Martin Larsen

Publications of Martin Larsen

• Antagonistic T-cell subsets in skin diseases.

  Authors: Laurent Arnaud, Martin Larsen, Guy Gorochov

  The New England journal of medicine. 10/2011; 365(15):1450-1; author reply 1451-2.

• Exhausted cytotoxic control of Epstein-Barr virus in human lupus.

  Authors: Martin Larsen, Delphine Sauce, Claire Deback, Laurent Arnaud, Alexis Mathian, Makoto Miyara, David Boutolleau, Christophe Parizot, Karim Dorgham, Laura Papagno, Victor Appay, Zahir Amoura, Guy Gorochov

  PLoS pathogens. 10/2011; 7(10):e1002328.

  Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virusSystemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8+ T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8+ T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1β) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8+ T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8+ T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8+ T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients.

• Multiparameter grouping delineates heterogeneous populations of human IL-17 and/or IL-22 T-cell producers that share antigen specificities with other T-cell subsets.

  Authors: Martin Larsen, Laurent Arnaud, Miguel Hié, Christophe Parizot, Karim Dorgham, Mohamed Shoukry, Mathilde Kemula, Stéphane Barete, David Derai, Delphine Sauce, Zahir Amoura, Jérôme Pène, Hans Yssel, Guy Gorochov

  European journal of immunology. 06/2011; 41(9):2596-605.

  The ontogenic relationship between pro-inflammatory populations of interleukin-17 (IL-17A)- and/or IL-22-producing T cells and other T-cell subsets is currently unclear in humans. To appreciate TThe ontogenic relationship between pro-inflammatory populations of interleukin-17 (IL-17A)- and/or IL-22-producing T cells and other T-cell subsets is currently unclear in humans. To appreciate T helper cell-lineage commitment, we combined cytokine production profiles of in vitro expanded T-cell clones with T-cell receptor (TCR) clonotypic signatures. Moreover, ex vivo cytokine production profiles at the single-cell level were analyzed using an original approach based on the hierarchical cluster analysis of multiparametric flow cytometry data. These combined approaches enabled the delineation of distinct functional T-cell subsets, including Th1, Th2, Tr1, Th17 cells and a highly polyfunctional IL-22-producing T-cell population. Cluster analysis highlighted that the IL-22-producing T-cell population should be considered independently from the Th17 and Th1 subsets, although it was more closely related to the former. In parallel, we observed extensive TCRαβ sharing across all five subsets defined. The strategy described here allows the objective definition of cellular subsets and an unbiased insight into their similarities. Together, our results underscore the ontogenic plasticity of CD4(+) T-cell progenitors, which can adopt a differentiation profile irrespective of antigen specificity.

• HIV disease progression despite suppression of viral replication is associated with exhaustion of lymphopoiesis.

  Authors: Delphine Sauce, Martin Larsen, Solène Fastenackels, Michèle Pauchard, Hocine Ait-Mohand, Luminita Schneider, Amélie Guihot, Faroudy Boufassa, John Zaunders, Malika Iguertsira [......] Anthony D Kelleher, Anne Simon, Laurence Meyer, Dominique Costagliola, Steven G Deeks, Olivier Lambotte, Brigitte Autran, Peter W Hunt, Christine Katlama, Victor Appay

  Blood. 03/2011; 117(19):5142-51.

  The mechanisms of CD4(+) T-cell count decline, the hallmark of HIV disease progression, and its relationship to elevated levels of immune activation are not fully understood. Massive depletion ofThe mechanisms of CD4(+) T-cell count decline, the hallmark of HIV disease progression, and its relationship to elevated levels of immune activation are not fully understood. Massive depletion of CD4(+) T cells occurs during the course of HIV-1 infection, so that maintenance of adequate CD4(+) T-cell levels probably depends primarily on the capacity to renew depleted lymphocytes, that is, the lymphopoiesis. We performed here a comprehensive study of quantitative and qualitative attributes of CD34(+) hematopoietic progenitor cells directly from the blood of a large set of HIV-infected persons compared with uninfected donors, in particular the elderly. Our analyses underline a marked impairment of primary immune resources with the failure to maintain adequate lymphocyte counts. Systemic immune activation emerges as a major correlate of altered lymphopoiesis, which can be partially reversed with prolonged antiretroviral therapy. Importantly, HIV disease progression despite elite control of HIV replication or virologic success on antiretroviral treatment is associated with persistent damage to the lymphopoietic system or exhaustion of lymphopoiesis. These findings highlight the importance of primary hematopoietic resources in HIV pathogenesis and the response to antiretroviral treatments.

• Systemic perturbation of cytokine and chemokine networks in Erdheim-Chester disease: a single-center series of 37 patients.

  Authors: Laurent Arnaud, Guy Gorochov, Frédéric Charlotte, Virginie Lvovschi, Christophe Parizot, Martin Larsen, Pascale Ghillani-Dalbin, Baptiste Hervier, Jean-Emmanuel Kahn, Claire Deback, Lucile Musset, Zahir Amoura, Julien Haroche

  Blood. 01/2011; 117(10):2783-90.

  Immunopathogenesis of Erdheim-Chester disease (ECD), a rare non-Langerhans cell histiocytosis, is poorly known. In previous studies, various cytokines were detected in ECD lesions, presumablyImmunopathogenesis of Erdheim-Chester disease (ECD), a rare non-Langerhans cell histiocytosis, is poorly known. In previous studies, various cytokines were detected in ECD lesions, presumably orchestrating lesional histiocyte recruitment. Because ECD lesions are frequently associated with systemic symptoms, we postulated that underlying global immune perturbations might also be revealed. We quantitatively analyzed 23 cytokines in serum samples obtained from a large single-center cohort of 37 patients with ECD, and studied the impact of treatment on cytokine production. IL-6, IL-12, interferon-α (IFN-α), and monocyte chemotactic protein-1 (MCP-1) levels were significantly higher in untreated patients than in controls, whereas interferon-γ (IFN-γ) inducible protein 10, IL-12, MCP-1, and IL-1 receptor antagonist were found significantly increased in IFN-α-treated patients. A biomathematical approach was used to rationalize multiparameter data, to generate new hypotheses, and identify global control pathways. Interestingly, cytokine profiles proved to be particularly stable at the individual level, and an "ECD signature" further distinguished patients from controls, based on their production of IFN-α, IL-12, MCP-1, IL-4, and IL-7. Altogether, our data underline the systemic immune Th-1-oriented perturbation associated with this condition and provide clues for the choice of more focused therapeutic agents in this rare disease with noncodified therapeutic management.

• Cytokine profiles in sepsis have limited relevance for stratifying patients in the emergency department: a prospective observational study.

  Authors: Virginie Lvovschi, Laurent Arnaud, Christophe Parizot, Yonathan Freund, Gaëlle Juillien, Pascale Ghillani-Dalbin, Mohammed Bouberima, Martin Larsen, Bruno Riou, Guy Gorochov, Pierre Hausfater

  PloS one. 01/2011; 6(12):e28870.

  Morbidity, mortality and social cost of sepsis are high. Previous studies have suggested that individual cytokines levels could be used as sepsis markers. Therefore, we assessed whether the multiplexMorbidity, mortality and social cost of sepsis are high. Previous studies have suggested that individual cytokines levels could be used as sepsis markers. Therefore, we assessed whether the multiplex technology could identify useful cytokine profiles in Emergency Department (ED) patients. ED patients were included in a single tertiary-care center prospective study. Eligible patients were >18 years and met at least one of the following criteria: fever, suspected systemic infection, ≥ 2 systemic inflammatory response syndrome (SIRS) criteria, hypotension or shock. Multiplex cytokine measurements were performed on serum samples collected at inclusion. Associations between cytokine levels and sepsis were assessed using univariate and multivariate logistic regressions, principal component analysis (PCA) and agglomerative hierarchical clustering (AHC). Among the 126 patients (71 men, 55 women; median age: 54 years [19-96 years]) included, 102 had SIRS (81%), 55 (44%) had severe sepsis and 10 (8%) had septic shock. Univariate analysis revealed weak associations between cytokine levels and sepsis. Multivariate analysis revealed independent association between sIL-2R (p = 0.01) and severe sepsis, as well as between sIL-2R (p = 0.04), IL-1β (p = 0.046), IL-8 (p = 0.02) and septic shock. However, neither PCA nor AHC distinguished profiles characteristic of sepsis. Previous non-multiparametric studies might have reached inappropriate conclusions. Indeed, well-defined clinical conditions do not translate into particular cytokine profiles. Additional and larger trials are now required to validate the limited interest of expensive multiplex cytokine profiling for staging septic patients.

• Distinct differentiation profiles of HIV-Gag and Nef-specific central memory CD8+ T cells associated with HLA-B57/5801 and virus control.

  Authors: Jing Xie, Wei Lu, Assia Samri, Dominique Costagliola, Aurélie Schnuriger, Bosco C M da Silva, Catherine Blanc, Martin Larsen, Ioannis Theodorou, Christine Rouzioux, Brigitte Autran

  AIDS (London, England). 09/2010; 24(15):2323-9.

  A superior capacity of controlling HIV has been attributed to CD8(+) T cells directed against HIV-Gag compared to Nef, particularly in the context of some protective human leukocyte antigen (HLA)A superior capacity of controlling HIV has been attributed to CD8(+) T cells directed against HIV-Gag compared to Nef, particularly in the context of some protective human leukocyte antigen (HLA) alleles. To further elucidate this protective effect, we compared the multifunctional and differentiation characteristics of CD8(+) T cells specific for HIV-Gag and Nef in HLA-B57/5801-positive and negative nonprogressors. A head-to-head comparison of CD8(+) T cells specific for HIV-Gag and Nef frequencies, cytokine production and differentiation was conducted, in 11 HLA-B57/5801 and 11 HLA-B57/5801 HIV-infected individuals selected from a cohort of 53 nonprogressors by using IFN-gamma-ELISpot assay and flow cytometry analysis of intracellular cytokine production and differentiation profile. Correlations with HIV parameters were studied. Frequencies of Gag-specific but not of Nef-specific CD8(+) T cells correlated with peripheral blood mononuclear cell (PBMC)-associated HIV-DNA. The HIV-Gag and Nef-specific CD8(+) T cells did not differ for IL-2 production in either HLA-B57/5801 or HLA-B57/5801 individuals. The IFN-gamma-producing Gag-specific CD8(+) T cells in HLA-B57/5801 individuals significantly differed from their Nef-specific counterparts by displaying higher proportions of central memory CD45RACCR7 cells positive for CD27(+). This differentiation pattern was not observed in HLA-B57/5801 individuals. Only these HLA-B57/5801-positive Gag-specific CD27(+) central memory CD8(+) T cells, but not their Nef-specific counterparts, negatively correlated with cell-associated HIV-DNA. HLA-B57/5801 drives a preferential CD27(+) differentiation of central memory CD8(+) T cells directed against HIV-Gag but not Nef that may contribute to the ability of Gag-specific CD8(+) T cells to better control HIV in HLA-B57/5801 nonprogressors.

• Evidence of premature immune aging in patients thymectomized during early childhood.

  Authors: Delphine Sauce, Martin Larsen, Solène Fastenackels, Anne Duperrier, Michael Keller, Beatrix Grubeck-Loebenstein, Christophe Ferrand, Patrice Debré, Daniel Sidi, Victor Appay

  The Journal of clinical investigation. 09/2009;

  While the thymus is known to be essential for the initial production of T cells during early life, its contribution to immune development remains a matter of debate. In fact, during cardiac surgeryWhile the thymus is known to be essential for the initial production of T cells during early life, its contribution to immune development remains a matter of debate. In fact, during cardiac surgery in newborns, the thymus is completely resected to enable better access to the heart to correct congenital heart defects, suggesting that it may be dispensable during childhood and adulthood. Here, we show that young adults thymectomized during early childhood exhibit an altered T cell compartment. Specifically, absolute CD4+ and CD8+ T cell counts were decreased, and these T cell populations showed substantial loss of naive cells and accumulation of oligoclonal memory cells. A subgroup of these young patients (22 years old) exhibited a particularly altered T cell profile that is usually seen in elderly individuals (more than 75 years old). This condition was directly related to CMV infection and the induction of strong CMV-specific T cell responses, which may exhaust the naive T cell pool in the absence of adequate T cell renewal from the thymus. Together, these marked immunological alterations are reminiscent of the immune risk phenotype, which is defined by a cluster of immune markers predictive of increased mortality in the elderly. Overall, our data highlight the importance of the thymus in maintaining the integrity of T cell immunity during adult life.

• Upregulation of IL-7 Receptor {alpha} and Programmed Death 1 marks an epitope-specific CD8+ T cell response that disappears following primary Epstein-Barr virus infection.

