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E D Williamson

Publications and source records attributed to E D Williamson.

At least 37 records · Page 2Linked to original sources
PubMed2001

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.

Animals↗
PubMed2001

Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach.

Using a combination of nanoflow-electrospray ionization and time-of-flight mass spectrometry we have analyzed the oligomeric state of the recombinant V antigen from Yersinia pestis, the causative agent of plague. The mass spectrometry results show that at pH 6.8 the V antigen in solution exists predominantly as a dimer and a weakly associated tetramer. A monoclonal antibody 7.3, raised against the V antigen, gave rise to mass spectra containing a series of well-resolved charge states at m/z 6000. After addition of aliquots of solution containing V antigen in substoichiometric and molar equivalents, the spectra revealed that two molecules of the V antigen bind to the antibody. Collision-induced dissociation of the antibody-antigen complex results in the selective release of the dimer from the complex supporting the proposed 1:2 antibody:antigen stoichiometry. Control experiments with the recombinant F1 antigen, also from Yersinia pestis, establish that the antibody is specific for the V antigen because no complex with F1 was detected even in the presence of a 10-fold molar excess of F1 antigen. More generally this work demonstrates a rapid means of assessing antigen subunit interactions as well as the stoichiometry and specificity of binding in antibody-antigen complexes.

Algorithms↗
PubMed2001

Macromolecular organization of the Yersinia pestis capsular F1 antigen: insights from time-of-flight mass spectrometry.

Mass spectrometry has been used to examine the subunit interactions in the capsular F1 antigen from Yersinia pestis, the causative agent of the plague. Introducing the sample using nanoflow electrospray from solution conditions in which the protein remains in its native state and applying collisional cooling to minimize the internal energy of the ions, multiple subunit interactions have been maintained. This methodology revealed assemblies of the F1 antigen that correspond in mass to both 7-mers and 14-mers, consistent with interaction of two seven-membered units. The difference between the calculated masses and those measured experimentally for these higher-order oligomers was found to increase proportionately with the size of the complex. This is consistent with a solvent-filled central cavity maintained on association of the 7-mer to the 14-mer. The charge states of the ions show that an average of one and four surface accessible basic side-chains are involved in maintaining the interactions between the 7-mer units and neighboring subunits, respectively. Taken together, these findings provide new information about the stoichiometry and packing of the subunits involved in the assembly of the capsular antigen structure. More generally, the data show that the symmetry and packing of macromolecular complexes can be determined solely from mass spectrometry, without any prior knowledge of higher order structure

Bacterial Capsules↗
PubMed2001

Tissue distribution of radioactivity following intranasal administration of radioactive microspheres.

The aim of this study was to increase understanding of the kinetics of microparticle distribution and elimination following intranasal application. To do this we investigated the in-vivo distribution of radioactivity following intranasal instillation of scandium-46 labelled styrene-divinyl benzene 7-microm-diameter microspheres. Groups of BALB/c mice received 0.250 mg (47.5 kBq) particles suspended in either 50-microL or 10-microL volumes of phosphate buffered saline. The in-vivo distribution of radioactivity was influenced by the volume of liquid that was used to instil the microsphere suspension. Comparatively large (50 microL) administration vehicle volumes resulted in substantial bronchopulmonary deposition (approximately 50% of administered dose). Intranasal instillation of microspheres suspended in 10-microL volumes tended to restrict particle deposition initially to the nasal cavity. For both administration vehicle volumes tested, the radioactivity per unit mass of excised nasal-associated lymphoid tissue (NALT) was found to be consistently elevated relative to other tissues. This corroborates the findings of other workers who have previously identified NALT as an active site of microparticle accumulation following intranasal application. Elimination via the alimentary canal was the principal fate of intranasally applied radiolabelled material. No significant concentration of radioactivity within excised gut-associated lymphoid tissue (GALT) (Peyer's patches) was noted. At latter time points we observed, in mice that received the 50-microL volume particle suspension nasally, accumulation of potentially relevant quantities of radioactivity in the liver (0.3% after 576 h) and spleen (0.04% after 576 h). Thus, our data corroborate the notion that epithelial membranes in the lung are probably less exclusive to the entry of microparticulates into systemic compartments than are those mucosae in the gastrointestinal tract or nasopharynx. This effect may contribute to the effectiveness of pulmonary delivered antigen-loaded microparticles as humoral immunogens.

Administration, Intranasal↗
PubMed2000

A single dose sub-unit vaccine protects against pneumonic plague.

In this study, the protection afforded against aerosolised Yersinia pestis by injection of a single dose of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine, prepared by admixing F1 antigen derived from a Y. pestis cell culture supernatant with recombinant V antigen derived from an E. coli cell lysate, fully protected an outbred strain of mouse against exposure to 10(6) CFU of virulent plague organisms (10(4) mouse lethal doses, MLD). In contrast, the whole cell vaccine provided only 16% protection against the same level of challenge. Furthermore, sub-unit vaccinees were able to clear the bacteria from their lungs post-challenge whereas bacteria were cultured from the lungs of a surviving KWC vaccinee post-challenge. In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable and the levels of F1-specific IgG in serum and in broncho-alveolar washings were significantly lower (p<0.05) compared with sub-unit vaccinees. In sub-unit vaccinees, an IgG titre to the F1 and V antigens was detected in serum where it was significantly higher (p<0.05) compared with broncho-alveolar washings suggesting that, at the time of challenge, protection is attributable mainly to the combined circulating IgG titre to the F1 and V sub-units. The enhanced protective efficacy of this sub-unit vaccine administered as a single dose compared with an existing vaccine has been demonstrated in an outbred animal model of pneumonic plague.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

Protection studies following bronchopulmonary and intramuscular immunisation with yersinia pestis F1 and V subunit vaccines coencapsulated in biodegradable microspheres: a comparison of efficacy.

We have compared the ability of intramuscularly and intratracheally administered recombinant F1 and V subunit antigens to safeguard mice from a lethal systemic challenge with plague. The combined subunits (1 microg V plus 5 microg F1) were inoculated either in the 'free' state as a solution, or entrapped within microspheres composed of a biodegradable polyester (Poly-L-lactide), on day 1 and 60 of the experiment. In comparison to the other regimens, introduction of microsphere suspensions into the respiratory tract resulted in statistically elevated levels of specific immunoglobulins in day 82 lung wash samples. A subcutaneous challenge with virulent Yersinia pestis bacteria on day 137, equivalent to more than 10(5) mouse LD(50)s, was comparatively well tolerated by all subunit treatment groups (with survival rates between 66 and 90%). In contrast, 80% of the mice injected intramuscularly with soluble F1 and V were defeated by a 10(7) MLD(50) subcutaneous challenge, whereas the group immunised intramuscularly with microparticles were significantly better protected (p<0.1) with 50% survival. Similarly, mice immunised intratracheally with microparticles were significantly better safeguarded (56% survival) compared with the group immunised with soluble subunits intramuscularly (p<0.01). Soluble sub-units delivered intratracheally afforded 33% protection against 10(7) MLD(50)s. These data indicate that bronchopulmonary administration of microsphere co-encapsulated recombinant F1 and V antigens elicits a similar level of protective immunity against systemic plague infection as that evoked by injecting co-encapsulated subunits into the muscle. Such findings corroborate the thesis that introduction of appropriately formulated F1 and V subunits into the respiratory tract may be an alternative to parenteral immunisation schedules for protecting individuals from plague.

Animals↗
PubMed2000

Generation of protective immune responses to plague by mucosal administration of microsphere coencapsulated recombinant subunits.

We have investigated noninvasive immunization to plague. Recombinant subunit antigens, F1 and V from Yersinia pestis, were coencapsulated in biodegradable poly(L100 LD(50's) inhalational challenge with virulent Y. pestis. These data expand on previous findings from our laboratories, providing further insight into the mechanics of safeguarding mice from plague through nasal immunization. Further, these results demonstrate that in a murine model, solid protection from pneumonic plague can be engendered by two intranasal administrations of appropriately formulated recombinant proteins.

Administration, Intranasal↗
PubMed2000

Biodegradable microparticles with different release profiles: effect on the immune response after a single administration via intranasal and intramuscular routes.

In the development of single-dose microparticulate vaccines, identification of the type of protein release profile required to elicit high and sustainable immune responses is important. Microparticles exhibiting different protein release profiles (continuous, pulsatile and plateau) were made by solvent evaporation or solvent extraction methods from biodegradable polymers encapsulating the model antigen, bovine serum albumin (BSA). The immune responses obtained after a single intranasal or intramuscular administration of microparticles were determined, and also after a subcutaneous boost after 11 months. Microparticles were manufactured with acceptable protein loading and average volume size ranging from 1 to 10 microm. The integrity of BSA extracted and released from microparticles after 2 months incubation was retained. Microparticulate preparations administered by either intranasal or intramuscular routes, evoked rapid, high titre and long-lived (up to 11 months after priming) specific serum IgG responses which were significantly greater than for free BSA. The type of protein release from microparticles had no significant effect on the systemic immune responses. Interestingly, a formulation exhibiting a plateau-release profile was the only microparticulate system capable of inducing significantly greater IgA responses than free BSA after intranasal immunization. This study shows the benefit of microencapsulation in inducing high and long-lasting systemic immune responses after a single dose by both parenteral and mucosal delivery. We conclude that of the microparticles tested, the longevity and magnitude of humoral responses was not effected by the type of in-vitro protein release profile.

