Maria Helena Bueno da Costa

Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity in vivo.

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed as one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration.

Silica-based cationic bilayers as immunoadjuvants.

BackgroundSilica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design.ResultsFirstly, the silica/DODAB assembly was characterized at 1 mM NaCl, pH 6.3 or 5 mM Tris.HCl, pH 7.4 and 0.1 mg/ml silica over a range of DODAB concentrations (0.001–1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.05 mM DODAB is enough to produce cationic bilayer-covered particles with good colloid stability. Secondly, conditions for maximal adsorption of bovine serum albumin (BSA) or a recombinant, heat-shock protein from Mycobacterium leprae (18 kDa-hsp) onto DODAB-covered or onto bare silica were determined. At maximal antigen adsorption, cellular immune responses in vivo from delayed-type hypersensitivity reactions determined by foot-pad swelling tests (DTH) and cytokines analysis evidenced the superior performance of the silica/DODAB adjuvant as compared to alum or antigens alone whereas humoral response from IgG in serum was equal to the one elicited by alum as adjuvant.ConclusionCationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum, better performance regarding cellular immune responses and employs very low, micromolar doses of cationic and toxic synthetic lipid.

Stabilisation of immunoconjugates by trehalose

Stable immunoconjugates were prepared in the presence of 400 mM trehalose. Their residual activity after freeze-drying, rehydration and incubation for 9 h at 40 °C was 35%. Freeze-dried conjugates containing 400 mM trehalose incubated at 40 °C for 4 days retained 80% of their original activity.

Conformational stability and antibody response to the 18kda heat- shock protein formulated into different vehicles

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp fromMycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

Dressing liposomal particles with chitosan and poly(vinylic alcohol) for oral vaccine delivery

Liposomes have been used as adjuvants since 1974. One major limitation for the use of liposomes in oral vaccines is the lipid structure instability caused by enzyme activities. Our aim was to combine liposomes that could encapsulate antigens (i.e., Dtxd, diphtheria toxoid) with chitosan, which protects the particles and promotes mucoadhesibility. We employed physical techniques to understand the process by which liposomes (SPC: Cho, 3:1) can be sandwiched with chitosan (Chi) and stabilized by PVA (poly-vinylic alcohol), which are biodegradable, biocompatible polymers. Round, smooth-surfaced particles of REVs-Chi (reversed-phase vesicles sandwiched by Chi) stabilized by PVA were obtained. The REVs encapsulation efficiencies (Dtxd was used as the antigen) were directly dependent on the Chi and PVA present in the formulation. Chi adsorption on the REVs surface was accompanied by an increase of ζ−potential. In contrast, PVA adsorption on the REVs-Chi surface was accompanied by a decrease of ζ−potential. The presence of Dtxd increased the Chi surface-adsorption efficiency. The PVA affinity by mucine was 2,000 times higher than that observed with Chi alone and did not depend on the molecule being in solution or adsorbed on the liposomal surface. The liberation of encapsulated Dtxd was retarded by encapsulation within REVs-Chi-PVA. These results lead us to conclude that these new, stabilized particles were able to be adsorbed by intestinal surfaces, resisted degradation, and controlled antigen release. Therefore, REVs-Chi-PVA particles can be used as an oral delivery adjuvant.

Evaluation of a Diphtheria and Tetanus PLGA Microencapsulated Vaccine Formulation without Stabilizers

Polymeric microspheres containing diphtheria and tetanus toxoids were prepared without protein stabilizers. A vaccine containing 2 Lf(tetanus) and 0.4 Lf(diphtheria) was injected either in BALB/c mice or in guinea-pigs. As control, a group received the alum-adsorbed unencapsulated toxoids. In mice, on day 44 one group and control received a booster and at day 111 the other group received the same booster dose. Before de booster, all groups had very low neutralizing antibodies, as determined by Toxin binding inhibition assay. One week after booster all groups had high antibody titers, especially those immunized with microencapsulated vaccine, which were at least 5 times higher than those immunized with alum vaccine for both antigens. Besides, guinea pigs receiving lower dose had antibodies titers as high as 60 UI/mL, and 30 times higher than those immunized with alum vaccine. Therefore by using an encapsulated vaccine without any kind of protein stabilizer we were able to induce in vivo protective responses irrespective of observed in vitro protein degradation by HPLC. Manipulating the vaccination schedule at the same time to the toxoids encapsulation does not only increase the antibody titers but also their specificity.

