scholarly journals Selection of Virus Variants and Emergence of Virus Escape Mutants after Immunization with an Epitope Vaccine

1998 ◽  
Vol 72 (2) ◽  
pp. 1403-1410 ◽  
Author(s):  
Lorenzo Mortara ◽  
Franck Letourneur ◽  
Helene Gras-masse ◽  
Alain Venet ◽  
Jean-Gerard Guillet ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mononuclear Cells ◽  
Cytotoxic T Lymphocyte ◽  
Target Cells ◽  
Epitope Vaccine ◽  
Peripheral Blood Mononuclear ◽  
Gag Proteins ◽  
Escape Mutants ◽  
Selection Of ◽  
Ctl Responses

ABSTRACT In this report, we assessed the evolution of the cytotoxic T-lymphocyte (CTL) response induced by an epitope vaccine. In two macaques immunized with a mixture of lipopeptides derived from simian immunodeficiency virus (SIV) Nef and Gag proteins, CTL responses were directed against the same, single epitope of the Nef protein (amino acids 128 to 137) presenting an alanine at position 136 (Nef 128-137/136A). However, after 5 months of SIV infection, peripheral blood mononuclear cells from both macaques lost their ability to be stimulated by autologous SIV-infected cells while still retaining their capacity to generate cytotoxic responses after specific Nef 128-137/136A peptide stimulation. The sequences of the pathogenic viral isolate used for the challenge showed a mixture of several variants. Within the Nef epitopic sequence from amino acids 128 to 137, 82% of viral variants expressed the epitopic peptide Nef 128-137/136A; the remaining variants presented a threonine at position 136 (Nef 128-137/136T). In contrast, sequence analysis of cloned proviral DNA obtained from both macaques 5 months after SIV challenge showed a different pattern of quasi-species variants; 100% of clones presented a threonine at position 136 (Nef 128-137/136T), suggesting the disappearance of viral variants with an alanine at this position under antiviral pressure exerted by Nef 128-137/136A-specific CTLs. In addition, 12 months after SIV challenge, six of eight clones from one macaque presented a glutamic acid at position 131 (Nef 128-137/131E+136T), which was not found in the infecting isolate. Furthermore, CTLs generated very early after SIV challenge were able to lyse cells sensitized with the Nef 128-137/136A epitope. In contrast, lysis was significantly less effective or even did not occur when either the selected peptide Nef 128-137/136T or the escape variant peptide Nef 128-137/131E+136T was used in a target cell sensitization assay. Dose analysis of peptides used to sensitize target cells as well as a major histocompatibility complex (MHC)-peptide stability assay suggested that the selected peptide Nef 128-137/136T has an altered capacity to bind to the MHC. These data suggest that CTL pressure leads to the selection of viral variants and to the emergence of escape mutants and supports the fact that immunization should elicit broad CTL responses.

scholarly journals Rapid Definition of Five Novel HLA-A∗3002-Restricted Human Immunodeficiency Virus-Specific Cytotoxic T-Lymphocyte Epitopes by Elispot and Intracellular Cytokine Staining Assays

2001 ◽  
Vol 75 (3) ◽  
pp. 1339-1347 ◽  
Author(s):  
Philip J. R. Goulder ◽  
, Marylyn M. Addo ◽  
Marcus A. Altfeld ◽  
Eric S. Rosenberg ◽  
Yanhua Tang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Cytotoxic T Lymphocyte ◽  
Intracellular Cytokine Staining ◽  
Initial Step ◽  
Peripheral Blood Mononuclear ◽  
Hla Restriction ◽  
Immunodeficiency Virus ◽  
Definition Of ◽  
Ctl Responses

