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D R Milich

Publications and source records attributed to D R Milich.

At least 73 records · Page 4Linked to original sources
PubMed1990

Differentiation-specific transcriptional regulation of the hepatitis B virus large surface antigen gene in human hepatoma cell lines.

The transcriptional activities of the four hepatitis B virus promoters were compared in three differentiated hepatoma cell lines, HepG2, Hep3B, and PLC/PRF/5; a dedifferentiated subline of HepG2, HepG2.1; a human cervical carcinoma cell line, HeLa S3; and a mouse fibroblast cell line, NIH 3T3. The plasmid constructs, which contain the complete hepatitis B virus genome directing the expression of the luciferase reporter gene, were analyzed by transient transfection assays. The relative orders of the levels of the transcriptional activities of the four promoters were similar in each of the cell lines. The major surface antigen and X-gene promoters displayed the highest activity levels, the core promoter activity level was less than or similar to the activity levels of these two promoters, and the large surface antigen promoter had the lowest activity level in all of the cell lines examined. The core promoter demonstrated an approximately 2- to 20-fold higher relative level of expression in the differentiated hepatoma cell lines, suggesting that this promoter might be preferentially active in these cells. The relative level of activity of the large surface antigen promoter in the differentiated hepatoma cell lines was approximately 5 to 90 times greater than that observed in the other cell lines, indicating that the activity of this promoter is highly specific for differentiation state and cell type. Deletion analysis of the large surface antigen promoter demonstrated that the sequence element responsible for the differentiation state-specific expression from this promoter is located between nucleotides 2719 and 2733 (-90 and -76). Within this sequence element is a binding site (GTTAATCATTACT) for the liver-specific transcription factor hepatocyte nuclear factor 1 (HNF1). This indicates that the preferential expression from the large surface antigen promoter in the differentiated hepatoma cell lines is probably mediated by HNF1 or an HNF1-related transcription factor.

Animals↗
PubMed1990

Synthetic peptides: prospects for vaccine development.

There is great interest in the use of synthetic peptides for vaccine purposes. Chemical synthesis guarantees an unlimited source of chemically stable protein at relatively low cost. Additionally, synthetic peptide antigens have been demonstrated to immunize T helper cells, cytotoxic T cells, and B cells which are reactive with representative native protein antigens. However, a number of obstacles to the use of peptide antigens have also been elucidated. The purpose of this review is to examine our current understanding of peptide antigens as immunogens, and the advantages and disadvantages of their use in vaccine design.

Animals↗
PubMed1989

Functional identification of agretopic and epitopic residues within an HBcAg T cell determinant.

Residues 120-131 within the hepatitis B core Ag (HBcAg) represent a dominant T cell recognition site for mice of the H-2S haplotype. This study was undertaken in order to identify residues within the p120-131 sequence which either interact with the TCR termed epitopic residues or interact with MHC class II molecules termed agretopic residues. For this purpose a panel of analogs of p120-131 composed of peptides containing single alanine substitutions for each residue was synthesized. These peptides were analyzed functionally for their ability to stimulate p120-131 or HBcAg-primed T cells and for their immunogenicity in B10.S or [B10.S X B10 (nonresponder)]F1 mice. Furthermore, analogs of p120-131 were used as stimulators and inhibitors of T cell activation in competitive inhibition experiments. Cumulatively these functional studies allowed us to identify residue 125 as a dominant epitopic residue and residues 127 and 129 as dominant agretopic residues. Furthermore, a p120-131 analog containing an alanine substitution for the dominant agretopic residue was immunogenic in B10.S mice, but was nonimmunogenic in (B10.S X B10)F1 mice indicating that T cell responsiveness is influenced by MHC class II gene dosage effects and can be inherited in an apparent recessive manner. In this study, critical residues involved in the immunogenicity of this dominant T cell determinant of HBcAg were defined, in a companion study, the influence of these residues on tolerogenicity was examined.

Amino Acid Sequence↗
PubMed1989

Distinction between immunogenicity and tolerogenicity among HBcAg T cell determinants. Influence of peptide-MHC interaction.

