Differential recognition of influenza A virus H1N1 neuraminidase by DNA vaccine-induced antibodies in pigs and ferrets.

Neuraminidase (NA) accounts for approximately 10-20% of the total glycoproteins on the surface of influenza viruses. It cleaves sialic acids on glycoproteins, which facilitates virus entry into the airways by cleaving heavily glycosylated mucins in mucus and the release of progeny virus from the surface of infected cells. These functions make NA an attractive vaccine target. To inform rational vaccine design, we define the functionality of influenza DNA vaccine-induced NA-specific antibodies relative to antigenic sites in pigs and ferrets challenged with a vaccine-homologous A/California/7/2009(H1N1)pdm09 strain. Sera collected pre-vaccination, post-vaccination and post-challenge were analyzed for antibody-mediated inhibition of NA activity using a recombinant H7N1CA09 virus. Antigenic sites were further identified with linear and conformational peptide microarrays spanning the full NA of A/California/04/2009(H1N1)pdm09. Vaccine-induced NA-specific antibodies inhibited the enzymatic function of NA in both animal models. The antibodies target critical sites of NA such as the enzymatic site, second sialic binding site and framework residues, shown here by high-resolution epitope mapping. New possible antigenic sites were identified that potentially block the catalytic activity of NA, including an epitope recognized solely in pigs and ferrets with neuraminidase inhibition, which could be a key antigenic site affecting NA function. These findings show that our influenza DNA vaccine candidate induces NA-specific antibodies that target known critical sites, and new potential antigenic sites of NA, inhibiting the catalytic activity of NA.

Alkaline pH has an unexpected effect on transcriptional pausing during synthesis of the Escherichia coli pH-responsive riboswitch.

Riboswitches are 5'-untranslated regions of mRNA that change their conformation in response to ligand binding, allowing post-transcriptional gene regulation. This ligand-based model of riboswitch function has been expanded with the discovery of a "pH-responsive element" (PRE) riboswitch in Escherichia coli. At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome-binding sequence, whereas at alkaline pH, the PRE adopts a translationally active structure. This unique riboswitch does not rely on ligand binding in a traditional sense to modulate its alternative folding outcomes. Rather, pH controls riboswitch folding by two possible modes that are yet to be distinguished; pH either regulates the transcription rate of RNA polymerase (RNAP) or acts on the RNA itself. Previous work suggested that RNAP pausing is prolonged by alkaline pH at two sites, stimulating PRE folding into the active structure. To date, there has been no rigorous exploration into how pH influences RNAP pausing kinetics during PRE synthesis. To provide that understanding and distinguish between pH acting on RNAP versus RNA, we investigated RNAP pausing kinetics at key sites for PRE folding under different pH conditions. We find that pH influences RNAP pausing but not in the manner proposed previously. Rather, alkaline pH either decreases or has no effect on RNAP pause longevity, suggesting that the modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding. These findings invite the possibility that the RNA itself actively participates in the sensing of pH.