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The rapid development of potency assays is critical in the development of life-saving vaccines. The traditional plaque assay or fifty percent tissue culture infectious dose (TCID 50 ) assay used to measure the potency of live virus vaccines is time consuming, labor intensive, low throughput and with high variability. Described here is the development and qualification of a cell-based reporter potency assay for two vaccines for respiratory viral infection, one based on the recombinant vesicular stomatitis virus (rVSV) backbone, termed Vaccine 1 in this paper, and the other based on the measles virus vector, termed Vaccine 2. The reporter potency assay used a Vero E6 cell line engineered to constitutively express NanuLuc ® luciferase, termed the VeroE6-NLuc or JM-1 cell line. Infection of JM-1 cells by a live virus, such as rVSV or measles virus, causes a cytopathic effect (CPE) and release of NanuLuc ® from the cytoplasm into the supernatant, the amount of which reflects the intensity of the viral infection. The relative potency was calculated by comparison to a reference standard using parallel line analysis (PLA) in a log-log linear model. The reporter assay demonstrated good linearity, accuracy, and precision, and is therefore suitable for a vaccine potency assay. Further evaluation of the Vaccine 1 reporter assay demonstrated the robustness to a range of deliberate variation of the selected assay parameters and correlation with the plaque assay. In conclusion, we have demonstrated that the reporter assay using the JM-1 cell line could be used as a potency assay to support the manufacturing and release of multiple live virus vaccines.

Described here is the evaluation of a luciferase (luc) and respiratory syncytial virus (RSV) messenger RNA / lipid nanoparticle (mRNA/LNP) vaccine using a Needle-free Injection System, Tropis®, from PharmaJet® (Golden, Colorado USA). Needle-free jet delivery offers an alternative to needle/syringe. To perform this assessment, compatibility studies with Tropis were first performed with a luc mRNA/LNP and compared to needle/syringe. Although minor changes in particle size and encapsulation efficiency were observed when using Tropis on the benchtop, in vitro luciferase activity remained the same. Next, the luc mRNA/LNP was administered to rats intramuscularly using Tropis or needle/syringe and tracking of the injection and distribution was performed. Lastly, an mRNA encoding a prefusion-stabilized F protein from RSV was delivered intramuscularly using both Tropis and needle/syringe at 1 and 5 mcg mRNA. An equivalent IgG response was observed using both Tropis and needle/syringe. The cell mediated immune (CMI) response was also evaluated, and responses to RSV-F were detected from animals immunized with needle/syringe at all dose levels, and from the animals immunized with Tropis in the 5 and 25 ug groups. These results indicated that delivery of mRNA/LNPs with Tropis is a potential means of administration and an alternative to needle/syringe., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Merck & Co. Inc. Rahway, NJ,USA and its affiliates. Published by Elsevier Ltd.)

Threats to public health and environmental quality from septic systems are more prevalent in areas with poorly draining soils, high water tables, or frequent flooding. Significant research gaps exist in assessing these systems' vulnerability and evaluating factors associated with higher rates of septic systems replacement and repair. We developed a novel GIS-based framework for assessing septic system vulnerability using a database of known septic system specifications and a modified Soil Topographic Index (STI) that incorporates seasonal high groundwater elevation to assess risks posed to septic systems in coastal Georgia. We tested the hypothesis that both the modified STI and septic system specifications such as tank capacity per bedroom and drainfield type would explain most of the variance in septic system repair and replacement using classification inference tree and generalized logistic regression models. Our modeling results indicate that drainfield type (level vs. mounded) is the most significant variable (p-value < 0.001) in predicting septic systems functionality followed by septic tank capacity per bedroom (p-value < 0.01). These show the importance of septic system design regulations such as a minimum requirement for horizontal separation distance between the bottom of trenches and seasonal water table, and adequate tank capacity design. However, for septic systems with a mounded drainfield and a larger tank capacity per bedroom, the modified STI representing hydrological characteristics of septic system location is a significant predictor of a high septic system repair and replacement rate. The methodology developed in this study can have important implications for managing existing septic systems and planning for future development in coastal areas, especially in an environment of rapid climatic change., Competing Interests: The authors have declared that no competing interests exist.

