Pneumonic Plague Pathogenesis and Immunity in Brown Norway Rats
2009; Elsevier BV; Volume: 174; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.071168
ISSN1525-2191
AutoresDeborah M. Anderson, Nancy Ciletti, Hanni Lee-Lewis, Derek Elli, Joshua Segal, Kristin L. DeBord, Katie A. Overheim, Maria Tretiakova, Robert R. Brubaker, Olaf Schneewind,
Tópico(s)Zoonotic diseases and public health
ResumoThe Brown Norway rat was recently described as a bubonic plague model that closely mimics human disease. We therefore evaluated the Brown Norway rat as an alternative small animal model for pneumonic plague and characterized both the efficacy and potency of vaccine candidates. When infected by intranasal instillation, these rats rapidly developed fatal pneumonic plague within 2 to 4 days of infection. Plague disease was characterized by severe alveolar edema and vascular hemorrhage in the lung in addition to fulminant necrotizing pneumonia caused by massive bacterial replication and inflammation. Twenty-four hours before death, animals developed systemic disease with an apparent delayed inflammatory response. We evaluated the ability of the protective antigen, LcrV, and a mutant derivative, V10, to protect these rats from pneumonic plague. Both were highly effective vaccines because complete protection was observed at challenge doses of 7500 LD50. Antibody analyses suggested stronger potency of V10 immune sera compared with LcrV in the passive transfer of immunity to bubonic plague, with multiple neutralizing epitopes in LcrV. Taken together, these data demonstrate the effectiveness of inhibiting type III secretion in the prevention of pneumonic plague in rats and reveal critical contributions from both the cellular and humoral immune systems. Thus, the Brown Norway rat is an appealing alternative small animal model for the study of pneumonic plague pathogenesis and immunity. The Brown Norway rat was recently described as a bubonic plague model that closely mimics human disease. We therefore evaluated the Brown Norway rat as an alternative small animal model for pneumonic plague and characterized both the efficacy and potency of vaccine candidates. When infected by intranasal instillation, these rats rapidly developed fatal pneumonic plague within 2 to 4 days of infection. Plague disease was characterized by severe alveolar edema and vascular hemorrhage in the lung in addition to fulminant necrotizing pneumonia caused by massive bacterial replication and inflammation. Twenty-four hours before death, animals developed systemic disease with an apparent delayed inflammatory response. We evaluated the ability of the protective antigen, LcrV, and a mutant derivative, V10, to protect these rats from pneumonic plague. Both were highly effective vaccines because complete protection was observed at challenge doses of 7500 LD50. Antibody analyses suggested stronger potency of V10 immune sera compared with LcrV in the passive transfer of immunity to bubonic plague, with multiple neutralizing epitopes in LcrV. Taken together, these data demonstrate the effectiveness of inhibiting type III secretion in the prevention of pneumonic plague in rats and reveal critical contributions from both the cellular and humoral immune systems. Thus, the Brown Norway rat is an appealing alternative small animal model for the study of pneumonic plague pathogenesis and immunity. Yersinia pestis is the causative agent of pneumonic plague, a disease that has killed more people in recorded history than any other bacterial pathogen. Pneumonic plague can arise either by inhalation of Y. pestis (primary) or as a result of septicemic spread of bacteria during bubonic plague (secondary). Both involve the congestion of the lower respiratory tract, severe hemorrhage, and edema caused by massive bacterial multiplication, and both progress rapidly to death.1Lien-Teh W A Treatise on Pneumonic Plague. League of Nations Health Organization, Geneva1926Google Scholar, 2Pollitzer R Plague. World Health Organization, Geneva1954Google Scholar Infection of the respiratory system by Y. pestis initially proceeds without a high degree of bacterial replication and the bacteria are generally undetectable in the sputum for at least 24 hours.1Lien-Teh W A Treatise on Pneumonic Plague. League of Nations Health Organization, Geneva1926Google Scholar During this time, the patient is noninfectious but may have a fever and rapid pulse. The disease then suddenly and quite rapidly progresses, together with the appearance of high bacterial titers in the sputum and blood. Lungs show severe edema and necrotizing pneumonia. Bacteria escape the lung likely via the vascular system and cause a systemic infection, with hemorrhages throughout the body, including liver, spleen, and heart.1Lien-Teh W A Treatise on Pneumonic Plague. 