Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68–1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68–1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68–1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68–1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68–1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post-transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence-activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three-fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin-opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines.

Varicella-zoster virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia.Reactivation from latency causes herpes zoster, which may be complicated by postherpetic neuralgia. Innate immunity mediated by interferon and proinflammatory cytokines represents the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways, including the central transcription factor NF-B. However, the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV) infection of rhesus macaques recapitulates key aspects of VZV pathogenesis, and this model thus permits examination of the role of immune evasion mechanisms in vivo. Here, we compare SVV and VZV with respect to interference with NF-B activation. We demonstrate that both viruses prevent ubiquitination of the NF-B inhibitor IB␣, whereas SVV additionally prevents IB␣ phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IB␣ ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with ␤-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IB␣. This interaction seems to inactivate SCF-mediated protein degradation in general, since the unrelated ␤-TrCP target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NF-B pathway, since SVV with ORF61 deleted still prevented IB␣ phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with tumor necrosis factor alpha (TNF-␣)-induced NF-B activation at multiple levels, which is consistent with the importance of these countermechanisms for varicella virus infection. IMPORTANCEThe role of innate immunity during the establishment of primary infection, latency, and reactivation by varicella-zoster virus (VZV) is incompletely understood. Since infection of rhesus macaques by simian varicella virus (SVV) is used as an animal model of VZV infection, we characterized the molecular mechanism by which SVV interferes with innate immune activation. Specifically, we studied how SVV prevents activation of the transcription factor NF-B, a central factor in eliciting proinflammatory responses. The identification of molecular mechanisms that counteract innate immunity might ultimately lead to better vaccines and treatments for VZV, since overcoming these mechanisms, either by small-molecule inhibition or by genetic modification of vaccine strains, is expected to reduce the pathogenic potential of VZV. Moreover, using SVV infection of rhesus macaques, it will be possible to study how increasing the vulnerability of varicella viruses to innate immunity will impact viral pathogenesis. V aricella-zoster virus (VZV) is a member of the subfamily Alphaherpesvirinae and is the causative agent of chickenpox and herpes zoster (HZ). Following primary infection, VZV establishes latency in ganglia. Reactivation from latency, which typically occurs late...

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In vitro and in vivo characterization of a recombinant1 rhesus cytomegalovirus containing a complete genome 2 3 Abstract (300 words) 42 Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species 43 specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models 44 using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has 45 been established as a representative model for infection of humans with HCMV due to the close 46 evolutionary relationships of both host and virus. However, the commonly used 68-1 strain of 47 RhCMV has been passaged in fibroblasts for decades resulting in multiple genomic changes due 48 to tissue culture adaptation that cause reduced viremia in RhCMV-naïve animals and limited 49 shedding compared to low passage isolates. Using sequence information from primary RhCMV 50 isolates we constructed a full-length (FL) RhCMV by repairing all presumed mutations in the 68-51 1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve 52 RM with the reconstituted virus resulted in significant replication in the blood similar to primary 53 isolates of RhCMV and furthermore led to extensive viremia in many tissues at day 14 post 54 infection. In contrast, viral dissemination and viremia was greatly reduced upon deletion of genes 55 also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-56 like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and 57 in vivo consistent with an important immunomodulatory function of the respective proteins. We 58 conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while 59 being amenable to genetic modifications through BAC recombineering techniques.60 61 4 Author Summary (150-200 word non-technical summary) 62 Human cytomegalovirus (HCMV) infections are generally asymptomatic in healthy 63 immunocompetent individuals, but HCMV can cause serious disease after congenital infection and 64 in individuals with immunocompromised immune systems. Since HCMV is highly species specific 65 and cannot productively infect immunocompetent laboratory animals, experimental infection of 66 rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related 67 animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting 68 cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines 69 against chronic and recurring infections with pathogens such as the human immunodeficiency 70 virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted 71 with derivatives of the 68-1 strain of RhCMV which has acquired multiple genomic alterations in 72 tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a 73 full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naïve 74 RM with FL-...

In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between “good” and “damaged” virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.