Iota-Carrageenan is a potent inhibitor of rhinovirus infection

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figures

Figure 1

Carrageenans promote cell viability of HRV2 infected HeLa cells and inhibit HRV2 replication in vitro. A. HeLa cells grown in 96-well plates were infected with HRV2 (0,1 TCID50/cell) in the presence of Carrageenans (different types are indicated at the x-axis) at a concentration of 200 μg/ml. Plates were incubated at 37°C until cells in the control (no polymer added) showed >90% damage. Cell proliferation was determined with an XTT-assay. OD values (492 nm) obtained from mock infected cells (compare x-axis) were set to 100%, and the viability of cells infected in the absence of polymer was set to 0% (y-axis). The bars represent the mean of a quadruplicate experiment, the standard deviation is indicated. B. HeLa cells in 24-well plates were infected with HRV2 (0,1 TCID50/cell) in the presence of Carrageenans (different types are indicated at the x-axis) at a concentration of 200 μg/ml. Viral infectivity in the supernatants was determined by TCID50 assay on HeLa cells (y-axis). Values represent the mean of six parallel titrations, standard deviation is indicated.

Figure 2

Iota-Carrageenan induced inhibition of HRV2 infected cells is dependent on the amount of virus. A. Preincubation of virus with polymer. HeLa cells grown in 96-well plates were infected with HRV2 in the presence of Iota-Carrageenan at concentrations as indicated at the x-axis. B. Treatment with polymer after infection. HeLa cells grown in 96-well plates were infected with HRV2. 30 minutes after infection medium containing Iota-Carrageenan at the concentrations indicated at the x-axis was added. Plates were incubated at 37°C until cells in the control (no polymer added) showed >90% damage. Cell proliferation was determined with an XTT-assay. OD values (492 nm) obtained from mock infected cells (compare x-axis) were set to 100%, and the viability of cells infected in the absence of polymer was set to 0% (y-axis). Black triangles indicate an amount of input virus of 0,01 TCID50/cell, black diamonds indicate 0,1 TCID50/cell and black squares indicate 1 TCID50/cell. A representative experiment is shown.

Figure 3

Iota-Carrageenan dose-dependently inhibits HRV2 replication in cell culture. (A) Preincubation of virus with polymer. HeLa cells grown in 12-well plates were infected with HRV2 (0,1 TCID50/cell) in the presence of Iota-Carrageenan at the concentration indicated at the x-axis. 30 minutes after infection the inoculum was removed and medium containing Iota-Carrageenan with the concentration indicated was added. Untreated cells were used as control (mock treated). B. Treatment with polymer after infection. HeLa cells grown in 24-well plates were infected with HRV2 (0,1 TCID50/cell). 30 minutes after infection the inoculum was removed and medium containing Iota-Carrageenan with the concentration indicated at the x-axis was added. Untreated cells were used as control (mock treated). Viral titers in the supernatants of infected cells were determined after 48 h by TCID50 assay on HeLa cells. Values are the means from six parallel titrations, standard deviation is indicated.

Figure 4

Iota-Carrageenan does not induce HRV2 escape mutants after 10 passages. A. HeLa cells in 6-well plates (8 * 104 cells per well) were infected with HRV2 in the presence of Iota-Carrageenan. After infection the cells were washed and medium containing polymer was added at concentrations between 2 μg/ml and 100 μg/ml. Plates were incubated at 37°C until cells in the control (no polymer added) showed >90% damage. Living cells were fixed and stained with crystal violet staining solution. B. Supernatants from infected wells with Carrageenan of 20 μg/ml were used for the next infection round. For the following infection rounds the supernatants of wells with 7 μg/ml or 20 μg/ml were used for the subsequent infection round. After ten repetitive infection experiments the sensitivity of the resulting virus (white bars) to different concentrations of Iota-Carrageenan (x-axis) was compared with that of the original virus (black bars). Cell proliferation was determined with an XTT-assay. Survival of mock infected cells was set to 100%, and that in the absence of polymer was set to 0% (y-axis). The bars represent means of six independent experiments standard deviation is indicated.

Figure 5

Effect of Iota-carageenan on HRV2 infected human nasal epithelial cells. A. Preincubation of virus with polymer. HNep cells were grown in 24-well plates were infected with HRV2 (0,1 TCID50/cell) in the presence of Iota-Carrageenan at the concentration indicated at the x-axis. 30 minutes after infection the inoculum was removed and medium containing Iota-Carrageenan with the concentration indicated was added. B. Treatment with polymer after infection. HNep cells were grown in 24-well plates were infected with HRV2 (0,1 TCID50/cell). 30 minutes after infection the inoculum was removed and medium containing Iota-Carrageenan with the concentration indicated at the x-axis was added. Viral titers in the supernatants of infected cells were determined after 48 h by TCID50 assay on HeLa cells (y-axis). Bars represent means of four parallel experiments, standard deviation is indicated.

Figure 6

Effect of Iota carageenan on the replication of HRVstrains 1A, 2, 8, 14, 16, 39, 83 and 84 on human nasal epithelial cells. HNep cells were grown in 96-well plates were infected with different HRV strains (indicated at the top of each panel; 0,1 TCID50/cell) in the presence of Iota-Carrageenan at the concentrations indicated at the x-axis. 30 minutes after infection the inoculum was removed and medium containing Iota-Carrageenan with the same concentration was added. Viral titers in the supernatants of infected cells were determined after 48 h by TCID50 assay on HeLa cells (y-axis). Bars represent means from four parallel experiments, standard deviations are indicated.