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This likely dampens innate and adaptive immune responses, but is thought to play only a minor role in virulence.36Reithmeier-Rost D Hill J Elvin S Williamson E Dittmann S Schmidt A Wilharm G Sing A The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis.Microbes Infect. 2007; 9: 997-1002Crossref PubMed Scopus (29) Google Scholar, 37Nakajima R Motin V Brubaker R Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization.Infect Immun. 1995; 63: 3021-3029Crossref PubMed Google Scholar Large amounts of LcrV circulating in the blood of mice are capable of disabling immune responses to some bacterial pathogens,37Nakajima R Motin V Brubaker R Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization.Infect Immun. 1995; 63: 3021-3029Crossref PubMed Google Scholar raising concerns that LcrV may be immune-suppressive when used as a vaccine. This concern is substantiated by the recently reported clinical application of LcrV to deliberately suppress mucosal immunity.38Foligne B Dessein R Marceau M Poiret S Chamaillard M Pot B Simonet M Daniel C Prevention and treatment of colitis with Lactococcus lactis secreting the immunomodulatory Yersinia LcrV protein.Gastroenterology. 2007; 133: 862-874Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar Nevertheless, inclusion of LcrV with F1 in a vaccine formulation produced a highly effective vaccine that protected against bubonic and pneumonic plague in mice and nonhuman primates and two formulations are currently under investigation for human use.39Williamson E Eley S Stagg A Green M Russell P Titball R A single dose sub-unit vaccine protects against pneumonic plague.Vaccine. 2001; 19: 566-571Crossref Scopus (80) Google Scholar, 40Williamson E Eley S Griffin K Green M Russell P Leary S Oyston P Easterbrook T Reddin K Robinson A Titball R A new improved sub-unit vaccine for plague: the basis for protection.FEMS Immunol Med Microbiol. 1995; 12: 223-230Crossref PubMed Google Scholar, 41Williamson E Flick-Smith H LeButt C Rowland C Jones S Waters E Gwyther R Miller J Packer P Irving M Human immune response to a plague vaccine comprising recombinant F1 and V antigens.Infect Immun. 2005; 73: 3598-3608Crossref PubMed Scopus (146) Google Scholar Both vaccines result in the production of high levels of antibodies that neutralize Y. pestis infections, and these antibodies correlate with vaccine efficacy. In addition, each of these vaccines can protect from aerosol challenge with F1 mutant Y. pestis in mice. In all cases, immunity correlates with in vitro assays that demonstrate the ability of LcrV antibodies to block macrophage apoptosis caused by the injection of Yops.14Weeks S Hill J Friedlander A Welkos S Anti-V antigen antibody protects macrophages from Yersinia pestis-induced cell death and promotes phagocytosis.Microb Pathog. 2002; 32: 227-237Crossref PubMed Scopus (67) Google Scholar, 18Heath D Anderson G Mauro M Welkos S Andrews G Adamovicz J Friedlander A Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine.Vaccine. 1998; 16: 1131-1137Crossref PubMed Scopus (237) Google Scholar, 41Williamson E Flick-Smith H LeButt C Rowland C Jones S Waters E Gwyther R Miller J Packer P Irving M Human immune response to a plague vaccine comprising recombinant F1 and V antigens.Infect Immun. 2005; 73: 3598-3608Crossref PubMed Scopus (146) Google Scholar, 42Williamson E Vesey P Gillhespy K Eley S Green M Titball R An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model.Clin Exp Immunol. 1999; 116: 107-114Crossref PubMed Scopus (131) Google Scholar, 43Welkos S Norris S Adamovicz J Modified caspase-3 assay indicates correlation of caspase-3 activity with immunity of nonhuman primates to Yersinia pestis infection.Clin Vaccine Immunol. 2008; 15: 1134-1137Crossref PubMed Scopus (15) Google Scholar Results of phase I clinical trials so far indicate that humans raise antibodies with similar potency and activity against Y. pestis in both animal and in vitro assays, although it has not been formally demonstrated that these antibodies indicate immunity to plague in humans.20Bashaw J Norris S Weeks S Trevino S Adamovicz J Welkos S Development of in vitro correlate assays of immunity to infection with Yersinia pestis.Clin Vaccine Immunol. 