Recently, the search for effective needle-free vaccination has escalated due to heightened concerns of pandemic diseases, bioterrorism, and specific disease eradication programs. Needlefree vaccination could assist withmass vaccination due toits easy and fast delivery, and by providing improved safety, decreasedcost, and reducedvaccination-associated pain, thus, improving compliance. There are two major hurdles in the development of a successful oral vaccine: (i) protection of the antigenof interest from low gastric pH and digestive enzymes, (ii) and delivery of the antigento dendritic cells (DCs) that are professional antigen presenting cells. To overcome these challenges, two approaches were taken. First, in order to protect the antigen from a harsh gastric environment, LactoBacillus species, including L. acidophilus and L. gasseri, bacteria that can survive and thrivein the gastrointestinal tract, were modified to secrete the antigen of interest in the gut. Secondly, to assist in the deliveryof antigen to professional antigen presenting cells, a twelve amino acid long “targeting” peptide was added tothe immunogenic vaccine subunit [1,2].

LactoBacillus Species as an Antigen Delivery Vehicle

Mucosal surfaces are the major route of entry for most of the pathogens that cause disease. Thus, vaccines capable of inducing a mucosal immune response can strengthen the defenses at the mucosal layer and protect against infection. Different species of LactoBacillus have been used as dietary componentsofhuman food for centuries and are considered safe for human consumption. Certain species of LactoBacillus are also a part of the normal gut microbiota [3]. Since lactobacillithrivein the low pH of the stomach, using this bacterium as a vaccine delivery vehicle protects the vaccine subunit from the harsh acidic environment and maintains vaccine bioavailability. Meanwhile, researchers have also developed both inducible and constitutive expression vectors effective in lactobacilli to deliver immunogenic antigens [1,4-6]. Although immunetolerance to a commensal inhabitant of the gastrointestinal mucosa was an initial concern with this strategy, certain lactobacilli such as L. gasseri, are able to overcome tolerance, likely via their strong innate adjuvant properties [7,8]. Using L. gasseri, our laboratory has been able to deliver the protective antigen (PA) of Bacillus anthracis to vaccinated miceto resist this potential agent of bioterrorism [2]. Additionally, another group has used L. gasseri for the successful delivery of Salmonella antigens [9].

Dendritic cells (DCs) are the most effective antigen presenting cells in humans and domestic animals, with the unique ability to present antigen and activate naïve T lymphocytes, thus, playinga criticalrole in the induction of specific primary immune responses. Expression of a varietyof surface receptors, such as C-type lectins (e.g., mannose R, DC-SIGN, DEC-205), Toll-like receptors (TLRs), receptors for the Fc portion of antibodies (FcRs) and complement receptors (CR3, CR4), allow these cells to efficiently bind antigens [10]. Captured antigensaresubsequently processed and efficiently presented to rare antigen specific T lymphocytesdue to the constitutive expression of class II MHC molecules and co-stimulatory/regulatory molecules, including CD40, CD86, and B7-H1 on mature DCs. Therefore, antigen delivered specifically to DCs, as opposed to B lymphocytes or macrophages that require activation to express co-stimulatory molecules,reduces the dose requirement of antigen for immune stimulationand has emerged as a potential vaccination tool to induce protective immune responses [10]. Utilization of traditional receptors (e.g., class II MHC molecules, CD11c, TLRs) for antigen targeting does not resultinefficient delivery of antigen specifically to DCs because many other cell types also express these receptors and compete with DCs for vaccine binding. With the goal to discover a specific DC-targeting peptide moiety, a phage display library was sequentially absorbed withmonocytes, T cells, B cells, and Langerhans cells and thenscreened for the ability to bindhuman myeloid-derived DCs [11]. The obtained sequence was then tested for its in vitro binding capacity and its application in antigen delivery in a murine model of oral vaccinationby fusing this DC-targeting peptide to PA of Bacillus anthracis [1,12].

Because of their ease of administration, oral vaccine strategies are currently under development or in use for Bordetellabronchiseptica, the primary causative pathogen of the contagious respiratory disease complex, infectious tracheobronchitis or “kennel cough”, which is commonly seen in dogs housed together in pet stores, kennels, and animal shelters. This disease can also affect cats and occasionally, immunocompromised humans [13]. Oral therapeutic strategies are also used in rabies control programs that target ownerless pet populations in enzootic areas by vaccinating via palatable bait [14]; for oral vaccination of foals against pneumonia caused by Rhodococcusequi [15]; to protect newly born calves from scours, diarrhea caused by E. coli; and via administration in the drinking water of poultry to prevent or mitigate Newcastle disease, fowl cholera, and avian encephalomyelitis. In fact, poultry are likely the most heavily vaccinated domestic species, with the intensity of production expected to dramatically increase in the next few decades as the economies of countries like China and India improve and poultry consumption increases [16]. To expand upon the applicability of this targeting peptide as an antigen delivery agent in domestic animals, we tested its ability to bind DCs from multiple species of veterinary importance. We found significantly higher binding of DC-peptide to the DCs isolated from peripheral blood from all species tested, including canine, feline, equine, bovine, porcine, and avian species when compared to a non-specific peptide, indicating that this peptide binds to highly conserved regions of its receptor, the identity of which is currently under investigation in our laboratory. These results favorably suggest future use of this particular strategy in the development of veterinarymultivalent vaccines.

Current vaccination methods employ needle-based administration, which has certain limitations that include the requirement of trained personnel, the risk of accidental needle sticks, and other constraints. To overcome these short comings, we have developed a combinatorial approach that delivers the DC-targeted antigenic protein to intestinal dendritic cells. This approach not only provides a platform for the development of an oral vaccine for human patients, but also allows for the possibility of the generation of oral vaccines against pathogens of veterinary importance.

This work was supported by NIH Grant 1R01AI098833-01.