Biomedical subjects

D B Boyle

Publications and source records attributed to D B Boyle.

At least 73 records · Page 4Linked to original sources

PubMed1988

A general method for the construction of recombinant vaccinia viruses expressing multiple foreign genes.

Plasmid vectors with multiple cloning sites adjacent to a vaccinia virus (VV) promoter were constructed and used to insert a protein coding sequence and a dominant selectable marker into a non-essential region of the VV genome. Recombinant viruses, selected on the basis of expression of the herpes simplex virus (HSV) thymidine kinase gene (tk), were shown to express in infected cells the model gene product, murine major histocompatibility complex (MHC) antigen H-2Kd, by cell-surface binding of antibody and by MHC-restricted recognition by cytotoxic T lymphocytes. Double recombinant VVs with insertions at two sites (in the VV tk gene and in the VV HindIII-F region) were constructed and shown to express influenza A/PR/8/34 haemagglutinin and H-2Kd antigen in addition to the HSV tk gene. The plasmids described allow the construction of recombinant VV expressing two genes of interest under the control of the same VV promoter. Such recombinant VVs can be used to study the interaction of immunologically important antigens simultaneously expressed.

Animals↗

PubMed1988

A dominant selectable marker for the construction of recombinant poxviruses.

Mycophenolic acid has been shown to be a potent inhibitor of vaccinia virus growth. By inserting the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt) into the vaccinia virus genome under control of the P-7.5 promoter this inhibition was overcome. When coupled in tandem with another gene of interest, recombinant vaccinia viruses can be positively selected carrying both genes. Since the gpt gene operates as a selectable marker in most mammalian cells it will be useful as a dominant selectable marker for the construction of recombinant viruses based on other host-specific poxviruses.

DNA Restriction Enzymes↗

PubMed1988

Class I MHC-restricted cytotoxic T cells efficiently recognize haemagglutinin that is defective in protein folding and cell surface expressions.

Cytotoxic T-cell recognition of an engineered variant of the influenza viral haemagglutinin (HA), expressed in vaccinia virus, was investigated. We show that the insertion of a Foot-and-Mouth Disease virus (FMDV) immunogenic peptide into the HA results in major disruption of its higher order structure with intracellular rather than cell surface localization accompanying the loss of conformational epitopes detected by antibody. In contradistinction to antibody, recognition of the chimaeric molecule by HA-specific class I-restricted cytotoxic T-cells was unimpaired, demonstrating that class I-specific T-cells recognize, in majority, continuous epitopes rather than conformational epitopes in the HA.

Animals↗

PubMed1988

Construction of recombinant fowlpox viruses as vectors for poultry vaccines.

Plasmid vectors have been constructed which allow the construction of infectious fowlpox virus (FPV) recombinants expressing foreign genes. The foreign genes were inserted within the thymidine kinase (TK) gene of FPV contained in these vectors. To facilitate the selection of recombinants the Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene was developed as a dominant selectable marker. This marker operates in a wide variety of cell types and obviates the need for TK- cell lines for selection of TK- recombinants when foreign genes have been inserted within the TK gene of FPV. The general approach adopted was to construct plasmid vectors in which the FPV TK was interrupted by the Ecogpt gene under the control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter. Selection of viruses expressing the Ecogpt gene simultaneously selects for recombinants carrying both the Ecogpt gene and the gene of interest. Using this approach a series of plasmid vectors was constructed in which the FPV TK gene was interrupted by the Ecogpt gene under the control of the P7.5 vaccinia virus promoter in tandem with the A/PR/8/34 haemagglutinin gene under the control of the PL11 vaccinia virus promoter. A recombinant FPV constructed using these plasmids had the expected genome arrangement, expressed influenza haemagglutinin, and induced haemagglutination-inhibiting antibodies when inoculated into chickens. These techniques should allow the construction of a variety of recombinant FPVs expressing poultry vaccine antigens. Such recombinants should be a very cost-effective means of delivering vaccines to poultry.

Animals↗

PubMed1987

Recognition by major histocompatibility complex class I-restricted cytolytic T lymphocytes of cells expressing vaccinia-encoded viral and class I proteins.

Target cells expressing influenza virus hemagglutinin (HA) could be recognized by cytotoxic T lymphocytes (CTL) in conjunction with the murine major histocompatibility complex class I antigen, H-2Kd, when both antigens were encoded by recombinant vaccinia virus. This recognition occurred if HA and H-2Kd were encoded by separate vaccinia viruses following dual infection of target cells or if HA and H-2Kd were encoded by a single recombinant virus. In contrast, target cells expressing nucleoprotein (NP) were only recognized by H-2Kd-restricted CTL if both NP and H-2Kd were encoded by the same vaccinia virus. These results show that the requirements for association of H-2Kd with different viral antigens derived from HA or NP can vary. Possible factors contributing to this difference are discussed.

Animals↗

PubMed1987

Fowlpox virus polypeptides: sequential appearance and virion associated polypeptides.

The polypeptides associated with fowlpox virus (FPV) infection of chicken embryo skin (CES) cells were examined by metabolic labelling with [35S]-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Polypeptide synthesis was followed over the first 48 hours post infection, as this was shown to be the period of viable virus production in CES cells. In contrast to infection with vaccinia virus (VV), which leads to a rapid total inhibition of host polypeptide synthesis in a number of cell lines, FPV infection of CES cells failed to cause a complete shut down of host polypeptide synthesis, with only a small number of host polypeptides being inhibited. A total of 21 FPV coded or induced polypeptides were resolved by metabolic labelling. As with VV, these polypeptides can be divided into two groups, the pre-replicative polypeptides containing a single member of 70,000 daltons, synthesised before viral DNA replication, and the post-replicative polypeptides, synthesised only after viral DNA replication has commenced. FPV DNA replication was shown to commence between 12 and 16 hours post-infection and to continue up to 48 hours post-infection. As also observed with VV, two temporally distinct classes of post-replicative polypeptides were identified based on their time of synthesis post-infection. The examination of purified FPV and VV by SDS-PAGE and coomassie blue staining allowed the resolution of 57 FPV particle associated polypeptides and 27 VV associated polypeptides.

Animals↗

PubMed1987

Fowlpox virus thymidine kinase: nucleotide sequence and relationships to other thymidine kinases.

The thymidine kinase (TK) gene of fowlpox virus (FPV) is located in a 2.2-kb HindIII-ClaI fragment derived from a 5.5-kb EcoR1 fragment of the FPV genome. The TK gene was mapped to the region of a 700-bp XbaI fragment contained within this HindIII-ClaI fragment. Nucleotide sequence analysis of this region revealed an open reading frame of 183 codons. Identification of this region as the FPV TK gene was confirmed by its homology with the vaccinia virus TK at both the nucleotide and amino acid levels. The derived FPV TK polypeptide has a calculated molecular weight of 20,380 and is six amino acids larger than the vaccinia virus TK gene product. We have reported previously that the FPV TK gene operates in vaccinia virus without the requirement for a vaccinia virus promoter. The sequence homologies between the two TK promoters substantiated this observation. Northern blot analysis of RNAs from cells infected with a vaccinia virus recombinant expressing the FPV TK gene showed major (700 nucleotide) and minor (1000 nucleotide) transcripts from the FPV TK gene. The deduced amino acid sequence of the FPV TK has significant homology with the TKs from chicken, man, and three other poxviruses, but shows no homology with herpes simplex virus TK. Comparisons of the homologous sequences indicated that the "core" of the enzyme has probably evolved in poxviruses four times as quickly as in vertebrates. Characterization of the FPV TK gene may facilitate the construction of recombinant FPVs as vehicles for the delivery of vaccine antigens to poultry and other avian species.

Amino Acid Sequence↗

PubMed1987

Effect of in vitro mutations in a vaccinia virus early promoter region monitored by herpes simplex virus thymidine kinase expression in recombinant vaccinia virus.

