Cytomegalovirus: from evasion to suppression?
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Animals↗
Biomedical subjects
G W Wilkinson
Publications and source records attributed to G W Wilkinson.
Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40.
The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.
Amino Acid Sequence↗
Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK.
Herpes simplex virus type 1-thymidine kinase (HSV1-TK) in combination with ganciclovir is an efficient and widely used strategy in brain tumour gene therapy. Recently, we have shown effective inhibition of glioma growth in a syngeneic rat model using recombinant adenoviruses expressing the full-length HSV1-TK and an N-terminus truncated variant, HSV1-DeltaTK in the presence of ganciclovir. We also showed active chronic brain inflammation in the long-term survivors (3 months) treated with HSV1-TK plus GCV. Furthermore, our results indicated loss of myelinated fibres, oedema and indices of ongoing axonal degeneration. In this study, we assessed the cytotoxicity of both HSV1-TK variants in the presence or absence of ganciclovir, in primary cultures of neurones and glia, and in the rat brain in vivo. Our results indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotoxicity for either neurones or glial cells grown in primary cultures, (2) on its own the full-length HSV1-TK is more cytotoxic than its truncated version HSV1-DeltaTK for a population of non-neuronal and non-glial cells within neocortical primary cultures, and (3) in vivo, when delivered into the striatum, RAds encoding HSV1-TK are more cytotoxic than RAds encoding HSV1-DeltaTK, after administration of ganciclovir. The effectiveness of HSV1-DeltaTK in preventing brain tumour growth in vivo, combined with its reduced cytotoxicity, both in vivo and in primary cultures of CNS cells, could represent an advantage for treatment of brain tumours using gene therapy.
Adenoviridae↗
Development of adenovirus vectors encoding rat complement regulators for use in therapy in rodent models of inflammatory diseases.
C activation has been implicated in the pathogenesis of numerous inflammatory human diseases and disease models. A therapy based on C inhibition might therefore be of benefit to reduce inflammation and ameliorate disease. C inhibition in vivo can be accomplished by the delivery of soluble recombinant C regulators either systemically or directly to a target site, but effects are transitory. We have developed a strategy for the efficient delivery of the membrane-bound rat C inhibitors, CD59, Crry, and decay-accelerating factor (DAF), using replication-deficient adenovirus vectors with the intention of treating rat models of disease in which C is implicated. The adenovirus recombinants(RAd), RAdCD59, RAdCrry, and RAdDAF, respectively, have been tested for expression and function of the transgene in vitro. Infection of human fetal foreskin fibroblasts resulted in high levels of expression of each of the rat inhibitors. The constructs were also tested for inhibition of rat C-mediated cell lysis and C3b deposition. In a cell lysis assay, each inhibited to varying degrees of efficiency in the order RAdCD59 = RAdDAF > RAdCrry. In a C3b deposition assay, RAdDAF caused a greater reduction in C3b deposition than RAdCrry and RAdCD59 was ineffective. These agents, individually or in combination, provide the tools for testing the effects of prolonged inhibition of C at a target site on the progress of experimental models of disease.
Adenoviridae↗
CTL epitopes identified with a defective recombinant adenovirus expressing measles virus nucleoprotein and evaluation of their protective capacity in mice.
Cytotoxic T-lymphocyte (CTL) responses to measles virus (MV) play an important role in recovery from infection, with one of the major target proteins for CTL activity being the nucleoprotein (Np). In this report, a replication-deficient adenovirus-5 recombinant, expressing for MV Np (Rad68) was tested for in vivo priming of MV Np-specific CTL responses in BALB/c and CBA mice. In both strains of mice strong Np-specific CTL responses were induced and these responses were shown to be MHC class I restricted. Using overlapping 15mer peptides spanning residues 1-505 of MV Np a single epitope comprising residues 281-295 was identified in BALB/c mice whereas, in CBA mice two epitopes comprising residues 51-65 and 81-95, were identified. These epitopes were found to contain class I motifs for H-2L(d) and H-2K(k) MHC molecules, respectively. Immunization of BALB/c and CBA mice with the respective CTL epitopes resulted in the in vivo induction of peptide-and MV Np-specific CTL responses. In addition, the identified H-2K(k) restricted CTL epitopes conferred some protection against encephalitis induced following intracerebral challenge with a lethal dose of canine distemper virus (the Np of which shares 70% sequence homology with MV Np). These findings highlight the potential of using well-defined CTL epitopes to control virus infection.
