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Saeeda Bobat,
Adriana Flores-Langarica,
Jessica Hitchcock,
Jennifer L Marshall,
Robert A Kingsley, Margaret Goodall,
Cristina Gil-Cruz,
Karine Serre,
Denisse L Leyton,
Shirdi E Letran,
Fabrina Gaspal,
Rebecca Chester,
Jayne L Chamberlain,
Gordon Dougan,
Constantino López-Macías,
Ian R Henderson,
James Alexander,
Ian C M MacLennan,
Adam F Cunningham
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ABSTRACT: Clearance of disseminated Salmonella infection requires bacterial-specific Th1 cells and IFN-γ production, and Th1-promoting vaccines are likely to help control these infections. Consequently, vaccine design has focused on developing Th1-polarizing adjuvants or Ag that naturally induce Th1 responses. In this study, we show that, in mice, immunization with soluble, recombinant FliC protein flagellin (sFliC) induces Th2 responses as evidenced by Ag-specific GATA-3, IL-4 mRNA, and protein induction in CD62L(lo) CD4(+) T cells without associated IFN-γ production. Despite these Th2 features, sFliC immunization can enhance the development of protective Th1 immunity during subsequent Salmonella infection in an Ab-independent, T-cell-dependent manner. Salmonella infection in sFliC-immunized mice resulted in augmented Th1 responses, with greater bacterial clearance and increased numbers of IFN-γ-producing CD4(+) T cells, despite the early induction of Th2 features to sFliC. The augmented Th1 immunity after sFliC immunization was regulated by T-bet although T-bet is dispensable for primary responses to sFliC. These findings show that there can be flexibility in T-cell responses to some subunit vaccines. These vaccines may induce Th2-type immunity during primary immunization yet promote Th1-dependent responses during later infection. This suggests that designing Th1-inducing subunit vaccines may not always be necessary since this can occur naturally during subsequent infection.
European Journal of Immunology 03/2011; 41(6):1606-18. · 5.10 Impact Factor
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Calman A MacLennan,
James J Gilchrist,
Melita A Gordon,
Adam F Cunningham,
Mark Cobbold, Margaret Goodall,
Robert A Kingsley,
Joep J G van Oosterhout,
Chisomo L Msefula,
Wilson L Mandala, [......],
Esther N Gondwe,
Saeeda Bobat,
Constantino López-Macías,
Rainer Doffinger,
Ian R Henderson,
Eduard E Zijlstra,
Gordon Dougan,
Mark T Drayson,
Ian C M MacLennan,
Malcolm E Molyneux
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ABSTRACT: Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibodies protect against Salmonella bacteremia. We report that high-titer antibodies specific for Salmonella lipopolysaccharide (LPS) are associated with a lack of Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from the target Salmonella or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus, our study indicates that impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whereas serum killing of Salmonella is induced by antibodies against outer membrane proteins.
Science 04/2010; 328(5977):508-12. · 31.20 Impact Factor
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Helen J Harris,
Christopher Davis,
Jonathan G L Mullins,
Ke Hu, Margaret Goodall,
Michelle J Farquhar,
Christopher J Mee,
Kitty McCaffrey,
Stephen Young,
Heidi Drummer,
Peter Balfe,
Jane A McKeating
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ABSTRACT: Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
Journal of Biological Chemistry 04/2010; 285(27):21092-102. · 4.77 Impact Factor
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ABSTRACT: Bacteremia caused by nontyphoidal strains of Salmonella is endemic among African children. Case-fatality rates are high and antibiotic resistance increasing, but no vaccine is currently available. T cells are important for clearance of Salmonella infection within macrophages, but in Africa, invasive Salmonella disease usually manifests in the blood and affects children between 4 months and 2 y of age, when anti-Salmonella antibody is absent. We have previously found a role for complement-fixing bactericidal antibody in protecting these children. Here we show that opsonic activity of antibody and complement is required for oxidative burst and killing of Salmonella by blood cells in Africans. Induction of neutrophil oxidative burst correlated with anti-Salmonella IgG and IgM titers and C3 deposition on bacteria and was significantly lower in African children younger than 2 y compared with older children. Preopsonizing Salmonella with immune serum overcame this deficit, indicating a requirement for antibody and/or complement. Using different opsonization procedures, both antibody and complement were found to be necessary for optimal oxidative burst, phagocytosis and killing of nontyphoidal Salmonella by peripheral blood cells in Africans. Although most strains of African nontyphoidal Salmonella can be killed with antibody and complement alone, phagocytes in the presence of specific antibody and complement can kill strains resistant to killing by immune serum. These findings increase the likelihood that an antibody-inducing vaccine will protect against invasive nontyphoidal Salmonella disease in African children.
