The Conception and Production of Conjugate Vaccines Using Recombinant DNA Technology

In recent years, the vaccine market has experienced significant growth following the introduction of several novel bacterial vaccines—more specifically conjugate vaccines—addressing unmet medical needs. These conjugate vaccines are safe and effective against bacterial diseases and have been used in humans for many years. Although several serious bacterial infections, such as Streptococcus pneumoniae and some Meningococcal strains, are prevented using conjugate vaccines, the underlying process of development and manufacture has limited their scope. The method used for developing and manufacturing conjugate bacterial vaccines is based on chemical conjugation technology. It is a complex chemistry-based process that, depending on the pathogen or serotype, is time-consuming and expensive. A new approach has been developed to conceive and produce conjugate vaccines by employing recombinant DNA technology. This technology enables the development and manufacture of conjugate vaccines, called bioconjugates, and addresses the limitations of the current chemical conjugation process.

BACTERIAL CONJUGATE VACCINES: AN IMPORTANT MARKET IN BACTERIAL INFECTIOUS DISEASE

The vaccine market experienced significant growth over the past decade, with global revenues forecast to exceed USD $24 billion in 2010 (1). Within the growing market, conjugate vaccines for the prevention of bacterial infections today account for over 25% of the total market. In 2009, two of the four leading vaccines by sales were the bacterial conjugate vaccines Prevnar (Pfizer) for pneumococcal disease and Menactra (Sanofi Pasteur) for meningitis serogroups A, C, W-135, and Y. Together, these two products alone accounted for 12% of global vaccine sales.

Despite the success of glycoconjugate vaccines, several important bacterial infections lack a vaccine. These pathogens are responsible for significant morbidity, mortality, and cost to healthcare systems. Key pathogens that lack vaccines include Staphylococcus aureus and Pseudomonas aeruginosa, both causing nosocomial infections; Neisseria meningitides type B; and many diarrheal pathogens such as Shigella sp., enterotoxigenic Escherichia coli (ETEC), and Salmonella sp.

THE LIMITATIONS OF CURRENT CONJUGATE VACCINE TECHNOLOGY

The conjugate is a large glycoprotein molecule consisting of a protein linked or conjugated to a polysaccharide. The sugars are surface-exposed bacterial antigens to which the body will develop an immune response. The protein carrier is responsible for eliciting a long-lasting immune response against the polysaccharide, leading to better protection against the target disease, especially in young children (2). In chemical conjugation, the bacteria producing the polysaccharide and the protein carrier are grown separately, then purified through multiple steps. The polysaccharide is then chemically bound to the protein carrier (see Figure 1). This method faces the following challenges and limitations:

• Because the polysaccharide is produced by toxic bacteria, specialized and costly containment facilities are required. Moreover, several purification steps are necessary to obtain an acceptable purity of the product, thus resulting in loss of material throughout the process and decreased yields.

• Chemical coupling between the polysaccharide and the protein carrier results in a heterogeneous product which may still contain some free polysaccharide that may interfere with the immune response to the conjugates. Any small change in the mixture affects the characteristics of the vaccine, so the same mixture must be maintained throughout scale up and production—a manufacturing and regulatory challenge.

• Chemical conjugation can change the structure of both the polysaccharide and the carrier protein, thus making them less immunogenic, or in some cases, not immunogenic. Toxic polysaccharides must be chemically detoxified, often leading to further loss of immunogenicity or increased safety concerns.

The net result is that chemical conjugate vaccines are restricted to certain targets, may induce suboptimal efficacy, are difficult to develop, and are costly to produce. In addition, the growing resistance to antibiotics, the ever-increasing standard of safety, and high development costs required to bring a product to market emphasize the need for new technologies to address these challenges and fulfill the worldwide need for new vaccines.

Figure 1: Chemical method currently used for production of conjugate vaccines. (ALL FIGURES ARE COURTESY OF THE AUTHOR)

NEW PROCESS FOR DEVELOPING AND MANUFACTURING CONJUGATE VACCINES

A new technology has been developed for the production of conjugate vaccines by an in vivo conjugation process. Instead of chemically conjugating polysaccharides to proteins, the conjugate is directly synthesized in appropriately engineered E. coli cells. Because E. coli is one of the fastest, least expensive, and highest product-to-volume systems available for the production of large molecules, the use of E. coli is appealing for the production of vaccines. However, until recently, it has not been possible to manufacture glycoprotein conjugates using bacterial cells.

