WO2002078717A2 - Compositions et methodes pour reduire la pathogenicite de virus a arn - Google Patents

Compositions et methodes pour reduire la pathogenicite de virus a arn Download PDF

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WO2002078717A2
WO2002078717A2 PCT/EP2002/003025 EP0203025W WO02078717A2 WO 2002078717 A2 WO2002078717 A2 WO 2002078717A2 EP 0203025 W EP0203025 W EP 0203025W WO 02078717 A2 WO02078717 A2 WO 02078717A2
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selenium
individual
mice
virus
influenza
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PCT/EP2002/003025
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WO2002078717A3 (fr
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Melinda Beck
Bruce German
Orville Levander
Peter Van Dael
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Societe Des Produits Nestle S.A.
University Of North Carolina At Chapel Hill
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Priority to AU2002308169A priority Critical patent/AU2002308169A1/en
Publication of WO2002078717A2 publication Critical patent/WO2002078717A2/fr
Publication of WO2002078717A3 publication Critical patent/WO2002078717A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to the treatment and prevention of diseases. More specifically, the present invention relates to compositions and methods for reducing the transmission of and treating diseases caused by RNA viruses.
  • viruses are known to cause a number of disease states. Viruses comprise intracellular molecular particles, having a central core of nucleic acid and an outer cover of protein, including sometimes lipid.
  • the nucleic acid core either RNA or DNA, represents the basic infectious material that, in many cases, can penetrate susceptible cells and initiate infection alone. Several hundred different viruses may infect man. In fact, many viruses have only recently been recognized. See, Merck Manual, 16 th Ed., p. 182.
  • Viral diseases are not susceptible to antibiotics. But, antibiotics are used to prevent complications, particularly in patients liable to superinfection with bacteria pathogens. The efficacy of such treatments is debatable, and indeed, indiscriminate use of antibiotics in viral infections (e.g., measles) may be harmful. Merck Manual, p. 183.
  • RNA viruses present an important pathogen to humans and animals. Diseases such as influenza, poliomyelitis, hepatitis, encephalitis, AIDS, hantaviruses, hemorrhagic fever, and many other diseases are known to be caused and transmitted through RNA viruses. Indeed, more than seventy percent (70%) of the known viruses either have RNA as genetic material or replicate via an RNA intermediate.
  • RNA viruses An issue with respect to RNA viruses is that the mutation rates during virus replication are much greater than those operating during replication of cellular DNA. This results in the generation of mutant genomes. Thus, due to the mutation rates it is difficult to treat RNA viruses. In addition, these mutation rates also make it difficult to provide an effective vaccine against at least certain RNA viruses.
  • RNA viruses are responsible for influenza. Infectious influenza viruses causes widespread morbidity and mortality. Each year over 20,000 deaths occur in the United States alone due to infectious influenza virus and from complications arising from post infection. Although vaccines have been developed with respect to certain strains of influenza viruses, due to the mutation rates of such viruses, often, such vaccines are not effective. Furthermore, methods of eradicating and treating diseases caused by RNA viruses are either non-existent for certain types of diseases or not entirely effective. For example, Acquired Immune Deficiency Syndrome (AIDS) for a number of years could not even be effectively controlled. Although today method of treatments are available that are effective in extending the lives of those that acquire HIV infection, methods of curing AIDS still are not existent.
  • AIDS Acquired Immune Deficiency Syndrome
  • the present invention provides methods and compositions for treating and/or preventing RNA viral diseases.
  • the present invention provides a method for treating influenza comprising the step of administering to an individual having influenza, a therapeutically effective amount of selenium.
  • the present invention provides a method for reducing the risk of influenza by reducing the mutations of the virus genome causing influenza comprising the step of administering to an individual a therapeutically effective amount of selenium.
  • the present invention provides a method of enhancing the efficacy of a viral vaccine comprising the step of administering to an individual receiving a viral vaccine a therapeutically effective amount of an antioxidant.
