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Biomedical subjects

G Sutter

Publications and source records attributed to G Sutter.

At least 19 recordsLinked to original sources
PubMed2006

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis.

Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DeltaE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DeltaF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.

Amino Acid Sequence↗
PubMed2004

Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen.

The immunogenicity and protective efficacy of a DNA and recombinant modified vaccinia Ankara (MVA) vaccine administered by two different routes were investigated. DNA expressing HIV-1 IIIB env, gag, RT, rev, tat and nef, and MVA expressing HIV-1 IIIB nef, tat and rev and simian immunodeficiency virus (SIV) macJ5 gag/pol and vaccinia HIV-1 env, were used as immunogens. Four cynomolgus macaques received DNA intramuscularly (i.m.) at month 0 and intrarectally (i.r.) and intra-orally (i.o.) at 2 months, followed by MVA i.m. at 4 months and i.r. and i.o. at 8 months. Another group of four monkeys received the same immunogens but only i.m. Overall, stronger cellular immune responses measured by ELISPOT and T-cell proliferation assay were detected in the group primed i.m. and boosted mucosally. Following homologous intravenous simian-human immunodeficiency virus (SHIV) challenge, one of eight vaccinated animals was completely protected. This monkey, immunized i.m. and i.r.+i.o., exhibited the highest levels of HIV Env, Nef and Tat antibodies, high HIV Tat cytotoxic T-lymphocyte activity and T-lymphocyte proliferative responses to HIV Env. Four weeks post-challenge none of the monkeys immunized i.m. and i.r.+i.o., and only two out of four animals immunized i.m., demonstrated detectable plasma viral RNA levels. In contrast, all eight control animals had demonstrable plasma viral RNA levels 4 weeks post-challenge. Thus, stronger cellular immune responses and reduction of challenge virus burden were demonstrated in animals immunized i.m. as well as mucosally, compared with animals immunized i.m. only. The breadth and magnitude of the induced immune responses correlated with protective efficacy.

Animals↗
PubMed2001

Immune escape of melanoma: first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway.

Sequence analyses of the transporter associated with antigen processing (TAP) in tumor cell lines with deficient MHC class I surface expression identified a bp deletion at position 1489 near the ATP-binding domain of Tap1, causing a frameshift in one melanoma cell line. The impaired TAP1 protein expression was associated with deficient TAP2 protein expression, peptide binding, translocation, and MHC class I surface expression. Stable TAP1 gene transfer reconstitutes the described defects, whereas lysis by HLA-A2-restricted CTLs was still abrogated. This was attributable to a 2-bp insertion at position 890 in the HLA-A2 gene and was corrected after HLA-A2 cotransfection. This study describes for the first time mutations in two distinct components of the MHC class I antigen processing pathway, suggesting an immune selection against CTLs recognizing both TAP-dependent and -independent T-cell epitopes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Enhanced simian immunodeficiency virus-specific immune responses in macaques induced by priming with recombinant Semliki Forest virus and boosting with modified vaccinia virus Ankara.

The immunogenicity of two vector-based vaccines, either given alone or in a prime-boost regimen, was investigated. Cynomolgus macaques were immunised with modified vaccinia virus Ankara (MVA) expressing simian immunodeficiency virus (SIV)macJ5 env, gag-pol, nef, rev, and tat genes (MVA-SIVmac) or primed with a Semliki forest virus (SFV) vaccine expressing the same genes (SFV-SIVmac) and boosted with MVA-SIVmac. Generally, antibody responses, T-cell proliferative responses and cytotoxic T-cell responses remained low or undetectable in vaccinees receiving MVA-SIVmac or SFV-SIVmac alone. In contrast, monkeys who first received SFV-SIVmac twice and then were boosted with MVA-SIVmac showed increased antibody responses as well as high T-cell proliferative responses. Three of these vaccinees had cytotoxic T-lymphocytes directed against three or four of the gene products. No evidence of protection was seen against an intrarectal heterologous SIVsm challenge given 3 months after the last immunisation. The study demonstrates a prime-boost strategy that efficiently induces both humoral and cellular immune responses.

AIDS Vaccines↗
PubMed2001

Functional evaluation of HIV/SIV Nef as superantigen.

