John R. Arrand

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.

Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments

A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned. Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned. Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA. Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells. In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322. The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases.

Sequence from early region of polyoma virus DNA containing viral replication origin and encoding small, middle and (part of) large T antigens

The sequence of about one third of the polyoma virus genome is presented. This sequence covers the origin of replication of two large plaque strains (A2 and A3) of polyoma virus. The two strains differ by 11 bp in the origin region. A model for replication is suggested. The sequence probably also covers the entire coding region of two of the polyoma virus early proteins--small and middle T antigens--as well as part of the coding region for large T antigen. Over a small region of the DNA, all three coding frames contain termination codons, which argues a need for spliced early messenger RNAs. In another region of the DNA, two coding frames can be used. Correlation with protein data suggests that one frame codes for part of middle T antigen and the other for part of large T antigen.

A new restriction endonuclease from Streptomyces albus G

A restriction endonuclease, SalI, has been partially purified from Streptomyces albus G. This enzyme cleaves adenovirus-2 DNA at three sites, bacteriophage λ DNA at two sites, but does not cleave simian virus 40 DNA or φX174 DNA. It recognizes the sequence and cuts at the sites indicated by the arrows. An endonuclease (XamI) with similar specificity has also been isolated from Xanthomonas amaranthicola.

Leaky Scanning Is the Predominant Mechanism for Translation of Human Papillomavirus Type 16 E7 Oncoprotein from E6/E7 Bicistronic mRNA

ABSTRACT Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5′ untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopusβ-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5′ end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5′ end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

The nucleotide sequences at the termini of adenovirus-2 DNA☆

The nucleotide sequence of the first 156 residues from the left end and the first 134 residues from the right end of adenovirus-2 DNA have been determined by direct DNA sequencing techniques. The inverted terminal repetition is 102 nucleotide pairs long. The 5′-ends of the intact DNA are resistant to the action of T4 polynucleotide kinase and the 5′ → 3′ exonucleases from phages lambda and T7. This resistance is most likely due to the covalent attachment of the 5′-terminal C residue to the terminal protein. No significant self-complementarity exists within the inverted terminal repetition, making terminal initiation of DNA replication via a self-priming mechanism unlikely. However, the terminal A + T-rich region followed immediately by a very G + C-rich region is consistent with other schemes for adenovirus-2 DNA replication. The left end of adenovirus-2 DNA contains extensive sequence repetition.

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.

Heterogeneity of HLA and EBER expression in Epstein-Barr virus-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an aggressive tumour of multifactorial aetiology that, although rare in most parts of the world, poses a significant mortality problem in its high incidence area of Southern China. Improved therapies are an urgent requirement and, towards this end, immunotherapeutic methods are being developed in several centres. Such strategies are dependent on the immune competence of the target tumour, in particular its expression of HLA class‐I. We examined HLA class‐I and ‐II expression in 27 primary NPC biopsies and found that 15% were extensively down‐regulated for class‐I expression with the majority of tumour cells appearing negative. Whilst HLA class‐II was expressed at high levels in the majority of tumours, 37% showed substantial down‐regulation. NPC is associated with Epstein‐Barr virus (EBV). Expression of the virus‐encoded EBER RNAs is accepted as a marker of EBV latency and is regarded as a valuable diagnostic criterion. EBER RNAs were expressed in all samples, but in some the level was remarkably heterogeneous, being barely detectable in many tumour cells. Our study reinforces the concept of extensive phenotypic variation in NPC. There are morphological differences between tumour cells. Some tumours express HLA class‐I and/or –II, whilst others are down‐regulated or negative. Individual tumours may or may not express the EBV‐encoded LMP‐1 protein, and individual tumour cells may express high levels of EBER, yet adjacent tumour cells express very little or none. Int. J. Cancer 88:949–955, 2000.

Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera

Existing assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens. These methods have had limited success in the detection of antibodies against the E6 protein. To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells. Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands. This protein reacted in Western blots with an antiserum raised against a purified E6 fusion protein produced in Escherichia coli. This antiserum, and several others raised against E. coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs). However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target. A RIPA was developed for the detection of anti-E6 protein antibodies in human sera. The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins. The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method. These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.

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.