Administration, Intranasal↗
PubMed1999

Gene gun mediated vaccination is superior to manual delivery for immunisation with DNA vaccines expressing protective antigens from Yersinia pestis or Venezuelan Equine Encephalitis virus.

Plasmids expressing the V antigen of Yersinia pestis or the E2 glycoprotein of Venezuelan Equine Encephalitis (VEE) virus were used to vaccinate mice by intra-dermal or intra-muscular injection, or by particle-mediated bombardment using the Helios gene gun. After two immunizations, groups of mice which had received 4 microg doses of plasmid DNA using the gene gun had IgG levels which were higher than in other groups manually immunised with 12-fold more plasmid DNA. The immunoglobulin isotype profile was predominantly IgG1 following inoculation with either plasmid. Our results indicate that gene gun mediated vaccination can be used to increase the magnitude of the immune response to both bacterial and viral antigens expressed by plasmid DNA.

Animals↗
PubMed1999

Immunological responses to nasal delivery of free and encapsulated tetanus toxoid: studies on the effect of vehicle volume.

In light of growing interest in the intranasal route as a non-invasive mode of immunisation, we have investigated the relationship between the volume of liquid instilled into the nasal passages and the development of subsequent immunological responses. Groups of six mice were intranasally immunised with soluble or microsphere encapsulated tetanus toxoid on days 1, 14 and 28 of the experiment. Microsphere suspensions and tetanus toxoid solutions were nasally instilled in two different volumes of buffer (10 or 50 microl). Nasal instillation of microspheres in 10 microl of buffer generated statistically depressed (P<0.001) tertiary serum anti-toxoid IgG responses in comparison to animals immunised with 10 or 50 microl of soluble vaccine, or 50 microl of microsphere suspension. Relative to other treatments, nasal inoculation of encapsulated toxoid suspended in 50 microl generated statistically (P<0.05) superior levels of specific IgG and IgA antibodies in day 49 lung wash samples. When radiolabelled microspheres were nasally instilled into mouse nares in 50-microl volumes of buffer, a significant portion of the dose (48%) entered the lungs (P<0.001), whereas more particles remained in the nasal passages when a smaller (10 microl) volume of suspension was given (P<0.001). These biodistribution and immunological data indicate that to generate optimal bronchopulmonary and systemic responses in concert following nasal administration, microparticulated vaccines should be administered with a delivery device that targets the formulation to distal regions of the nasal passages and the lower respiratory tract.

Administration, Intranasal↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Yersinia outer proteins (YOPS) E, K and N are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague.

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Presentation of protective antigen to the mouse immune system: immune sequelae.

Protective antigen (PA), the major protective component of the existing vaccine, is a potent immunogen. Protective antigen in alhydrogel induced a high serum IgG titre (> log10 4) in both the C57B16 and Balb/c mouse and the predominant subclass of antibody induced was IgG1, indicating that the response to PA was predominantly Th2 directed. When plasmid DNA encoding PA was used to immunize the Balb/c mouse, a low serum IgG titre was detected (</=log10 1), which was slightly increased by boosting with plasmid DNA. However, when mice immunized with plasmid DNA were later boosted with rPA, a significant and rapid increase in titre (up to threefold) was observed. Priming mice with PA-encoding plasmid DNA may be a mechanism of enhancing and accelerating the immune response to PA.

Animals↗
PubMed1999

The SCID/Beige mouse as a model to investigate protection against Yersinia pestis.

In this study, we have shown that severe combined immunodeficient/beige mice reconstituted with hyperimmune Balb/c lymphocytes can be used as a model to demonstrate adoptive and passive protection against plague infection. Reconstitution of severe combined immunodeficient/beige mice was successful in nine out of ten mice as demonstrated by spleen colonisation and sustained circulating immunoglobulin titres. Furthermore, an increase in antibody titre was evident after a booster immunisation of reconstituted mice. Presence of circulating antibody correlated with protection against a systemic plague challenge and indicated that in reconstituted mice adoptive transfer of a functional immune system had occurred. The severe combined immunodeficient/beige mouse was also used to demonstrate passive protection against inhaled and systemic plague infection. The reconstituted severe combined immunodeficient/beige mouse model demonstrating protective immunity against plague will be further developed to identify the immune cell subsets responsible for this protection.

Adoptive Transfer↗