Plague and the Fall of Baghdad (1258)

The recent suggestion that the late medieval Eurasian plague pandemic, the Black Death, had its origins in the thirteenth century rather than the fourteenth century has brought new scrutiny to texts reporting ‘epidemics’ in the earlier period. Evidence both from Song China and Iran suggests that plague was involved in major sieges laid by the Mongols between the 1210s and the 1250s, including the siege of Baghdad in 1258 which resulted in the fall of the Abbasid caliphate. In fact, re-examination of multiple historical accounts in the two centuries after the siege of Baghdad shows that the role of epidemic disease in the Mongol attacks was commonly known among chroniclers in Syria and Egypt, raising the question why these outbreaks have been overlooked in modern historiography of plague. The present study looks in detail at the evidence in Arabic sources for disease outbreaks after the siege of Baghdad in Iraq and its surrounding regions. We find subtle factors in the documentary record to explain why, even though plague received new scrutiny from physicians in the period, it remained a minor feature in stories about the Mongol invasion of western Asia. In contemporary understandings of the genesis of epidemics, the Mongols were not seen to have brought plague to Baghdad; they caused plague to arise by their rampant destruction. When an even bigger wave of plague struck the Islamic world in the fourteenth century, no association was made with the thirteenth-century episode. Rather, plague was now associated with the Mongol world as a whole.     

Acquisition of Maternal Antibodies both from the Placenta and by Lactation Protects Mouse Offspring from Yersinia pestis Challenge

Artificially passive immunization has been demonstrated to be effective against Yersinia pestis infection in animals. However, maternal antibodies'' protective efficacy against plague has not yet been demonstrated. Here, we evaluated the kinetics, protective efficacy, and transmission modes of maternal antibodies, using mice immunized with plague subunit vaccine SV1 (20 μg of F1 and 10 μg of rV270). The results showed that the rV270- and F1-specific antibodies could be detected in the sera of newborn mice (NM) until 10 and 14 weeks of age, respectively. There was no antibody titer difference between the parturient mice immunized with SV1 (PM-S) and the caesarean-section newborns (CSN) from the PM-S or between the lactating mice immunized by SV1 (LM-S) and the cross-fostered mice (CFM) during 3 weeks of lactation. The NM had a 72% protection against 4,800 CFU Y. pestis strain 141 challenge at 6 weeks of age, whereas at 14 weeks of age, NM all succumbed to 5,700 CFU of Y. pestis challenge. After 7 weeks of age, CFM had an 84% protection against 5,000 CFU of Y. pestis challenge. These results indicated that maternal antibodies induced by the plague subunit vaccine in mother mice can be transferred to NM by both placenta and lactation. Passive antibodies from the immunized mothers could persist for 3 months and provide early protection for NM. The degree of early protection is dependent on levels of the passively acquired antibody. The results indicate that passive immunization should be an effective countermeasure against plague during its epidemics.     

Impact of the Pla Protease Substrate α2-Antiplasmin on the Progression of Primary Pneumonic Plague

Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection.     

A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague

The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD₅₀. Intranasal infection of mice with 15 LD₅₀ of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24–72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of antimicrobial countermeasures in real time.     

Intranasal administration of an inactivated Yersinia pestis vaccine with interleukin-12 generates protective immunity against pneumonic plague

Inhalation of Yersinia pestis causes pneumonic plague, which rapidly progresses to death. A previously licensed killed whole-cell vaccine is presently unavailable due to its reactogenicity and inconclusive evidence of efficacy. The present study now shows that vaccination intranasally (i.n.) with inactivated Y. pestis CO92 (iYp) adjuvanted with interleukin-12 (IL-12) followed by an i.n. challenge with a lethal dose of Y. pestis CO92 prevented bacterial colonization and protected 100% of mice from pneumonic plague. Survival of the vaccinated mice correlated with levels of systemic and lung antibodies, reduced pulmonary pathology and proinflammatory cytokines, and the presence of lung lymphoid cell aggregates. Protection against pneumonic plague was partially dependent upon Fc receptors and could be transferred to naïve mice with immune mouse serum. On the other hand, protection was not dependent upon complement, and following vaccination, depletion of CD4 and/or CD8 T cells before challenge did not affect survival. In summary, the results demonstrate the safety, immunogenicity, and protective efficacy of i.n. administered iYp plus IL-12 in a mouse model of pneumonic plague.     

