Jaiver Rosas

Biodegradable PLGA microspheres as a delivery system for malaria synthetic peptide SPf66

SPf66 is the first chemically synthesised vaccine to elicit a partial protective immune response against malaria. The aluminium hydroxide (alum)-adsorbed SPf66 vaccine is weakly immunogenic and of poor to moderate efficacy in humans. To investigate the possibility of improving SPf66 vaccine immunogenicity, a delivery system based on poly-D,L-lactide-co-glycolide (PLGA) microspheres was developed and the immune response induced after its subcutaneous administration into mice was evaluated. Microspheres were prepared by a solvent extraction/double emulsion (w/o/w) method and characterised for morphology, size, peptide loading, release profile and peptide integrity. The in vitro and in vivo results obtained showed that there was no apparent effect of the encapsulation procedure on SPf66 integrity and immunogenicity. The subcutaneous administration of microspheres showed a significantly higher immune response (serum IgG levels) than that obtained with alum adsorbed SPf66 and it was comparable to that of SPf66 emulsified with Freunds adjuvant (FA). These observations illustrate the potential of PLGA microspheres as a delivery system for chemically synthesised antigens.

Evaluation of SPf66 malaria vaccine during a 22-month follow-up field trial in the Pacific coast of Colombia.

A double-blind randomized placebo-controlled field trial with the SPf66 malaria vaccine was carried out in an endemic area consisting of 14 small villages with exclusive fluvial access, in a rain forest area along the Rosario River, Colombia. A total of 1257 subjects completed the full three dose vaccination schedule on days 0, 30 and 180 (643 vaccinated group/623 placebo group) and were followed-up by passive and active surveillance over a period of 22 months. One hundred and thirty-four Plasmodium falciparum malaria episodes were detected (53 in vaccinated group/81 in placebo group), yielding an attack rate of 5.47 cases/100 person years of follow-up (pyears) in the vaccine group and 8.44/100 pyears in the placebo group. The estimated vaccine protective efficacy was 35.2% (95% CI 8.4-54.2%, P = 0.01). This result supports earlier findings that the SPf66 malaria vaccine diminishes the risk of infection by P. falciparum in endemic areas of South America.

γ-Irradiation effects on biopharmaceutical properties of PLGA microspheres loaded with SPf66 synthetic vaccine

Gamma-irradiation is currently the method of choice for terminal sterilization of drug delivery systems made from biodegradable polymers. However, the consequences of gamma-sterilization on the immune response induced by microencapsulated antigens have not yet been reported in the literature. The aim of the present work was to evaluate the effect of gamma-irradiation on the biopharmaceutical properties of PLGA microspheres containing SPf66 malarial antigen. Microspheres were prepared by a (w/o/w) double emulsion/solvent extraction method. Once prepared, part of the formulation was irradiated at a dose of 25 kGy using 60Co gamma as radiation source. The in vitro results obtained showed that the gamma-irradiation exposure had no apparent effect on SPf66 integrity and formulation properties such us morphology, size and peptide loading. Only the release rate of SPf66 was slightly faster after gamma-irradiation. Subcutaneous administration of irradiated and non-irradiated microspheres into mice induced a similar immune response (IgG, IgG1, IgG2a levels) and was comparable to that obtained with SPf66 emulsified with Freunds complete adjuvant. These observations illustrate the applicability of gamma-irradiation as a method of terminal sterilization of microparticulate delivery systems based on chemically synthesized antigens encapsulated into biodegradable PLGA microspheres.

Immune response after oral administration of the encapsulated malaria synthetic peptide SPf66.

The synthetic peptide SPf66 adsorbed on alum is one of the few Plasmodium falciparum vaccines which have been tested in field trials. We previously reported that subcutaneous administration of SPf66 loaded PLGA microparticles (MP) enhances the antibody response to this antigen compared to the conventional alum formulation. We now evaluate the suitability of polymeric formulations to obtain systemic immune responses by gastric intubation of Balb/c mice. Formulations composed of 1:1 mixtures of PLGA 50:50 and 75:25 (lactic:glycolic) microparticles were administered by the oral route, and when animals were boosted 3 weeks later significant systemic IgG antibody responses were elicited, comparable to alum triple shot and superior to the aqueous vaccine given by the oral route. The finding of IgG2a isotype for PLGA-vaccinated mice compared to the absent levels of this isotype for the alum-vaccinated group could be interpreted as a sign of Th1-like immune response and cellular immune response activation. Our results confirm that using the appropriate schedule the oral administration of PLGA particles is suitable to obtain systemic immune responses to the carried antigen.

