[A molecular basis of the plague vaccine development].

Molecular mechanisms of the Yersinia pestis pathogenicity and peculiarities of maturation of specific immunity to plague are reviewed. The history and modern state of the plague vaccine development are described. Special attention is focused on the prospects in the area of the plague vaccine development. The possible approaches to improvement of vaccine preparations are discussed.

Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

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

Plague in Guinea pigs and its prevention by subunit vaccines.

Human pneumonic plague is a devastating and transmissible disease for which a Food and Drug Administration-approved vaccine is not available. Suitable animal models may be adopted as a surrogate for human plague to fulfill regulatory requirements for vaccine efficacy testing. To develop an alternative to pneumonic plague in nonhuman primates, we explored guinea pigs as a model system. On intranasal instillation of a fully virulent strain, Yersinia pestis CO92, guinea pigs developed lethal lung infections with hemorrhagic necrosis, massive bacterial replication in the respiratory system, and blood-borne dissemination to other organ systems. Expression of the Y. pestis F1 capsule was not required for the development of pulmonary infection; however, the capsule seemed to be important for the establishment of bubonic plague. The mean lethal dose (MLD) for pneumonic plague in guinea pigs was estimated to be 1000 colony-forming units. Immunization of guinea pigs with the recombinant forms of LcrV, a protein that resides at the tip of Yersinia type III secretion needles, or F1 capsule generated robust humoral immune responses. Whereas LcrV immunization resulted in partial protection against pneumonic plague challenge with 250 MLD Y. pestis CO92, immunization with recombinant F1 did not. rV10, a vaccine variant lacking LcrV residues 271-300, elicited protection against pneumonic plague, which seemed to be based on conformational antibodies directed against LcrV.

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.

Current challenges in the development of vaccines for pneumonic plague.

Inhalation of Yersinia pestis bacilli causes pneumonic plague, a rapidly progressing and exceptionally virulent disease. Extensively antibiotic-resistant Y. pestis strains exist and we currently lack a safe and effective pneumonic plague vaccine. These facts raise concern that Y. pestis may be exploited as a bioweapon. Here, I review the history and status of plague vaccine research and advocate that pneumonic plague vaccines should strive to prime both humoral and cellular immunity.

Clinical and pathologic features of cynomolgus macaques (Macaca fascicularis) infected with aerosolized Yersinia pestis.

Since the anthrax attacks of 2001, the emphasis on developing animal models of aerosolized select agent pathogens has increased. Many scientists believe that nonhuman primate models are the most appropriate to evaluate pulmonary response to, vaccines for, and treatments for select agents such as Yersinia pestis (Y. pestis), the causative agent of plague. A recent symposium concluded that the cynomolgus macaque (Macaca fascicularis) plague model should be characterized more fully. To date, a well-characterized cynomolgus macaque model of pneumonic plague using reproducible bioaerosols of viable Y. pestis has not been published. In the current study, methods for creating reproducible bioaerosols of viable Y. pestis strain CO92 (YpCO92) and pneumonic plague models were evaluated in 22 Indonesian-origin cynomolgus macaques. Five macaques exposed to doses lower than 250 CFU remained free of any indication of plague infection. Fifteen macaques developed fever, lethargy, and anorexia indicative of clinical plague. The 2 remaining macaques died without overt clinical signs but were plague-positive on culture and demonstrated pathology consistent with plague. The lethal dose of plague in humans is reputedly less than 100 organisms; in this study, 66 CFU was the dose at which half of the macaques developed fever and clinical signs (ED(50)), The Indonesian cynomolgus macaque reproduces many aspects of human pneumonic plague and likely will provide an excellent model for studies that require a macaque model.

The anti-plague system and the Soviet biological warfare program.

The USSR possessed a unique national public health system that included an agency named "anti-plague system." Its mission was to protect the country from highly dangerous diseases of either natural or laboratory etiology. During the 1960s, the anti-plague system became the lead agency of a program to defend against biological warfare, codenamed Project 5. This responsibility grew and by the middle 1970s came to include undertaking tasks for the offensive biological warfare program, codenamed Ferment. This article describes the anti-plague system's activities relevant to both aspects of the Soviet Union's biological warfare program, offense and defense, and analyzes its contributions to each.

Immunisation against plague by transcutaneous and intradermal application of subunit antigens.

