Louise B. Lawson

Characterization of a Mutant Escherichia coli Heat-Labile Toxin, LT(R192G/L211A), as a Safe and Effective Oral Adjuvant

ABSTRACT Despite the fact that the adjuvant properties of the heat-labile enterotoxins of Escherichia coli (LT) and Vibrio cholerae (CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies. In vitro and in vivo data suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects.

Defending the mucosa: adjuvant and carrier formulations for mucosal immunity

A majority of infectious microorganisms either colonize or cross mucosal surfaces to enter the host. A major goal in vaccine design is to induce a protective, lasting immune response against potential pathogens at mucosal surfaces. In addition, mucosal vaccines can offer needle-free delivery, thereby improving accessibility, safety, and cost-effectiveness. Challenges to successful mucosal vaccination include poor induction of mucosal immunity, limited understanding of protective mechanisms and crosstalk between mucosal compartments, and the availability of safe, effective mucosal adjuvants and delivery systems. This review focuses on some key advances in the field of mucosal vaccinology within the past 2-3 years, including reports on promising new formulations and investigations into the mechanisms of established mucosal adjuvants and/or particulate carrier systems.

Hydration Effects on Skin Microstructure as Probed by High-Resolution Cryo-Scanning Electron Microscopy and Mechanistic Implications to Enhanced Transcutaneous Delivery of Biomacromolecules

Although hydration is long known to improve the permeability of skin, penetration of macromolecules such as proteins is limited and the understanding of enhanced transport is based on empirical observations. This study uses high-resolution cryo-scanning electron microscopy to visualize microstructural changes in the stratum corneum (SC) and enable a mechanistic interpretation of biomacromolecule penetration through highly hydrated porcine skin. Swollen corneocytes, separation of lipid bilayers in the SC intercellular space to form cisternae, and networks of spherical particulates are observed in porcine skin tissue hydrated for a period of 4-10 h. This is explained through compaction of skin lipids when hydrated, a reversal in the conformational transition from unilamellar liposomes in lamellar granules to lamellae between keratinocytes when the SC skin barrier is initially established. Confocal microscopy studies show distinct enhancement in penetration of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) through skin hydrated for 4-10 h, and limited penetration of FITC-BSA once skin is restored to its natively hydrated structure when exposed to the environment for 2-3 h. These results demonstrate the effectiveness of a 4-10 h hydration period to enhance transcutaneous penetration of large biomacromolecules without permanently damaging the skin.

The A Subunit of Escherichia coli Heat-Labile Enterotoxin Functions as a Mucosal Adjuvant and Promotes IgG2a, IgA, and Th17 Responses to Vaccine Antigens

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) produces both heat-labile (LT) and heat-stable (ST) enterotoxins and is a major cause of diarrhea in infants in developing countries and in travelers to those regions. In addition to inducing fluid secretion, LT is a powerful mucosal adjuvant capable of promoting immune responses to coadministered antigens. In this study, we examined purified A subunit to further understand the toxicity and adjuvanticity of LT. Purified A subunit was enzymatically active but sensitive to proteolytic degradation and unable to bind gangliosides, and even in the presence of admixed B subunit, it displayed low cyclic AMP (cAMP) induction and no enterotoxicity. Thus, the AB5 structure plays a key role in protecting the A subunit from proteolytic degradation and in delivering the enzymatic signals required for secretion. In contrast, the A subunit alone was capable of activating dendritic cells and enhanced immune responses to multiple antigens following intranasal immunization; therefore, unlike toxicity, LT adjuvanticity is not dependent on the AB5 holotoxin structure or the presence of the B subunit. However, immune responses were maximal when signals were received from both subunits either in an AB5 structure or with A and B admixed. Furthermore, the quality of the immune response (i.e., IgG1/IgG2 balance and mucosal IgA and IL-17 secretion) was determined by the presence of an A subunit, revealing for the first time induction of Th17 responses with the A subunit alone. These results have important implications for understanding ETEC pathogenesis, unraveling immunologic responses induced by LT-based adjuvants, and developing new mucosal vaccines.

Use of Nanocarriers for Transdermal Vaccine Delivery

Transdermal delivery is a safe, noninvasive method of administering vaccines directly onto bare skin, offering several potential advantages over traditional needle delivery. This technology is limited by the relative inefficiency of transport of large‐molecular‐weight vaccine antigens across intact skin. Recent evidence has shown that this barrier can be overcome by properly structured nanosized particles (nanocarriers). The specialized assembly of each type of nanocarrier gives each unique properties and different interactions within the stratum corneum. The use of nanocarriers for vaccine delivery is a platform technology, applicable to delivery of a variety of existing and potential vaccines.

In vitro degradation and release characteristics of spin coated thin films of PLGA with a "breath figure" morphology.

