Epidermal Powder Immunization Research Articles

A pilot study using a novel pyrotechnically driven prototype applicator for epidermal powder immunization in piglets

Epidermal powder immunization (EPI) is an alternative technique to the classical immunization route using needle and syringe. In this work, we present the results of an in vivo pilot study in piglets using a dried influenza model vaccine which was applied by EPI using a novel pyrotechnically driven applicator. A liquid influenza vaccine (Pandemrix®) was first concentrated by tangential flow filtration and hemagglutinin content was determined by RP-HPLC. The liquid formulation was then transformed into a dry powder by collapse freeze-drying and subsequent cryo-milling. The vaccine powder was attached to a membrane of a novel pyrotechnical applicator using oily adjuvant components. Upon actuation of the applicator, particles were accelerated to high speed as determined by a high-speed camera setup. Piglets were immunized twice using either the novel pyrotechnical applicator or classical intramuscular injection. Blood samples of the animals were collected at various time points and analyzed by enzyme-linked immunosorbent assay. Our pilot study shows that acceleration of a dried vaccine powder to supersonic speed using the pyrotechnical applicator is possible and that the speed and impact of the particles is sufficient to breach the stratum corneum of piglet skin. Importantly, the administration of the dry vaccine powder resulted in measurable anti-H1N1 antibody titres in vivo.

Hepatitis-B Surface Antigen (HBsAg) Powder Formulation: Process and Stability Assessment

The purpose of this study was to develop a hepatitis-B surface antigen (HBsAg) dry powder vaccine formulation suitable for epidermal powder immunization (EPI) via an efficient, scalable powder-formation process. Several HBsAg dry powder formulations were prepared using four different powder-formation methods: freeze-drying/compress/grind/sieve (FD/C/G/S), spray-drying (SD), agarose beads, and spray freeze-drying (SFD). Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Physical, chemical and biochemical stability of HBsAg was determined by dynamic light scattering, an enzyme immune assay, and immunogenicity in a mouse or hairless guinea pig model. Out of the four powder-formation methods evaluated SFD outperformed other methods in the following considerations: good process efficiency, flexible scalability, and desirable particle characteristics for skin penetration. The stress posed by SFD appeared to be mild as HBsAg in the dry form retained its potency and immunogenicity. Notably, the mechanism of fast freezing by SFD actually promoted the preservation of HBsAg nanoparticle size, in good correlation with long-term biochemical stability. Among several formulations screened, the formulation containing 10 microg HBsAg in 1-mg powder with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability performance. In conclusion, HBsAg dry powder formulations suitable for EPI were successfully prepared using SFD. Further, a systematic formulation development strategy allowed the development and optimization of an HBsAg dry powder formulation, demonstrating excellent long-term physical, biochemical, and immunological stability.

An antigen-independent but not antigen-specific TH1 response provides protection in the murine airway inflammation model

Atopic disorders are associated with an imbalanced T(H) cell response biased toward a strong T(H)2 type, resulting in excessive production of IgE antibodies, eosinophil recruitment and activation, and mast cell degranulation. Restoring the T(H)1-T(H)2 balance by increasing the antigen-specific T(H)1 response has been pursued for specific allergy immunotherapy. Synthetic oligodeoxynucleotides containing unmethylated CG dinucleotides (CpG) are strong T(H)1 adjuvants and are being investigated for allergy immunotherapy. This study was designed to investigate the protective role of antigen-specific T(H)1 responses induced by epidermal powder immunization with ovalbumin (OVA) and CpG in a murine airway inflammation model. An allergy model was used in which BALB/c mice were sensitized and then challenged with OVA. Mice received prophylactic or therapeutic immunizations with OVA, CpG, or both. After challenge, pulmonary inflammation and cell infiltration were measured on the basis of BAL cell counts and lung histology. Immune response was determined by measuring the levels of lavage cytokines and serum antibodies. Coadministration of OVA and CpG by means of subcutaneous injection or epidermal powder immunization, although inducing a strong T(H)1 response, neither suppressed T(H)2 cytokines nor offered protection against pulmonary eosinophilia and histopathology in a mouse challenge model. However, when CpG was used as a stand-alone treatment of previously sensitized animals, protection against allergic airway inflammation was observed. After challenge with OVA, eosinophilia was suppressed in the lungs of the CpG-treated mice. This finding argues against the approach of boosting an allergen-dependent T(H)1 response and favors induction of an antigen-independent T(H)1 response for allergy immunotherapy.

