Vaccinia Genome Research Articles

Abstract B101: Novel oncolytic vaccinia virus platform for systemic delivery of immunotherapeutic payloads

Abstract The treatment paradigm for patients with metastatic cancer has evolved rapidly with the approval of agents targeting CTLA-4 and the PD-1/L1 immune checkpoint axis. Despite the profound impact these agents have had, they are minimally effective in the majority of cancer patients. Rational combinations of complementary immune modulating agents have thus far not led to clear patient benefit, and newer technologies that are better able to safely combine multiple modes of action could well prove to be vital. Oncolytic viruses (OVs) have the capacity to be the ideal therapeutic partner for immune checkpoint therapeutics in several ways. First, on their own OVs can “heat-up” immunologically “cold” tumors by initiating a pro-inflammatory infection within the tumor microenvironment (TME). Second, some OVs can be engineered to strategically express one or more immune-modulating molecules. Finally, certain OVs have the capacity to be delivered systemically and thus enhance immune cell recruitment and activation in all metastatic sites. We have selected a novel vaccinia virus as our therapeutic OV platform and are using it to engineer multi-mechanistic cancer therapeutics. Previously it has been demonstrated that certain oncolytic vaccinia viruses can be delivered systemically and spread within metastatic lesions. These early clinical viruses, however, contain multiple potent immune suppressive genes and are not ideal for the generation of antitumor immune responses “in situ.” Furthermore, in clinical studies some of these therapeutics exhibited off-tumor infections (e.g., pox lesions), which may ultimately limit their ability to be used to deliver potent immune modulators. We used a combination of functional genomics and bio-selection strategies to optimize the vaccinia virus platform. Initially we developed a fitness assay to identify the vaccinia strain with the best ability to replicate in and kill both established cancer cell lines and cancer patient tumor explants. Next, we used a transposon insertion strategy and deep sequencing of viral populations to systematically examine the role of each vaccinia virus gene in its ability to be an anticancer therapeutic. Ultimately, we identified large regions (25Kb) of the vaccinia genome that when deleted, augment the oncolytic activity of a newly generated vaccinia backbone termed SKV. Our new best-in-class vaccinia, SKV, robustly stimulates anti-immune responses, rapidly spreads within and between tumors and has a substantially improved preclinical safety profile when compared to other vaccinia clinical candidates. As predicted, SKV synergizes well with immune checkpoint inhibitor antibodies and potently activates human immune cells. Due to the exquisite tumor selectivity of SKV, we have been able to engineer and express from the backbone a combination of very potent immune modulators that are safest and most effective when expressed within the TME. These include an immune checkpoint inhibitor, a membrane tethered cytokine and antigen-presenting cell activating ligand in a single virus. Ongoing toxicity and efficacy studies are being carried out to prepare our novel virus construct for clinical launch. Citation Format: John C. Bell, Adrian Pelin, Michael Huh, Matthew Tang, Fabrice Le Boeuf, Brian Keller, Jessie Duong, Katherine Clark-Knowles, Julia Petryk, Victoria A. Jennings, Alan Melcher, Mathieu Crupi, Larissa Pikor, Caroline Breitbach, Steven Bernstein, Michael Burgess. Novel oncolytic vaccinia virus platform for systemic delivery of immunotherapeutic payloads [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B101.

Generation of a More Immunogenic Measles Vaccine by Increasing Its Hemagglutinin Expression.

