Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells

The ability of vaccinia virus to accept and express cloned genes encoding immunologically important proteins of unrelated viruses and malarial parasites has suggested a novel approach to the development of live vaccines. Vaccinia virus recombinants retain infectivity and stimulate synthesis of specific antibodies to the cloned gene products in vaccinated animals. Moreover, animals inoculated with recombinants expressing the influenza virus haemagglutinin (HA), the hepatitis B virus surface antigen, and type 1 herpesvirus glycoprotein D were protected against subsequent challenge with the corresponding virus. For maximal effectiveness, vaccines should produce cellular as well as humoral immunity. We now report that a vaccinia virus recombinant, expressing the influenza HA, primes and stimulates a specific murine cytotoxic T-lymphocyte (CTL) response. Histocompatible cells infected with this recombinant also serve as targets for CTLs. These properties make vaccinia virus a unique tool for studying cell-mediated immunity and enhance the attractiveness of this vector for production of live vaccines.     

Recovery of immunodeficient mice from a vaccinia virus/IL-2 recombinant infection

Vaccinia virus recombinants that express cloned genes encoding antigens of unrelated infectious agents, such as hepatitis B virus and human immunodeficiency virus (HIV), provide a new approach to the development of live vaccines. Although there is evidence that genetically engineered vaccinia viruses have reduced pathogenicity a major obstacle to their use as vaccines is that severe complications can occur after vaccination, especially in immunodeficient individuals. We describe here a recombinant vaccinia virus expressing murine interleukin-2 (IL-2) and show that athymic nude mice infected with the recombinant virus resolve the virus infection rapidly whereas mice infected with control virus develop a progressive vaccinal disease. By incorporating the gene for IL-2 in live virus vaccines it may be possible to prevent the severe complications that arise in recipients with an impaired immune system.     

Tumour prevention and rejection with recombinant vaccinia

Tumour-specific antigens (TSA; ref. 1) have been exploited in the diagnosis and imaging of human cancer and anti-TSA antibodies have therapeutic potential. Vaccination with TSA or anti-idiotypic (TSA) antibodies has also been used to control tumour growth in model systems. An effective immune response nevertheless demands copresentation of antigen with host histocompatibility determinants. We therefore examined whether live vaccinia virus recombinants expressing TSA in cells of the vaccinated host might better elicit tumour immunity. Polyoma virus (PY) is tumorigenic in rodents; because killed PY-transformed cells can elicit tumour immunity, a PY-specific TSA has been postulated. Tumorigenesis involves expression of three early PY proteins, large-T (LT), middle-T (MT) and small-T (ST), but their role as TSAs is unclear. We therefore expressed the three T proteins in separate vaccinia recombinants. Rejection of PY tumours was observed in rats immunized with recombinants expressing either LT or MT. Further, tumour-bearing animals could be induced to reject their tumours by inoculation of recombinants.     

Large-scale eradication of rabies using recombinant vaccinia-rabies vaccine.

Rabies infection of domestic and wild animals is a serious problem throughout the world. The major disease vector in Europe is the red fox (Vulpes vulpes) and rabies control has focused on vaccinating and/or culling foxes. Culling has not been effective, and the distribution of five vaccine baits is the only appropriate method for the vaccination of wild foxes. Although some European countries have conducted field vaccination campaigns using attenuated rabies virus strains, their use has not been extensively approved because they retain pathogenicity for rodents and can revert to virulence. These strains cannot be used in North America because they are pathogenic for the striped skunk (Mephitis mephitis) and are ineffective in the raccoon (Procyon lotor). We have constructed a recombinant vaccinia virus, VVTGgRAB, expressing the surface glycoprotein (G) of rabies virus (ERA strain). The recombinant was a highly effective vaccine in experimental animals, in captive foxes and in raccoons. We report here the results of a large-scale campaign of fox vaccination in a 2,200 km2 region of southern Belgium, an area in which rabies is prevalent. After distribution, 81% of foxes inspected were positive for tetracycline, a biomarker included in the vaccine bait and, other than one rabid fox detected close to the periphery of the treated area, no case of rabies, either in foxes or in domestic livestock, has been reported in the area.     

