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Biomedical subjects

A R Neurath

Publications and source records attributed to A R Neurath.

At least 19 recordsLinked to original sources
PubMed1992

Cells transfected with human interleukin 6 cDNA acquire binding sites for the hepatitis B virus envelope protein.

Earlier studies revealed that human interleukin 6 (IL-6) contains recognition sites for the hepatitis B virus (HBV) envelope (env) protein, and that IL-6 and anti-IL-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for HBV with the preS(21-47) segment of the HBV env protein, encompassing the complementary attachment site for IL-6. This suggested that IL-6 mediates HBV-cell interactions. We report that: (a) Chinese hamster ovary cells transfected with human IL-6 cDNA and Spodoptera frugiperda ovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS(21-47) region of the HBV env protein, indicating that expression of IL-6 on the surface of cells is sufficient to endow them with receptors for HBV. (b) Among peptides covering the entire sequence of human IL-6 and the corresponding antipeptide antibodies, the peptide IL-6[35-66] and anti-IL-6[35-66] most effectively inhibited the interaction between human hepatoma HepG2 cells and the preS(21-47) ligand, suggesting that this region of the human IL-6 sequence encompasses a binding site for the HBV env protein. (c) Studies with replacement set peptides from the preS(21-47) sequence indicated that residues 21-25, 28, 31, 33-35, 39, and 43-45 can be replaced by alanine (serine) residues, while all the other residues are essential for maintaining the cell receptor/IL-6 binding activity. Further delineation of complementary sites on IL-6 and on the HBV env protein may contribute to the design of compounds inhibiting HBV replication.

Amino Acid Sequence↗
PubMed1992

Search for hepatitis B virus cell receptors reveals binding sites for interleukin 6 on the virus envelope protein.

The major target organ for hepatitis B virus (HBV) is the liver. However, cells other than hepatocytes, including peripheral blood lymphocytes and monocytes, may become infected with HBV. The cell receptor binding site was assigned to the preS(21-47) segment of the HBV envelope protein. HBV receptors were detected on human liver and hepatoma cells, on B lymphocytes, and, as shown here, on monocytes, and T cell lines, activated by Escherichia coli lipopolysaccharide and concanavalin A, respectively. The cell receptors for HBV have not been characterized until now. The detection of HBV receptors and their "activation antigen" characteristic on distinct cells suggested paths for identification of the receptors with already defined cell surface proteins. This search revealed that interleukin 6 contains recognition sites for the preS(21-47) sequence and mediates HBV-cell interactions. Thus, HBV belongs to a group of viruses utilizing cytokines or cytokine receptors for replication and interference with the host immune system.

Animals↗
PubMed1992

Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: the envelope of the human immunodeficiency virus (HIV-1).

Synthetic peptides and anti-peptide antibodies have been widely used as probes to map B- and T-cell epitopes on proteins. Such probes also have the potential to delineate contact sites involved generally in protein-protein interactions or in association of domains within a protein. We applied peptide/anti-peptide probes to define: (1) regions on the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41 involved in the association between these two glycoproteins; and (2) sites on gp120/gp41, essential for the association of HIV-1 with the CD4 cell receptor. Results of this examination suggested the following: (1) two segments on gp120, encompassing residues (102-126) and (425-452), contribute to the binding site for CD4 and are expected to be juxtaposed in the folded gp120 chain; (2) portions of immunodominant gp120 and gp41 epitopes, encompassing residues (303-338) and (579-611), respectively, appeared to be involved in the gp120-gp41 association, as suggested by direct binding studies and by the limited accessibility of these epitopes on HIV-1 virions: other portions of gp120 also appeared to contribute to the association between these two glycoproteins; (3) there is a partial overlap between gp41 and CD4 binding sites on gp120; (4) the fusion domain and a segment (637-666) of gp41 are not accessible to antibodies after oligomerization of gp41; and 5) the gp120-gp41 association was blocked by aurintricarboxylic acid, suggesting the possibility of developing antiviral compounds interfering with HIV-1 assembly.

