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Biomedical subjects

D R Milich

Publications and source records attributed to D R Milich.

At least 91 records · Page 5Linked to original sources
PubMed1987

Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences.

Amphotropic retroviral expression systems were used to synthesize hepatitis B virus surface antigen (HBsAg) and core antigen. The vectors permitted establishment of cell lines which expressed antigen from either the retroviral long terminal repeat or the mouse metallothionein-I promoter. HBsAgs were synthesized containing no pre-S sequences, pre-S(2) sequences alone, or pre-S(1) plus pre-S(2) sequences. Inclusion of pre-S(2) sequences did not affect the secretion or density of HBsAg particles but did reduce their mass by approximately 30%. Addition of pre-S(1) sequences almost completely abolished secretion of HBsAg and resulted in its localization in an aqueous-nonextractable pre- or early-Golgi cellular compartment. HBsAg was localized to the cytoplasm of the cell. This localization was unaffected by the presence of pre-S sequences in the antigen. Cell lines synthesizing hepatitis B antigens from core DNA fragments, containing or not containing precore sequences, secreted hepatitis B e antigen. However, the absence of precore DNA sequences resulted in additional synthesis of hepatitis core antigen, which was predominantly nuclear in localization.

Animals↗
PubMed1986

The nucleocapsid of hepatitis B virus is both a T-cell-independent and a T-cell-dependent antigen.

One characteristic of the immune response during hepatitis B virus (HBV) infection in humans is the vigorous production and subsequent persistence of antibodies of immunoglobulin (Ig) classes M and G to the nucleocapsid antigen (HBcAg). In this study HBcAg was shown to be similarly immunogenic in mice. When injected into athymic (nude) B10.BR and athymic BALB/c mice, HBcAg induced IgM and IgG class antibodies to HBc in spite of the absence of T cells in nude mice. In euthymic mice, HBcAg efficiently stimulated T-cell proliferation in vitro and helper T-cell function in vivo. The dual functions of HBcAg as a T-cell-independent and a T-cell-dependent antigen may explain its enhanced immunogenicity. Denaturation of HBcAg yields a nonparticulate antigen designated HBeAg; when HBeAg was used as the immunogen, antibody production required helper T-cell function. Although HBcAg and HBeAg are serologically distinct, they are structurally related, and in these experiments were highly cross-reactive at the T-cell level. These results suggest that the elevated levels of IgM antibodies to HBc and the enhanced immunogenicity of HBcAg during HBV infection in humans reflect the ability of HBcAg to directly activate B cells to produce antibodies to HBc in the presence or absence of HBcAg- or HBeAg-sensitized T cells.

Animals↗
PubMed1986

Two distinct but overlapping antibody binding sites in the pre-S(2) region of HBsAg localized within 11 continuous residues.

The fine specificity of the humoral immune response to the pre-S(2) region of the hepatitis B surface antigen was studied. It was demonstrated that the murine antibody response to the pre-S(2) region is focused on residues 133 through 143, and two distinct but overlapping epitopes were identified within 11 continuous residues. One epitope, defined by p133-139, is group specific, and the other epitope, defined by p137-143, is influenced by a subtype-dependent amino acid substitution at residue 141. However, the influence of residue 141 was "covert" in that it was only detected when synthetic antigens of 19 amino acids or smaller were used as the solid-phase ligand. The minimum size of both epitopes (p133-139 and p137-143) was seven amino acids. The physical and chemical form of the immunogen (i.e., protein vs peptide; conjugated vs free peptide) influenced antibody fine specificity. In quantitative antibody inhibition studies it was demonstrated that antibodies with nonoverlapping as well as overlapping fine specificities were capable of mutual inhibition. Finally, human HBV-infected, patient sera were shown to possess anti-pre-S(2) region antibodies that recognized sequences in common with the murine antisera. These results have implications relevant to the design of synthetic and recombinant second generation HBV vaccines and diagnostic reagents.

Amino Acid Sequence↗
PubMed1986

Nonoverlapping T and B cell determinants on an hepatitis B surface antigen pre-S(2) region synthetic peptide.

We have examined T cell recognition of a hepatitis B surface antigen (HBsAg), pre-S(2)-region synthetic peptide, p120-145, in terms of fine specificity, H-2-linked genetic influences, comparison to antibody binding, and relevance to T cell recognition of the native protein. We showed that the immune response to the synthetic peptide is regulated by H-2-linked genes, but that the pattern of H-2 restriction differed from that observed for the native anti-pre-S(2) response. Dominant and nonoverlapping T cell and B cell recognition sites were identified on the synthetic peptide p120-145. T cell recognition is focussed on the NH2-terminal sequence, and antibody (B cell) recognition is focussed on the COOH-terminal sequence. The fine specificity of T cell recognition of p120-145 was defined by a single, subtype-dependent amino acid substitution. With respect to the immunogenicity of p120-145, the synthetic peptide containing both T and B cell determinants is highly immunogenic in responder strains, whereas separate T or B cell peptide determinants are minimally immunogenic. Furthermore, the synthetic T cell recognition site can prime T cell help for antibody production to the synthetic B cell site, which is crossreactive with the native pre-S(2) region of HBsAg/p33 particles. This system provides evidence that totally synthetic T cell and B cell recognition sites can be combined to yield a functional immunogen.

Amino Acid Sequence↗
PubMed1986

Immune response to the pre-S(1) region of the hepatitis B surface antigen (HBsAg): a pre-S(1)-specific T cell response can bypass nonresponsiveness to the pre-S(2) and S regions of HBsAg.

Hepatitis B surface antigen (HBsAg) particles are composed of a major polypeptide, p25, and additional polypeptides of higher m.w., namely p33 and p39, are variably present. All three polypeptides share the 226 amino acid residues of the S region: p33 consists of the p25 sequence plus an NH2-terminal 55 residues (pre-S(2], and p39 consists of the p33 sequence plus an NH2-terminal 108-119 residues (pre-S(1). In previous studies we demonstrated the influence of two Ir genes on the humoral and cellular immune responses to the S region and identified nonresponder phenotypes (H-2f,s). Subsequent studies showed that the immune response to the pre-S(2) region was regulated by H-2-linked genes independently of the S region response, such that immunization of S region nonresponder, pre-(S2) region responder mice (H-2s) with HBsAg/p33 circumvented nonresponse to the S region. In the present study, we have extended this analysis to the pre-S(1) region of HBsAg, with the following results: 1) and pre-S(1) region is immunogenic at the T and B cell levels; 2) anti-pre-S(1) specific antibody production is regulated by H-2-linked genes and can be independent of anti-S and anti-pre-S(2) antibody production; 3) immunization of H-2f strains with HBsAg/p39 particles containing the pre-S(1) region can bypass nonresponsiveness to the S and pre-S(2) regions in terms of antibody production; 4) two synthetic peptides, p32-53 and p94-117, define murine and human antibody binding sites on the pre-S(1) region, and p1-21 and p12-32 define additional human antibody binding sites; 5) pre-S(1)-specific T cells can be elicited in S and pre-S(2) region nonresponder mice (H-2f) and provide functional T cell help for S-pre-S(2)-, and pre-S(1)-specific antibody production; and 6) a T cell recognition site in the pre-S(1) region, p12-32 was identified. These results are relevant to HBV vaccine development, and possibly to viral clearance mechanisms, since the higher m.w. polypeptides are preferentially expressed on intact virions.

Acute Disease↗
PubMed1986

Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice.

