The development of new oral vaccines using porous silica.

Ordered mesoporous silica (OMS) was proved to be an efficient oral adjuvant capable to deliver a wide in size variety of different antigens, promoting efficient immunogenicity. This material can be used in single or polivalent vaccines, which have been developed by a group of Brazilian scientists. The experiments performed with the model protein Bovine Serum Albumin (BSA) gave the first promissing results, that were also achieved by testing the virus like particle surface antigen of hepatitis B (HBsAg) and diphtheria anatoxin (dANA). Nanostructured OMS, SBA-15 type, with bi-dimensional hexagonal porous symmetry was used to encapsulate the antigens either in the mesoporous (pore diameter ∼ 10 nm) or macroporous (pore diameter > 50 nm) regions. This silica vehicle proved to be capable to create an inflammatory response, did not exhibit toxicity, being effective to induce immunity in high and low responder mice towards antibody production. The silica particles are in the range of micrometer size, leaving no trace in mice organs due to its easy expulsion by faeces. The methods of physics, usually employed to characterize the structure, composition and morphology of materials are of fundamental importance to develop proper oral vaccines in order to state the ideal antigen load to avoid clustering and to determine the rate of antigen release in different media mimicking body fluids.

Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions.

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56 °C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100 °C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20 °C). At frozen temperatures, large aggregates (>200 nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

Immunology in Brazil: historical fragments.

Inspired by the pioneers Adolfo Lutz, Vital Brazil and Carlos Chagas, Brazilian Immunology was born and became extremely broad and multidisciplinary, always in partnership with other disciplines such as Parasitology, Biochemistry, Physiopathology or Genetics, and constituting an important branch of what is now called Toxinology. Today, the general scenario of Immunology in Brazil represents an example that is perhaps unique in the world of integrations with other areas of knowledge.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis.

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H(III)) or low (L(III)) antibody response and for maximum (AIR(MAX)) or minimum (AIR(MIN)) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR(MIN) mice whereas in the other strains approximately 40% of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40 percent of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

The improvement of the therapeutic anti-Lachesis muta serum production in horses.

The main features associated with pit viper envenomations include the intense local lesions such as oedema, necrosis, acute renal failure and other effects. The severity of these reactions to snakebite depends on the degree of envenomation. Lachesis muta venom (LMV) has weak lethal activity, but due to the large amount often inoculated, the effects are extremely severe and demand anti-venom with a high neutralizing capacity. LMV had the lowest neutralizing antibody induction capacity in horses when compared with that of other venoms. For example, Bothrops anti-venom serum neutralizes 180 times the equivalent LD(50) to Bothrops venom; Crotalus anti-venom neutralizes 250 LD(50) of this venom, while Lachesis anti-venom neutralizes only five LD(50) of the Lachesis toxins. To examine the reasons for this low antibody induction, the H(GP) mouse line, genetically selected for high antibody production received, at different times during immunization with sheep erythrocytes (SE), whole LMV and isolated venom fractions I-VI eluted by gel-filtration chromatography on Superdex75. The specific antibody responsiveness showed a partial, but significant suppression of the anti-SE antibody responses during the kinetics of the primary and even the secondary immunizations, after 50-100 microg of fractions IV and V administration 72-48 h before the first antigen injections. Fraction IV was then applied in a Superose 12 column and three samples were obtained. The peak IVA containing a component of Mr 27 kDa was liable with the immunosuppressive effect as made evident by its effect on the H mice anti-SE responses. Horses receiving the LMV exempt of fractions IV and V produce highly significant anti-Lachesis sera with a 45 LD(50) neutralizing activity, providing, for the first time, an efficient specific therapeutic heterologous serum for human use.

Effects of Lonomia obliqua (lepidoptera, saturniidae) toxin on clotting, inflammatory and antibody responsiveness in genetically selected lines of mice.

