The live vector approach—viruses

Recombinant DNA technology has made it possible to express foreign genes in viruses and bacteria. This has led to the idea of engineering live attenuated vaccines to express protective antigens from foreign pathogens. Vaccinia virus, the smallpox vaccine, has been used to ploneer this approach. Several hundred recombinants have been recorded in the literature and many of them have been shown to protect animals against challenge with the appropriate pathogen. Although mixed results have been obtained with experiments in humans and primates, and there is some concern over complications associated with vaccination, recent advances in our under-standing of the basic biology of vaccinia virus should allow these difficulties to be overcome. Whatever the final outcome of proposals to use vaccinia recombinants in humans, vaccinia has proved to be a valuable general purpose expression system and is particularly useful for studying cellular immunity. The success of field trials with a vaccinia recombinant expressing the rabies virus glycoprotein may lead to widespread use of vaccinia recombinants as animal vaccines. Therefore it seems likely that vaccinia recombinants will continue to play a useful part in the development of new vaccines for infectious disease.This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 24–31 August 1990.     

Vaccinia virus: a versatile tool for molecular biologists.

Continued advances in genetic engineering have made possible the high-level expression of correctly processed cellular, viral and bacterial polypeptides. This article focuses on viral expression vectors and, more specifically, the vaccinia virus expression system. Vaccinia virus has been used to express a variety of proteins with useful immunogenic, catalytic or pharmaceutical properties. We discuss briefly the biology of vaccinia and its significance in the use of vaccinia as an expression vector, the variety of vaccinia systems currently in use and, finally, we summarize some recent developments which bode well for future applications of vaccinia virus technology.     

Biologically active recombinant prothrombin and antithrombin III expressed in a human hepatoma/vaccinia virus system.

Several systems have been devised that are based on viral promoters suitable for gene expression in eukaryotic cells. One such system, vaccinia virus, has been successfully used to express a variety of heterologous proteins following the construction of a recombinant virus. Indeed, because of the ability of vaccinia virus to infect a wide range of host cells, the expression system can be custom-designed so that a cell line can be instrumental in ensuring the correct processing of the recombinant polypeptide. We show that recombinant vaccinia virus and human hepatoma cells in culture are an efficient expression system with which to produce correctly modified and biologically active human prothrombin (15 micrograms/10(7) cells) and antithrombin III (40 micrograms/10(7) cells).     

Vaccinia virus infection during murine pregnancy: a new pathogenesis model for vaccinia fetalis

Vaccinia fetalis, the vertical transfer of vaccinia virus from mother to fetus, is a relatively rare but often fatal complication of primary vaccinia virus vaccination during pregnancy. To date there has been no attempt to develop an animal model to study the pathogenesis of this acute viral infection in vivo. Here we report that infection of gestating BALB/c mice by either intravenous or intraperitoneal routes with the Western Reserve strain of vaccinia virus results in the rapid colonization of the placenta and vertical transfer of virus to the developing fetus. Systemic maternal infections during gestation lead to the death of all offspring prior to or very shortly after birth. Using in situ hybridization for vaccinia virus mRNA to identify infected cells, we show that the virus initially colonizes cells lining maternal lacunae within the trophospongium layer of the placenta. The study of this model will significantly enhance our understanding of the pathogenesis of fetal vaccinia virus infections and aid in the development of effective treatments designed to reduce the risk of vaccinia virus-associated complications during pregnancy.     

Vaccinia and other poxvirus expression vectors.

Over the past year improvements have been made in recombinant vaccinia virus gene expression and a new method for inserting DNA into the poxvirus genome has been developed, along with alternative methods for selecting recombinant viruses. Attenuated and non-replicating vaccinia virus and avian poxvirus vectors are now being used successfully. Field trials of an oral, wild-life rabies vaccine and phase 1 testing of human vaccines derived from vaccinia virus are underway.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Recombinant vaccinia virus vaccines

Vaccinia viruses have been genetically engineered to express foreign antigens. Immunization with these chimeric viruses protects experimental animals against challenge with the relevant infectious agent. These results, together with the successful history of vaccinia virus as an immunizing agent against smallpox, provide the impetus for employing live recombinant vaccinia viruses for the immunoprophylaxis of infectious diseases of both human and veterinary importance.     

Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins

The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.     

In vivo dose-response of insects to Hz-2V infection

Background

Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus.

Results

We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP), and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells.

Conclusions

When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines.     

