Safety and immunogenicity of an influenza vaccine A/H5N1 (A/Vietnam/1194/2004) when coadministered with a heat-labile enterotoxin (LT) adjuvant patch.

BACKGROUND: The use of adjuvants to enhance the immune response to novel pandemic influenza vaccine candidates may overcome the poor immune responses seen in immunologically naïve populations. The confluence of a highly pathogenic H5N1 influenza virus and the widespread absence of pre-existing immunity has driven the search for effective strategies for immunization in the face of a lethal pandemic. The potent adjuvant, heat labile enterotoxin from E. coli (LT), placed over the immunization site in a patch, is a novel adjuvant strategy for immune enhancement, and was evaluated using an H5N1 injectable vaccine. METHODS: In this observer-blind, placebo-controlled clinical study, 500 healthy adults 18-49 years of age were randomized to receive two intramuscular doses of A/Vietnam/1194/2004 A/H5N1 vaccine (5microg, 15microg or 45microg) or placebo (saline) 21 days apart. For each of the influenza vaccine doses, a 50microg LT adjuvant patch was applied over the injection site at either the second or both immunizations and the HI responses (titers) were compared to H5N1 vaccine alone. The study's primary endpoint was safety, and secondary immunogenicity endpoints were evaluated using European (CHMP) licensure criteria. RESULTS: The vaccine was safe and well tolerated, and subjects generally lacked pre-existing H5N1 immunity. The single-dose injection 45microg HA/LT patch regimen met all CHMP licensure criteria, including a 73% seroprotection rate compared to 49% seroprotection without a patch. Significant adjuvant effects were seen at all HA doses on Day 21. By contrast, only modest adjuvant effects were observed with the boosting regimen in subjects first primed with H5N1 alone and given the adjuvant patch only on the second immunization. The two-injection/two-patch 45microg HA regimen achieved significantly higher titers and GMFR compared to injection alone (GMFR 33.1 vs. 16.9, HI 226 vs. 94, p<0.05) and a 94% seroprotection rate. CONCLUSIONS: The LT adjuvant patch placed over the injection site was safe, significantly enhanced the immune response to an H5N1 candidate vaccine, and achieved a 73% seroprotection rate after a single dose. The LT adjuvant patch has more modest benefits in recently primed populations similar to other candidate vaccine adjuvants, but a two-dose patch plus injection regimen resulted in robust HI responses.

Controlled, single-step, stratum corneum disruption as a pretreatment for immunization via a patch.

A Skin Prep System (SPS) has been developed to provide a well-tolerated and controlled method of stratum corneum disruption using mild abrasion as part of transcutaneous immunization (TCI). In this study, four groups (n=10) of volunteers were pretreated with the SPS using three different lengths of mild abrasive strips (13 mm, 25 mm and 38 mm), or a handheld applicator. They then received a vaccine patch containing 50 microg of the heat-labile enterotoxin from Escherichia coli (LT) at day 0 and day 21. Subsequent anti-LT IgG antibody responses were dependent on abrasive strip length, with highest immune responses seen after use of the longest strip. The development of a simple, single-use, disposable device that is well-tolerated and allows disruption to be modulated represents an important step forward in physical penetration enhancement for the skin.

Transcutaneous delivery and thermostability of a dry trivalent inactivated influenza vaccine patch.

A patch containing a trivalent inactivated influenza vaccine (TIV) was prepared in a dried, stabilized formulation for transcutaneous delivery. When used in a guinea pig immunogenicity model, the dry patch was as effective as a wet TIV patch in inducing serum anti-influenza IgG antibodies. When the dry TIV patch was administered with LT as an adjuvant, a robust immune response was obtained that was comparable with or better than an injected TIV vaccine. When stored sealed in a nitrogen-purged foil, the dry TIV patch was stable for 12 months, as measured by HA content, under both refrigerated and room temperature conditions. Moreover, the immunological potency of the vaccine product was not affected by long-term storage. The dry TIV patch was also thermostable against three cycles of alternating low-to-high temperatures of -20/25 and -20/40 degrees C, and under short-term temperature stress conditions. These studies indicate that the dry TIV patch product can tolerate unexpected environmental stresses that may be encountered during shipping and distribution. Because of its effectiveness in vaccine delivery and its superior thermostable characteristics, the dry TIV patch represents a major advance for needle-free influenza vaccination.

Transcutaneous immunization with heat-labile enterotoxin: development of a needle-free vaccine patch.

The skin is an attractive target for vaccine delivery. Adjuvants and antigens delivered into the skin can result in potent immune responses and an unmatched safety profile. The heat-labile enterotoxin (LT) from Escherichia coli, which acts both as antigen and adjuvant, has been shown to be delivered to human skin efficiently when used in a patch, resulting in strong immune responses. Iomai scientists have capitalized on these observations to develop late-stage products based on LT. This has encouraged commercial-level product development of a delivery system that is efficient, user-friendly and designed to address important medical needs. Over the past 2 years, extensive clinical testing and optimization has allowed the patch to evolve to a late-stage product. As a strategy for approval of a revolutionary vaccine-delivery system, the singular focus on optimization of LT delivery has enabled technical progress to extend patch-vaccine product development beyond LT. The field efficacy of the LT-based travelers' diarrhea vaccine has validated this approach. The discussion of transcutaneous immunization is unique, in that any consideration of the adjuvant must also include delivery, and the significant advances in a commercial patch application system are described. In this review, we integrate these concepts, update the clinical data and look to the future.

