A Study on the Application of Recombinant Factor C (rFC) Assay Using Biopharmaceuticals.

Gram-negative bacterial endotoxins can cause pathophysiological effects such as high fever when introduced into the bloodstream. Therefore, endotoxin testing is necessary when producing injectable pharmaceuticals. The pharmaceutical industry has widely used Limulus amebocyte lysate (LAL) to certify product quality. However, ethical concerns have been raised and the increasing scarcity of Limulus polyphemus necessitates the development of novel testing techniques. Recombinant factor C (rFC) was developed using genetic engineering techniques. The aim of this study was to investigate the validity of rFC testing and compare it with the LAL method. The specificity, linearity, accuracy, precision, and robustness of the rFC assay were evaluated. After validation, the rFC assay was found to be suitable for endotoxin detection. We compared the accuracy of the rFC and LAL assays using reference standard endotoxin. The rFC assay was as accurate as the LAL assay. We also compared the two assays using biopharmaceuticals. Greater interference occurred in some samples when the rFC assay was used than when the LAL assay was used. However, the rFC assay overcame the interference when the samples were diluted. Overall, we suggest that rFC can be applied to test biopharmaceuticals.

Suppression of inflammation by recombinant Salmonella typhimurium harboring CCL22 microRNA.

Atopic dermatitis (AD) is an inflammatory, chronically relapsing, puritic skin disorder. These syndromes result from multifactorial inheritance, with interaction between genetic and environmental factors. In particular, the macrophage-derived chemokine CCL22 is directly implicated in skin inflammatory reactions and its levels are significantly elevated in serum and correlated with disease severity in AD. We tested the suppression of the CCL22 gene by microRNA (miRNA) and observed the effects in mice with inflammation similar to AD. We used Salmonella as a vector to deliver miRNA. The recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) was prepared for in vivo knockdown of CCL22. ST-miRCCL22 was orally inoculated into mice and the CCL22 gene suppressed with CCL22 miRNA in the activated lymphocytes. IgE and interleukin-4 were inhibited and interferon-γ was induced after treatments with ST-miRCCL22 and CCL22 was suppressed. Further, Th17 cells were suppressed in the atopic mice treated with ST-miRCCL22. These results suggested that suppression of the CCL22 gene using Salmonella induced anti-inflammatory effects.

Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus.

Nucleic acid amplification techniques are used frequently for rapid diagnosis of viral diseases. In this study, a real-time polymerase chain reaction protocol that uses primers specific for the viral VP4 gene and the commercial SYBR Green reagent were evaluated for the quantitative measurement of human rotavirus (HRV) RNA in human stool specimens. SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. The assay resulted in a sensitive and reproducible detection of targets ranging from low (<10(2)rotavirus cDNA copies/reaction) to high numbers (>10(6)rotavirus cDNA copies/reaction). No cross-reaction was found with crude cell culture stocks of coxsackievirus, echovirus, poliovirus, hepatitis A virus and adenovirus. Analysis with the HRV cDNA standard demonstrated high reproducibility with a coefficient of variation (CV) of 0.2-0.9%. Daily performance among three different laboratories showed a CV no greater than 8%, indicating an intermediate level of variation. These results demonstrate the feasibility of this method for quantitative analysis of human rotavirus in clinical samples.

Evaluation of viral clearance in the production of HPV-16 L1 virus-like particles purified from insect cell cultures.

Biopharmaceutical products produced from cell cultures have a potential for viral contamination from cell sources or from adventitious introduction during production. The objective of this study was to assess viral clearance in the production of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs). We selected Japanese encephalitis virus (JEV), bovine viral diarrhea virus (BVDV), and minute virus of mice (MVM) as relevant viruses to achieve the aim of this study. A downstream process for the production of purified HPV-16 L1 VLPs consisted of detergent lysis of harvested cells, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. The capacity of each purification/treatment step to clear viruses was expressed as reduction factor by measuring the difference in log virus infectivity of sample pools before and after each process. As a result, detergent treatment (0.5% v/v, Nonidet P-40/phosphate-buffered saline) was effective for inactivating enveloped viruses such as JEV and BVDV, but no significant reduction (< 1.0 log(10)) was observed in the non-enveloped MVM. The CsCl equilibrium density centrifugation was fairly effective for separating all three relevant adventitious viruses with different CsCl buoyant density from that of HPV-16 L1 VLPs (JEV, BVDV, and MVM = 4.30, 3.10, > or = 4.40 log(10) reductions). Given the study conditions we used, overall cumulative reduction factors for clearance of JEV, BVDV, and MVM were > or = 10.50, > or = 9.20, and > or = 6.40 log(10) in 150 ml of starting cell cultures, respectively.

Surveillance study (2000 to 2001) of G- and P-type human rotaviruses circulating in South Korea.

Human rotavirus VP4 and VP7 gene sequences were amplified by reverse transcription-PCR from 53% (322 of 607) of fecal specimens collected from children with severe diarrhea who visited hospitals in six urban areas of South Korea in 2000 and 2001. G2 was the most frequently found G type (constituted 50.6%), followed by G1 (30.1%) and G4 (13.0%). Although the P types of high incidence were P[4] (53.1%) and P[8] (21.4%), a significant incidence of P[6] (20.2%) was also noticeable. The commonest G- and P-type combination found in this study was G2P[4], rather than G1P[8], the most prevalent type known worldwide.

Development of real-time RT-PCR for evaluation of JEV clearance during purification of HPV type 16 L1 virus-like particles.

Insect cell culture has greatly increased in part due to the widespread use of insect virus-based vectors for efficient expression of foreign proteins. Insect cells such as Sf9 cells are susceptible to arboviruses which may pose a safety concern by adventitious introduction during the production process. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived biotechnological products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses that are known to be infectious in insect cells. Here we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. The JEV specific primer was selected from the 3' untranslated region, and the expected band size was 323 base pairs (bp). The sensitivity of the assay was calculated to be approximately 15 TCID(50)per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. JEV clearance was determined during the purification process of rHPV-16 L1 VLPs by CsCl equilibrium density centrifugation. The comparative results obtained by real-time RT-PCR assay for JEV and infectivity titrations suggested that the real-time RT-PCR assay could have an additive effect on the interpretation and evaluation of virus clearance, especially during the virus removal process.