Adjuvanted Influenza Vaccine Administered Intradermally Elicits Robust Long-Term Immune Responses that Confer Protection from Lethal Challenge

The respiratory illnesses caused by influenza virus can be dramatically reduced by vaccination. The current trivalent inactivated influenza vaccine is effective in eliciting systemic virus-specific antibodies sufficient to control viral replication. However, influenza protection generated after parenteral immunization could be improved by the induction of mucosal immune responses.Transcutaneous immunization, a non-invasive vaccine delivery method, was used to investigate the quality, duration and effectiveness of the immune responses induced in the presence of inactivated influenza virus co-administered with retinoic acid or oleic acid. We observed an increased migration of dendritic cells to the draining lymph nodes after dermal vaccination. Here we demonstrate that this route of vaccine delivery in combination with certain immunomodulators can induce potent immune responses that result in long-term protective immunity. Additionally, mice vaccinated with inactivated virus in combination with retinoic acid show an enhanced sIgA antibody response, increased number of antibody secreting cells in the mucosal tissues, and protection from a higher influenza lethal dose.The present study demonstrates that transdermal administration of inactivated virus in combination with immunomodulators stimulates dendritic cell migration, results in long-lived systemic and mucosal responses that confer effective protective immunity

Enhanced Immunogenicity of Stabilized Trimeric Soluble Influenza Hemagglutinin

The recent swine-origin H1N1 pandemic illustrates the need to develop improved procedures for rapid production of influenza vaccines. One alternative to the current egg-based manufacture of influenza vaccine is to produce a hemagglutinin (HA) subunit vaccine using a recombinant expression system with the potential for high protein yields, ease of cloning new antigenic variants, and an established safety record in humans.We generated a soluble HA (sHA), derived from the H3N2 virus A/Aichi/2/68, modified at the C-terminus with a GCN4pII trimerization repeat to stabilize the native trimeric structure of HA. When expressed in the baculovirus system, the modified sHA formed native trimers. In contrast, the unmodified sHA was found to present epitopes recognized by a low-pH conformation specific monoclonal antibody. We found that mice primed and boosted with 3 microg of trimeric sHA in the absence of adjuvants had significantly higher IgG and HAI titers than mice that received the unmodified sHA. This correlated with an increased survival and reduced body weight loss following lethal challenge with mouse-adapted A/Aichi/2/68 virus. In addition, mice receiving a single vaccination of the trimeric sHA in the absence of adjuvants had improved survival and body weight loss compared to mice vaccinated with the unmodified sHA.Our data indicate that the recombinant trimeric sHA presents native trimeric epitopes while the unmodified sHA presents epitopes not exposed in the native HA molecule. The epitopes presented in the unmodified sHA constitute a "silent face" which may skew the antibody response to epitopes not accessible in live virus at neutral pH. The results demonstrate that the trimeric sHA is a more effective influenza vaccine candidate and emphasize the importance of structure-based antigen design in improving recombinant HA vaccines

Dissolving polymer microneedle patches for influenza vaccination

t e c h n i c a l r e p o r t s 9 2 0 VOLUME 16 | NUMBER 8 | AUGUST 2010 nature medicine also provides a new platform technology for simple administration of other vaccines and medicines to skin without the need for hypo dermic needles

Transdermal Influenza Immunization with Vaccine-Coated Microneedle Arrays

Influenza 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.Inactivated 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 x LD(50) 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.The 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

Effect of adjuvants on responses to skin immunization by microneedles coated with influenza subunit vaccine.

Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 µg of monovalent H1N1 subunit vaccine alone or with 1 µg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines

GCN4pII modification stabilizes the trimeric structure of A/Aichi/2/68 soluble HA.

<p><b>A.</b> BS₃ crosslinking was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012466#s4" target="_blank">Materials and Methods</a>. Lanes 1 and 2 – sHA, Lanes 3 and 4 – sHA.GCN4pII. Lane 1 and 3, no BS3, Lanes 2 and 4, 3 mM BS3. Trimeric HA corresponded to a band of ∼220 kDa, dimeric HA corresponds to a band of ∼140 kDa, and monomeric HA corresponded to a band of ∼75 kDA Western blot primary antibody, anti-histidine monoclonal antibody. <b>B.</b> Sandwich ELISA. 20 µg of sHA (red circle) or sHA.GCN4pII (blue square) were captured using guinea pig anti-A/Aichi/2/68 then binding affinity of HC67, LC89 or polyclonal sera was detected by absorbance at 450 nm. <b>C.</b> Hemagglutination test using 1 µg of recombinant protein (sHA or sHA.GCN4pII) or PBS. Proteins were diluted in PBS and incubated at room temperature for 30 minutes with 0.05% chicken red blood cells (washed).</p

Neutralizing antibody titers after vaccination.

<p>Virus-neutralizing antibody activities in sera collected 2 weeks after immunization. Dilutions of sera were incubated with approximately 100 plaque forming units of PR8 virus for 1 hr at room temperature, applied to monolayers of confluent MDCK cells, and a standard plaque reduction assay was performed. Representative data are shown from at least three independent experiments. Data shown are average and standard error of mean (SEM) from 6 mice per group. Groups are as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010897#pone-0010897-t001" target="_blank">Table 1</a>.</p

Protection of mice from lethal influenza virus challenge.

<p>Survival rates of immunized mice were monitored for 10 days after i.n. infection with mouse adapted PR8 virus. (A) Kaplan-Meier curve showing the percent of mice that survived a 5x LD₅₀ PR8 virus challenge. (B) Percent of mice that survived a 20x LD₅₀ PR8 virus challenge. (C) Percentage of body weight after i.n. challenge with 20x LD₅₀ PR8 virus. PBS, PR8, PR8-OA-RA, PR8-CT, PR8-CT-OA, and PR8-CT-RA groups are as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010897#pone-0010897-t001" target="_blank">Table 1</a>. Data represent the average of six mice per group for each challenge study.</p

Recombinant H3N2 A/Aichi/2/68 is expressed as the hemagglutinin precursor (HA0).

<p><b>A,B.</b> Western blot analysis of SDS-PAGE separated sHA (Lane 1) and sHA.GCN4pII (Lane 2) using polyclonal sera or anti-histidine tag monoclonal antibody (primary antibody). Protein bands were developed using goat anti-mouse HRP (secondary antibody) and ECL Plus (GE Healthcare). <b>C.</b> Coomassie blue stain of sHA (Lane 1) and sHA.GCN4pII (Lane 3) after nickel-bead column purification. Lane 2 - molecular weight marker. Each lane was loaded with 1 µg of recombinant protein.</p