Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines.

Influenza virus is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. The duration and quality of humoral immunity and generation of immunological memory to vaccines is critical for protective immunity. In the current study, we examined the long-lasting protective efficacy of chimeric VLPs (cVLPs) containing influenza HA and GPI-anchored CCL28 as antigen and mucosal adjuvant, respectively, when immunized intranasally in mice. We report that the cVLPs induced significantly higher and sustainable levels of virus-specific antibody responses, especially IgA levels and hemagglutination inhibition (HAI) titers, more than 8-month post-vaccination compared to influenza VLPs without CCL28 or influenza VLPs physically mixed with sCCL28 (soluble) in mice. After challenging the vaccinated animals at month 8 with H3N2 viruses, the cVLP group also demonstrated strong recall responses. On day 4 post-challenge, we measured increased antibody levels, ASCs and HAI titers with reduced viral load and inflammatory responses in the cVLP group. The animals vaccinated with the cVLP showed 20% cross-protection against drifted (Philippines) and 60% protection against homologous (Aichi) H3N2 viruses. Thus, the results suggest that the GPI-anchored CCL28 induces significantly higher mucosal antibody responses, involved in providing long-term cross-protection against H3N2 influenza virus when compared to other vaccination groups.

Influenza infection typically initiates at respiratory mucosal surfaces. Induction of immune responses at the sites where pathogens initiate replication is crucial for the prevention of infection. We studied the adjuvanticity of GPI-anchored CCL28 co-incorporated with influenza HA-antigens in chimeric virus-like particles (cVLPs), in boosting strong protective immune responses through an intranasal (i.n.) route in mice. We compared the immune responses to that from influenza VLPs without CCL28, or physically mixed with soluble CCL28 at systemic and various mucosal compartments. The cVLPs containing GPI-CCL28 showed in-vitro chemotactic activity towards spleen and lung cells expressing CCR3/CCR10 chemokine receptors. The cVLPs induced antigen specific endpoint titers and avidity indices of IgG in sera and IgA in tracheal, lung, and intestinal secretions, significantly higher (4–6 fold) than other formulations. Significantly higher (3–5 fold) hemagglutination inhibition titers and high serum neutralization against H3N2 viruses were also detected with CCL28-containing VLPs compared to other groups. The CCL28-containing VLPs showed complete and 80% protection, when vaccinated animals were challenged with A/Aichi/2/1968/H3N2 (homologous) and A/Philippines/2/1982/H3N2 (heterologous) viruses, respectively. Thus, GPI-anchored CCL28 in influenza VLPs act as a strong immunostimulator at both systemic and mucosal sites, boosting significant cross-protection in animals against heterologous viruses across a large distance.

Summary Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple United States cities have been attributed to a unique non-encapsulated meningococcal clade (the U.S. Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, N. gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64–256 μg/ml). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384–1024 μg/ml) and colistin (MIC 256 μg/ml) as well as enhanced LL-37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA-mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross-resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High-level resistance to AMPs may contribute to the pathogenesis of US_NmUC.