Imported measles virus (MV) outbreaks are maintained by poor vaccine responders and unvaccinated people. A convenient but more immunogenic vaccination strategy would enhance vaccine performance, contributing to measles eradication efforts. We report here the generation of alternative pediatric vaccines against MV with increased expression of the H protein in the background of the current MV vaccine strain. We generated two recombinants: MVvac2-H2, with increased full-length H expression resulting in a 3-fold increase in H incorporation into virions, and MVvac2-Hsol, vectoring a truncated, soluble form of the H protein that is secreted into the supernatants of infected cells. Replication fitness was conserved despite the duplication of the H cistron for both vectors. The modification to the envelope of MVvac2-H2 conferred upon this virus a measurable level of resistance to in vitro neutralization by MV polyclonal immune sera without altering its thermostability. Most interestingly, both recombinant MVs with enhanced H expression were significantly more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic after a single, human-range dose in genetically modified MV-susceptible mice. Measles incidence was reduced drastically following the introduction of attenuated vaccines, but progress toward the eradication of this virus has stalled, and MV still threatens unvaccinated populations. Due to the contributions of primary vaccine failures and too-young-to-be-vaccinated infants to this problem, more immunogenic measles vaccines are highly desirable. We generated two experimental MV vaccines based on a current vaccine's genome but with enriched production of the H protein, the main MV antigen in provoking immunity. One vaccine incorporated H at higher rates in the viral envelope, and the other secreted a soluble H protein from infected cells. The increased expression of H by these vectors improved neutralizing responses induced in two small-animal models of MV immunogenicity. The enhanced immunogenicity of these vectors, mainly from the MV that incorporates additional H, suggests their value as potential alternative pediatric MV vaccines.

Thymidine Kinase-Deleted, let7a-Regulated Vaccinia Virus Specifically Infects and Lyses Myeloma Cells in a Mouse Myeloma Model

Background: Vaccinia virus is a tool for a novel cancer therapy that infects and lyses cancer cells. Clinical trials targeting solid tumors are currently ongoing, and promising results are reported. Although in vitro studies suggest that hematological malignancies have only modest susceptibility in general, we found that multiple myeloma shows an exceptionally high susceptibility to vaccinia virus, therefore, we focused on myeloma. Because tumor specific infection is the key for a successful oncolytic therapy, we modified thymidine kinase (TK) gene and B5R gene in the vaccinia genome to make it more tumor-specific. Efficacy and safety was determined by the in vitro and in vivo mouse infection models.Methods: Vaccinia TK is required for viral replication, but it is dispensable in tumor cells because high level of TK produced by the tumor can substitute it. In order to delete TK gene, we generated a plasmid vector which have a homologous sequence of TK (pTK), and the firefly luciferase (Fluc) gene with the PSFL110 promoter was inserted into the middle of TK gene. By the co-transfection of 143B cells with the pTK-Fluc and parental vaccinia virus LC16mO (derived from Lister clone 16), a homologous recombination occurred and the TK-deleted vaccinia virus was generated. Vaccinia B5R gene is essential for the viral transmission from infected cells to adjacent cells. To regulate B5R gene expression and inhibit viral transmission in normal cells, we inserted a let-7a target sequence into the downstream of B5R in the pB5R plasmid. Let-7a is a micro RNA of which expression is lost in tumor cells but maintained in most of the normal cells, therefore, we hypothesized that endogenous let-7a in normal cells will inhibit B5R, inhibit viral transmission, and improve safety. By the co-transfection of RK13 cells with the pB5Rgfp-let7a and TK-deleted vaccinia virus, a homologous recombination occurred, and TK-deleted let-7a-regulated vaccinia virus was generated.Results: To find out a good target for this therapy, we first infected different cell lines including Kasumi-1, NB4, NALM6, Jurkat, OM9;22, K562 (these are from leukemia), FL218, Daudi (lymphoma), MT2, TLO-m1 (ATL), and RPMI8226, U266, NCI-H929 (myeloma) with a vaccinia virus. Very interestingly, myeloma-derived cell lines showed an exceptionally high susceptibility (10-100 times higher than others), and this susceptibility was as high as solid tumors; therefore we chose multiple myeloma as the target. To confirm that regulation of B5R by the let-7a improves myeloma-specific infection, we infected a myeloma cell line RPMI8226 as well as normal skin fibroblasts with the recombinant B5Rgfp-let7a vaccinia virus. In vitro infection of a RPMI8226 cells showed a significant infectivity even with a very low titer (MOI=0.1), while control normal cells (human skin fibroblasts) were not infected at all. Without the let7a-regulation, the vaccinia virus infected both myeloma cells and normal cells at the same titer, suggesting that let-7a-mediated regulation clearly works. As an in vivo infection model, 1x107 RPM8226-Rluc cells were injected into immunodeficient mice (CB.17-SCID) subcutaneously. Four weeks later, 1x106 pfu of virus (Parent; TK-deleted; TK and let7a double regulated) was administered via an intravenous injection, and tumor volume and the amount of virus were determined weekly by the in vivo imaging system with renilla and firefly luciferases. Without any regulation, vaccinia virus infected not only myeloma cells but also normal tissues, and mice developed pock lesions in the ear, nose, mouth, foot, and tail, resulting in death within 21 to 24 days. TK-deletion significantly alleviated viral toxicity but still caused death 42 days after infection. In contrast, infection with TK and let-7a double regulated vaccinia virus was clearly limited in myeloma, and all mice remained alive and the size of myeloma shrunk continuously.Conclusions: These data suggest that the TK-let7a-double regulated vaccinia virus infects and kills myeloma cells specifically, and will be a good candidate for the future clinical application. DisclosuresNo relevant conflicts of interest to declare.