The location of a promoter (PF) in the HindIII F region of the vaccinia virus genome was mapped by introducing deletions into this region of the DNA. Modified promoters were fused to the herpes simplex virus (HSV) thymidine kinase (TK) gene in plasmids facilitating the construction of recombinant vaccinia viruses, and promoter function was monitored by the ability of such plasmids to rescue TK+ vaccinia viruses from cells infected with TK- virus. Deletions from the 3' end of the promoter region produced mutants for which function was either not inhibited or abolished, allowing the 3' promoter boundary to be defined to within 13 nucleotides. As indicated by the presence of the PF transcript in early RNA and the kinetics of HSV TK expression in recombinant vaccinia viruses, transcription from PF occurred primarily at early times during infection. The major transcript was initiated at a site within 20 nucleotides of the 3' end of the promoter and nine bases upstream of the probable translation initiation codon. In one mutant for which a small but reproducible increase in promoter function was detected, the transcription start site was deleted. Nevertheless, transcription still appeared to begin at the equivalent position with respect to the promoter, despite the altered nucleotide sequence. The location of the start site for the PF transcript indicated that the HSV TK gene, inserted at the BamHI site following the promoter, was preceded by an initiation codon which could potentially attenuate expression of the inserted gene. Conversion of this ATG codon to TAG did not significantly improve HSV TK expression.

Base Sequence↗

PubMed1987

The roles of influenza virus haemagglutinin and nucleoprotein in protection: analysis using vaccinia virus recombinants.

Vaccinia virus recombinants expressing haemagglutinin (HA) or nucleoprotein (NP) from influenza virus A/PR/8/34 were used to investigate protective immunity in mice, with two protocols. Protection was assessed by mortality and morbidity rates and by lung virus titres after infection intranasally with A/PR/8/34. In the first protocol, mice immunized with vaccinia-HA recombinant virus and infected intranasally with A/PR/8/34 were almost totally protected, but mice immunized with vaccinia-NP virus were very poorly protected. In the second protocol, the recombinant viruses were used to stimulate in vitro T cells that are specific for HA and NP; both populations of T cells, when transferred to A/PR/8/34-infected mice, afforded good protection. The results indicate that an immune response specific for just HA provided protection that was almost indistinguishable from that provided by whole A/PR/8/34. On the other hand, immunization with vaccinia-NP provided poor protective immunity, despite the fact that transferred NP-specific T cells were very effective and vaccinia-NP immunization has previously been shown to stimulate cytotoxic T cells. These results demonstrate that a single viral antigen, delivered by live vaccinia virus, can provide effective protection, but that immunization for cross-protection against heterologous influenza virus remains elusive.

Animals↗

PubMed1987

Vaccinia virus recombinants expressing the SA11 rotavirus VP7 glycoprotein gene induce serotype-specific neutralizing antibodies.

A cDNA copy of the gene coding for the major outer neutralizing protein (VP7) of simian 11 rotavirus was incorporated into the vaccinia virus genome under the control of the vaccinia promoter (molecular weight, 7,500). A deletion mutant of this gene which codes for a secreted form of VP7 when expressed under the control of the simian virus 40 late promoter (M. S. Poruschynsky, C. Tyndall, G. W. Both, F. Sato, A. R. Bellamy, and P. H. Atkinson, J. Cell Biol. 101:2199-2209, 1985) was also inserted. Each recombinant vaccinia virus directed the synthesis of a rotavirus protein in infected cells, and the product encoded by the mutated gene was secreted. Rabbits immunized with the two types of recombinant vaccinia virus generated antibodies that were able both to recognize simian 11 rotavirus in an enzyme-linked immunosorbent assay and to neutralize the virus in a plaque-reduction test. Antibodies induced by the recombinant vaccinia viruses expressing either form of VP7 were serotype specific.

Antibodies, Viral↗

PubMed1986

Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response.

