Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections
The Journal of Biological Chemistry
Bacterial lipoproteins are structurally divided into two groups, based on their lipid moieties: diacylated (present in Gram-positive bacteria) and triacylated (present in some Gram-positive and most Gram-negative bacteria). Diacylated and triacylated lipid moieties differ by a single amide-linked fatty acid chain. Lipoproteins induce host innate immune responses by the mammalian Toll-like receptor 2 (TLR2). In this study, we added a lipid moiety to recombinant OMP26, a native nonlipidated (NL) membrane protein of Haemophilus influenzae, and characterized it extensively under different expression conditions using flow cytometry, LC/MS, and MALDI-TOF. We also investigated the ability of NL and lipidated (L) OMP26 to induce in vitro stimulation of HEK Blue-hTLR2-TR1 and hTLR-TLR6 cells. Our L-OMP26 was predominantly expressed in diacylated form, so we employed an additional gene copy of apolipoprotein N-acetyltransferase enzyme (Lnt)-rich Escherichia coli strain that further acylates the diacyl lipoproteins to enhance the production of triacylated L-OMP26. The diacyl and triacyl versions of L-OMP26, intended as a vaccine for use in humans, were characterized and evaluated as protein vaccine components in a mouse model. We found that the diacyl and triacyl L-OMP26 protein formulations differed markedly in their immune-stimulatory activity, with diacylated L-OMP26 stimulating higher adaptive immune responses compared with triacylated L-OMP26 and both stimulating higher adaptive immune response compared to NL-OMP26. We also constructed and characterized an L-OMP26φNL-P6 fusion protein, where NL-P6 protein (a commonly studied H. influenzae vaccine candidate) was recombinantly fused to L-OMP26. We observed a similar pattern of lipidation (predominantly diacylated) in the L-OMP26φNL-P6 fusion protein.
Medical Subject Headings
Mice; Animals; Humans; Bacterial Outer Membrane Proteins (genetics); Lipoproteins (genetics); Haemophilus Vaccines; Recombinant Proteins (genetics); Haemophilus Infections (prevention & control); Haemophilus influenzae (genetics); Mammals
Kaur, R., Mangiafesto, J., Pryharski, K., Rasam, S., Zagursky, R., & Pichichero, M. (2023). Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections. The Journal of Biological Chemistry, 299 (8), 105031. https://doi.org/10.1016/j.jbc.2023.105031