Human CD4 T cell epitopes selective for Vaccinia versus Variola virus

Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections.

HLA-DM Constrains Epitope Selection in the Human CD4 T Cell Response to Vaccinia Virus by Favoring the Presentation of Peptides with Longer HLA-DM–Mediated Half-Lives

HLA-DM (DM) is a nonclassical MHC class II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during Ag presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of DM in epitope selection is still unclear, especially in human response to infectious disease. In this study, we addressed this question in the context of the human CD4 T cell response to vaccinia virus. We measured the IC(50), intrinsic dissociation t(1/2), and DM-mediated dissociation t(1/2) for a large set of peptides derived from the major core protein A10L and other known vaccinia epitopes bound to HLA-DR1 and compared these properties to the presence and magnitude of peptide-specific CD4(+) T cell responses. We found that MHC II-peptide complex kinetic stability in the presence of DM distinguishes T cell epitopes from nonrecognized peptides in A10L peptides and also in a set of predicted tight binders from the entire vaccinia genome. Taken together, these analyses demonstrate that DM-mediated dissociation t(1/2) is a strong and independent factor governing peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability in the presence of DM.

In silico-accelerated identification of conserved and immunogenic variola/vaccinia T-cell epitopes

Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted from highly conserved open reading frames from seven complete vaccinia and variola genomes using EpiMatrix. Over 100 epitopes bearing low human sequence homology were selected and assessed in HLA binding assays and in T-cell antigenicity assays using PBMCs isolated from Dryvax-immunized subjects. This experimental validation of computational predictions illustrates the potential for immunoinformatics methods to identify candidate immunogens for a new, safer smallpox vaccine.

Vaccinia virus infection & temporal analysis of virus gene expression: Part 3

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia virus infection & temporal analysis of virus gene expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Comparative Analysis of Viral Gene Expression Programs during Poxvirus Infection: A Transcriptional Map of the Vaccinia and Monkeypox Genomes

BackgroundPoxviruses engage in a complex and intricate dialogue with host cells as part of their strategy for replication. However, relatively little molecular detail is available with which to understand the mechanisms behind this dialogue.Methodology/Principal FindingsWe designed a specialized microarray that contains probes specific to all predicted ORFs in the Monkeypox Zaire (MPXV) and Vaccinia Western Reserve (VACV) genomes, as well as >18,000 human genes, and used this tool to characterize MPXV and VACV gene expression responses in vitro during the course of primary infection of human monocytes, primary human fibroblasts and HeLa cells. The two viral transcriptomes show distinct features of temporal regulation and species-specific gene expression, and provide an early foundation for understanding global gene expression responses during poxvirus infection.Conclusions/SignificanceThe results provide a temporal map of the transcriptome of each virus during infection, enabling us to compare viral gene expression across species, and classify expression patterns of previously uncharacterized ORFs.

