3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report.

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled "3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities" was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.

A Recent Advancement in Nanotechnology Approaches for the Treatment of Cervical Cancer.

BACKGROUND: Cervical cancer is one of the leading causes of female death, with a mortality rate of over 200,000 per year in developing countries. Despite a decrease in cervical cancer occurrences in developed countries over the last decade, the frequency of the disease in developing nations continues to rise at an alarming rate, particularly when it is linked to the human papillomavirus (HPV). With just a few highly invasive conventional therapies available, there is a clear need for novel treatment options such as nanotechnology-based chemotherapeutic drug delivery. METHODS: Traditional anticancer therapy is limited by poor drug potency, non-specificity, unwanted side effects, and the development of multiple drug resistance (MDR), leading to a decrease in long-term anticancer therapeutic efficacy. An ideal cancer therapy requires a personalized and specialized medication delivery method capable of eradicating even the last cancer cell responsible for disease recurrence. RESULTS: Nanotechnology provides effective drug delivery mechanisms, allowing it to serve both therapeutic and diagnostic purposes. Nanotechnology-based formulations are widely used to accurately target the target organ, maintain drug load bioactivity, preferentially accumulate the drug at the target location, and reduce cytotoxicity. CONCLUSION: The key benefits of this drug delivery are that it improves pharmacological activity, solubility, and bioavailability and reduces toxicity in the target tissue by targeting ligands, allowing for new innovative treatment methods in an area that is desperately required. The goal of this review is to highlight possible research on nanotechnologybased delivery systems for cancer detection and treatment.

Graph Theoretical analysis, in silico modeling and molecular dynamic studies of (5-((2-chloropyridin-4-yl)oxy)-3-phenyl-1H-pyrazol-1-yl)-2-(4-substituted phenyl)-N,N-dimethylethen-1-amine derivatives for the treatment of breast cancer.

BACKGROUND: We synthesized a series of novel amide derivatives of (5-((2-chloropyridin-4-yl)oxy)-3-phenyl-1H-pyrazol-1-yl)-2-(4-substituted phenyl)-N,N-dimethylethen-1-amine [5a-5r] and assessed for their antiproliferative activity against human breast cancer cell line MCF7 by using MTT assay. Graph Theoretical analysis, in silico modeling, molecular dynamic studies, and ADME profile were screened for the synthesized compounds. Based on the observed report, the significant compounds were chosen for their anticancer activity. Graph Theoretical analysis, in silico modeling and molecular dynamic studies of (5-((2-chloropyridin-4-yl)oxy)-3-phenyl-1H-pyrazol-1-yl)-2-(4-substitutedphenyl)-N,N-dimethylethen-1-amine derivatives for the treatment of breast cancer. METHODS: 5-((2-chloropyridin-4-yl)oxy) (2-phenyl-1H-pyrazol-1-yl)-3-phenyl-1H-pyrazol-1-yl)-3-phenyl-1H-pyrazol-1- (4-substituted phenyl) -N,N-dimethylethen-1-amine [5a-5r] was synthesized using 2-bromo-1-phenylethanone and (5-(2-chloropyridin-4-yloxy)-3-phenyl-1H-pyrazol-1-yl)-N,N-dimethylmethanamine with different aromatic aldehydes and their characterization studies were evaluated by IR, NMR, and mass spectral analysis. RESULTS: The compound 2-(4-methylphenyl)-1-(5-((2-chloropyridin-4-yl)oxy)-3-phenyl-1H-pyrazol-1-yl)-N,N-dimethylethen-1-amine 5a and 2-(2-methylphenyl)-1-(5-((2-chloro pyridin-4-yl)oxy)-3-phenyl-1H-pyrazol-1-yl)-N,N-dimethylethen-1-amine 5c in the amide part exhibited promising cytotoxic activity against all cell lines with IC50 values of 3.3 mM for MCF-7 cells, and produced dramatic cell cycle arrest at EGFR phase as an indicator of apoptotic cell death induction. CONCLUSION: Based on their high potency in the cellular environment, these straightforward pyrazole-3-carboxamide derivatives may possess the potential to design more potent compounds for intervention with cancer cell proliferation.

