http://repub.eur.nl/res/aut/1295/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/3604/ 1997-01-01T00:00:00Z Peptides deduced from the central conserved region (residues 158 to 189) of protein G of human respiratory syncytial virus (HRSV) subtypes A and B were used as antigens in subtype-specific enzyme-linked immunosorbent assays (G-peptide ELISAs). These G-peptide ELISAs were compared with seven other serological assays to detect HRSV infection: ELISAs based on complete protein G, on fusion protein F, and on nucleoprotein N; a complement fixation assay; a virus neutralization test; and ELISAs for the detection of immunoglobulin A (IgA) or IgM antibodies specific for HRSV. In paired serum samples from patients with HRSV infection, more infections were diagnosed by the G-peptide ELISA (67%) than by all other serological tests combined (48%). Furthermore, for 16 of 18 patients (89%), the G-peptide ELISAs were able to differentiate between antibodies against HRSV subtypes A and B. This study shows that peptides corresponding to the central conserved region of the attachment protein G of HRSV can successfully be used as antigens in immunoassays. The G-peptide ELISA appeared to be more sensitive than conventional tests for the detection of HRSV antibody titer rises. http://repub.eur.nl/res/pub/3537/ 1995-01-01T00:00:00Z Viral progeny of two molecular clones of feline immunodeficiency virus (FIV), 19k1 and 19k32, were tested in a virus neutralization assay. In this assay the infection of thymocytes with FIV19k1 was neutralized by serum S1422, derived from an SPF cat 22 weeks after infection with FIV19k1. We previously reported that a point mutation at position 560 in hypervariable region-5 (HV-5) of 19k1 confers resistance to virus neutralization (Siebelink et al., 1993, J. Virol. 67:2202-2208). Viral progeny of the other molecular clone, FIV19k32, which differs in the envelope protein in only six amino acids from 19k1, was not neutralized. In order to map sites involved in virus neutralization we constructed chimeric clones by reciprocal exchange of 19k1 and 19k32 envelope gene fragments. Reciprocal exchange of a 1662 bp fragment, encoding almost the whole surface protein, which differs in five amino acids between these two clones, resulted in exchange of the phenotype. Amino acids of the envelope protein of 19k1 and 19k32, in which these clones differ, were substituted by point mutation. We demonstrated that one of these mutations, a substitution of leucine to serine at position 483 in HV-4, also conferred resistance of 19k1 to neutralization by serum S1422. http://repub.eur.nl/res/pub/3540/ 1995-01-01T00:00:00Z A type-specific human immunodeficiency virus type 1 (HIV-1)-neutralizing human monoclonal antibody (HuMAb MN215) is described that reacts with the V3 domain of a number of subtype B virus strains. Pepscan analysis indicated that amino acids at both sides of the tip of the V3 loop were involved in the binding of HuMAb MN215. The minimum epitope in a V3 sequence, obtained from the donor from whom the cell line originated, was 9 amino acids long and proved to be located at the C-terminal side of the tip of the loop. In a replacement Pepscan analysis, individual amino acids of the V3 loop important for binding of HuMAb MN215 were identified. Amino acids at positions 15 (H), 16 (I), 17 (G) and 18 (P) were found to be essential for binding of the antibody, whereas changes at positions 19 of G to N, 20 of R to K and 23 of F to L, as well as the addition of a negative charge at the C terminus, improved binding. Thus, amino acids involved in the binding of HuMAb MN215 are primarily located within highly variable regions of the V3 loop. HuMAb MN215 showed a higher affinity for the V3 domain sequences and recombinant envelope glycoproteins derived from non-syncytium-inducing strains than for those derived from syncytium-inducing strains. http://repub.eur.nl/res/pub/10644/ 1994-07-01T00:00:00Z A synthetic peptide vaccine which protects dogs against challenge with virulent canine parvovirus is described. The amino acid sequence used was discovered in previous studies on the immunogenic properties of previously mapped antigenic sites and represents the amino-terminal region of viral protein VP2. As with marker vaccines, it is possible to discriminate between vaccinated dogs that have not been exposed to the virus and dogs that have been infected with the virus. The protective mechanism can be