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B Moss

Publications and source records attributed to B Moss.

At least 307 records · Page 17Linked to original sources
PubMed1988

Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum.

Malaria is initiated by the inoculation of a susceptible host with sporozoites from an infected mosquito. The sporozoites enter hepatocytes and develop for a period as exoerythrocyte or hepatic stage parasites. Vaccination with irradiated sporozoites can provide protective immunity and a recent study shows that this can also be conferred by immunization with a recombinant salmonella expressing only the circumsporozoite protein that normally covers the sporozoites. Protection against infection is likely to be mediated by cytotoxic CD8+ cells, as depletion of CD8+ T cells in a sporozoite-immunized animal can completely abrogate immunity. Here we demonstrate directly the existence of CD8+ cytotoxic T lymphocytes (CTL) that recognize the circumsporozoite protein. B10.BR mice immunized with sporozoites or with recombinant vaccinia virus expressing the CS protein of Plasmodium falciparum contain CTL that specifically kill L cell fibroblasts transfected with the gene encoding the same CS protein. The peptide epitope from the CS protein that is recognized by CTL from this strain of mice is from a variant region of the protein.

Amino Acid Sequence↗
PubMed1988

Vaccinia virus poly(A) polymerase. Specificity for nucleotides and nucleotide analogs.

We have studied the nucleotide specificity of vaccinia virus poly(A) polymerase using a novel primer extension assay. Oligoribonucleotide primers labeled at the 5' end with 32P were elongated by the enzyme in the presence of ATP, leading to the 3' addition of greater than 1000 adenylate residues/primer molecule. In the presence of UTP, the enzyme catalyzed 3' polymerization of long poly(U) tails, albeit at a reduced rate of chain growth. In the presence of both ATP and UTP, 3' addition was selective for ATP. The transient accumulation of RNAs elongated by 10-16 residues suggested that polyadenylation (and polyuridylation) was a biphasic reaction. Quantitative 3' addition of GMP (from GTP) or CMP (from CTP) to the primer was also observed, although the rate of chain growth was so slow as to allow synthesis of only short oligo(G) or oligo(C) tails. The deoxynucleotides 3'-dATP (cordycepin triphosphate) and ddATP were markedly inhibitory to poly(A) polymerase. Primer elongation studies were consistent with inhibition due to 3' incorporation of inhibitor and chain termination. Incubation of enzyme with [alpha-32P] cordycepin triphosphate resulted in labeling of the Mr 57,000 enzyme subunit, apparently via formation of a covalent nucleotidyl-protein complex. These data are discussed in light of their implications for the catalytic mechanism of polyadenylation.

Adenosine Triphosphate↗
PubMed1988

Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA.

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.

Base Sequence↗
PubMed1988

HIV-1 reverse transcriptase is a target for cytotoxic T lymphocytes in infected individuals.

Characterization of the host immune response to human immunodeficiency virus type 1 (HIV-1) is critical to the rational design of an effective AIDS vaccine. In this study, cytotoxic T lymphocytes (CTL) specific for HIV-1 reverse transcriptase (RNA-dependent DNA polymerase) were found in blood samples from HIV-1-infected individuals. CTL targets were prepared by immortalizing B cells from ten seropositive and six seronegative individuals, and then infecting these cells with recombinant vaccinia viruses containing HIV-1 genes. CTL directed against autologous B lymphoblasts expressing HIV-1 reverse transcriptase were detected in fresh blood samples from eight HIV-1 seropositive subjects, but in no seronegative controls. The effector cells were identified as major histocompatibility complex-restricted CD3+CD8+ lymphocytes. Because the HIV-1 pol gene is highly conserved among different isolates and generates both humoral and cellular immune responses, it bears consideration for inclusion in a candidate AIDS vaccine.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Molecular cloning and sequence of the concatemer junction from vaccinia virus replicative DNA. Viral nuclease cleavage sites in cruciform structures.

