Jacobs’ shuttle system delivers jumping genes
Lots of bright stars in vaccine constellation
Gone are the days when vaccine development was a faint star on the horizon. Lately, it’s hard to turn the page of a journal or newspaper without seeing a report of another important finding in the field. Last month, much of the ink described research results from the lab of William R. Jacobs, Jr., PhD, associate investigator at the Howard Hughes Medical Institute of the Albert Einstein College of Medicine in the Bronx.1
There’s good reason for the commotion about vaccines in general, says Ann Ginsberg, MD, PhD, program officer for tuberculosis, leprosy, and other mycobacterial diseases in the Division of Microbiology and Infectious Diseases at the National Institutes for Health in Bethesda, MD.
"It shows the broader public health community, not just the basic research community, is beginning to feel a sense of urgency," says Ginsberg. Directly observed therapy, short-course, "is the best tool we have now, but it’s not doing the job adequately and we don’t want to be stuck with it 20 or 30 years from now."
In September, the International Union Against Tuberculosis and Lung Disease devoted the last two days of its Paris conference looking at current developments in the field; and last month, the topic of vaccine development was allotted a full day at the Centers for Disease Control and Prevention’s Advisory Council for the Elimination of Tuberculosis (ACET).
Researchers are tackling the vaccine challenge from a variety of angles.
Jacobs and his colleagues including Barry R. Bloom, PhD, also of Howard Hughes Medical Institute, and Graham F. Hatfull, PhD, of the University of Pittsburgh have compiled a mountain of research in the field of transposon mutagenesis.
Jacobs has found a way to insert transposons a sort of jumping gene, or piece of moveable DNA into the tuberculosis mycobacterium, without destroying it in the process. In crafting a delivery system for inserting transposons at random, Jacobs has arrived at a technique that will help him mutate each gene in the mycobacterium, eventually resulting in a complete library of mutated genes.
That "library," in turn, holds enormous significance: It can be used to determine what genes in the TB bug are responsible for virulence. Once researchers possess that key piece of knowledge, they will be on the road to making the ideal vaccine for TB that is to say, a TB bug that has had its virulent genes deactivated but maintains the ability to induce full immunity in the host.
"This is a major advance for the TB research community," says Anthony M. Fauci, MD, director of the NIH’s National Institute of Allergy and Infectious Diseases.
Jacobs’ delivery vehicle is a mycobacteriophage, a virus that infects M. tuberculosis and other related mycobacteria. In this case, Jacobs’ phage was equipped with a mutation that prevents it from replicating at a specific temperature: 37 degrees Celsius. In addition, the phages that were used carried a transposon containing a gene that confers resistance to the antibiotic kanamycin.
Jacobs took the phages, mixed them with M. tuberculosis, and incubated the mixture at the non-replicating temperature of 37 degrees Celsius.
Next, to see whether the process had worked, he attempted to grow out the infected mycobacteria in culture media that contained kanamycin. He was able to recover thousands of MTB mutants, all of them obviously resistant to kanamycin. That was proof that the process had worked as planned.
The same delivery vehicles, known as conditionally replicating shuttle plasmids, can now be used to mutate every gene in the tuberculosis mycobacterium, says Ginsberg. Colonies can be grown up from the separate mutations, and DNA isolated from the colonies. By examining the DNA, it will be possible to find out where the transposon has inserted itself.
The labor-intensive part of the process will be figuring out what function each mutation performs, she adds.
Ideally, the research community will go at the project in a collaborative fashion, with every lab taking a share of mutations and assaying them. "It’s far more than any one lab can do," she adds. Some assays for function presumably will be performed in vitro under certain conditions; others will involve animal models.
Using new "chip" and micro-array technologies, it will be possible to look for every mutant and determine which ones get switched on or off under a variety of conditions, Ginsberg says. Switching off the virulent genes can be accomplished by using another technique called allelic exchange. It too has been pioneered by Jacobs, working in this instance with researchers at France’s Pasteur Institute.2
That exchange process, which is sometimes dubbed "knockout mutagenesis," will let researchers selectively mutate any sequence they like, says Ginsberg. "If you figure out that Gene X or, as is more likely, a handful of genes are virulent but at the same time aren’t important in inducing protective immune response, then you can imagine producing a vaccine where you knocked them out." The result, she says, would be "the perfect vaccine."
On the other end of the host-pathogen equation, preliminary studies are also under way among several groups of researchers who are looking at which genes in humans make them more or less susceptible to TB, says Ginsberg.
Twin studies, for one, tend to support the idea that a genetic component is at work, Ginsberg says; so does everyday experience.
"Anecdotally, it’s clear that some humans can be exposed regularly to TB and not get sick," she says. "Others are exposed to a few organisms, and they get sick."
Most likely, more than just a single gene will eventually be implicated. Researchers looking for candidate genes often search out families whose members seem especially prone to getting TB; there, the starting point is to identify polymorphisms or differences between one person’s DNA and another’s at a particular site, says Ginsberg. As with other diseases, such as diabetes, that seem to be associated with a complex of genes, the variations "can be used as a marker, since the trait travels with that marker even though the group doesn’t necessarily [include] the relevant gene," she adds.
References
1. Bardarov S, Kriakov J, Carriere C , et al. Conditionally replicating mycobacteriophages: A system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci USA 1997; 94:10,961-10,966.
2. PelicicV, Jackson M, Reyrat, J-M, et al. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 1997; 94:10,955-10,960.
You have reached your article limit for the month. Subscribe now to access this article plus other member-only content.
- Award-winning Medical Content
- Latest Advances & Development in Medicine
- Unbiased Content