sequence.
  • a peptide or polypeptide corresponding to one or more antigenic determinants of the present invention should generally be at least five or six amino acid residues in length, and may contain up to about 10, about 15, about 20, about 25 about 30 ,about 35, about 40, about 45 or about 50 residues or so.
  • a peptide sequence may be synthesized by methods known to those of ordinary skill in the art, such as, for example, peptide synthesis using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA).
  • Longer peptides or polypeptides also may be prepared, e.g., by recombinant means.
  • a nucleic acid encoding an antigenic composition and/or a component described herein may be used, for example, to produce an antigenic composition in vitro or in vivo for the various compositions and methods of the present invention.
  • a nucleic acid encoding an antigen is comprised in, for example, a vector in a recombinant cell.
  • the nucleic acid may be expressed to produce a peptide or polypeptide comprising an antigenic sequence.
  • the peptide or polypeptide may be secreted from the cell, or comprised as part of or within the cell.
  • an immune response may be promoted by transfecting or inoculating an animal with a nucleic acid encoding an antigen.
  • One or more cells comprised within a target animal then expresses the sequences encoded by the nucleic acid after administration of the nucleic acid to the animal.
  • the vaccine may comprise "genetic vaccine" useful for immunization protocols.
  • a vaccine may also be in the fo ⁇ n, for example, of a nucleic acid (e.g., a cDNA or an RNA) encoding all or part ofthe peptide or polypeptide sequence of an antigen.
  • Expression in vivo by the nucleic acid may be, for example, by a plasmid type vector, a viral vector, or a viral/plasmid construct vector.
  • the nucleic acid comprises a coding region that encodes all or part of the NSP4, NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150, or an immunologically functional equivalent thereof.
  • the nucleic acid may comprise and/or encode additional sequences, including but not limited to those comprising one or more immunomodulators or adjuvants.
  • the nucleotide and protein, polypeptide and peptide encoding sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/).
  • the coding regions for these known genes may be amplified, combined with the sequences NSP4 (SEQ.ID.NO:16) , NSP4 114-135, NSP4 120- 147, NSP4 112-175 or NSP4 112-150 disclosed herein (e.g., ligated) and/or expressed using the techniques disclosed herein or by any technique that would be know to those of ordinary skill in the art (e.g., Sambrook et al, 1987).
  • a nucleic acid may be expressed in an in vitro expression system, in preferred embodiments the nucleic acid comprises a vector for in vivo replication and/or expression.
  • nucleic acids according to the present invention may encode an entire NSP4 gene, a domain of NSP4, or any other fragment of NSP4 as set forth herein.
  • the nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism.
  • sequences corresponding to NSP4 genes are sequences corresponding to NSP4 genes and are within the scope ofthe invention and are referenced with the corresponding GenBank Accession Numbers http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html): ALA(SEQ.ID.NO:17, AF144792); C-11 (SEQ.ID.NO:18, AF144793); R-2 (SEQ.LD.NO:19, AF144794); BAP-2 (SEQ. ID.
  • nucleic acid sequences for other rotavirus genes including, but not limiting to : VP6(SEQ.ID.NO:34, D00325); VP6(SEQ.ID.NO:35, K02086) and VP2(SEQ.ID.NO:36, X14949).
  • an antigenic composition of the invention may be combined with one or more additional components to form a more effective vaccine.
  • additional components include, for example, one or more additional antigens, immunomodulators or adjuvants to stimulate an immune response to an antigenic composition ofthe present invention and/or the additional component(s).
  • nucleic acids encoding antigens of the present invention may be transfected into plants, particularly edible plants, and all or part of the plant material used to prepare a vaccine, such as for example, an oral vaccine.
  • a vaccine such as for example, an oral vaccine.
  • immunomodulators can be included in the vaccine to augment a cell's or a patient's (e.g., an animal's) response.
  • Immunomodulators can be included as purified proteins, nucleic acids encoding immunomodulators, and/or cells that express immunomodulators in the vaccine composition.
  • the following sections list non- limiting examples of immunomodulators that are of interest, and it is contemplated that various combinations of immunomodulators may be used in certain embodiments (e.g., a cytokine and a chemokine).
  • Interleukins, cytokines, nucleic acids encoding interleukins or cytokines, and/or cells expressing such compounds are contemplated as possible vaccine components.
  • Interleukins and cytokines include but are not limited to interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, LL-12, IL-13, IL-14, IL-15, IL-18, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, angiostatin, thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH-1, METH-2, tumor necrosis factor, TGF ⁇ , LT and combinations thereof.
  • IL-1 interleukin 1
  • IL-2 interleukin-2
  • IL-3 IL-4
  • IL-5 IL-6
  • IL-7 IL-8
  • Chemokines nucleic acids that encode for chemokines, and/or cells that express such also may be used as vaccine components.
  • Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine coding sequence in combination with, for example, a cytokine coding sequence, to enhance the recruitment of other immune system components to the site of treatment.
  • chemokines include, for example, RANTES, MCAF, MIP1 -alpha, MIP1- Beta, IP- 10 and combinations thereof.
  • cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.
  • an antigenic composition's may be chemically coupled to a carrier or recombinantly expressed with a immunogenic carrier peptide or polypeptide (e.g., a antigen-carrier fusion peptide or polypeptide) to enhance an immune reaction.
  • a immunogenic carrier peptide or polypeptide e.g., a antigen-carrier fusion peptide or polypeptide
  • exemplary and preferred immunogenic carrier amino acid sequences include hepatitis B surface antigen, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as immunogenic carrier proteins.
  • Means for conjugating a polypeptide or peptide to an immunogenic carrier protein are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis- biazotized benzidine. It may be desirable to co-administer biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity.
  • BRM biologic response modifiers
  • Such BRMs include, but are not limited to, cimetidine (CM; 1200 mg/d) (Smith/Kline, PA); low- dose cyclophosphamide (CYP; 300 mg/m2) (Johnson/ Mead, NJ), or a gene encoding a protein involved in one or more immune helper functions, such as B-7.
  • Immunization protocols have used adjuvants to stimulate responses for many years, and as such adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation.
  • an adjuvant effect is achieved by use of an agent such as alum used in about 0.05 to about 0.1% solution in phosphate buffered saline.
  • the antigen is made as an admixture with synthetic polymers of sugars (Carbopol®) used as an about 0.25% solution.
  • Adjuvant effect may also be made by aggregation ofthe antigen in the vaccine by heat treatment with temperatures ranging between about 70° to about 101°C for a 30-second to 2-minute period, respectively.
  • Some adjuvants are certain organic molecules obtained from bacteria, act on the host rather than on the antigen.
  • An example is muramyl dipeptide (N- acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan.
