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D R Milich

Publications and source records attributed to D R Milich.

At least 37 records · Page 2Linked to original sources
PubMed1997

Interferon-alpha treatment induces delayed CD4 proliferative responses to the hepatitis C virus nonstructural protein 3 regardless of the outcome of therapy.

The proliferative responses to a hepatitis C virus (HCV) recombinant nonstructural protein 3 (rNS3) were analyzed in 9 patients with chronic HCV infection before, during, and after 24 weeks of treatment with interferon-alpha (IFN-alpha) alone or in combination with ribavirin. Regardless of the therapy and the subsequent outcome, all patients showed an increased rNS3-specific proliferative response in peripheral blood mononuclear cells in vitro within 48 weeks from the start of therapy (P < .01). The proliferating cell phenotype was CD4 and was dependent on HLA-DP/DQ/DR class II antigen presentation. rNS3 induced in vitro detectable interleukin (IL)-2, IL-10, and IFN-gamma production in some patients before or after therapy (or both). No significant differences existed between responders and relapsed responders plus nonresponders with respect to the NS3-specific CD4 T helper (Th) cell responses. Thus, IFN-alpha therapy induces HCV NS3-specific CD4 Th cell proliferation regardless of the outcome of therapy.

Adult↗
PubMed1997

Immune responses to the hepatitis C virus NS4A protein are profoundly influenced by the combination of the viral genotype and the host major histocompatibility complex.

The interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using synthetic full-length non-structural (NS) 4A proteins, residues 1658-1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688-1707. In immunized B10 H-2 congenic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670-1679 and 1683-1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restricted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.

Amino Acid Sequence↗
PubMed1997

Influence of T-helper cell subsets and crossregulation in hepatitis B virus infection.

Serological and biochemical studies indicate that acute HBV infection is resolved in the context of an efficient cell-mediated immune (CMI) response, whereas, chronic infection is characterized by weak to undetectable CMI responses and relatively efficient humoral immunity. Because humoral immunity and CMI are regulated by different TH subsets, factors which influence the induction of TH1 vs TH2 cells specific for the HBV nucleocapsid antigens (HBcAg, HBeAg) were examined in a murine model system. The factors which affected the HBc/HBeAg-specific TH1/TH2-cell balance included: (1) the structure of the antigen (i.e. HBcAg vs HBeAg); (2) the host MHC and T-cell site recognized; (3) crossregulation between TH1 and TH2 cells; (4) T-cell tolerance, which is more complete in TH1 than in TH2 cells; (5) secreted HBeAg, which preferentially depletes TH1 cells; (6) the HBV-specific subset response could be skewed towards either TH1 or TH2 predominance by cytokine treatment in vivo. The results suggest that the balance between TH1 and TH2 cells specific for the HBc/HBeAgs may be relevant in acute and chronic HBV infections. Importantly, HBeAg-specific TH2 cells preferentially evade tolerance induction as compared to their TH1-cell counterparts. Because HBeAg may act as a tolerogen during the vertical transmission of chronic HBV infection and preferentially depletes TH1 cells in the circulation, the predominance of HBeAg-specific TH2 cells may influence the initiation or maintenance of the chronic carrier state. In this case, cytokine therapy designed to shift a TH2-like response toward TH1 predominance (i.e. IL-12) may be beneficial in the treatment of chronic HBV infection.

Animals↗
PubMed1997

Antibodies to the hepatitis B e antigen (HBeAg) can be induced in HBeAg-transgenic mice by adoptive transfer of a specific T-helper 2 cell clone.

Production of antibody to hepatitis B e antigen (HBeAg); i.e., anti-HBe antibody,) in HBeAg-transgenic mice is believed to be mediated by T-helper 2 (Th2) cells. Injection of an HBeAg-specific Th2 clone into HBeAg-transgenic H-2k mice induced anti-HBe antibody production, confirming the function of Th2 cells in this model system.

Adoptive Transfer↗
PubMed1997

The hepatitis B virus core and e antigens elicit different Th cell subsets: antigen structure can affect Th cell phenotype.

Secretion of the hepatitis B virus (HBV) e antigen (HBeAg) has been conserved throughout the evolution of hepadnaviruses. However, the function of this secreted form of the viral nucleoprotein remains enigmatic. It has been suggested that HBeAg functions as an immunomodulator. We therefore examined the possibility that the two structural forms of the viral nucleoprotein, the particulate HBV core (HBcAg) and the nonparticulate HBeAg, may preferentially elicit different T helper (Th) cell subsets. For this purpose, mice were immunized with recombinant HBcAg and HBeAg in the presence and absence of adjuvants, and the immunoglobulin G (IgG) isotype profiles of anti-HBc and anti-HBe antibodies were determined. Second, in vitro cytokine production by HBcAg- and HBeAg-primed Th cells was measured. The immunogenicity of HBcAg, in contrast to that of HBeAg, did not require the use of adjuvants. Furthermore, HBcAg elicited primarily IgG2a and IgG2b anti-HBc antibodies, with a low level of IgG3, and no IgG1 anti-HBc antibodies. In contrast, the anti-HBe antibody response was dominated by the IgG1 isotype; low levels of IgG2a or IgG2b anti-HBe antibodies and no IgG3 anti-HBe antibodies were produced. Cytokine production by HBcAg- and HBeAg-primed Th cells was consistent with the IgG isotype profiles. HBcAg-primed Th cells efficiently produced interleukin-2 (IL-2) and gamma interferon (IFN-gamma) and low levels of IL-4. Conversely, efficient IL-4 production and lesser amounts of IFN-gamma were elicited by HBeAg immunization. The results indicate that HBcAg preferentially, but not exclusively, elicits Th1-like cells and that HBeAg preferentially, but not exclusively, elicits Th0 or Th2-like cells. Because HBcAg and the HBeAg are cross-reactive in terms of Th cell recognition, these findings demonstrate that Th cells with the same specificity can develop into different Th subsets based on the structural form of the immunogen. These results may have relevance to chronic HBV infection. Circulating HBeAg may downregulate antiviral clearance mechanisms by virtue of eliciting anti-inflammatory Th2-like cytokine production. Last, the influence of antigen structure on Th cell phenotype was not absolute and could be modulated by in vivo cytokine treatment. For example, IFN-alpha treatment inhibited HBeAg-specific Th2-mediated antibody production and altered the IgG anti-HBe isotype profile toward the Th1 phenotype.

Adjuvants, Immunologic↗
PubMed1997

Characterization of CD8+ cytotoxic T-lymphocyte responses after genetic immunization with retrovirus vectors expressing different forms of the hepatitis B virus core and e antigens.

Cytotoxic T-lymphocyte (CTL) activity appears to play an important role in resolving hepatitis B virus (HBV) infection, and the ability to induce such responses remains an important goal for developing effective immunotherapeutics. A panel of recombinant retrovirus vectors expressing different forms of the HBV core antigen (HBcAg) or e antigen (eAg) were found to induce antigen-specific major histocompatibility complex-restricted CTL responses in both mice and macaques. In addition, a novel retrovirus vector expressing an HBcAg-neomycin phosphotransferase II (HBc-Neo) fusion protein [LHBc-NEO(6A3)], which allows the measurement of the anti-Neo antibody response as a means of directly tracking biological activity of the vector, was generated. Doses greater than 10(7) CFU were necessary to induce CTL responses in H-2(k) mice. Intramuscular injections with 10(8) CFU of the LHBc-NEO(6A3) retrovirus vector into rhesus monkeys induced HBc/eAg-specific antibody production and CD8+ CTLs. The CTL response from one of the two responder rhesus monkeys was directed against a 9-residue peptide, GELMTLATW, at positions 63 to 71 of the HBc/eAg sequence. The CTL response is long lived, being detectable as late as 16 weeks after immunization, and can be boosted upon reimmunization. The potent ability of recombinant retrovirus vectors to induce HBcAg- and eAg-specific CTL responses may prove beneficial as a therapeutic treatment for chronic hepatitis B infection.