High level protein expression in mammalian cells using a safe viral vector: Modified vaccinia virus Ankara

Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of β-galactosidase, about 15 mg protein could be produced from 10 8 BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production.

Detection of monkeypox virus with real-time PCR assays

Background Human monkeypox, a zoonotic disease, was first reported outside of Africa during the 2003 US outbreak. Objectives We present two real-time PCR assays critical for laboratory diagnosis of monkeypox during the 2003 US outbreak. Study design A TaqMan-based assay (E9L-NVAR) targets the orthopoxvirus DNA polymerase gene and detects Eurasian orthopoxviruses other than Variola. A hybridization assay, utilizing a MGB Eclipse™ (Epoch Biosciences) probe, targets an envelope protein gene (B6R) and specifically detects monkeypox virus (MPXV). Assays were validated using coded orthopoxvirus DNA samples and used to evaluate lesion samples from five confirmed US monkeypox cases. Results E9L-NVAR did not detect variola (48 strains), North American orthopoxviruses (2), or DNA derived from non-poxviral rash illnesses. The assay reproducibly identified various concentrations of 13 Eurasian orthopoxvirus strains and was sensitive to 12.5 vaccinia genomes. The B6R assay recognized 15 different MPXV strains, while other orthopoxvirus (9) and bacteria (15) strains did not cross-react. Of the 13 human samples tested from confirmed cases, both assays identified 100% as containing MPXV DNA. Conclusions E9L-NVAR and B6R assays demonstrate 100% specificity for non-variola Eurasian orthopoxvirus and MPXV, respectively. Using two discrete viral gene targets, these assays together provide a reliable and sensitive method for quickly confirming monkeypox infections.

Interaction of F1L with the BH3 domain of Bak is responsible for inhibiting vaccinia-induced apoptosis

Apoptosis represents an important cellular defence mechanism against viral pathogens by virtue of its ability to remove infected cells. Consequently, many viruses have developed numerous strategies to prevent or delay host cell apoptosis in order to achieve productive replication. Here we report that deletion of the F1L gene from the vaccinia genome results in increased apoptosis during infection. We demonstrate that F1L, which has no sequence homology to Bcl-2 family members, inhibits apoptosis at the level of mitochondria by binding to Bak. As a consequence, F1L prevents Bak activation, oligomerization and interaction with active Bax, all critical steps in the induction of apoptosis. We demonstrate that residues 64-84 of F1L interact directly with the Bcl-2 homology domain 3 (BH3) domain of Bak. This region of F1L has limited sequence similarity to known Bak-interacting BH3 domains. We also find that such additional BH3-like domains exist in the vaccinia genome. We conclude that F1L uses this specific, BH3-like domain to bind and inhibit Bak at the mitochondria.

999. siRNA-Mediated Interference of Double-Stranded RNA Binding Protein [E3L] Is an Effective Strategy to Inhibit Vaccinia Virus