The concatemer junction from replicative forms of vaccinia virus DNA was cloned into plasmid vectors and shown to be a precise duplex copy of the viral terminal hairpin structure, with each strand corresponding to one of the alternative sequence isomers. The plasmids were relaxed circles with extruded cruciforms representing two copies of the vaccinia telomere hairpin structure. Head-to-head dimers containing two copies of the vaccinia virus concatemer junction were observed to contain only one set of stem-loop structures per molecule, suggesting that the initial formation of a small cruciform, and not branch migration, was the rate-limiting step in cruciform formation. The plasmids containing the concatemer junction were converted into nicked circular, linear and cross-linked linear molecules by a nuclease isolated from vaccinia virions. The region-specific cleavage near the border of the hairpin loop and the formation of DNA cross-links in some of the molecules is consistent with the nuclease acting as a nicking-closing enzyme that participates in the resolution of mature termini from replicative concatemer intermediates.

Base Sequence↗
PubMed1988

Characterization of human immunodeficiency virus gag/pol gene products expressed by recombinant vaccinia viruses.

Recombinant vaccinia viruses containing either the entire gag/pol gene or the reverse transcriptase (RT) domain of the human immunodeficiency virus (HIV) were constructed. In mammalian cells infected with the recombinant vaccinia virus containing the gag/pol gene, major and minor polypeptides of 55 and 41 kDa were made, but processed gag products (p24/p17/p15) were not detected. In addition, none of the products of the pol open-reading frame were seen. Both the 55- and 41-kDa gag proteins were post-translationally modified by addition of myristic acid residues in recombinant vaccinia-infected cells, and were immunoprecipitated by antiserum to p24 gag, as well as by antisera from HIV-infected patients. These results indicate that neither proteolytic processing nor other HIV proteins are required for myristilation, and suggest that the 55- and 41-kDa gag precursors share the same amino terminus as p17. Cells infected with a separate vaccinia recombinant containing a truncated piece of the gag/pol gene with added start and stop codons at the 5' and 3' ends of the RT reading frame synthesized a major 61-kDa and a minor 51-kDa protein product which reacted immunologically with both a monoclonal antibody to native HIV p66/51 and antisera from HIV-infected patients. These proteins were purified from recombinant vaccinia-infected mammalian cells, and their enzyme activity was found to be similar to that of authentic HIV RT. Cells infected with the vaccinia/RT vector contained approximately 200-fold more RT per milligram of protein than cells infected with HIV. Recombinant RT was inhibited by dideoxynucleoside triphosphates and should be useful in screening for specific inhibitors of this enzyme. Mice inoculated intradermally with 10(8) plaque-forming units of the vaccinia/RT vector developed specific antibodies to the p66/51 proteins of HIV, but anti-HIV antibodies were not detected in mice inoculated with the vaccinia/gag vector.

Antigens, Viral↗
PubMed1988

Use of vaccinia virus vectors to study the synthesis, intracellular localization, and action of the human immunodeficiency virus trans-activator protein.

A recombinant vaccinia virus that expresses the human immunodeficiency virus (HIV) trans-activator (tat) gene was constructed. The tat polypeptide migrated anomalously with an apparent molecular mass of 14 kDa on a sodium dodecyl sulfate-polyacrylamide gel and reacted with polyclonal anti-tat serum. The tat protein was localized predominantly in the cell nucleus despite the absence of other HIV proteins or intranuclear HIV DNA. Additional recombinant vaccinia viruses that contain the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under control of an early vaccinia promoter were constructed. Insertion of the HIV trans-activator-responsive (tar) sequence at the precise start of the CAT mRNA decreased CAT expression slightly. Trans-activation of vaccinia virus-encoded tarCAT failed to occur when CV-1 or HeLa cells were coinfected with the recombinant vaccinia virus expressing tat or when a HeLa cell line containing stably integrated copies of tat was used for infection, indicating the absence of transcriptional or translational effects under these conditions.