  • a method of enhancing the efficacy of an influenza vaccine is provided comprising the step of administering to an individual receiving an influenza vaccine a therapeutically effective amount of selenium.
  • an advantage of the present invention is to provide a improved method for preventing the transmission of an RNA virus.
  • an advantage of the present invention is to provide a composition for treating an RNA virus.
  • an advantage of the present invention is to provide a composition for improving the efficacy of a vaccine. Moreover, an advantage of the present is to provide a composition and method for reducing the mutations of an RNA virus in vivo. It is also an advantage of the present invention to provide a method for treating influenza.
  • An additional advantage of the present invention is to provide a method of reducing mutations in RNA viruses. Further, an advantage of the present invention is to provide a method for improving a vaccine used to prevent the transmission of an RNA viral disease.
  • the present invention applies for different populations of individuals which are all the populations having the risk of being in contact with an RNA virus or several RNA viruses.
  • the invention is intended for patients, infants, elderly, and pets.
  • Figure 1 illustrates graphically the pathology score for mice infected with influenza pursuant to Experiment No. 2.
  • Figures 2a and 2b illustrate photomicrographs of lungs of mice pursuant to Experiment No. 2.
  • Figure 3 illustrates the number of cells recovered from the bronchoalveolar lavage fluid from infected mice pursuant to Experiment No. 2.
  • Figures 4a and 4b illustrate the percents of CDA+ cells, macrophages, and NK cells of mice pursuant to Experiment No. 2 at day 5 post-infection and day 10 post- infection.
  • Figure 5 illustrates lung virus titers of mice pursuant to Experiment No. 2.
  • Figure 6 illustrates graphically percent change in cytokine mRNA levels pursuant to Experiment No. 2.
  • Figure 7 illustrates graphically mRNA expressions for chemokines pursuant to Experiment No. 2.
  • the present invention provides compositions and methods for treating viral infections. Specifically, the present invention provides compositions and methods for treating RNA viral infections. Additionally, the present invention provides compositions and methods for reducing the transmission of RNA viral infections. Broadly, it has been found that the use of selenium can reduce the pathogenicity of RNA viruses (e.g., influenza, coxsackie, acquired immune deficiency syndrome, etc.) This discovery provides a number of possible methods of treating and preventing the transmission of RNA viral diseases.
  • the selenium can be provided as a pharmaceutical, nutriceutical, supplement, nutritional product or in other forms, either alone or with other components.
  • RNA viral infection is involved in the reoxidation of reduced glutathione. It has a close metabolic interrelationship with vitamin E. It is part of the enzyme glutathione peroxidase, which is thought to destroy peroxides derived from unsaturated fatty acids. Selenium deficiencies are' known especially in patients receiving parenteral nutrition. As set forth in detail below, Applicants have found that by increasing selenium levels that the virulence of RNA viral infection can be reduced. As noted above, methods of treating RNA viral infections as well as methods of preventing RNA viral infections are provided. Specifically, pursuant to the present invention, a sufficient " amount of selenium is administered to apatient having an RNA viral infection.
  • sufficient selenium is administered to the individual to maintain the selenium plasma level of the individual at a level of at least 75 nanograms per mL of plasma and preferably at a level of at least 100 nanograms per mL of plasma.
  • theindividual's selenium levels are maintained at a level of at least 130 nanograms per mL of plasma.
  • the method of the present invention includes administering to an individual at least 100 micrograms of selenium per day. For a selenium deficient individual, the individual should receive at least 100 micrograms to about 400 micrograms per day of selenium. In an embodiment, the individual will receive approximately 100 to about 200 micrograms of selenium per day.
  • the selenium can be administered either alone or as part of a full nutritional regiment.
  • the selenium can be administered as a salt of selenium.
  • sodium selenite or sodium selenate can be utilized.
  • a variety of other selenium vehicles can be used.
  • the composition for treating a viral infection comprises administering capsules providing approximately 200 to about 400 micrograms of sodium selenate. This will provide the individual with approximately 100 to about 200 micrograms of selenium.
