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Skin vaccination by microneedle (MN) patch simplifies the immunization process to increase access to vaccines for global health. Lyophilization has been widely used to stabilize vaccines and other biologics during storage, but is generally not compatible with the MN patch manufacturing processes. In this study, our goal was to develop a method to incorporate lyophilized inactivated H1N1 influenza vaccine into MN patches during manufacturing by suspending freeze-dried vaccine in anhydrous organic solvent during the casting process. Using a casting formulation containing chloroform and polyvinylpyrrolidone, lyophilized influenza vaccine maintained activity during manufacturing and subsequent storage for 3 months at 40 [Image: see text] C. Influenza vaccination using these MN patches generated strong immune responses in a murine model. This manufacturing process may enable vaccines and other biologics to be stabilized by lyophilization and administered via a MN patch.

Vaccines prevent 2–3 million childhood deaths annually; however, low vaccine efficacy and the resulting need for booster doses create gaps in immunization coverage. In this translational study, we explore the benefits of extended release of licensed vaccine antigens into skin to increase immune responses after a single dose in order to design improved vaccine delivery systems. By administering daily intradermal injections of inactivated polio vaccine according to six different delivery profiles, zeroth-order release over 28 days resulted in neutralizing antibody titers equivalent to two bolus vaccinations administered one month apart. Vaccinations following this profile also improved immune responses to tetanus toxoid and subunit influenza vaccine but not a live-attenuated viral vaccine, measles vaccine. Finally, using subunit influenza vaccine, we demonstrated that daily vaccination by microneedle patch induced a potent, balanced humoral immunity with an increased memory response compared to bolus vaccination. We conclude that extended presentation of antigen in skin via intradermal injection or microneedle patch can enhance immune responses and reduce the number of vaccine doses, thereby enabling increased vaccination efficacy.

While the majority of influenza-infected individuals show no or mild symptomatology, pregnant women are at higher risk of complications and infection-associated mortality. Although enhanced lung pathology and dysregulated hormones are thought to underlie adverse pregnancy outcomes following influenza infection, how pregnancy confounds long-term maternal anti-influenza immunity remains to be elucidated. Previously, we linked seasonal influenza infection to clinical observations of adverse pregnancy outcomes, enhanced lung and placental histopathology, and reduced control of viral replication in lungs of infected pregnant mothers. Here, we expand on this work and demonstrate that lower infectious doses of the pandemic A/California/07/2009 influenza virus generated adverse gestational outcomes similar to higher doses of seasonal viruses. Mice infected during pregnancy demonstrated lower hemagglutination inhibition and neutralizing antibody titers than non-pregnant animals until 63 days post infection. These differences in humoral immunity suggest that pregnancy impacts antibody maturation mechanisms without alterations to B cell frequency or antibody secretion. This is further supported by transcriptional analysis of plasmablasts, which demonstrate downregulated B cell metabolism and post-translational modification systems only among pregnant animals. In sum, these findings corroborate a link between adverse pregnancy outcomes and severe pathology observed during pandemic influenza infection. Furthermore, our data propose that pregnancy directly confounds humoral responses following influenza infection which resolves post-partem. Additional studies are required to specify the involvement of plasmablast metabolism with early humoral immunity abnormalities to best guide vaccination strategies and improve our understanding of the immunological consequences of pregnancy.

Influenza virus causes life-threatening infections in pregnant women and their newborns. Immunization during pregnancy is the most effective means of preventing maternal and infant mortality/morbidity; however, influenza vaccination rates of pregnant women remain under 50%. Furthermore, the availability of vaccines in low-resource populations is limited. Skin immunization with microneedle patches (MN) is a novel and safe vaccination platform featuring thermostable vaccine formulations. Cold-chain independence and the potential for self-administration can expand influenza vaccination coverage in developing countries. In this study of pregnant BALB/c mice immunized with subunit H1N1 influenza vaccine, we demonstrate the advantage of skin vaccination over intramuscular delivery of a two-fold higher vaccine dose. MN vaccine induced superior humoral immune responses and conferred protective immunity against a lethal challenge dose of homologous influenza virus. Importantly, MN vaccination of mice at mid-gestation resulted in enhanced and long-lasting passive immunity of the offspring, measured by neutralizing antibody titers and survival rates after virus challenge. We conclude that skin vaccination using MN is a superior immunization approach with the potential to overcome immune tolerance observed in pregnancy, and lower vaccination costs through antigen dose-sparing, which is especially relevant in underserved countries.

