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Biomedical subjects

S Modrow

Publications and source records attributed to S Modrow.

At least 37 records · Page 2Linked to original sources
PubMed1996

Inhibition of cell adhesion to the virus by synthetic peptides of fiber knob of human adenovirus serotypes 2 and 3 and virus neutralisation by anti-peptide antibodies.

The fiber knob of adenovirus (Ad) causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s) several synthetic peptides derived from Ad2 and Ad3 fiber head sequences and their antisera were tested for interference with virus attachment to HeLa and FL cells and cell adhesion to viruses. The anti-peptide sera were also evaluated in ELISA and virus neutralisation test. Ad2 (of subgroup C) and Ad3 (of subgroup B) attachment was not significantly inhibited by peptides corresponding to the amino acid residues 535-554, 555-573, 562-582 of Ad2 fiber or 210-225, 267-283, 291-306 and 300-319 of Ad3 fiber. However, microplate pre-adsorbed Ad3 fiber residues 210-225 and 267-283 could bind FL and HeLa cells, and 1 mg/ml of Ad3 fiber residues 267-283 inhibited the cell adhesion to Ad3 virus to approximately 90%. This peptide may participate in the receptor binding site of Ad3 fiber. ELISA reactive anti-peptide antibodies against the homologous peptide and virus did not significantly reduce the cell adhesion to the immobilised virus or the virus attachment to cells, but in the neutralisation assay antibodies raised to Ad2 fiber residues 555-573 and 562-582 and Ad3 fiber residues 210-225 caused neutralisation of the homologous virus at serum dilutions of 1:500 and 1:32, respectively. The corresponding peptides and one further peptide of Ad2 fiber and two of Ad3 fiber seem to contain neutralisation epitopes.

Adenoviruses, Human↗
PubMed1996

Sequence variability among different parvovirus B19 isolates.

Parvovirus B19 is the causative agent of a variety of clinical manifestations, ranging from asymptomatic to severe infection. The basis for this complex pattern of B19-associated diseases is as yet poorly understood. In general there are two different possibilities: firstly, the infected individuals may have a genetic or acquired predisposition, which renders them susceptible for a certain course of infection; secondly, differences in the B19 genome may result in different outcomes of infection. In order to investigate this second possibility we have partially sequenced the genomes of 20 different B19 isolates derived from serum samples from patients with various B19-associated diseases. Four distinct regions, which cover nearly half of the genome and include parts of the coding regions of all three major B19 proteins-NS1, VP1 and VP2, were selected for sequencing. Comparisons between the different extracted virus isolates at the DNA and protein levels revealed that isolates from patients with persistent parvovirus B19 infection show a tendency towards higher genome variability with respect to isolates derived from persons with acute infection.

Base Sequence↗
PubMed1996

Lymphoproliferative responses after infection with human parvovirus B19.

Immunity after infection with the parvovirus B19 is assumed to be conferred by a humoral immune response with development of neutralizing antibody. In contrast, little is known about the nature of T-cell-mediated responses to parvovirus B19 infection in humans. We used recombinant proteins VP1, VP2, and NS1, as well as a recombinant VP1-specific amino-terminal sequence, to test the proliferative responses of peripheral blood mononuclear cells after infection of otherwise healthy individuals with parvovirus B19. These proteins were used as antigens for the stimulation of freshly isolated cells. The results show that a B19 virus-specific cellular immunity develops that is directed against the capsid proteins VP1 and VP2. We also demonstrate that viral determinants are presented to CD4+ T cells by HLA class II molecules.

Antigens, Viral↗
PubMed1996

Defined amino acids in the gag proteins of human immunodeficiency virus type 1 are functionally active during virus assembly.

A structurally highly ordered arrangement of the polyprotein precursor, Pr55gag is a necessary prerequisite for assembly, budding and maturation of the human immunodeficiency virus type 1 (HIV-1). In particular, distinct regions of the matrix protein (p17) and the capsid protein (p24) contained within Pr55gag are functionally active during these processes. In order to determine such regions we exchanged amino acid triplets within p17 (amino acids 46-61) and p24 (amino acids 341-352) for alanine residues and deleted the whole regions. Synthetic peptides derived from these regions had been shown previously to inhibit the production of infectious virus. The effect of the mutations on the release of viral particles was investigated by using recombinant baculoviruses for the expression of mutated Pr55gag as virus-like particles and by use of the respective HI proviruses for monitoring the production of infectious particles.

Amino Acids↗
PubMed1995

BiP binding sequences in antibodies.

During the process of folding and assembly of antibody molecules in the endoplasmic reticulum, immunoglobulin heavy and light chains associate transiently with BiP, a resident endoplasmic reticulum protein that is a member of the Hsp70 family of molecular chaperones. BiP is thought to recognize unfolded or unassembled polypeptides by binding extended sequences of approximately seven amino acids that include bulky hydrophobic residues not normally exposed on the surface of native proteins. We used a computer algorithm developed to predict BiP binding sites within protein primary sequences to identify sites within immunoglobulin chains that might mediate their association with BiP. Very few of the sequential heptapeptides in the heavy or light chain sequences were potential BiP binding sites. Analysis of the ability of synthetic heptapeptides corresponding to 24 potential sites in heavy chains to stimulate the ATPase activity of BiP indicated that at least half of them were authentic BiP binding sequences. These sequences were not confined to a single domain of the heavy chain but were distributed within both the VH and CH domains. Interestingly, when the BiP binding sequences were mapped onto the three-dimensional structure of the Fd antibody fragment, the majority involve residues that participate in contact sites between the heavy and light chains. Therefore, we suggest that in vivo BiP chaperones the folding and assembly of antibody molecules by binding to hydrophobic surface regions on the isolated immunoglobulin chains that subsequently participate in interchain contacts.

Adenosine Triphosphatases↗
PubMed1995

Antibodies to the nonstructural protein of parvovirus B19 in persistently infected patients: implications for pathogenesis.

Three patients with persistent parvovirus B19 infection, as documented by the prolonged presence of IgM directed to the viral capsid proteins and detection of viral DNA in serum by dot-blot hybridization or polymerase chain reaction (PCR), were investigated for the presence of antibodies to the nonstructural protein NS-1 of parvovirus B19. This was done by using an ELISA based on recombinant NS-1 protein. Whereas control sera displayed no reactivity, sera from persistently infected patients showed a strong specific antibody response to NS-1. Patients were followed for 3-18 months, during which IgM titers declined but IgG directed to the nonstructural protein remained detectable. The appearance of NS-1-specific antibodies might indicate an altered course of viral infection leading to the establishment of persistently active infection and subsequent destruction of cells of nonerythroid lineage.

Adult↗
PubMed1995

HLA-A0201 and HLA-B7 binding peptides in the EBV-encoded EBNA-1, EBNA-2 and BZLF-1 proteins detected in the MHC class I stabilization assay. Low proportion of binding motifs for several HLA class I alleles in EBNA-1.

