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D J Moss

Publications and source records attributed to D J Moss.

At least 55 records · Page 3Linked to original sources
PubMed1997

Selection of a diverse TCR repertoire in response to an Epstein-Barr virus-encoded transactivator protein BZLF1 by CD8+ cytotoxic T lymphocytes during primary and persistent infection.

We investigated the CD8+ cytotoxic T lymphocyte (CTL) repertoire to an HLA B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early protein, BZLF1. Repertoire selection was monitored by determining the TCR beta chain sequences of RAKFKQLLQ-specific CTL established from primary infected and healthy virus carriers. PCR analysis of spontaneous EBV-transformed lymphoblastoid cell lines (LCL) from three individuals with primary infection showed that two were infected with type A and one with type B EBV. Polyclonal and clonal CTL that were generated by stimulating peripheral blood mononuclear cells with an HLA B8+ homozygous LCL lysed T cell blasts pulsed with the peptide, RAKFKQLLQ; lysis of certain HLA B8+ LCL targets was associated with the abundance of BZLF1 transcripts. TCR beta analysis showed that while there was loop length restriction in the putative peptide contact site of all responding beta chains, diverse and unique (non-recurrent) TCR beta clonotypes were selected in individuals during primary infection and continued to emerge after long-term virus exposure. TCR-contact site heterogeneity was excluded as the selective force in diversity generation since the epitope-encoded sequences were found to be identical within endogenous virus isolates. In this first study of TCR repertoire selection for an EBV lytic antigen, a BZLF1-reactive component of diverse clonotypes was identified in primary type A or type B EBV infection which was sustained in the EBV-specific memory response throughout life-long infection. This diversity selection is likely to play a critical role in maintaining a balanced viral load throughout EBV persistence.

Amino Acid Sequence↗
PubMed1997

GP55 inhibits both cell adhesion and growth of neurons, but not non-neuronal cells, via a G-protein-coupled receptor.

There is compelling evidence for the role of inhibitory molecules in guiding neurons to their appropriate targets. Furthermore, continued expression of these molecules in the adult could explain why there is little regeneration of neurons in the central nervous system. We have previously identified a family of glycosyl phosphatidylinositol-linked glycoproteins (GP55) from adult chicken brain that has been shown to inhibit neurite outgrowth from dorsal root ganglion and forebrain neurons. GP55 consists of two or more glycoproteins and belongs to a subgroup of the lg superfamily which contains OBCAM, LAMP, neurotrimin and CEPU-1. We now show that GP55 is anti-adhesive, blocking the adhesion of neurons to normally adhesive substrata in a concentration dependent manner. The anti-adhesive effect can be blocked using antiserum raised against GP55 and pertussis toxin (PTX) but not the beta oligomer alone. In contrast, the adhesion of fibroblasts and Schwann cells to the substrata is not affected by GP55. Indeed, non-neuronal cells spread and grow normally. These results would suggest that both the anti-adhesive effect and the inhibition of outgrowth by GP55 is specific to neurons and is mediated by a PTX sensitive, G-protein-coupled receptor.

Animals↗
PubMed1997

Hierarchy of Epstein-Barr virus-specific cytotoxic T-cell responses in individuals carrying different subtypes of an HLA allele: implications for epitope-based antiviral vaccines.

Major histocompatibility complex class I-restricted Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) in healthy virus carriers constitute a primary effector arm of the immune system in controlling the proliferation of virus-infected B cells in vivo. These CTLs generally recognize target epitopes included within the latent antigens of the virus. For example, CTLs from HLA B44+ healthy virus carriers often recognize peptide EENLLDFVRF [corrected] from EBV nuclear antigen 6. However, the strength of this response directly correlates with the HLA B44 subtype expressed by the individual donor. Indeed, HLA B*4405+ virus carriers consistently show a very high frequency of CTL precursors for the EENLLDFVRF [corrected] epitope, while a much weaker response is seen in HLA B*4403+ and HLA B*4402+ individuals. This disparity is not due to an intrinsic difference in the CTLs generated by individuals carrying different subtypes of HLA B44. In fact, virus-specific CTLs recognize EENLLDFVRF [corrected] peptide-sensitized HLA B*4405+ target cells more efficiently than B*4402+ or B*4403+ target cells irrespective of the HLA B44 subtype expressed by the donors from whom these effectors were isolated. This effect is evident whether the CTL epitope is endogenously processed or exogenously presented. In addition, a comparison of the intracellular transport kinetics of different B44 subtypes revealed that the B*4405 allele is rapidly assembled and arrives in the trans-Golgi compartment at a faster rate than B*4402 or B*4403. Based on these results, we propose that HLA class I alleles that are capable of binding peptides more efficiently from the intracellular pool, and are rapidly assembled and transported, may confer a protective advantage against viral infection.

