RESUMEN

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.

Asunto(s)

Antígenos Bacterianos/biosíntesis , Proteínas Recombinantes/biosíntesis , Yersinia pestis/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Clonación Molecular , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Citotóxicas Formadoras de Poros

RESUMEN

The pathogenic Yersiniae produce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection with Y. pestis in murine models, and is considered to be a protective antigen. In this study, the protective efficacy of V antigen has been compared in the same model with that of other proteins (YopE, YopK and YopN), which are part of the contact-dependent virulence mechanism. Mice immunised with two intraperitoneal doses of V antigen or each of the Yops, administered with either Alhydrogel or interleukin-12, produced high antigen-specific serum IgG titres. As shown in previous studies, V+Alhydrogel was fully protective, and 5/5 mice survived a subcutaneous challenge with 90 or 9x10(3) LD50's of Y. pestis GB. In addition, these preliminary studies also showed that V+IL-12 was partially protective: 4/5 or 3/5 mice survived a challenge with 90 or 9x10(3) LD50's, respectively. In contrast, none of the mice immunised with the Yops survived the challenges, and there was no significant delay in the mean time to death compared to mice receiving a control protein. These results show that using two different vaccine regimens, Yops E, K and N, failed to elicit protective immune responses in a murine model of plague, whereas under the same conditions, V antigen was fully or partially protective.

Asunto(s)

Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Proteínas de la Membrana , Peste/prevención & control , Yersinia/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Especificidad de Anticuerpos , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Peste/microbiología , Peste/mortalidad , Proteínas Citotóxicas Formadoras de Poros , Organismos Libres de Patógenos Específicos , Yersinia pestis/inmunología

RESUMEN

Yersinia pestis, the aetiological agent of plague, has in the past caused social devastation on a scale unmatched by other infectious diseases. There is still a public health problem from plague, with at least 2000 cases reported annually. Most of these cases are of the bubonic form. Occasionally bubonic plague develops into pneumonic plague, and this form of the disease can spread rapidly between susceptible individuals. The recent outbreak of plague in India highlighted the potential for plague to explosively re-appear, and modern mass transport systems mean that there is the potential for the rapid spread of disease. Against this background, there is a need to ensure that vaccines and antibiotics are available to prevent and treat the disease. Progress has been made in devising a sub-unit vaccine, effective against bubonic and pneumonic plague. Antibiotics must be given in the early stages of disease to effect a cure.

Asunto(s)

Peste/epidemiología , Brotes de Enfermedades , Reservorios de Enfermedades , Humanos , Incidencia , Peste/prevención & control , Peste/terapia

RESUMEN

V antigen of Yersinia pestis is a multifunctional protein that has been implicated as a protective antigen, a virulence factor, and a regulatory protein. A series of V-antigen truncates expressed as glutathione S-transferase (GST) fusion proteins (GST-V truncates) have been cloned and purified to support immunogenicity and functionality studies of V antigen. Immunization studies with GST-V truncates have identified two regions of V antigen that confer protection against Y. pestis 9B (a fully virulent human pneumonic plague isolate) in a mouse model for plague. A minor protective region is located from amino acids 2 to 135 (region I), and a major protective region is found between amino acids 135 and 275 (region II). In addition, analysis of IgG titers following immunization suggested that the major antigenic region of V antigen is located between amino acids 135 and 245. A panel of monoclonal antibodies raised against recombinant V antigen was characterized by Western blotting against GST-V truncates, and epitopes of most of the monoclonal antibodies were mapped to region I or II. Monoclonal antibody 7.3, which recognizes an epitope in region II, passively protected mice against challenge with 12 median lethal doses of Y. pestis GB, indicating that region II encodes a protective epitope. This is the first report of a V-antigen-specific monoclonal antibody that will protect mice against a fully virulent strain of Y. pestis. The combined approach of passive and active immunization has therefore confirmed the importance of the central region of the protein for protection and also identified a previously unknown protective region at the N terminus of V antigen.

Asunto(s)

Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Peste/prevención & control , Proteínas Recombinantes de Fusión/inmunología , Vacunas Sintéticas/inmunología , Yersinia pestis/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Glutatión Transferasa/inmunología , Humanos , Inmunización Pasiva , Ratones , Ratones Endogámicos BALB C , Proteínas Citotóxicas Formadoras de Poros

RESUMEN

A novel approach to making fusions of F1 and V antigens, which may be incorporated into a live recombinant vaccine for plague, was developed. The nucleotide sequences encoding Yersinia pestis V antigen (lcrV) and the mature form of F1 antigen (caf1) were amplified by PCR with primers which included tails. At the 3' end of caf1 and the 5' end of lcrV, the tails encoded one of three six- or eight-amino acid linkers or their complementary sequences. The DNA overlap in each linker region was used to prime a second PCR to generate three F1/V fusions, which were cloned into pUC18. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-Ile-Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Arg. As shown by Western blotting of bacterial cell lysates with anti-V and anti-F1 sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenicity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Ile-Glu-Gly-Arg linker, was electroporated into the attenuated Salmonella typhimurium strain SL3261 (aroA). Mice receiving two intravenous doses of 5 x 10(6) cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG1:IgG2a isotype ratios, and T cell responses specific for V and F1 antigens. Six weeks after vaccination, mice were challenged subcutaneously with 7.4 x 10(2) or 7.4 x 10(4) LD50s of Y. pestis strain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressing Y. pestis antigens as fusion proteins to develop a live recombinant vaccine against plague.

