Influenza Virus N9 Neuraminidase Research Articles

Emerging from the Shadow of Hemagglutinin: Neuraminidase Is an Important Target for Influenza Vaccination

Gilchuk etal. (2019) and Zhu etal. (2019) provide detailed structural information about epitopes on influenza virus N9 neuraminidase and characterize breadth, potency, mechanism of action, and invivo protective effects of human antibodies targeting these epitopes. The findings highlight the importance of neuraminidase as a target for improved influenza virus vaccines.

Influenza H7N9 Virus Neuraminidase-Specific Human Monoclonal Antibodies Inhibit Viral Egress and Protect from Lethal Influenza Infection in Mice

H7N9 avian influenza virus causes severe infections and might have the potential to trigger a major pandemic. Molecular determinants of human humoral immune response to N9 neuraminidase (NA) proteins, which exhibit unusual features compared with seasonal influenza virus NA proteins, are ill-defined. We isolated 35 human monoclonal antibodies (mAbs) from two H7N9 survivors and two vaccinees. These mAbs react to NA in a subtype-specific manner and recognize diverse antigenic sites on the surface of N9 NA, including epitopes overlapping with, or distinct from, the enzyme active site. Despite recognizing multiple antigenic sites, the mAbs use a common mechanism of action by blocking egress of nascent virions from infected cells, thereby providing an antiviral prophylactic and therapeutic protection invivo in mice. Studies of breadth, potency, and diversity of antigenic recognition from four subjects suggest that vaccination with inactivated adjuvanted vaccine induce NA-reactive responses comparable to that of H7N9 natural infection.

Single-chain Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the VL domain of the scFv-0 (VL−VH) molecule is primarily responsible for formation of a tetramer–trimer equilibrium

Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the V(L) to V(H) orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the V(H)-V(L) isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between V(L)-V(H) trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55-L59 and L13-L17 from the two adjacent V(L) domains within the V(L)-V(H) trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer-trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.

Contacts between Influenza Virus N9 Neuraminidase and Monoclonal Antibody NC10

We are interested in studying how influenza virus escapes antibody inhibition. Based on the structure of the complex between N9 NA and monoclonal antibody NC10 Fab (R. L. Malby, W. R. Tulip, V. R. Harley, J. L. McKimm-Breschkin, W. G. Laver, R. G. Webster, and P. M. Colman, 1994, Structure 2, 733–746), we investigated the contribution made by individual amino acids to the stability of the complex. We made conservative changes in residues that are centrally located in the epitope and more drastic changes in peripheral contacts. The mutations made were N200L (removing an N-linked oligosaccharide), N329Q, N345Q, S370T, S372A, N400L, and K432M. Binding of each mutant to NC10 was quantitated by NA inhibition assays and ELISA. Except for N200L and N329Q, the mutants were inhibited by NC10 to the same extent as wild-type NA although with less affinity. The enzyme activity ( K cat ) of N200L is 80% reduced, indicating a defect in folding or assembly; therefore, the loss in binding activity due to the missing sugar residue cannot be assessed. The K d for N329Q is sixfold higher than for wild-type NA in the inhibition test, but the same as wild-type in ELISA, indicating a change in disposition of the antibody but no loss of affinity. The results show that the NC10 epitope can accommodate a change at any site and is not dominated by a few high-energy interactions as was found in the NC41 epitope. We propose that the difference lies in the contribution of buried water molecules to the NA-NC10 complex.

Molecular Modeling of Sialyloligosaccharide Fragments into the Active site of Influenza Virus N9 Neuraminidase

Molecular modeling studies have been carried out to investigate the interactions between substrate sialyloligosaccharide (SOS) fragments bearing different glycosidic linkages and influenza virus N9 neuraminidase, a surface glycoprotein of influenza virus subtype N9. The studies revealed that the allowed orientation for sialic acid (SA) is less than 1% in the Eulerian space at the active site. The active site of this enzyme has enough space to accommodate various SOS fragments, NeuNAcα(2–3)Gal, NeuNAcα (2–6)Gal, NeuNAcα (2–8)NeuNAc and NeuNAcα (2–9)NeuNAc, but on specific conformations. In the bound conformation, among these substrates there exists a conformational similarity leading to a structural similarity, which may be an essential requirement for the cleavage activity of the neuraminidases irrespective of the type of glycosidic linkage.

Analysis of the Binding of the Fab Fragment of Monoclonal Antibody NC10 to Influenza Virus N9 Neuraminidase from Tern and Whale Using the BIAcore Biosensor: Effect of Immobilization Level and Flow Rate on Kinetic Analysis

The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 μl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (KA = 6.3 ± 1.3 × 107 M−1) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (KA = 4.3 ± 0.7 × 106 M−1). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn200, which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.

The use of antibody fragments for crystallization and structure determinations.

We thank Andrew Prongay for his earlier contributions to the crystallization of the HIV p24 Fab complex, Jan McClure (Bristol Myers-Squibb Pharmaceutical Corporation, Seattle, WA) for providing the anti-p24 monoclonals, Lorna Ehrlich and Carol Carter (SUNY, Stony Brook, NY) for the recombinant HIV p24, and Tianwei Lin for discussions on antibody sequencing. We are grateful for an MRC and an NIH grant for MGR and for a research scholar award to LCK from the American Foundation for AIDS Research.

