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.     

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.     

Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype

Recent advances in molecular genetics have led to the possibility of using large DNA viruses, such as vaccinia virus, as a biological delivery system for immunizing man against unrelated disease-causing agents. When live vaccinia virus recombinants expressing the hepatitis B virus surface antigen (HBsAg), the influenza A virus haemagglutinin, the herpes simplex virus (HSV) type 1 D glycoprotein, the rabies virus G glycoprotein and the vesicular stomatitis virus G glycoprotein were used for immunization, animals were protected upon challenge with the appropriate pathogenic agent. A major concern with using such vaccines, however, stems from the previously documented vaccinia virus-associated post-immunizing complications. We present here experimental evidence that thymidine kinase-negative (TK-) vaccinia virus recombinants, constructed by inserting a variety of DNA coding sequences into the vaccinia virus tk gene, are less pathogenic for mice than wild-type virus.     

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.     

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.     

Identification of nuclear proteins encoded by viral and cellular myc oncogenes

The myelocytomatosis viruses are a family of replication-defective avian retroviruses that cause a variety of tumours in chickens and transform both fibroblasts and macrophages in culture through the activity of their oncogene v-myc. A closely related gene (c-myc) is found in vertebrate animals and is thought to be the progenitor of v-myc. Changes in the expression and perhaps the structure of c-myc have been implicated in the genesis of avian, murine and human tumours (for a review, see ref. 15). Elucidation of the mechanisms by which v-myc and c-myc might elicit tumorigenesis requires identification of the proteins encoded by these genes. To this end, we have expressed a portion of v-myc in a bacterial host and used the resulting protein to raise antisera that react with myc proteins. We report here that v-myc and c-myc encode closely related proteins with molecular weights (MWs) of approximately 58,000. Integration of retroviral DNA near or within c-myc in avian lymphomas apparently enhances expression of the gene. Here we have used cells from one such tumour to identify the protein encoded by c-myc and find that the coding domain for the gene is probably intact.     

Expression of the HTLV-III envelope gene by a recombinant vaccinia virus

The discovery that the aetiological agent of acquired immune deficiency syndrome (AIDS) is a retrovirus, referred to as human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) (for review see ref. 1), has raised the possibility of developing a vaccine. In this regard, the envelope (env) proteins of murine retroviruses can induce protective immunity in mice. The HTLV-III env gene specifies a primary polypeptide of approximately 860 amino acids that is glycosylated to form a precursor of relative molecular mass (Mr) 160,000 (gp160), which gives rise to mature membrane-associated proteins of Mr 120,000 (gp120) and 41,000 (gp41). The HTLV-III env gene has been expressed in Escherichia coli and by simian virus 40 (SV40) vectors but formation of the authentic proteins has not been demonstrated. Here, we describe the expression of the complete env gene by a vaccinia virus vector. Evidence is presented that synthesis, glycosylation, processing and membrane transport of the env polypeptide occurred without other HTLV-III gene functions; the env protein was recognized by sera from unrelated AIDs patients; and a single vaccination with the infectious recombinant vaccinia virus induced antibodies to gp120 in mice.     

Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum

Malaria is initiated by the inoculation of a susceptible host with sporozoites from an infected mosquito. The sporozoites enter hepatocytes and develop for a period as exoerythrocyte or hepatic stage parasites. Vaccination with irradiated sporozoites can provide protective immunity and a recent study shows that this can also be conferred by immunization with a recombinant salmonella expressing only the circumsporozoite protein that normally covers the sporozoites. Protection against infection is likely to be mediated by cytotoxic CD8+ cells, as depletion of CD8+ T cells in a sporozoite-immunized animal can completely abrogate immunity. Here we demonstrate directly the existence of CD8+ cytotoxic T lymphocytes (CTL) that recognize the circumsporozoite protein. B10.BR mice immunized with sporozoites or with recombinant vaccinia virus expressing the CS protein of Plasmodium falciparum contain CTL that specifically kill L cell fibroblasts transfected with the gene encoding the same CS protein. The peptide epitope from the CS protein that is recognized by CTL from this strain of mice is from a variant region of the protein.     

