TB Vaccines: Global Solutions for Global Problems

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Science  28 May 1999:
Vol. 284, Issue 5419, pp. 1479-1480
DOI: 10.1126/science.284.5419.1479

Tuberculosis remains one of the 10 most important causes of premature mortality worldwide, claiming over 2 million lives every year. In spite of dramatic successes in control of the disease in prosperous countries, geography is an unreliable correlate of protection. Recent sporadic increases in the incidence of tuberculosis (particularly drug-resistant forms of the disease)—most strikingly in New York City—attest to the ability of the causative pathogen, Mycobacterium tuberculosis, to exploit new opportunities for travel and immigration and to seek out the disadvantaged pockets in all societies. Control of tuberculosis must be at the global level, with the best prospects offered by improved diagnosis and treatment together with prevention by effective vaccination (1). The search for an effective tuberculosis vaccine has challenged and frustrated generations of scientists. In their report on page 1520 of this issue, Behr et al. (2) exploit state-of-the-art DNA microarray technology to provide new insights into this longstanding problem.

The current tuberculosis vaccine—the Bacille Calmette and Guérin, BCG—was derived from an isolate of Mycobacterium bovis (which causes bovine tuberculosis) that had been attenuated by laboratory passage in the early years of this century. It has had a chequered history in efficacy trials, providing more than 70% protection in trials in the United Kingdom, no significant protection in South India, and intermediate levels in a range of other studies in different countries. Behr and Small have previously suggested that differences in efficacy might have arisen as a result of changes in the vaccine strain over time (3). Their new study provides further support for this notion, demonstrating that the subculture of BCG in different laboratories resulted in a series of genetic deletions and the evolution of a number of BCG substrains. The authors compared the genomes of M. bovis and contemporary BCG strains with that of a virulent reference M. tuberculosis strain, using comparative hybridization to a DNA microarray. They found that Calmette and Guérin “lost” a 10-kilobase fragment from the M. bovis progenitor strain during the initial process of attenuation. A second fragment was deleted in the late 1920s, and three additional fragments were selectively deleted during subsequent passage in separate laboratories to generate the current diaspora of BCG substrains. Although there is as yet no direct evidence to confirm Behr and Small's hypothesis that these deletions are responsible for changes in vaccine efficacy, the results provide a rational starting point for attempts to generate—or perhaps regenerate—a better BCG vaccine.

These observations represent an important addition to knowledge of BCG deletions originally identified in previous groundbreaking publications (4, 5). A key aspect of the present study is that it provides the first such analysis at a whole-genome level. The authors have used information from the recently completed genome sequence of M. tuberculosis (6) to construct a DNA microarray in which almost every open reading frame is displayed. This has allowed a global analysis of genetic differences between M. tuberculosis, M. bovis, and the various BCG substrains. Almost 100 M. tuberculosis genes were “missing” from all of the M. bovis isolates that were examined. Earlier sequence-based analysis of a limited set of genes demonstrated a remarkably high degree of genetic conservation between M. bovis and M. tuberculosis (7). The new findings suggest that genetic diversity amongst members of the M. tuberculosis complex (comprising M. tuberculosis, M. bovis, M. africanum, and M. microti) may in fact be much greater than previously anticipated, and that gene deletion, rather than point mutation, may be a key source of this variation. Currently, information provided by the microarray approach is “one-way,” identifying M. bovis deletions relative to the framework provided by the M. tuberculosis genome. Sequence analysis of the genome of M. bovis is currently under way (8), and identification of regions present in M. bovis but absent from M. tuberculosis will also be of considerable interest in the context of understanding the evolution of the bovine and human pathogens. As in the case of the BCG vaccines, functional analysis of the “missing” genes may provide important insights into mycobacterial physiology.

In addition to the genotypic analysis described by Behr et al. the microarray approach is also applicable to the study of temporal changes in global patterns of gene expression. This is accomplished with the use of RNA isolated from organisms grown under different conditions and has been successfully applied to the investigation of global changes in gene expression associated with the shift from fermentation to respiration in yeast (9). This approach has also yielded information about gene expression patterns during different growth phases of Haemophilus influenzae and Streptococcus pneumoniae (10). This, however, is only the beginning. As microarrays and their associated technology get cheaper and easier to use, they will become a standard laboratory tool, considered in the same way as other hybridization methods, such as the blotting techniques pioneered by Ed Southern.

Microarrays present a wonderful opportunity for exploring the regulation of gene expression at the level of the whole cell. Operons are a hallmark of prokaryotes and are easy to spot in many bacteria simply by looking at the DNA sequence to identify multiple genes with a common promoter and terminator. However, in some organisms, including M. tuberculosis, such signatures are difficult to find; microarrays probed with RNA or complementary DNA from M. tuberculosis should highlight where a linear sequence of genes is cotranscribed. Survival of successful bacterial pathogens depends on their ability to alter global patterns of gene expression, using “regulons” and “modulons” to coordinate an overall response to the changing environments encountered during infection. Interestingly, Behr et al. (2) report that although none of the open reading frames deleted during attenuation of the BCG vaccine look like classical virulence determinants, there is an overrepresentation of genes classified as transcriptional regulators—both activators and repressors—emphasizing that virulence depends not only on the presence or absence of particular gene products, but also on the way that they are controlled.

Genome-based microarray technology offers an exciting new era in molecular microbiology. As with virulence, however, future success will be determined not just by what we've got, but also by the way that we use it.


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