NPL obtained in this work showed homogeneity with respect to shape and size, ranging from 50 to 70 nm of mean diameter, making them appropriate for antigen presentation to the immune system (Fig. 1A). Intramuscular administration of the NPL formulation was capable of inducing specific antimeningococcal-OMV-IgG antibody levels in immunized animals, at the same level as VA-MENGOC-BC®; its concentration was higher and different from those of the NC group, and from the non adjuvanted protein PD at 7, 14, 21 days after immunization. The subcutaneous footpad inoculation with PD at 21 days, challenging for the DTH assay, resulted in a very effective booster dose as demonstrated by the antibody level increase measured at 60 days. Antibody levels in animals immunized with NPL, VA-MENGOC-BC® and PD were not significantly different with each other, but only with those of the NC group.
These findings confirm the NPL and VA-MENGOC-BC® capacity for inducing immunological memory, since all the animals received an intradermal PD booster for the DTH assay. This reaction allowed in vivo confirmation of Th1 clone functioning after activation by their capacity to generate inflammatory processes when faced with a new antigenic challenge. There did not appear to be any immediate hypersensitivity reaction, proving that the inflammatory processes were provoked after 48 hours. Both NPL and PD-Tp induced higher responses than VA-MENGOC-BC®, and NC (Fig.1C).
SBA assays using pooled mouse sera from the 21 and 60 day time points after the last dose showed that those animals immunized with either NPL or VA-MENGOC-BC®, had the greatest bactericidal activity against the Cu385-83 epidemic strain. The sera from the groups immunized with the NPL and VA-MENGOC-BC® IM, (titres of 256 ± SD ≤ 0.5 log2 at 21 days and titre of 512 ± SD ≤ 0.5 log2 VA-MENGOC-BC® IM, and titre of 1024 ± SD ≤ 0.5 log2 NPL at 60 days), the PD-Tp sera at 21 days did not elicit bactericidal titres (SBT), while the bactericidal titre at 60 days was 8 ± SD ≤ 0.5log2. The IN route of immunization was less effective in inducing SBA titer sat this dosage since NPL only elicited titres of 64 ± SD ≤ 0.5log2 at 21 days and 128 ± SD ≤ 0.5log2 at 60 days while the OMV induced titres of 8 ± SD ≤ 0.5log2 at 21 days and 16 ± SD ≤ 0.5 log2 at 60 days (Fig.1D).
The SBA is a correlate for protective antibody responses against Neisseria meningitides. SBA titers above 8 or a 4 fold increase are considered as protective in many species . We found that IM immunization with NPL and VA-MENGOC-BC® yielded sera with greater bactericidal activity than the IN immunized groups with the same formulations. The observed differences between SBA for the different immunization routes IM vs. IN suggest that composition differences exist among antibody production, which motivated us to perform, in a separate study, an IgG subclass analysis. We found that those animals immunized IM with the NPL and VA-MENGOC-BC® elicited higher levels of IgG2a than did those animals immunized IN at 15 micrograms per dose (results not shown). In mice, the IgG2a isotype is the most efficient activator of complement, while the IgG1 isotype is the poorest. Thus, the lower IgG2a detected in IN treatment could in part explain the inferior bactericidal activity observed in these sera. Similar results were obtained by del Campo and coworkers  immunizing with OMV and its derivative cochleates at 50µg dose by the IN route.
Th1/Th2 patterns coexist in the immune response, but one predominates over the other, depending on the antigenic stimulation of the pathogen responsible for the disease and, in the case of vaccines, on the formulation used. The three assay formulations evaluated in this study stimulate IL-10, TGFβ, IFN-γ and TNF expression. These last pro-inflammatory cytokines, related to cellular response, significantly increase their expression in the groups vaccinated with PD-Tp and NPL, when they are administered by the IM, as well as IN route. Meanwhile the relative amounts of IL-10 and TGFβ messengers are reduced or maintained relatively equal to with respect to VA-MENGOC-BC®. These last cytokines favor more regulatory pressure on the immune response pattern.
In relation to these results, the new antimeningococcal formulation (NPL) without aluminum hydroxide prepared with components of defined molecular structure, and assembled in the form of nanoproteoliposomes, are equal in terms of immunogenicity to the classic formulation that uses aluminum hydroxide, even at a lower dosage, giving a more potent T-cell immune response with equivalent immunological memory, lower reactogenicity and better structural definition. This new formulation could be applicable to new vaccines against meningococcal disease, and eventually as modulators of the immune response against other diseases.