NeisVac-C is a polysaccharide-protein conjugate vaccine for use against 

 serogroup C infection. The Phase 1 clinical formulation consisted of physiological saline, thimerosal, and aluminum hydroxide. In an attempt to maintain pH for the commercial vaccine, phosphate buffered saline (PBS) replaced saline in a subsequent clinical formulation. In addition, thimerosal was eliminated and the vaccine was filled in single-dose syringes. Before the clinical trial began, the amount of group C meningococcal polysaccharide tetanus toxoid (GCMP-TT) conjugate adsorbed to aluminum hydroxide (percent adsorption) dropped from >90% to about 80% in 1–4 months. The PBS formulation was determined to be unacceptable and new lots were produced with the saline formulation. These lots were used for Phase 2–3 clinical trials. The saline formulation continued to be used for commercial product. The long-term stability data for both the PBS and saline formulations are presented in this article.

 NeisVac-C was introduced in the United Kingdom in 2000. Currently, it is licensed in 35 countries. The vaccine is used to prevent the invasive disease caused by 

 serogroup C. The active ingredient of the vaccine is a polysaccharide–protein conjugate. Each dose contains 10 μg of De-

-acetylated group C meningococcal polysaccharide (GCMP) conjugated to 10–20 μg of tetanus toxoid protein (TT) and adsorbed to 0.5 mg of aluminum in saline. This report summarizes the formulation studies performed during product development and demonstrates the importance of formulation in vaccine stability. 

The antigen component of the vaccine, De-

-acetylated GCMP, was purified from culture supernatant of 

 serogroup C, strain C11. The carrier protein, tetanus toxoid, was obtained from Statens Serum Institut, Denmark. The conjugation of GCMP to TT used reductive amination technology.

  The adjuvant, aluminum hydroxide, was obtained from Brenntag Biosector, Denmark.  

Percent adsorption by GCMP ELISA (method A):

 In a formulated sample, the aluminum hydroxide phase was removed by centrifugation and a competitive ELISA measured GCMP in the supernatant. In the competition assay, sample and serum containing antibody against the analyte were incubated together in a coated plate. The 96-well microtiter plates were coated with GCMP–human serum albumin conjugate and blocked with 2% casein. The plates were incubated with rabbit anti-GCMP–HSA antiserum, which was mixed with the supernatant from the formulation sample. Goat anti-rabbit IgG (H+L)–horseradish peroxidase conjugate was used as the probing antibody with TMB (3,3', 5,5'-tetramethylbenzidine) as the substrate. The color development was inversely proportional to the amount of GCMP in the sample. The parent lot of conjugate was used as the standard. Calculations were based on the maximum optical density (OD

), measured using wells containing no GCMP, and the OD from the standard. Figure 1 shows a typical standard curve. Percent adsorption was calculated as 100% – percent in the supernatant. 

Figure 1. Percent adsorption by competitive GCMP ELISA: standard curve  

Percent adsorption by resorcinol-HCl method (method B):

 This method measured GCMP monomer (sialic acid) by a colorimetric assay using N-acetylneuramic acid (sialic acid) as a standard.

  GCMP in the aluminum hydroxide phase (precipitate) and in the supernatant were separated by centrifugation. The amount of GCMP in the precipitate and in the supernatant and the whole formulated sample were measured separately. The interference from aluminum hydroxide was minimal because OD was measured by extracting the color substances into the organic phase while aluminum hydroxide was left in the aqueous phase and discarded. The percent adsorption was determined directly by dividing the amount of GCMP in the precipitate by the total GCMP. 

The protein content in the adsorbed supernatant was determined by the Bradford method using human immunoglobulin G (Pierce) as a standard.

Potency of the formulated vaccine was evaluated using Swiss Webster mice (Harlan, Indianapolis, IN). Mice, 10 in each group, were injected with 0.2 mL of the diluted vaccine sample at 10 μg/mL, a negative control, or a positive control, on days 0, 14, and 28. The animals were exsanguinated on day 38, and sera were collected and stored frozen until they were analyzed. The immune response of the murine serum was determined by serum bactericidal antibody titer, which reflected functional antibodies.

  The assay used baby rabbit complement (Pel-Freeze Biologicals, Rogers, AR) and strain C11 of 

. Test samples consisted of serum pools prepared from equal aliquots of individual mouse sera in each group.  

The Phase 1 formulation consisted of 20 μg/mL GCMP–TT (μg refers to GCMP) in 150 mM NaCl (saline), 0.01 % thimerosal and 1 mg/mL aluminum (added as aluminum hydroxide), and filled in single-dose vials with a deliverable dose of 0.5 mL. The 3 mL vial was Type 1 Flint glass and the closure was 13 mm chlorobutyl rubber stoppers. After four years of storage at 5 °C, the pH of the vaccine rose from 5.5 to 7.3. The slow rise in pH was caused by gradual leaching of alkaline from the glass vials.  

Phosphate buffered saline (PBS, 10 mM sodium phosphate and 150 mM NaCl, pH 7.4) was chosen for the Phase 2–3 clinical formulation because of pH stability concerns in the unbuffered vaccine.  

The adsorption kinetics were studied at small scale and GCMP ELISA (method A) was used to measure percent adsorption. Adsorption of GCMP–TT to aluminum hydroxide gel in saline reached completion instantaneously (>99.9% adsorbed). Adsorption in PBS went slowly and in an hour only 98% was adsorbed. For clinical production, we formulated in PBS for one hour and used a tentative specification of ≥90% for percent adsorption. The formulated vaccine was filled into 1-mL syringes with a 0.5-mL deliverable dose. The syringe barrels were silicon lubricated European and 

 type 1 borosilicate glass (Becton Dickinson). The closures were siliconized bromobutyl plunger stoppers. Medical grade DC 360 silicone helped stopper placement and movement and was consistent with US standards. Tip caps (chlorobutyl natural rubber blend) maintained sterility of the closure. Thimerosal preservative was not necessary for single-dose fills and was eliminated. The formulation consisted of 20 μg/mL GCMP–TT and 1 mg/mL aluminum in PBS.  