The human papillomavirus (HPV) 9-valent, recombinant vaccine (Gardasil™9) helps protect young adults (males and females) against anogenital cancers and genital warts caused by certain HPV genotypes (ref. Gardasil™9 insert). This vaccine is administered intramuscularly (IM). The aim of this study was to determine preclinically whether intradermal (ID) vaccination with an unadjuvanted 9-valent recombinant HPV vaccine using a first-generation ID delivery device, the Nanopatch™, could enhance vaccine immunogenicity compared with the traditional ID route (Mantoux technique). IM injection of HPV VLPs formulated with Merck & Co., Inc., Kenilworth, NJ, USA Alum Adjuvant (MAA) were included in the rhesus study for comparison. The Nanopatch™ prototype contains a high-density array comprised of 10,000 microprojections/cm 2 , each 250 µm long. It was hypothesized the higher density array with shallower ID delivery may be superior to the Mantoux technique. To test this hypothesis, HPV VLPs without adjuvant were coated on the Nanopatch™, stability of the Nanopatch™ with unadjuvanted HPV VLPs were evaluated under accelerated conditions, skin delivery was verified using radiolabelled VLPs or FluoSpheres®, and the immune response and skin site reaction with the Nanopatch™ was evaluated in rhesus macaques. The immune response induced by Nanopatch™ administration, measured as HPV-specific binding antibodies, was similar to that induced using the Mantoux technique. It was also observed that a lower dose of unadjuvanted HPV VLPs delivered with the first-generation Nanopatch™ and applicator or Mantoux technique resulted in an immune response that was significantly lower compared to a higher-dose of alum adjuvanted HPV VLPs delivered IM in rhesus macaques. The study also indicated unadjuvanted HPV VLPs could be delivered with the first-generation Nanopatch™ and applicator to the skin in 15 s with a transfer efficiency of approximately 20%. This study is the first demonstration of patch administration in non-human primates with a vaccine composed of HPV VLPs.