  • the selenium will be provided in at least two separate dosages during the day. Therefore, each capsule could contain 100 micrograms of sodium selenate and the individual would take 1 to 2 capsules twice a day.
  • a method of treating an RNA viral infection comprises the step of insuring that a patient having an RNA infection maintains a selenium plasma level of at least 75 nanograms per mL of plasma. In a preferred embodiment, the patient's selenium levels are maintained at a level of at least 100 nanograms per mL of plasma.
  • compositions and method of the present invention reduces mutations of RNA viruses
  • methods of preventing the transmission, or at least reducing the risk of transmission, of RNA viruses are provided.
  • the methods include the steps of administering to an individual at risk of an RNA viral infection 100 micrograms to 400 micrograms of selenium. Preferably 100 micrograms to 200 micrograms of selenium are administered to the individual.
  • the individual's plasma level of selenium should be at least .75 nanograms per mL of plasma. In an embodiment, the selenium levels are maintained at a level of at least 100 nanograms per mL of plasma.
  • an embodiment of the present invention provides for the supplementation of parenteral nutrition with a sufficient amount of selenium to reduce the risk that the patient will acquire a disease from an RNA virus. A sufficient amount is believed to be enough selenium to ensure the patient is not selenium deficient.
  • Selenium deficiency can be determined by checking the patient's selenium plasma levels. Selenium plasma levels below 75 nanograms/mL of plasma are considered to be demonstrative of a patient that is selenium deficient. However, it is believed that even at levels of greater than 75 nanograms of selenium per mL of plasma but less than 100 the patient may still be sufficiently selenium deficient or be at risk to RNA viral diseases. To avoid selenium deficiency the plasma selenium levels should be at least 75 nanograms per mL of plasma. It is believed that the composition and methods of the present invention can be used to enhance the efficacy of a vaccine used to prevent the transmission of RNA viral diseases.
  • an effective amount of selenium is administered to an individual receiving an RNA viral vaccine, e.g., influenza.
  • an RNA viral vaccine e.g., influenza.
  • approximately 100 to about 200 micrograms selenium is administered to an individual receiving a vaccine.
  • the selenium is administered at least 2 to 3 days prior to the vaccine and for at least 2 to 3 days after receiving the vaccine.
  • the individual receiving the vaccine is maintained at a plasma selenium level of at least 100 nanograms per mL of plasma.
  • the invention is intended for various populations, including all the populations at risk of acquireing a diosease from a RNA virus.
  • these populations are populations of infants, elderly, patients, and pets.
  • patient it has to be understood an individual having a disease, related or not with a RNA virus infection; for example, it can be an individual having a bacterial infection, or an individual submitted to surgery.
  • the genome of the influenza virus consists of 8 segments of RNA which code for both viral structural proteins and nonstructural proteins involved in viral synthesis.
  • the hemagglutinin (HA) and neuraminidase (NA) are both present on the surface of the virus and are involved in attachment to and entry into the host cells. Both the HA and the NA are associated with the antigenicity of the virus and changes in their structure are primarily responsible for the year-to-year antigenic variation of the virus.
  • Genomic variation in the HA and NA can occur through two different pathways, termed antigenic drift or antigenic shift.
  • Antigenic drift is the gradual accumulation of point mutations in the HA and NA over time.
  • Antigenic shift is a sudden complete change in the antigenic properties of either the HA and/or the NA. Antigenic shift often involves the complete replacement of one gene coding for the HA and/or NA for another.
  • Ml and M2 are associated with increased virulence of the influenza virus, and are also important targets for cytotoxic T lymphocytes, the immune cell chiefly responsible for clearance of influenza virus from the lungs.
  • Ml the most abundant polypeptide in the virion, is thought to be involved in influenza virulence by accelerating the viral growth cycle due to rapid uncoating of the Ml protein from the viral ribonucleoproteins (vRNP). This rapid uncoating leads to increased vR P transport into the nucleus of the host cell and subsequent onset of viral transcription.
