APUA One-on-One with Dr. Abraham Sonenshein
Abraham L. Sonenshein, Ph.D.
Molecular Biology and Microbiology
Tufts University School of Medicine
Q. What are some factors that make vaccines so expensive?
All currently available vaccines require refrigeration or freezing from the moment of manufacture until delivery to the patient. In addition, most are delivered by injection. As a result, they require technology and expertise for manufacture, storage and delivery that are not available to a very large fraction of the world’s population. In addition, most vaccines are purified protein antigens or purified, inactivated (or attenuated) pathogenic bacteria or viruses. Preparing purified antigens is expensive because large amounts of cell culture must be obtained and extracted, followed by rigorous elimination of all other proteins and other contaminating substances. Inactivating pathogens or using attenuated (mutant) pathogens requires the producer to verify rigorously that each batch of the pathogen has been truly inactivated or, if attenuated, that it has not reacquired virulence properties.
Q. You and Dr. Hanping Feng at Tufts are working on a vaccine against C. difficile infections that could potentially replace antibiotic treatments. Why are antibiotics an unsustainable treatment for C. difficile?
[Dr. Feng has moved to the University of Maryland. Although I had advised him on some of his work, he was the one developing novel vaccines against C. difficile. Our vaccine work at Tufts has been entirely in collaboration with Dr. Saul Tzipori.]
C. difficile infection is usually precipitated by treatment with antibiotics to deal with a previous infection. As a result, the intestinal microbial flora, which normally protects against C. difficile infection, becomes compromised and is unable to prevent colonization and growth of C. difficile. Current treatment depends on either vancomycin or metronidazole. Not only is neither fully effective in eradicating C. difficile infection, but they also prevent the re-establishment of the normal flora. In addition, recently appearing strains of C. difficile are not only more virulent but also resistant to several antibiotics, including fluoroquinolones, ruling out this class of antibiotics for future use. Moreover, the spore form of C. difficile is completely resistant to all antibiotics. Hence, many patients who appear to have been cured of C. difficile infection become re-infected within a few weeks. Some newer antibiotics appear to be somewhat more effective at preventing re-infection, but other, non-antibiotic treatments are also on the horizon. For instance, fecal transplants from healthy individuals have been used effectively to restore the flora of C. difficile patients who have not been treated successfully with antibiotics.
Q. Tell us about the work that your lab has done in mapping out the genes that bacteria express during nutritional stress, and the ones that they express during active growth.
Most Gram-positive bacteria use a common regulatory protein, CodY, to control the expression of dozens of genes that are turned on when the bacteria experience nutrient limitation. Most of these genes code for proteins involved in nutrient uptake and metabolism, but pathogenic bacteria use the same protein to control their major virulence genes. For instance, in C. difficile, the CodY protein represses during active growth the genes that code for the major toxin proteins TcdA and TcdB. It does so indirectly by repressing the synthesis of another protein, TcdR, that directs RNA polymerase to the toxin gene promoters. When cells experience nutrient limitation, CodY loses activity, TcdR is synthesized and the toxin genes are expressed.
Q. What made you think of using the dormant state of bacterial spores as a vehicle for delivering vaccine antigens?
The original idea came from Jerry Keusch, formerly Chief of Infectious Disease at Tufts Medical Center and now Professor of International Health at Boston University. In 1996, Jerry came back from a childhood vaccination meeting and asked me if it would be possible to use bacterial spores as vaccine delivery systems that wouldn’t need refrigeration. That phone call stimulated a collaborative project that continues to this day. In 2004 we enlisted Saul Tzipori to direct the animal studies and together were able to obtain a large grant from the Bill and Melinda Gates Foundation to move the project forward in a significant way.
Q. When you first engineered the B. subtilis vaccine, you envisioned the bacterium expressing a fragment of the tetanus toxin protein as an antigen on its surface, then being induced to take its dormant spore form in the lab, then being shipped all over the world to be taken as an oral vaccine, after which the bacterium would germinate in the gastrointestinal tract. In an article in Tufts Journal you say that “Almost every aspect of that plan turned out not to work.” What went wrong?