B lymphocytes immortalized with EBV in vitro, lymphoblastoid cell lines (LCL), express eight EBV-encoded proteins, EBV nuclear antigens -1 to -6 (EBNA-1 to -6), and latent membrane proteins 1 and 2 (LMP 1 and 2). After appropriate stimulations of blood lymphocytes from seropositive individuals, MHC-restricted cytotoxic T cells (CTL), which lyse LCL cells, can be generated in vitro. Such CTLs can recognize EBNA-2 to -6, and LMP 1 and 2, but not EBNA-1-derived peptides presented on the cell surfaces. We posed the question whether this exceptional feature of EBNA-1 is due to lack of MHC class I binding peptides. A computer search for 11 human leukocyte antigen (HLA) alleles showed that EBNA-1 has a lower number and lower proportion of relevant binding motifs to several alleles than EBNA-2 to -6 and LMP 1 and 2. The low motif numbers in EBNA-1 is in line with its apparent failure to generate a CTL response, and it may be the consequence of immunoselection allowing the existence of EBV genome-carrying B cells in the immunocompetent hosts. The binding capacities of synthetic peptides of EBNA-1 and -2 and of the immediate early lytic cycle protein BZLF-1 to HLA-A0201 (A2) and HLA-B7 molecules were tested in an MHC stabilization assay. The peptide transporter-deficient T2 line, which expresses a low level of HLA-A2 and its HLA-B7 transfectant subline, were used for this purpose because specifically bound peptides elevate the surface expression of these MHC molecules. Of five synthetic nonamer EBNA-1 peptides which include the relevant binding motif, four bound to A2. In a series of 20-amino acid-long overlapping EBNA-1 peptides none showed binding to A2, while eight peptides bound to B7. Two 20-amino acid-long EBNA-2 and seven BZLF-1 peptides were identified as A2 binders, and four EBNA-2 and eight BZLF-1 peptides bound to B7. Thus, we have exclude the possibility that the inability of the EBNA-1 protein to generate HLA-restricted CTLs could be due to the lack of HLA class I binding peptides in its sequence. The finding that several EBNA-1 peptides could bind to these two HLa molecules does not, however, necessarily reflect the natural situation because the peptides may not be processed and/or transported to the cell surfaces. We have stimulated lymphocytes of healthy donors with relevant HLA types with the autologous LCL.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗
PubMed1995

Antibodies to parvovirus B19 NS-1 protein in infected individuals.

Human parvovirus B19 is the aetiological agent of the common childhood disease erythema infectiosum (fifth disease). The infection is usually benign and self-limiting, but in adults cases of severe arthritis which may persist for years have been reported. Neutralizing antibodies directed against the structural proteins are usually produced shortly after the infection. The immune response against the third major protein, the non-structural protein NS-1, of parvovirus B19 has not been characterized so far. We cloned and expressed the full-length NS-1 protein and fragments thereof in Escherichia coli. The purified recombinant proteins were used to investigate the presence of antibodies to the NS-1 protein in sera from patients with parvovirus B19 infection. Specific antibodies could be detected in sera from patients suffering from severe parvovirus B19-associated arthritis using Western blot analysis and an ELISA. Sera from patients with acute or past infection without complications did not contain detectable levels of immunoglobulin to NS-1. The use of subfragments of the NS-1 protein allowed localization of the antigenic domains in the carboxy-terminal region of the protein.

Antibodies, Viral↗
PubMed1995

Specific cytotoxic T lymphocytes recognize the immediate-early transactivator Zta of Epstein-Barr virus.

We identified the immediate-early transactivator Zta of Epstein-Barr virus as a target for specific cytotoxic T lymphocytes (CTL). Cells pulsed with overlapping synthetic peptides representing the entire amino acid sequence of Zta proved to be efficient for the in vitro stimulation of Zta-specific CTL in several donors. With peptide-pulsed target cells, we found that CTL from several donors recognize a peptide comprising 15 amino acids. The immune response against this peptide exerted by CTL lines from different donors was found to be restricted by two different molecules of the major histocompatibility complex: HLA-B8 and HLA-Cw6. The latter molecule could for the first time be identified as a restricting element for a CTL response. The epitope of the HLA-B8-restricted CTL could be mapped to an octameric sequence between amino acid positions 190 and 197 of the Zta protein, whereas the minimal epitope of HLA-Cw6-restricted CTL consists of 11 to 15 residues between positions 187 and 201. Thus, the HLA-B8 and HLA-Cw6 epitopes widely overlap but are not completely identical. In vitro stimulation of blood lymphocytes from a panel of HLA-B8-positive or HLA-Cw6-positive virus carriers, using autologous cells pulsed with the Zta peptides comprising the HLA-B8 or HLA-Cw6 epitope, respectively, revealed in both cases that most of these donors developed a Zta-specific cytotoxic activity. These data, as well as the high spread of the major histocompatibility complex molecules HLA-B8 and HLA-Cw6 in most populations, suggest that an efficient CTL response directed against gene products of the immediate-early group of the lytic cycle exists in vivo in a considerable portion of virus carriers. A CTL response against proteins expressed immediately after the switch into the lytic cycle could eliminate lytically activated cells at an early stage and would thus efficiently prevent the production and release of progeny virions.

