Deletion of Major Nonessential Genomic Regions in the Vaccinia Virus Lister Strain Enhances Attenuation without Altering Vaccine Efficacy in Mice

Abstract

The vaccinia virus (VACV) Lister strain was one of the vaccine strains that enabled smallpox eradication. Although the strain is most often harmless, there have been numerous incidents of mild to life-threatening accidents with this strain and others. In an attempt to further attenuate the Lister strain, we investigated the role of 5 genomic regions known to be deleted in the modified VACV Ankara (MVA) genome in virulence in immunodeficient mice, immunogenicity in immunocompetent mice, and vaccine efficacy in a cowpox virus challenge model. Lister mutants were constructed so as to delete each of the 5 regions or various combinations of these regions. All of the mutants replicated efficiently in tissue culture except region I mutants, which multiplied more poorly in human cells than the parental strain. Mutants with single deletions were not attenuated or only moderately so in athymic nude mice. Mutants with multiple deletions were more highly attenuated than those with single deletions. Deleting regions II, III, and V together resulted in total attenuation for nude mice and partial attenuation for SCID mice. In immunocompetent mice, the Lister deletion mutants induced VACV specific humoral responses equivalent to those of the parental strain but in some cases lower cell-mediated immune responses. All of the highly attenuated mutants protected mice from a severe cowpox virus challenge at low vaccine doses. The data suggest that several of the Lister mutants combining multiple deletions could be used in smallpox vaccination or as live virus vectors at doses equivalent to those used for the traditional vaccine while displaying increased safety.Vaccinia virus (VACV) has displayed extraordinary efficacy as a live attenuated vaccine since it has enabled the eradication of smallpox, one of the most deadly infectious diseases for mankind. Although smallpox vaccination is no longer routinely employed, some countries continue to vaccinate selected populations because of the fear of biological warfare or bioterrorism. During the course of their use, the smallpox vaccines were responsible for numerous vaccine-associated accidents like eczema vaccinatum and progressive vaccinia in individuals with immune deficiencies or skin disorders. Encephalitis was also long recognized as a very serious vaccine-related risk with an unknown predisposition (35), and vaccine-induced myopericarditis has been documented as an adverse event (27, 32). It is therefore not surprising that a good deal of research has been devoted to designing strategies that reduce the risks associated with smallpox vaccination (52). Further attenuation of the smallpox vaccine has also been sought because VACV is an attractive live virus vector for vaccination against infectious diseases and displays promising activity as a vector for prophylaxis and therapy of cancer (25).Modified vaccinia virus Ankara (MVA) was one of the first VACV strains shown to be highly attenuated in small-animal models and safe in human trials (42,66). MVA was isolated during the 1960s fr...

CD8 T Cells Are Essential for Recovery from a Respiratory Vaccinia Virus Infection

“…7A ). To investigate the requirement for CD8 T cell immunity during a less virulent VACV infection we utilized two live-attenuated VACV variants that differ in their expression of several virulence factors that determine their replicative capacity and virulence in mice (37, 38). This reduced virulence is revealed by the need to utilize a significantly higher infectious dose (2 × 10 6 PFU/mouse) to provoke a measurable immune response and the lack of significant weight loss or outward signs of illness throughout infection.…”

Attenuation of monkeypox virus by deletion of genomic regions

“…Experimental deletion of individual and multiple genes in OPXV genomes has been used to study virulence factors as well as to improve the immunogenicity and safety of vaccine vectors (Dimier et al, 2011; Johnston and McFadden, 2004; Kochneva et al, 2005). These studies have shown that deletion of individual genes can either reduce (Johnston and McFadden, 2004; Senkevich et al, 1994) or enhance (Estep et al, 2011; Ng et al., 2001) replication and virulence.…”

The Vaccinia Virus O1 Protein Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase 1/2 and Promotes Viral Virulence

“…Over the course of the passages, six major deletions of 2.7 to 6.5 kbp and a large number of smaller deletions, insertions, and point mutations occurred in the genome (34). We and others have recently demonstrated that, in contrast to general belief, introduction of the six major deletions was not sufficient to create a severely host-range-restricted VACV with MVA-like properties (13,35). Host range restriction of MVA must therefore be governed by additional or completely independent mutations.…”

“…The samples were incubated with the DNase for 30 min at 37°C, and the DNase was subsequently inactivated by addition of 1.24 l of 0.5 M EDTA (pH 8.0) and heating to 75°C for 10 min. Reverse transcription was performed using an Omniscript reverse transcriptase (RT) kit (Qiagen) in the presence of RNase inhibitor (human; Sigma-Aldrich) at a final concentration of 10 U/l for 90 min at 37°C using oligo(dT) [12][13][14][15][16][17][18] primers (Invitrogen). A 1-l volume of RT reaction mixture was added to 20 l of the total PCR mixture for real-time quantitative PCR (qPCR).…”