Despite the ubiquitous presence of polysaccharides at the surface of bacterial cells, bacteria were thought to be unable to synthesize glycoproteins, and N-linked protein glycosylation was believed to be restricted to eukaryotes. The finding of N-linked glycoproteins in the human pathogen Campylobacter jejuni disproved this theory.

Various proteins of C. jejuni have been shown to be glycosylated by a heptasaccharide. This heptasaccharide is assembled on undecaprenyl pyrophosphate (UPP), the carrier lipid, at the cytoplasmic side of the inner membrane by the stepwise addition of nucleotide-activated monosaccharides catalyzed by specific glycosyltransferases. The lipid-linked oligosaccharide then flip-flops (i.e., diffuses transversely) into the periplasmic space by the flippase PglK. In the final step of N-linked protein glycosylation, the oligosaccharyltransferase PglB catalyzes the transfer of the oligosaccharide from the carrier lipid to Asn residues within the consensus sequence Asp/Glu-Xaa-Asn-Xaa-Ser/Thr, where Xaa can be any amino acid except Pro (3).

The gene cluster encoding this glycosylation machinery was functionally expressed in E. coli, allowing the heterologous production of Campylobacter glycoproteins in E. coli (4) and providing the first opportunity to produce N-linked glycoproteins in E. coli. In addition, the consensus amino acid sequence was introduced into different proteins that are not glycosylated in their original organism (see Figure 2).

Figure 2: Details of an engineered glycosylation pathway in Escherichia coli. Bacterial polysaccharide antigens are synthesized by stepwise action of glycosyltransferases at the cytoplasmic side of the membrane and polymerized after flipping. The oligosaccharyltransferase PglB is able to transfer a different polysaccharide from the carrier lipid to Asn within the consensus sequence because of its relaxed specificity.

The N-linked protein glycosylation biosynthetic pathway of Campylobacter has significant similarities to the polysaccharide biosynthesis pathway in bacteria (5). Because antigenic polysaccharides of bacteria and the oligosaccharides of Campylobacter are both synthesized on the carrier lipid, undecaprenyl pyrophosphate (UPP), the two pathways were combined in E. coli. The polysaccharide-synthesizing enzymes of different pathogens were expressed in the presence of the oligosaccharyltransferase PglB and a protein carrier (6, 7). The antigenic polysaccharides assembled on UPP are captured by PglB in the periplasm and transferred to a protein carrier. After fermentation of E. coli, the glycoconjugate is extracted from the periplasm and purified using simple and well-known manufacturing steps similar to those used for production and purification of recombinant proteins (see Figure 3).

Figure 3: In vivo glycosylation system for production of bioconjugates in Escherichia coli system. The bioconjugate is extracted from the periplasm and purified by column chromatography to high purity.

ADVANTAGES OF IN VIVO RECOMBINANT TECHNOLOGY

This in vivo technology to design and produce bioconjugates offers improved versatility, efficacy, safety, speed, and cost of development, partly resolving the challenges that the vaccine industry is currently facing. Some of the specific advantages of the technology are as follows:

• Bioconjugation is versatile, enabling the attachment of virtually any polysaccharide to virtually any protein. This versatility permits the development of novel conjugates that cannot be addressed with existing chemistry-based processes.

• Bioconjugates are engineered to have a specific structure optimized for efficacy. Bioconjugate vaccines can be designed to not only generate an immune response to the polysaccharide, but also to the protein from the target organism, thereby enhancing efficacy. No free polysaccharide is present during bioconjugate production that can inhibit the immune response.

• Bioconjugates are produced in a standard, nontoxic bacterial production system, with no risk of contamination by mammalian infectious organisms. Moreover, bioconjugates are engineered to a reproducible structure and final product, thus minimizing potential safety concerns. This design will lower the regulatory barriers and potentially accelerate clinical development.

• Bioconjugate process development and production are rapid and straightforward. Producing vaccine by recombinant methods in a standard E. coli expression system and using a conserved biosynthetic pathway that may differ slightly depending on serotypes is a well-understood and commonly used manufacturing method.