Gr1+ Cells Control Growth of YopM-Negative Yersinia pestis during Systemic Plague

YopM, a protein toxin of Yersinia pestis, is necessary for virulence in a mouse model of systemic plague. We previously reported YopM-dependent natural killer (NK) cell depletion from blood and spleen samples of infected mice. However, in this study we found that infection with Y. pestis KIM5 (YopM⁺) caused depletion of NK cells in the spleen, but not in the liver, and antibody-mediated ablation of NK cells had no effect on bacterial growth. There was no YopM-associated effect on the percentage of dendritic cells (DCs) or polymorphonuclear leukocytes (PMNs) in the early stage of infection; however, there was a YopM-associated effect on PMN integrity and on the influx of monocytes into the spleen. Ablation of Gr1⁺ cells caused loss of the growth defect of YopM⁻ Y. pestis in both the liver and spleen. In contrast, ablation of macrophages/DCs inhibited growth of both parent and mutant bacteria, accompanied by significantly fewer lesion sites in the liver. These results point toward PMNs and inflammatory monocytes as major cell types that control growth of YopM⁻ Y. pestis. Infection with fully virulent Y. pestis CO92 and a YopM⁻ derivative by intradermal and intranasal routes showed that the absence of YopM significantly increased the 50% lethal dose only in the intradermal model, suggesting a role for YopM in bubonic plague, in which acute inflammation occurs soon after infection.Yersinia pestis, the infective agent of bubonic and pneumonic plague, has caused widespread loss of human life during recurrent pandemics. Y. pestis still infects rodent populations in large geographic zones where Y. pestis is endemic, and there are cases of human plague reported annually (15, 19, 56). Y. pestis and the closely related food-borne pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica share a 70-kb plasmid carrying genes that encode a major set of proteins involved in pathogenic properties that compromise the host immune system (60). These include a type 3 secretion system (T3SS) that at mammalian body temperature delivers a set of six Yersinia outer protein (Yop) effector proteins into host cells once the bacteria contact host target cells. Enzymatic and cell biological mechanisms of five of the Yops, YopH, YopE, YopT, YpkA/YopO, and YopJ, have been elucidated. YopJ interferes with signal transduction through acetyltransferase activity but is not required for virulence in either a mouse model of systemic plague (57) or mouse and rat models of bubonic plague (28, 65). In tissue culture infection models, YopH, YopE, YopT, and YopO have been shown to antagonize focal complex formation and activity of Rho family GTPases and synergistically inhibit phagocytosis by mammalian cells. YopH and YopE have been shown to be crucial for lethality in a mouse model of systemic plague (intravenous [i.v.] infection), and a ΔyopH strain is attenuated for both bubonic and pneumonic plague (9). In addition, Y. pestis virulence proteins, such as the surface fibrils F1 and PsaA, have antiphagocytic effects and also have been found to contribute to virulence in systemic plague (7, 31). Accordingly, Y. pestis is believed to exist predominantly in an extracellular location in vivo, although initially the bacteria might invade resting tissue macrophages (Mφs) and dendritic cells (DCs), based on assays of mouse spleens in the systemic phase of bubonic plague (33). The intracellular versus extracellular locations of Y. pestis during the peripheral phases of plague on skin or in the lung have not yet been studied.It is believed that tissue Mφs, DCs, and polymorphonuclear leukocytes (PMNs) are early target cells for Yop delivery in vivo, because these cells are present before or soon after infection begins and function to initiate the innate defenses that are undermined by Yops. Consistent with this hypothesis, Y. pestis has been found in association with alveolar Mφs early during lung infection of mice (6) and likewise in association with Mφs, DCs, and PMNs in the spleens of mice infected i.v., and YopM can be injected into these cells (34). However, it is becoming clear that spleens and lungs present distinctly different inflammatory environments when infected by Y. pestis, with PMNs migrating rapidly into spleens infected by the i.v. route but not appearing in lungs until 36 h after intranasal (i.n.) infection (27, 58). Accordingly, some virulence properties required for lethality of systemic plague are not required in pneumonic plague. Examples are the capsular fibril F1 and the antiphagocytic adhesin PsaA (7, 11, 12). There is no information yet on the target cells or relative importance of Yops other than YopH in pneumonic or bubonic plague.The sixth effector Yop, YopM, is essential for virulence in the mouse model of systemic plague: in C57BL/6 mice, a YopM⁻ strain of Y. pestis KIM5 is reduced in lethality by at least 4 orders of magnitude (29). However, the function of YopM has not been defined. YopM is a 46.2-kDa acidic protein made up almost entirely of 15 repeats of a 19-residue leucine-rich repeat motif (30). The YopM monomer is horseshoe shaped and has the potential to form tetramers in which the monomers stack together to form a hollow cylinder; however, the form that YopM assumes within the mammalian cell is not known (16). After delivery to the host cell cytoplasm, YopM localizes to the nucleus in a process that is facilitated by vesicular trafficking (53). YopM was reported to form a complex with the serine/threonine kinases PRK2 (protein kinase C-related kinase 2) and RSK1 (90-kDa ribosomal S6 kinase) in HEK293 cells infected with Y. pseudotuberculosis (36), leading to activation of both kinases. However, the biological significance of this complex is not known. There is no visible effect of delivery of YopM into cultured cells, and microarray analysis of Mφ-like cell lines infected with Y. enterocolitica having or lacking YopM also has not yielded any clue to YopM''s mechanism of action (21, 50).Because these and other in vitro approaches to defining the pathogenic mechanism of YopM have not been fruitful, we have begun to characterize YopM''s effects in vivo. Previously we found that YopM was still required for lethality in i.v. infected SCID mice, showing that YopM''s virulence mechanism does not require B or T cells and indicating that early in systemic plague, YopM''s main function is to counteract a component of innate immunity (25). A striking YopM-specific effect during systemic plague in wild-type C57BL/6 mice was the depletion of natural killer (NK) cells from the spleen and a reduction of NK cell numbers in blood, suggesting that YopM might cause a loss of the NK cell compartment during systemic plague. Correlated with this effect, there was a YopM-associated loss of mRNA for gamma interferon (IFN-γ) by NK cells in infected spleens and diminished expression of mRNAs in splenic Mφs for cytokines that are required for viability and activation of NK cells (interleukin 15 [IL-15], IL-18, and IL-12). These findings supported the hypothesis that YopM may function to inhibit IFN-mediated activation of Mφs through the depletion of NK cells (25).In this study, we tested the hypothesis that NK cells are critical for controlling Y. pestis pathogenesis early in plague and that NK cells are the primary target of YopM. We characterized infection dynamics and leukocyte populations in both the liver and spleen during systemic plague in mice ablated for Mφs/DCs or for Gr1⁺ cells. The data point to PMNs and inflammatory monocytes as critical cells affected by YopM and to Mφs/DCs as an important early reservoir for bacterial growth. Consistent with a role in undermining acute inflammation, YopM was found to be important for the lethality of bubonic plague, but not pneumonic plague.     

Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1⁻ strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1⁻ mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1⁻ CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.     

Correlation between TS phenotype and pathogenicity of some animal viruses

Summary The pathogenicity of seven ts mutants of poliovirus belonging to 4 functional groups and of five ts mutants of fowl plague virus belonging to 5 complementation groups was studied. Among ts mutants of poliovirus five had lost their pathogenicity for monkeys, one showed a considerably reduced pathogenicity and one had retained it practically completely. Among ts mutants of fowl plague virus three had lost their pathogenicity for chickens to a considerable extent and two had retained it completely. The viruses isolated from the organs of the animals infected with the ts mutants retaining their pathogenicity had ts⁺ phenotype. No correlation has been found between the appurtenance of ts mutants to certain functional or complementation groups and degree of pathogenicity. It is suggested, however, that mutational changes in different cistrons of the genome of the viruses tested leading to development of the ts phenotype are, indeed, accompanied by reduction in pathogenicity and that retained pathogenicity in some mutants seems to be due to ts⁺ reversion.     

Deletion of Braun Lipoprotein and Plasminogen-Activating Protease-Encoding Genes Attenuates Yersinia pestis in Mouse Models of Bubonic and Pneumonic Plague

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4⁺ and CD8⁺ T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.     

SVM-based computer-aided diagnosis of the Alzheimer's disease using t-test NMSE feature selection with feature correlation weighting

This letter shows a computer-aided diagnosis (CAD) technique for the early detection of the Alzheimer's disease (AD) based on single photon emission computed tomography (SPECT) image feature selection and a statistical learning theory classifier. The challenge of the curse of dimensionality is addressed by reducing the large dimensionality of the input data and defining normalized mean squared error features over regions of interest (ROI) that are selected by a t-test feature selection with feature correlation weighting. Thus, normalized mean square error (NMSE) features of cubic blocks located in the temporo-parietal brain region yields peak accuracy values of 98.3% for almost linear kernel support vector machine (SVM) defined over the 20 most discriminative features extracted. This new method outperformed recent developed methods for early AD diagnosis.     

Allergen-specific pattern recognition receptor pathways

Allergic diseases continue to plague modernized societies, underscoring the need to identify the molecular basis for the propensity of a small number of environmental proteins to provoke maladaptive, allergic responses. Recent data suggest that the ability of allergenic proteins to drive allergic responses in susceptible hosts is driven by their unique innate immune activating capabilities. Although the identification of allergen-specific pattern recognition receptors is in its infancy, studies to date have shown that allergens drive Th2-biased immune responses via directly engaging C-type lectin receptors (dectin-2, DC-SIGN, and mannose receptor) on dendritic cells and/or mimicking toll-like receptor 4 signaling complex molecules expressed on airway structural cells. Elucidation of the specific innate immune pathways activated by allergens holds great promise in defining new therapeutic targets for the treatment of allergic diseases.     

The smpB-ssrA Mutant of Yersinia pestis Functions as a Live Attenuated Vaccine To Protect Mice against Pulmonary Plague Infection

