Purification and characterization of a recombinant Yersinia pestis V-F1 "Reversed" fusion protein for use as a new subunit vaccine against plague

We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS-PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.     

Vaccination with F1-V fusion protein protects black-footed ferrets (Mustela nigripes) against plague upon oral challenge with Yersinia pestis

Previous studies have established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion protein by subcutaneous (SC) injection protects the animals against plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected mouse, a probable route for natural infection. Eight black-footed ferret kits were vaccinated with F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits received placebos. The animals were challenged 6 wk after the boost by feeding each one a Y. pestis-infected mouse. All eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days after exposure. To determine the duration of antibody postvaccination, 18 additional black-footed ferret kits were vaccinated and boosted with F1-V by SC injection at 60 and 120 days-of-age. High titers to both F1 and V (mean reciprocal titers of 18,552 and 99,862, respectively) were found in all vaccinates up to 2 yr postvaccination, whereas seven control animals remained antibody negative throughout the same time period.     

Assessment of a recombinant F1-V fusion protein vaccine intended to protect Canada lynx (Lynx canadensis) from plague

As part of an ongoing restoration program in Colorado, USA, we evaluated adverse reactions and seroconversion in captive Canada lynx (Lynx canadensis) after vaccination with a recombinant F1-V fusion protein vaccine against Yersinia pestis, the bacterium that causes plague. Ten adult female lynx received the F1-V vaccine; 10 source- and age-matched lynx remained unvaccinated as controls. All of the vaccinated and control lynx remained apparently healthy throughout the confinement period. We observed no evidence of injection site or systemic reactions to the F1-V vaccine. Among vaccinated lynx, differences in log(10) reciprocal antibody titers measured in sera collected before and after vaccination (two doses) ranged from 1.2 to 5.2 for anti-F1 antibodies and from 0.6 to 5.2 for anti-V antibodies; titers in unvaccinated lynx did not change appreciably over the course of confinement prior to release, and thus differences in anti-F1 (P=0.003) and anti-V (P=0.0005) titers were greater among vaccinated lynx than among controls. Although our findings suggest that the F1-V fusion protein vaccine evaluated here is likely to stimulate antibody responses that may help protect Canada lynx from plague, we observed no apparent differences in survival between vaccinated and unvaccinated subject animals. Retrospectively, 22 of 50 (44%; 95% confidence interval 29-59%) unvaccinated lynx captured or recaptured in Colorado during 2000-08 had passive hemagglutination antibody titers >1:16, consistent with exposure to Y. pestis; paired pre- and postrelease titers available for eight of these animals showed titer increases similar in magnitude to those seen in response to vaccination, suggesting at least some lynx may naturally acquire immunity to plague in Colorado habitats.     

Protection against lethal subcutaneous challenge of virulent Y. pestis strain 141 using an F1-V subunit vaccine

In this study, we designed and engineered a two-component recombinant fusion protein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis. The recombinant F1-V pro-tein was formulated with Alhydrogel. A four-time injection with a dosage of 10, 20 and 50 μg/mouse in about two months was adopted for vaccination. Serum antibodies and subclass of T helper cells were measured and analyzed. After the final vaccination, the mice were challenged by 141 strain with 25― 600 LD50. In conclusion, the recombinant vaccine was capable of inducing protective immunity against subcutaneous challenge. The level of serum IgG was supposed to be a main factor that affected the final protection of challenge. 20 μg recombinant protein could induce an endpoint titre of serum IgG as high as 51200, which was enough to afford 100% protection against 400 LD50 virulent 141 challenge. The antibody isotype analysis showed that the vaccine induced predominantly an IgG1 rather than IgG2a response. Flow cytometric analysis revealed that Alhydrogel significantly helped induce a stronger humoral immunity instead of CTL cellular response. These findings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.     

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.     

人tPA信号肽和Yersinia Pestis保护性抗原F1-V融合基因的构建及其免疫效果观察

为获得含有鼠疫F1和V抗原编码基因以及人tPA信号肽基因的重组质粒tPA-pVAX1/F1-V,并测定其诱导特异性免疫应答的能力, 用PCR扩增鼠疫菌F1和V编码基因,分别与pGEM-T连接测序,构建pVAX1/F1-V融合重组质粒.PCR扩增tPA信号肽片段并将其插入到F1-V的上游,构建tPA-pVAX1/F1-V融合重组质粒;转染COS-7细胞,Western blot法鉴定目的蛋白的表达.重组质粒tPA-pVAX1/F1-V加GM-CSF佐剂免疫BALB/c小鼠,观察免疫效果.400个LD50强毒鼠疫菌皮下攻毒观察保护率.结果表明,tPA-pVAX1/F1-V在COS-7细胞中表达;免疫鼠体内产生特异性抗体;抗体亚型分析、细胞因子等指标的测定表明,所构建DNA疫苗以诱发Th1型免疫为主;&#61472;攻毒保护率达90%.结果提示,已成功构建F1-V融合蛋白真核表达载体tPA-pVAX1/F1-V,且具有诱导特异性细胞免疫和体液免疫应答的能力, 对强毒鼠疫菌皮下攻毒有一定的保护效力,为鼠疫菌新型疫苗研制奠定了基础.     

Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases

Despite the successes provided by vaccination, many challenges still exist with respect to controlling new and re-emerging infectious diseases. Innovative vaccine platforms composed of adaptable adjuvants able to appropriately modulate immune responses, induce long-lived immunity in a single dose, and deliver immunogens in a safe and stable manner via multiple routes of administration are needed. This work describes the development of a novel biodegradable polyanhydride nanoparticle-based vaccine platform administered as a single intranasal dose that induced long-lived protective immunity against respiratory disease caused by Yesinia pestis, the causative agent of pneumonic plague. Relative to the responses induced by the recombinant protein F1-V alone and MPLA-adjuvanted F1-V, the nanoparticle-based vaccination regimen induced an immune response that was characterized by high titer and high avidity IgG1 anti-F1-V antibody that persisted for at least 23 weeks post-vaccination. After challenge, no Y. pestis were recovered from the lungs, livers, or spleens of mice vaccinated with the nanoparticle-based formulation and histopathological appearance of lung, liver, and splenic tissues from these mice post-vaccination was remarkably similar to uninfected control mice.     

A new purification strategy for fraction 1 capsular antigen and its efficacy against Yersinia pestis virulent strain challenge

F1 antigen is an attractive candidate for the development of a subunit vaccine against plague. In previous study, the extraction of this antigen from Yersinia pestis is characterized by using organic solvents. In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. As revealed in this study, this purification procedure is simple and effective, and avoids potential adverse effect on the antigen by organic solvents. Highly purified F1 that adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to F1 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10(4) CFU of Y. pestis virulent strain 141.     

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.     

Prevention of bubonic and pneumonic plague using plant-derived vaccines

Yersinia pestis, the causative agent of bubonic and pneumonic plague, is an extremely virulent bacterium but there are currently no approved vaccines for protection against this organism. Plants represent an economical and safer alternative to fermentation-based expression systems for the production of therapeutic proteins. The recombinant plague vaccine candidates produced in plants are based on the two most immunogenic antigens of Y. pestis: the fraction-1 capsular antigen (F1) and the low calcium response virulent antigen (V) either in combination or as a fusion protein (F1–V). These antigens have been expressed in plants using all three known possible strategies: nuclear transformation, chloroplast transformation and plant-virus-based expression vectors. These plant-derived plague vaccine candidates were successfully tested in animal models using parenteral, oral, or prime/boost immunization regimens. This review focuses on the recent research accomplishments towards the development of safe and effective pneumonic and bubonic plague vaccines using plants as bioreactors.     

Antigenic profiling of yersinia pestis infection in the Wyoming coyote (Canis latrans)

Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.     

鼠疫溶菌疫苗免疫小鼠的体液免疫应答

为选择以F1抗原为主要有效成分的鼠疫溶菌疫苗(Whole cell lysate of Yersinia pestis vaccine,WCLY)的免疫程序,设计了这组试验。在37℃培养鼠疫EV菌,通过超声波裂解法制备鼠疫溶菌疫苗。设计(0,2周)、(0,4周)、(0,2,4周)三种免疫程序,以每剂总蛋白量7.9μg、31.5μg和126.0μg三个剂量皮下接种NIH小鼠。分别在第一针免疫后2、4、8、12周采集血清,通过间接ELISA检测抗鼠疫菌F1抗原和总抗原抗体。结果显示:免疫后血清抗体上升很快,2周内即可测出;无论哪种免疫程序,至12周时抗体滴度仍保持高水平;加强免疫后,抗体水平在4周或8周达到较高,可与活疫苗免疫者相比;溶菌疫苗的接种剂量为7.9μg时,动物只出现轻度不良反应。提示鼠疫溶菌疫苗需要两剂免疫,最短可间隔2周,接种剂量应不超过7.9μg,疫苗中应富含F1抗原。     

Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH₂-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced T_H1 and T_H2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.     

