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M Mach

Publications and source records attributed to M Mach.

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

Antibodies specific for the antigenic domain 1 of glycoprotein B (gpUL55) of human cytomegalovirus bind to different substructures.

Glycoprotein B (gB, gpUL55) is a major antigen for the induction of neutralizing antibodies against human cytomegalovirus, making it an attractive antigen for active and passive immunoprophylaxis. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure which requires a minimal linear amino acid sequence of more than 75 amino acids (aa 552-635) for antibody binding. We have analyzed the fine specificity of neutralizing and nonneutralizing AD-1-binding monoclonal antibodies. Point mutations were introduced into AD-1 and mutants were expressed as bacterial fusion proteins. The antigens were analyzed in immunoblots using a panel of 13 human and murine monoclonal antibodies. Complete loss of binding of all antibodies was observed with mutations at cysteine residues 573 and 610 as well as with a combinatorial exchange of prolines at position 577 and 613. The remaining mutations had different effects on antibody binding. Six individual recognition patterns were observed, indicating various antigenic substructures on AD-1. Changing the Fc portions of 3 murine monoclonal antibodies to human IgG1 showed that neutralization of AD-1-binding immunoglobulins is exerted by different mechanisms. Dependent on the recognized substructure within AD-1, avidity-dependent as well as Fc portion-mediated effects were observed.

Amino Acid Sequence↗
PubMed1996

Light chain shuffling of a high affinity antibody results in a drift in epitope recognition.

Human polyclonal and monoclonal antibodies against pathogens and toxins are potentially useful in the treatment of various diseases. A number of human monoclonal antibodies with protective capacity in vitro have been established by conventional hybridoma technology. However, with the development of phage-display technology, the possibility of specifically tailoring antigen-binding properties has improved substantially. We show here that the reactivity of a high affinity, virus-neutralizing human antibody against the AD-2 epitope of cytomegalovirus gB can be modified by introducing other Vkappa sequences together with the original VH sequence. The fine specificity, as determined by the requirement of particular amino acid residues in the epitope, is shifted in these new antibody fragments. It was also evident that the VH/Vkappa pairing was not promiscuous, since antibody fragments selected by phage display retained light chain sequences very similar to the original hybridoma-derived light chain, proving that a high affinity interaction was very dependent on a co-operativity between both variable domains. These findings show that phage display technology might modify the binding properties of pre-existing, high affinity antibodies.

Amino Acid Sequence↗
PubMed1996

Analysis of T helper cell response to glycoprotein H (gpUL75) of human cytomegalovirus: evidence for strain-specific T cell determinants.

The proliferative response of helper T cells against glycoprotein H (gH; gpUL75) of human cytomegalovirus (HCMV) was determined in T cell lines from 5 healthy HCMV-seropositive donors. A differential response in two lines was noted when gH from strain AD169 or Towne was used as antigen. T cell-reactive domains between aa 15 and 510 were identified using beta-galactosidase fusion proteins containing overlapping fragments of gH, and they were confirmed with synthetic peptides as stimulating antigen. T cell proliferation was observed with antigens containing aa 34-51, 111-142, 284-302, 324-342, and 454-510 of gH. None of the determinants stimulated all donors. The T cell epitope defined by aa 34-51 is located in close proximity to a strain-specific dominant B cell epitope; however, no strain dependence for this T cell determinant was observed. In contrast, the dominant T cell response against aa 284-302, which was observed in three T cell lines, was strain specific.

Amino Acid Sequence↗
PubMed1996

Glycoprotein H of human cytomegalovirus is a major antigen for the neutralizing humoral immune response.

A recombinant baculovirus expressing glycoprotein H (gpUL75) of human cytomegalovirus was used to examine the humoral immune response in naturally infected individuals. Recombinant baculovirus infected insect cells produced two forms of gH with molecular masses of 78-82 kDa and 94 kDa. The 94 kDa polypeptide was modified by high mannose oligosaccharide side-chains as shown by reduction in molecular mass after treatment with endoglycosidases H and F. The 78-82 kDa protein represented the non-glycosylated precursor which was resistant to the enzymes. In contrast to gH expressed in mammalian cells, the recombinant baculovirus expressed gH was transported to the cell surface. Glycoprotein H produced in insect cells was reactive with human convalescent sera and all tested neutralizing monoclonal antibodies recognizing either linear or conformational epitopes. Antibodies reacting with insect cell derived gH were detected in 96 percent of HCMV seropositive human sera. Using insect cells infected with the gH expressing recombinant baculovirus as immunoabsorbent, between 0 percent and 58 percent of the total virus neutralizing activity was removed from sera of individuals with a past HCMV infection. gH must therefore be considered a major antigen for the induction of neutralizing antibodies during natural infection.

Animals↗
PubMed1996

Human cytomegalovirus glycoproteins.

