Modeling amantadine treatment of influenza A virus in vitro

We analyzed the dynamics of an influenza A/Albany/1/98 (H3N2) viral infection, using a set of mathematical models highlighting the differences between in vivo and in vitro infection. For example, we found that including virion loss due to cell entry was critical for the in vitro model but not for the in vivo model. Experiments were performed on influenza virus-infected MDCK cells in vitro inside a hollow-fiber (HF) system, which was used to continuously deliver the drug amantadine. The HF system captures the dynamics of an influenza infection, and is a controlled environment for producing experimental data which lend themselves well to mathematical modeling. The parameter estimates obtained from fitting our mathematical models to the HF experimental data are consistent with those obtained earlier for a primary infection in a human model. We found that influenza A/Albany/1/98 (H3N2) virions under normal experimental conditions at rapidly lose infectivity with a half-life of , and that the lifespan of productively infected MDCK cells is . Finally, using our models we estimated that the maximum efficacy of amantadine in blocking viral infection is ∼74%, and showed that this low maximum efficacy is likely due to the rapid development of drug resistance.     

Removal of amantadine hydrochloride by dialysis in patients with renal insufficiency.

Two patients with Parkinson''s disease and renal insufficiency had excessively high concentrations of amantadine hydrochloride in the blood. The amounts of the drug removed by hemodialysis and peritoneal dialysis were small. However, since extrarenal elimination is negligible in such patients, frequently repeated dialysis may be required to remove the drug.     

Prevention and treatment of influenza with hyperimmune bovine colostrum antibody

Background

Despite the availability of specific vaccines and antiviral drugs, influenza continues to impose a heavy toll on human health worldwide. Passive transfer of specific antibody (Ab) may provide a useful means of preventing or treating disease in unvaccinated individuals or those failing to adequately seroconvert, especially now that resistance to antiviral drugs is on the rise. However, preparation of appropriate Ab in large scale, quickly and on a yearly basis is viewed as a significant logistical hurdle for this approach to control seasonal influenza.

Methodology/Principal Findings

In this study, bovine colostrum, which contains approximately 500 g of IgG per milking per animal, has been investigated as a source of polyclonal antibody for delivery to the respiratory tract. IgG and F(ab'')2 were purified from the hyperimmune colostrum of cows vaccinated with influenza A/Puerto Rico/8/34 (PR8) vaccine and were shown to have high hemagglutination-inhibitory and virus-neutralizing titers. In BALB/c mice, a single administration of either IgG or F(ab'')2 could prevent the establishment of infection with a sublethal dose of PR8 virus when given as early as 7 days prior to exposure to virus. Pre-treated mice also survived an otherwise lethal dose of virus, the IgG- but not the F(ab'')2-treated mice showing no weight loss. Successful reduction of established infection with this highly virulent virus was also observed with a single treatment 24 hr after virus exposure.

Conclusions/Significance

These data suggest that a novel and commercially-scalable technique for preparing Ab from hyperimmune bovine colostrum could allow production of a valuable substitute for antiviral drugs to control influenza with the advantage of eliminating the need for daily administration.     

Specific structural alteration of the influenza haemagglutinin by amantadine.

Amantadine hydrochloride specifically blocks the release of virus particles from H7 influenza virus infected cells. This appears to be the direct consequence of an amantadine induced change in the haemagglutinin (HA) to its low pH conformation. The effect is indirect and mediated via interaction of the drug with the M2 protein since mutants altered in this component alone are insensitive to amantadine. The timing of drug action, some 15-20 min after synthesis, and its coincidence with proteolytic cleavage indicates that the modifications to HA occur late during transport but prior to insertion into the plasma membrane. Reversal by mM concentrations of amines and 0.1 microM monensin indicates that amantadine action causes a reduction in intravesicular pH which triggers the conformational change in HA. We conclude, therefore, that the function of M2 inhibited by amantadine is involved in counteracting the acidity of vesicular compartments of the exocytic pathway in infected cells and is important in protecting the structural integrity of the acid-sensitive glycoprotein.     

Interaction of influenza virus proteins with planar bilayer lipid membranes. II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (delta U), transversal elasticity module (E1) and membrane conductivity (G0) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 X 10(5) M-1 and 1.3 X 10(4) M-1 for rimantadine and amantadine, respectively. The changes in G0 took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 micrograms/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

Interaction of influenza virus proteins with planar bilayer lipid membranes II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (ΔU), transversal elasticity module (E₁) and membrane conductivity (G₀) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 · 10⁵ M^?1 and 1.3 · 10⁴ M^?1 for rimantadine and amantadine, respectively. The changes in G₀ took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 μg/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

圈养大熊猫流行感冒的防治

大熊猫(Ailuropoda melanoleuca)是我国特有的珍稀濒危保护动物,具有极高研究价值和观赏价值。随着圈养种群的迅速增加,各种传染性疾病对大熊猫的威胁较大,有效的疾病防治便成了保证大熊猫种群安全的重要措施。中国保护大熊猫研究中心的圈养大熊猫种群于2003年出现了一次较大的     

Susceptibility of influenza A viruses to amantadine is influenced by the gene coding for M protein.