  Authors: Delphine Sauce, Martin Larsen, Rachel J. M. Abbott, Andrew D Hislop, Alison M Leese, Naeem Khan, Laura Papagno, Gordon J Freeman, Alan B Rickinson

  Journal of virology. 08/2009;

  In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8+ T cell response. However, in HLA-A*0201-positiveIn immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8+ T cell response. However, in HLA-A*0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, we noticed that the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVL, GLC and TLD, but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10-27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T cell fate, interleukin 7 receptor alpha (IL-7Ralpha, CD127) and programmed death 1 (PD1). Thus, following the antigen-driven down-regulation of IL-7Ralpha seen on all populations in acute IM, in every case the TLD-specific population recovered expression unusually quickly post-IM. Secondly, in four of six patients studied, TLD-specific cells showed very strong PD-1 up-regulation in the last bleed before the cells' disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen re-stimulation (rather than of IL-7-dependent signals), and (ii) is immediately preceded, and perhaps mediated, by PD-1 up-regulation to unprecedented levels.

• Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV suppressive activity.

  Authors: Jorge R Almeida, Delphine Sauce, David A Price, Laura Papagno, So Youn Shin, Arnaud Moris, Martin Larsen, Gianfranco Pancino, Daniel C Douek, Brigitte Autran, Asier Saez-Cirion, Victor Appay

  Blood. 05/2009;

  CD8(+) T-cells are major players in the immune response against HIV. However, recent failures in the development of T-cell-based vaccines against HIV-1 have emphasized the need to reassess our basicCD8(+) T-cells are major players in the immune response against HIV. However, recent failures in the development of T-cell-based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T-cell-mediated efficacy. CD8(+) T-cells from HIV-1 infected patients with slow disease progression exhibit potent polyfunctionality and HIV suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8(+) T-cell clonotypes isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV specific CD8+ T-cells from infected donors. We report that attributes of CD8(+) T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8(+) T-cells display polyfunctional profiles and potent HIV suppressive activity. These data provides news insights into the mechanisms underlying CD8+ T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T-cells with high levels of antigen sensitivity in order to elicit potent antiviral efficacy.

• Functionally fused antibodies-A novel adjuvant fusion system.

  Authors: Martin Larsen, Kim Bak Jensen, Peter Astrup Christensen, Eduardo Suarez, Dominique Paris, Laura Sanz, Peter Ravn, Delphine Sauce, Philippe Saas, Steffen Goletz, Luis Alvarez-Vallina, Peter Kristensen

  Journal of immunological methods. 11/2008;

  Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. TheseAntibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radioactivity- or effector-domain delivery. There is now a growing interest in using anti-idiotypic antibodies or other antigen mimics to induce potent immune responses against antigen structures in question. We have earlier reported on the functional rescue of antibodies that are active when fused to the phage, but inactive as soluble protein [Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F. and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566-73.]. The rescue was accomplished by maintaining the fusion between the antibody fragment and portions of the filamentous bacteriophage coat protein 3, as present in the original antibody-displaying phage. In the present study, we have applied this system in an attempt to improve immunogenicity of anti-idiotypic antibodies isolated by phage display. Here we demonstrate that by preserving linkage between phage antibody and the N-terminal domain of phage coat protein 3, we induce multimerization of the antibody fragments, and improve their immunogenicity. This immunization approach allows induction of anti-idiotypic antibodies in mice, and facilitates the use of antibodies that are non-functional as non-fused soluble protein.

• PD-1 expression on human CD8 T cells depends on both state of differentiation and activation status.

  Authors: Delphine Sauce, Jorge R Almeida, Martin Larsen, Laurine Haro, Brigitte Autran, Gordon J Freeman, Victor Appay

  AIDS (London, England). 11/2007; 21(15):2005-13.