Top of pageAbstract Background: The Vaccinia virus E3L Interferon resistance gene encodes a 25kDa polypeptide which is synthesized early during the viral infection cycle. The polypeptide binds dsRNA and thus competes with the N-terminal dsRNA binding domain of protein kinase. This prevents PKR activation. However, siRNA-mediated down-regulation of E3L is hypothesized to eliminate the competition for dsRNA in favor of protein kinase leading to its activation and the subsequent cascade that establishes the anti-viral state. Methods: RNA interference (RNAi), a natural biological phenomenon mediated by small interfering RNA (siRNA) molecules, has been demonstrated to be an effective anti-viral strategy. However, no study thus far has utilized this emerging technology for Vaccinia virus, which is a useful surrogate for the Smallpox virus. In this study, we have used 21bp chemically synthesized siRNA duplexes whose sequences are derived from the E3L region of the Vaccinia genome and are conserved in smallpox and related poxviruses. Viral replication was assessed by measuring the |[beta]|-galactosidase activity in extracts prepared from infected HeLa cells. A recombinant strain of Vaccinia WR virus carrying the indicator gene was used. Production of infectious virus particles was measured by a plaque assay. Sonicated extracts from infected HeLa cells were used to quantify the amount of plaques generated on BSC-1 cells. Results: E3L-specific siRNAs exerted a potent antiviral effect on Vaccinia virus [WR] replication. Viral levels were reduced to below detection limits [MOI=0.01pfu/cell] and 7.6% of control infection [MOI=0.1pfu/cell]. Strong inhibition was observed even at extreme viral loads [15-30% of control infection at MOI=0.1-0.4pfu/cell]. Of the four E3L-specific siRNAs, E3L-C siRNA had the strongest effect [3.5% of control infection at MOI=0.1pfu/cell] followed by E3L-A siRNA [17.2% of control at MOI=0.1pfu/cell]. Silencing of E3L leads to a reduced production of infectious viral particles as well. When all the four E3L-specific siRNAs were multiplexed, infectious viral production was reduced to 4.5% of control infection [MOI=0.1]. Conclusions: The immediate potential use of the E3L-specific siRNA is as a biodefense tool against smallpox. These molecules seem to have a therapeutic potential that might be superior to the existing vaccination alternative. The value of these molecules has to be assessed in light of the morbidity associated with the small pox vaccine and potential complications that might be of concern for the immunocompromised population. Since dsRNA plays a crucial role in the generation of the antiviral state, the above described siRNA will be useful for dissecting the molecular mechanisms associated with this phenomenon.

Resolution of a Holliday junction by vaccinia topoisomerase requires a spacer DNA segment 3' of the CCCTT/ cleavage sites.

Vaccinia virus DNA topoisomerase catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT/, that are opposed within a partially mobile four-way junction. Efficient resolution occurs on a junction with a 10 bp segment of branch mobility (5'-GCCCTTATCG) that extends 4 bp 3' of the scissile phosphate. Here we report that resolution is decreased when branch mobility is limited to an 8 bp segment extending 2 bp 3' of the cleavage site and then eliminated when branch mobility is confined to the 6 bp GCCCTT sequence 5' of the scissile phosphate. We surmise that a spacer region 3' of CCCTT is needed for simultaneous cleavage at two opposing sites at the junction. Branch mobility is not required for reaction chemistry at a junction, because topoisomerase cleaves a single CCCTT site in a non-mobile four-way junction where the scissile phosphate is at the crossover point. The junction resolvase activity of topo-isomerase may be involved in forming the hairpin telomeres of the vaccinia genome.

Expression of genes coding for animal virus glycoproteins in heterologous systems.

The outermost layers of animal viruses are usually composed of glycoproteins. They are responsible not only for the entrance of viruses into, and release from host cells but also for the initial interaction of a viral particle with immunological defense of the host. It is therefore not surprising that many laboratories devote a lot of effort to study viral glycoproteins at the molecular level. Very often such studies are possible only after the introduction of a glycoprotein gene into a heterologous system. Expression of glycoprotein genes is usually obtained in mammalian or insect cells. Expression in mammalian cells yields viral glycoproteins with glycan chains indistinguishable from the original counterparts in virion particles but the level of synthesis of glycoproteins is very low. Vaccinia virus is the most common vector for expression in mammalian cells. It is easy to grow, the introduction of foreign genes is relatively simple and, due to the size of the vaccinia genome, it can accept large pieces of foreign DNA. Glycosylation in insect cells is not as complex as in mammalian cells and usually glycoproteins produced in insect cells are of slightly lower molecular mass than those produced in mammalian cells. The most common vector for expression of glycoproteins in insect cells is a baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The great advantage of this system is a very high level of expression of foreign genes.

