Improvement of immunogenicity of meningococcal lipooligosaccharide by coformulation with lipidated transferrin-binding protein B in liposomes: implications for vaccine development

Abstract

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

Fig 1

HPAEC-PAD carbotyping (A) and structure (B) of L8 oligosaccharide (OS) from strain A1. Prior to carbotyping analysis, the OS was isolated from whole cells by mild acid hydrolysis with 1% acetic acid at 100°C for 1 h. β-d-Galp, β-d-galactopyranose; β-d-Glcp, β-d-glucopyranose; β-d-GlcpNAc, β-d-2-acetamido-2-deoxy-glucopyranose; LD-Hep, LD-heptose; KDO, 2-keto-3-deoxy-octulosonic acid; PEA, phosphoethanolamine.

Fig 2

LAL activity of L8 LOS incorporated into liposomes. Several molar ratios of lipid/LOS ranging from 25:1 to 400:1 were assessed for their capacity to reduce LAL activity of the LOS. LAL activity of purified L8 LOS was measured at 13,070 EU/μg.

Fig 3

Characterization of the lipidation of M982 rlip-TbpB after tryptic digestion of the protein. MALDI-TOF mass spectrum of the tryptic digests of rlip-TbpB. The sequence of the N-terminal fragment generated by tryptic digestion is CLGGGGSFDLDSVDTEAPRPAPK. au, arbitrary unit.

Fig 4

Anti-L8 LOS (A) and anti-rlip-TbpB (strain M982) (B) IgG responses in individual rabbits immunized with different formulations. Each group of four rabbits received two i.m. injections of one of the formulations at a 3-week interval. Protein and LOS doses were both 40 μg. There were only two rabbits in the negative-control groups. Postdose two serum samples were collected 2 weeks after the second injection.

Fig 5

Flow cytometry analysis of the binding of rabbit sera to live cells of N. meningitidis strain RH873 (A) or strain 8680 (B1 and B2). Binding of rabbit sera raised to empty liposomes was used as a negative control (black line on each graph). (A) Binding of rabbit sera to M982 rlip-TbpB (light gray), to liposomal L8 LOS (dark gray), or to M982 rlip-TbpB/liposomal L8 LOS (black). (B1) Binding of rabbit sera to M982 rlip-TbpB mixed with empty liposomes (light gray) or to M982 rlip-TbpB/liposomal L8 LOS (black). (B2) Binding of rabbit sera to B16B6 rlip-TbpB mixed with empty liposomes (light gray) or to B16B6 rlip-TbpB/liposomal L8 LOS (black).

Fig 6

IL-8 secretion evaluated in HEK293 cells expressing TLR1/2, TLR 2/6, or TLR4/MD2. Increasing doses of M982 rlip-TbpB versus nonlipidated M982 TbpB or synthetic TLR2 agonists (Pam2CSK4 and Pam3CAG) were used to stimulate cells, and IL-8 secretion was assessed after 24 h.