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J C Glorioso

Publications and source records attributed to J C Glorioso.

At least 19 recordsLinked to original sources
PubMed1992

Gene transfer to synoviocytes: prospects for gene treatment of arthritis.

Joints are difficult organs to target therapeutically. Intravenous, intramuscular, and oral routes of drug delivery provide poor access to the joint, and expose the body systemically to the therapeutic agent. Although intraarticular injection provides direct access to the joint, most injected materials have a short intraarticular half-life. We propose to circumvent these problems by introducing into the synovium gene(s) coding for proteins with antiarthritic properties. Two methods of gene delivery to synovium are under development. In the direct approach, in situ transduction of synoviocytes follows the injection of suitable vectors into the joint. In the indirect approach, synovium is removed from the joint, its synoviocytes are isolated, and the cells transduced in vitro. Genetically modified cells are subsequently transplanted back into the synovium. Using retroviral vectors, we have been able to express the lacZ and neo genes in lapine synovial fibroblasts in vitro. Following neoselection, all cells became LacZ+. Neo-selected cells carrying the lacZ marker gene were transplanted back into the knees of recipient rabbits to examine the persistence and expression of these genes in vivo. Islands of LacZ+, transplanted cells persisted in the recipient joints for at least 3 months. Furthermore, Neo+ cells could be grown from synovia recovered from these joints. Initial attempts to use retroviruses for the direct, in situ transduction of synovium have failed, probably because synoviocytes in the normal synovium are mitotically inactive. Present efforts are directed towards further development of our techniques for transferring genes to joints, and using these techniques to antagonize the intraarticular actions of interleukin-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

In vivo expression of beta-galactosidase in hippocampal neurons by HSV-mediated gene transfer.

Stereotactic inoculation of a herpes simplex virus (HSV) gene transfer vector into the hippocampus and caudate of rat brain resulted in limited and transient viral replication and the establishment of latency. Virus attenuation was achieved by insertional inactivation of a viral gene, Us3. Insertion of a lacZ reporter gene, under the control of the HSV glycoprotein C (gC) late gene promoter, allowed viral replication to be monitored in vivo. Unlike unattenuated virus, the Us3::pgC-lacZ recombinant caused little apparent damage to normal hippocampal morphology. Transient lacZ expression was detected in a considerable population of neurons of the dentate gyrus following hippocampal injection, whereas few positively staining neurons were present within the caudate after injection at that site. Latency-associated transcripts, the hallmark of latent infection, were detected in the brain 10 months after injection. This recombinant virus may be useful as a gene transfer vector for long-term expression of foreign genes in the central nervous system.

Animals↗
PubMed1992

Antigenic structure of the herpes simplex virus type 1 glycoprotein C: demonstration of a linear epitope situated in an environment of highly conformation-dependent epitopes.

A continuous epitope, situated within or in close proximity to antigenic site II of the herpes simplex virus type 1-specified glycoprotein C (gC-1), was identified. The continuous linear nature of the epitope, defined by a monoclonal antibody C2H12, was established by three independent lines of evidence: (i) The epitope was detectable by immunoblot under denaturing and reducing conditions. (ii) The epitope was detectable by RIPA of extracts from TM-treated HSV-infected cells, despite the malfolding caused by this treatment. (iii) The epitope was detected in an approximately 5,000-dalton papain fragment of gC-1. A mapping analysis, primarily based on use of mutant virus, expressing truncated gC-1 molecules, suggested that the mapping position of the epitope was delimited by amino acids 120 and 230. Other epitopes of this region of gC-1 are highly conformation-dependent, and the existence of a linear epitope, accessible on native gC-1, may facilitate the elucidation of the functional anatomy of gC-1.

Antibodies, Monoclonal↗
PubMed1992

Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.

