Reverse vaccinology for group B strep

Strategy results in successful mouse vaccine, and finds that bacterium contains pili structures

By | July 5, 2005

In a finding that further validates an emerging vaccine strategy, researchers in Italy and the United States have worked backward from genome to antigens to identify a protein cocktail that may confer global protection against group B streptococcus (GBS). The process, reported in this week's Science, also revealed that GBS contain pili structures on their surfaces, according to a Brevia in the same issue.

This discovery represents "one of the important ways in which the promise of genomics can be harnessed, in terms of developing rational and high-throughput screens for novel vaccines," said Victor Nizet of the University of California, San Diego, who did not participate in these studies. "This is a global, high-throughput approach that has no preconceptions or biases about the proteins," he said.

GBS is a particularly complex organism with nine different serotypes, Nizet said. The researchers compared the genomes of eight bacterial samples that represent the five most important disease-causing serotypes, according to the report. They used a computer algorithm to identify genes that were likely to be surface-exposed and successfully expressed 312 of these in E. coli. They then immunized adult female mice with these proteins, mated the mice, and challenged their pups with usually lethal doses of GBS. The ideal candidates would be "highly immunogenic and also broadly conserved [across strains]," said Nizet. Four antigens stood out that significantly increased their chances of survival.

As expected, the immunogenic success of individual antigens depended on whether or not the antigen was conserved in the mouse's challenge strain, the report said. Effectiveness was also correlated with the amount of surface expression, as revealed by in vitro fluorescence-activated cell sorting analysis.

Only one of the four targets–the previously identified Sip antigen–was found in all the strains. But when these four antigens were used in combination after challenge by 12 different strains representing all nine serotypes, 59% to 100% of the pups were protected. In vitro, the bacteria were killed more efficiently–through opsonophagocytosis–by the antibodies of mice exposed to the four-protein combination than to those corresponding to individual antigens.

"If you want to think about developing a vaccine with global coverage," vaccine study coauthor Lawrence Paoletti of Brigham and Women's Hospital in Boston, said, "you want to find a set of antigens, like the four we studied, that would give broad coverage."

In the past 30 years, GBS vaccine efforts have focused on the bacterium's capsular polysaccharide antigens, said Paoletti. When these antigenically distinct targets were found to be less immunogenic than they had hoped, researchers successfully conjugated them to proteins that would help provoke the immune system into action; several of these conjugates are now in phase 1 and 2 clinical trials.

This conjugation tactic is now being used on the protein targets identified in the study to develop a vaccine with a more global scope, using what Guido Grandi of Chiron Corporation in Siena, Italy, coauthor of the study and the Brevia, called "reverse vaccinology." Grandi and his colleagues at Chiron first developed this approach against the bacterium that causes cerebrospinal meningitis.

While the function of the four antigens described was not previously known, the Brevia reported that three of them are part of a previously undetected pilus-like structure extending from the GBS bacterium. Immunogold microscopy revealed two of the antigens–GBS80 and GBS104–on these long, skinny structures coming out of the bacterial wall. Furthermore, they found that two sortase genes on the same operon as the genes for these antigens are required to correctly assemble the structures.

According to Olaf Schneewind of the University of Chicago, who did not participate in these studies, it has long been known that pili help other bacteria adhere to eukaryotic cells. "The conclusion [here] is that these pili are important in pathogenesis. The principle is very much a fishing rod," he said.

"A vaccine antigen is doubly attractive if it's a surface expressed protein and plays a role in virulence," Nizet said, since antibodies have the potential to both recruit other immune system elements and directly inhibit pathogenesis.

Paoletti said that they would now focus on "understanding ways of optimizing the immune response" and how antigens are expressed differently under various environmental conditions.

Dele Davies of Michigan State University in Lansing, who did not participate in these studies, said that more work needs to be done to understand the differences in immune response between serotypes because "it's conceivable that there's something about the sugar coating that prevents the vaccine from getting at it."

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