[Adenoviral Vectors in Gene Therapy]. / Adenovírusové vektory v génovej terapii.

This review is focused on gene therapy, especially adenovirus vectors and their relationship with the immune system response. Adenovirus vectors belong to the most used gene delivery vehicles in gene therapy, study of gene expression or immunotherapy. One of the most important questions concerning their use is their influence on organism in vivo. Study of immunomodulating properties of the adenovirus vectors opens a way for further manipulation and their more effective practical use.

[Ananlysis of phosphoproteins and signalling pathways by quantitative proteomics]. / Analýza fosfoproteínov a signálnych dráh kvantitatívno proteomickými metódami.

Protein phosphorylation is a key regulator in cellular signaling pathways. It is involved in most cellular events in which interplay between phosphatases and kinases strictly controls bio-logical processes, such as differentiation, proliferation and apoptosis. Altered or defective signaling pathways often result in various diseases, emphasizing the importance of studying the phosphoproteome. The abundance of phosphoproteins in the proteome is often very low, which requires specific and highly sensitive approaches. By using quantitative proteomics methods, we are able to analyze changes in abundance of proteins and their posttranslational modifications and then changes in signaling pathways. In this review, we describe quantitative proteomics methods, which could be used for study of phosphoproteins and their connection in signaling pathways.

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.

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.

Viral evasion of natural killer cells during human cytomegalovirus infection.

Cytotoxic T cells are major players in the immune defence against human cytomegalovirus (HCMV). The virus has, however, developed several mechanisms to escape from this control. In particular, it down-regulates cell surface expression of HLA class I molecules. Because natural killer (NK) cells recognize and eliminate cells that lack HLA class I molecules, HCMV-infected cells could be more susceptible to NK lysis. In this review, we discuss the role played by NK cells in immune defence against HCMV and we describe potential strategies the virus has developed to escape from NK cell-mediated lysis. We focus in particular on a newly described protein, HCMV gpUL40, that induces cell surface expression of HLA-E, a non-classical class I molecule known to regulate NK cell functions.

HIV-1 indicator cell lines.

A simple quantitative bioassay for infectious HIV-1 has been developed. The assay is based on adherent CD4+ HeLa cell lines stably transfected with episomal vectors carrying the Escherichia coli beta-galactosidase gene under the control of the HIV long terminal repeat (LTR) promoter. HIV infection of these cell lines transactivates the LTR promoter inducing beta-galactosidase production. Infected cells and virus foci can be stained dark blue by the addition of the chromogenic substrate X-Gal. Alternatively, a readily automatable quantitative enzyme assay can be performed on the infected cultures. Because of its simplicity the bioassay may be useful for routine quantification of HIV-infected cultures, plaque purification, virus neutralization studies and for the screening of antiviral agents.

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.

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.

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.

Nucleotide sequence of the most abundantly transcribed early gene of human cytomegalovirus strain AD169.

Cytoplasmic poly(A+)RNA was isolated from human embryo fibroblast cells during the early phase of an infection with human cytomegalovirus strain AD169. These preparations contained a single abundant transcript of 2.7 kb which was derived from each of the repeat sequences flanking the long unique region of the virus genome. The gene was unspliced and poly(A+)RNA derived from it continued to accumulate in the cytoplasm of cells during the later stages of infection. This gene and the surrounding region was sequenced. A potential open reading frame of 170 amino acids was identified close to the 5'-terminus of the gene; the 2.7 kb early transcript therefore contains a long untranslated 3'-sequence. The promoter sequences of the 2.7 kb early gene show homology with corresponding regions of the HSV-1 gD and the human hsp 70 genes.

Transcription of the immediate early genes of human cytomegalovirus strain AD169.

Cloned sub-genomic fragments of human cytomegalovirus strain AD169 were used to analyse immediate-early (IE) transcription in virus-infected cells. Transcriptionally active regions of the HCMV genome were identified by hybridising cytoplasmic IE poly(A)+-RNA with dot blots and Southern transfers of restriction endonuclease digests of recombinant plasmids. The size, number and, in some cases, the orientation of transcription of IE RNA species were determined. The most abundant IE mRNA (IE-1.95) was mapped at 0.0764-0.0865 map units. The transcription of two middle abundant (1.7 and 2.15 kb) IE RNAs was initiated immediately downstream, and in the same orientation as the IE-1.95 gene. A second transcriptionally active area was identified at 0.593-0.619 map units. Three mRNA species (IE-1.75, IE-3.8 and IE-4.8) were derived from this region. Additional minor IE transcription was also observed from other regions of the HCMV genome. Hybrid-selected translation was used to identify the polypeptides encoded by the major IE RNA species.

The structure of the major immediate early gene of human cytomegalovirus strain AD169.

