Pestis F1 Antigen Research Articles

Characterization of two affinity matured Anti-Yersinia pestis F1 human antibodies with medical countermeasure potential.

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 μg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

The surveillance of plague among rodents and dogs in Western Iran.

The causative agent of plague, Yersinia pestis, is maintained in nature via a flea-rodent cycle. Western Iran is an old focus for plague, and recent data indicate that rodents and dogs in this region have serological evidence of Y. pestis infection. The purpose of this study was to conduct a large-scale investigation of Y. pestis infection in shepherd dogs, rodents, and their fleas in old foci for plague in Western Iran. This study was conducted in Hamadan province from 2014 to 2020. Rodents and fleas were collected from various locations throughout this region. Y. pestis was investigated in rodent spleen samples and fleas using culture, serology, and real-time PCR methods. Additionally, sera samples were collected from carnivores and hares in this region, and the IgG antibody against the Y. pestis F1 antigen was assessed using an ELISA. In this study, 927 rodents were captured, with Meriones spp. (91.8%) and Microtus qazvinensis (2.6%) being the most prevalent. A total of 6051 fleas were collected from rodents and carnivores, most of which were isolated from Meriones persicus. None of the rodents or fleas examined tested positive for Y. pestis using real-time PCR and culture methods. Meanwhile, IgG antibodies were detected in 0.32% of rodents. All serologically positive rodents belonged to M. persicus. Furthermore, none of the sera from the 138 carnivores (129 sheepdogs, five Vulpes vulpes, four Canis aureus), and nine hares tested positive in the ELISA test. This primary survey of rodent reservoirs shows serological evidence of Y. pestis infection. Western Iran is an endemic plague focus, and as such, it requires ongoing surveillance.

Enhancement of the Yersinia pestis EV NIIEG Vaccine Srain Immunogenic and Protective Activity under Cultivation with Azoxymer Bromide (Polyoxidonium)

Background. The live-attenuated vaccine based on the Yersinia pestis strain EV line NIIEG is still used in Russia, providing protective efficacy against plague. Nevertheless, there is an urgent need for developing new ways to increase the immunogenicity of the Y. pestis EV NIIEG vaccine strain. In this study, the ability of direct action of immunoadjuvant azoximer bromide (polyoxidonium, PO) on the immunobiological properties of vaccine strain Y. pestis EV NIIEG during cultivation on a dense nutrient medium was evaluated.Materials & Methods. Y.pestis EV NIIEG, cultivated at 28 °С for 48 h on LB agar, Miller pH 7.2 ± 0.1 (Sigma-Aldrich, USA) with the addition of PO and without. MALDI-TOF mass-spectrometry was deployed for the obtainment of mass-spectra of ribosomal proteins from Y. pestis EV NIIEG cells on the MicroflexTM LT mass spectrometer (Bruker Daltonics, Germany). Protective efficacy was evaluated under subcutaneously challenge guinea pigs and mice BALB's with 400 LD50 doses of the Y. pestis 231, Y. pestis P-13268 Vietnam (MLD=5 CFU). Antibody titers to F1 in serum were determined using an ELISA.Results. The addition of the therapeutic concentration of PO in the cultivation medium induced a significant increase in the immunogenicity of Y. pestis EV NIIEG that resulted in enhancement of serum antibody levels against Y. pestis F1 antigen and several times the growth of protective efficacy in the bubonic plague model on two types of experimental animals. ImD50 of the vaccine strain Y. pestis EV NIIEG, cultivated with PO, was significantly (p < 0,05) lower in comparison to ImD50 for Y. pestis EV NIIEG in standard cultivation conditions. One year of storage at a temperature of 4 °С did not alter the protective properties of the vaccine strain Y. pestis EV NIIEG, cultivated with PO.Conclusions. Morphological studies confirmed the absence of influence PO introduction into the cultivation environment on the safety of the vaccine strain. MALDI-TOF MS profile of the Y. pestis EV NIIEG, cultivated with PO, had peaks characteristic features. The mass peak at m/z 3,061 was significantly down-regulated and new mass peaks at m/z 2,759, m/z 3,533 were determined. These changes are accompanied by the increase of Y. pestis EV NIIEG immunogenicity.