Two lots (49801 and 49802) were produced for Phase 2–3 trials and placed on stability studies. The stability indicating methods included percent adsorption, pH, potency, sterility, general safety, and appearance. 

After completing formulation, the percent adsorption samples were taken from the formulated bulk and determined to be 97% and 98% (method A) for lots 49801 and 49802, respectively. Both lots passed specification. 

Before the start of clinical trials, percent adsorption dropped for both lots as determined by GCMP ELISA. Because of the decrease in percent adsorption, the supernatant contained detectable amounts of GCMP and protein that allowed us to confirm the percent adsorption by chemical assays measuring sialic acid and protein, respectively (Table 1).

Table 1. Desorption of conjugate vaccine from the aluminum hydroxide adjuvant in PBS 

The PBS formulation was determined to be unstable and unacceptable. New lots were formulated in saline and used for clinical trials. The same formulation has been used for commercial product. 

Table 2. Percent adsorption data (method B) for development lots 

For commercial product, a resorcinol method (method B) was used to determine percent adsorption. For the 12 development lots examined, the results ranged from 90% to 102%  (Table 2); this range is consistent with the variability of the sialic acid assay. Statistical analyses with 99% (one-sided) confidence limits suggested a specification of greater than or equal to 87%. Although the supernatant contained minimal amounts of GCMP, the recovery varied from 90% to 102% and the percent adsorption also varied from 90% to 102%. The change of percent adsorption specification reflected a change in assay method and not a decrease in percent adsorption. 

Commercial lots were filled in a Baxter facility in Vienna, Austria, and three lots were placed on long-term stability study.

Figure 2a. Percent adsorption for GCMPâTT in PBS

Figure 2c. Potency of GCMPâTT in PBS 

Figure 2d. Percent adsorption for GCMPâTT in saline 

Figure 2f. Potency of GCMPâTT in saline 

The stability data for PBS and saline formulations are presented in Figure 2. The time points for each study are summarized in Table 3. The specifications for saline and PBS formulations are presented in Table 4. The saline formulation provided stable adsorption with some drift in pH. The PBS formulation provided a stable pH, but, the percent adsorption dropped initially and stayed low. The vaccine remained potent in animals with both formulations. 

Table 3. Summary of the time points for the long-term stability studies at 2â8 Â°C (data shown in Figure 2) 

Desorption of antigen from aluminum hydroxide adsorbed vaccine by phosphate has been reported.

  Rinella, et al., reported desorption of negatively charged ovalbumin from aluminum hydroxide adjuvant by the addition of phosphate. Phosphate anions were adsorbed to aluminum hydroxide and lowered the isoelectric point of the adjuvant. This in turn decreased the electrostatic interaction with negatively charged antigen. The extent of desorption depended on the ionic strength, the phosphate concentration, and the age of the vaccine. As the antigen adjuvant complex aged, the ability of phosphate to desorb the antigen decreased. It was believed that the antigen might undergo conformational changes to optimize its interaction with adjuvant. Our data supported this hypothesis. The desorption of GCMP–TT from aluminum hydroxide occurred only in the first few months and leveled off during the two-year storage. 

Table 4. Specifications established for clinical and commercial lots 

Iyer, et al., treated aluminum hydroxide adjuvant with phosphate ion, which resulted in ligand exchange of phosphate for surface hydroxyl groups.

  This ligand exchange decreased the isoelectric point of the adjuvant. In addition, the adsorptive capacity and adsorptive coefficient for a negatively charged antigen were reduced. 

The antigen in the current study, GCMP–TT, is negatively charged because of functional groups on the sialic acid monomer. The lowering of percent adsorption in PBS was caused by the ligand exchange of phosphate for hydroxyl groups on the aluminum and decreasing electrostatic interaction between aluminum and GCMP–TT. In the adsorbed vaccine formulation, phosphate buffer should be avoided. If buffering capacity was required to maintain the stability of the vaccine, we would recommend using Tris buffer instead of phosphate buffer.  

The authors would like to thank Professor Stanley Hem for his interpretation of the PBS adsorption data. 

SHWU-MAAN LEE, PHD, is a technical director, BOB KRUSE, PHD, is aresearch scientist, and CHRIS DONALDSON is a research associate, all at Baxter Healthcare, Beltsville, MD, 301.419.8587, 

 1. Jennings HJ, Lugowski C. Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J Immunol. 1981;127:1011–8. 

 2. Svennerholm L, Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochimica et Biophysica Acta. 1957;24:604–11. 

 3. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. 

 4. Michon F, Huang CH, Farley EK, Hronowski L, Di J, Fusco PC. Structure activity studies on group C meningococcal polysaccharide-protein conjugate vaccines: effect of O-acetylation on the nature of the protective epitope. Dev Biol. 2000;103:151–60. 

 5. Rinella JV Jr, White JL, Hem SL. Effect of anions on model aluminum-adjuvant-containing vaccines. J Colloid Interface Sci. 1995;172:121–30. 

Articles

NeisVac-C is a polysaccharide-protein conjugate vaccine for use against Neisseria meningitidis serogroup C infection. The Phase 1 clinical formulation consisted of physiological saline, thimerosal, and aluminum hydroxide. The long-term stability data for both the PBS and saline formulations are presented in this article.

Formulation Studies of an Adsorbed Conjugate Vaccine

In a recent article, you say that vaccine manufacturers need to take into account the specific needs of vaccines, which are different from the manufacture of monoclonal antibodies. What are some of the vaccine-specific considerations? 

