Progress 10/01/04 to 09/30/09
Outputs
OUTPUTS: Modified vaccinia Ankara virus (MVA) is a 'next-generation' smallpox vaccine. Although practical application of MVA as a vaccine, or vaccine vector, is being actively pursued, the molecular biology of this virus remains poorly understood. MVA differs in many properties from 'wild-type' vaccinia virus (VAC) strains. The genetic basis of many of the distinct phenotypes of MVA remains unknown at this time. Prostaglandins (PGs) are lipid molecules derived from arachidonic acid that play important signaling roles in the innate immune system. As well as acting as inflammatory mediators, PGs are intimately involved in shaping the adaptive immune response. We have found recently that in vitro infection of C3HA cells, a mouse fibroblast cell line, with MVA, triggers the production of PGE2. In contrast, infection of C3HA cells with VAC did not cause the accumulation of PGE2. Because wild-type poxviruses encode a well-established repertoire of proteins that disrupt immune system signaling by cytokines, the failure of VAC to induce the production of PGs in vitro also may result from an active suppression of this response. In contrast, the attenuated virus MVA contains mutations in many viral genes and consequently may have lost the ability to suppress PG production by host cells. Traditionally, genetic mapping in poxviruses has relied on various techniques for marker rescue. Limitations of current genetic mapping techniques, particularly the need to use marker phenotypes that are selectable in cell culture, suggest that new strategies are required. This project is focused on the development of such an approach for MVA, and will demonstrate its utility by mapping viral genes that are required to induce PGE2 production from MVA-infected cells. Poxvirus genomes are large, and the viral DNA is not directly infectious when introduced into host cells by transfection. These factors have so far precluded the development of a genetic system by which infectious virus can be rescued entirely from cloned DNA. However, poxvirus genomic DNA can be replicated and packaged into infectious particles if it is transfected into cells that are already infected by another poxvirus that can act as a 'helper' virus. This phenomenon is referred to as genome reactivation. In the case of VAC, it also has been shown that infectious virus can be generated, in the presence of a suitable helper virus, from multiple, overlapping restriction fragments of the viral genome that are transfected together into host cells. This method has been termed 'recombination and reactivation' of poxvirus from DNA fragments. In this project, conditions will be established for recombination and reactivation of MVA from DNA fragments. The method will then be used to combine defined portions of the MVA and VAC genomes into specific recombinant, hybrid viruses. In this fashion, a panel of MVA-VAC hybrid poxviruses will be generated that can be used for rapid genetic mapping of any differential phenotype of interest. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Work on the project generated the following progress: (i) host cell lines that are permissive (BHK) or non-permissive (CHO) for the helper virus, Shope fibroma virus (SFV), were identified; (ii) a marker gene (CP77) that expands the useful host range of MVA in vitro to include CHO cells (non-permissive for the helper virus, SFV) was identified, and some properties of recombinant MVA carrying this marker gene (MVA/CP77) were characterized; (iii) an approach for engineering new restriction sites into the MVA genome to allow construction of specific MVA-VAC hybrids was demonstrated; (iv) infection of C3HA murine fibroblasts by MVA, but not VAC, was shown to induce PGE2 accumulation; (v) the accumulation of cyclooxygenase-2 was shown to increase during MVA infection of C3HA cells; (vi) MVA infection of murine bone marrow-derived dendritic cells (bmDCs) was shown to induce PGE2 accumulation; and, (vii) MVA that was inactivated by UV-irradiation was shown to induce PGE2 production by murine bmDCs. The information obtained during the course of this project will be used to make customized variants of MVA that can provoke qualitatively or quantitatively improved immune responses during vaccination.