The bacterial SmpB-SsrA system is a highly conserved translational quality control mechanism that helps maintain the translational machinery at full capacity. Here we present evidence to demonstrate that the smpB-ssrA genes are required for pathogenesis of Yersinia pestis, the causative agent of plague. We found that disruption of the smpB-ssrA genes leads to reduction in secretion of the type III secretion-related proteins YopB, YopD, and LcrV, which are essential for virulence. Consistent with these observations, the smpB-ssrA mutant of Y. pestis was severely attenuated in a mouse model of infection via both the intranasal and intravenous routes. Most significantly, intranasal vaccination of mice with the smpB-ssrA mutant strain of Y. pestis induced a strong antibody response. The vaccinated animals were well protected against subsequent lethal intranasal challenges with virulent Y. pestis. Taken together, our results indicate that the smpB-ssrA mutant of Y. pestis possesses the desired qualities for a live attenuated cell-based vaccine against pneumonic plague.Plague, a dangerous and often deadly disease, is caused by a Gram-negative bacterium, Yersinia pestis (38, 39). Depending on the route of entry, the disease can develop into a variety of forms, such as bubonic, pneumonic, or septicemic plague. Pneumonic plague is considered the most dangerous form of the disease since the organism can disseminate through aerosol droplets, resulting in high mortality. In fact, these features have led to the classification of Y. pestis as a category A agent of bioterrorism (24). Antibiotic therapy can be effective upon early diagnosis of plague. However, the appearance of multidrug-resistant Y. pestis strains in recent years presents a challenge for currently available antibiotic therapy (39). Therefore, there is a need for a safe and effective plague vaccine, which is currently not available.Animal infection studies have identified several antigens that could be used as recombinant subunit vaccines. These include the F1 antigen and the LcrV protein. Active or passive immunization of experimental animals with these antigens was shown to be protective against pneumonic plague (1-3, 18, 23). However, F1⁻ mutants of Y. pestis have been reported to retain full virulence in animal infection studies (15, 41, 52). Also, animals immunized with the LcrV protein can still be susceptible to Yersinia infections due to the variations in LcrV protein (44). Such strains could circumvent the effectiveness of subunit vaccines. Therefore, inclusion of additional elements, such as additional antigens or a library of antigens, could provide better protection against genetically engineered, fully virulent Y. pestis strains. One way to present many antigens at once is to utilize killed or live attenuated Y. pestis organisms. The use of heat-killed or formalin-fixed Y. pestis has a long history as a plague vaccine, and they were shown to be effective against bubonic plague (46). However, these vaccines have also caused significant adverse reactions, such as fever, malaise, headache, and lymphadenopathy. In addition, immunization with heat- or formalin-killed bacteria has generally failed to protect experimental animals against pneumonic plague (46). On the other hand, live attenuated plague vaccines, such as one based on the Y. pestis EV76 strain, appeared to be protective against pneumonic plague (46, 49, 53). Such genetically undefined strains can be unstable and retain significant virulence. Therefore, there is still a need to identify novel attenuated Y. pestis strains that can be used in production of safe and effective vaccines against all forms of plague.SsrA is a unique RNA molecule that performs an important quality control function in collaboration with its protein partner, SmpB (17). SsrA RNA functions as both tRNA and mRNA through its unique sequence and structural properties. The SmpB-SsrA function is required to deal with ribosomes stalled on defective mRNAs (27, 28). The smpB and ssrA genes are present in all bacteria examined to date (21, 28, 51). The SmpB-SsrA system is important for maintaining cellular homeostasis and for survival of bacteria under adverse conditions. Unfortunately, there are only a few studies examining the contribution of this system to bacterial pathogenesis. Previous reports showed that the SmpB-SsrA system plays a critical role in Salmonella pathogenesis through controlling the expression of virulence factors and improving the ability of this organism to survive within macrophages (6, 26). More recently we showed that the smpB-ssrA mutant of Yersinia pseudotuberculosis was avirulent in a mouse infection model (34). Based on this evidence, we investigated the importance of smpB-ssrA in Y. pestis pathogenesis and the possibility of using its mutants as a live cell-based plague vaccine. Our results show that the smpB-ssrA mutant of Y. pestis is severely attenuated in a mouse model of infection. Most importantly, mice vaccinated with this mutant are protected against pulmonary Y. pestis infection.     