Epidermal powder immunization against influenza

Epidermal powder immunization (EPI) can efficiently deliver powdered protein vaccines to the epidermis. A phase I clinical trial was conducted to evaluate powdered trivalent influenza vaccine delivered using the PowderJect ND5.2 delivery system. Subjects received either Fluvirin ® IM injection (15 μg of each influenza strain), a single EPI vaccination (15 μg of each influenza strain) or two adjacent EPI (total of 30 μg of each influenza strain). Systemic reactogenicity was similar between control and EPI vaccines. Site reactions following EPI were primarily mild and self-limiting. Seroconversions, titer increases and geometric mean titers to all strains were equivalent or higher in EPI-immunized groups than in controls. Powdered influenza vaccine delivered by EPI is safe and elicits humoral immune responses in humans.

Epidermal powder immunization: cellular and molecular mechanisms for enhancing vaccine immunogenicity

Epidermal powder immunization (EPI) of mice with an influenza vaccine elicited consistently a higher hemagglutination inhibition (HI) antibody titers than intramuscular (IM) injection using the same dose of vaccine. The epidermal Langerhans cells (LCs) at the site of EPI were found to play an important role in the immune responses. Indeed, depletion of LCs from the immunization site prior to EPI caused a significant reduction in the antibody response. Transfer of LCs isolated from the EPI sites to naive mice induced a robust antigen-specific antibody response. Cytokines produced by target site cells appear to be important for the augmented immune responses induced by EPI. LTR72, a genetically detoxified heat-labile toxin from Escherichia coli with a strong adjuvant effect in EPI, was found to bind the keratinocytes of the epidermis, but not the LCs, and caused the production of elevated TNF-α and IL-12 cytokines in emigrating epidermal cells. These results have important implications for the development of a more efficacious human influenza vaccine.

Influenza Vaccine Powder Formulation Development: Spray‐Freeze‐Drying and Stability Evaluation

The purpose of this study was to develop a spray‐freeze‐drying (SFD) process for preparing an influenza vaccine dry powder formulation suitable for epidermal powder immunization. After preformulation of two types of flu vaccines, their dry‐powder formulations were prepared by SFD. Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Chemical and biochemical stability of vaccine antigens was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, single radial immunodiffusion assay, and in vivo immunogenicity in a mouse model. We demonstrated that SFD could produce high‐density particles—a critical parameter for effective skin penetration. From the stability perspective, the stress posed by SFD was mild because the antigen in the dry powder retained its stability, potency, and immunogenicity. Among several formulations screened, we noted that formulation composition has a significant role in the powder's long‐term physical and biochemical stability. One formulation, in particular, containing sub‐unit vaccine (45 μg of antigen in 1 mg of powder) with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability, including acceptable biochemical stability after being exposed to a highly humid environment. After all, we have not only demonstrated the suitability of SFD to prepare powders for epidermal powder immunization but also developed a systematic formulation development strategy that allowed the optimization of an influenza vaccine dry powder formulation. More important, this study led to the selection of a formulation system that had been successfully tested in a human clinical study. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1912–1923, 2004

Pre-clinical and clinical studies of epidermal powder immunization with an influenza vaccine

Antigen presentation is a critical step for the immune system to mount an antigen-specific immune response to invading pathogens. Delivering antigens directly to antigen presenting cells (APCs) is a promising way to improve the immunogenicity and effectiveness of vaccines. Epidermal powder immunization (EPI) delivers vaccine to the epidermis, the superficial layer of skin containing a large number of APCs, and has been shown to elicit augmented antibody responses and cellular immunity to the split influenza vaccine in small animals, nonhuman primates, and humans. EPI is safe and effective and holds promise for future human immunization with influenza and other vaccines.

