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Biomedical subjects

E Diane Williamson

Publications and source records attributed to E Diane Williamson.

At least 19 recordsLinked to original sources
PubMed2006

Immunogenicity of a Yersinia pestis vaccine antigen monomerized by circular permutation.

Caf1, a chaperone-usher protein from Yersinia pestis, is a major protective antigen in the development of subunit vaccines against plague. However, recombinant Caf1 forms polymers of indeterminate size. We report the conversion of Caf1 from a polymer to a monomer by circular permutation of the gene. Biophysical evaluation confirmed that the engineered Caf1 was a folded monomer. We compared the immunogenicity of the engineered monomer with polymeric Caf1 in antigen presentation assays to CD4 T-cell hybridomas in vitro, as well as in the induction of antibody responses and protection against subcutaneous challenge with Y. pestis in vivo. In C57BL/6 mice, for which the major H-2(b)-restricted immunodominant CD4 T-cell epitopes were intact in the engineered monomer, immunogenicity and protective efficacy were preserved, although antibody titers were decreased 10-fold. Disruption of an H-2(d)-restricted immunodominant CD4 T-cell epitope during circular permutation resulted in a compromised T-cell response, a low postvaccination antibody titer, and a lack of protection of BALB/c mice. The use of circular permutation in vaccine design has not been reported previously.

Animals↗
PubMed2006

Mechanisms of major histocompatibility complex class II-restricted processing and presentation of the V antigen of Yersinia pestis.

We mapped mouse CD4 T-cell epitopes located in three structurally distinct regions of the V antigen of Yersinia pestis. T-cell hybridomas specific for epitopes from each region were generated to study the mechanisms of processing and presentation of V antigen by bone-marrow-derived macrophages. All three epitopes required uptake and/or processing from V antigen as well as presentation to T cells by newly synthesized major histocompatibility complex (MHC) class II molecules over a time period of 3-4 hr. Sensitivity to inhibitors showed a dependence on low pH and cysteine, serine and metalloproteinase, but not aspartic proteinase, activity. The data indicate that immunodominant epitopes from all three structural regions of V antigen were presented preferentially by the classical MHC class II-restricted presentation pathway. The requirement for processing by the co-ordinated activity of several enzyme families is consistent with the buried location of the epitopes in each region of V antigen. Understanding the structure-function relationship of multiple immunodominant epitopes of candidate subunit vaccines is necessary to inform choice of adjuvants for vaccine delivery. In the case of V antigen, adjuvants designed to target it to lysosomes are likely to induce optimal responses to multiple protective T-cell epitopes.

Animals↗
PubMed2006

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes.

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.

Animals↗
PubMed2006

Protection against heterologous Burkholderia pseudomallei strains by dendritic cell immunization.

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium which can cause either chronic infections or acute lethal sepsis in infected individuals. The disease is endemic in Southeast Asia and northern Australia, but little is known about the mechanisms of protective immunity to the bacterium. In this study, we have developed a procedure to utilize dendritic cells in combination with CpG oligodeoxynucleotides as a vaccine delivery vector to induce protective immune responses to various strains of B. pseudomallei. Our results show that strong cell-mediated immune responses were generated, while antibody responses, although low, were detectable. Upon virulent challenge with B. pseudomallei strain K96243, NCTC 4845, or 576, animals immunized with dendritic cells that were pulsed with heat-killed K96243 and matured in the presence of CpG 1826 showed significant levels of protection. These results show that a vaccine strategy that actively targets dendritic cells can evoke protective immune responses.

Animals↗
PubMed2005

Protection against bubonic and pneumonic plague with a single dose microencapsulated sub-unit vaccine.

Protection against virulent plague challenge by the parenteral and aerosol routes was afforded by a single administration of microencapsulated Caf1 and LcrV antigens from Yersinia pestis in BALB/c mice. Recombinant Caf1 and LcrV were individually encapsulated in polymeric microspheres, to the surface of which additional antigen was adsorbed. The microspheres containing either Caf1 or LcrV were blended and used to immunise mice on a single occasion, by either the intra-nasal or intra-muscular route. Both routes of immunisation induced systemic and local immune responses, with high levels of serum IgG being developed in response to both vaccine antigens. In Elispot assays, secretion of cytokines by spleen and draining lymph node cells was demonstrated, revealing activation of both Th1 and Th2 associated cytokines; and spleen cells from animals immunised by either route were found to proliferate in vitro in response to both vaccine antigens. Virulent challenge experiments demonstrated that non-invasive immunisation by intra-nasal instillation can provide strong systemic and local immune responses and protect against high level challenge. Microencapsulation of these vaccine antigens has the added advantage that controlled release of the antigens occurs in vivo, so that protective immunity can be induced after only a single immunising dose.