Proceedings of the National Academy of Sciences 02/2010; 107(7):3070-5. · 9.68 Impact Factor
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ABSTRACT: Anti-proteinase 3 antibodies are implicated in the pathogenesis of small vessel vasculitis. These are primarily immunoglobulin G (IgG), with different subclasses predominating at different stages of disease. However, little is known of their respective roles in pathogenesis. We have previously shown that patient IgG4 was able to induce superoxide release from human neutrophils. To circumvent difficulties in separating the subclasses and additional differences in polyclonal patient antibodies we have generated monoclonal mouse/human IgG1 and IgG4 anti-proteinase 3 antibodies. Using these antibodies we have compared effects of IgG1 and IgG4 on human neutrophils in terms of superoxide release, cytokine production, degranulation and adhesion. Additionally we have investigated the interaction of the subclasses with Fc receptors expressed by the neutrophil. Chimeric antibodies were generated using human constant regions of each subclass and a variable region taken from a monoclonal antibody directed against proteinase 3. Superoxide release from neutrophils was measured by the reduction of ferricytochrome C, degranulation by the conversion of a synthetic colour substrate, cytokine release by interleukin-8 enzyme-linked immunosorbent assay, and adhesion by a flow-based adhesion assay. Fc receptor binding was assessed using blocking antibodies. The IgG4 anti-proteinase 3 was able to induce a dose-dependent release of superoxide, degranulation and adhesion. The antibody was not able to stimulate the secretion of interleukin-8. Fc receptors were essential for neutrophil stimulation and the constitutive Fc receptors were necessary for different stimulatory pathways. The IgG4 anti-proteinase 3 antibodies are able to stimulate neutrophils to undergo a pro-inflammatory response and may play a role in the pathogenesis of small vessel vasculitis.
Immunology 11/2009; 128(2):236-44. · 3.32 Impact Factor
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ABSTRACT: Anti-proteinase 3 antibodies are implicated in the pathogenesis of small vessel vasculitis. These are primarily immunoglobulin G (IgG), with different subclasses predominating at different stages of disease. However, little is known of their respective roles in pathogenesis. We have previously shown that patient IgG4 was able to induce superoxide release from human neutrophils. To circumvent difficulties in separating the subclasses and additional differences in polyclonal patient antibodies we have generated monoclonal mouse/human IgG1 and IgG4 anti-proteinase 3 antibodies. Using these antibodies we have compared effects of IgG1 and IgG4 on human neutrophils in terms of superoxide release, cytokine production, degranulation and adhesion. Additionally we have investigated the interaction of the subclasses with Fc receptors expressed by the neutrophil. Chimeric antibodies were generated using human constant regions of each subclass and a variable region taken from a monoclonal antibody directed against proteinase 3. Superoxide release from neutrophils was measured by the reduction of ferricytochrome C, degranulation by the conversion of a synthetic colour substrate, cytokine release by interleukin-8 enzyme-linked immunosorbent assay, and adhesion by a flow-based adhesion assay. Fc receptor binding was assessed using blocking antibodies. The IgG4 anti-proteinase 3 was able to induce a dose-dependent release of superoxide, degranulation and adhesion. The antibody was not able to stimulate the secretion of interleukin-8. Fc receptors were essential for neutrophil stimulation and the constitutive Fc receptors were necessary for different stimulatory pathways. The IgG4 anti-proteinase 3 antibodies are able to stimulate neutrophils to undergo a pro-inflammatory response and may play a role in the pathogenesis of small vessel vasculitis.
Immunology 09/2009; 128(2):236 - 244. · 3.32 Impact Factor
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Charlotte Magdelaine-Beuzelin,
Séverine Vermeire, Margaret Goodall,
Filip Baert,
Maja Noman,
Gert Van Assche,
Marc Ohresser,
Danielle Degenne,
Jean-Michel Dugoujon,
Roy Jefferis,
Paul Rutgeerts,
Maire-Paule Lefranc,
Hervé Watier
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ABSTRACT: The chimeric anti-tumor necrosis factor-alpha antibody infliximab is known to induce antibodies-to-infliximab (ATI) in some treated patients. Immunogenicity in murine variable domains is expected; however, constant domains of its human heavy gamma1 chain may also be implicated as it expresses G1m1 and G1m17 allotypes. This allelic form may be immunogenic in patients that are homozygous for the G1m3 allotype commonly expressed in Caucasoid populations.
As G1m allotypic divergence may explain the presence of ATI or may influence their concentration, a genotyping method was developed and validated to determine antithetical (i.e. mutually exclusive) G1m3 and G1m17 allotypes (amino acid 120 of CH1 according to the international ImMunoGeneTics information system unique numbering) at the IGHG1 gene level (CH1 359g/a nucleotide polymorphism). Two hundred forty-five blood donors and 118 previously described patients suffering from Crohn's disease, treated with infliximab, and having developed ATI in 73 of them, were genotyped.
