Immunochemical characterization of Fraction I of Yersinia pestis strains by CAF1M gene

The role of caf1M gene in biogenesis of Yersinia pestis capsule was studied in natural strains of the agent with Fra+/- phenotypes and recombinant variants with ycaA (caf1+;caf1M;caf1A+;caf1R+) locus defect. These bacteria did not form a clearly discernible capsule stained by classical methods but synthesized Cafl, whose content in the cells was many times higher than in lysates, in external cell wall, and in the medium with reference Y. pestis EV NIIEG culture (caf1+;caf1M;caf1A+;caf1R+). However Caf1 was not detected on the surface or culture fluid of natural and mutant Y. pestis cells. Exclusive role of Caf1M in Caf1 delivery to Y. pestis cell surface, but not in F1 monomer folding, was proven. Retention of lipopolysaccharide (LPS), a typical SR-LPS configuration and epitope specificity of its components was demonstrated, ensuring similar reactivity in solid-phase enzyme immunoassay with a panel of monoclonal antibodies to Y. pestis LPS. Study of immunochemical properties of antigenic substances derived from caf1M-defective Y. pestis cells by isolation of F1 showed that these substances represent an envelope protein involved in the caf1+ strains (together with Caf1) in assembly of "mature" F1 molecule as a result of posttranslation modification of various genes products. Variants of identification of Y. pestis with Fra+ phenotype by means of monoclonal antibodies to F1, fibrinolysis/coagulase, or LPS in solid-phase enzyme immunoassay are discussed.     

Identification of the autoagglutination factor of Hms- cells of the plague agent

A search for cellular components responsible for autoagglutination (AA) in broth and salt solutions of Hms- cells of the plague agent Yersinia pestis was performed. The AA- mutants were obtained using vaccine strain Y. pestis EV76 derivative containing one species-specific plasmid pYP. The mutants were shown to differ from the parent strain by the decreased surface hydrophobicity, insensitivity to plague diagnostic L-413c bacteriophage and negative haemagglutination reaction with antibodies to F1 capsular substance of the plague agent. The mutants did not differ from the parent strain by electrophoretic mobility and immunochemical activity of LPS but were characterized by the absence of a 17 kDa protein on the cell surface. The AA+ cells that lost this protein after weak alkali extraction were less hydrophobic and failed to express AA in 0.5 M ammonium sulfate. After the extraction, the cells lost the ability to neutralize L-413c and to react with the anti-F1 antibodies, while both activities as well as 17 kDa protein were detected in the extracts. Thus, the 17 kDa protein is suggested to be a hydrophobic surface antigen which acts as a receptor of the L-413c bacteriophage and represents an AA factor of Hms- cells of Y. pestis.     

Expression of the envelope antigen F1 of Yersinia pestis is mediated by the product of caf1M gene having homology with the chaperone protein PapD of Escherichia coli.

The effective synthesis of the envelope antigen F1 of Y. pestis in E. coli HB101 is mediated by the expression of the caf1M gene. This gene was sequenced, and the protein encoded was found to have a significant homology with the chaperone protein PapD of uropathogenic E. coli. The data presented allow one to suppose Caf1M and PapD proteins perform similar functions in the biogenesis of the Y. pestis capsule and E. coli P-pili, respectively.     

The properties of the cellular surface of Yersinia pestis strain EV and its achromogenic variants

The comparative study of the properties of the surface of vaccine strain Y. pestis EV and its achromogenic variants (AV) differing from the initial strain by decreased immunogenicity and by the morphology of colonies, has been made. The achromogenicity of Y. pestis colonies has been shown to correlate with the loss of the outer membrane protein with a molecular weight 22 kD. The synthesis of this protein is determined by chromosomal genes. AV have been found to have different sensitivity to bacteriophages. The analysis of the electrokinetic potential of Y. pestis EV and its AV has revealed that in the latter have surface charge is considerably greater (1.4- to 1.5-fold). As shown in this study, the hemagglutinating activity of AV with respect to red blood cells of humans with blood group I (O) and guinea pigs is decreased by 1-2 orders and these strains do not agglutinate with sheep red blood cells. The low activity of the initial stage of the phagocytosis of AV by mouse macrophages has been shown. The possible role of the 22 kD proteins as an adhesion factor is discussed.     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

Influence of different F1-specific components of Yersinia pestis capsular antigen on cell-mediated immunity

The F1-specific components of Y. pestis capsular antigen, isolated by Baker's method, were shown to differ by their biological activity and the character of action on cell-mediated immunity factors, used in this study. Depending on the method of isolation, antigens could vary in the proportion of their components, which determined the specific features of the total preparations obtained in this investigation. Out of the four components under study having the same antigenic specificity, but different physico-chemical characteristics and genetically determined synthesis, only one component exhibited biological properties dominating in the secreted form of F1, more similar in their composition to protein Caf1. The other three components exhibited immunological activity on the level of unspecific protection in different ways, depending on the model, and influencing the outcome of the interaction "bacteria-macrophage". Lectin-like hemagglutinating and hemolyzing activity was shown by components not related to protein Caf1. The multi-component character of preparations F1 (Baker) and the individual activity of their components should be taken into consideration when using capsular antigen and different methods of its isolation for different aims.     