  • M2 is an integral membrane protein that acts as an ion channel.
  • M2 is a minor component of the virion, although part of the M2 protein is present on the surface of the virion, whereas the Ml protein is exclusively internal.
  • Previous studies have determined that the Ml protein is evolving very slowly while the M2 protein exhibits relatively rapid evoluntionary change in swine and human influenza viruses, but not in viruses recovered from avians. This difference in mutation rates is thought to be due to the exposure of M2 on the virion surface, thus subjecting the protein to immune pressure.
  • mice were fed a diet either adequate or deficient in selenium for four weeks prior to infection.
  • virus was isolated from the lungs of the selenium-adequate and selenium-deficient mice.
  • Five viral isolates from the selenium -deficient mice and five isolates from selenium -adequate mice were passed back into selenium -adequate mice.
  • mice infected with virus obtained from selenium -deficient mice developed severe pathology, whereas mice infected with virus isolated from selenium -adequate mice developed only mild lung pathology.
  • the genomic sequence of the viral matrix protein isolated from selenium -adequate animals had 1 nucleotide change compared with the stock virus that led to an amino acid change.
  • This nt change (no. 785) was found in only one isolate. The other isolate was identical to the stock virus.
  • the sequence of the matrix protein determined from virus that replicated in selenium - deficient animals had 29 nucleotide changes compared to the stock virus. Six of these nt changes resulted in amino acid changes.
  • the mutation rate of the influenza A virus has been calculated to be 10 "4 to 10 "5 mutations/nucleotide/replication cycle using a tissue culture system. Previous studies have reported that mutation rates of influenza virus will vary depending on the viral culture conditions. The growth of H3N2 viruses in eggs has a significant effect on the selection of antigenic variants of the virus compared to virus propagated in tissue culture. Only one amino acid change was found in egg-raised virus vs. 6 amino acid changes in tissue culture grown virus. All of these changes were found in the HA.
  • mice with normal selenium status were susceptible to the increased virulence of the virus.
  • the HA and the NA are exposed on' the surface of the virion, and are associated with antigenic changes of the virus.
  • our system did not involve the evolution of the influenza virus in a host over time, which is influenced by the immune pressure exerted by the host. Rather, our results reflect changes in the virus that occurred during its replication cycles in a single animal. It seems likely that the oxidative stress status of the host during the viral replication cycle contributed to the increased mutation rate of the matrix genome.
  • HA was sequenced from nt 181-810 and 855-1525 (Total nt number for HA is 1757).
  • NA was sequenced from nt 152-823 and 827-1304 (Total nt number for NA is 1392)
  • the purpose of this study was to determine if influenza infected selenium- deficient mice are at risk for increased pathology.
  • mice Three-week-old C57B1/6J male mice (Jackson Laboratories, Bar).
  • mice were housed 4/cage and provided with food and water daily. Mice were fed specified diets for 4 weeks prior to virus inoculation. Infection of mice with mouse-adapted strains of influenza virus induces an interstitial pneurnonitis, characterized by an influx of T and B cells and macrophages to the infected lung.
  • the mouse has a long history as a model system for influenza virus infection and is the most widely studied with respect to understanding the pathogenesis of infection with influenza virus.
  • mice were divided into 2 groups and fed either a diet adequate or deficient in selenium. Diets were purchased from Harlan Teklad (Indianapolis, IN). Selenium was added to the adequate diets as sodium selenite. The selenium level of the mouse diets was determined by continuous flow hydride generation atomic absorption spectrometry (HGAAS) after acid digestion.
  • HGAAS continuous flow hydride generation atomic absorption spectrometry
  • Virus Influenza A Bangkok/1/79 was propagated in 10-day old embryonated hen's egg. The virus was collected in the allantoic fluid and titered by both HA and TCID 50 on MDCK cells. Stock virus was aliquoted in 0.5 mL volumes and stored at -80° C until needed.