Maternal and neonatal tetanus claim tens of thousands lives every year in developing countries, but could be prevented by hygienic practices and improved immunization of pregnant women. This study tested the hypothesis that skin vaccination can overcome the immunologically transformed state of pregnancy and enhance protective immunity to tetanus in mothers and their newborns. To achieve this goal, we developed microneedle patches (MNPs) that efficiently delivered unadjuvanted tetanus toxoid to skin of pregnant mice and demonstrated that this route induced superior immune responses in female mice conferring 100% survival to tetanus toxin challenge when compared to intramuscular vaccination. Mice born to MNP-vaccinated mothers showed detectable tetanus-specific IgG antibodies up to 12weeks of age and complete protection to tetanus toxin challenge up at 6weeks of age. In contrast, none of the 6-week old mice born to intramuscularly vaccinated mothers survived challenge. Although pregnant mice vaccinated with unadjuvanted tetanus toxoid had 30% lower IgG and IgG1 titers than mice vaccinated intramuscularly with Alum®-adjuvanted tetanus toxoid vaccine, IgG2a titers and antibody affinity maturation were similar between these groups. We conclude that skin immunization with MNPs containing unadjuvanted tetanus toxoid can confer potent protective efficacy to mothers and their offspring using a delivery method well suited for expanding vaccination coverage in developing countries.

This study tested the hypothesis that optimized microneedle patch formulations can stabilize trivalent subunit influenza vaccine during long-term storage outside the cold chain and when exposed to potential stresses found during manufacturing and storage. Formulations containing combinations of trehalose/sucrose, sucrose/arginine, and arginine/heptagluconate were successful at retaining most or all vaccine activity during storage at 25°C for up to 24 months as determined by ELISA assay. The best formulation of microneedle patches contained arginine/heptagluconate, which showed no significant loss of vaccine activity during the study. To validate these in vitro findings, mice were immunized using trivalent influenza vaccine stored in microneedle patches for more than one year at 25°C, which elicited antibody titers greater than or equal to fresh liquid vaccine delivered by intradermal injection, indicating the retention of immunogenicity during storage. Finally, influenza vaccine in microneedle patches lost no significant activity during exposure to 60°C for four months, multiple freeze-thaw cycles, and electron beam irradiation. We conclude that optimally formulated microneedle patches can retain influenza vaccine activity during extended storage outside the cold chain and during other environmental stresses, which suggests the possibility of microneedle patch storage on pharmacy shelves without refrigeration.

The widely used influenza subunit vaccine would benefit from increased protection rates in vulnerable populations. Skin immunization by microneedle (MN) patch can increase vaccine immunogenicity, as well as increase vaccination coverage due to simplified administration. To further increase immunogenicity, we used granulocyte-macrophage colony stimulating factor (GM-CSF), an immunomodulatory cytokine already approved for skin cancer therapy and cancer support treatment. GM-CSF has been shown to be upregulated in skin following MN insertion. The GM-CSF-adjuvanted vaccine induced robust and long-lived antibody responses cross-reactive to homosubtypic and heterosubtypic influenza viruses. Addition of GM-CSF resulted in increased memory B cell persistence relative to groups given influenza vaccine alone and led to rapid lung viral clearance following lethal infection with homologous virus in the mouse model. Here we demonstrate that successful incorporation of the thermolabile cytokine GM-CSF into MN resulted in improved vaccine-induced protective immunity holding promise as a novel approach to improved influenza vaccination. To our knowledge, this is the first successful incorporation of a cytokine adjuvant into dissolvable MNs, thus advancing and diversifying the rapidly developing field of MN vaccination technology.

Prevention of seasonal influenza epidemics and pandemics relies on widespread vaccination coverage to induce protective immunity. In addition to a good antigenic match with the circulating viruses, the effectiveness of individual strains represented in the trivalent vaccines depends on their immunogenicity. In this study, we evaluated the immunogenicity of H1N1, H3N2, and B seasonal influenza virus vaccine strains delivered individually with a novel dissolving microneedle patch and the stability of this formulation during storage at 25 °C. Our data demonstrate that all strains retained their antigenic activity after incorporation in the dissolving patches as measured by single radial diffusion (SRID) assay and immune responses to vaccination in BALB/c mice. After a single immunization, all three antigens delivered with microneedle patches induced superior neutralizing antibody titers compared to intramuscular immunization. Cutaneous antigen delivery was especially beneficial for the less immunogenic B strain. Mice immunized with dissolving microneedle patches encapsulating influenza A/Brisbane/59/07 (H1N1) vaccine were fully protected against lethal challenge by homologous mouse-adapted influenza virus. All vaccine components retained activity during storage at room temperature for at least 3 months as measured in vitro by SRID assay and in vivo by mouse immunization studies. Our data demonstrate that dissolving microneedle patches are a promising advance for influenza cutaneous vaccination due to improved immune responses using less immunogenic influenza antigens and enhanced stability.

