Proteoliposome derived cochleate as novel adjuvant.

Cochleate structures (CS) consist in a highly stable lipid structures that have been reported to be a good antigen delivery system. The incorporation of pathogen associated molecular pattern (PAMP) from bacterial membranes into CS became in a promising approach to develop adjuvants, particularly mucosal adjuvants. Therefore, we prepare CS from proteoliposome (PL) obtained from Neisseria meningitidis B (PLCS) and evaluated it for its capability to stimulate the immune system as well as the adjuvant activity. The ability of PLCS to induce Thl polarization was also explored. The results and the easy capability for new antigen incorporation on CS support its use as adjuvant for immunization with a large variety of pathogen derived antigens and different routes of immunization.

The adjuvant potential of synthetic alkylglycerols.

Alkylglycerols (AGs) have shown immune stimulant and adjuvant activity in many studies, but natural sources are not so accessible and their extraction from them is very complicated. Therefore, a group of chemists at IFAL have synthesized AG analogs. The aim of this work was to evaluate the adjuvant potential of different synthetic AGs. A mix of ovoalbumin (Ova) and AGs increase anti-Ova IgG antibodies production in sera of immunized mice. The predominant subclass was IgG1 although higher levels of IgG2a were observed as the carbon chain length of AGs increased. AGs also induced the production of IL-12 and nitric oxide (NO) in the U937 human histiocyte and J774 mouse macrophage cell lines, respectively. These results indicate that synthetic AGs are effective adjuvants for the standardized antigen, Ova.

Immunological evaluation of bacterial derived Cochleate and proteoliposome as mucosal adjuvants.

We evaluated the potential of two bacterial derived compounds, Cochleate and Proteoliposome (PL), administrated to mice by nasal or oral routes on induction of antibody and cytokine responses. Anti PL IgG and IgA responses were measured by ELISA in saliva, sera or vaginal fluids of immunized mice. Productions of gammaIFN and IL-5 were determined in spleen cells of immunized mice following a recall in vitro with Cochleate or PL. Intranasal administration elicited a higher anti PL IgA response in both saliva and vaginal fluids as compared with oral route. Mice immunized with Cochleate or PL via intranasal or oral route-induced anti PL IgG and IgG2a antibody responses in their sera and vaginal fluids. Spleen cells from these immunized mice produced gammaIFN, but not IL-5, after a recall in vitro with Cochleate or PL. These results show that Cochleate and PL are capable of inducing both systemic and mucosal antibody responses as well as a Th1 type of immunity as evidenced by high gammaIFN and IgG2a antibody responses.

Proteliposome-derived Cochleate as an immunomodulator for nasal vaccine.

Proteoliposome (PL) has been recently used as a protective intramuscular (i.m.) anti-meningococcal BC vaccine. It induces a preferential Th 1 type of immune response. Nevertheless, mucosal protection is mainly mediated by IgA antibody response, which is not usually induced by i.m. vaccination route. IgA antibody production needs the stimulation of Th3 subpopulation, which is also related to the induction of small dose tolerance. We hypothesized that PL-derived Cochleate can induce a specific mucosal IgA and systemic IgG antibody responses. We could show that mice immunized with two or three intranasal doses of PL-derived Cochleate developed significantly increased levels of local anti PL IgA and systemic IgG antibody responses. Thus, our results suggest that PL-derived Cochleate can be used as a promising immunomodulator and delivery system for the development of mucosal, particularly nasal vaccines.

Adjuvant properties of lipopolysaccharide from Neisseria meningitidis serogroup B detoxified and conjugated with tetanus toxoid.

We evaluated the adjuvant properties and toxicity of purified Neisseria meningitidis serogroup B lipopolysaccharide (LPS) conjugated with tetanus toxoid (TT) using a new method of conjugation to obtain amine groups in the polysaccharide structure. The endotoxic activity of treated LPS was reduced 2400 times as determined by Limulus amoebocyte assay and no mortality was observed in Balb/c mice inoculated with detoxified LPS versus 100% mortality in native LPS inoculated mice. The conjugated LPS-TT elicited in mice higher anti-TT IgG2a and IgG1 than unconjugated TT. In addition, high levels of anti-LPS IgG and IgG subclasses were detected in sera. These results evidence the adjuvant activity of detoxified LPS and may suggest that the conjugation to TT changes the LPS immune response from thymus-independent to thymus-dependent.

Strategy for determination of an efficient Cochleate particle size.

Cochleate structures obtained from the outer membrane of Neisseria meningitidis serotype B have demonstrated to be high immunogenicity when administrated by intramuscular, oral or intranasal routes, and could be used as adjuvant and meningococcal nasal vaccine candidate. Due to the microparticulate nature of Cochleate it is necessary to control the particle size since it capture by cells of the immune system could be affected by this aspect. We combined optic microscopy and immunisation experiments to select the optimum particle size. Six different processes of producing Cochleate obtaining were evaluated and different mechanical stress conditions were carried out to homogenize and modulate the particles size. The more immunogenic particles were selected on the basis of the levels of specific IgA and IgG antibodies induced after intranasal immunisation in mice. The best treatment parameter for mechanical stress of the Cochleate was prolonged treatment with untrasonic low frequency waves.

Scale up of proteoliposome derived Cochleate production.

Cochleate are highly stable structures with promising immunological features. Cochleate structures are usually obtaining from commercial lipids. Proteoliposome derived Cochleate are derived from an outer membrane vesicles of Neisseria meningitidis B. Previously, we obtained Cochleates using dialysis procedures. In order to increase the production process, we used a crossflow system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The raw material and solutions used in the production process are already approved for human application. This work demonstrates that CFS is very efficient process to obtain Cochleate structures with a yield of more than 80% and the immunogenicity comparable to that obtained by dialysis membrane.

Novel adjuvant based on a proteoliposome-derived cochleate structure containing native lipopolysaccharide as a pathogen-associated molecular pattern.

Proteoliposomes (PL) from Neisseria meningitidis B have been widely used as a core antigen for antimeningococcal vaccination. PL contain major outer membrane proteins, LPS and phospholipids, and they induce a strong Th1 immune response, but they have low stability in solution. Attending to the need for new vaccine adjuvants, we developed a highly stable cochleate structure (CS) from PL using a technology that allows easy incorporation of new antigens. We explored the ability of PLCS to activate the immune system and its possible application as an adjuvant for parenteral and mucosal routes. Our results showed that PLCS were able to upregulate the expression of MHC class II and costimulatory molecules on human dendritic cells, as well as being able to stimulate the production of soluble mediators of a Th1 response, such as IL-12 and nitric oxide. High levels of anti-PL IgG were detected in serum after i.m. or mucosal (oral and nasal) administration, but also anti-PL secretory IgA was produced in saliva following nasal delivery. The immune response polarization to a Th1 pattern was confirmed by the induction of IgG2a antibodies, positive delayed type hypersensitivity reactions, and IFN-gamma production by splenocytes from immunized mice. The adjuvant potential was explored using PLCS containing ovalbumin (Ova). PLCS-Ova was able to elicit a substantial increase in anti-Ova IgG compared with Ova alone. In addition, a significant reduction in lesion size was observed in mice immunized with Leishmania major antigens in PLCS after challenge with virulent protozoa, suggesting at least partial modulation of the Th2 environment induced by this parasite. In conclusion, our results support the use of PLCS as a potent Th1 adjuvant for parenteral and mucosal vaccines.