MAB8261 Sigma-AldrichAnti-Influenza A Antibody, H1N1, clone 9B3.2

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.

Natural killer (NK) cells serve as a crucial first-line defense against tumors and virus-infected cells. We previously showed that lysis of influenza virus (IV)-infected cells is mediated by the interaction between the NK receptor, NKp46, and the IV hemagglutinin (HA) type 1 expressed by the infected cells. This interaction requires the presence of sialyl groups on the NKp46-T225 O-glycoforms. In the current study, we analyzed the O-glycan sequences that are imperative for the interaction between recombinant NKp46 (rNKp46) and IV H1N1 strains. We first showed that rNKp46 binding to IV H1N1 is not mediated by a glycoform unique to the Thr225 site. We then characterized the O-glycan sequences that mediate the interaction of rNKp46 and IV H1N1; we employed rNKp46s with dissimilar glycosylation patterns and IV H1N1 strains with different sialic acid alpha2,3 and alpha2,6 linkage preferences. The branched alpha2,3-sialylated O-glycoform Neu5NAcalpha2,3-Galbeta1,4-GlcNAcbeta1,6[Neu5NAcalpha2,3-Galbeta1,3]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for alpha2,3 linkage. In contrast, the linear alpha2,3-sialylated O-glycoform Neu5NAcalpha2,3-Galbeta1,3-GalNAc was not correlated with enhanced interaction between rNKp46 and IV H1N1 or a preference for alpha2,3 linkage. The branched alpha2,3- and alpha2,6-sialylated O-glycoform Neu5NAcalpha2,3-Galbeta1,3[Neu5NAcalpha2,6]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for alpha2,6 linkage. Previous viral HA-binding-specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells. This study shed light on the O-glycan sequences involved in the interaction of glycoprotein and viral hemagglutinins and may help in the design of agents inhibitory to hemagglutinin for influenza treatment.

Influenza pneumonitis causes severe systemic symptoms in mice, including hypothermia and excess sleep. The association of extrapulmonary virus, particularly virus in the brain, with the onset of such disease symptoms has not been investigated. Mature C57BL/6 male mice were infected intranasally with mouse-adapted human influenza viruses (PR8 or X-31) under inhalation, systemic, or no anesthesia. Core body temperatures were monitored continuously by radiotelemetry, and tissues (lung, brain, olfactory bulb, spleen, blood) were harvested at the time of onset of hypothermia (13 to 24 h post infection [PI]) or at 4 or 7 h PI. Whole RNA from all tissues was examined by one or more of three reverse transcriptase-polymerase chain reaction (RT-PCR) procedures using H1N1 nucleoprotein (NP) primers for minus polarity RNA (genomic or vRNA) or plus polarity RNA (replication intermediates). Selected cytokines were assayed at 4, 7, and 15 h in the olfactory bulb (OB). Minus and plus RNA strands were readily detected in OBs as early as 4 h PI by nested RT-PCR. Anesthesia was not required for viral invasion of the OB. Cytokine mRNAs were also significantly elevated in the OB at 7 and 15 h PI in infected mice. Controls receiving boiled virus expressed only input vRNA and that only in lung. Immunohistochemistry demonstrated localization of H1N1 and NP antigens in olfactory nerves and the glomerular layer of the OB. Therefore a mouse-adapted human influenza virus strain, not known to be neurotropic, was detected in the mouse OB within 4 h PI where it appeared to induce replication intermediates and cytokines.