Successful vaccination with a polyvalent live vector despite existing immunity to an expressed antigen

A global vaccination strategy must take into account production and delivery costs as well as efficacy and safety. A heat-stable, polyvalent vaccine that requires only one inoculation and induces a high level of humoral and cellular immunity against several diseases is therefore desirable. A new approach is to use live microorganisms such as mycobacteria, enteric bacteria, adenoviruses, herpesviruses and poxviruses as vaccine vectors. A potential limitation of live polyvalent vaccines, however, is existing immunity within the target population not only to the vector, but to any of the expressed antigens. This could restrict replication of the vector, curtail expression of antigens, and reduce the total immune response to the vaccine. Recently acquired immunity to vaccinia virus can severely limit the efficacy of a live recombinant vaccinia-based vaccine, so a strategy involving closely spaced inoculations with the same vector expressing different antigens may present difficulties. We have constructed a recombinant vaccinia virus that expresses surface proteins from two diverse pathogens, influenza A virus haemagglutinin and herpes simplex virus type 1 (HSV-1) glycoprotein D. Mice that had recently recovered from infection with either HSV-1 or influenza A virus could still be effectively immunized with the double recombinant.     

汉滩病毒囊膜糖蛋白g2基因重组腺病毒的构建与表达

获得汉滩病毒G2 基因 ,构建其重组腺病毒并在HEK2 93细胞中包装表达 ,为研究汉滩病毒基因疫苗提供了实验基础。设计引物采用PCR从含汉滩病毒 \|76 1 1 8株M基因的M5 6质粒扩增出糖蛋白G2 基因片段 ,并将其克隆入腺病毒载体Adeno XviralDNA ,筛选获得重组腺病毒DNA ,转染HEK2 93细胞 ,包装、扩增后得到汉滩病毒G2 基因重组腺病毒原种 ;并在感染细胞内初步表达 ,用ELISA检测表达产物。得到了含汉滩病毒G2 基因的重组腺病毒 ,其滴度约为 1 0 10 pfu/mL ,同时在感染的HEK2 93细胞中检测到汉滩病毒糖蛋白G2 的表达。含汉滩病毒糖蛋白G2 基因重组腺病毒的成功构建 ,为研究汉滩病毒基因疫苗提供了实验基础     

Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.     

Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor

Epidermal growth factor (EGF) and transforming growth factor type I (TGF) are polypeptides of 53 and 50 amino acid residues, respectively. Both bind to EGF receptor, a 1,200-residue transmembranous glycoprotein, leading to phosphorylation of the receptor, enhancement of its tyrosine-specific kinase activity and ultimately to stimulation of cell growth. We report here that a 140-residue polypeptide encoded by one of the early genes of vaccinia virus (VV) is related closely to EGF and TGF. The presence of putative signal and transmembranous sequences further suggests that the viral protein might be an integral membrane protein, but that, as in the case of EGF itself, the membrane-associated form may be the precursor of a soluble growth factor. Production of EGF-like growth factors by virally infected cells could account for the proliferative diseases associated with members of the poxvirus family such as Shope fibroma virus, Yaba tumour virus, and molluscum contagiosum virus (MCV).     

Virus-induced autoantibody response to a transgenic viral antigen

The induction of autoantibodies and their possible role in the pathogenesis of autoimmune disease are poorly understood. Involvement of infectious agents has been suspected, but direct evidence is sparse. Whether immunological unresponsiveness to self by antibody-forming B cells is maintained by clonal abortion, clonal anergy or suppression, or how the scenario of interactions between helper T cells, B cells and antigen-presenting cells is distorted in autoantibody responses, is being analysed and widely debated. To evaluate tolerance of neutralizing B-cell responses we used transgenic mice expressing the cell membrane associated glycoprotein (G) of vesicular stomatitis virus (VSV) as self-antigen. We show that autoantibodies to VSV-G cannot be induced by VSV-G in adjuvant or by recombinant vaccinia virus expressing VSV-G, but are triggered by infection with wild-type VSV. The data show that helper T-cell tolerance is crucial in maintenance of B-cell non-reactivity and that cognate T-B recognition is necessary to break tolerance of self-reactive B cells. These results may help to understand mechanisms of virus-induced autoimmunity.     

Cloning of a representative genomic library of the human X chromosome after sorting by flow cytometry

A library of 50,000 recombinants representative of the human X chromosome has been constructed. Human X chromosomes were physically separated using a fluorescence-activated cell sorter. The DNA was purified from the chromosomes, digested to completion with the restriction enzyme EcoRI and cloned into the phage lambda gtWES.lambda B. The X-derived nature of the recombinants was confirmed by hybridization to rodent/human cell line DNA containing only the human X chromosome. Such libraries will be particularly useful for the investigation of genetic diseases such as Duchenne muscular dystrophy, where the basic defect has not been elucidated, and of neoplasia, where several specific chromosomal anomalies, particularly for the leukaemias, have been identified.     