Background: The licensed live, attenuated varicella-zoster virus vaccine prevents herpes zoster in adults older than 50 years. We aimed to determine whether intradermal administration of zoster vaccine could enhance vaccine immunogenicity compared with conventional needle subcutaneous administration., Methods: In this randomised, dose-ranging study, adults aged 50 years or older who had a history of varicella or who had resided in a country with endemic varicella-zoster virus infection for 30 years or more were eligible. Participants received the approved full or a 1/3 dose of zoster vaccine given subcutaneously or one of four intradermal doses (full, 1/3, 1/10, or 1/27 dose) using the MicronJet600 device. The two subcutaneous doses and the four intradermal doses were randomised (1·5:1:1:1:1:1) by computer generated sequence with randomisation stratified by age (50-59 years or 60 years or older). The primary immunogenicity endpoint was the change from baseline in IgG antibody to varicella-zoster virus-specific glycoproteins (gpELISA) measured at 6 weeks. All patients were included in the primary and safety analyses. This study is registered with ClinicalTrials.gov, number NCT01385566., Findings: Between Sept 2, 2011, and Jan 13, 2012, 224 participants were enrolled from three clinics in the USA and 223 were randomly assigned: 52 to receive the full dose subcutaneous zoster vaccine, 34 to receive the 1/3 dose subcutaneous zoster vaccine, 34 to receive the full dose intradermal zoster vaccine, 35 to receive the 1/3 dose intradermal zoster vaccine, 34 to receive the 1/10 dose intradermal zoster vaccine, and 34 to receive the 1/27 dose intradermal zoster vaccine. Full dose zoster vaccine given subcutaneously resulted in a gpELISA geometric mean fold-rise (GMFR) of 1·74 (90% CI 1·48-2·04) at 6 weeks post-vaccination compared with intradermal administration which resulted in a significantly higher gpELISA GMFR of 3·25 (2·68-3·94; p<0·0001), which also remained high at 18 months. An apparent dose-response relation was observed with intradermal administration (1/3 dose subcutaneous GMFR 1·64 [90% CI 1·36-1·99], 1/3 dose intradermal 2·58 (2·13-3·13), 1/10 dose intradermal 2·22 [1·83-2·69], and 1/27 dose intradermal 1·64 [1·35-2·00]). Each partial dose of zoster vaccine given intradermaly had a gpELISA GMFR comparable to that of full dose zoster vaccine given subcutaneously. Transient erythema and induration were more common after intradermal administration (31% erythema for full subcutaneous dose and 77% for intradermal dose)., Interpretation: Intradermal zoster vaccine showed a greater increase in varicella-zoster virus gpELISA antibody compared with subcutaneous zoster vaccine at comparable doses. Larger and longer studies of intradermal administration of live, attenuated zoster vaccine are needed to provide convincing evidence of improved cell mediated immunity., Funding: Merck & Co Inc., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The amount, identity, and size distribution of particles in parenteral therapeutic protein formulations are of immense interest due to potential safety and efficacy-related implications. In this communication, we describe the use of a flow cytometer equipped with forward- and side-scattering as well as fluorescence detectors, to determine the number of subvisible particles in monoclonal antibody formulations. The method appears to detect particles of size 1 μ and larger, requiring relatively small sample volumes to estimate subvisible particle counts. Additionally, it facilitates differentiation of proteinaceous particles after staining with a fluorescent hydrophobic dye. The method is expected to be particularly well suited for pharmaceutical development, because it provides increased throughput due to the use of a 96-well autosampler., (Copyright © 2010 Wiley-Liss, Inc.)

The antimicrobial effectiveness or preservative effectiveness test is described in the tripartite compendia for sterile parenteral multi-dose formulated products. The execution of the test is essentially harmonized with respect to inoculum preparation and test execution but not the acceptance criteria. This article describes how a single test can be performed that procedurally satisfies all of the compendia and their acceptance criteria., (© 2011 American Association of Pharmaceutical Scientists)

Concern around the lack of monitoring of proteinaceous subvisible particulates in the 0.1-10 microm range has been heightened (Carpenter et al., 2009, J Pharm Sci 98: 1202-1205), primarily due to uncertainty around the potential immunogenicity risk from these particles. This article, representing the opinions of a number of industry scientists, aims to further the discussion by developing a common understanding around the technical capabilities, limitations, as well as utility of monitoring this size range; reiterating that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasizing that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. Measurement of subvisible particulates in the <10 microm size range has value as an aid in product development and characterization. Limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability, at the present time. Such particles constitute microgram levels of protein with currently monitored sizes >or=10 microm representing the largest fraction. These levels are well below what is detected or reported for other product quality attributes. Subvisible particles remain a product quality attribute that is also qualified in clinical trials., ((c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association)

The following review provides a comprehensive summary of antimicrobial preservatives that are commonly used in licensed parenteral products to date. The information reviewed includes the general properties of the preservatives, the doses and frequency of their use, the classes of the preserved products (peptide, protein, vaccine, and small molecule products), the interactions with other formulation components, and the criteria commonly used for their selection in parental product formulations. It was revealed that phenol and benzyl alcohol are the two most common antimicrobial preservatives used in peptide and protein products, while phenoxyethanol is the most frequently used preservative in vaccines. Benzyl alcohol or a combination of methylparaben and propylparaben are generally found in small molecule parenteral formulations. The key criteria for antimicrobial preservative selection are the preservative's dose, antimicrobial functionality, and effect on the active ingredient. Additionally, the use of spectroscopic techniques (circular dicroism (CD) and fluorescence) and differential scanning calorimetry (DSC) were identified as common techniques used in evaluating an antimicrobial preservative for its impact on the conformational stability of peptide, protein, and vaccine antigens. The future use of preservatives is also discussed, including antimicrobial agents such as peptides, and regulatory requirements for antimicrobial effectiveness testing., ((c) 2007 Wiley-Liss, Inc.)