Characteristics of an influenza mutant temperature-sensitive for viral RNA synthesis

A ts mutant of fowl plague virus has been isolated after mutagenesis with 5-fluorouracil, which is temperature-sensitive in viral RNA synthesis. By temperature shift experiments it was demonstrated that the synthesis of viral particle RNA as well as complementary RNA is inhibited in vivo at the nonpermissive temperature (40 °), although the viral RNA polymerase could be detected by an in vitro test and was active in vitro at the nonpermissive temperature. When infected cells were incubated at the permissive temperature (33 °) for 3 hr and then shifted to 40 °, almost normal yields of viral proteins and activities including infectious particles were found.     

Protective Immunity in Mice Achieved with Dry Powder Formulation and Alternative Delivery of Plague F1-V Vaccine

The potential use of Yersinia pestis as a bioterror agent is a great concern. Development of a stable powder vaccine against Y. pestis and administration of the vaccine by minimally invasive methods could provide an alternative to the traditional liquid formulation and intramuscular injection. We evaluated a spray-freeze-dried powder vaccine containing a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted spray-freeze-dried F1-V powder via intramuscular injection, microneedle-based intradermal delivery, or noninvasive intranasal administration. By intramuscular injection, the reconstituted powder induced serum antibody responses and provided protection against lethal subcutaneous challenge with 1,000 50% lethal doses of Y. pestis at levels equivalent to those elicited by unprocessed liquid formulations (70 to 90% protection). The feasibility of intradermal and intranasal delivery of reconstituted powder F1-V vaccine was also demonstrated. Overall, microneedle-based intradermal delivery was shown to be similar in efficacy to intramuscular injection, while intranasal administration required an extra dose of vaccine to achieve similar protection. In addition, the results suggest that seroconversion against F1 may be a better predictor of protection against Y. pestis challenge than seroconversion against either F1-V or V. In summary, we demonstrate the preclinical feasibility of using a reconstituted powder F1-V formulation and microneedle-based intradermal delivery to provide protective immunity against plague in a mouse model. Intranasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and a powder vaccine formulation may result in substantial health and economic benefits.Plague caused by gram-negative Yersinia pestis is one of the most deadly infectious diseases of animals and humans. Outbreaks of plague have caused the deaths of millions of people throughout human history. The pneumonic form of plague is the most dangerous, due to rapid onset and progression and the aerosol spread of the disease. Without proper early treatment, pneumonic plague can lead to mortality in close to 100% of cases (36).Throughout recorded human history, there were three plague pandemics that caused countless human deaths (20). The use of plague as a weapon also has a long history. Starting from the 12th century, there were many cases of using the bodies of plague victims to defeat enemies during wars. Most recently, during World War II, the Japanese army dropped Y. pestis-infected fleas over populated areas of China, causing plague outbreaks (20).Plague has been classified by the U.S. Centers for Disease Control and Prevention as a “category A” agent due to its potential threat to national security. Currently, however, there is no commercially available vaccine against plague approved for human use in the United States. The original plague vaccine licensed for use in the United States was a killed whole-cell bacterial vaccine. It provided some protection against bubonic plague, but not against aerosol exposure to Y. pestis (6, 10, 33, 40). Manufacture of the vaccine ceased in 1998 due to multiple side effects and short-term effectiveness.The potential use of Y. pestis as a bioweapon, combined with the threat of antibiotic-resistant plague (13), makes the development of a safe and effective human plague vaccine a high priority. Conventional vaccines are formulated as liquids, which generally require refrigeration for storage and distribution. In recent years, there has been growing interest in powder formulations for extended storage stability and increased shelf life (5, 12, 19). We previously reported intranasal (i.n.) delivery of dry powder influenza vaccine in a rat model (17). The influenza vaccine powder formulation was shown to be more stable than the liquid vaccine and to induce increased systemic and nasal mucosal immune responses. We also reported that rabbits immunized nasally with a spray-freeze-dried (SFD) anthrax recombinant protective antigen (rPA) powder vaccine formulation were completely protected against lethal inhalational anthrax, while a liquid preparation of the same vaccine provided only 63 to 67% protection by the nasal route (18, 28).The most common vaccine delivery route is intramuscular (i.m.) injection. Alternative delivery methods, such as the intradermal (i.d.) and i.n. routes, have attracted attention recently. The skin is a favorable site for vaccine delivery due to a rich population of antigen-presenting cells. I.d. delivery of vaccines has been shown to provide dose-sparing effects for rabies, hepatitis B, influenza, and rPA vaccines (1, 7, 8, 24, 28, 37). I.n. delivery has been shown to induce mucosal immunity (17, 31). In addition, it offers the advantages of noninvasive delivery and ease of use, reducing the need for highly trained health personnel to administer the vaccine.In the current study, we evaluated the immunogenicity and protective efficacy of an SFD powder vaccine formulation of a recombinant F1-V fusion protein of Y. pestis in a mouse model. The recombinant fusion protein was composed of the F1 subunit, a capsule protein of Y. pestis, and the low-calcium-response V subunit (LcrV), or V antigen. LcrV caps the tips of the injectisome needles of the type III secretion system (30).     

Cynomolgus macaque model for pneumonic plague

A recombinant vaccine (rF1V) is currently being developed for protection against pneumonic plague. An essential component in evaluating efficacy of the rF1V vaccine is the development of a well-understood animal model that shows similarity to human disease. The objective of this study was to determine the inhaled median lethal dose (LD₅₀), evaluate the pathophysiology of disease and identify appropriate study endpoints in a cynomolgus macaque (CM) model of pneumonic plague. Eighteen CMs were challenged by head-only aerosol exposure with seven dosages of Yersinia pestis CO92. An LD₅₀ of 24 colony forming units was estimated using Probit analysis. Disease pathology was evaluated by blood culture, clinical pathology, histopathology and telemetry. CMs that died became febrile following challenge and died 34–92 h after onset of fever. Bacteremia, increased respiration and heart rate, decreased blood pressure and loss of diurnal rhythm were also observed in conjunction with onset of fever. Histopathological examinations revealed significant findings in the lungs (intra-alveolar neutrophils and fibrinous pleuritis) consistent with pneumonic plague. These data indicate that the disease pathology observed in CMs following aerosol exposure to Y. pestis CO92 is similar to that of pneumonic plague in humans. Thus, the CM is an appropriate model to evaluate efficacy of a recombinant F1V vaccine candidate.     