The Use of an Organo-Selenium Peptide to Develop New Antimicrobials That Target a Specific Bacteria.

This study examines the use of a covalently selenium-bonded peptide and phage that binds to the Yersinia pestis F1 antigen for the targeting and killing of E. coli expressing this surface antigen. Using a Ph.D.-12 phage-display library for affinity selection of the phage which would bind the F1 antigen of Y. pestis, a phage displaying a peptide that binds the F1 antigen with high affinity and specificity was identified. Selenium was then covalently attached to the display phage and the corresponding F1-antigen-binding peptide. Both the phage and peptides with selenium covalently attached retained their binding specificity for the Y. pestis F1 antigen. The phage or peptide not labeled with selenium did not kill the targeted bacteria, while the phage or peptide labeled with selenium did. In addition, the seleno-peptide, expressing the F1 targeting sequence only, killed cells expressing the F1 antigen but not the parent strain that did not express the F1 antigen. Specifically, the seleno-peptide could kill eight logs of bacteria in less than two hours at a 10-µM concentration. These results demonstrate a novel approach for the development of an antibacterial agent that can target a specific bacterial pathogen for destruction through the use of covalently attached selenium and will not affect other bacteria.

Performance of plague rapid diagnostic test compared to bacteriology: a retrospective analysis of the data collected in Madagascar

BackgroundPlague is a highly fatal disease caused by Yersinia pestis. Late diagnosis hampers disease outcome and effectiveness of control measures, induces death and disease spread. Advance on its diagnosis was the use of lateral flow rapid diagnostic test (RDT).MethodsWe assessed the performance of the plague RDT based on Y. pestis F1 antigen detection more than 15 years after its deployment in Madagascar. We compared the RDT with bacteriological culture results, using data from plague notified cases collected during the periods for which both tests were performed independently and systematically.ResultsUsed with bubonic plague (BP) patient samples, RDTs had a sensitivity of 100% (95% CI: 99.7–100%), a specificity of 67% (95% CI: 64–70%) with a good agreement between bacteriology and RDT results (86%; κ = 0.70, 95% CI 0.67–0.73). For pneumonic plague (PP), RDT had a sensitivity of 100% (95% CI: 91–100%) and a specificity of 59% (95% CI: 49–68%) and concordance between the bacteriological and plague RDT results was moderate (70%; κ = 0.43, 95% CI 0.32–0.55). Analysis focusing on the 2017–2018 plague season including the unprecedented epidemic of PP showed that RDT used on BP samples still had a sensitivity of 100% (95% CI: 85–100%) and a specificity of 82% (95% CI: 48–98%) with a very good agreement with bacteriology 94% (κ = 0.86, 95% CI 0.67–1); for PP samples, concordance between the bacteriological and plague RDT results was poor (61%; κ = − 0.03, 95% CI -0.17 – 0.10).ConclusionsRDT performance appeared to be similar for the diagnosis of BP and PP except during the 2017 PP epidemic where RDT performance was low. This RDT, with its good sensitivity on both plague clinical forms during a normal plague season, remained a potential test for alert. Particularly for BP, it may be of great value in the decision process for the initiation of therapy. However, for PP, RDT may deliver false negative results due to inconsistent sample quality. Plague diagnosis could be improved through the development of next generation of RDTs.

