Larisa V. Gubareva

Influenza virus neuraminidase inhibitors

Neuraminidase promotes influenza virus release from infected cells and facilitates virus spread within the respiratory tract. Several potent and specific inhibitors of this enzyme have been developed, and two (zanamivir and oseltamivir) have been approved for human use. Unlike amantadine and rimantadine that target the M2 protein of influenza A viruses, these drugs inhibit replication of both influenza A and B viruses. Zanamivir is delivered by inhalation because of its low oral bioavailability whereas oseltamivir is administered by mouth. Early treatment with either drug reduces the severity and duration of influenza symptoms and associated complications. Both agents are effective for chemoprophylaxis. Because of a broader antiviral spectrum, better tolerance, and less potential for emergence of resistance than is seen with the M2 inhibitors, the neuraminidase inhibitors represent an important advance in the treatment of influenza.

Evidence for Zanamivir Resistance in an Immunocompromised Child Infected with Influenza B Virus

Zanamivir, a neuraminidase inhibitor, has shown promise as a drug to control influenza. During prolonged treatment with zanamivir, a mutant virus was isolated from an immunocompromised child infected with influenza B virus. A hemagglutinin mutation (198 Thr-->Ile) reduced the virus affinity for receptors found on susceptible human cells. A mutation in the neuraminidase active site (152 Arg-->Lys) led to a 1000-fold reduction in the enzyme sensitivity to zanamivir. When tested in ferrets, the mutant virus had less virulence than the parent; however, it had a growth preference over the parent in zanamivir-treated animals. Despite these changes, the sensitivity of the mutant virus to zanamivir assessed by a standard test in MDCK cells was unaffected. These data indicate that the current methods for monitoring resistant mutants are potentially flawed because no tissue culture system adequately reflects the receptor specificity of human respiratory tract epithelium.

INHALED ZANAMIVIR FOR THE PREVENTION OF INFLUENZA IN FAMILIES

BACKGROUNDnAs prophylaxis against influenza in families, amantadine and rimantadine have had inconsistent effectiveness, partly because of the transmission of drug-resistant variants from treated index patients. We performed a double-blind, placebo-controlled study of inhaled zanamivir for the treatment and prevention of influenza in families.nnnMETHODSnWe enrolled families (with two to five members and at least one child who was five years of age or older) before the 1998-1999 influenza season. If an influenza-like illness developed in one member, the family was randomly assigned to receive either inhaled zanamivir or placebo. The family member with the index illness was treated with either 10 mg of inhaled zanamivir (163 subjects) or placebo (158) twice a day for 5 days, and the other family members received either 10 mg of zanamivir (414 subjects) or placebo (423) once a day as prophylaxis for 10 days. The primary end point was the proportion of families in which at least one household contact had symptomatic, laboratory-confirmed influenza.nnnRESULTSnThe proportion of families with at least one initially healthy household contact in whom influenza developed was smaller in the zanamivir group than in the placebo group (4 percent vs. 19 percent, P<0.001); the difference represented a 79 percent reduction in the proportion of families with at least one affected contact. Zanamivir provided protection against both influenza A and influenza B. A neuraminidase-inhibition assay and sequencing of the neuraminidase and hemagglutinin genes revealed no zanamivir-resistant variants. Among the subjects with index cases of laboratory-confirmed influenza, the median duration of symptoms was 2.5 days shorter in the zanamivir group than in the placebo group (5.0 vs. 7.5 days, P=0.01). Zanamivir was well tolerated.nnnCONCLUSIONSnWhen combined with the treatment of index cases, prophylactic treatment of family members with once-daily inhaled zanamivir is well tolerated and prevents the development of influenza. In this study there was no evidence of the emergence of resistant influenza variants.

Selection of Influenza Virus Mutants in Experimentally Infected Volunteers Treated with Oseltamivir

Volunteers experimentally infected with influenza A/Texas/36/91 (H1N1) virus and treated with the neuraminidase (NA) inhibitor oseltamivir were monitored for the emergence of drug-resistant variants. Two (4%) of 54 resistant viruses were detected by NA inhibition assay among last-day isolates recovered from 54 drug recipients. They bore a substitution His274Tyr in the NA. Hemagglutinin (HA) variants detected in the placebo group differed from the egg-adapted inoculum virus by virtue of amino acid substitutions at residues 137, 225, or both. These variants had a higher affinity for Neu5Ac(alpha2-6)Gal-containing receptors, which are characteristic of human respiratory epithelium, than for Neu5Ac(alpha2-3)Gal-containing receptors, which are typical of chicken egg allantoic membrane. Although appearing to be more sensitive to oseltamivir in humans, the variants with increased affinity for Neu5Ac(alpha2-6)Gal receptors were less sensitive than the Neu5Ac(alpha2-3)Gal-binding variants in Madin-Darby canine kidney cells. Thus, HA affinity for receptors is an essential feature of influenza virus susceptibility to NA inhibitors, both in cell culture and in humans.