  OBJECTIVE AND DESIGN: PD-1 expression on HIV-specific CD8 T cells was recently reported to reflect functional exhaustion, resulting in uncontrolled HIV-1 replication. Assessing PD-1 expression on TOBJECTIVE AND DESIGN: PD-1 expression on HIV-specific CD8 T cells was recently reported to reflect functional exhaustion, resulting in uncontrolled HIV-1 replication. Assessing PD-1 expression on T cells may be highly relevant in T-cell immunology and vaccine monitoring. However, this requires us to gain further insights into the significance of PD-1 expression on CD8 T cells in humans. METHODS: We performed a detailed analysis of PD-1 expression pattern on various CD8 T cell subsets from healthy or HIV infected donors. RESULTS: PD-1 expression has two facets in vivo. On the one hand, it is linked to T-cell differentiation: PD-1 is up-regulated on early/intermediate differentiated subsets, which include HIV and Epstein-Barr virus-specific CD8 T-cell populations, but is down-regulated during late stages of differentiation. On the other hand, it is linked to T-cell activation: on PD-1 positive cells, PD-1 over-expression occurs along with the up-regulation of activation markers such as CD38 or HLA-DR. CONCLUSIONS: PD-1 expression on CD8 T cells, including those specific for HIV, can be related both to their differentiation stage and their activation status. It is important to consider these findings when assessing the expression of PD-1 on T cells.

• A role for intercellular antigen transfer in the recognition of EBV-transformed B cell lines by EBV nuclear antigen-specific CD4+ T cells.

  Authors: Graham S Taylor, Heather M Long, Tracey A Haigh, Martin Larsen, Jill Brooks, Alan B Rickinson

  Journal of immunology (Baltimore, Md. : 1950). 10/2006; 177(6):3746-56.

  The CD4+ T cell response to EBV may have an important role in controlling virus-driven B lymphoproliferation because CD4+ T cell clones to a subset of EBV nuclear Ag (EBNA) epitopes can directlyThe CD4+ T cell response to EBV may have an important role in controlling virus-driven B lymphoproliferation because CD4+ T cell clones to a subset of EBV nuclear Ag (EBNA) epitopes can directly recognize virus-transformed lymphoblastoid cell lines (LCLs) in vitro and inhibit their growth. In this study, we used a panel of EBNA1, 2, 3A, and 3C-specific CD4+ T cell clones to study the route whereby endogenously expressed EBNAs access the HLA class II-presentation pathway. Two sets of results spoke against a direct route of intracellular access. First, none of the clones recognized cognate Ag overexpressed in cells from vaccinia vectors but did recognize Ag fused to an endo/lysosomal targeting sequence. Second, focusing on clones with the strongest LCL recognition that were specific for EBNA2- and EBNA3C-derived epitopes LCL recognition was unaffected by inhibiting autophagy, a postulated route for intracellular Ag delivery into the HLA class II pathway in LCL cells. Subsequently, using these same epitope-specific clones, we found that Ag-negative cells with the appropriate HLA-restricting allele could be efficiently sensitized to CD4+ T cell recognition by cocultivation with Ag-positive donor lines or by exposure to donor line-conditioned culture medium. Sensitization was mediated by a high m.w. antigenic species and required active Ag processing by recipient cells. We infer that intercellular Ag transfer plays a major role in the presentation of EBNA-derived CD4 epitopes by latently infected target cells.

• EBV-associated mononucleosis leads to long-term global deficit in T-cell responsiveness to IL-15.

  Authors: Delphine Sauce, Martin Larsen, S John Curnow, Alison M Leese, Paul A H Moss, Andrew D Hislop, Michael Salmon, Alan B Rickinson

  Blood. 08/2006; 108(1):11-8.

  In mice, interleukin-7 (IL-7) and IL-15 are involved in T-cell homeostasis and the maintenance of immunologic memory. Here, we follow virus-induced responses in infectious mononucleosis (IM) patientsIn mice, interleukin-7 (IL-7) and IL-15 are involved in T-cell homeostasis and the maintenance of immunologic memory. Here, we follow virus-induced responses in infectious mononucleosis (IM) patients from primary Epstein-Barr virus (EBV) infection into long-term virus carriage, monitoring IL-7 and IL-15 receptor (IL-R) expression by antibody staining and cytokine responsiveness by STAT5 phosphorylation and in vitro proliferation. Expression of IL-7Ralpha was lost from all CD8+ T cells, including EBV epitope-specific populations, during acute IM. Thereafter, expression recovered quickly on total CD8+ cells but slowly and incompletely on EBV-specific memory cells. Expression of IL-15Ralpha was also lost in acute IM and remained undetectable thereafter not just on EBV-specific CD8+ populations but on the whole peripheral T- and natural killer (NK)-cell pool. This deficit, correlating with defective IL-15 responsiveness in vitro, was consistently observed in patients up to 14 years after IM but not in patients after cytomegalovirus (CMV)-associated mononucleosis, or in healthy EBV carriers with no history of IM, or in EBV-naive individuals. By permanently scarring the immune system, symptomatic primary EBV infection provides a unique cohort of patients through which to study the effects of impaired IL-15 signaling on human lymphocyte functions in vitro and in vivo.