A microchip developed for detecting antibodies against plaque-derived antigens

Currently available Russia-made preparations intended for serological plague diagnostics are usually aimed at detecting antibodies to single bacterial antigens in the blood serum. To improve reliability of the data obtained, it is rational to use test systems to simultaneously quantify antibodies to several immunodominant Y. pestis antigens. An opportunity of using biochip technology for quantifying specific antibodies to Yersinia pestis antigens was investigated. To do this, 5 commercially available sera, 35 blood sera obtained from individuals vaccinated with live plague vaccine collected 1, 4, 5, 18 months after immunization, as well as 5 sera obtained from healthy donors were analyzed. The objective of this work was to develop a biological microchip (immunochip) for detecting antibodies specific to Y. pestis-derived antigens. In particular, amino-modified slides were sensitized by immunodominant Yersinia pestis-derived antigens: capsule antigen F1, lipopolysaccharide (LPS), main somatic antigen (MSA), fibrinolysin, and pestin PP. Diagnostic specificity and sensitivity of the immunochip were assessed by using the approved homoand heterologous immune-biological preparations and experimental animal sera. It was found that the immunochip demonstrated a 100% diagnostic efficiency. An opportunity of applying this immunochip to determine specific antibody profile in individuals vaccinated with live plague vaccine was estimated. A commercially available ELISA-AB-F1 of Yersinia pestis kit was used for comparison that allowed to detect antibodies to Y. pestis F1 antigen in 77.1% of vaccinated individuals within the examined time period covering between 1 to 18 months post-vaccination, at titer 1:160–1:2560. In contrast, using the immunochip resulted in detecting F1 antigen-specific antibodies in 91.4% of samples post-vaccination at titer 1:320–1:2560. Moreover, such immunochip additionally allowed to detect antibodies specific to the remainder of Y. pestis-derived LPS, MSA, pestin PP in 54.3%, 20%, 42% of vaccinated individuals, respectively. The percentage of positive seroconversion in individuals vaccinated with live plague vaccine was 77.1% based on the ELISA data, 91.4% — to the F1 antigen according to the immunochip data, and 94.3% — by analyzing an extended antigen panel. Combining multiple antigenic markers in our immunochip allowed to identify greater seroconversion among vaccinated people compared to a standard ELISA. Thus, the data obtained suggest that the proposed immunochip technology might be promising in assessing developing humoral immunity.

Synthesis and expression of caf1 gene encoding F1 antigen of Yersinia pestis

Yersinia pestis is the etiologic agent of plague, one of the most deadly infectious diseases described in the history of humanity. It was responsible for millions of deaths all over the world. Yersinia pestis also can be used as a highly lethal biological potential weapon. For plague diagnosis in humans as well as to detect Y. pestis in the environment, fraction 1 capsular antigen (F1) of the bacteria was usually used as a good marker. The aim of this study is to produce Y. pestis F1 antigen to serve as a material for development of immunochromatographic test strips for rapid detection of Y. pestis. Because of the difficulty in Y. pestis culture for DNA extraction as well as F1 antigen production, we artificially synthesized the target caf1 coding for F1 antigen for expression in Escherichia coli. After the codon optimization step, caf1 was synthesized by “gapless” PCR using 22 overlaping oligonucleotides cover the complete sequence of this gene. The sequencing result showed that we successfully synthesized the target gene. In total 6 clones sequence, there are 2 clones sequence which were 100% identity with reference sequence. The target sequence was then introduced into pET-52b(+) vector and expressed in E. coli BL21 (DE3) in the form of (His)10 affinity tag fusion. As the result of SDS-PAGE, the recombinant protein Caf1 of 18 kDa was highly expressed in E. coli as inclusion body form and was purified by His-tag affinity chromatography. The recombinant Caf1 was then confirmed by Western blot with His-tag antibody.