The nucleotide sequence of the major immediate early (IE) gene of human cytomegalovirus strain AD169 was determined. The structure of the gene was examined by nuclease mapping and by sequence analysis of a cDNA clone made from IE mRNA. The gene encodes a spliced molecule of 1736 nucleotides, made up of four exon sequences of 121, 88, 185 and 1342 nucleotides. Three introns (827, 114 and 170n) were located near the 5' end of the gene. A single open reading frame starting in the second exon extends for 491 amino acids, corresponding to a protein of molecular weight 64000. The putative promoter region contains several short direct and inverted repeat sequences of 16, 18, 19 and 21 nucleotides, which extend 509n upstream from the transcription start site. The structure of the major IE gene and its protein product are discussed and compared with the corresponding IE gene from the Towne strain of HCMV.

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.

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.

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.

Therapeutic vaccines for cervical cancer: concept and clinical results.

Human papillomavirus (HPV) infection is associated with transformation and clonal expansion of infected epithelial cells, resulting in the production of a benign growth, i.e. a wart. Recently, however, HPV has emerged as the primary causative agent of cervical carcinoma, malignancy being associated with the presence of the viral genome (predominantly genotypes 16 and 18) in cancerous cells. The only HPV proteins reliably expressed in neoplastic lesions are the 'oncogenic' E6 and E7 proteins, that serve both as tumour-specific markers and potential targets for immunotherapeutic intervention. As intracellular (nuclear) proteins, the E6 and E7 gene products may be hidden from the humoral immune response. Attention has thus focused on the generation of a vaccine capable of inducing or stimulating a cellular immune response to HPV 16 and HPV 18 E6 and E7. Vaccine development has been constrained by the absence of an appropriate animal model, the oncogenic nature of E6 and E7 and technical difficulties associated with detection of cytotoxic T cell responses to these antigens. Despite these difficulties, vaccine strategies have now been devised based on immunisation with synthetic peptide, whole protein and a vaccinia virus recombinant. Phase I/II human clinical trials have been initiated, and preliminary results have demonstrated the induction of specific cellular immune responses after immunisation. The HPV-associated neoplasia in cervical cancer represents an excellent target for therapeutic intervention because the tumour-associated antigens are so clearly defined. As such, it provides an appropriate model for establishing the general principles of cancer immunotherapy in humans.

Production of replication-deficient adenovirus recombinants.

In essence, replication-deficient (RD) adenovirus (Ad) vectors can be considered to function as an extremely efficient DNA transfection system capable of providing transgene expression in up to 100% of cells both in vitro or in vivo. As researchers continue to realize the full potential of this vector system, discover novel applications, and further develop and enhance systems, the use of this vector system has increased exponentially. The exploitation of Ad recombinants in HCV research is encouraged by demonstration that the virus will efficiently infect and express transgenes in hepatocytes and that following iv inoculation, transgene expression can readily be detected in hepatocytes in the liver (1-5). A number of HCV proteins have now been expressed in Ad recombinants (6-8) whereas these vectors have also been used to deliver antisense and ribozyme molecules as prototype HCV therapeutics (9).

Cytomegalovirus destruction of focal adhesions revealed in a high-throughput Western blot analysis of cellular protein expression.

Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and post-translation regulation of cellular gene expression. High-throughput Western blotting (BD Biosciences Powerblot technology) with 1,009 characterized antibodies was therefore used to analyze and compare the effects of infection with attenuated high-passage strain AD169 and virulent low-passage strain Toledo at 72 hpi across gels run in triplicate for each sample. Six hundred ninety-four proteins gave a positive signal in the screen, of which 68 from strain AD169 and 71 from strain Toledo were defined as being either positively or negatively regulated by infection with the highest level of confidence (BD parameters). In follow-up analyses, a subset of proteins was selected on the basis of the magnitude of the observed effect or their potential to contribute to defense against immune recognition. In analyses performed at 24, 72, and 144 hpi, connexin 43 was efficiently downregulated during HCMV infection, implying a breakdown of intercellular communication. Mitosis-associated protein Eg-5 was found to be differentially upregulated in the AD169 and Toledo strains of HCMV. Focal adhesions link the actin cytoskeleton to the extracellular matrix and have key roles in initiating signaling pathways and substrate adhesion and regulating cell migration. HCMV suppressed expression of the focal-adhesion-associated proteins Hic-5, paxillin, and alpha-actinin. Focal adhesions were clearly disrupted in HCMV-infected fibroblasts, with their associated intracellular and extracellular proteins being dispersed. Powerblot shows potential for rapid screening of the cellular proteome during HCMV infection.

Gene therapy and viral vaccination: the interface.

Live viral vaccines have had a major impact on the incidence of acute virus infections world-wide. Virus infections recognised as future vaccine targets will require a modified approach based on a detailed understanding of the immunobiology of specific infections combined with the application of new technologies designed to generate specific and appropriate protective immunity. A similar vector technology directed at in vivo gene delivery is currently being exploited both for gene therapy and vaccination. The induction of an immune response to an expressed transgene represents a potential hazard for a gene therapy protocol but is the object of a vaccine strategy. In vivo gene delivery using replication-competent or replication-deficient viral vector systems and by direct transfer of naked DNA can generate an effective humoral, secretory and cell-mediated immune response to expressed transgenes.