b Brincidofovir (CMX001), a lipid conjugate of the acyclic nucleotide phosphonate cidofovir, is under development for smallpox treatment using "the Animal Rule," established by the FDA in 2002. Brincidofovir reduces mortality caused by orthopoxvirus infection in animal models. Compared to cidofovir, brincidofovir has increased potency, is administered orally, and shows no evidence of nephrotoxicity. Here we report that the brincidofovir half-maximal effective concentration (EC 50 ) against five variola virus strains in vitro averaged 0.11 M and that brincidofovir was therefore nearly 100-fold more potent than cidofovir.A lthough smallpox was declared eradicated by the World Health Organization in 1980, the etiologic agent (variola virus [VARV]) remains a category A select agent (subject to select agent regulations [42CFR, part 73]) or a "Highest Priority" biological threat due to its high mortality rate and ease of transmission (1). Vaccination using a closely related live orthopoxvirus (vaccinia virus) prevents smallpox but is associated with potentially severe complications and therefore is not recommended for routine use in the absence of an immediate threat of a VARV release or smallpox outbreak. The vaccine is also contraindicated in immunocompromised individuals, including the very young or old, pregnant women, and those receiving immunosuppressive therapies (2). In the event of a VARV release, there would be a need for antiviral drugs to treat individuals exposed to or infected with smallpox.Brincidofovir(BCV,CMX001,hexadecyloxypropyl-cidofovir[HDP-CDV]), a lipid-conjugated acyclic nucleotide phosphonate, has broad-spectrum in vitro activity against double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses (3). BCV has completed two phase 2 clinical trials for the prevention of clinically significant cytomegalovirus infection in hematopoietic stem cell transplant patients and is currently in phase 3 trials. Simultaneously, BCV has been in development for the treatment of smallpox under the Animal Rule, which states that when developing medical countermeasures for threat agents where human challenge studies are not ethical or feasible (e.g., VARV), FDA may grant approval based on animal model studies which demonstrate that the drug is reasonably likely to have clinical benefit in humans (http: //www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatory Information/Guidances/UCM078923.pdf) (3, 4).Upon entering a cell, the lipid moiety of BCV is cleaved to release free cidofovir, which is then phosphorylated to cidofovir diphosphate (CDV-PP). CDV-PP inhibits viral DNA polymerase by serving as an alternate substrate, resulting in the inhibition of viral DNA synthesis (5-7). Among the orthopoxviruses, BCV has proven activity in animal models against vaccinia virus, rabbitpox virus (a subspecies of vaccinia), and ectromelia virus and in vitro against monkeypox virus (3). BCV has also been shown to be active against VARV in vitro (8); however, due to regulations restricting its use, these...

Varicella-zoster virus (VZV; human herpesvirus 3) is the etiological cause of chickenpox and, upon reactivation from latency, zoster. Currently, vaccines are available to prevent both diseases effectively. A critical requirement for the manufacturing of safe and potent vaccines is the measurement of the biological activity to ensure proper dosing and efficacy, while minimizing potentially harmful secondary effects induced by immunization. In the case of live virus-containing vaccines, such as VZV-containing vaccines, biological activity is determined using an infectivity assay in a susceptible cellular host in vitro. Infectivity measurements generally rely on the enumeration of plaques by visual inspection of an infected cell monolayer. These plaque assays are generally very tedious and labor intensive and have modest throughput and high associated variability. In this study, we have developed a flow cytometry assay to measure the infectivity of the attenuated vaccine strain (vOka/Merck) of VZV in MRC-5 cells with improved throughput. The assay is performed in 96-well tissue culture microtiter plates and is based on the detection and quantification of infected cells expressing VZV glycoproteins on their surfaces. Multiple assay parameters have been investigated, including specificity, limit of detection, limit of quantification, range of linear response, signal-to-noise ratio, and precision. This novel assay appears to be in good concordance with the classical plaque assay results and therefore provides a viable, higher-throughput alternative to the plaque assay.Varicella-zoster virus (VZV; human herpesvirus 3) is a member of the Alphaherpesvirinae family. It is the etiological cause of two distinct and common diseases in humans: chickenpox and zoster. Exposure of immunologically naïve individuals to VZV results in chickenpox, a condition typically occurring during the first two decades of life. Chickenpox is usually a mild disease, although severe complications have been reported, especially in immune-compromised individuals or patients suffering from hematopoietic malignancies (29, 31). Resolution of the primary infection does not result in complete elimination of the virus, which subsists in a latent stage in sensory neural ganglia, despite sustained cellular and humoral immunity (1). This latent stage can be maintained for the remainder of the individual's life span. VZV reactivation from latency causes the symptoms of zoster which can be associated with severe and debilitating pain. A significant fraction of patients (up to 20%) will eventually suffer from long-term chronic neuralgia (postherpetic neuralgia) due to permanent nerve damage. The causes of reactivation are not fully understood, but a combination of fatigue, stress, and a declining level of cell-mediated immunity seems to be implicated. Indeed, there is a strong link between the rate of clinical reactivation and the increase in age of the affected patients (8).Several pediatric live attenuated vaccine formulations, which have proven very efficac...