One purpose of this study was to examine the concept of T cell immunodominance employing a neonatal tolerance model. The extent to which a single T cell recognition site can represent the total T cell response to hepatitis B core Ag (HBcAg) was examined in the B10.S and B10 murine strains. It was shown that the entire B10.S T cell response to HBcAg was focused on a single immunodominant site represented by residues 120-131. This was demonstrated by exposing B10.S neonatal mice to p120-140 or p120-131, which resulted in a state of T cell tolerance to the entire HBcAg. In contrast, p120-140 contained an immunogenic T cell site for B10 mice, p129-140, but this site was nontolerogenic. Similarly, injection of p120-140 into (B10.S X B10)F1 neonatal mice resulted in tolerization of p120-131-specific, I-As-restricted T cells, but not of p129-140-specific, I-Ab-restricted T cells. The second purpose of this study was to attempt to explain the immunologic basis of an immunogenic yet nontolerogenic T cell determinant. It was shown that the p120-131 T cell site, which is immunogenic and tolerogenic in B10.S mice, could be converted into an immunogenic/nontolerogenic T cell site by a single amino acid substitution in either residue 127 or 129. Residues 127 and 129 were previously shown to be involved in interaction with MHC class II molecules (agretopic). These results demonstrated that the relative avidity of a peptide-MHC interaction can influence T cell tolerance induction. Furthermore, the results suggest that a higher threshold of peptide-MHC avidity may be required to induce T cell tolerance as compared to the threshold of peptide-MHC avidity required to immunize T cells.

Amino Acid Sequence↗
PubMed1989

Molecular and genetic aspects of the immune responses to hepatitis B viral antigens.

In the absence of an inbred animal model of hepatitis B virus (HBV) infection, several laboratories have chosen to study the murine immune response to HBV-encoded proteins as immunogens as opposed to an infectious agent. This article reviews the immunogenicity, the fine specificity of T and B cell recognition of HBV antigens, and the genetic influences that regulate these responses. It is anticipated that this approach will increase our understanding of immune-mediated viral clearance mechanisms during HBV infection, and may provide the framework for the design of second and third generation HBV vaccines.

Animals↗
PubMed1989

Retroviral-mediated transfer and expression of hepatitis B e antigen in human primary skin fibroblasts and Epstein-Barr virus-transformed B lymphocytes.

Previously, an amphotropic retroviral expression system coding for the neomycin resistance gene was developed and used to synthesize hepatitis B e antigen (HBeAg) and hepatitis B core/e antigen (HBc/eAg) in transfected mouse NIH 3T3 fibroblasts (A. McLachlan et al., 1987, J. Virol. 61, 683-692). In the present study, these transfected cell lines were infected with a helper amphotropic murine leukemia virus resulting in the production of infectious recombinant retrovirus. The recombinant retrovirus was examined for its capacity to transmit resistance to the antibiotic, G418, and to express hepatitis B virus antigens in mouse NIH 3T3 fibroblasts, human primary skin fibroblasts, and Epstein-Barr virus (EBV)-transformed B lymphocytes. A mouse NIH 3T3 fibroblast clone was generated which produced recombinant retrovirus with the capacity to transmit HBeAg expression to these murine and human cell lines. In contrast, it was not possible to transmit HBc/eAg synthesis efficiently to these cell lines by recombinant retroviral infection. The difference between the efficiencies of transmission of HBeAg and HBc/eAg expression by recombinant retroviral-mediated infection was not predicted as the expression vector coding for HBc/eAg synthesis differs only by the deletion of approximately 90 nucleotides of HBV DNA sequence from the vector coding for HBeAg synthesis.

Adult↗
PubMed1989

Characterization of hepatitis B virus major surface antigen gene transcriptional regulatory elements in differentiated hepatoma cell lines.

The regulatory DNA sequence elements that control the expression of the hepatitis B virus major surface antigen gene in the hepatoblastoma cell line HepG2 were analyzed by using transient transfection assays. In this system, the hepatitis B virus enhancer increases transcription from the surface antigen promoter approximately twofold. The promoter elements regulating the expression of this gene are within a 200-nucleotide sequence located immediately upstream of the transcription initiation sites. The promoter consists of an 85-nucleotide distal element which increases transcription from the surface antigen gene by two- to fourfold and a proximal element of approximately 115 nucleotides which is essential for transcriptional activity. The proximal and distal promoter elements were shown to bind factors present in HepG2 nuclear extracts, which is consistent with the regulatory role demonstrated for these sequences. The regulatory role of these promoter sequences in the hepatocellular carcinoma cell lines PLC/PRF/5 and Hep3B was also demonstrated, indicating similar transcriptional regulation of the surface antigen gene in each of these differentiated hepatoma cell lines.