Protection against lethal subcutaneous challenge of virulent <Emphasis Type="Italic">Y. pestis</Emphasis> strain 141 using an F1-V subunit vaccine

In this study, we designed and engineered a two-component recombinant fusion protein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis. The recombinant F1-V protein was formulated with Alhydrogel. A four-time injection with a dosage of 10, 20 and 50 μg/mouse in about two months was adopted for vaccination. Serum antibodies and subclass of T helper cells were measured and analyzed. After the final vaccination, the mice were challenged by 141 strain with 25–600 LD₅₀. In conclusion, the recombinant vaccine was capable of inducing protective immunity against subcutaneous challenge. The level of serum IgG was supposed to be a main factor that affected the final protection of challenge. 20 μg recombinant protein could induce an endpoint titre of serum IgG as high as 51200, which was enough to afford 100% protection against 400 LD₅₀ virulent 141 challenge. The antibody isotype analysis showed that the vaccine induced predominantly an IgG1 rather than IgG2a response. Flow cytometric analysis revealed that Alhydrogel significantly helped induce a stronger humoral immunity instead of CTL cellular response. These findings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.     

Developing subunit immunogens using B and T cell epitopes and their constructs derived from the F1 antigen of Yersinia pestis using novel delivery vehicles

Yersinia pestis is the etiological agent of pneumonic and bubonic plague. As the currently licensed vaccines for plague have their own limitations, there is a need for a rational and more effective form of a subunit vaccine to combat both forms of the disease. Newer methods of antigen delivery coupled with adjuvant offer an alternative approach toward a plague vaccine. In order to develop a new generation vaccine against plague, we chose an immunodominant, outer membrane capsular protein, F1 of Y. pestis. The immunogenicity of the peptide sequences, predicted to possess B (three sequences, B1, B2 and B3) and T (two sequences, T1 and T2) cell determinants, was studied in a murine model with different genetic backgrounds, using alhydrogel and liposomes as delivery vehicles. All the peptide sequences are immunogenic in all mouse strains and showed primary and secondary immune response. B2 peptide was found to be most immunogenic, followed by B1 and B3 peptides. Chimeras made between B and T structures proved highly immunogenic and the antibody levels are comparable with native F1 antigen, thereby proving that T1 and T2 are helper sequences. Interestingly, the liposome mode of immunization was found to be more immunogenic and generated higher affinity antibodies than the alum-based preparation. Immunization using a mixture of all the peptides further proved B2 to be immunodominant. The IgG isotype profile showed predominance of IgG1, IgG2b followed by IgG2a for all the formulations irrespective of mode of antigen delivery. Lymphocyte proliferation of spleen cells primed in vivo with peptides, B-T conjugates and F1 antigen followed by in vitro stimulation with these antigens in soluble (medium) and particulate (liposome) form, showed dose-dependent stimulation of T cells, while B-T constructs showed a higher stimulation index, comparable to F1 antigen. The liposome mode of antigen presentation showed higher lymphoproliferation of spleen cells. Of all the peptides tested, T1 and T2 sequences showed the highest stimulation indices. The pattern of cytokine levels was in the following order: interferon-gamma>interleukin-2>interleukin-4. In vivo protective studies of the B-T conjugates revealed that B1T1 and a mixture of conjugates showed a survival rate of 10 days. Thus, the study highlights the importance of B and T cell epitopes as peptide-based immunogens, being a serious alternative for plague vaccine.     

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.     

Yersinia pestis Yop secretion protein F: purification, characterization, and protective efficacy against bubonic plague

Yersinia pestis is a gram-negative human pathogen that uses a type III secretion system to deliver virulence factors into human hosts. The delivery is contact-dependent and it has been proposed that polymerization of Yop secretion protein F (YscF) is used to puncture mammalian cell membranes to facilitate delivery of Yersinia outer protein effectors into host cells. To evaluate the potential immunogenicity and protective efficacy of YscF against Y. pestis, we used a purified recombinant YscF protein as a potential vaccine candidate in a mouse subcutaneous infection model. YscF was expressed and purified from Escherichia coli by immobilized metal-ion affinity chromatography and protein identity was confirmed by ion trap mass spectrometry. The recombinant protein was highly alpha-helical and formed relatively stable aggregates under physiological conditions. The properties were consistent with behavior expected for the native YscF, suggesting that the antigen was properly folded. Ten mice were inoculated subcutaneously, administered booster injections after one month, and challenged with 130 LD(50) of wild type Y. pestis CO92. Six animals in the vaccinated group but none in the control group survived the challenge. The vaccinated animals produced high levels of specific antibodies against YscF as determined by Western blot. The data were statistically significant (P = 0.053 by two-tailed Fisher's test), suggesting that the YscF protein can provide a protective immune response against lethal plague challenge during subcutaneous plague infection.     

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.