[Vaccinovigilance: Adverse reaction reports of animal vaccines in 2020]. / Vaccinovigilance: Gemeldete unerwünschte Arzneimittelwirkungen immuno­logischer Tierarzneimittel im Jahr 2020.

INTRODUCTION: The aim of the vigilance system in Switzerland is the evaluation and classification of reported suspected adverse reactions of immunological veterinary medicines (IVMP), including suspected lack of expected efficacy. The Institute of Virology and Immunology (IVI) is the competent authority for marketing authorizations of immunological veterinary medicinal products in Switzerland and responsible for the vaccinovigilance system. In 2020, 130 adverse reaction reports were received (5% less compared to 2019). The reports mainly concerned dogs (41%) and cats (25%) followed by cattle (18%) and horses (7%). Many of the reports in dogs involved the application of combined vaccines against canine distemper, hepatitis, parvovirosis and parainfluenza in combination with canine leptospira components, in cats against cat flu and feline panleukopenia in combination with feline leukaemia virus infection. Causality assessments were done according to the international ABON system. In 27% of the reported cases, the causality assessments between the vaccination and the reaction described were evaluated as being probable (ABON A), in 44% as possible (ABON B).

INTRODUCTION: L'objectif du système de vaccinovigilance en Suisse est l'évaluation et la classification des effets indésirables suspectés signalés en rapport à des médicaments vétérinaires immunologiques, y compris le manque présumé d'efficacité attendue. L'Institut de virologie et d'immunologie (IVI) est l'autorité compétente pour les autorisations de mise sur le marché des médicaments immunologiques vétérinaires en Suisse et est responsable du système de vaccinovigilance. En 2020, 130 déclarations d'effets indésirables ont été reçues (5% de moins par rapport à 2019). Les signalements concernaient principalement les chiens (41%) et les chats (25%) suivis des bovins (18%) et des chevaux (7%). Chez les chiens, ces annonces impliquaient principalement l'application de vaccins combinés contre la maladie de Carré, l'hépatite, la parvovirose et la parainfluenza en combinaison avec la leptospirose, chez les chats contre le coryza et la panleucopénie féline en association avec la leucose féline. Les évaluations de causalité ont été effectuées selon le système international ABON. Dans 27% des cas rapportés, les évaluations de causalité entre la vaccination et la réaction décrite ont été évaluées comme probables (ABON A), dans 44% comme possibles (ABON B).

Evaluation of the sensitivity of PCR methods for the detection of extraneous agents and comparison with in vivo testing.

In this study the sensitivity of polymerase chain reaction (PCR) methods for the detection of Newcastle disease virus (NDV), avian reovirus (ARV), avian influenza virus (AIV) and avian infectious bronchitis virus (IBV) was compared to the sensitivity of the corresponding serological tests described in the European Pharmacopoeia (Ph. Eur.). For this purpose, serial 10-fold dilutions of the respective inactivated vaccines were prepared and groups of SPF chickens were vaccinated with a double dose of the vaccine dilutions. After a period of 21 days, the animals were revaccinated with a single dose. Two weeks later, serum samples from each animal were tested for antibodies using an Idexx enzyme linked immunosorbent assay (ELISA). In parallel, samples of the diluted vaccines were tested by PCR. It was found that the sensitivity of the four PCR tests is comparable to or even slightly better than that of the corresponding serological tests. Thus these PCR tests fulfil the sensitivity requirements of the Ph. Eur. and could be used as alternative tests for the detection of extraneous agents in final batches of inactivated vaccines.

Pre-validation study for testing of avian viral vaccines for extraneous agents by PCR.

The biological nature of vaccines imposes a permanent risk for contamination with extraneous agents. Therefore, testing of vaccines for freedom from extraneous agents is essential in the manufacturing process and quality control. Relevant methods for testing for extraneous agents of avian viral vaccines are specified in the monographs of the European Pharmacopoeia (Ph. Eur.). Currently, most of these methods involve the use of embryonated eggs or chickens. Polymerase chain reaction (PCR) is a widely used and suitable tool for the amplification and detection of extraneous nucleic acids. Different PCR assays have been developed for the application in routine testing of veterinary vaccines. However, before introduction of new methods in monographs of the Ph. Eur., they must undergo validation. Here we report about a pre-validation study performed in Official Medicines Control Laboratories (OMCLs). Diluted samples of avian infectious laryngotracheitis, avian infectious bronchitis and avian infectious bursal disease viruses have been analysed using standardised procedures and reagents. The study demonstrated that PCR methods can be transferred to other laboratories. The results also show that further work is warranted for full validation of the method.

DNA vaccines: safety aspect assessment and regulation.

