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M Schroff

Publications and source records attributed to M Schroff.

8 recordsLinked to original sources
PubMed2004

DNA immunisation with minimalistic expression constructs.

The low efficacy obtained in large animals makes plasmid-based DNA vaccines commercially unviable. Another concern is the presence of antibiotic resistance markers on virtually all conventional plasmids. Here we describe the use of minimalistic, immunogenically defined gene expression (MIDGE) vectors for DNA vaccination. MIDGE are linear, covalently-closed vectors containing all the essential information for gene expression and none of the non-essential and potentially dangerous plasmid backbone sequences. MIDGE vectors can also be chemically modified on both ends at defined positions allowing targeting of the DNA to specific cell types or cellular compartments. Immunisation of mice with simple and end-modified MIDGE vectors showed that they are efficacious tools to generate and/or manipulate antigen-specific immune responses.

Animals↗
PubMed2002

Up-regulation by feline interleukin-4 and down-regulation by feline interferon-gamma of major histocompatibility complex class II on cat B-lymphocytes.

Interleukin-4 (IL-4) exhibits numerous biological and immunoregulatory functions on B- and T-lymphocytes, monocytes, and dendritic cells in both mice and humans. In the present study, we show that IL-4 also has a regulatory function in the cat species. Cells transfected with IL-4 DNA expressed a biologically active protein as demonstrated by the up-regulation of MHC class II molecules on B-lymphocytes (CD21(+)) in a flow cytometric assay. Increased levels of MHC class II expression on CD21(+) cells were seen in 11 out of 12 cats (p<0.05). In addition, 12 out of 12 cats showed up-regulation of MHC class II on CD21(-) cells, mainly consisting of T-lymphocytes (p<0.05). In contrast, concanavalin A (ConA)-induced culture supernatant from peripheral blood mononuclear cells (PBMCs) containing high levels of interferon-gamma (IFN-gamma) transcripts induced down-regulation of MHC class II molecules on CD21(+) cells of all samples (p<0.05). Variable results were observed for CD21(-) cells incubated with ConA-conditioned medium (p=0.71). The nature of the cytokine(s) responsible for these effects remains to be determined. However, the fact that down-regulation of MHC class II molecules on B cells occurred in all cats tested suggests that IFN-gamma may be involved. These data provide further insight into the mechanism by which MHC class II expression is regulated in feline lymphocytes, and suggest that the Th1/Th2 paradigm is also present in the cat.

Animals↗
PubMed2001

A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA.

Viral and plasmid vectors may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes contained in their sequence. Furthermore, replication-defective viral vectors carry the potential risk of recombination with wild-type viruses or activation of oncogenes. A new vector type for minimalistic, immunologically defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal-size gene transfer unit containing the expression cassette, including promoter, gene, and RNA-stabilizing sequence, flanked by two short hairpin oligonucleotide sequences. The resulting vector is a small, linear, covalently closed, dumbbell-shaped molecule. DNA not encoding the desired gene is reduced to a minimum. Here, we transfected colon carcinoma cell lines using cationic lipid, cationic polymer, and electroporation with several MIDGE vectors and corresponding plasmids containing transgenes encoding enhanced green fluorescent protein (eGFP) and human interleukin-2 (hIL-2). Transfection efficiency as measured qualitatively and quantitatively with eGFP was found to be comparable for both vector types. However, hIL-2 secretion and eGFP expression were approximately two- to fourfold higher in most cells transfected with these transgenes using MIDGE vectors compared to the plasmid control. This report demonstrates the advantages of this new vector type and its prospects for ex vivo gene therapy studies.

Cations↗
PubMed2001

Priming of immune responses to hepatitis B surface antigen with minimal DNA expression constructs modified with a nuclear localization signal peptide.

Nuclear localization signal (NLS) peptides conjugated to DNA increase transfection efficiency in vitro. We tested in mice whether conjugation of NLS peptides to DNA vaccines enhances their immunogenicity after intramuscular injection or gene gun mediated intradermal delivery. We constructed the plasmid pMOK-HBsAY that contains a transcription unit encoding hepatitis B surface antigen (HBsAg) and bacterial sequences for amplification of plasmid DNA. From this plasmid we derived the minimal expression construct pMOK-HBsAY-MIDGE, a covalently closed linear DNA that contains only the HBsAg transcription unit. Both constructs stimulated similar (predominantly IgG1) antibody response to HBsAg after gene gun immunization. In contrast, pMOK-HBsAY plasmid DNA was more efficient than pMOK-HBsAY-MIDGE DNA in priming predominantly IgG2a antibody responses to HBsAg after intramuscular injection. Both constructs efficiently primed cytotoxic T lymphocyte responses after intramuscular immunization. When a NLS peptide was coupled to the pMOK-HBsAY-MIDGE DNA, HBsAg transfection efficiency in vitro and priming of antibody responses to HBsAg after intramuscular (but not gene gun mediated) injection was enhanced 10- to 15-fold. These data show: (a) MIDGE constructs can be used as DNA vaccines indicating that bacterial sequences are not essential cofactors; and (b) in intramuscular (but not gene gun mediated) delivery the immunogenicity of a MIDGE-based vaccine is enhanced by coupling NLS peptides to the vector DNA.

Animals↗
PubMed2000

Protection against FIV challenge infection by genetic vaccination using minimalistic DNA constructs for FIV env gene and feline IL-12 expression.