From a technical perspective, the in vivo technology has the potential to provide uniform product, easily reproducible in a low-cost expression system, with an optimized safety and efficacy profile. These factors may decrease the regulatory barrier and the time to market and result in reduced development and manufacturing cost.

CHALLENGES OF IN VIVO RECOMBINANT TECHNOLOGY

The in vivo technology has the potential to overcome many issues that the chemical conjugation currently face in designing and producing conjugate vaccines. However, the following challenges are still unresolved.

• Because of the complexity of several bacterial pathogens, some vaccine candidates are still difficult to design and produce using in vivo recombinant technology. Bacterial pathogens such as N. meningitis B or Moraxella are challenging targets because the mechanism by which the antigenic sugar is assembled and expressed on the surface is less suitable for the in vivo glycoconjugation technology.

• The bioconjugate process is still early in development and its ultimate potential and limitations are not fully delineated. At this point, only data from preclinical and early clinical studies on a restricted number of pathogens are available. Additional work is required regarding process and assay development (i.e., scalability).

PROOF-OF-CONCEPT STUDIES USING THE BIOCONJUGATE PLATFORM

The process to create new and efficacious bioconjugate vaccines in a cost-effective and efficient manner has potential, but what is required is proof that such vaccines can be manufactured in commercial quantities, and that the vaccines produced are safe and effective. The following are examples that demonstrate the potential of in vivo bioconjugate technology:

A bioconjugate against Shigella sp. was produced under GMP conditions and tested for the first time in humans. Shigella is an important pathogen responsible for serious diarrhea and dysentery, so a vaccine to prevent infection in the emerging nations where it is present, as well as a vaccine for travellers, would provide a significant public health benefit. No vaccine exists for Shigella, despite ongoing research in many laboratories for several years. Attempts at vaccine development, both conjugate and live-attenuated bacteria, showed modest immunogenicity (8–11). Moreover, the technical hurdles to producing a conjugate vaccine with chemistry-based methods are very high. The bioconjugate produced against the serotype, Shigella dysenteriae, was tested in 40 healthy volunteers and found to be well tolerated. Importantly, the vaccine demonstrated a significant immunogenic response, and these immunogenicity data compare favorably to previous candidate vaccines tested against this pathogen. This promising Phase I data provide clinical proof-of-concept that the bioconjugate produced under GMP conditions by an recombinant DNA technology is safe and induces an immunogenic response in human.

The technology has been also applied for the development of a bioconjugate against Staphylococcus aureus. Nosocomial S. aureus infections represent up to 50% of all hospital infections. Moreover, methicillin-resistant S. aureus (MRSA) rates continue to increase dramatically. Despite significant research efforts undertaken by academic and pharmaceutical laboratories to develop a successful vaccine, there has been no recorded sustained effectiveness against S. aureus has been generated by the experimental vaccines tested (12, 13). More recently, the DNA recombinant in vivo technology was able to conjugate, for the first time, the main polysaccharides of S. aureus to a selected protein carrier of the same pathogen (i.e, antigen protein of S. aureus). This bioconjugate vaccine has been tested in animals and produced functional antibodies inducing protection in mice bacteremia and lethal pneumonia models (14). Although early, these results are promising considering recent clinical trial failures of S. Aureus candidate vaccines. The combination of polysaccharide and protein antigen against the pathogen will increase the immunogenicity of the vaccine at various stages and pathways of the infection, thus enhancing the possibility of protection.

These data demonstrate that this in vivo technology is a feasible approach for developing vaccines against challenging pathogens and offers the promise of improved efficiency in general.

SUMMARY

Antibacterial conjugate vaccines have become important tools for the public-health community to prevent serious bacterial infections. However, the complex development and manufacturing process has limited the potential of this important class of vaccine. This article describes a new in vivo process that incorporates a well-understood recombinant DNA technology in E. coli to manufacture bioconjugate vaccines. The process has demonstrated proof-of-concept in more than one bacterial pathogen, including a first-in-man study. Research is currently in progress to develop additional vaccine candidates and advance them into late-stage clinical trials.

Veronica Gambillara PhD is director of clinical and regulatory affairs at GlycoVacyn, Schlieren Switzerland, Veronica.Gambillara@glycovaxyn.com

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