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This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the co-application of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy.
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Vacuna contra la Peste , Yersinia pestis , Humanos , Animales , Ratones , Citocinas , Antígenos Bacterianos , Vacunas Sintéticas , AerosolesRESUMEN
The development of a safe and effective vaccine against Yersinia pestis, the causative organism for plague disease, remains an important global health priority. Studies have demonstrated effective immune-based protection against plague challenge that is induced by plague antigen subunit vaccination in an aqueous alhydrogel formulation; however, whether these candidate vaccines in this formulation and presentation, induce long-lasting immunological memory in the form of durable cellular and antibody recall responses has not been fully demonstrated. In this study, we analyzed germinal center T follicular helper and germinal center B cell responses following F1V and F1 + V plague subunit immunization of mice with vaccines formulated in various adjuvants. Our data demonstrate that recombinant plague protein immunization formulated with IL-2/GM-CSF cytokines bound to alhydrogel adjuvant drive an increase in the magnitude of the germinal center T follicular helper and germinal center B cell responses following primary immunization, compared to vaccines formulated with Alhydrogel adjuvant alone. In contrast, plague protein subunit immunization combined with CpG ODN bound to alhydrogel increased the magnitude and duration of the germinal center Tfh and B cell responses following booster immunization. Importantly, enhanced germinal center Tfh and B cell responses correlated with long-lasting and high F1V-specific antibody titers and more robust antibody recall responses to F1V re-exposure. These findings indicate that vaccine formulations that drive enhancement of the germinal center Tfh and B cell responses are critical for inducing durable plague-specific humoral immunity.
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Hidróxido de Aluminio , Centro Germinal , Ratones , Animales , Células T Auxiliares Foliculares , Vacunación , Adyuvantes Inmunológicos/farmacología , Vacunas de Subunidad , Inmunización Secundaria , Vacunas SintéticasRESUMEN
Background: The need for an updated plague vaccine is highlighted by outbreaks in endemic regions together with the pandemic potential of this disease. There is no easily available, approved vaccine. Methods: Here we have used a murine model of pneumonic plague to examine the factors that maximise immunogenicity and contribute to survival following vaccination. We varied vaccine type, as either a genetic fusion of the F1 and V protein antigens or a mixture of these two recombinant antigens, as well as antigen dose-level and formulation in order to correlate immune response to survival. Results: Whilst there was interaction between each of the variables of vaccine type, dose level and formulation and these all contributed to survival, vaccine formulation in protein-coated microcrystals (PCMCs) was the key contributor in inducing antibody titres. From these data, we propose a cut-off in total serum antibody titre to the F1 and V proteins of 100 µg/mL and 200 µg/mL, respectively. At these thresholds, survival is predicted in this murine pneumonic model to be >90%. Within the total titre of antibody to the V antigen, the neutralising antibody component correlated with dose level and was enhanced when the V antigen in free form was formulated in PCMCs. Antibody titre to F1 was limited by fusion to V, but this was compensated for by PCMC formulation. Conclusions: These data will enable clinical assessment of this and other candidate plague vaccines that utilise the same vaccine antigens by identifying a target antibody titre from murine models, which will guide the evaluation of clinical titres as serological surrogate markers of efficacy.
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Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.
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Carbunco/inmunología , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Antitoxinas/sangre , Toxinas Bacterianas/inmunología , Anticuerpos Neutralizantes/sangre , Bacillus anthracis/inmunología , Bacillus anthracis/patogenicidad , Humanos , Inmunidad Humoral , Inmunoglobulina G/sangre , Enfermedades Cutáneas BacterianasRESUMEN
Previously two capsule-specific monoclonal antibodies (4VA5 and 3VIE5) were identified as protective against Burkholderia pseudomallei in passive transfer experiments. Panning these antibodies against evolutionary phage libraries identified reactive peptides capable of inhibiting its parent monoclonal from binding to B. pseudomallei. Mice immunized with peptide conjugated to thyroglobulin developed serum antibodies capable of recognizing the immunizing peptide of which a subset recognized exopolysaccharide in the context of whole B. pseudomallei cells. These serum antibodies recognized protease treated B. pseudomallei but not B. thailandensis suggesting that these peptides are mimotopes of the B. pseudomallei capsular exopolysaccharide. In a murine model of acute melioidosis, immunization with the mimotope of the 4VA5 binding site extended the mean time to death to 8.00 days over the 2.18 days afforded by immunization with thyroglobulin alone. This mimotope may be of use in developing an antibody response against B. pseudomallei exopolysaccharide.
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Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/química , Epítopos/inmunología , Péptidos/inmunología , Polisacáridos Bacterianos/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Burkholderia pseudomallei/inmunología , Melioidosis/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos AnimalesRESUMEN
BACKGROUND: Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. RESULTS: More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 x 10(-5) - 8 x 10(-8)/cell). Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. CONCLUSION: The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production.
