WO2002078717A2

WO2002078717A2 - Compositions and methods for reducing rna virus pathogenicity

Info

Publication number: WO2002078717A2
Application number: PCT/EP2002/003025
Authority: WO
Inventors: Melinda Beck; Bruce German; Orville Levander; Peter Van Dael
Original assignee: Societe Des Produits Nestle S.A.; University Of North Carolina At Chapel Hill
Priority date: 2001-03-28
Filing date: 2002-03-26
Publication date: 2002-10-10
Also published as: WO2002078717A3; AU2002308169A1

Abstract

The present invention provides methods and compositions for treating and/or preventing RNA viral diseases. In an embodiment, the present invention provides a method for treating RNA viral infections comprising the step of administering to a individual having an RNA viral infection, a therapeutically effective amount of selenium.

Description

COMPOSITIONS AND METHODS FOR REDUCING RNA VIRUS PATHOGENICITY

Background of the invention

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. Of course, 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.

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.

As noted above, 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.

There is, therefore, a need for improved methods and compositions for treating and preventing viral infection.

Summary of the invention

The present invention provides methods and compositions for treating and/or preventing RNA viral diseases.

In an embodiment, 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.

In another embodiment, 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.

In a further embodiment, 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. In a still further embodiment, 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.

It is an advantage of the present invention to provide an improved method for treating a disease caused by an RNA virus.

Still further, an advantage of the present invention is to provide a improved method for preventing the transmission of an RNA virus.

Additionally, an advantage of the present invention is to provide a composition for treating an RNA virus.

Furthermore, 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. In particular, the invention is intended for patients, infants, elderly, and pets.

These and other advantages of the present invention will be apparent from and are described in the detailed description of the presently preferred embodiments set forth below.

Brief description of the figures

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.

Detailed description of the invention 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.

Selenium 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. In an embodiment, 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. In an embodiment, theindividual's selenium levels are maintained at a level of at least 130 nanograms per mL of plasma. In an embodiment, 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. In this regard, the selenium can be administered as a salt of selenium. For example, sodium selenite or sodium selenate can be utilized. However, a variety of other selenium vehicles can be used. In an embodiment, 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. Preferably 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.

In an embodiment of the present invention, a method of treating an RNA viral infection is provided. The method 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.

Moreover, because the composition 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.

Because patients receiving parenteral nutrition are susceptible both to selenium deficiency and RNA viruses, 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. To this end, an effective amount of selenium is administered to an individual receiving an RNA viral vaccine, e.g., influenza. In a preferred embodiment, approximately 100 to about 200 micrograms selenium is administered to an individual receiving a vaccine. Preferably, 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. In an embodiment, 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. In particular, these populations are populations of infants, elderly, patients, and pets. By 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.

Examples

By way of example and not limitation, examples of the present invention will now be provided.

Example 1

A study was carried out to determine if host selenium status could influence the genome of an influenza virus by increasing the rate of viral mutation. 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.

The matrix proteins, 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.

The Ml protein is also involved in preventing newly exported vRNPs from re- entering the nucleus of the infected cell. 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.

To determine if the increase in pathogenicity was due to a change in the viral genome, a passage experiment was designed. Mice were fed a diet either adequate or deficient in selenium for four weeks prior to infection. At day 5 post infection (the time of peak lung pathology in both groups of mice), 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. If the change in viral virulence- were due to host factors alone, then the selenium -adequate mice should not develop increased lung pathology when infected with virus obtained from selenium -deficient mice as compared with infection with virus from selenium -adequate mice. However, mice infected with virus from selenium -deficient mice developed severe pathology, whereas mice infected with virus isolated from selenium -adequate mice developed only mild lung pathology. These results strongly suggest that a change in the viral genome had occurred in the influenza virus that replicated in selenium -deficient mice. In order to determine if a genome change had occurred, we selected 2 viruses isolated from selenium -adequate mice and 3 viruses isolated from selenium -deficient mice for sequencing. We chose to sequence the HA and NA genes, due to their association with epidemics, as well as the genome of the Ml and M2 proteins, due to their involvement in viral virulence as well as their antigenic association with the cellular immune response.