Regulation of the expression of influenza A/PR/8/34 hemagglutinin (HA) by the vaccinia virus promoters PF (early), P7.5 (early and late) and PL11 (late) has been demonstrated using HA-vaccinia recombinant viruses VV-PR8-HA3, VV-PR8-HA6 and VV-PR8-HA, respectively. Levels of HA on the surface of VV-PR8-HA3 (PF)-infected cells were lower than with either VV-PR8-HA6 (P7.5) or VV-PR8-HA8 (PL11). Expression of HA under the control of the late promoter PL11 was inhibited in the absence of DNA replication. All three recombinant viruses stimulated a specific antibody response in mice which was dependent on the presence of infectious virus. Recognition of HA by cytotoxic T lymphocytes (CTL) was assessed by the ability of the viruses to stimulate naive precursors in vivo, to restimulate primed CTL in vitro and by target cell recognition. HA expressed under the control of either of the promoters with early function (PF or P7.5) was recognized by CTL when VV-PR8-HA3 or VV-PR8-HA6 were used to prime or restimulate splenocytes or to infect target cells. On the other hand, HA expressed by VV-PR8-HA8 (PL11) failed to prime for a CTL response in naive CBA/H mice, was ineffective at restimulation of primed splenocytes and failed to produce target cells for recognition by specific CTL. However, in BALB/c mice VV-PR8-HA8 did prime for a specific CTL response. These studies show that HA synthesized early in infection was recognized by both B and T cells while HA expressed after DNA replication was not generally recognized by T cells. The implications of the observations with the late promoter with respect to the use of late promoters in potential vaccinia virus-based vaccines are considered.

Animals↗

PubMed1986

Cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants.

Recombinant vaccinia viruses enable studies of immune recognition of antigens expressed from single viral genes. We have constructed recombinants expressing the haemagglutinin (HA) and nucleoprotein (NP) genes of the influenza virus A/PR/8/34 (H1N1). These recombinant viruses together with a recombinant expressing the HA from influenza virus A/JAP/305/57 (H2N2) have been used to examine the cytotoxic T lymphocyte (CTL) response to these influenza virus antigens. Both antigens are recognised by murine CTL and recognition of HA is influenza virus subtype-specific, whereas recognition of NP is crossreactive. In limiting dilution studies approximately 10% of the influenza CTL response is HA-specific, while approximately 30% of the response is NP-specific. Despite the ability of NP to stimulate a significant CTL response, mice immunised with the NP-vaccinia recombinant are not as well protected from subsequent lethal challenge with influenza virus, as mice immunised with the HA vaccinia recombinant. These studies demonstrate that viral antigens expressed from vaccine recombinants can provide protective immunity and that the influenza-poxvirus recombinants can provide data on protective immunity generated by individual viral proteins.

Animals↗

PubMed1986

Immune responses to H-2Kd antigen expressed by recombinant vaccinia virus.

A recombinant vaccinia virus (VV-H2Kd-6) containing the coding sequence for the murine major histocompatibility complex class I antigen H-2Kd has been constructed and used to express H-2Kd on the surface of infected cells. Vaccinia expressed H-2Kd has been shown to generate an H-2Kd-specific primary cytotoxic T-cell response in mice infected with the recombinant virus and to stimulate an H-2Kd-specific cytotoxic T-cell response in vitro. Cells infected with the recombinant virus acted as targets for specific lysis by appropriate alloreactive cytotoxic T cells, albeit relatively inefficiently when compared with alloreactive recognition and specific lysis of H-2Kd-containing P815 cells. However, H-2Kd expressed by the recombinant virus was recognized efficiently as a restricting element in association with vaccinia virus antigens, while lysis of VV-H2Kd-6-infected L929 (H-2k) cells by CBA/H (H-2k) anti-C3H.OH (H-2KdDk) cytotoxic T cells was comparatively weak. These data suggest that there are quantitative or qualitative differences, or both, between H-2Kd expressed by vaccinia virus and cells of the H-2d haplotype. Qualitative differences have not been demonstrated but cannot be excluded.

Animals↗

PubMed1986

Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia virus.

Using vaccinia virus as a selection and cloning vehicle, a thymidine kinase (TK) gene of fowlpox virus (FPV) has been identified. A plasmid, pF130, containing part of the HindIII-F region of vaccinia virus was used to shotgun clone EcoRI fragments of FPV DNA into TK- vaccinia virus and select for TK+ recombinants. The TK+ recombinant vaccinia virus contained a 5.5 kb EcoRI fragment of FPV. This FPV fragment was cloned into pUC9 and the presence of the TK gene in this fragment was confirmed by its ability to rescue TK+ vaccinia virus from TK- virus, when inserted into pF130. A recombinant vaccinia virus containing this FPV fragment induced TK enzyme activity in the cytoplasm of infected cells. The vaccinia virus RNA polymerase appeared able to recognize the FPV promoter sequences of the FPV TK gene since the fragment operated in the marker rescue, irrespective of its orientation to the vaccinia virus promoter in pF130. Using restriction enzyme analysis, insertion of subfragments of the 5.5 kb FPV fragment into pF130 and marker rescue, we were able to map the position of the TK gene in the 5.5 kb EcoRI fragment. This approach may facilitate identification and cloning of TK genes from other poxviruses.