  • MDP N- acetylmuramyl-L-alanyl-D-isoglutamine
  • the effects of MDP are not fully understood. MDP stimulates macrophages but also appears to stimulate B cells directly.
  • the effects of adjuvants therefore, are not antigen- specific. If they are administered together with a purified antigen, however, they can be used to selectively promote the response to the antigen.
  • Adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Patent 4,877,611).
  • hemocyanins and hemoerythrins may also be used in the invention.
  • the use of hemocyanin from keyhole limpet (KLH) is preferred in certain embodiments, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed.
  • Various polysacchari.de adjuvants may also be used.
  • the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et al, 1989).
  • the doses that produce optimal responses, or that otherwise do not produce suppression, should be employed as indicated (Yin et al, 1989).
  • Polyamine varieties of polysaccharides are particularly preferred, such as chitiii and chitosan, including deacetylated chitin.
  • muramyl dipeptide N-acetylmuramyl-L- alanyl-D-isoglutamine
  • bacterial peptidoglycans a group of bacterial peptidoglycans.
  • Derivatives of muramyl dipeptide such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated.
  • U.S. Patent 4,950,645 describes a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide which is described for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. It is the to be effective in activating human monocytes and destroying tumor cells, but is non-toxic in generally high doses.
  • the compounds of U.S. Patent 4,950,645 and PCT Patent Application WO 91/16347, are contemplated for use with cellular carriers and other embodiments ofthe present invention.
  • BCG Bacillus Calmette-Guerin, an attenuated strain of Mycobacterium
  • CWS BCG-cell wall skeleton
  • Trehalose dimycolate may be used itself. Trehalose dimycolate administration has been shown to correlate with augmented resistance to influenza virus infection in mice (Azuma et al, 1988). Trehalose dimycolate may be prepared as described in U.S. Patent 4,579,945.
  • BCG is an important clinical tool because of its immunostimulatory properties. BCG acts to stimulate the reticulo-endothelial system, activates natural killer cells and increases proliferation of hematopoietic stem cells. Cell wall extracts of BCG have proven to have excellent immune adjuvant activity. Molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG (Jacobs et al, 1987; Snapper et al, 1988; Husson et ⁇ /., 1990; Martin et al, 1990).
  • Live BCG is an effective and safe vaccine used worldwide to prevent tuberculosis.
  • BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly 2 billion immunizations, BCG has a long record of safe use in man (Luelmo 1982; Lotte et al, 1984). It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination.
  • An exemplary BCG vaccine is sold as TICE® BCG (Organon Inc., West Orange, NJ).
  • cells of Mycobacterium bovis-BCG are grown and harvested by methods known in the art. For example, they may be grown as a surface pellicle on a Sauton medium or in a fermentation vessel containing the dispersed culture in a Dubos medium (Dubos et al, 1947; Rosenthal 1937). All the cultures are harvested after 14 days incubation at about 37°C. Cells grown as a pellicle are harvested by using a platinum loop whereas those from the fermenter are harvested by centrifugation or tangential-flow filtration. The harvested cells are resuspended in an aqueous sterile buffer medium.
  • a typical suspension contains from about 2x10 ° cells/ml to about 2xl0 12 cells/ml.
  • a sterile solution containing a selected enzyme which will degrade the BCG cell covering material is added to this bacterial suspension.
  • the resultant suspension is agitated such as by stirring to ensure maximal dispersal of the BCG organisms.
  • a more concentrated cell suspension is prepared and the enzyme in the concentrate removed, typically by washing with an aqueous buffer, employing known techniques such as tangential-flow filtration.
  • the enzyme-free cells are adjusted to an optimal immunological concentration with a cryoprotectant solution, after which they are filled into vials, ampoules, etc., and lyophilized, yielding BCG vaccine, which upon reconstitution with water is ready for immunization.
  • Amphipathic and surface active agents e.g., saponin and derivatives such as QS21 (Cambridge Biotech) form yet another group of adjuvants for use with the immunogens of the present invention.
  • Nonionic block copolymer surfactants Roshanovich et al, 1994; Hunter et al, 1991
  • Oligonucleotides are another useful group of adjuvants (Yamamoto et al, 1988).
  • Quil A and lentinen are other adjuvants that may be used in certain embodiments ofthe present invention.
  • One group of adjuvants also contemplated for use in the invention are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Patent 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals.
  • the combination of detoxified endotoxins with trehalose dimycolate is also contemplated, as described in U.S. Patent 4,435,386.
  • Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Patent 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S.
  • Patents 4,436,727, 4,436,728 and 4,505,900 Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Patent 4,520,019.
  • NSP4 peptide (NSP4 114-135, NSP4 120- 147, NSP4 112-175 or NSP4 112-150) or toxoid thereof may be produced synthetically or by an expression vector.
  • Expression vectors that may be used include, but are not limited to mammalian, yeast, viral, bacterial, plant or insect.
  • the fusion protein comprises a NSP4 peptide linked to a protein that forms a virus-like particle.
  • the NSP4 peptide may include, but is not limited to, NSP4 114-135, NSP4 120-147, NSP4 112- 175 or NSP4 112-150.
  • the virus-like particle is a viral protein or peptide isolated from Caliciviridae or Reoviridae.
  • the viral protein or peptide isolated from Caliciviridae is a Norwalk virus protein or peptide and the viral protein or peptide isolated from Reoviridae is a rotavirus protein or peptide.
  • the Norwalk virus protein or peptide may be ORF2 or ORF3 or ORF2 plus ORF3 or a toxoid thereof.
  • the rotavirus protein or peptide may be VP2, VP4, VP5, NP6 and VP7. In specific embodiments, the rotavirus peptide is NP2.
  • virus-like particles consist of capsid proteins assembled into a shell-like structure without the presence of viral nucleic acid within the shell. These shells can display conformational epitopes that are not present on individual capsid proteins.
  • the use of NLPs offer several immunogenic advantages. First, NLPs present conformational epitopes to the immune system in such a way as native infectious particles so that neutralizing antibodies and other protective immune responses are induced effectively. Second, because NLPs are noninfectious, inactivation is not required. Thus, one skilled in the art realizes that the use of virus-like particles may be better immunogens than formalin- inactivated whole-virus vaccines or proteins. Thus, it can be appreciated that the fusion protein ofthe present invention may be a better immunogen than the NSP4 alone.
  • Another embodiment of the present invention comprises an expression vector comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a NSP4 peptide and a viral peptide that forms a virus-like particle.
  • the nucleic acid sequence is operatively linked to a promoter sequence.
  • vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • YACs artificial chromosomes
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
