Replication-deficient Vaccinia Virus Research Articles

Protective MVA-ST Vaccination Robustly Activates T Cells and Antibodies in an Aged-Hamster Model for COVID-19.

Aging is associated with a decline in immune system functionality. So-called immunosenescence may impair the successful vaccination of elderly people. Thus, improved vaccination strategies also suitable for an aged immune system are required. Modified Vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus that has been established as a multipurpose viral vector for vaccine development against various infections. We characterized a recombinant MVA expressing a prefusion-stabilized version of SARS-CoV-2 S protein (MVA-ST) in an aged-hamster model for COVID-19. Intramuscular MVA-ST immunization resulted in protection from disease and severe lung pathology. Importantly, this protection was correlated with a potent activation of SARS-CoV-2 specific T-cells and neutralizing antibodies. Our results suggest that MVA vector vaccines merit further evaluation in preclinical models to contribute to future clinical development as candidate vaccines in elderly people to overcome the limitations of age-dependent immunosenescence.

Use of JYNNEOS (Smallpox and Monkeypox Vaccine, Live, Nonreplicating) for Preexposure Vaccination of Persons at Risk for Occupational Exposure to Orthopoxviruses: Recommendations of the Advisory Committee on Immunization Practices - United States, 2022.

Certain laboratorians and health care personnel can be exposed to orthopoxviruses through occupational activities. Because orthopoxvirus infections resulting from occupational exposures can be serious, the Advisory Committee on Immunization Practices (ACIP) has continued to recommend preexposure vaccination for these persons since 1980 (1), when smallpox was eradicated (2). In 2015, ACIP made recommendations for the use of ACAM2000, the only orthopoxvirus vaccine available in the United States at that time (3). During 2020-2021, ACIP considered evidence for use of JYNNEOS, a replication-deficient Vaccinia virus vaccine, as an alternative to ACAM2000. In November 2021, ACIP unanimously voted in favor of JYNNEOS as an alternative to ACAM2000 for primary vaccination and booster doses. With these recommendations for use of JYNNEOS, two vaccines (ACAM2000 and JYNNEOS) are now available and recommended for preexposure prophylaxis against orthopoxvirus infection among persons at risk for such exposures.

Modified Vaccinia virus Ankara: Innate immune activation and induction of cellular signalling

Attenuated poxviruses are currently under development as vaccine vectors against a number of diseases including, influenza, HIV, malaria and tuberculosis. Modified Vaccinia virus Ankara (MVA) is an attenuated, replication deficient vaccinia virus (VACV) strain which, similar to replication competent VACV, is highly immunogenic. The lack of productive viral replication further improves the safety profile of MVA as a vector, minimizing the potential for reversion to virulent forms particularly if used in immunocompromised individuals. Despite its inability to replicate in most mammalian cells, MVA still efficiently expresses viral and recombinant genes making it a potent antigen delivery platform. Moreover, due to the loss of various immunomodulatory factors MVA infection leads to rapid local immune responses, fulfilling a requirement of an adjuvant. In this review we take a look at the immunostimulatory properties of MVA, paying particular attention to the signalling of the innate immune system in response to MVA and VACV infection. Understanding the cellular and molecular mechanisms modulated by VACV will help in the future design and engineering of new vaccines and may provide insight into previously unknown mechanisms of dominant virus–host interactions.

Viral Double-stranded RNAs from Vaccinia Virus Early or Intermediate Gene Transcripts Possess PKR Activating Function, Resulting in NF-κB Activation, When the K1 Protein Is Absent or Mutated

