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L Akerblom

Publications and source records attributed to L Akerblom.

46 records · Page 3Linked to original sources
PubMed1989

Serological responses in cats vaccinated with FeLV ISCOM and an inactivated FeLV vaccine.

Various approaches have been considered for generation of effective and safe vaccines against retroviruses, including HIV, with limited success. In the present vaccination study, encompassing 137 household cats, we have composed an experimental ISCOM subunit vaccine containing gp70 of feline leukaemia virus (FeLV)--the external glycosylated envelope protein, and the transmembrane protein p15E, with a commercial available inactivated FeLV vaccine (Leukocell). The two vaccines were estimated to contain approximately the same amount of gp70 antigen and the cats were immunized three times according to the recommendations of the commercial vaccine. A control preparation not containing gp70 or p15E was also included. During the observation period of 200 days all cats remained healthy and no virus was isolated during the isolation attempts. The serological responses were measured in ELISA, membrane immunofluorescence (MIF) and virus neutralization (VN) tests. In contrast to the cats in the other groups almost all ISCOM-vaccinated cats responded by seroconversion or increased titres in the three tests. The development of specific antibodies to gp70 and p15E were confirmed in Western blot. These results clearly illustrate the potential of the ISCOM structure for the development of safe and effective vaccines against retroviruses.

Animals↗
PubMed1989

Formation and characterization of FeLV ISCOMs.

Immunostimulating complexes (ISCOMs) have been prepared from feline leukaemia virus (FeLV) envelope proteins. The ISCOMs were characterized biochemically in SDS-polyacrylamide gel electrophoresis showing the presence of proteins of estimated molecular weights of 15,000, 27,000 and 70,000. Immunoblotting showed that both the transmembrane protein p15E and the external glycoprotein gp70 (making up the gp85 protein) were present in the ISCOM. Furthermore, a degradation product of gp70 with an estimated molecular weight of 32,000 was identified in the immunoblot. The FeLV ISCOM was shown by electron microscopy to have the characteristic cage-like structure of an ISCOM with a mean diameter of 37 nm. About 10% of the total amount of gp70 in the culture fluid was recovered in the ISCOMs. The largest loss was encountered during the sedimentation of the virus. In a preliminary immunization experiment in mice the FeLV ISCOMs elicited after a booster gave a clear-cut immune response against gp70.

Animals↗
PubMed1989

Immune response to immunostimulatory complexes (ISCOMs) prepared from human immunodeficiency virus type 1 (HIV-1) or the HIV-1 external envelope glycoprotein (gp120).

In mice, immunostimulatory complexes (ISCOMs) prepared from HIV-1 B external envelope glycoprotein (gp120) induced 10-fold higher antibody titres than gp120 emulsified in depot adjuvant, as measured by enzyme-linked immunosorbent assay (ELISA). Rhesus monkeys immunized with gp120 ISCOMs produced precipitating and virus neutralizing antibody titres equivalent to those seen in HIV-infected chimpanzees and humans. After multiple immunizations with HIV-1 B gp120 ISCOMs, a rhesus monkey developed a neutralizing response to the HIV-1 isolates RF and MN, but not to the CC isolate. Antisera from ISCOM-immunized rhesus monkeys recognized gp120 on the membranes of HIV-1 B-infected H9 cells, indicating the preservation of epitope structure in the ISCOMs matrix.

Adjuvants, Immunologic↗
PubMed1989

Free gp70 from FeLV: enrichment from cell culture fluid by ferric oxide-agarose chromatography.

A new chromatographic material based on beads of macroporous crosslinked agarose containing ferric oxide particles was used for enrichment of gp70--the envelope glycoprotein of feline leukemia virus (FeLV). Free gp70 was purified from cell culture fluid in one step with a recovery of 50 to 60% and a purification of about 60 times. The described procedure is a suitable first step for the purification of gp70 from large volumes of cell culture fluid.

Animals↗
PubMed1985

Azidocytidine is incorporated into RNA of 3T6 mouse fibroblasts.

Earlier work has shown that azidocytidine inhibits the growth and DNA synthesis of 3T6 mouse fibroblasts by inactivation of the enzyme ribonucleotide reductase. RNA synthesis, as measured by incorporation of [3H]cytidine was not affected. Here I show that azidocytidine is incorporated into RNA, but not into DNA. Incorporation of the analogue into RNA may under special circumstances contribute to the biological effect of the nucleoside.