We have investigated immunological responses in BALB/c mice following transcutaneous (TC) delivery of fraction 1 (F1) and V subunits from Yersinia pestis in conjunction with an enterotoxin-derived adjuvant (cholera toxin, CT). It was found that two or more TC applications of F1 and V subunits (admixed with cholera toxin) served to elicit significant levels of anti-F1 and V antibodies in the serum of immunised mice. IL-6 secretion from cultured splenocytes derived from immunised mice indicated that a single TC application of F1 and V subunits (admixed with cholera toxin) conferred a cell-mediated response. As compared with intranasal or direct intradermal injection of F1 and V, the numbers of F1/V-specific antibody-forming cells in the spleens of animals immunised by TC application of F1 and V (admixed with CT) was relatively low. It was noted that TC application of F1 and V admixed with CT was very effective for priming responses that were boosted by intranasal or intradermal routes. Similarly, it was found that TC application of F1 and V admixed with CT could be used to efficiently boost pre-existing responses engendered by intradermal injection or intranasal instillation of F1 and V. In order to assess if TC application of F1 and V admixed with CT could protect experimental animals from plague, immunised mice were injected with a virulent strain of Y. pestis. It was found that two TC applications of F1 and V admixed with CT conferred only limited protection against 10(2) MLDs. However, three TC applications of F1 and V admixed with CT conferred solid protection against 10(2) MLDs. Hence we have shown, for the first time, that TC application of F1 and V admixed with CT can protect animals against challenge with a virulent strain of plague causing bacteria. These data suggest that transcutaneous immunisation may be a simple and non-invasive method for immunising individuals against plague.

Protection against plague following immunisation with microencapsulated V antigen is reduced by co-encapsulation with IFN-gamma or IL-4, but not IL-6.

We have investigated intranasal delivery of novel vaccines for plague, based on poly-L-lactide (PLLA) microencapsulated recombinant V antigen (rV) of Yersinia pestis. Microspheres containing rV alone or co-encapsulated with the cytokines IFN-gamma, IL-4 or IL-6 were administered in a two-dose regimen and antibody responses and protective efficacy were monitored. All treatment groups stimulated high rV-specific antibody titres in serum, predominantly of the IgG1 isotype, which were maintained over several months. There was evidence of both IgG and IgA responses in lung samples from all groups. Formulations based on rV antigen alone or rV co-encapsulated with IL-6 provided complete protection against systemic challenge with Y. pestis strain GB; however protective efficacy was impaired by co-encapsulating either IFN-gamma or IL-4 with rV.

Vaccines for preventing plague.

BACKGROUND: Plague is endemic in China, Mongolia, Burma, Vietnam, Indonesia, India, large parts of Southern Africa, the United States and South America. There are three types of vaccines (live attenuated, killed and F1 fraction) with varying means of administration. OBJECTIVES: The objective of this review was to assess the effects of vaccines to prevent plague. SEARCH STRATEGY: We searched Medline, Embase, the Cochrane Controlled Trials Register and reference lists of articles. We handsearched the journal 'Vaccine' and contacted experts in the field. SELECTION CRITERIA: Randomised trials comparing live and killed plague vaccines against no intervention, placebo, other plague vaccines or vaccines against other disease (control vaccines). DATA COLLECTION AND ANALYSIS: Three reviewers assessed the eligibility of trials. MAIN RESULTS: No trials were included. REVIEWER'S CONCLUSIONS: There is not enough evidence to evaluate the effectiveness of any plague vaccine, or the relative effectiveness between vaccines and their tolerability. Circumstantial data from observational studies suggest that killed types may be more effective and have fewer adverse effects than attenuated types of vaccine. No evidence appears to exist on the long-term effects of any plague vaccine.

An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model.

The objective of this study was to identify an immunological correlate of protection for a two-component subunit vaccine for plague, using a mouse model. The components of the vaccine are the F1 and V antigens of the plague-causing organism, Yersinia pestis, which are coadsorbed to alhydrogel and administered intramuscularly. The optimum molar ratio of the subunits was determined by keeping the dose-level of either subunit constant whilst varying the other and observing the effect on specific antibody titre. A two-fold molar excess of F1 to V, achieved by immunizing with 10 micrograms of each antigen, resulted in optimum antibody titres. The dose of vaccine required to protect against an upper and lower subcutaneous challenge with Y. pestis was determined by administering doses in the range 10 micrograms F1 + 10 micrograms V to 0.01 microgram F1 + 0.01 microgram V in a two-dose regimen. For animals immunized at the 1-microgram dose level or higher with F1 + V, an increase in specific IgG1 titre was observed over the 8 months post-boost and they were fully protected against a subcutaneous challenge with 10(5) colony-forming units (CFU) virulent Y. pestis at this time point. However, immunization with 5 micrograms or more of each subunit was required to achieve protection against challenge with 10(7) CFU Y. pestis. A new finding of this study is that the combined titre of the IgG1 subclass, developed to F1 plus V, correlated significantly (P < 0.05) with protection. The titres of IgG1 in vaccinated mice which correlated with 90%, 50% and 10% protection have been determined and provide a useful model to predict vaccine efficacy in man.