Base Sequence↗
PubMed1988

Comparative immunogenicity of hepatitis B virus core and E antigens.

The nucleocapsid (hepatitis B core Ag (HBcAg] of the hepatitis B virus is a particulate Ag composed of a single polypeptide (p21). Although a non-particulate form of HBcAg designated hepatitis B e Ag (HBeAg) shares significant amino acid identity, the immune responses to these Ag appear to be regulated independently. This report describes the use of recombinant HBcAg and HBeAg to examine and compare murine T cell and B cell recognition of these related Ag. The HBcAg preparation was stable at pH 7.2 and 9.6 and expressed HBc antigenicity. However, the antigenicity of the HBeAg preparation was pH dependent. At pH 9.6 the HBeAg preparation was non-particulate and expressed HBe antigenicity exclusively; however, at pH 7.2 it was particulate and expressed both HBc and HBe antigenicities. Although this "hybrid" particle most likely does not exist naturally, it is a unique research reagent to investigate the interrelationship between HBcAg and HBeAg. HBcAg was significantly more immunogenic in terms of in vivo antibody production as compared to either the non-particulate or particulate forms of HBeAg. Nevertheless, in most murine strains HBcAg and HBeAg were equivalently immunogenic and crossreactive at the level of T cell activation. The disparity between anti-HBc and anti-HBe antibody production is best explained by the observation that HBcAg can function as a T cell-independent Ag whereas HBeAg is T cell dependent even when present within the same particulate structure as HBcAg. Furthermore, HBcAg was shown to function efficiently as an immunologic carrier moiety for the DNP hapten in athymic as well as euthymic mice in contrast to conventional carrier proteins. These results have implications relevant to the human immune responses to HBcAg and HBeAg during infection, and to vaccine development.

Animals↗
PubMed1988

Hepatitis B synthetic immunogen comprised of nucleocapsid T-cell sites and an envelope B-cell epitope.

Previous studies located T-cell recognition of the nucleocapsid of the hepatitis B virus (HBcAg) to residues 120-140 in mice bearing the H-2s or H-2b haplotypes. Herein, we demonstrate that B10.S (H-2s) and B10 (H-2b) H-2 congenic strains recognize distinct T-cell sites within the p120-140 (a synthetic peptide corresponding to residues 120-140 of HBcAg) sequence defined by p120-131 and p129-140, respectively. Peptide p120-131 stimulates B10.S HBcAg-primed T cells, and reciprocally p120-131-primed T cells recognize HBcAg. Similarly, the p129-140 sequence is a T-cell recognition site relevant to the native HBcAg in the B10 strain. It is also shown that these 12-residue peptides efficiently prime T-helper cells, which are capable of eliciting antibody production to HBcAg in vivo. These observations prompted us to examine the ability of the HBcAg-specific p120-140 sequence to function as a T-cell carrier moiety as a component of a totally synthetic hepatitis B vaccine. For this purpose a synthetic B-cell epitope from the pre-S(2) region (p133-140) of the viral envelope was chosen because this sequence represents a dominant antibody-binding site of the envelope. Immunization of B10.S and B10 strains with the synthetic composite peptide c120-140-(133-140) elicited anti-peptide antibody production, which was crossreactive with the native viral envelope. Furthermore, c120-140-(133-140) immunization primed p120-131-specific T cells in the B10.S strain and p129-140-specific T cells in the B10 strain, which recognized HBcAg and provided T-helper cell function for anti-envelope antibody production in vivo. These results demonstrate the feasibility of constructing complex synthetic immunogens that represent multiple proteins of a pathogen and are capable of engaging both T and B cells relevant to the native antigens.

Animals↗
PubMed1987

Immune response to hepatitis B virus core antigen (HBcAg): localization of T cell recognition sites within HBcAg/HBeAg.