Разработка реагента для оценки начальной стадии иммунного ответа на живую чумную вакцину

Objective of the study is to develop a reagent for the detection of lymphocytes with Yersinia pestis F1 antigen receptors. Materials and methods. Utilized have been: live plague vaccine based on the strain of Yersinia pestis EV NIIEG, formalin killed suspensions of microorganisms - Y. pestis , 3123, Y. enterocolitica O9 H-383 serovar, Y. pseudotuberculosis O1 2841 serovar; acetaldehyde-immobilized capsular antigen of Y. pestis F1 (obtained applying Baker methodology), lipopolysaccharide of Y. pestis K1, and bovine erythrocytes. Bovine erythrocyte F1 sensibilization has been performed using rivanol. Lymphocytes from blood have been isolated in density gradient ficoll-verografin 1.077. Lymphocytes with Yersinia pestis F1 antigen receptors have been detected by means of reagent adhesion onto the isolated lymphocytes. F1-free erythrocytes serve as controls. After the exposition, 7 evaluations of specificity to F1 and the lymphocytes, binding control reagent, have been carried out. Deployed have been 8 rabbits, immunized with live vaccine EV, and 2 rabbits, immunized with inactivated vaccine EV. Examined have been EV-vaccinated 5 persons. Results and conclusions. Identified is optimum sensibilizing dose of F1 antigen (250 µg/ml). Specificity of lymphocytes with receptors to F1 is demonstrated in inhibition experiments applying homologous and heterogeneous antigens. Lymphocytes with receptors to F1 (LRs) have been detected in peripheral blood of all rabbits and humans, immunized with vaccine EV. LRs have been registered since day 2 till day 35 in the rabbits, and in humans - since day 2 till day 14 after vaccination. It is shown that in case of revaccination of humans, LRs emerge and disappear earlier, than in case of primary immunization.

Complete Protection against Pneumonic and Bubonic Plague after a Single Oral Vaccination

BackgroundNo efficient vaccine against plague is currently available. We previously showed that a genetically attenuated Yersinia pseudotuberculosis producing the Yersinia pestis F1 antigen was an efficient live oral vaccine against pneumonic plague. This candidate vaccine however failed to confer full protection against bubonic plague and did not produce F1 stably.Methodology/Principal FindingsThe caf operon encoding F1 was inserted into the chromosome of a genetically attenuated Y. pseudotuberculosis, yielding the VTnF1 strain, which stably produced the F1 capsule. Given orally to mice, VTnF1 persisted two weeks in the mouse gut and induced a high humoral response targeting both F1 and other Y. pestis antigens. The strong cellular response elicited was directed mostly against targets other than F1, but also against F1. It involved cells with a Th1—Th17 effector profile, producing IFNγ, IL-17, and IL-10. A single oral dose (108 CFU) of VTnF1 conferred 100% protection against pneumonic plague using a high-dose challenge (3,300 LD50) caused by the fully virulent Y. pestis CO92. Moreover, vaccination protected 100% of mice from bubonic plague caused by a challenge with 100 LD50 Y. pestis and 93% against a high-dose infection (10,000 LD50). Protection involved fast-acting mechanisms controlling Y. pestis spread out of the injection site, and the protection provided was long-lasting, with 93% and 50% of mice surviving bubonic and pneumonic plague respectively, six months after vaccination. Vaccinated mice also survived bubonic and pneumonic plague caused by a high-dose of non-encapsulated (F1-) Y. pestis.SignificanceVTnF1 is an easy-to-produce, genetically stable plague vaccine candidate, providing a highly efficient and long-lasting protection against both bubonic and pneumonic plague caused by wild type or un-encapsulated (F1-negative) Y. pestis. To our knowledge, VTnF1 is the only plague vaccine ever reported that could provide high and durable protection against the two forms of plague after a single oral administration.

Surface plasmon resonance imaging of pathogens: the Yersinia pestis paradigm.

