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.     

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.     

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.     

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.     

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.     

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.     

Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus

The 'Spanish' influenza pandemic of 1918-19 was the most devastating outbreak of infectious disease in recorded history. At least 20 million people died from their illness, which was characterized by an unusually severe and rapid clinical course. The complete sequencing of several genes of the 1918 influenza virus has made it possible to study the functions of the proteins encoded by these genes in viruses generated by reverse genetics, a technique that permits the generation of infectious viruses entirely from cloned complementary DNA. Thus, to identify properties of the 1918 pandemic influenza A strain that might be related to its extraordinary virulence, viruses were produced containing the viral haemagglutinin (HA) and neuraminidase (NA) genes of the 1918 strain. The HA of this strain supports the pathogenicity of a mouse-adapted virus in this animal. Here we demonstrate that the HA of the 1918 virus confers enhanced pathogenicity in mice to recent human viruses that are otherwise non-pathogenic in this host. Moreover, these highly virulent recombinant viruses expressing the 1918 viral HA could infect the entire lung and induce high levels of macrophage-derived chemokines and cytokines, which resulted in infiltration of inflammatory cells and severe haemorrhage, hallmarks of the illness produced during the original pandemic.     

Recombinant vaccinia viruses as vaccines

The prospect of live vaccines consisting of genetically modified vaccinia virus expressing foreign genes is exciting, but important issues concerning safety and efficacy need to be resolved.     

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.     

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。     

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.     

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.     

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.     

病毒载体疫苗研究进展

从非复制性病毒（家禽痘、野鸟痘、痘苗和人类腺病毒）载体重组活疫苗、腺病毒载体重组口服活疫苗和杆状病毒载体重组亚单位疫苗三方面，评述了以病毒为表达载体的基因工程疫苗近年研究取得的重大进展。     

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.     

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.     

Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160

The development of a vaccine to provide protective immunity to human immunodeficiency virus type 1 (HIV-1), the virus causing AIDS, would be the most practical method to control its spread. Subunit vaccines consisting of virus envelope glycoproteins, produced by recombinant DNA technology, are effective in preventing viral infections. We have now used this approach in the development of a candidate AIDS vaccine. Chimpanzees were immunized with recombinant forms of the HIV-1 glycoproteins gp120 and gp160 produced in Chinese hamster ovary cells, and then challenged with HIV-1. The control and the two animals immunized with the gp160 variant became infected within 7 weeks of challenge. The two animals immunized with the gp120 variant have shown no signs of infection after more than 6 months. These studies demonstrate that recombinant gp120, formulated in an adjuvant approved for human use, can elicit protective immunity against a homologous strain of HIV-1.     

Evidence for host-cell selection of influenza virus antigenic variants

Extensive antigenic variability and a capricious epidemiology are characteristics of influenza A and B viruses of man. The haemagglutinin (HA) undergoes frequent and progressive antigenic drift as a result of selection, under immunological pressure, of viruses possessing alterations in the amino acid sequences at specific sites in the molecule. Here we present evidence for an additional selection mechanism for antigenic variants of influenza virus that depends on differing host cell tropisms of virus subpopulations. These studies were initiated after earlier observations of the occurrence of a marked degree of antigenic variation during passage of laboratory strains of influenza virus in eggs and cell cultures (J.C.J., in preparation). We have now shown that cultivation of influenza B viruses in eggs selects subpopulations which are antigenically distinct from virus from the same source grown in mammalian cell cultures. As antigenic characterization of influenza virus strains for epidemiological purposes and for the preparation of influenza vaccines conventionally relies on the cultivation of virus in eggs, our findings may have important practical implications for vaccine design and efficacy.     

Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs

To reduce the risks of immunization with killed or live attenuated virus vaccines, it may be advantageous to use a pure, defined antigen that contains determinants for both humoral and cellular immunity. However, although most non-living intact protein preparations induce antibodies and CD4+ major histocompatibility complex (MHC) class II-restricted helper and/or cytotoxic T lymphocytes (CTL), they do not elicit CD8+ MHC class I restricted CTL. Indeed, with a few exceptions, it has not so far been possible to induce CD8+ CTL by immunizing with intact soluble proteins. We show here that a single subcutaneous immunization in mice with immunostimulating complexes containing either purified intact gp160 envelope glycoprotein of the human immunodeficiency virus (HIV)-1 or influenza haemagglutinin results in reproducible and long-lasting priming of HIV specific or influenza-specific CD8+, MHC class I restricted CTL.