Animals↗
PubMed1997

Characterization of humoral and CD4+ cellular responses after genetic immunization with retroviral vectors expressing different forms of the hepatitis B virus core and e antigens.

The humoral and CD4+ cellular immune responses in mice following genetic immunization with three retroviral vectors encoding different forms of hepatitis B virus core antigen (HBcAg) and e antigen (HBeAg) were analyzed. The retroviral vectors induced expression of intracellular HBcAg (HBc[3A4]), secreted HBeAg (HBe[5A2]), or an intracellular HBcAg-neomycin phosphoryltransferase fusion protein (HBc-NEO[6A3]). Specific antibody levels and immunoglobulin G isotype restriction were highly dependent on both the host major histocompatibility complex and the transferred gene. Humoral and CD4+ cellular HBcAg and/or HBeAg (HBc/eAg)-specific immune responses following retroviral vector immunization were of a lower magnitude but followed the same characteristics compared with those after immunization with HBc/eAg in adjuvant. Two factors influenced the humoral responses. First, in vivo depletion of CD8+ cells in HBc-NEO[6A3]-immunized H-2k mice abrogated both HBcAg-specific antibodies and in vitro-detectable cytotoxic T lymphocytes. Second, priming of H-2b mice with an HBc/eAg-derived T-helper (Th) peptide in adjuvant prior to retroviral vector immunization greatly enhanced the HBc/eAg-specific humoral responses to all three vectors, suggesting that insufficient HBc/eAg-specific CD4+ Th-cell priming limits the humoral responses. In conclusion, direct injection of retroviral vectors seems to be effective in priming HBc/eAg-specific CD8+ but comparatively inefficient in priming CD4+ Th cells and subsequently specific antibodies. However, the limited HBc/eAg-specific CD4+ cell priming can effectively be circumvented by prior administration of a recombinant or synthetic form of HBc/eAg in adjuvant.

Animals↗
PubMed1997

Immunization with hybrid hepatitis B virus core particles carrying circumsporozoite antigen epitopes protects mice against Plasmodium yoelii challenge.

The hepatitis B virus nucleocapsid antigen (HBcAg) was investigated as a carrier moiety for circumsporozoite protein (CS) repeat B cell epitopes of the rodent malaria agent Plasmodium yoelii. A vector expressing a hybrid gene coding for the dominant CS repeat epitope (QGPGAP)4 was constructed and transformed into avirulent Salmonella typhimurium. The resulting hybrid HBcAg-CS polyproteins were purified from recombinant Salmonella typhimurium. They purified as particles and displayed HBc as well as P. yoelii CS antigenicity. To investigate immunogenicity and protective efficacy, BALB/c mice were immunized with the hybrid HBcAg-CS particles. Immunization resulted in high titered antinative CS serum IgG antibody litres. BALB/c mice immunized with hybrid HBcAgCS particles were between 90-100% protected against subsequent P. yoelli challenge. Protective immunity persisted for a minimum of three months. These data confirm the previous suggestion (Schödel et al., 1994), that hybrid HBcAg particles could become a useful component of future human malaria vaccines.

Amino Acid Sequence↗
PubMed1996

Immunogenicity and antigenicity of the ATPase/helicase domain of the hepatitis C virus non-structural 3 protein.

