Vaccine Strategy Beats Listeria at Its Own Game

Developing vaccines for pathogens that replicate while hidden inside human cells is tricky business. Antibodies that work in the bloodstream cannot reach these intracellular pathogens, so the body relies on its cellular immune system to develop lasting protection. But this system does not respond until the pathogens cause infection, making a healthy person sick and putting an immunocompromised individual in extreme jeopardy. A study in the March 20 online edition of Proceedings of the National Academy of Sciences describes a novel vaccine strategy that stimulates the adaptive cellular immune system to promote lasting immunity to intracellular pathogens without incurring the risk of illness.

Image courtesy of Darren Higgins

Safe steps toward protective immunity. An antigen presenting cell takes up genetically modified cytoLLO Listeria monocytogenes (Lm) and degrades it in the vacuole. Released LLO lyses the vacuole, allowing Lm antigens into the cytoplasm. The proteasome processes the antigens into peptides that are transported to the endoplasmic reticulum and assembled onto MHC Class I molecules. The Golgi transports each peptide/MHC complex to the cell surface, where it can be detected by a cytotoxic T cell.

The study builds on previous research from 2002 in which Darren Higgins, HMS associate professor of microbiology and molecular genetics, and colleagues developed their initial “directed antigen delivery” strategy. They crafted nonpathogenic Escherichia coli bacteria to act as messengers, delivering a model antigen, ovalbumin, directly to the cytoplasm of antigen-presenting cells (APCs), where it stimulated adaptive T cell immunity. Though promising, this strategy requires the knowledge of which antigens trigger immunity for each pathogen—information that, for most pathogens, simply does not exist.

Higgins and colleagues decided to try using a selected pathogen to deliver its own antigens to the APC, starting with Listeria monocytogenes. As part of the body’s first line of defense, the APC encapsulates L. monocytogenes in a vacuole. The bug beats this barrier by perforating the vacuole wall with a secreted protein, listeriolysin O (LLO) and escaping into the cytoplasm, where it replicates and stimulates T cell immunity, but also causes illness and sometimes death.

One L. monocytogenes mutation allowed the researchers to deliver the bacteria’s antigens without risking illness. Co-authors Christine Alberti-Segui, research fellow, and Nathan Berkowitz, research assistant, both in Higgins’s lab, eliminated the secretion signal for LLO, creating a cytoLLO strain. Since a cytoLLO bacterium cannot secrete LLO, the pathogen remains trapped in the vacuole. The bacterial cell wall dissolves, exposing the LLO inside to the vacuole membrane. The LLO lyses the membrane, releasing the bacterium’s contents, including all of its antigens, into the cytoplasm. The APC then acts as a sacrificial lamb, presenting these antigens on its surface to alert cytotoxic T cells to fight the pathogen; the T cells kill the APC and turn into memory T cells that provide protective immunity by becoming “like an army you’ve recruited, just waiting for battle,” said Higgins.

Challenge tests showed that mice vaccinated twice with cytoLLO L. monocytogenes developed immunity half as strong as those vaccinated with wild-type L. monocytogenes. These mice, when challenged with wild-type bacteria 28 days later, survived as well as those vaccinated with the wild-type bug, even at high dosages. Severe combined immunodeficient (SCID) mice survived vaccination with cytoLLO, confirming its safety.

Though the vaccine is ready for human safety trials, “there is no real need for a Listeria vaccine,” said Higgins. “Rather, this is a proof of concept that allows us to develop a [vaccine] strategy transferrable to any intracellular pathogen we can genetically manipulate.” To apply this method, researchers need only disable a pathogen’s ability to replicate within host cells, then genetically introduce a nonsecreted LLO protein. Higgins is now working on developing a strategy that applies to pathogens that are not genetically modifiable, such as Chlamydia.

—Elizabeth Dougherty

Natural Mineral Fibers in Turkey Cause Extreme Cancer Risk

The landscape of the Cappadocia region of central Turkey seems otherworldly, with rugged peaks and wind-carved spires. Ancient inhabitants shaped churches out of the area’s tuff, the soft rock formed from volcanic ash, and modern villagers use tuff bricks for construction. But some of the tuff contains erionite, a fibrous and woolly mineral that crumbles when touched. When inhaled, its effects are even more deleterious than asbestos.

