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Matthieu Carrière,
Véronique Pène,
Adrien Breiman,
Filoména Conti,
Sandrine Chouzenoux,
Eliane Meurs, Muriel Andrieu,
Patrick Jaffray,
Lilia Grira,
Olivier Soubrane,
Philippe Sogni,
Yvon Calmus,
Stanislas Chaussade,
Arielle R Rosenberg,
Philippe Podevin
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ABSTRACT: The detection of negative-strand hepatitis C virus (HCV) RNA is a hallmark of replication. A highly sensitive and specific method is required to quantify the very low level of replication inherent to in vitro infection systems. Based on reverse transcription with a tagged primer in the 5' non-coding region of the HCV genome, followed by a nested PCR with a second round of real-time PCR, a novel method is described with improved sensitivity for negative-strand HCV RNA quantification. The lower detection level was 25 copies per reaction of negative-strand HCV RNA, even in the presence of 1 x 10(5) copies of positive-strand HCV RNA. This protocol was applied to the detection of negative HCV strand RNA in the liver of HCV-infected patients as well as in primary human hepatocytes infected in vitro. In both models, and particularly in each of three, independent in vitro infection experiments, this assay permitted the quantitation of HCV replication.
Journal of Medical Virology 03/2007; 79(2):155-60. · 2.82 Impact Factor
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ABSTRACT: We showed that an anti-HIV lipopeptide vaccine injected to HIV-uninfected volunteers was well tolerated and able to induce a specific CD4(+) and CD8(+) T cell responses. The same vaccine was injected in HIV-1 chronically infected patients controlled by HAART to evaluate its immunogenicity. In this trial, 24 patients were immunized three times with a mixture of six lipopeptides (Nef 66-97, Nef 117-147, Nef 182-205, Gag 183-214, Gag 253-284, and Env 303-335) at 0, 3, and 6 weeks. We studied the HIV-1-specific CD4(+) T cell proliferative responses. The IFN-gamma secretion by activated CD8(+) T cells was evaluated, using an ex vivo ELISpot assay and 60 CD8(+) T cell epitopes derived from the vaccine. Before immunization (W0), anti-HIV CD4(+) T cell responses to Gag, Nef, and Env large peptides were detected in 7/23 (30%) analyzable patients. After three injections, 17/23 (74%) patients had a proliferative response and 16 of them induced new specific CD4(+) T cell responses. At W0, CD8(+) T cell responses to HIV-1 epitopes were detected in 6/23 (26%) patients. After vaccination, 16/23 (70%) patients showed CD8(+) T cell responses and 13 of these patients induced new T cell responses to 25 different HIV-1 epitopes. These HIV-1 epitopes were detected in patients with various HLA class I molecules (HLA-A2, -A3/A11, -A24, -B7 superfamily, -B8), as found in the majority of the white population. Lipopeptides induce new anti-HIV T cell responses in vaccinated infected patients and could be used as a new immunotherapy strategy. The majority of these responders induced specific new CD4(+) and CD8(+) T cell responses.
AIDS Research and Human Retroviruses 08/2006; 22(7):684-94. · 2.25 Impact Factor
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Marina Guille, Muriel Andrieu,
Cécile Braudeau,
Catherine Ruiz,
Nathalie Daniel,
Annaïck Pallier,
Bénédicte Charmeteau,
Joelle Veziers,
Neal Oden,
Nelly Bonilla,
Sophie Brouard,
Jean-Gérard Guillet,
Jean-Paul Soulillou
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ABSTRACT: In this article, we studied the T cell receptor (TCR)beta chain transcript mobilization in peripheral blood lymphocytes harvested from HIV-1-infected patients before and after vaccination with a mixture of six lipopeptides and at the moment and serially after highly active antiretroviral therapy (HAART) interruption. This study was performed by using a combined qualitative and quantitative assessment of Vbeta mRNA alterations at the level of complementary determining region 3 length distribution (CDR3-LD) of the TCR. Whereas healthy individuals displayed both stable CDR3-LD profiles and Vbeta transcript accumulations over time, the four HIV-1-infected patients in a quiescent disease phase under HAART have a highly significantly biased CDR3-LD. In addition, they displayed a significant further increase of alterations of their beta CDR3-LD profile after vaccination and both a more altered CDR3-LD (p < 0.05) and an increased transcript accumulation of some Vbeta families after HAART interruption. These modifications mostly concerned the CD8(+ve) T cells. Such a global approach of TCR alterations may help to follow the immune response of these patients and allow targeting of more complex in vivo studies by identifying the T cells with a selected repertoire.
