Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence suggests that they activate dendritic cells (DCs) via Toll-like receptors (TLRs)1,2. For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed3, activates DCs via multiple TLRs to stimulate pro-inflammatory cytokines4,5. Triggering specific combinations of TLRs in DCs can induce synergistic production of cytokines6, which results in enhanced T cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that programs such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus Toll-like receptor (TLR) ligands 4 + 7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with a single TLR ligand. Consistent with this there was enhanced persistence of germinal centers (GCs), and of plasma cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma cell response, relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated early programming towards B cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells (DCs), as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.

Although scarce after annual influenza vaccination, B cells producing antibodies capable of neutralizing multiple influenza strains are abundant in humans infected with pandemic 2009 H1N1 influenza.

Influenza prophylaxis would benefit from a vaccination method enabling simplified logistics and improved immunogenicity without the dangers posed by hypodermic needles. Here, we introduce dissolving microneedle patches for influenza vaccination using a simple patch-based system that targets delivery to skin’s antigen-presenting cells. Microneedles were fabricated using a biocompatible polymer encapsulating inactivated influenza virus vaccine for insertion and dissolution in the skin within minutes. Microneedle vaccination generated robust antibody and cellular immune responses in mice that provided complete protection against lethal challenge. Compared to conventional intramuscular injection, microneedle vaccination resulted in more efficient lung virus clearance and enhanced cellular recall responses after challenge. These results suggest that dissolving microneedle patches can provide a novel technology for simpler and safer vaccination with improved immunogenicity that could facilitate increased vaccination coverage.

BackgroundInfluenza is a contagious disease caused by a pathogenic virus, with outbreaks all over the world and thousands of hospitalizations and deaths every year. Due to virus antigenic drift and short-lived immune responses, annual vaccination is required. However, vaccine coverage is incomplete, and improvement in immunization is needed. The objective of this study is to investigate a novel method for transdermal delivery using metal microneedle arrays (MN) coated with inactivated influenza virus to determine whether this route is a simpler and safer approach than the conventional immunization, capable to induce robust immune responses and confer protection against lethal virus challenge.Methodology/Principal FindingsInactivated A/Aichi/2/68 (H3N2) influenza virus was coated on metal microneedle arrays and applied to mice as a vaccine in the caudal dorsal skin area. Substantial antibody titers with hemagglutination inhibition activity were detected in sera collected two and four weeks after a single vaccine dose. Challenge studies in mice with 5×LD50 of mouse adapted Aichi virus demonstrated complete protection. Microneedle vaccination induced a broad spectrum of immune responses including CD4+ and CD8+ responses in the spleen and draining lymph node, a high frequency of antigen-secreting cells in the lung and induction of virus-specific memory B-cells. In addition, the use of MN showed a dose-sparing effect and a strong Th2 bias when compared to an intramuscular (IM) reference immunization.Conclusions/SignificanceThe present results show that delivery of inactivated influenza virus through the skin using metal microneedle arrays induced strong humoral and cellular immune responses capable of conferring protection against virus challenge as efficiently as intramuscular immunization, which is the standard vaccination route. In view of the convenience of delivery and the potential for self-administration, vaccine-coated metal microneedles may provide a novel and highly effective immunization method.

Palese. 2010. Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio. 1:e00018-10.