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Microstructured transdermal systems can deliver a vaccine in close proximity to the antigen-presenting cells in the epidermis.
Prophylactic vaccines are one of the most cost-effective ways of preventing illness and death from infectious disease. The public health benefit of this approach, however, is dependent on the proportion of the at-risk population that is vaccinated. The frequent need for more than one dose of vaccine to offer protection and the unfriendly method of administration—commonly needle and syringe—means that compliance with vaccination programs is an ongoing problem. In addition, the use of sharps can lead to public health problems, particularly where safe disposal of hazardous waste is not readily available. There is an increasing interest in alternative delivery mechanisms for antigens, and many companies are developing needle-free approaches. This article will review some of the technologies that aim to revolutionize vaccine administration by focusing on oral vaccine delivery using live bacteria.
The demand for new and improved vaccines against human diseases is rising and the interest in developing new delivery mechanisms for antigens is increasing concurrently.1–4 Needle-free vaccinations offer the advantage of convenience, and remove the need for sharps, which can cause public health problems. Many companies, therefore, are focusing on developing alternative transdermal and oral vaccination methods.
Mechanisms for transdermal delivery include needle-free devices that deliver solid or liquid formulations of vaccines. Oral delivery of protein antigens presents major challenges because of the strong natural barriers along the gastrointestinal (GI) tract, including stomach acid and proteases that can degrade these molecules. Moreover, the rate of absorption of proteins along the GI tract is often poor. Encasing protein antigens with lipid molecules to deliver them through the hostile environment of the stomach and releasing them in the intestine is one approach to address these problems. An alternative approach is the development of live bacterial vaccines that carry foreign antigens.2,5,6 Live bacterial vaccines are relatively inexpensive to manufacture, room temperature stable, easy to administer, and offer the added benefit of being inherently immunogenic.
The skin immune system arises from cellular and humeral components of the epidermis and the dermis. Among the most important components of the skin immune system are the Langerhans cells, which are specialized antigen presenting cells found in the viable epidermis. The Langerhans cells detect and internalize antigens, and carry them to draining lymph nodes where they are presented to T-cells, invoking an immune response.
Jet injectors, such as Antares Pharma's Medi-Jector VISION, deliver medication through high-speed, pressurized liquid penetration of the skin without a needle. These have been developed as single-use devices and multiuse systems. A high peak pressure behind the liquid is required so it can drill a hole in the skin, and then the pressure is reduced to allow the rest of the liquid to enter the skin. These pressures need to be carefully controlled. The difficulty in doing this and the cost attached to the various solutions are some of the reasons why jet injectors are not used more widely.7
Other transdermal approaches deliver the antigen in a solid form. These approaches have the added benefit that the therapeutic agent is more stable and therefore may not need cold storage. PowderMed, which was acquired by Pfizer in October 2006, uses its PMED delivery device to propel gold particles coated in a DNA vaccine into the skin using high-pressure helium. This approach delivers the DNA directly to Langerhans cells, thereby stimulating immunity. The device is pain free, and reportedly can be administered without the need for trained medical personnel. This is particularly important given that one of the vaccines in development is against pandemic avian influenza.8
A third approach uses the pharmaceutical formulation itself to puncture the skin. Glide Pharma has developed a low-velocity, spring-powered administrator that pushes a pointed rod of pharmaceutical material through the skin in a fraction of a second. This administrator enables constant, reliable delivery of a solid dosage form and could be applied to various vaccines. The low cost of manufacturing the device is an added bonus, which further supports its use in developing countries.7
Microstructured transdermal systems (MTS) are also suitable for vaccine delivery because the vaccine can be delivered in close proximity to the antigen-presenting cells in the epidermis, and thus it is possible to achieve excellent immune response with smaller quantities of antigen than used in subcutaneous injection. The small size of microstructures causes comparatively less mechanical trauma to the skin and these are painless upon insertion. Arrays of microstructures are typically held within a patch that secures them against the skin. In the solid-coated microstructures the vaccine coats their exterior surface and is released once these penetrate the skin.9
Electroporation is increasingly used for DNA vaccine administration in clinical trials, although, this is not a method of needle-free delivery as it uses an electric shock to increase the efficiency of uptake of injected DNA. Applying an electric pulse is a common technique in the laboratory for getting DNA into a variety of cells, and this approach has been scaled up in portable machines for use on humans and livestock. Portable devices have been developed consisting of an array of electrodes that deliver an electric shock to the skin at the point where a DNA vaccine solution has been injected, resulting in up to 100-fold improvement in efficiency over using a needle alone to deliver naked DNA.
