By David N. Leff
Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.
For tuberculosis (TB) - the world's leading cause of death from a single infectious organism, Mycobacterium tuberculosis - the only vaccine extant has its drawbacks.
It was developed early this century at the Pasteur Institute by two French bacteriologists, Leon Calmette (1863-1933) and Camille Guirin (1872-1961). Their vaccine, named BCG ("Bacille de Calmette-Guirin") works, when it does, on the same principle as smallpox immunization, which is based on an attenuated live pathogen from bovines infected with cowpox.
Cattle acquire TB from the bovine bacillus, Mycobacterium bovis, which - like cowpox - provided a non-virulent vaccine target against the human disease. In fact, in the years before BCG, physicians advised their tubercular patients to move into the cowshed and inhale the breath of cows.
To make their BCG vaccine safe against infection but efficacious for immunization, the two Pasteur scientists rendered their M. bovis strains avirulent by passaging them from one culture dish to another 230 times between 1908 and 1921. Some of these were lost during World War I, but afterwards, the first recipients to be inoculated with the new vaccine were calves. However, by 1928, 116,000 French children had been vaccinated, with equivocal results.
In subsequent worldwide trials, BCG's efficacy varied wildly, from 70 percent in the U.K. to zero in South India.
Today, the World Health Organization recommends the vaccine for under-developed countries where TB is a major menace. It currently infects two billion people worldwide, and kills more adults each year than AIDS, malaria and tropical diseases combined.
In prosperous nations such as the U.S., TB has been kept at bay until very recent years by expensive antibiotics, so BCG vaccine, while licensed, is rarely used. Its main employment is as a skin test to determine if an individual has antibodies against the organism.
In recent years, the wily Mycobacterium tuberculosis deploys resistance to each new antibiotic as it comes along, and the disease is making a comeback in American inner cities. Hence, experts are taking a fresh look at the spurned BCG vaccine to see whether and how it might be remobilized as multi-drug resistance overcomes the beleaguered TB-specific antibiotics. (See BioWorld Today, March 3, 1995, p. 1.)
The latest such effort is a report in the current issue of Science, dated May 28, 1999. It bears the title: "Comparative genomics of BCG vaccines by whole-genome DNA microarray." The paper's lead author is bacteriologist M. A. Behr, at McGill University in Montreal.
As the Science co-authors point out, "[T]his live vaccine required continued passage, eventually resulting in a profusion of phenotypically different daughter strains that are collectively known as BCG." They added that by the 1960s, "these vaccines had been separately propagated through about 1,000 additional passages." It was high time for the real BCG to stand up - or be recreated.
Last year, Mycobacterium tuberculosis became the 15th organism to have its genome totally sequenced. At 4.40 megabases (mb), the bulky bacillus comes in right behind Escherichia coli's 4.60 mb. It was that newly revealed panoply of the pathogen's gene complement that the McGill team exploited in their microchip array. It comprised 4,896 spots, representing 3,902 (or 99.4 percent) of the organism's 3,924 open reading frames.
They comparatively hybridized the many and varied contemporary BCG strains, plus the genome of M. bovis, with the newly sequenced M. tuberculosis gene endowment, as a virulent reference. In the process, they reported that during Calmette and Guirin's 230 initial attenuation passages, they had "lost" a 10-kilobase fragment of their original B. bovis strain. Four more fragments have been deleted since the vaccine went public in the 1920s.
An editorial accompanying Behr's paper concludes: "[T]he results provide a rational starting point for attempts to generate - or perhaps regenerate - a better BCG vaccine."
Electrical Vector Galvanizes Mouse Muscles Into Gene Therapy Sites For Prolonged EPO Expression
Recombinant erythropoietin (rEPO) - one of biotechnology's clinical and commercial triumphs - has two prime customers, one licit, the other illicit. Lawful users are patients on kidney dialysis, a blood-cleansing process that shears red blood cells and causes anemia. Clandestine consumers are athletes, who shoot up the product as an energy-enhancing blood booster.
Patients on dialysis need several EPO injections a week to bolster their hematocrit - the serum level of red blood cells needed for wellbeing. If gene therapy could release the body's own EPO as demanded by declining hematocrit, this would spare kidney-failure sufferers one added expense and inconvenience.
An Italian proof-of-principle study in vivo describes an unusual gene therapy approach that supplied mice with hematocrit-sustaining rEPO for at least six months. The paper, in the current Proceedings of the National Academy of Sciences, dated May 25, 1999, is titled: "Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation." Its senior author is molecular biologist Elena Fattori, at the Molecular Biology Research Institute in Rome.
The co-authors' gene delivery construct consisted of the complete murine EPO coding region harnessed to a cytomegalovirus promoter. They injected this package into surgically exposed thigh muscles of mice, then positioned ultra-thin steel electrode wires parallel to the muscle fibers. These delivered one-second pulses of high-frequency, 45-volt electrical stimulation every other second.
In the course of their in vivo experiments, the team demonstrated that, unlike viral gene-delivery vectors, the electro-muscle route produced no immune reaction. Moreover, readministration 56 days after the initial treatment led to higher hematocrit levels than in the single-session-only animals.
"The 100-fold improvement in muscle transduction over naked DNA injection alone," their paper stated, "makes muscle electroporation one of the most efficient methods of nonviral gene delivery described so far [and] provides a potentially safe and low-cost treatment for serum protein deficiencies."