Iota-Carrageenan is a potent inhibitor of rhinovirus infection
© Grassauer et al; licensee BioMed Central Ltd. 2008
Received: 23 July 2008
Accepted: 26 September 2008
Published: 26 September 2008
Human rhinoviruses (HRVs) are the predominant cause of common cold. In addition, HRVs are implicated in the worsening of COPD and asthma, as well as the loss of lung transplants. Despite significant efforts, no anti-viral agent is approved for the prevention or treatment of HRV-infection.
In this study we demonstrate that Iota-Carrageenan, a sulphated polysaccharide derived from red seaweed, is a potent anti-rhinoviral substance in-vitro. Iota-Carrageenan reduces HRV growth and inhibits the virus induced cythopathic effect of infected HeLa cells. In addition, Iota-Carrageenan effectively prevents the replication of HRV1A, HRV2, HRV8, HRV14, HRV16, HRV83 and HRV84 in primary human nasal epithelial cells in culture. The data suggest that Iota-Carrageenan acts primarily by preventing the binding or the entry of virions into the cells.
Since HRV infections predominately occur in the nasal cavity and the upper respiratory tract, a targeted treatment with a product containing Iota-Carrageenan is conceivable. Clinical trials are needed to determine whether Iota-Carrageenan-based products are effective in the treatment or prophylaxis of HRV infections.
The family Picornaviridae comprises some notable members, including human rhinovirus (HRV), which infects humans more frequently than any other virus. Infections with HRV lead to the common cold with symptoms such as sore throat, rhinitis, nasal congestion, and cough . The National Institutes of Health (NIH) estimates that there are more than a billion cases of common colds in the USA each year. Besides the self-limiting infection, HRV is implicated as a cause or predisposing agent for otitis media, sinusitis and exacerbations of asthma, as well as other lower respiratory tract disorders [1–4].
Despite significant efforts no anti-viral agent is approved for the prevention or treatment of HRV-infection. A number of anti-viral compounds have been evaluated for the management of HRV induced colds, including the capsid binders pirodavir and Pleconaril [3, 5–7]. Studies with biologicals such as intranasal Tremacamra a soluble intercellular adhesion molecule 1 (ICAM-1) and alpha interferon have shown that targeting HRV is possible especially when the drugs are applied prophylactically or the intervention is early. [8–10].
Another approach targets the HRV proteases 2A and 3C with small molecules. Protease 3C is an enzyme necessary for the posttranslational cleavage of viral precursor polyproteins. Studies with experimental HRV infection showed promising results for Ruprintrivir a compound developed by (Agouron/Pfizer) . Development of Tremacamra and Ruprintrivir has not advanced to phase III clinical trials until today.
To effectively inhibit the HRV induced inflammatory cascade of the common cold the treatment needs to be initiated rapidly after the first symptoms or even before. Since the HRV infection is self limiting and not life threatening in most cases a potential therapy has to be safe and effective with an almost unrecognizable level of side effects.
Polymers from various sources are substances that might bear these desired safety properties. In particular sulphated polysaccharides including Carrageenan, a sulphated polysaccharide extracted from red seaweed has an excellent safety profile and has shown anti-viral efficacy against several viruses. The anti-HIV-1 activity of Lambda-, Kappa- and Iota-Carrageenan and other sulphated polymers has been described previously [11, 12]. In a review, Gonzalez M.E. et al.  report an anti-viral efficacy of different sulphated polysaccharides including Iota-Carrageenan against several animal viruses. Iota-Carrageenan showed anti-viral activity against the enveloped viruses Herpes simplex virus type 1 and type 2, Semliki Forest virus (SFV), vaccinia virus, African swine fever virus (ASF), and against encephalomyocarditis (EMC) virus. Iota-Carrageenan had no effect on vesicular stomatitis virus (VSV), measles virus, polio virus type 1 (member of the picornaviridae) and adenovirus type 5. Carlucci et al. [11, 13] demonstrated a protective effect of Lambda-Carrageenan on genital herpes simplex virus infection in mice. Pujol et al.  showed the anti-viral activity of a Carrageenan isolated from Gigartina skottsbergii against intraperitoneal murine herpes simplex virus infection.
Carrageenan has been generally recognized as safe by the FDA. In addition, Carrageenan has been extensively used in the food, cosmetic and pharmaceutical industry as a thickener and gelling agent. In this report we show that Iota-Carrageenan inhibits the replication of HRV in tissue culture. Therefore Iota-Carrageenan might be a promising candidate for the evaluation of efficacy against HRV in clinical trials in humans.
