Forskningsresultater 1999

Two new members of the emerging KDWK family of combinatorial transcription modulators bind as a heterodimer to flexibly spaced PuCGPy half-sites

Christensen J, Cotmore SF, Tattersall P. Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA. Mol Cell Biol. 1999 Nov;19(11):7741-50

Initially recognized as a HeLa factor essential for parvovirus DNA replication, parvovirus initiation factor (PIF) is a site-specific DNA-binding complex consisting of p96 and p79 subunits. We have cloned and sequenced the human cDNAs encoding each subunit and characterized their products expressed from recombinant baculoviruses. The p96 and p79 polypeptides have 40% amino acid identity, focused particularly within a 94-residue region containing the sequence KDWK. This motif, first described for the Drosophila homeobox activator DEAF-1, identifies an emerging group of metazoan transcriptional modulators. During viral replication, PIF critically regulates the viral nickase, but in the host cell it probably modulates transcription, since each subunit is active in promoter activation assays and the complex binds to previously described regulatory elements in the tyrosine aminotransferase and transferrin receptor promoters. Within its recognition site, PIF binds coordinately to two copies of the tetranucleotide PuCGPy, which, remarkably, can be spaced from 1 to 15 nucleotides apart, a novel flexibility that we suggest may be characteristic of the KDWK family. Such tetranucleotides are common in promoter regions, particularly in activating transcription factor/cyclic AMP response element-binding protein (ATF/CREB) and E-box motifs, suggesting that PIF may modulate the transcription of many genes.

Theilade MD, Gram GJ, Jensen PB, Cianfriglia M, Rorth M, Hansen JES. Laboratory for Infectious Diseases, Department 144, Hvidovre Hospital, Denmark. APMIS 1999 Sep;107(9):851-8

Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC cells, and that MLV-A as well as GALV-1 retroviral vectors are suitable for further development of gene therapy in SCLC.

Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

Schønning K, Lund O, Lund OS, Hansen JES. Laboratory for Infectious Diseases 144, Hvidovre Hospital, DK-2650 Hvidovre, Denmark. J Virol 1999 Oct;73(10):8364-70

In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit  to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.

Transmission of a minor variant of transcriptionally active HIV-1

Machuca R, Schonning K, Hansen JES, Bruun L, Fomsgaard A, Nielsen C. Department of Virology, Statens Serum Institut, Copenhagen, Denmark. Scand J Infect Dis 1999;31(3):243-9

We investigated the genotypic variation of pro-viral human immunodeficiency virus type 1 (HIV-1) DNA in a virus donor-recipient pair by comparing sequences from the HIV-1 V3 region of the gp120 gene and the p17gag gene. We found that the transmitted virus was a minor variant in the donor's virus  population in blood. Possible selection mechanisms within the host (neutralization by antibodies) were studied in order to investigate whether antibodies could explain the conserved HIV genotype found in the recipient. In conclusion, our data indicate that a minor variant of pro-viral transcriptionally active HIV-1 found in PBMC was transmitted from donor to recipient. Development of a homogeneous genotype regarding the V3 region (V3d-B1(1)) of pro-viral DNA in the recipient's PBMC and a partially homogeneous genotype regarding the HIV-RNA was possibly caused by an envelope
associated selection that is not dependent upon neutralizing antibodies.

The N-linked glycan of the V3 region of HIV-1 gp120 and CXCR4-dependent multiplication of a human immunodeficiency virus type 1 lymphocyte-tropic variant

Losman B, Biller M, Olofsson S, Schonning K, Lund OS, Svennerholm B, Hansen JE, Bolmstedt A. FEBS Lett 1999 Jul 2;454(1-2):47-52

We have previously shown that an N-glycosylation site of N306 of HIV-1 gp120 is not necessary for the HIV-1 infectivity but protects HIV-1 from neutralising antibodies. In contrast Nakayama et al. [FEBS Lett. (1998) 426, 367-372], using a virus with an identical V3 region, suggested that elimination of this particular glycan reduced the ability of T-tropic HIV to bind to CXCR4 and hence its ability to infect T cell lines. We therefore re-examined the ability of a mutant virus, lacking the N306 glycan, to replicate in various types of cells and found no change in co-receptor usage for mutant virus. The ability of mutant virus to replicate or to induce syncytia in infected cells was similar to that of wild type virus. These results corroborate our original observation, confirming that the induced mutation in the N306 glycosylation site neither impairs nor improves the ability of mutant virus to replicate in permissive cells.

Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus

Martinez-Torrecuadrada, J. L.; Langeveld, J. P.; Venteo, A.; Sanz, A.; Dalsgaard, K.; Hamilton, W. D.; Meloen, R. H.; Casal, J. I. Virology 1999; 257, 449-59

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151- 200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179- 185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.

Production of a highly immunogenic subunit ISCOM vaccine against Bovine Viral Diarrhea Virus

Kamstrup, S.; Roensholt, L.; Jensen, M. H.; Dalsgaard, K. Vaccine 1999; 17, 1057-64

Bovine Viral Diarrhea Virus (BVDV) is a major pathogen of cattle in most countries. The main reservoir of virus in herds are BVDV persistently infected animals, which arise as a result of infection of the bovine fetus early in gestation. The spread of virus to the unborn fetus may be prevented by vaccination of the dam. We describe in this report the production and initial testing of an inactivated subunit vaccine against BVDV. The vaccine is based on production of antigen in primary bovine cell cultures, extraction of antigens from infected cells with detergent, chromatographic purification, concentration, and insertion of antigens into immune stimulating complexes (ISCOMs). Vaccines based on two different Danish strains of BVDV were injected into calves and the antisera produced were tested for neutralising activity against a panel of Danish BVDV strains. The two vaccines induced different neutralisation responses, which seem to partly complement each other. The implication of these observations for successful vaccination against BVDV is discussed.

Induction of cytotoxic T-cell responses by gene gun DNA vaccination with minigenes encoding influenza A virus HA and NP CTL-epitopes.

Fomsgaard A, Nielsen HV, Kirkby N, Bryder K, Corbet S, Nielsen C, Hinkula J, Buus S. Department of Virology, Statens Serum Institut, 5 Artillerivej, DK-2300, Copenhagen, Denmark. afo@ssi.dk Vaccine 1999 Nov 12;18(7-8):681-91

Cytotoxic T-lymphocyte (CTL) response is an important component of anti-viral immunity. CTLs are specific to short peptides presented by MHC-I molecules and immunisation with the exact peptide sequence introduced in the cytosol is therefore a minimal approach, which potentially affords a high degree of controllability. We have examined the induction of murine CTL's by this approach using DNA plasmid minigene vaccines encoding known mouse K(k) minimal CTL epitopes (8 amino acids) from the influenza A virus hemagglutinin and nucleoprotein. We here report that such an approach is feasible and that wild type influenza virus flanking amino acid sequences can influence the CTL response but are not essential for optimal CTL induction. We also examined the effect of different new amino acid sequences flanking the CTL epitopes. In one version, two CTL epitopes were linked together as 'string of beads'. This did not improve CTL induction. In another version, one CTL epitope was inserted into a known T-helper protein (HBsAg). This did significantly augment the response probably due to immunological help from HBsAg Th epitopes. Finally, the CTL inducing minigene DNA vaccines were compared with Flu-induced CTL responses and tested for their protective effect against a lethal influenza A virus infection in mice and no effect was found. We conclude that a specific and highly directed CTL induction is possible by unlinked minigene DNA immunisation, but that CTL induction solely is not always sufficient to provide protection.

Mutations in CCR5-coding sequences are not associated with SIV carrier status in African nonhuman primates.

Muller-Trutwin MC, Corbet S, Hansen J, Georges-Courbot MC, Diop O, Rigoulet J, Barre-Sinoussi F, Fomsgaard A. Unite de Biologie des Retrovirus, Institut Pasteur, Paris, France, Department of Virology, Statens Serum Institut, Copenhagen, Denmark. AIDS Res Hum Retrovi-ruses 1999 Jul 1;15(10):931-9

African monkeys can be naturally infected with SIV but do not progress to AIDS. Since mutations in the human CCR5 gene have been shown to influence susceptibility to HIV infection and disease progression, we have now investigated whether mutations in CCR5-coding sequences in African nonhuman primates can explain species-specific differences in susceptibility to lentiviral infection. The animals studied comprise chronically infected monkeys corresponding to four natural hosts of SIV (Cercopithecus aethiops, Cercopithecus pygerythrus, Cercopithecus sabaeus, and Cercopithecus tantalus), noninfected animals from three species that are known to be susceptible to SIV infection (Cercopithecus patas, Cercopithecus Ihoesti, and Pan troglodytes), and monkeys of six species that do not carry SIV in the wild (Cercocebus galeritus, Cercocebus aterrimus, Cercopithecus ascanius, Cercopithecus nictitans, Cercopithecus neglectus, and Cercopithecus cephus). We observed a high degree of genetic divergence among the species. The rate of accumulation of amino acid mutations was, however, not higher in SIV carriers than in other nonhuman primates. No homozygous premature stop codons, deletions, or frameshift mutations were detected. In at least two animals, one infected AGM (Cercopithecus tantalus) and one noninfected monkey (Cercocebus aterrimus), the CCR5 alleles identified encode functional proteins, as they were identical in terms of amino acid sequence to that of functional CCR5 reported in the literature. We found no other consistent differences in the genetic variability of CCR5-coding sequences between the nonhuman primates that are carriers of SIV and those that are not.