Administration, Intranasal↗
PubMed2005

Immunological responses after immunisation of mice with microparticles containing antigen and single stranded RNA (polyuridylic acid).

Certain toll-like receptor (TLR) agonists, e.g. CpG DNA, can be used as potent vaccine 'adjuvants'. It is known that some sequences of single stranded (ss) RNA stimulate proinflammatory and antiviral responses following interaction with TLR 7 and 8. We have encapsulated ovalbumin (OVA) in the presence and absence of polyuridylic acid (poly-U) inside polylactide microparticles. In comparison to microparticles containing only OVA, bulk cultures of bone marrow-derived plasmacytoid and myeloid dendritic cells produced more (P<0.05) IL-12 and interferon (IFN)-alpha when stimulated with microparticles containing OVA and poly-U. Subcutaneous injection of comicroencapsulated OVA and poly-U resulted in statistically elevated levels of serum anti-OVA IgG1 (P<0.05 versus naïve mice). Conversely, anti-OVA IgG1 levels in C57 BL6 mice immunised with OVA loaded microparticles (without RNA) were statistically indifferent to naïve animals. Furthermore, injection of coencapsulated OVA and poly-U resulted in (P<0.05) greater numbers of OVA specific IFN-gamma secreting T-cells as compared with mice injected with OVA loaded microparticles. A similar trend was seen in mice immunised with OVA loaded microparticles decorated with CpG or solutions of admixed OVA and CpG (P<0.05). These data demonstrate, for the first time, that appropriately formulated ssRNA can act as a potent adjuvant and modulator of adaptive immunological responses.

Adjuvants, Immunologic↗
PubMed2005

Mucosal delivery of microparticle encapsulated ESAT-6 induces robust cell-mediated responses in the lung milieu.

ESAT-6 from Mycobacterium tuberculosis is an important T-cell antigen for cell-mediated immunity in the early phase of tuberculosis infection. Since the lung is the organ in which infection is initiated, immune responses in the lung play a significant role in restricting the initial infection with M. tuberculosis. The aim of the present study was to assess whether efficient cell-mediated immune responses in the lung and draining mediastinal lymph nodes could be stimulated by pulmonary administration of ESAT-6 encapsulated in poly(lactide) (PLA) microspheres. BALB/c mice were immunised intranasally on days 1, 28 and 56 with 2 microg microencapsulated ESAT-6. Cellular responses in the lungs, spleen and mediastinal lymph nodes (MLN) were characterised using ELISPOT and proliferation assays. Fluorescence activated cell sorting (FACS) was used to assess the expression of CD44 on CD4+ and CD8+ cells derived from the MLN of immunised animals. For comparison, groups of mice were immunised intranasally with soluble 'free' ESAT-6 or intramuscularly with ESAT-6 in Alhydrogel. Intranasal instillation of microencapsulated ESAT-6 induced greatest numbers of ESAT-6 specific IFN-gamma and IL-4 secreting cells in the lung and MLN (P<0.05). Similarly, ESAT-6 specific recall responses were strongest following intranasal immunisation of mice with microsphere encapsulated antigen (P<0.05). FACS demonstrated a higher proportion of T cells expressing CD44 in the MLN from mice immunised intranasally with microencapsulated ESAT-6. These data support the notion that the immune system is compartmentalised and responses are often strongest in compartments proximal to the site of vaccine application. Furthermore, our data indicate that, for efficient activation of cell-mediated responses, antigens must be presented to the immune system in an appropriate formulation.

Administration, Intranasal↗
PubMed2005

Activation of dendritic cells by microparticles containing Bacillus anthracis protective antigen.