HPV16、18型E6/E7融合基因痘苗病毒表达载体的构建

目的:构建一种含人乳头瘤病毒(HPV)16型、18型E6/E7融合基因痘苗病毒表达载体.方法:PCR扩增HPV16型、18型E6、E7基因,克隆到pSC-A载体中,通过定点突变方法,分别构建成含E6/E7融合基因的质粒,即pSC-A.HPV16 E6/E7和pSC-A-HPV18 mE6/E7,并将二者连接成载体pSC-A-HPV16 E6/E7-HPV18mE6/E(pSC-T,HPV16 E6/E7-HPV18mE6/E7:T),最后以痘苗病毒表达载体pJ38为转移载体,构建质粒pJ38-HPV16 E6/E7-HPV18mE6/E7(pJ38-T),对所有重组质粒进行酶切鉴定和测序分析.结果:E6/E7融合基因成功克隆到pJ38上.结论:成功构建表达HPV E6/E7融合基因重组痘苗病毒载体,为研制宫颈癌治疗性疫苗奠定基础.     

onc sequences (v-fes) of Snyder-Theilen feline sarcoma virus are derived from noncontiguous regions of a cat cellular gene (c-fes)

Type C sarcoma viruses are genetic recombinants containing portions of replication-competent helper viruses linked to sarcoma virus-specific sequences (generically designated onc genes) which are thought to be required for acute fibroblast transformation. The onc elements of different avian and mammalian sarcoma viral isolates are each homologous to subsets of cellular DNA sequences which have no well-defined role in normal cells. Because of the lack of significant homology between helper viral genes and cellular onc sequences, the recombinational mechanisms which facilitate the formation of sarcoma viral genomes remain unclear. In Moloney murine sarcoma virus, viral onc (or v-mos) and cellular onc (or c-mos) sequences exhibit complete and uninterrupted homology as determined by heteroduplex and restriction enzyme analyses of molecularly cloned DNA. By contrast, the cellular counterparts of the onc elements of Rous sarcoma virus (G. Cooper and R. Parker, personal communication), avian erythroblastosis virus (B. Vennstrom, personal communication), Abelson leukaemia virus (D. Baltimore, personal communication), Harvey sarcoma virus (E. Scolnick, personal communication) and simian sarcoma virus (R. Gallo, personal communication) are now known to contain intervening sequences which do not appear in the respective viral genomes. Here we report the use of the Southern blot technique to examine cat cellular DNA sequences (c-fes) homologous to the onc gene (v-fes) of Snyder-Theilen feline sarcoma virus (ST-FeSV). We used cloned DNA 'probes' containing defined portions of the ST-FeSV genome to show that v-fes sequences originate from at least four noncontiguous sequences in cat cellular DNA, separated from each other by intervening sequences.     

Detecting putative recombination events of hepatitis B virus: An updated comparative genome analysis

An updated collection of 791 human hepatitis B virus (HBV) genomes and 38 non-human primate HBV genomes was analyzed for identifying putative recombination events and their recombinants by using two bioinformatics software tools: Simplot and RDP3 with five algorithms (RDP, GENECONV, MaxChi, Chimaera, and SiScan). A total of 61 recombinants from nine putative recombination events were detected with RDP3, especially the breakpoints of six events which have both two parental sequences that can be determined precisely with Simplot. To our knowledge, 53 recombinants were found for the first time. Our study also suggests that a relatively high recombination frequency occurs in the PreC/C gene region and the position near gene boundaries.     

Similarity between the vaccinia virus 19K early protein and epidermal growth factor

An analysis of the 1,217-amino acid residue sequence of the precursor of mouse epidermal growth factor (mEGF) revealed regions of considerable similarity with bovine factor X, a blood coagulation factor. Similarities of mEGF itself with factor X, pancreatic secretory trypsin inhibitor and, most strikingly, transforming growth factor I (TGF-I) have been observed. On the basis of the comparisons described here, it seems that the presumptive 140-residue 19K early protein (relative molecular mass (Mr) 19,000) of vaccinia virus from residues 40-91 shows an overall identity of 36% (19/53 residues) with both mEGF and urogastrone (human epidermal growth factor, hEGF); a single deletion is assumed for vaccinia virus 19K protein which allows the six Cys residues (positions 45-80) to be aligned with those of mEGF or hEGF. This protein is encoded in the 10.3-kilobase (kb) inverted terminal repeat. Because it is an early protein with an EGF-like central portion, the 19K vaccinia virus protein may have an autocrine function and may be required for DNA synthesis.     