Interleukin-6 (IL-6) family of cytokines are multifunctional proteins that play an important role in host defenses, acute phase reactions, immune responses, hematopoiesis, and tumorigenesis. The cytokines are produced by various lymphoid and nonlymphoid cells and mediate their biological activity through initial low-affinity binding to cell surface receptors, which are specific for their respective ligands. Ligand-specific receptor binding results in the receptor heterodimerization with ubiquitously expressed signal-transducing transmembrane component gp130 followed by activation of the gp130-associated Janus kinase, which, in turn, phosphorylates signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 (pSTAT3) dimerizes and translocates to the nucleus, where it activates gene transcription. Activation of STAT3 is essential to IL-6 family-associated physiological effects. Therefore, the ability to assess STAT3 phosphorylation is important for drug discovery efforts targeting IL-6 family cytokines. Various reagents and technologies are available to detect the effect of IL-6 type cytokines in treated cells. The present study describes the development of two pSTAT3 detection assays: the high-throughput screening assay based on Meso-Scale Discovery technology, which utilizes electrochemoluminescent signal measurements for the detection of pSTAT3 in treated cell extracts, and the secondary characterization assay based on fluorescent imaging analysis, which monitors pSTAT3 nuclear translocation in cells after activation. We have successfully utilized these assays to screen a small library of secreted proteins and identified inducers of STAT3 phosphorylation. The results obtained in this study demonstrate that both assays are robust, reliable, and amenable to high-throughput screening applications.

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

Bordetella pertussis infection has been increasing in the US, with reported cases reaching over 50,000 in 2012, a number last observed in the 1950s. Concurrently, B. pertussis lacking the pertactin protein, one of the immunogens included in the acellular vaccine formulations, has rapidly emerged since 2010, and has become the predominant circulating phenotype. Monitoring the production of the remaining acellular vaccine immunogens, such as pertussis toxin (Pt), is a critical next step. To date, methods for screening Pt have been either through genomic sequencing means or by conventional ELISAs. However, sequencing limits detection to the DNA level, missing potential disruptions in transcription or translation. Conventional ELISAs are beneficial for detecting the protein; however, they can often suffer from poor sensitivity and specificity. Here we describe a rapid, highly sensitive and specific electrochemiluminescent capture ELISA that can detect Pt production in prepared inactivated bacterial suspensions. Over 340 isolates were analyzed and analytical validation parameters, such as precision, reproducibility, and stability, were rigorously tested. Intra-plate and inter-plate variability measured at 9.8% and 11.5%, respectively. Refrigerated samples remained stable for two months and variability was unaffected (coefficient of variation was 12%). Interestingly, despite the intention of being a qualitative method, the assay was sensitive enough to detect a small, but statistically significant, difference in protein production between different pertussis promoter allelic groups of strains, ptxP1 and ptxP3. This technology has the ability to perform screening of multiple antigens at one time, thus, improving testing characteristics while minimizing costs, specimen volume, and testing time.

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