Aug 19, 2005 (CIDRAP News) – Two research reports on plague were recently released, one describing the mechanism that the plague bacterium uses to evade the body's immune system and the other describing a potential vaccine that was tested successfully in mice.
Senior author Olaf Schneewind led the two research teams at the University of Chicago, whose results were published online Jul 28 in Science and in the August issue of Infection and Immunity.
The bacterium Yersinia pestis causes different forms of plague, depending on the route of exposure. Bubonic plague, infamous as the "Black Death" of the Middle Ages, is transmitted by flea bites. The disease persists in its natural reservoir of wild animals, primarily rodents, passing to humans via fleas that first bite an infected animal. Inhalation of the bacteria causes pneumonic plague, a disease that is seen less often in nature but could develop if aerosolized plague bacteria were released in an act of bioterrorism.
Today, about 1,000 to 3,000 cases of plague (primarily bubonic) occur worldwide each year, including approximately a dozen in the western and southwestern United States. Although plague killed hundreds of millions of people in past pandemics, no human plague vaccine is currently licensed in the United States.
Without prompt antibiotic treatment, the case-fatality rate of plague approaches 100%. A major reason for this high mortality is that the invading bacteria suppress the body's immune response. The plague bacterium uses a system that injects toxins directly into host cells. Previous studies have indicated that Y pestis evades the immune system by resisting phagocytosis and suppressing the inflammatory response.
The authors of the Science paper wanted to know exactly which immune cells are targeted by plague. The authors engineered a protein to identify the cell types injected with toxin by the plague bacterium. They created a piece of DNA containing genes for both Yersinia outer proteins (Yop) effectors and beta-lactamase (Bla). Yops are the toxins normally injected by plague into host cells; Bla is an enzyme that cuts a dye called CCF2-AM, making it fluoresce blue instead of green. The team inserted this piece of DNA into Y pestis strains.
Mice were first infected with the modified plague strains and then sacrificed and their spleen tissues separated into specific cell populations. Staining with CCF2-A dye allowed visualization of cells injected with Yop-Bla, because only cells containing this engineered protein glowed blue. Despite the predominance of CD4 and CD8 cells in the spleen, the majority of cells targeted by the modified Y pestis were macrophages, neutrophils, and dendritic cells. This strategy, the authors wrote, allows the bacterium to "destroy cells with innate immune function that represent the first line of defense, thereby preventing adaptive responses and precipitating the fatal outcome of plague."
Prior studies have pointed to the role of the bacterial protein LcrV as a critical element of plague virulence. LcrV acts a molecular "needle" to inject toxins into host cells, and it also suppresses the immune response through two mechanisms. First, it stimulates the host to release interleukin-10 (IL-10), which dampens the innate immune response. Second, it prevents the release of two pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). This immunosuppressive activity makes native LcrV a poor vaccine candidate, despite its immunogenicity.
In the Infection and Immunity article, the researchers describe how they investigated modified LcrV proteins for three criteria of a successful vaccine: lack of IL-10 stimulation, preservation of pro-inflammatory cytokine secretion, and robust antigenicity. The authors deleted sequential 30-amino-acid portions of LcrV to create 12 candidate deletion proteins. Two of these, rV7 and vV10, failed to induce IL-10 secretion in mouse macrophages. Further testing showed that the rV10 peptide was not capable of significantly suppressing TNF-alpha secretion.
Finally, the authors tested the rV10 protein in a mouse immunization experiment. The protein elicited high titers of immunoglobulin G (mean, 112,500 by ELISA), which were comparable to those induced by native LcrV. Additionally, two intramuscular doses of rV10 (100 mcg each, with alhydrogel adjuvant) protected 100% of mice challenged with heavy doses of Y pestis.
Thus rV10, the authors explained, "satisfied our experimental criteria and displayed significant defects in immune suppression without reducing the protective properties of plague vaccines." They conclude, "It appears that LcrV variants with reduced immune modulatory properties could be used as a human vaccine to generate protective immunity against plague."
Both studies received support from the National Institute of Allergy and Infectious Diseases.
Marketon MM, Depaolo RW, Debord KL, et al. Plague bacteria target immune cells during infection. Science 2005 (published online Jul 28) [Abstract]
Overheim KA, Depaolo RW, Debord KL, et al. LcrV plague vaccine with altered immunomodulatory properties. Infect Immun 2005 Aug;73(8):5152-9 [Abstract]
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