AIDS Research and Human Retroviruses 07/2006; 22(7):648-56. · 2.25 Impact Factor
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André Inwoley,
Patricia Recordon-Pinson,
Marion Dupuis,
Jessintha Gaston,
Mathieu Genête,
Albert Minga,
Franck Letourneur,
François Rouet,
Jeannine Choppin,
Hervé Fleury,
Jean-Gérard Guillet, Muriel Andrieu
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ABSTRACT: Most HIV vaccine trials in the world are conducted with clade B while most circulating viral strains in Africa are non-B subtypes. We determined whether CD8+ T cells from HIV-1 intersubtype CRF02_AG-infected Ivorian individuals were able to recognize clade B epitopes. CD8+ T cell responses of nine HIV-1 intersubtype CRF02_AG-infected Ivorian patients and nine HIV-1 subtype B-infected French patients were studied using pools of HIV-1 clade B peptides (110 well-defined HIV CD8+ T cell epitopes) in an ELISPOT IFN-gamma assay. There was no difference in the number of recognized peptide pools between Ivorian and French cohorts (mean of four pools in both cases). Ivorian individuals had generated CD8+ T cell responses cross-reactive against HIV-1 subtype B and some individual peptides had been identified. Furthermore, sequence analysis of nef HIV genes of the Ivorian patients and nef cloning in two patients revealed very few variations between HIV- 1 intersubtype CRF02_AG and subtype B in nef immunodominant regions included in HIV clade B lipopeptide vaccines, currently tested in France.
AIDS Research and Human Retroviruses 08/2005; 21(7):620-8. · 2.25 Impact Factor
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Nathalie Daniel,
Bénédicte Charmeteau,
Sophie Grabar,
Gilles Pialoux,
Dominique Salmon,
Nelly Bonilla,
Marion Dupuis,
Christelle Troadec,
Willy Rozenbaum,
Hanne Gahéry-Ségard,
Jean-Gérard Guillet, Muriel Andrieu
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ABSTRACT: Highly active antiretroviral therapy (HAART) is associated with a dramatic clinical benefit to HIV-infected patients through significant plasma viremia reduction and CD4(+) T cells increase. In previous reports, HIV-specific CD4(+) and/or CD8(+) T cell responses have been studied separately during HAART; therefore the relationship between these two virus-specific populations is currently not well understood. In this study, both HIV-specific CD4(+) and CD8(+) T cell responses were investigated using a large panel of well-defined T cell epitope peptides in 24 HIV-1-infected patients undergoing HAART, with undetectable viral load and CD4(+) T cell count >/= 350/mm(3). One-third of the patients had CD4(+) T cells able to proliferate when exposed to HIV-1 protein fragments but only two patients displayed polyclonal responses. In addition the majority (78%) of HAART-treated patients displayed no or monospecific CD8(+) T cell responses and the phenotypic analysis of these HIV-specific CD8(+) T cells demonstrated the absence of terminally differentiated effectors. In conclusion, the experimental approach used in this study shows that CD4(+) T cell responses may persist during HAART but are not associated with strong CD8(+) T cell responses
AIDS Research and Human Retroviruses 08/2004; 20(8):827-35. · 2.25 Impact Factor
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Helene Rouard,
Bernard Klonjkowski,
Jeanine Marquet,
Chantal Lahet,
Stephanie Mercier, Muriel Andrieu,
Patrick Maison,
Valerie Molinier-Frenkel,
Marc Eloit,
Jean-Pierre Farcet,
Pierre Langlade-Demoyen,
Marie-Helene Delfau-Larue