We've seen an enormous upswing, both in the physical number and types of vaccines that we're seeing brought into our organization for development and manufacturing. These include not only prophylactic vaccines, but also therapeutic vaccines—a vast, different array of novel vaccines. It's a very exciting time out there. 

What does that mean? Well, they all need different production processes. There is really no standard platform for producing many of these novel vaccines. With monoclonal antibodies (MAbs), we've got to the stage where MAbs are well-defined products; we're able to characterize them properly. The regulators are comfortable with that of course, it makes it easier to make changes in the process because we can prove we're developing the same product. Also, the actual production of MAbs has become a platform nowadays. Most companies do it exactly the same way, working with perhaps a CHO cell or NS0, similar bioreactors, Protein A purification, and low pH hold for viral clearance.  

For vaccines it's very different, because these products are tremendously diverse. So really we are almost stuck in what I consider a 1980s paradigm with vaccines. We're back to the situation where the process really defines the product. That's the way it was with monoclonals and a lot of other recombinants probably 10 to15 years ago. 

 Do the specific needs of vaccine manufacture cause particular concerns when outsourcing vaccine production, for example, in terms of tech transfer?

Absolutely. The process is really seen by the regulators as defining the product at this point, and therefore, every time you make a change or you scale up or change facilities, there are particular regulatory concerns for many of these novel vaccine products.  

Because immunogenicity is hugely important for vaccines, you run the risk of making changes during tech transfer which could change your entire product and its clinical efficacy. So you got to be extremely careful. And it's much more important when you are  outsourcing manufacture, to look for a service provider who can stay with you longer and has the scale you may need for future production, because then you can avoid future tech transfers.   

 Can you give us some examples of the different types of vaccines you've worked on? And how different are they from one another? 

We have both a service business and  a consultancy business, so we have seen a wide range of products. In our UKfacilities, we're doing some work on a virus-like particle (VLP), which we're producing in 

 in suspension culture. We also have some interesting technology that we've bought on and applied ourselves for some adenoviral work for vaccine use; it's also in suspension culture rather than having to rely on adherent cell lines. We also worked with whole-cell microbial  and recombinant protein vaccines. In our consulting group, we work with live attenuated vaccines. Our management group has particular skills and expertise in this area. They're actually the group that launched in Europe a third generation hepatitis B vaccine, which was expressed in mammalian cells. Because we see a huge range, we have to look at production technologies that vary—cell factories, disposable bioreactors, roller bottles, conventional bioreactors, and fermenters.  

 For your vaccine clients, do you usually carry out the process development as well, or do the clients often do the process development and transfer it to you?

For the most part, we do the development. That's actually really what our business is—we're fundamentally in process development, although we do transfer in products as well.  

We're doing upstream development, whether it's expressed in a mammalian system or a microbial cell, that's pretty conventional. We do have some particular expertise in doing suspension culture for some of these products, which would be somewhat unusual. On the downstream side, we're developing purification processes. We're getting away from the old days of cesium gradients and trying to put together something which approaches a platform of chromatographic techniques for many of these vaccines.  

The key part of it, I believe, is actually the analytical side. We've seen some tremendous advances there. We no longer have to wait three days for a plaque assay result when we're changing a filter in a process. So we can apply some of the newer techniques such as anion exchange and HPLC, and get answers in minutes or hours instead of days.   

The other thing to be considered in these days of  pandemics and bioterrorism is that although it is difficult to transfer these processes, sometimes we 

 to transfer them. We may be developing them for developing markets such as the Chinese or Indian markets. So we've got to make the processes more robust and transferable. 

President and group commercial director, Eden Biodesign, Inc.

Interview with Roger Lias, president and group commercial director at Eden Biodesign, Inc.

Bridging the Gap in Translational Research for Vaccines

This article discusses the production process of the major influenza antigen, hemagglutinin (HA), by rDNA methods in 

 Fusing the gene for HA to the gene for flagellin, a Toll-like receptor (TLR) ligand, yields a bi-functional protein. The HA moiety contains the structures recognized by the immune system as it generates neutralizing antibody, and the flagellin targets the HA antigen into the appropriate compartment of an antigen presenting cell.  Having the antigen and the TLR ligand physically connected drives a robust antibody and cellular immune response. Producing this protein in 

provides the additional benefit of high yield per culture volume and global portability. These vaccines could help immunize the global population rapidly during an influenza pandemic. 

Next to clean water and sanitation, vaccines have had the greatest impact on public health. Early vaccines were based on animal pathogens related to human pathogens (cowpox versus smallpox, 

), killed pathogens (pertussis, poliovirus, typhoid fever), attenuated viruses (yellow fever, measles), or components of pathogens (tetanus toxoid, diphtheria toxoid).

  Although these vaccines were considered to be quite effective in protecting against disease, their use was limited, in some cases, because of poor tolerability. In addition, not all infectious diseases could be addressed by these limited vaccine approaches. 

COMPOSITE_TOSHIHIDE GOTOH/GEORGE DOYLE/GETTY IMAGES 

The advent of molecular biology in the late 1970s brought the promise of vaccines made of highly purified, well-characterized subunits of pathogens, which could be produced by cloning and expressing pathogen genes using recombinant DNA techniques. There have been two spectacular successes in this realm: the hepatitis B vaccines licensed in the mid 1980s, and the human papillomavirus vaccines licensed in the past year. These two successful vaccines are based on virus-like particles, which are made up of self-assembling virus capsid proteins. These virus-like particles display hundreds of arrayed viral epitopes on their surface, which allows the particles to be picked up, processed, and presented by the antigen-presenting cells (APCs) of the immune system in a very efficient manner.

Despite these successes, the fact is that the vast majority of viral antigens do not self assemble into regular arrays and thus are not presented efficiently by APCs. For the past two decades, researchers have been cloning, expressing, and purifying proteins from pathogens and testing them as vaccines. Overall, these monomeric proteins have been found to be poorly immunogenic. The collective experience has shown that while natural infection generally results in a robust and durable immune response to a variety of components of the pathogen, vaccination with the same components as purified proteins does not. Clearly, something was missing from vaccine candidates based solely on purified recombinant viral proteins. 