The following study was conducted to determine the effect of different preservatives commonly used in the biopharmaceutical industry on the product-specific bubble point of sterilizing-grade filters when used to filter product processed with different types of tubing. The preservatives tested were 0.25% phenol, m-cresol, and benzyl alcohol. The tubing tested was Sani-Pure (platinum-cured silicone tubing), Versilic (peroxide-cured silicone tubing), C-Flex, Pharmed, and Cole-Parmer (BioPharm silicone tubing). The product-specific bubble point values of sterilizing grade filters were measured after the recirculation of product through the filter and tubing of different types of materials for a total contact time of 15 h. When silicone tubing was used, the post-recirculation product-specific bubble point was suppressed on average 13 psig when compared to the pre- recirculation product-specific bubble point. Suppression was also observed with C-Flex, but to a much lesser extent than with silicone tubing. Suppression was not observed with Pharmed or BioPharm tubing. Alcohol extractions performed on the filters that experienced suppressed bubble points followed by Fourier transform infrared spectroscopy analysis indicated the filters contained poly(dimethylsiloxane). Direct addition of poly(dimethlysiloxane) to solutions filtered through sterilizing-grade filters suppressed the filter bubble points when tested for integrity. Silicone oils most likely reduced the surface tension of the pores in the membrane, resulting in the ability of air (or nitrogen) to pass more freely through the membrane, causing suppressed bubble point test values. The results of these studies indicate that product-specific bubble point of a filter determined with only product may not reflect the true bubble point for preservative-containing products that are recirculated or contacted with certain tubing for 15 h or greater. In addition, tubing material placed in contact with products containing preservatives should be evaluated for impact to the product-specific bubble point when being utilized with sterilizing-grade filters.

Electron paramagnetic resonance and Hall measurements show consistently the presence of two donors ( D1 and D2) in state-of-the-art, nominally undoped ZnO single crystals. Using electron nuclear double resonance it is found that D1 shows hyperfine interaction with more than 50 shells of surrounding 67Zn nuclei, proving that it is a shallow, effective-mass-like donor. In addition D1 exhibits a single interaction with a H nucleus ( a(H) = 1.4 MHz), thus H is the defining element. It is in agreement with the prediction of Van de Walle [Phys. Rev. Lett. 85, 1012 (2000)] that H acts as a donor in ZnO. The concentration of D1 is 6x10(16) cm(-3) emphasizing its relevance for carrier statistics and applications.

The aryl hydrocarbon receptor (AhR) is a ligand-inducible transcription factor that mediates biological responses to halogenated aromatic hydrocarbons. The unliganded AhR is a cytoplasmic, tetrameric complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2). The role of XAP2 as a member of the AhR core complex is poorly understood. XAP2 shares significant homology with the immunophilins FKBP12 and FKBP52, including a highly conserved, C-terminal, tetratricopeptide repeat (TPR) domain. XAP2 forms a complex with hsp90 and the AhR but can also bind to both independently. This binding is mediated by the conserved TPR domain. Single-point mutations in this region are sufficient to disrupt the association of XAP2 with both the AhR and hsp90 in cells. Cotransfection of the AhR and XAP2 in COS-1 cells results in increased AhR levels compared with cells transfected with the AhR alone. In contrast, coexpression of the AhR with the TPR containing proteins FKBP52, protein phosphatase 5 (PP5), or XAP2 TPR-mutants deficient in binding to the AhR and hsp90 does not affect AhR levels and coexpression of the AhR with the TPR domain of PP5 results in AhR down-regulation. These results demonstrate that XAP2 is apparently unique among hsp90-binding proteins in its ability to enhance AhR levels. A yellow fluorescent protein (YFP)-XAP2-FLAG was constructed and biochemically characterized, and no loss of function was detected. YFP-XAP2-FLAG was transiently transfected into NIH 3T3 and was found to localize in both the nucleus and the cytoplasm when visualized by fluorescence microscopy. Treatment of Hepa-1 cells with the hsp90-binding benzoquinone ansamycin, geldanamycin, and the macrocyclic antifungal compound radicicol resulted in AhR but not XAP2 or FKBP52 turnover. Taken together, these results suggest that XAP2/hsp90 and FKBP52/hsp90 complexes are similar yet exhibit unique functional specificity.

