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MICROBIOLOGY Combo Vector Delivers Blow Against Melanoma Bacterial Construct Makes for Elegant Vaccine For the last four decades, researchers have poked and prodded the bacterium Listeria monocytogenes to discover how it interacts with the immune system, invades cells, robs them of nutrients, and blossoms within other cells, eventually shutting down key bodily functions. Darren Higgins and colleagues have created a vaccine vector system that exploits the best features of live and killed bacterial vaccines. The vector, loaded with a model tumor antigen, prevented the development of melanoma in mice. (Photo by Pam Murray) Building on this knowledge, investigators from HMS and London's Hammersmith Hospital have found a way to use Listeria along with Escherichia coli to fight disease instead of causing it. In the November Gene Therapy, Darren Higgins, HMS assistant professor of microbiology, and Hammersmith co-authors report that by modifying E. coli to express a Listeria protein, they have created a vaccine that protects mice against melanoma. The study, conducted at the Molecular Oncology Unit of Cancer Research UK at Hammersmith, is the first in vivo test of this new vaccine approach developed by Higgins and colleagues. Breaking and Entering In a 1999 study, published in Molecular Microbiology, Higgins and collaborators at the University of California, Berkeley, showed that they could deliver proteins to the cytosol of macrophages using an E. coli vector expressing the Listeria protein listeriolysin O (LLO). LLO's natural function is to lyse phagosomes in infected cells, allowing the bacterium to enter the cytosol. To create the vector, they stripped out the virulence components from killed E. coli, leaving a framework that remains attractive to the macrophages and dendritic cells at the front lines of the immune system. In the current study, prior to injecting the killed bacteria into mice, they added two proteins to the E. coli shell: LLO to lyse the phagosome and ovalbumin, which served as a model tumor antigen. Then they also injected the mice with a strain of melanoma tagged with ovalbumin. In the animals, the killed E. coli cannot replicate and are quickly digested by patrolling macrophages or dendritic cells. Once inside, the Trojan horse E. coli is taken within a phagosome to be destroyed. But unlike the case with normal E. coli, the vector's LLO subsequently lysed the phagosome, spilling its contents into the cytosol. This LLO-mediated lysis frees ovalbumin to travel to the cell surface, where it is presented via the MHC class I pathway. As an antigen, ovalbumin tells T cells what to search for and eliminate within the body--in this case, the melanoma cells coded with ovalbumin. The image at left shows a macrophage with a fluorescent protein delivery vector inside its phagosome. With the background light removed (middle), the phagosome is clearly illuminated. At right, the phagosome is lysed and its fluorescent contents pour into the macrophage cytosol. (Image courtesy of Darren Higgins) The vaccination resulted in complete protection in six of eight mice, which remained tumor-free for more than 90 days, and a significant delay in tumor growth in the other two. By contrast, all mice in the control groups had tumors that reached maximum volume within 16 days. "The results of this study are very positive," said Higgins. "They suggest we could utilize this killed bacterial formulation to prime the immune system against diseases like cancer or viral and bacterial pathogens." Broader Use "We are now moving toward the insertion of numerous pathogen-specific antigens into the vector to elicit protective responses," he added. The technique was first conceived as a fast method for identifying new cytotoxic T cell antigens to prime the immune system. By screening genomic libraries expressed in the E. coli- LLO vectors, Higgins and his colleagues identified the antigens of intracellular pathogens that are most likely to generate an effective immune response. Then they put the antigens into vectors that would channel them into the correct presentation pathway in human cells without having to infect cells with the whole dangerous organism. This method sidesteps a central dilemma in vaccinology: live attenuated vaccines are more effective than dead or protein vaccines, but they may cause disease. The new approach is an attempt to give identified protein antigens the efficiency of whole pathogens but not the risk. The vector is now being tested against several additional infectious disease models. In addition, the researchers continue working to develop their technique as an antigen-screening system. --John Lacey and Tom Reynolds