The IGHG1 CH1 359g/a polymorphism does not depart from the Hardy-Weinberg equilibrium in the control population, and allele frequencies were similar in controls and patients. No association was found between the patient G1m allotypes and the presence of ATI or their concentration. It remains possible that anti-Gm1 antibodies are not well detected by the enzyme-linked immunosorbent assays used for ATI detection and/or that the G1m allotypes are minor antigens on IgG1.
The IGHG1 polymorphism does not seem to play a major role in the induction of ATI. Further analyses will be required to determine whether it is also the case for humanized or fully human antibodies bearing the same G1m allotypes.
Pharmacogenetics and Genomics 04/2009; 19(5):383-7. · 3.48 Impact Factor
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ABSTRACT: The CTLA-4 pathway is recognized as a major immune inhibitory axis and is a key therapeutic target for augmenting antitumor immunity or curbing autoimmunity. CTLA-4-deficient mice provide the archetypal example of dysregulated immune homeostasis, developing lethal lymphoproliferation with multiorgan inflammation. In this study, we show that surprisingly these mice have an enlarged population of Foxp3(+) regulatory T cells (Treg). The increase in Treg is associated with normal thymic output but enhanced proliferation of Foxp3(+) cells in the periphery. We confirmed the effect of CTLA-4 deficiency on the Treg population using OVA-specific Treg which develop normally in the absence of CTLA-4, but show increased proliferation in response to peripheral self-Ag. Functional analysis revealed that Ag-specific Treg lacking CTLA-4 were unable to regulate disease in an adoptive transfer model of diabetes. Collectively, these data suggest that the proliferation of Treg in the periphery is tuned by CTLA-4 signals and that Treg expression of CTLA-4 is required for regulation of pancreas autoimmunity.
The Journal of Immunology 02/2009; 182(1):274-82. · 5.79 Impact Factor
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Calman A MacLennan,
Esther N Gondwe,
Chisomo L Msefula,
Robert A Kingsley,
Nicholas R Thomson,
Sarah A White, Margaret Goodall,
Derek J Pickard,
Stephen M Graham,
Gordon Dougan,
C Anthony Hart,
Malcolm E Molyneux,
Mark T Drayson
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ABSTRACT: Nontyphoidal strains of Salmonella (NTS) are a common cause of bacteremia among African children. Cell-mediated immune responses control intracellular infection, but they do not protect against extracellular growth of NTS in the blood. We investigated whether antibody protects against NTS bacteremia in Malawian children, because we found this condition mainly occurs before 2 years of age, with relative sparing of infants younger than 4 months old. Sera from all healthy Malawian children tested aged more than 16 months contained anti-Salmonella antibody and successfully killed NTS. Killing was mediated by complement membrane attack complex and not augmented in the presence of blood leukocytes. Sera from most healthy children less than 16 months old lacked NTS-specific antibody, and sera lacking antibody did not kill NTS despite normal complement function. Addition of Salmonella-specific antibody, but not mannose-binding lectin, enabled NTS killing. All NTS strains tested had long-chain lipopolysaccharide and the rck gene, features that resist direct complement-mediated killing. Disruption of lipopolysaccharide biosynthesis enabled killing of NTS by serum lacking Salmonella-specific antibody. We conclude that Salmonella-specific antibody that overcomes the complement resistance of NTS develops by 2 years of life in Malawian children. This finding and the age-incidence of NTS bacteremia suggest that antibody protects against NTS bacteremia and support the development of vaccines against NTS that induce protective antibody.
Journal of Clinical Investigation 05/2008; 118(4):1553-62. · 15.39 Impact Factor
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ABSTRACT: Antineutrophil cytoplasmic antibodies (ANCA) are associated with small-vessel vasculitis and have been implicated in its pathogenesis. The subclass distribution of ANCA IgG deviates from normal patterns, and it has been suggested that the IgG3 subclass may have pathogenic potential over the IgG1 subclass and may be more likely to be associated with active disease and renal involvement.
To deal with potential pathogenicity, chimeric antibodies were constructed of IgG1 and three subclasses with human IgG1 or three constant regions and a murine-derived variable region that binds an epitope within the ANCA antigen proteinase 3 (PR3) that is recognised by human autoantibodies.
The antibodies were characterised for binding to PR3, including affinity and avidity, before being used as tools to explore their ability to activate human neutrophils for superoxide release, cytokine release, degranulation and ability to induce neutrophil adhesion under flow.
Both subclass antibodies elicited similar neutrophil responses for superoxide release, degranulation and interleukin (IL) 8 production, although quantitative responses showed that the IgG1 subclass favoured degranulation and the IgG3 subclass favoured IL8 production. Both antibodies were able to convert neutrophils from selectin-dependent rolling adhesion to integrin-dependent stationary adhesion in a flow assay.
