The biotechnology era has experienced significant changes in the number of companies involved in vaccine manufacturing as well as in the production systems they use. Nevertheless, challenges in this area are multiple. In the current vaccine-manufacturing environment, time to market and cost effectiveness are key issues that need to be addressed in addition to smooth R&D and clinical studies. Furthermore, scale up and safety are important for maintaining a successful manufacturing process.  

As a result, state-of-the-art technologies to simplify vaccine development and manufacturing are becoming ever-more crucial. In this article, the authors discuss these challenges and evaluate the technologies and process parameters that need to be considered when developing a safe, effective, and economically efficient vaccine. The article primarily deals with prophylactic vaccines.  

Vaccines are a group of biologics considered to be the lifeline of the human race. It has been said about vaccines that, "With the exception of safe water, no other modality, not even antibiotics, has had such a major effect on mortality reduction..." (1).  

A good vaccine is one that elicits an appropriate immune response for the particular pathogen, which could either be a cell-mediated response to tuberculosis or an antibody response to a bacterial or viral infections. It should be safe to use in a variety of patients and the vaccine itself should not cause disease or induce adverse effects. A vaccine should offer long-term protection (ideally lifelong) with one dose and retain its immunogenicity despite adverse storage conditions before administration. Furthermore, it must be inexpensive to make and buy (2).  

 The development and manufacturing of vaccines must follow four ground rules (3):  

Vaccines must be developed, produced, and delivered in large volumes. 

Development and delivery times must be short. 

Patient and employee safety should not be compromised.

The largest market for vaccines is found in the developing world, where doses need to be less expensive than those sold in the developed world in order to have any chance of getting to the patients who need them (4). Companies, therefore, need to weigh the risks of killed or weakened whole organisms against the costs of recombinant vaccine production for products meant to help a large percentage of the economically weak population.  

As part of the "Elements of Biopharmaceutical Production" series (see sidebar), this article presents an overview of the various challenges encountered during process development for the manufacture of prophylactic vaccines. 

Elements of Biopharmaceutical Production 

Process development for vaccines can pose unique obstacles for manufacturers. Because most vaccines are new products, there is no history or experience to rely on with regard to how subjects will respond to the drug. Furthermore, promising preclinical results in animal models are generally not duplicated when the therapy is tested in humans. Often, it is challenging to develop robust manufacturing processes and to validate quality control assays for these products because there is a need for a specific biological assay for each product (5). 

One important difference between the production of vaccines and other biopharmaceuticals is the risk-safety consideration related to working with pathogens and pathogenic antigens. As with all biomolecules purified from crude biological material, the removal of contaminants (e.g., derivatives from host cell such as DNA, protein, or leachables), must be documented. However, the removal or inactivation of adventitious viruses remains a unique challenge (3).  

NEW AND EXISTING TRENDS IN VACCINE MANUFACTURING 

The process of vaccine development begins with a good understanding of the underlying biology involved.  

There has been much debate regarding whether the egg-based method for manufacturing a viral vaccine is more effective than the animal cell-culture based method. A third method using virus-like particles (VLPs) is also in use. This method is similar to the recombinant-vaccine manufacturing process in terms of cloning and expressing recombinant proteins.  

Although the egg-based viral vaccine manufacturing process is an old and familiar technology that has been practiced for more than 60 years, it has its own complex issues. Egg-based viral vaccine manufacturing, for example requires a large number of specific pathogen free (SPF) eggs because the virus needs to be propagated inside the fertilized chicken eggs. However, typcially, one can only produce one or two doses of vaccine per egg. Here, the nutrition status of the poultry is also important because changes in their diet can dramatically affect the virus yield from the eggs. The process is labor intensive and highly susceptible to bacterial contamination. In addition, individuals who are allergic to eggs might have allergic reactions to such vaccines.  

On the contrary, animal cell-culture driven viral vaccine processes are being encouraged by regulatory authorities, particularly because the process reduces the lead time from laboratory to market. Furthermore, during an emergency, chances of scaling up production capacity of an egg-based vaccine is dismal considering the reliance on SPF eggs, compared with cell culture, which can be propagated multiple times from frozen cellbanks. In addition, the footprint for cell-culture-based production is considerably smaller, and processing takes place in closed systems, thereby reducing chances of contamination. 