Our stock virus of influenza A/Bangkok/1/79 (Flow Laboratories, McLean VA) was propagated in the allantoic fluid of 10-day old embryonated hen's eggs. Sequence analysis of the HA, NA, Ml and M2 of our stock virus and comparison to published sequences of influenza A/Bangkok/ 1/79 (GenBank accession numbers KOI 140, JO2092, AF201843, AF008901, AF008899, KOI 150) revealed one nucleotide difference in the HA (nt930: G-»A, no aa change), no changes in the NA, and 5 differences in the matrix protein (ntl47: C-»T, aa change A-»V; nt510: T-»A, aa change L- H; nt688: C-»T, no aa change; nt756 and 757: TG-→-AT, aa change M-»N). Thus, our virus stock was 99% homologous with previously published sequence data. Sequencing of the HA region of the influenza virus obtained from selenium - deficient and selenium -adequate mice demonstrated 2 nt changes in the virus from selenium -deficient mice and 2 nucleotide changes in the virus from selenium -adequate mice as compared to our stock virus (see Table 1 below). One nt change was identical in both viruses. One nt change in the virus from the selenium -adequate mice led to a change in an amino acid. The other nt changes were silient mutations.

Sequencing of the NA region revealed no changes between the stock virus and selenium -adequate virus. The NA genome isolated from the selenium -deficient animals had one nt change that led to a change in an amino acid (see Table 1 below).

As illustrated in Table 2 below, 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. However, 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.

This result indicates that the rate of mutation in the matrix protein was accelerated in the selenium -deficient mice compared with the selenium -adequate mice. The fact that the genome for the matrix protein is generally stable among influenza A strains makes these results even more intriguing. 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.

Our study examined virus isolated in eggs and expanded in tissue culture prior to sequencing. The introduction of mutations by growing the virus in vitro is not responsible for the large number of nucleotide changes in the virus grown in the selenium-deficient animals. Virus grown in selenium-adequate mice differed by only 3 nt changes vs 29 nt changes in the virus isolated from the selenium-deficient mice. Thus, the changes in the virus grown in the selenium-deficient mice were due to the conditions within the host, rather than to culture conditions.

The fact that the genome for the matrix protein was significantly altered in the virus isolated from the selenium-deficient mice when compared with the selenium- adequate mice demonstrates that a deficiency in selenium can have a profound effect on the genome of influenza virus. Infection with influenza virus increases the oxidative stress status of the host and infection with influenza virus also upregulates the expression of NF-κB, a redox sensitive nuclear transcription factor. We hypothesize that the influenza viral infection, coupled with a lack of antioxidant protection from selenium, leads to a state of heightened oxidative stress in the selenium-deficient animals. This increase in oxidative stress may have led to direct oxidative damage to the viral RNA, resulting in an altered genome with increased pathogenicity. Significantly, once these changes in the genome had occurred, even mice with normal selenium status were susceptible to the increased virulence of the virus. • The fact that the nucleotide changes were predominantly found in the matrix protein, rather than the HA or the NA, was an unexpected finding. The HA and the NA are exposed on' the surface of the virion, and are associated with antigenic changes of the virus. However, 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.

The results of this experiment demonstrate the considerable influence that selenium status of the host may exert on a viral pathogen and suggests that many RNA viruses may be susceptible to oxidative damage. The results demonstrate that the selenium levels of the host should be considered when exploring mutations and mutation rates of viruses. Finally, the results demonstrate a unique mechanism by which viruses can mutate and demonstrates the importance of selenium against viral disease. Table 1

Comparison of nucleotide sequences of influenza A/Bangkok/1/79 HA and NA proteins in stock virus vs. virus isolated from selenium-adequate and selenium-deficient mice.

Nucleotide # Virus Isolated From:

(3'-5') Stock selenium + selenium - Amino acid change

HA: 435 C C A No change

456 C T T No change

583 T A T F→Y

NA: 925 G G A V→l

Sequences based on isolates from two individual infected selenium-adequate mice and three individual infected selenium-deficient mice. Sequences within groups matched 100%. 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)

Table 2

Comparison of nucleotide sequences of influenza A/Bangkok/1/79 matrix protein in stock virus and virus isolated from selenium-adequate and selenium-deficient mice.

Nucleotide # Virus Isoli ated From:

(3'-5') Stock selenium + selenium - Amino acid change

136 A A C No change

205 G G A No change

309 G G A R→K

322 A A G No change

325 C C T No change

328 A A G No change

331 A A G No change Nucleotide # Virus Isolated From:

(3 '-5') Stock selenium + selenium - Amino acid change

334 T T C No change

370 A A C No change

371 G G T A→S

406 C C T No change

439 A A G No change

454 C C A No change

502 C C T No change

503 A A C No change

524 G G A A→T

544 A A C No change

566 C C T No change

568 G G A No change

610 A A G No change

619 G G A No change

652 C C T No change

655 G G A No change

667 G G A No change

670 A A G No change

677 G G A A→T

712 A A G No change

716 G G A D→N

740 A A G T→A

785 C A C P→T

Sequences based on isolates from three individual selenium-adquate mice and three individual infected selenium-deficient mice. Sequences shown in the table were found in all samples sequenced. The selenium-adequate genomes differed from each other by only one nt (785). For the three selenium-deficient samples, two samples matched 100% to each other, and differed from the third sample which had five additional nucleotide changes, all silent. Matrix genome was sequenced from nt 119-952. (Total number of nts of matrix protein gene: 1028). Ml sequence is nt 26-784 and M2 sequence is 26-51 and 740-1007.