A recombinant vaccinia virus containing the papilloma E2 protein promotes tumor regression by stimulating macrophage antibody-dependent cytotoxicity

Human papillomavirus infection is associated with cervical cancer. The E6 and E7 papillomavirus proteins are normally required for the maintenance of the malignant phenotype. Expression of these proteins in infected cells is negatively regulated by the binding of the papilloma E2 protein to the long terminal control region of the papilloma virus genome. The E2 protein can also promote cell arrest and apoptosis in HeLa cells. Therefore, it is clear that this protein has the potential of inhibiting the malignant phenotype. Because, anticancer vaccines based in vaccinia viruses have recently been shown to be an effective way to treat and to eradicate tumors, a recombinant vaccinia virus expressing the E2 gene of bovine papilloma virus (Modified Vaccinia Ankara, MVA E2) was created, to explore further the antitumor potential of the E2 protein. A series of rabbits, containing the VX2 transplantable papilloma carcinoma, were treated with MVA E2. An impressive tumor regression, up to a complete disappearance of tumor, was observed in most animals (80%). In contrast, very little or no regression was detected if the normal vaccinia virus was used. Lymphocytes isolated from MVA E2-treated rabbits did not show cytotoxic activity against tumor cells. However, in these animals a humoral immune response against tumor cells was observed. These antitumor antibodies were capable of activating macrophages to destroy tumor cells efficiently. These data indicate that injecting the MVA E2 recombinant vaccinia virus directly into the tumor results in a robust and long-lasting tumor regression. Data also suggest that antitumor antibodies are responsible, at least in part, for eliminating tumors by activating macrophage antibody-dependent cytotoxicity. Received: 23 November 1999 / Accepted: 12 April 2000     

Incomplete but Infectious Vaccinia Virions Are Produced in the Absence of Oncolysis in Feline SCCF1 Cells

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.     

IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress.

A novel method has been developed to study the functional roles of individual vaccinia virus gene products that is neither limited by the possible essentiality of the target gene nor by the availability of conditional lethal mutants. The system utilises the E. coli lac repressor protein, the operator sequence to which it binds and the specific inducer IPTG. It allows the generation of recombinant viruses in which the expression of any chosen gene, and hence virus replication, can be externally controlled. In principle, this system is broadly applicable to the functional analysis of genes in any large DNA virus. This approach has demonstrated that the gene encoding the 14 kDa membrane protein of vaccinia virus is non-essential for the production of infectious intracellular virus particles, but essential for the envelopment of intracellular virions by Golgi membrane and for egress of mature extracellular viral particles. This is the first vaccinia virus protein shown to be specifically required for these processes. In vivo this system may prove useful as a means of attenuating recombinant vaccinia virus vaccines by preventing virus spread without reducing the amount of the foreign antigen expressed in each infected cell. Attenuation of other live virus vaccines may be developed in a similar way.     

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.     

Therapeutic effect of a vaccinia colon oncolysate prepared with interleukin-2-gene encoded vaccinia virus studied in a syngeneic CC-36 murine colon hepatic metastasis model

Vaccinia CC-36 murine colon oncolysate (VCO) prepared with interleukin-2-gene encoded recombinant vaccinia virus (IL-2VCO) was used in the treatment of a syngeneic murine colon adenocarcinoma (CC-36) hepatic metastasis to test the beneficial effect of the interleukin-2-gene encoded vaccinia virus over a control recombinant vaccinia virus in producing a vaccinia oncolysate tumor cell vaccine. Results suggest that the IL-2VCO treatment significantly reduced the hepatic tumor burden in comparison with the controls that received either IL-2-gene-encoded recombinant vaccinia virus or a plain recombinant vaccinia virus or vaccinia oncolysate prepared with the plain recombinant virus. The survival of mice treated with IL-2VCO was also improved in comparison with mice treated with other preparations. The induction of a cytolytic T lymphocyte response was examined to elucidate the mechanism of the induction of antitumor responses in IL-2VCO-treated mice. Fresh peripheral blood lymphocytes (PBL) isolated from IL-2VCO-treated mice showed a higher cytolytic activity against CC-36 tumor cell target when compared to PBL from the mice of other treatment groups, suggesting that the IL-2VCO induced an antitumor cytolytic T lymphocyte response. These results suggest that a vaccinia oncolysate, prepared with recombinant vaccinia virus encoding an immunomodulating cytokine gene will enhance antitumor responses in the host.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses

Plasmid vectors containing multiple cloning sites suitable for the rapid insertion of protein-coding sequences into poxviruses have been constructed. They are based on pUC plasmids and carry the thymidine kinase (TK) gene of vaccinia virus interrupted by a vaccinia virus promoter. Six unique restriction enzyme sites (BamHI, SalI/HincII, PstI, HindIII, EcoRI), located within 40 bp of vaccinia virus promoters transposed from the HindIII-F or HindIII-C fragment of the vaccinia virus genome, allow rapid insertion of foreign-protein-coding sequences into these plasmids. Such plasmids can be used to construct recombinant poxviruses expressing foreign proteins using marker-rescue recombination techniques and selection for TK negative viruses. Vaccinia viruses expressing the haemagglutinin (HA) gene of swine influenza virus, A/NJ/11/76 (H1N1), have been constructed.     

The use of vaccinia virus for the construction of recombinant vaccines

Recombinant DNA technology has been used to engineer vaccinia virus genetically into a eukaryotic expression vector. An exciting outcome of these gene-splicing techniques is that after the insertion of one or more genes which encode the information for antigens responsible for conferring immunity toward an infectious disease, vaccinia can be adapted for the development of live recombinant vaccines. This review discusses recombinant vaccinia design and the feasibility of using these vaccines for disease protection.