Carrageenan promotes cell survival after HRV2 infection
Iota-Carrageenan reduces production of HRV particles
HeLa cells were seeded in 24-well plates (2 * 104 cells per well) and infected with HRV2 (0,1 TCID50/cell) in the presence of Iota-Carrageenan at a concentration of 200 μg/ml. When cell lysis was observed in the untreated control, supernatants were harvested. Viral titers were determined by TCID50 assays on HeLa cells. HRV2 replication in untreated control cells resulted in the generation of 108 TCID50/ml after 48 h (Figure 1B). Lambda- and Kappa-Carrageenan reduced HRV2 titers in cell supernatants by two orders of magnitude. Iota-Carrageenan exceeded the activity of Lambda- and Kappa-Carrageenan and prevented viral titer production for at least 6 orders of magnitude when compared with the untreated control (Figure 1B). Since, the detection limit was 102 TCID50 in this test an even higher effect cannot be excluded.
The anti-viral effect of Iota-Carrageenan is dependent on the amount of input virus
Search for Iota-Carrageenan resistant variants
Iota-carageenan blocks replication of HRV2 in primary human nasal epithelial cells (HNep)
Iota-carrageenan inhibits replication of HRV serotypes 1A, 8, 14, 16, 83, 84 on primary human epithelial cells
In this report we demonstrate that Iota-Carrageenan, a commercial thickening agent derived from seaweed, is a potent inhibitor of rhinovirus infectivity in vitro. Two other related polymers Lambda- and Kappa-Carrageenan show moderate effects and did not fully inhibit virus induced cell death in HRV2 infected HeLa cells (Figure 1).
Protection of HeLa cells from virus induced cell death was dependent on the amount of input virus in both cases, when cells were infected in the presence or absence of Iota-Carrageenan (Figure 2). When cells were treated after infection, protection was observed only for the lowest input amount tested (0,01 TCID50/cell; Figure 2B). Therefore we conclude that Iota-Carrageenan most likely inhibits binding or entry of the virus into the cells and not a later stage of viral replication. These findings are consistent with previous studies with other viruses that have shown that Carrageenan is active against several viruses in vitro and in vivo [11, 16, 18–24].
Iota-Carrageenan has been shown to be a potent inhibitor of papillomavirus infection with 50% inhibitory doses in the low ng/ml range . However, when tested against rhinoviruses Iota-Carrageeenan appears to be effective against HRV at concentrations several orders of magnitude higher in the low μg/ml range (Figure 3). This result is comparable with in-vitro data of other viruses such as HIV-1 and Herpes virus [15, 25].
Repeated passage of the HIV virus in the presence of polyanions can lead to resistance mediated by mutations in the envelope glycoprotein gp120, particularly in the V3 loop (K269E, Q278H, N293D), as originally shown for dextran sulphate, and subsequently for Zintevir and negatively charged albumins [26, 27]. While resistant variants emerge relatively fast with HIV-1 we were not able to detect a difference in an in-vitro test between the original virus stock and a HRV2 virus after 10 subsequent passages in the presence of Iota-Carrageenan at concentrations between 7 μg/ml and 20 μg/ml (Figure 4). Although the potential emergence of resistant variants deserves detailed and extensive studies we conclude that Iota-Carrageenan resistant variants do not occur with a high frequency. This result supports the hypothesis that Iota-Carrageenan prevents HRV virions from cell attachment or cell entry in a less specific manner when compared to the results that were obtained by Buck and co-workers for papillomavirus . However, it cannot be excluded that resistant variants of HRV2 may occur at later passages and further studies are needed.
In situ hybridization studies have revealed that the airway epithelial cell is the primary site of HRV infection in vivo [28, 29] and there is growing evidence that virally induced alterations in epithelial cell biology may contribute to disease pathogenesis [30, 31]. Thus we selected HNep cells as target cells for rhinovirus infection studies. Again Iota-Carrageenan was found to be effective against HRV2 on primary human epithelial cells with similar results when compared to the studies on HeLa cells (Figure 5). Our study also shows that viral titers in supernatants of infected HNep cells can vary by several orders of magnitude dependent on the strain (Figure 6). Replication studies with three Type A viruses HRV1A, HRV8, HRV16 and three Type B viruses HRV14, HRV83 and HRV84 revealed that Iota-Carrageenan is effectively inhibiting replication of Type A and Type B rhinoviruses when the polymer is present during infection (Figure 6). Differences between batches of HNep cells resulted in a variation of titers of HRV strains tested. However the anti-viral activity of Iota-Carrageenan was comparable in all tested HNep batches (data not shown).
Our data on primary cells are consistent with the data from infected HeLa cells and thereby support the hypothesis that Iota-Carrageenan interferes with viral replication at a very early stage of viral infection. Most likely, the binding of virions to the cells is hindered. It is not clear whether Carrageenan exerts any additional effects. The inhibitory effect of Iota-Carrageenan might be due to the occlusion of virion surfaces involved in binding to cellular receptors. Alternatively, obligatory conformational changes in the virus may be blocked and in addition, a post-attchment inhibitory effect may exist as described recently for papillomaviruses [12, 32].
