Study Questions Need for Potential Vaccine Additive

By Jeff Worley

A team of scientists at The Scripps Research Institute has published a study that questions the need for incorporating an ingredient—TLR ligands—in vaccines to increase their effectiveness. Excluding TLR ligands would help keep down manufacturing costs and would avoid this ingredient's potential side effects, such as inflammation and autoimmune syndromes.

The study appears in the December 22 issue of Science.

In the new research, the Scripps Research team discovered that mice lacking all Toll-like receptor (TLR) signaling still respond robustly to antigens, foreign substances that cause the immune system to mount specific immune responses. These findings suggest that TLR ligands, currently a major thrust of commercial and basic science vaccine research, may be excluded from vaccine adjuvants—those ingredients added to make the vaccine more effective.

If these results are confirmed, the study could redirect the course of future research. "One conclusion that could be drawn from our study is that perhaps in the future we need to put less emphasis on stimulating Toll-like receptors and spend more time trying to understand and characterize other pathways," said Assistant Professor Amanda Gavin of the Scripps Research Department of Immunology who led the project, which was funded by the National Institutes of Health.

Toll-like receptors belong to a family of proteins in the innate immune system—the body's first line of defense against foreign pathogens—that can detect parts of microbes and are critical in providing protection against microbial infection.

"Many researchers believe that the innate immune system is critical for initiation of the adaptive immune system, which involves T cells and B cells," said Gavin. "While that is true, our work shows that you can generate good antibody responses to a T cell-dependent antigen without Toll-like receptor signals."

Prior to this study, researchers had known that TLR ligand recognition leads to the activation of transcription factors that drive cytokine expression, proliferation, survival, and inflammatory mediator expression. Cytokines are proteins secreted by lymph cells that affect cellular activity and control inflammation. It was also well established that TLR signaling is initiated by four adapters: MyD88, TRIF, TIRAP/Mal, and TRAM. MyD88 also contributes to signaling in B cells, and is required for maximal B-cell responses to foreign proteins when present in the vicinity of TLR ligands.

Mice genetically deficient in both MyD88 and TRIF (MyD88/TRIF double mutant mice were identified by Scripps Research investigators Kasper Hoebe and Bruce Beutler) have a complete lack of known TLR signaling, a condition that allows TLR dependence of antibody responses to be assessed. "We took advantage of this to explore more precisely the role of TLR signaling in antibody responses to immunization and the augmenting roles of adjuvants in this response," explained Gavin.       

Her team mixed a protein, an experimental T cell dependent antigen, with one of four typical adjuvants—alum, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and "Ribi" adjuvant—and injected it into groups of four to six mutant mice. Four groups were given one of the enhanced proteins; a fifth group, the control, was given the protein and saline solution. The mice were tested every seven days for two weeks to a month to see how much antibody the animals were making against the protein.

The most surprising finding from this study was that the mutant mice with MyD88 and TRIF deficiencies were able to make all of the antibody types that wild-type mice make, and in similar amounts.

"We thought that, especially in the case of complete Freund's adjuvant, an oily compound with heat-killed Mycobacterium tuberculosis [a pathogen known to cause tuberculosis in mammals], the mice without Toll-like receptor signaling would not be able to respond," Gavin said. "We believed the mycobacterial component would be very strong and dramatic, and that there would be a significant difference in the antibody response between the wild type and the mutant mice."

To the contrary, the mutant mice made a variety of antibodies as robustly as the wild type mice in the control group.

David Nemazee, a professor in the Department of Immunology at Scripps Research who was a member of the research team, said, "Often in science, attractive ideas grow to become 'common sense' before being rigorously tested. A paradigm had emerged, based on less direct evidence, that TLR signaling is critical to getting antibody responses going. This led to concepts in vaccine design incorporating TLR ligands as adjuvants. Amanda's work shows that TLR ligands are a minor and dispensible component of adjuvants. The importance of this work is that it will redirect the field to elucidating TLR independent pathways of adjuvant action. This may have direct consequences for human health if major improvements in vaccine efficacy result."

In addition to Gavin and Nemazee, other authors of the paper, "Adjuvant-enhanced Antibody Responses Occur in the Absence of Toll-like Receptor Signaling," include Kasper Hoebe, Bao Duong, Takayuki Ota, Christopher Martin, and Bruce Beutler. See the journal Science at: http://www.sciencemag.org/cgi/content/summary/314/5807/1859a.

Send comments to: mikaono[at]scripps.edu

Immunologists Amanda Gavin (left) and David Nemazee have pubished a paper in Science that may redirect future research on vaccines. Photo by Kevin Fung.