We have carried out an in vitro investigation into the mechanism by which microencapsulation enhances the immunogenicity of recombinant protective antigen (rPA) from Bacillus anthracis. Murine bone marrow derived dendritic cells (DC) were cocultured with soluble and microencapsulated rPA and the activation status of the cells monitored using FACS. As compared with soluble rPA, it was found that coculture of DC with rPA-loaded microparticles stimulated higher levels of MHC II, CD54, CD80 and CD86 expression (p<0.05). To investigate the longevity of antigen presentation, splenocytes from naïve mice were pulsed overnight with (3)H-thymidine following 1, 3 or 6 days coculture with DC transiently exposed to soluble or microencapsulated rPA. Splenocyte proliferation was more pronounced, and continued for a more protracted period, if the 'feeder' cells were exposed to microencapsulated antigen as compared with soluble antigen or 'empty' microspheres. To this end, our findings indicate that microsphere uptake increases the surface expression of MHC and co-stimulatory molecules on DC and can facilitate prolonged presentation of antigen to T-cells, possibly by acting as an intracellular depot.

Animals↗
PubMed2005

Antibiotic-free plasmid stabilization by operator-repressor titration for vaccine delivery by using live Salmonella enterica Serovar typhimurium.

Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella enterica serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacO sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestis F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonella expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.

Animals↗
PubMed2005

Humoral and cell-mediated adaptive immune responses are required for protection against Burkholderia pseudomallei challenge and bacterial clearance postinfection.

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacillus endemic to areas of southeast Asia and northern Australia. Presently, there is no licensed vaccine for B. pseudomallei and the organism is refractive to antibiotic therapy. The bacterium is known to survive and multiply inside both phagocytic and nonphagocytic host cells and may be able to spread directly from cell to cell. Current vaccine delivery systems are unlikely to induce the correct immune effectors to stimulate a protective response to the organism. In this study, we have developed a procedure to utilize dendritic cells as a vaccine delivery vector to induce cell-mediated immune responses to B. pseudomallei. Dendritic cells were produced by culturing murine bone marrow progenitor cells in medium containing granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha. Purified dendritic cells were pulsed with heat-killed whole-cell B. pseudomallei and used to immunize syngeneic mice. Strong cellular immune responses were elicited by this immunization method, although antibody responses were low. Booster immunizations of either a second dose of dendritic cells or heat-killed B. pseudomallei were administered to increase the immune response. Immunized animals were challenged with fully virulent B. pseudomallei, and protection was demonstrated in those with strong humoral and cell-mediated immunity. These results indicate the importance of both cell-mediated and humoral immune mechanisms in protection against intracellular pathogens.

Adoptive Transfer↗
PubMed2004

Evolutionary genetics: Ambiguous role of CCR5 in Y. pestis infection.

Mecsas and colleagues suggest that a deficiency in the chemokine receptor CCR5 in humans is unlikely to confer protection against plague, based on their study of Yersinia pestis infection in Ccr5-deficient mice. They were testing the hypothesis that a mutation in the CCR5 gene, frequently found in Caucasians, may have been selected for in the past because it provided protection against (bubonic) plague; the mutation, called CCR5Delta32, is characterized by a 32-base-pair deletion. We have also tested this hypothesis by using Y. pestis infection in mice and, in addition, we have done phagocytosis experiments with macrophages from wild-type and Ccr5-deficient mice. Although, like Mecsas et al., we did not see any difference in the survival of the two groups of mice, we did find that there was a significantly reduced uptake of Y. pestis by Ccr5-deficient macrophages in vitro. Our results indicate that the role of Ccr5 in Y. pestis infection may therefore be more complex than previously thought.

Animals↗
PubMed2004

Induction of protective immunity against lethal anthrax challenge with a patch.

BACKGROUND: Transcutaneous immunization (TCI) is a needle-free technique that delivers antigens and adjuvants to potent epidermal immune cells. To address critical unmet needs in biodefense against anthrax, we have designed a novel vaccine delivery system using a dry adhesive patch that simplifies administration and improves tolerability of a subunit anthrax vaccine. METHODS: Mice and rabbits were vaccinated with recombinant protective antigen of Bacillus anthracis and the heat-labile toxin of Escherichia coli. Serologic changes, levels of toxin-neutralizing antibodies (TNAs), and pulmonary and nodal responses were monitored in the mice. A lethal aerosolized B. anthracis challenge model was used in A/J mice, to demonstrate efficacy. RESULTS: The level of systemic immunity and protection induced by TCI was comparable to that induced by intramuscular vaccination, and peak immunity could be achieved with only 2 doses. The addition of adjuvant in the patch induced superior TNA levels, compared with injected vaccination. CONCLUSIONS: Anthrax vaccine patches stimulated robust and functional immune responses that protected against lethal challenge. Demonstration of responses in the lung suggests that a mechanism exists for protection against challenge with aerosolized anthrax spores. A formulated, pressure-sensitive, dry adhesive patch, which is stable and can be manufactured in large scale, elicited comparable immunoglobulin G and TNA responses, suggesting that an anthrax vaccine patch is feasible and should advance into clinical evaluation.