The complex biology of human cytomegalovirus (HCMV) necessarily begins with an initial interaction between the envelope of the infectious virion and the host cell. Understanding the initial events of infection will require a further analysis of the glycoprotein components of the virion envelope as well as their expression in the membranes of the infected cell. This experimental goal has been hindered by the large genome of HCMV, which may encode over 65 unique glycoproteins. Protein homologs of only 4 herpes simplex virus (HSV) glycoproteins, gB, gH, gL and gM, have been identified, and potential functions have been postulated based on studies of specific glycoprotein null mutants of HSV and other herpesviruses. Additional glycoproteins have been analyzed but to date their function in the replicative cycle of this virus is unknown. Several of the envelope glycoproteins elicit strong host immune responses, including the production of virus-neutralizing antibodies. This response is felt to be a key component of host immunity and represents a goal of vaccine development. Finally, recent findings have also provided evidence that HCMV glycoproteins may contribute to evasion of host cellular immune responses by limiting viral antigen presentation.

Animals↗
PubMed1995

Comparative analysis of fourteen individual human cytomegalovirus proteins for helper T cell response.

The potential of selected proteins of human cytomegalovirus (HCMV) to induce a helper T (Th) cell immune response was investigated in healthy HCMV-seropositive donors. Recombinant derived glycoproteins B (gpUL55), H (gpUL75), integral membrane protein (pUL100), the US6-US11 glycoprotein family (pUS6-US11), the matrix proteins pp65 (ppUL83), pp28 (ppUL99) and the immediate early proteins IE1 (pUL123), IE2 (pUL122) and UL69 (pUL69) were used as stimulating antigens in a lymphocyte proliferation assay. The antigen-specific proliferative response was measured in HCMV-specific T cell lines (phenotype CD4+ CD8-) generated from five donors by stimulation of peripheral blood mononuclear cells with purified HCMV or HCMV-infected fibroblasts. A proliferative T cell response was induced by pp65, gB, gH, IE1, IE2 and UL69, with a dominant response to pp65 in all donors. Three T cell lines responded to gB and gH, respectively. For IE1, IE2 and UL69 a T cell stimulation could be demonstrated in single cell lines generated with lysate of HCMV-infected fibroblasts.

Antigens, Viral↗
PubMed1995

Searching for antibodies specific for human cytomegalovirus: is it diagnostically useful? When and how.

Diagnosis of human cytomegalovirus (CMV) infection can be obtained by direct demonstration of the virus or virus components in pathological materials or indirectly through serology. Serological diagnosis gives only indirect evidence of the presence of the virus, and is problematic because of the immunological disorders occurring in most patients at risk of developing a CMV infection. Furthermore, antigenic reagents used in commercially available kits are not standardized and discordant results are often obtained. However, serology is cheaper than the other diagnostic tests, requires a short execution time, is safe and can be completely automated. Therefore, it is worthwhile exploring the possible application fields in which the use of serology is justified nowadays. This is what this review will attempt to do.

Antibodies, Viral↗
PubMed1994

Epitope-specific distribution of IgG subclasses against antigenic domains on glycoproteins of human cytomegalovirus.

The IgG subclass pattern against linear antibody binding sites on glycoproteins of human cytomegalovirus (HCMV) was investigated in HCMV-positive healthy blood donors, human immunodeficiency virus-infected persons, sera from mothers with primary HCMV infection during pregnancy and their children, and sequential sera from transplant recipients. As antigens, three immunodominant domains capable of inducing neutralizing antibodies during natural infection were selected on glycoproteins gp58/116 (gB) and gp86 (gH). Bacterial fusion proteins representing these regions were used as antigens in a subclass-specific ELISA. Reactivity against the antibody binding site on gp86 was detected in both the IgG1 and IgG3 subclasses. In contrast, exclusively IgG1 antibodies were found against both linear domains on glycoprotein complex gp58/116 and also against full-length gp58/116 expressed in insect cells. The data demonstrate a differential regulation of the antibody response to envelope components of HCMV.

AIDS-Related Opportunistic Infections↗
PubMed1993

An ELISA using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus.

Two prokaryotically expressed fusion proteins encompassing amino acids 484-650 (AD-1) and 27-100 (AD-2) of glycoprotein gp58/116 of human cytomegalovirus (HCMV) were purified from E. coli lysates and used in ELISA to determine antibody levels in human sera. The specificity of the test was established by comparison of 116 randomly selected sera with commercially available HCMV-ELISA tests. The recombinant polypeptides were then used for the analysis of antibody titers in 112 human sera and were compared to the capacity to neutralize HCMV. A strong correlation between the neutralization titer and antibody levels against AD-1 and a weaker correlation for AD-2 was observed. Of 29 sera with a high neutralization titer (> 1:128), 96% and 62% were positive for AD-1 and AD-2, respectively, while 44% and 19% were positive in sera with low neutralization titer (< 1:8). Serum pools prepared from human sera selected on the basis of recognition of the recombinant antigens had a 10-fold higher neutralization capacity than pools prepared from sera with a high titer in commercially available HCMV tests. A synchronous increase in neutralization capacity and titer against recombinant antigens was observed in transplant patients.