Red Cells Bound to Influenza Virus N9 Neuraminidase Are Not Released by the N9 Neuraminidase Activity

Influenza virus neuraminidase (NA) of the N9 subtype also possesses hemagglutinin activity and the hemagglutinating, or hemabsorbing (HB), site is distinct from the catalytic site. Previous results suggested that the NA was binding to sialic acid on the red cell surface, but we now report that the HB receptor is not sensitive to N9 influenza neuraminidase activity. Cell lines that constitutively express N9 Or N2 neuraminidase have been used to further investigate the specificity of red blood cell binding to the HB site. The results suggest that the ligand is N-acetylneuraminic acid in a linkage or environment that is not sensitive to influenza virus neuraminidase, but which is released by the broadly specific bacterial sialidases from Micromonospora viridifaciens or Arthrobacter ureafaciens.

Binding affinity of influenza virus N9 neuraminidase with Fab fragments of monoclonal antibodies NC10 and NC41

Sedimentation equilibrium centrifugation has been applied to determine the affinity and stoichiometry of the interaction between Fab fragments, derived from monoclonal antibodies NC10 and NC41, with influenza virus neuraminidase N9 isolated from either tern or whale. Although the two neuraminidase epitopes recognized by NC10 and NC41 Fab overlap, crystallographic studies have shown that the modes of binding of each Fab are different. The sedimentation equilibrium experiments described here reveal that the binding affinities are also different, with NC10 Fab binding more strongly to each neuraminidase. Furthermore, comparison of the affinity of binding of each antibody fragment reveals a stronger interaction with tern neuraminidase than with whale neuraminidase. Although the respective epitopes recognized by each antibody on the two antigens are similar, this technique shows that they do nevertheless possess sufficient differences to affect significantly the binding of antibody.

Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.

Mechanism of antigenic variation in an individual epitope on influenza virus N9 neuraminidase

Monoclonal antibodies which inhibit influenza virus neuraminidase (NA) and which therefore indirectly neutralize virus infectivity bind to epitopes located on the rim of the active-site crater. The three-dimensional structure of one of these epitopes, recognized by monoclonal antibody NC41, has previously been determined (W. R. Tulip, J. N. Varghese, R. G. Webster, G. M. Air, W. G. Laver, and P. M. Colman, Cold Spring Harbor Symp. Quant. Biol. 54:257-263, 1989). Nineteen escape mutants of influenza virus A/tern/Australia/G70c/75 (N9) NA selected with NC41 were sequenced. A surprising restriction was seen in the sequence changes involved. Ten mutants had a Ser-to-Phe change at amino acid 372, and six others had mutations at position 367. No escape mutants with changes at 369 or 370 were found, although these mutations were selected with other antibodies and rendered the epitope unrecognizable by antibody NC41. Another N9 NA, from A/ruddy turnstone/NJ/85, which differs by 14 amino acids from the tern virus NA, still bound antibody NC41. Epitope mapping by selecting multiple escape mutants with antibody NC41 thus identified only three of the five polypeptide loops on NA that contact the antibody. Escape mutants selected sequentially with three different monoclonal antibodies showed three sequence changes in two loops of the NC41 epitope. The multiple mutants were indistinguishable from wild-type virus by using polyclonal rabbit antiserum in double immunodiffusion tests, but NA inhibition titers were fourfold lower. The results suggest that although the NC41 epitope contains 22 amino acids, only a few of these are so critical to the interaction with antibody that a single sequence change allows selection of an escape mutant. In that case, the variety of amino acid sequence changes which can lead to polyclonal selection of new epidemic viruses during antigenic drift might be very limited.

Antigenic structure and variation in an influenza virus N9 neuraminidase.

We previously determined, by X-ray crystallography, the three-dimensional structure of a complex between influenza virus N9 neuraminidase (NA) and the Fab fragments of monoclonal antibody NC-41 [P. M. Colman, W. G. Laver, J. N. Varghese, A. T. Baker, P. A. Tulloch, G. M. Air, and R. G. Webster, Nature (London) 326:358-363, 1987]. This antibody binds to an epitope on the upper surface of the NA which is made up of four polypeptide loops over an area of approximately 600 A2 (60 nm2). We now describe properties of NC-41 and other monoclonal antibodies to N9 NA and the properties of variants selected with these antibodies (escape mutants). All except one of the escape mutants had single amino acid sequence changes which affected the binding of NC-41 and which therefore are located within the NC-41 epitope. The other one had a change outside the epitope which did not affect the binding of any of the other antibodies. All the antibodies which selected variants inhibited enzyme activity with fetuin (molecular weight, 50,000) as the substrate, but only five, including NC-41, also inhibited enzyme activity with the small substrate N-acetylneuramin-lactose (molecular weight, 600). These five probably inhibited enzyme activity by distorting the catalytic site of the NA. Isolated, intact N9 NA molecules form rosettes in the absence of detergent, and these possess high levels of hemagglutinin activity (W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, and G.M. Air, Virology 137:314-323, 1984). The enzyme activity of N9 NA was inhibited efficiently by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, whereas hemagglutinin activity was unaffected. The NAs of several variants with sequence changes in the NC-41 epitope lost hemagglutinin activity without any loss of enzyme activity, suggesting that the two activities are associated with separate sites on the N9 NA head.