Inactivated rabies vaccines: Standardization of an in vitro assay for residual viable virus detection.

Human rabies, a neglected viral zoonosis, is preventable through domestic animals vaccination and post-exposure prophylaxis using inactivated rabies vaccines. During vaccine production, several mandatory in vivo quality control trials, such as potency, live virus, and safety, are responsible for the use of large numbers of laboratory animals. Over the years, global organizations encouraged the development of alternative methods to reduce, replace and refine the use of animals in the pharmaceutical industry. In this study we standardized an in vitro assay for determination of residual live virus combining viral isolation techniques with direct immunofluorescence detection and viral quantification by a molecular method. Standardization of viral recovery steps and quantification by RT-qPCR were performed and the combined method was shown to be 3 fold more sensitive than the in vivo assay. It was possible to identify viral suspensions cultures, which still had residual viable rabies virus particles, evidencing the importance to implement this method in quality control schemes of rabies vaccine production. In addition, this developed assay is more practical, inexpensive and less time consuming, producing results in just 4 days, which may allow greater agility in the internal quality control of the vaccine. The in vitro method may reduce 2/3rd of laboratory animals numbers used for this purpose, since it can be applied in the intermediate quality control of inactivated rabies vaccine production.

Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines.

Rabies is an infectious viral disease, characterized as a neglected zoonosis, responsible for nearly 60,000 deaths annually. The virus is transmitted mainly by dogs in Africa and Asia, and wildlife in Europe and the Americas, to all mammals' species, causing severe encephalitis almost always fatal after the onset of neurological symptoms. Human rabies can be prevented through extensive vaccination of dogs and pre/post-prophylaxis treatments in humans with inactivated rabies vaccines. The vaccine manufacture involves a series of quality control assays using laboratory animals, which are mandatory to exclude the presence of viable residual virus. The quality controls must be carried out in various steps during the vaccine production, which demands the use of a large number of animals. In this study, we standardized a real-time quantitative RT-PCR duplex assay to be used during intermediate stages of the vaccine production. This assay was done for the quantification of vaccine strain rabies virus, targeting rabies nucleoprotein, and ß-actin mRNA of BHK-21 cells as an internal endogenous control. The results showed specific amplification, with the analytical sensitivity ranged from 101 to 106 TCID50/mL with high repeatability rate for the quantification of rabies virus in inactivated vaccine samples. Global organizations are engaged to develop new approaches to determine viable residual virus, and this assay can be applied in combination with traditional in vitro methods for the release of intermediate batches of vaccines during the production process, keeping the in vivo tests only for final release.

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1.

Prion diseases involve the conversion of the endogenous cellular prion protein, PrP(C), into a misfolded infectious isoform, PrP(Sc). Several functions have been attributed to PrP(C), and its role has also been investigated in the olfactory system. PrP(C) is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp(-/-) mice showed impaired behavior in olfactory tests. Given the high PrP(C) expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP(C)-binding partners. Ten different putative PrP(C) ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP(C) with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP(C)-Stub1 interaction are under investigation. The PrP(C)-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP(C) is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.

Human neutrophil migration and activation by BJcuL, a galactose binding lectin purified from Bothrops jararacussu venom.

BACKGROUND: Neutrophil migration to an inflamed site constitutes the first line of the innate immune response against invading microorganisms. Given the crucial role of endogenous lectins in neutrophil mobilization and activation, lectins from exogenous sources have often been considered as putative modulators of leukocyte function. Lectins purified from snake venom have been described as galactoside ligands that induce erythrocyte agglutination and platelet aggregation. This study evaluated human neutrophil migration and activation by C-type lectin BJcuL purified from Bothrops jararacussu venom. RESULTS: Utilizing fluorescence microscopy, we observed that biotinylated-BJcuL was evenly distributed on the neutrophil surface, selectively inhibited by D-galactose. Lectin was able to induce modification in the neutrophil morphology in a spherical shape for a polarized observed by optical microscopy and exposure to BJcuL in a Boyden chamber assay resulted in cell migration. After 30 minutes of incubation with BJcuL we found enhanced neutrophil functions, such as respiratory burst, zymozan phagocytosis and an increase in lissosomal volume. In addition, BJcuL delays late apoptosis neutrophils. CONCLUSION: These results demonstrate that BJcuL can be implicated in a wide variety of immunological functions including first-line defense against pathogens, cell trafficking and induction of the innate immune response since lectin was capable of inducing potent neutrophil activation.