Cutting up frogs is a rite of passage for kids taking grade-school or high-school biology. They douse their dissected specimens in formaldehyde to fix, or "freeze," the tissues and organs.
Formaldehyde does this job of rendering proteins inert by cross-linking their strands. On a larger scale than frogs, formaldehyde is the prime ingredient in solutions used for funeral-home embalming of the departed for burial.
Virologist Jack Nunberg, at the University of Montana in Missoula, has put this cross-linking process to a novel use - capturing and immobilizing an ephemeral vaccine target in the AIDS virus.
"What formaldehyde does," Nunberg explained, "is cross-link all the proteins, stitches or staples them together, making them hold still in the structure that they were in at the time that we fixed it. These protein complexes can be immobilized, like a stop-motion frame on a movie film. We captured the HIV structures that were present at one moment in time, and kept them that way."
Nunberg is director of the Montana Biotechnology Center, and professor of biological sciences at the university.
The occult, elusive HIV antigen that he entrapped is the most baffling player in the virus's weirdly complex light fantastic dance by which it fuses with its target human cell - T lymphocyte or macrophage.
Until two or three years ago, HIV researchers pictured this dance as a one-step. The viral envelope's glycoprotein, gp120, simply coupled with the CD4 receptor perched seductively on the T cell's surface membrane.
Then in 1996, HIV's dance card suddenly filled up:
* CXCR4, the first T-cell-targeting viral co-receptor discovered after CD4, served as a co-conspirator, honchoing the fusion of the HIV virion's gp120 envelope with the membrane enwrapping those target cells, enabling the infective retrovirus to break and enter.
* CCR5 was the next link in the intricate daisy chain of the viral ballet.
* CCR3, the rarest HIV co-receptor, occurs on certain brain cells incident to AIDS dementia.
All along, efforts to contrive an AIDS vaccine focussed on gp120 as the one and only antigenic bull's eye game in town.
"It was definitely the recognition of co-receptors," Nunberg recalled, "and of the multiple protein interactions and conformational changes that the envelope protein underwent during infection, that crystallized the new vaccine approach for me."
The concept behind his breakaway prototype vaccine, Nunberg told BioWorld Today, "arose out of the historic failure of gp120 vaccines to elicit neutralizing antibodies, while in the same assays infected patient serum had the ability to neutralize primary isolates. What's unique about our vaccine is that it's able to elicit antibodies that neutralize primary viral isolates, that is, street virus obtained from HIV-positive individuals. The gp120 vaccines just hit the HIV laboratory strains. They had no immune-response effect on primary isolates."
Nunberg is senior author of a paper in Science, dated Jan. 15, 1999, titled "Fusion-competent vaccines: Broad neutralization of primary isolates of HIV."
"We show in our paper," he said, "that if you just present a nonfunctioning viral envelope, that gp120, you target these outer, more exposed but also more variable antigenic determinants, which for some reason are able to neutralize lab viruses. But it's only in targeting the cryptic, fusion-dependent structure captured by our formaldehyde cross-linking that we got broadly neutralizing antibodies to primary isolates."
His present demo-model vaccine consists of "a mixture of two whole cells. One is a primate line expressing HIV envelope; the other [is] human glioma cells, expressing CD4 and co-receptors. We've also made a successful vaccine with just human embryo kidney cells - but that's later, unpublished work."
One of the paper's co-authors, Dan Littman, of New York University, contributed to the Montana project a transgenic mouse immunization model that expresses human CD4 and the HIV co-receptors. "Therefore," Nunberg observed, "when we neutralize with these complex vaccines now on our drawing board, we don't have to worry about an antibody response to CD4 and co-receptors."
Though fixed and fleetingly exposed, the cryptic antigen has so far been identified only by its unique immunogenic effect. The co-authors are now trying to isolate these cross-linked complexes from their fusion cells. "One thing we know about them," Nunberg said, "is that whatever the determinant is, it's very conserved in the virus, because our antibodies were able to neutralize it." They did so by eliciting an immune response that blocked cell infection in 23 of 24 genetically diverse HIV subtypes from the U.S., Europe, Africa, India and Thailand.
Kicking HIV's Shifty, Variable-Strain Hang-Up
"When you think about it," Nunberg said, "all strains of HIV, regardless of their genetic sequence variation, have to do the same thing. They have to bind CD4 plus co-receptors, and they have to initiate fusion. So it's apparently this common functional structure, which perhaps is not exposed in the nonfunctioning envelope gp120, that we've been able to target by freezing our envelope in mid-function.
"So, you can imagine that it would make sense for the virus to hide that common Achilles heel until the last minute. Otherwise, it would be very easy for antibodies to block its activity."
Nunberg cites "the many hurdles - of manufacturing, safety, regulation - in developing a formaldehyde-fixed, whole-cell vaccine for human testing. So, with Therion Biologics Corp. [of Cambridge, Mass.], we're trying to convert the concept to using recombinant vaccinia viruses, instead of the cells.
"One vaccinia strain expresses envelope; another, CD4 and co-receptors. We're hoping that the recombinant vaccinia viruses will infect the skin in your arm, for instance. Then, hopefully, the fusion process will occur in your arm, and elicit the same kind of response as the cell-based vaccine did in our mice. We should know shortly whether it's a home run or requires more research."
Nunberg concluded: "Our thrust with Therion is to find a formulation that encompasses the entire concept, but is easier to develop into a clinical vaccine candidate. Finally, the question remains, 'OK, we can for the first time elicit these antibodies that neutralize the primary isolates, [but] are they sufficient to protect against actual HIV infection?'" n