BackgroundYersinia pestis, causing deadly plague, is classified as a group A bioterrorism bacterium. Some recent DNA-based methods were used for detection of bioterrorism agents.ResultsY. pestis was used as a model organism to develop an immunosensor based on surface plasmon resonance imaging (SPRi) using monoclonal antibody against Y. pestis F1 antigen. The experimental approach included step-by-step detection of Y. pestis membrane proteins, lysed bacteria, intact bacteria, mock-infected powder and mock-infected clinical specimens. SPRi detected on average 106 intact Y. pestis organisms in buffer, in mock-infected powder and in a 1:4 mixture with HEL cells.ConclusionsThis study offers the proof-of-concept of the SPRi-based detection of a human pathogen in both environmental and clinical specimens.

Surveillance and response: Integrated indigenous health systems-based sentinel detection of human plague in a plague endemic West Nile region of Uganda (2008-2013)

Background: Bacterial plague, caused by Yersinia pestis, is a zoonotic disease predominantly rodent flea-borne. Approximately 2000 suspected cases of plague were reported from the plague endemic West Nile region of Uganda through the past decade. Case mortality rates were high due to delays in seeking appropriate medical treatment. Community health workers (CHWs), private for profit (PFP) clinics and drug-shops provide immediate alternative source of health care to those suffering from illness. Besides, an estimated 40-60% of the study population seeks the service of traditional healers (THs). This study piloted an innovative one heath tailored community-based surveillance aimed at enabling the indigenous health care providers to recognize primary symptoms of plague among their clients and refer suspects to the nearest health center (HC); and to determine the number of patients referred by the participating THs, the proportion that actually arrive in the HCs, and the diagnostic outcome of referred patients. Methods & Materials: Participating THs were provided with easy to use patient referral kits whilst the catchment HCs received rapid diagnostic tests (RDT) for on-spot detection of Y. pestis F1 antigen. Samples that tested positive by RDT at the HCs or negative but presented with clinical signs suggestive of Y. pestis infection were re-subjected to serial testing by RDTs direct fluorescent assay (DFA), bacterial culture and serology. Results: Out of 562 patients referred to HCs by THs, 1 patient (0.18%) was confirmed with Y. pestis infection whilst a significant proportion tested positive to other febrile infections such as malaria (51.0%), pneumonia (18.9%), tuberculosis (4.3%) and others (25.8%). We summerized in table below human plague incidance and case mortality (2008-2012) in the study area.Tabled 1YearConfirmedNo. of deaths due to Y. pestis20085825200921201011201122201296 Open table in a new tab Conclusion: Although the proportion of referred patients confirmed with Y. pestis is minimal, it's clearly evident that the involving THs has saved many lives from several illnesses. The integration of CHWs, clinics and drug-shops increased sensitivity of the surveillance system. Such community-based surveillance one health models that innovatively integrate indigenous health systems and scientific research are likely to have significant impact on mitigating public health crises in report settings.

Development of Yersinia pestis F1 antigen-loaded microspheres vaccine against plague

Yersinia pestis F1 antigen-loaded poly(DL-lactide-co-glycolide)/polyethylene glycol (PEG) (PLGA/PEG) microspheres were produced using a water-in-oil-in-water emulsion/solvent extraction technique and assayed for their percent yield, entrapment efficiency, surface morphology, particle size, zeta potential, in vitro release properties, and in vivo animal protect efficacy. The Y. pestis F1 antigen-loaded microspheres (mean particle size 3.8 μm) exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (38.1%), and presented a controlled in vitro release profile with a low initial burst (18.5%), then continued to release Y. pestis F1 antigen over 70 days. The distribution (%) of Y. pestis F1 on the microspheres surface, outer layer, and core was 3.1%, 28.9%, and 60.7%, respectively. A steady release rate was noticed to be 0.55 μg Y. pestis F1 antigen/mg microspheres/day of Y. pestis F1 antigen release maintained for 42 days. The cumulative release amount at the 1st, 28th, and 42nd days was 8.2, 26.7, and 31.0 μg Y. pestis F1 antigen/mg microspheres, respectively. The 100 times median lethal dose 50% (LD50) of Y. pestis Yokohama-R strain by intraperitoneal injection challenge in mice test, in which mice received one dose of 40 μg F1 antigen content of PLGA/PEG microspheres, F1 antigen in Al(OH)3, and in comparison with F1 antigen in Al(OH)3 vaccine in two doses, was evaluated after given by subcutaneous immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with one dose of F1 antigen-loaded PLGA/PEG microspheres, and two doses of F1 antigen in Al(OH)3 vaccine (100%). In vivo vaccination studies also demonstrated that F1 vaccines microspheres had a protective ability; its steady-state IgG immune protection in mice plasma dramatic increased from 2 weeks (18,764±3,124) to 7 weeks (126,468±19,176) after vaccination. These findings strongly suggest that F1-antigen loaded microspheres vaccine offer a new therapeutic strategy in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.