T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses

There is much interest in developing vaccines against acquired immune deficiency syndrome (AIDS), which is caused by a retrovirus termed human immunodeficiency virus (HIV). Isolates of this virus include human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Several approaches towards the development of an AIDS vaccine result in the production of antibodies in subprimates. These methods involve the use of: antigens isolated from the AIDS virus; viral antigens expressed by transfected cells or by recombinant vaccinia viruses; and particular synthetic peptides of viral antigens. Because T-cell-mediated immunity (in addition to antibodies) is involved in resistance to diseases and death caused by various enveloped viruses, we sought to determine whether potential AIDS vaccines can induce T-cell responses against the AIDS virus. Here we report that immunization of non-human primates, Macaca fascicularis (macaques), with recombinant vaccinia viruses that express LAV envelope glycoproteins gp41 and gp110 results not only in the production of antibodies against the LAV envelope antigens but also in the generation of T-cells that proliferate and produce the lymphokine interleukin-2 (IL-2), in response to stimulation with purified LAV. We believe this is the first report demonstrating T-cell-mediated immunity to the virus that causes AIDS.     

Immunization with a synthetic T-cell receptor V-region peptide protects against experimental autoimmune encephalomyelitis

T cells expressing the alpha beta T-cell receptor (TCR) for antigen can elicit anti-idiotypic antibodies specific for the TCR that regulate T-cell function. Defined sequences of the TCR, however, have not been used to elicit specific antibodies and the role of cellular immunity directed against TCR determinants has not been studied. We immunized Lewis rats with a synthetic peptide representing a hypervariable region of the TCR V beta 8 molecule. Subsequent induction of experimental autoimmune encephalomyelitis, a paralytic disease of the central nervous system mediated primarily by V beta 8+ T cells specific for myelin basic protein was prevented. T cells specific for the TCR V beta 8 peptide conferred passive protection against the disease to naive rats, apparently by shifting the predominant T-cell response away from the major encephalitogenic epitope of basic protein. This is the first report demonstrating the use of a synthetic TCR V-region peptide to induce specific regulatory immunity and has important implications for the regulation of human disease characterized by common TCR V-gene usage.     

Drosophila RNAi screen identifies host genes important for influenza virus replication

All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.     

重组痘苗病毒对不同哺乳动物细胞株的感染效率及EGFP表达水平研究

研究重组痘苗病毒对不同哺乳动物细胞的感染效率及表达水平，可为痘苗病毒表达系统宿主细胞的正确选择提供依据．本研究利用重组绿色荧光蛋白基因的痘苗病毒WR—EGFP同时感染不同的哺乳动物细胞株，利用流式细胞仪检测EGFP的表达强度．共使用20种哺乳动物细胞株，其中10种人类组织细胞，2种猴组织细胞。8种小鼠组织细胞．结果表明，重组痘苗病毒wR-EGFP对鼠细胞系BHK21和人细胞系A-549的感染效率和表达效率最佳；整体看，痘苗病毒对多数灵长类动物细胞的感染效率和表达效率优于鼠细胞；对贴壁细胞的感染效率和表达效率明显优于悬浮细胞；但没有特别的组织偏嗜性。     

A group specific anamnestic immune reaction against HIV-1 induced by a candidate vaccine against AIDS

The first experimental immunization of humans against the AIDS retrovirus, HIV-1, was started in a series of HIV seronegative, healthy volunteers in November 1986. For the primary vaccination recombinant vaccinia virus (V25) expressing the complete gp160 env protein of the HTLV-IIIB strain of HIV-1 was introduced by scarification. This elicited a weak primary response which we subsequently attempted to enhance by additional immunizations (boosting), using four different immunization protocols. We report here that intravenous injection of paraformaldehyde-fixed autologous cells infected in vitro with V25 (individual D.Z.) gave the best results. This individual received second and third boosts of intramuscular gp160 derived from an HTLV-IIIB clone using the hybrid vaccinia virus/bacteriophage T7 expression system. An anamnestic humoral and cellular immune reaction was achieved for over one year after the original vaccination, with high levels of antibodies to the viral envelope, and neutralizing antibodies against divergent HIV-1 strains such as HTLV-IIIB and HTLV-IIIRF (also called HTLV-III HAT) after the first boost. In addition, group-specific cell-mediated immunity and cell-mediated cytotoxicity against infected T4 cells were obtained after the primary vaccine and enhanced by the boosts. Finally, skin tests showed both immediate and delayed hypersensitivity to gp160 in vivo. Although this protocol is not practical for a large scale vaccine trial, our results show for the first time that an immune state against HIV can be obtained in man.     