Humoral and Cellular Immune Responses to Yersinia pestis Infection in Long-Term Recovered Plague Patients

Plague is one of the most dangerous diseases and is caused by Yersinia pestis. Effective vaccine development requires understanding of immune protective mechanisms against the bacterium in humans. In this study, the humoral and memory cellular immune responses in plague patients (n = 65) recovered from Y. pestis infection during the past 16 years were investigated using a protein microarray and an enzyme-linked immunosorbent spot assay (ELISpot). The seroprevalence to the F1 antigen in all recovered patients is 78.5%. In patients infected more than a decade ago, the antibody-positive rate still remains 69.5%. There is no difference in the antibody presence between gender, age, and infected years, but it seems to be associated with the F1 antibody titers during infection (r = 0.821; P < 0.05). Except F1 antibody, the antibodies against LcrV and YopD were detected in most of the patients, suggesting they could be the potential diagnostic markers for detecting the infection of F1-negative strains. Regarding cellular immunity, the cell number producing gamma interferon (IFN-γ), stimulated by F1 and LcrV, respectively, in vitro to the peripheral blood mononuclear cells of 7 plague patients and 4 negative controls, showed no significant difference, indicating F1 and LcrV are not dominant T cell antigens against plague for a longer time in humans. Our findings have direct implications for the future design and development of effective vaccines against Y. pestis infection and the development of new target-based diagnostics.     

Biosafety Level 2 Model of Pneumonic Plague and Protection Studies with F1 and Psa

