Complete Protection Against Yersinia pestis in BALB/c Mouse Model Elicited by Immunization With Inhalable Formulations of rF1-V10 Fusion Protein via Aerosolized Intratracheal Inoculation.

Pneumonic plague, caused by Yersinia pestis, is an infectious disease with high mortality rates unless treated early with antibiotics. Currently, no FDA-approved vaccine against plague is available for human use. The capsular antigen F1, the low-calcium-response V antigen (LcrV), and the recombinant fusion protein (rF1-LcrV) of Y. pestis are leading subunit vaccine candidates under intense investigation; however, the inability of recombinant antigens to provide complete protection against pneumonic plague in animal models remains a significant concern. In this study, we compared immunoprotection against pneumonic plague provided by rF1, rV10 (a truncation of LcrV), and rF1-V10, and vaccinations delivered via aerosolized intratracheal (i.t.) inoculation or subcutaneous (s.c.) injection. We further considered three vaccine formulations: conventional liquid, dry powder produced by spray freeze drying, or dry powder reconstituted in PBS. The main findings are: (i) rF1-V10 immunization with any formulation via i.t. or s.c. routes conferred 100% protection against Y. pestis i.t. infection; (ii) rF1 or rV10 immunization using i.t. delivery provided significantly stronger protection than rF1 or rV10 immunization via s.c. delivery; and (iii) powder formulations of subunit vaccines induced immune responses and provided protection equivalent to those elicited by unprocessed liquid formulations of vaccines. Our data indicate that immunization with a powder formulation of rF1-V10 vaccines via an i.t. route may be a promising vaccination strategy for providing protective immunity against pneumonic plague.

Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague.

Based on our improved novel Salmonella vaccine delivery platform, we optimized the recombinant attenuated Salmonella typhimurium vaccine (RASV) χ12094 to deliver multiple Yersinia pestis antigens. These included LcrV196 (amino acids, 131-326), Psn encoded on pYA5383 and F1 encoded in the chromosome, their synthesis did not cause adverse effects on bacterial growth. Oral immunization with χ12094(pYA5383) simultaneously stimulated high antibody titers to LcrV, Psn and F1 in mice and presented complete protection against both subcutaneous (s.c.) and intranasal (i.n.) challenges with high lethal doses of Y. pestis CO92. Moreover, no deaths or other disease symptoms were observed in SCID mice orally immunized with χ12094(pYA5383) over a 60-day period. Therefore, the trivalent S. typhimurium-based live vaccine shows promise for a next-generation plague vaccine.

Effect of nanovaccine chemistry on humoral immune response kinetics and maturation.

Acute respiratory infections represent a significant portion of global morbidity and mortality annually. There is a critical need for efficacious vaccines against respiratory pathogens. To vaccinate against respiratory disease, pulmonary delivery is an attractive route because it mimics the route of natural infection and can confer both mucosal and systemic immunity. We have previously demonstrated that a single dose, intranasal vaccine based on polyanhydride nanoparticles elicited a protective immune response against Yersinia pestis for at least 40 weeks after immunization with F1-V. Herein, we investigate the effect of nanoparticle chemistry and its attributes on the kinetics and maturation of the antigen-specific serum antibody response. We demonstrate that manipulation of polyanhydride nanoparticle chemistry facilitated differential kinetics of development of antibody titers, avidity, and epitope specificity. The results provide new insights into the underlying role(s) of nanoparticle chemistry in providing long-lived humoral immunity and aid in the rational design of nanovaccine formulations to induce long-lasting and mature antibody responses.

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 ß-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the ß-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 TH1 and TH2 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.

Inhibition of Yersinia pestis DNA adenine methyltransferase in vitro by a stibonic acid compound: identification of a potential novel class of antimicrobial agents.