Publications
- Lynch, H.E., Oie, K.L., Pollara, J.J., Petty, I.T.D., Sadler, A., Williams, B.R.G. and Pickup, D.J. 2009. Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication. Virology 391:177-186.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Prostaglandins (PGs) are eicosanoids, lipid molecules derived from arachidonic acid, that play important signaling roles in the innate immune response. As well as acting as inflammatory mediators, PGs are intimately involved in shaping the adaptive immune response. We have found recently that in vitro infection of C3HA cells, a mouse fibroblast cell line, with a poxvirus vaccine vector, modified vaccinia Ankara virus (MVA), triggers the production of PGE2. In contrast, infection of C3HA cells with a wild-type strain of vaccinia virus (VAC) did not cause the accumulation of PGE2. A similar effect was seen in mouse bone marrow-derived dendritic cells (bmDCs). Infection of mouse bmDCs with MVA resulted in the production of PGE2, whereas infection of these cells with wild-type cowpox virus (CPX) did not. Because wild-type poxviruses encode a well-established repertoire of proteins that disrupt immune system signaling by cytokines, the failure of VAC and CPX to induce the production of PGs in vitro also may result from an active suppression of this response. In contrast, the attenuated virus MVA contains mutations in many viral genes and consequently may have lost the ability to suppress PG production by host cells. In this project, these hypotheses will be tested through a combination of in vitro approaches using C3HA cells and murine bmDCs. In addition, the ability of MVA infection to induce PGE2 production in vitro from human monocyte-derived dendritic cells, as well as a selection of human primary cells, will be used to evaluate whether induction of PGs may have a role during vaccination. Finally, to investigate the relative importance of PG-mediated signaling in the immune response to the vaccine vector MVA, a well-established mouse model of infection in vivo will be employed. The goals of this research project are two-fold: (i) Establish the significance of PGE2 production by virus-infected cells in shaping the adaptive immune response to the vaccine vector, MVA. (ii) Define the molecular mechanism through which wild-type VAC avoids PGE2 production. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Work on the project so far has generated the following progress: (i) infection of C3HA murine fibroblasts by MVA, but not VAC, has been shown to induce PGE2 accumulation. (ii) The accumulation of cyclooxygenase-2 (COX-2) has been shown to increase during MVA infection of C3HA cells. (iii) MVA infection of murine bone marrow-derived dendritic cells has been shown to induce PGE2 accumulation. And, (iv) MVA, or CPX, that had been inactivated by UV-irradiation was shown to induce PGE2 production by murine bmDCs. The information obtained during the course of this project will be used to make customized variants of MVA that can provoke qualitatively or quantitatively improved immune responses during vaccination.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
Modified vaccinia Ankara virus (MVA) is a 'next-generation' smallpox vaccine that recently completed phase I/Ib clinical trials in the United States. Although practical application of MVA as a vaccine, or vaccine vector, is being actively pursued, the molecular biology of this virus remains poorly understood. MVA differs in many properties from other, 'wild-type' vaccinia virus (VAC) strains. The genetic basis of many of the distinct phenotypes of MVA remains unknown at this time. Traditionally, genetic mapping in poxviruses has relied on various techniques for marker rescue. In this approach, the unknown gene to be mapped, the marker, must confer a dominant, selectable phenotype. Transfer of the marker gene, by homologous recombination, into a recipient virus that normally does not express the phenotype will allow only the recombinant virus to grow. In this fashion, markers can be transferred between poxvirus genomes in mixed infections, or from isolated restriction
fragments of a donor virus genome. Limitations of current genetic mapping techniques, particularly the need to use marker phenotypes that are selectable in cell culture, suggest that new strategies are required. This project is focused on the development of such an approach for MVA, and will demonstrate its utility by mapping viral genes that are required to induce PGE2 production from MVA-infected cells. Poxvirus genomes are large, and the viral DNA is not directly infectious when introduced into host cells by transfection. These factors have so far precluded the development of a genetic system by which infectious virus can be rescued entirely from cloned DNA. However, poxvirus genomic DNA can be replicated and packaged into infectious particles if it is transfected into cells that are already infected by another poxvirus that can act as a 'helper' virus. This phenomenon is referred to as genome reactivation. In the case of VAC, it also has been shown that infectious virus can be
generated, in the presence of a suitable helper virus, from multiple, overlapping restriction fragments of the viral genome that are transfected together into host cells. This method has been termed 'recombination and reactivation' of poxvirus from DNA fragments. In this project, conditions will be established for recombination and reactivation of MVA from DNA fragments. The method will then be used to combine defined portions of the MVA and VAC genomes into specific recombinant, hybrid viruses. In this fashion, a panel of MVA-VAC hybrid poxviruses will be generated that can be used for rapid genetic mapping of any differential phenotype of interest. This approach differs in several ways from classical marker rescue techniques: (i) The recombination points in hybrid genomes will be precisely engineered. (ii) Multiple fragments can be joined simultaneously to construct complex hybrid genomes in a single step. (iii) Importantly, no selection for the trait(s) being mapped need be applied
during construction of the hybrid viruses. This will allow recessive traits, or those that do not lend themselves to selection in cell culture, to be mapped genetically.