Adenoviridae↗
Human cytomegalovirus infection downregulates expression of the cellular aminopeptidases CD10 and CD13.
During the course of a productive infection, human cytomegalovirus (HCMV) has a sophisticated relationship with its host cell. An increasing number of virus-encoded genes are being identified which act specifically to usurp or modulate functions in the host cell associated with transcriptional control, cell signalling, and protein synthesis. While HCMV infection is associated with a general upregulation of cellular gene expression, the expression a small subset of cellular proteins, including the MHC-1 heavy chain and fibronectin, is downregulated. This study now identifies two additional cellular proteins, aminopeptidase N (CD13) and neutral endopeptidase (CD10), that are downregulated during HCMV infection. While aminopeptidase N and neutral endopeptidase exhibit no significant sequence homology, both are expressed on the cell surface and have very similar enzymatic properties. HCMV infection was associated with reduced surface expression and enzyme activity of CD13 and CD10, an apparent decrease in the rate of synthesis of both proteins in metabolic-labelling experiments, and inhibited glycosylation of the nascent CD13 and CD10 polypeptide chains that were synthesized. Levels of CD10 poly A+ RNA were suppressed efficiently at all stages of virus infection; however, the reduction in CD13 poly A+ RNA levels was much less pronounced. This differential effect suggests that HCMV may be downregulating expression of CD10 and CD13 by independent mechanisms. Indeed, treatment of cells with an inhibitor of viral DNA synthesis blocks downregulation of CD13, whilst downregulation of CD10 is unaffected. While it is not yet clear what advantage is bestowed on the virus by downregulating expression of CD13 and CD10, aminopeptidases are known to have a role in peptide processing in both the MHC class I the MHC class II antigen presentation pathways.
CD13 Antigens↗
Disruption of PML-associated nuclear bodies mediated by the human cytomegalovirus major immediate early gene product.
The PML gene product is associated with a defined nuclear structure (10-20 per cell) known variously as PML-bodies, ND10, PODs or Kr bodies. Certain conditions are known to compromise the integrity of PML-bodies; these include environmental stress (e.g. heat shock), a chromosomal translocation-associated acute promyelocytic leukaemia, and infection with certain viruses [including human cytomegalovirus (HCMV), herpes simplex virus type 1 and adenovirus]. Expression of the HCMV major immediate early (IE) protein (IE1(491aa)) is by itself sufficient to cause disruption of PML-bodies, resulting in the dispersal of the PML antigen uniformly throughout the nucleus. In uninfected cells undergoing mitosis PML is excluded from chromatin. However, both IE1(491aa) and PML were observed to associate with mitotic chromosomes in cells infected with HCMV or transfected with the IE1 gene. A series of in-frame IE1 deletion mutants was used in DNA transfection experiments to identify two large sequence elements (aa 132-274 and the C-terminal aa 347-491) not required for dispersal of the PML antigen. However, a putative leucine-zipper domain (aa 105-139), a putative zinc-finger domain (aa 267-286) and exon 2 and 3 coding sequences (aa 6-85) were required. The association of the IE1 gene product with chromatin required an acidic domain near the C terminus (aa 421-486). The interaction of IE1(491aa) with chromatin was therefore not required for the disruption of PML-bodies. Exon 2 (aa 1-24) was shown to encode a nuclear localization signal.
Animals↗
An outbreak of epidemic keratoconjunctivitis caused by adenovirus type 37.
An outbreak of acute keratoconjunctivitis in an ophthalmology department affected 15 patients and seven members of staff and necessitated temporary closure of the unit. Adenovirus (Ad) was isolated from eye swabs taken during the outbreak, but typing of the isolates by virus neutralisation assay proved unsatisfactory, wrongly assigning the isolate to serotype 10. A robust and rapid method for preparing microgram amounts of Ad DNA from infected cells was devised and used as the basis of a non-radioactive method for routine genotyping of adenovirus clinical isolates. All isolates associated with the outbreak, and one from a patient presenting at a nearby hospital during the outbreak, were found to have restriction endonuclease digestion patterns characteristic of Ad 37.