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ABSTRACT: Therapeutic vaccination aims at a strong stimulation of antigen-specific CD8(+) T-cells, so that they differentiate into effectors active in vivo against antigenic targets. Two adenovirus vectors (Ad) encoding two HLA-A*0201-restricted HIV epitope sequences (pol 476 and pol 589) were constructed. The Ad differ by the presence or absence of a ubiquitin monomer sequence (AdUb(+) and AdUb(-)). The effect of transgene product ubiquitination was analyzed on (1) in vivo, the immunization of Ad vaccinated HLA-A*0201 humanized HHD mice and (2) in vitro, the presentation of the transgene encoded peptides by transduced human dendritic cells (DC). In vivo, we found that immunization of humanized HHD mice with AdUb(+) elicited a transgene product-specific interferon (INF)-gamma CD8(+) T-cell response detectable by enzyme-linked immunospot (ELISPOT), whereas the AdUb(-) construction did not. Antigen-specific cytotoxic T lymphocytes (CTL) were also generated in HHD mice immunized with AdUb(+) and not with AdUb(-). In vitro, using human AdUb(+)-transduced DC, a sizeable expansion of pol 476 and pol 589 tetramer positive CD8(+) T cells as well as CD8(+) CTL were obtained in healthy donors. Compared to AdUb(-)-transduced DC, AdUb(+)-transduced DC triggered a higher number of pol 476-specific IFN-gamma-secreting CD8(+) T cells. In agreement, AdUb(+) transduced DC, used as target in a (51)Cr-release assay, were more efficiently lysed by peptide-specific CTL than AdUb(-)-transduced DC. In conclusion, the addition of an ubiquitin sequence to the adenoviral transgene, used as an antigen source, resulted in both in vivo enhanced CD8(+) T-cell immunogenicity in HHD mice and in vitro increased HLA class I-restricted presentation of encoded peptides by human DC.
Human Gene Therapy 10/2003; 14(14):1319-32. · 4.22 Impact Factor
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Ulrike Seifert,
Concepción Marañón,
Ayelet Shmueli,
Jean-François Desoutter,
Lisa Wesoloski,
Katharina Janek,
Peter Henklein,
Susanne Diescher, Muriel Andrieu,
Henri de la Salle,
Toni Weinschenk,
Hansjörg Schild,
Diego Laderach,
Anne Galy,
Gaby Haas,
Peter-M Kloetzel,
Yuval Reiss,
Anne Hosmalin
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ABSTRACT: Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73-82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73-82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73-82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.
Nature Immunology 05/2003; 4(4):375-9. · 26.01 Impact Factor
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ABSTRACT: An efficient vaccine against human immunodeficiency virus (HIV) must induce good cellular immune responses. To do this, it must be processed and presented by dendritic cells, which are required for primary T-lymphocyte stimulation. We have previously shown that a model lipopeptide containing a short epitopic peptide from HIV-1 was endocytosed and presented in association with major histocompatibility complex class I molecules by human dendritic cells to specific CD8(+) T lymphocytes, but the cross-presentation pathway needed to be precisely determined. We have studied a longer lipopeptide (Pol(461-484)) and another lipopeptide (Nef(66-97)) currently being used in vaccine trials. Like the shorter lipopeptide, the rhodamine-labeled Pol(461-484) lipopeptide was internalized by endocytosis, as assessed by confocal microscopy. The lipopeptides were processed by dendritic cells and presented to CD8(+) T cells specific for the HLA-A*0201-restricted Pol(476-484) and the HLA-A*0301-restricted Nef(73-82) epitope, respectively. Presentation of both lipopeptides was inhibited by brefeldin A. Presentation of the Pol lipopeptide was inhibited by epoxomycin, a proteasome-specific inhibitor, but not by monensin. This shows that it gained access to the cytosol to be digested by the proteasome. In contrast, presentation of the Nef lipopeptide was not inhibited by epoxomycin but was inhibited by monensin, a classical inhibitor of acid-dependent endosomal enzyme activity, indicating an endocytic processing pathway yielding to major histocompatibility complex class I-restricted presentation. Therefore, the two lipopeptides followed different cross-presentation pathways, both resulting in efficient presentation to CD8(+) T lymphocytes.
