Well-defined and potent liposomal meningococcal B vaccines adjuvated with LPS derivatives.

Potent liposomal PorA formulations containing various lipopolysaccharide (LPS) derivatives were developed. The following adjuvants were compared: the commonly used aluminum phosphate (AlPO(4)), and three LPS like adjuvants: monophosphoryl lipid A (MPL), lipopolysaccharide (galE LPS) and the less toxic LPS mutant lpxL1. The immunogenicity in mice was evaluated and compared with that against an outer membrane vesicle (OMV) vaccine. The IgG isotype distribution and bactericidal activity were determined. Furthermore, PorA specific proliferation of lymph node cells after immunization and restimulation in vitro was studied with selected formulations. Both AlPO(4) and MPL were unable to improve the functional immunogenicity (i.e. bactericidal response) of liposomal PorA. Besides, when these adjuvants were used, the percentage of responders in the groups did not reach 100%. This was also observed with non adjuvated PorA-liposomes or OMV. Of the adjuvants studied, only galE LPS and lpxL1 LPS were capable of increasing the immunogenicity and avoid non responsiveness against PorA-liposomes. Importantly, the adjuvant activity of lpxL1 LPS was accompanied by an improved PorA specific proliferation of lymph node cells and a concomitant increase in IL-2 production. In conclusion and considering its lower toxicity, lpxL1 LPS adjuvated liposomes are superior to other formulations tested.

Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition.

Vaccine efficacy might be improved by exploiting the potent antigen presenting properties of dendrite cells (DCs), since their ability to stimulate specific major histocompatibility complex-restricted immune responses has been well documented during the recent years. In that light, we investigated how the interaction of antigen-containing liposomes with DCs was affected by the bilayer composition. Monocyte-derived human DCs and murine bone marrow-derived DCs were analysed and compared upon in vitro incubation with liposomes by flow cytometry and confocal microscopy. Anionic liposomes with a bilayer composition of phosphatidylcholine, cholesterol and phosphatidylglycerol or phosphatidylserine interacted with a limited fraction of the total DC population in case of both DC types. Inclusion of mannosylated phosphatidylethanolamine (Man-PE) for targeting to the mannose receptor (MR) increased the interaction of negatively charged liposomes with both human and murine DCs. This increase could be blocked in human DCs by addition of the polysaccharide mannan indicating that uptake might be mediated by the mannose receptor. Cationic liposomes containing trimethyl ammonium propane interacted with a very high percentage of both DC types and could be detected in high amounts intracellularly. In conclusion, liposome bilayer composition has an important effect on interaction with DCs and might be critical for the vaccination outcome.

Immunogenicity of meningococcal PorA formulations encapsulated in biodegradable microspheres.

The purpose of our study was to investigate the possibility to microencapsulate liposomes and meningococcal outer membrane vesicles (OMV), both containing neisserial pore protein A (PorA), in biodegradable dextran- and mannan-based microspheres and to study the immunogenicity of the encapsulated PorA formulations. PorA-liposomes and OMV were encapsulated in dextran- or mannan-based microspheres by using an aqueous two-phase system consisting of a polyethylene glycol solution and a methacrylated dextran or mannan solution. The formulations were characterized for size distribution, PorA structure and antigen recovery after release. Calcein-containing model liposomes were used to establish the encapsulation efficiency and release profiles from both types of microspheres. The immunogenicity of the PorA-containing formulations was determined in mice after subcutaneous immunization. Liposomes were encapsulated in dextran and mannan microspheres with a high efficiency (70-90%). Calcein liposomes, after a 5-day lag period, exhibited apparent zero-order release kinetics from both types of microspheres between Days 5 and 10 of incubation in vitro. The total release was 80 and 100% from mannan and dextran microspheres, respectively. The trimeric PorA conformation was preserved in the released liposomes and OMV and the antigen was partly recovered. The immunogenicity of PorA-liposomes and OMV encapsulated in dextran or mannan microspheres was preserved. In conclusion, PorA-liposomes and OMV could be encapsulated in dextran- and mannan-based microspheres with high efficiency. The immunogenicity of encapsulated antigen was preserved.

Stability of mono- and trivalent meningococcal outer membrane vesicle vaccines.

The stability during storage of outer membrane vesicles (OMVs) of Neisseria meningitidis group B was studied. Three types of OMVs were compared for their stability, containing either one (monovalent) or three different PorA subtypes (trivalent), the latter with and without class 4 outer membrane protein (OMO, RmpM). Aqueous formulations were stored freeze-dried (4 degrees C), frozen (-70 degrees C) and in liquid form at 4, 37 and 56 degrees C. Physico-chemical properties and immunogenicity of the OMVs as well as PorA conformation and antigenicity (P1.7-2,4, the subtype present in all formulations) were monitored during 1 year. At -70 or 4 degrees C, the structure and immunogenicity of OMVs was preserved. Storage of OMVs at high temperatures (37 or 56 degrees C) induced destruction of the OMV structure and denaturation of PorA, followed by chemical degradation. Immunogenicity decreased or was lost completely. Changes observed in the fluorescence spectra of degraded OMVs were also seen in tryptophan (Trp) and tyrosine (Tyr) derivatives incubated at 56 degrees C, indicating the occurrence of chemical degradation of tryptophan and tyrosine residues in PorA. Trivalent OMVs were slightly more stable at 37 degrees C than monovalent OMVs as assessed by in vitro methods, but these differences did not result in differences in the immunogenicity. The stability of trivalent OMVs was not affected by the presence of RmpM. Both trivalent and monovalent OMVs could be freeze-dried with preservation of their immunogenicity. In conclusion, OMVs are sensitive to elevated temperatures, but are stable in the frozen or freeze-dried state or when stored at 4 degrees C in the liquid state.

Liposomal meningococcal B vaccination: role of dendritic cell targeting in the development of a protective immune response.

The effect of targeting strategies for improving the interaction of liposomal PorA with dendritic cells (DC) on the immunogenicity of PorA was investigated. PorA, a major antigen of Neisseria meningitidis, was purified and reconstituted in different types of (targeted) liposomes, i.e., by using mannose or phosphatidylserine as targeting moieties, or with positively charged liposomes. We studied the efficiency of liposome uptake and its effect on the maturation of and interleukin 12 (IL-12) production by murine DC. Moreover, mice were immunized subcutaneously to study the localization and immunogenicity of PorA liposomes. Uptake of liposomes by DC was significantly increased for targeted liposomes and resulted in the maturation of DC, but to various degrees. Maturation markers (i.e., CD80, CD86, major histocompatibility complex class II, and CD40) showed enhanced expression on DC incubated with targeted PorA liposomes relative to those incubated with nontargeted PorA liposomes. Moreover, only the uptake of targeted PorA liposomes induced production of IL-12 by DC, with levels similar to those produced by lipopolysaccharide (LPS)-pulsed DC. Mannose-targeted PorA liposomes administered subcutaneously had an increased localization in draining lymph nodes compared to nontargeted PorA liposomes. Liposomes in draining lymph nodes interacted preferentially with antigen-presenting cells, an effect that was enhanced with targeted PorA liposomes. Immunization studies showed an improvement of the bactericidal antibody response (i.e., increased number of responders) generated by targeted PorA liposomes compared to that generated by nontargeted ones or LPS-containing outer membrane vesicles. In conclusion, the use of targeted PorA liposomes results in an improved uptake by and activation of DC and an increased localization in draining lymph nodes. These effects correlate with an enhanced immune response toward the vaccine.

Restored functional immunogenicity of purified meningococcal PorA by incorporation into liposomes.

The impact of the conformation, lipooligosaccharide (LOS)-depletion and the presentation form of outer membrane protein PorA from Neisseria meningitidis (PorA) subtype P1.7-2,4 on the immune response in mice was studied. Native PorA was purified from outer membrane vesicles (OMVs) derived from meningococci and reconstituted into liposomes. The conformation of PorA after purification from OMVs and reconstitution in liposomes was monitored by use of electrophoretic and spectroscopic techniques and compared with the conformation of PorA in outer membrane complexes (OMCs) and heat-denatured PorA. The antigenicity of the PorA formulations was measured by ELISA by using a bactericidal anti-P1.4 monoclonal antibody. Immunogenicity was determined in Balb/c mice. PorA-specific IgG, isotype distribution and bactericidal activity were measured after subcutaneous immunization. In all formulations except in heat-denatured OMVs, PorA was present as trimers. The lipooligosaccharide (LOS) content was reduced by 96% in the purified protein with respect to the original OMVs. The antigenicity of purified PorA (i.e. ELISA response) was substantially higher as compared to PorA in liposomes, OMVs or OMCs. The results of the immunogenicity studies showed that all formulations were able to induce comparable IgG titers. However, whereas the antibodies raised by OMVs were bactericidal, the antibodies elicited by immunization with purified PorA were unable to kill meningococci. Remarkably, the ability to induce bactericidal antibodies was fully recovered by incorporation of the purified PorA into liposomes, in the absence of other adjuvants, as compared to LOS-containing OMVs.