Protection of cottontop tamarins against Epstein-Barr virus-induced malignant lymphoma by a prototype subunit vaccine

Epstein-Barr (EB) virus is one of the five herpesviruses of man. Strong links between this agent and the chain of events causing two human cancers, endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, have long been evident (reviewed in ref. 1). Because of this, and because of the very high incidence of nasopharyngeal carcinoma in certain large populations, it was suggested in 1976 that a vaccine should be developed against EB virus to prevent infection and thereby reduce tumour incidence amongst those at risk. The virus-determined membrane antigen (MA) was proposed as immunogen because it was known to elicit naturally occurring virus-neutralizing antibodies in man and because analogous antigens had been shown to act as effective experimental vaccines for preventing the herpesvirus-induced lymphomas of Marek's disease in chickens. Progress has been achieved in defining, quantifying and preparing MA molecules, and in enhancing their immunogenicity; a sensitive assay for antibodies to MA has been elaborated. Here we report that isolated cell membranes expressing MA, or purified MA glycoprotein of relative molecular mass (Mr) 340,000 (gp340), have been used to vaccinate cottontop tamarins (Saguinus oedipus oedipus), and that animals receiving either preparation were protected against the effects of a 100% tumour-inducing challenge dose of EB virus.     

Deactivation of macrophages by transforming growth factor-beta

Macrophage activation--enhanced capacity to kill, in a cell that otherwise mostly scavenges--is essential for host survival from infection and contributes to containment of tumours. Both microbes and tumour cells, therefore, may be under pressure to inhibit or reverse the activation of macrophages. This reasoning led to the demonstration of macrophage deactivating factors from both microbes and tumour cells. In some circumstances the host itself probably requires the ability to deactivate macrophages. Macrophages are essential to the healing of wounds and repair of tissues damaged by inflammation. Yet the cytotoxic products of the activated macrophages can damage endothelium, fibroblasts, smooth muscle and parenchymal cells (reviewed in ref. 6). Thus, after an inflammatory site has been sterilized, the impact of macrophage activation on the host might shift from benefit to detriment. These concepts led us to search for macrophage deactivating effects among polypeptide growth factors that regulate angiogenesis, fibrogenesis and other aspects of tissue repair. Among 11 such factors, two proteins that are 71% similar proved to be potent macrophage deactivators: these are transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2.     

Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin

Anthrax poses a clear and present danger as an agent of biological terrorism. Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death. Anthrax lethal toxin (LT) is a critical virulence factor of B. anthracis, which occurs as a complex of protective antigen and lethal factor. Here we demonstrate that LT severely impairs the function of dendritic cells--which are pivotal to the establishment of immunity against pathogens--and host immune responses by disrupting the mitogen-activated protein (MAP) kinase intracellular signalling network. Dendritic cells exposed to LT and then stimulated with lipopolysaccharide do not upregulate co-stimulatory molecules, secrete greatly diminished amounts of proinflammatory cytokines, and do not effectively stimulate antigen-specific T cells in vivo. Furthermore, injections of LT induce a profound impairment of antigen-specific T- and B-cell immunity. These data suggest a role for LT in suppressing host immunity during B. anthracis infections, and represent an immune evasion strategy, where a microbe targets MAP kinases in dendritic cells to disarm the immune response.     

Bv8 regulates myeloid-cell-dependent tumour angiogenesis

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.     

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.     

Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen

The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting alpha-helices to expand/constrict the channel, producing an 'iris' effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.