Spring Conference 2011 - Society for General Microbiology

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Please note: Abstracts are published as received from the authors and are not subject to editing. 23 Spring Meeting 11–14 April 2011 Harrogate – www.sgmharrogate2011.org.uk SESSION AbSTrACTS ↑Contents HA04 Cont. We have developed solid lipid nanoparticles and oil-filled nanocapsules (NPs) and have demonstrated enhanced humoral and cellular immune responses to a number of different HiV antigens (Tat, Nef, Env, Gag p24, Gag p41) and model antigens (OVA, β-Gal), with codelivery of selected adjuvants (i.e., CpG) that activate specific innate response pathways. Among other attributes, the NPs are antigen dose sparing, enhance MHC i presentation, and enhance the production of neutralizing antibodies. This talk will highlight engineering aspects of the NPs and how the surface properties, size, zeta-potential, and composition can be controlled to advantageously affect the biological properties in terms of (lack of) cell and gross toxicity and inflammation, and enhance antigen-specific immune responses. Current approaches are focusing on the engineering of lipid-based NPs with a small amount of surface accessible Ni for high affinity binding to multiple his-tagged HiV proteins. Are mucosal adjuvants needed to stimulate protective anti-HIV responses? Martin Cranage Centre for Infection and Immunity, St George’s University of London, London SW17 0RE The development of an effective vaccine against HiV is hampered by a series of critical issues not least of which is how can protective responses be induced at the mucosal portals of entry. This may be crucial for HiV as the virus infects the immune system directly and is rapidly spread from the mucosal portals of entry. A further significant factor is avoidance of local pro-inflammatory responses that are often induced by adjuvants, since this may facilitate primary infection. We have used trimeric gp140 of HiV-1 clade C to explore its mucosal vaccination potential with particular respect to the generation of antibodies. Non-adjuvanted gp140 administered intravaginally to rabbits was shown to be highly immunogenic; however, the same preparation administered to macaques was considerably less effective. Prior or subsequent systemic immunization with AS01-adjuvanted gp140 considerably enhanced the efficacy of vaginal immunization. Alternative mucosal-adjuvanted prepartions have so far failed enhance the responses seen with systemic vaccination, suggesting that access to antigen after mucosal administration may be a rate-limiting factor. Conjugation of transferrin to trimeric gp140 was shown, in a murine model, to enhance bio-availability after mucosal administration and to stimulate production of anti-gp140 antibodies. Offered paper In vitro selection and characterization of recombinant human antibodies from HIV-1-positive donors JiE TANG1 , Anatoliy Markiv1 , Hanna Dreja2 , Áine McKnight2 , Mingyue He3 , Angray Kang1 1 2 3 University of Westminster, London, Queen Mary, University of London, London, The Babraham Institute, Cambridge Human single-chain antibodies reacting with recombinant HiV-1 surface glycoprotein gp120 have been selected by in vitro ribosome display from two combinatorial antibody libraries.The immunoglobulin libraries were prepared from two long-term survivors whose serum showed cross-clade neutralizing activity against HiV-1. Total rNA was extracted from isolated peripheral blood mononuclear cells (from as little as 20ml fresh blood). Members of 8 heavy chain families, 4 κ light chain families and 9 λ light chain families were amplified by rT-PCr. Single-chain antibody libraries of igG1234 isotypes in the format of Vl-link–VH-CH were constructed, with all possible combinatorial pairs generated and analysed by gel electrophoresis. Both libraries were subjected to a cell-free eukaryotic ribosome display selection. Analysis of selected sequences reveals enrichment during selection and the isotypes of these antibodies (mostly igG1, with some igG2 and 4). More than 300 recombinant antibodies have been isolated. Seven recombinant antibodies from the heterologous library and three from autologous library exhibited significant binding to recombinant gp120 in EliSA. interestingly, similar CDr3 sequences of light and heavy chins were observed from the two libraries, suggesting the selection worked by ribosome display. Offered paper Assessment of novel tuberculosis vaccine antigens using MVA rOBErT WATSON, Yper Hall, James McCowen, Karen Buttigieg, Ann Williams, Miles Carroll Health Protection Agency, Porton Down The proven clinical safety record, induction of cellular antigen-specific immune responses and ability to boost a BCG prime, make Modified Vaccinia virus Ankara (MVA) a promising viral vector for delivery of novel tuberculosis vaccine candidates. We have identified 5 novel TB vaccine antigens that are potentially effective against multiple stages of disease. These were shown to give protection as plasmid DNA and adjuvanted protein against Mycobacterium tuberculosis infection in a guinea-pig aerosol challenge model. recombinant MVAs expressing these antigen candidates have been generated using transfer plasmids supplied by B. Moss (NiH) and assessed for immunogenicity in vivo. Following vaccination, antigen-specific iFN-g responses were observed as measured by ex vivo splenocyte EliSpot assay. Peptide pools spanning each whole protein were used to re-stimulate cells for 18 hours prior to enumeration of cells secreting cytokine, expressed as spot-forming units. These data demonstrated successful induction of a Th-1 cellular immune response by each of the antigens assessed. Furthermore, construction and evaluation of novel recombinant MVA was investigated with regards to the vaccinia promoter driving s o c i e t y f o r g e n e r a l Microbiology