However, an important issue with cell-culture processes is the tumorigenic and oncogenic potential of the cells in which the virus is propagated. Although highly tumorigenic cell substrates have never been used in vaccine manufacturing, it is important to keep in mind that some Madine-Darby Canine Kidney epithelial cell (MDCK) sublines are highly tumorigenic. Such cell substrates can pose significant regulatory challenges (e.g., if the cell line harbors oncogenic or tumorigenic viruses, that virus could integrate into the recipient's genome and cause a tumor). Other cell-culture concerns include the oncogenic potential of virus producing cells due to the presence of contaminating DNA in the product. Additionally, the use of animal-derived components in the cell-culture media—whether serum-sourced from calf or trypsin-sourced from porcine—could be potential sources of animal viruses. The International Conference on Harmonization (ICH) Q5A guideline on the quality of biotechnology products, therefore, recommends using serum-free, chemically defined media for cell culture and genetically engineered trypsin to ensure the absence of any animal-derived component in the raw materials used in manufacturing. 

During the past few years, the use of VLPs for the manufacturing of vaccines is becoming more popular. For example, the H1N1 influenza vaccine developed jointly by Novovax and Cadila Pharmaceuticals uses VLPs. The advantages of a VLP-based vaccine platform is that it uses a recombinant-vaccine technology with a baculovirus expression system and, as a result, one does not need to handle a live pathogenic virus. This process rules out the need for a containment facility. There are no safety concerns and the process is easily scalable to large quantities and more economical in terms of facility, materials, labor, and utility costs (6). When using recombinant technology, one can select the exact genetic match for hemagglutinin,  neuraminidase, and matrix proteins of the circulating virus strain. The entire process of manufacturing, from cloning to development and release, takes about 12 weeks (6). Other VLP-derived vaccines include Merck's Gardasil, which protects against human papillomavirus types 6, 11, 16, and 18. 

PROCESS DEVELOPMENT SCHEME FOR A CELL-CULTURE BASED VACCINE

Current technologies for vaccine manufacturing can help ensure quality and reduce time. Some of these technologies include analytical methods such as surface plasmon resonance, upstream technologies such as microcarrier beads for adherent cell lines, high-throughput screening methods, and downstream technologies such as membrane chromatography and cross-flow filtration. Figure 1 outlines the schematic flow for complete vaccine-process development.  

Figure 1: Process-development scheme for vaccine manufacturing.  

The productivity of large-scale cell culture can be increased either by scaling up to larger volumes with cell densities of 2–3 x 10

 /mL, or by intensifying the process in smaller volumes but with higher cell densities (up to 2 x 10

  cells/mL). When intensifying cell densities, more frequent media changes are needed and perfusion is eventually applied (7). 

Many alternative technologies are also available. Cross-linked dextran beads (i.e., microcarriers), for instance, provide an extended surface and a stable environment for optimal cell growth. Microcarrier culture of anchorage-dependent or entrapped cells reduces volume and thus belongs to the latter of the options cited above (8). The technique, in general, has many advantages for the commercial manufacturer. It can be operated in batch or perfusion modes during cell culture and it is well-suited to efficient process development and smooth scale-up. Washing and changing culture media just before viral infection is easier because there is no virus inside and one does not have to follow additional precautions as required while handling a virus. The reactors can also be modified to grow other organisms.  

As evident from Figure 1, the downstream processing and purification steps occupy a major portion of the vaccine-manufacturing process. As a result, the use of robust and economical steps to develop and optimize parameters for purification is beneficial. For example, in general, downstream steps are more expensive to carry out than upstream steps because of the use of chromatography columns and membranes. The onus for generating a product that is absolutely free of pathogens and other contaminants such as host cell protein (HCP), host cell DNA, or endotoxin, relies on downstream steps. If this cost escalates, the manufacturer could run into business risks.  

The current industry trend for purification of biological therapeutics, including vaccines, involves the use of membrane chromatography to purify viruses as well as for polishing applications. Membrane chromatography offers many benefits compared with density gradient ultracentrifugation, including the removal of HCPs, contaminating DNA and endotoxins. Membrane chromatography compared with centrifugation offers many advantages (e.g., processing time is faster, virus yield is higher, cleaning validation is not needed, the carbon footprint is smaller) (see Table I). This method can therefore  bring greater effectiveness to the key stages of bioprocessing.  

Table I: Comparison between the use of membrane chromatography and density gradient ultracentrifugation for virus purification.  

The implementation of membrane chromatography for virus purification, in place of density gradient ultracentrifugation, is gaining prominence in many areas, even though the latter remains the gold standard among traditional vaccine manufacturers.  

Membrane chromatography has been successfully used in multiple applications for virus purification. In a recent study, human and equine influenza A virus in cell-culture supernatant (i.e., serum-free and serum-containing cultivation) was directly adsorbed onto Sartobind Q 75 anion-exchanger, and eluted out by displacement with sodium chloride (up to 1.5 M, pH 7.0), which resulted in average yields of 86% based on hemagglutinin activity (9). In another instance a virulent wild type NIA3 strain of 

, grown in porcine kidney epithelial (PK15) cell monolayers was purified using Sartobind S cation exchanger membranes (10).  

 In another study, Sartobind D membrane adsorber was used in a larger scale downstream process, for the purification of rotavirus VLPs to a clinical grade at 46% global recovery yield, and with nearly 100% removal of host bulk DNA together with approximately 98% of HCPs (11). Sartobind D was also used for purification of recombinant baculoviruses that are widely used as vectors for the production of recombinant proteins in insect cells (12). 

Another robust technology is cross-flow filtration (using tangential-flow filters), which is best suited for higher solid contents, more viscous feed solutions, or in cases where concentration, recovery, and purification of cells or target species is desired. This technology is largely used for concentrating and washing feed streams before chromatography (3).  

For vaccine manufacturing, particularly virus purification, fully scalable macrovoid-free hollow-fiber technology applied to ultrafiltration and microfiltration offers great advantages in the virus-purification process due to its open porous structure. The open-flow path design of hollow fibers gently processes cell suspensions and other particulate feed streams and reduces shear forces, thereby maintaining the integrity of the virus. Hence, recovery rates of the target virus and overall process economics are improved (13). 

Figure 2: A generic process for egg-based vaccine manufacturing using platform technologies. SPF is specific pathogen free. UF is ultrafiltration. DF is diafiltration.  

PLATFORM TECHNOLOGIES IN VACCINE MANUFACTURING

One way to eliminate bottlenecks, especially in downstream-process development, is to use purification platforms. The biopharmaceutical manufacturing industry has communicated timesavings of 3–8 months using a fast-track development approach when technology platforms are applied in all key aspects of development. This includes upstream as well as downstream processing, such as cell-line development, cell culture, downstream processing, analytical concepts, and filling (3). Major considerations when designing a manufacturing platform include the infrastructure, resources and manufacturing capacity available, the degree of scalability and productivity desired, the time available for producing the first dose and the dosing regime. Figures 2 and 3 illustrate some platform processes for vaccine manufacturing in eggs and in animal cell culture. A complete platform for downstream processing as noted in one study includes three steps: depth filtration, ultra/diafiltration (UF/DF), and membrane adsorption for purification of recombinant baculoviruses. Global recovery yields reached 40% at purities over 98% (12).  

Figure 3: A generic process for animal cell culture based vaccine manufacturing using platform technologies. HCP is host cell protein.  

VACCINE STABILITY AND SUPPLY-CHAIN MANAGEMENT 

Another important parameter to consider when manufacturing a vaccine is its thermostability. All vaccines lose potency over time, and the rate of potency loss is temperature dependent. Hence, there is a need to develop, monitor, and maintain cold-chain systems to ensure that the potency of vaccines is maintained under refrigerated conditions (mostly between 2–8 °C) until the point of use. The World Health Organizaion (WHO) recommends that reconstituted vaccines be kept cold and that any unused vaccine from a multidose vial be discarded after 6 h (14). This is primarily due to the instability of reconstituted vaccines and also due to the chances of bacterial contamination because live vaccines do not contain preservatives (15). New approaches to develop thermostable vaccine formulations that are resistant to damage caused by freezing or overheating are necessary to eliminate dependence on a cold chain. Such approaches could have great economic benefits in terms of reducing vaccine wastage and preventing adverse health consequences of administering damaged vaccines to recipients. Furthermore, such approaches would improve the effectiveness of vaccines and enable delivery of the vaccine to remote populations (15).  

In addition to ensuring vaccine stability, optimizing the vaccine supply chain through quality management in vaccine manufacturing plays a key role in safe and effective vaccine manufacturing. In this context, performing a risk analysis for the manufacturing-process parameters that affect product quality, as well as an assessment of each validation step, including cleaning protocols, monitoring of the air filters, and assurance of sterility of the source materials, are also crucial to a quality management process (16). 

Articles

Features

An evaluation of the technologies needed to develop a safe, effective, and economically efficient vaccine. This article is part of a special section on vaccines.

Challenges and Trends in Vaccine Manufacturing

 that contains an antigen gene controlled by a promoter that functions only in animal cells, usually the cytomegalovirus promoter. The DNA vaccine plasmid must be delivered to antigen-presenting cells (APCs) of the host, where the antigenic protein is expressed, processed, and presented to the immune system. This approach works efficiently in mice and other animal models when DNA is delivered by intramuscular injection, and DNA vaccines have been licensed for veterinary applications, yet no DNA vaccine has been approved for use in humans. A key problem is achieving the delivery of sufficient plasmid to the APCs. This article discusses the approaches designed to achieve delivery, including a focus on the use of live bacterial vectors. 

Since DNA vaccination was developed in the early 1990s, the most common method for immunization has been intramuscular injection of DNA. The DNA is usually dissolved in water or an isotonic saline solution, with the inclusion of an adjuvant if necessary. The aim is to get the plasmid into APCs, which in muscle tissue are primarily dendritic cells. There, the DNA is transported to the nucleus for expression, and the resulting polypeptide is processed and presented on the cell surface in a major histocompatibility complex (MHC). DNA vaccination can therefore produce a protective CD8+ cytotoxic T lymphocyte (CTL) response via MHC Class I (1). Also important are bystander effects, whereby antigens expressed and released from adjacent myocytes (i.e., muscle cells) are taken up by APCs, thus triggering antibody production through MHC Class II. This effect enables DNA vaccines to be developed to target a wide range of bacterial, viral, and parasitic diseases, in addition to the rapidly expanding field of cancer immunotherapy. 

The first licensed DNA vaccine was West Nile-Innovator DNA, approved in 2005 for the immunization of horses against West Nile Virus. It was given in two intramuscular doses 2–4 weeks apart, then as a single dose annually (2). However, this vaccine has now been discontinued by Pfizer following their acquisition of the developers, Wyeth's Fort Dodge Animal Health. Also in 2005, Novartis Animal Health (Basel, Switzerland) gained approval for Apex-IHN, a vaccine against infectious hematopoietic necrosis virus in salmon, which encodes a viral glycoprotein and is administered by a single intramuscular dose of 10 µg DNA (3). 

Despite promising results in animals, DNA vaccine efficacy has been disappointing in human clinical trials. This is generally because of a much lower specific immune response generated by injected plasmid DNA alone, and has led to the adoption of a prime-boost vaccination strategy whereby the plasmid DNA injection is followed by a boost with the same antigen gene in a viral vector. This heterologous boosting strategy has significantly increased the specific immune response. Commonly-used attenuated viral vectors include those based on modified vaccinia ankara (derived from the smallpox vaccine), lentivirus (mainly derived from HIV), and adenoviruses of primate origin. Viral vectors are expensive and complex to manufacture compared with DNA, thus eliminating the advantages of the DNA vaccine approach such as lower cost and simple, generic production processes. An additional disadvantage is the potential of a host immune response directed against the vector. Development of the ideal DNA vaccine therefore requires the optimization of delivery strategies and plasmid vectors. 

A traditional approach for inserting DNA into microbial and animal cells in culture has been by the application of an electric pulse, which creates transient pores in the cell membrane and allows entry of the negatively charged DNA molecules. Electroporation has been adapted from its 

 delivery to tissues following intramuscular injection, and enables a significant increase in transfection efficiency and immune response over injection alone. The disadvantages include pain at the injection site due to the electric pulse, and the requirement for specialized electroporation devices. The most common approaches use a disposable grid consisting of injection and electrode needles attached to the device that provides the electric pulse. The DNA solution is injected, needle electrodes are inserted through the skin and into muscle tissue, and an electric field applied to facilitate DNA entry into dendritic cells and myocytes. 

Ichor Medical Systems (San Diego, CA) has developed the TriGrid Delivery System (TriGrid) a hand-held electroporation device that has been demonstrated to increase DNA vaccine delivery efficiency by as much as 1000-fold over injection alone. Four electrodes are arranged in a diamond shape around the central needle, which is a conventional single-use hypodermic syringe that is inserted into the device, and an electric pulse is administered upon injection (see Figure 1a). Ichor has demonstrated greatly enhanced potency of DNA vaccines delivered with the TriGrid in several animal models using DNA vaccines that encode antigens such as anthrax, hepatitis B virus, and tumor-associated antigens. The device is being evaluated in several clinical trials, and results demonstrating enhanced responses to a prophylactic HIV DNA vaccine delivered with TriGrid compared with conventional needle injection have been published recently (4). In addition, Ichor has shown in animal models that delivery of DNA encoding the protein therapeutics erythropoietin and interferon-β may be a viable alternative to injection of the recombinant proteins. 

Figure 1: Devices for plasmid DNA delivery indluding (a) TriGrid and (b) CELLECTRA with applicator (for intramuscular electroporation), (c) DermaVax (for transdermal electroporation) and (d) ZetaJet (for subcutaneous or intramuscular high-pressure injection).  

A therapeutic plasmid DNA application (although not a vaccine) that was licensed for pigs in 2008 is LifeTide SW, developed by VGX Animal Health (The Woodlands, TX) (5). The plasmid produces growth hormone-releasing hormone and is administered to sows of breeding age to increase the number of piglets weaned, and requires only a single treatment. Plasmid delivery is by direct injection into skeletal muscle, followed by electroporation using a portable electrokinetic device.  The device, called CELLECTRA, was developed by Inovio (see Figure 1b) and uses a sterile, disposable electroporation needle array. Inovio has also demonstrated 1000-fold greater DNA delivery compared with injection alone. The device is equally suited to DNA vaccine applications, and users of CELLECTRA have targeted a range of pathogens including HIV, 

, hepatitis C virus, and malaria parasites. Inovio has an ongoing Phase II clinical trial for cervical-cancer therapy (human papilloma virus therapeutic vaccine) using the device. Additional ongoing human clinical trials include Phase I trials for prophylactic vaccines for influenza (i.e., avian and seasonal) and HIV, and a Phase I clinical trial for HIV therapy. 

While ample evidence indicates electroporation enhances the delivery and immunogenicity of injected DNA vaccines, it has the drawback of increased discomfort at the administration site due to the electrical pulses. However, initial human clinical-trial data with intramuscular delivery indicates that the electroporation procedure is tolerable for therapeutic and even prophylactic applications. Furthermore, continued improvements to device design are likely to result in increased acceptability of the procedure. A novel approach to delivering electrical pulses within target tissue that may increase tolerability is the generation of electrical fields by piezoelectricity, as used in a contactless, noninvasive device developed by Inovio. 

Another interesting alternative to penetrative electrodes has been developed by MagneGene (Lake Forest, CA), which uses contactless magnetic electroporation. This technique relies on a strong, rapidly changing magnetic field generated by a paddle-shaped device to induce rotating electric fields in the host tissue. This device has been evaluated in guinea pigs following injection with reporter plasmid, where enhanced 

 gene expression was demonstrated. Although the magnetic field causes muscle spasms, these subside immediately after use of the device, and there is no pain following the procedure (6). 

TRANSDERMAL MICRONEEDLES AND ELECTROPORATION

Transdermal DNA immunization involves the use of arrays of microneedles, each a few hundred microns long, to pierce the barrier of the stratum corneum (i.e., the skin's outer layer, typically 10–20 µm thick) and deliver the vaccine (7). The skin has a high concentration of APCs called Langerhans cells, which makes it an attractive target. The advantages of microneedles are that they are easy to administer and cause less pain at the injection site than does a conventional needle. There are various strategies to achieve vaccination using microneedles. The simplest involves arrays of microneedles that are used to pierce or scarify the stratum corneum, thus increasing permeability to a topically applied DNA solution. This approach is suitable for preclinical studies, but is not scalable. Alternatively, the microneedles can be coated with the dried vaccine which dissolves following administration. Hollow microneedles can be used to inject solutions containing the vaccine. These can be submillimeter arrays or scaled-down needles in the range of 1–2 mm. An interesting alternative to fixed, disposable microneedles is a solid, soluble microneedle array that is either formulated or coated with the vaccine. This array is inserted into the skin, where the microneedles dissolve or degrade, leaving only the backing to be disposed of and thus eliminating contaminated sharps. 

Smallpox DNA vaccination of mice was demonstrated using the Easy Vax device supplied by Cellectis therapeutics (Paris), and was the first example of microneedle-mediated electroporation (8). The gun-shaped Easy Vax combines the electroporation and microneedle approaches, with cutaneous Langerhans cells as the targeted APCs. The plasmid DNA was dried onto the tips of the eighty-microneedle array and inserted into the skin to enable the DNA to dissolve, followed by the delivery of six electric pulses. 

While the microneedle approach is gaining interest for delivery of therapeutic and antigenic recombinant proteins, only a few studies in mice have shown immune protection against microneedle-administered DNA vaccines. An additional problem when translating animal results to humans relates to the amount of DNA that can be loaded onto a microneedle array. A typical human clinical trial injected dose of 2–4 mg DNA is already relatively small compared with the murine equivalent from which it is extrapolated, yet this would be prohibitively high to expect to dissolve intradermally from a dried formulation on a microneedle array, which leaves liquid DNA solution injection as the only viable strategy. 

Cellectis therapeutics also supplies the DermaVax electroporator (see Figure 1c), which requires a DNA solution to be injected sub-dermally using a hypodermic syringe, followed by electroporation through a series of pulses from 2 mm needle electrodes attached to the bench-top DermaVax apparatus. The DermaVax system induces a 100- to 1000-fold greater increase in gene expression over injection alone and is being used for the delivery of DNA vaccines against HIV-1, as well as vaccines against prostate and colorectal cancers in human clinical trials (9). 

Inovio Pharmaceuticals has developed a transdermal electroporation device called CELLECTRA-3P. In pilot studies in nonhuman primates, Inovio demonstrated that intradermal administration of a smallpox vaccine or an H5N1 vaccine successfully protected animals from a lethal challenge of monkeypox or an H5N1 infection, respectively (10, 11). The device is now in Phase I clinical trials for a universal influenza vaccine. Inovio has also developed a prototype minimally invasive electroporation device that has been demonstrated for influenza DNA vaccine delivery. 

Gene guns have been used since the advent of DNA vaccination for high-pressure transdermal delivery of microbeads coated with DNA (biolistics). PowderMed (Oxford, UK, acquired by Pfizer) developed a handheld particle-mediated epidermal delivery (PMED) device that uses a high-pressure helium minicylinder to fire DNA-coated gold microbeads into the epidermis. Because the gold beads enter the cell cytoplasm and target more APCs than does needle injection, greater CTL responses can be generated for the same amount of DNA (11). In one example, PMED was used to deliver 2 µg of two plasmids to rhesus macaques: one with an H1N1 hemagglutinin gene, the other expressing the adjuvant GM–CSF. This method generated good antibody and CTL responses (12). However, limitation to the microbead approach is the amount of DNA that can be coated onto beads, which could restrict its ability to be scaled up for human applications. 

Merial (Duluth, GA) developed the pioneering canine melanoma DNA vaccine Oncept, which was fully approved in 2010. It contains the human tyrosinase gene, delivered (following surgical tumor removal) in four doses at two-week intervals, followed by a single dose every six months. Oncept is administered using a needle-free Canine Transdermal Device developed by Bioject Medical Technologies (Portland, OR) (13). Bioject specializes in the development of needle-free injection systems that use high-pressure injection to fire a stream of the DNA vaccine solution through the epidermis. Their latest device, ZetaJet, is small and spring-activated for ease of use in the field, and fires up to 0.5 mL into skin or muscle tissue. 

ORAL DELIVERY USING LIVE BACTERIAL VECTORS

The oral route for vaccination avoids needles and reduces the logistical issues and costs of implementing vaccination programs requiring trained healthcare professionals, which would be particularly beneficial in developing countries. Oral vaccination also stimulates a mucosal immune response, which is important because mucosal surfaces are a more common route of entry for many pathogens than is the skin. Much of the research in oral DNA vaccine delivery has focused live bacterial vectors (LBVs) derived from attenuated bacterial pathogens 

, enteric species that are able to replicate the high copy-number DNA vaccine plasmids normally produced in 

. These attenuated pathogens carry mutations of biosynthetic or invasive genes that eliminate their pathogenicity and ability to persist in host tissues or the environment. 

Figure 2: Oral recombinant vaccine delivery using live bacterial vectors. (a) The live bacterial vectors (LBVs) are ingested and travel into the small intestine, where they invade the lining of the ileum through M cells and enter Peyer’s patches. (b) The LBVs invade antigen presenting cells and are phagocytosed. Shigella can escape the phagosome and enter the cytoplasm, while Salmonella modifies the phagosome and persists until it is digested by fusion with a lysosome. MHC is major histocompatibility complex.  

LBVs that have been ingested travel from the stomach into the small intestine. Here, 

 invade microfold (M) cells and enter lymphatic nodules called Peyer's patches. (see Figure 2). Once inside the Peyer's patches, the bacteria become targets for (or actively enter) macrophages, where they are internalized in membrane-bound phagosomes. At this point, one of two strategies is employed: 

 escapes from the phagosome into the cytoplasm, while 

alters the composition of the phagosome to survive and replicate (14). However, the precise mechanism of DNA vaccine delivery using bacterial vectors is not understood. For 

, the cells are thought to eventually lyse in the cytoplasm, releasing the DNA vaccine plasmids which are then transported to the nucleus by the host cell. Attenuating mutations that increase the rate of cell lysis have been shown to improve DNA vaccine delivery using 

, the potential mechanism is less obvious, but could be due to a bystander effect in which DNA released from host cells that have undergone apoptosis due to 

 invasion is carried to APCs in membrane blebs (14). As with other DNA delivery routes, once the plasmid enters the nucleus, the antigen gene is expressed, and the protein processed for MHC presentation. Another advantage of bacterial vectors is potent immunostimulatory properties, due mainly to the lipid A (endotoxin) in the cell membranes, which eliminates the need for an adjuvant. The direct targeting of the mucosal immune system means that vector-directed immune responses are not likely to inhibit the repeated use of live bacterial vectors.  

Drug development suffers from the high attrition rate in translating promising preclinical results in animal models into success in human clinical trials. One variable in DNA vaccine development is the relative dose between preclinical and clinical subjects. For example, a DNA vaccination by conventional needle injection of a 20 g mouse typically requires 100 µg of DNA to generate a protective immune response. To scale this to a 70-kg human would involve injecting 350 mg, which is prohibitive in terms of both the volume required and the associated cost (15). A typical vaccine dose used in human clinical trials is 2–4 mg DNA, which represents only 0.5–1% of the scaled dose. The extent to which the lack of scalability has affected human DNA vaccine development is debatable, as the first vaccine approved was effective in an even larger animal: the horse. But in contrast, 

-based approaches use the same dose in terms of bacteria and plasmid DNA in humans as in mice. This similarity allows the murine dose to be representative, because 

 serovar Typhimurium in mice mimics the invasion and pathogenicity of 

 has the ability to invade M cells and escape from the phagosome, and has been used to demonstrate DNA vaccine delivery, although it cannot replicate the high copy-number plasmids that are universally used as DNA vaccines. 

 strains have also been used as DNA vaccine vectors, having been genetically modified to express the invasion and phagosomal escape proteins from its pathogenic relatives. 

 and other pathogen-based vectors is important for this to be a viable strategy, so it is encouraging that a current licensed typhoid vaccine is a live attenuated 

 serovar Typhi marketed as Vivotif (Crucell, The Netherlands), which has been safely administered to more than 200 million patients over 25 years (16). Other attenuated enteric bacteria have also shown good safety profiles in recent clinical trials, and the increasing use of probiotics has spread the concept of ingesting beneficial bacteria. However, several issues must be overcome before live bacterial vectors can be commercialized. The most convenient oral delivery for adults would use a capsule containing lyophilized bacteria. An enteric coating protects against the stomach acid, but lyophilized bacteria are susceptible to bile when released into the small intestine. To mitigate this susceptibility, Cambridge University in collaboration with Cobra Biologics developed a bile-adsorbing resin formulation that allows water to penetrate, but adsorbs the bile acids (17). This formulation enables the bacteria to rehydrate for the few crucial minutes that are required to increase their resistance to the effects of bile, before their release as the capsule dissolves (see Figure 3). 

Figure 3: Bile-adsorbing resin for oral capsular delivery of lyophilized enteric bacteria.  

Another key issue to address is plasmid instability in bacterial cells. The antibiotic-resistance genes normally used to select and maintain plasmid-containing cells are strongly discouraged by regulatory authorities for reasons of biosafety when used in LBVs. FDA requires a valid justification and proof that these genes cannot be transferred to commensal bacteria (18). As there is arguably no longer a credible justification for their use, antibiotic resistance genes are not used by companies developing LBV strategies. Even if they were permitted, they contribute to a significant metabolic burden on the bacterial cell due to their constitutive expression and the high plasmid copy-number—the major factor in plasmid loss in the first place—and it is not acceptable practice to dose a vaccinee with antibiotics. Therefore, antibiotic-free plasmid selection and maintenance systems are essential for DNA vaccine delivery using LBVs, but most systems retain the metabolic burden of an alternative expressed selectable marker gene.  

To address this problem, Cobra Biologics applied its operator–repressor titration (ORT) technology to 

 as ORT–VAC. ORT requires an essential bacterial chromosomal gene to be controlled by a promoter such as 

. The cell is unable to grow in the absence of the chemical inducer because the repressor protein binds to the chromosomal operator and prevents gene expression. However, when the cell is transformed with a plasmid that also possesses the operator (a short, nonexpressed sequence), multiple copies of the plasmid titrate the repressor and enable expression of the essential gene and cell growth (see Figure 4). 

Figure 4: Operatorârepressor titration for stable maintenance of selectable marker gene-free plasmids in live bacterial vectors. (a) The natural promoter of an essential gene is replaced with an inducible promoter and repressor gene, such that a repressor protein binds to an operator sequence and prevents essential gene expression and cell growth. (b) When the cell is transformed with a multicopy plasmid that also contains the operator sequence, the repressor protein is titrated by binding to the operator, thus enabling essential gene expression and cell growth.  

ORT–VAC has recently been applied for successful immunization using a tuberculosis DNA vaccine expressing the antigen mpt64 from 

, generating greater T-cell responses than the injected DNA vaccine in mice (19). This technique reduced the pulmonary count of infecting 

 following a challenge. The positive control was the standard murine dose of 100 µg of intramuscularly-injected DNA, but ORT–VAC achieved better results despite delivering only 0.01–0.1% of this plasmid dose in 10

THE CURRENT AND FUTURE DNA VACCINE MARKET

Only two DNA vaccines (i.e., Apex–IHN and Oncept) and one DNA therapy (i.e., LifeTide) are currently on the market, all for veterinary applications. But despite a slow start, the DNA vaccine market is growing steadily, and the approval of a human DNA vaccine in the next few years would be a significant shot in the arm for the sector. There is no fundamental reason why DNA vaccines will not work in humans, and the success of the Oncept canine melanoma vaccine is significant, because it is one of only two licensed immunotherapeutic cancer vaccines. Overall sales in the DNA vaccine market totaled $141 million in 2008, and with a compound annual growth rate (CAGR) of 69.5%, are forecast to increase to $2.7 billion by 2014 (20). The value of human DNA vaccines was only $12 million in 2008, but it doubled a year later and is predicted to reach $2.3 billion by 2014 (149.6% CAGR) (20). In 2008 there were 95 open clinical trials involving plasmid DNA; the majority of these were for anticancer applications (see Figure 5) (13). 

Figure 5: The number of open Phase IâIII clinical trials involving plasmid DNA in 2008 (13). 

For all recombinant vaccines, the selection of an antigen that is able to stimulate a protective, long-lasting immune response is the key to success. In addition to  the antigen choice, effective delivery technologies are essential for realizing the promise of DNA vaccines, because there is a limit to what can be achieved through engineering of plasmids and adjuvant formulation. The fact that two out of the three plasmid-based medicines on the market use relatively novel approaches (i.e., electroporation and high-pressure transdermal delivery) demonstrates the capacity for innovation in this field. Effective oral delivery would represent a significant advance. The cost of DNA vaccines would be greatly reduced by a simple manufacturing process and no requirement  for a viral boost or specialized delivery device. 

, is head of molecular biology at Cobra Biologics, Stafforshire UK,  

 2. "West Nile-Innovator DNA", Fort Dodge Animal Health. E0257D 15M AP (Nov. 2008).  

 3. N.C. Simard, "The Road to Licensure of a DNA Vaccine for Atlantic Salmon", 

www.bion.no/filarkiv/2010/07/vaccine_simard.pdf

 6. T. Kardos. "Contactless Magnetic Electroporation" presented at the DNA Vaccines Conference (San Diego, CA, 2011). 

Development of the ideal DNA vaccine requires the optimization of delivery strategies and plasmid vectors.

BioPharm International-10-02-2011

Challenges and Trends in Vaccine Manufacturing

DNA Vaccine Delivery

BioPharm International, October 2011 Supplement