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Biomedical subjects

Anders Fomsgaard

Publications and source records attributed to Anders Fomsgaard.

6 recordsLinked to original sources
PubMed2003

Routine genotyping of human papillomavirus samples in Denmark.

In order to examine a sensitive unbiased consensus PCR with routine sequencing for HPV typing, we analysed Danish male and female patients suspected of having an HPV infection. We used the well-characterised nested PCR setting with MY09/MY11 and GP5+/GP6+ primers, followed by routine cycle sequencing. Of 1283 clinical samples from female patients based on suspected HPV infection, we found 379 (29%) negatives and 894 (70%) positives. Samples containing >5000 HPV copies/ml were genotyped by sequencing. Of the 552 HPV genotyped samples from women >15 years of age, 398 were characterised as high-risk types and the remaining 154 as low-risk types. The most commonly found high-risk types were HPV-16, HPV-31, HPV-33, HPV-18, HPV-58, and HPV-52, and the most commonly found low-risk types were HPV-6, HPV-53 and HPV-11. In addition, we observed that other typing assays could not perform as sensitively or accurately as the nested PCR/cycle sequencing method used in this study. For instance, 87 out of 552 genotyped samples could not have been typed correctly in the Hybrid Capture II assay. Of these 87 samples, 46 (53%) were considered as high-risk types.

Adolescent↗
PubMed2003

Immunogenicity in Mamu-A*01 rhesus macaques of a CCR5-tropic human immunodeficiency virus type 1 envelope from the primary isolate (Bx08) after synthetic DNA prime and recombinant adenovirus 5 boost.

Envelopes of primary R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates may be particularly relevant for vaccine purposes and should be evaluated for immunogenicity in animals including macaques before carrying out human vaccine trials. In the present study, the immunogenicities of synthetic HIV-1 env DNA vaccines, which had been derived from the early primary isolate Bx08 and contain humanized codons, were evaluated in mice, guinea pigs and rhesus macaques. Neutralization sensitivity of the HIV-1(Bx08) isolate was found to resemble that of other primary isolate prototypes. Immunogenicity of gp120 delivered as codon-optimized DNA vaccine was comparable to that of recombinant gp120 protein plus adjuvant in mice. Similarly, DNA vaccination of guinea pigs with synthetic gp140(Bx08) and gp150(Bx08) DNA induced a strong antibody response independent of the gene construct and DNA immunization route. Mamu-A*01 rhesus macaques were DNA vaccinated with synthetic gp150(Bx08) or gp140(Bx08) DNA and boosted with a replication-deficient recombinant human adenovirus type 5 expressing a synthetic gp120(Bx08) gene. DNA-vaccinated rhesus macaques developed specific CD8+ T lymphocyte responses and anti-rgp120(IIIb) antibody responses. Both the humoral and cellular responses were significantly improved following intramuscular boosting with the recombinant adenovirus. The demonstrated humoral and cellular immunogenicities of these HIV Bx08 Env vaccines in non-human primates encourages their further development as one component in candidate HIV vaccines for humans.

AIDS Vaccines↗
PubMed2003

Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes.

MHC-I-restricted cytotoxic responses are considered a critical component of protective immunity against viruses, including human immunodeficiency virus type 1 (HIV-1). CTLs directed against accessory and early regulatory HIV-1 proteins might be particularly effective; however, CTL epitopes in these proteins are rarely found. Novel artificial neural networks (ANNs) were used to quantitatively predict HLA-A2-binding CTL epitope peptides from publicly available full-length HIV-1 protein sequences. Epitopes were selected based on their novelty, predicted HLA-A2-binding affinity and conservation among HIV-1 strains. HLA-A2 binding was validated experimentally and binders were tested for their ability to induce CTL and IFN-gamma responses. About 69 % were immunogenic in HLA-A2 transgenic mice and 61 % were recognized by CD8(+) T-cells from 17 HLA-A2 HIV-1-positive patients. Thus, 31 novel conserved CTL epitopes were identified in eight HIV-1 proteins, including the first HLA-A2 minimal epitopes ever reported in the accessory and regulatory proteins Vif, Vpu and Rev. Interestingly, intermediate-binding peptides of low or no immunogenicity (i.e. subdominant epitopes) were found to be antigenic and more conserved. Such epitope peptides were anchor-optimized to improve immunogenicity and further increase the number of potential vaccine epitopes. About 67 % of anchor-optimized vaccine epitopes induced immune responses against the corresponding non-immunogenic naturally occurring epitopes. This study demonstrates the potency of ANNs for identifying putative virus CTL epitopes, and the new HIV-1 CTL epitopes identified should have significant implications for HIV-1 vaccine development. As a novel vaccine approach, it is proposed to increase the coverage of HIV variants by including multiple anchor-optimized variants of the more conserved subdominant epitopes.

Adult↗
PubMed2003

Hepatitis C virus subtyping by a core-envelope 1-based reverse transcriptase PCR assay with sequencing and its use in determining subtype distribution among Danish patients.

A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5'-untranslated region (5'-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5'-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5'-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5'-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.

5' Untranslated Regions↗
PubMed2002

A plasmid selection system in Lactococcus lactis and its use for gene expression in L. lactis and human kidney fibroblasts.

We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363Deltathr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363Deltathr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.

Cell Line↗