The unliganded aryl hydrocarbon receptor (AhR) exists in the cytoplasm in a tetrameric 9S core complex, consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2), an immunophilin-related protein sharing homologous regions with FKBP12 and FKBP52. Interactions between the recently identified XAP2 subunit and other members of the unliganded AhR complex and its precise role in the AhR signal transduction pathway are presently unknown. Mapping studies indicate that XAP2 requires the PAS, hsp90, and ligand binding domain(s) of the AhR for binding, and that both proteins directly interact in the absence of hsp90. XAP2 is also able to interact with hsp90 complexes in the absence of the AhR, and C-terminal sequences of XAP2 are required for this interaction. XAP2 binds to the C-terminal end of hsp90, which contains a tetratricopeptide repeat domain acceptor site, whereas the AhR binds to a domain in the middle of hsp90. XAP2 was not found to be associated with the AhR-Arnt heterocomplex either in vitro or in nuclear extracts isolated from Hepa 1 cells treated with TCDD. Transient expression of XAP2 in COS-1 cells resulted in enhanced cytosolic AhR levels, suggesting a role for XAP2 in regulating the rate of AhR turnover.

Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an approximately 38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element-luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.

Discs of rabbit tibia, 5 mm thick, were utilized to study the adherence of Staphylococcus aureus to the bone surface in the presence and absence of clindamycin. Bacteria were grown in broth media containing the bone slices and varying concentrations of clindamycin. In the absence of the antibiotic, S. aureus adhered extensively to bone surfaces and formed large microcolonies which were surrounded by an amorphous matrix. In the presence of 0.025 micrograms/ml of clindamycin (0.1 MIC), S. aureus adhered less to bone surfaces, forming smaller and fewer microcolonies. In the presence of 0.0625 micrograms/ml of clindamycin (0.25 MIC), S. aureus adhered to the bone surfaces only sparsely, forming small microcolonies with very little matrix holding them together, and leaving very large areas of the bone surface uncolonized. In the presence of 0.125 micrograms/ml of clindamycin (0.5 MIC), bone surfaces were basically clean, with only one or two cells (no microcolonies) found in crevices and indentations of the bone surface. In the presence of 0.25 micrograms/ml (1 MIC) no bacteria adhered to the bone surfaces.

Daily autoclaving of drinking-water bottles or daily replacement of their contents resulted in drinking water hygienically acceptable for laboratory rats. However, daily autoclaving of the bottles imposes an additional workload which many institutions cannot afford. The daily replacement of the drinking water is not desirable, since with the usual routines it is virtually impossible to guarantee a bottle is returned to the same cage. A reliable method of preventing bacterial growth for more then 1-2 days in the drinking water of conventional laboratory rats is its acidification with hydrochloric acid to pH 2.3-2.5.

The reaction of rats and rabbits to long-term application of acidified drinking water (pH 2.3-2.5) was observed over a 7-months period. The following parameters were studied: growth curves initiated at weaning, haematology, blood glucose, total serum protein, creatinine, inorganic phosphate, sodium, potassium, calcium, alkaline phosphatase, creatinine kinase, serum glutamate oxalacetate transaminase, and serum glutamate pyruvate transaminase in serum as well as the acid-base status in arterial blood; in addition in rabbits gamma-glutamyl transferase and lactate dehydrogenase were examined in the serum. No significant changes were seen in comparison to the control groups.