PKR is a potent antiviral molecule that can terminate infection by inhibiting protein synthesis and stimulating NF-κB activation and apoptosis. Originally, it was thought that only intermediate and late gene transcription produced double-stranded (ds) RNA to activate PKR during vaccinia virus (VACV) infection. The VACV E3 or K3 proteins squelch this effect by binding to either dsRNA or PKR. However, in the absence of the K1 protein, VACV infection activates PKR at very early times post-infection and despite the presence of E3 and K3. These data suggest that VACV infection induces PKR activation by a currently unknown mechanism. To determine this mechanism, cells were infected with K1L-containing or -deficient VACVs. By using conditions that limited the progression of the poxvirus replication cycle, we observed that early gene transcripts activated PKR in RK13 cells, identifying a new PKR-activating mechanism of poxvirus infection. Using a similar approach for HeLa cells, intermediate gene transcription was sufficient to activate PKR. RNA isolated from infected RK13 or HeLa cells maintained PKR-activating properties only when dsRNA was present. Moreover, viral dsRNA was directly detected in infected cells either by RT-PCR or immunofluorescent microscopy. Interestingly, dsRNA levels were higher in infected cells in which the K1 protein was nonfunctional. Only K1 proteins with PKR inhibitory function prevented downstream NF-κB activation. These results reveal a new PKR activation pathway during VACV infection, in which the K1 protein reduces dsRNA levels early in VACV infection to directly inhibit PKR and several of its downstream antiviral effects, thereby enhancing virus survival.

Intradermal Delivery of Recombinant Vaccinia Virus Vector DIs Induces Gut‐Mucosal Immunity

Antigen-specific mucosal immunity is generally induced by the stimulation of inductive mucosal sites. In this study, we found that the replication-deficient vaccinia virus vector, DIs, generates antigen-specific mucosal immunity and systemic responses. Following intradermal injection of recombinant DIs expressing simian immunodeficiency virus gag (rDIsSIVgag), we observed increased levels of SIV p27-specific IgA and IgG antibodies in faecal extracts and plasma samples, and antibody-forming cells in the intestinal mucosa and spleen of C57BL/6 mice. Antibodies against p27 were not detected in nasal washes, saliva, and vaginal washes. The enhanced mucosal and systemic immunity persisted for 1 year of observation. Induction of Gag-specific IFN-gamma spot-forming CD8(+) T cells in the spleen, small intestinal intraepithelial lymphocytes, and submandibular lymph nodes was observed in the intradermally injected mice. Heat-inactivated rDIsSIVgag rarely induced antigen-specific humoral and T-helper immunity. Moreover, rDIsSIVgag was detected in MHC class II IA antigen-positive (IA(+)) cells at the injection site. Consequently, intradermal delivery of rDIs effectively induces antigen-specific humoral and cellular immunity in gut-mucosal tissues of mice. Our data suggest that intradermal injection of an rDIs vaccine may be useful against mucosally transmitted pathogens.

Preclinical Evaluation of the Immunogenicity of C-Type HIV-1-Based DNA and NYVAC Vaccines in the Balb/C Mouse Model

As part of a European initiative (EuroVacc), we report the design, construction, and immunogenicity of two HIV-1 vaccine candidates based on a clade C virus strain (CN54) representing the current major epidemic in Asia and parts of Africa. Open reading frames encoding an artificial 160-kDa GagPolNef (GPN) polyprotein and the external glycoprotein gp120 were fully RNA and codon optimized. A DNA vaccine (DNA-GPN and DNA-gp120, referred to as DNA-C), and a replication-deficient vaccinia virus encoding both reading frames (NYVAC-C), were assessed regarding immunogenicity in Balb/C mice. The intramuscular administration of both plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial T-cell responses against both antigens as well as Env-specific antibodies. Whereas low doses of NYVAC-C failed to induce specific CTL or antibodies, high doses generated cellular as well as humoral immune responses, but these did not reach the levels seen following DNA vaccination. The most potent immune responses were detectable using prime:boost protocols, regardless of whether DNA-C or NYVAC-C was used as the priming or boosting agent. These preclinical findings revealed the immunogenic response triggered by DNA-C and its enhancement by combining it with NYVAC-C, thus complementing the macaque preclinical and human phase I clinical studies of EuroVacc.