Gene gun DNA vaccination with Rev-independent synthetic HIV-1 gp160 envelope gene using mammalian codons.

Vinner L, Nielsen HV, Bryder K, Corbet S, Nielsen C, Fomsgaard A. Department of Virology, Statens Serum Institut, Copenhagen, Denmark. Vaccine 1999 Apr 23;17(17):2166-75

DNA immunization with HIV envelope plasmids induce only moderate levels of specific antibodies which may in part be due to limitations in expression influenced by a species-specific and biased HIV codon usage. We compared antibody levels, Th1/Th2 type and CTL responses induced by synthetic genes encoding membrane bound gp160 versus secreted gp120 using optimized codons and the efficient gene gun immunization method. The in vitro expression of syn.gp160 as gp120 + gp41 was Rev independent and much higher than a classical wt.gp160 plasmid. Mice immunized with syn.gp160 and wt.gp160 generated low and inconsistent ELISA antibody titres whereas the secreted gp120 consistently induced faster seroconversion and higher antibody titres. Due to a higher C + G content the numbers of putative CpG immune (Th1) stimulatory motifs were highest in the synthetic gp160 gene. However, both synthetic genes induced an equally strong and more pronounced Th2 response with higher IgG1/IgG2a and IFNgamma/IL-4 ratios than the wt.gp160 gene. As for induction of CTL, synthetic genes induced a somewhat earlier response but did not offer any advantage over wild type genes at a later time point. Thus, optimizing codon usage has the advantage of rendering the structural HIV genes Rev independent. For induction of antibodies the level of expression, while important, seems less critical than optimal contact with antigen presenting cells at locations reached by the secreted gp120 protein. A proposed Th1 adjuvant effect of the higher numbers of CpG motifs in the synthetic genes was not seen using gene gun immunization which may be due to the low amount of DNA used.

Multicenter quality assessment of PCR methods for detection of enteroviruses.

Muir P, Ras A, Klapper PE, Cleator GM, Korn K, Aepinus C, Fomsgaard A, Palmer P, Samuelsson A, Tenorio A, Weissbrich B, van Loon AM. Department of Virology, Guy's, King's College & St Thomas' Hospitals' School of Medicine, London, United Kingdom. p.muir@umds.ac.uk J Clin Microbiol 1999 May;37(5):1409-14

We conducted a multicenter evaluation of commercial and in-house PCR methods for the detection of enteroviruses. Three coded panels of test and control RNA samples, artificial clinical specimens, and representative enterovirus serotypes were used to assess amplification methods, RNA extraction methods, and reactivities with different enterovirus serotypes. Despite several differences between PCR methods, there was good agreement, although some variation in sensitivity was observed. Most PCR methods were able to detect enterovirus RNA derived from 0.01 50% tissue culture infective dose (TCID50) and were able to detect at least 1 TCID50 of enterovirus in cerebrospinal fluid, stool, or throat swab specimens. Most were also able to detect a wide range of enterovirus serotypes, although serotypic identification was not possible. Some laboratories experienced false-positive results due to PCR contamination, which appeared to result mainly from cross-contamination of specimens during RNA extraction. Provided that this problem is overcome, these PCR methods will prove to be a sensitive and rapid alternative to cell culture for the diagnosis of enterovirus infection.

Improved immunogenicity of HIV-1 epitopes in HBsAg chimeric DNA vaccine plasmids by structural mutations of HBsAg.