  • mice were lightly anesthetized with an intraperitoneal injection of ketamine (2.2 mg/mL) and xylazine (1.56 mg/mL). Following anesthesia, 0.05 mL of influenza A/Bangkok 1/79 (10 HAU) was instilled intranasally, and the mice were allowed to recover from the anesthesia.
  • Liver and serum selenium and GSH-Px levels Liver and serum selenium levels were determined by continuous flow HGAAS and graphite furnace AAS with longitudinal Zeeman background correction, respectively. The analysis was validated against NIST 1577b bovine liver (NIST, Gaithersburg, MD) and a commercial serum quality control material.
  • Serum glutathione peroxidase (GSH-Px) activity was determined according to Belsten and Wright, European Community — Flair Common Assay for whole-blood glutathione peroxidase (GSH-Px), Europ. J. Clin. Nutr. 49:921- 927.
  • mice were killed and their lungs removed for study.
  • the right lobe of the lung was removed, inflated with OCT diluted in PBS and embedded in OCT (Sigma, MO) and immediately frozen on dry ice. Sections (6 ⁇ m) were cut on a cryostat and fixed and stained with hematoxylineosin. The extent of infiammation was graded without knowledge of the experimental variables by the investigators. Grading was performed semiquantitatively according to the relative degree (from lung to lung) of inflammatory infiltration.
  • the scoring was as follows: 0, no inflammation; 1+ mild influx of inflammatory cells with cuffing around vessels; 2+ increased inflammation with approximately 25-50% of the total lung involved; 3+ severe inflammation involving 50-75% of the lung; and 4+ almost all lung tissue contains inflammatory infiltrates.
  • Determination of lung virus titers One quarter of the left lobe of the lung was removed immediately after the mice were killed and frozen in liquid nitrogen. The lung section was weighed and ground in a small volume of RPMI 1640 using a Tenbroeck tissue grinder (Fisher Scientific, Pittsburgh, PA). Ground tissues were then centrifuged at 2000 x g for 15 minutes and the supernate recovered and grown in the allantoic fluid of 10-day old embryonated hen's eggs. The allantoic fluid was further titered by TCID 50 on MDCK cells.
  • Serum neutralizing antibody titers were measured by inhibition of viral cytopathic effects (CPE).
  • Bronchoalveolar lavage Mice were killed and the thorax was opened. Lungs were lavaged with 1 mL PBS using a tracheal cannula. The recovered lavage fluid was subsequently centrifuged and the cell pellet was collected for analysis.
  • FACS analysis Cell suspensions from the bronchoalveolar lavage (BAL) fluid of infected (or uninfected control) mice were stained with the following anti-mouse monoclonal antibodies: PE anti-CD3, FITC anti-CD4 or FITC anti-CD8, FITC-Mac-3 and PE NK cells marker (Pharmingen, San Diego, CA). After staining, the cells were sorted and counted by FACS analysis on a FACScan machine using LYS YS II, Version 1.1 software (Becton Dickinson, San Jose, CA).
  • RNAse Protection Assay Total RNA from the mediastinal lymph nodes (which drain the lung) of uninfected and infected mice at each time period were prepared using TRIzol Reagent (GIBCO BRL, Grand Island, NY). Chemokine and cytokine levels were determined using the "RiboQuant Multipurpose Ribonuclease Protection Assay (RPA) System" with the mCK-5 probe set and the mCK-1 probe set (Pharmingen). The mCK-1 probe set contains probes for IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6 and IFN ⁇ .
  • RPA Ribonuclease Protection Assay
  • the mCK-5 probe set contains probes for Ltn, RANTES, Eotaxin, MlP-l ⁇ , MlP-l ⁇ , MIP-2, IP-10, MCP-1 and TCA-3.
  • the dried gel was exposed to X-ray film and developed for 24 hours at -70° C. Bands were detected and densitometrically quantitated using RiboQuant software. All chemokine and cytokine values were normalized to the housekeeping gene GAPDH.
