Forskningsresultater 2000

FLT3 Ligand Preserves the Uncommitted CD34+CD38- Progenitor Cells During Cytokine Prestimulation for Retroviral Transduction.

Nielsen SD, Husemoen LL, Sorensen TU, Gram GJ, Hansen J-ES. Laboratory for Infectious Diseases, H:S Hvidovre Hospital, 2650 Hvidovre, Denmark. J Hematother Stem Cell Res 2000 Oct;9(5):695-701

Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this study, a retroviral vector carrying the genes for the enhanced version of green fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used for transduction of CD34(+) cells. The effect of cytokine prestimulation on transduction efficiency and the population of uncommitted CD34(+)CD38(-) cells was determined. CD34(+) cells harvested from umbilical cord blood were kept in suspension cultures and stimulated with combinations of the cytokines stem cell factor (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 prior to transduction. Expression of the two genes was assessed by flow cytometry and determination of neomycin-resistant colonies in a selective colony-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed in a higher percentage of cells than the EGFP gene, but there seemed to be a positive correlation between expression of the two genes. The effect of cytokine prestimulation was therefore monitored using EGFP as marker for transduction. When SCF was compared to SCF in combination with more potent cytokines, highest transduction efficiency was found with SCF and IL-3 and IL-6 (5.05% +/- 0.80 versus 2.66% +/- 0.53 with SCF alone, p = 0.04). However, prestimulation with SCF in combination with IL-3 and IL-6 also reduced the percentage of CD34(+) cells (p = 0.02). Then, prestimulation with SCF and FLT3 ligand was compared. Significant difference in transduction efficiency was not found. Interestingly, FLT3 ligand seemed to preserve the population of CD34(+)CD38(-) cells compared to SCF (16.56% +/- 2.02 versus 9.39% +/- 2.35, p = 0.03). In conclusion, prestimulation with potent cytokine combinations increased the transduction efficiency, but reduced the fraction of CD34(+) cells. Importantly, the use of FLT3 ligand seemed to preserve the population of uncommitted cells.

Construction, biological activity, and immunogenicity of synthetic envelope DNA vaccines based on a primary, CCR5-tropic, early HIV type 1 isolate (BX08) with human codons.

Corbet S, Vinner L, Hougaard DM, Bryder K, Nielsen HV, Nielsen C, Fomsgaard A. Department of Virology Statens Serum Institute, DK-2300 Copenhagen, Denmark. AIDS Res Hum Retroviruses 2000 Dec 10;16(18):1997-2008

So far codon-optimized HIV-1 envelope genes have been investigated for the T cell line-adapted strain MN, which differs in several aspects from primary isolates. Envelopes of primary isolates may be more relevant for vaccine purposes. This article describes for the first time the engineering and characterization of four "humanized" genes encoding the secreted gp120/gp140, or the membrane-bound gp150/gp160, of a primary CCR5 tropic, clade B, clinical isolate HIV-1(BX08). The genes were built in fragments for easy cassette exchange of regions important for immunogenicity, function, and expression. The transcription and expression of the synthetic genes in mammalian cell lines were Rev independent and highly increased. Increased expression of membrane-bound gp160 induced a high cytopathic effect in U87.CD4.CCR5 cells. Gene gun and intramuscular DNA vaccination in mice induced a strong specific cytotoxic T lymphocyte response independent of the gene construct, expression level, or DNA immunization route. In contrast, the highest anti-gp120 antibody levels were induced by synthetic genes encoding the secreted glycoproteins followed by gp160/gp150. Unlike HIV-1(MN), HIV-1(BX08) V3 was not immune dominant. Despite the high antibody response only low and inconsistent neutralizing titers to the homologous HIV-1 isolate were measured. However, neutralization of SHIV89.6P could be obtained. Thus, the neutralizing epitopes on the cell line-adapted SHIV89.6P and HIV-1(MN) may be more antigenically available for the cross-neutralizing antibodies induced. In conclusion, complete "humanization" of the DNA vaccine genes failed to induce a consistent neutralizing antibody response, albeit expression and immunogenicity of the primary HIV-1 glycoproteins were greatly improved. Optimization in terms of improving neutralization may require further modifications of the DNA vaccine gene. The synthetic cassette construct described is a convenient tool developed to investigate this further.

Two widely spaced initiator binding sites create an HMG1-dependent parvovirus rolling-hairpin replication origin

Cotmore SF, Christensen J, Tattersall P. Departments of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA. J Virol. 2000 Feb;74(3):1332-41

Minute virus of mice (MVM) replicates via a linearized form of rolling-circle replication in which the viral nickase, NS1, initiates DNA synthesis by introducing a site-specific nick into either of two distinct origin sequences. In vitro nicking and replication assays with substrates that had deletions or mutations were used to explore the sequences and structural elements essential for activity of one of these origins, located in the right-end (5') viral telomere. This structure contains 248 nucleotides, most-favorably arranged as a simple hairpin with six unpaired bases. However, a pair of opposing NS1 binding sites, located near its outboard end, create a 33-bp palindrome that could potentially assume an alternate cruciform configuration and hence directly bind HMG1, the essential cofactor for this origin. The palindromic nature of this sequence, and thus its ability to fold into a cruciform, was dispensable for origin function, as was the NS1 binding site occupying the inboard arm of the palindrome. In contrast, the NS1 site in the outboard arm was essential for initiation, even though positioned 120 bp from the nick site. The specific sequence of the nick site and an additional NS1 binding site which directly orients NS1 over the initiation site were also essential and delimited the inboard border of the minimal right-end origin. DNase I and hydroxyl radical footprints defined sequences protected by NS1 and suggest that HMG1 allows the NS1 molecules positioned at each end of the origin to interact, creating a distortion characteristic of a double helical loop.

Application of the polymerase chain reaction in determination of recombinant retrovirus titers as fifty percent endpoints

Husemoen LL, Gram GJ, Hansen JES. Laboratory for Infectious Diseases, Hvidovre Hospital, Denmark. APMIS 2000 Jan;108(1):38-44

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. One of the most laborious routine assays in the application of retroviral-mediated gene transfer is the determination of viral titers of vector producer cell lines. Traditionally, determination of virus titer involves the testing of culture medium from individual packaging cell lines for the ability to transfer drug resistance to susceptible cells - a process that can easily take up to 14 days. It is generally agreed that this method is cumbersome. We sought to develop PCR-based protocols that would significantly simplify and shorten this procedure. Using PCR and primers specific for the Neoregion of the MLV-derived vector LeGSN, we determined 1. the proviral integration in target cells, and 2. the viral nucleic acid (RNA or DNA) content of the vector stock. Results were compared with those using the conventional method. We found that these specific PCR-based procedures were indeed useful for rapid determination of viral titers as well as for quick screening for high-titer vector-producing cell clones and successful transduction of target cells.

(c) 1999, 2000
Siden er opdateret: 05. juni 2008