When law enforcement agents get a search warrant to impound a suspect’s documents before he can shred them, nowadays they also confiscate his computer hard drive. This electronic memory bank has its counterpart in human blood. That life fluid’s white cells record all past encounters with infectious antigens, and the resulting memory antibodies are evidence.
So it was that in recent months immunochemist Kim Janda, at the Scripps Research Institute in La Jolla, Calif., drew blood from 20 healthy human volunteers. He was looking for signs that these donors had at some time in their lives met up with and repelled a relatively harmless bacterium, Bacillus subtilis by name. It is the prototype of a dozen Bacillus species, of which B. anthracis is now notorious as the inflicter of anthrax.
Besides taking the lives of about five Americans, and sickening many more, bioweaponized anthrax spores have disrupted the U.S. Postal Service, wreaked ongoing societal havoc and pose a grim threat of random terrorist sorties or all-out biowarfare.
Members of the Bacillus genus, being soil bacteria, form spores to preserve their lifestyle. When nutrients are scarce, the germs roll up into rock-hard, spherical packets, 1.5 microns in diameter. Thus ensconced underground, or in the hairy skins of the domestic animals they infest, the spores avoid environmental hardships, from temperature extremes to man-made disinfectants. When times ease up, the spores explode into life again and go about their rampages infecting man and beast. (See BioWorld Today, March 6, 2002, and Oct. 16, 2001, both p. 1.)
Janda, an endowed professor of chemistry at Scripps, is senior author of a paper in the latest Proceedings of the National Academy of Sciences (PNAS), dated April 16, 2002. It’s titled: “Human antibodies against spores of the genus Bacillus: A model study for detection of and protection against anthrax and the bioterrorist threat.”
B. subtilis Leads Bacillus Laundry List
B. subtilis makes an ideal research whipping boy for B. anthracis performing all its sporogenic strategies while avoiding the danger of anthrax infection.
The Scripps group tested a dozen strains of Bacillus, to wit: B. subtilis, B. licheniformis (two strains), B. cereus (two strains), B. megaterium, B. pumilus, B. polymyxa, B. circulans, B. sphaericus, B. globigii and B. thuringiensis.
“Our PNAS paper reports,” Janda told BioWorld Today, “that we could find antibodies able to provide us with signatures for spores. That is, they could differentiate between classes of Bacillus spores.
“Typically, what people do to obtain antibodies,” Janda pointed out, “is immunize a mouse or rat or rabbit with the particular antigen, and get monoclonal antibodies that way. We didn’t need to immunize them. We just took this na ve library for the human volunteer’s blood, and made the antibodies. The novel strategy, which no one had done before, was to screen against whole live spores vs. just the toxin they release, which people have always focused on.
“What we were trying to do, using the phage-display monoclonal antibodies” he added, “was see if there were different signatures for each one of these spores.” He explained: “Your signature for you would be as an individual, whereas my signature would be for myself like handwriting.
“We panned against whole spores,” Janda recounted. “That is, we displayed antibodies on the surface of a bacteriophage. It’s like sifting for gold. In a river you have a panful of water, and you sift through it. You search up the gold dust and nuggets, and everything else stays behind. That’s similar to what we did.”
Janda continued: “We affixed the antigen of interest in this case, the bacterial spore onto a plate, and ran a phage library across it. The ones that stuck, stayed behind; the others washed out. We can quickly differentiate and tell you whether we have a spore there or not, determine what particular class of spores it comes from and what the spore potentially is. What makes this technique significant,” he went on, “is the speed and the ability to quickly ascertain what we have there. Also sensitivity; we could detect just one spore.
“The single-chain antibody, which we ourselves constructed,” Janda related, “is just a smaller version of Immunoglobulin G. It’s more useful with regard to potential human therapy because it doesn’t have the large, normal size and can be cleared quickly from the blood. So its active and constant sites instead of 150,000 molecular weight total only 25,000.”
Snaring One Single Spore In A Zillion
To single out that one spore among many millions, Janda and his co-authors affixed a fluorescein molecule to their antibodies in solution. Fluorescent microscopy revealed the bright green, glowing marker that quickly determined whether a powder sample contained any spores.
To simulate the mysterious powder-laced envelopes that bedeviled the anthrax scare, the team bound antibody-phage to a selection of half a dozen common powders of similar consistency wheat and green bean starch, tooth whitening paste, yeast, baking powder and after-bath baby powder.
Janda pictures his biopanned human antibodies as an anti-anthrax vaccine. “It could be a passive vaccine,” he observed, “so the monoclonal antibodies obtained from panning could then be grown up in large quantities, and injected into people. Because they are already human, we wouldn’t have to humanize them. It’s not a bad thing,” he commented, “because you could keep the antibodies circulating for weeks, so the vaccine would be potentially useful for going into combat.”
However, he and his team have moved on to other things. “We are now working on a different biological threat, so we’ve set this vaccine project aside,” Janda said. This study was done a year and a half ago, before 9/11 or any of that stuff,” he noted, “but I couldn’t publish it because one of the government agencies that funded us had to give us clearance first.
“If someone approached me now,” he pointed out, “we could go back and look at this again. The methodology we developed would be applicable to any type of spore that we’re interested in. Yes,” Janda concluded, “we could develop an anthrax vaccine if we wanted to.”