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.

ELISA and modified toxin-binding inhibition test for quality control of the clostridial vaccine processes / ELISA direto e ToBI-test modificado para o controle de qualidade de processos de vacinas clostridiais

This study aimed to assess and standardize the ELISA and modified ToBI test in vitro methods in order to verify the potency of epsilon toxicoid in comparison with the in vivo TCP method. The following epsilon toxoids were used: NIBSC standard from batches 375/07, 532/08, 551/08, 373/07 and 378/07. These were evaluated using a TCP test, ELISA and ToBI tests. The results indicate that the correlation ratio between the dilutions of standard NIBSC toxicoid and absorbance values of 89.44% obtained with the ELISA method support the use of the curve to evaluate epsilon toxoids. However, it was observed that the absorbance values were similar for all toxoids, thus presenting no significant difference between higher and lower concentration toxoids. For the ToBI test, the correlation ratio of 96.76, obtained in the curve pattern, demonstrates the effectiveness of the curve to be used in the epsilon toxoid evaluation. The correlation ratio between the titration degrees of toxoids obtained through TCP and ToBI tests was higher than 90%. It is concluded that the type of ELISA test used does present discriminative power for toxoids with different concentrations, which does not support the use of this technique for such a purpose. The ToBI test can be used as a screening method for it is sensitive and effective to detect epsilon toxicoid produced by C. perfringens type D...

Teve-se por objetivo avaliar e padronizar as metodologias in vitro, ELISA e ToBI-test modificado, para a análise de toxoide épsilon, em comparação com a metodologia in vivo TCP. Foram utilizados os seguintes toxoides épsilon: padrão NIBSC e os lotes 375/07, 532/08, 551/08, 373/07 e 378/07, os quais foram avaliados por métodos in vivo, TCP, e in vitro, ELISA e ToBI-test. A análise do título de toxoide épsilon por meio dos métodos in vitro foi realizada a partir de uma curva-padrão, estabelecida previamente. Os principais resultados mostram que os valores de absorbância foram semelhantes para todos os toxoides, não apresentando diferença significativa entre os toxoides mais concentrados e menos concentrados. No ToBI-test, o coeficiente de correlação de 96,76%, obtido na curva-padrão, demonstra a eficiência da curva para avaliação do toxoide épsilon. O coeficiente de correlação entre os títulos de toxoide obtidos pelo TCP e ToBI-test foi superior a 90%. Conclui-se que o tipo de ELISA utilizado não apresenta poder discriminativo para toxoides com diferentes concentrações, inviabilizando a técnica para esse fim. O ToBI-test pode ser utilizado como um método de triagem sensível e eficaz para a detecção de toxoide épsilon de C. perfringens tipo D...

Passive acquisition of protective antibodies reactive with Bordetella pertussis in newborns via placental transfer and breast-feeding.

Although acquisition of anti-pertussis antibodies by the newborn via placental transfer has been demonstrated, a subsequent recrudescence of pertussis infection is often observed, particularly in infants. The present study investigated the passive transfer of anti-pertussis IgG and IgA antibodies to term newborns and their ability to neutralize bacterial pathogenicity in an in vivo experimental model using mice intracerebrally challenged with viable Bordetella pertussis. Forty paired samples of maternal/umbilical cord sera and colostrum were obtained. Anti-pertussis antibodies were analysed by immunoenzymatic assay and by Immunoblotting. Antibody neutralizing ability was assessed through intracerebral B. pertussis challenges in mice. Anti-pertussis IgG titres were equivalent in both maternal and newborn sera (medians = 1:225 and 1:265), with a transfer rate of 118%. The colostrum samples had variable specific IgA titres (median = 1:74). The immunoblotting assays demonstrated identical recognition profiles of paired maternal and newborn serum pools but different bacterial recognition intensities by colostrum pools. In the animal model, significant differences were always observed when the serum and colostrum samples and pools were compared with the positive control (P < 0.05). Unlike samples with lower anti-pertussis titres, samples with high titres showed protective capacities above 50%. Pertussis-absorbed serum and colostrum pools protected 30% of mice and purified IgG antibodies protected 65%. Both pooled and single-sample protective abilities were correlated with antibody titres (P < 0.01). Our data demonstrated the effectiveness of anti-pertussis antibodies in bacterial pathogenesis neutralization, emphasizing the importance of placental transfer and breast-feeding in protecting infants against respiratory infections caused by Bordetella pertussis.

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

Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice.

The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M. fortuitum beta-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B. pertussis infection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.

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.

Large unilamellar vesicles as trehalose-stabilised vehicles forvaccines storage time and in vivo studies

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

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.

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.

Microquantification of proteins with low chromophore content

The advantages of the intrinsic fluorescence of the tryptophan and the absorbance of the methionine residues of the 18 kDa-hsp - a recombinant protein from Mycobacterium leprae - was exploited here to develop a sensitive and low costs method for protein assaying. They presented linearity between 3 and 1000 ìg of protein. The correlations between intrinsic fluorescence or absorbance at 230 nm and protein contents were both superiors to 0.99. These methods can be extended to others proteins with low aromatic residue contents