ABSTRACT Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) play a major role in control of viral replication. To understand the contribution of this antiviral response, an initial step is to fully define the specific epitopes targeted by CTL. These studies focused on CTL responses restricted by HLA-A∗3002, one of the HLA-A molecules most prominent in African populations. To avoid the time-consuming effort and expense involved in culturing CTL prior to defining epitopes and restricting alleles, we developed a method combining Elispot assays with intracellular gamma interferon staining of peripheral blood mononuclear cells to first map the optimal epitopes targeted and then define the HLA restriction of novel epitopes. In two A∗3002-positive subjects whose CTL responses were characterized in detail, the strongest response in both cases was to an epitope in p17 Gag, RSLYNTVATLY (residues 76 to 86). Using this method, CTL epitopes for which there were no motif predictions were optimized and the HLA restriction was established within 48 to 72 h of receipt of blood. This simple and convenient approach should prove useful especially in the characterization of CTL responses specific to HIV and other viruses, particularly in localities where performing cytotoxicity assays would be problematic.

scholarly journals Identification of Dominant Optimal HLA-B60- and HLA-B61-Restricted Cytotoxic T-Lymphocyte (CTL) Epitopes: Rapid Characterization of CTL Responses by Enzyme-Linked Immunospot Assay

2000 ◽  
Vol 74 (18) ◽  
pp. 8541-8549 ◽  
Author(s):  
Marcus A. Altfeld ◽  
Alicja Trocha ◽  
Robert L. Eldridge ◽  
Eric S. Rosenberg ◽  
Mary N. Phillips ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
Mononuclear Cells ◽  
Cytotoxic T Lymphocyte ◽  
Hla Class I ◽  
Peripheral Blood Mononuclear ◽  
Antiviral Immune Response ◽  
Ctl Epitopes ◽  
Hla Class I Molecules ◽  
Hiv 1 ◽  
Ctl Responses

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1.

scholarly journals CD4+ Cytotoxic T-Lymphocyte Activity against Macrophages Pulsed with Bovine Herpesvirus 1 Polypeptides

1998 ◽  
Vol 72 (9) ◽  
pp. 7040-7047 ◽  
Author(s):  
Chong Wang ◽  
Gary A. Splitter
Keyword(s):  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Cytotoxic T Lymphocyte ◽  
Bovine Herpesvirus ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Bovine Herpesvirus 1 ◽  
Effector Cells ◽  
Herpesvirus 1

ABSTRACT Bovine herpesvirus 1 (BHV-1) induces immune suppression, but the mechanisms for suppression are not well identified. We examined the induction and activity of BHV-1-specific cytolytic CD4+ T lymphocytes (CTL) by stimulating peripheral blood mononuclear cells (PBMC) of cattle immunized with attenuated live BHV-1. Cytolytic effector cells were primarily CD4+ T lymphocytes and lysed autologous, but not allogeneic, macrophages infected with BHV-1 or pulsed with BHV-1 polypeptides. Apoptosis of BHV-1-expressing target cells was observed in CD4+ CTL assays by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. To determine if apoptosis was mediated by a perforin- or Fas-mediated pathway, EGTA, a known selective inhibitor of the perforin pathway, was used. EGTA did not inhibit CD4+-T-cell-mediated cytotoxic activity, but it did limit the NK cell cytotoxicity of virus infected cells. These findings support the concept that CD4+ CTL lyse macrophages pulsed with BHV-1 polypeptides through a Fas-mediated lytic pathway by inducing apoptosis in the target cells. The prominent cytotoxicity mediated by CD4+ CTL suggests a mechanism of selective removal of viral antigen-associated antigen-presenting cells.

Investigation On Exosomes Carrying TSA Derived From Healthy Human White Buffy Coat with Positive HLA-A2 and PolyI:C On Subcellular Antitumor Vaccination.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4747-4747
Author(s):  
Weina Ren ◽  
Chunkang Chang ◽  
Xiao Li
Keyword(s):  
Antitumor Effect ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cytotoxic T Lymphocyte ◽  
Human Leukocyte ◽  
K562 Cells ◽  
Buffy Coat ◽  
Target Cells ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear

Abstract Abstract 4747 Aim Use human leukocyte antigen –A2 (HLA-A2) positive human dendritic cell (DC) derived DEX to support NY-ESO-1 antigen and polyI:C to increase the proliferation of specific cytotoxic T lymphocyte (CTL) in transgenic mice, and increase its anti-tumor effect. Methods Mature dendritic cells derived from peripheral blood mononuclear cells (PBMC) are isolated from the blood from healthy adults with positive HLA-2A. By centrifugation and membrane ultrafiltration, EXO is extracted from the supernatant of DC secretions. Transgenic C57 mice were immunized by human derived tumor testis antigen NY-ESO-1/EXO with or without polyI:C. Mice were sacrificed four weeks after immunization, and spleen cells were isolated and underwent function test. The experiments include: antigen specific CTL proliferation efficiency was tested by dimerization experiment; the antitumor effect for K562 cells and melanoma were tested under different ratio between effecter cells and target cells (0:1, 10:1, 50:1 and 100:1). Results Dimerization experiment indicated that the effect of DEX/TSA+PolyI:C was (1.98±0.79)%. The effect of DEX/TSA was (1.61±0.58)%. The antitumor effect for the ratio of 0:1, 10:1, 50:1 and 100:1 by DEX/TSA:PolyI:C were (11.14±1.36%) A (14.17±0.62%) A (15.71±2.48%) A (24.31±2.91%) for K562 cells; The antitumor effect for DEX/TSA group (12.23±2.25%)A(13.10±1.57%)A(15.27±2.93%)A(19.87±2.72%)for K562 cells; With ratio 10:1 and 100:1, the antitumor effect of DEX/TSA +PolyI:C is better than DEX/TSA group (P<0.05). Whereas, with increased ratio between effecter cells and target cells, there is no significant improvement on antitumor effect for control cells. Conclusion It is promising to combine DEX/TSA derived from healthy human blood with positive HLA-A2 and PolyI:C as a new subcellular antitumor vaccination. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL.

1996 ◽  
Vol 184 (2) ◽  
pp. 485-492 ◽  
Author(s):  
M A Alexander-Miller ◽  
G R Leggatt ◽  
A Sarin ◽  
J A Berzofsky
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocyte ◽  
Target Cells ◽  
High Dose ◽  
High Avidity ◽  
Apoptotic Pathway ◽  
Peptide Antigen ◽  
Kinetics Of ◽  
Selection Of

Experimental data suggest that negative selection of thymocytes can occur as a result of supraoptimal antigenic stimulation. It is unknown, however, whether such mechanisms are at work in mature CD8+ T lymphocytes. Here, we show that CD8+ effector cytotoxic T lymphocytes (CTL) are susceptible to proliferative inhibition by high dose peptide antigen, leading to apoptotic death mediated by TNF-alpha release. Such inhibition is not reflected in the cytolytic potential of the CTL, since concentrations of antigen that are inhibitory for proliferation promote efficient lysis of target cells. Thus, although CTL have committed to the apoptotic pathway, the kinetics of this process are such that CTL function can occur before death of the CTL. The concentration of antigen required for inhibition is a function of the CTL avidity, in that concentrations of antigen capable of completely inhibiting high avidity CTL maximally stimulate low avidity CTL. Importantly, the inhibition can be detected in both activated and resting CTL. Blocking studies demonstrate that the CD8 molecule contributes significantly to the inhibitory signal as the addition of anti-CD8 antibody restores the proliferative response. Thus, our data support the model that mature CD8+ CTL can accommodate an activation signal of restricted intensity, which, if surpassed, results in deletion of that cell.

scholarly journals Sendai virus-specific, H-2-restricted cytotoxic T lymphocyte responses of nude mice grafted with allogeneic or semi-allogeneic thymus glands.

1980 ◽  
Vol 152 (6) ◽  
pp. 1805-1810 ◽  
Author(s):  
J P Lake ◽  
M E Andrew ◽  
C W Pierce ◽  
T J Braciale
Keyword(s):  
T Lymphocyte ◽  
Nude Mice ◽  
Sendai Virus ◽  
Cytotoxic T Lymphocyte ◽  
Target Cells ◽  
Self Recognition ◽  
Parental Haplotype ◽  
Lymphocyte Responses ◽  
Ctl Responses