The outer membrane of the hepatitis B virus consists of host lipid and the hepatitis B virus major (p25, gp28), middle (gp33, gp36), and large (p39, gp42) envelope polypeptides. These polypeptides are encoded by a large open reading frame that contains three in-phase translation start codons and a shared termination signal. The influence of the large envelope polypeptide on the secretion of hepatitis B surface antigen (HBsAg) subviral particles in transgenic mice was examined. The major polypeptide is the dominant structural component of the HBsAg particles, which are readily secreted into the blood. A relative increase in production of the large envelope polypeptide compared with that of the major envelope polypeptide led to profound reduction of the HBsAg concentration in serum as a result of accumulation of both envelope polypeptides in a relatively insoluble compartment within the cell. We conclude that inhibition of HBsAg secretion is related to a hitherto unknown property of the pre-S-containing domain of the large envelope polypeptide.

Animals↗
PubMed1985

A transgenic mouse model of the chronic hepatitis B surface antigen carrier state.

In an attempt to establish a model of the healthy carrier state in hepatitis B virus (HBV) infections, transgenic mice expressing HBV genes were produced. Fertilized one-cell eggs were microinjected with subgenomic fragments of HBV DNA containing the coding regions for the HBV surface antigen (HBsAg) and pre-S and X antigens. Either the normal (HBV) or metallothionein promoters were used to obtain expression of the HBV genes. There was no evidence of viral replication or tissue pathology. The integrated HBV DNA sequences were inherited in a normal Mendelian fashion. Three of 16 transgenic mice expressed HBV-encoded gene products to which they were immunologically tolerant. Expression was not tissue specific and may be influenced by the genomic integration site and cellular factors. Both HBsAg and pre-S antigen were detectable within the cytoplasm of hepatocytes and renal tubular epithelial cells. High serum concentrations of HBsAg were detectable and the secreted product appeared authentic as judged by mean density, morphology, mean particle diameter, polypeptide composition, and antigenicity. The absence of tissue pathology in these immunologically tolerant animals supports the hypothesis that cellular injury under these conditions is not a direct consequence of expression of the pre-S or HBs regions of the HBV genome.

Animals↗
PubMed1985

Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen.

The 55 codons upstream of the gene sequence encoding the hepatitis B surface antigen (HBsAg) are called the pre-S(2) region. It has been proposed that polypeptides of high molecular weight that contain the pre-S(2) region should be included in future hepatitis B virus (HBV) vaccines. The pre-S(2) region and the S gene product [25 kilodalton (kD)] together compose a polypeptide of high molecular weight (33 kD). As an initial attempt to determine the relevance of the 33-kD polypeptide to development of an HBV vaccine, the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide was examined. The results indicate (i) the pre-S(2) region is significantly more immunogenic than the S region of HBsAg, (ii) the 26 amino acid residues at the NH2-terminus of the 33-kD polypeptide represent a dominant antibody binding site on the pre-S(2) region, (iii) the immune response to the pre-S(2) region is regulated by H-2-linked genes distinct from those that regulate the response to the S region, and (iv) immunization of an S region nonresponder strain with HBV envelope particles that contain both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region.

Amino Acid Sequence↗
PubMed1985

Distinct H-2-linked regulation of T-cell responses to the pre-S and S regions of the same hepatitis B surface antigen polypeptide allows circumvention of nonresponsiveness to the S region.

Recently, additional polypeptide components of the surface envelope of hepatitis B virus (HBV) have been identified. The pre-S(1) and pre-S(2) regions of the HBV genome encode NH2-terminal amino acid residues that together with the S-gene product (25 kDa) comprise polypeptides of 33 kDa and 39 kDa. The possible immunopathologic significance of these larger polypeptides and their relevance to vaccine development prompted us to examine the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide. Previous work showed that the pre-S(2) region elicits greater antibody production in vivo than does the S region of hepatitis B surface antigen. In this study, we examined immunogenicity of the pre-S(2) region at the T-cell level, H-2- and non-H-2-linked genetic influences on the pre-S(2) response, and the effect of the immune response to one region on the immune response to the other region. The results indicate that (i) the pre-S(2) region is significantly more immunogenic than the S region at the T-cell level; (ii) pre-S(2)-region-specific T-cell activation is regulated by H-2-linked genes and correlates with the H-2 restriction of in vivo antibody production to the pre-S(2) region; (iii) the H-2 restriction of the T-cell response to the pre-S(2) region is distinct from the H-2 restriction of the T-cell response to S-region determinants; (iv) non-H-2-linked and non-Igh-linked genes also influence the humoral immune response to the pre-S(2) region; and (v) immunization of an S-region-nonresponder, pre-S(2)-region T-cell-responder strain with HBV envelope particles containing both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region through pre-S(2)-specific T-cell helper function.