Amino Acid Sequence↗
PubMed1992

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

This report describes a novel method for complementation studies of defective herpes simplex virus (HSV) genes. Viral test gene and nonviral reporter gene cassettes were rapidly integrated into the HSV genome in a site-specific and reversible manner by using the P1 phage-based Cre-lox recombination system. Shuttle plasmids contained a functional loxP recombination site, an expressible form of the bacterial lacZ gene, and a copy of the wild-type glycoprotein B (gB) gene or double mutant gB allele containing both a temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viral genome, K delta T::lox1, was constructed from the HSV type 1 (syn) gB-deficient mutant virus, K delta T, by marker transfer of the loxP recombination site into the viral thymidine kinase locus. Shuttle plasmids of up to 12.9 kb in length were recombined with high efficiency (11 to 20%) into the K delta T::lox1 genome in cell-free, Cre-mediated recombination reactions. Expression of a functional wild-type or double mutant gB polypeptide complemented the nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production of either wild-type or Syn- plaques on Vero cells. The latter recombinant virus was also ts for growth. The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.

Animals↗
PubMed1991

Protection against zosteriform spread of herpes simplex virus by monoclonal antibodies.

The in vivo protective role of herpes simplex virus (HSV-1)-specific antibody was analyzed using monoclonal antibodies (MAbs) reactive with discrete antigenic sites on glycoproteins B, C, and D (gB, gC, gD) in the murine zosteriform spread model of HSV-1. All of the anti-gC and anti-gD MAbs, and one of four anti-gB MAbs (B6) were protective. The in vitro abilities of the MAbs to neutralize HSV-1 and mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-1-infected cells were examined as potential mechanistic correlates to in vivo protection. All animals given MAbs at high ADCC unit doses were protected. Some but not all mice given MAbs at high ADCC unit doses were protected. These studies designate specific epitopes recognized by protective antibodies and indicate that protection from the neurologic spread of HSV may be related to neutralization, ADCC, or both. The actual contribution of ADCC and neutralization to in vivo antibody-mediated protection remains unclear.

Animals↗
PubMed1991

Endogenously produced interferon alpha protects mice from herpes simplex virus type 1 corneal disease.

Intravenous (i.v.) injection of u.v. light-inactivated herpes simplex virus type 1 (UV HSV-1) at the time of HSV-1 corneal infection reduced the cytotoxic T lymphocyte (CTL) response to HSV-1, and significantly reduced the incidence of HSV-1-induced corneal stromal disease in A/J mice. The spread of HSV-1 through the eye after corneal infection, detected using engineered HSV-1 (US3::Tn5-lacZ) with the lacZ gene under the transcriptional control of the viral late gene promoter for glycoprotein C, was also markedly reduced by i.v. UV HSV-1 injection. The restriction of HSV-1 corneal invasiveness in i.v. UV HSV-1-injected mice preceded the onset of a detectable specific cell-mediated or humoral immune response to HSV-1, and was accompanied by an elevated serum titre of interferon (IFN-alpha), reversed by anti-IFN-alpha/beta antibody, and mimicked by systemic IFN-alpha treatment. IFN-alpha-treated mice developed a normal CTL response to HSV-1 after corneal infection, but the corneal invasiveness of the virus was markedly reduced and none of the treated mice developed corneal stromal disease. Together with our previous findings that HSV-1-specific CTLs participate in the pathogenesis of corneal stromal disease, these results indicate that i.v. injection of UV HSV-1 at the time of corneal infection may prevent stromal disease by the combined effects of IFN-mediated reduction of the spread of virus in the cornea and inhibition of the activity of the HSV-specific T lymphocytes that induce tissue destruction in the corneal stroma.

Animals↗
PubMed1991

Selective induction of discrete epitopes of herpes simplex virus type 1-specified glycoprotein C by interference with terminal steps in glycosylation.