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Fagos de Bacillus/genética , Bacillus anthracis/genética , Cromosomas Bacterianos/genética , Profagos/genética , Activación Viral/fisiología , Sitios de Ligazón Microbiológica/genética , Bacillus/clasificación , Bacillus/genética , Fagos de Bacillus/fisiología , Bacillus anthracis/clasificación , Secuencia de Bases , Cartilla de ADN/química , ADN Viral/química , Electroforesis en Gel de Agar/métodos , Orden Génico , Microscopía Electrónica de Transmisión/métodos , Mitomicina/farmacología , Reacción en Cadena de la Polimerasa/métodos , Profagos/fisiología , Activación Viral/efectos de los fármacosRESUMEN
We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication (discrepancy rate of 7.4 x 10(-7)) and by comparison to independently generated shotgun sequence (discrepancy rate < 2.5 x 10(-6)). Population genomics studies of microbial pathogens using rapid resequencing technologies such as resequencing arrays are critical for recognizing newly emerging or genetically engineered strains.
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Bacillus anthracis/genética , Bacillus anthracis/aislamiento & purificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADN/métodos , Bacillus anthracis/clasificación , Bacillus anthracis/patogenicidad , Cromosomas Bacterianos/genética , Variación Genética/genética , Genoma Bacteriano , Datos de Secuencia Molecular , Recombinación Genética/genética , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/economía , Análisis de Secuencia de ADN/normas , Factores de TiempoRESUMEN
A DNA encoding the 27-kDa domain I of anthrax lethal factor protein (LF), was linked to the carboxyl terminus of the cholera toxin B-subunit (CTB-LF). The CTB-LF fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated in vivo transformation methods and antibiotic-resistant plants were regenerated. The CTB-LF fusion gene was detected in transformed potato leaf genomic DNA by polymerase chain reaction (PCR)-mediated DNA amplification. Immunoblot analysis with anti-CTB and anti-LF primary antibodies verified the synthesis and assembly of biologically active CTB-LF fusion protein oligomers in transformed plant tuber tissues. Furthermore, the binding of CTB-LF fusion protein pentamers to intestinal epithelial cell membrane receptors measured by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) indicated that the CTB-LF fusion protein made up approx 0.002% of the total soluble tuber protein. Synthesis of CTB-LF monomers and their assembly into biologically active CTB-LF fusion protein pentamers in potato tuber tissues demonstrates the feasibility of using edible plants for production and delivery of adjuvanted LF protein for CTB-mediated immunostimulation of mucosal immune responses against anthrax toxin.
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Antígenos Bacterianos/genética , Toxinas Bacterianas/genética , Toxina del Cólera/genética , Proteínas Recombinantes de Fusión/síntesis química , Secuencia de Bases , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genéticaRESUMEN
DNA vaccination is vaccination at its simplest. Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development. DNA vaccination against anthrax is an active area of research showing promising results at present, which in the short-term and in the future could form the basis for new advances in multi-agent vaccine development. The anthrax 'model' constitutes an important experimental system for genetic immunisation technology development.
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Vacunas contra el Carbunco , Carbunco/prevención & control , Vacunas de ADN , Animales , Vacunas contra el Carbunco/inmunología , Bacillus anthracis/genética , Bacillus anthracis/inmunología , Bioterrorismo , Bovinos , Enfermedades de los Bovinos/transmisión , ADN Bacteriano/inmunología , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Macaca mulatta , Masculino , Ratones , Plásmidos/administración & dosificación , Plásmidos/genética , Conejos , Vacunación , Vacunas de ADN/inmunologíaRESUMEN
Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.
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Carbunco , Antígenos Bacterianos , Bacillus cereus/genética , Bacillus cereus/patogenicidad , Toxinas Bacterianas/genética , Animales , Carbunco/etiología , Bacillus anthracis/clasificación , Bacillus anthracis/citología , Bacillus anthracis/genética , Bacillus cereus/clasificación , Bacillus cereus/citología , Genoma Bacteriano , Genómica , Humanos , Ratones , Plásmidos/genéticaRESUMEN
The outer membrane protein F gene (oprF) of Pseudomonas aeruginosa was recently shown by us to protect mice from P. aeruginosa chronic pulmonary infection when used as a DNA vaccine administered by three biolistic (gene gun) intradermal inoculations given at 2-week intervals. In the present study, we used two different strategies to improve the protective efficacy of the DNA vaccine. In the first strategy, mice were primed with two biolistic intradermal inoculations with the oprF vaccine and then were given a final intramuscular booster immunization containing either a synthetic peptide-keyhole limpet hemocyanin (KLH) conjugate or a chimeric influenza virus. Both the synthetic peptide conjugate and the chimeric virus contained peptide 10, a previously identified immunoprotective epitope of protein F. The second strategy involved the addition of a second outer membrane protein to the vaccine. DNA encoding a fusion protein comprised of the C-terminal half of protein F fused to OprI was administered by three biolistic intradermal inoculations. Challenge with P. aeruginosa in a chronic pulmonary infection model demonstrated that boosting with the chimeric virus (but not with peptide-KLH) or adding oprI to the DNA vaccine significantly enhanced protection as compared to that afforded by the oprF vaccine given alone. Thus, both strategies appear to augment the protection afforded by an oprF-only DNA vaccine.