Biphasic lipid vesicles as a subcutaneous delivery system for protein antigens and CpG oligonucleotides.

One of the major drawbacks to the development of novel vaccines has been the lack of safe yet effective adjuvants. Biphasic lipid vesicles are formulations suitable for the delivery of proteins, peptides and oligo/polynucleotides. They constitute a new class of delivery system into which antigens and adjuvants can be incorporated. The purpose of these studies was to investigate the ability of biphasic lipid vesicles (Vaccine-Targeting Adjuvants--VTA) to induce immune responses to bacterial antigens and to enhance the adjuvant activity of CpG ODNs. Immunization of mice with bacterial antigen and CpG ODNs in saline was not as effective at inducing immune responses as formulation in VTA vesicles. Results showed that formulation of CpG ODN in VTA significantly enhanced its adjuvanticity.

Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines.

One potential reason for the enhancement of immune responses to DNA vaccines following electroporation is increased gene expression. However, the inflammatory response and accompanying cellular infiltration stimulated by electroporation may also be essential for enhancing immune responses to DNA vaccines. These parameters were investigated in pigs, using different electroporation conditions to induce different levels of gene expression and inflammation. Results indicated that the least effective strategy was conventional intramuscular injection where there was low gene expression and low inflammatory cell infiltration. The most efficacious strategy was plasmid administration immediately followed by electroporation. This latter set of conditions elicited a combination of high gene expression and high cellular infiltration. This indicates that electroporation enhances immune responses to DNA vaccines through increased gene expression and inflammatory cell infiltration.

Biphasic lipid vesicles (Biphasix) enhance the adjuvanticity of CpG oligonucleotides following systemic and mucosal administration.

CpG oligonucleotides (ODNs) are potent mucosal and systemic adjuvants. For practical applications however, improvements in delivery need to be developed. A mouse model was used to determine if the biological activity of CpG ODNs could be enhanced using a novel delivery system of biphasic lipid vesicles (Biphasix Vaccine-Targeting Adjuvant; VTA). Immunization studies were performed to evaluate the potential of VTA formulations to enhance the immunoadjuvant activity of CpG ODNs following systemic or mucosal administration with gD. Immune responses following immunization were assessed by protection from HSV-1 viral challenge and characterization of serum gD-specific antibody responses using ELISA. VTA formulations in combination with CpG and glycoprotein D (gD) were able to increase gD-specific IgG in serum compared to gD alone, and protect from a lethal HSV-1 challenge following subcutaneous immunization. Following mucosal immunization, VTA formulations in combination with CpG and antigen enhanced mucosal IgA responses compared to CpG and antigen administered in PBS.

Needle-free topical electroporation improves gene expression from plasmids administered in porcine skin.

Electroporation has been shown to increase the potency of DNA vaccines that have demonstrated significant potential in mice. However, there is a need to develop noninvasive or minimally invasive vaccination methods. In pigs, in vivo gene expression was assessed to compare intradermal needle injection to a needle-free dermal BioJect as a means of delivery of plasmids. Each administration method was further tested with and without surface electroporation. Experiments with plasmid DNA encoding luciferase demonstrated that needle-free administration results in higher gene expression levels than needle injection. Electroporation enhanced gene expression for both intradermal delivery methods. Needle-free plasmid injection in combination with electroporation led to a more rapid induction of immune responses compared to other methods of plasmid administration. It was concluded that needle-free topical electroporation significantly enhances gene expression, possibly by improving cellular uptake of plasmid DNA.

Electroporation improves the efficacy of DNA vaccines in large animals.

It is generally recognized that DNA vaccines are often less effective in large animals than in mice. One possible reason for this reduced effectiveness may be transfection efficiency and the low level of expression elicited by plasmid vectors in large animals. A possible way to improve plasmid gene expression in vivo is electroporation. To determine whether we could enhance immune responses in pigs by electroporation, we used plasmids encoding two different genes (bovine herpesvirus glycoprotein D (gD) and hepatitis B surface antigen (HBsAg)) and two different electrodes, a single-needle electrode and a six-needle electrode. Electroporation significantly enhanced immune responses to both antigens. In addition, we demonstrated that co-administration of plasmids coding for two different antigens (pgD and pHBsAg) did not result in significant interference between the plasmids. We also incorporated a DNA prime/protein boost strategy to examine the effect of DNA priming with electroporation on the immune response after a protein boost.

The haemopoietic growth factor, Flt3L, alters the immune response induced by transcutaneous immunization.

Topical application of antigen induces antigen-specific humoral and cellular immune responses. In this study we examined whether expansion of dendritic cells (DC) by Flt3 ligand (Flt3L) treatment influences the induction of immune responses following transcutaneous immunization. Mice were treated intraperitoneally with Flt3L or phosphate-buffered saline (PBS) and immunized transcutaneously with hen egg lysozyme (HEL). Flt3L-treated mice developed lower HEL-specific cellular and humoral immune responses than PBS-treated mice. However, in the presence of cholera toxin (CT), a potent adjuvant for mucosal and transcutaneous immunization, Flt3L-treated mice developed significantly higher cellular and humoral immune responses to HEL when compared to PBS-treated mice. We assessed whether the immunomodulatory effects of CT were a result of activation of epidermal dendritic cells (Langerhans' cells; LC). Our results indicate that within 8-12 hr of topical application of CT, epidermal LC cells lose their dendritic morphology and become rounder in appearance. In addition, we observed enhanced expression of major histocompatibility complex (MHC) class II, and of adhesion molecules CD11c and intracellular adhesion molecule-1 (ICAM-1). Our observations support the concept that the state of activation of DC in the skin is central to the regulation of immune responses. This information is relevant to the design of effective transcutaneous vaccination strategies.

Topical delivery of plasmid DNA using biphasic lipid vesicles (Biphasix).

The development of non-invasive methods for the delivery of vaccines through the skin will greatly improve the safety and the administration of human and veterinary vaccines. In this study we examined the efficiency of topical delivery of plasmids by assessing the localization of gene expression using luciferase as a reporter gene and induction of immune responses using a plasmid encoding for the bovine herpesvirus type-1 glycoprotein D (pgD). Topical administration of plasmids in a lipid-based delivery system (biphasic lipid vesicles--Biphasix) resulted in gene expression in the lymph node, whereas with intradermal injection, antigen expression was found in the skin. Following administration of plasmid with the gene gun, antigen expression was observed in both the skin as well as in the draining lymph nodes. Transcutaneous immunization with pgD formulated in biphasic lipid vesicles elicited gD-specific antibody responses and a Th2-type cellular response. In contrast, immunization by the intradermal route resulted in the stimulation of a Th1-type response. These findings have implications for both vaccine design and tailoring of specific immune responses.