For years, researchers have known that one can improve the immunogenicity of an antigen by adding an adjuvant. Freund's Complete Adjuvant (FCA), has been the benchmark for laboratory work. FCA, an oily emulsion containing killed mycobacteria, can raise both robust antibody and cytotoxic T-cell immune responses in vaccinated animals. But this comes at the expense of a high frequency of sterile abcesses, making FCA unsuitable for human use. The adjuvant that is extensively used in man is alum, a generic term for a range of particulate forms of aluminum sulfate mixed with sodium hydroxide and varying amounts of phosphate. Aluminum-containing adjuvants work both as depots—most antigens can be made to stick to it—and as irritants, drawing cells of the immune system to the site of injection. Antigens formulated on aluminum-containing adjuvants tend to elicit good antibody responses, but not cytotoxic T-cell responses. However, for most naturally caused infectious diseases, both antibody and cytotoxic T-cell responses are required to control the disease and protect against future infection. This suggests that naturally infecting pathogens must carry some kind of FCA-like adjuvant activity. 

In the mid-1980s, the Toll protein of the fruit fly, 

  In Drosophila, Toll controls, among other things, the release of peptides that are active against infectious agents, a major line of defense.

  In subsequent years, a related set of Toll-like receptors (TLRs) were found on or in mammalian APCs, including dendritic cells, macrophages, endothelial cells, and some plasmacytoid cells.

  Together with neutrophils, eosinophils, basophils, and mast cells, these constitute the "innate immune system." These cells mount the first attack against incoming pathogens, resulting in an inflammatory response that is the necessary predecessor of the T-cell and B-cell "adaptive immune response." 

In 1997, Medzhitov, Janeway, and others demonstrated that TLRs are the mediators of the innate immune response.

  TLRs collectively recognize families of structures that are unique to pathogens and not part of mammalian biology. These structures are collectively termed pathogen-associated molecular patterns (PAMPs), and they provide the primary signal to the mammalian host that a pathogenic insult has occurred.

  Figure 1 illustrates the known TLRs and their ligands, or PAMPs.

  Each TLR binds a specific class of pathogen-related molecules. For example, TLR4 binds to lipopolysaccharides (LPS) found on the surface of bacteria but not in the mammalian host.

  TLR3 binds to double-stranded RNA (dsRNA), a non-mammalian nucleic acid conformation that is the genome structure or replication intermediate of many viruses.

  TLR5 binds to flagellin, a protein that polymerizes to form the bacterial flagella.

  Mammalian TLRs are expressed either on the surface or in an internal compartment inside of an APC. In general, cell surface TLRs tend to recognize bacterial components (e.g., lipoproteins, lipopolysaccharides, flagellin) whereas internal TLRs tend to recognize nucleic acids and analogs (e.g., dsRNA, CpG oligonucleotides, or nucleotide derivatives). 

Figure 1.  The known Toll-like receptors (TLRs) and their ligands. Pathogen-associated molecules are recognized by TLRs. 

Binding of a PAMP to a TLR results in a cascade of events inside an APC (Figure 2, unpublished data). First, the PAMP-pathogen complex binds to a TLR and is internalized to an endocytic vesicle. The vesicle now contains an activated TLR that sends a series of signals to the nucleus. These signals drive the fusion of the vesicle with a lysosome where the contents are degraded into peptides for presentation in the groove of the MHC class I or class II complex.

  TLR signaling triggers expression of genes for cytokines, which stimulate neighboring T cells as well as the expression of co-stimulatory molecules (CD80/86) that bind to the T cell and promote its activation.

  This triad of peptide processing and presentation, cytokine secretion, and T-cell co-stimulation satisfies the criteria for activating a T cell and initiating a productive immune response. 

Figure 2.  Toll-like receptors' mechanism of action: the stimulation of Toll-like receptors activates pathways that 1) promote antigen processing and presentation and 2) provide the second signal to T cells, informing them that the antigens are proper subjects for vigorous immune response. 

How does this relate to vaccines? First, it explains why FCA works so well. Mycobacteria, the active ingredient of FCA, are also pathogens, and therefore, contain numerous PAMPs. More importantly, it suggests that using a TLR as a portal of entry for an antigen into an APC might result in more efficient antigen processing and presentation. This, in turn, should elicit a more robust immune response. 

Multiple PAMP–TLR pairs are available for use as a portal of entry (Figure 1).

  However, only one of these pairs, flagellin-TLR5, involves a purely protein ligand and a cell- surface TLR. Coupling the DNA sequence for flagellin in frame with the DNA sequence for a protein antigen and expressing this chimeric assembly in 

 yields a protein that contains the cell entry and immunostimulatory activity of both a TLR ligand and an antigen in a single molecule. This ensures that both components enter not only the same cell but also the same endocytic vesicle within that cell. Thus, the target antigen is present within an endosome that contains an activated TLR, ensuring that both components are trafficked through the same endosome-lysosome-processing and presentation cycle in close proximity. Elegant confocal microscopy studies using other PAMPs, including LPS, have shown that this is the case at the cellular level.

Co-Delivering Antigens and TLR Ligands to APCs

We have built our platform technology on the concept of co-delivering vaccine antigens and TLR ligands to APCs. Using this technology, we have demonstrated that coupling flagellin to a model antigen, ovalbumin, can elicit a rapid IgG antibody response in mice one week after a single dose; ovalbumin alone on aluminum hydroxide takes three weeks to reach the same level of antibody.

  The same flagellin–ovalbumin fusion elicits a classical T-helper 1 antibody and CD8+ T-cell response, while ovalbumin alone, or mixed with flagellin, yields a classical T-helper 2 response.

