Specific and cross-reactive immune response against Mycobacterium tuberculosis antigens in mice immunized with proteoliposomes from Mycobacterium bovis BCG

Objective: To characterize the immunogenicity and the induction of cross-reactive responses against Mycobacterium tuberculosis(M. tuberculosis) of a proteoliposome(PL)from Mycobacterium bovis Bacillus Calmette–Guerin(BCG) with and without alum hydroxide(AL) as adjuvant(PLBCG-AL and PLBCG, respectively) in BALB/c mice.Methods: BALB/c mice were inoculated with phosphate buffer solution, BCG, PLBCG and PLBCG-AL. The humoral immunogenicity was determined by ELISA [immunoglobulin G(Ig G), Ig G1 and Ig G2a] and the cellular immunogenicity was evaluated in vivo by delayed type hypersensitivity. The humoral cross-reactive response against M. tuberculosis was determined by Western blot.Results: Sera from animals immunized with PLBCG-AL and PLBCG showed significant increase in specific total Ig G and Ig G1 antibodies and the presence of cross-reactive antibodies against M. tuberculosis antigens, which were more intense with the use of alum as adjuvant. Mice immunized with PLBCG and PLBCG-AL also showed a specific cellular response in vivo.Conclusions: The cellular and humoral immunogenicity of PLBCG and the capacity to induce cross-reactive responses against M. tuberculosis is in agreement with the protective capacity previously demonstrated by this vaccine candidate and supports the continuation of its evaluation in further stages.

Proteoliposome and Polysaccharide-Based Meningococcal Vaccine Are Immunogenic in Infants and Toddlers and Primes for Memory against Serogroup C Polysaccharide

Neisseria meningitidis capsular polysaccharides are the main target of the protective immune response against bacterial meningitis. They are thymus-independent type 2 (TI-2) antigens that are poorly immunogenic and not protective in young children, and their administration may impair subsequent challenge with the same polysaccharide. These problems have been addressed using three different vaccines consisting of 1) polysaccharide alone, 2) polysaccharide covalently conjugated to a carrier protein, and 3) polysaccharide with Proteoliposome (PL) adsorbed onto Al(OH)3. VA-MENGOC-BC? is one of the third types of vaccine. It contains PL (detergent-extracted external membrane proteins forming vesicles) and polysaccharide (Ps) from N. meningitidis serogroups B and C (PsC), respectively. Nevertheless, there is a concern that to overcome the TI-2 nature of Ps the covalently conjugation to a carrier is mandatory. Therefore, we evaluated the immune response induced by VA-MENGOC-BC? in infants and toddlers in order to determine whether it stimulates the response against the PsC. High IgG anti PsC and anti PL responses were seen following the administration of two doses in infants and toddlers, after a 3rd dose in pre-teenagers, and after confirmed carrier stages in young adults. The anti PL IgG response persisted longer than anti PsC IgG response and IgM response against both antigens was maintained. An IgG1 anti PL response predominated, as well as IgG4 > IgG3 > IgG1 anti PsC responses. These results suggest that non-covalent incorporation of PsC onto Al(OH)3 containing?PL as adjuvant is immunogenic, primes for memory, and induces long-lasting specific antibody response. The improved PsC immunogenicity of this vaccine may be due to the preferential and potent Th1 response induced in mice and human by the PL as adjuvant.

Glutamine transporters as pharmacological targets: From function to drug design

Among the different targets of administered drugs,there are membrane transporters that play also a role in drug delivery and disposition.Moreover,drug-transporter interactions are responsible for off-target effects of drugs underlying their toxicity.The improvement of the drug design process is subjected to the identification of those membrane transporters mostly relevant for drug absorption,delivery and side effect production.A peculiar group of proteins with great relevance to pharmacology is constituted by the membrane transporters responsible for managing glutamine traffic in different body districts.The interest around glutamine metabolism lies in its physio-pathological role;glutamine is considered a conditionally essential amino acid because highly proliferative cells have an increased request of glutamine that cannot be satisfied only by endogenous synthesis.Then,glutamine transporters provide cells with this special nutrient.Among the glutamine transporters,SLC1A5,SLC6A14,SLC6A19,SLC7A5,SLC7A8 and some members of SLC38 family are the best characterized,so far,in both physiological and pathological conditions.Few 3D structures have been solved by CryoEM;other structural data on these transporters have been obtained by computational analysis.Interactions with drugs have been described for several transporters of this group.For some of them,the studies are at an advanced stage,for others,the studies are still in nuce and novel biochemical findings open intriguing perspectives.

GM3-containing nanoparticles in immunosuppressed hosts:Effect on myeloid-derived suppressor cells

Cancer vaccines to date have not broadly achieved a significant impact on the overall survival of patients. The negative effect on the immune system of the tu-mor itself and conventional anti-tumor treatments such as chemotherapy is, undoubtedly, a key reason for these disappointing results. Myeloid-derived suppres-sor cells （MDSCs） are considered a central node of the immunosuppressive network associated with tumors. These cells inhibit the effector function of natural killer and CD8＋ T cells, expand regulatory T cells and can differentiate into tumor-associated macrophages within the tumor microenvironment. Thus, overcoming the suppressive effects of MDSCs is likely to be criti-cal for cancer immunotherapy to generate effective anti-tumor immune responses. However, the capacity of cancer vaccines and particularly their adjuvants to overcome this inhibitory population has not been well characterized. Very small size proteoliposomes （VSSP） is a nanoparticulated adjuvant specifcally designed to be formulated with vaccines used in the treatment of immunocompromised patients. This adjuvant contains immunostimulatory bacterial signals together with GM3 ganglioside. VSSP promotes dendritic cell maturation, antigen cross-presentation to CD8＋ T cells, Th1 polar-ization, and enhances CD8＋ T cell response in tumor-free mice. Currently, four cancer vaccines using VSSP as the adjuvant are in Phase？Ⅰ？and Ⅱ clinical trials. In this review, we summarize our work characterizing the unique ability of VSSP to stimulate antigen-specifc CD8＋ T cell responses in two immunocompromised sce-narios; in tumor-bearing mice and during chemother-apy-induced leukopenia. Particular emphasis has been placed on the interaction of these nanoparticles with MDSCs, as well as comparison with other cancer vac-cine adjuvants currently in preclinical or clinical studies.

EFFECT OF TRANSMEMBRANE Ca^(2+) GRADIENT ON CONFORMATION AND ENZYME ACTIVITY OF RECONSTITUTED ADENYLATE CYCLASE

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca^(2+) gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca^(2+)concentration (≈10^(-6) mol/L, similar to thephysiological condition). If the transmembrane Ca^(2+) gradient was in the inverse direction (i.e. outside higher Ca^(2+) concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca^(2+) ionophore A23187. Proteoliposomes without transmembrane Ca^(2+) gradient exhibited intermediate activities.The conformation difference of adenylatecyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.