Impacts
Work on the project so far has generated the following progress: (i) identification of host cell lines that are permissive (BHK) or non-permissive (CHO) for the helper virus, Shope fibroma virus (SFV); (ii) identification of a marker gene that expands the useful host range of MVA in vitro to include CHO cells (non-permissive for the helper virus, SFV); and, (iii) demonstration of an approach for engineering into the MVA genome new restriction sites to allow construction of specific MVA-VAC hybrids.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Modified vaccinia Ankara virus (MVA) is an attenuated orthopoxvirus being evaluated as a possible replacement for the current smallpox vaccine. Although testing indicates that MVA is safe and fairly effective, the location and nature of its attenuating mutations are unknown. In cell culture, MVA has a restricted host range. Whether this contributes to its attenuation also remains to be determined. To facilitate genetic analysis of MVA, an approach to engineer its host range in cell culture is being evaluated. It has long been known that cowpox virus (CPX) can infect the Chinese hamster ovary (CHO) cell line, while vaccine (VAC) strains cannot. Furthermore, transfer of single CPX gene, called CHOhr (for CHO cell host-range), to VAC allows the recombinant virus to grow on CHO cells. Comparative analysis reveals that nucleotide sequences homologous to CHOhr are defective in MVA. To evaluate the impact of a functional CHOhr gene on the host range of MVA, the defective
allele was repaired by homologous recombination with the functional CHOhr gene from CPX. In overview the experimental design was as follows. Susceptible host cells were infected with MVA at a low multiplicity of infection (MOI). A fragment of CPX DNA carrying the CHOhr gene was then transfected into the cells, and the virus infection was allowed to proceed. During this time, some homologous recombination events occurred between the MVA genome and the CPX DNA fragment, which resulted in repair of CHOhr. The small number of CHOhr+ viruses generated were separated from excess parental MVA (CHOhr-) and amplified by passage in CHO cells, which only the former viruses could infect. Uncloned CHOhr+ virus was amplified by sequential passages in CHO cells, and viral genomic DNA was prepared for analysis. Restriction digestion of genomic DNA and polymerase chain reaction products revealed the presence of several viruses within the population. One of the viruses was the MVA/CHOhr+ product
expected from precise, homologous recombination between MVA and the CPX CHOhr gene DNA fragment. However, some parental MVA, as well as CHOhr+ viruses from which various amounts of additional sequence had been deleted through illegitimate recombination of the CPX DNA fragment, were also detected. In conclusion, the results of these experiments showed that adding the CHOhr gene can extend the host range of MVA such that it includes CHO cells, at least. Plaque purification of MVA/CHOhr+, and the other CHOhr+ viruses, generated in the experiment will now be carried out so that the properties of individual virus isolates can be quantified and compared.
Impacts
The results of these studies are expected to provide important information for the further development of MVA, and related orthopoxviruses, as vaccine vectors for protection against smallpox and for other applications.
Publications
- No publications reported this period
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