Amino Acid Sequence↗
PubMed1991

Enhancement of human immunodeficiency virus type 1 infection by antisera to peptides from the envelope glycoproteins gp120/gp41.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (gp120 and gp41) elicit virus-neutralizing antibodies (VNAB) and also antibodies enhancing HIV-1 infection (EAB). Several epitopes eliciting VNAB have been defined, the principal virus-neutralizing determinant being assigned to the V3 loop of gp120. To provide a background for a rational design of anti-HIV vaccines, it also appears important to define domains eliciting EAB. This was accomplished by screening antisera against synthetic peptides covering almost the entire sequence of gp120/gp41 for their enhancing effects on HIV-1 infection of MT-2 cells, a continuous T cell line. Many (16/30) of the antisera significantly enhanced HIV-1 in the presence of human complement. Antibodies to complement receptor type 2 (CR2) abrogated the antibody-mediated enhancement of HIV-1 infection. Antisera to V3 hypervariable loops of 21 distinct HIV-1 isolates were also tested for their enhancing effects on HIV-1IIIB infection. 11 of these sera contained VNAB and 10 enhanced HIV-1IIIB infection. All antisera with virus-enhancing activity contained antibodies crossreactive with the V3 loop of HIV-1IIIB, and the virus-enhancing activity increased with increasing serological crossreactivity. These results suggest that immunization with antigens encompassing V3 loops may elicit EAB rather than protective antibodies if epitopes on the immunogen and the predominant HIV-1 isolate infecting a population are insufficiently matched, i.e., crossreactive serologically but not at the level of virus neutralization.

Animals↗
PubMed1991

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef.

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.

Animals↗
PubMed1991

Peptides mimicking selected disulfide loops in HIV-1 gp120, other than V3, do not elicit virus-neutralizing antibodies.

The positions of all 9 intrachain disulfide bonds within the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1) have been established recently. Peptides expected to mimic some of the disulfide-bonded domains [(120-133)-(203-221); (133-138)-(164-203); (224-254); (391-425) and (385-392)-(425-452)] were synthesized. All peptides, except (120-133)-(203-221), elicited in immunized rabbits relatively high levels of antibodies reacting with gp120 in enzyme-linked immunosorbent assay (ELISA) and/or Western immunoblot assays. However, these antibodies failed to neutralize the infectivity of HIV-1. Combined with earlier reports concerning other gp120 loop peptides, these results confirm the uniqueness of the V3 (303-338) loop in encompassing a principal determinant(s) involved in virus neutralization.

Amino Acid Sequence↗
PubMed1991

Antibody responses of chimpanzees immunized with synthetic peptides corresponding to full-length V3 hypervariable loops of HIV-1 envelope glycoproteins.

Immunization of primates or humans with human immunodeficiency virus type 1 (HIV-1) glycoproteins usually elicited moderate immune responses to the principal neutralizing determinant (PND) located within the V3 hypervariable loop of gp120. Since an antibody response to the PND appears to be protective, experiments were carried out to determine the responsiveness of chimpanzees to immunization with synthetic peptides corresponding to the full-length V3 loop. Seven chimpanzees (4 preimmunized with gp160, 2 preimmunized with HIV-1 antigens unrelated to gp160, and 1 unimmunized) were vaccinated with a mixture of full-length V3 loop peptides from 21 distinct HIV-1 isolates (clones) either in unconjugated form or linked to carrier proteins from HIV-1 nef and gag P18, respectively. Six chimpanzees developed high levels of antibodies to the peptides (dilution endpoints 1: greater than 25,000), and 5 had high levels of antibodies to gp120 from HIV-1IIIB (endpoint titers 1: greater than 500,000). Chimpanzees immunized with peptide-carrier conjugates (4) had antibodies to the carrier proteins nef and gag P18, respectively (endpoint titers 1: greater than or equal to 35,000). Virus-neutralizing (VN) antibodies were detected in sera of 5 of 7 chimpanzees, but were present at titers of 1: greater than or equal to 400 only in sera of 2 chimpanzees. One of these was challenged with HIV-1 and was protected against infection, as reported elsewhere. The antibodies were primarily specific for the HIV-1 isolate used for primary immunization before boosting with peptides. The relatively low dilution endpoints of VN antibodies as compared with endpoints determined by site-specific immunoassays probably can be ascribed to imperfect mimicry of conformational epitopes by synthetic peptides. Nevertheless, sequential or simultaneous immunization with recombinant envelope glycoproteins of HIV-1 and selected synthetic peptides offers an approach for eliciting protective immunity against HIV-1.