Alleles↗
PubMed1997

Evolutionary dynamics of genetic variation in Epstein-Barr virus isolates of diverse geographical origins: evidence for immune pressure-independent genetic drift.

The question whether immune pressure exerted by cytotoxic T lymphocytes (CTLs) can influence the long-term evolution of genetically stable viruses such as Epstein-Barr virus (EBV) has generated considerable scientific interest, primarily due to its important implications for the overall biology of the virus. While arguing for a role of CTLs in the evolution of viruses, it is important to differentiate between genetic variation in virus and immune recognition of these variant virus by CTLs. To assess the role of genetic selection in the long-term evolution of EBV, we have analyzed a large panel of type 1 EBV isolates from African, Southeast Asian, Papua-New Guinean (PNG), and Australian Caucasian individuals. Seven different regions of the EBV genome, which include nine CTL epitopes restricted through a range of HLA class I alleles, were sequenced and compared. Although numerous nucleotide changes were identified within these isolates, comparison of synonymous and nonsynonymous substitutions in the CTL epitope indicated that the genetic variation was generated mostly independently of immune selection pressure. Surprisingly, an inverse correlation between genetic variation within certain CTL epitopes and the frequency distribution of HLA alleles that present the CTL epitopes was seen, suggesting that the evolutionary pressures on the CTL epitopes of the virus may be toward their conservation rather than their inactivation. Furthermore, molecular evolutionary genetic analysis of nucleotide sequences revealed that viral isolates from PNG are evolving as a lineage distinct from isolates from African, Southeast Asian, and Australian Caucasian individuals.

Amino Acid Sequence↗
PubMed1997

Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection.

Epstein-Barr virus (EBV) provides one of the most informative systems with which to study cytotoxic T lymphocyte (CTL) responses in humans. The virus establishes a highly immunogenic growth-transforming infection of B lymphocytes, associated with the coordinate expression of six virus-coded nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, -LP) and two latent membrane proteins (LMPs 1 and 2). This elicits both primary and memory CT8+ CTL responses that are markedly skewed toward HLA allele-specific epitopes drawn from the EBNA3A, 3B, 3C subset of latent proteins, with reactivities to other antigens being generally much less frequent. This hierarchy of immunodominance among the different latent proteins may at least partly reflect their differential accessibility to the HLA class I-processing pathway. Furthermore, CTLs to some of the immunodominant epitopes involve highly conserved T cell receptor (TCR) usage, a level of focusing which evidence suggests could have immunopathological consequences from cross-reactive recognition of other target structures. EBV is associated with a range of human tumors, and there is increasing interest in the possibility of targeting such malignancies using virus-specific CTLs. The dramatic reversal of EBV-driven lymphoproliferations in bone marrow transplant patients following CTL infusion demonstrates the potential of this approach, and here we discuss prospects for its extension to other EBV-positive tumors in which the immunodominant EBNA3A, 3B, 3C proteins are not expressed.

Amino Acid Sequence↗
PubMed1996

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus-specific CTL response was shown to include specificities for two HLA-B8-restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR-beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease.

Acute Disease↗
PubMed1996

Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing.

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.

Amino Acid Sequence↗
PubMed1996

Recombinant polyepitope vaccines for the delivery of multiple CD8 cytotoxic T cell epitopes.

Development of epitope-based CD8 alpha beta CTL vaccines requires effective strategies for codelivery of large numbers of individual epitopes. We have designed an artificial "polyepitope" protein containing 10 contiguous minimal CTL epitopes, which were restricted by five MHC alleles and derived from five viruses, a parasite, and a tumor model. A recombinant vaccinia virus coding for this protein was capable of inducing MHC-restricted primary CTL responses to all 10 epitopes. Mice immunized with this recombinant vaccinia showed protection against murine cytomegalovirus, Sendai virus, and a tumor model. This simple generic approach to multiepitope delivery should find application in CTL-based vaccine design.

Amino Acid Sequence↗
PubMed1996

Recruitment during infectious mononucleosis of CD3+CD4+CD8+ virus-specific cytotoxic T cells which recognise Epstein-Barr virus lytic antigen BHRF1.

Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis (IM) which is a common sequel to primary EBV infection. Thereafter, the virus is maintained as a lifetime latent infection. Although the proteins expressed during the latent EBV infection provide a rich source of immunogenic epitopes, very little is known about cytotoxic T lymphocyte (CTL) control of primary EBV infection. The present report is based on an analysis of CTL clones derived from a patient suffering from acute IM. An intriguing feature of six CTL clones that displayed an HLA-restricted pattern of cell lysis was their initial coexpression of the T cell markers CD3, CD4, and CD8. Detailed analysis of one of these clones, which was restricted through the class II MHC antigen DR2, revealed reactivity with an epitope within the EBV lytic cycle early antigen, BHRF-1, which corresponds to the C-terminal region of the protein (AGLTLSLLVICSYLFISRG) (residues 171-189). There have been no previously published reports describing a CTL response during acute IM directed against an EBV lytic antigen. Interestingly, the coexpression of CD4 and CD8 by these CTLs during acute IM suggests that CD3+CD4+CD8+ cortical thymocytic precursor cells are recruited in order to overcome the EBV infection.

Acute Disease↗
PubMed1996

Cytotoxic T-lymphocyte clones specific for an immunodominant epitope display discerning antagonistic response to naturally occurring Epstein-Barr virus variants.

In the present study we have identified Epstein-Barr virus isolates which encode variant sequences within an HLA B35-restricted immunodominant cytotoxic T-lymphocyte (CTL) epitope that act as natural antagonists and can inhibit CTL activity on the wild-type epitope. This effect can be demonstrated if the wild-type epitope is presented as a synthetic peptide or when processed from a full-length Epstein-Barr virus protein expressed by recombinant vaccinia constructs. However, this antagonistic effect was only selectively seen with some CTL clones, while a strong agonistic effect was evident for other clones in the presence of the same variant peptide. The data presented in this study strongly suggest that it is unlikely that the variant viruses can completely antagonize a virus-specific CTL response by this mechanism since the host immune response is capable of generating CTLs expressing a diverse array of T-cell receptors. Moreover, many of these CTLs can recognize the variant sequences as efficiently as wild-type epitope.

Antigens, Viral↗
PubMed1996

Identification of type B-specific and cross-reactive cytotoxic T-lymphocyte responses to Epstein-Barr virus.

Persistent Epstein-Barr virus (EBV) infection is primarily controlled by HLA class I-restricted memory cytotoxic T-cell (CTL) responses which can be reactivated in vitro by stimulation of peripheral blood lymphocytes with autologous lymphoblastoid cell lines. During an investigation of a donor infected by both type A and type B EBV, CTL specific for type B EBV were isolated. The CTL were found to recognize an epitope encoded by the EBNA-6B gene. The minimal epitope sequence was identified as QNGALAINTF, corresponding to residues 213 to 222 in the EBNA-6B protein, and presentation of this epitope was shown to be via HLA B62 (B15). This is the first report of the characterization of an epitope that is EBV type B specific. CTL recognizing sequences common to type A and type B EBV were identified as well. A cross-reactive epitope recognized by these CTL was encoded within the EBNA-6 gene of both type A and type B. This minimal sequence for this epitope was LLDFVRFMGV (residues 284 to 293 in both types), and the epitope was restricted through HLA A*0201. This second epitope sequence overlaps with a published EBV B44-restricted epitope (EENLLDFVRF). The implications of these findings are discussed with respect to the design and efficacy of epitope-based vaccines.

Adolescent↗
PubMed1996

Unusually high frequency of Epstein-Barr virus genetic variants in Papua New Guinea that can escape cytotoxic T-cell recognition: implications for virus evolution.

Cytotoxic T lymphocytes (CTLs) which recognize viral antigens in association with human leukocyte antigens (HLAs) play an important role in controlling persistent virus infections. These viruses use several mechanisms to evade the immune response, including mutations that affect either T-cell receptor recognition or binding of viral epitopes to the HLA. It has recently been proposed that the distribution of HLA frequencies and the specific CTL response may influence the long-term evolution of Epstein-Barr virus (EBV) by selecting variants which lack immunodominant CTL epitopes. To test this hypothesis, we have studied EBV isolates from two genetically distinct Papua New Guinea (PNG) populations, residing in coastal and highland regions, for polymorphism within seven viral CTL epitope sequences restricted through several class I HLAs. Surprisingly, all EBV isolates analyzed displayed identical amino acid substitutions within HLA A11-, B35- and B8-restricted CTL epitope sequences which completely abrogated CTL recognition and binding of synthetic peptides to HLA molecules. Furthermore, these substitutions revealed no correlation with the contemporary distribution of HLAs in the different PNG populations, which argues for a minimal influence of immune pressure. The sequence homology between EBV isolates from coastal and highland PNG suggests that the virus may have had a single origin and, more importantly, that these isolates are genetically distinct from those present in a Caucasian population.

Amino Acid Sequence↗
PubMed1996

Peptide transporter (TAP-1 and TAP-2)-independent endogenous processing of Epstein-Barr virus (EBV) latent membrane protein 2A: implications for cytotoxic T-lymphocyte control of EBV-associated malignancies.

