, the causative agent of plague, has accounted for the deaths of millions of people throughout recorded history. The second pandemic (the Black Death) is thought to have killed an estimated 17 to 28 million Europeans between the 14th and 17th centuries. The third pandemic, believed to have started in the Yunan Province of China in the 1850s, has led to the worldwide spread of plague, which is now endemic to several regions, including Africa, India, and the southwestern states of the United States (25
). Despite the current low incidence of plague, the bacterium resides in natural animal reservoirs, and regular, although relatively small, outbreaks of plague occur (7
). Improvements in transport links between areas of endemicity and large population centers bring with them the potential for large-scale plague outbreaks, highlighted by the recent outbreak in India (33
). There is therefore a need for effective disease surveillance to reduce the risk of plague transmission to new areas and subsequent outbreaks of disease. Vaccination is recommended for research scientists and other professionals who come into contact with the bacterium, but fast-acting treatments are also required for individuals exposed to Y. pestis
in areas of endemicity or through their work. In addition, after a major outbreak, there would be a need to protect health care workers and first responders.
At present, protection against plague can be mediated through vaccination or antibiotic treatment. Antibiotics are used both to treat plague victims and as prophylaxis to control the spread of the disease (25
). The incidence of antibiotic resistance in Y. pestis
is low, but recent plague isolates in Madagascar have been found to have multiple drug resistance, conferred by a transferable plasmid (10
). Although the bacteria were resistant to the frontline antibiotics streptomycin and tetracycline, they were susceptible to additional antibiotics.
Existing plague vaccines include killed whole-cell preparations, and efforts to develop new vaccines are in progress (39
). Problems associated with whole-cell vaccines include relatively low levels of protection, adverse side effects, slow time to immunity, and a need for regular booster immunizations (30
). Although whole-cell vaccines are thought to be effective against the most common form of plague (bubonic plague), which develops following a bite from an infected insect, their efficacy against pneumonic plague has been questioned. Consequently, whole-cell vaccines are no longer licensed for use in the United States. Next-generation plague subunit vaccines, based on the recombinant F1 and V (LcrV) antigen proteins derived from Y. pestis
, are being developed. Immunization with either protein provides protection against pneumonic or bubonic disease in animal models of infection (12
), but greater-than-additive protection is achieved when F1 and LcrV are combined, with protection against up to 109
median lethal doses (MLD) of Y. pestis
). Such vaccines must be administered prior to exposure, and multiple doses are required. Although strategies to reduce the time to immunity and the number of vaccine doses have shown promise (41
), it is unlikely that vaccination will provide postexposure protection against disease. There is therefore a need for alternative fast-acting antiplague treatments to provide rapid protection, particularly to combat drug-resistant strains of Y. pestis.
Because antisera have been used widely to treat a range of diseases caused by other pathogens (15
), we considered monoclonal antibodies (MAbs) as a treatment for plague. Previously, F1-04-A-G1, a mouse MAb raised against F1, was shown to protect mice in models of bubonic and pneumonic plagues (1
). Also, preliminary studies showed that an LcrV-specific MAb (MAb 7.3) protected mice in a bubonic plague model (13
). In this study, we considered the prophylactic and therapeutic properties of MAb 7.3, when administered alone and in combination with F1-04-A-G1, to determine whether antibodies could be used as a postexposure therapy for plague.
MAb 7.3 and F1-04-A-G1 were purified by ammonium sulfate precipitation from hybridoma supernatants. An equal volume of saturated ammonium sulfate solution was added slowly to tissue culture supernatants, followed by overnight stirring at 4°C and then centrifugation at 3,000 × g for 30 min. The pellets were drained and resuspended in phosphate-buffered saline (PBS; GIBCO, Paisley, United Kingdom) in 0.1 volume of the original volume, which was then dialyzed against three changes of PBS. Disposable Econopak columns (BioRad, Hemel Hempstead, United Kingdom) were packed with protein G-Sepharose beads (Sigma, Poole, United Kingdom), and antibody solution was passed through the column. The beads were washed with PBS, and then antibody was eluted with 50 mM glycine (pH 3) and stored in fractions containing 150 μl of Tris HCl (pH 9.1) per 3 ml of eluate. Protein fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10 to 15% Phastgels (Pharmacia, Milton Keynes, United Kingdom), and fractions containing antibody were dialyzed against three changes of PBS. Antibody concentration was determined by bicinchoninic acid assay (Perbio, Tattenhall, United Kingdom) with a bovine serum albumin standard as recommended by the manufacturers. Antibody purity was assessed by SDS-PAGE analysis.
Antibodies were tested in murine models of bubonic and pneumonic plagues. Six- to 8-week-old BALB/c mice were used (Charles River, Ltd., Margate, United Kingdom). Animal experiments were performed in accordance with United Kingdom legislation relating to animal experimentation (Animals [Scientific Procedures] Act 1986).
Mice received antibody by intraperitoneal (i.p.) injection in 100 μl of PBS prior to or after infection as indicated. Y. pestis
strain GB, a fully virulent human isolate, with an estimated MLD of 1 CFU via the subcutaneous (s.c.) route (30
), was used in all challenge experiments. In the bubonic plague model, mice received approximately 10 to 105
MLD resuspended in 100 μl of PBS, by s.c. injection. In the pneumonic plague model, mice were exposed to approximately 100 MLD of airborne bacteria, as described previously (42
). Animals were checked at least twice daily, and deaths were recorded over a 14-day period.