Controlled Covalent Conjugation of a Tuberculosis Subunit Antigen (ID93) to Liposome Improved In Vitro Th1-Type Cytokine Recall Responses in Human Whole Blood

摘要

Tuberculosis (TB) remains a foremost poverty-related disease with a high rate of mortality despite global immunization with Bacille Calmette–Guérin (BCG). Several adjuvanted recombinant proteins are in clinical development for TB to protect against the disease in infants and adults. Nevertheless, simple mixing of adjuvants with antigens may not be optimal for enhancing the immune response due to poor association. Hence, co-delivery of adjuvants with antigens has been advocated for improved immune response. This report, therefore, presents a strategy of using chemical conjugation to co-deliver an adjuvanted recombinant protein TB vaccine (ID93 + GLA-LSQ). Chemical conjugation involving glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was used to associate the antigen (ID93) to the modified liposome (mGLA-LSQ). The physicochemical stability of the formulations was evaluated using high-performance liquid chromatography (HPLC) (adjuvant content), dynamic light scattering (DLS, particle size analysis), and sodium dodecyl sulfate-polyacrylamide gel (SDS) electrophoresis (protein analysis). The bioactivity was assessed by cytokine stimulation using fresh whole blood from 10 healthy donors. The conjugates of ID93 + mGLA_LSQ maintained liposomal and protein integrity with the two protein chemistries. The GLA and QS21 content of the vaccine were also stable for 3 months. However, only the glutaraldehyde conjugates provoked significant secretion of interleukin-2 (210.4 ± 11.45 vs 166.7 ± 9.15; p = 0.0059), interferon-gamma (210.5 ± 14.79 vs 144.1 ± 4.997; p = 0.0011), and tumor necrosis factor alpha (2075 ± 46.8 vs 1456 ± 144.8; p = 0.0082) compared to simple mixing. Conjugation of recombinant protein (ID93) to the liposome (mGLA_LSQ) through chemical conjugation resulted in a stable vaccine formulation, which could facilitate co-delivery of the subunit vaccine to promote a robust immune response.