We have described two types of oligosaccharide modification influencing the antigenicity of the herpes simplex virus type 1 (HSV-1)-specified glycoprotein C (gC-1). First, the expression of several epitopes belonging to antigenic site II of gC-1 is dependent on the peripheral galactose of N-linked oligosaccharides. We have also shown that treatment of HSV-1-infected cells with 5-n-propyl-2'-deoxyuridine (PdU) under certain circumstances results in other modifications of peripheral carbohydrate determinants, which are associated with increased antigenic activity of gC-1. In the present study we have mapped and characterized the epitopes susceptible to PdU induction by analysing the reactivity to a number of monoclonal antibodies defining several epitopes of antigenic sites I and II. The results indicate that the strict galactose dependence of epitopes and the PdU-induced increase of antigenic activity are independent and unrelated phenomena. Thus, we identified galactose-dependent epitopes that were not PdU-inducible and vice versa, and some epitopes were both galactose-dependent and PdU-inducible. The results support a model where PdU treatment blocks synthesis of an antigen-masking carbohydrate determinant. In addition, PdU treatment of HSV-1-infected cells seemed to increase the antigenic activity of other HSV-1 glycoproteins.

Animals↗
PubMed1991

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.

Amino Acid Sequence↗
PubMed1990

Antigenic and functional analysis of a neutralization site of HSV-1 glycoprotein D.

Herpes simplex virus glycoprotein D is a component of the virion envelope and appears to be involved in attachment, penetration, and cell fusion. Monoclonal antibodies (MAbs) against this protein can be arranged in groups, on the basis of a number of biological and biochemical properties. Group I antibodies are type-common, have high complement-independent neutralization titers, recognize discontinuous (conformational) epitopes, and block each other in a binding assay. The sum of their epitopes constitutes antigenic site I of gD. Using a panel of neutralization-resistant mutants, we previously found that group I MAbs can be divided into two subgroups, Ia and Ib, such that mutations selected with Ia antibodies have little or no effect on binding and neutralization by Ib antibodies, and vice versa. Antigenic site I therefore consists of two parts, Ia and Ib. We have now identified the point mutations which prevent neutralization. Two Ib MAbs (DL11 and 4S) selected a Ser to Asn change at residue 140; this alteration creates a new N-linked glycosylation site, which is used. A third Ib MAb (D2) selected a Gln to Leu change at 132. The mutation selected by the Ia MAb HD1 (Ser to Asn at residue 216) is identical to that selected by MAb LP2, another Ia antibody. By using oligonucleotide-directed mutagenesis, we have produced gD genes with combinations of the above mutations. Attempts to recombine these genes into the virus genome were unsuccessful, suggesting that the combinations are lethal. This was confirmed by a complementation assay which measures the ability of gD transiently expressed in transfected Vero cells to rescue the production of infectious virus by the gD-minus mutant F-gD beta.

Base Sequence↗
PubMed1990

Cross-linking studies show that herpes simplex virus type 1 glycoprotein C molecules are clustered in the membrane of infected cells.

Chemical cross-linking using ethylene glycol succinimidyl succinate (EGS) and dithiobispropionimidate (DTBP) was performed to determine the association of herpes simplex virus type 1 glycoprotein C (HSV-1 gC) with its nearest neighbours. Human embryonic lung (HEL) cells were infected with HSV-1 strain KOS, treated with EGS, lysed with Nonidet P40, immunoprecipitated with monoclonal antibodies specific for gC, and analysed by SDS-PAGE. These analyses demonstrated the presence of cross-linked complexes that migrated with an apparent Mr in the range 150,000 to 260,000. Two-dimensional SDS-PAGE (non-reduced and then reduced) analyses of HSV-1-infected HEL cells treated with the cleavable cross-linker DTBP demonstrated that molecules that comigrated with gC were the only components of these high Mr complexes. Immunoelectroblot (Western blot) analyses using polyclonal rabbit antiserum specific for gC verified that the high Mr complexes contained gC. These results indicated that gC molecules may be localized in the infected cell membrane as dimers.

Blotting, Western↗
PubMed1990

Recombinogenic properties of herpes simplex virus type 1 DNA sequences resident in simian virus 40 minichromosomes.

