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Biomedical subjects

G Utter

Publications and source records attributed to G Utter.

At least 19 recordsLinked to original sources
PubMed1992

Four distinct antigenic regions are present in the primary structure of HIV-1 and HIV-2 proteinases.

OBJECTIVE: The purpose of this study was to assay reactivity of antibody-positive sera to different parts of the HIV-1 and HIV-2 proteinase (PR) proteins. DESIGN: Since the majority of HIV-1-antibody-positive sera react to the proteinase, but the antigenic determinants on the protein have not been identified, we attempted to identify these determinants. INTERVENTIONS: We synthesized 18 peptides representing the PR of HIV-1 and HIV-2 in order to map serum reactivity to the PR protein. RESULTS: Both HIV-1- and HIV-2-antibody-positive sera recognized four distinct antigenic regions in the HIV-1 and HIV-2 PR. CONCLUSIONS: Correlation between our results and the crystallographic structure of the protein revealed that the antigenic regions are positioned at the surface of the HIV-1 PR. Although the structure of HIV-2 PR has not yet been characterized, our results indicate that the folding of the HIV-1 and HIV-2 PR may be very similar.

Amino Acid Sequence↗
PubMed1991

Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.

Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys.

Amino Acid Sequence↗
PubMed1991

Comparison of linear antigenic sites in the envelope proteins of human immunodeficiency virus (HIV) type 2 and type 1.

The occurrence of dominant linear antigenic sites in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2) was evaluated. Twenty-five peptides representing different regions of HIV-2, strain SBL-6669, were synthesized. For comparison the corresponding peptides of HIV-1, strain BRU, were also prepared. The peptides were tested in enzyme-linked immunosorbent assay (ELISA) with human sera from individuals with proven HIV-1 or HIV-2 infection and simian sera from animals infected with HIV-2 or simian immunodeficiency virus of sooty mangabay monkey origin (SIVsm). Four major antigenic regions were identified. Peptides representing parts or the whole V3 (neutralizing loop) region and an additional stretch of amino acids located at the carboxy terminal of this region showed considerable reactivity. This reaction was predominantly type specific, but some heterotypic reactivity was also seen. Peptides representing the carboxy terminal 21 amino acids of the V3 region of the type-related viruses HIV-2 and SIVsm allowed selective identification of strain-specific antibodies. A second major antigenic region was found close to the carboxy terminal end of the large glycoproteins. This region was cross-reactive between the two types. The two additional dominating antigenic regions were located in the amino terminal region of the transmembrane glycoprotein. One region has previously been shown to be a uniquely antigenic type-specific site. The other region was also type-specific, but was identified only in HIV-2, amino acids Glu634-Lys649. Excellent facilities are available for the design of not only type-unique site-specific serological tests but potentially also type-cross-reactive and strain-specific assays.

Animals↗
PubMed1991

Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid-phase DNA sequencing.

To study the molecular basis for the emergence of human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to neutralization by autologous sera, the DNA sequence of the envelope V3 loop was determined in HIV-1 isolates derived from four patients with primary HIV-1 infection and sequentially thereafter. The gene fragment encoding the V3 loop of gp120 was amplified by a nested polymerase chain reaction (PCR) and subsequently sequenced by a novel solid phase DNA sequencing approach allowing direct sequencing of the viral genome without the need for previous cloning. The results show that all patients have HIV-1 with unique primary sequence of the V3 loop and antibodies to this structure are produced at seroconversion. The structural analysis also demonstrates that the mechanism for virus escape from neutralization in vivo is complex. Thus, in one patient the emergence of neutralization-resistant viruses coincided with the introduction of several amino acid changes in the V3 loop, while in two other patients the V3 loop remained completely unchanged. These findings suggest that an understanding of the interaction between the humoral immune response and HIV-1 may require detailed structural studies of the entire envelope.

Amino Acid Sequence↗
PubMed1991

Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.

Amino Acid Sequence↗
PubMed1991

Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies.

The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Mapping of linear epitopes of human papillomavirus type 16: the L1 and L2 open reading frames.