  • a “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference.
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • a specific initiation signal also may be required for efficient translation of coding sequences.
  • Exogenous translational control signals include the ATG initiation codon or adjacent sequences.
  • Exogenous translational control signals including the ATG initiation codon, may need to be provided.
  • One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • the exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art.
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously replicating sequence
  • the cells contain nucleic acid construct ofthe present invention
  • a cell may be identified in vitro or in vivo by including a marker in the expression vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • host cell refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
  • a host cell can, and has been, used as a recipient for vectors.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication ofthe vector or expression of part or all ofthe vector-encoded nucleic acid sequences.
  • Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).
  • ATCC American Type Culture Collection
  • An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
  • a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
  • Bacterial cells used as host cells for vector replication and/or expression include DH5 ⁇ , JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACKTM Gold Cells (STRATAGENE®, La Jolla).
  • bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
  • Examples of eukaryotic host cells for replication and/or expression of a vector include
  • a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression ofthe vector.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patent No. 5,871,986,
  • CONTROLTM Inducible Mammalian Expression System which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
  • INVITROGEN® which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
  • INVITROGEN® also provides a yeast expression system called the Pichia methanohca Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanohca.
  • a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
  • gastroenteritis viruses including rotaviruses, caliciviruses, astroviruses, enteric adenoviruses, coronoviruses and parvoviruses, including viral enterotoxins which induce the diarrhea associated with viral infection.
  • Pharmaceutical compositions and administration including administering viruses, viral proteins or peptides thereof to one or more of three new animal models for diarrheal virus infections, the CDl mouse, the Balb/C mouse and the Sprague-Dawley rat.
  • Aqueous compositions of the present invention comprise an effective amount of NSP4 (NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150) protein, polypeptide, peptide, expression vector, or cells containing the expression vector, antibodies, epitopic core region, inhibitor, and/or such like, dissolved and/or dispersed in a pharmaceutically acceptable carrier and/or aqueous medium.
  • NSP4 NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150
  • polypeptide polypeptide
  • peptide expression vector
  • cells containing the expression vector, antibodies, epitopic core region, inhibitor, and/or such like
  • Aqueous compositions of gene therapy vectors expressing any ofthe foregoing are also contemplated.
  • phrases "pharmaceutically and/or pharmacologically acceptable” refer to molecular entities and/or compositions that do not produce an adverse, allergic and/or other untoward reaction when administered to an animal as appropriate.
  • pharmaceutically acceptable carrier includes any and/or all solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and/or absorption delaying agents and/or the like.
  • the use of such media and/or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media and/or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • preparations should meet sterility, pyrogenicity, general safety and/or purity standards as required by FDA Office of Biologies standards.
  • the biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophihzed for more ready formulation into a desired vehicle, where appropriate.
  • the active compounds may generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and/or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and/or even intraperitoneal routes.
  • the preparation of an aqueous compositions that contain an effective amount of the agent as an active component and/or ingredient will be known to those of skill in the art in light of the present disclosure.
  • compositions can be prepared as injectables, either as liquid solutions and/or suspensions; solid forms suitable for using to prepare solutions and/or suspensions upon the addition of a liquid prior to injection can also be prepared; and/or the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions and/or dispersions; formulations including sesame oil, peanut oil and/or aqueous propylene glycol; and/or sterile powders for the extemporaneous preparation of sterile injectable solutions and/or dispersions.
  • the form must be sterile and/or must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and/or storage and/or must be preserved against the contaminating action of microorganisms, such as bacteria and/or fungi.
  • Solutions of the active compounds as free base and/or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and/or in oils. Under ordinary conditions of storage and/or use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the active compounds of the present invention can be formulated into a composition in a neutral and/or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and/or which are formed with inorganic acids such as, for example, hydrochloric and/or phosphoric acids, and/or such organic acids as acetic, oxalic, tartaric, mandelic, and/or the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, and/or ferric hydroxides, and/or such organic bases as isopropylamine, trimethylamine, histidine, procaine and/or the like.
  • the carrier can also be a solvent and/or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and/or liquid polyethylene glycol, and/or the like), suitable mixtures thereof, and/or vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and/or the like.
  • isotonic agents for example, sugars and/or sodium chloride.
  • Prolonged absorption ofthe injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and/or gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation are vacuum-drying and/or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparation of more, and/or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and/or in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and/or the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and/or the liquid diluent first rendered isotonic with sufficient saline and/or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and/or intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure (see for example, "Remington's Pharmaceutical Sciences” 15th Edition). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the active peptides and/or agents may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, and/or about 0.001 to 0.1 milligrams, and/or about 0.1 to 1.0 and/or even about 10 milligrams per dose and/or so. Multiple doses can also be administered.
  • other pharmaceutically acceptable forms include, e.g., tablets and/or other solids for oral administration; liposomal formulations; time release capsules; and/or any other form currently used, including cremes.
  • Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops and/or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, the aqueous nasal solutions usually are isotonic and/or slightly buffered to maintain a pH of 5.5 to 6.5.
  • antimicrobial preservatives similar to those used in ophthalmic preparations, and/or appropriate drug stabilizers, if required, may be included in the formulation.
  • Various commercial nasal preparations are known and/or include, for example, antibiotics and/or antil istamines and/or are used for asthma prophylaxis.
  • vaginal suppositories are solid dosage forms of various weights and/or shapes, usually medicated, for insertion into the rectum, vagina and/or the urethra. After insertion, suppositories soften, melt and/or dissolve in the cavity fluids.
  • traditional binders and/or carriers may include, for example, polyalkylene glycols and/or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1 %-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and/or the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations and/or powders.
  • oral pharmaceutical compositions will comprise an inert diluent and/or assimilable edible carrier, and/or they may be enclosed in hard and/or soft shell gelatin capsule, and/or they may be compressed into tablets, and/or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and/or used in the form of ingestible tablets, buccal tables, troches, capsule ' s, elixirs, suspensions, syrups, wafers, and/or the like.
  • Such compositions and/or preparations should contain at least 0.1 %> of active compound.
  • the percentage of the compositions and/or preparations may, of course, be varied and/or may conveniently be between about 2 to about 75% of the weight of the unit, and/or preferably between 25-60%.
  • the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and/or the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, and/or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and/or the like; a lubricant, such as magnesium stearate; and/or a sweetening agent, such as sucrose, lactose and/or saccharin may be added and/or a flavoring agent, such as peppermint, oil of wintergreen, and/or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, and/or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and/or the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as
  • NSP4 was purified from recombinant-baculo virus pAC461-G10 infected Spodoptera frugiperda (Sf9) cells expressing gene 10 by FPLC on a QMA anion exchange column as previously described (Tian et al, 1994 and Tian et al, 1995), and with an additional affinity purification step on a column containing anti-NSP4 antibodies.
  • Different NSP4 preparations of >70% and 90% purity gave the same biologic results.
  • the protein was sterile based on bacteriologic culturing in L-broth incubated at 37 C for one week, and lacked endotoxin based on testing by the limulus amebocyte lysate (LAL) assay (Levin 1968 and Novitsky 1984).
  • LAL limulus amebocyte lysate
  • VP6 was purified to >95% purity from recombinant-baculoviras pAc461/SAll-G6 infected Sf9 cells by gradient centrifugation as previously described (Zeng et al, 1996). Both proteins were lyophihzed and diluted in sterile PBS to a final volume of 50 ⁇ l per dose, regardless of the route of administration.
  • Synthetic NSP4-specific and control peptides utilized in this study were originally selected based on algorithms which predict surface potential (Parker et al, 1986), turn potential (Pt) (Chou 1978), and amphipathic structure (Margolit et al, 1987). A block length of 11 was used and an amphipathic score (AS) of 4 was considered significant.
  • Sequences were selected based on the high predicted propensities for folding into amphipathic helices and reverse turns, because small peptides which typically lack any folding pattern in an aqueous environment can fold into an ordered secondary structure resembling the nascent protein if the structural propensity is high (Dyson et al, 1988; Dyson et al, 1991; Dyson et al, 1988; Dyson et al, 1995; Yao et al, 1994; Waltho et al, 1993; Dyson et al, 1992; Wright et al, 1988).
  • NSP4 114-135 peptide, mNSP4 13 IK (DKLTTREIEQVELLKRIKD KLT, SEQ.ID.NO:4) AS 31 ; and a peptide from the COOH- terminus of the Norwalk virus capsid protein having a centrally located tyrosine residue (Jiang et al, 1990), NV 464-483
  • NSP4 114-135 DKLTTREffiQVELLKRIYDKLT 35 1.12 (YDKL) 2705
  • NSP4 2-22 EKLTDLNYTLSVITLMNNTLH 13.9 1.12
  • TDLN 2434
  • TKDE 4092
  • NSP4 ml31K DKLTTREIEQVELLKRI ⁇ K DKLT 31.4 • 1.08 (KDKL) 2669 NV 464-483 DTGRNLGEFKAYPDGFLTCV 41.4 1.58 (YPDG) 2204
  • NSP4 sequence from rotavirus SAll (Both et al, 1983).
  • Norwalk virus (NV) sequence from Jiang et al, 1990.
  • Underlined sequence is the region of the NSP4 2-22 peptide which overlaps with the NSP4 114-135 peptide. This substitution decreases the trun potential from 1.12 to 1.08.
  • the mutated tyrosine to lysine residue is shown in bold and in parentheses.
  • AS amphipathic score.. A block length of 11 was used with an AS of 4 considered significant (Margolit et al., 1987).
  • PT turn potentials greater than 1.0 within the selected peptide based on the algorithm of Chou and Fasman (1974, 1978).
  • Mr Theoret ⁇ cal mass.
  • peptides Prior to use, peptides were further purified either by HPLC on a semi-preparative, reverse-phase C18 column (uBondapak, Waters) or by multiple elutions from a conventional gel filtration column (1.5 mm X 40 mm). Peptide purity was confirmed prior to inoculations by gel filtration chromatography (Protein-Pak 60 column, lO ⁇ m, Waters) on a Waters HPLC unit. The elution profiles were monitored by UN absorption (Lambda-Max LC-spectrophotometer, Waters) at 220nm and recorded by a 745 Data Module (Waters). The elution buffer was PBS, pH 7.2, and the flow rate 0.5 ml/min. Sterility was confirmed as described for ⁇ SP4 protein.
  • KLH keyhole limpet hemocyanin
  • NSP4 114-135 peptide-specific antiserum was generated in CDl mice and New
  • emulsified antigen 100 mnol of peptide
  • mice were immunized every three weeks by the IM, SC and IP routes.
  • Preimmunization and postimmunization sera were evaluated by peptide ELISAs (titer of 400-3200) as previously described (Ball et al, 1994) and by Western blot analyses.
  • Purified NSP4 protein, peptide alone, or cross-linked to itself, were administered to young (6-10 days) and older (11-25 days) outbred CDl or inbred Balb/C mice, and outbred Sprague-Dawley rats by the intraperitoneal (IP), intraileal (IL), intramuscular (IM), subcutaneous and oral routes.
  • IP intraperitoneal
  • IL intraileal
  • IM intramuscular
  • the peptide or protein inocula were diluted in sterile PBS to a final volume of 50 ⁇ l per dose, regardless ofthe route of administration or inoculum.
  • a 30 G needle was employed for the IP and IL delivery of the inocula.
  • Peptide was delivered orally to young mice by gavage using a PE-10 polyethylene flexible tubing (Intramedic, Becton Dickinson) and food coloring.
  • animals were anesthetized with isofurane (Anaquest), a small incision was made below the stomach, the inocula were directly injected into the upper ileum, and the incision was sealed with polypropylene sutures (PROLENE 6-0, Ethicon). The pups were isolated, kept warm, and closely monitored for a minimum of 2 hrs prior to returning them to their cage.
  • Diarrhea induction by the NSP4 protein and peptides was carefully monitored for 24 hrs following the inoculations. Each pup was examined every 1-2 hr for the first 8 hr and at 24 hr post inoculation by gently pressing on the abdomen. Diarrhea was noted and scored from 1 to 4 with a score of 1 reflecting unusually soft, loose, yellow stool, and a score of 4 being completely liquid stool. A score of 2 (mucous with liquid stool, some loose but solid stool) and above was considered diarrhea. A score of 1 was noted, but was not considered as diarrhea. The scoring was done by a single person and the pups were coded during analysis of diarrhea. Other symptoms monitored included lethargy, coldness to the touch, and ruffled coats in older animals.