• Enhancement of DNA vaccine potency through linkage of antigen to filamentous bacteriophage coat protein III domain I.

  Authors: Angel M Cuesta, Eduardo Suárez, Martin Larsen, Kim Bak Jensen, Laura Sanz, Marta Compte, Peter Kristensen, Luis Alvarez-Vallina

  Immunology. 05/2006; 117(4):502-6.

  Although DNA-based cancer vaccines have been successfully tested in mouse models, a major drawback of cancer vaccination still remains, namely that tumour antigens are weak and fail to generate aAlthough DNA-based cancer vaccines have been successfully tested in mouse models, a major drawback of cancer vaccination still remains, namely that tumour antigens are weak and fail to generate a vigorous immune response in tumour-bearing patients. Genetic technology offers strategies for promoting immune pathways by adding immune-activating genes to the tumour antigen sequence. In this work, we converted a model non-immunogenic antigen into a vaccine by fusing it to domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion generated antigen-specific T helper 1-type cellular immune responses. These results demonstrate that the incorporation of protein III into a DNA vaccine formulation can modulate the gene-mediated immune response and may thus provide a strategy for improving its therapeutic effect.

• Multivalent scFv display of phagemid repertoires for the selection of carbohydrate-specific antibodies and its application to the Thomsen-Friedenreich antigen.

  Authors: Peter Ravn, Antje Danielczyk, Kim Bak Jensen, Peter Kristensen, Peter Astrup Christensen, Martin Larsen, Uwe Karsten, Steffen Goletz

  Journal of molecular biology. 11/2004; 343(4):985-96.

  The Thomsen-Friedenreich disaccharide (TF) is a promising target antigen for tumor immunotherapy, since it is almost exclusively expressed in carcinoma tissues. The TF-specific antibodies generatedThe Thomsen-Friedenreich disaccharide (TF) is a promising target antigen for tumor immunotherapy, since it is almost exclusively expressed in carcinoma tissues. The TF-specific antibodies generated so far are IgMs of mouse origin with limited therapeutic potential. Phage-displayed scFv repertoires are an established source for recombinant antibodies; however, we were unable to identify scFvs binding to TF when applying libraries in the standard monovalent display format of phagemid systems. Here, we report on the successful selection of TF-specific antibody fragments using a multivalent scFv phagemid library format based on shortened linkers (one amino acid residue). The libraries were constructed from mice immunized with asialoglycophorin and selected using TF displayed on two different carrier molecules in combination with the proteolytically cleavable helper phage KM13. All isolated clones encoded the same framework genes and the same complementarity-determining regions. After affinity maturation only scFv with the founder sequence were selected from secondary repertoires. This indicates a very narrow sequence window for TF-specific antibodies. Investigating other linker-length formats revealed a clear inverse correlation between linker length and binding activity both as soluble proteins and displayed on phages. The highest affinity was obtained with the tetrameric format. The selected scFv was specific for TF on various carrier molecules and tumor cells and performed well in ELISA and immunohistochemistry. We postulate that scFv phagemid library formats with short linkers (i.e. multimeric scFvs) may, in general, be advantageous in selections for the generation of scFvs against carbohydrate epitopes or other epitopes associated with low intrinsic affinity per binding site), and expect that they will be superior in applications for diagnosis or therapy.

• Functional improvement of antibody fragments using a novel phage coat protein III fusion system.

  Authors: Kim Bak Jensen, Martin Larsen, Jesper Søndergaard Pedersen, Peter Astrup Christensen, Luis Alvarez-Vallina, Steffen Goletz, Brian F C Clark, Peter Kristensen

  Biochemical and biophysical research communications. 12/2002; 298(4):566-73.

  Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have beenFunctional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.

• Functional improvement of antibody fragments using a novel phage coat protein III fusion system

  Authors: Kim Bak Jensen, Martin Larsen, Jesper Søndergaard Pedersen, Peter Astrup Christensen, Luis Álvarez-Vallina, Steffen Goletz, Brian F.C Clark, Peter Kristensen

  Biochemical and Biophysical Research Communications.

  Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have beenFunctional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.