Lines of mice genetically selected for high (H) or low (L) antibody response and for maximal (AIRMAX) or minimal (AIRMIN) acute inflammatory reaction, in which the opposite extreme potentialities have been clearly defined, offer an appropriate model for investigating the environmental and genetic factors acting on innate and adaptative immunobiological functions. This model has been successfully employed to study the resistance or susceptibility against pathogens and/or toxins. It had been demonstrated that the skin contact with Lonomia obliqua caterpillar bristles induces local inflammation and may elicit severe hemorrhagic disorders. In the present study, blood coagulation time, and the acute inflammatory reaction were scored 24 h after injection of the Lonomia bristles crude extract in a subcutaneous dorsal air pouch. The acute inflammation was determined by the leukocyte concentration in the local exudates. The highest interline differences were observed between the AIRMAX (10(6) cells/ml) and AIRMIN (2 x 10(5) cells/ml) and this distinct expression involves the number of monocytes, eosinophils and mainly neutrophils. Regarding coagulation, the highest interline difference was observed between the HIII and LIII mice, and the F1)[LIII x HIII] hybrids showed the overdominance of the fast clotting character. The adaptative immune response was evaluated by comparing the anti-Lonomia bristle extract IgG titer among the lines: the antibody titers were higher in the H lines than in the L ones and equivalent in the AIRMAX and AIRMIN mice, in accordance to the phenotype profiles generated by the distinct selective processes. The genetically selected mice lines-AIRMAX, AIRMIN, HI, HIII, HG, LIII and LG-showed an almost continuous distributions for inflammation, coagulation time and IgG antibody titers, being the interline variances always higher than the intraline ones for the individually measured phenotypes. Altogether, these results suggest the independent polygenic regulation of these traits, being indicative of the genetic control to Lonomia toxin innate and adaptative sensitivity in humans.

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.

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 percent of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4 percent and decreased its ß-T1 structure by 2 percent. 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

Independent genetic control of B- and T-cell tolerance in strains of mouse selected for extreme phenotypes of oral tolerance.

Two strains of mice were genetically selected for susceptibility (TS-Ab/HetS strain) or resistance (TR-Ab/HetS strain) to oral tolerance of the humoral response by using ovalbumin (OVA). The progressive interstrain divergence produced by bi-directional selective breeding during 15 generations demonstrated the polygenic nature of oral tolerance. This paper shows the humoral and delayed-type hypersensitivity (DTH) responses, after intragastric administration of OVA and subsequent immunization with that immunogen in complete Freund adjuvant (CFA). Only the TS-Ab/HetS mice were tolerant for immunoglobulin (Ig)G production with its tolerance degree being the same as that obtained when Al ((OH)(3)) was employed. The DTH reactivity was not correlated to the antibody responsiveness, because both the TS-Ab/HetS and TR-Ab/HetS strains had their DTH reactions suppressed. The cyclophosphamide (Cy) pretreatment prevented DTH suppression on TR-Ab/HetS but do not in TS-Ab/HetS mice. Interstrain difference was also observed for the splenic index in the Cy-treated groups, although the number of splenocytes was the same. Flux cytometry cell analysis showed the recovery of CD3(+) cell numbers in both strains, but only the TR-Ab/HetS mice had their CD4/CD8 pattern restored. These results suggest: firstly, the independent control of DTH and humoral tolerance responsiveness; secondly no support for the clonal anergy concept; and thirdly the matrix proteins neo-synthesis after Cy treatment may facilitate the tolerance abrogation.

Large unilamellar vesicles as trehalose-stabilised vehicles for vaccines: storage time and in vivo studies.

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein from Mycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with large unilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehalose avoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrapped protein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on the liposomal membrane integrity.

Schistosoma mansoni: continuous variation in susceptibility of the vector snail of schistosomiasis, Biomphalaria tenagophila I. Self-fertilization-lineage.

Mascara, D., Kawano, T., Magnanelli, A. C., Silva, R. P. S., Sant' Anna, O. A., and Morgante, J. S. 1999. Schistosoma mansoni: continuous variation in susceptibility of the vector snail of schistosomiasis, Biomphalaria tenagophila I. Self-Fertilization-Lineage. Experimental Parasitology 93, 133-141. Artificial selection of Biomphalaria tenagophila snails for susceptibility to infection by Schistosoma mansoni (Brazilian SJ strain) was carried out from natural populations. After five self-fertilization generations, two lineages were isolated and were designated as SUSC (highly susceptible 93-100%) and RES (nonsusceptible 5-0%). Length of the prepatent period, cercarial production, and mortality of the hosts in postexposure were determined in all generations (F(1)-F(8)) and were analyzed as quantitative traits related to host susceptibility. Distribution patterns of frequencies were observed within snail families (samples derived from one F(0) snail), these traits showing a significant influence by selection applied to susceptibility. The multiple quantitative classes were described in terms of continuous variation. During the selection of SUSC lineage, classes with higher values of prepatent length and lower cercarial production were eliminated, and the heritability calculated for these two traits was 0.811 and 0.709, respectively. Experimental results were correlated with an increase in the level of susceptibility in the generations selected and are discussed in relation to inheritance patterns as well as the quantitative variation of susceptibility.

