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Research holds up genomes and finds lots of holes
A new series of genetic analyses of BCG holds out hope that scientists will someday understand what’s so baffling about the bacille of Calmette and Guerin: That is, why did it perform well in one trial, so badly in another, and at middling points in the rest?
Researchers recently announced they had found that in its journey through time, the BCG vaccine or, more precisely, the vaccine’s 13 "daughter" strains lost lots of genetic baggage.
Whether something important and protective was tucked into the missing bags isn’t certain, says Marcel Behr, MD, assistant professor of medicine at McGill University in Montreal. But clearly, some baggage has been lost.
For example, Behr found that BCG-Pasteur — the strain maintained at the Pasteur Institute in France — has undergone four genetic deletions, on four occasions, between 1908 and 1961. Shortly thereafter, a way to freeze-dry the strain was perfected, a development which presumably put an end to subsequent large-scale losses of genetic material.
Extrapolating from that kind of evidence, Behr says most "younger" daughter strains that existed earlier in time would have lost less genetic material than their "older" sibling strains used in later trials.
For example, it’s known that one of the daughter strains used in the American Indian trial, where the vaccine performed excellently, also was used in Chingleput, where the vaccine disgraced itself. "We know the BCG-Pasteur strain of the 1930s had changed by the time the Chingleput trials took place," Behr says.
As for the "mother" strain, it has vanished in a fire. Little can be said for certain about this departed family matriarch, Behr adds. "You can read the studies, but the way studies were carried out in the 20s, 30s, and 40s is not the way they are done today. And in some ways, even the people who got the vaccines in the 30s and 40s are different from people today."
Working with colleagues, among them Peter Small, MD, assistant professor of medicine in the division of infectious diseases at Stanford University Medical Center, Behr accomplished two notable feats in his latest round of work on BCG. First, he compared the genome of BCG to that of M. tuberculosis; second, he compared the 13 daughter strains available today with M. bovis, the member of the complex that was attenuated to form the original culture.
In their comparisons, the researchers turned up many differences: genes present here but missing there, genes absent here but present there. "We’ve only shown changes in some of the 13 BCG strains," Behr adds. "But one can only suspect that each strain has undergone its own evolution."
If true, that wouldn’t necessarily cancel the long-cherished supposition that cross-reactions from ambient tropical mycobacteria make BCG lazier, if you like, as it draws closer to the equator, the so-called "southern hemisphere/northern hemisphere disconnect." It only sets forth a second hypothesis, this one backed by a new study’s evidence.
To bring about BCG’s genetic evolution, two kinds of forces were probably at work, Behr says. On one hand, the bug may have adapted passively to laboratory conditions; on the other, active pressures from manufacturers and consumers of the vaccine may have forced certain genetic changes.
One variety of active forces known to have been at work was the demand for a vaccine that reliably converts the tuberculin skin test. Why hope for an outcome that renders the skin test incapable of saying clearly whether or not recent infection has occurred? Well, says Behr, because tuberculin reactivity, along with relative lack of virulence, are the two traits prospective buyers got in the habit of wanting in their BCG vaccines.
BCG batches that failed the tuberculin reactivity test could be counted on to be returned to their manufacturer, perhaps with a nasty note enclosed; that meant the pressure was on to make sure that whatever else it might do, a BCG vaccination would cause a skin test to react positively.
The same goes for a tolerable level of virulence: Countries that found their BCG shipments provoked big suppurations on the arms of recipients typically sent back their products and asked for a refund. The end result, Behr adds, is a bad joke of a vaccine — one that usually messes up the tuberculin skin test and is mild-acting enough to offer what appears to be little protection.
On what he calls a "micro" level, individual labs probably also tweaked their seed lots in various ways. For sure, scientists are known to have fiddled with one strain to make it less apt to die in the process of being freeze-dried. Did that alter the protective ability of the lots? "There’s no way to know," he says. "All we can say is they were selecting for mutants that were more tolerant of being frozen and dried."
The genetic variations Behr and Small turned up suggest a number of starting points for how BCG might be restored to its best-ever levels of usefulness, which, after all, have never been measured at more than somewhere between 70% and 80%, Behr adds. The work also suggests places in the genome to go looking for genes for virulence and attenuation. "By learning about the differences, we also might find out whether one of the currently available BCG strains is better than the others," he says. "So we could at least recommend using the best BCG."
Behr acknowledges his indebtedness to the scientists at the Pasteur Institute, who carried out the mammoth genome-sequencing project and then shared it with the world. He also pays homage to Stanford researchers who devised the technology for the DNA micro-array.
"When you do a Southern blot," he explains, "you take a couple of micrograms of DNA and you ask, Is this gene present or not?’ But when you make a micro-array, you make 5,000 small [representations], one for each gene of the genome, and ask: Which of these 3,924 genes is absent? Which is present?’"