Enhanced immune responses by skin vaccination with influenza subunit vaccine in young hosts.

Skin has gained substantial attention as a vaccine target organ due to its immunological properties, which include a high density of professional antigen presenting cells (APCs). Previous studies have demonstrated the effectiveness of this vaccination route not only in animal models but also in adults. Young children represent a population group that is at high risk from influenza infection. As a result, this group could benefit significantly from influenza vaccine delivery approaches through the skin and the improved immune response it can induce. In this study, we compared the immune responses in young BALB/c mice upon skin delivery of influenza vaccine with vaccination by the conventional intramuscular route. Young mice that received 5 µg of H1N1 A/Ca/07/09 influenza subunit vaccine using MN demonstrated an improved serum antibody response (IgG1 and IgG2a) when compared to the young IM group, accompanied by higher numbers of influenza-specific antibody secreting cells (ASCs) in the bone marrow. In addition, we observed increased activation of follicular helper T cells and formation of germinal centers in the regional lymph nodes in the MN immunized group, rapid clearance of the virus from their lungs as well as complete survival, compared with partial protection observed in the IM-vaccinated group. Our results support the hypothesis that influenza vaccine delivery through the skin would be beneficial for protecting the high-risk young population from influenza infection.

Targeting the skin for microneedle delivery of influenza vaccine.

Influenza infection represents a major socioeconomic burden worldwide. Skin represents a new target that has gained much attention in recent years for delivery of influenza vaccine as an alternative to the conventional intramuscular route of immunization. In this review we describe different microneedle vaccination approaches used in vivo, including metal and dissolving microneedle patches that have demonstrated promising results. Additionally we analyze the immunological basis for microneedle skin immunization and targeting of the skin's dense population of antigen presenting cells, their role, characterization, and function. Additionally we analyze the importance of inflammatory signaling in the skin after microneedle delivery.

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.

ZBTB32 is an early repressor of the CIITA and MHC class II gene expression during B cell differentiation to plasma cells.

CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.

Delivery of subunit influenza vaccine to skin with microneedles improves immunogenicity and long-lived protection.

Influenza infection represents a major socio-economic burden worldwide. Novel delivery methods can render influenza vaccination easier and more acceptable by the public, and importantly confer protection equal or superior to that induced by conventional systemic administration. An attractive target for vaccine delivery is the skin. Recent studies have demonstrated improved immune responses after transdermal delivery of inactivated influenza virus with microneedle patches. Here we show that immunization with a licensed influenza subunit vaccine coated on metal microneedles can activate both humoral and cellular arms of the immune response and confer improved long-term protection in the mouse model when compared to the conventional systemic route of delivery. These results demonstrate the promising potential of microneedle delivery of licensed influenza subunit vaccines, that could be beneficial in increasing vaccine coverage and protection and reducing influenza-related mortality worldwide.

Local response to microneedle-based influenza immunization in the skin.

UNLABELLED: Microneedle patches (MN) provide a novel method of vaccine delivery to the skin with the objective of targeting the large network of resident antigen-presenting cells to induce an efficient immune response. Our previous reports demonstrated that cutaneous delivery of inactivated influenza virus-coated MN to mice protects against lethal infection. Protection is correlated with sustained levels of anti-influenza virus serum antibodies, hemagglutination inhibition titers, and robust cellular responses that are often stronger than those generated by intramuscular vaccination. Here we dissect the early events occurring in murine skin after microneedle delivery of inactivated influenza virus. We demonstrate correlation of immunization against influenza virus with a local increase of cytokines important for recruitment of neutrophils, monocytes and dendritic cells at the site of immunization. We also observed prolonged antigen deposition, and migration of matured dendritic cells bearing influenza virus antigen from the skin. IMPORTANCE: The immunological mechanisms by which MN vaccination confers protective immunity are not well understood. The present study provides a first analysis of the early immune events after microneedle-based vaccination.

Serological memory and long-term protection to novel H1N1 influenza virus after skin vaccination.

BACKGROUND: A major goal in influenza vaccine development is induction of serological memory and cellular responses to confer long-term protection and limit virus spread after infection. Here, we investigate induction of long-lived immunity against the 2009 H1N1 virus after skin vaccination. METHODS: BALB/c mice received a single dose of 5 µg inactivated A/California/04/09 virus via coated metal microneedles (MN) applied to skin or via subcutaneous injection. RESULTS: MN or subcutaneous vaccination elicited similar serum IgG and hemagglutination inhibition titers and 100% protection against lethal viral challenge 6 weeks after vaccination. Six months after vaccination, the subcutaneous group exhibited a 60% decrease in functional antibody titers and extensive lung inflammation after challenge with 10 × LD(50) of homologous virus. In contrast, the MN group maintained high functional antibody titers and IFN-γ levels, inhibition of viral replication, and no signs of lung inflammation after challenge. MN vaccination conferred complete protection against lethal challenge, whereas subcutaneous vaccination induced only partial protection. These findings were further supported by high numbers of bone marrow plasma cells and spleen antibody-secreting cells detected in the MN group. CONCLUSIONS: A single skin vaccination with MN induced potent long-lived immunity and improved protection against the 2009 H1N1 influenza virus, compared with subcutaneous injection.

Enhanced immunogenicity of stabilized trimeric soluble influenza hemagglutinin.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

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 new technology for simpler and safer vaccination with improved immunogenicity that could facilitate increased vaccination coverage.

Immunity to pre-1950 H1N1 influenza viruses confers cross-protection against the pandemic swine-origin 2009 A (H1N1) influenza virus.

The 2009 H1N1 influenza virus outbreak is the first pandemic of the twenty-first century. Epidemiological data reveal that of all the people afflicted with H1N1 virus, <5% are over 51 y of age. Interestingly, in the uninfected population, 33% of those >60 y old have pre-existing neutralizing Abs against the 2009 H1N1 virus. This finding suggests that influenza strains that circulated 50-60 y ago might provide cross-protection against the swine-origin 2009 H1N1 influenza virus. To test this, we determined the ability of representative H1N1 influenza viruses that circulated in the human population from 1930 to 2000, to induce cross-reactivity to and cross-protection against the pandemic swine-origin H1N1 virus, A/California/04/09. We show that exposure of mice to the 1947 virus, A/FM/1/47, or the 1934 virus, A/PR/8/34, induced robust cross-protective immune responses and these mice were protected against a lethal challenge with mouse-adapted A/California/04/09 H1N1 virus. Conversely, we observed that mice exposed to the 2009 H1N1 virus were protected against a lethal challenge with mouse-adapted 1947 or 1934 H1N1 viruses. In addition, exposure to the 2009 H1N1 virus induced broad cross-reactivity against H1N1 as well as H3N2 influenza viruses. Finally, we show that vaccination with the older H1N1 viruses, particularly A/FM/1/47, confers protective immunity against the 2009 pandemic H1N1 virus. Taken together, our data provide an explanation for the decreased susceptibility of the elderly to the 2009 H1N1 outbreak and demonstrate that vaccination with the pre-1950 influenza strains can cross-protect against the pandemic swine-origin 2009 H1N1 influenza virus.

Adjuvanted influenza vaccine administered intradermally elicits robust long-term immune responses that confer protection from lethal challenge.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Transdermal influenza immunization with vaccine-coated microneedle arrays.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.