  • Unstripped intestinal mucosal sheets from 19-22 and 35 day old mice were analyzed for chloride secretory responsiveness to NSP4 114-135.
  • Short-circuit currents (Isc) were measured across unstripped intestinal mucosal sheets from 19-22 and 35 day old CDl mice using an automatic voltage clamp (Bioengineering, Univ. of Iowa) as described previously (Sears et al, 1995 and Morris et al, 1994). The mid-ileum of the mouse intestines was utilized.
  • the mucosal bath contained sodium-free (N-methyl-D- glutamine) substituted Ringers to minimize the effects on Isc of cAMP stimulated electrogenic Na + glucose co-transport across the small bowel (Grubb 1995).
  • basal Isc measurements were taken and intestinal mucosal sheets were challenged with cross-linked peptide (either NSP4 114-135, NSP4 2-22, or mNSP4 13 IK), the calcium-elevating agonist carbachol (Cch), or the cAMP-agonist forskolin (FSK). Bumetamide sensitivity was tested and confirmed the chloride secretory response.
  • NSP4 protein induces age-dependent diarrhea in mice Whether administration was IP or intraileal (IL), diarrhea was observed within 1 to 4 hr post inoculation, typically continued for up to 8 hr, but occasionally persisted for 24 hr.
  • Purified NSP4 (0.1-5nmol) was administered by the IP route to 6-7 and 8-9 day old CDl pups. In 6-7 day old CDl pups, IP administration of 0.1 nmol of NSP4 induced diarrhea in 60%) of the mice, whereas no disease was induced in 8-9 day old mice with the same concentration of protein (FIG. 1). IP administration of 1 nmol of NSP4 resulted in 100% of the 6-7 day pups with diarrhea, and 60%> ofthe 8-9 day old mice with disease.
  • NSP4 NSP4-induced diarrhea in 90% of the older (8-9 day) mice. Additional clinical symptoms included lethargy and coldness to the touch, which were observed in the majority of treated animals with diarrhea of all ages. The induction of diarrhea by NSP4 was shown to be specific for this protein as administration of the same volume of buffer or VP6 had no effect.
  • mice 11-12 X-linked NSP4 114-135 10 2/6
  • mice 15-17 X-linked NSP4 114-135 10 0/6
  • CDl mice 25 X-linked NSP4 114-135 100-200 0/8
  • FIG. 2 Additional data showing a dose response in 6-7 day old CDl pups is presented in FIG. 2.
  • the amount of peptide administered is shown in nanomoles and micrograms.
  • 0.04- 1.0 nmols (1-25 ⁇ g) of purified NSP4 was administered to 6-7 day old CD-I pups by the IP route.
  • a correlation between increasing incidence of diarrhea and increasing dose was seen (FIG. 2) over the range tested.
  • the NSP4 114-135 peptide has an AS of 35, is localized in the cytoplasmic domain of NSP4, and mobilizes intracellular calcium in eukaryotic cells (Tian et al, 1994 and Tian et al, 1995).
  • NSP4 114-135 peptide Doses exceeding 50 nmol (lmM) of NSP4 114-135 peptide were sufficient to induce diarrhea in the majority of young mice when administered by the IP route. The diarrhea was observed within 1 to 4 hr post inoculation and typically continued for up to 8 hr, but occasionally was present for 24 hr. The severity of diarrhea typically increased with time. That is, a mouse with a diarrhea score of 1 in the first hr post inoculation would have a diarrhea score of 4 in the next hr. Various degrees of lethargy were noted following the administration of peptide and this was most pronounced at 3 to 4 hr post inoculation. The lethargy was accompanied by the pups being cold to the touch and was age-dependent. The severity of the induced diarrhea was greater in the Balb/C pups. No symptoms were noted with control peptides (NSP4 2-22, NV C-terminus) or PBS administered to the same age and species of mice.
  • NSP4 120-147 peptide induces diarrhea in mice
  • a peptide corresponding to amino acid residues 120-147 of NSP4 was prepared and tested in 5-7 day old pups. When a dose of 100 nmols was administered, all (5 of 5) animals exhibited severe diarrhea. A dose of 5 nmols induced diarrhea in 7 out of 8 animals (88%).
  • One skilled in the art can readily appreciate that both the length of the peptides, and the number of residues that overlap in adjacent peptides, can be varied at the discretion of the practitioner without deviating from the spirit ofthe present invention.
  • Diarrhea induction in CDl and Balb/C mice by cross-linked NSP4 114-135 The NSP4 114-135 peptide was cross-linked to itself by glutaraldehyde and administered to young mouse pups by the IP route to determine if the diarrhea induction was affected by structure or oligomerization. Diarrhea was induced in the majority of the CDl pups at a lower dose of NSP4 114-135 when the peptide was cross-linked to itself when compared to the peptide alone (FIG. 7A and FIG. 7B).
  • the NSP4 114-135 peptide was tested in a second species, the Sprague-Dawley rat to determine whether the disease response induced by this peptide was only effective in young mice.
  • IP inoculation of 100-250 nmol of cross-linked peptide induced diarrhea in 78%> of young (6 days) rat pups and in none of the older (10 day) rat pups (FIG. 5).
  • the response in rats was slower than that observed in mice, taking from 6 to 12 hr before the onset of diarrhea was noted, compared to 2 to 4 hours post inoculation for the mice, and required a higher concentration of peptide to observe disease.
  • the induced diarrhea and lethargy in the young rats frequently persisted for up to 48 hr. These differences may reflect the difference in size and intestinal transit time between the rat and mouse or species (genetic) variation.
  • Diarrhea induction is age dependent
  • NSP4 114-135 peptide was administered by the IP route to different age outbred mice and rats. Diarrhea was observed in the young mice within 2 to 4 hr post inoculation, whereas reduced or no symptoms were seen in older (11-12 or 15-17 days) animals (FIG. 5 and FIG. 6). With IP administration of peptide alone, disease was induced in 60% of the 6-7 day old CDl pups with no symptoms noted in the 11-12 and 15-17 day old mice. IP administration of cross-linked peptide resulted in 90%) diarrhea induction in 6-7 day old CDl pups, 30%> disease in 11-12 day old pups, and only 10%) disease in 15-17 day old mice.
  • NSP4 90-123 which overlaps the 114-135 peptide by 9 residues, induced diarrhea in only 20%> (2/10) ofthe mice tested (Table 3). The percentage of diarrhea induction increased to 50% when the NSP4 90-123 peptide was crosslinked.
  • NSP4 antibodies protect against virus-induced disease
  • NSP4 antibodies to protect against virus-induced disease was tested by challenging pups, born to dams which were immunized with the NSP4 114-135 peptide or a control peptide, with a high dose of infectious SAl 1 virus, FIG. 8, left hand side. Diarrheal disease in pups born to dams immunized with the NSP4 114-135 peptide was significantly (Fisher's exact test) reduced in severity, duration, and in the number of pups with diarrhea (Table 4). The NSP4 2-22 peptide was used as a control peptide, as it does not induce diarrhea in pups. Table 4.
  • NSP4 2-22 100% 63% 25% 3.5+ (16/16) (10/16) (4/16)
  • NSP4 antiserum or control antiserum was orally administered every 4-6 hours for 60 hr, FIG. 8, right hand side.
  • the pups administered NSP4-specific antibody had significantly reduced diarrheal disease compared to animals given no treatment, rabbit pre-immune serum or normal rabbit serum (NRS), (Table 5). These data show the potential of NSP4 antibodies to block rotavirus-induced disease.