The original idea was to engineer bacteria to express antigens on the surface of vegetative cells, induce the cells to form spores and ship the spores around the world. A patient would drink the spores, which would germinate in the intestinal tract and display the antigens. The main problem turned out to be that B. subtilis spores don’t germinate very well in the GI tract. As a result, there was very little display of antigen and very little immunity.
Q. Then you re-engineered the vaccine so that the tetanus antigen was expressed on the surface of the dormant spore instead. Did that eliminate those setbacks?
Our current approach sounds similar but is different in many fundamental ways. We engineer the bacteria to express the antigens on the surface of the spores and vaccinate by adding a few drops into the nose or under the tongue. Germination of the spores isn’t necessary since the cells of the immune system interact directly with the surface-exposed antigens in the nose or mouth. By freeze-drying the spores, we can store them at any temperature between -20°C and 45°C (113°F) for more than a year without any loss of potency. Thus, we now have needle-free vaccines that don’t need to be refrigerated. In addition, since growing B. subtilis industrially is relatively inexpensive and no purification is needed, the vaccine can be produced at minimal cost.
Q. What experimental work with animals have you conducted so far with the B. subtilis tetanus vaccine?
The tetanus vaccine, our test case, has been given to mice and piglets either intranasally or sublingually. In both cases, the animals developed high levels of antibodies against tetanus. No deleterious effects of the vaccine have been seen in the hundreds of animals we have tested so far.
Q. The next step in the regulatory process for the approval of the B. subtilis tetanus vaccine and rotavirus vaccine is to demonstrate that they are not toxic to humans. What are your opinions on the duration and complexity of the FDA regulatory process for vaccines?
We are working with a group at MGH headed by Elizabeth Hohmann and Patricia Hibberd (formerly at Tufts and a collaborator on the Gates-funded project). They are seeking FDA approval for a Phase I clinical trial of the tetanus vaccine. Even a small trial is very expensive to carry out and the Phase II and Phase III trials require a huge investment. Therefore, we are trying to find governmental and industrial partners in the U.S. and in the developing world who would be interested in helping support the trials as well as collaborate on development of additional vaccines.
Q. How does the cost of a vaccine delivered via a dormant bacterial spore compare to the cost of an injected vaccine today?
We estimate that the spore vaccines can be produced for less than 40 cents per dose.
Q. What other approaches are you working on to block C. difficile infections without resorting to antibiotics?
A few years ago, Joseph Sorg, then a postdoc here, found that certain bile acids are required to activate germination of C. difficile spores whereas other bile acids inhibit germination by competing with the pro-germinant bile acids for binding to an apparent receptor. The inhibitory bile acids could potentially be useful in blocking infection but they are metabolized by the normal intestinal flora and recirculated from the intestine to the liver. In collaboration with Joe (now an Assistant Professor at Texas A&M) and Med-Chem Partners, a medicinal chemistry company, we are designing analogs of the inhibitory bole acids that are neither metabolized nor reabsorbed. We hope that these compounds can be sued either to prevent initial C. difficile infections or to block recurrence. They would have a very narrow spectrum of activity (as far as we can tell only C. difficile germinates in response to bile acids).
We have also been studying for many years the bacterial regulatory protein called CodY that controls many aspects of metabolism in Gram-positive bacteria. In pathogens, it also regulates the expression of key virulence genes. For instance, in C. difficile, CodY is the predominant regulatory protein (repressor) for the toxin genes. CodY is activated as a DNA-binding protein by interaction with isoleucine, valine and leucine, the so-called branched-chain amino acids (BCAAs). We are searching for analogs of the BCAAs that activate CodY but are not metabolized by bacteria or human cells. If given orally, such compounds would, in principle, repress toxin gene expression in C. difficile cells in the intestinal tract. Since these compounds are expected to affect gene expression but not growth, they will probably not give rise to resistant mutants.
[Please note that the C. difficile work described above and the B. subtilis vaccine project are not linked at the moment. Dr. Hanping Feng has begun to attempt to use the B. subtilis system as one of his approaches to creating new vaccines, but he is not very far along and it is not his major effort in the vaccine field.]
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