Poly (lactic-co-glycolic acid) (PLGA) coatings on implant materials are widely used in controlled drug delivery applications. Typically, such coatings are made with non-porous films. Here, we have synthesized a thin PLGA film coating with a highly ordered microporous structure using a simple and inexpensive water templating “breath figure” technique. A single stage process combining spin coating and breath figure process was used to obtain drug incorporated porous thin films. The films were characterized by scanning electron microscope (SEM) to observe the surface and bulk features of porosity and also, degradation pattern of the films. Moreover, the effect of addition of small amount of poly (ethylene glycol) (PEG) into PLGA was characterized. SEM analysis revealed an ordered array of ~2 µm sized pores on the surface with the average film thickness measured to be 20 µm. The incorporation of hydrophilic poly (ethylene glycol) (PEG) enhances pore structure uniformity and facilitates ingress of water into the structure. A five week in vitro degradation study showed a gradual deterioration of the breath figure pores. During the course of degradation, the surface pore structure deteriorates to initially flatten the surface. This is followed by the formation of new pinprick pores that eventually grow into a macroporous film prior to film breakup. Salicylic acid (highly water soluble) and Ibuprofen (sparingly water soluble) were chosen as model drug compounds to characterize release rates, which are higher in films of the breath figure morphology rather than in non-porous films. The results are of significance in the design of biodegradable films used as coatings to modulate delivery.

The novel adjuvant dmLT promotes dose sparing, mucosal immunity and longevity of antibody responses to the inactivated polio vaccine in a murine model

One option for achieving global polio eradication is to replace the oral poliovirus vaccine (OPV), which has the risk of reversion to wild-type virulence, with the inactivated poliovirus vaccine (IPV) vaccine. Adjuvants and alternate routes of immunization are promising options that may reduce antigen dose in IPV vaccinations, potentially allowing dose sparing and cost savings. Use of adjuvants and alternate routes of immunization could also help promote mucosal immunity, potentially mimicking the protection against intestinal virus shedding seen with OPV. In the current study, we examined the impact of combining the novel adjuvant dmLT with trivalent IPV for dose sparing, induction of mucosal immunity and increasing longevity of anti-poliovirus (PV) responses in a mouse model following either intradermal (ID) or intramuscular (IM) delivery. We found that non-adjuvanted ID delivery was not superior to IM delivery for fractional dose sparing, but was associated with development of mucosal immunity. Vaccination with IPV+dmLT promoted serum anti-PV neutralizing antibodies with fractional IPV doses by either IM or ID delivery, achieving at least five-fold dose sparing above non-adjuvanted fractional doses. These responses were most noticeable with the PV1 component of the trivalent vaccine. dmLT also promoted germinal center formation and longevity of serum anti-PV neutralizing titers. Lastly, dmLT enhanced mucosal immunity, as defined by fecal and intestinal anti-PV IgA secretion, when included in IPV immunization by ID or IM delivery. These studies demonstrate that dmLT is an effective adjuvant for either IM or ID delivery of IPV. Inclusion of dmLT in IPV immunizations allows antigen dose sparing and enhances mucosal immunity and longevity of anti-PV responses.

Undulating Tubular Liposomes through Incorporation of a Synthetic Skin Ceramide into Phospholipid Bilayers

Nonspherical liposomes were prepared by doping L-alpha-phosphatidylcholine (PC) with ceramide VI (a skin lipid). Cryo-transmission electron microscopy shows the liposome shape changing from spherical to an undulating tubular morphology, when the amount of ceramide VI is increased. The formation of tubular liposomes is energetically favorable and is attributed to the association of ceramide VI with PC creating regions of lower curvature. Since ceramides are the major component of skin lipids in the stratum corneum, tubular liposomes containing ceramide may potentially serve as self-enhanced nanocarriers for transdermal delivery.

Liposomes in double-emulsion globules.

Tubular liposomes containing a hydrophilic model compound (fluorescein sodium salt, FSS) were entrapped inside the internal aqueous phase (W(1)) of water-in-oil-in-water (W(1)/O/W(2)) double-emulsion globules. Our hypothesis was that the oil membrane of double emulsions can function as a layer of protection to liposomes and their contents and thus better control their release. Liposomes were prepared in bulk, and their release was observed microscopically from individual double-emulsion globules. The liposomes containing FSS were released through external coalescence, and the behavior of this system was monitored visually by capillary video microscopy. Double-emulsion globules were stabilized with Tween 80 as the water-soluble surfactant, with Span 80 as the oil-soluble surfactant, while the oil phase (O) was n-hexadecane. The lipids in the tubular liposomes consist of L-alpha-phosphatidylcholine and Ceramide-VI. Variations of Tween 80 concentration in the external aqueous phase (W(2)) and Span 80 concentration in the O phase controlled the release of liposomes from the W(1) phase to the W(2) phase. The major finding of this work is that the sheer presence of liposomes in the W(1) phase is by itself a stabilizing factor for double-emulsion globules.

Mucosal immune responses induced by transcutaneous vaccines.

The skin has been investigated as a site for vaccine delivery only since the late 1990s. However, much has been discovered about the cell populations that reside in the skin, their active role in immune responses, and the fate of trans- cutaneously applied antigens. Transcutaneous immunization (TCI) is a safe, effective means of inducing immune responses against a number of pathogens. One of the most notable benefits of TCI is the induction of immune responses in both systemic and mucosal compartments. This chapter focuses on the transport of antigen into and beyond intact skin, the cutaneous sentinel cell populations that play a role in TCI, and the types of mucosal immune responses that have been generated. A number of in vivo studies in murine models have provided information about the broad responses induced by TCI. Cellular and humoral responses and protection against challenge have been noted in the gastrointestinal, reproductive, and respiratory tracts. Clinical trials have demonstrated the benefits of this vaccine delivery route in humans. As with other routes of immunization, the type of vaccine formulation and choice of adjuvant may be critical for achieving appropriate responses and can be tailored to activate specific immune-responsive cells in the skin to increase the efficacy of TCI against mucosal pathogens.