Genetics of immunological tolerance: I. Bidirectional selective breeding of mice for oral tolerance.

The polygenic nature of oral tolerance regulation was elucidated by the method of bidirectional selective breeding of mouse strains for tolerance susceptibility (TS) and resistance (TR) starting from a genetically heterogeneous population achieved by the equilibrated intercrossing of eight inbred mouse strains (A/J, DBA/2J, P/J, SWR/J, SJL/J, CBA/J, BALB/cJ and C57BL/6J). Seven days after intragastric administration of 5 mg OVA or BSA, mice were intraperitoneally immunized with 100 microg of the corresponding antigen. The individual antibody titres were measured by haemagglutination. The phenotypes at the highest and lowest extremes were selected for assortative mating, avoiding consanguinity. The second litter of each mating couple was intraperitoneally immunized only to evaluate the immunocompetence of the corresponding generation and to ascertain the non-selection of non-responder mice. A normal distribution of agglutinin titres ranging from 4 to 14 log2 was observed in the F0 population. In the F12 generation, TR and TS strains showed highly significant differences for agglutinin titres (TR=15.06+/-1.80 and TS=8.35+/-2.44), and IgG responses by ELISA. Up to the F12 generation, the mean realized heritability was 0.14+/-0.02. The response to the selection was 0.43 log2 and the selection differential 3.10 log2/generation. A provisional estimation indicated that oral tolerance may be influenced by eight or nine independent loci.

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 from Mycobacterium 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.

Effect of genetic modification of acute inflammatory responsiveness on tumorigenesis in the mouse.

Two distinct bidirectional selective breedings for quantitative traits were initiated from identical genetically heterogeneous mouse populations. The resulting lines are characterized by maximal or minimal acute inflammatory responsiveness (AIR): AIRmax and AIRmin lines, respectively, and by resistance or susceptibility to chemical skin tumorigenesis: Car-R and Car-S lines, respectively. The AIR response to s.c. injection of polyacrylamide microbeads, measured by cell content in the local exudate, was 10 times higher in AIRmax than in AIRmin mice. The response to selection was asymmetrical: the realized heritability was 0.26 in AIRmax and 0.008 in AIRmin, and resulted from the additive effect of 7-11 quantitative trait loci (QTL). Low responsiveness was globally dominant in F1 and 48% of F2 segregant variance was found to be due to genetic factors. These findings are the first demonstration of innate regulation of AIR by germ line genes. Susceptibility to skin tumorigenesis induced by a two-stage initiation (DMBA)-promotion (TPA) protocol was lower in AIRmax mice than in AIRmin mice, a 6-fold difference in tumor induction rate. Intense AIR was found to be associated with resistance, and low AIR with susceptibility to tumorigenesis, in F2 segregants chosen for extreme AIR phenotypes. At least some of the AIR QTLs therefore contain genes controlling tumorigenesis. Tumor phenotypes differed more in Car-R and Car-S than in AIRmax and AIRmin lines, indicating that QTLs unrelated to AIR, contribute to the host response to tumorigenesis. The extreme phenotypes/genotypes of the four selected lines and the known genetic constitution of their foundation population, offer new possibilities to discriminate the genes/mechanisms controlling two important traits: AIR and response to chemical tumorigenesis. Collaborative projects will be favorably considered. The description of tumor resistance genes in AIRmax and Car-R mice may be helpful for epidemiology and therapy of human cancer.