Rhinovirus infections yearly account for huge economic losses in terms of lost school and working days. Moreover, recent evidence points towards rhinoviruses as a major cause in exacerbating asthma and COPD. For a review see Drescher et al 2007 . A number of molecules and strategies have been examined in order to combat rhinovirus and the whole family of picornaviruses [5, 12]. Despite this, no therapy has been approved for the treatment of rhinovirus infections yet, and patient care remains symptomatic.
Moreover, the efforts in therapeutic development are hampered by the fact that more than 100 distinctive HRV serotypes are circulating in humans. Our studies on primary HNep cells demonstrate that Iota-Carrageenan potently inhibits the replication of the seven distinct rhinovirus strains HRV1, 2, 8, 14, 16, 83 and 84 (Figure 5 and Figure 6). Although we are convinced that the result can be extrapolated for the whole family of human rhinoviruses further experiments are needed proof the efficacy on all strains of HRV.
The primary site of infection and replication of HRV in humans is the nasal mucosa. It is tempting to speculate that a targeted treatment of the nasal mucosa with Iota-Carrageenan might create a hostile environment for HRV and thereby block viral entry and replication. Carrageenan is generally recognized as safe for use in food and topical applications. Given the sensitivity and anti-viral effectiveness against several strains of HRV in primary human epithelial cells Iota-Carrageenan deserves consideration as a candidate for clinical trials for the prophylaxis and treatment of rhinovirus induced common cold.
Lambda carrageen, Kappa carrageen and Iota carrageen were purchased from FMC Biopolymers (Philadelphia, PA). The dry polymer powders were dissolved in cell culture water (PAA, Austria) to a final concentration of 0.4%. This stock solution was sterile filtered through a 0.45 μm filter (Sarstedt, Germany) and stored at 4°C until use.
Viruses, cell lines and media
HRV serotypes (HRV 1A, 2, 8, 14, 16, 83, 84) were obtained from the American Type Culture Collection (Manassas, VA) and grown on HeLa cells. The stocks were frozen at -80°C and virus titers were determined by TCID50 assay. The human cervical epithelial carcinoma cell line (HeLa) was obtained from the American Type Culture Collection. The cells were cultivated in Dulbecco's minimal essential medium (PAA) supplemented with 10% fetal bovine serum (PAA) and 1% antibiotic-antimycotic mix (PAA) in a 37°C incubator (Sanyo, Japan; CO2: 5%, relative humidity: >95%). During virus infection and viral experiments a medium containing 2% fetal bovine serum and 1% antibiotic-antimycotic mix was used.
Human nasal epithelial cells were obtained from PromoCell GesmbH (Heidelberg, Germany) and cultivated in airway epithelial cell growth media (PromoCell).
For determination of anti-viral activity a CPE inhibition assay was performed. HeLa cells were seeded in tissue culture plates 24 hours prior the experiments. At 80% confluence cells were infected with inoculums at indicated amounts of input virus (TCID50/cell). In order to test whether the polymers can inhibit viral replication the cells were infected with virus in the presence or absence of polymer. For determination of the CPE plates were washed with PBS (PAA) and stained with 1% crystal violet (Sigma, St. Louis, MO) in 20% ethanol (?) and 3.7% formaldehyde (?). Cell damage was quantified with respect to intensity of the stain retained by living cells in a plate reader (Labsystems, Finland) at 630 nm. Alternatively, the cell metabolism was measured with an XTT reagent kit (Roche, Switzerland). Cell survival in the presence of inhibitors was calculated by setting mock infected cells to 100% survival and cells infected without inhibitor to 0% survival. Virus titers in 50% tissue culture infectious doses (TCID50)/ml were determined according to Reed and Muench .
Search for resistant variants
HeLa cells (8 * 104 cells per well) were seeded in a 6-well plate and infected with HRV2. The cells were infected with a polymer virus mixture at final concentrations of 200, 20, 7 and 2 μg/ml with an amount of input virus of 0,1 TCID50/cell. As control, one well was mock infected with medium and one well was infected with mock treated virus. After an infection time of 30 minutes the plates were washed twice and an overlay with infection medium containing polymer or control medium was added. Samples were taken from each well when a clear cytopathic effect was visible in the untreated control well. For the next selection round the wells showing a clear CPE and difference to the control infection was used.
The infection cycle was repeated 10 times and the resulting virus sample was compared in a CPE inhibition test with the original virus stock.
We would like to thank Sekina Sadovsky-Sherif for excellent laboratory assistance. This work was supported in part by the Austrian Research Promotion Agency (FFG) grant numbers 813886 and 818252.
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