The immunogenicity and antigenicity of an enzymatically functional (ATPase/helicase) recombinant protein encompassing residues 1207-1612 of the hepatitis C virus (HCV) non-structural 3 (NS3) protein was characterized using B10 congenic mice. Previous studies have indicated a high frequency of NS3-specific antibodies in HCV-infected humans. Similarly, all six immunized murine haplotypes were antibody responders to the NS3 ATPase/helicase domain, with the H-2k and H-2s haplotypes as high responders. As also observed in HCV-infected humans, the murine NS3 antibodies were predominantly directed to conformational determinants. Irrespective of the murine haplotype, IgG1 predominated in the primary anti-NS3 response, whereas IgG1 and IgG2b predominated in the secondary response. The antibody responder hierarchy was reiterated at the T cell level, with the H-2k and the H-2s haplotypes as the best responders. In both the H-2d and H-2k haplotypes ATPase/helicase-primed T cells secreted interleukin 2 and interferon gamma, corroborating observations from HCV-infected humans. In the H-2d, H-2k and H-2s haplotypes the fine specificity of the T cell recognition of the ATPase/helicase domain was further characterized. Multiple, although generally weak, T cell recognition sites were found for all three haplotypes. The large size of the NS3 protein together with the presence of multiple class II binding motifs explain the high prevalence of NS3 antibodies in immunized mice and predict a similar explanation for the observed high frequency of NS3-specific antibodies in HCV-infected humans.

Adenosine Triphosphatases↗
PubMed1995

Interleukin 12 suppresses autoantibody production by reversing helper T-cell phenotype in hepatitis B e antigen transgenic mice.

Helper T (Th) cells are classified as Th1 or Th2 cells by virtue of cytokine secretion and function as mediators of cellular or humoral immunity, respectively. Cytokines also regulate the differentiation of Th cells. For example, interleukin (IL)-12 promotes Th1 and suppresses Th2 cell development, suggesting that IL-12 may be useful therapeutically in Th2-mediated autoimmune and allergic disorders. Therefore, the effect of systemic IL-12 treatment on in vivo autoantibody synthesis in hepatitis B e antigen (HBeAg)-expressing transgenic mice, which is dependent on self-reactive Th2 cells, was examined. Low-dose IL-12 significantly inhibited autoantibody production by shifting the Th2-mediated response toward Th1 predominance. Additionally, previous studies suggest that a predominance of HBeAg-specific Th2-type cells may contribute to chronicity in hepatitis B virus infection. Therefore, IL-12 may also prove beneficial in modulating the HBeAg-specific Th response to favor viral clearance in chronic hepatitis B virus infection.

Animals↗
PubMed1995

The hepatitis nucleocapsid as a vaccine carrier moiety.

The "carrier effect," defined as the provision of T cell recognition sites physically linked to B cell epitopes in order to provide Th cell function for antibody synthesis, is well known. Peptides, proteins, and more recently particulate protein antigens have been used for this purpose. The hepatitis B core antigen represents a highly immunogenic antigen in humans as well as in experimental animal models. Studies in mice have provided insight into this enhanced immunogenicity. For example, HBcAg directly activates B cells (i.e., T cell independence), HBcAg elicits strong T cell responses, and HBcAg is efficiently processed and presented by antigen presenting cells (APCs). These characteristics suggested that HBcAg may be an ideal carrier moiety for B cell epitopes requiring additional Th cell function. Therefore, a number of HBV and non-HBV B cell epitopes have been chemically linked or fused by recombinant methods to HBcAg as a method to increase immunogenicity with significant success. We have designed bacterial expression vectors that allow insertion of heterologous B cell epitopes at various positions within HBcAg particles and permit efficient purification of hybrid HBcAg particles. Studies of positional effects have demonstrated that an internal insertion into a dominant HBcAg-specific B cell site represents a superior location for enhanced antibody production. Immunogenicity studies have been extended to protection against experimental challenge in several systems. For example, a malaria CS repeat sequence derived from P. berghei was inserted into HBcAg at the internal site, and purified hybrid HBcAg/CS particles were highly immunogenic and protected 100% of experimentally challenged BALB/c mice. This system has also been exploited for purposes of oral vaccination by expressing genes coding for hybrid HBcAg particles in live, avirulent vaccine strains of Salmonella species.