Amino Acid Sequence↗
PubMed1994

Identification of wild-type and mutant p53 peptides binding to HLA-A2 assessed by a peptide loading-deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay.

Mutations of the p53 gene are the most frequently observed genetic changes in human cancers; often leading to an overexpression of the wild-type (wt) p53 protein. Demonstrable T cell reactivity against tumor cells overexpressing wt or mutant p53-derived peptides could support the application of such epitopes in cancer immunotherapies. As the binding of peptide to MHC class I molecules is a prerequisite for antigen-specific T cell recognition, we evaluated the ability of wt and mutant p53 peptides to bind to HLA-A2.1 using two independent flow cytometry-based assay systems, the T2 major histocompatibility complex (MHC) class I peptide stabilization assay (stabilization assay) and the peptide-induced MHC class I reconstitution assay (reconstitution assay). The twenty selected wt sequences each conformed to the previously reported HLA-A2.1 peptide binding motif. Seven of the wt p53 and 2/13 mutant p53 peptides derived from the previously chosen wt peptides bound to HLA-A2.1 in both the stabilization and the reconstitution assays. An additional six wt and six mutant p53 peptides, presumably exhibiting lower affinity for HLA-A2.1, were identified only in the reconstitution assay. Those p53 peptides binding HLA-A2.1 may provide useful immunogens for the generation of HLA-A2.1-restricted cytolytic T lymphocytes in vitro and in vivo.

Amino Acid Sequence↗
PubMed1994

Flow-cytometric determination of peptide-class I complex formation. Identification of p53 peptides that bind to HLA-A2.

A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.

Amino Acid Sequence↗
PubMed1994

Inhibition of infectious human immunodeficiency virus type 1 particle formation by Gag protein-derived peptides.

Sequential overlapping Gag protein-derived oligopeptides of human immunodeficiency virus type 1 (HIV-1) 22 to 24 amino acids long, were synthesized and tested in vitro for antiviral activity. Two synthetic peptides, one derived from the matrix protein p17 (NPGLLETSEGCRQ, amino acids 47 to 59) and one located in the capsid protein p24 (PAATLEEMMTA, amino acids 339 to 349) inhibited the production of infectious virus when added to HIV-1-infected cultures when used in the range of 20 to 200 micrograms/ml. As shown by thin section electron microscopy, peptide treatment resulted in the release of immature, deformed virus particles suggesting that the two peptides interfered with assembly and maturation. Other Gag protein-derived oligopeptides had little or no influence on virus production. To characterize further the functionally active regions we synthesized peptide derivatives with three consecutive amino acids substituted by alanine; they did not cause inhibition. Therefore the regions responsible for inhibition were located between amino acids 50 to 61 in p17, and 342 to 350 in p24. These observations might lead to the development of a new antiviral strategy affecting the late stage of virus replication.

Amino Acid Sequence↗
PubMed1994

Monoclonal antibodies directed against tick-borne encephalitis virus with neutralizing activity in vivo.

Monoclonal antibodies (MoAbs) were raised against the tick-borne encephalitis (TBE) virus, strain K23. The reactivities of 14 selected MoAbs were characterized by ELISA, Western blot analysis, haemagglutination inhibition, immunoprecipitation, in vivo protection and in vitro neutralization tests. All MoAbs reacted only with the glycoprotein E. The binding epitope of one MoAb could be delimited by a synthetic peptide to amino acids 306-339 representing one immunodominant loop structure of the glycoprotein E. The MoAbs exhibited individual reactivities against 13 different TBE virus isolates in ELISA and immunoblot test ranging from type-specific reactions to a broad reactivity with all isolates. Four MoAbs also showed a cross-reaction with other flaviviruses like West Nile virus and/or Yellow fever virus in immunoblot analysis. By competition ELISA the MoAbs could be divided into five different reaction patterns. Four MoAbs showed neutralizing activity with titers in the range 1:140 to 1:5,000 in an in vitro assay. These neutralizing activities could be confirmed by an in vivo mouse challenge model. The MoAbs are useful for diagnostic purposes and for differentiation of TBE virus strains and other flaviviruses.