Prevention of vaccinia virus infection in immunodeficient mice by vector-directed IL-2 expression

Recombinant vaccinia viruses have been proposed as live vaccines against a variety of infectious diseases, including AIDS (acquired immune deficiency syndrome). Objections have been concerned primarily with side effects of the vaccinia virus vector itself. Recently it has been shown that inactivation of the vaccinia virus thymidine kinase gene or deletion of certain other non-essential genes is associated with a marked reduction in pathogenicity. Nevertheless, the ability of vaccinia virus to produce a progressive infection in immunodeficient individuals remains a most serious problem. Indeed, an incident of this type in a vaccinated man seropositive for human immunodeficiency virus was recently reported. We have used immunodeficient athymic nude mice to establish a model of disseminated vaccinia virus infection, and to demonstrate a novel approach to virus attenuation which involves insertion of a gene encoding human interleukin-2 into the genome of vaccinia virus vectors.     

A novel selective broad-spectrum anti-DNA virus agent

A new compound has been found, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine ((S)-HPMPA), that has potent and selective activity against a broad spectrum of DNA viruses, including herpes simplex virus (types 1 and 2); varicella zoster virus; thymidine kinase-deficient (TK-) mutants of herpes simplex and varicella zoster virus; human cytomegalovirus; phocid, simian, suid, bovid and equid herpesviruses; African swine fever virus; vaccinia virus; and human adenoviruses. It is also active against retroviruses. We also report that, in mice and rabbits in vivo, the compound is effective against both local and systemic infections with herpes simplex virus type 1, including herpetic keratitis caused by a TK- mutant which is resistant to the classical anti-herpes drugs.     

AcNPV增强子hr5增强HBsAg基因表达的研究

用形成包涵体（ＯＯＣ＋）并能利用人工合成启动序列和多角体ＸＩＶ启动子表达外源基因的转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３将多角体基因、乙型肝炎病毒表面抗原（ＨＢＳＡｇ）基因和苜蓿丫纹夜蛾核型多角体病毒（ＡｃＮＰＶ）的增强子ｈｒ５部分序列同时插入无包涵体的粉纹夜蛾核型多角体病毒ＴｎＮＰＶ－ＳＶＩ－Ｇ基因组中，得到两株高效表达ＨＢｓＡｇ基因又形成包涵体的重组病毒ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋．对重组病毒的酶切鉴定、ＤＮＡ斑点杂交和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证实，外源基因及其相应的启动子和增强子序列已正确插入病毒基因组中．插入顺序中，ｈｒ５增强子是插入ＨＢｓＡｇ基因下游，多角体基因与ＨＢｓＡｇ基因方向相反．１２５Ⅰ－固相放射免疫检测和Ｗｅｓｔｅｒｎｂｌｏｔ结果表明，ＨＢｓＡｇ基因在昆虫离体细胞中得到高效表达并保留了抗原活性．ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋表达的ＨＢｓＡ吕蛋白与没有插入增强子序列的重组病毒ＴｎＮＰＶ—ＨＢｓ８５－ＯＣＣ＋的比较，分别提高了４０％和４６％．     

肿瘤基因治疗载体的研究进展

对在肿瘤基因治疗中较常见的载体--病毒载体(痘苗病毒载体、腺病毒与腺病毒相关载体、单纯疱疹病毒和逆转录病毒)和非病毒载体(脂质体和稳定质粒--脂质颗粒)进行了介绍,同时对其存在的问题作了简要阐述,并对该研究前景进行了展望.     

Genetic retrieval of viral genome sequences from herpes simplex virus transformed cells

Oncogenic transformation of cultured cells by inactivated herpes simplex virus (HSV) types 1 and 2 has been demonstrated. Expression of HSV information in these transformed cells has been shown by immunofluorescence studies, detection of HSV neutralizing antibody in sera from tumour-bearing animals and by hybridization of HSV-specific RNA. Molecular hybridization studies of DNA from HSV-2 transformed hamster cells have detected up to 40% of the HSV genome present in several copies. Complementation of three HSV-2 temperature-sensitive mutants when superinfecting the RE1 rat embryo cell line (transformed by the HSV-2 temperature-sensitive mutant ts1) suggests that resident viral genes can be expressed. Brown et al. used a similar approach to detect HSV information latent in human ganglia. We report here retrieval of intertypic HSV recombinants from HSV transformed cells after superinfection with ts mutants of the alternative serotype of HSV. Restriction enzyme analysis which clearly differentiates between HSV-1 and HSV-2 DNA has demonstrated the isolation of recombinants spanning the genome and of virus indistinguishable from the original transforming virus.