Cell Compartmentation↗
PubMed1988

Cell proliferative response to vaccinia virus is mediated by VGF.

VGF, a polypeptide encoded by vaccinia virus, shares amino acid sequence homology and functional properties with cellular growth factors EGF and TGF-alpha. The availability of a VGF minus (VGF-) virus mutant has enabled us to examine the role of VGF in the replication of virus in vitro and in vivo. Studies in vitro with A431 cells (high EGF receptor density) showed that VGF+ wild-type virus induced the rapid formation of a focus of infection (not a plaque) which could be blocked by a monoclonal antibody to the EGF receptor. In vivo experiments with chicken embryos indicated that VGF+ virus stimulated the growth of ectodermal and entodermal cells of the chorioallantoic membrane. At early times, the majority of proliferating cells contained no detectable virus antigen, indicating that cell growth preceded infection and was a consequence of VGF secretion. Relative to VGF- virus, VGF+ virus produced lesions which contained more proliferating cells, more virus antigen, and increased amounts of infectious progeny. Secretion of VGF thus explains the conundrum of a nontransforming, strongly cytopathic virus inducing a hyperplastic cell response.

Amino Acid Sequence↗
PubMed1988

Live viral vaccines for respiratory and enteric tract diseases.

In its programme for accelerated development of vaccines for viral respiratory and enteric tract diseases the WHO has assigned a very high priority to respiratory syncytial virus (RSV), parainfluenza viruses and rotaviruses. There is also some interest in alternative approaches to immunization against influenza viruses because of the failure of inactivated vaccines to provide complete and reasonably durable immunity. Current attempts to develop satisfactorily attenuated viruses for use in prevention of disease caused by the above viral pathogens are described.

Gastrointestinal Diseases↗
PubMed1988

Roles of vaccinia virus in the development of new vaccines.

Vaccinia virus is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. With increased levels of gene expression, obtained either with stronger vaccinia promoters or through incorporation of the bacteriophage T7 RNA polymerase gene into the vaccinia genome, proteins may be synthesized in mammalian cells for use as subunit vaccines. For use as a live recombinant vaccine, efforts are being made to attenuate vaccinia virus further, either by inactivating genes contributing to virulence or by introducing human lymphokine genes into the vaccinia genome.

Genetic Vectors↗
PubMed1988

Group-specific, major histocompatibility complex class I-restricted cytotoxic responses to human immunodeficiency virus 1 (HIV-1) envelope proteins by cloned peripheral blood T cells from an HIV-1-infected individual.

Freshly separated unfractionated peripheral blood mononuclear cells (PBMC) and cloned cell lines from a healthy human immunodeficiency virus 1 (HIV-1)-seropositive individual were examined for cytotoxic responses to HIV proteins expressed by recombinant vaccinia viruses. It was found that freshly isolated PBMC recognize variant envelope proteins of HIV-1 but not a more distantly related envelope protein derived from the simian immunodeficiency virus (SIVmac). Although the effector cells were predominantly CD8+, both MHC-matched and -unmatched target cells were lysed. Cytotoxic T lymphocyte (CTL) clones were found to lyse cells expressing HIV-1 envelope or reverse transcriptase. In contrast to the cytotoxic response detected with PBMC, the cloned CTLs were major histocompatibility complex (MHC) class I restricted. Our finding that a cloned CTL line lysed cells expressing highly divergent HIV envelopes strongly suggested that a conserved epitope was recognized. Identification of these shared epitopes may assist in designing a vaccine for HIV-1 that could stimulate MHC-restricted cytotoxic responses.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Binding region for human immunodeficiency virus (HIV) and epitopes for HIV-blocking monoclonal antibodies of the CD4 molecule defined by site-directed mutagenesis.

