ABSTRACT
To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. 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 influenza A virus H3 subtype (A[H3]) viruses display a 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 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), 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 have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.
Subject(s)
Dog Diseases/diagnosis , Influenza A Virus, H3N2 Subtype/isolation & purification , Influenza, Human/diagnosis , Orthomyxoviridae Infections/diagnosis , Animals , Antiviral Agents/pharmacology , Cell Line , Dog Diseases/drug therapy , Dog Diseases/virology , Dogs , Enzyme-Linked Immunosorbent Assay/methods , Hemagglutination Inhibition Tests/methods , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza A Virus, H3N2 Subtype/drug effects , Influenza, Human/drug therapy , Influenza, Human/virology , Madin Darby Canine Kidney Cells , Netherlands , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Viral Core Proteins/chemistryABSTRACT
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.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/immunology , Influenza, Human/virology , Adolescent , Adult , CD8-Positive T-Lymphocytes/chemistry , Female , Histocompatibility Antigens Class I/immunology , Humans , Interferon-gamma/biosynthesis , Male , Middle Aged , Staining and Labeling , Time Factors , Young AdultABSTRACT
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.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/immunology , Vaccination/methods , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Body Weight , CD8-Positive T-Lymphocytes/immunology , Disease Models, Animal , Female , Ferrets , Flow Cytometry , Hemagglutination Inhibition Tests , Histocytochemistry , Immunohistochemistry , Lung/pathology , Lung/virology , Microscopy , Neutralization Tests , Orthomyxoviridae Infections/prevention & control , Pharynx/virology , Vaccines, Inactivated/immunologyABSTRACT
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.
Subject(s)
Birds/virology , Influenza A Virus, H5N1 Subtype/immunology , Influenza A virus/immunology , T-Lymphocytes, Cytotoxic/immunology , Alleles , Amino Acid Sequence , Animals , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens/chemistry , Histocompatibility Antigens/genetics , Histocompatibility Antigens/immunology , Humans , Lymphocyte Count , Sequence Homology, Amino Acid , T-Lymphocytes, Cytotoxic/chemistry , T-Lymphocytes, Cytotoxic/cytologyABSTRACT
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.
Subject(s)
Cytotoxicity, Immunologic/immunology , Immunodominant Epitopes/immunology , Influenza A virus/immunology , Interferon-gamma/biosynthesis , T-Lymphocytes, Cytotoxic/immunology , Adult , Cells, Cultured , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HLA-B Antigens/immunology , HLA-B27 Antigen , HLA-B35 Antigen , Humans , Immunodominant Epitopes/genetics , Influenza A virus/physiology , Lymphocyte Activation/immunology , Middle Aged , Mutagenesis, Site-Directed , Virus ReplicationABSTRACT
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.
Subject(s)
HIV Infections/virology , HIV-1/physiology , Macrophages/virology , T-Lymphocytes, Cytotoxic/immunology , Animals , Disease Models, Animal , Gene Products, rev/immunology , Graft vs Host Disease/immunology , Graft vs Host Disease/virology , HIV Infections/immunology , HIV-1/genetics , Humans , Leukocytes, Mononuclear/virology , Mice , Mice, Inbred CBA , Mutation , Virus Replication , rev Gene Products, Human Immunodeficiency VirusABSTRACT
The evolution of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV-infected macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain reaction-detectable provirus in peripheral blood. Although proviral DNA persisted in the spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings were associated with the detection of CTLps directed against both structural and regulatory SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to reinfection. These findings bear implications for vaccination strategies aimed at inducing protective CTLs against lentiviruses.
Subject(s)
Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/physiology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/virology , Animals , Coculture Techniques , Humans , Kinetics , Lymph Nodes/immunology , Macaca fascicularis , Polymerase Chain Reaction , Simian Immunodeficiency Virus/classification , Simian Immunodeficiency Virus/isolation & purification , Spleen/immunology , T-Lymphocytes/immunology , Time Factors , Viral Load , Virus ReplicationSubject(s)
Acquired Immunodeficiency Syndrome/prevention & control , Gene Products, rev/immunology , Gene Products, tat/immunology , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Animals , Humans , Macaca fascicularis , Pilot Projects , T-Lymphocytes, Cytotoxic/immunology , Vaccination , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency VirusABSTRACT
The antiviral activity of a CD8(+) cytotoxic T-lymphocyte (CTL) clone (TCC108) directed against a newly identified HLA-B14-restricted epitope, human immunodeficiency virus type 1 (HIV-1) Rev(67-75) SAEPVPLQL, was analyzed with respect to its kinetics of target cell lysis and inhibition of HIV-1 production. Addition of TCC108 cells or CD8(+) reverse transcriptase-specific CTLs to HLA-matched CD4(+) T cells at different times after infection with HIV-1 IIIB showed that infected cells became susceptible to CTL-mediated lysis before peak virus production but after the onset of progeny virus release. When either of these CTLs were added to part of the infected cells immediately after infection, p55 expression and virus production were significantly suppressed. These data support a model in which CTLs, apart from exerting cytolytic activity which may prevent continued virus release, can interfere with viral protein expression during the eclipse phase via noncytolytic mechanisms. TCC108-mediated inhibition of virus replication in peripheral blood mononuclear cells caused rapid selection of a virus with a mutation (69E-->K) in the Rev(67-75) CTL epitope which abolished recognition by TCC108 cells. Taken together, these data suggest that both cytolytic and noncytolytic antiviral mechanisms of CTLs can be specifically targeted to HIV-1-infected cells.