Recovery of Drug-Resistant Influenza Virus from Immunocompromised Patients: A Case Series

Influenza virus with resistance to antiviral drugs emerges with increased frequency in immunocompromised patients and can limit the benefit of M2 and neuraminidase (NA) inhibitors. We document 3 cases of influenza in severely immunocompromised patients from whom virus variants with molecular markers of resistance to anti-influenza drugs were recovered. Virus variants recovered from 2 patients had mutations in the M2, NA (with a previously recognized Glu119Val NA substitution), and hemagglutinin genes. We describe a novel Asp198Asn NA mutation in an influenza B virus and its decreased susceptibility to both oseltamivir and zanamivir.

Characterization of Influenza A/HongKong/156/97 (H5N1) Virus in a Mouse Model and Protective Effect of Zanamivir on H5N1 Infection in Mice

A recent outbreak of influenza in Hong Kong was caused by a highly virulent virus of avian origin. Concern that the appearance of such a virus in the human population may be a harbinger of a new pandemic has brought increased attention to the issue of antivirals available for treatment of influenza. A/HongKong/156/97 (H5N1), the first virus of H5N1 subtype isolated from a human host, is highly virulent in the mouse model and can infect mouse lungs without requiring adaptation. High mortality and evidence of systemic disease, including spread to the brain after intranasal inoculation, are observed. Zanamivir, a novel neuraminidase inhibitor, is effective at decreasing replication of the virus in vitro. In a model of lethal challenge in mice, zanamivir reduces lung titers of the virus and decreases morbidity and mortality.

Detection of influenza virus resistance to neuraminidase inhibitors by an enzyme inhibition assay

We previously characterized influenza viruses whose selection in the presence of neuraminidase (NA) inhibitors resulted in a substituted residue (position 119,152,274, or 292) in the NA active center. To identify the most favorable conditions for detecting NA inhibitor-resistant viruses we compared the results of four modifications of the NA inhibition assay utilizing a fluorogenic substrate. The IC50 values were highly dependent upon assay conditions, and most mutant enzymes were more sensitive to changes in assay conditions (e.g. addition of PO4(3-), Ca2+, DMSO, or EDTA) than wild-type enzymes or a mutant NA with an Arg292-->Lys substitution. Although the levels of resistance to zanamivir, oseltamivir carboxylate, and BCX-1812 (RWJ-270201) for each mutant varied among assays, mutants with substitutions at framework residues 119 or 274 exhibited sensitivity to at least one inhibitor. Viruses with substitutions at catalytic residues 152 or 292 were resistant to each inhibitor in all assays. Monitoring resistance in a clinical setting will require a panel of resistant viruses to ensure that assay conditions are favorable for detecting variants with substituted residues in the NA active center. Because variants selected in the presence of one NA inhibitor could be variably resistant to other inhibitors, all three inhibitors should be used in drug susceptibility testing.

Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N.

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at -4 or +6 h, but was significantly reduced at +12h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control-75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 microg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2-7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.

Comparative Activities of Oseltamivir and A-322278 in Immunocompetent and Immunocompromised Murine Models of Influenza Virus Infection

We developed an immunocompromised murine model of influenza virus infection and demonstrated comparable efficacy of oral oseltamivir and A-322278 (both given at dosages of 10 mg/kg/day) in reducing viral replication, decreasing weight loss, and prolonging survival. Once the treatment was discontinued, severe combined immunodeficient (SCID) mice had progressive viral replication and clinical decline. Drug-resistant variants were detected in 4 (29%) of 14 and 2 (13%) of 15 mice (both BALB/c and SCID) treated with oseltamivir or A-322278, respectively; no resistant variants were detected in placebo-treated mice. Amino acid substitutions in the hemagglutinin receptor-binding site at aa 137 or 225 were detected in cloned resistant isolates. A substitution in the neuraminidase (NA) active site (Arg292Lys) was detected in the cloned virus recovered from an oseltamivir-treated mouse. This model would be useful for elucidation of the molecular mechanisms of resistance to NA inhibitors and for testing of anti-influenza therapy options that might prevent the emergence of resistant variants.

Accumulation of Defective Neuraminidase (NA) Genes by Influenza A Viruses in the Presence of NA Inhibitors as a Marker of Reduced Dependence on NA

With the use of neuraminidase (NA) inhibitors (BCX-1812, oseltamivir, or zanamivir), drug-resistant variants of influenza A viruses were generated that lacked characteristic markers of resistance, such as substitutions in the NA active center or in the hemagglutinin. Drug resistance was associated with the accumulation of defective (Delta) RNA segments encoding NA. This phenomenon could be explained by reduced dependence of the virus on its NA activity. Analysis of the last isolates recovered from 11 volunteers, experimentally infected with influenza virus and treated with BCX-1812, revealed that they maintained full susceptibility to the drug in the NA inhibition assay (50% inhibitory concentration, 0.35-0.5 nM). The presence of DeltaRNA segments was detected in 1 of these isolates but was not found in the isolates recovered from placebo recipients (n = 8). Because of a lack of cell culture-based assays for susceptibility testing of human influenza viruses, detection of DeltaRNA segments should be considered an additional assay for monitoring of NA inhibitor resistance.