Search

Human papillomavirus (HPV) infection is a leading cause of morbidity and mortality in women worldwide. The virus is associated with benign warts and a broad spectrum of malignancies, including cervical cancer, considered a disease of high clinical relevance, especially in developing countries. In this study we developed the production of recombinant proteins HPV16 L1 and HPV16 L2 in human cells in suspension (293-F), which were transiently co-transfected with the pUF3/L1h and pUF3/L2h vectors. Expressions of recombinant HPV16 L1 and L2 capsid proteins was detected by laser scanning confocal microscopy and flow cytometry. Both proteins were identified intracellularly in the nucleus and cytoplasm of cells. The presence of these heterologous proteins and VLPs formation were detected by transmission electron microscopy (TEM) through colloidal gold immunolabeling and negative staining. Cell extracts containing recombinant proteins were purified by affinity chromatography and immunization of Balb/c mice with the formulation HPV16 L1/L2 VLPs containing adjuvant was able to induce higher titer of anti-HPV16 L1, when compared to HPV16 L2 antibodies by indirect ELISA assay. These data indicate that transient expression in 293-F cells was efficiently established. The results are promising for obtain recombinant proteins of the HPV capsid for future studies involving human papillomavirus, as well as to contribute for the development of other vaccine strategies for prevention against HPV.

O câncer cervical é a consequência mais séria da infecção por Papilomavírus humano - HPV, constituindo uma das principais causas de morte entre mulheres no mundo, remetendo a um importante desafio para a saúde pública mundial. Desenvolvemos a produção de VLPs contendo as proteínas L1 e L2 de HPV16, utilizando células epiteliais humanas 293-F em suspensão e em meio isento de soro fetal bovino. As células possuem metabolismo intenso, adequado para a expressão de proteínas heterólogas. Apresentam vantagens como a facilidade de cultivo, transfecção e ocorrência de processos como a glicosilação de proteínas, fosforilação, formação de pontes dissulfeto e outras modificações pós-traducionais essenciais para a função das proteínas, facilitando a produção similar às condições in vivo. O sistema de produção de VLPs L1L2 de HPV16 foi estabelecido de maneira eficiente, em células epiteliais humanas em suspensão e com resultados promissores, podendo contribuir para o desenvolvimento de uma vacina profilática de amplo espectro de proteção, com custo reduzido de produção. Cervical cancer is the most serious consequence of infection by human papillomavirus - HPV, constituting one of the leading causes of death among women worldwide that points to a major challenge to global public health. We have developed the production of VLPs containing L1 and L2 proteins of HPV16, using human epithelial cells 293-F suspended in medium without fetal bovine serum. The cells have intense metabolism, suitable for expressing heterologous proteins. Present advantages such as ease of culture, transfection and occurrence of processes such as protein glycosylation, phosphorylation, formation of disulfide bonds and other essential post-translational modifications to protein function, facilitating the production similar to the conditions in vivo. The VLPs L1L2 of HPV16 production system was established efficiently in human epithelial cells in suspension and with promising results, which may contribute to the development of a prophylactic vaccine broad protection spectrum with reduced production cost.

This paper investigated the porcine surfactant protein A (pSP-A) immunogenicity in murine model. Many elegant stu-dies about SP-A therapeutic applications are available however specific studies about its exogenous immunogenicity were not easily assumed. Therefore, we investigated the immunogenicity of this porcine protein in mice. The mice re-ceived pSP-A subcutaneously on days 0 and 7. The animals were observed during 90 days and the blood was collected on days 30, 60 and 90 for assessment the immunogenic potential of pSP-A. Some animals showed circulating antibodies above the screening cut point, which was calculated based on control mice sera signals. However, those antibodies were considered false positive read-outs by the performed competitive inhibition assay. Also no neutralizing antibodies were detected able to avoid the porcine protein ability to promote lipid aggregation. So far in this model, porcine surfactant protein-A could be considered not immunogenic.

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of approximately 7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 microg porcine aprotinin inhibited 6 microg trypsin whereas 1 microg commercial soybean inhibitor inhibited only 1 microg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.

Samples from 20 lots of dT vaccine and from 20 lots of DTP vaccine were used to standardize and validate the Vero cell and the toxin binding inhibition (ToBI) tests for the potency control of diphtheria component. For the Vero cell method, violet crystal solution was used to stain the cells and estimate the endpoint of diluted diphtheria antitoxin. Diphtheria anatoxin was used for performing the ToBI test instead of toxin. The results obtained by both in vitro tests were similar to those obtained by in vivo toxin neutralization test in guinea pigs. The various analysis and the chi(2) test applied to evaluate the reproducibility and homogeneity, respectively, among in vitro tests and in vivo toxin neutralization test did not detect statistical significant difference for both analysed vaccines. An excellent correlation among in vitro tests and in vivo neutralization test was observed by Spearman's correlation coefficient.

As colectinas pulmonares SP-A e SP-D são marcadores específicos de doenças pulmonares. A determinação destas proteínas por ensaios imunoenzimáticos permite aos clínicos correlacionarem seu papel funcional durante o desenvolvimento do processo patológico baseado nas anormalidades de suas concentrações. Como o lavado broncoalveolar (BAL) é uma amostra que permite estudar as proteínas secretadas pelo epitélio pulmonar em quaisquer circunstâncias, foi proposto o desenvolvimento de um ELISA indireto capaz de detectar SP-A no BAL humano usando um anti-soro policlonal contra SP-A suína produzido em coelho. SP-A suína foi purificada por protocolo que acopla precipitação ácida do extrato pulmonar suíno e cromatografia de afinidade. O anti-soro reagiu com as duas espécies de SP-A testadas: humana e suína. A curva padrão foi otimizada utilizando como calibrador a SP-A purificada de pacientes com artrite reumatóide. A faixa selecionada da curva de calibração foi de 0,312 to 5,0 mg/mL usando a diluição 1:1000 do anticorpo. O limite de detecção da curva foi de 0,625 mg/mL. The lung collectins SP-A and SP-D are specific markers for lung diseases. Determination of amounts of these proteins using polyclonal and monoclonal antibodies on ELISA assays enabled clinicians to predict their role in the course of the lung disease process based on abnormalities on their concentrations. As the bronchoalveolar lavage (BAL) is a sample that permits to study the proteins secreted by the lung epithelium at any conditions, we proposed to develop an indirect ELISA able to detect SP-A in the BAL using polyclonal rabbit antiserum, raised against porcine SP-A. Porcine SP-A was purified by a protocol that includes an acid precipitation of the porcine pulmonary extract before affinity chromatography. The antiserum reacted with the two tested species porcine and human. The calibration curve were optimized, using human SP-A purified from patients with rheumatoid arthritis as human antigen calibrator. The selected calibration curve range was 0.312 to 5.0 mg/mL using the antiboby dilution 1:1.000. The detection limit of the standard curve was 0.625 mg/mL.

International audience; In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.