An ELISA-based antigenicity test of rabies recombinant glycoprotein cannot predict its protective potency in vivo.

The potency of human and veterinary rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, variable, and requires sacrifice of large numbers of mice. ELISA-based methods quantifying rabies glycoprotein (rGP) are being developed as potential alternatives to the NIH potency test for release of rabies vaccines. The aim of the current study was focused on the evaluation of in vitro- and in vivo-based assays in order to assess their concurrence for adequate and reliable assessment of immunogenicity and protective potency of a plant-derived recombinant rGP. The recombinant rGP of strain ERA.KK was engineered, expressed and purified from Nicotiana benthamiana plants. The recombinant rGP excluded the transmembrane and intracytoplasmic domains. It was purified by chromatography (≥90%) from the plant biomass, characterized, and mainly presented as high molecular weight forms, most likely soluble aggregates, of the rGP ectodomain. It was well-recognized and quantified by an ELISA, which utilizes two mouse monoclonal antibodies, D1-25 and 1112-1, and which should only recognize the native trimeric form of the rGP. However, in mice, the recombinant rGP did not induce the production of anti-rabies virus neutralizing antibodies and did not confer protection after intracerebral viral challenge. Similar immunogenicity was observed in guinea pigs and rabbits. Our results demonstrate that use of the ELISA method described here is not predictive of performance in vivo. These data highlight the critical need to develop in vitro potency assays that reliably define the antigen content that can induce a protective response.

Global harmonization of vaccine testing requirements: Making elimination of the ATT and TABST a concrete global achievement.

This one-day symposium organized by Humane Society International (HSI) brought together 18 international experts from Argentina, Brazil, China, Europe, India, Russia, South Africa and the United States to discuss the elimination of the abnormal toxicity test (ATT) from the testing requirements for human vaccines as well as the target animal batch safety test (TABST) and the laboratory animal batch safety test (LABST) for veterinary vaccines. Participants reported on country-specific regulatory requirements and, where present, the perspectives on waiver and elimination of those tests. In addition, the attendees, with HSI in the role of facilitator, moved to define the barriers to the complete elimination or waiving of these tests. This report expounds the outcomes of the symposium, and introduces a proposed roadmap - populated with country specific activities - for the elimination of these tests.

Achieving scientific and regulatory success in implementing non-animal approaches to human and veterinary rabies vaccine testing: A NICEATM and IABS workshop report.

This two-day workshop, co-sponsored by NICEATM and IABS-NA, brought together over 60 international scientists from government, academia, and industry to advance alternative methods for human and veterinary Rabies Virus Vaccine (RVV) potency testing. On day one, workshop presentations focused on regulatory perspectives related to in vitro potency testing, including recent additions to the European Pharmacopoeia (5.2.14) that provide a scientific rationale for why in vivo methods may be less suitable for vaccine quality control than appropriately designed in vitro methods. Further presentations reviewed the role of the consistency approach to manufacturing and vaccine batch comparison to provide supportive data for the substitution of existing animal-based methods with in vitro assays. In addition, updates from research programs evaluating and validating RVV glycoprotein (G) quantitation by ELISA as an in vitro potency test were presented. On the second day, RVV stakeholders participated in separate human and veterinary vaccine discussion groups focused on identifying potential obstacles or additional requirements for successful implementation of non-animal alternatives to the in vivo potency test. Workshop outcomes and proposed follow up activities are discussed herein.

Re-evaluating the LD50 requirements in the codified potency testing of veterinary vaccines containing Leptospira (L.) serogroup Icterohaemorrhagiae and L. serogroup Canicola in the United States.