  Similar results have been obtained by a fusion of flagellin to antigens from 

The same strategy has recently been applied for developing a West Nile virus vaccine that is based on the conformation-dependent structure of a subunit of the envelope of the virus, EIII. EIII contains a disulfide bond that is essential for proper folding of the domain and presentation of epitopes that are recognized by virus-neutralizing antibodies.

  Genetic fusion of the sequence for EIII to the sequence for flagellin yields a protein that retains TLR5 activity and properly folds the EIII domain such that it is recognized by neutralizing monoclonal antibodies and induces protective immune response in mice.

  The ability to express such a conformation-dependent protein in 

suggested that this strategy could be applied to other  vaccine targets.

This technology has also been applied to both seasonal and pandemic influenza vaccines. Two distinct, but complementary, vaccines are currently in development. The first is based on the highly conserved ectodomain of the viral M2 ion channel protein (M2e). M2e is a 24 amino acid extension of M2 protruding through the membrane of the virus and the infected cell. Antibodies raised against the M2e sequence have been shown to reduce the rate of spread of influenza virus in culture and to protect mice from a lethal challenge of virus. The M2e sequence is highly conserved across the H1, H2, and H3 serotype viruses that have infected humans over the past 90 years.

  It is also well-conserved in the avian serotypes, suggesting that a cocktail of three or four M2e vaccines could provide broad protection against any influenza virus. A significant advantage of such an approach would be that the vaccine would not have to change on an annual basis, unlike the current influenza vaccine strategies. 

Figure 3.  Influenza M2e vaccine: mice vaccinated with 0.3 or 3.0 Î¼g of globular head fusion protein are protected 100% from death following a challenge with virus that kills 90% of naÃ¯ve mice 

M2e as a peptide is poorly-immunogenic and ineffective, even when delivered at high doses on aluminum-containing adjuvants. Four copies of M2e head-to-tail mixed with flagellin, rather than fused, are also ineffective. However, fusing four head-to-tail copies of the human consensus M2e sequence to the C-terminus of flagellin creates a highly potent M2e vaccine. Doses of 0.3 μg provide 90–100% protection against a challenge dose of virus that kills 90% of naïve mice (Figure 4, unpublished data),

  reinforcing the value of coupling a TLR ligand to an antigen. The first study of M2e in man, funded by the Gates Foundation, has been completed and it was found that the vaccine is highly immunogenic at low doses. 

Figure 4a.  Influenza STF2.1HA vaccine efficacy: 0.3 Î¼g provide 90â100% protection against a challenge dose of virus that kills 90% of naÃ¯ve mice 

Another application of this technique has been in the major antigen of the virus, hemagglutinin (HA), one of the two predominant proteins on the viral surface. HA binds to sialic acid sugars on the surface of cells, and therefore, is a prime target for the immune system. Virus-neutralizing antibody raised by natural infection is known to be directed to the area of HA around the sialic acid binding site, and can thus prevent binding and block infection.

  Viruses that have mutations in the HA gene in the region of the sialic acid binding site have an infectious advantage over their neighbors, and thus can dominate the population (a primary reason the influenza vaccine needs to be updated every year). 

Figure 4b and 4c.  Low doses of PR8 STF2.HA subunit protect mice against challenge with homologous virus 

Current inactivated influenza vaccines are made by growing the virus in embryonated eggs. The allantoic fluid is harvested, and the virus in it is killed with formalin. The killed virus is enriched by centrifugation and then disrupted with detergent, and is generally followed with a second formalin kill step. The result is the bulk vaccine, of which HA comprises up to 20% of the protein component.

We have discovered a means of producing just the "globular head" of HA, the segment of the protein that contains all of the structures recognized by neutralizing antibodies. This globular head can be produced as a fusion protein at the C-terminus of flagellin in 

, and can be purified to endotoxin-free homogeneity. Although the globular head moiety is a complex structure with disulfide bonds that must be configured and folded correctly for optimum immunogenicity, we have found that the purified protein reacts strongly with serum from convalescent animals as well as with monoclonal antibodies directed at the neutralizing epitopes, suggesting that it is appropriately structured.  

In addition, vaccinating mice with two doses of the HA globular head fusion can raise antibodies that recognize native HA on infected cells and also have the ability to block the virus's ability to agglutinate red blood cells, a classic assay for quality of HA antigens. Mice vaccinated with 0.3 or 3.0 μg of globular head fusion protein are protected 100% from death following a challenge with virus that kills 90% of naïve mice (Figure 3).

  This vaccine is also highly immunogenic in rabbits and ferrets (unpublished data). As seen with other proteins, the globular head alone, or mixed with flagellin, is not immunogenic. In the last 12 months, we have made a variety of HA vaccines with different serotypes, including H1 PR8, H1 New Caledonia, H1 Solomon Islands, H3 Wisconsin, H5 Vietnam, H5 Indonesia, H5 Anhui, B Malaysia, B Lee, and B Shanghai. All are well-expressed in 

 (20–25% of total cell protein on induction) and the first clinical trial is underway. A new vaccine seed strain may be made and initial protein produced in a month, providing a rapid response to an emerging virus. 

The speed, simplicity, and efficiency of production of these vaccines in 

 provides an attractive alternative to the present egg-derived influenza vaccine. A typical protein yield is >2 g/L of fermentation broth, and this may be optimized further. Compared to the average of about 6–8 mg of HA recovered per liter of allantoic fluid of infected eggs, and 0.6 to 6 mg of HA per liter from mammalian cell culture, an 

-based vaccine approach is highly attractive (unpublished data). 

