These layers were then infected with diluted IAV preparations for 45 min find more at 37 °C in PBS and tested for presence of IAV infected cells after 7 h using a monoclonal antibody directed against the influenza A viral nucleoprotein (provided by Dr. Nancy Cox, CDC, Atlanta, GA, USA) as previously described [8, 15]. IAV was pre-incubated for 30 min at 37 °C with collectins or control buffer, followed by addition of these viral samples to the MDCK cells. Where indicated, collectins were first incubated with mAb prior to adding them to IAV. In addition to MBL, three
serum collectins have been identified in bovidae: conglutinin, CL-43 and CL-46. We have previously reported that hSP-D-NCRD has minimal binding to IAV. Using identically prepared trimeric NCRD fusion proteins, which have S-protein binding sites on the N-terminal tags, we were able to directly compare binding activity LY2157299 research buy of the NCRD of conglutinin and CL-43 to IAV. Both
of these bovine collectin NCRD bound significantly more strongly to IAV than hSP-D-NCRD, with the strongest binding obtained with CL-43 (Fig. 1A). We next compared neutralizing activity of NCRD using each at a concentration of 20 μg/ml (Fig. 1B). Results obtained on viral neutralization assays were generally consistent with the binding results. As we have previously shown, the hSP-D-NCRD lacks neutralizing activity at this concentration; however, NCRD of conglutinin and CL-43
had strong activity. Again, CL-43 had significantly stronger activity than conglutinin. We also tested a preparation of the NCRD of CL-46 that was generated in Pichia pistoris as previously described [23]. Because this preparation lacks a fusion tag, we could not directly compare binding affinity to IAV; however, the NCRD of CL-46 also had substantial neutralizing activity (Fig. 1C). In addition, the CL-46 NCRD inhibited HA activity of various strains of IAV (Table 1). As for SP-D and CL-43, the activity was dependent on glycosylation of the viral strain and the presence Montelukast Sodium of calcium. The Braz7/BS and Phil82/BS strains were derived from the wild-type parental strains by Dr. E. Margot Anders through growth in the presence of bovine serum β inhibitors (subsequently shown to be principally conglutinin) [18, 27]. These strains differ from the parental strains in lacking a single high mannose oligosaccharide positioned close to the sialic acid binding site of the HA, and they are partially or fully resistant to inhibition by SP-D, MBL, conglutinin and CL-43. The PR-8 strain lacks all high mannose attachments on its envelope proteins and was highly resistant to CL-46. Amino acid residues 325 and 343 define ridges on either side of the primary calcium coordination (lectin) site of SP-D (Fig. 2). These residues have a major impact on binding properties of SP-D [20, 28, 29].