Iota-Carrageenan Is a Potent Inhibitor of Influenza A Virus Infection

The 2009 flu pandemic and the appearance of oseltamivir-resistant H1N1 influenza strains highlight the need for treatment alternatives. One such option is the creation of a protective physical barrier in the nasal cavity. In vitro tests demonstrated that iota-carrageenan is a potent inhibitor of influenza A virus infection, most importantly also of pandemic H1N1/2009 in vitro. Consequently, we tested a commercially available nasal spray containing iota-carrageenan in an influenza A mouse infection model. Treatment of mice infected with a lethal dose of influenza A PR8/34 H1N1 virus with iota-carrageenan starting up to 48 hours post infection resulted in a strong protection of mice similar to mice treated with oseltamivir. Since alternative treatment options for influenza are rare, we conclude that the nasal spray containing iota-carrageenan is an alternative to neuraminidase inhibitors and should be tested for prevention and treatment of influenza A in clinical trials in humans

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model

<div><p>Background</p><p>Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated <i>in-vitro</i> and <i>in-vivo</i>.</p><p>Principal Findings</p><p>We show <i>in-vitro</i> that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection.</p><p>Conclusion</p><p>A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.</p></div

Therapeutic efficacy in influenza H1N1(09)pdm lethally infected mice.

<p>Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan and Zanamivir (1 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant.</p

Isobologram of compound interaction.

<p>Comparison of the combination of different doses of both compounds necessary to reach 50% replication inhibition of (A) H7N7 and (B) H1N1(09)pdm (◆) to a model of dose additivity that would represent a curve progression of 1 (□). Dose response was tested with an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. On the x-axis the concentration of Zanamivir and on the y-axis the concentration of carrageenan is presented. The concentrations (determined as mean of 3 replicates) are given as the fraction of the respective IC₅₀ values of the different viruses with the particular compound (IC₅₀ = 1).</p

IC₅₀ values of carrageenan and Zanamivir for influenza A viruses of human and animal origin.

<p>^a IC₅₀ values were calculated in comparison to untreated infected cells. Each value represents the mean IC₅₀ of 6 replicates/assay and their standard deviation.</p><p>IC₅₀ values of carrageenan and Zanamivir for influenza A viruses of human and animal origin.</p

Binding of H1N1 influenza virus to iota-carrageenan.

<p>(A)-(F). Alexa Fluor 488-conjugated H1N1 influenza virus (H1N1-A488) was incubated with iota-carrageenan-coated agarose beads (iota-beads) or control beads for 30 min at room temperature and visualized microscopically. (A) Bright field picture of iota-beads, showing no green auto-fluorescence (B). (C) Control agarose beads incubated with H1N1-A488 do not facilitate unspecific virus binding. (D) Iota-beads incubated with H1N1-A488 demonstrates binding of virus to iota-carrageenan as evidenced by bright green staining of iota-beads. (E) Binding of H1N1-A488 to iota-beads is inhibited in the presence of iota-carrageenan (400 µg/ml), but is not abolished in the presence of CMC (400 µg/ml) (F). Scale bar  = 100 µm. (G) FACS analysis of MDCK cells incubated with H1N1-A488 in the presence of iota-carrageenan (400 µg/ml) (H) or control polymer CMC (400 µg/ml) (I) showing that binding of H1N1-A488 to cognate receptors is inhibited by iota-carrageenan but not CMC.</p

Effect of iota-carrageenan on pandemic H1N1/2009 virus.

<p>Confluent monolayers of MDCK cells in 6-well plates were washed free of protein-containing growth medium before use. An equal volume of virus suspension mixed with iota-carrageenan, containing 50 to 150 plaque-forming units, was added 5 to 10 min later, and plates were incubated at room temperature for 60 min with frequent shaking. The inoculum was removed and covered with an overlay medium consisting of 0.6% agarose (3 ml) in Eagle minimal essential medium and trypsin (2 µg/ml). Plates were incubated at 37°C in a humidified atmosphere with 5% CO₂. After 36 to 48 h, plaques were stained with crystal violet and counted. The percentage of plaque inhibition relative to infected control plates (y-axis) was determined for each drug concentration (x-axis). The standard deviation of three independent experiments is indicated.</p

Iota-carrageenan promotes cell viability and reduces viral titer of influenza A-infected MDCK cells.

<p>MDCK cells grown in 96-well plates were infected with H1N1 A/PR/8/34 virus (A) and H3N2 A/Aichi/2/68 virus (B) (0.01 PFU/cell) in the presence of carrageenans (iota-carrageenan black diamonds, kappa-carrageenan black squares) at concentrations as indicated on the x-axis in µg/ml. Plates were incubated at 37°C until cells in the control (no polymer added) showed >90% damage. Cell proliferation was determined with a Resazurin-based in vitro toxicology assay. Samples were measured fluorometrically by monitoring the increase in fluorescence at a wavelength of 590 nm using an excitation wavelength of 544 nm. Values obtained from mock-infected cells were set to 100%, and the values of cells infected in the absence of polymer were set to 0% (y-axis). (C)-(F) MDCK cells were infected with H1N1 A/PR/8/34 as before and further kept in the presence of iota-carrageenan (circles) or the control polymer CMC (squares) at indicated concentrations and 24 (C), 48 (D), 72 (E), and 96 hours (F) post infection, respectively. Supernatants were harvested, pooled, and subsequently used to determine the TCID50/ml according to the method of Reed and Muench <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014320#pone.0014320-Reed1" target="_blank">[59]</a>. The points represent the mean of a quadruplicate experiment, the standard deviation is indicated.</p

Effect of iota-carrageenan on influenza A-infected primary human nasal epithelial cells.

<p>Primary human nasal epithelial cells (HNep) cells grown in 96-well plates were infected with A/PR/8/34 virus (5 PFU/cell) in the presence of iota-carrageenan at concentrations indicated on the x-axis in µg/ml. 30 minutes after infection, the inoculum was removed and medium containing iota-carrageenan (black bars) or CMC (white bars) in indicated concentrations added. Cell proliferation was determined with a Resazurin-based in vitro toxicology assay. Samples were measured fluorometrically by monitoring the increase in fluorescence at a wavelength of 590 nm using an excitation wavelength of 544 nm. Values obtained from mock-infected cells were set to 100%, and the values of cells infected in the absence of polymer were set to 0% (y-axis). The bars represent the mean of a quadruplicate experiment, the standard deviation is indicated.</p

Therapeutic efficacy of iota-carrageenan against H1N1 influenza virus in a lethal mouse infection model.

<p>(A) Ten mice per group were intranasally infected with 8.7×10² PFU H1N1 A/PR/8/34 viral particles at day 0. Intranasal therapy twice daily with 60 µg iota-carrageenan in 0.5% NaCl or placebo (blue) started on the same day as infection (black), 24 h post infection (poi) (orange), or 48 h poi (green), and was performed twice daily for the entire experiment. P values were calculated by a Log-rank (Mantel-Cox) test. Asterisk, p<0.05, double asterisk p<0.01. (B)-(C). Determination of viral titers from nose (B) and lung (C) specimens. Five mice per group were intranasally infected at day 0 as before. The group receiving placebo (blue) was compared to groups receiving intranasal therapy with iota-carrageenan or oral therapy with oseltamivir (10 mg/kg/day in 5% sucrose) starting 24 (orange or light grey) and 48 hours (green or dark grey) post infection until groups of mice were sacrificed at day 2 and 5 days, respectively. Subsequently, nose and lung specimens of animals from each experimental group and time point were pooled and viral titers determined by plaque assays on MDCK cells at two different dilutions. Bars represent the mean±SEM.</p