Elsevier

Research in Veterinary Science

Volume 132, October 2020, Pages 386-392
Research in Veterinary Science

Basic information for the development of a toxicity assay in inactivated bacterial vaccines

https://doi.org/10.1016/j.rvsc.2020.07.020Get rights and content

Highlights

  • Bacterial antigens toxicity can be assessed in vitro.

  • Both MTT and IL-1beta release assays can depict the toxicity of bacterial antigens.

  • The results of MTT and IL-1beta release assays are positively correlated.

  • Toxicity of bacterial antigens is correlated with extracellular protein concentration.

Abstract

This study dealt with the toxicity of inactivated bacteria intended for veterinary autogenous vaccines toward a suitable control assay. Two in vitro methods were used. The [3-(4, 5 -dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide] (MTT) test, based on the metabolic reaction of a tetrazolium salt in vital cells, was adopted on the basis of previous positive results. The Interleukin (IL)-1 beta release assay on monocyte-derived pig macrophages was carried out for comparative purposes, to evaluate the possible role of the inflammatory response. MTT and IL-1 beta responses showed a significant correlation (P < 0.05) at defined test dilutions of bacterial antigens, whereas no correlation was demonstrated using MTT responses normalized on bacterial cell concentration. Furthermore, the toxic effects shown in the MTT test were positively correlated to the extracellular protein content. On the whole, the above results could be a useful basis for the development of a toxicity assay on inactivated bacterial vaccines. Also, our data point at bacterial autolysis as a major component underlying toxicity.

Introduction

Autogenous veterinary vaccines are immunizing products based on pathogenic microorganisms isolated from sick animals of one herd and only usable in the same farm following a veterinary prescription. Therefore, autogenous vaccines are considered as a useful and economical tool for improving animal health and welfare alike, protecting public health and food safety. They are produced and applied case by case if traditional vaccines are not available on the market or are not formulated as requested on farm. Most important, autogenous vaccines usually have to be available in a short time; hence, they are not submitted to lengthy procedures to assess potency and safety, as requested for conventional vaccines (Attia et al., 2013).

Directive 2001/82/EC, 2020 (https://ec.europa.eu/health//sites/health/files/files/eudralex/vol-5/dir_2001_82_cons2009/dir_2001_82_cons2009_en.pdf) requires that conventional vaccines obtain an official market approval. On the contrary, autogenous veterinary vaccines are exempted from this legislation, and there are no binding legal provisions for EU member Countries regarding this type of vaccines (Directive 2001/82/EC, 2020). In accordance with Articles 3 and 4 of Directive 2001/82/EC, 2020, EU Member States are authorized to manufacture autogenous vaccines outside the regulatory framework of the directive.

In Italy, an in vivo toxicity test is carried out for each batch of autogenous vaccine produced. The test is based on the observation of five mice inoculated subcutaneously with the final product obtained from the vaccine production process. To guarantee the suitability of the product, there must be no abnormal reactions, neither local nor general.

The datasets of the above toxicity control show that the animal test is not indicative of vaccine safety, as adverse reactions are very rare and not correlated with the reactions observed in target animals on farm. The unsatisfactory results, the waiting time of test results and the sacrifice of a large number of animals underlie a substantial need for methods measuring in vitro reliable correlates of vaccine toxicity (Roth and Henderson, 2001; Directive 2010/63/EU, 2020).

A major attention to the issues of production and control of autogenous vaccines is also stressed by the European Medicine Agency (EMA), which encourages member states to define alternatives to the use of animals (EMA/CMDv, 2017). Accordingly, new studies are being carried out for the evaluation of bacterial endotoxins in autogenous vaccines, as a possible toxicity index of these products (Eu. Ph. 2.6.14, Ed.2009; Di Paolo et al., 2018). Yet, our experience on the Limulus amebocyte lysate (LAL) test for bacterial endotoxin, carried out as currently prescribed (Eu. Ph. 01/2018:20614), was inconclusive. Residues of endotoxin from Gram-negative bacterial cultures were generally very high, as expected, and not correlated to the profiles of vaccine toxicity observed in vivo (Supplementary Table 1). This is likely due to the dramatic reduction of toxicity of bacterial endotoxin in the presence of vaccine adjuvants (Park et al., 2005). Accordingly, adjuvants like alum strongly inhibit the LAL reaction and demand very high dilutions for testing, whereas oil adjuvants are not compatible at all with the LAL test procedure adopted in our laboratory (Colella E.M., unpublished results).

Owing to the above, this study dealt with possible alternative in vitro procedures for the evaluation of abnormal toxicity, based on cell culture technologies. Such an approach is likely to give considerable ethical advantages in accordance to the 3Rs principle (Replacement, Reduction, Refinement), and to practical convenience. In particular, the use of in vitro toxicity assays is likely to shorten the delivery time of autogenous veterinary vaccines on farm (Roth and Henderson, 2001; Directive 2010/63/EU, 2020).

The aim of this study was to investigate the main toxicity features of bacteria, as a basis for the future development of a control assay. This study involved the application of two in vitro methods based on [3-(4, 5 -dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide, MTT] and Interleukin (IL)-1 beta responses to the inactivated bacterins.

Our working hypothesis implied that toxicity of bacterins could be correlated to the extent of tissue damage and the inflammatory response in myeloid cells. In turn, we surmised that inflammasome formation and IL-1beta release (Martinon et al., 2002) could be useful indicators of such an inflammatory response, and related to MTT test results, considered as a “golden standard” of toxicity assays in other research areas (UNI EN ISO 10993-5, 2009).

Section snippets

Preparation of the samples

In this study, the samples used for in vitro tests consisted of inactivated bacterial antigens (bacterins) collected before mixing with the adjuvants of autogenous veterinary vaccines. These are produced in our vaccine department on the basis of isolated and typed bacterial strains, propagated to a high antigenic mass in biofermenters over 16–24 h in a suitable medium. Once the fermentation time has elapsed, the bacterial culture is inactivated by adding in the biofermenter formalin (37%

MTT test results

Samples were examined up to the 1: 2048 dilution. The antigens showing the highest cytotoxicity had titers of 1:128 according to our 70% threshold value, in agreement with previous results on whole bacterial vaccines (Profeta et al., 2019); those with the least toxicity were MTT-negative at the 1:8 dilution (Table 1). Please notice that 1:4-diluted bacterial antigens still appear viscous and turbid; as such, they may non-specifically interfere with the spectrophotometric reading (Tolosa et al.,

Discussion

The radical changes in the European regulatory framework in recent years toward alternatives to animal testing has stimulated the development of new in vitro methods in different areas. In particular, this aspect of the research also focused on alternative in vitro methods for the evaluation of autogenous vaccines. This trend is due to ethical reasons, but there is also a need for shorter delivery times of vaccines. Last but not least, thein viv test was not deemed as representative of the true

Declaration of Competing Interest

The authors declare that they have no conflict of interests.

Acknowledgements

The collaboration of Dr. I.L. Archetti (IZSLER, Brescia, Italy) is gratefully acknowledged. This work was financially supported by the Italian Ministry of Health, project MIN_SAL_3R_2016.

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