Epidermal powder immunization: cellular and molecular mechanisms for enhancing vaccine immunogenicity

Epidermal powder immunization (EPI) of mice with an influenza vaccine elicited consistently a higher hemagglutination inhibition (HI) antibody titers than intramuscular (IM) injection using the same dose of vaccine. The epidermal Langerhans cells (LCs) at the site of EPI were found to play an important role in the immune responses. Indeed, depletion of LCs from the immunization site prior to EPI caused a significant reduction in the antibody response. Transfer of LCs isolated from the EPI sites to naive mice induced a robust antigen-specific antibody response. Cytokines produced by target site cells appear to be important for the augmented immune responses induced by EPI. LTR72, a genetically detoxified heat-labile toxin from Escherichia coli with a strong adjuvant effect in EPI, was found to bind the keratinocytes of the epidermis, but not the LCs, and caused the production of elevated TNF-α and IL-12 cytokines in emigrating epidermal cells. These results have important implications for the development of a more efficacious human influenza vaccine.

Spray-coating for biopharmaceutical powder formulations: beyond the conventional scale and its application.

Fluid-bed spray-coating process is widely used to prepare non-protein pharmaceutical solid dosage forms using macro-size seed particles (200-1000 microm) at kilogram batch sizes. In this study we developed a small-scale fluid-bed spray-coating process (20 g) to produce micro-sized vaccine powder formulations (40-60 microm) for epidermal powder immunization (EPI) METHODS: A bench-top spray coater was used to spray two vaccines, diphtheria toxoid (dT) and alum-adsorbed hepatitis-B surface antigen (Alum-HBsAg), onto crystalline lactose particles of 40-60 microm in diameter. Particle properties such as particle size, surface morphology, and degree of particle agglomeration were determined. Protein stability was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenicity of the vaccine was evaluated in vivo by needle injection and epidermal powder immunization (EPI) of mice or guinea pigs. Coating feasibility was demonstrated for both vaccine formulations containing different excipients. However, the nature of the vaccine antigen appeared to affect coating feasibility in terms of particle agglomeration considerably. Delivery of spray-coated dT and alum-HBsAg through EPI to mice and guinea pigs, respectively, generated significant antibody responses, at a level comparable to liquid formulation delivered subcutaneously through needle/syringe injection. The new spray-coating process represents an important technical advance and may provide a useful tool for developing high-valued biopharmaceutical powder formulations for novel applications. The strong in vivo performance of the coated dT and alum-HBsAg powders by EPI further demonstrated that spray-coating is a viable dry powder formulation process and the skin's epidermal layer presents an efficient vaccine delivery route.

A protective effect of epidermal powder immunization in a mouse model of equine herpesvirus-1 infection

To evaluate the protective effect of epidermal powder immunization (EPI) against equine herpesvirus-1 (EHV-1) infection, we prepared a powder vaccine in which formalin-inactivated virions were embedded in water-soluble, sugar-based particles. A PowderJect device was used to immunize mice with the powder vaccine via their abdominal skin. We found that twice-immunized mice were protected against challenge with the wild-type virus. This protective effect was equivalent to or better than that observed in mice immunized with other types of vaccines, including a gene gun-mediated DNA vaccine containing the glycoprotein D (gD) gene or conventional inactivated virus vaccines introduced via intramuscular or intranasal injections. These findings indicate that the powder vaccine is a promising approach for the immunological control of EHV-1 infection, either alone or as a part of prime-boost vaccination strategies.