Amino Acid Sequence↗
PubMed1985

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). VI. T cell fine specificity.

In the companion paper it was demonstrated that the T cell proliferative response to HBsAg was controlled by I region genes as was previously shown for in vivo anti-HBs production. In this paper, the structural requirements for T cell recognition of HBsAg were compared with B cell (antibody) recognition of HBsAg. Secondly, we attempted to map determinants on HBsAg required for activation of HBsAg-primed T cells, and we examined the influence of I region genotype on the observed T cell antigenic fine specificity. The results of these studies indicate clear differences between T cell and B cell recognition of HBsAg. T cell activation required significantly less native structure as compared with antibody binding to HBsAg. Reduced and alkylated HBsAg, the subunit polypeptide P25, tryptic fragments of P25, and synthetic peptide analogues of HBsAg were all capable of eliciting a T cell proliferative response, whereas these "denatured" forms of the antigen bind anti-HBs marginally or not at all. Furthermore, the results suggest that T cell recognition sites on HBsAg do not necessarily overlap with B cell recognition sites. Examination of T cell fine specificity in a series of H-2 congenic strains, with the use of HBsAg, P25, tryptic fragments of P25, and synthetic peptides, revealed multiple T cell recognition sites on HBsAg, and the particular site(s) recognized is dependent on the H-2 genotype of the responding strain. Finally, preliminary results indicate that the specificity of human, HBsAg-primed T cells appear to be variable among individuals.

Animals↗
PubMed1985

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). V. T cell proliferative response and cellular interactions.

We previously demonstrated that in vivo antibody production to HBsAg in the mouse is regulated by at least two immune response (Ir) genes mapping in the I-A (HBs-Ir-1) and I-C (HBs-Ir-2) subregions of the H-2 locus. To confirm that H-2-linked Ir genes regulate the immune response to HBsAg at the T cell level and to determine if the same Ir genes function in T cell activation as in B cell activation, the HBsAg-specific T cell responses of H-2 congenic and intra-H-2 recombinant strains were analyzed. HBsAg-specific T cell proliferation, IL 2 production, and the surface marker phenotype of the proliferating T cells were evaluated. Additionally, T cell-antigen-presenting cell (APC) interactions were examined with respect to genetic restriction and the role of Ia molecules in HBsAg presentation. The HBsAg-specific T cell proliferative responses of H-2 congenic and intra-H-2 recombinant strains generally paralleled in vivo anti-HBs production in terms of the Ir genes involved, the hierarchy of responses status among H-2 haplotypes, antigen specificity, and kinetics. However, the correlation was not absolute in that several strains capable of producing group-specific anti-HBs in vivo did not demonstrate a group-specific T cell proliferative response to HBsAg. The proliferative responses to subtype- and group-specific determinants of HBsAg were mediated by Thy-1+, Lyt-1+2- T cells, and a possible suppressive role for Lyt-1-2+ T cells was observed. In addition to T cell proliferation, HBsAg-specific T cell activation could be measured in terms of IL 2 production, because anti-HBs responder but not nonresponder HBs-Ag-primed T cells quantitatively produced Il 2 in vitro. Finally, the T cell proliferative response to HBsAg was APC dependent and genetically restricted in that responder but not nonresponder parental APC could reconstitute the T cell response of (responder X nonresponder)F1 mice, and Ia molecules encoded in both the I-A and I-E subregion are involved in HBsAg-presenting cell function.