Administration, Oral↗
PubMed1995

Characterization of self-reactive T cells that evade tolerance in hepatitis B e antigen transgenic mice.

Previous studies of hepatitis B e antigen (HBeAg)-expressing transgenic (Tg31e) mice have indicated that the degree of T cell tolerance was epitope specific. For example, T cells specific for residues 120-131 of HBeAg are profoundly tolerant, whereas a proportion of T cells specific for residues 129-140 escape tolerance induction in B10. S x B10-Tg31e mice. To understand the basis for differential tolerance towards two T cell sites on the same self antigen, we characterized T cell recognition of HBeAg by primary T cells and T cell hybridomas derived from HBeAg-Tg and non-Tg mice. The self-reactive T cells surviving in B10-Tg31e mice exhibited a unique fine specificity, albeit still focussed on HBeAg residues 129-140, which could be distinguished from the HBeAg-specific T cell repertoire in non-Tg B10 mice. Further, self-reactive T cells were comprised predominantly of Th2-type cells that preferentially evaded tolerance induction as compared to their Th1 counterparts. Because HBeAg may act as a tolerogen during the vertical transmission of chronic hepatitis B virus (HBV) infection, these results suggest that a predominance of HBeAg-specific Th2 cells expressing a limited repertoire may influence the initiation or the maintenance of the HBV chronic carrier state.

Animals↗
PubMed1995

Preferential recognition of hepatitis B nucleocapsid antigens by Th1 or Th2 cells is epitope and major histocompatibility complex dependent.

Regulatory T-helper (Th) cells have been categorized into two functional subsets, Th1 and Th2 cells, which produce distinct lymphokines. In general, Th1 cells mediate cellular immune responses and Th2 cells mediate humoral immunity. Recent serological studies suggest that the Th1-Th2 balance may be relevant in acute and chronic hepatitis B virus (HBV) infections. The purpose of this study was to determine the potential of the nucleocapsid antigens (Ags) (hepatitis B core and e Ags [HBc/eAg]) of HBV to preferentially elicit either a Th1 or a Th2 dominant response. For this purpose, H-2 congenic B10.S and B10 mice were immunized with HBc/eAg, and Ag-specific T-cell proliferative responses, T-cell helper function, and T-cell cytokine production were analyzed. The results indicated that B10.S mice preferentially develop a Th1-like response whereas B10 mice preferentially develop a Th2-like response after immunization with HBc/eAg. Furthermore, the preferential Th1 and Th2 response patterns were reproduced when 12-residue peptides representing the dominant HBc/eAg-specific T-cell sites for B10.S (peptide 120-131) and B10 (peptide 129-140) mice were used as immunogens. Therefore, the combination of the T-cell site recognized and the major histocompatibility complex restricting element can in large part determine the Th phenotype of the HBc/eAg-specific T-cell response. Other factors that influenced Th phenotype were the presence of exogenous cytokines, Ag structure, and tissue distribution.

Amino Acid Sequence↗
PubMed1994

Soluble CTLA-4 can suppress autoantibody production and elicit long term unresponsiveness in a novel transgenic model.

Activation of Ag-specific T cells often requires costimulatory signals in addition to the primary signal mediated through the TCR. The CD28-B7 interaction provides one important costimulatory signal. Previous studies have shown that a soluble CD28 homologue, CTLA4lg, binds B7 with high affinity and can inhibit CD28-B7-mediated costimulation in vitro and in vivo. In this study we examined the ability of soluble human CTLA4lg to inhibit autoantibody production in vivo. For this purpose we used a novel transgenic (Tg) model of autoantibody production. Hepatitis B eAg-expressing Tg mice can be induced to produce autoantibody to the circulating autoantigen (HBeAg) by the injection of a T cell recognition site that fails to elicit T cell tolerance in these mice. Autoimmunity in this model is quantitative because serum autoantibody and autoantigen concentration are inversely correlated and easily measurable by ELISA. In this system a single regimen of CTLA4lg treatment significantly suppressed primary autoantibody production and variably led to long term unresponsiveness. Furthermore, in vivo treatment with CTLA4lg inhibited both T cell activation and T cell-B cell interactions.