It is speculated that a virus-encoded superantigen is involved in the pathogenesis of human and simian immunodeficiency virus infections and that the accessory protein Nef might be that superantigen. We are able to show, using a murine superantigen screening system, that Nef does not display features characteristic of a superantigen. Upon transfection into MHC class II expressing antigen-presenting cells, it is expressed, but fails to induce Vbeta-specific expansion of peripheral T lymphocytes, which is a characteristic feature of superantigens in mixed lymphocyte culture. Therefore, we cannot support the hypothesis that Nef is a superantigen. The observations in favor of that hypothesis must be explained by other mechanisms.

Animals↗
PubMed2001

Vaccination with recombinant modified vaccinia virus Ankara protects against measles virus infection in the mouse and cotton rat model.

Modified vaccinia virus Ankara (MVA) has been used as an experimental vaccine vector against respiratory infections. We have tested the safety and immunogenicity of a recombinant virus expressing the hemagglutinin of measles virus (MVA-MV-H) using the mouse model of measles virus induced encephalitis and the cotton rat model for respiratory infection. MVA-MV-H proved to induce a TH1 response, neutralizing antibodies and conferred protection against both encephalitis and lung infection. The cotton rat is very sensitive to infection with replication competent vaccinia virus. In these animals MVA-MV-H proved to be a very well tolerated vaccine. However, the efficiency in the presence of MV specific maternal antibodies was low (even using a prime-boost strategy) and therefore might have to be improved.

Animals↗
PubMed2000

A randomized, double blind study in young healthy adults comparing cell mediated and humoral immune responses induced by influenza ISCOM vaccines and conventional vaccines.

Although current influenza vaccines have been shown to reduce influenza-related morbidity and mortality, there is a desire to develop more efficacious products. Vaccines which can induce CD8(+) cytotoxic T cell (CTL) responses in addition to strong antibody responses may be more effective in preventing disease since it has been demonstrated that CTL contribute to protective immunity, even against drift variants of influenza A viruses. The immunogenicity of two types of experimental influenza vaccines, which were based on immune stimulating complexes (ISCOM), were evaluated and compared with a conventional non-adjuvanted inactivated split virion vaccine, after immunization of human volunteers. In this randomized, double blind study, it was shown that the ISCOM vaccines altered the kinetics of the serum antibody response, resulting in more rapid titer rises against the vaccine strains. This accelerated antibody response coincided with enhanced in vitro proliferative T cell responses, which were observed shortly after vaccination. In addition, CTL responses were observed in a higher proportion of the vaccinees receiving an ISCOM vaccine, than in vaccinees receiving the conventional influenza vaccine.

Adjuvants, Immunologic↗
PubMed2000

Transporter (TAP)- and proteasome-independent presentation of a melanoma-associated tyrosinase epitope.

The melanosomal protein tyrosinase is considered as a target of specific immunotherapy against melanoma. Two tyrosinase-derived peptides are presented in association with HLA-A2.1 [Wölfel et al., Eur. J. Immunol., 24, 759-764 (1994)]. Peptide 1-9 (MLLAVLYCL) is generated from the putative signal sequence. The internal peptide 369-377 is posttranslationally converted at residue 371, and its presentation is dependent on functional TAP transporters and proteasomes [Mosse et al., J. exp. Med.187, 37-48 (1998)]. Herein, we report on the processing and transport requirements for the signal sequence-derived peptide 1-9 that were studied in parallel to those for peptide 369-377. After infection of TAP-deficient (T2) and TAP-positive (T1) cells with a Modified Vaccinia Ankara construct carrying the human tyrosinase gene (MVA-hTyr), we found that recognition by CTL against peptide 1-9 did not require TAP function as opposed to recognition by CTL against peptide 369-377. When target cells with intact processing and transport functions were infected with MVA-hTyr, lysis by CTL against peptide 1-9 was not impaired by lactacystin, a specific inhibitor for the proteasome, whereas lysis by CTL against peptide 369-377 was completely abrogated. Taken together, peptide 1-9 derived from the signal sequence of tyrosinase is presented in a TAP-independent fashion and does not require proteasomes for processing. Cellular immune responses against this hydrophobic peptide can be monitored with lymphokine spot assays as documented in the case of a patient with metastatic melanoma, in whom we observed a preferential T-cell response against tyrosinase peptide 1-9 subsequent to chemoimmunotherapy. Independence of cytosolic processing and transport pathways and potentially enhanced expression levels make signal sequence-derived peptides and their carrier proteins important candidates for specific immunotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Human tumor growth is inhibited by a vaccinia virus carrying the E2 gene of bovine papillomavirus.

