Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague

Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine.     

A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.     

Concomitant administration of Yersinia pestis specific monoclonal antibodies with plague vaccine has a detrimental effect on vaccine mediated immunity

Antibodies can be used to confer rapid immunity against infectious agents for short periods of time. By comparison, vaccine induced immunity is more protective, but takes a relatively long time to develop. Concomitant administration of antibody and vaccine by different routes was evaluated as a means of providing both rapid and long-term protection against plague. BALB/c mice were treated intraperitoneally with monoclonal antibodies, with specificities for Yersinia pestis LcrV and F1 antigens. A cohort of these mice was simultaneously vaccinated with rF1 and rLcrV by the intramuscular route. Antibody co-administration with vaccine reduced the level of vaccine mediated protection afforded against a high level Y. pestis challenge. Conversely, antibody-mediated protection was unaffected by vaccine co-administration and lasted for at least 8 weeks post administration. We also evaluated the effect of administering vaccine intradermally and antibody intratracheally and observed that, irrespective of administration route, concomitant administration of antibody reduced the effectiveness of vaccine mediated immunity. The results of passive transfer experiments supported the thesis that the development of protective antibody responses following vaccination is impaired by the presence of circulating monoclonal antibodies with specificities for important B-cell epitopes in the vaccine. We also noted that intradermal injection of LcrV antigen and cholera toxin adjuvant afforded good levels of protection against systemic and aerosol challenge with Y. pestis: intradermal injection might therefore be considered as a potential minimally invasive method of plague vaccine administration. These data have implications for the design of therapeutic strategies against plague infection.     

Relative immunogenicity and protection potential of candidate Yersinia Pestis antigens against lethal mucosal plague challenge in Balb/C mice

Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine.     

Developing live vaccines against plague

Three great plague pandemics caused by the gram-negative bacterium Yersinia pestis have killed nearly 200 million people and it has been linked to biowarfare in the past. Plague is endemic in many parts of the world. In addition, the risk of plague as a bioweapon has prompted increased research to develop plague vaccines against this disease. Injectable subunit vaccines are being developed in the United States and United Kingdom. However, the live attenuated Y. pestis-EV NIIEG strain has been used as a vaccine for more than 70 years in the former Soviet Union and in some parts of Asia and provides a high degree of efficacy against plague. This vaccine has not gained general acceptance because of safety concerns. In recent years, modern molecular biological techniques have been applied to Y. pestis to construct strains with specific defined mutations designed to create safe, immunogenic vaccines with potential for use in humans and as bait vaccines to reduce the load of Y. pestis in the environment. In addition, a number of live, vectored vaccines have been reported using attenuated viral vectors or attenuated Salmonella strains to deliver plague antigens. Here we summarize the progress of live attenuated vaccines against plagu.     

Antibodies and cytokines independently protect against pneumonic plague

Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFalpha) and gamma-interferon (IFNgamma) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFalpha and IFNgamma confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFalpha and IFNgamma upon exposure to Y. pestis.     

A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs

The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.     

Neutralization of Yersinia pestis-mediated macrophage cytotoxicity by anti-LcrV antibodies and its correlation with protective immunity in a mouse model of bubonic plague

Plague is a life-threatening disease caused by Yersinia pestis, for which effective-licensed vaccines and reliable predictors of in vivo immunity are lacking. V antigen (LcrV) is a major Y. pestis virulence factor that mediates translocation of the cytotoxic Yersinia protein effectors (Yops). It is a well-established protective antigen and a part of currently tested plague subunit vaccines. We have developed a highly sensitive in vitro macrophage cytotoxicity neutralization assay which is mediated by anti-LcrV antibodies; and studied the potential use of these neutralizing antibodies as an in vitro correlate of plague immunity in mice. The assay is based on a Y. pestis strain with enhanced cytotoxicity to macrophages in which endogenous yopJ was replaced by the more effectively translocated yopP of Y. enterocolitica O:8. Mice passively immunized with rabbit anti-LcrV IgG or actively immunized with recombinant LcrV were protected against lethal doses of a virulent Y. pestis strain, in a mouse model of bubonic plague. This protection significantly correlated with the in vitro neutralizing activity of the antisera but not with their corresponding ELISA titers. In actively immunized mice, a cutoff value for serum neutralizing activity, above which survival was assured with high degree of confidence, could be established for different vaccination regimes. The impact of overall findings on the potential use of serum neutralizing activity as a correlate of protective immunity is discussed.     

Fatal laboratory-acquired infection with an attenuated Yersinia pestis Strain--Chicago, Illinois, 2009

On September 18, 2009, the Chicago Department of Public Health (CDPH) was notified by a local hospital of a suspected case of fatal laboratory-acquired infection with Yersinia pestis, the causative agent of plague. The patient, a researcher in a university laboratory, had been working along with other members of the laboratory group with a pigmentation-negative (pgm-) attenuated Y.?pestis strain (KIM D27). The strain had not been known to have caused laboratory-acquired infections or human fatalities. Other researchers in a separate university laboratory facility in the same building had contact with a virulent Y.?pestis strain (CO92) that is considered a select biologic agent; however, the pgm- attenuated KIM D27 is excluded from the National Select Agent Registry. The university, CDPH, the Illinois Department of Public Health (IDPH), and CDC conducted an investigation to ascertain the cause of death. This report summarizes the results of that investigation, which determined that the cause of death likely was an unrecognized occupational exposure (route unknown) to Y.?pestis, leading to septic shock. Y. pestis was isolated from premortem blood cultures. Polymerase chain reaction (PCR) identified the clinical isolate as a pgm- strain of Y. pestis. Postmortem examination revealed no evidence of pneumonic plague. A postmortem diagnosis of hereditary hemochromatosis was made on the basis of histopathologic, laboratory, and genetic testing. One possible explanation for the unexpected fatal outcome in this patient is that hemochromatosis-induced iron overload might have provided the infecting KIM D27 strain, which is attenuated as a result of defects in its ability to acquire iron, with sufficient iron to overcome its iron-acquisition defects and become virulent. Researchers should adhere to recommended biosafety practices when handling any live bacterial cultures, even attenuated strains, and institutional biosafety committees should implement and maintain effective surveillance systems to detect and monitor unexpected acute illness in laboratory workers.     

Intranasal delivery of a protein subunit vaccine using a Tobacco Mosaic Virus platform protects against pneumonic plague

Yersinia pestis, one of history’s deadliest pathogens, has killed millions over the course of human history. It has attributes that make it an ideal choice to produce mass casualties and is a prime candidate for use as a biological weapon. When aerosolized, Y. pestis causes pneumonic plague, a pneumonia that is 100% lethal if not promptly treated with effective antibiotics. Currently, there is no FDA approved plague vaccine. The current lead vaccine candidate, a parenterally administered protein subunit vaccine comprised of the Y. pestis virulence factors, F1 and LcrV, demonstrated variable levels of protection in primate pneumonic plague models. As the most likely mode of exposure in biological attack with Y. pestis is by aerosol, this raises a question of whether this parenteral vaccine will adequately protect humans against pneumonic plague. In the present study we evaluated two distinct mucosal delivery platforms for the intranasal (IN) administration of LcrV and F1 vaccine proteins, a live bacterial vector, Lactobacillus plantarum, and a Tobacco Mosaic Virus (TMV) based delivery platform. IN administration of L. plantarum expressing LcrV, or TMV-conjugated to LcrV and F1 (TMV-LcrV+TMV-F1) resulted in the similar induction of high titers of IgG antibodies and evidence of proinflammatory cytokine secretion. However, only the TMV-conjugate delivery platform protected against subsequent lethal challenge with Y. pestis. TMV-LcrV+TMV-F1 co-vaccinated mice had no discernable morbidity and no mortality, while mice vaccinated with L. plantarum expressing LcrV or rLcrV+rF1 without TMV succumbed to infection or were only partially protected. Thus, TMV is a suitable mucosal delivery platform for an F1-LcrV subunit vaccine that induces complete protection against pneumonic infection with a lethal dose of Y. pestis in mice.     

Intranasal Protollin/F1-V vaccine elicits respiratory and serum antibody responses and protects mice against lethal aerosolized plague infection

F1-V is a recombinant plague antigen comprising the capsular (F1) and virulence-associated (V) proteins. Given intramuscularly with Alhydrogel, it protects mice against challenge, but is less effective in non-human primates against high-dose aerosolized Yersinia pestis challenge, perhaps because it fails to induce respiratory immunity. Intranasal immunization of mice with F1-V formulated with a Proteosome-based adjuvant (Protollin), elicited high titers of specific IgA in lungs whereas intranasal F1-V alone or intramuscular Alhydrogel-adsorbed F1-V did not. The Protollin-adjuvanted F1-V vaccine also induced high serum titers of specific IgG, comparable to those induced by intramuscular Alhydrogel-adsorbed F1-V. Mice immunized intranasally with Protollin-F1-V were 100% protected against aerosol challenge with 170 LD50 of Y. pestis and 80% against 255 LD50.     

Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.

Yersinia pestis, the causative organism of plague, produces V antigen (LcrV), a bifunctional protein with regulatory and virulence roles that has been shown to be highly protective against a plague challenge. A combined sub-unit vaccine, comprising recombinant V and Fraction 1 antigens is currently being developed. We report here the expression and purification of recombinant V antigen (rV) using three different expression systems: the N-terminal GST fusion pGEX-5X-2 and pGEX-6P-2 systems from Pharmacia Biotech, and the C-terminal CBD fusion (IMPACT I) system from New England Biolabs. After cleavage from the carrier protein, the yields of rV were 25 mg l(-1) (pGEX-5X-2), 31 mg l(-1) (pGEX-6P-2) and 0.75 mg l(-1) (IMPACT I). All of the recombinant proteins were immunogenic in mice, although there were some differences in their protective efficacy against subcutaneous challenge with Y. pestis. Whilst rV antigen derived from the IMPACT I and pGEX-6P-2 systems and given in two immunising doses protected fully against challenge with 1 x 10(7) colony forming units (cfu) of Y. pestis, there was breakthrough in protection against 1 x 10(5) cfu of Y. pestis in animals immunised twice with rV from the pGEX-5X-2 system. From this study, the pGEX-6P-2 has been selected for the production of rV as a vaccine component. The pGEX-6P-2 system utilises a GST tagged PreScission Protease (a recombinant human rhinovirus 3C protease) to cleave the fusion protein, thereby allowing efficient removal of the enzyme from the final product. In addition, the enzyme is not of animal origin, therefore making it suitable for vaccine production.     

A live attenuated strain of Yersinia pestis ΔyscB provides protection against bubonic and pneumonic plagues in mouse model

To develop a safe and effective live plague vaccine, the ΔyscB mutant was constructed based on Yersinia pestis biovar Microtus strain 201 that is avirulent to humans, but virulent to mice. The virulence, immunogenicity and protective efficacy of the ΔyscB mutant were evaluated in this study. The results showed that the ΔyscB mutant was severely attenuated, elicited a higher F1-specific antibody titer and provided protective efficacy against bubonic and pneumonic plague in mouse model. The ΔyscB mutant could induce the secretion of both Th1-associated cytokines (IFN-γ, IL-2 and TNF-α) and Th2-associated cytokines (IL-4 and IL-10). Taken together, the ΔyscB mutant represented a potential vaccine candidate based on its ability to generate strong humoral and cell-mediated immune responses and to provide good protection against both subcutaneous and intranasal Y. pestis challenge.     

Vaccination against bubonic and pneumonic plague.

Yersinia pestis is the etiological agent of bubonic and pneumonic plague, diseases which have caused over 200 milllion human deaths in the past. Plague still occurs throughout the world today, though for reasons that are not fully understood pandemics of disease do not develop from these outbreaks. Antibiotic treatment of bubonic plague is usually effective, but pneumonic plague is difficult to treat and even with antibiotic therapy death often results. A killed whole cell plague vaccine has been used in the past, but recent studies in animals have shown that this vaccine offers poor protection against pneumonic disease. A live attenuated vaccine is also available. Whilst this vaccine is effective, it retains some virulence and in most countries it is not considered to be suitable for use in humans. We review here work to develop improved sub-unit and live attenuated vaccines against plague. A sub-unit vaccine based on the F1- and V-antigens is highly effective against both bubonic and pneumonic plague, when tested in animal models of disease. This vaccine has been used to explore the utility of different intranasal and oral delivery systems, based on the microencapsulation or Salmonella delivery of sub-units.     