Animals↗
PubMed1985

Azidocytidine is a specific inhibitor of deoxyribonucleotide synthesis in 3T6 cells.

2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication.

Animals↗
PubMed1982

Effects of azidocytidine on DNA synthesis and deoxynucleotide pools of mouse fibroblast cell lines.

2'-Deoxy-2'-azidocytidine inhibits DNA synthesis of cells in tissue culture. In vitro, the diphosphate of azidocytidine was earlier shown to inhibit mammalian ribonucleotide reductase, while the triphosphate did not inhibit DNA replication in isolated cell nuclei. In intact cells, however, deoxyribonucleoside triphosphate pools were not decreased by azidocytidine in a manner expected from an inhibitor of ribonucleotide reductase. In order to differentiate between inhibition of precursor synthesis and DNA replication we studied in the present investigation the effects of azidocytidine on pool sizes of deoxynucleotides as well as on the incorporation of labeled cytidine or deoxycytidine into deoxycytidine triphosphate pools and DNA of cultured growing mouse fibroblasts. Azidocytidine was transformed to an active metabolite by phosphorylation with deoxycytidine kinase. Cell lines deficient in this enzyme showed an increased resistance against the analogue. Azidocytidine had a dual effect on the incorporation of labeled nucleosides. At low concentrations, the incorporation of cytidine into DNA was severely inhibited without a decrease of the specific activity of the dCTP pool or the size of the other three dNTP pools, suggesting a direct effect on DNA replication. At high concentrations of azidocytidine, incorporation into dCTP was also affected, demonstrating inhibition of CDP reduction. Our data also show that reduction of CDP is not necessarily tightly coupled to DNA replication since a rapid turnover of the dCTP pool occurred at the low concentrations of azidocytidine which severely inhibited DNA synthesis.

Animals↗
PubMed1981

Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells.

Hydroxyurea inhibits the activity of ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxy-ribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) in bacteria and mammalian cells. The reductase from Escherichia coli consists of two nonidentical subunits (B1 and B2) and hydroxyurea acts by specifically destroying a tyrosine free radical of B2 required for enzyme activity. The mammalian enzyme also consists of two nonidentical subunits (M1 and M2), only one of which (M1) has been obtained in pure form. By continuous culture at stepwise increasing drug concentrations, we have now obtained a 3T6 mouse fibroblast cell line with a 100-fold increased resistance to hydroxyurea. Extracts from resistant cells showed a 3- to 15-fold increase in reductase activity. The amount of M1 protein was not increased. The amount of M2 protein could not be measured directly, but the M2 activity in extracts from resistant cells (but not normal cells) showed an EPR spectrum very similar to that of the tyrosine radical of the bacterial B2 subunit. We propose that resistance to hydroxyurea is caused either by overproduction of the complete M2 subunit or by increased generation of the tyrosine radical within the M2 protein. It seems that either alternative mirrors a possible normal regulatory mechanism for the activity of the reductase.

Animals↗
PubMedDate not supplied

The role of type-1 and type-2 T-helper immune responses in HIV-1 vaccine protection.

The dichotomy of type-1 and type-2 T-helper (Th) immune responses is thought to be an obstacle to develop Human immunodeficiency virus-type- (HIV-1) vaccines capable of inducing effective cellular as well as humoral immune responses. Macaca mulatta were immunized using two different HIV-1sf2 envelope vaccine strategies, based on either immune-stimulating complexes (ISCOM) or chimeric Fowlpox (FP) vaccines. One month following the third immunization all animals were heterologously challenged with simian/human immunodeficiency virus (SHIVsf13). Vaccinated monkeys, which were protected had the highest levels of both type-1 and type-2 HIV-1 specific T-helper cell (Th) responses in addition to the highest homologous and heterogenous virus neutralizing antibodies. To determine how long Th responses persisted and if they correlated with protection, animals were rechallenged after waiting for four months without re-boosting. Macaques which maintained the highest gp120-specific type-1 (IFN-gamma) responses were protected, while there was evidence of viral clearance in two others. These findings demonstrate the importance of both or mixed type-1 and type-2 Th responses in HIV-1 vaccine induced immunity while suggesting a possible role of persistent type-1 responses in maintaining protective immunity over time.

AIDS Vaccines↗