Abstracts
Abstracts from the 3rd Annual Infection and Immunity Meeting A meeting of the BSI Infection & Immunity Affinity Group Held on Thursday 11 April 2013 at Park Crescent Conference Centre, 229 Great Portland Street, London W1W 5PN Abstracts A1 Innate immune responses in acute HIV-1 infection: protective or pathogenic? P. Borrow Nuffield Dept of Clinical Medicine, University of Oxford, UK
The importance of events in acute HIV-1 infection (AHI) in determining the subsequent disease course prompts a need to understand the virus-immune system interactions in this phase of infection that impact on concurrent and ensuing viral replication and pathogenesis. The speed with which innate responses can be mobilized following pathogen exposure suggests they may play critical roles in AHI. We sought to characterise the innate responses activated during AHI and identify components of the response that contribute to control of viremia or conversely promote immune activation and virus replication. Peripheral blood samples cryopreserved at serial timepoints post-infection were used to characterise the dynamics of systemic up-regulation of cytokines/ chemokines and activation of innate cell subsets in AHI and address the impact of these factors on viral control. The increase in viremia in AHI was associated with rapid activation of systemic innate responses, evidenced by a reduction in the circulating dendritic cell frequency, elevations in plasma levels of type 1 interferon (IFN) and other soluble factors and natural killer (NK) cell activation and proliferation. Some components of the innate response, e.g. strong NK cell responses, correlated with establishment of lower set-point viral loads. However the association between any one component of the innate response and set-point viremia was confounded by other interlinked innate variables. To seek evidence that type 1 IFN exerts selective pressure on HIV replication during the establishment of infection, the IFN-resistance of HIV isolates derived from AHI subjects and
founder virus infectious molecular clones (IMCs) was compared to that of virus isolates/IMCs generated from the same subjects in chronic infection. Founder viruses were found to be more IFN-resistant than matched chronic viruses, suggesting that type I IFN plays an important role in HIV control during the initial stages of infection and may drive selection for IFN-resistant founder viruses from the transmitted virus pool. Identification of type 1 IFN-mediated antiviral activity and NK effector activity as mechanisms that contribute to HIV-1 control during the initial stages of infection could enable harnessing of these activities to complement the protection afforded by vaccine-elicited adaptive responses.Supported by funding from the NIH (CHAVI AI-067854 and CHAVI-ID AI-100645)
A2 Controlled viral infection of humans to analyse natural killer cell activation in vivo G. P. Cook Leeds Institute of Molecular Medicine, University of Leeds, UK
NK cells are critical for antiviral immunity, but studying their activation kinetics during infection is fraught with logistical and ethical problems. Use of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten cancer patients received large repeated doses of oncolytic Retrovirus. NK activation was rapid and bore the hallmarks of a type I interferon response which was supported by in vitro data. These results define the kinetics of human NK cell activation in vivo, providing information that could previously only be inferred from studies in animal models.
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
1
Abstracts
A3 Host-pathogen interactions in the lung; alveolar macrophage killing games D. Dockrell Department of Infection and Immunity, The University of Sheffield Medical School, University of Sheffield, UK
Alveolar macrophages (AM) play an essential role in clearing bacteria from the lower airway. As the resident phagocyte AM must both phagocytose and kill bacteria and if unable to do this completely must co-ordinate an inflammatory response. The decision to escalate the inflammatory response represents the transition between sub-clinical infection and the development of pneumonia. Alveolar macrophages are well equipped to phagocytose bacteria and have a large phagolysosomal capacity in which ingested bacteria are killed. The rate-limiting step in bacterial control is the capacity of AM to kill ingested bacteria and AM complement canonical microbicidal strategies with an additional level of apoptosis-associated killing to help kill ingested bacteria. Susceptibility to pneumonia can be predicted by the extent to which this aspect of host defence is engaged. Conversely pathogenic micro-organisms have developed a range of adaptations to circumvent this level of innate immune control.