explained by a humoral response against the peptide aided by T-cell epitopes contained in the carrier protein used for peptide coupling. This is the first example of a synthetic peptide vaccine that induces protection in target animals. http://repub.eur.nl/res/pub/3499/ 1994-01-01T00:00:00Z A synthetic peptide vaccine which protects dogs against challenge with virulent canine parvovirus is described. The amino acid sequence used was discovered in previous studies on the immunogenic properties of previously mapped antigenic sites and represents the amino-terminal region of viral protein VP2. As with marker vaccines, it is possible to discriminate between vaccinated dogs that have not been exposed to the virus and dogs that have been infected with the virus. The protective mechanism can be explained by a humoral response against the peptide aided by T-cell epitopes contained in the carrier protein used for peptide coupling. This is the first example of a synthetic peptide vaccine that induces protection in target animals. http://repub.eur.nl/res/pub/3460/ 1993-01-01T00:00:00Z We infected a specific-pathogen-free cat (cat 14) with molecularly cloned feline immunodeficiency virus clone 19k1 (FIV19k1 [K. H. J. Siebelink, I. Chu, G. F. Rimmelzwaan, K. Weijer, A. D. M. E. Osterhaus, and M. L. Bosch, J. Virol. 66:1091-1097, 1992]). Serum of this cat obtained 22 weeks postinfection (serum 1422) neutralized FIV19k1 but not FIV19k32, which is 99.3% identical to FIV19k1 in the envelope gene. Serum 1422 also neutralized virus isolated from cat 14 at weeks 2 and 32 postinfection. We then cultured FIV19k1 in the continuous presence of serum 1422, which resulted in a delay in virus replication of 6 weeks. The resulting virus population appeared to be resistant to virus neutralization by serum 1422. Nucleotide sequencing of the env open reading frame of this presumed escape mutant revealed the presence of one silent and two substitution mutations, both of the latter in hypervariable region 5. Through the construction of chimeric viruses and site-directed mutagenesis, we demonstrated that one of these mutations, the substitution of lysine to glutamine at amino acid position 560 in hypervariable region 5, was sufficient to allow the escape of FIV19k1 from neutralization by serum 1422. http://repub.eur.nl/res/pub/3478/ 1993-01-01T00:00:00Z The core of a unique linear neutralization epitope (G5) on the glycoprotein of rabies virus, recognized by a virus-neutralizing mouse monoclonal antibody (MAb 6-15C4), was determined by Pepscan analysis. The G5 epitope was defined as an octapeptide (LHDFRSDE). The contribution of the individual amino acids of the G5 epitope to the binding of MAb 6-15C4 was analysed with a set of synthetic peptides in which the individual amino acids had been replaced in turn by each of the other 19 naturally occurring amino acids. Five amino acids of the octapeptide proved to be essential for the binding of MAb 6-15C4. The conservation of the G5 epitope within the glycoprotein of the different rabies virus strains sequenced to date proved to be absolute at the amino acid level. Studies concerning the immunodominance of the G5 epitope were carried out by determining the presence of G5 epitope-specific serum antibodies in vaccinated human and mice, and by determining the frequency of G5 epitope-specific B lymphocytes in the blood of vaccinated humans. These studies indicated that antibodies to the G5 epitope constitute a minor population of the rabies virus-specific serum antibodies induced by rabies vaccination. http://repub.eur.nl/res/pub/3408/ 1990-01-01T00:00:00Z Three synthetic peptides derived from the amino acid sequence of VP2 of canine parvovirus (CPV) which were recently shown to represent three distinct T cell epitopes for BALB/c mice could prime BALB/c mice for a CPV-specific proliferative T cell response upon immunization. Proliferative responses of peripheral blood mononuclear cells (PBMC) from CPV-immunized dogs upon stimulation with these and other peptides, covering the major part of the sequence of VP2', identified the presence of T cell epitopes for this species. Most of these epitopes were recognized by PBMC from only a minority of the dogs tested. With three newly generated canine Thyl+ T cell clones, which recognized CPV antigen in association with major histocompatibility complex class II molecules, two distinct T cell epitopes were identified within the unique sequence of VP1.