Future of TABST and LABST in the Indian Pharmacopoeia Monographs A Humane Society International/India Workshop Report.

Humane Society International India (HSI India) organized and facilitated a workshop on the 'Future of Target Animal Batch Safety Test (TABST) and Laboratory Animal Batch Safety Test (LABST) in the Indian Pharmacopoeia (IP) Monographs'. The workshop hosted key Indian regulators from the Indian Pharmacopoeia Commission (IPC) and the Central Drugs Standard Control Organization (CDSCO), industry representatives from the Indian Federation of Animal Health Companies (INFAH), Asian Animal Health Association (AAHA), and international experts representing the European Directorate for the Quality of Medicines (EDQM), the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH), and multinational veterinary products manufacturers. The workshop was organized to encourage a bidirectional flow of information and to discuss the deletion of TABST and LABST from the veterinary vaccine monographs in the IP. This workshop was built from the symposium held by Humane Society International on the 'Global Harmonization of Vaccine Testing Requirements' held in 2019. This report details the outcomes of the workshop with proposed activities to be taken up as part of the next steps for the elimination or waiving of these tests.

Impact of metabolic indices of 18F-fluorodeoxyglucose positron emission tomography/computed tomography on post transplantation recurrence of hepatocellular carcinoma.

BACKGROUND: Tumor recurrence is the leading cause of death after liver transplantation in patients with hepatocellular carcinoma. There is an ongoing debate as to whether metabolic indices such as tumor to liver standardized uptake value ratio in 18F-fluorodeoxyglucose positron emission tomography/computed tomography of the primary tumor can identify patients outside the Milan criteria with as low recurrence rates as patients inside Milan and thus should be added to the established prognostic factors. METHODS: This retrospective study analyzes 103 consecutive patients who underwent 18F-fluorodeoxyglucose positron emission tomography/computed tomography before liver transplantation for hepatocellular carcinoma using data of clinical tumor registry. Primary endpoints were overall survival and 10-year cumulative recurrence rates. RESULTS: Tumor to liver standardized uptake value ratio of the primary tumor was statistically significant higher in Milan out tumors, "up-to-seven" out tumors, grade 3 tumors, α- fetoprotein level >400 ng/ml and lesions > 5cm in diameter. Factors with statistically significant influence on the 10- year overall survival in the univariate analysis were Milan, up-to-seven" criteria, number of lesions and pT-category. COX regression analysis did not show independently statistically significant factors for 10-year overall survival. Milan, "up-to-seven" criteria, grade, pV, number of lesions, size of lesion, pT-category, tumor to liver standardized uptake value ratio influenced 10-year cumulative recurrence rates statistically significant. Tumor to liver standardized uptake value ratio, grade and pT-category proved to be independently statistically significant factors for 10-year cumulative recurrence rates. CONCLUSIONS: Our study suggests that tumor to liver standardized uptake value standardized uptake value ratio in 18F-fluorodeoxyglucose positron emission tomography/computed tomography is an independent prognostic factor in transplanted patients with hepatocellular carcinoma. If we focus on preoperative findings, such as tumor size, tumor number and AFP value adding the information given by TLR of 18F-FDG PET/CT allows to estimate the risk of tumor recurrence more accurate than the established classifications Milan and UTS. Therefore, it may add valuable information to other preoperative findings, such as tumor size, tumor number and AFP level.

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.

Flexible and broadly tunable infrared light source based on shaped sub-10-fs pulses for a multimodal microscopy setup.

We present a versatile approach for mid-infrared spectroscopy through the flexible control of a difference-frequency-generation (DFG) process by femtosecond (fs) pulse shaping and spectral focusing. Based on a broadband sub-10-fs oscillator, the spectral position and spectral resolution can be independently selected within the molecular fingerprint region of more than 2000 cm-1. A spectral resolution better than 20 cm-1 can be achieved, which depends solely on the pulse shaper configuration. An absorption experiment on a polystyrene reference sample finally validates the concept and opens the door for an additional modality in nonlinear multimodal microscopy setups.

Enhancement of coherent anti-Stokes Raman signal via tailored probing in spectral focusing.

A novel approach for spectral focusing using a single-beam coherent anti-Stokes Raman scattering setup with a pulse shaper controlling the phase and amplitude is presented. By identifying the frequencies acting as the pump, Stokes, and probe, the high degree of control can be exploited in order to specifically and independently tailor the spectral region to act only as probe to achieve the highest signal intensity. While maintaining the optimal excitation of the vibrational coherence, a signal increase by a factor of six in comparison with usual spectral focusing schemes is readily obtained. The signal improvement and contrast is demonstrated on human skin tissue.

Multimodal nonlinear optical microscopy with shaped 10 fs pulses.

We demonstrate the use of shaped 10 fs pulses for multimodal microscopy. The combination of a broadband oscillator and a pulse shaper provides a flexible light source that can be optimized for various nonlinear effects produced in the sample, either for signal intensity or for selectivity. While the highest nonlinear generation efficiency is achieved with the shortest pulses, more complex waveforms address specific transitions in the sample for better contrast. This is shown experimentally with the imaging of a moss leaf and of human skin biopsies using coherent anti-Stokes Raman scattering, two-photon fluorescence and second harmonic generation signals.

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.

European regulatory framework and practices for veterinary Leptospira vaccine potency testing.