A4 An in vitro T cell killing assay to assess new Plasmodium falciparum vaccine candidates R. J. Longley, K. Ewer, M. G. Cottingham, K. Bauza, A. Williams, A. J. Spencer and A. V. S. Hill Jenner Institute, University of Oxford, UK
The development of an efficacious Plasmodium falciparum malaria vaccine remains a top priority. Research undertaken in our laboratory has demonstrated the ability of prime-boost virally vectored sub-unit vaccination regimes to produce extremely high levels of T cells. The liver-stage of the life-cycle is the main target of T cell mediated immunity, yet a major challenge in identifying an immunogenic and protective liver-stage antigen has been the lack of a suitable pre-clinical assay. Our aim is to screen a number of new candidate liver-stage vaccines by developing an in vitro T cell killing assay. We developed an in vitro killing assay by infecting the murine hepatoma cell line Hepa1-6 with P. berghei-GFP sporozoites, followed by the addition of antigen-specific CD8+ enriched splenocytes. On average, 3–5% of hepatocytes were infected as measured by flow cytometry. We utilized P. berghei TRAP as a model antigen as 20–30% of circulating CD8+ T cells secrete IFNc following a prime-boost regime in mice. We found that P. berghei TRAP CD8+ T cells were able to specifically kill P. berghei infected Hepa1-6 cells in a
2
dose-dependent manner, with up to 60% inhibition observed at high E:T ratios. We expressed nine candidate P. falciparum liver-stage antigens in the viral vectors ChAd63 and MVA, and analysed immunogenicity in Balb/ c, C57BL/6 and HLA-A2 transgenic mice. Following an 8 week prime-boost interval, candidate liver-stage antigens LSA1, LSA3, LSAP1, LSAP2, PFE1590w, PFI0580c, PfUIS3, Exp1 and CelTOS generated positive ELISpot responses with medians of 200–1000 SFC per million splenocytes. We plan to further develop the in vitro assay using human hepatocytes and P. falciparum, and to test the potential of the nine candidate vaccines in this system via vaccination of HLA transgenic mice to induce antigen-specific MHC matched CD8+ T cells.
A5 The filarial nematode product, ES-62 targets IL-22 to mediate protection in collagen-induced arthritis M. A. Pineda, L. Al-Riyami, W. Harnett and M. M. Harnett Institute of Infection, Immunity and Inflammation, University of Glasgow and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, UK
Parasitic worms skew immune responses to a Th2-like phenotype, inhibiting Th1/Th17 pathways and expanding regulatory cells such as Tregs. Worms can achieve such immunoregulation by secreting specific products like ES62, a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae. We have previously shown ES-62 to be protective in collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis, and that the mechanism associated with protection involves a down-regulation of the cytokine IL-17. We have now analyzed the effects of ES-62 on other Th17-related cytokines focusing on IL-22, a cytokine that has been reported to exhibit both pro and anti-inflammatory actions in animal models of inflammatory disease. In CIA, administration of ES-62 appears to target the IL17/IL-22 balance in lymph nodes and joints of animals resulting in modulation of the number and inflammatory phenotype of infiltrating and resident cells in the joint, and consequent attenuation of inflammation. Interestingly, using a combination of recombinant IL-22 and blocking antibody in in vivo studies, the precise type of effect of IL-22 on CIA progression was shown to depend largely both on the location of IL-22 action and also the stage at which the cytokine is produced during disease progression. These findings have allowed us to identify a novel mechanism used by the helminth product ES-62 to reduce autoimmune arthritis and they also contribute to our understanding of the complex biology of IL-22
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
Abstracts
A6 Live vaccination for the prevention of melioidosis A. E. Scott1, T. R. Laws1, P. Tan2, J. L. Prior1 and T. P. Atkins1 1
Defence Science and Technology Laboratory, Porton Down, Salisbury;
and 2Genome Institute of Singapore, 60 Biopolis Street, 138672 Singapore
Burkholderia pseudomallei is a Gram negative bacterium which can be readily isolated from soil and standing water in tropical regions. It is a facultative intra-cellular pathogen and is the etiological agent of melioidosis, a disease associated with a high case-mortality rate in endemic regions. Exposure through the inhalation route leads to particularly acute disease. B. pseudomallei is highly refractory to antibiotics; even with appropriate therapy the mortality rate in endemic areas is over 40%. The development of a licensed vaccine has been a focus of our group for several years. We have previously demonstrated that immunisation with live strains of B. pseudomallei offers protection in animal models, but concerns over safety and the ability to licence such a vaccine have limited this approach. Burkholderia thailandensis is a close relative of B. pseudomallei and is essentially avirulent in humans, but the lack of key protective antigens renders this species less attractive as a vaccine candidate. Recently, we have identified a strain of B. thailandensis which contains these protective antigens. This strain has demonstrably low virulence and offers excellent protection against experimental melioidosis, even with extremely high challenge doses. Further analysis indicates that responses within the first 24 hours of the infection are key to providing longer term immunity, and that the bulk of these responses are directed against the key protective antigen. © Crown Copyright 2013. Published with the permission of the Defence Science and Technology Laboratory on behalf of the controller of HMSO.
