Innate and adaptive immune responsiveness to Leptospira in dogs: In vitro alternatives to animal testing in Leptospira vaccine manufacturing

Abstract

Quality control of vaccine batches before their release onto the market is mandatory to demonstrate that they are safe and efficacious. For inactivated vaccines, this is often achieved using immunization challenge tests, requiring large numbers of laboratory animals. Ethical concerns about animal use and the questionable relevance of many animal models for the target species urged for implementation of the 3R’s principle (replacement, reduction and refinement of animal use) in vaccine manufacturing and quality control. Increased knowledge of vaccine antigens, implementation of in-process testing, Good Manufacturing Practice (GMP) and Quality Assurance in vaccine manufacturing enabled standardized manufacturing processes. Together, these implementations facilitate production of better defined and less variable vaccine batches. Therefore, demonstration of batch-to-batch consistency has been proposed as a new approach for batch release testing. Once consistency in production has been demonstrated by showing that new batches do not differ from previous batches of sufficient quality by measuring a set of crucial parameters, testing of subsequent batches could rely on assessment of these same parameters, ideally by a panel of in vitro tests. This testing strategy is known as the consistency approach which has the potential to abolish the need for animal tests for batch release onto the market. Potency assessment of inactivated canine Leptospira vaccine batches still relies, to varying degrees among vaccine manufacturers, on the use of in vivo hamster challenge experiments. In view of the 3R principle, there is a clear need for development of novel host specific in vitro tests to study vaccine potency and/or immune activation. Pathogenic Leptospira are the causative agents of leptospirosis, an emerging bacterial zoonosis, potentially affecting all vertebrates, including humans and dogs. Leptospira spp. are Gram-negative spirochetes found in water and soil and comprise of saprophytic and pathogenic species. Numerous Leptospira serovars have been identified based on the carbohydrate structure of the leptospiral LPS. Vaccination of dogs against Leptospira is recommended to protect the animals against potentially fatal infection and to prevent transmission to the environment. Commercially available Leptospira vaccines consist of inactivated whole bacteria of different serovars. Monoclonal antibodies (mAbs) recognising LPS epitopes of specific Leptospira serovars were developed for quantification of Leptospira antigen in vaccines using an enzyme-linked immunosorbent assay (ELISA), for quality control purposes. Although this LPS-based ELISA is a reproducible and affordable alternative to the animal batch release test, it is serovar specific, which means that mAbs need to be developed for each serovar. Furthermore, this method does not provide any information about the protective immune responses induced by inactivated Leptospira vaccines. The purpose of the studies described in this thesis was to develop cell-based assays to determine the immunostimulatory properties of inactivated canine Leptospira vaccines and individual serovars in vitro, and to assess the applicability of these assays for future routine quality control testing. Aspects of innate immunity and its response to inactivated Leptospira vaccines in the dog were investigated and described in Chapters 2, 3 and 4, while Chapter 5 addresses the examination of adaptive immune responses induced by these vaccines.