Rabies infection and specific effect of vaccination in mice selected for high and low immunobiological parameters

Innate and acquired resistance to rabies infection was investigated in mice genetically selected for high(H) or low(L) antibody responsiveness from selections I, III and IV and in mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction. These mouse lines were infected intramuscularly with different virus dilutions and the LD50 was determined. The HIII and HIV mouse lines were more susceptible than the LIII and LIV lines and the HI line showed a discrete but higher resistance than the LI line. Analysis of the interline (H x L) F1 hybrids from selections III and IV indicated different dominance effects on the "resistant" and "susceptible" phenotypes when the route of vaccination was changed. No differences were observed between the AIRmax and AIRmin mice, suggesting that inflammation plays a minor role in the resistance to rabies virus. The comparison of LD50 in mice vaccinated by distinct routes showed that the highest interline difference occurred after intramuscular vaccination (250-fold between H and L and 800-fold between F1 and L). These results indicate that different mechanisms may participate in acquired antirabies resistance.

Rabies infection and specific effect of vaccination in mice selected for high and low immunobiological parameters.

Innate and acquired resistance to rabies infection was investigated in mice genetically selected for high (H) or low (L) antibody responsiveness from selections I, III and IV and in mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction. These mouse lines were infected intramuscularly with different virus dilutions and the LD50 was determined. The HIII and HIV mouse lines were more susceptible than the LIII and LIV lines and the HI line showed a discrete but higher resistance than the LI line. Analysis of the interline (H x L) F1 hybrids from selections III and IV indicated different dominance effects on the "resistant" and "susceptible" phenotypes when the route of vaccination was changed. No differences were observed between the AIRmax and AIRmin mice, suggesting that inflammation plays a minor role in the resistance to rabies virus. The comparison of LD50 in mice vaccinated by distinct routes showed that the highest interline difference occurred after intramuscular vaccination (250-fold between H and L and 800-fold between F1 and L). These results indicate that different mechanisms may participate in acquired antirabies resistance.

Incomplete dominance of the low antibody response to Cryptosporidium parvum antigens in mice selected for high and low antibody responsiveness.

The humoral antibody response to Cryptosporidium was investigated in mice genetically selected for high (H) and low (L) antibody responsiveness. Groups of 4-5 mice from two different selections, general primary (GP) and general secondary (GS), were studied. Following immunization with Cryptosporidium parvum antigens, the maximum levels of IgG in the HGP (X +/- SD = 1.13 +/- 0.35, N = 5) in the HGS (0.42 +/- 0.15, N = 4) lines, and of IgM in the HGP line (0.86 +/- 0.53, N = 5) were significantly higher than those in their L counterparts (0.04 +/- 0.02, N = 5; 0.05 +/- 0.02, N = 4 and 0.24 +/- 0.07, N = 5, respectively). These findings were similar to those reported for other immunogens. However, the IgG (0.22 +/- 0.05, N = 4) and the IgM (0.33 +/- 0.08, N = 4) responses to immunization of F1 (LGP x HGP) hybrids indicated an incomplete dominance of the low response, in contrast to the incomplete dominance of the high response described for many other antigens and representing an important exception. In addition, the H, L and F1 mice did not develop detectable infections when inoculated with live Cryptosporidium oocysts, supporting the view that a reduced or zero antibody production itself is not enough to permit the establishment of Cryptosporidium infection in adult mice.

Incomplete dominance of the low antibody response to Cryptosporidium parvum antigens in mice selected for high and low antibody responsiveness

The humoral antibody response to Cryptosporidium was investigated in mice genetically selected for high (H) and low (L) antibody responsiveness. Groups of 4-5 mice from two different selections, general primary (GP) and general secondary (GS), were studied. Following immunization with Cryptosporidium parvum antigens, the maximum levels of IgG in the HGP, (X ñ SD = 1.13 ñ 0.35, N = 5) and in the HGS (0.42 ñ 0.15, N = 4) lines, and of IgM in the HGP line (0.86 ñ 0.53, N = 5) were significantly higher than those in their L counterparts (0.04 ñ 0.02, N = 5;0.05 ñ 0.02, N = 4 and 0.24 ñ 0.07, N = 5, respectively). These findings were similar to those reported for other immunogens. However, the IgG (0.22 ñ 0.05, N = 4) and the IgM (0.33 ñ 0.08, N = 4) responses to immunization of F1 (LGP x HGP) hybrids indicated an incomplete dominance of the low response, in contrast to the incomplete dominance of the high response described for many other antigens and representing an important exception. In addition, the H, L and F1 mice did not develop detectable infections when inoculated with live Cryptosporidium oocysts, supporting the view that a reduced or zero antibody production itself is not enough to permit the establishment of Cryptosporidium infection in adult mice.