A7 Bacillus subtilis spores as a vaccine adjuvant for tuberculosis L. Sibley Bacillus subtilis produces spores that are resistant to heat and chemical assault. These spores are immunogenic and can be genetically modified or bind proteins to deliver antigens on the spore surface. These characteristics make them an attractive potential vaccine adjuvant. In this study spores were investigated for their potential as a nasal vaccine. Interactions of spores with lung cells is not fully understood, but are thought to interact with macrophages. Mice were dosed intranasally with GFP expressing spores to track where spores were delivered. There was
progression of spores from the bronchioles into the tissue, followed by phagocytosis and cellular infiltration. In vitro techniques demonstrated phagocytosis of spores by macrophages. Spores were investigated for their potential as a vaccine adjuvant for tuberculosis TB. One third of the world’s population is reported to be infected with TB. BCG is the only licensed vaccine, which has variable efficacy. TB antigens MPT64, Ag85B-Acr were tested. They were delivered adsorbed to the surface of the spores dosed intranasally to mice with and without prime boost with BCG. Inactivated recombinant spores expressing MPT64 rHU58MPT64) were also tested. Th1 responses were induced, as shown using IFNc ELISPOT and polyfunctional T-cell analysis. Mice were challenged with Mycobacterium tuberculosis to test protective efficacy. The results showed that spores carrying antigen were able to reduce the CFU to a comparable level to BCG and boosting reduced CFU further.
A8 Mechanism(s) involved in Clostridium difficilemediated Inflammasome activation M. J. Songane1, S. Kuehne2, H. Hussain3, N. Klein1, N. Minton2, E. Allan3 and M. Bajaj-Elliott1 1
Institute of Child Health, University College London, London, UK, NIHR Biomedical Research Unit, University of Nottingham,
2
Nottingham, UK; and 3Eastman Dental Institute, University College London, London, UK
Background: Emerging epidemic Clostridium difficile (CD) strains are responsible for the recent increase in antibiotic-associated diarrhoea. CD toxin A (TcdA) and TcdB are known potent activators of the ‘Infammasome’ complex that culminates in cytokine (IL-1b/IL-18) production and pyroptosis. Studies utilising recombinant toxins implicate a role for NOD-like receptor (NLR) NALP3 in CD toxin-mediated inflammasome activation. At present the potential contribution of toxin-independent Inflammasome function and its interaction with the autophagy machinery during CD infection unknown. Methods: Phorbol myristate acetate (PMA)-differentiated THP-1 macrophages and human blood monocyte-derived macrophages (MDM) were infected with two virulent wild-type (WT) R20291 and 630 Derm CD strains and their respective toxin-deficient isogenic mutants, in the presence of inhibitors targeting inflammasome and autophagy. Results: R20291 and 630Derm WT strains mediated a significant increase in bioactive IL-1b in a caspase 1-dependent manner. Interestingly, substantial amounts of IL-1b secretion were also recorded in response to toxin-deficient isogenic mutants. IL-1b secretion was preceded by ASC speck formation accompanied by NALP3 cellular re-locali-
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
3
Abstracts
sation. Inhibition of NALP3 through high intracellular K+ or blocking K+ channel activity led to diminished cytokine secretion. In addition, inhibition of autophagy caused a marked increase in IL-1b and ASC specks, while induction of this process caused the opposite effect. Conclusion: Our data suggests that TcdA and TcdB are equipotent inflammasome activators. Toxin-mediated modulation of NALP3 function was partly dependent on low intracellular K+, and autophagy limits CD-mediated inflammasome activation. Our current hypothesis is that the toxin-independent inflammasome activation is NLRC4 mediated.
