Biomedical subjects

H C Ertl

Publications and source records attributed to H C Ertl.

At least 55 records · Page 3Linked to original sources

PubMed1995

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression.

Adenoviruses, Human↗

PubMed1995

Immune responses to nucleic acid vaccines to rabies virus.

A plasmid vector expressing the full-length rabies virus glycoprotein (G protein) under the control of the simian virus 40 (SV40) promoter has previously been shown to induce upon intramuscular (i.m.) inoculation into mice a specific B- and T-cell-mediated immune response and protection against challenge with a virulent strain of the virus. Here we tested two parameters that might affect the efficacy of this DNA vaccine. First, we replaced the SV40 promoter of the original vector with the early promoter derived from cytomegalovirus leaving all other parameters of the plasmid intact. Although upon transfection in vitro the two vectors showed a striking difference in their ability to cause stable expression of the rabies virus G protein, upon i.m. inoculation into mice both constructs induced comparable immune responses. Second, we constructed a vector that induces expression of a secreted form of rabies G protein by inserting a stop codon just upstream of the transmembrane domain of the rabies G protein gene. The immune responses to the DNA vaccines expressing the two different forms of the G protein, secreted and membrane bound, were compared and found to be similar in magnitude. The long-term effect of DNA vaccination was also investigated especially with regard to adverse immunological reactions such as the induction of unresponsiveness against rabies virus and the development of antibodies to DNA. DNA vaccination was found to induce long-lasting immunity to rabies virus without apparent negative side effects such as development of T cell tolerance or generation of anti-DNA antibodies.

Animals↗

PubMed1995

Comparison of the effects of amino acid substitutions and beta-N- vs. alpha-O-glycosylation on the T-cell stimulatory activity and conformation of an epitope on the rabies virus glycoprotein.

The first potential N-glycosylation site of the rabies virus glycoprotein, the antigen that carries epitopes for glycoprotein-specific T-cells and virus neutralizing antibodies, is glycosylated inefficiently. Recently, we showed that addition of a beta-N-acetyl-glucosamine moiety to the asparagine residue in the corresponding synthetic fragment V V E D E G C T N L S G F (amino acids 29-41), significantly diminished the T-cell stimulatory activity and reduced the characteristic alpha-helicity of the peptide. The amino acid sequence of the glycoprotein in this region exhibits some degree of variability among different rabies virus and rabies virus related strains, including the replacement of the asparagine residue with aspartic acid or threonine. In the current study, stimulation of a specific T-cell clone by various viral strains and appropriate tridecapeptide sequences and their analogs was investigated. The T-cell recognition pattern of the rabies and rabies-related viruses was identical to that of the synthetic peptides representing the respective epitope sequences. While the asparagine could be replaced without complete loss of T-cell stimulatory activity, amino acid modifications at the C-terminus of the peptide were not tolerated. In contrast to glycosylation of the asparagine, coupling of an N-acetyl-galactosamine moiety at the serine, or galactosyl-N-acetyl-galactosamine moieties at the threonines preceding or replacing the asparagine (all O-linked sugars in the natural alpha-anomeric configuration) resulted in epitopes that lowered rather than abolished the T-cell stimulatory activity. All non-glycosylated peptides assumed a low-to-medium helicity in trifluoroethanol. O-glycosylation was more efficient than N-glycosylation in breaking the helical conformation of the peptides to result in the formation of reverse-turns or unordered structure.

Amino Acid Sequence↗

PubMed1995

Stable secretion of a soluble, oligomeric form of rabies virus glycoprotein: influence of N-glycan processing on secretion.

Rabies virus glycoprotein (RGP) is a 505 amino acid type I transmembrane glycoprotein that is important in the pathogenesis of rabies virus infection. RGP also stimulates the development of neutralizing antibodies by the host. N-linked glycosylation is required for both cell surface expression and immunogenicity of RGP. In the current study, a soluble form of RGP, constructed by insertion of a stop codon external to the transmembrane domain, was expressed in transfected Chinese hamster ovary cells. The soluble form of RGP was found to be appropriately antigenic and immunogenic. Similar to full-length RGP, the soluble form was assembled into homodimers and homotrimers. Core glycosylation was required for secretion of soluble RGP and cell surface expression of full-length RGP. In addition, initial glucose trimming of the N-glycans was necessary and sufficient for secretion of soluble RGP and cell surface expression of full-length RGP. Further N-glycan processing was not required for secretion or cell surface expression of soluble or full-length RGP, respectively.

Animals↗

PubMed1995

Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines.