青海省三江源地区鼠疫病原学分析

目的 研究青海省三江源地区鼠疫菌株生物学特性并确定疫源地空间结构及性质.方法 对1954-2007年青海省三江源地区分离的鼠疫菌株进行生物学表型鉴定及分子生物学研究.结果 411株代表性菌株中,12株脱氮(-)阿胶糖(-)甘油(+)属田鼠型,399株为脱氮(+)阿胶糖(+)甘油(+)属古典型.411株均能产生F1和Pst I;vw+菌株占95.13%(391/411),VW-菌株占4.87%(20/411);Pgm+菌株占80.78%(332/411)、Pgm+菌株占9%(37/411)、Pgm-菌株占10.22%(42/411).220株代表株中96.82%(213/220)的菌株对小白鼠表现为强毒株,3.18%(7,220)为中等毒力,说明绝大多数具高致病性,其毒力很强.90.02%(307/341)菌株携带6×106 45×106 x65×106质粒.80株代表株包括8个基因组型,其中6个基因型与原分型相同,另有2个新的基因组型.结论 三江源地区鼠疫菌株具备青藏高原鼠疫病原体特性,人类一旦感染可导致发病急、病情重、传染性强、病死率高等特点.     

Protection conferred by a fully recombinant sub-unit vaccine against Yersinia pestis in male and female mice of four inbred strains

In this paper, we describe for the first time the use of a fully recombinant sub-unit vaccine for plague. We have compared the protection afforded by the recombinant vaccine against Yersinia pestis in male and female mice of four inbred strains. We also determined the in vivo cellular memory and antibody response after one year. The recombinant vaccine was capable of inducing protective immunity, against subcutaneous and aerosol challenge, in mice from all four strains. Although, there was some breakthrough in the CBA males challenged with 10(7) median lethal dose (MLDs) the other mice regardless of sex or strain were well protected even at this extreme challenge dose. In both male and female mice, the specific IgG titres to both antigens peaked at day 28 and 35 and in female mice these titres were maintained for >1 year.     

Inducing systemic and mucosal immune responses to B-T construct of F1 antigen of Yersinia pestis in microsphere delivery

Plague is a zoonotic disease caused by Yersinia pestis, an etiological agent of pneumonic and bubonic plague. There is a need for an improved plague vaccine that may overcome the limitation of presently available whole cell vaccine. An alternative approach described here, is the use of protective epitopes from immunodominant antigen of Y. pestis. One such antigen is the F1 antigen, a major envelope and virulent protein that possess antiphagocytic and anti-microbial properties. The present study was aimed to develop a peptide-based vaccine, based upon the constructs made between B and T cell epitopes of F1 antigen of Y. pestis. The immunogenicity, IgG subclass pattern, affinity, avidity and in vivo protective efficacy of the antibodies generated for different B-T constructs were studied in murine model using microsphere as the delivery vehicle. The mode of immunization was both intranasal and intramuscular, with single and multiple doses of immunization, respectively. Intranasal immunization generated consistent high titre and long lasting immune response both for IgG and IgA in sera and sIgA in washes while intramuscular route generated peak IgG levels in sera only. The IgG isotypic levels pattern showed higher IgG2a/IgG2b levels in intranasal route while mixed isotypic levels of IgG1, IgG2a/IgG2b were observed in intramuscular route. The affinity and relative avidity of antibodies showed best results with intranasal route as compared to the intramuscular route. The specific activity measurement (IgG/IgA content) in sera and washes were well correlated with the antibody levels. Finally, in vivo protective studies showed that B1T1 and B2T1 conjugates protected the mice till day 15 while rest of the conjugates showed poor protection.     