A9 Yersinia pestis: Towards a next generation vaccine D. Williamson Dstl Porton Down, Salisbury Wilts
Yersinia pestis is the causative agent of plague and has evolved from an enteropathogenic lifestyle with moderate virulence, to become a flea-vectored pathogen with enhanced virulence. As such, it has acquired plasmids which encode an array of virulence factors, including a type three secretion (T3S) system which enables the bacterium to translocate cytotoxic factors into host cells with which it makes close contact in vivo. Pivotal to this process is the V antigen which has a role in regulating T3S within the bacterium and also tips the physical injectisome. Once in vivo, the bacterium also surrounds itself with a proteinaceous capsule comprising the F1 antigen and with a fibrillar adhesin comprising pH6 antigen.
4
Combined with the effect of other genome –encoded adhesins, these surface components are strongly antiphagocytic and complement-resistant. As an understanding of these virulence mechanisms has developed, so has a next generation vaccine strategy. Previously used formulations such as the Plague vaccine USP comprised a suspension of killed Y. pestis in a preservative and were thought to be effective in preventing bubonic, but experimental and clinical evidence suggested they were ineffective against pneumonic plague. On the other hand, a combination of the F1 and V antigens in recombinant form has been demonstrated to protect a range of animal models, including non-human primates against pneumonic plague. A genetic fusion of these two proteins is being developed as a next generation vaccine and is in advanced clinical trials. The vaccine will be licensed under the FDA’s Animal Rule which requires the identification of an immune correlate(s) of protection in animal models which authentically replicate the human syndrome. The mechanisms of immune protection include the development of antibodies to F1 and V which enhance opsonophagocytosis of Y. pestis and neutralise T3S effectors, respectively. However, the findings of our own and other groups indicate that the specific priming and engagement of CD4+ T-cells is critical for survival and the clearance of Y. pestis from tissues following exposure. This has been demonstrated by the adoptive transfer of F1/V primed CD4+ T-cells into B-cell deficient or severe combined immunodeficient (SCID/bge) mice to achieve survival following exposure. An HLADR1 restricted T-cell epitope has been identified in the C-terminal region of F1 antigen, whilst T-cell epitopemapping of the V antigen is ongoing.
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
Abstracts from the 3rd Annual Infection and Immunity Meeting A meeting of the BSI Infection & Immunity Affinity Group Held on Thursday 11 April 2013 at Park Crescent Conference Centre, 229 Great Portland Street, London W1W 5PN Abstracts A1 Innate immune responses in acute HIV-1 infection: protective or pathogenic? P. Borrow Nuffield Dept of Clinical Medicine, University of Oxford, UK
The importance of events in acute HIV-1 infection (AHI) in determining the subsequent disease course prompts a need to understand the virus-immune system interactions in this phase of infection that impact on concurrent and ensuing viral replication and pathogenesis. The speed with which innate responses can be mobilized following pathogen exposure suggests they may play critical roles in AHI. We sought to characterise the innate responses activated during AHI and identify components of the response that contribute to control of viremia or conversely promote immune activation and virus replication. Peripheral blood samples cryopreserved at serial timepoints post-infection were used to characterise the dynamics of systemic up-regulation of cytokines/ chemokines and activation of innate cell subsets in AHI and address the impact of these factors on viral control. The increase in viremia in AHI was associated with rapid activation of systemic innate responses, evidenced by a reduction in the circulating dendritic cell frequency, elevations in plasma levels of type 1 interferon (IFN) and other soluble factors and natural killer (NK) cell activation and proliferation. Some components of the innate response, e.g. strong NK cell responses, correlated with establishment of lower set-point viral loads. However the association between any one component of the innate response and set-point viremia was confounded by other interlinked innate variables. To seek evidence that type 1 IFN exerts selective pressure on HIV replication during the establishment of infection, the IFN-resistance of HIV isolates derived from AHI subjects and
founder virus infectious molecular clones (IMCs) was compared to that of virus isolates/IMCs generated from the same subjects in chronic infection. Founder viruses were found to be more IFN-resistant than matched chronic viruses, suggesting that type I IFN plays an important role in HIV control during the initial stages of infection and may drive selection for IFN-resistant founder viruses from the transmitted virus pool. Identification of type 1 IFN-mediated antiviral activity and NK effector activity as mechanisms that contribute to HIV-1 control during the initial stages of infection could enable harnessing of these activities to complement the protection afforded by vaccine-elicited adaptive responses.Supported by funding from the NIH (CHAVI AI-067854 and CHAVI-ID AI-100645)
A2 Controlled viral infection of humans to analyse natural killer cell activation in vivo G. P. Cook Leeds Institute of Molecular Medicine, University of Leeds, UK
NK cells are critical for antiviral immunity, but studying their activation kinetics during infection is fraught with logistical and ethical problems. Use of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten cancer patients received large repeated doses of oncolytic Retrovirus. NK activation was rapid and bore the hallmarks of a type I interferon response which was supported by in vitro data. These results define the kinetics of human NK cell activation in vivo, providing information that could previously only be inferred from studies in animal models.
