Mike Mackett

BIOLOGICAL CHARACTERIZATION OF RECOMBINANT VACCINIA VIRUSES IN MICE INFECTED BY THE RESPIRATORY ROUTE

A murine model based on infection by the respiratory route has been used to study the pathogenesis of recombinant vaccinia viruses. The neurovirulent Western Reserve (WR) strain and the Wyeth smallpox vaccine strain were used as vectors. Recombinant viruses were constructed by insertion of the Epstein-Barr virus membrane glycoprotein 340 gene into the thymidine kinase (TK) gene of each vaccinia virus. Intranasal inoculation of DBA/2 mice with 10(6) pock-forming units (pk.f.u.) of the WR strain was lethal but mice survived similar infection with the WR recombinant virus. Each virus was recovered from lung, blood and brain but, unlike wild-type virus, the recombinant virus was subsequently cleared. No deaths occurred after similar infection with the Wyeth strain or the Wyeth recombinant virus. There was limited growth of the Wyeth strain in the respiratory tract, low levels of virus in the blood and only sporadic recovery in brain extracts. The Wyeth recombinant virus was cleared rapidly with little viraemia or detectable infection of the central nervous system. No phenotypic character determined in vitro could be related consistently to the virulence of wild-type and recombinant viruses. Although the lethal character of the WR strain was affected by its TK+ phenotype, mice survived infection by intranasal inoculation with 10(6) pk.f.u. of WR TK+ recombinant viruses which either expressed the human interleukin 2 gene or had a deficient vaccinia virus growth factor gene.

Recombinant vaccinia virus induces neutralising antibodies in rabbits against Epstein-Barr virus membrane antigen gp340.

The Epstein‐Barr virus membrane antigen gene gp340 was isolated, inserted into several strains of vaccinia virus and expressed under the control of a vaccinia virus promoter. The EBV‐derived protein which was produced by the recombinant vaccinia viruses was heavily glycosylated, readily labelled with threonine, could be detected at the surface of infected cells and had a mol. wt. of approximately 340 kd, all of which are properties of the authentic gp340. Polyclonal rabbit antisera against gp340 and an EBV‐neutralising anti‐gp340 monoclonal antibody both recognised cells infected with the recombinant vaccinia viruses. Moreover, rabbits vaccinated with one of the recombinants produced antibodies that recognised EBV‐containing lymphoblastoid cells and neutralised EBV.

Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68

Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpesviruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, 9p150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus salmiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.

Protective immunization against Epstein-Barr virus-induced disease in cottontop tamarins using the virus envelope glycoprotein gp340 produced from a bovine papillomavirus expression vector

Inoculation with Epstein-Barr virus (EBV) induces malignant lymphomas in the cottontop tamarin (Saguinus oedipus oedipus). This provides an experimental animal model for assessing the efficacy of candidate EBV vaccines which are intended to reduce the incidence of human tumours associated with EBV infection. Previous work has shown that experimental vaccines based on the major virus envelope glycoprotein gp340 prepared from the membranes of EBV-infected cells are effective in protecting cottontop tamarins against EBV-induced disease. However, not all purified gp340 preparations induce protective immunity against EBV lymphoma in the tamarin. In this work, cottontop tamarins were immunized with recombinant gp340, produced using a bovine papillomavirus (BPV) expression vector, and a threonyl muramyl dipeptide adjuvant formulation. Although the recombinant-derived gp340 lacked the membrane anchor sequence of authentic gp340 and was expressed in mouse cells, it was immunogenic and induced virus-neutralizing antibodies. Healthy vaccinated tamarins were protected against EBV-induced disease. The demonstration that a recombinant gp340 product is able to elicit protective immunity in the cottontop tamarin is a significant step in the development of an EBV vaccine because previously it had not been clear whether a recombinant product would have the exact tertiary structure, including the necessary carbohydrate components, to induce protective immunity. A recombinant gp340 vaccine offers various advantages over production of the authentic molecule by laborious biochemical separation, including lower cost and the absence of potentially oncogenic EBV DNA. Therefore, recombinant gp340 produced using the BPV expression vector is suitable for development as a candidate EBV vaccine for a human Phase I trial and beyond.

Immunization of cottontop tamarins and rabbits with a candidate vaccine against the Epstein-Barr virus based on the major viral envelope glycoprotein gp340 and alum.

The Epstein-Barr virus (EBV) is associated with a range of life-threatening diseases in humans. Development of an effective vaccine has therefore been an important objective. One problem in the development of a subunit vaccine for human administration is the selection of a satisfactory adjuvant since the only one currently licensed for human use is alum, although this is not considered to be very effective. The present study demonstrated that a subunit vaccine composed of the EBV envelope glycoprotein gp340 with alum as the adjuvant did elicit protective immunity against EBV-induced lymphoma in three out of five cottontop tamarins. Furthermore, rabbits immunized with gp340/alum developed the same range of antibody responses as rabbits immunized with gp340/SAF-1, an experimental adjuvant claimed to be more effective than alum. Therefore, these results indicate that alum should be evaluated as an adjuvant as part of a human trial of a gp340-based subunit vaccine.

Herpesvirus papio encodes a functional homologue of the Epstein-Barr virus apoptosis suppressor, BHRF1

The human tumour virus Epstein-Barr virus (EBV) encodes a 17 kDa protein, BHRF1, which is a member of the BCL:-2 family and has been shown to suppress apoptosis. The role of this gene in the life-cycle of EBV has not been fully elucidated. In order to identify motifs conserved in herpesviruses and possibly shed light on its function we isolated a BHRF1 homologue from herpesvirus papio (cercopithecine herpesvirus-12) a closely related gammaherpesvirus of baboons. The gene, hvpBHRF1, also encodes a 17 kDa protein which shares 64% identity and 79% similarity with EBV BHRF1 at the amino acid level. In biological assays, hvpBHRF1 and BHRF1 conferred similar levels of protection on human keratinocytes induced to apoptose with cis-platin.

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV

Epstein‐Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life‐threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95‐8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton‐top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild‐type strains of EBV in the general population than does the standard B95‐8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95‐8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

Purification and characterization of Epstein-Barr virus gp340/220 produced by a bovine papillomavirus virus expression vector system

Our initial results with a bovine papilloma virus (BPV) vector expression system indicated that we could produce significant amounts of Epstein-Barr virus (EBV) gp340/220 in the supernatant of a mouse fibroblast cell line. We have now extended these findings to show that the truncated version of gp340/220, where the membrane anchor sequence is deleted, is produced even after extended passage of the cells, at a level of approximately 1 mg/4 x 10(8) cells. A simple purification protocol using Sephacryl S300HR and gelatin agarose gives a product which is greater than 90% pure. This product is recognized by anti-gp340 monoclonal antibodies from five different epitope groups and induces antibody that recognizes the authentic gp340/220 and neutralizes EBV in vitro. The purified gp340/220 can be used in ELISA and stimulates the proliferation of T-cell clones specific for gp340/220. These characteristics, together with the fact that BPV-transformed lines have been utilized for the production of pharmaceuticals for use in humans, suggest that this gp340/220 is suitable as a source of antigen for vaccination to prevent EBV infection and related diseases.