In Europe, the legal basis for requirements for medicinal products is described in the European Pharmacopoeia (Ph. Eur.) In the European Union, the Ph. Eur. is supplemented by several guidelines issued by the European Medicines Agency. Immunological veterinary products must comply with the Ph. Eur. monograph on veterinary vaccines and the accompanying texts, as well as specific monographs. The Ph. Eur. includes monographs on canine leptospirosis and bovine leptospirosis vaccines (inactivated). Both monographs require that an immunogenicity test be performed once in the target species during the life of a vaccine. The hamster challenge test is applied for batch potency testing of canine vaccines. Alternatively, serological tests or suitable validated in vitro tests to determine the content of one or more antigenic components indicative of protection may be performed. Vaccines for use in cattle are tested in a serological test in guinea pigs. The acceptance criteria in alternative tests are set with reference to a batch of vaccine that has given satisfactory results in the immunogenicity test. At a January 2012 European workshop, the suitability of the hamster potency test was questioned and unanimous agreement was reached that moving toward complete in vitro testing is possible and should be promoted.

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: state of the science and planning the way forward.

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.

Three Rs Approaches in the Production and Quality Control of Fish Vaccines.

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants - experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry - agreed that efforts should be undertaken to replace the vaccination-challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum.

The consistency approach for the quality control of vaccines.

Current lot release testing of conventional vaccines emphasizes quality control of the final product and is characterized by its extensive use of laboratory animals. This report, which is based on the outcome of an ECVAM (European Centre for Validation of Alternative Methods, Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, Italy) workshop, discusses the concept of consistency testing as an alternative approach for lot release testing. The consistency approach for the routine release of vaccines is based upon the principle that the quality of vaccines is a consequence of a quality system and of consistent production of lots with similar characteristics to those lots that have been shown to be safe and effective in humans or the target species. The report indicates why and under which circumstances this approach can be applied, the role of the different stakeholders, and the need for international harmonization. It also gives recommendations for its implementation.

Application of RT-PCR for the detection of avian reovirus contamination in avian viral vaccines.

An efficient procedure for the detection of avian reovirus (ARV)-specific RNA sequences in veterinary immunological medicinal products using reverse transcriptase polymerase chain reaction (RT-PCR) is described. Four ARV vaccine strains (1133, 1733, 2408 and Olson WVU2937), two ATCC strains (VR826 and VR856) as well as several ARV field isolates obtained from domestic, wild and pet birds could be easily detected with S2- and S4-specific primers. The identity of the amplified fragments was confirmed by restriction endonuclease analysis and sequence analysis. Furthermore, not only the starting substrates used for the production of a vaccine, but also additives and the manufacturing process (matrix effects) can influence the results. These RT-PCRs have been useful for screening poultry vaccines for extraneous ARV in the quality control of biologicals used in veterinary medicine.

Early DNA vaccination of puppies against canine distemper in the presence of maternally derived immunity.

Canine distemper (CD) is a disease in carnivores caused by CD virus (CDV), a member of the morbillivirus genus. It still is a threat to the carnivore and ferret population. The currently used modified attenuated live vaccines have several drawbacks of which lack of appropriate protection from severe infection is the most outstanding one. In addition, puppies up to the age of 6-8 weeks cannot be immunized efficiently due to the presence of maternal antibodies. In this study, a DNA prime modified live vaccine boost strategy was investigated in puppies in order to determine if vaccinated neonatal dogs induce a neutralizing immune response which is supposed to protect animals from a CDV challenge. Furthermore, a single DNA vaccination of puppies, 14 days after birth and in the presence of high titers of CDV neutralizing maternal antibodies, induced a clear and significant priming effect observed as early as 3 days after the subsequent booster with a conventional CDV vaccine. It was shown that the priming effect develops faster and to higher titers in puppies preimmunized with DNA 14 days after birth than in those vaccinated 28 days after birth. Our results demonstrate that despite the presence of maternal antibodies puppies can be vaccinated using the CDV DNA vaccine, and that this vaccination has a clear priming effect leading to a solid immune response after a booster with a conventional CDV vaccine.

[The establishment of endotoxin limits which satisfy animal welfare considerations in the testing of porcine vaccines]

Endotoxin (lipopolysaccharide, LPS) is a constituent of the cell walls of gram-negative bacteria and is found in many vaccines produced from these bacteria. High levels of endotoxin can give rise to a range of pathophysiological reactions, and adverse reactions tend to be seen in animals following vaccination. In this study, pigs of various ages and weights were vaccinated with licensed porcine vaccines and the endotoxin content of the vaccines was determined with the LAL, conducted according to DAB 10. The experiments followed the DAB guidelines relating to the safety testing of veterinary vaccines. The animals were monitored for 48 h after vaccination, their body temperatures were measured, and blood samples were taken for analysis and for the determination of plasma endotoxin levels. There was a clear relationship between vaccine endotoxin content and changes in blood cell counts and in the clinical picture. Elevated plasma endotoxin levels correlated with the occurrence of initial leucopenia followed by leucocytosis as well as with clinical symptoms ranging from refusal of food and depression to shock-like symptoms. After 24 h, normal physiological values were regained. Young animals weighing between 10 and 40 kg were found to be very sensitive to elevated endotoxin content in vaccines. The differences in individual reactions could be due not only to differences in vaccine endotoxin content, but also to differences in the reactivity of the organism, and in the type of bacteria used or in the composition of the vaccine.