Attenuated Yersinia pestis pgm strains, such as KIM5, lack the siderophore yersiniabactin. Strain KIM5 does not induce significant pneumonia when delivered intranasally. In this study, mice were found to develop pneumonia after intranasal challenge with strain KIM5 when they were injected intraperitoneally with iron dextran, though not with iron sulfate. KIM5-infected mice treated daily with 4 mg iron dextran died in 3 days with severe pneumonia. Pneumonia was less severe if 4 mg iron dextran was administered only once before infection. The best-studied experimental vaccine against plague currently consists of the Yersinia pestis capsular antigen F1 and the type 3 secreted protein LcrV. The F1 antigen was shown to be protective against KIM5 infections in mice administered iron dextran doses leading to light or severe pneumonia, supporting the use of an iron dextran-treated model of pneumonic plague. Since F1 has been reported to be incompletely protective in some primates, and bacterial isolates lacking F1 are still virulent, there has been considerable interest in identifying additional protective subunit immunogens. Here we showed that the highly conserved Psa fimbriae of Y. pestis (also called pH 6 antigen) are expressed in murine organs after infection through the respiratory tract. Studies with iron dextran-treated mice showed that vaccination with the Psa fimbrial protein together with an adjuvant afforded incomplete but significant protection in the mouse model described. Therefore, further investigations to fully characterize the protective properties of the Psa fimbriae are warranted.Yersinia pestis, a Gram-negative bacterium belonging to the family Enterobacteriaceae, is the causative agent of plague, a disease that affects a variety of mammalian hosts and that can be transmitted by various arthropod vectors. Humans are susceptible to Y. pestis, whether transmitted by aerosol or by infected fleas, causing the highly lethal pneumonic plague or bubonic plague, respectively (53). Aerosol infections by Y. pestis remain a concern for public health (12). The only currently available tools for combating Y. pestis in the United States are antibiotics, since manufacture of the previously licensed formalin-killed Y. pestis vaccine that was used by the U.S. military during the Vietnam War (41) stopped in 1998. Although effective against bubonic plague, this vaccine was not efficiently protective against pneumonic plague and caused significant side effects (11). The emergence of multiple antimicrobial resistance in Y. pestis (72) and the lack of a safe and effective vaccine have been the impetus for the search for new inhibitory drugs as well as a better vaccine (48, 49).Y. pestis has been classified as a Category A select agent (8) requiring special precautions, particularly because of the risk of pneumonic plague due to bacterial aerosolization. One of the major impediments to studying the biology and pathogenesis of Y. pestis is that many laboratories do not have access to the required biosafety level 3 (BSL-3) facilities and therefore must work with attenuated strains that were excluded from the CDC select-agents list. Such strains lack either the pgm locus (e.g., KIM5) or the virulence plasmid pCD1 (51). A major virulence component of the pgm locus is the yersiniabactin (Ybt siderophore)-dependent iron transport system, which is essential for plague infection from peripheral sites (52). The pCD1 plasmid encodes a type III secretion system (T3SS, or Yops regulon) that, upon contact with host cells (or in low calcium concentrations in vitro), produces an elaborate injection machinery used to transfer a set of effector proteins with potent antiphagocytic and/or anti-inflammatory effects (2, 67) directly to the cytosol of the eukaryotic cell. Mutants lacking pgm or pCD1 are safer to manipulate, allowing experimental studies to be carried out in BSL-2 laboratories. However, the disease progression elicited by these strains does not recapitulate that observed with fully virulent strains (44), and in comparison to the wild-type strain, these strains are highly attenuated by any natural route of infection. Specifically, the Δpgm strain KIM5 is attenuated when given subcutaneously (s.c.) (52). Similarly, another Δpgm derivative of the virulent strain KIM, strain D27 (KIM