Amino Acid Sequence↗
PubMed1990

Antigenic mimicry of an immunoglobulin A epitope by a hepatitis B virus cell attachment site.

The preS(21-47) sequence of the hepatitis B virus (HBV) envelope protein is involved in binding of the virus to cell receptors. A protein similarity search revealed a partial homology between this sequence and a segment of the human immunoglobulin A (IgA) heavy chain constant region, suggesting that the cell attachment site for HBV might be located on secretory component representing a receptor for polymeric IgA. Data presented herein do not support this hypothesis but provide evidence for immunological cross-reactivity between IgA and the preS(21-47) region of the HBV env protein.

Amino Acid Sequence↗
PubMed1990

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1).

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.

Amino Acid Sequence↗
PubMed1990

Search for epitope-specific antibody responses to the human immunodeficiency virus (HIV-1) envelope glycoproteins signifying resistance to disease development.

It is essential for the development of strategies for prevention and therapy of human immunodeficiency virus (HIV-1) infections to define host factors playing a dominant role in determining the clinical outcome of infection. Antibodies directed against restricted regions of the HIV-1 glycoproteins gp120 and gp41 are likely to represent important factors involved in host defense against HIV-1. Definition of qualitative and quantitative differences in the spectrum of anti-gp120 and anti-gp41 antibodies between two vastly different groups of HIV-1-infected individuals, long-term asymptomatic carriers, and individuals with acquired immunodeficiency syndrome (AIDS) who died, might reveal the epitope specificity of antibodies contributing to prevention of clinical disease. To accomplish this goal, sera from both groups were assayed for antibodies recognizing synthetic peptides from gp120/gp41 which were shown in earlier experiments to mimic epitopes on the two HIV-1 glycoproteins. None of the sera recognized all of the distinct 27 peptides from gp120 and gp41. The spectrum of antibodies was distinct for each of the sera from both groups of HIV-1-infected individuals. Nevertheless, antibody responses distinguishing the two groups from each other were discerned. In particular, it was possible to predict the unfavorable outcome of disease by comparative measurements of levels of antibodies to a peptide (303-338), corresponding to the entire V3 hypervariable loop of gp120 and/or by providing evidence for declining levels of these antibodies during the course of infection. Antibodies recognizing additional peptides [(219-245), (280-306), (425-452), (658-682), (729-758), (808-845), and (845-862)] were significantly less prevalent in AIDS patients than in asymptomatic carriers. It appears possible that maintenance of high levels of the respective antibodies would contribute to preventing AIDS in HIV-1-infected individuals. Active immunization with antigens containing epitopes defined by the respective peptides and/or administration of the corresponding antibodies may be considered as a modality for therapy of HIV-1 infections.

Epitopes↗
PubMed1990

B cell epitope mapping of human immunodeficiency virus envelope glycoproteins with long (19- to 36-residue) synthetic peptides.

Envelope glycoproteins, gp 120 and gp41, of the human immunodeficiency virus type 1 (HIV-1) elicit immune responses, including virus-neutralizing antibodies, which are expected to play a role in the defence against HIV-1 infection. Subregions of the gp120/gp41 sequence have immunosuppressive effects or may be implicated in autoimmune responses. Some of the immunodominant epitopes of gp120/gp41 do not contribute to protective immunity and act as immunological decoys. These circumstances emphasize the need to select from gp120/gp41 regions inducing protective responses. Towards this goal, 30 peptides covering approximately 87% of the HIV-1 strain BH10 gp120/gp41 sequence were synthesized. Antibodies in rabbit and human anti-HIV-1 sera recognized 28 and nine of the peptides, respectively, indicating that most of the gp120/gp41 sequence is immunogenic and secondly, that the antibody response to HIV-1 is restricted in infected humans. Most of the peptides, without conjugation to carriers, elicited high levels of anti-peptide (endpoints 1: greater than 10(4] and anti-gp120/gp41 (endpoints 1: greater than or equal to 10(3] antibodies. The highest levels of virus-neutralizing antibodies were elicited by peptide 306 to 338 from a hypervariable loop of gp120. Additional peptides from the full-length hypervariable loop (303 to 338) of HIV-1 BH10 and from 20 additional HIV-1 isolates were recognized differentially by human anti-HIV, suggesting that success of passive immunization may depend on a match between administered antibodies and the challenging HIV-1 strain, and also that active immunization with selected peptides from a hypervariable region of distinct HIV-1 isolates should be explored further as a method for prophylaxis against infection.