Could the ‘Black Death’ Become a Re-Emerging Infectious Disease?

During the optimal time of the ‘Black Death’, in the fourteenth century, around 75 million people globally perished, mainly through lymphadenitis. This manifestation of the plague continued to be a disease of major importance until the seventeenth century and the main regions affected were Europe (where 30–60% of the European population was wiped out) and Asia [2]. The ‘Black Death’ is conventionally known within the medical field as the Bubonic plague (named after the buboes commonly found in the armpits, upper femoral, groin and neck region). Together with the septicemic plague and the pneumonic plague, these infections are caused by the bacterium Yersinia pestis (formerly described as Pasteurella pestis). The bacterium seemingly evolved several thousand years ago from a far more benign, gut dwelling bug called Y. pseudotuberculosi (one of a group of relatively benign intestinal diseases). Y. pestis is a facultative anaerobic Gramnegative rod-shaped bacterium. It is unknown if Y. pestis caused all causes of plague during this period, although it stands as the main the etiologic agent (many of the skeletons exhumed from ‘plague pits’) have been tested using a rapid diagnostic test for the detection of Y. pestis F1 antigen to confirm the cause of their death. The bacterium is what is known as a zoonotic disease, indicating its ability to be transferred between different species. In terms of animal to human transmission, the role of rats and fleas (like Pulex irritans) and their detailed role in the transmission of plague has been discovered and experimentally verified [3]. The disease affects the lungs and is highly contagious, leading to mass outbreaks across populations. Without treatment, the bubonic plague kills about two thirds of infected humans within four days [4]. Those infected with the bacteria develop symptoms that can include swollen, tender lymph glands, fever, headache, chills, and weakness. Other symptoms may include muscle pain and seizures. The human body is generally unsuccessful in fighting the disease because cells of Y. pestis can resist phagocytosis. Other major plague events have occurred around the world over the past centuries and similar discoveries of the remains of some of the victims have been made. In 2012 an archaeological discovery of the last remnants of a “lazaretto” or “lazaret” was made in the city of Marseille. This was a place equipped with an infirmary and destined to isolate ship passengers quarantined for health reasons. It was a major site of a plague outbreak.

Black Death in the rural cemetery of Saint-Laurent-de-la-Cabrerisse Aude-Languedoc, southern France, 14th century: immunological evidence

An excavation carried out in 2007 in Saint-Laurent-de-la-Cabrerisse (Aude-Languedoc, southern France), revealed a medieval rural cemetery used during the 8th–14th centuries. One hundred and forty nine graves were identified. Amongst those, three burials radiocarbon dated to the 14th century contained the remains of several individuals. No paleotraumatological evidences of violence due to warfare or to a civil massacre were identified in the exhumed skeletons. Therefore, we hypothesized that the simultaneous inhumations could have occurred during the “Black Death” outbreak or during one of its resurgences. A rapid diagnostic test for the detection of Yersinia pestis F1 antigen was applied to the nine putative plague victims to authenticate the cause of their death. Seven of the nine individuals tested from the three graves were positive to Y. pestis F1 antigen. We additionally tested fourteen skeletons from single graves archaeologically dated to the 13th–14th centuries. Four of them were positive to Y. pestis F1 antigen, thus showing a higher mortality rate due to plague than originally expected. The Saint-Laurent-de-la-Cabrerisse plague victims are the oldest samples from which Y. pestis F1 antigen has been, hitherto, identified. Few rural French cemetery containing plague victims have previously been identified and amongst them, only one dates to the 14th century. In the present report, we provide suggestive evidence of a second rural cemetery from southern France where victims of Y. pestis infection were buried. Our findings provide new information about the management of the plagued corpses during the “Black Death” in small rural communities.