Asunto(s)

Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Peste/prevención & control , Salmonella typhimurium/genética , Yersinia pestis/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Femenino , Expresión Génica , Inmunización , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peste/inmunología , Vacuna contra la Peste/genética , Vacuna contra la Peste/inmunología , Vacuna contra la Peste/farmacología , Reacción en Cadena de la Polimerasa , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/farmacología , Yersinia pestis/genética

RESUMEN

The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.

Asunto(s)

Antígenos Bacterianos/inmunología , Vacuna contra la Peste , Peste/prevención & control , Vacunas Sintéticas , Yersinia pestis/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/sangre , Cápsulas Bacterianas/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Ratones , Peste/microbiología , Vacuna contra la Peste/inmunología , Proteínas Citotóxicas Formadoras de Poros , Vacunación , Vacunas Sintéticas/inmunología

RESUMEN

The effect of buffer composition on simultaneous PCR amplification of 16S rRNA gene fragments of five bacterial species was examined using a number of different buffer systems. Tris-based PCR buffers at final concentrations of 10 mM proved unreliable. However, when the final concentration of Tris was increased to 75 mM, all five samples were routinely detected. The use of other buffers, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO) and 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO), resulted in PCR amplification of five products even at low final concentrations (10 mM). The presence of certain proteins in the amplification reaction could overcome an inhibitory effect seen when soil suspension was present in the reaction, as might occur when testing field samples for the presence of bacteria. Bovine serum albumin was found to be the most effective additive tested in overcoming inhibition.

Asunto(s)

Bacterias/aislamiento & purificación , Etanolaminas , Reacción en Cadena de la Polimerasa/métodos , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Microbiología del Suelo , Ácidos Alcanesulfónicos , Animales , Bacterias/genética , Tampones (Química) , Anhidrasas Carbónicas/farmacología , Bovinos , Ciclohexilaminas , Etanolamina , Datos de Secuencia Molecular , Miosinas/farmacología , Ovalbúmina/farmacología , Alineación de Secuencia , Albúmina Sérica Bovina/farmacología , Suspensiones , Trometamina

RESUMEN

In this study, we have determined the limit of protection achievable by immunisation with sub-units of Yersinia pestis against the development of plague in an experimental animal model. Co-immunisation with the purified culture-derived F1 and the recombinant V sub-units afforded a greater level of protection than with either sub-unit alone. The protection given by the combined sub-units was several orders of magnitude greater than that afforded by the whole cell killed (Cutter USP) vaccine and was equivalent to that achieved by vaccination with EV76, the live attenuated Y. pestis vaccine strain. However, the combined sub-unit vaccine has clear advantages over the live vaccine in terms of safety of use and absence of side-effects.

Asunto(s)

Vacuna contra la Peste/administración & dosificación , Peste/prevención & control , Vacunas Sintéticas/inmunología , Yersinia pestis/inmunología , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Western Blotting , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Proteínas Citotóxicas Formadoras de Poros , Organismos Libres de Patógenos Específicos , Linfocitos T/inmunología , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología , Vacunas de Productos Inactivados/efectos adversos , Vacunas de Productos Inactivados/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/normas

RESUMEN

The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.

Asunto(s)

Antígenos Bacterianos/inmunología , Peste/prevención & control , Yersinia pestis/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Secuencia de Bases , Cartilla de ADN/química , Femenino , Genes Bacterianos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes de Fusión , Vacunas Sintéticas

RESUMEN

An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S. typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence. The recombinant was used to vaccinate mice intragastrically and intravenously. The immunity induced was able to protect mice against challenge with a virulent strain of plague. Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.

Asunto(s)

Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Peste/prevención & control , Yersinia pestis/inmunología , Animales , Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Femenino , Genes Bacterianos , Inmunidad Celular , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Salmonella typhimurium , Bazo/inmunología , Vacunas Atenuadas , Vacunas Sintéticas/inmunología

Asunto(s)

Antígenos Bacterianos/genética , Vacuna contra la Peste/aislamiento & purificación , Peste/prevención & control , Yersinia pestis/genética , Yersinia pestis/inmunología , Animales , Clonación Molecular , Femenino , Genes Bacterianos , Humanos , Ratones , Peste/inmunología , Vacuna contra la Peste/genética , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Salmonella typhimurium/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/aislamiento & purificación