A DNA vaccine producing LcrV antigen in oligomers is effective in protecting mice from lethal mucosal challenge of plague

There is an urgent need to develop effective vaccines against pneumonic plague, a highly lethal and contagious disease caused by the Gram-negative bacterium Yersinia pestis. Here we demonstrate that a novel DNA vaccine expressing a modified V antigen (LcrV) of Y. pestis, with a human tissue plasminogen activator (tPA) signal sequence, elicited strong V-specific antibody responses in BALB/c mice. This tPA-V DNA vaccine protected mice from intranasal challenge with lethal doses of Y. pestis. In comparison, a DNA vaccine expressing the wild type V antigen was much less effective. Only tPA-V formed oligomers spontaneously, and elicited a higher IgG2a anti-V antibody response in immunized mice, suggesting increased TH1 type cellular immune response. Our data indicate that antigen engineering is effective in inducing high quality protective immune responses against conformationally sensitive antigens. These results support that optimized DNA vaccines have the potential to protect against bacterial pathogens than is generally recognized.     

Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines

Yersinia pestis causes plague, a disease with high mortality in humans that can be transmitted by fleabite or aerosol. A US Food and Drug Administration (FDA)-licensed plague vaccine is currently not available. Vaccine developers have focused on two subunits of Y. pestis: LcrV, a protein at the tip of type III secretion needles, and F1, the fraction 1 pilus antigen. F1-V, a hybrid generated via translational fusion of both antigens, is being developed for licensure as a plague vaccine. The rV10 vaccine is a non-toxigenic variant of LcrV lacking residues 271-300. Here we developed Current Good Manufacturing Practice (cGMP) protocols for rV10. Comparison of clinical grade rV10 with F1-V did not reveal significant differences in plague protection in mice, guinea pigs or cynomolgus macaques. We also developed cGMP protocols for rV10-2, a variant of rV10 with an altered affinity tag. Immunization with rV10-2 adsorbed to aluminum hydroxide elicited antibodies against LcrV and conferred pneumonic plague protection in mice, rats, guinea pigs, cynomolgus macaques and African Green monkeys. The data support further development of rV10-2 for FDA Investigational New Drug (IND) authorization review and clinical testing.     

Identification of encapsulated and non-encapsulated Yersinia pestis by immunofluorescence tests using polyclonal and monoclonal antibodies

Rabbit polyclonal hyperimmune antibodies to Yersinia pestis, and a mouse monoclonal antibody against the capsular antigen fraction 1 (F1) were compared in immunofluorescence (IF) tests. Fluorescent antibody conjugates were prepared from polyclonal antisera to four F1 positive Y. pestis strains; the conjugated antibody to strain A1122 gave the strongest IF staining of F1 positive and F1 negative Y. pestis strains. Indirect assays were rejected in favour of direct assays utilizing polyclonal and monoclonal reagents because the increased background staining reduced the effective contrast of bacterial visualisation. Polyclonal conjugates gave fairly homogeneous staining of Y. pestis bacterial populations, but in monoclonal assays a skew distribution of fluorescence intensity was observed, the majority of bacteria being poorly stained. The proportion of cells stained well by the monoclonal sufficed for easy identification of Y. pestis of the F1 positive phenotype however, and staining was not affected by washing the bacteria or treating them with formaldehyde. Y. pestis strains of the F1 positive genotype reacted with the monoclonal if bacteria were grown at 37 degrees C but not if the growth temperature was reduced to 25 degrees C thus preventing capsule production. The polyclonal conjugate reacted with bacteria of these strains that had been grown at either temperature. Strains of F1 negative genotype grown at either temperature reacted with the polyclonal conjugate but not with the monoclonal. Cross reactions between the polyclonal reagents and Y. enterocolitica biovar 2, serovar O 8 could not be removed by selective absorption; however, the monoclonal antibody gave no cross reaction. The F1 phenotypic status of bacterial preparations was verified by ELISA measurement of the fraction 1 antigen concentration. Antigen levels for F1 positive and F1 negative phenotypes differed by about three logs for suspensions of Y. pestis harvested from solid media. The polyclonal and monoclonal direct IF tests applied to spleen and blood smears of laboratory mice infected with Y. pestis were able to differentiate between lethal infection with an F1 positive strain carrying all four classical virulence determinants, an F1 positive vaccine strain, and an F1 negative strain.