A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model.

The median lethal dose (MLD) of a pathogenic strain of Yersinia pestis was established by three routes of administration in three strains of mouse. There was no significant difference between the MLDs in the different strains of mouse. The MLD by the subcutaneous route in Balb/C and an outbred line was approximately 1 c.f.u.; the MLD following intraperitoneal administration was tenfold higher. There were significant differences in the mean times to death after administration of the challenge by different routes. The relative efficacy of a live attenuated vaccine strain of Y. pestis (EV76) was compared with that of the formaldehyde-killed vaccine (Plague vaccine, USP). EV76 protected against high challenge doses (up to 5.75 x 10(6) MLD), though immunized animals showed side effects of varying severity. The killed vaccine was less effective in terms of dose-protection (deaths occurred after challenge with 4000 MLD) and several of the vaccinated animals suffered sub-lethal, plague-related sequelae to the challenge.

[Duration and intensity of postvaccinal immunity to plague in experimental animals]. / Dlitel'nost' i napriazhennost' postvaktsinal'nogo immuniteta k chume v eksperimente na zhivotnykh.

Experiments were conducted on guinea pigs and Papio hamadryas; it was shown that a reduction of the intensity of postvaccinal immunity occurred at various periods after a single vaccination. In inhalation method of immunization in guinea pigs it decreased in 6 months 135 times, in monkeys in one year--133 times. However, at the mentioned periods vaccination provided protection of 50% of the animals from infection with Past. pestis in a dose constitutin 20 to 25 aerosol LD50 for nonimmunized animals. Despite the more pronounced (57--640 times) reduction of the intensity of immunity than in the animals vaccinated by inhalation, in the subcutaneously vaccinated guinea pigs in subcutaneously infected with Past. pestis protection level remained high (resistance index in 3 and 6 months constituted 37.10(6) and 3-3-10(6), respectively).

Immunity in plague: protection induced in Cercopithecus aethiops by oral administration of live, attenuated Yersinia pestis.

A live, attenuated Yersinia pestis vaccine, designated EV76 (Paris) F, was pathogenic in Cercopithecus aethiops when administered parenterally. Although an oral dose of 10(9) colone-forming units of the vaccine was nonfatal to vervets, a transient but severe gastrointestinal disturbance resulted in four of 12 animals. Seven immunized vervets were protected against intradermal challenge. The remaining five vervets without serologic reactions and five untreated controls died from typical bubonic (septicemic) plague within seven days. General reactions after vaccination were not predictive of later-developing immunity, which appeared to correlate with the production of Fraction I-specific antibodies. Marked differences in immunity were noted with two methods of vaccine administration (incorporation into bananas and dropping into the mouth). This finding supported the prior observation that the oral mucosa is the major portal of entry for this strain of vaccine. Local reactions in the buccal cavity after feeding were negligible. Thus this strain of Y. pestis is a nonlethal, effective weapon against bubonic plague in a susceptible species.

Effectiveness of live or killed plague vaccines in man.

While the safety of the available live plague vaccine EV 76 (Paris) continues to be the subject of further study, the USP formol-killed, virulent Pasteurella pestis (Yersinia pestis) suspension capable of protecting 60% of non-human primates, particularly Hanuman langurs (Presbytis entellus), warrants further clinical tests and field trials. Inoculated in a dosage of 2x10(9) killed plague bacilli (1 ml), followed by a booster of 400 million organisms (0.2 ml) in 1-3 months, this vaccine stimulates the appearance of passive mouse-protection antibodies (below an index of 10) and passive haemagglutinins in 60%-65% of human subjects. Recent experiences in Viet-Nam demonstrate that personnel vaccinated with the USP vaccine, although frequently exposed, enjoy almost complete freedom from the disease. One of the 4 known and confirmed cases of bubonic plague in North Americans occurred in an unvaccinated individual. Among individuals inoculated with the USP vaccine, 2 confirmed cases of pneumonic plague and 1 case of asymptomatic pharyngeal plague have been recorded. The incidence of plague in the Republic of Viet-Nam during the past 3 years is estimated at 13 263 cases in a population in part vaccinated with a live plague which exhibited inadequate immunogenic efficacy in experimental tests.