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 5μg 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.

Systemic use of epidermal growth factor receptor inhibitors (EGFRIs) has been shown to alter MHC expression and that of several chemokines and to enhance immune cell recruitment into human skin. We hypothesized that EGFRIs may have value as cutaneous immune response modifiers and determined the effects of topical application of an irreversible EGFRI on a well-established murine model of influenza vaccination. We found that a single topical application of an EGFRI led to increased levels of antibodies that inhibit influenza mediated hemagglutination and viral cytopathic effects. The topically applied EGFRI significantly enhanced the generation of vaccine-specific IL-4 and IFN-γ producing cells within skin-draining lymph nodes as early as one week following vaccination. The EGFRI/vaccine group showed a twelve-fold reduction in detectable pulmonary viral load four days after infection as compared to the vaccine alone control group. The reduction in the lung viral titers correlated with the survival rate, which demonstrated 100% protection in the EGFRI/vaccine immunized group but only 65% protection in the mice immunized with vaccine alone. These findings are significant because they demonstrate that inhibition of defined signaling pathways within the skin using small molecule kinase inhibitors provides a novel approach to enhance immune responses to vaccines.

Increased susceptibility to influenza virus infection during pregnancy has been attributed to immunological changes occurring before and during gestation in order to “tolerate” the developing fetus. These systemic changes are most often characterized by a suppression of cell-mediated immunity and elevation of humoral immune responses referred to as the Th1-Th2 shift. However, the underlying mechanisms which increase pregnant mothers’ risk following influenza virus infection have not been fully elucidated. We used pregnant BALB/c mice during mid- to late gestation to determine the impact of a sub-lethal infection with A/Brisbane/59/07 H1N1 seasonal influenza virus on completion of gestation. Maternal and fetal health status was closely monitored and compared to infected non-pregnant mice. Severity of infection during pregnancy was correlated with premature rupture of amniotic membranes (PROM), fetal survival and body weight at birth, lung viral load and degree of systemic and tissue inflammation mediated by innate and adaptive immune responses. Here we report that influenza virus infection resulted in dysregulation of inflammatory responses that led to pre-term labor, impairment of fetal growth, increased fetal mortality and maternal morbidity. We observed significant compartment-specific immune responses correlated with changes in hormonal synthesis and regulation. Dysregulation of progesterone, COX-2, PGE2 and PGF2α expression in infected pregnant mice was accompanied by significant remodeling of placental architecture and upregulation of MMP-9 early after infection. Collectively these findings demonstrate the potential of a seasonal influenza virus to initiate a powerful pro-abortive mechanism with adverse outcomes in fetal health., Author summary Maternal immunology is finely balanced to maintain a tolerant and supportive molecular environment for the developing fetus while continuing surveillance against foreign microbial threats. Influenza viral infection during pregnancy is a significant clinical risk for mothers and their newborns, increasing hospitalization, preterm birth, low birth weight, and maternal and neonatal deaths worldwide. In a mouse pregnancy model, we show how influenza virus infection disrupts the delicate and interconnected cytokine and hormonal signaling pathways that respond to respiratory pathogens. The health of mothers and offspring was impacted in our study, after pregnant mothers’ lung and placental architecture was compromised by infection. Influenza virus infection increased the stress on the mother’s body already present due to pregnancy, or reversed the hormonal environment required to establish and maintain healthy pregnancy. By dissecting the effects of inflammation post-infection throughout the mother’s anatomy, we can tailor anti-inflammatory treatments for the pregnant population. Also, thorough knowledge of immune responses will assist in tailoring vaccine design and dosage for this delicate period of women’s immunological and reproductive health.

Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly definedin vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouse-adapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50× 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM+) virus-specific plasma cells than IgG+plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did.

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.