Amino terminal peptides of the ring infected erythrocyte surface antigen of Plasmodium falciparum bind specifically to erythrocytes.

The Ring-Infected Erythrocyte Surface Antigen (Pf155/RESA) sequence was chemically synthesized in fifty four 20-mer sequential peptides, covering the entire protein, each of which was tested in erythrocyte binding assays. Peptides 6671 and 6673, corresponding to residues 141-160 and 181-200, respectively, presented a high specific binding activity to erythrocytes with affinity constants of 190 nM and 105 nM respectively. Their binding was sensitive to previous enzymatic treatment of erythrocytes. A region of peptide 6673 has been identified, very recently, as a B-cell epitope, target of neutralizing antibodies (Siddique AB, Iqbal J, Ahlborg N, Wâhlin FB, Perlmann P, Berzins K. Antibodies to nonrepeat sequences of antigen Pf155/RESA of Plasmodium falciparum inhibit parasite growth in vitro. Parasitol Res 1998;84:485-91). The critical residues for erythrocyte binding for peptide 6671 (MTDVNRYRYSNNYEAIPHIS) and for peptide 6673 (LGRSGGDIIKKMQTLWDEIM) were recognized. Based on these data, the presence of five functional regions of RESA is postulated.

Enhancing Immunogenicity and Reducing Dose of Microparticulated Synthetic Vaccines: Single Intradermal Administration

AbstractPurpose. Our purpose was to evaluate the ability of a polymeric vehicle to release a model synthetic vaccine to the skin in order to reach a potent activation of the specific immune response. Methods. The peptide-loaded poly-d,l-lactide-co-glycolide acid (PLGA) microparticles were prepared by a double emulsion technique and administered to Balb/c mice. The immune response (antibody and T cell activation) obtained by the intradermal (i.d.) and the subcutaneous (s.c.) routes was tested. Results. When similar doses of peptide-loaded microparticles were injected s.c. or i.d. in mice, the antipeptide IgG antibody immune response was found to be significantly higher after i.d. injection into the skin. We could also reduce the dose of antigen 10 times by the i.d. route and find a similar antibody response to that obtained by the s.c. immunization. At the lowest i.d. dose level, the IgG2a/IgG1 ratio was also incremented and the IgE production decreased. The i.d. microparticles induced, at both dose levels, a marked IFN-γ secretion by peptide-stimulated splenocytes and lymph node cells and a significant T cell proliferation in spleen cell cultures. Conclusions. The results demonstrate that peptide-loaded microparticles were efficiently administered by the i.d. route because lower doses were required and powerful antibody and T cell responses were obtained compared to the conventional s.c. administration.

Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells.

Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complexs strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54‐kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an α‐helical structure. HABP–target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus–target cell interactions and implications for developing strategies for controlling this disease.

Structural modifications enable conserved peptides to fit into MHC molecules thus inducing protection against malaria.

Developing a rational methodology for obtaining vaccines against P. falciparum malaria (the disease’s most lethal form, afflicting more than 250 million people around the world per year and killing about 2 million of them) has become one of the main objectives of public health authorities around the world. A multiantigenic vaccine, containing molecules from the parasite’s different developmental stages, is required due to the parasite’s remarkable complexity and adaptability. The first such approach (the SPf66 synthetic vaccine), 5] which used peptides from molecules from different parasite stages, conferred limited protective efficacy in Aotus monkey studies and in field trials carried out on human volunteers around the world.

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells

Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasites in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.

Comparison of the adjuvanticity of two different delivery systems on the induction of humoral and cellular responses to synthetic peptides.

The aim of this work was to test, evaluate, and compare the immunogenicity of the S3 malarial short synthetic model peptide in Balb/c mice when it was delivered with different adjuvants. Specifically, it studied the adjuvanticity of two different particulate delivery systems, human compatible Montanide® ISA 720 w/o emulsion and poly-lactide-co-glycolide acid microparticles, in terms of the enhancement and sub-set type of the immune response elicited following immunization. Aditionally, conventional aluminum hydroxide gel adjuvant was included as a reference. Aluminum adjuvant failed to improve the lack of immunogenicity of this antigenic peptide on its own. On the other hand, Montanide and microparticles given subcutaneously resulted in effective adjuvants and revealed mixed Th1/Th2 immune responses, with moderate antibody and lymphoproliferative responses, and higher IFN-γ secretion for Montanide. Hence, microparticles administered intradermally (not possible with Montanide) elicited superior and potent antibody levels, including higher cytophilic isotype (IgG2a), and the greatest limphoproliferation and IFN-γ levels. The results here presented support the capability and suitability of microparticle delivery systems to reach the adequate adjuvanticity necessary for future malaria vaccine development.