Influenza A virus hemagglutinin specific antibodies interfere with virion neuraminidase activity via two distinct mechanisms

Abstract

We studied the ability of monoclonal Abs (mAbs) recognizing the major hemagglutinin (HA) antigenic sites to inhibit neuraminidase (NA) cleavage of sialic acids on fetuin. We show that virion associated-NA activity in the enzyme linked lectin assay (ELLA) is largely dependent on HA-mediated attachment of virions to immobilized fetuin. For a Sb-specific mAb, there is a nearly perfect correlation between neuraminidase inhibition and blocking virus attachment to immobilized fetuin. By contrast, Sa-, Ca-, and Cb- specific mAbs block NA activity in ELLA or the traditional thiobarbituric acid assay by sterically interfering with NA access to substrate. We conclude first, that ELLA with intact virus can only be used to measure anti-NA Abs if serum lacks HA-specific Abs, and second, that anti-HA Abs block NA activity by both limiting virion interaction with sialic acid containing surfaces and by sterically limiting NA access to sialic acids attached to macromolecules.

Neuraminidase Inhibition Contributes to Influenza A Virus Neutralization by Anti-Hemagglutinin Stem Antibodies

“…As with globular domain specific mAbs (Kosik and Yewdell, 2017), NAI with stem binding Abs was abrogated using detergent-treated virus, illustrating the steric nature of inhibition ( Fig. S1B).…”

“…The commonly used ELLA method for measuring Ab-mediated NA inhibition (NAI) is complicated by blockade of viral access to the plate-bound substrate by anti-HA head Abs that block viral attachment (Kosik and Yewdell, 2017). This does not, however, apply to stem-binding Abs which do not block virus attachment even at saturating concentrations ( Figure S1A).…”

“…We produced the human IgG1 bnHA stem mAbs 310-16G8, 310-18F8, FI6, CR8020 (Ekiert et al, 2011;Whittle et al, 2014) by transient plasmid (kindly provided by Dr. Adrian McDermott) co-transfection in the Expi293 Expression System (Thermo Fisher) following manufacturer recommendations. The IC5-4F8 Sb specific, H2-6A1 Sa specific, HA2 specific mAb RA5-22, NP specific mAb HB65, rabbit anti C-terminus NP polyclonal serum 2364 (487-498aa), NA specific NA2-1C1 mAb, rabbit anti C-terminus NA polyclonal serum, TW1.3 vaccinia specific mAb were prepared in the lab as published previously (Kosik and Yewdell, 2017;Yewdell et al, 1981). We prepared the polyclonal anti-HK68 mouse serum by single dose intramuscular immunization (5 μg) of whole purified UV-inactivated virus.…”

“…We performed AI assay as described previously (Kosik and Yewdell, 2017). Briefly, we coated black, half area high binding 96-well plates (Nunc) with 50 μl fetuin in DPBS (25 μg/ml) overnight at 4°C.…”

“…In vivo, anti-stem Abs may largely exert protection via Fc mediated activation of humoral and cellular innate immune functions (Cox et al, 2016;DiLillo et al, 2014a) HA is typically present on virions at more than 5-fold higher molar amounts than NA. Abs specific for the HA globular as well as for stem domain can sterically interfere with NA activity against large protein substrates as long as virus remains intact (Kosik and Yewdell, 2017;Rajendran et al, 2017;Russ et al, 1974), suggesting a possible mechanism for stem-Ab mediated inhibition of viral release from infected cells. Here we show that NA inhibition can be a major contributor of BN activity of stem-specific Abs in vitro and in vivo, in the latter case likely by interfering with Fc-receptor activated innate immune cell anti-viral effector activity.…”