A Vaccine Factory Inside Each Cell

By Jason Socrates Bardi

When Edward Jenner fired his magic bullet into the arm of eight year-old James Phipps, it kept traveling. Inoculation, a new paradigm for controlling diseases, had arrived. He had lifted a cowpox scab from the hand of milkmaid Sarah Nemes to see if it would protect the Phipps boy against smallpox. It worked. And in the 200 years since, nothing has been as effective at combating, controlling, and in some cases eradicating infectious diseases as vaccines.

Smallpox, tetanus, measles, polio, diphtheria, pertussis, and several other once pandemic viral and bacterial pathogens are now gone or all but eliminated—destroyed through widespread application of Jenner’s basic technique of stimulated immune response. Inoculations have become widespread—in some cases universal. It’s no secret that vaccines are the best way to treat many epidemics.

And it’s certainly no secret for Bruce Torbett, associate professor of molecular medicine, who says his scientific outlook is deeply influenced by his background in public health. Trained in epidemiology, Torbett has seen first-hand how successful vaccines can be, and he regards vaccines as the logical approach to controlling those epidemics that rage in our society today—AIDS and cancer.

Only, the “vaccines” on which he is working aren’t really vaccines at all.

At least, they’re not vaccines in the sense of being shots that challenge the immune system to produce specific antibodies against something bad or foreign—such as an HIV virion or a cancer cell—and target it for destruction. His vaccines work by changing genotypes that make cells susceptible in the first place.

Torbett’s laboratory is developing and testing a gene delivery technique that may someday be used to deliver genes into cells, providing a high level of protection against HIV or cancer. The technique involves treating hematopoietic stem cells (HSC). These are the pluripotent granddaddy of all blood cells, located in the bone marrow, that develop into lymphocytes, platelets, erythrocytes, and red blood cells.

The basic idea is to give these cells genes that will allow them to resist an HIV infection, then implant them into tissue where they can freely grow, develop, and resist HIV infection. The same approach may be used to inhibit cells from becoming cancerous.

"I view [the therapy] as a general vaccine—something that provides protection for particular cells," says Torbett.

"Protection is prevention," he adds. "And prevention is the best form of disease control."

Take Them Out, Change Them, and Put Them Back

Several years ago, researchers noticed that against the apparent odds, some people who had had unprotected sex or shared needles on numerous occasions with multiple HIV-positive partners continued to test negative for the virus. Somehow, despite repeated high-risk exposures, they had remained uninfected.

Even more stunning was the result that cells isolated from these individuals were resistant to HIV in vitro, even though the cells had normal expression of the CD4 surface receptor that HIV envelope glycoproteins bind to and use to gain entry.

It turned out that HIV virions require a coreceptor for entry into a cell—the chemokine receptors CCR5 and, to some extent, CXCR4, present on the cell surface. Individuals who are homozygous for a mutation that knocks out the CCR5 receptor are highly resistant to infection with HIV, and individuals who are heterozygous for this mutation produce less CCR5 and tend to be among the more healthy people with HIV—the so-called "slow progressors."

Because the mutation confers some level of resistance from initial infection, provides a better prognosis for the course of their infection, and has no otherwise ill effect, Torbett felt that the coreceptor would make a good candidate for a gene therapy vaccine approach.

To do this he enlisted Carlos Barbas's support. Barbas has experience producing specific immunoglobulins, or antibodies, which are normally released into the bloodstream by B-cells to target antigens during an adaptive immune response.

Barbas and his group isolated an antibody, and its gene, that is specific for CCR5 and then designed a peptide "anchor" on one end that keeps it retained in the endoplasmic reticulum in the cell. These intracellular antibodies have been termed "intrabodies." Once the intrabody gene was incorporated in a cell, the cell would express the intrabody that would then grab the CCR5 and keep it from getting to the surface of the cell.

Thus, the cells would then be protected from infection.

This sort of therapy could prove useful as a way of treating people who are already infected with HIV. Torbett and Barbas would like to be able to deliver it into patients’ stem cells,"

However, the trick is to effectively "deliver" the intrabody genes, or for that matter any therapeutic gene, into stem or selected cells. For this approach Torbett has been working with the very virus that causes the disease—HIV.

Using a crippled version of HIV as a gene delivery vector/vehicle that can no longer spread in human cells and cause disease, Torbett’s group has shown that human stem cells can be given the gene for green fluorescent protein from jelly fish, and all cells developed from these stem cells express this protein.

"Our first generation gene delivery vectors were useful for proof of concept to get a gene into stem cells and show that it made a product, but we are now developing newer generation gene delivery vectors," says Torbett. The newer vectors may be useful for targeting stem and other selected cell populations.

One would take out the patient’s bone marrow, remove the stem cells and infect them with the intrabody gene using the HIV vector, then return the cells to the patient. The stem cells would then develop into dendritic cells and blood cells, including cells that HIV infects, such as macrophages and T-cells. These new cells would be phenotypically equivalent to CCR5 negative cells since the intrabody would prevent the coreceptors from reaching the cellular surfaces.