BACKGROUND: Papillomavirus is the etiologic agent associated with cervical carcinoma. The papilloma E2 protein is able to regulate negatively the expression of E6 and E7 papilloma oncoproteins. Therefore, a new, highly attenuated vaccinia virus known as modified vaccinia virus Ankara (MVA), which carries the papillomavirus E2 gene, was used for the treatment of tumors associated with human papillomavirus. METHODS: Analysis of expression of the E2 gene from the recombinant vaccinia virus was performed by reverse transcription-polymerase chain reaction of RNA isolated from infected cells. Detection of the E2 protein was done by immunoprecipitation from proteins labeled with [(35)S]-methionine, isolated from infected cells. The therapeutic effect of the MVA E2 recombinant virus over human tumors was tested in nude mice bearing tumors generated by inoculation of HeLa cells. Series of 10 nude mice with tumors of different sizes were injected with MVA, MVA E2, or phosphate-buffered saline. Tumor size was monitored every week to assess growth. RESULTS: The MVA E2 recombinant virus efficiently expressed the E2 protein in BS-C-1 cells. This protein was able to repress, in vivo, the papillomavirus P105 promoter, which controls the expression of the E6 and E7 oncoproteins. In nude mice the MVA E2 virus reduced tumor growth very efficiently. In contrast, tumors continued to grow in mice treated with MVA or PBS. The life expectancy of MVA E2-treated mice was also increased three- to fourfold compared with that of animals that received MVA or PBS. CONCLUSIONS: The growth of human tumors was efficiently inhibited by the MVA E2 recombinant vaccinia virus. The absence of side effects in treated animals suggested that the MVA E2 virus is a safe biologic agent that could in the future be used in humans for the treatment of cervical carcinoma.

3T3 Cells↗
PubMed2000

A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens.

A large number of recombinant of viral and bacterial systems have been engineered as vectors to express foreign genes for vaccination and/or gene therapy. A common problem is the immune response to the vector itself. The presence of anti-vector immune responses may preclude sufficient 'priming' or immunogenicity if pre-existing immune responses are present, or they may impair optimal 'boosting' upon repeated immunization or delivery with the same vector. To circumvent this problem we developed a strategy using different chimeric vectors which share only the expression of common specific antigens desired for immunization. This approach not only has the advantage of avoiding increased anti-vector responses, but allows the use of combinations of vectors which could subsequently present the same or related antigen differently to the immune system as well as at alternative sites to induce the optimal type of immunity against the pathogen of interest.

Animals↗
PubMed2000

Transient host range selection for genetic engineering of modified vaccinia virus Ankara.

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.

Animals↗
PubMed1999

Stable expression of HIV-1 Nef induces changes in growth properties and activation state of human astrocytes.

OBJECTIVE: Nef was shown to be the predominant viral protein expressed in HIV-1-infected astrocytes in vivo and in vitro suggesting a distinct role of Nef in this cell type. Nef-induced activation of T cells is well described, whereas the functional activities of Nef in astrocytes are unknown. Our aim was to examine the effect of Nef on growth properties and activation of astrocytes. DESIGN: Human Nef-expressing astrocytic cell lines were established by stable transfection with different wild-type and mutant nef genes derived from laboratory isolates and brain tissue. METHODS: Nef-expressing astrocytes were characterized in terms of growth properties (proliferation, growth in soft agar, focus formation) and morphology. Apoptotic cell death and expression of activation markers were determined by fluorescent antibody cell sorting. RESULTS: Astrocytic cell lines revealed persistent Nef expression--detectable at the levels of mRNA and protein--and showed altered growth properties and morphology. Elevated expression of activation markers such as glial fibrillary acidic protein and CD88 (complement receptor C5a) was observed; these are regarded as markers for inflammatory processes in the brain. This effect was independent of the nef type or the expression level of the Nef protein. In contrast with previous reports no evidence for increased apoptotic cell death was found in astrocytes expressing Nef stably. CONCLUSIONS: Our findings suggest that Nef changes the cellular properties of astrocytes, thus contributing to astrocyte activation and induction of astrogliosis in the central nervous system of individuals with AIDS.