Subject(s)
HIV Infections/immunology , HIV-1/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Cell Line , Cytotoxicity Tests, Immunologic , Gene Products, rev/immunology , HIV Infections/blood , HIV Reverse Transcriptase/immunology , HIV-1/growth & development , HIV-1/physiology , HLA-A Antigens/immunology , HLA-B Antigens/immunology , Humans , Kinetics , Molecular Sequence Data , Virus Replication , rev Gene Products, Human Immunodeficiency VirusABSTRACT
Immunological correlates of AIDS-free survival after human immunodeficiency virus type 1 (HIV-1) infection are largely unknown. Cytotoxic T lymphocyte (CTL) responses are generally believed to be a major component of protective immunity against viral infections. However, the relationship between HIV-1-specific CTL responses and disease progression rate is presently unclear. Here we show in twelve HIV-1-infected individuals that detection of Rev-specific CTL precursors (CTLp) early in the asymptomatic stage, as well as detection of Rev- and Tat-specific CTLp later during follow-up, inversely correlate with rapid disease progression. No such correlation was found for detection of CTLp against Gag, RT or Nef. Further studies are required to determine whether a protective mechanism is indeed the basis of the observed correlation. The data presented are in agreement with the hypothesis that CTL against proteins that are important for early viral transcription and translation are of particular importance in protection from rapid disease progression.
Subject(s)
Acquired Immunodeficiency Syndrome/physiopathology , Gene Products, rev/immunology , Gene Products, tat/immunology , HIV Seropositivity/physiopathology , HIV-1/immunology , HLA-A Antigens/immunology , HLA-B8 Antigen/immunology , T-Lymphocytes, Cytotoxic/immunology , Acquired Immunodeficiency Syndrome/immunology , Alleles , Amino Acid Sequence , CD4 Lymphocyte Count , DNA Primers , Disease Progression , Follow-Up Studies , Gene Products, rev/chemistry , HIV Seropositivity/immunology , HLA-A Antigens/genetics , HLA-A1 Antigen/genetics , HLA-A1 Antigen/immunology , HLA-A2 Antigen/genetics , HLA-A2 Antigen/immunology , HLA-B8 Antigen/genetics , Histocompatibility Testing , Humans , Polymerase Chain Reaction , Time Factors , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Detailed analyses of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses in vaccinated and infected macaques may help to clarify the role of CTL immunity in protection against lentiviruses. Here, the optimal conditions for the measurement of SIV Gag-specific CTL were investigated by bulk and limiting dilution assays of peripheral blood mononuclear cells (PBMC) from naive and vaccinated cynomolgus macaques (Macaca fascicularis) infected with SIVmac32H(J5). In vitro restimulation was generally required for CTL detection. Selective activation of CD8+ and MHC-restricted SIV Gag-specific CTL was induced by stimulation with autologous para-formaldehyde-fixed B-lymphoblastoid cell lines infected with a recombinant vaccinia virus expressing SIV Gag. Applied to limiting dilution assays, antigenic stimulation reproducibly demonstrated SIV Gag-specific CTL precursors (CTLp) in PBMC of all animals studied, including those lacking significant responses in standard bulk CTL assays.
Subject(s)
Antigens, Viral , CD8-Positive T-Lymphocytes/immunology , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Animals , Antibody Specificity , Cytotoxicity Tests, Immunologic , Gene Products, gag/immunology , Indicator Dilution Techniques , Kinetics , Macaca fascicularis , VaccinationABSTRACT
A detailed analysis of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses and the identification of the proteins and epitopes they target may improve the design of immunotherapeutic interventions and provide insights into AIDS pathogenesis. Here, we identified a new CTL epitope in the SIV Gag protein, recognized by CD8+ and MHC class I-restricted CTL clones from a long-term asymptomatic cynomolgus macaque (Macaca fascicularis) infected with SIVmac32H-J5. Using overlapping synthetic peptides, the optimal minimal epitope was characterized as a nine amino acid peptide representing amino acids 242-250 of p26 (SVDEQIQWM). CTL recognition was shown to be abolished by amino acid substitutions observed within homologous human immunodeficiency virus (HIV)-1 and HIV-2 sequences.
Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Gene Products, gag/immunology , Macaca fascicularis/virology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Animals , CD8-Positive T-Lymphocytes/virology , Epitopes/immunology , Humans , Immunity, Cellular , Macaca fascicularis/immunologyABSTRACT
Human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) responses were studied in seven seropositive long-term asymptomatic individuals (CDC A1) with stable CD4 counts for more than 8 years. Using a set of partially overlapping peptides covering the whole Gag, five 15-20-mer peptides were found to contain CTL epitopes. Further characterization of these epitopes revealed a new HLA-A25-restricted CTL epitope in p24, p24(203-212) ETINEEAAEW. This region of Gag is highly conserved in clades B and D of HIV-1. Naturally occurring amino acid sequences, containing p24(203)D (consensus HIV-1 clades A, C, F, G and H) or p24(204)I (HIV-2ROD) were not recognized by CTL recognizing the index peptide. No virus variants with mutations in this sequence were found in peripheral blood mononuclear cells from the HIV-1-infected individual concerned during the 8 year observation period, indicating that the virus had not escaped from the observed CTL response.
Subject(s)
Conserved Sequence , Epitopes, T-Lymphocyte , Gene Products, gag/immunology , HIV-1/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Epitope Mapping , Genetic Variation , HIV Core Protein p24/immunology , HIV Infections/immunology , HLA-A Antigens/immunology , Histocompatibility Antigens Class I , Humans , Leukocytes, Mononuclear/immunology , Molecular Sequence Data , SurvivorsABSTRACT
The frequencies of human immunodeficiency virus type 1 (HIV-1) Gag- and Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte precursors (CTLp) were studied longitudinally in peripheral blood mononuclear cells from 9 HIV-1-infected persons. By antigen-specific stimulation, HIV-1 Gag-specific CTLp were detected in vitro throughout the course of HIV-1 infection, even after the onset of overt disease. In 4 patients, however, HIV-1 Gag-specific CTLp frequencies declined over time in the presence of maintained EBV-specific CTLp. This decline was correlated with decreasing CD4 (r = .38; P < .05) and CD8 (r = .75; P < .001) cell numbers. The maintenance of EBV-specific CTLp in patients with low CD4 cell numbers indicated that EBV-specific CTL-mediated immunity may remain longer unaffected by HIV-1-induced immune dysfunction. Consistent with this observation, the growth of EBV-specific CTL could be supported in vitro by EBV-infected lymphoblastoid B cell lines, independent of both CD4 cells and exogenous cytokines.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , HIV-1/immunology , Herpesvirus 4, Human/immunology , Lymphocyte Activation/immunology , T-Lymphocytes, Cytotoxic/immunology , CD4 Antigens , CD8 Antigens , Cytotoxicity Tests, Immunologic , Cytotoxicity, Immunologic , Disease Progression , Gene Products, gag , Genes, MHC Class I/immunology , HIV-1/genetics , Herpesvirus 4, Human/genetics , Humans , Kinetics , Longitudinal StudiesABSTRACT
To gain more insight into the role of HIV-1-specific cytotoxic T lymphocytes (CTL) in the pathogenesis of AIDS, we investigated temporal relations between HIV-1 Gag-specific precursor CTL (CTLp), HIV-1 viral load, CD4+ T cell counts, and T cell function. Six HIV-1-infected subjects, who were asymptomatic for more than 8 yr with CD4+ counts > 500 cells/mm3, were compared with six subjects who progressed to AIDS within 5 yr after HIV-1 seroconversion. In the long-term asymptomatics, persistent HIV-1 Gag-specific CTL responses and very low numbers of HIV-1-infected CD4+ T cells coincided with normal and stable CD4+ counts and preserved CD3 mAb-induced T cell reactivity for more than 8 yr. In five out of six rapid progressors Gag-specific CTLp were also detected. However, early in infection the number of circulating HIV-1-infected CD4+ T cells increased despite strong and mounting Gag-specific CTL responses. During subsequent clinical progression to AIDS, loss of Gag-specific CTLp coincided with precipitating CD4+ counts and severe deterioration of T cell function. The possible relationships of HIV-1 Gag-specific CTLp to disease progression are discussed.