Characterization of an alternate form of Newcastle disease virus fusion protein

The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes and infected COS-7 cells (J. Virol. 77:1951-1963, 2003). One form is the classical type 1 glycoprotein, while the other is a polytopic form in which approximately 200 amino acids of the amino-terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein-expressing cells, and antibodies specific for these sequences inhibited red blood cell fusion to hemagglutinin-neuraminidase and F protein-expressing cells, suggesting a role for surface-expressed CT sequences in cell-cell fusion. Extending these findings, we have found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of the F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single-cycle infections, as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed.     

Interleukin-1-inducing activity of the polysaccharide-containing antigens of the cell wall in Yersinia pestis

The induction of the synthesis of interleukin-1 (IL-1) in human monocytes under the influence of the endotoxic preparations (LPS) and Y. pestis basis somatic antigen has been experimentally studied. The results obtained in this study make it possible to come to the conclusion that the capacity of the endotoxin of Y. pestis cell wall, consisting of LPS of type R, for inducing the synthesis of IL-1 in human monocytes is not different from the corresponding capacity of Salmonella and Shigella LPS, type S. Y. pestis O-specific polysaccharide in a discrete state has considerably greater IL-1-inducing activity in comparison with other preparations used in this experimental study. Such typical changes, characteristic of the initial stage of Y. pestis infection, as a sharp rise in temperature, transitory neutropenia, significant primary suppression of the oxygen-dependent metabolism of polymorphonuclear neutrophils are probably due to the induction of the synthesis of IL-1 by the polysaccharide-containing antigen of Y. pestis cell wall (LPS, basic somatic antigen) in cells of the mononuclear phagocytizing system.     

Lectin of Yersinia pestis vaccine strain EV and its immunological properties

As the result of the chromatographic separation of Y. pestis EV membrane proteins, a protein fraction with hemagglutinating activity was obtained. The isolated preparation was glycoprotein with a molecular weight of 22 kD, contained 16% of carbohydrates and exhibited thermolabile properties. The determination of the carbohydrate specificity of this glycoprotein revealed that it belonged to the class of lectins. Changes in the content of 11 corticosteroids and the population composition of lymphocytes, as well as the detection of specific antibodies in the blood serum of guinea pigs immunized with lectin, were indicative of the fact that the preparation was sufficiently immunogenic and induced the activation of the processes of proliferation and activation of lymphocytes during immunogenesis. The lectin isolated from Y. pestis EV outer membrane may be regarded as an additional factor ensuring the contact of the pathogen with the cells of the body and as a promising component of combined plague vaccine.     

Constructing a test system for the identification of plague microbe based on genetic probes]

DNA probes for detection of the plague agent Yersinia pestis were made on a basis of its three typical extrachromosomal replicons. The recombinant plasmid pBS2 including pBR327 vector and SalGI-BspRI fragment of the plasmid pFra was constructed. The above fragment is connected with synthesis of Y. pestis capsular antigen and it is a 400 bp species-specific DNA probe called F1 which is suitable for identification of Y. pestis species that bears the 60 mdal plasmid. The DNA probes called P1 was made on a basis of the plasmid pPst; it is the 460 BglII-BamHI fragment of the fibrinolysin-coagulase gene suitable for species-specific detection of Y. pestis species that bears the 60 mdal plasmid. The P1 fragment was cloned into the pAT153 vector and the constructed recombinant plasmid was called pEK7. The recombinant plasmid pCL1, including the pBR325 vector and the 6th BamHI fragment of Y. pestis EV plasmid pCad was constructed. The above fragment includes the replication origin of the pCad and it is hybridized to the pCad-bearing strains of Y. pestis and Y. tuberculosis only. Thus, it may be a basis for a bi-species-specific DNA probe making. These three recombinant plasmids are considered as a test-system for detection of both typical and atypical strains of Y. pestis.     

Antigen biosynthesis during the limitation of Yersinia pestis growth

The effect of the ingredients of a semisynthetic culture medium on the synthesis of Y. pestis antigens (F1, LPS, "mouse" toxin) under the conditions of batch cultivation at 28 degrees C was studied. The study revealed that the amount of antigens produced by bacterial cells depended on the character of the limitation of growth.     

The effect of the adhesion pili of Yersinia pestis on the physiological activity of the leukocytes and macrophages in experimental animals

The data on the influence of the preparation of Y. pestis adhesion pili on peritoneal macrophages in white mice and guinea pigs are presented. Y. pestis adhesion pili have been found to induce the dose-dependent increase of cell chemiluminescence. They have also been found to induce a number of biochemical changes in target cells: the secretion of myeloperoxidase, an increase in the activity of cAMP-dependent protein kinases.     

