Failure of TB Vaccine Traced to Lost Genes

The recent rise in cases of tuberculosis, and the anticipation of 80 million more cases in the next decade, is causing people to take a close look at the TB vaccine. The old stalwart BCG vaccine, in use for over 80 years, varies considerably in human trials, and further, it doesn't distinguish between people who have had the disease and those who have been immunized against it. Using DNA microarray technology, Stanford researchers have discovered that the strains used in vaccine production have suffered wholesale losses of genes, which might account for the ineffectiveness of the vaccine. This work, reported in the May 28 issue of Science, could lead to better vaccines and more accurate diagnostics for the disease.

The BCG (or Bacille Calmitte-Guerin) vaccine developed in the early part of the century, is a live, attenuated strain of Mycobacterium bovis. Scientists at the Pasteur Institute in Paris created the vaccine in the early part of this century by culturing a cattle bacterium that resembled the TB bacterium for more than 200 generations. This so-called domestication process of the bovine strain was done to reduce pathogenicity while preserving immunogenicity.

At the time of development of BCG, methods for preserving strains were not available. Hence, the stocks were maintained in culture for some 1,000 additional generations until seed lots of BCG strains were finally made in the 1960s. "The early versions of the vaccine were probably quite good at stimulating the immune system to make defenses against TB," said Peter M. Small, assistant professor of medicine at Stanford University School of Medicine and leader of the research effort. "However, the vaccine has lost this ability in the years since," he said.

The availability of the sequence of the entire genome of M. tuberculosis provided the researchers with a tool for comparing the sequence of the vaccine strains with the virulent tuberculosis strain. Using a whole genome microarray of a reference M. tuberculosis strain, H37Rv, parallel comparative hybridization were conducted with virulent strains of M. bovis, and various BCG daughter strains.

The researchers found that eleven regions, comprising some 91 open reading frames that are present in the reference M. tuberculosis strain are missing from one or more virulent M. bovis strains. In addition, five regions with 38 open reading frames present in virulent M. bovis were missing from some or all of the BCG strains.

Furthermore, the researchers were able to construct a genealogy of BCG strains by comparing the genetic composition of vaccines obtained from different sources worldwide. For example, regions found in the genome of the virulent strain that are absent from all BCGs likely were lost in the original attenutation. On the other hand, some genes that were found to be missing only in particular isolates indicate that the deletion occurred after dissemination of BCG from the Pasteur Institute. The superposition of the genetic profile with the historical record provides a unique use of DNA microarray technology to describe bacterial evolution.

"Bacteria that had lost so many genes probably would not survive outside the lab," Small said. "But it's a pretty cushy life for a bacterium to grow in a laboratory," he said. With every need met, the bacteria can survive without some formerly indispensable genes.

"Knowing what's missing from BCG may help scientists craft a better TB vaccine. But the first application from these results will likely be a more accurate test for TB infection," Small said. Vaccination with BCG is known to stimulate a positive reaction on the most commonly used test for TB infection, the PPD test, making it difficult to distinguish people who have TB from those who have merely been vaccinated against it.

Small's co-authors include Marcel M. Behr, an associate professor of infectious diseases and medical microbiology at McGill University (Montreal, Canada), and Michael A. Wilson, Wendy P. Gill, Hugh Salamon, Gary K. Schoolnik, and Sangeeta Rane, all of Stanford University.

For more information: Peter M. Small, Assistant Professor of Medicine/Infectious Diseases, Stanford University School of Medicine, 300 Pasteur Dr., S-143, Stanford, CA 94305-5119. Tel: 650-725-7908. Fax: 650-725-2395. Email: peter@molepi.stanford.edu.