By David N. Leff

One fine day in May 1796, an English country doctor named Edward Jenner found a young dairy-maid named Sarah Nelmes. He noticed her fingers were decorated with scabby, pus-filled blisters known as the cowpox. This was a mild occupational hazard afflicting people who milked cows.

Jenner, among others, had noticed that individuals with cowpox lesions seldom came down with smallpox, a frequently fatal disease, endemic throughout Europe. Playing a carefully nurtured hunch, he collected pus from Sarah Nelmes' hands, and inoculated the material into an 8-year-old boy named James Phipps. The lad developed a low fever and some skin lesions. Then on July 1, Jenner vaccinated James with the real thing - pus from a smallpox victim.

This time, Phipps suffered no symptoms at all, and became the first documented person to be immunized against the smallpox virus.

In some ways, but perhaps not quite all, immunization today has become a deal more sophisticated than in Edward Jenner's 18th century.

From adenovirus to Yellow fever, 30 licensed vaccines are available in the U.S. today. All but one consist of weakened strains of the live or inactivated virus or bacterium, with all their immunity-eliciting antigens on board. The exception is hepatitis B vaccine, which deploys a single inactive viral antigen subunit.

"There are many subunit vaccines in development," observed bio-organic chemist Alexey Margolin, "but, despite all the promises of subunit vaccines, the only one used widely is hepatitis B." He added, "There's a list of five or 10 potential antigens for the several subunit AIDS vaccines under development." Margolin is vice president of research and development at Altus Biologics Inc. in Cambridge, Mass. He is senior author of a paper in the current Proceedings of the National Academy of Sciences (PNAS) dated Aug. 17, 1999. Its title, "Cross-linked protein crystals for vaccine delivery," describes Altus' effort to redeem the immunity-promoting promise of subunit vaccines.

"Our whole rationale," Margolin told BioWorld Today, "is to enhance the immune response. For this, you would like to have particulate protein antigens rather than soluble ones. Right now, vaccinologists normally use adjuvants, such as alum. One of the presumed reasons alum works is just because these are particles of aluminum salts, on which the vaccine is deposited. And of course," he continued, "protein crystals are particles. Moreover, you can change their shape and size at will.

"The second point," Margolin went on, "is that in many cases, when you need an enhanced immune response, you would like to have a so-called depot effect, where you deposit your antigen, from which it leaches out over time, in a controlled-release mode. And because of this depot leaching, you obtain an initial booster effect."

In Search Of A One-Shot Vaccine

"What you do normally," Margolin explained, "is get a shot of vaccine - of antigen, basically. Then immune-system macrophage cells come in and attack this antigen, and it's gone. Ten days later, say, you have a second shot. The first one was just to prime the immune system. The second one ensures that you have a sustained immune response. That's how vaccination works.

"Now the idea has always been," he pointed out, "that it would be great to be immunized in a single shot. Some people are developing polymeric microspheres. What they do is mix a polymer with an antigen, and instead of having a first and then a second injection, you may need only one, and the microcarrier releases its protein antigen over time. So it primes the immune system and then delivers the antigen, all in one shot. It's a very significant area of research."

Instead of polymers, Altus uses a very high concentration of particulate antigens in crystalline form, released over time. "What the protein crystals can give that nothing else can give," Margolin pointed out, "is a highly repetitive antigenic structure. And this PNAS paper, to the best of our knowledge is the first that reports cross-linking protein crystals. The crystals are not cross-linked to each other; it's the molecules inside the crystals that are. If you look at the crystal structure, as depicted in this paper, it just jumps out at you, because it's so repetitive, resembling virus-like particles."

He made the point, "An advantage of having protein crystals is thermostability. So in a sense you solve the problem of storing and delivering vaccines, which is very acute, especially in Third World countries, where you don't have refrigeration and so on. Besides stability, another advantage will be providing for combination vaccines."

To test their putative vaccine in vivo, Margolin and his co-authors set up six groups of rats. They vaccinated the first two cohorts with crystalline human serum albumin as a model antigen, in two degrees of cross-linking. The third set of animals received the protein in normal soluble form. Then they repeated this trial design in the remaining three rodent samples, but mixing the antigen to a powerful adjuvant.

Cross-Linked Crystallized Proteins Work Antigen-Free

"The most important result," Margolin recounted, "was that groups receiving cross-linked protein crystals without an adjuvant produced significantly higher immune responses than soluble-antigen recipients. The magnitude of this effect was very similar to what we normally see when using a soluble antigen and adjuvant. For six months," he added, "we monitored these immune responses up to 30-fold higher than what we got with soluble material. We measured these results by titers of circulating antibodies against the albumin."

Margolin suggested the major benefit of this novel subunit vaccine approach "will come not for vaccines that are available today but for those under development." He cited as potential candidates "human papillomavirus, which has several antigens on its surface; the AIDS vaccine, with its gp120 envelope protein - in clinical trials right now; and Helicobacter pylori, the ulcer bacterium, on which people are working toward a subunit vaccine based on its urease antigen.

"In this specific context," he indicated, "quite frankly, we are now in the course of negotiating with several vaccine companies. Altus is basically not a vaccine producer. Our major technology is protein crystals for many different applications. Not only vaccines, but several therapeutic programs. So we are now looking for a partner," Margolin concluded, "who will provide us with real pathogenic antigen, so we can develop the vaccine further, and show immune protection as well."