These findings indicate that humanised antibodies directed against a single epitope of PR3 can recapitulate the effects of polyclonal human ANCA, which recognises multiple PR3 epitopes. Further, PR3-ANCA of both IgG1 and IgG3 subclasses can activate neutrophils, although the more potent IL8 response by IgG3 PR3-ANCA may encourage further neutrophil recruitment and amplify injury.
Annals of the Rheumatic Diseases 06/2007; 66(5):676-82. · 8.73 Impact Factor
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ABSTRACT: The characterisation of monoclonal antibodies (MAbs) and their epitopes is important prior to their application as molecular probes. In this study, Western blotting using IgG1 Fc and pFc' fragments was employed to screen seven MAbs before pepscan analysis to determine their reactivity to potentially linear epitopes. MAbs PNF69C, PNF110A, X1A11 and MAbs WC2, G7C, JD312, 1A1 detected epitopes within the C(H)3 and C(H)2 domains, respectively. However, only four MAbs showed pepscan profiles that highlighted likely target residues. In particular, MAbs PNF69C and PNF110A that have previously been characterised with pan-IgG and anti-G3m(u) specificity, detected the peptide motif 338-KAKGQPR-344 which was located within the N-terminal region of the C(H)3 domain. Furthermore the majority of residues were present in all four IgG subclasses. Consequently the peptide identified was consistent with the pan-IgG nature of these antibodies. By using PCImdad, a molecular display programme, this sequence was visualised as surface accessible, located in the C(H)2/C(H)3 inter-domain region and proximal to the residue arginine(435). It is speculated that this residue may be important for phenotypic expression of G3m(u) and specificity of these reagents. Pepscan analysis of MAbs G7C and JD312 (both pan-IgG) highlighted the core peptide sequence 290-KPREE-294, which was present in the C(H)2 domain and was common to all four IgG subclasses. PCImdad also showed this region to be highly accessible and was consistent with previous bioinformatic and autoimmune analysis of IgG. Overall these MAbs may serve as useful anti-IgG or anti-G3m(u) reagents and probes of immunoglobulin structure.
Immunology Letters 08/2003; 88(1):77-83. · 2.53 Impact Factor
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ABSTRACT: Bullous pemphigoid (BP) is a sub-epidermal autoimmune blistering disease associated with autoantibodies to the dermal-epidermal junction (DEJ). Patients' autoantibodies induce dermal-epidermal separation when co-incubated with cryosections of human skin and leucocytes from healthy volunteers. IgG autoantibodies trigger complement and/or leucocyte activation resulting in specific pathology in several autoimmune conditions. In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage. The capacity of IgG4 autoantibodies to mediate tissue damage has not yet been demonstrated. In this study, we isolated IgG1 and IgG4 autoantibodies from bullous pemhigoid patients'serum and analysed their blister-inducing potential in our cryosection assay. As expected, complement-fixing IgG1 autoantibodies induced sub-epidermal splits in this experimental model. Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal-epidermal separation. The potential of IgG4 autoantibodies to induce Fc-dependent dermal-epidermal separation was significantly lower compared to IgG1. Our results demonstrate that IgG4 autoantibodies are able to activate leucocytes and point to a hitherto less recognized function of IgG4. Moreover, for the first time, we clearly demonstrate that BP IgG4 autoantibodies have the capacity to induce leucocyte-dependent tissue damage.
Journal of Cellular and Molecular Medicine 11(5):1117-28. · 4.13 Impact Factor
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ABSTRACT: Human IgG subclass proteins exhibit more than 95% primary amino acid sequence homology in their Fc regions, but each has a unique profile for recognition by the 3 human Fcγ receptors. The FcγRs are themselves highly homologous members of the immunoglobulin supergene family. Consistent with these data we have proposed that FcγRI, FcγRII and FcγRIII recognise overlapping non-identical interaction sites in the lower hinge region of the CH2 domain of the IgG molecule. Evidence in support was provided by protein engineering effecting single amino acid replacements in the proposed site. Alternatively, we have demonstrated that the primary amino acid sequence alone is not sufficient for IgG molecules to fold with the generation of FcγR interaction sites and that glycosylation of Asn 297 of the CH2 domain is essential. We have further defined a ‘core’ oligosaccharide structure that provides for the generation of FcγR interaction sites which suggests that the addition of outer-arm sugar residues does not affect this primary activity; although in vivo it could influence other essential biological activities.These findings have opened up a new approach to engineering antibody function — by protein engineering of amino acid residues that form contacts with the oligosaccharide moiety. In the present report we demonstrate that replacement of contact residues for galactose on the α(1–6) arm does not affect FcγRI and FcγRII recognition while replacement of Asp 265, a contact for a ‘core’ N-acetylglucosamine residue, results in a loss of FcγRI and FcγRII recognition.
Immunology Letters.