Antibody Specificity↗
PubMed1979

Radioimmunoassay of hepatitis B e-antigen (HBeAg): identification of HBeAg not associated with immunoglobulins.

A radioimmunoassay for hepatitis B e-antigen (HBeAg) is described. Polystyrene beads coated with IgG prepared from a human serum containing antibodies to HBeAg (anti-HBe) and anti-HBe IgG labelled with 125I-p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester were used in the test. The radioimmunoassay was approximately 1,000-fold more sensitive than immunodiffusion. At least a transient presence of HBeAg in serum appears to be a common feature of infections by hepatitis B virus. The radioimmunoassay was instrumental in establishing conditions for identification of apparently free monomeric HBeAg. The HBeAg has an approximate mol. wt.of 35,000 and was recovered after isoelectric focusing in fractions with a pH between 4.25 and 4.8. Polyacrylamide gel electrophoresis revealed the presence in HBeAg of a polypeptide with an apparent mol. wt. of 17,000.

Hepatitis B Antigens↗
PubMed1979

Association of hepatitis B e-antigen (HBeAg) determinants with the core of Dane particles.

Immunoprecipitates obtained by reacting Dane particle cores with human antibodies to hepatitis B core antigen (HBcAg) were chromatographed on columns of Sepharose 4B CL using 3 M-NaSCN as eluant. An antigen having the size and immunological specificity of monomeric e-antigen (HBeAg) was separated from HBcAg by this method. Antisera from animals immunized with HBcAg or HBeAg reacted not only with the antigen used for immunization but also with HBeAg and HBcAg, respectively. This indicates that HBeAg determinants are associated with the core of Dane particles.

Animals↗
PubMed1979

Radioimmunoassay for albumin-binding sites associated with HBsAg: correlation of results with the presence of e-antigen in serum.

A radioimmunoassay for albumin-binding sites associated with hepatitis B surface antigen (HBsAg) is described. Polystyrene beads coated with glutaraldehyde-polymerized human serum albumin were incubated with diluted specimens of HBsAg-positive sera which contained either e-antigen (HBeAg) or anti-HBe or were negative for both HBeAg and anti-HBe as determined by rheophoresis. The quantity of HBsAg attached to the beads, determined by measuring the amount of 125I-labeled anti-HBs adsorbed during a subsequent incubation step, was related to the presence of HBeAg in the specimens.

Adsorption↗
PubMed1979

Immunohistologic demonstration of hepatitis B viral antigens in liver with reference to its significance in liver injury.

Indirect immunoperoxidase stainings for hepatitis B core and surface antigens (HBc&s Ag) were applied to formalin-fixed paraffin sections in 113 liver specimens from 56 patients. Many cytomorphologic staining characteristics of HBc&s Ag were illustrated, and the percentages of the cellular population positive for HBc&s Ag were estimated for all specimens in order to provide the basis for general analyses. The quantitative expressions and the topographic distribution of HBc&s Ag were assessed with respect to their significance and implication in histopathologic reactions. A definitive relationship or relevance was neither established nor completely excluded due to the size of samples. However, cytomorphologically the membranous expression of HBc&s Ag was shown often in association with acute lobular hepatitis and chronic active hepatitis. This observation supports the concept that the membranous expression may be the prerequisite for immune mediated hepatitic injury in viral hepatitis. In this study we also carried out indirect immunoferritin and immunoperoxidase electron microscopy for HBc&s Ag on formalin-fixed liver specimens. The results assured the validity of the light microscopic immunohistologic procedures. The immunoelectron microscopy confirmed the presence of core antigen in the Dane particles formation that takes place in the cisternae of endoplasmic reticulum. The significance of the cytoplasmic core antigen and the possible role of immunoelectron microscopy in the elucidation of mechanisms of hepatitic injury were discussed.

Acute Disease↗