Development and validation of multiplex assays for mouse and human IgG and IgA to Neisseria gonorrhoeae antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

Tertiary Plasticity Drives the Efficiency of Enterocin 7B Interactions with Lipid Membranes.

The ability of antimicrobial peptides to efficiently kill their bacterial targets depends on the efficiency of their binding to the microbial membrane. In the case of enterocins, there is a three-part interaction: initial binding, unpacking of helices on the membrane surface, and permeation of the lipid bilayer. Helical unpacking is driven by disruption of the peptide hydrophobic core when in contact with membranes. Enterocin 7B is a leaderless enterocin antimicrobial peptide produced from Enterococcus faecalis that functions alone, or with its cognate partner enterocin 7A, to efficiently kill a wide variety of Gram-stain positive bacteria. To better characterize the role that tertiary structural plasticity plays in the ability of enterocin 7B to interact with the membranes, a series of arginine single-site mutants were constructed that destabilize the hydrophobic core to varying degrees. A series of experimental measures of structure, stability, and function, including CD spectra, far UV CD melting profiles, minimal inhibitory concentrations analysis, and release kinetics of calcein, show that decreased stabilization of the hydrophobic core is correlated with increased efficiency of a peptide to permeate membranes and in killing bacteria. Finally, using the computational technique of adaptive steered molecular dynamics, we found that the atomistic/energetic landscape of peptide mechanical unfolding leads to free energy differences between the wild type and its mutants, whose trends correlate well with our experiment.

Vaccination with antigenically complex hemagglutinin mixtures confers broad protection from influenza disease.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.