[Show abstract][Hide abstract] ABSTRACT: To assess the efficacy of novel antiviral drugs against influenza in clinical trials it is necessary to quantify infectious virus titers in patients' respiratory tract samples. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens, and detecting the production of progeny virus by hemagglutination since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating A(H3) influenza viruses display reduced capacity to agglutinate erythrocytes.Here, we report the magnitude of this problem by analyzing the frequency of HA deficient A(H3) viruses detected in the Netherlands from 1999-2012. Furthermore, we report development and validation of an alternative method to monitor the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on detection of viral nucleoprotein (NP) in virus culture plates by ELISA, and it produced similar results compared to the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2) and other seasonal A(H1N1), A(H1N1)pdm09 and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that circulate since 2010 failed to display HA activity and infectious virus titers could only be determined by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which non-hemagglutinating influenza A viruses circulate.
Full-text · Article · Mar 2014 · Journal of clinical microbiology
[Show abstract][Hide abstract] ABSTRACT: This study aimed to evaluate HIV sequence evolution in whole genes and in CD8 T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates.
HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI.
HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8 T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution.
Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8 T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes.
Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8 T-cell epitope level.
No preview · Article · Oct 2013 · AIDS (London, England)
[Show abstract][Hide abstract] ABSTRACT: In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses.
Full-text · Article · Nov 2011 · Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: The 2009 H1N1 influenza pandemic provided an opportunity to study human virus-specific T cell responses after infection with
a novel influenza virus against which limited humoral immunity existed in the population. Here we describe the magnitude,
kinetics, and nature of the virus-specific T cell response using intracellular gamma interferon (IFN-γ) staining and fluorochrome-labeled
major histocompatibility complex (MHC) class I-peptide complexes. We demonstrate that influenza virus-infected patients develop
recall T cell responses that peak within 1 week postinfection and that contract rapidly. In particular, effector cell frequencies
declined rapidly postinfection in favor of relatively larger proportions of central memory cells.
Full-text · Article · Sep 2011 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes
(heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of
heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which
correlated with the absence of virus-specific CD8+ T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination
on the development of the virus-specific CD8+ T cell immunity in children is currently unknown. Here we compared the virus-specific CD8+ T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza
A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic
fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4+ T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8+ T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results
indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific
CD8+ T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity
to seasonal and future pandemic influenza viruses.
Full-text · Article · Aug 2011 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 microg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.
Full-text · Article · Aug 2010 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Here we describe a flowcytometric assay that measures the defining function of virus-specific 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 cell-mediated 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.
[Show abstract][Hide abstract] ABSTRACT: The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.
[Show abstract][Hide abstract] ABSTRACT: Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.
Full-text · Article · Jul 2008 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: In the present study, we examined the effect of the loss of the human leucocyte antigen (HLA)-B*3501-restricted nucleoprotein (NP)(418-426) epitope on interferon (IFN)-gamma-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 NP(418-426) epitope has led to repeated evasion from specific CTL. We generated recombinant influenza viruses with variants of the NP(418-426) epitope, 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 NP(418-426) epitope resulted in a significant reduction of IFN-gamma-expressing CD8+ T cells, similar to that observed previously after the loss of the HLA-B*2705-restricted NP(383-391) epitope. In addition, the effect of the loss of the NP(418-426) epitope 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 NP(383-391) epitope, 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.
[Show abstract][Hide abstract] ABSTRACT: Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine
production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigen–transfected
target cell–CTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection
with DNA vectors encoding antigen–green fluorescent protein (GFP) fusion proteins. After coculture at various effector:target
(E:T) cell ratios, viable and dead GFP-positive cells were quantified by flow cytometry, and antigen-specific target-cell
elimination was calculated. The assay was validated with human immunodeficiency virus (HIV)– and influenza virus–specific
CTL clones and revealed cytotoxicity at lower E:T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex vivo within 1 day in peripheral blood mononuclear
cells from HIV-infected individuals. The FATT-CTL assay provides a versatile tool that will advance our understanding of cell-mediated
Preview · Article · Nov 2005 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Recent studies indicate that the time required for virus-infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV-1-specific CTL, we measured virus production in cultures containing CD4(+) T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev-specific CTL prevented at least 2 log(10) more HIV-1 production, in 10 days, than similar numbers of RT-specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL-target cell interactions during successive infection cycles. The results show that substantially higher CTL-mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT-specific CTL were at least as effective as the Rev-specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.
Full-text · Article · Oct 2002 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: The immune response against early regulatory proteins of simian- and human immunodeficiency virus (SIV, HIV) has been associated with a milder course of infection. Here, we directly compared vaccination with Tat/Rev versus Pol/Gag. Challenge infection with SIVmac32H (pJ5) suggested that vaccination with Tat/Rev induced cellular immune responses that enabled cynomolgus macaques to more efficiently control SIV replication than the vaccine-induced immune responses against Pol/Gag. Vaccination with Tat/Rev resulted in reduced plasma SIV loads compared with control (P=0.058) or Pol/Gag-vaccinated (P=0.089) animals, with undetectable plasma viral loads in two of the four Tat/Rev-vaccinated animals. Therefore, the results warrant further investigation of the early regulatory proteins and their potential for vaccination against HIV.
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence indicates that cytotoxic T-lymphocytes (CTL) play an important role in the clearing of primary and control of chronic human immunodeficiency virus (HIV) infection. Here, we discuss recent findings that indicate that the timing of target cell recognition critically contributes to CTL effectiveness. In this light several problems that have troubled CTL research are discussed. The use of early proteins like Tat and Rev is proposed for future vaccines design.
[Show abstract][Hide abstract] ABSTRACT: In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.
Full-text · Article · Feb 2002 · Journal of Virological Methods
[Show abstract][Hide abstract] ABSTRACT: Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found,
even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual
transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are
relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where
the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse.
With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human
peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic
T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth
of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells.
Full-text · Article · Apr 2001 · Journal of Virology