David C. Flyer

Safety and immunogenicity of an enterotoxigenic Escherichia coli vaccine patch containing heat-labile toxin: Use of skin pretreatment to disrupt the stratum corneum

ABSTRACT Transcutaneous immunization allows safe delivery of native heat-labile enterotoxin (LT) from Escherichia coli via application of a simple patch. Physical disruption of the stratum corneum can improve the efficiency of delivery. In the current study, the stratum corneum was disrupted using an electrocardiogram prep pad prior to patch application. The effects were quantified using transepidermal water loss (TEWL) and were correlated with the immune responses. Sixty adults received 50 μg of LT from three lots of LT (20 adults per group) administered in a patch on days 0 and 21. The immunizations were well tolerated. There were no differences in the anti-LT immunoglobulin G (IgG) titers between the three LT lots; the seroconversion rate was 100%, and the mean anti-LT IgG titer was 12,185 enzyme-linked immunosorbent assay units (EU) (a 24-fold increase). TEWL measurements obtained at the time of the second immunization were found to correlate with the day 42 individual increases in the anti-LT IgG titer (r = 0.59, P < 0.001). In a comparative assessment of the immune responses, sera after an LT+ ST+ (E2447A) oral ETEC challenge, which induced moderate to severe diarrhea in 81% of the recipients, had anti-LT IgG titers of 3,245 EU (a 10.8-fold increase). Similarly, the anti-LT IgG titer after administration of an oral cholera toxin B subunit-containing cholera vaccine, which cross-reacts with LT and protects against LT and LT/heat-stable toxin ETEC disease in the field, was 6,741 EU (a 3.3-fold increase). This study confirmed that a well-tolerated regimen for stratum corneum disruption before vaccine patch application results in robust immunity comparable to natural immunity and vaccine-induced immunity and that the magnitude of stratum corneum disruption correlates with the immune response.

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 Echerichia 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.

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.

A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models

Antibodies targeting the Clostridium difficile toxin A and toxin B confer protective immunity to C. difficile associated disease in animal models and provided protection against recurrent C. difficile disease in human subjects. These antibodies are directed against the receptor binding domains (RBD) located in the carboxy-terminal portion of both toxins and inhibit binding of the toxins to their receptors. We have constructed a recombinant fusion protein containing portions of the RBD from both toxin A and toxin B and expressed it in Escherichia coli. The fusion protein induced high levels of serum antibodies to both toxins A and B capable of neutralizing toxin activity both in vitro and in vivo. In a hamster C. difficile infection model, immunization with the fusion protein reduced disease severity and conferred significant protection against a lethal dose of C. difficile spores. Our studies demonstrate the potential of the fusion protein as a vaccine that could provide protection from C. difficile disease in humans.

Identification by Mass Spectrometry of CD8+-T-Cell Mycobacterium tuberculosis Epitopes within the Rv0341 Gene Product

ABSTRACT Identification of Mycobacterium tuberculosis proteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes from M. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a single M. tuberculosis protein that is encoded by the M. tuberculosis Rv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8+ T cells from healthy blood donors in vitro and capable of recognizing and lysing M. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification of M. tuberculosis proteins that can be evaluated as potential vaccine candidates.

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 studys 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.

Transcutaneous immunization with Intercell's vaccine delivery system.

Transcutaneous immunization (TCI) has become an attractive alternate route of immunization due to increase understanding of the skin immune system and to recent technical innovations in skin patch delivery systems. Basic principles of TCI have been demonstrated in animal and human studies, covering a variety of bacterial, viral, and cancer diseases. At Intercell, we have advanced two major platforms of TCI: 1) a needle-free vaccine delivery patch (VDP) and 2) a vaccine enhancement patch (VEP). Simplified, the VDP contains an antigen with or without an adjuvant that is administered on the skin; while the VEP contains only the adjuvant and is used in combination with an injected vaccine. In many of our TCI studies, the VDP or VEP is routinely applied on pretreated skin, in which the stratum corneum has been partially removed by mild abrasion. Recently, we have achieved technical breakthroughs in formulating and stabilizing vaccines in a dry patch format. For instance, a microplate-based screening process has been implemented to rapidly identify excipients, singularly or in combination, to stabilize biological macromolecules in patch blend formulations. A second technical innovation is our nonwoven (patch) disc matrix-supported drying technology, which allows efficient drying of our patch formulation blend to produce dry stable dosage forms of VDP or VEP. The low cost and the facileness in the manufacturing of VDP (or VEP) combined with the development of thermostable dry patches should improve the supply chain efficiency and reduce the dependence on cold chain.

Improved Titers against Influenza Drift Variants with a Nanoparticle Vaccine

Improving the Influenza Vaccine Many approaches are being sought to improve the vaccine against influenza virus. In this report, data on the immunogenicity of a nanoparticle vaccine with a baculovi...