  • Selenium content of mouse diets The selenium content of the commercial chow was determined to be 154 +8 ⁇ g selenium /kg for the selenium-adequate diet and below the instrumental detection limit of 2.7 ⁇ g selenium /kg for the selenium-deficient diet.
  • liver and serum selenium status In order to determine if feeding the selenium-deficient diet was able to significantly lower the selenium level as well as glutathione peroxidase activity (as a biomarker for selenium status), liver and serum samples were tested for selenium levels at days 4, 5 and 6 post infection. As shown in Table 3 below, the liver and serum selenium level was significantly decreased in mice fed the selenium-deficient diet as compared with mice fed the selenium-adequate diets. Similarly, glutathione peroxidase activity was also significantly decreased in the selenium-deficient mice (see Table 3).
  • Lung Pathology Lungs from infected mice were examined for histopathologic changes at days 4, 5, 6, 10 and 21 days post inoculation. As shown in Figure 1, mice fed the selenium-deficient diet had significantly more inflammation at days 4 and 6 post infection. The differences in inflammation were not significant between groups at day 5 post inoculation. For both groups of mice, the pathology peaked at day 6 post infection. The lung pathology in the selenium-adequate mice began to diminish after day 6, whereas the selenium-deficient mice still had severe pathology even at 21 days post infection. The infiltrate in both selenium-deficient and selenium-adequate mice was characterized as an interstitial pneumonitis, which is typical for an influenza infection in mice.
  • Figures 2a and 2b are photomicrographs of the lungs of a mouse fed a diet deficient (Figure 2a) or adequate (Figure 2b) in selenium at day 6 post infection. The figures demonstrate the increase in inflammation in the lungs of selenium-deficient animals.
  • BAL bronchoalveolar lavage
  • the phenotype of the infiltrating cells was also assessed for the selenium-adequate and selenium-deficient mice.
  • selenium-deficient mice have increased percentages of CD8+ cells, macrophages and NK cells 5, days post infection when compared with the selenium-adequate mice.
  • the percentage of CD8+ cells dropped in the selenium-deficient animal when compared with the selenium-adequate mice, suggesting an impairment of the immune response against the virus as illustrated in Figure 4b.
  • Antibody responses The development of an antibody response is a critical component of the immune response against influenza virus. Neutralizing antibodies protect against reinfection with the same strain of virus. In addition, a functioning T cell response is required in order for B cells to produce antibody. Thus, a defect in either B or T cell immunity can affect the secretion of virus-specific antibody.
  • serum from mice at 5 and 10 days post infection were analyzed for the presence of influenza-specific neutralizing antibody. As shown in Table 4, neutralizing antibody titers against influenza were similar in both the selenium-adequate as well as the selenium-deficient animals, suggesting that there was no impairment in the ability of B cells to produce antibody.
  • CD8+ T cells are believed to be primarily responsible for viral clearance in influenza infected lungs. Because the level of CD 8+ cells in the lungs of influenza infected selenium-deficient mice was decreased when compared with the selenium-adequate mice, an increase in viral titer of the selenium-deficient mice might be expected. However, as illustrated in Figure 5, lung virus titers of selenium-deficient mice were equivalent to the lung virus titers of the selenium-adequate mice, although there was wide variation within groups. All mice were able to clear the virus by day 10 post infection. RNAse Protection Assay: Both cytokines and chemokines are important mediators in the inflammatory response to influenza virus infection.
  • RNAse protection assay we looked at a number of cytokines and chemokines involved in inflammatory responses. We found differences in both cytokine and chemokine mRNA expression in mediastinal lymph nodes between selenium-adequate and selenium-deficient mice.
  • Figure 6 illustrates the percent change in cytokine mRNA levels from selenium-deficient mice as compared with selenium-adequate mice. At all time points, mRNA for ⁇ -IFN was much more abundant in the selenium-adequate mice when compared with the selenium-deficient mice. Similarly, mRNA IL-2 levels were also higher in the selenium-adequate mice.