For licensing purposes, besides the immunogenic aspects, deoxyribonucleic acid (DNA) vaccines present safety considerations that must be critically assessed during preclinical or/and clinical safety studies. The major concerns with regard to safety are integration of the plasmid DNA into the host genome, adverse immunopathological effects, the formation of anti-DNA antibodies resulting in auto-immune disease and the use of novel molecular adjuvants. Moreover, for veterinary vaccines intended to be used in husbandry animals, food safety aspects will become an important issue. All new vaccine candidates should therefore be thoroughly tested in target animals, keeping in mind that for food producing animals, the products will be consumed. Finally, a further safety aspect of interest concerns the possible spread of genetic material to the environment, by the potential transformation of the environmental microflora with only a few copies of complete or fragmented plasmid. These are issues that need to be considered in the final scientific decisions underpinning the registration of vaccines. Thus, to establish criteria for guidance and regulations for industry and licensing authorities, a project has been initiated to assess such risks of plasmid DNA vaccinations. Major emphasis will be placed on aspects such as the biodistribution of plasmid in vaccinated animals. This paper is intended as a contribution to the debate on the use of biotechnology in the future and should facilitate further discussions on the various safety aspects of DNA-based immunisations.

Monitoring veterinary vaccines for contaminating viruses.

The detection of extraneous agents (EA) has always been critical for assessing the safety associated with vaccines. Vaccines and other biological medicines are derived from living substrates. This poses particular problems with regard to assuring their efficacy and safety due to the inherent variability of starting materials, the production processes, and the complex nature of the products themselves. For viral vaccines and other biological medicines, the key to effective quality control is rigorous testing and validation applied to the seed lots and also to the final batches. The quality assurance requirements for marketing products in Europe are given in the European Pharmacopoeia (EP). With the advent of molecular biotechnology, there are increasing uses of molecular methods for the characterization and quality control of vaccines. PCR for extraneous agent testing for live and inactivated poultry vaccines has been recognized to be a valid alternative testing method and is now being used extensively as a diagnostic tool in assuring the freedom from EAs. A number of generic PCR assays for product testing have been developed and validated. The current status and issues of the application of nucleic acid testing (NAT) to the standardization and control of vaccines will be reviewed.

Serological responses of adult dogs to revaccination against distemper, parvovirus and rabies.

Serum antibody titres to canine distemper virus (CDV), canine parvovirus (CPV) and rabies were measured in dogs that had not been revaccinated annually and compared with the titres in a control group of regularly vaccinated animals; 83 per cent (171 of 207) of the dogs vaccinated against CDV one or more years earlier had serum neutralising antibody titres equal to or greater than 16; 64 per cent (136 of 213) of the dogs vaccinated against CPV one or more years earlier had haemagglutination inhibiting titres equal to or greater than 80; and 59 per cent (46 of 78) of the dogs vaccinated against rabies two or more years earlier had serum neutralising antibody titres equal to or greater than 0.5 iu/ml. Three weeks after a single booster vaccination the dogs' antibody titres against CDV had increased above the threshold level in 94 per cent of the dogs, against CPV in 68 per cent, and against rabies in 100 per cent.

Use of PCR for extranous agent testing in avian vaccines.

Quality control of biologicals, in particular avian vaccines, includes certification of freedom from extraneous agents (EA). Adventitious viruses have always represented a major concern as a potential risk associated with the use of biologicals. Extensive testing for the presence of potential extraneous agents is therefore required to ensure the safety of the vaccines. To replace the use of laboratory animals (the Three Rs-approach) we developed and implemented a series of alternative PCR tests for detection of extraneous agents in avian viral vaccines (the PCR-approach). The application of PCR to screen poultry vaccines for viral contaminants is an example of how PCR can be usefully employed in quality control assays. The object of this work is to show how the use of in vitro methods can contribute to reduction in animal testing in the quality control of biologicals.

Use of polymerase chain reaction (PCR) for the detection of vaccine contamination by infectious laryngotracheitis virus.

Quality control of biologicals for veterinary use includes certification of freedom from extraneous agents. Contamination of vaccines may originate from various materials used for production and during manufacturing process. Requirements for avian virus vaccines to demonstrate freedom of adventitious agents are stated in the European Pharmacopoeia and include monitoring for infectious laryngotracheitis virus (ILTV). ILTV is an avian herpesvirus belonging to the alphaherpesvirus subfamily causing acute respiratory disease. To date the methods to detect ILTV contaminating biologicals consist of demonstration of antibody induction in chicken after immunization or virus cultivation in embryonated eggs. These methods are time consuming and laborious. Therefore, a specific, simple and sensitive in vitro polymerase chain reaction (PCR) for the detection of ILTV contamination in avian virus vaccines was developed. Primers were designed to amplify part of the p32 gene. Four different ILTV vaccine strains could be unequivocally detected. The identity of the amplified fragment was confirmed by restriction endonuclease analysis.

Use of reverse transcriptase polymerase chain reaction for detection of vaccine contamination by avian leukosis virus.