Influenza A virus recombinants derived from "resistant" and "sensitive" parental viruses were examined for susceptibility to inhibition by amantadine. Correlation of gene constellation and amantadine susceptibility revealed that the gene coding for M protein influences sensitivity or resistance to amantadine. All recombinants which derived an M protein from an amantadine-resistant parent were found to be resistant to amantadine. All amantadine-sensitive recombinants derived an M gene from the amantadine-sensitive parent. However, a few amantadine-resistant recombinants which derived an M gene from the sensitive parent were also isolated, suggesting that the expression of amantadine sensitivity in these recombinants may be influenced by other genes.     

Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block.

The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.     

Inhibition of influenza virus uncoating by rimantadine hydrochloride.

In freeze-thaw lysates of MDCK cells infected with 32P-labeled influenza virus A/WSN in the presence of added RNase, acid-precipitable radioactivity diminished to about 50% of initial values within 90 min after a 1-h virus adsorption period. A similar preparation containing rimantadine at a concentration of 50 micrograms/ml exhibited only a 10% reduction in acid-precipitable radioactivity. These findings suggest that rimantadine interferes with uncoating of influenza virus in infected cells.     

Efficacy of combined therapy with amantadine, oseltamivir, and ribavirin in vivo against susceptible and amantadine-resistant influenza A viruses

The limited efficacy of existing antiviral therapies for influenza--coupled with widespread baseline antiviral resistance--highlights the urgent need for more effective therapy. We describe a triple combination antiviral drug (TCAD) regimen composed of amantadine, oseltamivir, and ribavirin that is highly efficacious at reducing mortality and weight loss in mouse models of influenza infection. TCAD therapy was superior to dual and single drug regimens in mice infected with drug-susceptible, low pathogenic A/H5N1 (A/Duck/MN/1525/81) and amantadine-resistant 2009 A/H1N1 influenza (A/California/04/09). Treatment with TCAD afforded >90% survival in mice infected with both viruses, whereas treatment with dual and single drug regimens resulted in 0% to 60% survival. Importantly, amantadine had no activity as monotherapy against the amantadine-resistant virus, but demonstrated dose-dependent protection in combination with oseltamivir and ribavirin, indicative that amantadine's activity had been restored in the context of TCAD therapy. Furthermore, TCAD therapy provided survival benefit when treatment was delayed until 72 hours post-infection, whereas oseltamivir monotherapy was not protective after 24 hours post-infection. These findings demonstrate in vivo efficacy of TCAD therapy and confirm previous reports of the synergy and broad spectrum activity of TCAD therapy against susceptible and resistant influenza strains in vitro.     

pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus

The M2 proton channel from the influenza A virus is a small protein with a single transmembrane helix that associates to form a tetramer in vivo. This protein forms proton-selective ion channels, which are the target of the drug amantadine. Here, we propose a mechanism for the pH-dependent association, and amantadine binding of M2, based on studies of a peptide representing the M2 transmembrane segment in dodecylphosphocholine micelles. Using analytical ultracentrifugation, we find that the sedimentation curves for the peptide depend on its concentration in the micellar phase. The data are well-described by a monomer-tetramer equilibrium, and the binding of amantadine shifts the monomer-tetramer equilibrium toward tetrameric species. Both tetramerization and the binding of amantadine lead to increases in the magnitude of the ellipticity at 223 nm in the circular dichroism spectrum of the peptide. The tetramerization and binding of amantadine are more favorable at elevated pH, with a pK(a) that is assigned to a His side chain, the only ionizable residue within the transmembrane helix. Our results, interpreted quantitatively in terms of a reversible monomer and tetramer protonation equilibrium model, suggest that amantadine competes with protons for binding to the deprotonated tetramer, thereby stabilizing the tetramer in a slightly altered conformation. This model accounts for the observed inhibition of proton flux by amantadine. Additionally, our measurements suggest that the M2 tetramer is substantially protonated at neutral pH and that both singly and doubly protonated states could be involved in M2's proton conduction at more acidic pHs.     

A model for influenza with vaccination and antiviral treatment

Compartmental models for influenza that include control by vaccination and antiviral treatment are formulated. Analytic expressions for the basic reproduction number, control reproduction number and the final size of the epidemic are derived for this general class of disease transmission models. Sensitivity and uncertainty analyses of the dependence of the control reproduction number on the parameters of the model give a comparison of the various intervention strategies. Numerical computations of the deterministic models are compared with those of recent stochastic simulation influenza models. Predictions of the deterministic compartmental models are in general agreement with those of the stochastic simulation models.