The US Department of Health and Human Services has stated that its goal is to have within the US the capacity to make enough monovalent pandemic influenza vaccine to give two doses to the entire US population, or 600 million doses. Current egg-based systems are unable to meet this demand, and even with recent enormous investments in expanded egg facilities and new cell-culture efforts, there will be a shortfall. As described here, an 

-based system could produce the required vaccine in one 5,000-L fermenter cycle. Moreover, because the methodologies are standard, the production process could easily be transferred to other countries or regions. Given the projected worldwide impact of a pandemic, local or regional manufacture of this vaccine may be able to provide countries or regions without a current flu vaccine, the means to be self-sufficient. With this kind of production capability, one can contemplate vaccinating the global population effectively and rapidly, if the necessary public health infrastructure could be developed. 

, is the president and CEO of VaxInnate, Cranbury, NJ, 609.860.2260, 

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 8. McDonald WF, Huleatt JW, Foellmer HG, Hewitt D, Tang J, Desai P, Price A, Jacobs A, Takahashi VN, Huang Y, Nakaar V, Alexopoulou L, Fikrig E, Powell TJ. A West Nile virus recombinant protein vaccine that co-activates innate and adaptive immunity. J Infect Dis. 2007;195:1607–17. 

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This article discusses the production process of the major influenza antigen, hemagglutinin (HA), by rDNA methods in E. coli.

Vaccines Incorporating Toll-Like Receptor Ligands

 We know that vaccines developed using chicken eggs have many drawbacks. What do plasmid DNA vaccines offer that traditional cell and egg-based vaccines do not?

 Plasmid-DNA vaccines are much easier to manufacture. They are made in a circular DNA that is grown in bacteria. It is such an easy process compared to typical vaccines. The manufacture takes weeks versus many months. They are easy to characterize and easy to store. They have many advantages in terms of manufacturing and cost. 			

 I understand that plasmid DNA vaccines can be developed faster than traditional vaccines. How much faster can they be developed, especially when it comes to production for pandemics? What can plasmid DNA vaccines offer in terms of manufacturing scale-up and development times?

Like I said before, we are talking weeks as compared to many months here. You are also talking about using technologies that are readily available—fermenters for bacterial fermentation that can be used very readily for this production. So there is really nothing new that needs to be put together for this production. Also, another great advantage is storage. These vaccines can be stored at room temperature because they can be lyophilized, so of course, that's a major advantage.  

 Are there particular challenges to formulating plasmid DNA vaccines? Are there safety concerns that are different from the safety concerns of traditional vaccines?

 In terms of safety concerns, none, really. FDA requires many studies for safety and they are extremely safe. The plasmid DNA vaccines do not integrate into the body so they are very safe. This is a major advantage. In terms of formulation, I think one of the challenges currently for DNA vaccines is really to make them better, to increase the protection, to make sure they work as well or better than traditional vaccines. Because of that, there are still many challenges in terms of formulation. The magic formulation hasn't been found yet.  

What types of delivery systems are being developed for plasmid DNA vaccines? Are there challenges to delivering plasmid DNA vaccines to their targets in the body?

Yes, absolutely; the main challenge is delivery. The delivery systems for DNA vaccines go from very simple, naked DNA—just a plasmid in the buffer. In some cases, lipids can be added, some polymers can be added, and also there are systems such as electroporation that are being used and tested at the clinical site right now.  

There are no plasmid DNA vaccines on the market today. Are there any that are close to being approved?

Interestingly, there are DNA vaccines for animals that are working on right now and are on the market. There is a DNA vaccine for salmon and there is a DNA vaccine for horses, and they work really well. In terms of DNA vaccines for human use, I think that there is a lot of hope and a lot of enthusiasm. Several of them are in advanced clinical trials but none of them have been approved yet.  

 Why are plasmid DNA vaccines sometimes used in conjunction with traditional subunit vaccines?

The reason they're used in conjunction with other vaccines is really to increase efficacy, and clinical trials have shown that by using this method you get better protection, at least in animal studies. This is the reason why they are used in this manner.  

After delivery, the plasmid exists independently in the host. Why isn't the plasmid killed off by the host's immune system? 

 killed by the human system. The way it works is: You deliver the plasmid DNA into the body, it gets into the cell, the protein is expressed, and then the plasmid is eventually destroyed by the host.  

Have effectiveness studies been conducted? If so, how effective are plasmid DNA vaccines when compared to traditional vaccines?

I think that plasmid DNA vaccines have a lot of advantages in terms of cost, manufacturing, the mechanism of action, which is very simple. But we haven't really found a way that is really optimal for protection in terms of DNA vaccines. But I think it is very hopeful because there are DNA vaccines that are working in some animal species and I think this is very encouraging. 

co-CEO and co-president, Althea Technologies

Interview with Magda Marquet, co-CEO and co-president of Althea Technologies

Developing Plasmid DNA Vaccines

There are a number of specific characteristics to be considered when developing and manufacturing vaccines. This article discusses specific requirements to be fulfilled for three attenuated live bacterial vaccines (LBVs) including Mycobacterium bovis BCG vaccine against tuberculosis, Salmonella typhi Ty21a vaccine against typhoid fever, and Vibrio cholerae CVD 103-HgR vaccine against cholera. Special characteristics for these vaccines comprise the appropriate level of attenuation, the balance between safety and immunogenicity, the genetic stability of the organisms combined with environmental risk assessment, the challenge of old-fashioned upstream and downstream methods in combination with quality control of the final product, and the release requirements.

Vaccination has proven to be the most efficient, cost-effective means for preventing a wide variety of infectious diseases. Vaccine development and manufacturing, however, poses several challenges. Inherent to all biological systems is the difficulty to achieve robust production processes guaranteeing reproducible efficacy and safety of the products. Highly automated bioprocess systems or advanced analytical systems used for closed-loop control may be a solution to overcome this.

  Yet these techniques are still not standard in the industrial environment or cannot be applied to vaccine production processes because of historical reasons, as some of the vaccines have been developed early in the previous century.  