Animals↗
PubMed1984

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). IV. Distinct H-2-linked Ir genes control antibody responses to different HBsAg determinants on the same molecule and map to the I-A and I-C subregions.

We have previously demonstrated that the murine humoral immune responses to the group-specific a and subtype-specific d/y determinants of hepatitis B surface antigen (HBsAg) are controlled by H-2-linked immune response (Ir) genes. High responder (H-2d,q), intermediate responder (H-2a greater than b greater than k) and nonresponder (H-2f,s) haplotypes have been identified (8, 9). The kinetics and specificity of in vivo antibody production after HBsAg immunization in congeneic, H-2-recombinant strains was analyzed to further define relevant Ir genes and their influence on the immune response to distinct antigenic determinants. These studies indicate that the humoral anti-HBs response is regulated by at least two Ir genes, one in the I-A subregion (Ir-HBs-1) and one in the I-C subregion (Ir-HBs-2) of the murine H-2 complex. Ir-HBs-1 regulates the primary responses to all HBsAg determinants, whereas the influence of Ir-HBs-2 is determinant specific, affecting the responses to the d or y determinants. The anti-a response is regulated exclusively by Ir-HBs-1. Strains possessing only the Ir-HBs-2 gene [B10.S(9R) and B10.HTT] produce no anti-a response and a subtype-specific antibody response is detected only after secondary or tertiary immunization. In contrast, the influence of Ir-HBs-2 in the presence of Ir-HBs-1 is detected upon primary immunization and is additive rather than exclusive. There is also suggestive evidence that the presence of the Ek molecule, at least in the context of I-Ak, may have a suppressive influence on the anti-HBs response. Additionally, HBsAg-specific, T cell proliferative responses were H-2 restricted and the kinetics and specificity of T cell proliferative responses paralleled in vivo antibody production. These data indicate that, although the I-A subregion exerts a dominant influence, distinct Ir-HBs genes, mapping in separate I subregions, control immune responses to alternate HBsAg determinants on the same protein molecule.

Animals↗
PubMed1983

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). II. Qualitative characteristics of the humoral immune response to the a, d, and y determinants of HBsAg.

It was previously demonstrated that the murine humoral immune responses to the common a and subtype-specific d determinants of HBsAg are H-2 restricted. The H-2q haplotype confers high responsiveness and the H-2s haplotype low responsiveness to nonresponsiveness to both determinants. We have now demonstrated that the H-2s haplotype also confers nonresponsiveness to the subtype-specific y determinant as well. Studies of H-2 congenic (nonresponder X responder)F1 and backcross mice indicated that responsiveness was inherited as a dominant trait, with no gene dosage effects observed. Qualitative characteristics of the humoral anti-a and anti-d responses were evaluated with respect to strain variation, kinetics, antigen specificity and antibody titer, affinity, and subclass distribution. Unique immune response patterns were observed for each H-2 haplotype studied. On the basis of these patterns, it was possible to construct a hierarchy of responsiveness to HBsAg of the ad subtype as follows: high responders, H-2q and H-2d; intermediate responders, H-2a greater than H-2b greater than H-2k; and nonresponders, H-2s.

Animals↗
PubMed1982

Immune response to synthetic peptide analogues of hepatitis B surface antigen specific for the a determinant.