D27), was unable to grow to high numbers in the lungs of intranasally infected mice, suggesting that bacterial growth was contained by the host''s innate immune system. Moreover, these mice did not develop significant pneumonia, as they would if infected with wild-type Y. pestis strains (33). However, strain KIM D27 was still able to spread and grow in spleens and livers. This suggested that the iron uptake system of the pgm locus was needed to counteract iron restriction in the lungs. Interestingly, the results of earlier studies suggested that parenteral administration of iron sulfate to mice could bypass the need for yersiniabactin, rendering a parenterally administered Y. pestis Δpgm mutant fully virulent (10). However, a recent study showed that the use of the same approach to induce pneumonic plague after intranasal (i.n.) administration of pgm strain KIM D27 was not successful (33). This study also highlighted the toxicity of ferrous chloride, limiting administration doses to 0.5 mg per mouse. The latter result was consistent with the early toxicity data for inorganic iron at 30 to 60 mg/kg of body weight (26).The toxicity problem of inorganic iron and the need for iron treatments for various medical conditions led to the development of less-toxic colloids consisting of various carbohydrates with ferric (oxy)hydroxide (16). In agreement with the results of earlier studies with inorganic iron, intraperitoneal (i.p.) administration of colloidal iron to mice subsequently infected subcutaneously with a Y. pestis pgm strain resulted in death, with histological lesions in the livers and spleens resembling those resulting from infection with a virulent strain (68). A variety of more-recent models of bacterial infections were developed by taking advantage of the extremely low toxicity of the colloid iron dextran, administrations of which in the milligram range were shown to be nontoxic (29, 62, 75). Based on the success of these models, we undertook experiments to determine whether the administration of high doses of iron dextran would permit Y. pestis to grow in the lungs of i.n. infected mice and cause local inflammation. Here we describe the development of a BSL-2 pneumonic plague model in mice based on the administration of iron dextran. In contrast to the negative results obtained with iron chloride, the use of iron dextran has allowed us to bypass toxicity problems and to take advantage of its favorable pharmacokinetic properties, such as slower clearance and a longer half-life for iron than those with other colloidal or inorganic iron preparations (16).Our model was tested by confirming the expected protective property of F1 in pneumonic plague. We also used the model to evaluate the potential protective property of the Psa fimbriae (pH 6 antigen). Both F1 and Psa are surface structures that share the structural properties of homopolymeric fimbriae, each formed by a protein subunit that requires its own chaperone and usher proteins for export. Structural studies have classified F1 and Psa within a family of fimbrial polyadhesins assembled by FGL chaperones (for their long F1 and G1 β-strands) (76). The F1 capsular antigen is an efficient immunogen that is protective by itself in a murine model of pneumonic plague with F1⁺ strains (4). The protective property of the F1 immunogen can be attributed in great part to its polymeric structure and in vivo surface exposure. Similarly, mixtures of fimbriae and surface-exposed adhesins of other pathogens have been used successfully as commercial vaccine components for both humans and animals (17, 43, 54). Surface-expressed antigens such as F1 induce opsonizing and/or antivirulence antibodies, and such antibodies by themselves are protective (28). A role for opsonization by antibodies directed toward Y. pestis surface antigens is suggested by studies that showed that neutrophil depletion (15) or inhibition of cytokines that boost the antimicrobial activity of macrophages (18, 50) interfered with antibody-mediated protection. Properties of Psa similar to those of F1, such as its polymeric structure and bacterial surface location, make it an attractive vaccine candidate. Therefore, we evaluated Psa for its in vivo expression, immunogenicity, and protective properties by using the pneumonic plague model that we developed.