Acute Disease↗
Human papillomavirus-specific cytotoxic T lymphocytes in patients with cervical intraepithelial neoplasia grade III.
Human papillomaviruses (HPVs), especially types 16 and 18, are strongly associated with the development of cervical intraepithelial neoplasia (CIN) and invasive carcinoma. The HPV E6 and E7 proteins are expressed constitutively in the majority of CIN lesions and carcinomas. Therefore, they are targets for the immune response against HPV and candidates for active immunotherapy. We have previously detected HPV-specific CTLs from the peripheral blood mononuclear cells from a cervical cancer patient following immunization with a recombinant vaccinia using in vitro restimulation with adenovirus recombinants expressing HPV16 or HPV18 E6/E7 fusion proteins. In this study, we used a similar protocol to determine the prevalence of CTL responses against HPV16 and HPV18 E6/E7 in the peripheral blood mononuclear cells from 10 CIN III and 10 normal subjects. HPV-specific CTL responses were detected in 6 of 10 CIN III subjects. These CTL lines recognized HPV16 E6/E7 proteins presented by at least three MHC class 1 HLA alleles and by HPV-transformed CaSki cells. No HPV-specific CTLs were detected in normal subjects. This study demonstrates the presence of naturally occurring HPV-specific memory CTLs in a majority of CIN III patients and provides an approach for further study of their role in modulating cervical malignancy.
Adult↗
The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation.
In its attempt to evade cytotoxic T cell recognition, human cytomegalovirus encodes several genes that target MHC class I molecules at different points in their assembly pathway. We show here that the human cytomegalovirus US6 gene encodes a 22-kDa glycoprotein that binds the transporter-associated with antigen processing (TAP)/class I complex and inhibits translocation of peptide from the cytosol to the endoplasmic reticulum. Major histocompatibility complex class I molecules are therefore unable to load TAP-dependent peptides, resulting in the retention of MHC class I molecules in the endoplasmic reticulum, with a consequent reduction in class I at the cell surface. Interferon-gamma treatment of US6 transfected cells overcomes this inhibition of peptide translocation and restores class I at the cell surface to wild type levels. The functional consequence of TAP inhibition is that US6 transfected cells are unable to present endogenous antigen to cytotoxic T lymphocytes and are therefore resistant to cytotoxic T lymphocyte lysis.
Antigen Presentation↗
Cell type-specific expression in brain cell cultures from a short human cytomegalovirus major immediate early promoter depends on whether it is inserted into herpesvirus or adenovirus vectors.
Expression from a short human cytomegalovirus (HCMV) major immediate early (IE) promoter-enhancer was tested in three different virus vectors: recombinant adenovirus (Ad), recombinant herpes simplex virus type 1 (HSV-1) and HSV-1-derived amplicon vectors. The HCMV major IE promoter-enhancer within a replication-deficient recombinant Ad vector was shown to produce cell-specific expression in rat nervous system cell cultures. Recombinant Ad entered all cell types examined but the HCMV major IE promoter was silent in primary cultures of neocortical neurons and Schwann cells, although it drove transgene expression in astrocytes and fibroblasts. Moreover, in neurons and Schwann cells, expression from the HCMV major IE promoter-enhancer in the replication-deficient Ad vector was activated by superinfection with HSV-1, replication-competent Ad and HCMV. The HCMV major IE promoter-enhancer was active in neurons when inserted into HSV-1 recombinant vectors. Further experiments with HSV-1-derived amplicons strongly suggested that an IE protein was responsible for the activation of HCMV major IE-induced expression in neurons. This demonstrates that the activity of the HCMV major IE promoter-enhancer element can depend on the expression of other genes encoded in the virus vector backbone within which it is inserted, and that it can function in a neuronal cell type-specific manner when inserted into a replication-deficient Ad vector.
Adenoviridae↗
A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer.