Because vaccines are  administered to healthy people, the efficacy and safety of vaccines are most important. Regulatory cGMP requirements are increasing to guarantee the safety of vaccines being developed and produced.  Using raw material of animal origin is avoided in the early-stages of research and development. Only few techniques such as nanofiltration and chromatography steps are applicable to viral removal. For viral inactivation, only formaldehyde treatment and beta-propiolactone inactivation are approved. The worldwide ban of preservatives such as thiomersal raised the standard for sterile production and supported the trend to monodose presentations.  

To date, vaccines based on three different technologies are registered for human use: (1) whole inactivated vaccines containing entire killed bacteria or viruses, (2) subunit vaccines, containing only the relevant antigens of the pathogens in a highly purified form, and (3) live attenuated vaccines. The quality and safety requirements are even higher for live attenuated vaccines than for the killed and subunit vaccines. In this article, we will describe how vaccine developers and manufacturers solve the challenges related to manufacturing live attenuated vaccines.  

Live attenuated vaccines are among the most widely used vaccination technologies. Attenuated vaccines consist of bacterial or viral strains, which are weakened by stable mutations that allow the bacteria or viruses to infect humans only transiently. This transient infection elicits immune responses, while the vaccine strains are designed in such a way that they will not cause the symptoms of natural infection by the wildtype pathogen. There are a number of advantages of live attenuated vaccines in comparison to killed and subunit vaccines: (a) they mimic natural infection, therefore eliciting immune responses that are highly specific, effective, and long-lasting (b) they can prevent infection by the pathogen, not just disease symptoms, (c) in comparison to highly purified subunit vaccines, they are relatively cheap to produce and administer, and do not require sophisticated downstream processing or formulation with adjuvants, and (d) several live attenuated vaccines can be administered orally, which has a higher acceptance and better safety profile than injection with syringe and needle, and mimics natural infection better.  

Three attenuated live bacterial vaccine (LBV) strains are currently licensed for human use: Mycobacterium bovis strain Bacille Calmette-Guerin (BCG), Salmonella typhi Ty21a, and Vibrio cholerae CVD 103-HgR. M. bovis BCG is the vaccine strain in the parenteral vaccine against human tuberculosis, S. typhi Ty21a represents the only oral vaccine against typhoid fever, and Vibrio cholerae CVD 103-HgR is the orally administered live vaccine against cholera (Table 1).

Table 1.  Attenuated live bacterial vaccines licensed for human use 

While the advantages of LBVs are widely accepted, there are several key considerations associated with their successful development. Most importantly, researchers developing  an LBV must generate a vaccine strain that meets a delicate balance. The strain must reach an appropriate level of attenuation to be safe and be sufficiently immunogenic to ensure protective efficacy. Traditionally, live attenuated vaccines were developed by passing the pathogens under 

 conditions until they had lost virulence for humans. This empirical approach was taken in the case of the M. bovis BCG vaccine strain. The BCG strain was attenuated by Calmette and Guerin between 1908 and 1920 by 231 serial passages of a virulent M. bovis strain through bile salts.

 passage, the M. bovis microbes became attenuated because of the loss of numerous gene complexes, as was demon-strated by a recent genome analysis of the vaccine strain.

The second example, S. typhi Ty21a, underwent a more targeted attenuation approach. Germanier and FFCrer reasoned that a 

 strain, which is sensitive to galactose and could not express a polysaccharide coat (which protects the bacteria from immune responses), should be attenuated.

  They generated the vaccine strain Ty21a in the early 1970s by chemical mutagenesis of wildtype 

 using nitrosoguanidine and screening for clones that had a phenotype, which is negative in the enzyme galactose epimerase, resulting in galactose sensitivity, and which is also unable to express the Vi-polysaccharide capsule.

  As a result of the chemical mutgenesis method, the strain was also mutated in genes responsible for amino acid biosynthesis and stress resistance, making it auxotrophic and less resistant to environmental stresses.  

Finally, Kaper and Levine used genetic engineering technology in the 1980s to generate the 

  They reasoned that the main virulence factor of 

 is the expression of the cholera toxin. CVD 103-HgR was derived from a wildtype 

 strain by the targeted deletion of 95% of both chromosomal copies of the ctxA gene, which encodes the toxic A subunit of the cholera toxin while keeping the expression of the nontoxic but immunogenic B subunit, leading to intermediary strain CVD 103. Subsequently, they inserted a mercury resistance marker into the genome to readily allow for identifying the vaccine strain and its differentiation from wildtype organisms on vaccination.

 The safety and immunogenicity of vaccines are tested in animal models before they enter clinical trials. However, in the case of LBVs this is only possible if appropriate animal models exist, which is the case only for some bacteria. We would like to refer interested readers to a detailed article by Passetti, et al., which describes the challenges when developing an animal model that allows meaningful vaccine testing for 

There are general differences in developing therapeutic agents for vaccines. All vaccines licensed to date have a prophylactic effect against infectious diseases. For some infectious pathogens, clear correlates of protection are known, for example, for threshold antibody titers. In such cases, the clinical development may be quite smooth. However, for many diseases such correlates of protection do not exist. In such cases, vaccines are administered to healthy individuals in the pivotal Phase-3 trials, and researchers then have to wait for the trial participants to get infected by the pathogen the vaccine should protect against and for this pathogen to cause disease. Differences in the incidence of infection or disease between the vaccine and placebo group then allow the researchers to calculate the protective efficacy of the vaccine. For some infectious diseases with high incidence, such a clinical trial may take just a few months. However, for diseases like tuberculosis, for which clinical symptoms may occur only more than a decade after initial infection, Phase-3 trials may take 10–20 years. Furthermore, the incidence of a disease may be low, requiring enormous numbers of clinical trial participants to get significant data. Therapeutics are administered to ill people to cure them or control disease whereas vaccines are administered to healthy individuals to protect them from becoming ill. The acceptance of side effects and safety risks is therefore much lower for vaccines in comparison to therapeutics. Hence, vaccines have to undergo careful pre- and postlicensure safety studies.  