The Orthopoxvirus 68-Kilodalton Ankyrin-Like Protein Is Essential for DNA Replication and Complete Gene Expression of Modified Vaccinia Virus Ankara in Nonpermissive Human and Murine Cells

Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus (VACV) that is being evaluated as replacement smallpox vaccine and candidate viral vector. MVA lacks many genes associated with virulence and/or regulation of virus tropism. The 68-kDa ankyrin-like protein (68k-ank) is the only ankyrin repeat-containing protein that is encoded by the MVA genome and is highly conserved throughout the Orthopoxvirus genus. We showed previously that 68k-ank is composed of ankyrin repeats and an F-box-like domain and forms an SCF ubiquitin ligase complex together with the cellular proteins Skp1a and Cullin-1. We now report that 68k-ank (MVA open reading frame 186R) is an essential factor for completion of the MVA intracellular life cycle in nonpermissive human and murine cells. Infection of mouse NIH 3T3 and human HaCaT cells with MVA with a deletion of the 68k-ank gene (MVA-Delta68k-ank) was characterized by an extensive reduction of viral intermediate RNA and protein, as well as late transcripts and drastically impaired late protein synthesis. Furthermore, infections with MVA-Delta68k-ank failed to induce the host protein shutoff that is characteristic of VACV infections. Although we demonstrated that proteasome function in general is essential for the completion of the MVA molecular life cycle, we found that a mutant 68k-ank protein with a deletion of the F-box-like domain was able to fully complement the deficiency of MVA-Delta68k-ank to express all classes of viral genes. Thus, our data demonstrate that the 68k-ank protein contains another critical domain that may function independently of SCF ubiquitin ligase complex formation, suggesting multiple activities of this interesting regulatory protein.

Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection

The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.

Mucosal Administration of Completely Non‐Replicative Vaccinia Virus Recombinant Dairen I strain Elicits Effective Mucosal and Systemic Immunity

We studied the immunogenicity of completely replication-deficient vaccinia virus Dairen I strain recombinant encoding simian immunodeficiency virus (SIV) gag/pol (rDIs) in both mucosal and systemic compartments. When administered either intranasally or intragastrically, rDIs elicited enhanced levels of both SIV Gag p27-specific IgA antibodies and specific plasma antibodies, and the enhanced immunity persisted for the 1-year of observation by intranasal immunization. Increases were observed in antigen-specific IgA antibody-forming cells (AFC) in intestinal mucosal tissues and in IgG AFC in spleens. Furthermore, induction of type 1 and 2 helper cytokines in CD4+ spleen T cells and of CD8+ IFN-gamma spot-forming cells in mucosal tissues was observed in the intranasally immunized mice. Moreover, not even high-dose rDIs generated an SIV gene signal in the brain tissues of immunized mice. These findings suggest that mucosal immunization with the DIs recombinant hold promise as a safe mucosal vector.

Rapid protection in a monkeypox model by a single injection of a replication-deficient vaccinia virus

The success of the World Health Organization smallpox eradication program three decades ago resulted in termination of routine vaccination and consequent decline in population immunity. Despite concerns regarding the reintroduction of smallpox, there is little enthusiasm for large-scale redeployment of licensed live vaccinia virus vaccines because of medical contraindications and anticipated serious side effects. Therefore, highly attenuated strains such as modified vaccinia virus Ankara (MVA) are under evaluation in humans and animal models. Previous studies showed that priming and boosting with MVA provided protection for >2 years in a monkeypox virus challenge model. If variola virus were used as a biological weapon, however, the ability of a vaccine to quickly induce immunity would be essential. Here, we demonstrate more rapid immune responses after a single vaccination with MVA compared to the licensed Dryvax vaccine. To determine the kinetics of protection of the two vaccines, macaques were challenged intravenously with monkeypox virus at 4, 6, 10, and 30 days after immunization. At 6 or more days after vaccination with MVA or Dryvax, the monkeys were clinically protected (except for 1 of 16 animals vaccinated with MVA), although viral loads and number of skin lesions were generally higher in the MVA vaccinated group. With only 4 days between immunization and intravenous challenge, however, MVA still protected whereas Dryvax failed. Protection correlated with the more rapid immune response to MVA compared to Dryvax, which may be related to the higher dose of MVA that can be tolerated safely.

Induction of Positive Cellular and Humoral Immune Responses by a Prime-Boost Vaccine Encoded with Simian Immunodeficiency Virusgag/pol

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.