BACKGROUND: Human papillomavirus (HPV) infection, especially with type 16 or 18, is associated with cervical cancer. Two HPV proteins, E6 and E7, are consistently expressed in tumour cells. The objectives of the study were to examine the clinical and environmental safety and immunogenicity in the first clinical trial of a live recombinant vaccinia virus expressing the E6 and E7 proteins of HPV 16 and 18 (TA-HPV). METHODS: The study was an open label phase I/II trial in eight patients with late stage cervical cancer. The patients were vaccinated with a single dose of TA-HPV and kept in strict isolation to monitor local and systemic side-effects, environmental spread, and anti-E6/E7 immune responses. FINDINGS: Vaccination resulted in no significant clinical side-effects and there was no environmental contamination by live TA-HPV. Each patient mounted an antivaccinia antibody response and three of the eight patients developed an HPV-specific antibody response that could be ascribed to the vaccination. HPV-specific cytotoxic T lymphocytes, the effector mechanism most likely to be of therapeutic benefit, were detected in one of three evaluable patients. INTERPRETATION: Further studies to investigate the use ot TA-HPV for immunotherapy of cervical cancer are warranted.
Adult↗
Agonist-stimulated free calcium in subcellular compartments. Delivery of recombinant aequorin to organelles using a replication deficient adenovirus vector.
Changes in the concentration of calcium ions ([Ca2+]) within cellular organelles play a central role in controlling cellular function. We have engineered the Ca2+ sensitive photoprotein aequorin to monitor selectively [Ca2+] within defined subcellular compartments, namely the cytosol, nucleus and endoplasmic reticulum. DNA encoding the engineered aequorins have been inserted into a replication deficient adenovirus (Ad) type 5 E1-vector, under control of the cytomegalovirus (CMV) major immediate early promoter. The Ad vector provides a simple and efficient method to express the photoproteins in a wide variety of mammalian cell types. Efficient targeting of the photoproteins to the appropriate cellular compartment was established immunocytochemically in COS7 cells, where it was expressed in up to 100% of the target population. Levels of expression could be controlled by virus dose and chemical agents which affect the activity of the CMV promoter. In HeLa cells expressing nuclear targeted aequorin or cytosolic aequorin, ATP or histamine induced immediate biphasic elevations of both nuclear and cytosolic [Ca2+]; subsequent challenge with agonist evoked similar responses. In addition to epithelial type adherent cell lines (COS7 and HeLa), aequorin expression was also readily detected in non-adherent cells of myeloid lineage (K562 and HL60) and non-adherent primary cells polymorphonuclear leucocytes (neutrophils). The Ad vectors can, therefore, be used to express targeted aequorin in a range of different cell types and represents a novel method to monitor changes in free [Ca2+] in cellular organelles.
Adenosine Triphosphate↗
Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells.
Remodelling of the extracellular matrix resulting from increased secretion of metalloproteinase enzymes (MMPs) is implicated in many pathological conditions, including rheumatoid arthritis, restenosis following balloon angioplasty, atherosclerosis and cancer cell invasion and metastasis. Clear definition of the normal and pathological function of individual MMPs will benefit from approaches that use gene transfer to produce increases in MMP levels that mimic those observed in pathological conditions. Similarly, gene transfer methods leading to controlled increases in levels of the tissue inhibitor of metalloproteinases (TIMPs) will help to define the function of MMPs both in vitro and in vivo. Gene transfer of TIMPs may also have therapeutic potential in pathological conditions where inhibition of MMP activity may be beneficial. We have used the adenovirus serotype 5 vector system to generate replication-deficient recombinant adenoviruses capable of expressing the MMP-9, TIMP-1 or -2 genes. High level expression is driven by the cytomegalovirus major immediate early promoter (CMV IEP). Efficient and selective over-production of each recombinant protein was shown by immunofluorescence in either rabbit smooth muscle cells (SMC) or human MCF-7 adenocarcinoma cells. High level secretion directly dependent on the multiplicity of infection (MOI) was observed for each functional transgene by gelatin zymography. Using a quantitative ELISA assay, levels of recombinant TIMP-1 were detected when SMC were infected with as low as three plaque forming units (pfu) of virus per cell in vitro. A linear increase in TIMP-1 secretion was observed up to 1000 pfu/cell of virus (0.75 ng/10(4) cells/24 h at 3 pfu/cell to 1243 ng/10(4) cells/24h at 1000 pfu/cell). Similar levels of secretion of MMP-9 and TIMP-2 were observed by Western blot analysis using the same MOI of adenovirus. Thus, recombinant adenoviruses are an efficient and flexible system for high level expression of MMPs and TIMPs and will be useful tools in the study of matrix remodelling in vivo and in vitro.