BACKGROUND AND PURPOSE: Multiple antibiotic resistant strains of plague are emerging, driving a need for the development of novel antibiotics effective against Yersinia pestis. DNA adenine methylation regulates numerous fundamental processes in bacteria and alteration of DNA adenine methlytransferase (Dam) expression is attenuating for several pathogens, including Y. pestis. The lack of a functionally similar enzyme in humans makes Dam a suitable target for development of novel therapeutics for plague. EXPERIMENTAL APPROACH: Compounds were evaluated for their ability to inhibit Dam activity in a high-throughput screening assay. DNA was isolated from Yersinia grown in the presence of lead compounds and restricted to determine the effect of inhibitors on DNA methylation. Transcriptional analysis was undertaken to determine the effect of an active inhibitor on virulence-associated phenotypes. KEY RESULTS: We have identified a series of aryl stibonic acids which inhibit Dam in vitro. The most active, 4-stibonobenzenesulfonic acid, exhibited a competitive mode of inhibition with respect to DNA and a K(i) of 6.46 nM. One compound was found to inhibit DNA methylation in cultured Y. pestis. The effects of this inhibition on the physiology of the cell were widespread, and included altered expression of known virulence traits, including iron acquisition and Type III secretion. CONCLUSIONS AND IMPLICATIONS: We have identified a novel class of potent Dam inhibitors. Treatment of bacterial cell cultures with these inhibitors resulted in a decrease in DNA methylation. Expression of virulence factors was affected, suggesting these inhibitors may attenuate bacterial infectivity and function as antibiotics.

[Current state of problem of improving tools for plague vaccine prophylaxis].

Literature data on main immunobiological characteristics of 1st generation plague vaccines as well as ways of development of new tools for specific prophylaxis of plague: recombinant live, chemical, antiidiotypic, and DNA vaccines are presented in the review. Their expected advantages and disadvantages, perspectives of development and practical use in system of antiepidemic measures are assessed.

Biophysical characterization and formulation of F1-V, a recombinant plague antigen.

The recombinant plague antigen, F1-V, was studied for its structural characteristics using several biophysical techniques. A larger apparent molecular weight relative to its calculated molecular weight obtained from size exclusion chromatography, an unusually large R(g) obtained from MALS, and ANS dye binding studies which indicate that all hydrophobic regions of the protein are exposed to solvent demonstrated that F1-V exists like a disordered protein with a worm-like conformation. The pH-solubility profile of F1-V showed a solubility minimum at pH 5, close to its pI, consistent with the lack of repulsive forces that result in aggregation. Thus, in contrast to most globular proteins that exhibit a secondary and a tertiary structure, F1-V seems to lack tertiary structure and like an unfolded protein is more prone to aggregation via hydrophobic interactions. Despite this, when renatured gradually using descending guanidine hydrochloride concentration dialysis, in the presence of Mg+2, a surfactant and arginine hydrochloride at a pH of 7.5, F1-V appears to populate predominantly in its monomeric state.

Lipid A mimetics are potent adjuvants for an intranasal pneumonic plague vaccine.

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1 degrees /2 degrees dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (TH1) immune response. This intranasal vaccine showed 90% protection in Sprague-Dawley rats challenged with 1000 LD50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

Yersinia pestis (plague) vaccines.

Live attenuated and killed whole cell vaccines against disease caused by Yersinia pestis have been available since the early part of the last century. Although these vaccines indicate the feasibility of protecting against disease, they have a number of shortcomings. The live attenuated vaccine is highly reactogenic and is not licensed for use in humans. The killed whole cell vaccine, also reactogenic, provides poor protection against pneumonic plague and immunisation requires multiple doses of the vaccine. Against this background, a range of candidate vaccines, including rationally attenuated mutants, subunit vaccines and naked DNA vaccines have been described. Of these, an injected subunit vaccine is likely to offer the best near-term solution to the provision of a vaccine that protects against both bubonic and pneumonic plague.

[Lectin of Yersinia pestis vaccine strain EV and its immunological properties]. / Lektin vaktsinnogo shtamma Yersinia pestis EV i izuchenie ego immunobiologicheskikh svoistv.

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.