2007; 14: 605-616Crossref PubMed Scopus (55) Google Scholar, 44Williamson E Flick-Smith H Waters E Miller J Hodgson I LeButt C Hill J Immunogenicity of the rF1+rV vaccine for plague with identification of potential immune correlates.Microb Pathog. 2007; 42: 11-21Crossref PubMed Scopus (53) Google Scholar Recently we sought to improve the safety and potency of LcrV vaccines by creating mutant variants with decreased immune-suppressive activities. We described a novel candidate called V10, which is a deletion of amino acids 271 to 300 near the carboxyl terminus of LcrV, and this deletion protein induces little to no interleukin-10 secretion from mouse and human phagocytic cells.45Overheim K DePaolo R DeBord K Morrin E Anderson D Green N Brubaker R Jabri B Schneewind O LcrV plague vaccine with altered immunomodulatory properties.Infect Immun. 2005; 73: 5152-5159Crossref PubMed Scopus (90) Google Scholar This derivative was nevertheless found to be highly antigenic and elicited high titers of neutralizing IgG in the blood of vaccinated mice.46DeBord K Anderson D Marketon M Overheim K DePaolo R Ciletti N Jabri B Schneewind O Immunogenicity and protective immunity against bubonic plague and pneumonic plague by immunization of mice with the recombinant V10 antigen, a variant of LcrV.Infect Immun. 2006; 74: 4910-4914Crossref PubMed Scopus (51) Google Scholar In fact, V10 was found to be more potent than LcrV in both protection and antibody titers in the mouse pneumonic plague model. We therefore decided to further investigate its mechanism of action, and to ask whether similar findings would be made in other animal models. We focused our attention on the Brown Norway rat because of close similarities between rat and human bubonic plague.47Sebbane F Gardner D Long D Gowen B Hinnebusch B Kinetics of disease progression and host response in a rat model of bubonic plague.Am J Pathol. 2005; 166: 1427-1439Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar In this study, we describe the Brown Norway rat as a model for pneumonic plague and its use in the evaluation of LcrV plague vaccines. For challenge experiments, Y. pestis CO92 was streaked from frozen stock onto heart infusion agar with Congo Red and grown for 48 hours at 26°C.48Surgalla M Beesley E Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis.Appl Microbiol. 1969; 18: 834-837PubMed Google Scholar A single red colony, indicative of expression of the pgm locus was inoculated in 10 ml of heart infusion broth supplemented with 2.5 mmol/L CaCl2 and grown for 18 to 24 hours at 37°C.49Fetherston J Schuetze P Perry R Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102kb of chromosomal DNA which is flanked by a repetitive element.Mol Microbiol. 1992; 6: 2693-2704Crossref PubMed Scopus (210) Google Scholar Bacteria were diluted in sterile phosphate-buffered saline (PBS) to the desired dose just before use in challenge studies. All experiments performed with Y. pestis CO92 were performed in compliance with the select agent regulations at biosafety level 3. E. coli BL21 (Novagen, San Diego, CA) was used for the overexpression and purification of recombinant LcrV and V10 as previously described.45Overheim K DePaolo R DeBord K Morrin E Anderson D Green N Brubaker R Jabri B Schneewind O LcrV plague vaccine with altered immunomodulatory properties.Infect Immun. 2005; 73: 5152-5159Crossref PubMed Scopus (90) Google Scholar Six- to eight-week-old (200 g) female Brown Norway rats were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed in BSL3 containment at least 3 days before challenge. For challenge, groups of four to six animals were anesthetized with ketamine/xylazine, and bacteria prepared as described above, were instilled by pipetting into the left naris of the anesthetized animal. Infected animals were observed for recovery from anesthesia and for the development of signs of lethal plague disease for a period of 14 days. After the observation period, remaining animals were humanely euthanized by CO2 asphyxiation, a method approved by the Panel on Euthanasia of the American Veterinary Medical Association. Euthanized animals were necropsied and blood drawn by cardiac puncture. For histochemical analyses, lungs were perfused in 10% formalin and tissues were fixed in 10% formalin for 48 hours. Sections of the left lower lobe were submitted for tissue sectioning, staining with hematoxylin and eosin (H&E), and pathological analysis. For bacterial enumeration, whole tissues were homogenized in sterile PBS and plated on heart infusion agar with Congo Red. All studies conformed to guidelines set forth by the National Institutes of Health and