These progeny cells, then, would be effectively resistant to HIV. Having a selective advantage over the wild type cells, they would repopulate the body. The intrabodies would then do what antiretroviral drugs have done for years: keep the virus in check. "It’s no different from protease, really," says Torbett. "The idea is to keep the viral level low, protect the T-cells, and allow the immune system to do its job and control the infection."

There are many basic questions that need to be addressed before this type of therapy is ready for human trials, though. One needs to understand how efficiently the gene inserts and the intrabody expresses, what regulation machinery is involved, and how well the intrabody protects the cell. Another unanswered question is just how fast the virus will mutate to adapt to the intrabody treatment, just as it does to defeat drugs like AZT and protease inhibitors. "These concerns are critical for the success of gene therapy in general and for an HIV treatment," says Torbett. "My group and our colleagues, Carlos Barbas and Daniel Salomon, are currently working on these areas."

Fighting the Virus Without Drug Holidays

One of the major problems with current HIV antiretroviral therapy is adherence to the drug regimens that are very demanding. There are so many pills to keep track of—those to take with food, and those on an empty stomach; some once a day, others throughout the day—and some patients just cannot maintain a constant dosage.

Other patients on therapy go through healthy periods, and some patients consciously take "drug holidays" to reward themselves for a special occasion they would like to enjoy while free from the toxicity of their medicine.

Once they stop, the virus may rebound. Worse, when it comes back, it may have become a mutated form that is highly resistant to various drugs. This gene therapy approach could be used to create a stable immune response without having to take a rigorous daily regiment of drugs. Instead of an oral compound—or, more realistically, a combination of compounds—the intrabody would be given to a patient once.

Although this approach could one day be used as a vaccine to protect uninfected people against infection, the picture here is much more cloudy.

Vaccines are the best hope for controlling HIV in parts of the world where the epidemic is the most problematic, such as sub-Saharan Africa and parts of Asia. But besides the efficacy question for gene delivery, there are unresolved basic science questions of how to regulate the genes placed into a cell, and how to keep the immune response from reacting to products made by the new genes. Those issues notwithstanding, nobody knows if a treatment like this would be feasible as a traditional vaccine.

"It’s a big ‘if’," says Torbett. "Right now, given the cost, this kind of treatment would be beyond the scope of many parts of the world. But it could very well protect people from being infected."

Epilogue: A Technique to Treat HIV and Cancer Both

Inserting HIV intrabody genes is only one of several applications of Torbett’s work to control cellular function. Another promising application is the treatment of a certain type of cancer called acute myeloid leukemia (AML), a common form of acute leukemia in adults.

Myeloid cells, which constitute about 60 percent of bone marrow cells, are the non-differentiated progenitors of such diverse blood cells as monocytes, neutrophils, and platelets. Myeloid cells, in turn, derive from hematopoietic stem cells during hematopoiesis; a process that relies on a properly functioning transcription factor called PU.1.

PU.1 is a small, winged helix-turn-helix protein that binds to a purine rich sequence containing the GGAA motif of DNA. This protein is a major player in normal or abnormal myeloid development because it regulates transcription of those genes that control the lineage and differentiation of myeloid cells—ones expressing cytokine receptors and their signaling pathways, adhesion molecules, and other key cellular proteins. Knocking out PU.1 halts myeloid development but does not halt cell division, a state that may contribute to development of AML.

Aberrant or loss of expression of key genes controlled by PU.1, such as those that code for cell adhesion molecules and cytokine receptor signaling pathways, may result in cells that do not finish their development, yet still can grow, allowing additional molecular changes that may promote cancer. The malignant population of immature cells derived from a single malfunctioning myeloid founder cell may promote AML.

Torbett is looking at ways of controlling AML by controlling PU.1 transcription. He uses in vivo models that mimic what happens in normal myeloid development in humans and he manipulates PU.1 to gain insight into normal and pathological states and to understand gene regulation. "Our group is looking at this gene as a paradigm for hematopoietic and myeloid development," he says.

Medicines in the future may move away from targeting gene products with some compounds to turning on and off the genes involved in cell functions that are relevant to health and disease. These gene-based drugs could be compounds that mimic regulatory proteins or even pieces of DNA that, once inserted into cells, would control regulatory regions.

"The current drugs are useful," he says, "but the future of molecular medicine comes down to understanding and controlling the regulation of genes and their products."

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Bruce Torbett is developing and testing a gene delivery technique that may someday be used to deliver genes into cells, providing a high level of protection against HIV or cancer.

Human cells developed from stem cells transduced with HIV vectors contain a surrogate marker. In this case, a green fluorescent protein gives a qualitative measurement of expression. The cell on the right flouresces, the one on the left does not. "You can easily distinguish between cells that have the gene of interest and those that don’t," says Torbett.