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
1
Abstracts
A3 Host-pathogen interactions in the lung; alveolar macrophage killing games D. Dockrell Department of Infection and Immunity, The University of Sheffield Medical School, University of Sheffield, UK
Alveolar macrophages (AM) play an essential role in clearing bacteria from the lower airway. As the resident phagocyte AM must both phagocytose and kill bacteria and if unable to do this completely must co-ordinate an inflammatory response. The decision to escalate the inflammatory response represents the transition between sub-clinical infection and the development of pneumonia. Alveolar macrophages are well equipped to phagocytose bacteria and have a large phagolysosomal capacity in which ingested bacteria are killed. The rate-limiting step in bacterial control is the capacity of AM to kill ingested bacteria and AM complement canonical microbicidal strategies with an additional level of apoptosis-associated killing to help kill ingested bacteria. Susceptibility to pneumonia can be predicted by the extent to which this aspect of host defence is engaged. Conversely pathogenic micro-organisms have developed a range of adaptations to circumvent this level of innate immune control.
A4 An in vitro T cell killing assay to assess new Plasmodium falciparum vaccine candidates R. J. Longley, K. Ewer, M. G. Cottingham, K. Bauza, A. Williams, A. J. Spencer and A. V. S. Hill Jenner Institute, University of Oxford, UK
The development of an efficacious Plasmodium falciparum malaria vaccine remains a top priority. Research undertaken in our laboratory has demonstrated the ability of prime-boost virally vectored sub-unit vaccination regimes to produce extremely high levels of T cells. The liver-stage of the life-cycle is the main target of T cell mediated immunity, yet a major challenge in identifying an immunogenic and protective liver-stage antigen has been the lack of a suitable pre-clinical assay. Our aim is to screen a number of new candidate liver-stage vaccines by developing an in vitro T cell killing assay. We developed an in vitro killing assay by infecting the murine hepatoma cell line Hepa1-6 with P. berghei-GFP sporozoites, followed by the addition of antigen-specific CD8+ enriched splenocytes. On average, 3–5% of hepatocytes were infected as measured by flow cytometry. We utilized P. berghei TRAP as a model antigen as 20–30% of circulating CD8+ T cells secrete IFNc following a prime-boost regime in mice. We found that P. berghei TRAP CD8+ T cells were able to specifically kill P. berghei infected Hepa1-6 cells in a
2
dose-dependent manner, with up to 60% inhibition observed at high E:T ratios. We expressed nine candidate P. falciparum liver-stage antigens in the viral vectors ChAd63 and MVA, and analysed immunogenicity in Balb/ c, C57BL/6 and HLA-A2 transgenic mice. Following an 8 week prime-boost interval, candidate liver-stage antigens LSA1, LSA3, LSAP1, LSAP2, PFE1590w, PFI0580c, PfUIS3, Exp1 and CelTOS generated positive ELISpot responses with medians of 200–1000 SFC per million splenocytes. We plan to further develop the in vitro assay using human hepatocytes and P. falciparum, and to test the potential of the nine candidate vaccines in this system via vaccination of HLA transgenic mice to induce antigen-specific MHC matched CD8+ T cells.