Priming-Boosting Vaccination with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin and a Nonreplicating Vaccinia Virus Recombinant Leads to Long-Lasting and Effective Immunity

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.

A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity.

To evaluate immunity induced by a novel DNA prime-boost regimen, we constructed a DNA plasmid encoding the gag and pol genes from simian immunodeficiency virus (SIV) (SIVgag/pol DNA), in addition to a replication-deficient vaccinia virus strain DIs recombinant expressing SIV gag and pol genes (rDIsSIVgag/pol). In mice, priming with SIVgag/pol DNA, followed by rDIsSIVgag/pol induced an SIV-specific lymphoproliferative response that was mediated by a CD4+-T-lymphocyte subset. Immunization with either vaccine alone was insufficient to induce high levels of proliferation or Th1 responses in the animals. The prime-boost regimen also induced SIV Gag-specific cellular responses based on gamma interferon secretion, as well as cytotoxic-T-lymphocyte responses. Thus, the regimen of DNA priming and recombinant DIs boosting induced Th1-type cell-mediated immunity, which was associated with resistance to viral challenge with wild-type vaccinia virus expressing SIVgag/pol, suggesting that this new regimen may hold promise as a safe and effective vaccine against human immunodeficiency virus type 1.

Cloning and expression of the complement receptor glycoprotein C from Herpesvirus simiae (herpes B virus): protection from complement-mediated cell lysis.

Simian herpes B virus (SHBV) is the herpes simplex virus (HSV) homologue for the species MACACA: Unlike in its natural host, and unlike other animal herpesviruses, SHBV causes high mortality in accidentally infected humans. SHBV-infected cells, like those infected with HSV-1 and equine herpesvirus types 1 and 4, express complement C3 receptor activity. To study immunoregulatory functions involved in susceptibility/resistance against interspecies transmission, the SHBV glycoprotein C (gC(SHBV)) gene (encoding 467 aa) was isolated. Sequence analysis revealed amino acid identity with gC proteins from HSV-2 (46.9 %), HSV-1 (44.5 %) and pseudorabies virus (21.2 %). Highly conserved cysteine residues were also noted. Similar to gC(HSV-2), gC(SHBV) is less glycosylated than gC(HSV-1), resulting in a molecular mass of 65 kDa if expressed in replication-deficient vaccinia virus Ankara. Stable transfectants expressing full-length gC(SHBV) on the cell surface induced C3 receptor activity and were substantially protected from complement-mediated lysis; no protection was observed with control constructs. This suggests that expression of the gC homologues on infected cell surfaces might also contribute to the survival of infected cells in addition to decreased virion inactivation. Interestingly, soluble gC(SHBV) isolated from protein-free culture supernatants did not interfere with the binding of the alternative complement pathway activator properdin to C3b, which is similar to our findings with gC(HSV-2) and could be attributed to major differences in the amino-terminal portion of the protein with extended deletions in both gC(SHBV) and gC(HSV-2). Binding of recombinant gC(SHBV) to polysulphates was observed. This, together with the heparin-sensitivity of the gC(SHBV)-C3 interaction on the infected cell surface, suggests a role in adherence to heparan sulphate, similar to the gC proteins of other herpesviruses.

A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs, both by themselves unable to complete the virus life cycle

The replication-deficient vaccinia virus (VV) MVA-T7 produces large amounts of T7 RNA polymerase and permits efficient protein expression from cDNA of T7-promoted genes. Yet, unlike recombinant VV vTF7-3, (VV) MVA-T7 produces no cytopathic effect in primate cells, thus allowing the study of processes with slow kinetics. We have applied MVA-T7 to aid genome expression of HAV, a representative of the Picornaviridae family that is well known for its inefficient replication in mammalian cell cultures. After cDNA transfection and MVA-T7 infection, empty capsids and mature HAV particles were formed with different kinetics and were characterized by their morphology, protein content, and infectivity. The data suggests that HAV genome replication is initiated from RNA, which was transcribed in vivo by the MVA-T7-encoded T7 RNA polymerase. HAV genome replication was also demonstrated in a recombination assay. After co-expression of two subgenomic HAV cDNAs, both by themselves unable to complete the viral life cycle, infectious HAV was rescued, indicating that replication-dependent genetic recombination has occurred. We propose that the high-level genome expression mediated in vivo by the VV-encoded T7 RNA polymerase augments the amount of viral RNA, such that replication of viruses poorly replicating in cell cytoplasm is detectable.

