http://repub.eur.nl/res/aut/7179/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/14281/ 2008-11-01T00:00:00Z Here we describe a flowcytometric assay that measures the defining function of virusspecific cytotoxic T lymphocytes (CTL), i.e., killing viral protein expressing cells. The fluorescent antigen-transfected target cell (FATT)-CTL assay requires no viruses, recombinant viral vectors, or radioactive isotopes to generate CTL target cells that present naturally processed epitopes. It facilitates developing standardized applications in clinical trial settings. Plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used directly to nucleofect immortalized B cells or peripheral blood mononuclear cells (PBMCs). Elimination of antigen-GFP expressing cells by cloned CTL, in vitro sensitized PBMC, or ex vivo PBMC was quantified following a 4-18-h coculture period by flowcytometry. This technology successfully detected cellmediated cytotoxicity in studies involving human PBMC and various viral antigens, including structural proteins of influenza A virus, and structural and nonstructural HIV proteins. Standardized protocols are currently being developed in the framework of a clinical immunotherapy trial in HIV-infected individuals. The FATT-CTL assay principles facilitate standardized flowcytometric detection of antigenic protein-specific cell-mediated cytotoxicity in many different basic research and clinical trial settings. By measuring their defining function, the FATT-CTL assay contributes to a more complete assessment of antigen-specific CTL responses to infection and vaccination. http://repub.eur.nl/res/pub/35795/ 2007-06-01T00:00:00Z Influenza virus-specific CD4+T-helper cells were cloned that recognized a virus strain isolated in 1981, but that failed to recognize more recent strains. The HLA-DR*1601-restricted epitope recognized was located in the hemagglutinin (HA99-113) and the naturally occurring A → V substitution at position 106 was responsible for abrogating the recognition by HA99-113-specific CD4+T-cells. This amino acid substitution was found in influenza A/H3N2 viruses that circulated between 1999 and 2005 and did not affect recognition by virus-specific antibodies. It was speculated that influenza viruses could evade recognition by virus-specific CD4+T-cells, at least temporarily. http://repub.eur.nl/res/pub/35453/ 2007-05-01T00:00:00Z In the present study, we examined the effect of the loss of the human leucocyte antigen (HLA)-B*3501-restricted nucleoprotein (NP)418-426epitope on interferon (IFN)-γ-production and lytic activity of the human cytotoxic T lymphocyte (CTL) response in vitro. Extensive amino acid variation at T cell receptor contact residues of the NP418-426epitope has led to repeated evasion from specific CTL. We generated recombinant influenza viruses with variants of the NP418-426epitope, which were used to stimulate peripheral blood mononuclear cells obtained from six HLA-B*3501-positive study subjects in order to expand virus-specific CTL. Loss of the NP418-426epitope resulted in a significant reduction of IFN-γ-expressing CD8+T cells, similar to that observed previously after the loss of the HLA-B*2705-restricted NP383-391epitope. In addition, the effect of the loss of the NP418-426epitope on the lytic activity of the virus-specific CTL response was assessed. Also this functional property of the virus-specific CTL response was affected significantly by the loss of this and the NP383-391epitope, as determined using the newly developed fluorescent antigen-transfected target cell (FATT)-CTL assay. These findings indicate that the loss of single immunodominant epitopes affects the functionality of the virus-specific CTL response significantly. http://repub.eur.nl/res/pub/8841/ 2007-02-28T00:00:00Z Abstract : Cellular immunity plays an important role in the control of viral infections, including those caused by influenza viruses. However, viruses can exploit a variety of strategies to evade cellular immunity, like the accumulation of amino acid substitutions in CTL epitopes. It was unclear to what extent these amino acid substitutions affect the influenza virus-specific CTL response. In this thesis, this issue was addressed by assessing the effect of the loss of immunodominant epitopes on the human influenza A virus-specific CTL response in vitro. To this end, recombinant influenza viruses with and without the HLA-B*2705-restricted epitope NP383-391 or the HLA-B*3501-restricted epitope NP418-426 were generated, which were used to induce IFN-gamma production or lytic activity in clonal and polyclonal virus-specific CD8+ T-cell populations (chapter 2 and 