Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
1998-05-26
2001-04-03
Bui, Phuong T. (Department: 1638)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S139100, C424S147100, C424S159100, C424S184100, C424S185100, C424S186100, C435S069100, C435S069300, C435S235100, C435S320100, C530S300000, C530S326000, C530S324000, C530S325000, C536S023720
Reexamination Certificate
active
06210682
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Related Technology
Rotaviruses are the leading cause of severe, life-threatening viral gastroenteritis in infants and animals (1) and are associated with sporadic outbreaks of diarrhea in elderly (2) and immunocompromised patients (3). These viruses have a limited tissue tropism, with infection primarily being restricted to cells of the small intestine (4). Rotavirus infections also cause morbidity and mortality in many animal species. Moreover, 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.
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 (5). 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 (6). Treatment of 8 day old mice with cortisone acetate which promotes premature maturation of intestinal epithelial cells, results in a reduced susceptibility to rotavirus-induced diarrhea, although the mice can still be infected (7). These data were interpreted to suggest that the capacity of murine rotaviruses to induce diarrhea in young, but not adult mice, is due to the quantity of rotavirus-binding receptors on the surface of villus epithelial cells in the young mouse intestine.
When compared to rotavirus infections in other species, 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 (8). 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.
Despite the prevalence of rotavirus infections and extensive studies in several animal models and many advances in understanding rotavirus immunity, epidemiology, replication and expression, rotavirus pathogenesis, specifically, the mechanism of diarrhea induction, remains poorly understood. Proposed pathophysiologic mechanisms by which rotaviruses induce diarrhea following viral replication and viral structural protein synthesis include malabsorption secondary to the destruction of enterocytes (9), disruption of transepithelial ion homeostasis resulting in fluid secretion (10), and local villus ischemia leading to vascular damage and diarrhea (11). However, these proposed mechanisms do not explain cases of rotavirus-induced diarrhea observed prior to, or in the absence of, histopathologic changes (12).
On the other hand, the pathophysiology of bacterial-induced diarrhea based on interactions with intestinal receptors and bacterial enterotoxins is well understood (13). The heat-stable toxin A and the heat-labile toxin of
E. coli
, and guanylin (an endogenous, 15 amino acid intestinal ligand originally isolated from rat jejunum) induce diarrhea by binding a specific intestinal receptor, increasing cAMP or cGMP, and activating a cyclic nucleotide signal transduction pathway (14). The net effect of these bacterial toxins is to increase Cl
−
secretion, and decrease Na
+
and water absorption.
Previous studies in insect cells indicated that a receptor-mediated phospholipase C pathway is associated with the increases in [Ca
2+
]
i
, following exogenous treatment of cells with NSP4 or NSP4 114-135 peptide (15). The rotavirus nonstructural ER glycoprotein, NSP4, has been shown to have multiple functions including the release of calcium from the endoplasmic reticulum (ER) in SF9 insect cells infected with recombinant baculovirus containing the NSP4 cDNA (15, 16). In addition, NSP4 disrupts ER membranes and may play an important role in the removal of the transient envelope from budding particles during viral morphogenesis (unpublished data). 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 (15, 16), and (ii) destabilize liposomes. (unpublished data).
Expression of NSP4 in insect cells increased [Ca
2+
]
i
levels from a subset of the thapsigargin-sensitive store (ER) (15). The [Ca
2+
]
i
mobilized by NSP4 or the NSP4 114-135 peptide was blocked by a phospholipase C inhibitor, the U-73122 compound, suggesting that a receptor-mediated pathway is responsible for the calcium release from the ER induced by NSP4 (15).
BRIEF DESCRIPTION OF THE INVENTION
This invention stems from the discovery of the 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.
This paper reports 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 wellknown 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.
It is also reported here that a synthetic peptide corresponding to aa 114-135 of SA11 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, we studied the response to the peptide 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. Because the entire protein possesses more poten
Ball Judith
Estes Mary K.
Tien Peng
Baylor College of Medicine
Bui Phuong T.
Fulbright & Jaworski L.L.P.
LandOfFree
Rotavirus enterotoxin NSP4 and methods of using same does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Rotavirus enterotoxin NSP4 and methods of using same, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Rotavirus enterotoxin NSP4 and methods of using same will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2544877