Coincorporation of LpxL1 and PagL Mutant Lipopolysaccharides into Liposomes withNeisseria meningitidis Opacity Protein: Influence on Endotoxic and Adjuvant Activity

Abstract

Wild-type lipopolysaccharide (LPS) of Neisseria meningitidis normally contains six acyl chains. Pentaacylated LPS forms were generated through inactivation of the lpxL1 gene or through the expression of the Bordetella bronchiseptica pagL gene in N. meningitidis. The resulting LPS species, designated LpxL1 LPS and PagL LPS, respectively, display reduced endotoxic activity compared to wild-type LPS. Here, we determined the adjuvant potential of PagL LPS by comparison with the broadly used LpxL1 LPS. We also investigated the potential benefit for adjuvanticity of coincorporating these LPS species, together with the meningococcal opacity-associated protein OpaJ as a model antigen, in a liposomal delivery system. PagL LPS showed a higher endotoxic activity than LpxL1 LPS, and their incorporation into liposomes significantly reduced their endotoxic activity as determined by measuring the induction of interleukin-6 (IL-6) production in a murine macrophage cell line. To determine the adjuvant effect, BALB/c mice were immunized with OpaJ-containing liposomes and either free LPS or LPS coincorporated into the proteoliposomes. OpaJ-containing liposomes adjuvanted with AlPO 4 or not adjuvanted at all were included as control groups. In the appropriate dose, PagL LPS showed a superior adjuvant effect compared with LpxL1 LPS, and for both LPS types, free LPS showed a higher adjuvant effect than when coincorporated into the liposomes, as evidenced by higher titers of IgG2a and IgG2b antibodies against OpaJ ؉ meningococci and higher bactericidal titers. In conclusion, PagL LPS is a better adjuvant than LpxL1 LPS, but coincorporation of either LPS into proteoliposomes did not improve their adjuvant activity.

Lipopolysaccharide Engineering in Neisseria meningitidis

“…However, the lpxL1 Ϫ LPS was more strongly reduced in its ability to activate human as compared with murine TLR4, as measured by the expression of the NF-B transcription factor and various MyD88-dependent cytokines (40). For PagL-deacylated meningococcal LPS, adjuvant activity has also been demonstrated in mice, but it should be noted that in this animal species there is very little difference in endotoxic activity compared with wild-type hexaacylated LPS (41). In this study, we have determined that the lpxL1 Ϫ -pentaacylated LPS structure containing a fully phosphorylated lipid A, as determined by MS analysis, at the concentrations used is unable to induce the production of not only MyD88-dependent cytokines (IL-6 and IL-10) but also of the TRIF-dependent chemokine IP-10 in human monocytic MM6 cells.…”

Expression of human CEACAM1 in transgenic mice limits the Opa-specific immune response against meningococcal outer membrane vesicles

“…Considering on the one hand that Opa proteins can be effective targets for bactericidal antibodies [25,26], but on the other hand may suppress or at least decrease an effective immune response against OMV antigens by this inhibition of CD4 T cell proliferation, it is important to establish whether these in vitro observations are relevant for in vivo immunogenicity. Until now it has not been possible to answer this question by immunization experiments in mice or other animals because the Opa-CEACAM1 interaction is strictly species-specific.…”

Improvement of Immunogenicity of Meningococcal Lipooligosaccharide by Coformulation with Lipidated Transferrin-Binding Protein B in Liposomes: Implications for Vaccine Development

“…We thus combined the two antigens in the same liposomes, with the aim of delivering them together to the same antigenpresenting cells and to study how this would affect immune responses. Although previous studies reported the coincorporation of meningococcal proteins with LOS into liposomes, with LOS acting as an adjuvant on antiprotein responses (3,4), none of these studies ever demonstrated that the meningococcal protein could act as an adjuvant on the anti-LOS response. Surprisingly, we found that rlip-TbpB was a potent adjuvant for anti-LOS responses when it was coformulated with LOS in liposomes, but we did not observe an adjuvant effect of LOS on the anti-TbpB response.…”