Subject(s)
Gene Products, gag/immunology , HIV Infections/immunology , HIV-1/immunology , T-Lymphocytes, Cytotoxic/immunology , Acquired Immunodeficiency Syndrome/immunology , Adult , CD4 Lymphocyte Count , Cytotoxicity, Immunologic , Disease Progression , HIV Infections/virology , HIV-1/isolation & purification , Humans , Kinetics , Longitudinal Studies , Viremia/immunologyABSTRACT
Peripheral blood mononuclear cells from 36 asymptomatic HIV-1 seropositive individuals were tested longitudinally for in vitro T-cell proliferation and IL-2 production in response to synthetic peptides spanning the entire gp120 of HIV-1. At baseline, significant T-cell proliferative to pooled and individual peptides was observed in 15 of the 36 donors. After 12 months, proliferative responses to peptide pools were lost or decreased significantly in most donors. Responses appeared to fluctuate over time: at 12 months new recognition sites were detected in four of 10 donors showing T-cell proliferation at baseline, as well as in five of 15 donors with no previous proliferative responses. IL-2 production appeared to be a more sensitive and longer preserved parameter of T-helper cell function: at baseline the majority of donors with no T-cell proliferation produced IL-2 in response to pooled peptides. This response was not decreased significantly after 12 months. The overall patterns of response to both pooled and individual peptides were heterogeneous among donors. Multiple recognition sites were detected in both variable and conserved regions of gp120, but no pool or individual peptide was recognized by all responders. Functional T-cell responses were not statistically correlated to CD4+ cell percentile and absolute numbers.
Subject(s)
HIV Envelope Protein gp120/immunology , HIV Infections/immunology , HIV-1/immunology , T-Lymphocytes, Helper-Inducer/immunology , Adult , CD4-Positive T-Lymphocytes , Female , HIV Infections/blood , Humans , In Vitro Techniques , Interleukin-2/biosynthesis , Kinetics , Leukocyte Count , Lymphocyte Activation , Male , Middle Aged , Peptide Fragments/immunology , Time FactorsABSTRACT
OBJECTIVE: To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor frequencies of HIV-1 Gag-specific CTL in the blood of seropositive individuals. METHODS: B-lymphoblastoid cell lines (B-LCL) infected with recombinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-presenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL activity was determined in 51Cr-release assays using B-LCL as targets after infection with rVV-Gag or after pulsing with partially overlapping peptides spanning the Gag sequence. RESULTS: In vitro stimulation resulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DR+ cells. Gag-specific cytotoxicity, mediated predominantly by HLA class I-restricted CD8+ CTL, was observed in all seven individuals studied. Multiple HLA-restricted CTL epitopes were identified with a single culture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine CTL precursor (CTLp) frequencies. CONCLUSION: PFA-fixed rVV-Gag-infected autologous B-LCL can be used as stimulator cells in bulk PBMC cultures to identify CTL epitopes and to determine CTLp frequencies. This method will facilitate the analysis of HIV-1-specific CTL responses in HIV-infected and vaccinated individuals.
Subject(s)
Gene Products, gag/immunology , HIV-1/immunology , HLA Antigens/immunology , Leukocytes, Mononuclear/immunology , T-Lymphocytes, Cytotoxic/immunology , Antigen-Presenting Cells/immunology , B-Lymphocytes/immunology , Cell Differentiation , Cell Line , Cells, Cultured , Epitopes/immunology , Humans , Leukocyte Common Antigens/immunology , Lymphocyte Activation , Peptide Fragments/immunology , Recombinant Proteins/immunology , Vaccinia virus/geneticsSubject(s)
Cell Separation/methods , T-Lymphocyte Subsets/cytology , CD3 Complex/analysis , CD8 Antigens/analysis , Cytotoxicity, Immunologic , HIV/immunology , Humans , Immunity, Cellular , Immunoglobulin Fab Fragments , Immunophenotyping , In Vitro Techniques , Magnetics , Measles virus/immunology , T-Lymphocyte Subsets/immunologyABSTRACT
The routes used by antigen-presenting cells (APC) to convert the transmembrane fusion glycoprotein (F) of measles virus (MV) to HLA class I and class II presentable peptides have been examined, using cloned cytotoxic T lymphocytes in functional assays. Presentation by Epstein-Barr virus-transformed B lymphoblastoid cell lines was achieved using live virus, ultraviolet light-inactivated virus, and purified MV-F delivered either as such or incorporated in immunostimulating complexes (MV-F-ISCOM). Only live virus and MV-F-ISCOM allow presentation by class I molecules, while all antigen preparations permit class II-restricted presentation. We observe presentation of MV-F from live virus and as MV-F-ISCOM by class II molecules in a fashion that is not perturbed by chloroquine. Our studies visualize novel presentation pathways of type I transmembrane proteins.