7). During this study, it was found that recombinant influenza viruses with a single amino acid substitution at! position 384 of the NP (R384G) could not be rescued. We hypothesized that one or more co-mutations were required to compensate for the detrimental effect of the R384G mutation, which is described in chapters 3 and 4. In line with these results, we hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTL. The inability to overcome these functional constraints may explain the highly conserved nature of most identified influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes (chapter 5). Since examples of evading mutations in influenza A virus CTL epitopes are limited, we assessed the extent of variation in CTL epitopes using virus-specific CD8+ T-cell clones (chapter 6). In addition to CD8+ T-cell clones, we also observed CD4! + T-cell clones that recognized a variable epitope. To date, no evadin g activities from CD4+ T-cells have been described in influenza virus infection. The variable CD4+ T-cell epitope was located and the amino acid substitution responsible for abrogation of CD4+ T-cell recognition was identified (chapter 8). The studies described in this thesis are discussed in the light of evasion of influenza A virus from human T-cell immunity. http://repub.eur.nl/res/pub/35591/ 2007-02-01T00:00:00Z The influenza A virus nucleoprotein (NP) and matrix protein are major targets for human virus-specific cytotoxic T-lymphocyte (CTL) responses. Most of the CTL epitopes that have been identified so far are conserved. However, sequence variation in CTL epitopes of the NP has recently been demonstrated to be associated with escape from virus-specific CTLs. To assess the extent of variation in CTL epitopes during influenza A virus evolution, 304 CTL clones derived from six study subjects were obtained with specificity for an influenza A/H3N2 virus isolated in 1981. Subsequently, the frequency of the CTL clones that failed to recognize a more recent influenza virus strain isolated in 2003 was determined. In four of six study subjects, CTLs were found to be specific for variable epitopes, accounting for 2.6 % of all CTL clones. For some of these CTL clones, the minimal epitope and the residues responsible for abrogation of T-cell recognition were identified. http://repub.eur.nl/res/pub/13889/ 2005-09-01T00:00:00Z Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M1(58-66). We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M1(58-66) epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes. http://repub.eur.nl/res/pub/8466/ 2005-01-01T00:00:00Z Amino acid substitutions have been identified in the influenza A virus nucleoprotein that are associated with escape from recognition by virus-specific cytotoxic T lymphocytes (CTLs). One of these is the arginine-to-glycine substitution at position 384 (R384G). This substitution alone, however, is detrimental to viral fitness, which is overcome in part by the functionally compensating co-mutation E375G. Here, the effect on viral fitness of four other co-mutations associated with R384G was investigated by using plasmid-driven rescue of mutant viruses. Whilst none of these alternative co-mutations alone compensated functionally for the detrimental effect of the R384G substitution, the M239V substitution improved viral fitness of viruses containing 375G and 384R. The nucleoprotein displays unexpected flexibility to overcome functional constraints imposed by CTL epitope sequences, allowing influenza viruses to escape from specific CTLs. http://repub.eur.nl/res/pub/13474/ 2004-08-01T00:00:00Z Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs. http://repub.eur.nl/res/pub/10337/ 2004-01-01T00:00:00Z Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B*2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP(383-391)]; SRYWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP(383-391) epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B*2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using (51)Cr release assays with a T-cell clone specific for the NP(383-391) epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B*2705-positive target cells pulsed with the original NP(383-391) peptide. The proportion of virus-specific CD8+ gamma interferon (IFN-gamma)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-gamma staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B*2705. The proportion of virus-specific CD8+ T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B*2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP(383-391) epitope is the most important HLA-B*2705-restricted epitope in the nucleoprotein of influenza A viruses.