In a previous work, it was demonstrated that the bacterial transposon Tn5 is capable of undergoing sequence inversion via recombination between its duplicated IS50 elements when replicated by the herpes simplex virus type 1 (HSV-1) origin oris but not by the simian virus 40 (SV40) origin orisv. Further analysis of the latter phenomenon indicated that this lack of recombination was the result of topological constraints imposed by the SV40 minichromosome, such that recombination events could be readily detected in Tn5 derivatives in which the IS50 elements were arranged in a direct rather than inverted orientation. With this information, a second set of experiments were carried out to examine how the highly recombinogenic sequences which mediate the inversion of the long (L) and short (S) components of the HSV-1 genome behave in an SV40 minichromosome. Tandem copies of the L-S junction of the HSV-1 genome were observed to promote deletions in an SV40 shuttle plasmid at a frequency that was considerably greater than that of duplicated bacterial plasmid vector DNA. However, the presence of superinfecting HSV-1 did not enhance the frequency of these recombination events. These results support our previous findings that HSV-1 genome isomerization is mediated by a homologous recombination mechanism which is intimately associated with the act of viral DNA synthesis. Moreover, they demonstrate that the sequences which comprise the L-S junction appear to be inherently recombinogenic and, therefore, do not contain specific signals required for HSV-1 genome isomerization.

Animals↗
PubMed1990

Herpes simplex virus type 1-specific immunity induced by peptides corresponding to an antigenic site of glycoprotein B.

Herpes simplex virus (HSV) envelope glycoproteins are the prime targets of adaptive antiviral immunity. Previous investigation identified a protective, neutralizing, glycoprotein B1 (gB-1)-reactive monoclonal antibody (MAb B6) and localized the linear epitope recognized by the MAb to residue 84 of gB-1. Three overlapping peptides (two 20-mers and one 18-mer), together spanning amino acids 63 to 110 of the wild-type sequence of gB-1, were synthesized and analyzed for their ability to stimulate immunity which cross-reacts with HSV-1. All stimulated some level of response. Two peptides, the gB 18-mer and 20.1-mer, were recognized by MAb B6 and HSV-immune antibody but were unable to stimulate virus-neutralizing antibody or serum able to protect against zosteriform spread in vivo. The 20.2-mer peptide, however, which was not recognized by MAb B6 or HSV-generated immune antibody, stimulated the production of neutralizing antibody and serum able to protect against zosteriform spread. Immunization with all of the peptides was able to enhance viral clearance of a low dose of HSV-1 in an ear challenge model and induce antibody reactive in antibody-dependent complement-mediated lysis of HSV-1-infected cells in vitro. These results are the first report of HSV immunity induced by peptides corresponding to gB and indicate that the best immunogen, in terms of stimulating neutralizing antiserum able to protect in vivo against HSV-1, was a peptide not recognized by HSV-immune mechanisms or by the MAb used to localize it.

Amino Acid Sequence↗
PubMed1990

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.

Animals↗
PubMed1990

Localization on the herpes simplex virus type 1 genome of a region encoding proteins involved in adsorption to the cellular receptor.

We have previously shown that aminoglycosides such as neomycin and the polyamino acids polylysine and polyarginine selectively inhibit the binding of herpes simplex virus type 1 (HSV-1) to the cellular receptor, whereas HSV-2 infection is unaffected. In the present study we took advantage of this difference between HSV-1 and HSV-2 by using HSV(-1)-HSV(-2) intertypic recombinants to locate a region on the HSV-1 genome encoding proteins affecting the binding of the virion to the cellular receptor. The results were consistent with those obtained by marker rescue experiments. The identified region, which mapped between coordinates 0.580 and 0.687, contains two partial and eight complete genes, including the glycoprotein C (gC) gene and two others with potential transmembrane sequences. Various gC monoclonal antibody-resistant mutants of HSV-1 as well as a mutant completely lacking gC were found to be fully sensitive to neomycin, suggesting that gC is not the site of drug sensitivity and is not essential for adsorption of virus to the cellular receptor. However, the rate of adsorption was reduced in the absence of gC, indicating a facilitating function of the glycoprotein. The universal nature of this HSV-1 receptor binding was revealed by the similarity in drug sensitivity of infectivity in four different cell lines from various tissues and species.

Adsorption↗