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Biomedical subjects

T R Fuerst

Publications and source records attributed to T R Fuerst.

At least 19 recordsLinked to original sources
PubMed1997

In vitro infection and replication of hepatitis E virus in primary cynomolgus macaque hepatocytes.

An in vitro model was developed to replicate hepatitis E virus (HEV) in normal primary cynomolgus macaque hepatocytes using a hormonally defined, serum-free medium formulation. Primary hepatocytes were infected in tissue culture following isolation by collagenase treatment of liver wedge biopsy material. Viral replication was monitored by a highly strand-specific reverse transcription-polymerase chain reaction (RT-PCR) assay, which could detect the positive- and negative-strands of HEV RNA independently in a sensitive and specific manner. Several infectious HEV (Burma strain) inocula were titered by this RT-PCR assay, and a minimum effective infectious dose was determined. Appearance of newly replicated virus was demonstrated by detection of both strands of HEV RNA in experimentally infected hepatocytes as well as the genomic positive-strand viral RNA in the culture medium. Infectivity of the virus particles present in the media was confirmed by serial passage and replication of the virus in culture. Using this in vitro infection system, a neutralization assay was developed to assess the ability of anti-HEV antibodies to block virus infection of liver cells. Results presented in this report represent the first in vitro demonstration of a neutralizing anti-HEV antibody directed against the ORF2-encoded putative capsid protein.

Animals↗
PubMed1996

Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry.

A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.

Amino Acid Sequence↗
PubMed1996

In vitro propagation and production of hepatitis E virus from in vivo-infected primary macaque hepatocytes.

Hepatitis E virus (HEV) is responsible for sporadic cases as well as large epidemics of acute viral hepatitis in many developing countries. The nucleotide sequence of HEV appears to be unique among known viruses and thus may represent a prototype human pathogen in a novel class of single-stranded, positive-sense RNA viruses. To facilitate further studies of the biology of HEV, a tissue culture system using a serum-free medium formulation has been developed to propagate the virus in vitro. Hepatocytes were isolated from livers of cynomolgus macaques experimentally infected with a HEV (Burma strain) inoculum and maintained in long-term cultures. Using a highly strand-specific RT-PCR assay, both the positive-sense and the negative replicative strands of HEV RNA were detected in these hepatocytes throughout the course of the experiments. Positive-strand genomic RNA was also detected in the culture medium, suggesting the production and secretion of HEV virus particles. The virus particles were successfully concentrated 200-fold from the medium using ultrafiltration, and they could be observed by immunoelectron microscopy using anti-HEV-positive immune serum. These results demonstrate the capacity of this hepatocyte culture system to replicate HEV in vitro, thus providing an experimental means to study the replicative process of the virus.

Animals↗
PubMed1996

Purification of a soluble hepatitis E open reading frame 2-derived protein with unique antigenic properties.

The second open reading frame (ORF2) of hepatitis E virus (HEV) is predicted to encode a 73-kDa capsid protein (1). When full-length ORF2 was expressed in insect cells (Spodoptera frugiperda (Sf9)) using a recombinant baculovirus, two distinct HEV polypeptides were observed: a full-length insoluble 73-kDa protein, and a soluble 56.5-kDa protein. Following purification and sequence analysis, it was determined that the 56.5-kDa protein was derived from endoproteolytic cleavage site that was between the Thr and Ala residues located at amino acids 111 and 112 in the ORF2 sequence with the carboxy terminus corresponding to residue 636 of the ORF2 sequence. Comparative ELISA data using human acute-phase antisera demonstrated that the 56.5-kDa protein served as a highly reactive antigen in detecting anti-HEV antibodies. These data suggest that the 56.5-kDa protein may serve as a particularly useful antigen for both diagnostic and vaccine purposes.

Amino Acid Sequence↗
PubMed1996

Host range restricted, non-replicating vaccinia virus vectors as vaccine candidates.

Three model systems were used to demonstrate the immunogenicity of highly attenuated and replication-defective recombinant MVA. (1) Intramuscular inoculation of MVA-IN-Fha/np induced humoral and cell-mediated immune responses in mice and protectively immunized them against a lethal respiratory challenge with influenza virus. Intranasal vaccination was also protective, although higher doses were needed. (2) In rhesus macaques, an immunization scheme involving intramuscular injections of MVA-SIVenv/gag/pol greatly reduced the severity of disease caused by an SIV challenge. (3) In a murine cancer model, immunization with MVA-beta gal prevented the establishment of tumor metastases and even prolonged life in animals with established tumors. These results, together with previous data on the safety of MVA in humans, suggest the potential usefulness of recombinant MVA for prophylactic vaccination and therapeutic treatment of infectious diseases and cancer.