Adjuvants, Immunologic↗
PubMed2004

Stat 4 but not Stat 6 mediated immune mechanisms are essential in protection against plague.

The Caf1 and LcrV sub-unit vaccine for plague has been shown to be highly protective against challenge with virulent Yersinia pestis in a mouse model. Production of large amounts of IgG1 in response to the vaccine correlates with protection against aerosol and parenteral infection. In this study the effect of genetic mutation in the immune system on protection was addressed. Stat 6(-/-) mice which are unable to utilise the type 2 cytokines IL-4 and IL-13 and so should have reduced IgG1 responses were utilised in order to determine whether an immune system biased towards the type 1 axis could mount an effective response to the vaccine. Conversely in the Stat 4(-/-) mouse model, IL-12 and interferon-gamma-mediated immune mechanisms are inactive and the immune response should be biased towards the type 2 axis. Serum antibody responses to vaccination in both the knockout strains and their wild type controls revealed little difference in levels of IgG and isotype profiles. Elispot analysis of cytokine production at the single cell level did however reveal a functional defect in the Stat 4(-/-) mice which had low levels of IFN-gamma producing cells. Following virulent challenge, the Stat 6(-/-) mice showed high levels of protection, while the Stat 4(-/-) mice were poorly protected, indicating a fundamental defect in their immune systems which could not be overcome even by the passive transfer of CD4(+) cells from immunised BALB/c donors. It appears therefore that type 1 immune mechanisms, activated following Stat 4 phosphorylation, are essential in protection against plague.

Animals↗
PubMed2004

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.

Animals↗
PubMed2003

Differential processing of CD4 T-cell epitopes from the protective antigen of Bacillus anthracis.

We have mapped CD4+ T-cell epitopes located in three domains of the recombinant protective antigen of Bacillus anthracis. Mouse T-cell hybridomas specific for these epitopes were generated to study the mechanisms of proteolytic processing of recombinant protective antigen for antigen presentation by bone marrow-derived macrophages. Overall, epitopes differed considerably in their processing requirements. In particular, the kinetics of presentation, ranging from 15 (fast) to 120 min (slow), suggested sequential liberation of epitopes during proteolytic processing of the intact PA molecule. Pretreatment of macrophages with ammonium chloride or inhibitors of the major enzyme families showed that T-cell responses to an epitope presented with fast kinetics were unaffected by raising endosomal pH or inhibiting cysteine or aspartic proteinases, suggesting presentation independent of lysosomal processing. In contrast, responses to epitopes presented with slower kinetics were dependent on low pH and the activity of cysteine or aspartic proteinases indicating a requirement for lysosomal processing. In addition, responses to all epitopes, whether their presentation was dependent on low pH or not, were prevented by treatment of macrophages with broad spectrum serine proteinase inhibitors. Thus, our data are consistent with a model of sequential antigen processing within the endosomal system, beginning with a pre-processing step mediated by serine or metalloproteinases prior to further processing by lysosomal enzymes. Rapidly presented epitopes seemed to require only limited proteolysis at earlier stages of endocytosis, whereas the majority of epitopes required more extensive processing by neutral proteinases followed by lysosomal enzymes.

Ammonium Chloride↗
PubMed2003

The use of live attenuated bacteria as a delivery system for heterologous antigens.

Live attenuated mutants of several pathogenic bacteria have been exploited as potential vaccine vectors for heterologous antigen delivery by the mucosal route. Such live vectors offer the advantage of potential delivery in a single oral, intranasal or inhalational dose, stimulating both systemic and mucosal immune responses. Over the years, a range of strategies have been developed to allow controlled and stable delivery of antigens and improved immunogenicity where required. Most of these approaches have been evaluated in Salmonella vaccine vectors and, as a result, several live attenuated recombinant Salmonella vaccines are now in human clinical trials. In this review, these strategies and their use in the development of a delivery system for the Yersinia pestis V antigen are described.

Animals↗