The binding region for human immunodeficiency virus (HIV) and epitopes for a panel of HIV-blocking anti-CD4 monoclonal antibodies of the CD4 molecule were defined by using in vitro site-directed mutagenesis. Codons for two amino acid residues (Ser-Arg) were inserted at selected positions within the region encoding the first and second immunoglobulin-like domains of CD4. A vaccinia virus-based expression system was used to produce soluble full-length extracellular CD4 fragments containing the insertions. The mutant proteins were tested for direct binding to soluble gp120 (the CD4-binding subunit of the viral envelope glycoprotein) and to a series of HIV-blocking anti-CD4 monoclonal antibodies. Impaired gp120 binding activity resulted from insertions after amino acid residues 31, 44, 48, 52, 55, and 57 in the first immunoglobulin-like domain. The epitopes for two HIV-blocking monoclonal antibodies, OKT4A and OKT4D, were also mapped in the gp120-binding region in the first domain. Insertions after amino acid residues 21 and 91 in the first domain had no effect on gp120 binding but impaired the binding of OKT4E, suggesting that this antibody recognizes a discontinuous epitope not directly involved in gp120 binding. Moderate impairment of gp120 binding resulted from the insertion after amino acid residues 164 in the second immunoglobulin-like domain, where the epitopes for monoclonal antibodies MT151 and OKT4B were also mapped.

Amino Acid Sequence↗
PubMed1988

A soluble recombinant polypeptide comprising the amino-terminal half of the extracellular region of the CD4 molecule contains an active binding site for human immunodeficiency virus.

Infection of helper T lymphocytes by human immunodeficiency virus is initiated by a specific interaction of the viral envelope glycoprotein with CD4, an integral membrane glycoprotein of the target cell. We have adapted a vaccinia virus-based mammalian cell expression system to produce variants of the CD4 molecule for structure-function studies. In this report we demonstrate that a truncated 180-amino acid fragment representing approximately the N-terminal half of the extracellular region of CD4 is found primarily in soluble form in the extracellular medium. Epitope analysis with a panel of anti-CD4 murine monoclonal antibodies indicates that the fragment reacts with those antibodies known to block the interaction between CD4 and the human immunodeficiency virus envelope glycoprotein but reacts poorly or not at all with those antibodies that do not block this interaction. We also show that the fragment forms a specific complex with a soluble form of gp120, the CD4-binding subunit of the viral envelope glycoprotein. These results indicate that this soluble CD4 fragment contains an active binding site for human immunodeficiency virus.

Amino Acid Sequence↗
PubMed1988

An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes.

Because cytotoxic T lymphocytes (CTL) may be important for preventing direct cell-to-cell transmission of human immunodeficiency virus (HIV), the agent responsible for acquired immunodeficiency syndrome, we have begun to investigate the epitope specificity and immune response (Ir) gene control of anti-HIV CTL responses in experimental animals. Mice were infected with a recombinant vaccinia virus expressing the HIV gp160 envelope gene, and the primed lymphocytes were restimulated in vitro with a transfected histocompatible cell line expressing the same gene. Our results show that H-2d mice are CTL high responders and H-2k mice are low responders to the HIV gp160 envelope protein under these conditions. Moreover, the H-2d mice respond predominantly to a single immunodominant site represented by a 15-residue synthetic peptide conforming to the amphipathic alpha-helix model of T-cell epitopes and seen by CD4- CD8+ CTL in association with the Dd class I major histocompatibility complex (MHC) molecules. The facts that CTL responses were detected in the context of only one of four class I MHC molecules tested and that the response was limited predominantly to a single epitope indicate that the CTL repertoire elicited by the HIV envelope protein in association with murine class I MHC molecules may be very limited. In addition, this epitope occurs in a highly variable segment of the envelope protein. This puts constraints on the use of a single peptide sequence from this region in a vaccine, as such a vaccine would have to be polyvalent. Nevertheless, this same variability suggests that this region may be under selective pressure from human CTL, and therefore that this site may be immunodominant in humans as well as mice and so of clinical importance in vaccine development.

Acquired Immunodeficiency Syndrome↗