Correlates of GLA family adjuvants' activities.

Lipopolysaccharide (LPS) is a well-defined agonist of Toll-like receptor (TLR) 4 that activates innate immune responses and influences the development of the adaptive response during infection with Gram-negative bacteria. Many years ago, Dr. Edgar Ribi separated the adjuvant activity of LPS from its toxic effects, an effort that led to the development of monophosphoryl lipid A (MPL). MPL, derived from Salmonella minnesota R595, has progressed through clinical development and is now used in various product-enabling formulations to support the generation of antigen-specific responses in several commercial and preclinical vaccines. We have generated several synthetic lipid A molecules, foremost glucopyranosyl lipid adjuvant (GLA) and second-generation lipid adjuvant (SLA), and have advanced these to clinical trial for various indications. In this review we summarize the potential and current positioning of TLR4-based adjuvant formulations in approved and emerging vaccines.

Nanobiotechnological approaches to delivery of DNA vaccine against fungal infection.

Vaccines play an essential role in keeping humans healthy. Innovative approaches to their use include the utilization of plasmid DNA encoding sequences to express foreign antigens. DNAhsp65 from Mycobacterium leprae is suitable for this purpose due to its ability to elicit a powerful immune response. Controlled release systems represent a promising approach to delivering vaccines. In this work, we used liposomes or PLGA systems to deliver DNAhsp65 to treat the pulmonary fungal infection Paracoccidioidomycosis. Both formulations modulated a protective immune response and reduced the pulmonary fungal burden even in the groups receiving less than four times the amount of the DNAhps65 entrapped within the nanoparticles. Although both systems had the same effective therapeutic results, the advantage of the liposome formulation was that it was administered intranasally, which may be more easily accepted by patients. These systems are a great alternative to be considered as adjuvant vaccine therapy for systemic mycosis.

Liposomal delivery of Mycobacterium leprae antigen(s) with murabutide and Trat peptide inhibits Fas-mediated apoptosis of peripheral blood mononuclear cells derived from leprosy patients.

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose capped lipoarabinomannan) may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with particulate mode of antigen presentation led to both decreased percentage of propidium iodide (PI) positive cells and T cells expressing Fas-FasL, as well as decreased caspase-8/-3 activities in the lepromatous patients, thereby inhibiting apoptosis, while converse was true with stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-X(L) in the lepromatous patients, thereby inhibiting apoptosis. Thus, the liposomal formulation of antigen promoted proliferation of anergized T cell by inhibiting apoptosis through decreased expression of death receptors and caspase activities and increased expression of anti-apoptotic protein Bcl-X(L) in these patients.

Encapsulation and stability of clofazimine liposomes.

The entrapment of clofazimine (CLO) in a liposomal delivery system for topical application can protect it from absorption into the blood circulation and increase its residence time within the skin. This may reduce the very long mean period of leprosy treatment, as well as the side effects due to the long term administration of large doses of the drug. This investigation deals with critical parameters controlling the formulation and stabilization of liposomes with encapsulated CLO. The entrapment efficiency of CLO in liposomes was increased by altering the proportion of phosphatidyl choline (PC) and cholesterol (CHOL) in liposomes. The stability of liposomal suspensions and the liposomal gels (HPMC K4M) in terms of retention of CLO was measured at refrigeration temperature (2-8 degrees C), room temperature (25 +/- 2 degrees C) and body temperature (37 degrees C) for a period of 3 months. The results show that entrapment of CLO in liposomes can be increased by increasing the proportion of PC. However, the optimum encapsulation and retention of CLO was achieved only with a specific PC:CHOL molar ratio (5.13:1.00). An almost identical value of the entrapment efficiency was obtained when gel filtration and ultracentrifugation methods were used to separate the CLO-carrying liposomes from free drug. The effect of vortexing and sonication on the entrapment efficiency gave similar results, although the mean particle size was different. CLO liposomal gels were found to be stable at room temperature for up to 3 months.

Interactions between cationic liposomes and an antigenic protein: the physical chemistry of the immunoadjuvant action.

The 18 kDa antigenic protein from Mycobacterium leprae (P) or its N-acyl derivative (AP) was incorporated in dioctadecyldimethylammonium bromide (DODAB) liposomes in water or in phosphate-buffered saline (PBS). In water, 100% P incorporation in liposomes contrasts with 65% in PBS. There is 75-80% AP incorporation to liposomes in water against 55-65% in PBS, showing that attachment of hydrophobic residues to the protein, instead of increasing, further decreases incorporation to the liposomes. From protein adsorption on latex, P affinity is larger than AP affinity for the latex surface whereas limiting adsorption for AP is much larger than that obtained for P, possibly due to AP aggregation in solution. P-induced rupture of liposomes containing [14C]sucrose was evaluated from dialysis of protein/liposomes mixtures. In water, P incorporation to the liposomes causes leakage of radioactive contents contrasting with the absence of leakage for P incorporation in PBS. Immunization tests for delayed type hypersensitivity indicate a enhancement of cell-mediated immunological response towards P/DODAB complexes that is not obtained for the isolated protein. Absence of leakage for P in PBS is associated with a P "lying-over" on the liposome and optimization of protein presentation to the immunological system.