Abatacept↗
PubMed1994

Extrathymic expression of the intracellular hepatitis B core antigen results in T cell tolerance in transgenic mice.

We previously developed a transgenic (Tg) murine lineage (B10.S-Tg31e), which secretes the hepatitis B e Ag (HBeAg) into the serum at a concentration of 10 ng/ml. This serum concentration was sufficient to render B10.S-Tg31e mice functionally tolerant at the T cell but not B cell level. To determine the tolerogenic potential of an intracellular form of this Ag, namely the hepatitis B core Ag (HBcAg), expressed outside the thymus, the B10.S-Tg10c lineage was developed. In B10.S-Tg10c mice the HBcAg is expressed as an intracellular "self"-Ag predominantly in the liver, and cannot be detected in the serum, the thymus or in nonthymic lymphoid tissue. Despite the liver-specific and intracellular location of this transgenic self-protein, B10-STg10c mice demonstrate a significant degree of HBcAg-specific T cell tolerance at the level of T cell proliferation. Similarly, in vivo anti-HBc antibody production after HBcAg immunization is significantly reduced as compared with non-Tg littermate controls. No spontaneous anti-HBc antibody is produced in B10.S-Tg10c mice, however, adoptive transfer of HBcAg-specific T cells from non-Tg B10.S mice elicits anti-HBc specific "autoantibody" production. Interestingly, antibodies with specificity for the HBeAg as well as the HBcAg are produced. Antibody production in B10.S-Tg10c mice adoptively transferred with T cells indicates that sufficient native HBcAg can gain access to the extracellular compartment to engage HBcAg-specific B cells that are clearly not tolerant in this model. No liver injury was observed as a consequence of HBcAg expression, even in B10.S-Tg10c mice adoptively transferred with HBcAg-specific T cells. Unless HBcAg is unique in this regard, these results suggest that organ-specific, intracellular self-Ag may be released during normal cell turnover in sufficient concentrations to elicit systemic T cell tolerance. B10.S-Tg10c mice also serve as an immunologic model system for chronic infection with the HBeAg-negative mutant of the hepatitis B virus.

Animals↗
PubMed1994

Hepatitis B virus infection, the immune response and hepatocellular carcinoma.

More than 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and have a 200-fold increased risk of developing hepatocellular carcinoma (HCC). This is one of the most common cancers in the world with a geographical distribution highest in areas where HBV is endemic. A number of molecular mechanisms have been proposed to explain this correlation including an acutely transforming viral oncogene; chromosomal aberrations due to HBV integration (i.e. deletions, translocations, duplications); activation of cellular proto-oncogenes; inactivation of cellular anti-oncogenes; and transactivation of cellular genes by HBV gene products. HCC usually develops only after 20-30 years of persistent HBV infection accompanied by hepatocyte necrosis, inflammation and regenerative hyperplasia. Because HBV is not directly cytopathic, liver injury must be immune mediated. Factors that predispose HBV-infected individuals to develop HCC are chronicity, an immune response and liver injury rather than a direct genetic event. Hepatic injury and continuous hepatocyte regeneration may allow an accumulation of multiple mutational events sufficient for the emergence of HCC. Pathways which lead to chronicity, the immune response during HBV infection, mechanisms of pathogenesis and methods to prevent HBV infection are all relevant to the development of HCC. Recent studies characterizing the humoral and cellular immune responses in patients chronically infected with HBV and transgenic mouse models of HBV-specific immune tolerance and pathogenesis are providing new insights into the complex association between HBV infection and HCC.

Animals↗