Amino Acid Sequence↗
PubMed1993

Structural dissection of the multidomain kininogens. Fine mapping of the target epitopes of antibodies interfering with their functional properties.

Kininogens, the large precursor molecules of the vasoactive kinin peptides, are prototypic multidomain proteins serving numerous functions. To investigate their structure-function relationships, we have raised a panel of monoclonal antibodies against human H-kininogen and L-kininogen and fragments thereof and characterized them with respect to their target epitopes. Of 35 antibodies, 12 were directed to the amino-terminal domains (D1 to D3) of cystatin-like structure, 3 recognized domain D4 bearing the kinin segment, 17 bound to the carboxyl-terminal domains of H-kininogen (D5H and D6H), and 3 bound to the carboxyl-terminal domain D5L of L-kininogen. At least 14 distinct epitopes spread over the kininogen molecules were identified: 9 epitopes located on L-kininogen and 13 epitopes harbored by H-kininogen. Of these, 8 are shared by the two types of kininogens. Fine mapping of the epitopes by proteolytic fragments, recombinant proteins, and anti-idiotypic antibodies demonstrated that most but not all of the antibodies recognize liner epitopes. Synthesis of 28 peptides covering more than one-third of the entire kininogen sequences allowed us to narrow down seven major epitopes to 7-31 residues. Functional analyses identified 14 antibodies interfering with specific biological roles of the kininogens, i.e. cysteine proteinase inhibition, platelet attachment, cofactor binding, contact activation, and kinin delivery. Cross-reactivity studies indicated that three of the epitopes are present throughout the mammalian kininogens and further identified a unique epitope characteristic for the H/L-type of kininogens not present in their T-type. The panel of mapped antibodies provides powerful tools for the characterization of relevant interaction sites exposed by the pleiotropic kininogens and for the development of molecular surrogates mimicking these functional loci.

Amino Acid Sequence↗
PubMed1993

Identification of a region in the Pr55gag-polyprotein essential for HIV-1 particle formation.

The pr55gag polyprotein of HIV-1 plays a critical role in the formation of immature virus particles in the cell and during the budding process. We investigated the influence of amino acid substitutions in the p24CA- region of the gag polyprotein on the viral assembly process. Deletion of the amino acids 341-352 in the carboxy terminal part of the p24CA resulted in a loss of the capacity of the gag polyprotein to form virus-like particles when expressed in eucaryotic cells by recombinant vaccinia virus. In further experiments it turned out that the amino acids 341-346 and 350-352 are important for the ability of the pr55gag to form virus-like particles. Because these stretches are conserved among HIV-1, HIV-2, SIV, and FIV, we conclude that these amino acids form a domain highly important for the assembly of these lentiviruses.

Amino Acid Sequence↗
PubMed1993

Epitope mapping with peptides of Chi t I component III and immunomodulation of the Chi t immune response.

BACKGROUND: Hemoglobins of the Diptera family are known as aggressive inhalant allergens. METHODS: The whole sequence of the insect allergen Chi t I component III was studied for T-cell epitope activity in cell cultures of 15 sensitized patients with a panel of 17 overlapping synthetic peptides and 12 peptides obtained by tryptic cleavage. RESULTS: All but one of the peptides tested were found to be immunogenic. The nonstimulatory sequence 102-109 and two other tryptic peptides (69-90 and 110-135) were used in inhibition experiments. The Chi t I-induced proliferation was downregulated by all tested peptides when compared with stimulation with Chi t I alone. CONCLUSIONS: Because stimulation with phytohemagglutinin or tetanus toxoid was not influenced by the above-mentioned peptides, this mechanism seems to be specific for the antigen. Specific immunomodulation with nonstimulating peptides may offer new therapy approaches.

Adjuvants, Immunologic↗