Apoptosis↗
PubMed1999

Humoral and cellular immune responses to HIV-1 nef in mice DNA-immunised with non-replicating or self-replicating expression vectors.

OBJECTIVE: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an effective immunogen in humoral and cellular immune responses. We have used two different self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their efficiency in raising humoral and cellular immune responses. DESIGN AND METHODS: The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. RESULTS: Efficient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-specific cytotoxic T lymphocyte (CTL) responses within four weeks. CONCLUSIONS: We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

AIDS Vaccines↗
PubMed1999

Modified vaccinia virus Ankara for delivery of human tyrosinase as melanoma-associated antigen: induction of tyrosinase- and melanoma-specific human leukocyte antigen A*0201-restricted cytotoxic T cells in vitro and in vivo.

Vaccination with tumor-associated antigens is a promising approach for cancer immunotherapy. Because the majority of these antigens are normal self antigens, they may require suitable delivery systems to promote their immunogenicity. A recombinant vector based on the modified vaccinia virus Ankara (MVA) was used for expression of human tyrosinase, a melanoma-specific differentiation antigen, and evaluated for its efficacy as an antitumor vaccine. Stable recombinant viruses (MVA-hTyr) were constructed that have deleted the selection marker lacZ and efficiently expressed human tyrosinase in primary human cells and cell lines. Tyrosinase-specific human CTLs were activated in vitro by MVA-hTyr-infected, HLA-A*0201-positive human dendritic cells. Importantly, an efficient tyrosinase- and melanoma-specific CTL response was induced in vitro using MVA-hTyr-infected autologous dendritic cells as activators for peripheral blood mononuclear cells derived from HLA-A*0201-positive melanoma patients despite prior vaccination against smallpox. Immunization of HLA-A*0201/Kb transgenic mice with MVA-hTyr induced A*0201-restricted CTLs specific for the human tyrosinase-derived peptide epitope 369-377. These in vivo primed CTLs were of sufficiently high avidity to recognize and lyse human melanoma cells, which present the endogenously processed tyrosinase peptide in the context of A*0201. Tyrosinase-specific CTL responses were significantly augmented by repeated vaccination with MVA-hTyr. These findings demonstrate that HLA-restricted CTLs specific for human tumor-associated antigens can be efficiently generated by immunization with recombinant MVA vaccines. The results are an essential basis for MVA-based vaccination trials in cancer patients.

Animals↗
PubMed1999

Feline calicivirus capsid protein expression and capsid assembly in cultured feline cells.

The capsid protein of feline calicivirus (FCV) was expressed by using plasmids containing cytomegalovirus, simian virus 40, or T7 promoters. The strongest expression was achieved with the T7 promoter and coinfection with vaccinia virus expressing the T7 RNA polymerase (MVA/T7pol). The FCV precursor capsid protein was processed to the mature-size protein, and these proteins were assembled in to virus-like particles.

Animals↗
PubMed1998

Highly attenuated modified vaccinia virus Ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells.

Although desirable for safety reasons, the host range restrictions of modified vaccinia virus Ankara (MVA) make it less applicable for general use. Propagation in primary chicken embryo fibroblasts (CEF) requires particular cell culture experience and has no pre-established record of tissue culture reproducibility. We investigated a variety of established cell lines for productive virus growth and recombinant gene expression. Baby hamster kidney cells (BHK), a well-characterized, easily maintained cell line, supported MVA growth and as proficient expression of the E. coli lacZ reporter gene as the highly efficient CEF, whereas other cell lines were non-permissive or allowed only very limited MVA replication. Importantly, no virus production occurred in patient-derived infected primary human cells. These results emphasize the safety and now improved accessibility of MVA for the development of expression vectors and live recombinant vaccines.

Animals↗