Major histocompatibility [correction of histocampatability] complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) recognizing Epstein-Barr virus (EBV) latent antigens play a pivotal role in restricting the proliferation of EBV-infected normal B cells. However, it is now well established that most of the EBV-associated malignancies escape this potent CTL response in vivo. This resistance to immune surveillance is not due to an obvious CTL dysfunction but has been partly attributed to the down-regulation of the peptide transporters, TAP-1 and TAP-2, thus restricting the endogenous loading of MHC class I molecules with peptides derived from viral nuclear antigens. In the present study we have explored the possibility that EBV latent membrane protein 2A (LMP2A), which is often expressed in many of the EBV-associated malignancies, such as nasopharyngeal carcinoma and Hodgkin's disease tumors, can be endogenously processed through an alternative, TAP-1- and TAP-2-independent pathway. The data presented in this study clearly demonstrate not only that LMP2A can be processed by a TAP-independent mechanism but also that tumor cells with down-regulated TAP expression can be efficiently recognized by LMP2A-specific T cells following infection with recombinant vaccinia virus encoding this protein. We propose that since LMP2A is a membrane protein, it is directly translocated into the secretory pathway and the processing enzymes present in the endoplasmic reticulum are capable of generating the relevant peptide epitopes for MHC binding. The present finding of TAP-1- and TAP-2-independent presentation of LMP2A epitopes suggests a novel mechanism for immune targeting of EBV-positive malignancies, such as nasopharyngeal carcinoma and Hodgkin's disease tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1.

We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a recently discovered chick protein CEPU-1. Our results suggest molecules within this family are capable of acting as cell adhesion molecules and inhibitors of neurite outgrowth.

Amino Acid Sequence↗
PubMed1996

Expression of p53, bcl-2, bax, bcl-x2 and c-myc in radiation-induced apoptosis in Burkitt's lymphoma cells.

Apoptosis, a form of physiological cell death, is a genetically determined program essential for normal development and maintenance of tissues, which has been linked to a variety of gene products. We have examined the susceptibility to radiation-induced apoptosis of cell lines derived from the human B cell tumour, Burkitt's lymphoma (BL), displaying a variety of phenotypic characteristics and expressing genes implicated in apoptosis at different levels. The susceptibility to apoptosis following gamma radiation varied significantly amongst the lines. Cell lines with wild type p53 were susceptible to radiation-induced apoptosis but two of five BL lines with only mutant p53 allele also displayed similar susceptibility. Some BL cell lines that expressed bcl-2 at levels comparable with Epstein-Barr virus (EBV) transformed normal B cells were highly susceptible to gamma radiation-induced apoptosis, whereas others expressing low levels were resistant. When these lines were analysed for bax and bcl-X(L) expression again no correlation was observed with susceptibility or resistance to apoptosis. Two BL cell lines having deregulated expression of c-myc were resistant to the induction of apoptosis while two others which had regulated c-myc expression were susceptible. Thus the status of p53, c-myc, bcl-2, bcl-X(L) and bax is not sufficiently informative in BL lines to predict susceptibility to radiation-induced apoptosis.

Journal Article↗
PubMed1995

Isolation of cytotoxic T lymphocytes from healthy seropositive individuals specific for peptide epitopes from Epstein-Barr virus nuclear antigen 1: implications for viral persistence and tumor surveillance.

The question of whether Epstein-Barr nuclear antigen 1 (EBNA1) includes cytotoxic T lymphocyte (CTL) epitopes has generated considerable scientific interest, primarily due to its important implications for the overall biology of Epstein-Barr virus (EBV). Earlier studies have suggested that EBV-associated malignancies that express only EBNA1 escape virus-specific immune surveillance since this antigen is not a target for CTL recognition. In the present report we have used a modified protocol to demonstrate that EBNA1 includes sequences which can be recognized by both polyclonal and clonal CTLs. CD4+ CTL clones were isolated from a healthy, seropositive donor that recognized the peptide epitope TSLYNLRRGTALA from EBNA1 in association with HLA DR1. Interestingly, these CTLs are unable to lyse EBV-infected B cells suggesting that EBNA1 may not be endogenously processed and/or presented to the host CTL response. Despite recent suggestions that glycine-alanine repeat sequences within EBNA1 can inhibit endogenous processing, target cells infected with recombinant vaccinia vectors encoding truncated EBNA1 proteins, without these repeat sequences, were not recognized by this CTL clone. Thus it seems that the presence of glycine-alanine repeats is not responsible for inhibiting the processing of the class II-restricted epitope defined in this study. These results substantiate the view that EBV-infected normal and malignant cells, where viral gene expression is limited to EBNA1, can resist CTL-mediated immune surveillance in vivo.

Amino Acid Sequence↗