Powder and particle-mediated approaches for delivery of DNA and protein vaccines into the epidermis

The epidermis of the skin is both a sensitive immune organ and a practical target site for vaccine administration. However, administration of vaccines into the epidermis is difficult to achieve using conventional vaccine delivery methods employing a needle and syringe. A needle-free vaccine delivery system has been developed that efficiently delivers powdered or particulate DNA and protein vaccines into the epidermal tissue. The delivery system can be used to directly transfect antigen presenting cells (APCs) by formulating DNA or protein vaccines onto gold particles (particle-mediated immunization). Antigen can be directly presented to the immune system by the transfected APCs. Antigen can also be expressed and secreted by transfected keratinocytes and picked up by resident APCs through the exogenous antigen presentation pathway. Alternatively, protein antigens can be formulated into a powder and delivered into the extracellular environment where they are picked up by APCs (epidermal powder immunization). Using any of these formulations, epidermal immunization offers the advantage of efficiently delivering vaccines into the APC-rich epidermis. Recent studies demonstrate that epidermal vaccine delivery induces humoral, cellular, and protective immune responses against infectious diseases in both laboratory animals and man.

Epidermal powder immunization of mice and monkeys with an influenza vaccine

Epidermal powder immunization (EPI) with an influenza vaccine and an adjuvant such as QS-21, LTR72, or cholera toxin elicited augmented serum and mucosal antibody responses in mice. Rhesus macaques, which have an immune system and skin structure similar to humans, were used to further evaluate the immunogenicity of the influenza vaccine following EPI. EPI of rhesus macaques with an influenza vaccine and QS-21 adjuvant elicited significantly higher serum hemagglutination inhibition (HI) titers than antigen alone administered by EPI or by intramuscular (IM) injection using a needle and syringe. In the absence of QS-21, EPI and IM injection elicited comparable HI titers in the monkeys. This study suggests that EPI is a promising technique for administering human vaccine and that QS-21 augments the immunogenicity of co-administered influenza vaccine.

Immune responses to hepatitis B surface antigen following epidermal powder immunization.

Langerhans cells in the epidermis of skin are potent antigen-presenting cells that trigger the immune system to respond to invading microorganisms. We have previously shown that epidermal powder immunization with a powdered inactivated influenza virus vaccine, by targeting the Langerhans cell-rich epidermis, was more efficacious than deeper tissue injection using a needle and syringe. We now report enhanced humoral and cellular immune responses to recombinant hepatitis B surface antigen following epidermal powder immunization. We observed that epidermal powder immunization with unadjuvanted hepatitis B surface antigen elicited an antibody titre equivalent to that induced by the alum-adjuvanted vaccine delivered by intramuscular injection, suggesting that epidermal powder immunization can overcome the need for adjuvantation. We demonstrated that synthetic CpG oligonucleotides (CpG DNA) could be coformulated with hepatitis B surface antigen and delivered by epidermal powder immunization to further augment the antibody response and modulate T helper cell activities. Epidermal powder immunization of hepatitis B surface antigen formulated with CpG DNA formulations resulted in 1.5-2.0 logs higher IgG antibody titres than alum-adjuvanted commercial vaccines administered by intramuscular injection. Formulation of hepatitis B surface antigen with CpG DNA elicited an augmented IgG2a antibody response and increased frequency of IFN-gamma secreting cells. In addition, CpG DNA was found to activate epidermal Langerhans cells and stimulate the production of TNF-alpha and IL-12 cytokines by epidermal cells, explaining its strong adjuvant activity following epidermal powder immunization. These results show that epidermal powder immunization is a safe and effective method to deliver hepatitis B surface antigen and the addition of new adjuvants, such as CpG DNA, may further enhance the efficacy of this vaccine.

Stabilization of Alum-Adjuvanted Vaccine Dry Powder Formulations: Mechanism and Application

