Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.

Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient.

Selection of Vaccinia Virus Recombinants Using CRISPR/Cas9.

The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate.

Viperin has species-specific roles in response to herpes simplex virus infection.

Viperin is a gene with a broad spectrum of antiviral functions and various mechanisms of action. The role of viperin in herpes simplex virus type 1 (HSV-1) infection is unclear, with conflicting data in the literature that is derived from a single human cell type. We have addressed this gap by investigating viperin during HSV-1 infection in several cell types, spanning species and including immortalized, non-immortalized and primary cells.

Direct Priming of CD8 T Cells Persists in the Face of Cowpox Virus Inhibitors of Antigen Presentation.

Vaccinia virus (VACV) was the vaccine used to eradicate smallpox and is being repurposed as a vaccine vector. CD8 T cells are key anti-viral mediators, but require priming to become effector or memory cells. Priming requires an interaction with dendritic cells that are either infected (direct priming), or that have acquired virus proteins but remain uninfected (cross priming).

Surprisingly Effective Priming of CD8 T Cells by Heat-Inactivated Vaccinia Virus Virions.

Robust priming of CD8 T cells by viruses is considered to require infection and expression of viral antigens. A corollary of this is that inactivated viruses are thought of as being inevitably poor vaccines for eliciting these responses. In contrast to this dogma, we found that some antigens present in vaccinia virus (VACV) virions prime strong CD8 T cell responses when the virus was rendered noninfectious by heat.

Modified Vaccinia Virus Ankara Can Induce Optimal CD8 T Cell Responses to Directly Primed Antigens Depending on Vaccine Design.

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8 T cell immunity. While much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as modified vaccinia virus Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required.

Most viral peptides displayed by class I MHC on infected cells are immunogenic.

CD8 T cells are essential effectors in antiviral immunity, recognizing short virus-derived peptides presented by MHC class I (pMHCI) on the surface of infected cells. However, the fraction of viral pMHCI on infected cells that are immunogenic has not been shown for any virus. To approach this fundamental question, we used peptide sequencing by high-resolution mass spectrometry to identify more than 170 vaccinia virus pMHCI presented on infected mouse cells.

Effective Priming of Herpes Simplex Virus-Specific CD8 T Cells Does Not Require Infected Dendritic Cells.

Resolution of virus infections depends on the priming of virus-specific CD8 T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8 T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV).

Simultaneous Quantification of Viral Antigen Expression Kinetics Using Data-Independent (DIA) Mass Spectrometry.

The generation of antigen-specific reagents is a significant bottleneck in the study of complex pathogens that express many hundreds to thousands of different proteins or to emerging or new strains of viruses that display potential pandemic qualities and therefore require rapid investigation. In these instances the development of antibodies for example can be prohibitively expensive to cover the full pathogen proteome, or the lead time may be unacceptably long in urgent cases where new highly pathogenic viral strains may emerge. Because genomic information on such pathogens can be rapidly acquired this opens up avenues using mass spectrometric approaches to study pathogen antigen expression, host responses and for screening the utility of therapeutics.

Systemic toll-like receptor ligation and selective killing of dendritic cell subsets fail to dissect priming pathways for anti-vaccinia virus CD8⁺ T cells.

CD8⁺ T cell responses can be generated by direct or cross-priming mechanisms, and several mouse models have been used to reveal which of these is the most important pathway for various viruses. Among these models is systemic treatment of mice with a CpG-containing oligodeoxynucleotide (CpG) to mature all dendritic cells (DCs), rendering them incapable of cross-presentation. A second is the use of cytochrome c (cytc) as a selective poison of the subsets of DCs able to cross-present antigen.

Kinetics of antigen expression and epitope presentation during virus infection.

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored.

An intradermal model for vaccinia virus pathogenesis in mice.

Intradermal injection of vaccinia virus in the ear pinnae of mice provides a model of dermal infection and vaccination. The key features of this model are the appearance of a lesion on the surface of the ear that can be measured as a clinical sign of disease and substantial growth of virus in the infected skin in the absence of systemic spread. In addition, infected ears can be easily removed to allow virological, histological, and cellular analyses.

Engineering recombinant poxviruses using a compact GFP-blasticidin resistance fusion gene for selection.

Recombinant poxviruses are important tools for research and some are candidate vaccines. To make these viruses a simple, small vector that can be used to engineer multiple strains of vaccinia virus and other model poxviruses, including ectromelia virus is of value. Here a set of plasmids and methods for making these viruses that uses an enhanced green fluorescent protein-blasticidin resistance (GFP-bsd) fusion gene as a transient selectable marker are described.

Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis.

Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest.

Growth inhibitory retinoid effects after recruitment of retinoid X receptor beta to the retinoic acid receptor beta promoter.

Nuclear retinoid receptors mediate retinoid effects through tissue-specific, ligand-receptor interactions and subsequent transcriptional regulation of secondary target genes. Retinoic acid receptor beta (RARbeta) is itself a retinoid target gene with a retinoic acid response element (betaRARE) in the 5' untranslated region of the RARbeta2 gene. Altered transcriptional regulation of RARbeta may play a role in human carcinogenesis and the retinoid-responsiveness of malignant cells.

Combined RAR alpha- and RXR-specific ligands overcome N-myc-associated retinoid resistance in neuroblastoma cells.

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels.