Limitations of the rabbit pyrogen test for assessing meningococcal OMV based vaccines

Abstract

SummaryThe rabbit pyrogen test was developed in the early 1900's to detect contaminating pyrogens in parenteral medicines. Since its conception alternative methods with improved sensitivity, relevancy and which are ethically more acceptable have been developed. However, the test is still a current pharmacopeial method and is used to evaluate the pyrogen content of some vaccines. In this article the limitations and pitfalls of using the test to measure pyrogenicity of vaccines containing outer membrane vesicles are described. The method is unsuitable as a safety test for these products due to the high levels of endotoxin present in the vaccine which generate a pyrogenic response in rabbits when delivered intravenously without dilution. Its use as a consistency test is also ambiguous as the test gives a qualitative rather than quantitative response, and the rabbit models are highly variable. In addition there is evidence that measuring the temperature rise of the animals over three hours does not capture the maximum fever response. Finally the article considers the use of alternative methods and the validity of animal models when applying a consistency based approach for assessing the quality of licensed products.

Outer membrane vesicles: moving within the intricate labyrinth of assays that can predict risks of reactogenicity in humans

“…By this adaptation, a vaccine was accepted if not inducing fever in rabbit when diluted (several hundred-fold) at an equivalent of 1 (for MenBvac 69 ) or 0.214 μg/per kg (for MenZB 70 ). According to Pharmacopoeias (EU, US, and JP), rabbits' temperature needs to be monitored for 3 h; although this timeframe is optimal to detect the peak in temperature rise in response to purified endotoxin (happening between 1 and 2 h), the maximum temperature rise recorded in rabbits following vaccination with meningococcal dOMV was at 4.5 h. 71 These results indicate that the response to endotoxin alone is different from the response when it is presented in an OMV, further suggesting that the classical RPT is not suitable for testing vaccines like OMVs which intrinsically contain endotoxin.…”

“…81,82 MAT (method C) has been validated as the most appropriate assay for measuring the pyrogenicity of the 4CMenB vaccine (Bexsero), especially for lots release. 71,82 In the validated MAT, batches equally pyrogenic or less pyrogenic than those batches shown to be safe in a clinical trial can be also certified as safe (i.e. if results fall in a specified range of relative pyrogen units).…”

Evaluation of protective immunity responses against pneumococcal PhtD and its C-terminal in combination with outer-membrane vesicles as adjuvants

“…PBS was injected in five mice as the negative control group, while 500 µg of OMVs was injected to each of the five mice in the test group. Weight reduction and other abnormal indications, such as fever, weakness and mortality, were recorded for 7 days [29].…”

Optimization of the monocyte-activation-test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine

“…4B) of the total variability, while contribu- (1:100, 1:200 and 1:400 Nowadays, regarding vaccines, the MAT has been successfully implemented only for an outer membrane vesicle-based meningococcal B vaccine, an intrinsically pyrogenic product (Vipond et al, 2019;Valentini et al, 2019), thus avoiding the high rate of false positive results obtained by using RPT. Those frequent false positives were probably due to the necessity to perform high dilutions of the vaccine before intravenous administration in rabbit, and the LAL limitation in detecting NEPs (Vipond et al, 2016).…”