Hepatitis B virus nucleocapsid particles (HBcAg) can function as a T cell-independent antigen when injected into athymic mice. However, immunization of euthymic mice with HBcAg results in dramatically increased anti-HBc titers. Therefore we have examined the murine T cell response to HBcAg in terms of immunogenicity, the influence of H-2-linked genes, and the fine specificity of T cell recognition using synthetic peptide analogs. The HBcAg was shown to be an extremely efficient immunogen in terms of T cell activation as measured by the in vivo dose required to induce T cell sensitization (1.0 microgram), and the minimal in vitro concentration required to elicit interleukin 2 (IL 2) production (0.03 ng/ml). The degree of T cell immunogenicity of HBcAg and its ability to directly activate B cells most likely explain the enhanced humoral response to HBcAg in euthymic mice and HBV-infected patients. The influence of H-2-linked genes on the humoral response to HBcAg was discernable, and high responder (H-2k,s,d), intermediate responder (H-2b,f), and low responder (H-2p) haplotypes were identified. The H-2-linked regulation of the T cell response correlated with in vivo anti-HBc production. Examination of the fine specificity of T cell recognition revealed HBcAg-specific T cells from a variety of strains recognize multiple but distinct sites within the HBcAg/HBeAg sequence. T cell recognition sites were defined by small (16 to 21 residue) synthetic peptides. Each strain recognized a predominant T cell determinant, and the fine specificity of this recognition process was dependent on the H-2 haplotype of the responding strain. For example H-2s,b strains recognized p120-140, H-2f,q strains recognized p100-120, and H-2d mice recognized p85-100 predominantly. Because these sequences are common to both HBcAg and a nonparticulate form of the antigen termed HBeAg, these results indicate that HBcAg and HBeAg are highly cross-reactive at the T cell level although they are serologically distinct. These findings may have clinical relevance, because T cell sensitization to HBeAg and the subsequent seroconversion to anti-HBe status correlates with viral clearance during hepatitis B infection.

Animals↗
PubMed1987

A single 10-residue pre-S(1) peptide can prime T cell help for antibody production to multiple epitopes within the pre-S(1), pre-S(2), and S regions of HBsAg.

The purpose of this study was to identify and characterize T cell and B cell recognition sites within the pre-S(1) region of HBsAg/p43, and to then analyze functional T cell-B cell interactions at the level of in vivo antibody production. The results indicate: three peptide sequences within the pre-S(1) region of HBsAg were identified which can induce and elicit HBsAg/p43-specific T cell proliferation; a 10-amino acid peptide, p12-21, defines one pre-S(1)-specific T cell recognition site, and residues 18 and 19 are critical to the recognition process; the p12-21 sequence can function as a T cell carrier for a synthetic B cell epitope within the pre-S(2) region; the p94-117 sequence contains at least two T cell recognition sites; five distinct, pre-S(1)-specific antibody binding sites were identified; synthetic pre-S(1) region T cell determinants can prime in vivo antibody production to multiple B cell epitopes within the pre-S(2) and S regions, as well as within the pre-S(1) region; the specificity of the primed T cell population can influence the specificity of the B cell response; and T cell recognition of pre-S(1) region peptides is regulated by H-2-linked genes.

Animals↗
PubMed1987

T-cell recognition of pre-S regions of HBsAg can bypass nonresponse to the S region.

The objective of the studies reported herein was to identify and characterize T cell and B cell recognition sites within the pre-S regions of HBsAg/p39, and to analyze functional T-cell-B cell interactions at the level of in vivo antibody production. The results indicate: (1) several peptides within the pre-S(1) region of HBsAg were identified which can induce and elicit HBsAg/p39-specific T-cell proliferation; (2) a 10 amino acid peptide, p12-21, and the 94-117 sequence define pre-S(1)-specific T-cell recognition sites; (3) five distinct, pre-S(1)-specific antibody binding sites and 2 pre-S(2)-specific antibody binding sites were identified; (4) synthetic pre-S(1) region T-cell determinants can prime in vivo antibody production to multiple B-cell epitopes within the pre-S(2) and S regions, as well as within the pre-S(1) region; and (5) specificity of the primed T cell population can influence the specificity of the B-cell response.

Animals↗