A5 The filarial nematode product, ES-62 targets IL-22 to mediate protection in collagen-induced arthritis M. A. Pineda, L. Al-Riyami, W. Harnett and M. M. Harnett Institute of Infection, Immunity and Inflammation, University of Glasgow and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, UK
Parasitic worms skew immune responses to a Th2-like phenotype, inhibiting Th1/Th17 pathways and expanding regulatory cells such as Tregs. Worms can achieve such immunoregulation by secreting specific products like ES62, a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae. We have previously shown ES-62 to be protective in collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis, and that the mechanism associated with protection involves a down-regulation of the cytokine IL-17. We have now analyzed the effects of ES-62 on other Th17-related cytokines focusing on IL-22, a cytokine that has been reported to exhibit both pro and anti-inflammatory actions in animal models of inflammatory disease. In CIA, administration of ES-62 appears to target the IL17/IL-22 balance in lymph nodes and joints of animals resulting in modulation of the number and inflammatory phenotype of infiltrating and resident cells in the joint, and consequent attenuation of inflammation. Interestingly, using a combination of recombinant IL-22 and blocking antibody in in vivo studies, the precise type of effect of IL-22 on CIA progression was shown to depend largely both on the location of IL-22 action and also the stage at which the cytokine is produced during disease progression. These findings have allowed us to identify a novel mechanism used by the helminth product ES-62 to reduce autoimmune arthritis and they also contribute to our understanding of the complex biology of IL-22
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
Abstracts
A6 Live vaccination for the prevention of melioidosis A. E. Scott1, T. R. Laws1, P. Tan2, J. L. Prior1 and T. P. Atkins1 1
Defence Science and Technology Laboratory, Porton Down, Salisbury;
and 2Genome Institute of Singapore, 60 Biopolis Street, 138672 Singapore
Burkholderia pseudomallei is a Gram negative bacterium which can be readily isolated from soil and standing water in tropical regions. It is a facultative intra-cellular pathogen and is the etiological agent of melioidosis, a disease associated with a high case-mortality rate in endemic regions. Exposure through the inhalation route leads to particularly acute disease. B. pseudomallei is highly refractory to antibiotics; even with appropriate therapy the mortality rate in endemic areas is over 40%. The development of a licensed vaccine has been a focus of our group for several years. We have previously demonstrated that immunisation with live strains of B. pseudomallei offers protection in animal models, but concerns over safety and the ability to licence such a vaccine have limited this approach. Burkholderia thailandensis is a close relative of B. pseudomallei and is essentially avirulent in humans, but the lack of key protective antigens renders this species less attractive as a vaccine candidate. Recently, we have identified a strain of B. thailandensis which contains these protective antigens. This strain has demonstrably low virulence and offers excellent protection against experimental melioidosis, even with extremely high challenge doses. Further analysis indicates that responses within the first 24 hours of the infection are key to providing longer term immunity, and that the bulk of these responses are directed against the key protective antigen. © Crown Copyright 2013. Published with the permission of the Defence Science and Technology Laboratory on behalf of the controller of HMSO.
A7 Bacillus subtilis spores as a vaccine adjuvant for tuberculosis L. Sibley Bacillus subtilis produces spores that are resistant to heat and chemical assault. These spores are immunogenic and can be genetically modified or bind proteins to deliver antigens on the spore surface. These characteristics make them an attractive potential vaccine adjuvant. In this study spores were investigated for their potential as a nasal vaccine. Interactions of spores with lung cells is not fully understood, but are thought to interact with macrophages. Mice were dosed intranasally with GFP expressing spores to track where spores were delivered. There was
progression of spores from the bronchioles into the tissue, followed by phagocytosis and cellular infiltration. In vitro techniques demonstrated phagocytosis of spores by macrophages. Spores were investigated for their potential as a vaccine adjuvant for tuberculosis TB. One third of the world’s population is reported to be infected with TB. BCG is the only licensed vaccine, which has variable efficacy. TB antigens MPT64, Ag85B-Acr were tested. They were delivered adsorbed to the surface of the spores dosed intranasally to mice with and without prime boost with BCG. Inactivated recombinant spores expressing MPT64 rHU58MPT64) were also tested. Th1 responses were induced, as shown using IFNc ELISPOT and polyfunctional T-cell analysis. Mice were challenged with Mycobacterium tuberculosis to test protective efficacy. The results showed that spores carrying antigen were able to reduce the CFU to a comparable level to BCG and boosting reduced CFU further.