  • Cystic Fibrosis is caused by a defect in the gene that codes for the cAMP-activated chloride channel called CFTR.
  • the CFTR channel is defective and chloride secretion ⁇ and hence water secretion — is greatly diminished. Without sufficient secretion of water, membranes accumulate excessive amounts of mucous and eventually become obstructed.
  • HIV gpl20 causes diarrhea in mice
  • HIV Human immunodeficiency virus
  • gpl20 HIV glycoprotein 120
  • gpl20 enterotoxin
  • Other proteins of HIV or other retrovirus or other proteins of other viruses may be found to have similar functional activity - i.e., to directly induce diarrhea.
  • Example 23 Identification of small molecule inhibitors of NSP4/receptor interaction.
  • the above data demonstrate that effective treatment of rotavirus-induced diarrhea can be accomplished through inhibition of NSP4's interaction with its receptor.
  • Identification of small molecule inhibitors of NSP4 is well within the ability of the ordinary practitioner according to known techniques.
  • Small molecule inhibitors are known in the art to refer to any ligand which can bind to a target molecule with sufficient affinity to inhibit the target molecule's activity.
  • Libraries of small molecules such as random peptide libraries, random oligonucleotide libraries, and pharmaceutical drug libraries, are available either according to known techniques or commercially, and may be quickly and easily screened against a purified target molecule for small molecules that bind with high affinity to a target molecule. Examples include the "FliTrx Peptide Library,” (Invitrogen) and the SELEX technology.
  • NSP4 protein from each of these two strains has been cloned and expressed in a baculovirus expression system and purified.
  • the purified NSP4 proteins were tested for their ability to induce diarrhea in mouse pups.
  • the NSP4 protein from the virulent strain causes increased intracellular calcium concentration and induced diarrhea while that of the attenuated strain did not.
  • comparisons can be made using virulent and avirulent pairs of amino acid sequences.
  • identification of NSP4 sequences that correlate to an attenuated phenotype makes it a routine matter to construct attenuated reassortment viruses that include such an NSP4 sequence, using techniques that are well known to those skilled in the art. This permits the construction of rotaviruses for use as vaccines that retain the antigenicity of the virulent strain yet display an attenuated phenotype as a result of the incorporation into the genome of the virus a nucleic acid coding for an NSP4 protein having a selected sequence.
  • Vaccines comprising NSP4 in the form of a toxoid may be prepared from purified NSP4 protein, NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150.
  • the purified protein can be chemically treated, using known techniques, to inactivate the biological activity ofthe NSP4 protein while retaining the immunogenicity.
  • the purified protein may be treated with a 10%o solution of formaldehyde at about 37 . C for about an hour.
  • One skilled in the art will recognize that other equivalent protocols to produce a toxoid may be employed without deviating from the spirit of the invention.
  • the toxoid will typically be washed with buffer, for example phosphate buffered saline or the like, and formulated into a vaccine.
  • the toxoid may be in solid form such as adsorbed to alum or the like. Alternatively, the toxoid may be in solution in any pha ⁇ naceutically acceptable liquid.
  • the toxoid may be administered as a vaccine in the absence of adjuvant.
  • a vaccine formulated with the toxoid may include adjuvants including but not limited to alum, Freund's complete and incomplete adjuvants, Ribi's adjuvant, bacterial and mycobacterial cell wall components and derivatives thereof, liposomes and any other adjuvant formulation known in the art.
  • Vaccines thus formulated may be administered using parenteral or mucosal routes such as by intraperitoneal, intranasal, intragastric, subcutaneous, intramuscular, or rectal application.
  • Example 26 Characterization of the receptor for NSP4
  • the human intestinal cell line HT29 was assayed for sensitivity to NSP4.
  • these cells showed an increase in intracellular calcium levels.
  • trypsin the response is ablated.
  • the binding of radiolabeled NSP4 protein to responsive cells is dose-dependent and saturable as would be expected for a receptor dependent phenomenon.
  • these two results demonstrate that NSP4 binds to a protein receptor.
  • Recent tests with respiratory epithelial cells have demonstrated that these cells do not respond to NSP4 and do not bind radiolabeled NSP4 protein.
  • the mRNA from a responsive cell can be isolated using standard techniques and reverse transcribed into cDNA. This cDNA can then be inserted into a vector and then used to transform the nonresponsive cell line.
  • exemplary vectors include, but are not limited to, insect expression vectors, bacteria expression vectors, mammalian expression vectors or viral expression vectors.
  • the genomic DNA from the responsive cells may be inserted into a vector and used to transform the nonresponsive cell line.
  • the fransformed cells will be screened for the expression of the receptor using routine techniques, for example, by screening for cells capable of binding radiolabeled NSP4. Cells that express the receptor will be isolated.
  • NSP4 112-175 was produced in an insect cell suspension culture system and purified by immunoaffinity chromatography as reported elsewhere (Zhang et al, 2000). The purified NSP4 112-175 was lyophihzed, and stored at 4 C in a dessicator until used. SDS-15% PAGE/silver staining and Western blot were used to evaluate protein purity.
  • NSP4 production and purification were conducted in an insect cell suspension system, purified by FPLC and immunoaffinity chromatography (Zhang et al, 1998 and Tian et al, 1996).
  • VP6 was expressed in SF9 cells grown in optimized serum-free media, SF90011 SFM (Gibco, Grand Island, NY), and purified by CsCl isopycnic centrifugation (Zeng et al, 1996).
  • TMB peroxidase substrate tetramethylbenzidine and H 2 O 2
  • chromogenic reagent Kirkegaard and Perry Laboratories, Gaithersburg, MD
  • Optical densities (O.D.) at 450 nm were measured with an ICN Flow Titertech Multiscan Plus MK11 plate reader (McLean VA).
  • Antibody titers were defined as the reciprocal of the highest dilution giving a net O.D. value (O.D. value of detected serum minus O.D. value of pooled pre-immune serum) higher than 0.1.
  • the level of replication of SAll in the intestine of suckling mice was determined by titration of infectious virus by plaque assay (Ramig 1988). Similar experiments could not be performed with ECwt challenged mice because the ECwt virus does not replicate efficiently in vitro. Additional mock-infected (PBS) mice were also included in this experiment as controls. The entire intestinal tract was removed from one SAll- and mock-infected mouse pup from 1-7 days post infection (DPI). The experiment was repeated once, for a total of two mouse pups for every timepoint.
  • Each intestinal tract was homogenized separately in 1 ml of serum-free medium 199, extracted with an equal volume of Freon (l,l,2-Trichloro-l,2,2- frifluoroethanol, Fisher Scientific, Springfield, NJ), the water soluble phase was collected and freated with 20 ⁇ g/ml of trypsin for 30 min at 37 C. Each intestinal sample was tested in duplicate for virus titer in plaque assays with MA10 4 cells.
  • S.frugiperda insect cells were grown and maintained in TNM-FH (Hinks) medium (Gibco, Grand Island, NY) containing 10%> fetal bovine serum (FBS).
  • TNM-FH Hinks