Approximately one-third of the reportable USDA Category D and E laboratory animals in the United States are expended on the potency testing of leptospiral vaccines by the codified hamster vaccination-challenge assay. Valid tests require ≥80% of challenge controls to succumb to disease and an LD50 between 10 and 10,000. This work evaluates the risk associated with the removal of LD50 limits; thereby, eliminating back-titration hamsters from in vivo potency assays for Leptospira (L.) serogroups Canicola and Icterohaemorrhagiae. The impact was assessed through 1) retrospective analysis of industry and CVB serial release data from July 2011-April 2015 and 2) evaluation through vaccination-challenge assays. For the initial vaccination-challenge assays (n = 3/serogroup), one group received potent bacterin (PB) and six groups received subpotent bacterins (SB1-SB6). PB and SB1 were challenged with a single dilution of Leptospira between 10 and 10,000 LD50. SB2-SB6 received serial dilutions of more concentrated challenge. Based on the retrospective analysis and in vivo assays, 80% of the challenge controls succumbing to disease reasonably ensured the minimal LD50 was administered. Subpotent vaccines were not at increased risk for being deemed potent when challenged with >10,000 LD50, but potent vaccines were at risk of being deemed subpotent when challenged with >10,000 LD50.

Report on the international workshop on alternative methods for Leptospira vaccine potency testing: state of the science and the way forward.

Routine potency testing of Leptospira vaccines is mostly conducted using a vaccination-challenge test that involves large numbers of hamsters and unrelieved pain and distress. NICEATM, ICCVAM, and their international partners organized a workshop to review the state of the science of alternative methods that might replace, reduce, and refine the use of animals for veterinary Leptospira vaccine potency testing and to identify ways to advance improved alternative methods. Vaccine manufacturers were encouraged to initiate or continue product-specific validation using in vitro enzyme-linked immunosorbent assays as replacements for potency testing of four common Leptospira serogroups. Participants discussed the potential for eliminating the back-titration procedure in the hamster challenge assay, which could reduce animal use by 50% for each individual potency test. Further animal reduction may also be possible by using cryopreserved Leptospira stock to replace continual passaging through hamsters. Serology assays were identified as a way to further reduce and refine animal use but should be considered only after attempting in vitro assays. Workshop participants encouraged consideration of analgesics and use of earlier humane endpoints when the hamster vaccination-challenge potency assay is used. International harmonization of alternative potency methods was recommended to avoid duplicative potency testing to meet regionally different requirements.

International regulatory requirements for Leptospira vaccine potency testing. Roundtable: current requirements and opportunity for harmonization.

Progress continues to be made in the ongoing efforts to replace, reduce, or refine the use of laboratory animals for Leptospira vaccine potency testing in certain markets/regions. Leptospira-containing vaccines, as with many veterinary vaccines, are manufactured and distributed both on a regional basis by local manufacturers and internationally by large multinational firms. Three general scenarios exist for the international testing and distribution of veterinary vaccines including: 1) the importing country recognizes the country of origin's testing and batch release data with no additional testing; 2) the importing country requires the manufacturer to conduct a specific potency assay based on the current importing market's regulations for the importing country or 3) the importing country requires retesting of the product in country prior to distribution. Scenarios 2 and 3 both have the potential to significantly increase the usage of laboratory animals for what may be considered redundant testing. Specific requirements for the importation of Leptospira vaccines in the United States, Europe, and Mexico were presented as well as efforts to reduce the use of laboratory animal testing through the availability of internationally recognized tests.

Non-animal replacement methods for veterinary vaccine potency testing: state of the science and future directions.