Procedures for scaling up the recombinant 18kDa-hsp lepra protein production

We present here one systematic strategy to optimize the preliminary purification of the recombinant 18kDa-hsp from Mycobacterium leprae at laboratory level in order to design a scaling up process. In a few steps a pure protein-as determined by western blot- was obtained. The overall process recovered 33% of the 18kDa-hsp.

HEAT SHOCK PROTEIN MICRO-ENCAPSULATION AS A DOUBLE TOOL FOR THE IMPROVEMENT OF NEW GENERATION VACCINES

ABSTRACT The modern vaccinology encompasses the recombinant DNA technology, protein and carbohydrate chemistry to obtain safe molecularly defined vaccines. Nevertheless most of the vaccines are poorly immunogenic because a large number of antigens are membrane proteins and consequently they are not present in their active conformation in the vaccine. Others are not as potent because they contain only B epitopes and therefore, cannot stimulate cellular memory. We have been studying the characteristics of the recombinant heat shock protein 18kDa-hsp from Mycobacterium leprae as an alternative carrier protein with a T epitope source to enhance the activity of these second generation vaccines. Here we proved that the 18kDa-hsp acted as carrier, without masking the activity of the carried antigen, with similar immune stimulatory effect when compared with ODN1668. Supramolecular aggregates of 18kDa-hsp and Mice serum albumin (MSA) were obtained using glutaraldehyde as cross linker. The Neisseria meningitides serogroup C polysaccharide (PSC, a B epitope) and the carrier protein 18kDa-hsp were co-encapsulated within Soybean phosphatidylcholine liposomes (SPC: Cho : α-Toc, 22 : 5 : 0.18 molar ratio, respectively). These liposomes were prepared in MPB buffer (20 mM phosphate, 295 mM mannitol pH 7.2) in the presence or absence of the ODN1668, TCCATGACGTTCCTGATGCT. When mice were injected with 18kDa-hsp-MSA no antibody against the MSA was observed. This means that the 18kDa-hsp acted as carrier, without masking the carried protein immune activity. Stable liposomes of 150 nm were obtained using mannitol as a cryoprotector. Genetically selected mice when injected with liposomes containing PSC and 18kDa-hsp displayed an antibody titer of 12. In contrast, in those mice injected with free PSC there was no response. The 18kDa-hsp adjuvant effect on the PSC liposomal formulation was comparable to that observed when ODN1668 was co-encapsulated with PSC. Confirming our expectations we observed that the formulation containing 18kDa-hsp conferred a memory response to the carried antigen—the Neisseria meningitidis serogroup C polysaccharide.

Enhanced liposomal vaccine formulation and performance: simple physicochemical and immunological approaches

The Dtxd (Diphtheria toxoid) was the first antigen encapsulated within liposomes, their adjuvant properties were discovered (their capacity to enhance the vaccine immunogenicity). The point here is not to propose a new method to prepare this lipossomal vaccine. The central idea is to give new dresses for old vaccines by using classical and well-established liposome preparation method changing only the encapsulation pH and the immunization protocol. The most appropriate method of Dtxd encapsulation within liposome was based on lipid film hydration in 100 mM citrate buffer, pH 4.0. This was accompanied by changes on protein hydrophobicity, observed by CD and fluorescence spectroscopies. Whenever the Dtxd exposed its hydrophobic residues at pH 4.0, it interacted better with the lipossomal (observed by electrophoretic mobility) film than when its hydrophobic residues were buried (pH 9.0). The Dtxd partition coefficient in Triton-X114 and the acrylamide fluorescence quenching were also pH dependent. Both were bigger at pH 4.0 than at pH 9.0. The relationship protein structure and lipid interaction was pH dependent and now it can be easily maximized to enhance encapsulation of antigens in vaccine development. Mice were primed with formulations containing 5 μg of Dtxd within liposomes prepared in pH 4.0 or 7.0 or 9.0. The boosters were done 38 or 138 days after the first immunization. The IgM produced by immediate response of all lipossomal formulations were higher than the control (free protein). The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. The IgG1 titers produced by both formulations at pH 4.0 and 7.0 were at least 22 higher than those produced by mice injected lipossomal formulation at pH 9.0. When the boosters were done, 138 days after priming the mice produced a IgG2a titer of 29 and the group that received the booster 30 days after priming produced a titer of 25. The strongest antibody production was the neutralizing antibody (245 higher than the control) produced by those mice injected with lipossomal formulation at pH 4.0 with the booster done 138 days after priming. The simple change on lipossomal pH formulation and timing of the booster enhanced both antibody production and selectivity.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80 degrees C for 20 min). N-Acylation increased its ordered structure by 4% and decreased its beta-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.