Licensing Requirements for Avian Vaccines Within the European Union.

Avian vaccines are a key factor when it comes to ensuring the availability of products derived from healthy poultry and preventing the transmission of infections from domestic and wildlife birds to humans. A marketing authorization for veterinary vaccines is granted after the product's quality, safety, and efficacy have been confirmed. During the licensing procedure, the manufacturing process is assessed to guarantee consistent quality and stability of the vaccine components. Furthermore, both the safety for the target species and the risk for the user, the consumer, and the environment must be demonstrated. In addition, specific tests and studies are required to support the efficacy of the vaccine. The authorization procedures and related licensing requirements for avian vaccines to be marketed in the European Union (EU) based on the requirements of Regulation (EU) 2019/6 Article 8 and the Commission Delegated Regulation (EU) 2021/805 amending Annex II to Regulation (EU) No. 2019/6 are explained in the paper.

Requisitos de licencia para vacunas aviares dentro de la Unión Europea. Las vacunas aviares son un factor clave a la hora de garantizar la disponibilidad de productos derivados de aves sanas y prevenir la transmisión de infecciones de aves domésticas y silvestres a los humanos. La autorización de comercialización de vacunas veterinarias se concede una vez confirmada la calidad, seguridad y eficacia del producto. Durante el procedimiento de concesión de licencia, se evalúa el proceso de fabricación para garantizar una calidad y estabilidad constantes de los componentes de la vacuna. Además, se debe demostrar tanto la seguridad para las especies a las que dicha vacuna está destinada, así como el riesgo para el usuario, el consumidor y el medio ambiente. Además, se requieren pruebas y estudios específicos que respalden la eficacia de la vacuna. En este documento se explican los procedimientos de autorización y los requisitos de licencia relacionados para las vacunas aviares que se comercializarán en la Unión Europea (U.E.) con base en los requisitos de la Regulación (U.E.) 2019/6 Artículo 8 y la Regulación Delegada de la Comisión (U.E.) 2021/805 que modifica el Anexo II del Reglamento. (U.E.) No. 2019/6.

[Regulatory aspects of the development, licensing, and availability of immunological veterinary medicinal products]. / Regulatorische Aspekte der Entwicklung, Zulassung und Verfügbarkeit von immunologischen Tierarzneimitteln.

Vaccines constitute an essential tool for animal health management. In addition to applying traditional vaccine development strategies to new or changing pathogens, the use of innovative approaches in this area is increasing. Recombinant vaccines often have superior safety and efficacy profiles, due to their specific attenuation and the ability to serologically distinguish vaccinated from infected animals. The more detailed understanding of the immune system has also enabled the development of immune-based therapeutics, ranging from immunological castration to tumor therapy, and cell-based therapies for the treatment of cartilage or tendon injuries are increasingly used. In many of these cases, veterinary medicine applications provide important insights for future use in human patients. The associated regulatory challenges lie on the one hand in the integration of the innovative approaches in the existing national and EU legislative framework, and on the other hand in the development of appropriate criteria to evaluate their safety and efficacy. The promotion of innovative products has to be balanced with the safety of the target population as well as contact species and, in the case of food-producing animals, food safety. From the regulatory perspective, simplified licensing procedures represent an attractive alternative on the national level.

Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain.

Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini-Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram-negative bacteria. However, integration of multiple mini-Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7). Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity. Integration of multiple copies of a mini-Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini-Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini-Tn7, subsequent chromosomal insertion of a-attTn7 and a second round of transposition with lux-mini-Tn7. Mini-Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria.

Development and validation of a serological potency test for the release of Leptospira vaccines--requirements in the European Union.

Both European Pharmacopoeia Monograph 01/2008:0447 "Canine Leptospirosis vaccine (inactivated)" and the more recent Monograph 01/2008:1939 "Bovine Leptospirosis vaccine (inactivated)" explicitly allow for a sero-response test to assess batch potency. Test setup and requirements for in vivo and in vitro validation are described. Furthermore, the two main strategies to assess batch potency and their specific demands are addressed.

Replacement of the in vivo neutralisation test for efficacy demonstration of tetanus vaccines ad us. vet.

The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horse and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms. Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium fuer Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELISA for horses and sheep separately. Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project. 564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of antibody responses were recorded. The in vitro DAE method seems to be suitable to replace the mouse neutralisation test used for the detection of tetanus antitoxin in sera of target animal species. The comparison of some sera in the ELISA and the TNT showed good equivalence of results. Nevertheless, before an ELISA titre in horse and sheep sera indicating unambiguous protection against tetanus can be fixed, further comparative assays of low titre sera in the TNT and the DAE will have to be performed.

Quality assurance of C. perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test.