NICEATM and ICCVAM convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing methods and to identify opportunities to advance new and improved methods that can further reduce, refine, and replace animal use. Six topics were addressed in detail by speakers and workshop participants and are reported in a series of six reports. This workshop report, the second in the series, provides recommendations for current and future use of non-animal methods and strategies for veterinary vaccine potency testing. Workshop participants recommended that future efforts to replace animal use give priority to vaccines (1) that use large numbers of animals per test and for which many serials are produced annually, (2) that involve significant animal pain and distress during procedures, (3) for which the functional protective antigen has been identified, (4) that involve foreign animal/zoonotic organisms that are dangerous to humans, and (5) that involve pathogens that can be easily spread to wildlife populations. Vaccines identified as the highest priorities were those for rabies, Leptospira spp., Clostridium spp., Erysipelas, foreign animal diseases (FAD), poultry diseases, and fish diseases. Further research on the identification, purification, and characterization of vaccine protective antigens in veterinary vaccines was also identified as a priority. Workshop participants recommended priority research, development, and validation activities to address critical knowledge and data gaps, including opportunities to apply new science and technology. Recommendations included (1) investigations into the relative impact of various adjuvants on antigen quantification assays, (2) investigations into extraction methods that could be used for vaccines containing adjuvants that can interfere with antigen assays, and (3) review of the current status of rabies and tetanus human vaccine in vitro potency methods for their potential application to the corresponding veterinary vaccines. Workshop participants recommended enhanced international harmonization and cooperation and closer collaborations between human and veterinary researchers to expedite progress. Implementation of the workshop recommendations is expected to advance alternative in vitro methods for veterinary vaccine potency testing that will benefit animal welfare and replace animal use while ensuring continued protection of human and animal health.

Comparative analysis of the extracellular proteomes of two Clostridium sordellii strains exhibiting contrasting virulence.

Clostridium sordellii is a toxin-producing anaerobic bacillus that causes severe infections in humans and livestock. C. sordellii infections can be accompanied by a highly lethal toxic shock syndrome (TSS). Lethal toxin (TcsL) is an important mediator of TSS. We recently obtained a clinical strain of C. sordellii (DA-108) lacking the TcsL-encoding tcsL gene, which was not fatal in rodent models of infection, in contrast to a tcsL(+) reference strain (ATCC9714). Protein preparations derived from cell-free, stationary phase cultures obtained from ATCC9714 were lethal when injected into mice, while those obtained from DA-108 were not, a difference that was attributed to the unique presence of TcsL in the ATCC9714-derived proteins. We questioned whether there were other major differences between the extracellular proteomes of these two strains, apart from TcsL. Two-dimensional gel electrophoresis was conducted using crude cell-free supernatants from these strains and 14 differentially expressed proteins were subjected to mass spectrometric analysis. Nine of these 14 proteins were more highly expressed by DA-108 and 5 by ATCC9714. Twelve of the 14 proteins isolated from the 2-D gels were putatively identified by mass spectrometry. Several of these proteins were identical, possibly reflecting enzymatic cleavage, degradation, and/or post-translational modifications. Excluding identical sequences, only 5 unique proteins were identified. Four proteins (ferredoxin-nitrite reductase; formate acetyltransferase; Translation Elongation Factor G; and purine nucleoside phosphorylase) were over-expressed by DA-108 and 1 (N-acetylmuramoyl-l-alanine amidase) by ATCC9714. These results support the concept that TcsL is the major determinant of C. sordellii TSS during infection.

Lethal toxin is a critical determinant of rapid mortality in rodent models of Clostridium sordellii endometritis.

The toxigenic anaerobe Clostridium sordellii is an uncommon but highly lethal cause of human infection and toxic shock syndrome, yet few studies have addressed its pathogenetic mechanisms. To better characterize the microbial determinants of rapid death from infection both in vitro and in vivo studies were performed to compare a clinical strain of C. sordellii (DA-108), isolated from a patient who survived a disseminated infection unaccompanied by toxic shock syndrome, to a virulent reference strain (ATCC9714). Rodent models of endometrial and peritoneal infection with C. sordellii ATCC9714 were rapidly lethal, while infections with DA-108 were not. Extensive genetic and functional comparisons of virulence factor and toxin expression between these two bacterial strains yielded many similarities, with the noted exception that strain DA-108 lacked the tcsL gene, which encodes the large clostridial glucosyltransferase enzyme lethal toxin (TcsL). The targeted removal by immunoprecipitation of TcsL protected animals from death following injection of crude culture supernatants from strain ATCC9714. Injections of a monoclonal anti-TcsL IgG protected animals from death during C. sordellii ATCC9714 infection, suggesting that such an approach might improve the treatment of patients with C. sordellii-induced toxic shock syndrome.