Biomedical subjects
Publications and source records attributed to Jim E Eyles.
Intratracheal delivery of aerosolized monoclonal antibodies with specificity for Yersinia pestis LcrV and F1 antigens protected mice in a model of pneumonic plague. These data support the utility of inhaled antibodies as a fast-acting postexposure treatment for plague.
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.
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.
Expanding identification of potentially protective subunit antigens and correlates of protection has provided a basis for the introduction of safer vaccines. Despite encouraging results in animal models, the significant potential of particulate delivery systems in vaccine design has not yet translated into effective vaccines available for use in humans. This review article will focus on the current status of the development of particulate vaccines, mainly liposomes and bio-degradable polymers, against potential agents for biowarfare: plague, anthrax, botulinum, and smallpox; and filoviruses: Marburg and Ebola.
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.
We hypothesized that the addition of polymer to the surface of liposome/DNA complexes may potentially enhance in vivo delivery of plasmid DNA to antigen-presenting cells and thereby facilitate enhanced immune responses to encoded protein. BALB/c mice were immunized subcutaneously or intramuscularly three times with a total of 50 microg of the plasmid pRc/CMV-HBs(S) (ayw subtype) encoding for the hepatitis B surface antigen. We measured transgene-specific total immunoglobulin G (IgG), IgG2a, IgG2b and IgG1 antibody responses as well as splenocyte and T-cell proliferation and cytokine production upon re-stimulation following immunization. Modification of lipid/DNA complexes by the polymer precipitation method used here for the addition of poly(d,l-lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal DNA or naked DNA in eliciting transgene-specific immune responses to plasmid-encoded antigen when administered by the subcutaneous route. In addition, the polymer-modified formulations delivered by this route were more effective than naked DNA delivered by the intramuscular route in inducing antibody responses (n=5, P<0.03). Our observations provide 'proof of principle' for the use of these multicomponent formulations, which offer potential for manipulation and increased transfection efficiency in vivo for the purposes of genetic immunization.
Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly-L-lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 10(3) median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.