Cloning, Molecular↗

PubMed1986

Responses of cattle, sheep and poultry to a recombinant vaccinia virus expressing a swine influenza haemagglutinin.

Groups of cattle, sheep and poultry were inoculated with a recombinant vaccinia virus expressing the haemagglutinin of the swine influenza virus A/NJ/11/76. No adverse clinical responses were recorded and none of the animals developed a viraemia when inoculated with the recombinant or wild-type vaccinia virus. Recombinant virus reisolated from lesions in cattle was stable, maintaining its thymidine kinase negative phenotype and ability to express the swine influenza haemagglutinin. Antibodies to the influenza haemagglutinin were detected in cattle, sheep and poultry inoculated with the recombinant virus. While no animals inoculated with wild-type virus developed these antibodies, there was no detectable spread of either recombinant or wild-type virus from the inoculation sites or to in-contact uninoculated animals. The results indicate that recombinant vaccinia viruses can induce immune responses in cattle, sheep and poultry demonstrating their potential as vaccine vectors in a variety of important veterinary species.

Animals↗

PubMed1985

Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses.

Plasmid vectors containing multiple cloning sites suitable for the rapid insertion of protein-coding sequences into poxviruses have been constructed. They are based on pUC plasmids and carry the thymidine kinase (TK) gene of vaccinia virus interrupted by a vaccinia virus promoter. Six unique restriction enzyme sites (BamHI, SalI/HincII, PstI, HindIII, EcoRI), located within 40 bp of vaccinia virus promoters transposed from the HindIII-F or HindIII-C fragment of the vaccinia virus genome, allow rapid insertion of foreign-protein-coding sequences into these plasmids. Such plasmids can be used to construct recombinant poxviruses expressing foreign proteins using marker-rescue recombination techniques and selection for TK negative viruses. Vaccinia viruses expressing the haemagglutinin (HA) gene of swine influenza virus, A/NJ/11/76 (H1N1), have been constructed.

Base Sequence↗

PubMed1984

A possible molecular basis for strain specific immunity to malaria.

A 250 kDa antigen implicated in the induction of protective immunity to Plasmodium chabaudi was examined with a panel of 11 monoclonal antibodies in cloned parasite lines. 2 antibodies cross-reacted with the different parasite lines while 9 were specific for one line. This antigenic diversity was correlated with major differences in one dimensional peptide maps between the purified antigen from different lines of parasites. The peptide maps also revealed some apparently conserved structure which may have been responsible for the antigenic cross reactivity. Using cloned lines of P. falciparum and a second series of monoclonal antibodies, similar antigenic and structural diversity was evident in the equivalent antigen from the important human pathogen. These findings are discussed with relationship to the induction of protective immunity to malaria.

Animals↗

PubMed1983

Maturation of the intracellular parasite and antigenicity.

The protein antigens synthesized by the malarial parasite change as the parasite matures, with a number of proteins showing strict stage-specificity. A detailed correlation between the stage-specificity of protein synthesis and parasite structure has yet to be established, but a number of proteins synthesized in the cycle are lost selectively during merozoite escape and reinvasion. These antigens are presumably associated with structures utilized and ultimately lost during this process. Particular interest has focused on some greater than 200K proteins identified as being on the surface of infected erythrocytes and internally and on the surface of merozoites. Smaller parasite proteins have also been identified in the erythrocyte membrane. The erythrocyte itself, including its membrane, is much modified by parasite growth. Changes include the presence of new cytoplasmic structures and differences in the surface labelling and isoantigenic characteristics of the surface membrane. An appreciation of the variability and specificity of exposed parasite antigens, and their relationship to newly exposed isoantigens, is central to our understanding of protective immunity to malaria.

Animals↗