The TriGrid delivery system developed by Ichor Medical Systems combines the injection and electric pulse in a single device. The MedPulser DNA electroporation therapy system from Inovio Biomedical Corporation is used to deliver the pulse following an injection. These systems have been evaluated successfully using a range of DNA vaccines. However, the continued reliance on needles, the additional requirement for a source of electricity, and the discomfort of administration is likely to restrict electroporation to DNA vaccine applications.
Oral delivery of therapeutics and vaccines is a promising approach to improve compliance with vaccination regimes. However, because of the hostile environment of the stomach and the GI tract, antigens—particularly protein antigens—must be protected to enable them to reach the site of absorption. One strategy is to encase the protein antigens in lipid molecules or lectins. Elan Pharmaceuticals has demonstrated the use of a ligand (Ulex europaeus agglutinin I) that binds specifically to oligosaccharides on M cells in the GI tract to deliver polystyrene microparticles and polymerized liposomes in a mouse gut loop model.10 In principle, these can be loaded with an antigen for targeted oral vaccine delivery.
An alternative method of oral vaccine delivery is to develop live bacterial vaccines that can carry foreign antigens, either as recombinant protein or DNA vaccines.2,5,6 The bacteria used for this naturally invade the gut and target inductive sites of the host immune system, such as mucosal surfaces and antigen-presenting cells. These vaccines can induce cellular immunity and humoral responses to the heterologous antigens that they carry (See Figure 1).
Using live attenuated bacteria as the basis of a vaccine is not a new concept. Attenuated Salmonella enterica serovar Typhi, administered as acid resistant capsules, is used as a vaccine against typhoid, and attenuated Vibrio cholorae forms the basis of a vaccine against cholera. However, the possibility of using attenuated bacteria as a vehicle for delivering antigens against pathogens other than themselves is still being researched.5,6,11
The benefits of this approach are wide ranging. Live bacterial vaccines are relatively inexpensive to manufacture as the problems and costs associated with antigen purification are avoided. Also, they are easy to administer and are stable at room temperature, eliminating the need for cold storage. In addition, the bacteria are naturally immunogenic, removing the need to include an adjuvant in any vaccine formulation. Another benefit of administering live bacterial vaccines is that they have some ability to reproduce as they travel through the digestive system, thus increasing the dose beyond what is initially delivered.
Currently, derivatives of both pathogenic and nonpathogenic bacteria are being evaluated as the basis of live vaccines. These include Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Vibrio cholorae, and Escherichia coli.2,3 An international consortium led by the Royal Holloway, University of London, and including Cobra Biomanufacturing was recently set up to develop Bacillus subtilis for delivery of foreign antigens. This nonpathogenic bacterium is naturally found both in the soil and as a transient component of the gut flora. Its spores, which are currently taken as a probiotic to aid digestive health, are stable for long periods of time and across a wide range of temperatures, properties which would aid distribution of any vaccine.
Once they reach the intestine, many of these bacterial species can translocate through the M-cells of the gut wall. In the gut cell wall they are phagocytosed by antigen presenting cells (APCs) within the Peyer's patches (Figure 1). Salmonella, Listeria, and Shigella are all able to replicate following phagocytosis. After internalization, Salmonella remain in the phagosome, but Listeria and Shigella can escape into the cytoplasm of the APC.
Antigens secreted by bacteria, either in the phagosome or the cytoplasm, are displayed by MHC class I molecules on the surface of the APC, thus stimulating a CD8+ T-cell response. As the phagosome contents are degraded, the killed bacteria and their contents are presented via MHC class II molecules, inducing a CD4+ T-cell response. Salmonella are also able to induce strong mucosal (secretory IgA) antibody responses and Vibrio cholerae can elicit the production of strong systemic (serum IgG) and mucosal antibody responses, even though these bacteria are not invasive.