  • the levels of mRNA for IL-4 and IL-5 were both increased at day 4in selenium-adequate mice compared with selenium-deficient mice, then decreased relative to selenium-deficient mice at days 14 and 21.
  • selenium-deficient mice had greatly increased levels of mRNA for IL-10 and IL-13 at day 6 post infection and for IL-4, IL-5, IL-10 and IL-13 at day 14 post infection when compared with selenium-adequate mice.
  • Chemokine responses were also affected by a deficiency in selenium. As illustrated in Figure 7, we found that mRNA expression for chemokines in the selenium-deficient mice occurred beginning at day 6 post infection, with the greatest increases occurring at day 10 and 14 post infection.
  • mRNA for chemokines expressed in the selenium-adequate mice was highest on days 4 and 5 post infection, and then sharply declined thereafter.
  • mRNA levels for chemokines occurs early in the selenium-adequate mice, which corresponds with an early increase in lung inflammation.
  • the chemokine response declines, at which time the lung pathology is also resolving.
  • the selenium-deficient mice have increased lung pathology at later time points, which also corresponds with the increase in mRNA for chemokines.
  • CD8+ T cells are capable of lysing viral-infected cells and are known to be a major factor in influenza viral clearance.
  • CD4+ T cells which secrete cytokines, are also capable of clearing influenza virus in the absence of CD8+ T cells.
  • CD4+ T cells can be further divided into two subsets: T helper 1 (THI) and T helper 2 (TH2).
  • THI responses are characterized by the release of ⁇ -IFN and IL-2, whereas TH2 responses are characterized by a release of IL-4 and IL-10.
  • the THI response generates cytokines which increase CD8+ T cells, whereas TH2 responses generally suppress CD 8+ T cell generation.
  • a THI response is thought to be important for recovery from viral infection.
  • Chemoattractant cytokines are inducible pro-inflammatory molecules involved in the recruitment of inflammatory cells to sites of injury or infection. Chemokines are also important in the trafficking of leukocytes to both lymphoid and nonlymphoid tissues. For example, mice that are deficient in the chemokine macrophage inflammatory protein la (MlP-la) develop much less lung inflammation post influenza virus infection when compared with normal mice. This finding points to the importance of the chemokine response for the development of inflammation post influenza infection.
  • MlP-la macrophage inflammatory protein la
  • Influenza infected selenium-deficient mice had an overexpression of mRNA for chemokines later in infection when compared .with the selenium-adequate mice.
  • the increase in RANTES, MlP-l ⁇ , MlP-l ⁇ , MIP-2,' IP-10 and MCP-1 all suggest that the inflammatory response was upregulated in these; mice
  • the continuing inflammation noted, in the selenium-deficient animals at a time when ! the pathology was resolving in the selenium-adequate mice suggests that the overexpression of the pro-inflammatory chemokines contributed to the continued influx of inflammatory cells in the lungs.
  • a deficiency in selenium would lead to increase in chemokine expression in influenza infected animals.
  • a deficiency in selenium a co-factor for the antioxidant enzyme glutathione peroxidase, would have led to an impaired oxidative stress defense system, which in turn would lead to increased oxidative stress in these animals.
  • the increase in oxidative stress would likely be most pronounced in the infected lung tissue, where the viral infection itself would contribute to the oxidative load.
  • the nuclear factor, NF- ⁇ B is thought to be upregulated by an increase in oxidative stress.
  • NF- ⁇ B once activated, will upregulate the production of mRNA for a number of genes, including the chemokines RANTES and MCP-1.
  • the activation of NF- ⁇ B has also been associated with influenza viral infection.
  • the increased lung pathology in the selenium-deficient animals may be due to an excess activation of NF- ⁇ B (due to the influenza virus infection itself and the increased oxidative stress due to a lack of glutathione peroxidase activity) which in turn upregulates the expression of chemokines.
  • These overexpressed chemokines induce in influx of inflammatory cells to the infected lung tissue.
  • a second possibility for the increased in lung pathology of the influenza infected selenium-deficient mice is the possibility of changes in the virus.