Animals↗
PubMed1996

Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara.

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.

Animals↗
PubMed1995

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Animals↗
PubMed1994

Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR.

Analysis of hepatitis C virus (HCV) replication has been hampered due to the difficulty encountered in in vitro cultivation of the virus in conventional tissue culture systems. In this study, primary chimpanzee hepatocyte cultures maintained in a serum-free medium formulation were susceptible to in vitro infection with HCV. In order to document infection, two new methods of reverse transcription/polymerase chain reaction were developed that permit accurate distinction between positive and negative strand HCV RNA. One method relied upon the use of a tagged cDNA primer, while the second method employed a thermostable reverse transcriptase. Following inoculation of chimpanzee hepatocytes with HCV, intracellular positive and negative strand HCV RNA were detectable 4 days postinfection and throughout the remainder of the experimental period, 25 days. Analysis of HCV-inoculated baboon hepatocytes revealed a total absence of negative strand HCV RNA, while residual positive strand RNA from the inoculum could be detected for up to 11 days. The in vitro replication of HCV RNA in chimpanzee hepatocytes could be suppressed by alpha-interferon. This system should be amenable to the study of HCV replication, antiviral compounds, and the development of neutralization assays.

Animals↗
PubMed1993

Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys.

Because the transmission of HIV is likely to occur through cell-associated virus, an effective HIV vaccine should be capable of eliciting HIV-specific CTL. We have employed the simian immunodeficiency virus (SIV)/rhesus monkey model to explore the use of the attenuated tuberculosis bacillus, Calmette Guérin bacillus (BCG), as a vaccine vehicle to elicit AIDS virus-specific CTL. BCG was engineered to express SIVmac gag under the control of hsp70 regulatory sequences. Immunization with this rBCG-SIVmac gag elicited MHC class I-restricted, CD8+ SIVmac gag-specific CTL in rhesus monkeys. In fact, SIVmac gag-specific CTL could be cloned readily from peripheral blood lymphocytes of these immunized monkeys. These findings provide further evidence for the power of BCG as a vaccine vector and its continued exploration as a vehicle for eliciting HIV-specific immunity.

Amino Acid Sequence↗
PubMed1993

Expression of human recombinant 72 kDa gelatinase and tissue inhibitor of metalloproteinase-2 (TIMP-2): characterization of complex and free enzyme.

The human 72 kDa gelatinase/type IV collagenase is a metalloproteinase that is thought to play a role in metastasis and angiogenesis. The 72 kDa progelatinase can be isolated from conditioned media as a complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2). To investigate 72 kDa gelatinase-TIMP-2 interactions and to compare the activity of the complex versus that of the free enzyme, we have expressed and purified human 72 kDa progelatinase and TIMP-2 as single proteins in a recombinant vaccinia virus mammalian cell expression system. The recombinant 72 kDa progelatinase was able to bind TIMP-2, and it digested gelatin and collagen type IV after activation by p-aminophenylmercuric acid (APMA). The specific activity of the recombinant free enzyme was 20-fold higher than the activity of an APMA-treated stoichiometric complex of recombinant 72 kDa progelatinase and TIMP-2. Also, TIMP-2 caused an 86% inhibition of activity when added to the activated enzyme at a 1:1 molar ratio. Activation of the free recombinant 72 kDa progelatinase yielded the 62 kDa species and two fragments of 46 and 35 kDa that cross-reacted with monoclonal antibodies to the 72 kDa proenzyme. TIMP-2 inhibited the conversion of the recombinant proenzyme to the 62 kDa species and the appearance of the 45 and 35 kDa bands. These results suggest that TIMP-2 is not only a potent inhibitor of the activated enzyme but also prevents the generation of low-molecular-mass species and full enzymic activity from the zymogen.

Blotting, Western↗
PubMed1993

Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro.

It has been proposed that proteases are important in endothelial cell behavior. We examined the contribution of the gelatinase/type IV collagenase system in an in vitro model of endothelial differentiation. Human umbilical vein endothelial cells rapidly align and form networks of tubes when cultured on a basement membrane preparation, Matrigel. Zymograms of culture supernates demonstrate a 72-kD and a 92-kD gelatinase activity; the cells produce most of the 72-kD gelatinase, whereas the 92-kD activity is derived entirely from the Matrigel. Addition of antibodies against type IV gelatinase/collagenase decreases the area of the tube network. Both tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, similarly decrease tube formation when added to cultures. Conversely, exogenous recombinant 72-kD gelatinase increases tube-forming activity. The effects of the anti-gelatinase antibodies and the TIMPs are not additive. Inhibition by either antibodies or TIMPs is greatest when they are added at culture initiation, suggesting that the protease activity is important in the early steps of morphogenesis. However, culture of the cells on Matrigel does not increase early expression of mRNA for the 72-kD gelatinase. Expression of message for the enzyme actually decreases during the course of the assay, while transcription of mRNAs for TIMPs increases, further supporting the concept that collagenases facilitate an early event in tube formation. These data demonstrate that gelatinase/type IV collagenase activity is important in endothelial cell morphogenesis on Matrigel, and suggest a role for collagenases in formation of new capillaries in vivo.