OBJECTIVE: To evaluate the efficacy of a genetic vaccination protocol based on minimalistic, immunogenic defined gene expression (MIDGE) vectors coding for domains of the feline immunodeficiency virus (FIV) env gene and feline IL-12. METHODS: Three groups of four cats each were immunized three times within 6 weeks by the ballistic transfer of gold particles coated with MIDGE vectors. Group 1 received non-coated gold beads, groups 2 and 3 MIDGE vectors expressing FIV surface plus part of the transmembrane protein. In addition, group 3 received feline IL-12 DNA. All cats were challenged by intraperitoneal injection of 25 TCID50 of infectious FIV Z2. The following criteria were monitored: clinical signs, antibodies to transmembrane protein, antibodies to whole FIV, haematological parameters and kinetics of CD4 and CD8 cells, FIV proviral load (determined by quantitative polymerase chain reaction; PCR) and cytotoxic T lymphocyte (CTL) activity (in selected cats). RESULTS: None of the cats developed a detectable antibody response during immunizations. Four weeks after challenge exposure, all cats in group 1 (control) and group 2 (FIV surface-transmembrane protein) had seroconverted and showed a high proviral load until week 19 (end of experiment). In contrast, only one of four cats in group 3 (surface-transmembrane protein and IL-12) showed antibodies; it was provirus positive at reduced virus load. Short-lived CTL activity was found in two cats in group 3. CONCLUSION: Genetic vaccination using a MIDGE-based construct for the expression of the surface-transmembrane protein domain of FIV env and feline IL-12 DNA led to protection against homologous virus challenge in three out of four vaccinated cats.

Animals↗
PubMed2000

Immunization of cats against feline immunodeficiency virus (FIV) infection by using minimalistic immunogenic defined gene expression vector vaccines expressing FIV gp140 alone or with feline interleukin-12 (IL-12), IL-16, or a CpG motif.

Four groups of cats, each containing four animals, were immunized at 0, 3, and 6 weeks with minimalistic immunogenic defined gene expression vector (MIDGE) vaccines containing the gene(s) for feline immunodeficiency virus (FIV) gp140, FIV gp140 and feline interleukin-12 (IL-12), FIV gp140 and feline IL-16, or FIV gp140 and a CpG motif. MIDGEs were coated onto gold beads and injected intradermally with a gene gun. A fifth group of four cats were immunized in an identical manner but with blank gold beads. All cats were challenge exposed to virulent FIV 4 weeks following the final immunization, and the course of infection was monitored. The two groups of cats immunized with the FIV gp140 gene alone or with blank gold particles became highly viremic and seroconverted as early as 4 weeks after infection. In contrast, three of four cats immunized with FIV gp140 in combination with feline IL-12 failed to become viremic or seropositive, as has been shown elsewhere (F. S. Boretti, C. M. Leutenegger, C. Mislin, et al., AIDS 14:1749-1757, 2000). Here we show the effect of IL-12 when used as an adjuvant on the viral RNA and DNA load and on the cytokine profile. In addition, the two groups of cats immunized either with gp140 and IL-16 or with gp140 and the CpG had greatly reduced viremia. Protection correlated weakly with cytotoxic T-lymphocyte activity and increased cytokine transcription of IL-12, gamma interferon, and IL-10 by peripheral blood mononuclear cells in the postchallenge period. This study extends the data on IL-12 and provides new results on CpG motifs and IL-16 used as adjuvants in the FIV cat model.

Animals↗
PubMed1998

Vaccination with IL-7 gene-modified autologous melanoma cells can enhance the anti-melanoma lytic activity in peripheral blood of patients with a good clinical performance status: a clinical phase I study.

Recently, cytokine gene transfer into tumour cells has been shown to mediate tumour regression in animal models via immunomodulation. Consequently, a number of clinical protocols have been developed to treat cancer patients with cytokine gene-modified tumour cells. Here, we report the results of a clinical phase I trial using for the first time autologous, interleukin 7 gene-modified tumour cells for vaccination of ten patients with disseminated malignant melanoma. Melanoma cells were expanded in vitro from surgically removed metastases, transduced by a ballistic gene transfer technique and were then injected after in vitro irradiation s.c. at weekly intervals. Clinically, there was no major toxicity except for mild fever, and no major clinical response towards vaccination was observed. Eight of ten patients completed the initial three s.c. vaccinations and were eligible for immunological evaluation. Post vaccination, peripheral mononuclear cells (PBMCs) were found to contain an increased number of tumour-reactive proliferative as well as cytolytic cells, as determined by a limiting dilution analysis. In three of six patients, the frequencies of anti-melanoma cytolytic precursor cells increased between 2.6- and 28-fold. Two of these patients showed a minor clinical response. Analysis of the autologous tumour cell vaccines regarding IL-7 secretion after gene transfer, HLA class I and class II cell surface expression, secretion of immunosuppressive mediators (TGF-beta1, IL-10) and various melanoma-associated tumour antigens revealed a very diverse expression profile. In conclusion, vaccination using gene-modified autologous melanoma cells induced immunological changes in a group of advanced, terminally ill patients. These changes can be interpreted as an increased anti-tumour immune response. However, immunological modulation was most pronounced in patients in good physical condition. Therefore, patients with minimal tumour load or minimal residual disease might preferentially benefit from tumour cell vaccination in further studies. In order to evaluate the effects of the cytokine gene-modified tumour cell vaccines more precisely, an antigenically better defined vaccine is needed.

Adult↗