In addition to regulations governing all vaccines, LBVs face additional regulatory hurdles. First, they must achieve the right balance between safety and immunogenicity. Additional issues include the potential for genetic reversion to partial or full pathogenicity, gene transfer into and out of the vaccine cells, and the potential risks for humans and the environment. For many vaccine candidates, the appropriate balance of attenuation and immunogenicity could not be met, and development of such candidates was consequently discontinued at an early stage of clinical evaluation. For the trivalent oral polio vaccine, which has been licensed for decades, it is known that reversion to virulence occurs quite often in the gastrointestinal tract of vaccinated individuals. This reversion to wildtype is a common cause of disease when a virus is shed and transmitted to nonimmune household contacts. Hence, these safety issues have to be addressed very carefully during preclinical and clinical development. 

In the case of Ty21a, safety and efficacy of the vaccine were demonstrated in a large number of clinical trials, with over 500,000 vaccinated children and adults in the US, Europe, Africa, Latin America, and Asia. Excellent tolerability and an overall protective efficacy of 67–80% were demonstrated for up to seven years in large field trials.

  The safety and tolerability profile of Ty21a was further confirmed in more than 200 million vaccinees during its more than 25-years of use worldwide. Recent postmarketing surveillance has identified only mild and infrequent adverse events associated with Ty21a. From 1990 to 2000, more than 38 million people were vaccinated with Ty21a with only 743 spontaneous reports of adverse events, an incidence of 0.002%. The most common adverse events reported with Ty21a were mild and transient gastrointestinal disturbances, followed by general symptoms such as pyrexia.  

As mentioned above, the most important safety feature is to demonstrate that the vaccine strain is unable to revert to a virulent phenotype during production, inside the human body and after potential excretion of the vaccine strain, if applicable. For Ty21a, reversion to virulence has not been observed 

 during the more than 30 years since the strain was developed. No mutations were found in master and working seed lots produced over a 25-year period in genetic stability studies, nor was any reversion found in clinical trials. Clinical trials have also shown either a limited and transient level or a complete lack of shedding in the stools of volunteers depending on the administered dose of Ty21a. With a 10-fold overdose, mainly on day one post-vaccination, a low rate of excretion was observed. Further studies showed a lack of fecal excretion of Ty21a upon administration of the commercial formulation. The reason for the low excretion rate is probably the limited ability of the vaccine strain to proliferate 

. However, if there is excretion, additional studies need to be performed. In the case of Ty21a, these included an analysis of vaccine strain transmission to household contacts, which was not observed for Ty21a. Furthermore, Ty21a demonstrated a limited ability to survive in the environment because of its auxotrophy and reduced stress resistance, therefore not posing environmental risks. 

The clinical program was somewhat different for CVD 103-HgR. For 

, a human challenge model exists. This means that after vaccination, humans can be infected intentionally with wildtype, fully virulent 

 bacteria to determine whether the vaccine is indeed efficacious against infection and disease. In the volunteer challenge model, significant efficacy of CVD 103-HgR against wildtype 

 challenge infection was demonstrated in North American adults as early as eight to 10 days after vaccination and for at least six months.

  Hence, oral vaccination with CVD 103-HgR is highly suitable to elicit protective immunity in North Americans, as shown in trials that are comparatively easy to perform. However, challenge trial results in North American adults may have limited relevance for the vaccines' protective efficacy in cholera-endemic areas of the world. Hence, a field trial was performed in Indonesia with CVD 103-HgR. Unfortunately, protective efficacy of CVD 103 HgR could not be demonstrated in this field trial. More than 60,000 children and adults were given either vaccine or placebo. However, cholera was virtually absent in Indonesia after initiating the clinical trial, and therefore, there were almost no cholera cases in the vaccine and the placebo group. This made it almost impossible to calculate a protective efficacy for the vaccine in this particular setting. This exemplifies the challenges vaccine field trials can pose.  

In many controlled studies and after a decade of postmarketing experience, CVD 103-HgR has been shown to be safe and well tolerated. In diverse populations, no adverse reactions occurred significantly more often in vaccinees than in placebo recipients.

Genetic Stability and Environmental Risks

Several tasks were performed to determine the genetic stability and environmental risks of CVD 103-HgR including (1) global and local genetic characterization and stability of the vaccine strain with emphasis on the modified gene loci, (2) identifying natural cryptic prophages and plasmids,  (3) confirming the absence of DNA sequences from the various plasmids used during construction, and (4) evaluating biosafety aspects pertaining to the rate of excretion from vaccinees, the potential for vaccine survival in various ecosystems, and the potential for acquisition or export of genetic material. The strain was shown to be stable in a number of studies. For stability studies, two vaccine production lots manufactured three years apart were compared to the CVD 103-HgR master seed lot (MSL)after prolonged storage in a lyophilized state and outgrowth for 16 to 17 generations. In addition, fecal isolates from immunized volunteers were also compared to the MSL. This is important because DNA rearrangements may conceivably occur during transit of the vaccine organisms through the human gastrointestinal tract.

Studies on American adult volunteers revealed that CVD 103-HgR is only excreted at low levels (2 x 10

  CFU/g of feces) by 20 –30% of the volunteers for a maximum of seven days with a peak on day four. In people living in endemic countries, the excretion rate is even lower. However, since it is excreted, CVD 103-HgR had to be compared to wildtype 

There are a number of specific characteristics to be considered when developing and manufacturing live bacterial vaccines.

BioPharm International-10-02-2008

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Bridging the Gap in Translational Research for Vaccines

Vaccines Incorporating Toll-Like Receptor Ligands

Developing Plasmid DNA Vaccines

Developing and Manufacturing Attenuated Live Bacterial Vaccines

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