title: Variation in the divalent cation requirements of influenza a virus N2 neuraminidases.
abstract: Influenza virus N2 neuraminidases were chromatographically purified from several vaccine candidate strains from 1957 to 1994. Enzymatic kinetic parameters and immunogenicity were tested for each strain. For each NA tested, with ionic strength held constant, Ca(2+) or Mg(2+) increased the initial rate of enzymatic activity. Earlier N2-NA strains had the highest initial velocity, V(max)/K(m) and V(max). There were significant differences among the influenza virus strains in enzymatic activity before and after addition of Ca(2+) or Mg(2+): V(max)/K(m) varied from 0.54 M(-1) s(-1) to 0.88 M(-1) s(-1) and V(max) varied from 2.45 s(-1) to 4.3 s(-1) before the addition of a divalent cation; and increased approximately 2-fold each of these kinetic parameters for each strain after the addition of exogenous Ca(2+) or Mg(2+). Exhaustive dialysis with EDTA reduced the initial velocity of each strain with significant differences found among strains, with a range of 0.1% to 8% of original activity. Activity was partially restored by the addition of exogenous Ca(2+) or Mg(2+), varying from 8% to 60% of pre-dialysis levels, but original rates were not achieved. This reduction in enzymatic activity for the tested strains (i.e., A/Japan/57 and A/Johannesburg/94) was accompanied by a parallel decrease in NA-immunogenicity, with antibody response decreasing by as much as 76% as measured by NI titer, and ELISA titer decreasing by as much as 68%. The addition of Ca(2+) or Mg(2+) to the post-dialysis sample restored immunogenicity to as much as 80% of pre-dialysis NI titers and as much as 78% of pre-dialysis ELISA titers. Dialysis had the least effect on early strains as measured by enzymatic kinetic parameters and immunogenicity studies. Zn(2+) had a slight inhibitory effect on the activity of all tested strains. Review of the nucleic acid sequence of each of these strains could not predict their enzymatic activity, immunogenicity or response to dialysis. If immunity against neuraminidase is desirable in vaccination against influenza, selection of vaccine candidate strains must include not only analysis of antigenic changes and sequence analysis but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.
authors.affiliation: Department of Pediatrics, Division of Critical Care Medicine, Weill Medical College of Cornell University, 525 East 68th St., Box 437, New York, NY 10021, USA. bertjoh@pol.net
authors.forename: Bert E
authors.initials: BE
authors.lastname: Johansson
authors.forename: Ian C
authors.initials: IC
authors.lastname: Brett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D003956:Dialysis; D004355:Drug Stability; D005260:Female; D007114:Immunization; D009980:Influenza A virus / Q000201:enzymology* / Q000276:immunology; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011897:Random Allocation; D015032:Zinc / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D009439:Neuraminidase; D008274:Magnesium; D015032:Zinc; D002118:Calcium