Inoculation of plasmid vectors encoding a viral protein into muscle tissue was shown to result in expression of the transantigen and, consequently, an antiviral immune response. Here, we show that coinoculation of a plasmid expressing the glycoprotein of rabies virus with plasmids encoding mouse cytokines modulated the immune response to the viral protein. Coinoculation with a vector expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the B and T helper cell activity to rabies virus, while coinoculation with a plasmid expressing interferon-gamma (IFN gamma) resulted in a decrease of the immune response to the viral antigen.

Animals↗

PubMed1995

Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.

Recombinant adenoviruses are an attractive vehicle for gene therapy to the lung in the treatment of cystic fibrosis (CF). First-generation viruses deleted of E1a and E1b transduce genes into airway epithelial cells in vivo; however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. In this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive transfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. Our studies indicate that major histocompatibility complex class I-restricted CD8+ cytotoxic T lymphocytes are activated in response to newly synthesized antigens, leading to destruction of virus infected cells and loss of transgene expression. Major histocompatibility complex class II-associated presentation of exogenous viral antigens activates CD4+ T-helper (TH) cells of the TH1 subset and, to a lesser extent, of the TH2 subset. CD4+ cell-mediated responses are insufficient in the absence of cytotoxic T cells to completely eliminate transgene containing cells; however, they contribute to the formation of neutralizing antibodies in the airway which block subsequent adenovirus-mediated gene transfer. Definition of immunological barriers to gene therapy of cystic fibrosis should facilitate the design of rational strategies to overcome them.

Adenoviridae↗

PubMed1995

Computer design of T-cell agonist or antagonist glycopeptides: the effect of sugar identity and anomeric configuration on MHC binding.

The improved chemical and biological properties of synthetic glycopeptides over peptides suggest their use as T cell agonists or antagonists. Recently, we prepared glycopeptide analogues of major T helper cell epitopic peptides corresponding to rabies virus proteins, and experimentally characterized their ability to bind to MHC class II proteins and stimulate T cell clones to rabies virus. In the current study, we investigated these MHC: peptide interactions by molecular modeling. We obtained structural support for our finding concerning the anomeric specificity of MHC with binding. While alpha-linked glycopeptides can bind to MHC without major alterations in the spatial arrangements and hydrogen bonding pattern of class II-peptide binding, the binding of beta-linked glycopeptides is considerably less favorable due to steric and columbic conflicts. Depending on where the saccharides are positioned along the peptide sequence, the MHC: glycopeptide complex may or may not produce the surface profile required for successful T cell receptor interaction. Application of this approach to other antigenic stimuli offers a good model to "dial in" the necessary sugar identity, length and anomeric configuration, as well as promising amino acid mutation sites, for successful design of T cell agonist or antagonist glycopeptides.

Amino Acid Sequence↗

PubMed1994

Glycosylation of synthetic T helper cell epitopic peptides influences their antigenic potency and conformation in a sugar location-specific manner.

The immunodominant T helper cell epitopes 31D and VF13N of rabies virus nucleoprotein and glycoprotein, respectively, correspond to peptide sequences AVYTRIMMNGGRLKR and VVEDEGCTNLSGF, and are expressed between amino acids 404-418 and 29-41, of the appropriate proteins. We investigated how internal or external glycosylation affects the biological activity and conformation of the peptides 31D and VF13N. Mid-chain incorporation of maltobiose or N-acetylglucosamine moieties into the asparagine residues greatly diminished the T-cell stimulatory activity in vitro (due to the diminished ability of the glycopeptides to bind to major histocompatibility complex determinants) and reduced the characteristic alpha-helicity of the peptides in aqueous trifluoroethanol solutions. In contrast, addition of maltobiose- or N-acetylglucosamine-coupled asparagines to the N-termini of peptides 31D and VF13N resulted in unchanged T-cell activity. Furthermore, N-terminal glycosylation of peptide 31D, as indicated by the functional assay, decreased the sensitivity of the peptide to degradation in human serum and did not affect the alpha-helical conformation. These data indicate that glycosylation of T-cell epitopes is not a preferable method for the preparation of antagonists, but incorporation of the sugars to appropriate positions may be advantageous in the design of T-cell agonists and peptide-based vaccines.

Amino Acid Sequence↗

PubMed1994

Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus.

A plasmid vector, termed pSG5rab.gp, that expresses the rabies virus glycoprotein under the control of an SV40 early promoter, was tested in C3H/He mice for induction of rabies virus-specific immune responses. Mice immunized intramuscularly with the pSG5rab.gp vector developed rabies virus glycoprotein-specific cytolytic T cells, lymphokine-secreting T helper cells of the TH1 subset, and rabies virus-neutralizing antibodies. Mice vaccinated with the pSG5rab.gp vector were fully protected against a subsequent challenge with rabies virus.

Animals↗

PubMed1994

A simple method to test the ability of individual viral proteins to induce immune responses.