Comparison of Vaccine Strategies Using Recombinant env–gag–pol MVA with or without an Oligomeric Env Protein Boost in the SHIV Rhesus Macaque Model

Rhesus macaques were immunized with a replication-deficient vaccinia virus (MVA) expressing human immunodeficiency virus type 1 89.6 envelope (env) and SIV gagpol (MVA/SHIV89.6) with or without a protein boost consisting of soluble 89.6 env (gp140). Immunization with MVA/SHIV89.6 alone elicited binding antibodies in all animals and neutralizing antibodies in 5 of 15 animals. Both types of antibodies were enhanced by protein boosting. In addition, CD8 cells exhibiting CM9 tetramer binding were detected in the subset of animals that were Mamu-A*01 positive. Animals were challenged intravenously with either SHIV-89.6 (Study 1) or the more pathogenic derivative SHIV-89.6P (Study 2). In Study 1, all control and vaccinated animals except one became infected. However, the levels of viremia were as follows: controls > rMVA alone > rMVA + protein. The differences were statistically significant between immunized and control groups but not between the two immunized groups. In Study 2, all animals became infected; however, the vaccinated group exhibited a 5-fold reduction in peak viremia and a 10-fold reduction in the postacute phase viremia in comparison to the controls. All of the controls required euthanasia by 10 months after challenge. A relationship between vaccine-induced antibody titers and reduction in virus burden was observed in both studies. Thus, immunization with MVA/SHIV89.6 alone or with a protein boost stimulated both arms of the immune system and resulted in significant control of viremia and delayed progression to disease after challenge with SHIV-89.6P.

Replication-deficient vaccinia virus gene therpay vector: evaluation of exogenous gene expression mediated by PUV-inactivated virus in glioma cells.

Mild psoralen and UV (PUV) treatments inactivate viral DNA replication, but the virus retains its ability to infect cells. Thus, PUV treatment of vaccinia virus (VV) vectors may increase the safety of gene delivery and extend the duration of gene expression. Although the first studies on PUV-inactivated VV (PUV-VV) for the delivery of suicide or cytokine genes to cancer cells were promising, the efficiency and kinetics of exogenous gene expression have not been fully evaluated. Furthermore, these studies should be extended to other gene therapy strategies, e.g. tumor suppressor genes. We constructed VV recombinants carrying the luciferase (luc) gene, or the tumor suppressor p53 gene, to analyze exogenous gene expression after PUV treatment. Apoptosis induction and antitumor effects were examined in glioma cell culture and in an animal model, respectively. PUV-VV induced efficient PE/L-driven expression of luc and p53 exogenous genes in infected cells. A surprising prolonged p53 protein production was measured in glioma cells infected with PUV-VV expressing p53 (VV-TK-53) on Days 5-7 post-infection, reaching a maximal level of 9 microg/ml. VV-TK-53 induced apoptosis in 88% and 77.6% of infected C6 and 9L glioma cells, respectively. In contrast, 80% of cells infected with the PUV-inactivated control virus remained viable. Finally, ex vivo infection of C6 glioma cells with PUV-inactivated VV-TK-53 significantly reduced subsequent tumor growth in nude mice. Replication-deficient PUV-VV is safe and very efficient in prolonged foreign gene expression. Therefore PUV-VVs are recommended as vectors for applications in cancer gene therapy and recombinant vaccine development.

Mechanisms of Replication-Deficient Vaccinia Virus/T7 RNA Polymerase Hybrid Expression: Effect of T7 RNA Polymerase Levels and α-Amanitin

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995).Virology210, 202–205) stimulated high levels of expression from a T7 promoter–chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by α-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an α-amanitin-sensitive eukaryotic factor(s).