Neuraminidase is one of the two glycoprotein spikes protruding from the influenza virus membrane. We have determined by X-ray crystallography the native structure of B/Lee/40 neuraminidase (NA) and the structures of its crystals soaked with a substrate, N-acetylneuraminyllactose (NANL), and an inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) at 1.8-A resolution. NANL was hydrolyzed by the crystalline NA to generate the product N-acetylneuraminic acid (NANA, also known as sialic acid), which is still able to bind to NA. In the difference Fourier map of the presumed NA-NANA complex, the moiety bound in the active site had a distorted boat conformation of NANA, but there is no significant electron density for O2. The structure of the bound moiety is not identical to that of chemically synthesized DANA soaked into NA crystals. Prolonged incubation of NANA with NA in solution at room temperature produced only a trace amount of DANA as detected by NMR. On the basis of our studies, a mechanism is proposed for the enzymatic hydrolysis by influenza virus neuraminidase.

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A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes.