An influenza pandemic is a global health risk and many solutions are being developed to attempt to address this serious threat. Vaccination is thought to be a preferred solution, although access to a timely and sufficient supply of vaccine against a new emerging pandemic strain is inadequate in every country around the world. A new approach makes it possible to provide vaccine within 10 to 12 weeks of identification of a new viral strain, with an exact match to the wild-type influenza strain, by using disposables-based manufacturing facilities that can be built more quickly and cheaply than traditional vaccines plants.

The threat of an influenza pandemic is well recognized. Many thought leaders in the field believe it is a matter of when, not if, a pandemic event will occur. The threat has triggered emergency preparedness efforts in many developed countries and the result has been the purchase of multiple medical countermeasures including stockpiles of medical supplies (e.g., masks, gloves), antiviral drugs (e.g., Tamiflu), and the development and stockpiling of vaccines against multiple prepandemic strains of the H5N1 influenza virus (e.g., A/Vietnam, A/Indonesia, A/Anhui). Because the total supply and cross-reactivity of these stockpiled vaccines is insufficient for mass immunization, adjuvants are also stockpiled, to stretch the available antigen supply and to improve the cross-reactivity of the vaccines we can make today.

Virus-like particles (VLPs) mimic a natural virus in size, structure and surface proteins, but do not have the nuclear material necessary for infection. They trigger an immune response as if a natural infection takes place. (Novavax, Inc.) 

As a result of this government-sponsored procurement and development of these medical countermeasures, there is clearly an improved state of preparedness today in some specific countries, particularly the US. However, vulnerabilities remain even in the best-prepared countries, and the majority of the global population remains unprotected. No country has a supply of vaccine for a novel influenza strain (e.g., H2, H7, H9), as the focus of development efforts to date has been on various strains of H5N1. No country has a sufficient supply of vaccine without adjuvants, and the adjuvants have not been proven safe in large-scale clinical studies. Most countries do not have influenza manufacturing capabilities within their national borders, and because borders will close on the declaration of a pandemic to control the spread of disease, vaccine distribution will also be hindered. In addition, it will be politically difficult to ship precious vaccine out of a country until all citizens are protected. In the end, most countries would be left without a supply of pandemic vaccine.

The Challenges of Ensuring Sufficient Supply

Today, the vast majority of influenza vaccines are made by growing the target influenza virus in fertile chicken eggs. The eggs are infected with the virus, the virus is allowed to grow for several days, and then the virus is removed from the egg, purified, inactivated, and treated with detergent to remove most egg and influenza proteins other than the hemagglutinin (HA) protein. (Antibody to HA from immunization will neutralize a similar virus on infection and is thereby correlated with protection from disease risk).

Newer methods are being developed to replace the chicken egg with a cell line developed from various mammalian species, although the purification and inactivation are managed in a similar manner.

The cost of developing new vaccine production capacity by traditional means is prohibitively expensive. New facilities in the US have been reported to cost $150 million for large-scale egg-based (100 million doses/season) and to $600 million for mammalian cell–based (50 million doses/season) facilities. These high costs have resulted in large centralized facilities in developed countries where the market value is high, with  excess supply being exported to other markets.

In addition, the capacity needed for a rapid and complete pandemic response is far greater than the seasonal influenza vaccine demand. Building capacity for the pandemic need would result in overcapacity for seasonal needs. Such new capacity would be severely under-utilized, or the market for seasonal vaccine would be overserved, lowering prices and margins and hence diminishing the financial returns required to build and maintain the operational infrastructure for manufacturing under current good manufacturing practices (CGMPs). This may already be the case in the US, where new facility projects (e.g., Solvay's cell culture plant) are being shelved because of poor economics, even with government cost-sharing for new facilities.

Many governments are aware of these problems and the need to create supply within their borders. However, the initial high capital requirements for traditional approaches have left them with a list of expensive alternatives that are difficult to justify given the uncertainty of the pandemic need. Likewise, a facility built to react to a pandemic event would only be effective if it were in a constant state of readiness for production, because equipment needs to be exercised and maintained, and people must be trained. This state of readiness can be accomplished only by making other products in the facility and then switching to pandemic flu vaccine production when needed. The best fit would be seasonal influenza production for the local market, but this has important implications for global influenza vaccine oversupply, competition, and the utilization of capital, as the facility would likely not be well suited for other vaccines made by traditional means.

An Alternative Approach: Baculovirus Production in Disposable Equipment

Novavax, Inc. (Rockville, MD), is developing a novel, recombinant vaccine using cell culture to rapidly produce three proteins specific to an emerging influenza strain that self-assemble into an enveloped, non-infectious particle (virus-like particle or VLP) resembling the influenza virus. The approach provides vaccine within 10 to 12 weeks of identification of the new strain and provides an exact match to the wild-type influenza strain. Yields are significantly higher than egg and mammalian cell culture production. The high yields and use of a nonpathogenic baculovirus allows production in disposable manufacturing equipment. The use of disposables radically lowers facility costs, making the solution affordable to many governments. These advantages are explained below.

The time required to manufacture this insect-cell based vaccine is 10–12 weeks, as shown in Figure 1. This time includes four weeks to make recombinant baculovirus production seed stock from the native influenza virus DNA or from the DNA sequence identified by the relevant health agencies, such as the Centers for Disease Control or the World Health Organization. These seed stocks are used to make manufacturing lots starting in week 5. In parallel, recombinant hemagglutinin (HA), neuraminidase (NA), and matrix (M1) protein are made for the generation of reagents needed for lot-release testing. During weeks 8 to 10, vaccine lots, which have been in production since week 5, are formulated, filled, and packaged for distribution, with release expected in weeks 10 to 12. This is approximately the same time that the first pandemic wave of disease will peak, so having vaccine available this quickly offers the potential to halt the pandemic in its tracks. Traditional manufacturing processes, in contrast, require preparation of a nonpathogenic virus (because an avian virus would kill the fertile egg used in production), and then production of materials used to make reagents. Only when these reagents are available can vaccine be released. The traditional process in this case takes at least 20 to 24 weeks, which means that vaccine is not available until the second wave of pandemic disease, after much of the damage has been done.

Figure 1. The insect-cell based vaccine can be ready in 10â12 weeks, compared to 24 for a traditional method. Part of the time savings is achieved by preparing the reagants for lot-release testing while the vaccine is in production. 

The insect-cell production system also has a very high productivity compared to traditional approaches (Figure 2). Yields in the production of HA are 7–10 times higher (in grams of protein per volume of production lot) than with traditional methods. These high yields make it possible to use small disposable reactors (1,000–2,000 L) instead of 20,000-L reactors needed for mammalian cell culture production. The system developed for making Phase 1 and Phase 2 clinical lots uses 200-L Wave reactors (GE Healthcare, Uppsala, Sweden) for the production of VLPs, followed by a series of purification steps that are also executed in disposable equipment. The current high yields are expected to increase as high-performance disposable stirred reactors (Xcellerex Inc., Waltham, MA) are used to scale up the process to 1,000 L for commercial production. Higher gas transfer rates are expected to support higher insect cell culture densities, thus providing higher volumetric productivity. Further, clinical testing to date has shown that much smaller doses of the VLP vaccine are needed to match the effectiveness of licensed pandemic vaccines (without the use of adjuvants). This means that the yield advantage is further magnified to >40-fold higher productivity in terms of doses of vaccine per liter of cell culture.

Figure 2. Relative yield from traditional vaccine production compared to baculovirus production of a virus-like particle (VLP) based vaccine. Because the VLP vaccine is effective at a much lower dose, the effective yield of the baculovirus production system is 42 times higher. 

Novavax contracted Jacobs Engineering (Conshohocken, PA) to provide a conceptual design and cost estimate of a facility for making influenza vaccine using the VLP manufacturing technology in disposables. The plant, which would be capable of producing 75 million doses per season, would be one-third the size of a facility of similar capacity designed for vaccine production in eggs or mammalian cell culture (assuming the same dose).

The cost of the new facility is estimated at $40 million, one-twelfth the cost of the mammalian cell culture facility at 50 million doses per season. The savings result in part from the higher yield, but mainly from the use of disposable manufacturing systems. Instead of large, fixed stainless-steel equipment, connected by a maze of piping and process controls, with automated cleaning and sterilization systems, the disposable approach uses free-standing skids with removable liners, flexible tubing, and disposable filters and chromatography columns. The result is the avoidance of the ever increasing cost of stainless steel, as well as the need for the clean-in-place and sterilize-in-place equipment, parts washers, and clean steam generators required for traditional processes. Further, other facility systems, like contaminated waste collection and kill systems and water-for-injection (WFI) systems are small, because all product contact parts arrive presterilized and are discarded after one use without cleaning. (In a traditional manufacturing process using stainless-steel equipment,  cleaning processes contribute the vast majority of the volumes of purified water). This disposables-based approach also requires much less labor. An apparent waste issue of the disposable approach is the volume of plastics that are discarded. However, these high-energy plastic side streams can be incinerated with the cogeneration of power to minimize their environmental impact.

The smaller, less-complicated facility also can be built, commissioned, qualified, and validated much more quickly. The sequential validation of multiple water and steam systems is avoided; as well as the cleaning and sterilization validation required to begin process execution. The final Novavax facility will use manufacturing systems identical to those used in the pilot plant, so no scale-up is required, only the confirming validation runs duplicating what has already been intensely validated during the process development. The result is project execution in about two years, compared to four to six years for a traditional facility. The shorter time supports earlier availability of product for a pandemic, as well as faster recovery of the investment permitted by the shorter and less expensive project.

From a cost of goods sold (COGS) perspective, the cost of insect-cell based VLP vaccine is equivalent to the egg-based influenza manufacturing process based on current yields (Figure 3). However, the mix of costs is quite different. Although eggs are a relatively expensive raw material, the remaining raw material costs of egg-based flu vaccines are low. By comparison, the disposable equipment in the Novavax process (e.g., reactor bags, chromatography resin/columns) are more costly. The 7-to 10-fold higher yields of the Novavax system make the per-dose costs reasonable, however, and additional yield gains should provide a COGS advantage for insect-cell based production. One key advantage of the disposable approach is the low labor requirements, because equipment is not cleaned and because the early steps of the egg-based system (i.e., the handling of the eggs) is very labor intensive. Thus, these factors cancel each other out , making the overall variable costs of the two approaches very similar (per gram of HA).

Figure 3. Comparison of the relative variable and fixed costs of traditional vaccine production facilities and insect cell cultureâbased production. 

On the fixed cost side, however, the disposable approach has a significant advantage. Lower capital costs mean less depreciation as a fixed cost. Likewise, the lower energy consumption of disposables (no cleaning/sterilization, lower water use) also reduces fixed costs in the facility. The more important implication of low fixed costs, however, is that high facility capacities are not needed to reduce the unit cost impact of the fixed costs. That is, you loose the need for economies of scale and hence you can effectively build a smaller plant, suited for the local population, rather than a large plant to cover many regions.

To demonstrate this concept, Novavax has built a pilot plant and commercial launch facility in Rockville, MD, with a fully disposable process capability. The facility houses a single 1,000-L Xcellerex Bioreactor for VLP production, supported by two Wave bioreactors and a 200-L Xcellerex bioreactor for seed preparation. At current yields, the existing process equipment can produce up to 30 million doses of H5N1 vaccine in six months (at 15 mcg/dose). We expect yield to increase such that the facility can support up to 80 million doses in six months, once the new bioreactor conditions are optimized. The facility was built for less than $6 million. The facility is a prototype of what we could place "in-border" to support a government's desire to secure its supply of pandemic vaccine. The overall cost for a facility would be higher than the quoted price, as the Rockville facility does not include formulation, filling, packaging, or media and buffer preparation, which will be contracted out. The cost also does not include quality control or quality assurance space. It still represents a good order-of-magnitude savings over what would be possible with traditional flu vaccine manufacturing approaches and provides countries with a proof of concept for the approach. The construction and qualification was completed in 120 days, which shows the time savings of the disposable approach as well. The facility became fully operational at the end of 2008.

In addition to securing pandemic influenza vaccine supply, this technology approach offers other advantages. First, this technology makes it possible to customize vaccine for a given region. Some regions do not get great benefit from the recommended vaccine formulation. When influenza strains emerge in Southeast Asia, they mutate as they migrate across Asia into Europe and North America, then Africa and South America. The strains recommended for Europe and North America have already passed through Southeast Asia and a different vaccine formula would be advantageous in that region.

Further, additional antigens may be warranted in a region, because multiple B strains can circulate and choosing just one for the seasonal formula has the potential to reduce a vaccine's effectiveness if the alternate strain were to become prominent. Therefore, having local capacity gives governments an opportunity to add an additional strain (even a prepandemic avian strain) to the formula to add value to the seasonal vaccination program.

Next, there are sometimes 'late-breaking' strains of influenza that are antigenically variant from the seasonal formula. The speed and yield of the process in insect cells affords the local government the opportunity to make an additional vaccine in a timely manner to address this risk as needed.

Finally, the process that Novavax has developed for influenza vaccine production is also effective at making other VLP vaccines. The company is developing other VLP vaccine candidates in collaboration with the US National Institute of Health (NIH) that use the same process as influenza manufacturing in insect cells. A change of the clone alone is required to make alternate VLPs. As these and other similar vaccines are developed and approved, pandemic facilities can be used to manufacture these products for local markets. This increases facility utilization and provides a "warm base" operation so the facility is always ready to switch to pandemic influenza vaccine manufacturing mode.

Because Novavax is a 100-person vaccine development company, the ability of this company alone to help the world create a better solution to pandemic influenza is limited. In December 2007, Novavax signed a collaboration with GE Healthcare to expand its ability to provide a pandemic solution to governments around the world. GE is a large company with a history of innovation and a track record for delivering large projects, and the company already serves many governments by constructing hospitals and utility plants. GE will provide the facility engineering, project management, and government access to support the Novavax technology application in delivering in-border or regional vaccine manufacturing capabilities to those that have recognized the need to create this valuable resource.

An influenza pandemic is a recognized global health risk and many solutions are being developed to attempt to address this serious threat. To date, most solutions fall short of providing an effective response to protect the global population. Vaccination is thought to be a preferred solution, although access to a timely and sufficient supply of vaccine against a new emerging pandemic strain is inadequate in every country around the world. Novavax, Inc., teamed with GE Healthcare, can offer a solution that answers the call for a fast vaccine response to a novel influenza vaccine strain using an affordable technology for many developed and emerging countries. With the help of government and nongovernmental organizations, the technology can also be available to developing countries. This approach is economically rational and its implementation may actually be capable of halting a pandemic in the countries where it is most likely to start, where those populations have no defense today.

 is vice-president of technical and quality operations at Novavax, Inc., Rockville, MD 20850, 240.268.2019, 

Articles

With virus-based production, vaccines can be available in 10-12 weeks.

An Alternative to the Scale-up and Distribution of Pandemic Influenza Vaccine

To provide effective patient protection, many traditional vaccines require multiple injections, which results in a costly and inconvenient regimen. These disadvantages have spurred the development of single-shot vaccines that can provide protection against infection with only one injection. This article describes the manufacture and early results of a prototype single-shot vaccine combining a prime and booster dose in one injection. The development process is based on the example of a hepatitis B vaccine. 

Vaccines contribute significantly to improving world health by prevent the debilitating, and in some cases, fatal effects of infectious diseases by inducing a protective immune response against the causative agent. Vaccines' success is limited, however, by the need for multiple injections, and because this costly and inconvenient regimen often leads to logistical challenges and poor patient compliance. Recent advances in the use of biodegradable microsphere-based systems show the potential of developing single-shot vaccines to overcome this limitation. Based on the example of hepatitis B vaccination, this article describes the development process of a prototype single-shot vaccine combining a prime and booster dose in one injection.

Figure 1. Illustration of single-shot (B) versus traditional (A) vaccination schemes 

The single-shot vaccine is a combination product of a prime component—antigen with an appropriate adjuvant—and a microsphere component that encapsulates antigen and provides the booster immunizations by delayed release of the antigen (Figure 1). Many aspects need to be taken into consideration when developing such controlled release technology-based vaccines (Table 1).

Table 1. Important determinants for single-shot vaccine development 

The microsphere component uses OctoPlus's proprietary OctoVAX microsphere technology, which is based on cross-linked modified dextran polymers. Dextrans are ideal polymers to form biocompatible hydrogels. Two major advantages of dextran microspheres as protein delivery systems are that the particles are prepared in the absence of organic solvents, and that degradation of the microspheres does not result in a pH drop.  Both exposure to organic solvents and an acidic environment are known to negatively affect protein stability.

  Several different dextrans have been developed for hydrogel formation. One of these dextran-based polymers is derivatized with hydroxy-ethyl methacrylate (dex-HEMA, Figure 2), which introduces hydrolytically sensitive carbonate ester groups that ensure biodegradation under physiological conditions.

  Studies have shown that protein therapeutics developed with this polymer retain the activity of the encapsulated protein following encapsulation and release.

Figure 2. Chemical structure of dex-HEMA, the building block of the hydrogel microspheres. The carbonate ester site that confers hydrolytic sensitivity to the dex-HEMA is indicated. 

Formulation and Manufacturing of Single-Shot Vaccines

Important factors in the manufacture of a microsphere-based vaccine are high encapsulation efficiency and a consistent particle-production process. Several formulation parameters play an important role in obtaining a robust process. Below, we discuss the processes and equipment used to manufacture several formulations. 

Figure 3.  Schematic representation of the microsphere preparation process 

Dex-HEMA has been shown to be very suitable for the formation of the hydrogel that facilitates controlled release of encapsulated proteins. A microsphere formulation process has been developed based on this polymer (Figure 3).

  In this process, an emulsion of aqueous dex-HEMA solution is formed in an aqueous polyethylene glycol (PEG) solution, by mixing them in a bioreactor vessel (Figure 4). To ensure consistently high encapsulation efficiencies, the protein to be encapsulated is added to the dex-HEMA solution before adding the PEG solution. Subsequently, microspheres are obtained by polymerizing the HEMA groups using potassium persulfate (KPS) as initiator and N,N,N',N'-tetramethylethylenendiamine (TEMED) as the catalyst. After extensive washing, the final microsphere suspension can be filled into vials and freeze-dried to stabilize the product. 

Figure 4. Bioreactor used during scale-up of the production process 

Several factors are critical parameters for the formulation of consistent microspheres. First, the size distribution of the microspheres can be controlled by the shear force applied during the emulsification step in the bioreactor vessel. Factors that have been identified to influence this shear force are the mechanical stirring speed in the bioreactor vessel and the viscosity of the PEG solution, which is determined by the concentration and molecular weight of the PEG. Second, the presence of excipients in the starting composition can influence the matrix density and encapsulation efficiency of the microsphere product, either by a direct effect on the microsphere formation or on the protein characteristics.

  Finally, polymerization conditions such as KPS concentration, pH, and temperature, can influence the strength of the formed hydrogel matrix.

Controlling Particle Size During Process Scale-up

The dextran microsphere preparation method, described by Stenekes et al., 

 was initially performed on a 5-g scale (containing 120 mg of microspheres), and used vortexing as a means to emulsify the dex-HEMA phase in the continuous PEG phase. However, vortexing is not practical at large scale. Therefore,  we evaluated the feasibility of stirring, a process that is relatively easy to scale up, as a means of emulsification, ultimately at a 500-g scale.

A direct correlation was observed between stirring speed and mean particle diameter of the microspheres (Figure 5), thus confirming that the particle size of the dextran emulsion is dependent on the energy input during emulsification. It is important to note that, despite the larger mean diameter of microspheres prepared at the 500-g scale, more than 90% of the resulting particles had a size below 90 μm, a size suitable for subcutaneous injection. 

Figure 5. Microscopy pictures of microspheres produced at a high stirring speed of 700 rpm (A) or a low stirring speed of 60 rpm (B). Pictures were taken at 1,000 x magnification; bars indicate 30 Î¼m. 

To optimize the stirring process, the production set-up was transferred from regular laboratory equipment and glassware to an autoclavable 2-L jacketed bioreactor unit equipped with baffles and a stirring assembly. The production process has now been scaled up to 1,500 g with a production of miscrospheres  averaging 40 μm in size.

Delayed Antigen Release from Dex-HEMA Microspheres

Once the freeze-dried microsphere product is rehydrated by reconstitution in an aqueous solution, hydrolysis of the carbonate ester groups in the dex-HEMA will be initiated. This will increase the mesh size in the hydrogel network. The encapsulated protein will be released when the mesh size exceeds the hydrodynamic diameter of the protein. The actual release profile of the encapsulated protein is primarily determined by the matrix density.

It has been shown that the release of proteins and liposomes can be tailored from days to months by varying the main factors influencing the matrix density, which are the relative number of HEMA groups in dex-HEMA (degree of substitution, DS) and the initial water content of the microspheres.

 In Figure 6, the effects of different DS and initial water content on 

 release profiles of a model protein, IgG, are presented.

Figure 6. The effect of dex-HEMA DS and initial water content on in vitro release of IgG. In vitro release of proteins is measured in time during incubation at physiological conditions. Release is expressed as percent of the maximal released amount of protein. 

Most other microsphere delivery technologies, such as the poly-lactic-co-glycolic acid (PLGA)–based delivery technology, comprise process steps that expose the protein to potentially detrimental organic solvents. In contrast, the OctoVAX microsphere formulation is a completely aqueous process, which limits the effects of manufacturing on the integrity of the encapsulated proteins. Furthermore, because of the open matrix of the microspheres, hydrolytic degradation does not induce local acidification that may affect the protein's activity. As part of our single-shot vaccine development program, we have recently shown in a mouse model that the potency to induce an antibody response is comparable between encapsulated HBsAg and freely injected HBsAg (Figure 7). These data suggest that the HBsAg particle domains that induce these antibody responses are not affected by the formulation process or by the release process.

Figure 7. Antibody titers induced by HBsAg administered as a protein solution, admixed with placebo microspheres (MSP), or encapsulated in OctoVAX microspheres (MSP). Antibody titers at day 84 are represented as geometric mean titers per group of 10 animals in relation to an international reference standard. 

 Study Results from a Prototype Single-Shot Hepatitis B Vaccination

To show the potential of the single-shot approach, prototypes were tested in 

 studies. The results reveal the impact of the composition of the formulation on the immune response.

The OctoVAX microsphere technology is applied with the objective to reduce the number of injections to a single-shot vaccination that confers full protection against a specific infection for a period at least similar to the conventional regimen. For proof of concept of the single-shot vaccine approach, prototype vaccines were developed using the HBsAg antigen. Current practice for hepatitis B vaccination is either a two- or a three-injection regimen, consisting usually of a prime injection followed by two booster injections at 1–2 months and 3–12 months after the first administration. This vaccine consists of aluminum-adjuvanted HBsAg.

 release profiles, prototype microsphere formulations were selected for further investigation of the concept. Proof of concept studies were performed by immunizing Balb/c mice with prototype microsphere vaccine formulations that combine a prime dose with a single microsphere component. This microsphere component represents the booster dose with a delayed antigen release around day 30. In these studies, the prototype single-shot vaccines were compared with two injections at day 0 and 28, representing the prime and booster dose. The potency of the vaccines to induce an antibody immune response was determined by analyzing the development, over time, of serum anti-HBsAg immunoglobulin concentration using a commercial immunoassay.

The results of these animal studies show that the single-shot HBsAg vaccines based on the OctoVAX controlled release technology induce responses that are similar to those induced by the representative two-injection control vaccination by subcutaneous administration. Figure 8 shows that vaccines with differences in physical aspects and adjuvant presence induce antibody responses with different kinetics. Formulation A induced the desired levels of antibodies without a prime component, in contrast to formulation B, which needed an adjuvanted prime. However, even with formulation B, the data show that OctoVAX microspheres can provide a booster dose on initiation of an immune response by a prime component, providing proof of concept for the single-shot controlled delivery principle. In additional experiments, it has been shown that intramuscular application results in similar or better immune responses, which may ultimately be the preferential choice for route of administration (data not yet published).

Figure 8. Antibody titers induced by prototype single-shot HBsAg vaccines based on microspheres of formulation A or B, which differ in their physical characteristics. Microspheres were either administered by themselves or mixed with aluminum hydroxide (AlOH)-adjuvanted HBsAg. Control mice received two injections of AlOH-adjuvanted HBsAg. Antibody titers are represented as geometric mean titers per group of 10 animals in relation to an international reference standard at 28, 56, and 84 days after injection. 

In addition to the efficacy data, animal studies have revealed that OctoVAX microspheres are well-tolerated and induce no systemic toxicity effects.

 Furthermore, local reactogenicity was limited to minor foreign body reactions, as to be expected for any biodegradable implanted material. Therefore, it can be concluded that OctoVAX microspheres are well-tolerated and safe controlled-release vehicles that can be applied for the development of single-shot vaccines.

The development of a single-shot vaccine technology could contribute to a significant increase in vaccination coverage worldwide by improving patient compliance and lowering administration costs. To achieve this innovation, single-shot vaccines must  be rationally designed. To facilitate this, complex factors in the development and manufacturing process that have a significant influence on the efficacy of the immune response must be controlled precisely during the development and manufacturing phases.

The concept of single-shot vaccination is applicable to various types of antigens and vaccines. Further research is required to investigate the influence of adjuvants and release characteristics in various vaccination regimens.

, is a scientist in  vaccine development, 

 is a manager of corporate  communications, and 

 is a director of preclinical research and  development, all at OctoPlus NV, Leiden, the Netherlands, +31.71.5241071, 

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The disadvantages of the traditional vaccine regime (prime plus boost) have spurred the development of single-shot vaccines. This article describes the development and manufacture of a prototype single-shot vaccine that uses microspheres made from cross-linked modified dextran polymers for controlled release of the antigen.

Advances in Single-Shot Vaccine Development

There is accelerating interest in the use of nonspecific immunostimulants or adjuvants as a means of enhancing or inducing nonspecific immunity. A further requirement for the development of successful therapeutic vaccines is the upregulation or induction of intrinsic innate immune defenses that have the capacity to destroy the infectious agent or tumor cell directly, or to synergize with existing cytotoxic or immunologic treatment regimens. This article discusses the potential of MIS416 adjuvant, a novel vaccine adjuvant and immunogen co-delivery system, to fulfill this requirement. 

There is a great need for the development of therapeutic vaccines for patients with cancer or acquired chronic infections, such as HIV, hepatitis, tuberculosis, and malaria. Therapeutic vaccines aim to enhance the inherent capability of the patient's immune system to fight the disease, leading to a reduction in severity or complete elimination of the disease. To date, developments have fallen short of expectations. This is caused, in part, by the failure of chronically infected or immunosuppressed patients to respond adequately to the vaccine adjuvant component, which itself may not have the appropriate breadth of immunostimulatory activity.  

A requirement underpinning the successful development of prophylactic and therapeutic vaccines lies in the ability to target adjuvant or antigen complexes to dendritic cells with the simultaneous induction of a coordinated and targeted inflammatory response in the secondary lymphoid tissues. In addition to providing adequate immunostimulation to overcome host factors that may limit the success of therapeutic vaccines, we believe a further requirement for the development of successful therapeutic vaccines is the upregulation or induction of intrinsic innate immune defenses that have the capacity to destroy the infectious agent or tumor cell directly, or to synergize with existing cytotoxic or immunologic treatment regimens.  

MIS416 adjuvant, a novel vaccine adjuvant and immunogen co-delivery system designed by Virionyx Corporation, is well positioned to fulfill this requirement, acting to induce anti-infective mechanisms such as the production of anti-viral IFN-α, and activation of oxidative microbiocidal pathways, in addition to the activation of broad spectrum anti-tumor responses. MIS416 adjuvant-mediated co-stimulation of dendritic and accessory cell immune crosstalk, concomitant with MIS416-enhanced anti-infective or anticancer cytotoxic activity is likely to facilitate uptake and presentation of autologous viral, bacterial, or tumor antigens, enhancing the generation of vaccine induced immunity. The additional activation of broad spectrum host immune defenses by MIS416 vaccine adjuvant therefore presents an exciting strategy for developing a broad range of therapeutic vaccines.  

MIS416 comprises multiple immunostimulatory TLR9 and NOD-2 ligands that are cross linked in a manner that also permits the covalent attachment of a broad range of immunogens (Figure 1). MIS416 vaccine candidates incorporating a broad range of immunogens have been shown to act in a well-defined manner and have demonstrated significant potency for induction of protective, long-lived humoral immunity, and cellular adaptive immunity with no adverse events noted, even following multiple immunizations. 

Figure 1. Graphical representation of MIS416. MIS416 is a semi-synthetic, nontoxic, nonimmunogenic, cage structured microparticle (2 microns) composed of muramyl dipeptide repeats covalently attached to a poly amino acid backbone principally composed of lysine and glycine amino acid repeats. N-acetyl glucosamine is incorporated as an oxidizable carbohydrate for use in linking amino containing ligands. Bacterial DNA is incorporated through amino linkages covalently attached to the poly amino backbone. Microparticle components are isolated from the cell wall of Propionibacterium acnes and ligands or immunogens are produced synthetically with recombinant technology or isolated from natural sources to produce the desired vaccine formulation or activate the desired immune pathway. Insertion and covalent linkage of ligands or immunogens is achieved using bifunctional cross linkers, which link through primary and secondary amino groups present in the microparticle or through carbohydrate moieties following oxidation. (IMAGE CREDIT: RUSSELL KIGHTLEY MEDIA)  

Activating Host Antimicrobial Defense Mechanisms

The release of inflammatory mediators by first-line immune cells is followed by cell activation. These mediators act, in part, through autocrine and paracrine signaling loops to enhance innate immune activities, such as phagocytosis and intracellular killing. A key example of this is the ability of MIS416 to induce reactive oxygen species (ROS) and nitrogen monoxide (NO) production by monocytes and granulocytes (Figure 2).  

Figure 2.  MIS416 is a potent activator of reactive oxygen species and nitric monoxide in granulocytes and monocytes. (A) Human blood was cultured with 10, 5, or 1 Âµg/mL MIS416 for three hours at 37Â°C. Cells were loaded with 1 ÂµM of cell permeant fluorescent ROS indicator 3Â´-(p-aminophenyl) fluorescein (APF) for the last 30 minutes. Reactions were quenched by the addition of excess ice-cold 1.5 mM EDTA and cells were labelled with anti-CD14-PerCP for identification of monocytes and PI viability dye (1 Âµg/mL). Cells were analyzed by flow cytometry of gated viable monocytes and granulocytes and the percent of cells expressing upregulated APF fluorescence was determined. (B) Whole diluted blood was incubated for two hours with 10, 5, or 1 Âµg/mL MIS416. 5 ÂµM nitric oxide (NO) cell permeant indicator dye (4-amino-5-methylamino-2, 7-difluorofluorescein diacetate; DAF-FM) was added and cells incubated for a further 30 minutes to allow for dye loading. Cells were then washed and re-incubated for 20 minutes at 37Â°C to allow for de-esterification fo the dye, then samples were analyzed by flow cytometry. The percent of cells showing DAF-FM positive fluorescence are indicated. 

The functional potential of MIS416 microbiocidal activity has been demonstrated in a mouse therapeutic vaccine model studying the ability of MIS416 and Titermax adjuvant to induce adaptive immunity against drug resistant Mycobacterium tuberculosis (MTb) and reduce the infectious bioburden (Figure 3). Immunization of mice three times over a period of 14 days led to a significant reduction of infectious MTb in the MIS416-treated group, which was greater than that observed in the Titermax treated group. 

Figure 3.  MIS416 enhances the immunogenicity of ESAT-6 and clearance of drug-resistant M. tuberculosis in mice. C57Bl/6 mice were intravenously infected with drug-resistant strain of MTb at 105 CFU per mouse and infection allowed to progress for 14 days. Mice were then treated with 25 Âµg per mouse of recombinant MTb ESAT-6 protein in either Titermax adjuvant, MIS416 adjuvant, or no adjuvant, given on days 14, 18, and 21 (3 doses per mouse). Mice were sacrificed on day 28 and lung CFU counts performed by plating organ homogenates on 7H11 agar. 3-mice per time point were analyzed  (* denotes significance by t test). 

Several innate immune responses are considered to be able to contribute to the control of viral infections. These effector mechanisms are multifaceted and include direct antiviral activities and immunomodulatory effects on infected host immune cells that contribute to the elimination of these cells. Direct antiviral activity may comprise soluble factors, such as CD8 antiviral factor (CAF) and IFN-α, which have the capacity to directly affect viral transcription. Although a wide range of other innate cytokines can mediate biologic functions regulating aspects of antiviral immunity, high levels of IFN-α/β appear to be dominant in the context of viral infections and act to regulate other innate responses. The clinical use of IFN-α in treating a variety of viral disorders such as chronic hepatitis B and C, and a broad range of human cancers, lies in its ability to induce a dominant array of antiviral genes that drive pleiotropic host defense pathways that prevent viral replication. Virionyx's MIS416 has been designed to induce both high levels of IFN-α and other key pro-inflammatory cytokines that are clinically relevant to the induction of broad-spectrum innate antiviral immunity. Plasamocytoid dendritic cells demonstrate potent induction of IFN-α following MIS416 uptake (Figure 4). Preclinical studies demonstrate MIS416 is able to inhibit mortality and ameliorate morbidity in a mouse influenza A model when administered following viral exposure (Figure 5).  

Figure 4.  MIS416 induces pDC IFN-Î± production. Human pDCs were purified from PBMCs using magnetic bead selection of BDCA-2+ cells to high purity and viability. Sorted pDC were cultured at 2.6 x 105 cells/mL in the presence of assay positive control (TLR9 ligand (CpG A ODN 2216; 1 Âµm) or a dose-response of MIS416 for 24 hours. Supernatants were assayed for IFN-Î± content using flow cytometry cytokine bead array methodology.  

Activating Broad-Spectrum, Tumor-Killing Mechanisms

Therapeutic vaccines administered to cancer patients are designed to treat cancer by stimulating the immune system to recognize and attack human cancer cells without harming normal cells, and to overcome the immune system's tolerance to cancer antigens. 