Adenocarcinoma↗
Immunization of mice with plasmid DNA expressing the measles virus nucleoprotein gene.
The measles virus (MV) nucleocapsid (N) protein gene has been inserted into a plasmid vector so as to place the gene under the control of the strong constitutive human cytomegalovirus major immediate early promoter. On intramuscular injection of pMV64 DNA into C3H/He mice, seroconversion with increasing titers of N-specific serum IgG antibodies was observed over a period of 3 months. However, when 3-week-old mice were immunized by intramuscular injection of pMV64 in a two-dose schedule, and challenged intracranially with a rodent-adapted measles virus strain (CAM/RB) at 5 weeks of age, no significant protective response was seen. The lack of effective protection evoked by DNA immunization in this model, where MV challenge must take place before 8 weeks of age, may be due to inefficient induction of cell-mediated immunity resulting from expression in muscle tissue, compounded by a relatively slow rise in immune response compared with that seen with the recombinant adenovirus.
Animals↗
A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.
While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.
Adenoviridae↗
Expression of the measles virus nucleoprotein gene in Escherichia coli and assembly of nucleocapsid-like structures.
To investigate the use of fusion systems to aid the purification of recombinant proteins for structure/function studies and potential uses as diagnostic reagents, the measles virus (MV) gene encoding the nucleoprotein was cloned and expressed in Escherichia coli in three forms: as a full-length intact protein and as two fusion proteins. Expression of the intact N gene under the control of the tac promoter in the pTrc99c plasmid produced a protein of the correct size (60 kDa) which represented approx. 4% of the total cellular protein, and was recognised by known measles positive human sera. 'Herringbone' structures characteristic of paramyxovirus nucleocapsids (NuC) were identified in fractured cells examined by electron microscopy. The production of NuC-like structures in a prokaryotic cell indicates folding of the nucleoprotein can occur in the absence of MV genomic RNA, other MV-encoded gene products and eukaryotic cell proteins or RNA, to produce structures which are morphologically and antigenically similar to those seen in virus-infected cells. Conversely, synthesis of N protein as a fusion protein with either E. coli beta-galactosidase or the E. coli maltose-binding protein resulted in the production of fused proteins which could not be assembled into NuC-like structures or readily used as diagnostic reagents. However, the ability of MV N protein to form NuC-like structures in E. coli will facilitate structure/function and mutational analysis of the NuC protein.
ATP-Binding Cassette Transporters↗
High-level expression of the measles virus nucleocapsid protein by using a replication-deficient adenovirus vector: induction of an MHC-1-restricted CTL response and protection in a murine model.
Replication-deficient adenovirus (Ad) vectors provide an efficient technology for direct DNA delivery to cells both in vitro and in vivo. We have inserted the measles virus nucleoprotein (N) gene under the control of the strong constitutive CMV major IE promoter into an Ad type 5 E1- vector to produce the recombinant virus RAd68. Following infection of human fibroblasts with RAd68 in vitro, recombinant N protein was synthesized as a 60-kDa protein that represented up to 20% total soluble cell protein. Long filamentous structures were produced in both the nucleus and the cytoplasm that were similar in appearance to measles virus nucleocapsids. These "nucleocapsid-like" structures were readily purified by density gradient centrifugation. Murine immunization with RAd68 elicited (i) a humoral immune response to N, (ii) a major histocompatibility complex class I-restricted, antigen-specific cytotoxic T cell response, and (iii) protection against challenge with the measles virus CAM/RB strain in mice. This study demonstrates the capacity of replication-deficient Ad recombinants both to induce and to characterize cell-mediated immune responses.
Adenoviruses, Human↗