Please note: Abstracts are published as received from the authors and are not subject to editing. 24 Spring Meeting 11–14 April 2011 Harrogate – www.sgmharrogate2011.org.uk SESSION AbSTrACTS ↑Contents HA04 Cont. transgene expression and vaccination route. Protection studies will follow to determine efficacy against M. tuberculosis challenge and the most promising candidates will be taken forward for further develoment. Adjuvants to enhance effector T cell responses Kingston H.G. Mills Immunology Research Centre, School of Biochemistry & Immunology, Trinity College Dublin, Ireland The design of effective subunit vaccines is dependent on identifying effective adjuvants to generate appropriate protective immune responses against the pathogen. Most licensed vaccines work by inducing protective neutralizing antibody responses. Although effective for prophylactic vaccination against virus and toxin-mediated bacterial diseases, antibodies alone are not adequate for many intracellular pathogens and may be ineffective for therapeutic vaccination. Here cellular immune responses mediated by different population of effector T cells may be more important. The induction of effector T cells is directed by innate immune cells, activated by pathogen-associated molecular patterns (PAMPs) or adjuvants. iFN-γ-secreting Th1 cells function to activate macrophages and enhance complement fixing antibodies in immunity to intracellular pathogens. immunity to extracellular pathogens required Th2 cells, which promote antibody production, but also Th17 cells that recruit neutrophils. Finally, regulatory T cells (Treg cells), can promote pathogen persistence by suppressing host protective immune responses. Therefore, a major obstacle to therapeutic vaccination for chronic infections, such as HiV, hepatitis C virus and Mycobacterium tuberculosis is to overcome the immunosuppressive effects of Treg cells. Targeting innate immune cells with designer adjuvants that turn off immunosuppressive molecules and Treg cells but promote effector T cells is one approach to achieve this objective. bacterial toxins as mucosal adjuvants Gill Douce IBLS, Division of Infection & Immunity, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ induction of immunity at mucosal surfaces is thought to be an essential feature in protection of the host against the many pathogens that gain access through these surfaces. Of the many different antigens tested, the most effective immunogens at these surfaces appear to be bacterially derived components and in particular bacterial toxins. Of those proteins studied to date, the highly homologous enterotoxins, Cholera toxin (CT) from Vibrio cholerae and heat labile toxin from enterotoxigenic Escherichia coli (lT) stimulate strong local and systemic anti-toxin response following mucosal presentation. in addition, co-administration of normally non-immunogenic antigens results in the production of antibody to these proteins. Whilst the high toxicity has made their use impractical for human vaccine development, generation and testing of site directed mutants has shown toxicity and adjuvant activity are not linked. Subsequent testing of these mutants in human trials demonstrated limited success. This talk will focus on the potential for the use of toxins in vaccine design, including the advantages and pitfalls that have been identified to date. This will include discussion of how our knowledge has and continues to allow rational design of recombinant proteins that more effectively stimulate immune responses at these surfaces. Understanding the mechanism of action of alum MirJAM KOOl1,2,3 , Monique M. Willart1 , Kaat Fierens1 , Menno van Nimwegen3 , Hamida Hammad1 , Bart N. lambrecht1,3 1Laboratory of Immunoregulation & Mucosal Immunology, Dept of Pulmonary Medicine, University Hospital Gent, Belgium; 2 3 Dept for Molecular Biomedical Research, Flemish Institute for Biotechnology (VIB), Gent, Belgium; Dept of Pulmonary Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Aluminum containing adjuvants (alum) are the most widely used adjuvants in human vaccines, however only recently we started to unravel the mechanisms by which they potentiate the immune system. it is common knowledge that alum predominantly induces humoral immunity. Surprisingly, in vitro studies showed no stimulatory effects on nature’s adjuvant, the dendritic cell (DC). However, in vivo after injection of alum the antigen was taken up, processed and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response that re-circulated to other lymphoid organs. The stimulating effects of alum on both cellular and humoral immunity were completely abolished when CD11c + monocytes and DCs were conditionally depleted during immunization in CD11c-DTr transgenic mice. Strikingly, alum adjuvant induced high levels of uric acid. When uric acid crystals were injected as an adjuvant, they induced the same strong adjuvant effect as seen with alum adjuvant. One of the targets of uric acid is the Nlrp3 inflammasome. When DCs are exposed to alum adjuvant or uric acid crystals they release il-1β, and this is abrogated in cells lacking various Nlrp3 inflammasome components. The Nlrp3 inflammasome is also partially required in vivo for the innate immune response to ovalbumin in alum. The early production of il-1β and the influx of inflammatory cells into the peritoneal cavity is reduced in Nlrp3 deficient s o c i e t y f o r g e n e r a l Microbiology
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Spring Conference 2011 - Society for General Microbiology

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