Figure 5.  MIS416 therapy suppresses weight loss and mortality after infection with type A influenza. (A) C57BL/6 mice (n = 10 per group) were infected with 0.3 LD-50 influenza A strain A/PR/8/34 (H1N1). The following day, animals received vehicle control (saline), 50 Âµg MIS416, or 250 Âµg of MIS416 intravenously. Weights were monitored daily. Where indicated (*), MIS416 therapy significantly reduced weight loss. (B) Mice were infected as described above. Animals that lost more than 30% of their initial weight were considered moribund and euthanized. Data are pooled from two independent experiments (n = 15 total mice per group). The reduced morbidity of animals treated with MIS416 was statistically significant (p = 0.035 by Log rank test). 

Figure 6.  Enhancement of purified human NK spontaneous killing activity following stimulation with MIS416.  Human CD56+ cells were purified from whole blood to 99% and cultured for 40 hours with no stimulus, known NK activating agents IL-2 (500 U/mL) and IL-12 (50 ng/mL) or with MIS416 (40, 20, 10, and 5 Âµg/mL). Stimulated NK cells were subsequently tested for cytotoxicity against fluorescently labelled NK sensitive K562 tumor targets at effector:target ratios of 5:1, 2:1, and 1:1. Tumor cell killing was determined after four hours by flow cytometric analysis of viability dye uptake (propidium iodide) of gated, fluorescent K562 targets.   

Tumor-specific killing that results in the induction of apoptosis leads to clearance of these cells through the phagocytic pathway, resulting in uptake and cross-presentation of apoptotic tumor cell antigens by dendritic cells, which is required for the development of cytotoxic CD8+ antitumor responses. The induction of antitumor activity by immunostimulatory compounds is a clinically relevant therapeutic approach to treating neoplastic disease, as is demonstrated by the usefulness of BCG in preventing recurrence of bladder cancer that has been surgically removed at early stages of cancer development. Although natural killer (NK) cells play a central role in tumor cell surveillance and destruction, NKTs and monocytes or macrophages represent additional nonredundant arms of innate antitumor responses. Together these cell subsets are known to kill tumour targets by several mechanisms including, but not limited to, granule, FAS L and tumor necrosis factor-alpha (TNFα)-mediated pathways. MIS416 activates both NK cell granule-mediated cytotoxicity (Figure 6) and the secretion of cytotoxic soluble factors such as FAS L (Figure 7). When MIS416-stimulated pooled NK/NKT cells are tested against a range of tumors that have known differential sensitivity to these mechanisms, reliable killing is detected (Figure 8).  

Figure 7.  Induction of FAS L secretion by MIS416 stimulated NK/NKT cells. Human NK and NKT cells were purified from whole blood to 99% and cultured for 24 hours with no stimulus, known NK/NKT activating agents IL-2 (500 U/mL) and IL-12 (50 ng/mL), or with MIS416 (20, 10, 5, and 1 Âµg/mL). Cell-free supernatants were assayed for FAS L content using flow cytometric bead array technology.  

Figure 8.  MIS416 activates PBMC NK granule and FAS L cytotoxicity. MIS416-activated PBMC were cultured with MIS416 at 20 and 5 Âµg/ml.  Known NK cell-activating agents, IL-2 (500 U/ml), IL-12 (50 ng/mL), and TLR3 ligand, poly I:C (50 Âµg/ml) served as assay positive controls. Following a 46-hour culture, activated PBMCs were then washed into fresh medium and tested for cytotoxicity against fluorescently labelled tumor target cells at an effector:target ratio of 100:1. Tumor cell killing was determined after four hours by flow cytometric determination of viability dye uptake live/dead discrimination of gated, fluorescent tumor targets. It can be seen that MIS416 microparticle enhances human PBMC spontaneous killing activity against NK sensitive K562 (erythroleukemia) and DU-145 (prostate), as well as FAS L sensitive Daudi (Burkitt lymphoma) and T47D (breast) tumor cell targets. 

The molecular and cellular basis for adjuvancy and immune-boosting therapeutics is overlapping. There is accelerating interest in the use of nonspecific immunostimulants or adjuvants as a means of enhancing or inducing nonspecific immunity, which is a key to successful therapeutic vaccine development. The rationale behind this approach exploits several aspects associated with the innate immune system such as its rapid activation, the diverse activated array of broad-spectrum innate defenses are less likely to suffer the development of resistance, and the coherent activation of innate immune responses promotes normal activation of adaptive immune responses, which prevents or limits recurrent or progressive infections. The potential of the innate immune system as a therapeutic target has been demonstrated in several infectious and cancer models using TLR based and nonTLR-based approaches of selectively boosting the innate antiviral and antibacterial immune responses

 .  As such, incorporation of this activity into vaccine designs is highly desirable from a therapeutic vaccine standpoint, and MIS416 is well placed to fulfill this role.  

, is founding scientist at Virionyx Corporation Ltd., Auckland, New Zealand, +64 (9) 6362500, 

1. Brown KL, Cosseau C, Gardy JL, Hancock REW. Complexities of Targeting Innate Immunity to Treat Infection. Trends in Immun. 2007;28:6. 

This article discusses the potential of MIS416 adjuvant, a vaccine adjuvant and immunogen co-delivery system, to provide adequate immunostimulation to overcome host factors that may limit the success of therapeutic vaccines.

BioPharm International-01-02-2009

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