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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

A genetically humanized mouse model for hepatitis C virus infection

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.     

Effect of immunization with a vaccinia-HIV env recombinant on HIV infection of chimpanzees

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV) and is now recognized as a worldwide epidemic for which there is no cure or vaccine. Chimpanzees are the only other animals that can be infected by HIV, and therefore the chimpanzee-HIV model system is useful for testing potential HIV vaccines. However, with one exception, there have been no reports of clinical manifestations of AIDS in chimpanzees. We report here results of an HIV vaccine trial in which nine chimpanzees were first immunized with either a recombinant vaccinia virus expressing the envelope glycoproteins of HIV strain LAV-1 (v-env5) or a control recombinant vaccinia virus and were then challenged with a high or low dose of LAV-1. Although HIV-specific antibody and T-cell responses were elicited by immunization, virus was isolated from lymphocytes of all challenged chimpanzees, indicating that immunization did not prevent infection by HIV. Among the animals that received a higher dose of LAV-1, one of two control chimpanzees, but none of the four v-env5-immunized chimpanzees developed substantial and persistent lymphadenopathy.     

TritonX-100裂解流感病毒的研究

 在流感病毒高浓度条件下,寻找TritonX-100裂解流感病毒的适宜条件.在不同条件下用TritonX-100裂解WHO推荐用2007～2008年度流感病毒疫苗毒株制备的3个亚型的流感病毒,用电镜观察裂解程度,通过蔗糖密度梯度离心纯化抗原后免疫动物,以检测疫苗的免疫效果.结果表明当流感病毒的血凝效价是1:16382,裂解时间为24h时,TritonX-100在质量分数为1%可以完全地裂解B型,质量分数达到2%才可以完全裂解流感病毒H1N1和H3N2,这说明TritonX-100对A,B亚型毒株的裂解效果不同.由于每年都要更换毒株工人生产新的流感疫苗,因此应该对使用TritonX-100的裂解不同亚型的流感病毒条件进行研究,以保证裂解疫苗的质量.     

Ⅰ型单纯疱疹病毒包膜糖蛋白L基因片段的克隆及高效表达

 采用PCR方法扩增HSV-1病毒型特异性包膜糖蛋白L(gL)基因片段并克隆至原核表达载体pGEX-5X-1获得重组质粒pGEX-5X-1-gL,将重组质粒转化E.coli BL21表达菌后经IPTG诱导表达目的蛋白.SDS-PAGE蛋白检测表明,在分子质量56 ku处有HSV-1 GST-gL融合蛋白的高效表达,通过IPTG浓度筛选和诱导前表达菌扩增培养时间的比较分析对诱导条件进行了优化,GST-gL融合蛋白表达量可达到菌体蛋白总量的48.65%.Western blot中利用HSV-1灭活病毒获得的多克隆抗体确证所表达蛋白为HSV-1病毒组分.这一表达系统的建立和优化对进一步探讨HSV-1 gL蛋白功能及其免疫原性提供了有利条件.     

单价流感疫苗脂质体干粉细胞免疫研究

从细胞免疫水平考察流感疫苗脂质体干粉肺部免疫的免疫原性,以验证在稳定性提高的同时,流感疫苗脂质体干粉肺部免疫原性不低于现行应用的流感疫苗原液腹腔注射免疫。将实验小鼠分为2个大组,每组分为阴性对照组、疫苗脂质体冻干粉组、非脂质体流感疫苗原液组和阳性对照组(n=5)。非脂质体流感疫苗原液组和疫苗脂质体冻干粉组分别以每只6μg血凝素(以H1N1计)肺部灌注免疫,同时以每只6μg非脂质体流感疫苗原液组腹腔免疫作为阳性对照。分别免疫14 d和28 d后,用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测脾淋巴细胞增殖情况,以考察其细胞免疫原性。脂质体肺部免疫可以诱导细胞免疫,且其免疫原性明显高于流感疫苗原液传统腹腔注射免疫组。与流感疫苗原液腹腔注射免疫相比,流感疫苗脂质体干粉通过肺部免疫,细胞免疫效果明显提高。     

Antigenic peptides recognized by T lymphocytes from AIDS viral envelope-immune humans

T-lymphocyte immunity is likely to be an important component of the immune defence against the AIDS virus, because helper T cells are necessary for the antibody response as well as the cytotoxic response. We have previously predicted two antigenic sites of the viral envelope protein gp120 likely to be recognized by T lymphocytes, based on their ability to fold as amphipathic helices, and have demonstrated that these are recognized by T cells of mice immunized with gp120 (ref. 1). A peptide corresponding to one of these sites can also be induce immunity in mice to the whole gp120 protein. Because many clinically healthy seropositive blood donors have already lost their T-cell proliferative response to specific antigen, we tested the response to these synthetic peptides of lymphocytes from 14 healthy human volunteers who had been immunized with a recombinant vaccinia virus containing the AIDS viral envelope gene and boosted with a recombinant fragment. Eight of the 14 responded to one peptide, and four to the other peptide, not included in the boost. These antigenic sites recognized by human T cells may be useful components of a vaccine against AIDS. We also found a correlation between boosting with antigen-antibody complexes (compared to free antigen) and higher stimulation indices, suggesting a more effective method of immunization.     

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.     

H5亚型禽流感病毒HA基因昆虫/杆状病毒系统的表达及对BALB/c小鼠的免疫保护

经RT-PCR扩增了禽流感病毒A/Goose/Guangdong/1／96 H5N1亚型1．7kb HA基因的cDNA,将其克隆到pMD18-T中并测序。亚克隆到杆状病毒转移载体pMelBacA的蜜蜂蜂毒素分泌信号下游中,测序正确后与线性化的杆状病毒DNA(Bac-N-BlueTM DNA)共转染Sf9昆虫细胞。将重组杆状病毒感染HFive细胞,72h左右收获细胞,超声波裂解,SDS—PAGE结果表明HA基因在重组杆状病毒感染的HFive细胞中获得表达。蛋白胶薄层扫描分析显示:表达的HA蛋白占重组杆状病毒感染细胞总蛋白含量的17．1％。Western-blot 及血凝实验结果显示,表达的禽流感H5N1亚型病毒HA蛋白具有生物学活性。表达的H5 HA蛋白定量乳化后,皮下多点注射免疫SPF 级BALB/c雌性小鼠,免疫后产生了H5 HA特异抗体,并在三免前后达到并保持较高水平。用致死剂量的HPAIV H5N1攻击小鼠,免疫组小鼠提供了100％的保护力,而对照组小鼠先后发病且死亡:为研制禽流感H5N1亚型病毒亚单位疫苗,防制禽流感奠定了基础。     

Humoral and cell-mediated immune responses to live recombinant BCG-HIV vaccines

Several viral and bacterial live recombinant vaccine vehicles are being developed to produce a new generation of vaccines against a broad spectrum of infectious diseases. The human tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) has features that make it a particularly attractive live recombinant vaccine vehicle. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly two billion immunizations, BCG has a long record of safe use in man. It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination. Recently developed molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG. Here we report that a variety of human immunodeficiency virus type 1 polypeptides can be expressed in BCG recombinants under the control of the mycobacterial hsp70 promoter and that the foreign polypeptides produced in BCG can induce antibody and T-cell responses. These results demonstrate that BCG can be used as a live recombinant vaccine vehicle to induce immune responses to pathogen proteins produced by the bacillus.     

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.     

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.