The in vitro secondary cytotoxic T lymphocyte (CTL) response to Sendai virus-treated stimulator cells by primed spleen cells from thymus gland-grafted nude mice was examined. BALB/c (H-2d) nude mice grafted with allogeneic C57BL/10 (H-2b) thymus glands developed CTL responses directed exclusively to Sendai virus-infected H-2d target cells. (C57BL/6 X BALB/c)F1 nude mice grafted with thymus glands of either parent developed CTL responses preferentially against infected target cells expressing the MHC antigens present in the parental thymus graft, but also had detectable activity for infected target cells of the parental haplotype not expressed in the thymus. These results provide evidence against the concept that self recognition by MHC-restricted CTL is directed exclusively by the MCH type of the thymus.

scholarly journals Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response toCandida albicans

2000 ◽  
Vol 68 (12) ◽  
pp. 6777-6784 ◽  
Author(s):  
Roberto La Valle ◽  
Silvia Sandini ◽  
Maria Jesus Gomez ◽  
Francesca Mondello ◽  
Giulia Romagnoli ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mononuclear Cells ◽  
Opportunistic Pathogen ◽  
Inducible Promoter ◽  
Protein Band ◽  
Signal Peptidase ◽  
Pulse Field ◽  
Expression Library ◽  
Peripheral Blood Mononuclear ◽  
Glycosylation Sites

ABSTRACT A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

scholarly journals Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors

2015 ◽  
Vol 90 (5) ◽  
pp. 2316-2331 ◽  
Author(s):  
Nadeene E. Riddick ◽  
Fan Wu ◽  
Kenta Matsuda ◽  
Sonya Whitted ◽  
Ilnour Ourmanov ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Rhesus Macaques ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Sooty Mangabeys ◽  
Natural Hosts

ABSTRACTAfrican green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIVin vivo, while human-derived CXCR6 and GPR15 also appear to be usedin vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptorsin vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4+T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4+T cells and are potential alternative coreceptors for SIVagm usein vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals.IMPORTANCEAfrican green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cellsin vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptorsin vitro. In this study, we investigated whether SIV from vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of the CCR5 antagonist maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm entry. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entryin vitroand may serve as entry coreceptors for SIVagmin vivo, since their mRNAs were detected in AGM memory CD4+T cells, the preferred target cells of SIV.

scholarly journals Minor H Antigen HA-1–specific Regulator and Effector CD8+ T Cells, and HA-1 Microchimerism, in Allograft Tolerance

2004 ◽  
Vol 199 (7) ◽  
pp. 1017-1023 ◽  
Author(s):  
Junchao Cai ◽  
Junglim Lee ◽  
Ewa Jankowska-Gan ◽  
Richard Derks ◽  
Jos Pool ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Renal Allograft ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Severe Combined Immunodeficiency ◽  
Interferon Γ ◽  
T Cell Subsets ◽  
Delayed Type Hypersensitivity ◽  
Peripheral Blood Mononuclear

The role of the hematopoietic lineage-restricted minor histocompatibility (H) antigen HA-1 in renal allograft tolerance was explored. We obtained peripheral blood samples from three recipients of histocompatibility leukocyte antigen (HLA)–matched, HA-1–mismatched renal transplants, one of which had discontinued immunosuppression &gt;30 yr ago while sustaining normal kidney function. Peripheral blood mononuclear cells (PBMCs) were injected into the footpads of severe combined immunodeficiency mice to measure human delayed type hypersensitivity (DTH) responses. All three patients manifested regulated DTH responses to HA-1H peptide. By differential tetramer staining intensities, we observed two distinct minor H antigen HA-1–specific CD8+ T cell subsets. The one that stained dimly had the characteristics of a T regulatory (TR) cell and produced interleukin (IL) 10 and/or transforming growth factor (TGF) β. These HA-1–specific TR cells coexisted with bright tetramer-binding CD8+ T effector (TE) cells. The CD8+ TE cells mediated HA-1–specific DTH and produced interferon-γ. Suppression of these TE functions by TR cells was TGFβ, IL-10, and cytotoxic T lymphocyte–associated antigen 4 dependent. In addition, HA-1 microchimerism was detected in two recipients, primarily in the dendritic cell fraction of the PBMCs. This is the first demonstration of coexisting CD8+ memory TR and TE cells, both specific for the same HA-1 antigen, in the context of renal allograft tolerance.

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