Sutter, G. & Moss, B. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc. Natl Acad. Sci. USA 89, 10847-10851

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/1992; 89(22):10847-51. DOI: 10.1073/pnas.89.22.10847
Source: PubMed


Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

Download full-text


Available from: Gerd Sutter, Jun 12, 2015
  • Source
    • "Severely attenuated, transgenes inserted (Sutter and Moss, 1992; Kochneva et al., 2012) Various cancer model (Drexler et al., 1999; Carroll et al., 1997) Various tumors (Larocca and Schlom, 2011; Amato et al., 2012; Gómez et al., 2013) MVA-5T4*, MVAhup53 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Naturally occurring oncolytic viruses are live, replication-proficient viruses that specifically infect human cancer cells while sparing normal cell counterparts. Since the eradication of smallpox in the 1970s with the aid of vaccinia viruses, the vaccinia viruses and other genera of poxviruses have shown various degrees of safety and efficacy in pre-clinical or clinical application for human anti-cancer therapeutics. Furthermore, we have recently discovered that cellular tumor suppressor genes are important in determining poxviral oncolytic tropism. Since carcinogenesis is a multi-step process involving accumulation of both oncogene and tumor suppressor gene abnormalities, it is interesting that poxvirus can exploit abnormal cellular tumor suppressor signaling for its oncolytic specificity and efficacy. Many tumor suppressor genes such as p53, ATM, and RB are known to play important roles in genomic fidelity/maintenance. Thus, tumor suppressor gene abnormality could affect host genomic integrity and likely disrupt intact antiviral networks due to accumulation of genetic defects, which would in turn result in oncolytic virus susceptibility. This review outlines the characteristics of oncolytic poxvirus strains, including vaccinia, myxoma, and squirrelpox virus, recent progress in elucidating the molecular connection between oncogene/tumor suppressor gene abnormalities and poxviral oncolytic tropism, and the associated preclinical/clinical implications. I would also like to propose future directions in the utility of poxviruses for oncolytic virotherapy.
    The Journal of Microbiology 04/2015; 53(4):209-18. DOI:10.1007/s12275-015-5041-4 · 1.44 Impact Factor
  • Source
    • "Clonal virus isolate F6 at passage 584 on primary chicken embryo fibroblasts (CEFs) was used for this study. Recombinant MVA constructs have been described previously and virus stocks were generated by standard methods (Sutter and Moss, 1992; Kremer et al., 2012). In brief, viruses were propagated on CEF, concentrated and purified by ultracentrifugation through sucrose, and titrated on CEF to determine infectious units (IU). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ectopic lymphoid tissue, such as bronchus-associated lymphoid tissue (BALT) in the lung, develops spontaneously at sites of chronic inflammation or during infection. The molecular mechanisms underlying the neogenesis of such tertiary lymphoid tissue are still poorly understood. We show that the type of inflammation-inducing pathogen determines which key factors are required for the formation and maturation of BALT. Thus, a single intranasal administration of the poxvirus modified vaccinia virus Ankara (MVA) is sufficient to induce highly organized BALT with densely packed B cell follicles containing a network of CXCL13-expressing follicular DCs (FDCs), as well as CXCL12-producing follicular stromal cells. In contrast, mice treated with P. aeruginosa (P.a.) develop BALT but B cell follicles lack FDCs while still harboring CXCL12-positive follicular stromal cells. Furthermore, in IL-17-deficient mice, P.a.-induced BALT largely lacks B cells as well as CXCL12-expressing stromal cells, and only loose infiltrates of T cells are present. We show that Toll-like receptor pathways are required for BALT induction by P.a., but not MVA, and provide evidence that IL-17 drives the differentiation of lung stroma toward podoplanin-positive CXCL12-expressing cells that allow follicle formation even in the absence of FDCs. Taken together, our results identify distinct pathogen-dependent induction and maturation pathways for BALT formation.
    Journal of Experimental Medicine 03/2014; 211(4). DOI:10.1084/jem.20131737 · 12.52 Impact Factor
  • Source
    • "Following the demonstration of high levels of recombinant protein production from recombinant MVA [12] it was proposed that recombinant MVA could be employed as a safe but highly immunogenic vaccine in humans, and initial pre-clinical development showed promise [13]. The block in viral replication in human cells occurs after DNA synthesis, and in the majority of mammalian cells both early and late gene expression takes place, such that recombinant antigen is expressed inside the infected cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The smallpox vaccine Vaccinia was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one or two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is a replication-deficient and attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. Many clinical trials of these new vaccines have been conducted, and the findings of these trials are reviewed here. The safety of MVA is now well documented, immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate.
    Vaccine 03/2013; 31(39). DOI:10.1016/j.vaccine.2013.03.020 · 3.62 Impact Factor
Show more