Vaccines Incorporating Toll-Like Receptor Ligands

Attaching the HA antigen to a TLR can produce a strong immunogenic response and can be produced quickly and easily in E. coli.
Oct 02, 2008


This article discusses the production process of the major influenza antigen, hemagglutinin (HA), by rDNA methods in E. coli. 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 E. coli 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, Mycobacterium bovis versus Mycobacterium tuberculosis), killed pathogens (pertussis, poliovirus, typhoid fever), attenuated viruses (yellow fever, measles), or components of pathogens (tetanus toxoid, diphtheria toxoid).1 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.

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.1

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.

The Role of Adjuvants

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.

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