Adolescent↗
PubMed1993

Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies.

The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-specific antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gp116, potently neutralized the virus at 1 to 2 micrograms of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58.

Antibodies, Monoclonal↗
PubMed1992

The DNA-binding protein P52 of human cytomegalovirus reacts with monoclonal antibody CCH2 and associates with the nuclear membrane at late times after infection.

Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.

Amino Acid Sequence↗
PubMed1992

Glycoprotein gp116 of human cytomegalovirus contains epitopes for strain-common and strain-specific antibodies.

Glycoprotein gp116 of human cytomegalovirus (HCMV) is a target for neutralizing antibodies. Gp116 is a component of the gCI complex which consists of gp58 and gp116. Like its homologue, glycoprotein B of herpes simplex virus type 1, gp116 contains a highly antigenic region in the N-terminal part of the molecule, between amino acids 28 and 84. Prokaryotic expression plasmids and synthetic peptides were used to define binding sites for mouse and human monoclonal antibodies (MAbs) as well as HCMV convalescent sera. Site I, located between amino acids 68 and 77, contains an epitope recognized by the human MAb C23, which is capable of neutralizing HCMV independently of complement and the site is conserved between HCMV strains. Of HCMV-positive human sera, 53% recognized site I. Site II was mapped using mouse MAbs as well as human sera. It is located between residues 50 and 54, an area which is not conserved between strains AD169 and Towne, the two laboratory strains of known sequence. Strain-specific antibodies were detected in 25% of human sera. Site II-specific antibodies, purified from human sera by affinity chromatography, were found to be incapable of neutralizing HCMV in tissue culture.

Amino Acid Sequence↗
PubMed1992

The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific.

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.

Amino Acid Sequence↗
PubMed1992

A continuous sequence of more than 70 amino acids is essential for antibody binding to the dominant antigenic site of glycoprotein gp58 of human cytomegalovirus.

Antigenic domain 1 (AD-1) on glycoprotein gp58 of human cytomegalovirus was characterized in detail, using mouse and human monoclonal antibodies as well as human convalescent sera. Series of procaryotically expressed fusion proteins and synthetic peptides of various lengths were used as sources of antigen. Binding of antibodies was found to depend on a continuous sequence of more than 70 amino acids between residues 552 and 635 of gp58. The fine specificities for sequences involved in antibody binding were (i) amino acids 557 to 635 for neutralizing as well as nonneutralizing mouse monoclonal antibodies, (ii) amino acids 552 to 630 for a neutralizing human monoclonal antibody, and (iii) amino acids 557 to 630 for antibodies present in human sera. Experiments involving fragments of AD-1, presented either as procaryotically expressed fusion protein or as synthetic peptides, indicated that the intact structure was required for recognition of AD-1 by antibodies.

Amino Acid Sequence↗
PubMed1991

Cleavage and purification of prokaryotically expressed HIV gag and env fusion proteins for detection of HIV antibodies in the ELISA.

Parts of the gag p24 and the gp41 transmembrane protein of the human immunodeficiency virus HIV-1 were expressed as fusion proteins in Escherichia coli, using an expression vector carrying aa 1-375 of the lac-Z gene linked to the recognition sequence for the blood coagulation factor Xa. Fusion proteins were cleaved into the bacterial and viral portion and the viral polypeptide was purified by a molecular sieve column. The purified viral antigens were tested with 288 human sera in the enzyme-linked immunosorbent assay (ELISA) technique. Comparison with commercially available tests showed comparable sensitivity and a higher specificity of the gag/env-ELISA for borderline reactive sera.

Chromatography, Gel↗
PubMed1991

Comparative sequence analysis of human cytomegalovirus strains.

Three regions of DNA from five low-passage clinical isolates of human cytomegalovirus were amplified by polymerase chain reaction. The DNA sequences as well as the predicted amino acid sequences were compared with those of the laboratory strains AD169 and Towne. The genomic regions consisted of (i) three regions from the major glycoprotein (gp58/116, unique long [UL]55), (ii) three regions from the integral membrane protein (IMP, UL100), and (iii) a region from the major immediate-early 1 and 2 (IE-1/2) enhancer/promoter. Homologies ranged from 75.8 to 100.0% on the nucleotide level and from 47 to 100% on the amino acid level. The following two patterns were observed. (i) There are regions with a high degree of conservation with few scattered point mutations (mainly in the IE-1/2 enhancer/promoter and in the IMP gene). (ii) There are clusters of highly variable regions (parts of the gp58/116 gene and of the IMP gene). Within the areas of high variability, the strains could be classified into a limited number of subtypes.

Amino Acid Sequence↗
PubMed1991

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.

Animals↗