In vitro alternative for reactogenicity assessment of outer membrane vesicle based vaccines.

Intrinsic or added immune activating molecules are key for most vaccines to provide desired immunity profiles but may increase systemic reactogenicity. Regulatory agencies require rabbit pyrogen testing (RPT) for demonstration of vaccine reactogenicity. Recently, the monocyte activation test (MAT) gained popularity as in vitro alternative, yet this assay was primarily designed to test pyrogen-free products. The aim was to adjust the MAT to enable testing of pyrogen containing vaccines in an early stage of development where no reference batch is yet available. The MAT and RPT were compared for assessing unknown safety profiles of pertussis outer membrane vesicle (OMV) vaccine candidates to those of Bexsero as surrogate reference vaccine. Pertussis OMVs with wild-type LPS predominantly activated TLR2 and TLR4 and were more reactogenic than Bexsero. However, this reactogenicity profile for pertussis OMVs could be equalized or drastically reduced compared to Bexsero or a whole-cell pertussis vaccine, respectively by dose changing, modifying the LPS, intranasal administration, or a combination of these. Importantly, except for LPS modified products, reactogenicity profiles obtained with the RPT and MAT were comparable. Overall, we demonstrated that this pertussis OMV vaccine candidate has an acceptable safety profile. Furthermore, the MAT proved its applicability to assess reactogenicity levels of pyrogen containing vaccines at multiple stages of vaccine development and could eventually replace rabbit pyrogen testing.

Performance of monocyte activation test supplemented with human serum compared to fetal bovine serum.

The monocyte activation test (MAT) is used to detect pyrogens in pharmaceutical products and serves as replacement of the rabbit pyrogen test. The peripheral blood mononuclear cell-based MAT assay requires the addition of serum to the medium and is performed with either fetal bovine serum (FBS) or human serum (HS). Since the capacity to detect non-endotoxin pyrogens (NEPs) in a sensitive manner is an important strength of MAT compared to the bacterial endo­toxin test, the performance of the MAT using FBS and HS was compared using endotoxin and several NEPs. The MAT was more sensitive for endotoxin when FBS was used, however for most NEPs the MAT was more sensitive when per­formed in HS. Furthermore, heat-inactivation of FBS affected the performance of the MAT for endotoxin to some extent but not for the NEPs. Interestingly, heat-inactivation of HS led to an almost complete loss of reactivity towards endotoxin, reduced the response towards heat-killed Staphylococcus aureus and peptidoglycan, but had minor or no effects on the responses towards R848, flagellin, and Pam3CSK4. Product testing of a human blood-derived product in MAT using HS was beneficial since endotoxin spike recoveries were improved. This product is therefore currently batch released with the HS-based MAT assay. Overall, to guarantee optimal performance of MAT, heat-inactivated serum should be avoided. The HS-based MAT appears to be the first choice to replace the rabbit pyrogen test, while in some cases the FBS-based MAT may be favored.