Immune responses to a mouse fibroblast line, transfected with a plasmid that causes expression of the rabies virus glycoprotein under the control of a simian virus 40 early promoter, were studied. Transfected cells were shown to be recognized in vitro by a panel of monoclonal antibodies directed to conformational epitopes of the different antigenic sites of the glycoprotein. They stimulated rabies virus glycoprotein-specific T helper cells in the presence of antigen-presenting cells and were, furthermore, recognized by rabies virus-induced cytolytic T cells. In vivo, immunization of H-2-compatible mice with the transfected cell line led to a rabies virus glycoprotein-specific antibody response, and to protection against a subsequent challenge with live virus. We propose this procedure, i.e. use of cell lines transfected with plasmids expressing a viral protein under a mammalian promoter, as a simple and inexpensive method to screen individual viral proteins for their ability to elicit immune responses, including T helper cells, cytolytic T cells, antibodies, and protection against viral challenge.

Animals↗

PubMed1994

MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses.

The use of E1-deleted recombinant adenoviruses in gene therapy has consistently been associated with transient gene expression and inflammation due to immune-based destruction of the infected cells. We have used murine models of adenovirus-mediated gene transfer to liver to investigate these immunologic mechanisms. Adoptive transfer experiments, as well as studies involving genetic knockout mice, confirmed the original hypothesis that cell-mediated immunity induced by E1-deleted adenovirus destroyed trans-gene-expressing hepatocytes and defined MHC class I-restricted CD8+ cytolytic lymphocytes as the primary immune effectors for hepatocyte destruction. Responses mediated by CD4+ cells per se were insufficient to mediate destruction of hepatocytes in vivo, despite the activation of virus-specific T helper cells of Th1 subsets. A better understanding of the response of the host to in vivo gene therapy is important in evaluating its usefulness in humans.

Adenoviridae Infections↗

PubMed1992

Immune response to the nominal phosphoprotein of rabies virus.

The immune response to the nominal phosphoprotein (NS protein) of rabies virus was investigated with the use of a vaccinia recombinant virus that expressed the NS protein of a fixed rabies virus strain. Mice of the H-2k haplotype that were injected with either live rabies virus or the vaccinia recombinant virus developed a strong cytolytic T-cell response specific for the NS protein. This response was under immune response (Ir) gene control. The NS protein as presented by the vaccinia recombinant virus was a poor inducer of rabies virus-specific T-helper (Th) cells and B cells in the H-2k background. Furthermore, mice of the H-2k haplotype could not be protected by vaccination with the vaccinia recombinant virus expressing the NS protein, although protection in outbred mice was partial and incomplete. These data indicate that cytolytic T cells to the NS protein of rabies virus are insufficient to protect mice against a challenge with rabies virus.

Animals↗

PubMed1992

Transfer of maternal antibodies results in inhibition of specific immune responses in the offspring.

A potentially detrimental consequence of maternally transferred antibodies was demonstrated in a mouse model for rabies virus, where pups from rabies virus-immune dams showed a decrease in the generation of specific B- and T-cell responses to immunization with rabies virus antigen, resulting in vaccine failures. The degree and duration of the vaccine failures was inversely correlated with the amounts of maternally transferred antibodies, and exceeded the time when maternal antibodies provided reliable protection against a viral challenge. The low responsiveness to vaccination, measured by serum antibody titers and by lymphokine release upon in vitro restimulation of in vivo-primed lymphocytes, was specific for the target virus of the maternal antibodies and was also observed in pups from Sendai virus-immune dams upon vaccination with the homologous virus. In addition, an inhibition of the specific immune responses was demonstrated upon passive immunization of newborn mice with monoclonal antibodies to rabies virus. Although the mechanism(s) that causes the observed inhibition in the offspring of immune dams or in pups that were inoculated with antibodies postnatally is currently unknown, data presented in this manuscript indicate that the observed effect on B- and T-cell responses might not be solely caused by removal of the antigenic load due to residual maternal antibodies.

Animals↗

PubMed1992

Inhibition of immune responses against rabies virus by monoclonal antibodies directed against rabies virus antigens.

Treatment of mice with a cocktail of murine anti-rabies monoclonal antibodies (mAb-C) interfered with the ability of these animals to mount a virus-neutralizing antibody response to rabies vaccine. Administered mAb-C did not affect the induction of rabies virus-specific T-helper cells. The magnitude of the inhibition of rabies virus-specific B-cell response was dependent on the concentration of the mAb-C and the duration of the mAb-mediated interference was inversely proportional to the biological half-life of the mAb. As long as the serum titres were above a critical threshold, the suppression could not be overcome even by multiple vaccinations. Since injection of mice with immunocomplexes consisting of inactivated rabies virus and mAb rendered the animals non-responsive to a subsequent vaccination with inactivated rabies virus, it is concluded that the mAb-induced suppression might be caused by the formation of antigen-antibody complexes which exert a negative signalling effect to premature B cells.