Recovery from a natural infection with hepatitis B virus or vaccination with purified envelope protein leads to production of antibodies against the hepatitis B surface antigen (HBsAg). Such physiologic response in man is generally directed against the a determinant of HBsAg common to all serotypes of the virus. To define the immunochemical specificity of this determinant, the secondary structure of HBsAg was derived from its sequence of 226 amino acids. Hydrophilic stretches expected to contain the antigenic determinants were located between residues 32 and 76 and between residues 110 and 156. Loss of the antigenic activity after chemical modification of lysine residues of HBsAg indicated their critical importance in antigenicity. Because all lysines are located between residues 121 and 160, we selected this region for localization of HbsAg determinants. Solid-phase synthesis was used to prepare seven peptide analogues of HBsAg (PsAs): 122-137, 128-134, 139-147, 139-158, 140-158, 145-158, and 150-158. For experimental immunization of rabbits the synthetic peptides were coupled to keyhole limpet hemocyanin. We studied the antigenicity of each peptide analogue by serologic neutralization of human antibodies specific for the a determinant of HBsAg. Analogues 139-147, 139-158, and 140-158 showed antigenicity as well as function of anti-HBsAg. The rabbit antibodies were inhibited with each of the three peptide analogues and all serotypes of natural HbSag, having only the a determinant in common. These results indicate that the nonapeptide sequence 139-147 represents the total or an essential part of the a determinant of HBsAg.

Amino Acid Sequence↗
PubMed1982

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). I. H-2 restriction of the murine humoral immune response to the a and d determinants of HBsAg.

In hepatitis B virus (HBV) infection correlation of disease activity with major histocompatibility complex (MHC) antigens has been suggested. However, no data are available regarding the nature and regulation of the immune response to the surface viral protein (HBsAg). This consideration and the current emphasis on production of an HBV vaccine led us to investigate the humoral immune response to HBsAg in inbred strains of mice to assess genetic factors that may regulate the humoral anti-HBs response. Studies with H-2 congenic and noncongenic inbred strains revealed the T-dependent humoral immune responses to the group-specific a and subtype-specific d determinants of HBsAg are regulated by gene(s) mapping to the murine MHC. The H-2q haplotype conferred high responsiveness, the H-2s,f haplotypes conferred low to nonresponsiveness, and the H-2a,b,d,k haplotypes were intermediate. Studies of H-2 recombinant strains allowed tentative mapping of regulation of the anti-a response and possibly the anti-d response to the K/I-A, I-B region of the MHC. The H-2-restricted regulation of the humoral immune response to HBsAg was circumvented by immunization with 10-fold higher HBsAg doses. These findings demonstrate a clear linkage between the murine MHC and the regulation of the immune response to specific determinants on HBsAg.

Animals↗
PubMed1981

Interactions between polymerized human albumin, hepatitis B surface antigen, and complement: I. Binding of polyalbumin to Clq.

There is considerable evidence that substances other than immunoglobulins can bind human Clq. Utilizing purified human Clq immobilized on polystyrene beads, we have demonstrated that polymerized human albumin (PHALB-125I) binds to human Clq in a direct binding assay. This interaction required a high degree of albumin polymerization as the percentage of binding was proportional to the polymer size and monomeric albumin was unreactive. Binding was species specific in the human Clq bound only human, and not xenogeneic, polyalbumins. Similarly, polymers of various other human plasma proteins were unreactive. To demonstrate that this interaction was not unique to immobilized Clq, soluble Clq was shown to inhibit PHALB-125I binding to solid phase Clq. Because aggregated IgG, poly(I):poly(C), dextran sulfate, polyglutamic acid, and polylysine have been previously shown to bind Clq, we used them in further blocking experiments and found them also to inhibit the interaction between Clq and PHALB. Anti-human Clq and, to a lesser extent, anti-PHALB antibodies inhibited the interaction. The Clq-PHALB binding was pH, ionic strength, and temperature dependent. In addition, human Clq was not observed to bind hepatitis B surface antigen (HBsAg) directly; however, in the presence of sufficiently polymerized human albumin a Clq-PHALB-HBsAg complex was formed. These interactions may be implicated in hepatocyte-HBsAg receptor function as well as in the host defense mechanisms involved in hepatitis B virus infection.

Animals↗