Studies were performed to elucidate the mechanism of alum gel coagulation upon freezing and drying and its relationship to vaccine potency loss and to develop a novel freeze-drying process for the production of stable alum-adjuvanted vaccine formulations suitable for conventional needle injection and epidermal powder immunization (EPI). The alum hydroxide-adjuvanted hepatitis-B surface antigen (Alum-HBsAg) and the alum phosphate-adjuvanted diphtheria and tetanus toxoids (Alum-DT) were dehydrated by freeze drying (FD), spray drying (SD), air drying (AD), or spray freeze drying (SFD). After drying by FD, SD, or AD, alum gels coagulated when examined by optical microscopy and particle size analysis. In addition, desorption of antigen molecules from the coagulated when examined by optical microscopy and particle size analysis. In addition, desorption of antigen molecules from the coagulated alum gel upon reconstitution appeared to be difficult, as indicated by attenuated band intensity on SDS-PAGE. In contrast, SFD alum gels turned a homogenous suspension upon reconstitution, suggesting minimal alum coagulation. In the mouse model, the in vivo immunogenicity of SFD Alum-HBsAg was preserved, whereas the FD Alum-HBsAg suffered significant immunogenicity loss. Grinding of coagulated FD Alum-HBsAg into smaller particles could partially recover the immunogenicity. In a guinea pig study using EPI, the SD Alum-DT formulation was not immunogenic, but the SFD Alum-DT formulations had a vaccine potency comparable to that of the untreated DT administered by I.M. injection. Overall, the relationship of coagulation of alum gel upon reconstitution and the loss of vaccine potency was established in this study. Alum gels became highly coagulated after dehydration by spray drying and traditional freeze-drying processes. However, freezing rate played a critical role in preserving the adjuvant effect of alum and fast freezing decreased the tendency of alum coagulation. Spraying the alum gel into liquid nitrogen represents the fastest freezing rate achievable and resulted in no discernible alum coagulation. Therefore, SFD presents a novel and effective drying process for alum-adjuvanted vaccine formulations and is particularly valuable for dry powder applications such as EPI.

Optimization of an alum-adsorbed vaccine powder formulation for epidermal powder immunization.

To develop stable and effective aluminum salt (alum)-adsorbed vaccine powder formulations for epidermal powder immunization (EPI) via a spray freeze-drying (SFD) process. Powder properties were determined using particle size analysis, tap density, and scanning electron microscopy. Alum coagulation was monitored via optical microscopy and particle sedimentation. Protein analysis was determined by the BCA protein assay, SDS-PAGE, and an enzyme immunoassay. In vivo immunogenicity and skin reactogenicity were performed on hairless guinea pigs and pigs, respectively. SFD of hepatitis B surface antigen (HBsAg) adsorbed to aluminum hydroxide or aluminum phosphate using an excipient combination of trehalose/mannitol/dextran produced vaccine powders of dense particles and satisfactory powder flowability and hygroscopicity. This formulation also offered excellent long-term stability to the powder and the antigen. The two most important factors influencing alum particle coagulation are the freezing rate and the concentration of aluminum in the liquid formulation for SFD. The SFD vaccines, when delivered to hairless guinea pigs by EPI or injected intramuscularly after reconstitution, were as immunogenic as the original liquid vaccine. A further study showed that EPI with SFD alum-adsorbed diphtheria-tetanus toxoid vaccine was well tolerated, whereas needle injection of the liquid formulation caused persistent granuloma. Stabilization of alum-adsorbed vaccine by SFD has important implications in extending vaccination to areas lacking a cold chain for transportation and storage and may also accelerate the development of new immunization technologies such as EPI.

Epidermal powder immunization with a recombinant HIV gp120 targets Langerhans cells and induces enhanced immune responses.

The recombinant envelope gp120 (rgp120) of human immunodeficiency virus (HIV) is a weak immunogen when administered by intramuscular (IM) injection. In the present study, we report that epidermal powder immunization (EPI) elicits robust antibody responses to the rgp120. EPI of mice with a dose 0.2-5 microg of rgp120 protein elicited geometric mean antibody titers that were 18- to 240-fold higher than that elicited by IM injection using a 5.0 microg dose. Targeting antigen to and mobilization of Langerhans cells (LCs) by EPI may explain the enhanced immunogenicity of the rgp120. EPI with rgp120 using sugar and gold particles as carrier resulted in differential antigen entry into the LCs and differential IgG subclass antibody and cellular immune responses. EPI may serve as a useful tool to evaluate vaccine potential of the rgp120 protein.

Targeting epidermal Langerhans cells by epidermal powder immunization.

Immune reactions to foreign or self-antigens lead to protective immunity and, sometimes, immune disorders such as allergies and autoimmune diseases. Antigen presenting cells (APC) including epidermal Langerhans cells (LCs) play an important role in the course and outcome of the immune reactions. Epidermal powder immunization (EPI) is a technology that offers a tool to manipulate the LCs and the potential to harness the immune reactions towards prevention and treatment of infectious diseases and immune disorders.