A study of three villages in Cappadocia in the March 15 Journal of the National Cancer Institute found an extremely high risk of mortality from pleural mesothelioma in erionite-exposed areas compared with unexposed areas. “Only the pipe fitters in the United States from the 1940s and ’50s, with the highest and longest exposures to asbestos, even approach this risk,” said co-author Philippe Grandjean, adjunct professor of environmental health at HSPH.

In the 1970s, first author Y. Izzettin Baris of Hacettepe University in Ankara set out to investigate the high mortality rates in Cappadocia. Many deaths there had been attributed to liver cirrhosis, an unusual disease for a nondrinking population, and tuberculosis. Using X-rays, he instead found high rates of peritoneal and pleural mesothelioma. Other investigators observed that dust and lung tissue samples from the exposed villages contained erionite fibers, which other research found to be highly carcinogenic in lab animals. Erionite causes pathological effects similar to asbestos, such as pleural thickening, plaques, and calcifications.

Acting on these findings, Baris tracked 891 adults from three Cappadocian villages from 1979 to 2003, 661 from villages exposed to erionite and the rest from an unexposed village. During the study period, 372 participants died, 102 from pleural mesothelioma. Of those cases, only one was from the unexposed village. For the two exposed villages, Grandjean calculated a standardized pleural mesothelioma mortality ratio of 485, indicating that this population shows nearly 500 times the risk of a typical, unexposed population. “We often worry about a doubled risk,” he said. “This is truly a mind-boggling number.”

Linking cancer to an environmental cause is not easy because unstable populations make long-term follow-up complicated. “This setting, where a son takes over the family farm, marries a woman from the same village, where they stay and their children stay, has been very conducive to the research,” said Grandjean.

Turkish public health authorities have responded to this emergency by building new houses with safe materials, yet many villagers in Cappadocia remain at risk. Because so many residents already have erionite in their lungs, said Grandjean, new mesothelioma cases will likely appear for decades.

—Elizabeth Dougherty

Prayer Found Ineffective at Reducing Complications After Heart Surgery

Researchers in the Study of the Therapeutic Effects of Intercessory Prayer (STEP), the largest study to examine the effects of intercessory prayer, have evaluated the impact of such prayer on patients recovering from coronary artery bypass graft (CABG) surgery.

The team, composed of investigators at multiple institutions, including Beth Israel Deaconess Medical Center and the Mind/Body Medical Institute, found that intercessory prayer had no significant benefit on recovery from surgery and that patients who knew they were receiving intercessory prayer fared worse. The paper appears in the April American Heart Journal.

“The primary goal of STEP was to evaluate whether intercessory prayer or the knowledge of receiving it would influence recovery after bypass surgery,” said co-author Jeffery Dusek, HMS instructor in medicine and associate research director at the Mind/Body Medical Institute.

STEP investigators enrolled 1,802 bypass surgery patients from six hospitals and randomly assigned each to one of three groups. The 604 patients in group 1 received intercessory prayer after being informed that they may or may not receive it; the 597 patients in group 2 did not receive prayer after being informed that they may or may not receive it; and the 601 patients in group 3 received intercessory prayer after being informed that they would receive it. Caregivers and independent auditors comparing case reports to medical records were unaware of the patients’ assignments.

Among people who were unsure whether they would receive prayer, complications occurred in 52 percent of those who did receive it (group 1) versus 51 percent of those who did not (group 2). In contrast, complications occurred in 59 percent of patients who knew that they would receive intercessory prayer (group 3). Major complications and 30-day mortality were similar across the three groups.

“Our study was never intended to address the existence of God or the presence or absence of intelligent design in the universe. The study did not endeavor, either, to compare the efficacy of one prayer form over another or to assess participants’ understanding of the nature and purpose of prayer. Finally, it was not our objective to discover whether prayers from one religious group work better than prayers from another,” said co-author Father Dean Marek, director of chaplain services at the Mayo Clinic. In addition to the clinic and the two Harvard-affiliated institutions, co-authors came from Baptist Memorial Hospital in Memphis; Integris Baptist Medical Center in Oklahoma City; St. Joseph’s Hospital in Tampa; and Washington Hospital Center in Washington, D.C.

“One caveat is that with so many individuals receiving prayer from friends and family, as well as personal prayer, it may be impossible to disentangle the effects of study prayer from background prayer,” said co-author Manoj Jain of Baptist Memorial Hospital.

“Each study builds on others, and STEP advanced the design beyond what had been previously done,” said Dusek. “The findings, however, could well be due to the study limitations.”

—Elizabeth Dougherty

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