In Mice, CpG Outstrips Classic Vaccine Adjuvant 5-Fold; Clinical Trial Coming So

DNA Motif Gooses Immune Antigenicity Response

In Mice, CpG Outstrips Classic Vaccine Adjuvant Five-Fold; Clinical Trial To Begin Shortly

By David N. Leff

Plumbers may not rank highest on the blue-collar totem pole, but their stock in trade - pipes, tubes and hoses - propels the lifeblood of modern machinery, from cars to space shuttles.

These cylindrical conduits are lined for smoothness, to minimize friction between their inner surface and the fluids that flow along them. The same is true of the epithelial and endothelial cells that line the lumens of tubing, from capillaries to colon, which deliver the human body's blood, air and nourishment.

The mucous membranes that line these internal tubular surfaces cover some 200 times more area than the skin that encloses the outer body itself. If spread out in a single seamless sheet, a body's total mucous membranes might pave one-and-a-half standard tennis courts.

But there's much more to mucous membranes than mere inner linings. For one thing, they are happy hunting grounds for infectious pathogens. And they are, by that token, powerful ramparts of the body's immune defenses.

So, it would seem that vaccines administered by mouth or nostril should be a natural for clobbering infection. Standing in the way of this logical strategy is the fact that most antigenic vaccines don't work well, if at all, when administered naked to the mucous membrane (the oral polio vaccine being an exception).

It takes a vaccine adjuvant - like a seeing-eye dog guiding its blind master - to make mucosal immunization a practical proposition.

"Adjuvants have been developed that work for mucosal vaccination," observed immunologist Arthur Krieg, a professor of internal medicine at the University of Iowa, in Iowa City. "One of the prime examples is cholera toxin [CT]. But," he pointed out, "the problem is that CT is very toxic. Even a tiny dose is enough to make a human being very ill. CT does, however, work well in mice - in moderate doses. Detoxified versions of CT don't work as adjuvants any more, at least not well enough."

Four years ago, the Iowa immunologist discovered in E. coli's bacterial genome DNA sequences that apparently get around CT's Catch-22. Their active ingredient consists of two bases, cytosine and guanine, connected by a phosphate bond. So, Krieg christened this molecule CpG - cytosine-phosphate-guanine.

He and his colleagues determined that the immune system detects invading bacteria and viruses not only by their protein coats - classical vaccine targets - but by immunostimulatory motifs in their microbial DNA, notably CpG. "Its mode of action," he explained, "is based on a receptor in the immune-system cell that detects foreign DNA and scans it to see if it has this pattern of bacterial DNA. If it does, that triggers an alarm signal that sets off a cascade of protective immune responses."

The university has patents pending on the technology Krieg developed, and licensed them to a virtual company he created on campus, called CpG ImmunoPharmaceuticals Inc. (See BioWorld Today, Oct. 15, 1997, p. 1.)

This year the firm moved its headquarters to Wellesley, Mass., and acquired a president and CEO, Robert Bratzler. Krieg serves as chief scientific officer. CpG's director of vaccine development is vaccinologist Heather Davis, a professor at the University of Ottawa.

Davis is senior author of a preclinical research report in the current issue of the twice-monthly Journal of Immunology, dated Nov. 1, 1998. Under the fast-track rubric "Cutting Edge," it's titled: "CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice."

In Vitro Trial Proved CpG's Enhancement Power

Inhalation of the viral antigen alone failed to induce antibodies in any of 15 mice tested. But all animals raised immunoglobulin G antibodies to the hepatitis protein when either CpG or CT was added to the antigen as an adjuvant. And a second booster dose of these enhanced vaccines showed CpG yielding 500 times the initial antibody titer, versus less than 100 times in the CT.

"The desirability of mucosal vaccines has long been recognized," Davis commented, "but their development has been severely hampered by lack of a safe and effective adjuvant. These data suggest that CpG DNA may be a suitable candidate for this role. In about two months we should be enrolling our first human subjects in our first clinical trial of CpG as a vaccine adjuvant. This Phase I study, overseen by Davis, will be done at the University of Toronto."

It will test the CpG-enhanced commercial recombinant hepatitis-B (HBV) vaccine Engerix-B, from London-based SmithKline Beecham plc, on 48 healthy volunteers at various dose levels of the adjuvant. However, administration will be by conventional intramuscular injection rather than the mucosal route.

The endpoint of this first human study, Krieg explained, "will be the antibody level that we get. The conventional HBV vaccine requires three injections, in order to achieve successful immunization in 90 percent of subjects. One of our goals is to get these antibody levels after two injections instead of three.

"We hope to move on quickly to mucosal human trials," Krieg said, "but we may need to get sponsorship, or establish a partnership with a pharmaceutical company, before committing ourselves to this. Within the next few years we'd like to have trials ongoing with mucosal vaccination."

Fighting Infection, Cancer, Allergies

Krieg's CpG-derived products take the form of synthesized oligonucleotides, rather than the native bacterial DNA. "The technology itself," he said, "is the DNA sequences. And these synthetic oligonucleotides - which can be as short as eight bases long - will be used in one marketable drug that we will sell either as a stand-alone product or combined with a vaccine. This would be true for vaccines against infectious diseases, cancer and allergies. The concept is the same for all of those."

Krieg cited another prospective product "that is really the flip side of this one. It may not be intuitively obvious, but in gene-therapy applications, where people are using plasmids for delivering gene sequences, you don't want to induce an immune response.

"So, we also have a proprietary position on ways to avoid this reaction, by getting rid of the stimulatory CpG sequences, and cloning in other DNA motifs that we've discovered, which block the stimulatory effect."

Following a paper in the Proceedings of the National Academy of Sciences last month describing these inhibitory sequences, Krieg added, "already, two of the leading gene therapy companies have contacted us, because they want help in avoiding this problem." n