Certain types of human papillomavirus (HPV), notably HPV type 16, are associated with flat or inverted proliferative lesions of the cervix uteri that can progress to malignancy. As a first step towards the serological study of the epidemiology of HPV, we have synthesized the entire amino acid sequences of the 2 major viral capsid proteins of HPV type 16, L1 and L2, as a set of 66 synthetic 20-residue peptides with an overlap of 5 amino acids. The peptides were tested for reactivity with IgA, IgG and IgM antibodies in the sera of 30 patients with HPV-16-carrying cervical neoplasms. Both IgG and IgM antibody responses were detected, but most of the reactivity found was of the IgA class. The most immunoreactive peptides were further analyzed for reactivity with sera from 22 patients with parotid gland tumors and with sera from 38 healthy individuals. The L2-encoded protein contained only one major linear epitope, which was not specific for HPV-16-carrying neoplasms. In contrast, the L1-encoded protein contained several epitopes that were regularly immunoreactive with antibodies present in the sera of patients with HPV-16-carrying cervical neoplasms, but only rarely so in the sera of patients with other tumors or of healthy individuals.

Amino Acid Sequence↗
PubMed1990

A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop.

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.

Amino Acid Sequence↗
PubMed1989

Immunochemistry of the dominating antigenic region Ala582 to Cys604 in the transmembranous protein of simian and human immunodeficiency virus.

The immunochemistry of two homologous uniquely antigenic peptides representing Ala582 to Cys604 in the transmembrane proteins of simian immunodeficiency virus of rhesus macaque origin, SIVmac (closely related to HIV-2) and HIV-1 (strain HTLV-IIIB) was characterized at the resolution of single amino acids. Five different antigenic sites were identified in the SIVmac peptide by use of 34 mAb against this peptide and two different sites were similarly demonstrated in the HIV-1 peptide by use of 10 peptide-specific mAb. Within some sites the mAb could be subgrouped to show a progressively more narrow epitopic dependence on amino acids in the central part of the site. Three SIVmac peptide mAbs had a remarkably narrow amino acid dependence, Glu584 and Tyr586. Anti-peptide mAbs reacting with the site Trp596 to Gln602 effectively blocked the capacity of the peptide to react with human postinfection HIV-2 antibodies previously demonstrated to have a restricted reactivity involving this site. No similar blocking was seen when mAb specific for Leu587 to Gln590 were used except with a single broadly reacting HIV-2 serum, which depended on an amino acid at a distance of only 6 residues, Trp596. A cross-reacting site involving amino acids Ala582 to Glu588/Lys588 was identified with mAb and rabbit hyperimmune sera against the two peptides. This site was not accessible in the intact transmembrane proteins as determined by ELISA and Western blot tests. Antipeptide mAb against other sites as well as rabbit sera reacted strongly in these tests and can be used as type-specific, component-unique reagents.

Alanine↗
PubMed1989

Analysis of a subclass-restricted HIV-1 gp41 epitope by omission peptides.

To define the amino acids involved in IgG subclass reactivity to two overlapping HIV-1 gp41 (E34/32; amino acid positions 582-613) peptides, sera from 18 HIV-infected individuals were studied. Peptides mimicking E34 but with single amino acid deletions or glycine substitutions were used to define the amino acid residues necessary for antibody binding. Two dominating immunogenic epitopes, containing highly hydrophilic amino acids, were found on the original peptide. Further analysis was undertaken with two corresponding omission sets of dodecapeptides representing halves of the complete E34 plus a terminal cystein peptide. The subclass reactivities usually differed between the patients with regard to the epitopes with which the different IgG subclasses reacted and also to the importance of different amino acids in antibody binding. The 600 glycine and the 601 lysine were involved in the binding of all IgG1, 2 and 4 and most IgG3. The development of E34/32-reactive IgM and IgG subclasses showed different patterns in four patients with primary HIV infections, contradicting the existence of a general pattern for the development of IgG subclasses to this peptide. The findings suggest that different progenitor clones are selected for synthesis of the different subclasses.