Epidermal powder immunization using non-toxic bacterial enterotoxin adjuvants with influenza vaccine augments protective immunity

The non-toxic B subunit of cholera toxin (CTB) and E. coli heat-labile toxin mutant proteins with reduced toxicity (LTR72) or no toxicity (LTK63) were used as adjuvants for epidermal powder immunization (EPI) with an influenza vaccine. When administered by EPI, CTB, LTR72 and LTK63 significantly augmented antibody responses to the influenza vaccine and protection against a lethal challenge in a mouse model. The antigen dose could be reduced by 125-fold. These adjuvants were well-tolerated both locally and systemically following EPI. These results suggest that EPI with influenza vaccine and a non-toxic bacterial enterotoxin hold promise for human vaccination.

Epidermal powder immunization induces both cytotoxic T-lymphocyte and antibody responses to protein antigens of influenza and hepatitis B viruses.

Cytotoxic T lymphocytes (CTL) play a vital role in host defense against viral and intracellular bacterial infections. However, nonreplicating vaccines administered by intramuscular injection using a syringe and needle elicit predominantly humoral responses and not CTL responses. Here we report that epidermal powder immunization (EPI), a technology that delivers antigens on 1.5- to 2.5-microm gold particles to the epidermis using a needle-free powder delivery system, elicits CTL responses to nonreplicating antigens. Following EPI, a majority of the antigen-coated gold particles were found in the viable epidermis in the histological sections of the target skin. Further studies using transmission electron microscopy revealed the intracellular localization of the gold particles. Many Langerhans cells (LCs) at the vaccination site contained antigen-coated particles, as revealed by two-color immunofluorescence microscopy, and these cells were found in the draining lymph nodes 20 h later. Immune responses to several viral protein antigens after EPI were studied in mice. EPI with hepatitis B surface antigen (HBsAg) and a synthetic peptide of influenza virus nucleoprotein (NP peptide) elicited antigen-specific CTL responses as well as antibody responses. In an in vitro cell depletion experiment, we demonstrated that the CTL activity against HBsAg elicited by EPI was attributed to CD8(+), not CD4(+), T cells. As controls, needle injections of HBsAg or the NP peptide into deeper tissues elicited solely antibody, not CTL, responses. We further demonstrated that EPI with inactivated A/Aichi/68 (H3N2) or A/Sydney/97 (H3N2) influenza virus elicited complete protection against a mouse-adapted A/Aichi/68 virus. In summary, EPI directly delivers protein antigens to the cytosol of the LCs in the skin and elicits both cellular and antibody responses.

Serum and mucosal immune responses to an inactivated influenza virus vaccine induced by epidermal powder immunization.

Both circulating and mucosal antibodies are considered important for protection against infection by influenza virus in humans and animals. However, current inactivated vaccines administered by intramuscular injection using a syringe and needle elicit primarily circulating antibodies. In this study, we report that epidermal powder immunization (EPI) via a unique powder delivery system elicits both serum and mucosal antibodies to an inactivated influenza virus vaccine. Serum antibody responses to influenza vaccine following EPI were enhanced by codelivery of cholera toxin (CT), a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs (CpG DNA), or the combination of these two adjuvants. In addition, secretory immunoglobulin A (sIgA) antibodies were detected in the saliva and mucosal lavages of the small intestine, trachea, and vaginal tract, although the titers were much lower than the IgG titers. The local origin of the sIgA antibodies was further shown by measuring antibodies released from cultured tracheal and small intestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays. EPI with a single dose of influenza vaccine containing CT or CT and CpG DNA conferred complete protection against lethal challenges with an influenza virus isolated 30 years ago, whereas a prime and boost immunizations were required for protection in the absence of an adjuvant. The ability to elicit augmented circulating antibody and mucosal antibody responses makes EPI a promising alternative to needle injection for administering vaccines against influenza and other diseases.