Use of protein microarray to identify gene expression changes of Yersinia pestis at different temperatures

Yersinia pestis is a bacterium that is transmitted between fleas, which have a body temperature of 26 °C, and mammalian hosts, which have a body temperature of 37 °C. To adapt to the temperature shift, phenotype variations, including virulence, occur. In this study, an antigen microarray including 218 proteins of Y. pestis was used to evaluate antibody responses in a pooled plague serum that was unadsorbed, adsorbed by Y. pestis cultivated at 26 °C, or adsorbed by Y. pestis cultivated at 26 and 37 °C to identify protein expression changes during the temperature shift. We identified 12 proteins as being expressed at 37 °C but not at 26 °C, or expressed at significantly higher levels at 37 °C than at 26 °C. The antibodies against 7 proteins in the serum adsorbed by Y. pestis cultivated at 26 and 37 °C remained positive, suggesting that they were not expressed on the surface of Y. pestis in LB broth in vitro or specifically expressed in vivo. This study proved that protein microarray and antibody profiling comprise a promising technique for monitoring gene expression at the protein level and for better understanding pathogenicity, to find new vaccine targets against plague.     

A rapid diagnostic test for plague detects Yersinia pestis F1 antigen in ancient human remains

A rapid diagnostic dipstick test (RDT) that detects Yersinia pestis F1 antigen has been recently applied on 18 putative plague victims exhumed from four archaeological burial sites in southeastern France dating back to the 16(th), 17(th) and 18(th) centuries. The Y. pestis antigen F1 was detected in 12 ancient samples out of 18 (67%). Negative controls confirmed their negativity (100%). Our results emphasize that the detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a first retrospective diagnosis of Y. pestis infection in ancient remains, and confirm the high specificity and sensitivity of the assay. Double-blind analyses performed by using two different techniques (RDT and 'suicide PCR') led us to the identification of the Y. pestis F1 antigen and the Y. pestis pla and gplD genes. These data provide clear evidence of the presence of Y. pestis in the examined specimens.     

Detection of viable Yersinia pestis by fluorescence in situ hybridization using peptide nucleic acid probes

A successful method has been developed for the detection of live Yersinia pestis, the plague bacillus, which incorporates nascent RNA synthesis. A fluorescent in situ hybridization (FISH) assay using peptide nucleic acid (PNA) probes was developed specifically to differentiate Y. pestis strains from closely related bacteria. PNA probes were chosen to target high copy mRNA of the Y. pestis caf1 gene, encoding the Fraction 1 (F1) antigen, and 16S ribosomal RNA. Among Yersinia strains tested, PNA probes Yp-16S-426 and Yp-F1-55 exhibited binding specificities of 100% and 98%, respectively. Y. pestis grown in the presence of competing bacteria, as might be encountered when recovering Y. pestis from environmental surfaces in a post-release bioterrorism event, was recognized by PNA probes and neither hybridization nor fluorescence was inhibited by competing bacterial strains which exhibited faster growth rates. Using fluorescence microscopy, individual Y. pestis bacteria were clearly differentiated from competing bacteria with an average detection sensitivity of 7.9x10(3) cells by fluorescence microscopy. In the current system, this would require an average of 2.56x10(5) viable Y. pestis organisms be recovered from a post-release environmental sample in order to achieve the minimum threshold for detection. The PNA-FISH assays described in this study allow for the sensitive and specific detection of viable Y. pestis bacteria in a timely manner.     

Antibody profiling in plague patients by protein microarray

A protein microarray containing 144 known or putative virulence-related proteins of Yersinia pestis was used to evaluate the antibody responses of plague patients. Forty-two proteins were found to be expressed in vivo and antibodies against 14 of them were detected in all patients analyzed, providing potential candidates for novel protective antigens and novel serodiagnostic markers in Y. pestis. Moreover, the lack of antibody to LcrV in the five patients in Focus F might be a challenge to our understanding of the pathogenesis of Y. pestis.     

Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines

A number of antibodies generated during human respiratory syncytial virus (RSV) infection have been cloned by the phage library approach. Antibodies reactive with an immunodominant epitope on the F glycoprotein of this virus have a high affinity for affinity-purified F antigen. These antibodies, however, have a much lower affinity for mature F glycoprotein on the surface of infected cells and are nonneutralizing. In contrast, a potent neutralizing antibody has a high affinity for mature F protein but a much lower affinity for purified F protein or F protein in viral lysates. The data indicate that at least two F protein immunogens are produced during natural RSV infection: immature F, found in viral lysates, and mature F, found on infected cells or virions. Binding studies with polyclonal human immunoglobulin G suggest that the antibody responses to the two immunogens are of similar magnitudes. Competitive binding studies suggest that overlap between the responses is relatively limited. A mature envelope with an antigenic configuration different from that of the immature envelope has an evolutionary advantage in that the infecting virus is less subject to neutralization by the humoral response to the immature envelope that inevitably arises following lysis of infected cells. Subunit vaccines may be at a disadvantage because they most often resemble immature envelope molecules and ignore this aspect of viral evasion.