Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. The traditional thiobarbituric acid (TBA) method to quantify NA inhibiting antibodies is cumbersome and not suitable for routine serology. An enzyme-linked lectin assay (ELLA) described by Lambre et al. (1990) is a practical alternative method for measuring NA inhibition (NI) titers. This report describes optimization of the ELLA for measuring NI titers in human sera against influenza A viruses, using H6N1 and H6N2 viruses as antigens. The optimized ELLA is subtype-specific and reproducible. While the titers measured by ELLA are somewhat greater than those measured by a miniaturized TBA method, seroconversion rates are the same, suggesting similarity in assay sensitivity under these optimized conditions. The ELLA described in this report provides a practical format for routine evaluation of human antibody responses to NA.
MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 ؋ 10 5 cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10 6 cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10 6 cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs.anchorage-independent ͉ hemagglutinin ͉ sialyltransferase ͉ vaccine
Single-cell clones have been established from the MDCK cell line, characterized for their morphology and evaluated for their suitability for influenza virus research. Three discrete cell morphotypes were identified using light microscopy. Besides morphological features, the cell types can be distinguished by the level of expression of surface glycans recognized by peanut agglutinin (PNA). All clones were susceptible to infection by influenza viruses of different subtypes of influenza A virus (H1N1, H1N1pdm09, H3N2, H5N1) and influenza B virus, and all possessed on their surface terminally sialylated glycans with both types of glycosidic linkage (α2–3 and α2–6). The Type-1 cell lines were able to support a multicycle replication of influenza A and B viruses without help of an exogenous trypsin. In contrast, cell lines exhibiting Type-2 morphology were unable to support multicycle replication of influenza A viruses without trypsin supplementation. Western blot analysis of the hemagglutinin of H1N1 strains demonstrated that Type-2 cells were deficient in production of proteolytically activated hemagglutinin (no cleavage between HA1/HA2 was observed). HA1/HA2 cleavage of influenza B viruses in the Type-2 cells was also significantly impaired, but not completely abrogated, producing sufficient amount of activated HA to support efficient virus replication without trypsin. In contrast, all clones of Type-1 cells were able to produce proteolytically activated hemagglutinin of influenza A and B viruses. However, the growth kinetics and plaque size of influenza A viruses varied significantly in different clones. Influenza B virus also showed different plaque size, with the biggest plaque formation in the Type-2 cells, although the growth kinetics and peak infectivity titers were similar in all clones. Taken together, the study demonstrates that the population of original MDCK cells is represented by various types of cells that differ in their capacities to support replication of influenza A and B viruses.
Aims and Methods To facilitate antigenic characterization of the influenza A 2009 pandemic H1N1 [A(H1N1)pdm09] hemagglutinin (HA), we generated a panel of murine monoclonal antibodies (mAbs) using as the immunogen mammalian‐derived virus‐like particles containing the HA of the A/California/04/2009 virus. The antibodies were specific for the A/California/04/2009 HA, and individual mAbs suitable for use in several practical applications including ELISA, immunofluorescence, and Western blot analysis were identified. Results and Conclusions As the panel of mAbs included antibodies with hemagglutination inhibition (HI) and virus neutralizing activities, this allowed identification and characterization of potentially important antigenic and neutralizing epitopes of the A/California/04/2009 HA and comparison of those epitopes with the HAs of other influenza viruses including seasonal H1N1 viruses as well as the A/South Carolina/1918 and A/New Jersey/1976 H1N1 viruses. Three mAbs with the highest HI and neutralizing titers were able to provide passive protection against virus challenge. Two other mAbs without HI or neutralizing activities were able to provide partial protection against challenge. HA epitopes recognized by the strongest neutralizing mAbs in the panel were identified by isolation and selection of virus escape mutants in the presence of individual mAbs. Cloned viruses resistant to HI and antibody neutralization were sequenced to identify mutations, and two unique mutations (D127E and G155E) were identified, both near the antigenic site Sa. Using human post‐vaccination sera, however, there were no differences in HI titer between A/California/04/2009 and either escape mutant, suggesting that these single mutations were not sufficient to abrogate a protective antibody response to the vaccine.
Selection of high-growth virus variants of strain B/Victoria/504/2000 by serial passage in eggs resulted in three amino acid substitutions, G141E, R162M, and D196Y, in the vicinity of the receptor-binding pocket of viral hemagglutinin. Virus variants containing the identified amino acid substitutions, individually or in various combinations, were constructed using reverse genetics and analyzed for their receptor-binding properties using glycan microarray platform. Three different patterns of virus binding were revealed. A low-growth virus variant, corresponding to the original egg-derived virus B/Victoria/504/2000 prior to acquisition of amino acid changes G141E, R162M, and D196Y, had a clear preference for the oligosaccharide chains terminated with alpha2-6-linked sialic acid with very weak binding of the glycans terminated with alpha2-3-linked sialic acid. Amino acid substitutions R162M and D196Y had similar effects, resulting in viruses that bound with high efficiency almost all terminally sialylated glycans represented on the array regardless of the type of glycosidic linkage. In contrast, substitution of G141E alone, or in combinations with the other two amino acid substitutions, significantly restricted virus glycan-binding capabilities. All virus variants possessing this substitution lost the ability to bind glycans with alpha2-6 glycosidic linkage as well as most of the glycans with alpha2-3 glycosidic linkage. Linear penta- and heptasaccharide chains represented at the non-reducing end by alpha2-3 sialylated Type-II motif (LacNAc) were the only structures bound with high affinity by the virus variants with G141E substitution. In all cases when the effects on virus binding of individual amino acid substitutions differed, the effect of R162M was subordinate to the effect of either G141E or D196Y.
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