Example 2

The purpose of this study was to determine if influenza infected selenium- deficient mice are at risk for increased pathology.

Methods and Materials:

Mice: Three-week-old C57B1/6J male mice (Jackson Laboratories, Bar

Harbor, ME) 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.

Diets: 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.

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 TCID₅₀ on MDCK cells. Stock virus was aliquoted in 0.5 mL volumes and stored at -80° C until needed.

Infection of mice: 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.

Histopathology of lungs: At days 4, 5, 6, 10 and 21 post infection, 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 μm) 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 TCID₅₀ on MDCK cells.

Measurement of antibody titer: 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 IFNγ. The mCK-5 probe set contains probes for Ltn, RANTES, Eotaxin, MlP-lβ, MlP-lα, 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.

Results

Selenium content of mouse diets: The selenium content of the commercial chow was determined to be 154 +8 μg selenium /kg for the selenium-adequate diet and below the instrumental detection limit of 2.7 μg 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.

Total number and phenotype of cells infiltrating the lungs: In order to further characterize the histopathology post influenza infection, cells recovered from the bronchoalveolar lavage (BAL) fluid of selenium-adequate and selenium-deficient mice were counted and stained for various cell surface markers. Stained cells were then analyzed by FLOW cytometry. As shown in Figure 3, the total number of cells recovered from the BAL fluid were significantly higher in the mice fed the diets deficient in selenium. This finding correlated with the increased histopathology found in the selenium-deficient mice.

The phenotype of the infiltrating cells was also assessed for the selenium-adequate and selenium-deficient mice. As shown in Figure 4a, selenium-deficient mice have increased percentages of CD8+ cells, macrophages and NK cells 5, days post infection when compared with the selenium-adequate mice. However, at day 10 post infection, 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. To determine if the deficiency in selenium affected the ability of the host to secrete neutralizing 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.

Viral Titers: 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. Using an 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 γ-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. Expression of mRNA for chemokines in the selenium-adequate mice was highest on days 4 and 5 post infection, and then sharply declined thereafter. Thus, mRNA levels for chemokines occurs early in the selenium-adequate mice, which corresponds with an early increase in lung inflammation. At later time points, the chemokine response declines, at which time the lung pathology is also resolving. However, the selenium-deficient mice have increased lung pathology at later time points, which also corresponds with the increase in mRNA for chemokines.

Conclusions:

This experiment was designed to determine if a deficiency in selenium could influence the pathogencity of a virus. Our data demonstrated that, infection of selenium-deficient mice with Influenza A/Bangkok/1/79 induces increased pathology when compared with selenium-adequate mice. Selenium-adequate mice infected with Influenza A/Bangkok develop a relatively mild inflammatory response. However, the inflammation is much more severe in the selenium-deprived animals. The severity of the inflammation is reflected in the increased number of inflammatory cells obtained by BAL as well as the pathology score.

The increase in total number of inflammatory cells present in the lungs of selenium-deficient influenza infected mice might have been expected to clear the virus earlier and/or reduce the viral titer when compared with selenium-adequate mice. However, clearance rates and viral titers were equivalent between selenium-deficient and selenium-adequate mice, suggesting that the increased number of inflammatory cells was unable to respond adequately to the virus.

Because of the importance of the CD8+ T cells in viral clearance, we determined the phenotype of the lung infiltrating cells of both selenium-adequate and selenium-deficient influenza infected mice. We found that although the percentage of CD4+, CD 8+ and NK cells was increased early during the infection in the selenium-deficient mice, the percentage of the cytotoxic T cells infiltrating the lungs late during infection were decreased. This result suggested that the selenium-deficient mice had impaired recruitment of CD8+ cells to the lung. Although the total amount'of inflammatory cells was increased in the selenium-deficient mice, a deficiency in total numbers of CD 8+ T cells likely contributed to the inability of the selenium-deficient mice to clear the virus faster than the selenium-adequate animals.

Recovery from influenza virus is mediated predominantly by cellular immune responses. CD8+ T cells are capable of lysing viral-infected cells and are known to be a major factor in influenza viral clearance. However, 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 γ-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. Thus, a THI response is thought to be important for recovery from viral infection.