Animals↗

PubMed1992

Posttranslational side chain modification of a viral epitope results in diminished recognition by specific T cells.

A stretch of 16 amino acid residues within the nominal phosphoprotein of rabies virus was shown to carry an immunodominant epitope for class I- and class II-restricted T cells. The nominal phosphoprotein of rabies virus is thought to be heterogeneously phosphorylated at multiple serine and threonine residues. The synthetic peptide that expressed the T-cell epitope contained a single serine residue corresponding to position 196 of the protein. Phosphorylation of this serine within the synthetic peptide caused a significant decrease of the antigenic potency of the peptide. A similar effect was seen if the serine was replaced by an alanine or if the peptide was glycosylated at its acidic residues. These data suggest that T-cell-mediated recognition of antigen presented by major histocompatibility complex class I- or II-positive cells is impaired not only by point mutations but also by posttranslational side chain modifications of residues within viral epitopes.

Amino Acid Sequence↗

PubMed1991

Rabies virus nucleoprotein expressed in and purified from insect cells is efficacious as a vaccine.

A cDNA copy of the RNA gene that encodes the nucleoprotein N of rabies virus Evelyn-Rokitnicki-Abelseth strain was cloned into baculovirus. The recombinant baculovirus expressed the N protein abundantly in Spodoptera frugiperda cells. The N protein was extracted from infected Spodoptera frugiperda cells and purified to near homogeneity by affinity chromatography. The purified N protein reacted with 31 of 32 monoclonal antibodies that recognize native rabies virus ribonucleoprotein. Like the ribonucleoprotein, the purified N protein was a major antigen capable of inducing virus-specific helper T cells. Priming of mice with the purified N protein prior to a booster inoculation with inactivated Evelyn-Rokitnicki-Abelseth virus vaccine resulted in a 20-fold increase in the production of virus-neutralizing antibodies. After immunization with the purified N protein, mice developed a strong anti-ribonucleoprotein antibody response and were protected against a lethal challenge of rabies virus. These data indicate that the N protein expressed in insect cells is antigenically and immunogenically comparable to the authentic rabies virus ribonucleoprotein and therefore represents a potential source of an effective and economical vaccine for large-scale immunization of humans and animals against rabies.

Animals↗

PubMed1991

T helper cell epitope of rabies virus nucleoprotein defined by tri- and tetrapeptides.

T cell clones and subsequently hybridomas were generated from rabies virus-immune C3H/He mice to an immunodominant epitope of the viral nucleoprotein, termed 31D, that had previously been identified by a 15-amino acid-long synthetic peptide. T cells to this epitope that by phenotypical and functional characteristics belonged to the T helper cell subset were shown to respond to most rabies and rabies-related viruses. In order to define the minimal sequence needed to elicit a response from 31D-specific T cell clones or hybridomas, a number of peptides of varied lengths, i.e. 3-32 amino acids long, were tested. The ability of the peptides to induce a response was inversely correlated in their lengths, i.e., short peptides (3-5 amino acids long) had to be used at 10(6) times higher concentrations as compared to long peptides (15 or 32 amino acids long). Conversely, the specificity of the T cell response was directly correlated to the length of the peptides, i.e., while the response to 15-amino acid-long peptides exhibited a high degree of specificity, the response to 3- to 5-amino acid-long peptides showed a high degree of flexibility. The long as well as the short peptides had to be presented in association with I-Ek. We speculate that in this system the T cell receptor interacts predominantly with a peptide-induced modification of the I-Ek molecule.

Amino Acid Sequence↗

PubMed1991

Identification of an immunodominant epitope within the phosphoprotein of rabies virus that is recognized by both class I- and class II-restricted T cells.

Immunization of H-2k mice with live rabies virus induces cytolytic T lymphocytes to the phosphoprotein of rabies virus. The antigenic determinant responsible for stimulating this class I-restricted cytolytic response was mapped to 50 amino acids (residues 180 to 229) of the phosphoprotein by using vaccinia virus recombinants expressing either the full-length phosphoprotein or C-terminal truncations of the phosphoprotein. The epitope was more finely mapped to residues 191 to 206 by using synthetic peptides. Several CD4+, class II-restricted T-cell lines were isolated from splenocytes of H-2k mice immunized with the vaccinia virus-rabies virus phosphoprotein recombinant virus. These lines were specifically stimulated by the phosphoprotein, and in addition, each line proliferated and released lymphokines in response to the same synthetic peptide shown to stimulate phosphoprotein-specific, class I-restricted cytolytic T cells.

Amino Acid Sequence↗