Clostridium (C.) perfringens is a Gram-positive anaerobic spore-forming bacterium. Disease caused by C. perfringens infection is called enterotoxaemia. C. perfringens strains are classified on the basis of the lethal exotoxins formed by the bacteria. Epsilon toxin is one of the major lethal toxins and is formed by C. perfringens types B and D. C. perfringens is an ubiquitous bacterium. Infection occurs via food, water, animal litter or soil. Affected animals include mainly sheep, pigs and cattle. C. perfringens infection manifests as pulpy kidney disease and diarrhoea in suckling lambs. Enterotoxaemia development is peracute in most cases. Animals die suddenly while grazing on the pasture, without any prior signs of disease. Therefore, treatment is possible only in very rare cases. Suitable immunoprophylactic measures are the treatment of choice to combat the disease: Vaccines and immunosera have therefore been used extensively for a long time. The requirements for quality, efficacy and safety testing of the inactivated vaccines are laid down in the Ph. Eur. in the monograph: Clostridium perfringens vaccines for veterinary use. After a marketing authorisation is attained, the product batches must be tested in laboratory animal models for their potency against all vaccine components (Pharmeuropa, 1997). For potency testing (batch control) of C. perfringens types B and D, the induction of specific antibodies against epsilon toxin in rabbits must be verified. For this purpose, 10 rabbits are immunised twice with the product to be tested. Their blood is taken 14 days after the last immunisation and the serum is pooled. The pooled serum is then tested for its protective effect. This is done by means of the toxin neutralisation test in mice (optionally also in guinea pigs) in comparison with an international reference serum. The evaluation criterion is the death rate of the mice in the test and reference groups after administration of lethal doses of epsilon toxin. The exact efficacy of the test serum is given in International Units (IU). The tested serum must show a minimum content of 5 IU. This in vivo method requires a very high number of experimental animals. Approximately 400 mice (or 50 guinea pigs) are used per vaccine batch. The monograph for C. perfringens vaccines, which has recently been revised, expressly indicates that a validated serological method may be used for batch testing. In addition, a reference serum known as clostridium multicomponent serum has been available since 2000. The objective is to test vaccine batches against this reference and by means of a competitive ELISA developed in the precursor project, using a monoclonal antibody for direct determination of specific antitoxins in rabbit sera. This ELISA method was subjected to an international validation to verify whether the protocol and the precision can be transferred within and between the participating laboratories.

[Development and prevalidation of alternative methods for the potency testing of C. perfringens vaccines]

The requirements for the quality control of C. perfringens vaccines for veterinary use are described in the monograph 363 of the European Pharmacopoeia (Ph. Eur.). In the current used potency test neutralising antibodies against C. perfringens beta- and epsilontoxin are determined in a mouse neutralisation test (MNT). Two ELISA methods were developed for the replacement of the MNT. Both methods use monoclonal antibodies to determine the quantity of specific antibodies against beta toxin (Capture-ELISA) and epsilon toxin (Competitive-ELISA) in vitro. In parallel to the routine batch potency test in mice, the beta- and epsilonantitoxin levels in 523 samples were estimated in the ELISA procedures. A high specificity and a good reproducibility are evident for both test systems. An interlaboratory prevalidation study was carried out to evaluate the relevance and the transferability of the ELISA procedures. It is concluded that both ELISA systems seems to be suitable alternative methods for assessing the potency of beta- and epsilontoxoid in batches of vaccines for veterinary use.

[An in vitro alternative to the mouse neutralisation assay for potency testing of betatoxoid containing Clostridium, perfringens type B and type C vaccines for veterinary use]

The quality control of Clostridium (C.) perfringens type B and type C vaccines requires animal experiments according to European Pharmacopoiea monograph 363. For potency estimation, the vaccine is first administered to rabbits. In a second step antibodies from these rabbits against C. perfringens betatoxin are measured quantitatively in a mouse neutralisation assay using lethal doses of betatoxin for the challenge. We report about the development of an in vitro assay enabling specific and reproducible measurement of antibodies against C. perfringens betatoxin in rabbit sera. A Capure-Enzyme Linked Immuno Sorbent Assay (ELISA) using a monoclonal antibody against betatoxin as catching antibody was used. A rabbit serumpool freeze dried in 3500 aliquots was always used as reference. This reference serum can be supplied for further national or international collaborative studies. The estimation of relative potency of unknown sera in a parallel line assay was calculated with a computer programme provided by the World health organisation (WHO). The capture-ELISA did not show unspecific reactivity with pre-vaccination sera of cross-reactivity with sera from rabbits immunised with other clostridial antigens e.g. C. perfringens type D, C. chavoei or C. tetani. Reproducibility studies focused on the linear parts of the dose-response curves resulted in intra-assay coefficient of variations of less then 10%. The inter-assay coefficient varied between 12-25% depending on the serum dilutions used. Correlation studies between the result of the animal experiment (only one test) and the capture-ELISA (10 repetitions) from four rabbit serum pools revealed a coefficient of correlation of 0.81-0.84 depending on the basis for calculation of r (Mean or Median from ELISA repetitions). Therefore this test may be a suitable alternative for the currently required mouse neutralisation assay. For acceptance of this test by the European Pharmacopoiea further validation studies are necessary.