AFCo1 Induces Mucosal Cross Response Against Polysaccharides Of Neisseria Meningitidis Serogroups In Mice

Neisseria meningitidis is a causal agent of human meningitis. Twelve serogroups exist base in their polysaccharide compositions, six of them cause meningococcal disease, plane and covalently conjugated parenteral vaccines are available. The serum IgG cross reactivity between pathogenic and non-pathogenic strains is a fact. It is consequence of their high protein homology. Nevertheless, the mucosal IgA cross response against polysaccharides are unknown. So, the response induce by polysaccharide C from N. meningitidis alone of adjuvantated with AFCo1 adjuvant in Balb/c mice was addressed. Three intranasal doses did not induce serum IgG responses against A, Y nor W135N. meningitidis serogroups. Contrary, polysaccharide C alone and better when it is formulated with AFCo1 induce mucosal


Short Communication
Neisseria is a mucosal obligate pathogen or non-pathogen species where N. meningitidis has 13 serogroups. This classification is based in its polysaccharide expression and they are: A, B, C, D, E, H, I, K, L, W135, X, Y y Z. Of them, the A, B, C, W135 and Y serogroups are the most disease causing agents. As polysaccharides are thymus-independent antigens the current approach for vaccines purpose is the covalent conjugation to some proteins and theirs use as parenteral vaccines [1]. Nevertheless, in not immunodeficient human the occurrence of a disease by N. meningitidis infection is only one time and it arrives by mucosal route. In addition, the mucosal IgA response induce by N. meningitidis infections have been less explored. The IgA response could be induced as a thymus-dependent and -independent manner [2]. The former is mainly induced against proteins antigens and it is more specific. The latter is not totally clear which antigen is the main responsible for its induction and might be more cross reactive.
Consequently, we would like to explore the possible cross reactivity induce by polysaccharides applied by mucosal route in mice.
Although N. meningitidis infects through mucosal surfaces where IgA is the main protecting antibody, systemic vaccination is currently used which did not induce mucosal response. At present antigen delivery to mucosal surfaces is very interesting especially for protection against infectious agent that are mediated by local immune response. However, mucosal immunization has been limited by important aspects as safety and immunogenicity. An approach to improve this route of vaccination is the use of safe and effective adjuvants. Cross reactivity at serum IgG level between species of Neisseria genus, in particular with N. gonorrhoeae have been described [3]. However, its main explanation is by their structural homology between commons proteins in those species.
One of the shortcomings of parenteral immunization is its relatively poor ability to induce specific IgA [4,5]. Nevertheless, cross reactivity at mucosal IgA level against polysaccharides between N. meningitidis serogroups have been less explored [6]. The first vaccine against N. meningitidis serogroups B and C is base in the outer membrane detergent extractions of a Proteoliposome (PL) form the B serogroup [7] which contains several proteins [8,9]. It is bivalent as it contains also the C polysaccharide adsorbed onto Al(OH)3 (Alum). Our group have been demonstrated that PL in addition to the protective antigens function as a potent adjuvant [10,11], and using this polysaccharide covalent conjugation, the hallmark of polysaccharide vaccines, is not required [12][13][14]. This vaccine is use by parenteral route overcoming the polysaccharide C thymus-independence also in human toddlers [15]. Nevertheless, as PL is absorbed onto alum it could not be use as mucosal adjuvant.
AFCo1 is a multilayer microparticle formed by the interaction of the lipid bilayer of the PL and Ca2+, which contains similar proteins and pathogen-associated molecular pattern as the PL [10,11] inducing systemic specific IgG and in addition mucosal specific IgA. AFCo1 applied by nasal route induces mucosal at regional and distal places (specific IgA) and systemic immune responses (serum specific IgG, specific IgG2a subclass, and IFN-γ) polarizing to a Th1 pattern [16]. The mucosal approach has being applied to several antigens [17,18].

Animals
Female Balb/c mice, six to eight weeks old, supplied by Cenpalab, Cuba were used for all the experiments. All the experiments were performed with the approval from the Ethical Committee for Laboratory at Finlay Institute, Cuba. The studies were performed at least twice.

Preparation of Proteoliposome (PL) and AFCo1
PLs from outer membrane vesicles detergent-extracted from N. meningitidis serogroup B were prepared and supplied as an ethanol precipitate, as well as the C polysaccharide, by the vaccine production unit of the Finlay Institute, Havana, Cuba, for N. meningitidis serogroup B vaccine as previously described. PLderived AFCo1 was prepared as previously described [11].

Animal Immunization and Sample Collection
Mice were immunized intranasal (i.n) with 3 doses of AFCo1 + C polysaccharide; polysaccharide alone or naive as negative control with 7 days intervals. In all groups the blood was obtained from the retrorbital vein at 21 days after the last dose. Saliva samples were collected at 7 days after the last immunization by Pilocarpine 0.5% intraperitoneal stimulation. These were immediately inactivated at 56ºC for 15 min and the supernatants after centrifugation (9000 g) were stored as well the sera at -20°C.

Statistical Análisis
Statistical analyses were done by two way ANOVA followed by Bonferroni post test using Graph Pad Prism 5 software (CA, USA).

Neither C Polysaccharide Alone nor Adjuvantated With AFCo1 by Nasal Route Induce Cross Reactive Serum Igg Against Heterologous Neisseria Meningitidis Polysaccharides
Low serum anti Polysaccharide C IgG was obtained immunizing mice with it alone. This response was significantly increase by the adjuvantation with AFCo1. No serum specific IgG responses were obtained against heterologous polysaccharides with both formulation (Figure 1). These results are in agreement with the strong adjuvant capacity of AFCo1 against proteins [18,19].and non-covalently conjugated polysaccharides [19,20]. Our previous
We are exploring this strategy for the induction of both mucosal and systemic cross reactive responses agains differents Neisseria meningitidis serogroups.