A functional measles virus replication and transcription machinery encoded by the vaccinia virus genome

Measles virus encodes three proteins required for the encapsidation, transcription and replication of viral genomes. The genes for these proteins have been inserted into the vaccinia virus genome together with the gene for the bacteriophage T7 RNA polymerase. Cells infected with this recombinant virus were able to encapsidate, transcribe and replicate a CAT gene positioned in the negative polarity behind a T7 promoter and flanked by measles virus genomic termini. Inhibition of the accumulation of the nucleocapsid proteins by actinomycin D led to an increase in CAT expression. Thus the measles virus polymerase activity, encoded by the vaccinia genome, was regulated by the level of measles proteins just as the authentic polymerase. The recombinant vaccinia described in this study could be useful for the production of measles virus-like particles encoding foreign genes and employed in vaccination or gene therapy strategies.

Construction and Characterization of Vaccinia Direct Ligation Vectors

Poxvirus vectors are extensively used as expression vehicles for protein and antigen expression in eukaryotic cells. Customarily, the foreign DNA is introduced into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been used to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We describe the construction and characterization of a new set of direct ligation vectors designed to be universally applicable for the generation of chimeric vaccinia genomes. These vectors contain the pair of unique restriction sitesNotI andApaI to eliminate religation of poxvirus arms and fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters. The insertion cassette has been placed at the beginning of the thymidine kinase gene in vaccinia to use drug selection in the isolation of recombinants. These viruses provide a set of universally applicable direct ligation poxvirus cloning vectors, extending the utility of poxvirus vectors for construction and expression of complex libraries.

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase.

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.

Therapeutic Antitumor Response After Immunization with an Admixture of Recombinant Vaccinia Viruses Expressing a Modified MUC1 Gene and the Murine T-Cell Costimulatory Molecule B7

Tumor-associated antigens have considerable promise not only as diagnostic or prognostic markers but also as targets for active or passive immunotherapy. DF3/MUC1 is a tumor-associated antigen that is overexpressed with an abnormal glycosylation pattern in breast, ovarian, lung, and pancreatic cancers. The major extracellular portion of MUC1 is composed of tandem repeat units of 20 amino acids. Recombinant vaccinia viruses encoding mucin molecules have been constructed by several groups. However, these recombinants have met with limited success in protecting animals from MUC1-expressing tumors because of the vaccinia genome being subject to high-frequency homologous recombination, therefore being unstable in expression of the tandem repeats. In light of these studies, two concurrent strategies were used to improve immune responses to MUC1: a recombinant vaccinia virus was constructed containing a modified "mini" MUC1 gene containing only 10 tandem repeat sequences to minimize vaccinia-mediated rearrangement (designated rV-MUC1); and an admixture was used containing rV-MUC1 and a recombinant vaccinia virus containing the gene for the murine T-cell costimulatory molecule B7-1 (rV-B7). The rV-MUC1 gene product maintained a consistent molecular weight throughout several passages, indicating stability of the inserted gene. Mice inoculated with rV-MUC1 demonstrated MUC1-specific cytolytic responses that were further enhanced by admixture with rV-B7. In a MUC1-expressing pulmonary metastases prevention model, mice inoculated two times with rV-MUC1 were protected from the establishment of metastases. No additive effect on antitumor immunity (> 90% with rV-MUC1 alone) was observed in mice primed with an admixture of rV-MUC1 and rV-B7 and boosted with rV-MUC1. When rV-MUC1 was used to treat established MUC1 positive metastases, however, three administrations of rV-MUC1 were not sufficient to confer antitumor effects. In contrast, when tumor-bearing mice were primed with an admixture of rV-MUC1 and rV-B7, followed by two boosts with rV-MUC1, there was a significant reduction in pulmonary metastases (p = < 0.0001), which correlated to 100% survival. Coexpression of the B7 molecule, although not necessary for the induction of an immune response of sufficient magnitude to prevent MUC1 tumors, was thus essential in a treatment setting.