A reverse transcriptase polymerase chain reaction (RT-PCR) for avian leukosis virus (ALV) was developed for the detection of contamination of vaccines produced in embryonated eggs and cell cultures derived from chicken. ALV is highly pathogenic and induces a wide spectrum of disease in infected animals. ALV can be divided into five subgroups (A-E). The envelope glycoprotein (env gp85) is the main antigen determinant and responsible for subgroup classification. Viral RNA of all subgroups (A-E) was isolated and amplified using three sets of primers. Subsequently, restriction endonuclease analysis confirmed the product identity and discriminated between subgroups. In specific pathogen free (SPF) eggs experimentally inoculated with ALV, viral RNA was found in allantoic fluids, as well as in vaccines spiked with different subgroups of ALV. No adventitious virus was detected in commercially available preparations. This system provides a rapid and specific in vitro method for the detection of ALV RNA as an extraneous agent and may be applied for quality control of avian vaccines.

Molecular cloning and differential expression patterns of avian glutamate receptor mRNAs.

We have identified and studied potential ionotropic glutamate receptor genes in pigeon brain. Three cDNA clones exhibit significant amino acid sequence identity to members of a rodent ligand-gated ion channel family. One of them, GluP-II, encodes a full-length AMPA-sensitive glutamate receptor GluR2 (GluR-B) homologue, whereas the other two partial clones, designated as GluP-III and -IV, are nearly identical to rodent GluR3 (GluR-C) and GluR4 (GluR-D) receptor subunits. Northern analysis demonstrated that the avian genes are widely expressed in the brain. Within the brain regions analyzed by in situ hybridization histochemistry, the three avian GluR subunits showed distinct and regionally specific mRNA expression patterns in the adult. Most of the differences in their expression were observed in cell types of the telencephalon, certain thalamic nuclei, the optic tectum, and the cerebellar cortex. A particularly striking finding was the expression of GluP-II in Golgi epithelial/Bergmann glial cells. In contrast, Bergmann glial cells in rat cerebellum do not express GluR2 (GluR-B) subunit genes. Immunoreactivity for a monoclonal sequence-specific antipeptide antibody was widespread and most prominent in Purkinje cell perikarya and their dendrites, neuronal cell bodies of the ectostriatum, and the deep optic tectum. These results demonstrate the existence of multiple subunits of the ionotropic glutamate receptor channel family in avians. Excitatory amino acid receptor genes appear to be highly conserved during evolution.

Primary structure of mouse secretogranin III and its absence from deficient mice.

The rat brain- and pituitary-specific 1B1075 mRNA encodes a chromogranin/secretogranin-like protein, called secretogranin III (SgIII), that is a component of intracellular dense core vesicles. In order to study the function of this gene product in a mouse model system, we have isolated the murine homolog of the rat 1B1075 mRNA. This mRNA contains 2163 bp encoding a putative protein of 421 amino acids. Cleavage of the strong putative signal sequence would yield a mature protein of 51 kDa. The sequence of the encoded murine protein preserves the structural features that suggest SgIII is a member of the granin family, and allowed us to recognize and correct errors in our published rat sequence.

1B1075: a brain- and pituitary-specific mRNA that encodes a novel chromogranin/secretogranin-like component of intracellular vesicles.

The rat 1B1075 mRNA encodes a 533-residue novel chromogranin/secretogranin-like acidic protein that contains an apparent secretion signal, several pairs of tandem basic residues, and internally repeated sequence elements. 1B1075 transcripts are detected, by blotting and in situ hybridization, at the highest levels in the neocortex, hippocampus, cerebellar cortex, selected pontine and diencephalic nuclei, and presumptive pituitary corticotrophs, at lower levels in specific nuclei in most other brain regions, but in none of several other tissues. Utilizing antisera to several nonoverlapping synthetic peptide fragments of the predicted protein sequence, we detect a brain- and pituitary-specific 57-kDa protein in cellular processes and fiber tracts, generally consistent with axonal transport from the cell bodies identified by in situ hybridization. Ultrastructural studies demonstrate that this protein is a component of intraneuronal vesicles in axons and vesicle-like structures in dendrites. Based on these data, we suggest the name Secretogranin III for the 1B1075 gene product. In related collaborative studies, a mouse deleted for the 1B1075-homologous gene has been produced that should allow assessment of its physiological role.

Genetic ablation of a mouse gene expressed specifically in brain.

The 1B1075 gene was initially identified from a cDNA clone of a rat brain messenger RNA expressed in particular subsets of CNS neurons and pituitary cells. Although the protein encoded by this gene is of unknown function, its sequence suggests that it may be related to secretogranin proteins, which are found in association with secretory granules in a variety of peptidergic endocrine and neuronal cells. Here we show that the mouse 1B1075 gene is located between the dilute (d) and short ear (se) genes on chromosome 9. Many different deletion mutations have previously been isolated in the genetic region that includes these genes. By producing mice carrying two deletions that overlap at the 1B1075 locus, the gene for this brain-specific message can be completely eliminated from otherwise viable animals. The animals missing the 1B1075 gene provide an important new tool for determining the function of this gene in the brain. In addition, these results provide a new molecular entry point for detailed characterization of other genes in the d-se region.

Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork.

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.