  • FBS fetal bovine serum
  • Baculovirus recombinants encoding the following rotavirus proteins were used: pFastBac/SAl l-10 112-
  • NSP4 aa 112-175 was expressed in and purified from the medium of insect Sf9 cells.
  • Purified NSP4 112-175 contained a single band with an apparent molecular weight of 7,000 identified by SDS-15% PAGE/silver staining (FIG. 11 A, arrow). A single band was also visualized by Western blot using rabbit anti-NSP4 peptide 114-135 (FIG. 11B, arrow), rabbit anti-NSP4 full-length antiserum, or a rabbit anti-NSP4 peptide 120-147 antiserum.
  • Seronegative female mice were subcutaneously and intramuscularly immunized with 14 ⁇ g NSP4 112-175 plus 20 ⁇ g Quillaja saponaria [adjuvant QS-21 (Kensil, et al, 1991)] (NSP4-dam) or with 20 ⁇ g QS-21 alone (QS-21 -dam).
  • the first inoculation was followed by a booster two weeks later at which time breeding was begun. A second booster was given on the tenth day of breeding. Each dam was tail-bled on the indicated days for determination of antibodies by ELISA.
  • the control group (QS-21-dam) was inoculated with QS-21 alone. In the initial experiments, pups were nursed by their own dams. The serum antibody titers in NSP4-dams, QS-21 -dams and all pups were determined on 0, 7, and 16 DPP. The serum antibody titers to NSP4 aal 12-175 and to NSP4 full-length detected by ELISA were essentially the same (FIG. 12A and FIG. 12B).
  • 13 A and 13B show that (a) sera from the NSP4-pups cross-nursed by QS-21 -dams maintained their low antibody status (GMT, 3x10 ) during the experimental period, at levels similar to their antibody level at birth (white bars); and (b) by 7 DPP, QS-21-pups cross-nursed by NSP4-dams acquired antibody titers in the sera (GMT, 9xl0 4 ) similar to those of their nursing NSP4-dams which were maintained throughout the experimental period (gray bars). Cross-nursing experiments confirmed that 97% of antibody in pups was acquired from lactogenic transfer and only 3%o from transplacental transfer.
  • each pup was challenged by stomach gavage with either 20 diarrhea dose 50% (DD50) of SAl l or 10 DD50 of ECwt in 50 ⁇ l of endotoxin-free PBS.
  • DD50 diarrhea dose 50%
  • SAl l DD50
  • ECwt endotoxin-free PBS.
  • Virus inocula were not trypsin-activated prior to inoculation. All cages were coded and individual mice were checked for diarrhea daily for 7 or 8 days after inoculation by gentle palpation of their abdomen.
  • Stool classification was: 0, no stool; 1, normal stool; 2, normal stool accompanied with yellow pasty stool; 3, all yellow pasty stool; 4, milky-liquid stool.
  • the pups with a stool score >2 were considered to have diarrhea.
  • NSP4 112-175 was administered parenterally with the saponin adjuvant QS-21, rather than Freund's adjuvant, because QS-21 may be licensed for use in humans and is more potent than aluminum phosphate (Ciarlet et al, 1998).
  • NSP4 112-175 administered parentally induced protection of suckling mice against diarrhea induced by heterotypic murine ECwt rotavirus.
  • Rotavirus replication is reduced in pups delivered to and nursed by the dams iimnunized with NSP4 112-175 plus QS-21.
  • the infectious virus titers of SAl 1 in the intestines of a subset of pups from NSP4-dams and QS-21 -dams were determined in two separate experiments. No virus was detected from the intestines of either of the mock- infected control pups (FIG. 14A, FIG. 14B and FIG. 14C). The yields of infectious SAll were consistently higher and were detected for a longer period of time in the two QS-21 - pups, compared to virus titers in the two NSP4-pups.
  • the serum antibody titers to VP6 in the NSP4-pups were significantly higher than those in the QS-21-pups (GMT, 3xl0 2 ) (PO.001).
  • a VP2-NSP4 fusion protein was constructed such that the coding region of gene 10, amino acids 112-175 without the stop codon, was cloned at the 3'-end of gene 2, amino acids 94-881 (SEQ.ID.NO:39 or SEQ.ID.NO:37).
  • a linker composed of 3 alanine residues followed by one alanine and one serine residues were included between the two coding regions.
  • This construct was cloned behind the plO promoter of the baculovirus transfer vector pBAC4x-l.
  • the entire coding region for VP6 (SEQ.ID.NO:38) was cloned behind the polyhedrin promoter in the pBAC4x-l vector containing NP2-NSP4 fusion gene.
  • Virus-like particles were expressed, purified and analyzed by Western blot and electron microscopy.
  • NP2- ⁇ SP4 and NP6 were detected by Western blot analysis using a polyclonal antibody against SAl 1 virus (FIG. 16). The presence of NP2- ⁇ SP4 was confirmed using an antiserum against NSP4.
  • the linker used above is one possibility of making such a fusion protein with VP2.
  • the fusion protein can contain one or more than one copy of a NSP4 protein.
  • a similar method could be used to make NLPs carrying proteins or peptides from other pathogens.
  • Antiserum to the NSP4 peptide from the rotavirus strain SAl 1 reacts with NSP4 from at least one other strain, the porcine rotavirus strain OSU that is classified in a separate genetic * group from the SAl 1 NSP4.
  • An in vitro assay was developed to measure "biologic neutralizing activity" of antisera to regions of NSP4 that would block enterotoxin or signaling of the enterotoxin.
  • NSP4 was tested to determine whether it can affect the transepithehal resistance (TER) of polarized epithelial cells grown on filters. It was found that polarized epithelial cells displayed high resistance and this resistance begins to decrease about 16 hours after the cells were treated with NSP4.
  • TER transepithehal resistance
  • NSP4 120-147 In contrast, antibodies to NSP4 114-135, NSP4 120-147 and to the full-length protein blocked the drop in TER in cells treated with NSP4 (FIG. 17 and Table 10). Thus, these data indicate that antibodies to NSP4 120-147 behave in a manner similarly to antibodies to NSP4 114-135. Since the 114-135 peptide causes diarrhea in mice and antibodies to NSP4 114-135 have also been shown to prevent diarrhea in mice, and the NSP4 120-147 peptide causes diarrhea in mice, these in vitro" neutralization data" support the claim that immunization with the peptide NSP4 120- 147 will result in protection from diarrhea. This data demonstrates that if a peptide causes diarrhea in mice, antibody to that peptide will protect against diarrhea. The data indicate that the diarrhea is caused by activation by the peptide (or protein) of a cell signaling pathway that results in diarrhea. Antibody to these peptides likely stops this signaling process.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention porte sur la glycoprotéine non structurelle de Rotavirus, NSP4, laquelle remplit différentes fonctions dans le cycle de réplication des virus, notamment au cours de la morphogenèse virale. Plus précisément, la NSP4 est un récepteur intracellulaire qui intervient dans le développement d'une enveloppe membranaire transitoire pendant la phase de bourgeonnement de nouvelles particules sous-virales dans le réticulum endoplasmique (ER). Ladite invention porte sur la NSP4 et ses méthodes d'utilisation. Elle a plus particulièrement trait à l'utilisation de la NSP4 et de fragments de celle-ci (NSP4 114-135, NSP4 120-147, NSP4 112-174, ou NSP4 112-150) dans la prévention ou le traitement de maladies à Rotavirus.
EP01992588A 2000-11-03 2001-11-02 Enterotoxine nsp4 de rotavirus et ses methodes d'utilisation Withdrawn EP1349571A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/705,621 US6673355B1 (en) 1995-06-14 2000-11-03 Rotavirus enterotoxin NSP4 and methods of using same
US705621 2000-11-03
PCT/US2001/045255 WO2002036172A2 (fr) 2000-11-03 2001-11-02 Enterotoxine nsp4 de rotavirus et ses methodes d'utilisation