Amino Acid Sequence↗
PubMed1992

Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.

The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.

Base Sequence↗
PubMed1992

Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor.

The gene encoding bacteriophage T7 RNA polymerase (T7gene1) was placed under the control of regulatory elements from the Escherichia coli lac operon to construct an inducible vaccinia virus expression system consisting entirely of prokaryotic transcriptional machinery. Regulated expression of T7 RNA polymerase was necessary to construct a stable recombinant vaccinia virus harboring a T7 promoter; otherwise, uncontrolled expression led to interference with endogenous virus replication. To this end, the gene encoding the repressor protein of the lac operon was fused to a viral early/late promoter so that it was expressed constitutively, and the lac operator was interposed between a viral major late promoter and T7gene1. Greater than 99% repression of T7 RNA polymerase, which was relieved approximately 80-fold in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), was obtained. An expression cassette containing a T7 promoter-controlled beta-galactosidase reporter gene was recombined into a different region of the viral genome containing T7gene1. A stable, double recombinant virus was isolated and grown to a high titer. In the absence of inducer, beta-galactosidase expression was substantially repressed. Addition of increasing amounts of IPTG induced expression of beta-galactosidase to the point of suppression of viral replication. This hybrid vaccinia virus system (Vac/Op/T7) has potential applications for the efficient bioproduction of a wide variety of gene products.

Base Sequence↗
PubMed1991

Specific lysis of human immunodeficiency virus type 1-infected cells by a HLA-A3.1-restricted CD8+ cytotoxic T-lymphocyte clone that recognizes a conserved peptide sequence within the gp41 subunit of the envelope protein.

A HLA-A3.1-restricted CD8+ cytotoxic T-cell clone, E7.20, that lyses cells infected with human immunodeficiency virus type 1 was isolated from an infected individual. The epitope was localized to amino acids 768-778 (RLRDLLLIVTR, NL43 env sequence) of the cytoplasmic domain of gp41 by successive use of a panel of recombinant vaccinia viruses that express truncated env genes and synthetic peptides. The epitope is conserved on 7 (NL43, BRU, HXB2, BRVA, SC, JH3, and JFL) of 13 human immunodeficiency virus type 1 isolates from North America. Synthetic peptides of this region of strains RF and CDC4 are also recognized by E7.20 despite a nonconservative Thr----Val or Thr----Ala change at amino acid 777; however, an MN peptide, which has four amino acid substitutions, was not reactive. The epitope recognized by E7.20 has a predicted hydrophobic alpha-helical structure, with three contiguous Leu residues followed by Ile and Val at amino acids 772-776. Cytotoxicity was restricted by HLA-A3.1 using allogeneic target cells that shared HLA class I antigens with the donor and an HLA-A and -B negative human plasma cell line transfected with the HLA-A3.1 gene. The transfected cells were infectable by human immunodeficiency virus type 1 strains IIIB and MN but only the former virus sensitized them to killing by E7.20. The ability of E7.20 to specifically lyse a human lymphocyte line infected with a human immunodeficiency virus type 1 strain carrying the conserved epitope is consistent with an important role for cytotoxic T cells in controlling infection.

Amino Acid Sequence↗
PubMed1991

New use of BCG for recombinant vaccines.

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.

Animals↗
PubMed1991

Molecular cloning and analysis of the chromosomal region 26A of Drosophila melanogaster.

Region 26A of the second chromosome of Drosophila melanogaster has been extensively characterized at the genetic level. We report here the cloning of virtually the entire 26A region via a bidirectional chromosome walk. Deletion and translocation breakpoints in the 26A interval have been localized at the molecular level by both chromosomal in situ hybridization and Southern analysis. The locations of the genetically defined loci in this chromosomal region have also been correlated with transcriptional units mapped onto the DNA of the proximal region of the chromosomal walk. The position of the alpha-glycerophosphate dehydrogenase (alpha-Gpdh) gene in 26A5-7 has been confirmed and a putative transcriptional unit for the beta-galactosidase-1 (beta-Gal-1) gene has been identified in the 26A7-9 interval.

Animals↗