were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Chicago. A 50% lethal dose of 171 cfu, as calculated according to the method of Reed and Muench,50Reed L Muench H A simple method of estimating fifty percent endpoints.Am J Hyg. 1938; 27: 493-497Google Scholar was used to calculate challenge dose for disease and vaccine experiments. Tissues that had been fixed in 10% formalin as described above were sectioned on slides for immunohistochemical analysis. Slides were stained with mouse anti-rat CD68 (MorphoSys, Oxford, UK) or rabbit anti-cleaved caspase-3 (Trevigen, Gaithersburg, MD) according to the manufacturer's protocol. Detection of CD68 was achieved by secondary staining with biotinylated polyclonal rabbit anti-mouse IgG (DAKO, Carpinteria, CA), and detection of activated caspase-3 was achieved by secondary staining with polyclonal anti-rabbit IgG conjugated to horseradish peroxidase (HRP). Staining and detection were performed according to the manufacturer's guidelines. Fifty μg of recombinant protein was emulsified in 25% Alhydrogel (Brenntag Biosector, Frederikssund, Denmark) (v/v) and delivered by intramuscular injection into the leg of the animals. Animals were boosted with an equal dose of antigen emulsified in 25% Alhydrogel on day 21. On day 42, five animals per study group were deeply anesthetized and blood for antibody analysis was withdrawn by cardiac puncture followed by euthanizing the animals by CO2 asphyxiation. Immune sera from five rats per group were collected by cardiac puncture from animals immunized as described above. Individual serum was pooled, and IgG purified by filtration through the Melon purification kit (Pierce, Rockford, IL). Purified IgG, 400 μg, 200 μg, or 100 μg, was delivered by intraperitoneal injection to each of 10 BALB/c mice (female, 6 to 8 weeks of age). After 60 minutes, mice were challenged with 20 MLD of Y. pestis CO92 by subcutaneous injection (corresponds to 20 cfu bacteria and ∼90 to 95% lethality). Infected mice were monitored for 14 days for signs of lethal bubonic plague, including severe weight loss of >25% body mass and immobility when stimulated. The mouse protection index (MPI) was calculated as the number of susceptible mice divided by their mean time to death. These data were then normalized by dividing the sample MPI by the PBS control MPI. Neutralizing activity was concluded if the normalized value was less than 0.8 because this was the maximum variability observed in control mice between experiments. Statistical significance of time to death was evaluated by unpaired Student's t-test; significance was concluded when P < 0.05. Micro wells were coated with recombinant antigen (LcrV, V10, or CaF1) at 1 μg/ml in carbonate buffer and incubated overnight at 4°C. After washing three times with PSB-Tween, plates were incubated in PBS/1% bovine serum albumin (BSA) for 1 hour at room temperature to block nonspecific binding to wells. Dilutions of rat sera were made in PBS/1% BSA solution and 100 μl of each sample was added to the well in triplicate and incubated overnight at 4°C. Plates were washed three times with PBS/BSA. Biotinylated secondary anti-rat IgG, IgG1, or IgG2a (BD Biosciences, San Jose, CA) was then diluted in PBS/1% BSA, added to the wells, and incubated for 1 hour. Detection was performed after incubation with streptavidin-alkaline phosphatase according to the manufacturer's recommendations, and binding measured at 450 nm in a plate reader. For antibody epitope mapping, peptides conjugated to keyhole-limpet hemocyanin were used at 1 μg/ml to coat ELISA plates. Sera from immunized rats were used as primary antibodies and incubated with antigen for 2 hours at room temperature. Anti-rat IgG conjugated to HRP was used to detect binding to the peptides according to the manufacturer's recommendations. One μg/ml of recombinant LcrV was bound to a 96-well plate and incubated overnight at 4°C. After washing, plates were incubated in blocking buffer (PBS with 5% BSA); then 0.2 μg/ml of LcrV mAb BA-5 was added to each well and incubated at 4°C for 16 hours. Rat immune sera or rat serum from adjuvant-only immunized animals were diluted 1:3 in PBS with 5% BSA in duplicate. Diluted serum (0.1 ml) was added to the plates and incubated for 1 hour at 37°C and then washed three times in PBS, followed by the addition of HRP-goat anti-mouse IgG. Binding of LcrV mAb BA-5 was detected according to the manufacturer's instructions and measured in a plate reader at 450 nm. Absorbance was adjusted for background binding and per
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