Bronx, NY, Oct 28, 2003 -- In a finding that could lead to better methods for preventing tuberculosis, researchers at the Albert Einstein College of Medicine have helped to solve a nearly century-old medical mystery: What caused the bacteria in the TB vaccine to become weakened and therefore safe for human use? Their findings, published in the October 14th issue of the Proceedings of the National Academy of Sciences, could lead to a better vaccine for treating TB, which kills an estimated two million to three million people worldwide each year. Dr. William Jacobs, the study’s senior author, is a Howard Hughes investigator and professor of microbiology and immunology at Einstein. In the early 1900’s, French scientists Léon Calmette and Camille Guérin were working with Mycobacterium bovis, the major cause of TB in cattle and other animals—and capable of infecting humans who drank the milk of TB-infected cows. They wanted to produce a weakened form of M. bovis that might serve as a TB vaccine. So they grew successive generations of M. bovis in culture media and tested each new generation on guinea pigs to assess toxicity. After the 39th generation, or “passage,” the researchers noticed that M. bovis suddenly was no longer fatal to guinea pigs. This weakened, or attenuated, strain of M. bovis formed the basis for the Bacillus Calmette-Guérin (BCG) vaccine used ever since for vaccinating people against TB. Some three billion doses of this “live” vaccine have been administered over the years. But until now, the reason for the attenuation of M. bovis has remained elusive. Dr. Jacobs and his colleagues worked with M. tuberculosis, which causes human TB and is closely related to M. bovis. They wanted to identify the genetic changes that cause mycobacteria to loss their virulence and to learn how those mutations actually weakened the microbes. Earlier work had identified a region of 10 genes, known as RD1, that is normally present in both M. bovis and M. tuberculosis but is absent from the BCG strain. Focusing on genes in this region, the researchers made a series of “targeted” gene deletions in normal M. tuberculosis bacteria and then (after infecting mice with the mutant bacteria and adding the bacteria to human lung cells in tissue culture) observed how each mutation affected the toxicity of M. tuberculosis. The study proved conclusively that deletion of the RD1 region causes attenuation of M. tuberculosis (and, by extension, of the closely related M. bovis). In addition, the researchers identified how this weakening occurs. Virulence, they found, depends on three RD1 genes, all involved in producing or secreting a protein that destroys lung cells that the bacteria have infected. Deletion of one or more of these genes meant that infection caused much less tissue destruction—explaining the sudden loss of toxicity that Calmette and Guerin observed nearly 100 years ago. After creating the targeted mutations of M. tuberculosis, the researchers injected them into mice that were then exposed to virulent M. tuberculosis bacteria. These M. tuberculosis vaccines protected the mice against toxic effects of infection in a manner similar to the BCG vaccine. “We’re hopeful that learning why tuberculosis bacteria become attenuated will lead to better TB vaccines that can save more lives,” says Dr. Jacobs. In addition to Dr. Jacobs and his Einstein colleagues, other researchers involved in this study were from the University of California, Los Angeles; the U.S. Food and Drug Administration; and the Emory School of Medicine.