Evaluation of the THP-1 lineage in the monocyte activation test for quality control of Meningococcal C Conjugate and Yellow Fever Vaccines

Introduction: Vaccine production must be carried out under GMP, which requires monitoring of pyrogenic activity due to public health risks.Methods such as the rabbit pyrogen test and LAL are used to detect pyrogens, the latter being a recognized alternative to animal testing, following the 3Rs principles.However, the monocyte activation test (MAT) is more sensitive and complete and allows the detection of non-endotoxin pyrogens (NEPs) but faces challenges like the use of cryopreserved human blood, which presents heterogeneity.Objectives: Evaluate of the THP-1 lineage in the MAT for quality control of Meningococcal C Conjugate (MenCC) and Yellow Fever (VFA) vaccines produced by Bio-Manguinhos.Methodology: THP-1 cells were cultured using RPMI-1640 with 10% and 2% of Fetal Bovine Serum for cell maintenance and for the assays, respectively.The cells were stimulated with Gram-negative (LPS; E. coli) and Gram-positive (lipoteichoic acid; LTA) stimuli and incubated for 16-20h.The expression of pro-inflammatory cytokines IL-1 and IL-6 was evaluated by ELISA.Tests were performed with three batches of each vaccine after incubation with THP-1 cells.Results: A working bank of the THP-1 lineage with viable cells was produced, documented and validated.Responsiveness assays showed that the THP-1 cell was stimulated by LPS, resulting in the production and detection of cytokines.The maximum valid dilution (MDV) determined according to the European Pharmacopoeia for VFA considering IL-6 was 1:215 and 1:131 for IL-1, while for MenCC, the MDV was 1:5 for IL-6 and 1:2 for IL-1.Interference tests showed VFA did not interfere with the MAT system, since LPS recovery was within the limits (50-200%).For MenCC, LPS recovery was adequate for IL-6, but only one assay met acceptability parameters for IL-1.THP-1 lineage was also responsive to LTA treated with Polymyxin B, a compound that neutralizes endotoxic activity, allowing the identification of NEPs.The cell line showed good performance in detecting IL-6, up to the twelfth generation of cells, with a cell concentration of 2.5 x 10 6 /mL. Conclusion:THP-1 lineage showed promise for the detection of pyrogens in VFA, while for MenCC further studies will be required.The cells also showed sensitivity to the stimulation of pyrogens associated with Gram-positive and Gram-negative microorganisms (LTA and LPS).After validation process, the test could be implemented as a quality control method for the studied and other vaccines produced by Bio-Manguinhos.