An auto-biotinylated bifunctional protein nanowire for ultra-sensitive molecular biosensing

In order to obtain an ultra-sensitive molecular biosensor, we designed an auto-biotinylated bifunctional protein nanowire (bFPNw) based on the self-assembly of a yeast amyloid protein, Sup35, to which protein G and a biotin acceptor peptide (BAP) were genetically fused. These auto-biotinylated bFPNws can transfer hundreds of commercially available diagnostic enzymes to an antigen–antibody complex via the biotin–avidin system, greatly enhancing the sensitivity of immune-biosensing. Compared to our previously reported seeding-induced bFPNws ( Men et al., 2009), these auto-biotinylated bFPNws gave greater signal amplification, reduced non-specific binding and improved stability. The auto-biotinylated self-assembled bFPNw molecular biosensors were applied to detect Yersinia pestis ( Y. pestis) F1 antigen and showed a 2000- to 4000-fold increase in sensitivity compared to traditional immunoassays, demonstrating the potential use of these self-assembling protein nanowires in biosensing.

Protection against pneumonic plague following oral immunization with a non-replicating vaccine

Yersinia pestis is a dangerous bacterial pathogen that when inhaled can rapidly induce fatal pneumonic plague. Thus, there is a need for stable, safe, and easily administered mucosal vaccines capable of eliciting effective protection against pulmonary Y. pestis infections. Cationic liposome–nucleic acid complexes (CLDC) have been shown previously to be effective vaccine adjuvants for parenteral immunization, but have not been previously evaluated for use in oral immunization. Therefore, we investigated the ability of an orally administered CLDC adjuvanted vaccine to elicit protective immunity against lethal pneumonic plague. C57Bl/6 mice were vaccinated orally or subcutaneously using 10 μg Y. pestis F1 antigen combined with CLDC and immune responses and protection from challenge was assessed. We found that oral immunization elicited high titers of anti-F1 antibodies, equivalent to those generated by parenteral immunization. Importantly, orally immunized mice were protected from lethal pulmonary challenge with virulent Y. pestis for up to 18 weeks following vaccination. Vaccine-induced protection following oral immunization was found to be dependent primarily on CD4+ T cells, with a partial contribution from CD8+ T cells. Thus, CLDC adjuvanted vaccines represent a new type of orally administered, non-replicating vaccine capable of generating effective protection against pulmonary infection with virulent Y. pestis.

Seeding-Induced Self-assembling Protein Nanowires Dramatically Increase the Sensitivity of Immunoassays

Aiming to build a supersensitive and easily operable immunoassay, bifunctional protein nanowires were generated by seeding-induced self-assembling of the yeast amyloid protein Sup35p that genetically fused with protein G and an enzyme (methyl-parathion hydrolase, MPH), respectively. The protein nanowires possessed a high ratio of enzyme molecules to protein G, allowing a dramatic increase of the enzymatic signal when protein G was bound to an antibody target. As a result, a 100-fold enhancement of the sensitivity was obtained when applied in the detection of the Yersinia pestis F1 antigen.