Publications (2)

Publication Number Publication Date
EP1349571A2 true EP1349571A2 (fr) 2003-10-08
EP1349571A4 EP1349571A4 (fr) 2005-02-16

Family

ID=24834264

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01992588A Withdrawn EP1349571A4 (fr) 2000-11-03 2001-11-02 Enterotoxine nsp4 de rotavirus et ses methodes d'utilisation

Country Status (4)

Country Link
EP (1) EP1349571A4 (fr)
AU (1) AU2002220025A1 (fr)
CA (1) CA2427809A1 (fr)
WO (1) WO2002036172A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1576877A1 (fr) * 2004-03-04 2005-09-21 Bioprotein Technologies Production de protéines recombinantes de rotavirus dans le lait d'animaux transgéniques non-humains
WO2007081447A2 (fr) 2005-11-22 2007-07-19 Novartis Vaccines And Diagnostics, Inc. Antigènes de norovirus et de sapovirus
HUE033858T2 (en) * 2012-02-14 2018-01-29 Merial Inc Rotavirus unit vaccines and method for their preparation and use
CN104661675B (zh) * 2012-05-16 2019-04-30 阿德莱德研究创新私人有限公司 细胞疫苗和诱导受试者免疫反应的方法
EA201791683A1 (ru) * 2015-01-23 2017-12-29 Медикаго Инк. Получение ротавирусоподобных частиц в растениях
CN108504718A (zh) * 2018-03-27 2018-09-07 东莞市第三人民医院 一种CaSR在RV、NSP4感染后的表达水平分析方法
CN117866858B (zh) * 2023-11-30 2024-06-21 吉林农业大学 一种表达猪轮状病毒抗原的重组植物乳杆菌及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007941A1 (fr) * 1990-10-25 1992-05-14 University Of Saskatchewan Particules virales assemblees et leur utilisation dans un vaccin contre les maladies causees par rotavirus
WO2001027335A1 (fr) * 1999-10-14 2001-04-19 Baylor College Of Medicine Adjuvant
WO2001066566A2 (fr) * 2000-03-07 2001-09-13 Institut National De La Recherche Agronomique (Inra) Particules pseudovirales de rotavirus et leur utilisation pour vectoriser des proteines ou des acides nucleiques

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5186933A (en) * 1986-12-30 1993-02-16 Baylor College Of Medicine Synthesis and immunogenicity of rotavirus genes using a baculovirus expression system
AU6387196A (en) * 1995-06-14 1997-01-15 Baylor College Of Medicine Rotavirus enterotoxin nsp4 and methods of using same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007941A1 (fr) * 1990-10-25 1992-05-14 University Of Saskatchewan Particules virales assemblees et leur utilisation dans un vaccin contre les maladies causees par rotavirus
WO2001027335A1 (fr) * 1999-10-14 2001-04-19 Baylor College Of Medicine Adjuvant
WO2001066566A2 (fr) * 2000-03-07 2001-09-13 Institut National De La Recherche Agronomique (Inra) Particules pseudovirales de rotavirus et leur utilisation pour vectoriser des proteines ou des acides nucleiques

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CRAWFORD S E ET AL: "Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells" JOURNAL OF VIROLOGY 1994 UNITED STATES, vol. 68, no. 9, 1994, pages 5945-5952, XP002289212 ISSN: 0022-538X *
See also references of WO0236172A2 *
ZENG C Q-Y ET AL: "IMMUNIZATION OF MICE WITH THE VIRAL ENTEROTOXIN INDUCES HOMOTYPIC AND HETEROTYPIC PROTECTION AGAINST ROTAVIRUS DIARRHEA" GASTROENTEROLOGY, W.B.SAUNDERS COMPANY, PHILADELPHIA, US, vol. 120, no. SUPPL 5, April 2001 (2001-04), page A214, XP009035735 ISSN: 0016-5085 *
ZHANG MINGDONG ET AL: "A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells" JOURNAL OF VIROLOGY, vol. 74, no. 24, December 2000 (2000-12), pages 11663-11670, XP002303656 ISSN: 0022-538X *

Also Published As

Publication number Publication date
WO2002036172A3 (fr) 2002-09-19
AU2002220025A1 (en) 2002-05-15
CA2427809A1 (fr) 2002-05-10
EP1349571A4 (fr) 2005-02-16
WO2002036172A2 (fr) 2002-05-10

Similar Documents

Publication Publication Date Title
Dalsgaard et al. Plant–derived vaccine protects target animals against a viral disease
US5690938A (en) Oral immunization with multiple particulate antigen delivery system
Horie et al. Diarrhea induction by rotavirus NSP4 in the homologous mouse model system
US20220313812A1 (en) Senecavirus a immunogenic compositions and methods thereof
KR20190110605A (ko) 돼지 코로나바이러스 백신
US6673355B1 (en) Rotavirus enterotoxin NSP4 and methods of using same
NO341881B1 (no) Polypeptid, protein, metode for fremstilling av salmonid alphavirus nøytraliserende antistoffer, nukleinsyre, bærer, vaksine og diagnostisk sett
US20100034845A1 (en) Avian hepatitis e virus, vaccines and methods of protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis e
US8685411B2 (en) Rotavirus antigens
US20220160866A1 (en) Fusion protein useful for vaccination against rotavirus
JPH06504190A (ja) アセンブルしたウイルス粒子およびロタウイルス疾患ワクチンにおけるその使用
US6210682B1 (en) Rotavirus enterotoxin NSP4 and methods of using same
WO2002036172A2 (fr) Enterotoxine nsp4 de rotavirus et ses methodes d'utilisation
CN108431214B (zh) 人轮状病毒g9p[6]毒株和作为疫苗的用途
US20030175303A1 (en) Production method of recombinant rotavirus structural proteins and vaccine composition
Viaplana et al. Antigenicity of VP60 structural proteinof rabbit haemorrhagic disease virus
AU768605B2 (en) Rotavirus enterotoxin NSP4 and methods of using same
KR101671528B1 (ko) 돼지 유행성 설사병 바이러스의 에피토프와 점막면역보조제를 발현하는 형질전환체 및 이를 포함하는 백신 조성물
US20240050555A1 (en) Immunogenic composition useful for vaccination against rotavirus
JP2023544403A (ja) サーコウイルス科カプシドタンパク質を含む融合タンパク質、及びそれから構成されるキメラウイルス様粒子
CN117959420A (zh) 一种猪流行性腹泻病毒二价亚单位疫苗及制备方法
Ren Epitope mapping of the non-structural protein 2 (NSP2) of rotavirus
Kim Detection of group C Rotaviruses in Feeder Pigs, production of group a/c rotavirus-like particles (VLPS) and use of group A VLP Vaccines in cattle
Both et al. Analysis of Rotavirus Proteins by Gene Cloning, Mutagenesis, and Expression
JPH03503359A (ja) 外来シグナルペプチド及び任意のトランスメンブレンアンカー配列を包含する遺伝子発現系（特にロタウイルスｖｐ７タンパク質）

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030603

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

A4 Supplementary search report drawn up and despatched

Effective date: 20050103

17Q First examination report despatched

Effective date: 20050401

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060601