We found that selenium-deficient mice had decreased γ-IFN and IL-2 compared with selenium-adequate mice. In addition, the selenium-deficient mice produced more IL-10, IL-13, IL-4 and IL-5 than selenium-adequate mice. Taken together, this suggests that the immune response in the lungs of influenza infected selenium-deficient mice was skewed towards a TH2 response rather than a THI response. A deficiency of IL-10 or IL-13 has been associated with autoimmune diseases in which an overactive immune response is responsible for the pathology. Thus, our results suggest that a deficiency in selenium led to increased inflammatory responses due to the overproduction of the pro-inflammatory cytokine IFN-γ and the decreased production of the anti-inflammatory cytokines IL- 10 and IL- 13.

Chemoattractant cytokines, or chemokines, 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.

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-lβ, MlP-lα, 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.

Although not wanting to be bound to any theory, one possibility of why a deficiency in selenium would lead to increase in chemokine expression in influenza infected animals is an increase in oxidative stress in the deficient 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-κB, is thought to be upregulated by an increase in oxidative stress. NF-κB, once activated, will upregulate the production of mRNA for a number of genes, including the chemokines RANTES and MCP-1. The activation of NF-κB has also been associated with influenza viral infection. Thus, the increased lung pathology in the selenium-deficient animals may be due to an excess activation of NF-κB (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. In summary, mice fed a diet deficient in selenium develop much more severe lung pathology post influenza virus infection when compared with selenium-adequate mice. Although 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. Furthermore, 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.

selenium-adequate * selenium-deficient

Day PI** Serum (μg/L) Liver (ng Se/g) Serum Liver

4 433+A35 ND# 40 +/-8.54 ND

5 489 +/-55 686.8 +/-41 32 +A8.3 80.77 +/-16

*Mice were fed the diets for 4 weeks prior to infection. **PI: Post infection. #ND: Not done.

Table 4

Neutralizing antibody titers post influenza A infection. Geometric Mean Titers Day post infection selenium-adequate selenium-deficient

4 20 +/-0 26.3 +/- 11

5 20 +/-0 20 +/-0 14 47.5 +/-23.1 54.6 +/-20

Titers shown are geometric mean titers +/- S.D. of 5 samples

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1. A method of treating influenza comprising the step of administering to an individual having influenza a therapeutically effective amount of selenium.

2. The method according to claim 1 wherein the individual is administered a sufficient amount of selenium to maintain the individual's selenium levels at a level of at least 75 nanograms per mL of plasma.

3. The method according to claim 1 or claim 2 wherein the individual is administered approximately 100 micrograms to about 400 micrograms of selenium per day.

4. The method according to one of claims 1 to 3 wherein the selenium is administered as part of a pharmaceutical.

5. The method according to one of claims 1 to 4 wherein the selenium is part of a complete nutritional product.

6. The method according to one of claims 1 to 5 wherein the individual is receiving parenteral nutrition.

7. A method of reducing the risk of contracting influenza comprising the step of administering to an individual a therapeutically effective amount of selenium.

8. The method according to claim 7 wherein the individual is administered a sufficient amount of selenium to maintain the individual's plasma level of selenium at a level of at least 75 nanograms per mL of plasma.

9. The method according to claim 7 or claim 8 wherein the individual is administered approximately 100 micrograms to about 400 micrograms of selenium per day.

10. The method according to one of claims 7 to 9 wherein the selenium is administered as a nutritional supplement.

11. The method according to one of claims 7 to 10 wherein the selenium is part of a complete nutritional product.

12. The method according to one of claims 7 to 11 wherein the individual is receiving parenteral nutrition.

13. A method of enhancing the efficacy of a viral vaccine comprising the step of administering to a individual receiving a viral vaccine a therapeutically effective amount of an antioxidant.

14. The method according to claim 13 wherein the antioxidant is selenium.

15. The method according to claim 13 or claim 14 wherein the selenium is administered prior to the individual receiving the vaccine.

16. The method according to one of claims 13 to 15 wherein the selenium is administered after the individual receives the vaccine.

17. A method of enhancing the efficacy of a viral vaccine compring the steps of administering to a individual that has received the viral vaccine a sufficient amount of selenium to maintain the individual's plasma level of selenium at a level of at least 75 nanograms per L of plasma.

18. The method according to claim 17 wherein the individual is administered approximately 100 micrograms to about 400 micrograms of selenium per day.

19. A method of enhancing the efficacy of an influenza vaccine comprising the step of administering to a individual receiving an influenza vaccine a therapeutically effective amount of selenium to maintain the individual's plasma selenium levels at a level of at least 100 nanograms per mL of plasma.

20. A method according to any of the preceeding claims wherein the the individual is an infant, an elderly, a patient or a pet.