Amino Acid Sequence↗
PubMed1986

Detection of antigen in immune precipitates by silver staining of SDS-polyacrylamide gels.

The silver staining technique of Merril et al. (1981) was used to identify antigens separated by SDS-PAGE after dissociation of immune complexes purified by absorption with staphylococcus A protein. The five and four dominating structural components of measles and RS virus (Mr range 28,000 to 79,000) respectively precipitated with monoclonal antibodies were readily identified, except when the Mr of the virus-specific antigen was in the same range as immunoglobulin heavy and light chains. Specific antigens were identified less effectively after precipitation with polyclonal sera because of a larger background of stainable proteins. This method, which combines the sensitivity and high resolution of silver staining and the specificity of immune complexing with monoclonal antibodies, is rapid and inexpensive.

Antibodies, Monoclonal↗
PubMed1986

Protection against canine distemper virus in dogs after immunization with isolated fusion protein.

Canine distemper virus attachment (hemagglutinin [H] equivalent) and fusion (F) antigens were purified by affinity chromatography with monoclonal antibodies. The purified antigens were used to immunize groups of three dogs. Radioimmune precipitation assays with sera from these animals showed that the F antigen preparation was pure and induced only an F polypeptide-specific antibody response but that the H antigen preparation had a slight contamination by the F antigen. Immunized animals were challenged with virulent canine distemper virus. Two animals in each group developed pronounced humoral and cellular immune responses after challenge. Among these infected animals, only the dogs immunized with H antigen developed symptoms, albeit mild. In contrast, three nonimmunized control animals developed severe disease, with a fatal outcome in two cases. The complete resistance against challenge in two dogs was interpreted to reflect in one case anti-F immunity and in the other case most likely a high level of anti-H immunity. It is suggested that the F antigen may be of particular interest for the development of morbillivirus and possibly other paramyxovirus subunit or synthetic vaccines, because it can induce immunity capable of blocking virus infection and in situations of virus replication prevent the emergence of symptoms.

Animals↗
PubMed1979

F-Actin-depolymerizing activity of human serum.

Non-heated human and animal sera contain a factor which exhibited an inhibiting activity on the staining of actin-containing structures by anti-actin antibodies in indirect immunofluorescence experiments. The presence of this factor lowered the viscosity of F-actin preparations and caused, as studied by electron-microscopy, a depolymerization of F-actin filaments as well as inhibition of filament formation of G-actin. The factor was, after its reaction with F-actin, liberated seemingly unaffected, indicating an enzymatic activity. The factor tentatively termed 'F-actin depolymerizing factor' was heat-sensitive and trypsin sensitive but resisted reduction. It was Ca2+ dependent and the staining inhibiting reaction was faster at 30 degrees C and 37 degrees C than at lower temperatures. Gel filtration experiments on Sephadex G-200 suggested a molecular size of the actin depolymerizing factor slightly higher than that of albumin. The electrophoretic mobility was that of gamma 2 globulin. The physiological role of the factor might be to prevent the presence of F-actin filaments within the circulation.

Actins↗
PubMed1978

Purification of hepatitis B surface antigen by affinity chromatography.

Hepatitis B surface antigen (HBsAg), was purified from plasma by affinity chromatography on matrix-bound sulphated carbohydrates such as heparin or dextran sulphate. Further purification by precipitation with polyethylene glycol (PEG) 6000 resulted in a highly purified HBsAg preparation. The overall recovery amounted to about 70% of the total antigen content of the starting plasma. Electron microscopic data revealed mainly 22 nm spherical particles accompanied by few or no filaments. The process is simple, rapid and lends itself readily to large-scale applications.

Chromatography, Affinity↗
PubMed1977

The reaction of cells with anti-actin sera in relation to the amount of cellular actin.

The staining pattern of anti-actin sera on various cells smeared on glass was compared to the relative amount of cellular actin estimated by SDS-polyacrylamide-gel electrophoresis with subsequent scanning of the gel. Although the cells showed a varying stainability the actin content was fairly constant. Thus, the staining differences reflected changes in the organization of cellular actin rather than actual differences in the amount of actin.

Actins↗