  • a normally benign coxsackievirus B3 (CVB3) becomes virulent in selenium-deficient mice due to a change in the viral genome, changing an avirulent virus to a virulent one.
  • the newly mutated virus is able to induce heart pathology in selenium-deficient animals. Once the mutations occur in the virus, even mice with normal selenium levels were also susceptible to cardiac pathology. It is possible that the genome of the influenza virus in the selenium-deficient mice changed to a more virulent genotype. This possibility awaits further study.
  • mice fed a diet deficient in selenium develop much more severe lung pathology post influenza virus infection when compared with selenium-adequate mice.
  • the increase in lung pathology was not associated with an increase in viral titer, it was associated with an increase in the mRNA expression of pro-inflammatory cytokines and chemokines and a decrease in the expression of anti-inflammatory cytokines.
  • the immune response in the lung infected tissue was shifted away from a THI response and towards a TH2 response in the selenium-deficient animals.
  • the experiment demonstrates the importance of adequate selenium levels for protection against viral infection.
  • the experiment demonstrates that selenium-dependent glutathione peroxidase may play an important role during an influenza induced inflammatory process.
  • Table 3 Serum and Liver selenium status for mice fed selenium-adequate or selenium- deficient diets.
  • Titers shown are geometric mean titers +/- S.D. of 5 samples

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  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Pulmonology (AREA)
  • Molecular Biology (AREA)
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  • General Chemical & Material Sciences (AREA)
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  • Inorganic Chemistry (AREA)
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Abstract

La présente invention concerne des méthodes et des compositions de traitement et/ou de prévention de maladies virales à ARN. Dans un mode de réalisation, la présente invention concerne une méthode de traitement d'infections virales à ARN comprenant l'étape consistant à administrer à un individu souffrant d'une infection virale à ARN, une dose thérapeutique efficace de sélénium.
PCT/EP2002/003025 2001-03-28 2002-03-26 Compositions et methodes pour reduire la pathogenicite de virus a arn WO2002078717A2 (fr)

Priority Applications (1)

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AU2002308169A AU2002308169A1 (en) 2001-03-28 2002-03-26 Compositions and methods for reducing rna virus pathogenicity

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US81938701A 2001-03-28 2001-03-28
US09/819,387 2001-03-28

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WO2002078717A2 true WO2002078717A2 (fr) 2002-10-10
WO2002078717A3 WO2002078717A3 (fr) 2003-08-28

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111568921A (zh) * 2020-04-20 2020-08-25 奥格生物技术（六安）有限公司 一种促进冠状病毒患者康复的新型硒制剂配方及其制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0596717A1 (fr) * 1992-11-05 1994-05-11 Chandra Consultants Complément nutritif pour personne agée
US5770217A (en) * 1997-07-02 1998-06-23 Atlatl, Inc. Dietary supplement for hematological, immune and appetite enhancement
WO1998030228A1 (fr) * 1997-01-13 1998-07-16 Emory University Composes et combinaisons de ces composes destines au traitement de l'infection grippale

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0596717A1 (fr) * 1992-11-05 1994-05-11 Chandra Consultants Complément nutritif pour personne agée
WO1998030228A1 (fr) * 1997-01-13 1998-07-16 Emory University Composes et combinaisons de ces composes destines au traitement de l'infection grippale
US5770217A (en) * 1997-07-02 1998-06-23 Atlatl, Inc. Dietary supplement for hematological, immune and appetite enhancement

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
F.GIRODON E.A.: "Impact of trace elements and vitamin supplementation on immunity and infections in institutionalized elderly patients" ARCHIVES ON INTERNAL MEDICINE, vol. 159, no. 7, 1999, pages 748-754, XP008013803 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111568921A (zh) * 2020-04-20 2020-08-25 奥格生物技术（六安）有限公司 一种促进冠状病毒患者康复的新型硒制剂配方及其制备方法

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AU2002308169A1 (en) 2002-10-15

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