Journal of Virology 02/2003; 77(2):1564-70. · 5.40 Impact Factor
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ABSTRACT: Trypanosoma cruzi, the etiological agent of Chagas' disease, may persist for many years in its mammalian host. This suggests escape from the immune response and particularly a suboptimal CD8(+) T cell response, since these cells are involved in infection control. In this report, we show that T. cruzi inhibits the lipopolysaccharide (LPS)-induced up-regulation of MHC class I molecules at the surface of human dendritic cells (DC). To further investigate the functional consequences of this inhibition, a trypomastigote surface antigen-derived peptide (TSA-1(514-522) peptide) was selected for its stable binding to HLA-A*0201 molecules and used to generate a primary T. cruzi-specific human CD8(+) T cell line in vitro. We observed that DC infected with T. cruzi or treated with T. cruzi-conditioned medium (TCM) had a weaker capacity to present this peptide to the specific CD8(+) T cell line as shown in an IFN-gamma ELISPOT assay. Interestingly, T. cruzi or TCM also reduced the antigen presentation capacity of DC to CD8(+) T cell lines specific for the influenza virus M(58-66) or HIV RT(476-484) epitopes. This dysfunction appears to be linked essentially to reduced MHC class I molecule expression since the stimulation of the RT(476-484) peptide-specific CD8(+) T cell line was shown to depend mainly on the MHC class I-TCR interaction and not on the co-stimulatory signals which, however, were also inhibited by T. cruzi. This impairment of DC function may represent a novel mechanism reducing in vivo the host's ability to combat efficiently T. cruzi infection.
International Immunology 11/2002; 14(10):1135-44. · 3.41 Impact Factor
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ABSTRACT: CD8+ T lymphocytes, which are major immune effectors, require primary stimulation by dendritic cells (DC) presenting MHC class I molecule-bound epitopes. Sensitization to exogenous protein epitopes that are not synthesized in DC, such as cross-priming, is obtained through pathways leading to their association with MHC class I. To follow class I-restricted pathways in human DC, we have tracked a lipopeptide derived from the conserved HLA-A*0201-restricted HIV-1 reverse transcriptase 476-484 epitope, by N-terminal addition of an Nϵ-palmytoyl-lysine. Indeed, lipopeptides elicit cytotoxic responses from CD8+ T lymphocytes, whereas peptides without a lipid moiety do not. The lipopeptide and its parent peptide were labeled unequivocally by rhodamine to study their entry into immature monocyte-derived human DC by confocal microscopy. The lipid moiety induced endocytosis of the lipopeptide, assessed by rapid entry into vesicles, colocalization with Dextran-FITC and dependence on energy. Internalization occurred even when actin filaments were depolymerized by Cytochalasin B. This internalization induced functional stimulation of specific CD8+ T lymphocytes in IFN-γ ELISPOT assays. The peptide alone was not visualized inside the DC and was presented through direct surface association to HLA-A*0201. Therefore, lipopeptides are a unique opportunity to define precisely the pathways that lead exogenous proteins to associate with MHC class I molecules in DC. The results will also be useful to design lipopeptide vaccines.
European Journal of Immunology 10/2000; 30(11):3256 - 3265. · 5.10 Impact Factor
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ABSTRACT: The HIV-1 Nef protein down-modulates surface expression of MHC class I proteins. Primary infected T lymphocytes thus escape lysis by cytotoxic T lymphocytes (CTL). In contrast, during HIV-1 infection there are strong CTL responses to several HIV proteins, and there is mounting evidence that CTL are critical for controlling the virus. The present study was carried out to assess Nef protein-cell interaction as it occurs in naturally infected antigen-presenting cells. To evaluate the presentation of peptides derived from viral antigen to CTL, we transfected nef genes obtained from peripheral blood mononuclear cells of HIV-1-seropositive subjects into dendritic cells isolated from monocytes of healthy donors. We demonstrate that expression and subsequent processing of Nef by transfected dendritic cells did not alter the presentation of an immunodominant epitope of Nef to CTL of HIV + subjects. However, mutations in nef gene sequences from primary isolates may abolish this presentation by a mechanism that probably interferes with protein processing.
European Journal of Immunology 12/1998; 29(1):196 - 202. · 5.10 Impact Factor