To act as an efficient vaccine, the protective antigen must be stably expressed at high levels in the live attenuated bacterial vector. One approach is to insert the antigenic genes directly into the bacterial chromosome. However, this will result in only one copy of the gene being present within each cell. Stability is increased, but the amount of antigenic protein to be produced from a single copy of the gene tends to be too limited to stimulate an adequate immune response. Vaccine developers, therefore, tend to use multicopy plasmids to express their antigen of interest.
Unfortunately, plasmids are frequently lost during cell divisions. The principle approach to overcome this problem involves complimenting a mutation in one of the host's essential genes by introducing a plasmid with a functioning copy of the gene. In theory this seems promising, but in practice the transformed bacteria end up producing far more essential protein from the plasmids than they need for survival. This overexpression results in a metabolic burden that reduces the fitness of the organism. In addition, because of the excessive amounts of protein expressed the selective pressure to maintain plasmids is significantly reduced. Both these factors contribute to plasmid loss.
An alternative approach—a 'post-segregational killing' mechanism (e.g., hok-sok)—uses a plasmid possessing genes that encode a toxin and an anti-toxin. The toxin is highly stable; the anti-toxin is less stable. To keep the cell alive, the anti-toxin must be continuously produced so that it can counteract the toxin's effects. In cases where the plasmid is lost, the rapid breakdown of the anti-toxin will lead to the cell being killed. Unfortunately, this can be ineffective for maintaining plasmids during prolonged culture, where plasmid-free cells that have escaped the killing effect of the toxin eventually predominate. Also, there is no associated selection mechanism for the initial transformation of the plasmid into the bacterial cell.
Cobra Biomanufacturing has developed a plasmid maintenance system that can overcome the problems described above. The operator-repressor titration (ORT) technology eliminates the requirement for antibiotic resistance markers and genes for stable plasmid maintenance, but retains the advantage of being a plasmid system, i.e., high levels of antigen expression. It also reduces metabolic pressure on the cell, as metabolic genes are only present as a single copy.
The ORT-VAC system involves the use of modified bacterial strains in which an essential bacterial gene, dapD, has been engineered to be under the control of the lac operator. In the absence of an inducer such as lactose, the LacI repressor protein binds to the operator site, blocking transcription of dapD. Lack of the dapD protein eventually causes cell death. However, if these bacterial cells are transformed with a high copy number plasmid containing the lac operator and for the development of vaccines, the antigen of interest, the lac operator sequences in the plasmids titrate LacI away from the chromosomal lac operator. The presence of plasmid thus enables dapD to be expressed again, so cells can survive for as long as plasmids are maintained (see Figure 2).
The strains that have been most extensively evaluated in preclinical and early phase clinical studies are attenuated mutants of Salmonella enterica serovars Typhi and Typhimurium.3 Researchers have tested strains expressing in excess of 50 different bacterial, viral, and protozoan antigens. Cobra used a plasmid expressing the F1 antigen of Yersinia pestis to convey immunity against bubonic plague in mice in an ORT-VAC Salmonella Typhimurium strain, in conjunction with the Defence Science and Technology Laboratory.12 This provided single-dose protection against a challenge with Y. pestis. The ORT-VAC strain was also able to maintain a high copy number plasmid that was rapidly lost from a control strain. This demonstrates the potential of the plasmid system to provide a method of increasing the vaccine dose, and therefore, creating a more potent vaccine.
The benefits of needle-free delivery of vaccines are clear. Not only are the public health problems from use of sharps reduced, but vaccination programs that make use of these new systems are also likely to be easier to implement, and thus be more effective. Much effort is focused on developing novel mechanisms for delivering vaccines in both solid and liquid form through the skin. However, interest is also growing in oral vaccine technologies. In particular, using live bacteria to deliver vaccines orally has a number of added advantages, including the relatively simple downstream processing and the increase in immunogenicity afforded by the vector doubling up as an adjuvant. To take this approach forward, new technologies have been developed to overcome maintenance issues of plasmids and studies with a range of bacterial species and antigens have already shown promising results. There are still hurdles to overcome, such as maintaining the balance between strain attenuation and efficacy, and dealing with the issue of immunity against the vector itself in subsequent vaccinations. Nonetheless, perhaps the end of the needle is nigh?
ROCKY CRANENBURGH, PHD, is head of research at Cobra BioManufacturing plc, Keele, UK, +44 0 1782 714181, email@example.com
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