Rotaviruses are the most important viral agents causing gastroenteritis in children living in both developing and developed countries (Yolken et al, "Human Milk Mucin Inhibits Rotavirus Replication and Prevents Experimental Gastroenteritis", Journal of Clinical Investigation 90, 1984-1991, 1992). Rotaviruses also cause diarrhea in nursing homes and day care centers, among travelers, in adults who have contact with children, and in immunocompromised patients.
Rotaviruses cause 35-50% of severe diarrheal episodes in infants and young children in both developed and developing countries, and are the most important etiological agents of severe diarrhea in this age group. Rotaviruses infect over 90% of humans by age 3 in both developed and developing countries regardless of hygiene standards. In developing countries rotaviruses are estimated to cause 18 million cases of moderately severe or severe diarrhea and over 870,000 deaths annually in infants and young children under 5 years old (Hoshino et al., "Rotavirus Vaccine Development for the Prevention of Severe Diarrhea in Infants and Young Children", Trends in Microbiology 2:242-9, 1994). Thus, there is an urgent need to develop methods of preventing infection of humans, especially infants, from the consequences of infection by rotavirus.
Rotaviruses are included among the Reoviridae. They are nonenveloped viruses with an icosahedral morphology and a double-layered protein capsid containing 11 segments of a double-stranded ribonucleic acid (RNA) genome. The viruses are stable over wide pH and temperature ranges and in airborne aerosols. The virion (virus particle) delivers the nucleocapsid core through the acidic environment of the gastrointestinal tract and across the intestinal lumen to the target tissue. The complete virion is partially digested in the G.I. tract and the viruses are adsorbed to columnar epithelial cells that cover the villi of the small intestine. Upon entry into the cell, the outer capsid releases the core into the cytoplasm, and the enzymes in the core initiate mRNA production. The virus causes permeability changes and cell lysis. Rotaviruses are assumed to be passed from person to person by the fecal-oral route and the virus survives on hands and on inanimate objects.
It is known that sulfated polysaccharides specifically interfere with the adsorption process of enveloped viruses (De Clerq, "Antiviral Agents:Characteristic Activity Spectrum Depending on the Molecular Target with Which They Interact", Advances in Virus Research 42:p.2, 1993; De Clerq, "Selective Virus Inhibitors", Microbiologica 13:165-178, 1990). The group of sulfated polysaccharides contains a number of compounds including dextran sulfate and iota- (.iota.-), kappa- (.kappa.-), and lambda- (.lambda.-) carrageenans. The antiviral activity of dextran sulfate and carrageenans has been previously demonstrated against particular viruses. De Clerq in the 1993 review article refers (p.9) to a variety of enveloped viruses including retroviruses, herpesviruses, poxviruses, togaviruses, paramyxoviruses, and rhabdoviruses whose replication is inhibited by these sulfated polysaccharides. De Clerq, however, claims, contrary to the disclosure of the instant invention, that nonenveloped viruses, included reoviruses, are insensitive to inhibition by the sulfated polysaccharides.
The inhibitory property of sulfated polysaccharides, including the carrageenans, against various viruses has been described in the relevant literature. Gonzalez et al. ("Polysaccharides as Antiviral Agents: Antiviral Activity of Carrageenan", Antimicrobial Agents and Chemotherapy 31(9):1388-1393, 1987) found that iota-carrageenan showed strong antiviral activity against herpes simplex virus type I (HSV-1). Contrary to the results reported in De Clerq (1993) that sulfated polysaccharides inhibit virus attachment to the cell surface, Gonzalez et al. found, using labeled virion particles, that HSV-1 virions are internalized even in the presence of high concentrations of iota-carrageenan. This suggests that, at least for HSV-1, carrageenan inhibits a step in virus replication subsequent to viral attachment and internalization.
Sulfated homopolysaccharides, including cellulose sulfate, dextran sulfate and carrageenans, were shown by Mizumoto et al. ("Sulfated Homopolysaccharides with Immunomodulating Activities are More Potent Anti-HTLV-III Agents than Sulfated Heteropolysaccharides", Japanese Journal of Experimental Medicine 58(3): 145-151, 1988) to inhibit the growth of human T cell lymphotropic virus type III. Nakashima et al. ("Purification and Characterization of an Avian Myeloblastosis and Human Immunodeficiency Virus Reverse Transcriptase Inhibitor, Sulfated Polysaccharides Extracted from Sea Algae", Antimicrobial Agents and Chemotherapy 31(10):1524-1528, 1987) described a member of the lambda carrageenan family that had an inhibitory effect on reverse transcriptase activity of Human Immunodeficiency Virus (HIV) and suppressed replication of the virus in vitro. Girond et al. ("Antiviral activity of carrageenan on hepatitis A virus replication in cell culture", Research in Virology 142: 261-270, 1991) found that sulphated polysaccharides such as iota-, kappa-, and lambda-carrageenan showed a potent inhibitory effect on the replication of hepatitis A virus (HAV), a non-enveloped virus, in a human hepatoma cell line. Iota- and lambda-carrageenan were shown to be especially effective at inhibiting HAV antigen expression and infectivity.
WO 94/15624 discloses the use of sulphated polysaccharides, including carrageenan and dextran sulfate, to inhibit the transmission of HIV, the causative agent of AIDS. WO 88/06396 discloses a method for treating retroviral infections, including infection with the AIDS virus, by administering a carrageenan or a mixture of carrageenans. In EPA 293,826 sulfated polysaccharides, including dextran sulfate and carrageenans, are disclosed as in vitro inhibitors of HIV-1. GB 2,262,531 A disclosed that sulphated polysaccharides produced by a class of marine algae showed antiviral activity against both DNA and RNA viruses including the retroviruses and particularly against HIV.
In view of the different responses by different viruses to sulfated polysaccharides described above, it is clear that the response of a particular virus to carrageenan cannot be predicted with certainty without experimentation. The mechanism by which sulfated polysaccharides, particularly the carrageenans, inhibit viral replication and infectivity may not be uniform as different investigators reported contradictory findings when working with different viruses and cells types. It would not be obvious to one skilled in the art that a substance such as a sulfated polysaccharide that is an effective inhibitor of one virus would demonstrate similar efficacy against another virus. None of the cited references disclosed the use of carrageenans or other sulfated polysaccharides to inhibit infection of animal cells with rotavirus. The preferred sulfated polysaccharides of the present invention are the carrageenans or dextran sulfate, with the most preferred being lambda-carrageenan. Lambda-carrageen was shown in experiments to be described below to be the most efficacious in inhibiting rotavirus infection in vitro.
The prevalence of rotavirus infection in groups at risk, especially infants and children, and the seriousness of its effects attest to the need for developing effective treatments. The present invention demonstrates that carrageenan, and particularly .lambda.-carrageenan, is an effective inhibitor of rotavirus infection in animal cells. Animal studies are being conducted to further evaluate the efficacy of .lambda.-carrageenan in the treatment of rotavirus infection.