Plague immunodetection in remains of religious exhumed from burial sites in central France

The skeletons of four Benedictine nuns and two priests were exhumed from two burial sites in central France. The corpses were dated to the 16th– 18th centuries (archaeological dating). Thick layers of lime were associated with their burials. During the Second Plague Pandemic, lime was associated with plague burials. We hypothesize that Yersinia pestis infection might have been the cause of death of these religious. A rapid diagnostic test for plague (RDT), that detects Y. pestis F1 antigen, was applied to all six putative plague victims. Y. pestis F1 antigen was detected in all six skeletons (100%). The F1 antigen concentrations ranged from 2.5 to 0.625 ng/ml. The eight samples used as negative controls yielded negative results. Soils samples taken from archaeological sites related to both positive and negative samples tested negative for F1 antigen. In earlier reports we provided suggestive evidence that the RDT for plague might be diagnostic for the detection of Yersinia pestis in ancient human remains. This putative evidence was based on four French and one German reputed plague sites in which RDT for plague and PCR sequenced-based analyses were performed. Both tests gave results consistent with our interpretation that RDT is effective in identifying plague in skeletal remains. Several different European putative plague burial sites are currently under investigation. We hope to obtain additional material to allow statistical validation of our results obtained thus far. We now confirm that the detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a retrospective diagnosis of plague in ancient skeletal remains. This method supplements the historical record with independent biologic evidence. Thus, our hypothesis that the six religious were infected by Y. pestis during the plague outbreaks which occurred in the two sites, Poitiers and La Chaize-le-Vicomte, between 1587 and 1632, has independent biologic support.

Technical note: A rapid diagnostic test detects plague in ancient human remains: An example of the interaction between archeological and biological approaches (southeastern France, 16th–18th centuries)

A rapid diagnostic test (RDT) that detects Yersinia pestis F1 antigen was applied to 28 putative plague victims exhumed from seven burial sites in southeastern France dating to the 16th-18th centuries. Yersinia pestis F1 antigen was detected in 19 of the 28 (67.9%) samples. The 27 samples used as negative controls yielded negative results. Soil samples taken from archeological sites related to both positive and negative samples tested negative for F1 antigen. The detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a preliminary retrospective diagnosis of Y. pestis infection in human remains. The high specificity and sensitivity of the assay were confirmed. For two sites positive to F1 antigen (Lambesc and Marseille), Y. pestis-specific DNA (pla gene) had been identified previously by PCR-sequence based analyses. Specifically, the positive results for two samples, from the Lambesc cemetery and the Marseille pit burial, matched those previously reported using PCR. Independent analyses in Italy and France of different samples taken from the same burial sites (Draguignan and Martigues) led to the identification of both Y. pestis F1 antigen and Y. pestis pla and gplD genes. These data are clear evidence of the presence of Y. pestis in the ancient human remains examined in this study.

IMMUNIZATION OF BLACK-TAILED PRAIRIE DOG AGAINST PLAGUE THROUGH CONSUMPTION OF VACCINE-LADEN BAITS

Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis and, along with other wild rodents, are significant reservoirs of plague for other wildlife and humans in the western United States. A recombinant raccoon poxvirus, expressing the F1 antigen of Y. pestis, was incorporated into a palatable bait and offered to three groups (n = 18, 19, and 20) of black-tailed prairie dogs (Cynomys ludovicianus) for voluntary consumption, either one, two, or three times, at roughly 3-wk intervals. A control group (n = 19) received baits containing raccoon poxvirus without the inserted antigen. Mean antibody titers to Y. pestis F1 antigen increased significantly in all groups ingesting the vaccine-laden baits, whereas the control group remained negative. Upon challenge with virulent Y. pestis, immunized groups had higher survival rates (38%) than the unimmunized control group (11%). The mean survival time of groups ingesting vaccine-laden baits either two or three times was significantly higher than that of animals ingesting vaccine-laden baits just one time and of animals in the control group. These results show that oral immunization of prairie dogs against plague provides some protection against challenge at dosages that simulate simultaneous delivery of the plague bacterium by numerous (3-10) flea bites.