GM1 binding-deficient exotoxin is a potent noninflammatory broad spectrum intradermal immunoadjuvant.

Intradermal (i.d.) immunization is a promising route of vaccine administration. Suitable i.d. adjuvants are important to increase vaccine efficacy in poorly responding populations such as the elderly or for dose-sparing strategies in the face of vaccine shortages. Bacterial exotoxins, such as Escherichia coli heat-labile enterotoxin (LT), exert strong immunostimulatory effects through binding to monosialoganglioside (GM1) cell surface receptors; however, injection is hampered by local inflammation. We demonstrate that the injection of LT formulations deficient in GM1 binding by mutation (LT(G33D)) or in vitro ligand coupling does not cause localized edema and inflammation in mice, yet these formulations retain potent adjuvant activity by enhancing functional Ab and cellular immune responses to coadministered Ags. Complete protection against in vivo lethal tetanus toxin challenge and the induction of Ag-specific CTL responses capable of killing target cells in vivo indicated in vivo efficacy of the induced immune responses. LT(G33D) proved superior to standard alum adjuvant regarding the magnitude and breadth of the induced immune responses. Immunizations in complex ganglioside knockout mice revealed a GM1-independent pathway of LT adjuvanticity. Immunostimulation by i.d. LT(G33D) is explained by its ability to induce migration of activated APCs to the proximal draining lymph nodes. LT(G33D) is a promising candidate adjuvant for human trials of parenteral vaccines in general and for current i.d. vaccine development in particular.

Induction of protective immunity against lethal anthrax challenge with a patch.

BACKGROUND: Transcutaneous immunization (TCI) is a needle-free technique that delivers antigens and adjuvants to potent epidermal immune cells. To address critical unmet needs in biodefense against anthrax, we have designed a novel vaccine delivery system using a dry adhesive patch that simplifies administration and improves tolerability of a subunit anthrax vaccine. METHODS: Mice and rabbits were vaccinated with recombinant protective antigen of Bacillus anthracis and the heat-labile toxin of Escherichia coli. Serologic changes, levels of toxin-neutralizing antibodies (TNAs), and pulmonary and nodal responses were monitored in the mice. A lethal aerosolized B. anthracis challenge model was used in A/J mice, to demonstrate efficacy. RESULTS: The level of systemic immunity and protection induced by TCI was comparable to that induced by intramuscular vaccination, and peak immunity could be achieved with only 2 doses. The addition of adjuvant in the patch induced superior TNA levels, compared with injected vaccination. CONCLUSIONS: Anthrax vaccine patches stimulated robust and functional immune responses that protected against lethal challenge. Demonstration of responses in the lung suggests that a mechanism exists for protection against challenge with aerosolized anthrax spores. A formulated, pressure-sensitive, dry adhesive patch, which is stable and can be manufactured in large scale, elicited comparable immunoglobulin G and TNA responses, suggesting that an anthrax vaccine patch is feasible and should advance into clinical evaluation.

Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice.

Improvement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination. We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin of Escherichia coli (LT) enhances immune responses to injected vaccines. We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice. LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses. The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone. These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination. Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.

Transcutaneous immunization and immunostimulant strategies: capitalizing on the immunocompetence of the skin.

The skin is an attractive target for vaccine delivery. Topical application of adjuvants results in potent immune responses and good safety profiles. Adjuvants can be coadministered in a patch with vaccine antigens (transcutaneous immunization) or similar delivery format, or administered separately with an injection or IS patch (Iomai), leading to enhanced immune responses. These observations have moved into the clinic, highlighting the likelihood that skin delivery of vaccines will play an important future role in vaccine applications.

Transcutaneous immunization and immunostimulant strategies.

The skin provides an attractive immune environment for vaccine delivery and a safe and confined anatomic space for the use of potent adjuvants. It has been presumed that LCs as a class of dendritic cells should stimulate potent immune responses when activated by adjuvants, and this theory is beginning to be validated. Progress on simple pretreatment of the skin has led to well-developed, simple-to-use protocols that are not dissimilar from current protocols used to cleanse the skin before injection. Antigen and adjuvant formulation optimization has progressed, leading to phase 2 testing of the technology in formulated, manufacturable patches. Although delivery optimization and product testing is challenging, the major biologic observations underlying TCI and the IS patch have been established clearly in that large protein antigens have been delivered clinically, resulting in robust immune responses in a safe manner. During the next 5 years, the challenge will be to conduct a development program that leads to safe and effective vaccination in the context of specific applications.