A8 Mechanism(s) involved in Clostridium difficilemediated Inflammasome activation M. J. Songane1, S. Kuehne2, H. Hussain3, N. Klein1, N. Minton2, E. Allan3 and M. Bajaj-Elliott1 1
Institute of Child Health, University College London, London, UK, NIHR Biomedical Research Unit, University of Nottingham,
2
Nottingham, UK; and 3Eastman Dental Institute, University College London, London, UK
Background: Emerging epidemic Clostridium difficile (CD) strains are responsible for the recent increase in antibiotic-associated diarrhoea. CD toxin A (TcdA) and TcdB are known potent activators of the ‘Infammasome’ complex that culminates in cytokine (IL-1b/IL-18) production and pyroptosis. Studies utilising recombinant toxins implicate a role for NOD-like receptor (NLR) NALP3 in CD toxin-mediated inflammasome activation. At present the potential contribution of toxin-independent Inflammasome function and its interaction with the autophagy machinery during CD infection unknown. Methods: Phorbol myristate acetate (PMA)-differentiated THP-1 macrophages and human blood monocyte-derived macrophages (MDM) were infected with two virulent wild-type (WT) R20291 and 630 Derm CD strains and their respective toxin-deficient isogenic mutants, in the presence of inhibitors targeting inflammasome and autophagy. Results: R20291 and 630Derm WT strains mediated a significant increase in bioactive IL-1b in a caspase 1-dependent manner. Interestingly, substantial amounts of IL-1b secretion were also recorded in response to toxin-deficient isogenic mutants. IL-1b secretion was preceded by ASC speck formation accompanied by NALP3 cellular re-locali-
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
3
Abstracts
sation. Inhibition of NALP3 through high intracellular K+ or blocking K+ channel activity led to diminished cytokine secretion. In addition, inhibition of autophagy caused a marked increase in IL-1b and ASC specks, while induction of this process caused the opposite effect. Conclusion: Our data suggests that TcdA and TcdB are equipotent inflammasome activators. Toxin-mediated modulation of NALP3 function was partly dependent on low intracellular K+, and autophagy limits CD-mediated inflammasome activation. Our current hypothesis is that the toxin-independent inflammasome activation is NLRC4 mediated.
A9 Yersinia pestis: Towards a next generation vaccine D. Williamson Dstl Porton Down, Salisbury Wilts
Yersinia pestis is the causative agent of plague and has evolved from an enteropathogenic lifestyle with moderate virulence, to become a flea-vectored pathogen with enhanced virulence. As such, it has acquired plasmids which encode an array of virulence factors, including a type three secretion (T3S) system which enables the bacterium to translocate cytotoxic factors into host cells with which it makes close contact in vivo. Pivotal to this process is the V antigen which has a role in regulating T3S within the bacterium and also tips the physical injectisome. Once in vivo, the bacterium also surrounds itself with a proteinaceous capsule comprising the F1 antigen and with a fibrillar adhesin comprising pH6 antigen.
4
Combined with the effect of other genome –encoded adhesins, these surface components are strongly antiphagocytic and complement-resistant. As an understanding of these virulence mechanisms has developed, so has a next generation vaccine strategy. Previously used formulations such as the Plague vaccine USP comprised a suspension of killed Y. pestis in a preservative and were thought to be effective in preventing bubonic, but experimental and clinical evidence suggested they were ineffective against pneumonic plague. On the other hand, a combination of the F1 and V antigens in recombinant form has been demonstrated to protect a range of animal models, including non-human primates against pneumonic plague. A genetic fusion of these two proteins is being developed as a next generation vaccine and is in advanced clinical trials. The vaccine will be licensed under the FDA’s Animal Rule which requires the identification of an immune correlate(s) of protection in animal models which authentically replicate the human syndrome. The mechanisms of immune protection include the development of antibodies to F1 and V which enhance opsonophagocytosis of Y. pestis and neutralise T3S effectors, respectively. However, the findings of our own and other groups indicate that the specific priming and engagement of CD4+ T-cells is critical for survival and the clearance of Y. pestis from tissues following exposure. This has been demonstrated by the adoptive transfer of F1/V primed CD4+ T-cells into B-cell deficient or severe combined immunodeficient (SCID/bge) mice to achieve survival following exposure. An HLADR1 restricted T-cell epitope has been identified in the C-terminal region of F1 antigen, whilst T-cell epitopemapping of the V antigen is ongoing.
ª 2013 The Authors Clinical and Experimental Immunology ª 2013 British Society for Immunology, Clinical and Experimental Immunology, 173 (Suppl. 1), 1–4