Bryder K, Sbai H, Nielsen HV, Corbet S, Nielsen C, Whalen RG, Fomsgaard A. Department of Vi-rology, Statens Serum Institut, Copenhagen, Denmark. DNA Cell Biol 1999 Mar;18(3):219-25

To improve the immunogenicity of epitopes from the envelope protein of HIV-1, we have developed gene gun-delivered subunit DNA vaccines by inserting the sequences encoding the V3 region into the hepatitis B virus (HBV) envelope gene, often called the surface antigen (HBsAg). We have examined the possibility of modifying the immune response to V3 by introducing modifications into the carrier HBsAg in gene gun DNA immunization of mice. In some plasmid constructions, the V3 sequence was introduced into the preS2 region of the HBsAg. Although this region is not present in all protein subunits of the HBsAg particles produced, abolishing the internal translational initiation site for the S protein had no effect on the immune response to V3. Expression of V3 at the N-terminal or C-terminal part of the HBsAg protein resulted in equal anti-V3 antibody and cytotoxic T-lymphocyte (CTL) responses. However, elimination of secretion by single amino-acid mutations in the HBsAg decreased the anti-HBsAg antibody response but enhanced the anti-V3 antibody response. In contrast, the CTL response to V3 was independent of the structural mutations but could be improved by a total deletion of the HBsAg sequence part. Thus, the immune response to heterologous epitopes can be altered by modifications in the carrier HBsAg protein. Modifications of the HBsAg carrier might interfere with the dominant immune response to the HBsAg epitopes, allowing better antibody induction to less immunogenic foreign epitopes. However, for induction of CTL responses, the expression of minimal epitopes may be advantageous.

HIV-1 DNA vaccines.

Fomsgaard A. Department of Virology, Statens Serum Institut, Copenhagen, Denmark. afo@ssi.dk Immunol Lett 1999 Jan;65(1-2):127-31

HIV-1 was among the original DNA vaccine targets and HIV DNA vaccines are now in human trials. Lack of strong correlates of protective immunity makes vaccine design difficult; however, DNA vaccines have the potential to be an ideal vaccine and therapeutic approach against HIV-1. DNA vaccines induce conformational-dependent antibodies, mimic live vaccines but without the pathogenic potential, and can easily be made polyvalent. Genes which encode important CTL and antibody epitopes can be included while those that confer pathogenicity, virulence, antibody enhancement or represent non-conserved epitopes can be excluded. In our hands pre-treatment of muscles with bupivacaine or cardiotoxin did not offer any advantage over no muscle pre-treatment or gene gun inoculation of skin although gene gun immunization seem to favour a Th2 type response. As DNA vaccine candidates we have compared vaccines encoding native HIV MN gp160 with Rev-independent synthetic genes encoding MNgp160 and MNgp120 using mammalian high expression codons. In these experiments the gene encoding secreted gp120 gave highest antibody neutralizing titers. High and fast antibody responses could also be obtained by transferring the HIV-1 MN V3 loop to the secreted HBsAg as a fusion gene vaccine. Thus, in the case of HIV-1 MN genes encoding secreted surface glycoproteins may be preferred instead of membrane bound envelopes. CTL responses were induced in all cases. However, in order to meet the high diversity of HIV and HLA types our approach is to include many CTL epitopes in a multivalent minigene vaccine. We found that gene gun DNA vaccination with minimal epitopes could induce specific CTL. Flanking sequences influenced the CTL response but was not needed. DNA vaccines encoding known and computer predicted CTL epitopes are now being developed.

Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice

Brennan, F. R.; Bellaby, T.; Helliwell, S. M.; Jones, T. D.; Kamstrup, S.; Dalsgaard, K.; Flock, J. I.; Hamilton, W. D. J Virol 1999; 73, 930-8

The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or in the presence of ISCOM matrix, primed CPMV-specific T cells and generated high titers of CPMV- and FnBP-specific immunoglobulin G (IgG) in sera. Furthermore, CPMV- and FnBP-specific IgA and IgG could also be detected in the bronchial, intestinal, and vaginal lavage fluids, highlighting the ability of CVPs to generate antibody at distant mucosal sites. IgG2a and IgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal IgA was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.

Virus, bakterier og toksiner som biologiske kampstoffer

Biologiske våben er i dag en væsentlig del af det internationale trusselsbillede. Den teknologiske udvikling har gjort fremstilling af biologiske våben relativt simpel og billig, og selvom en FN-konvention fra 1972 forbyder biologiske våben, har såvel stater som ikke-statslige organisationer eller enkeltpersoner i de senere år udviklet biologiske våben til offensivt brug til krig eller terrorvirksomhed. Oversigtsartiklen gennemgår den offentligt tilgængelige litteratur på området, og sygdomsbilledet og de terapeutiske muligheder ved de gængse biologiske kampstoffer skitseres. Mulighederne for beskyttelse af militære og civile grupper beskrives, og behovet for en national beredskabsplan understreges.

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