Production of HPV16 capsid proteins in suspension cultures of human epithelial cells

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 pUF3L1h and pUF3L2h 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.

Behavior of alfa-tomatine and tomatidine against several genera of trypanosomatids from insects and plants and Trypanosoma cruzi / Comportamento de alfa-tomatina e tomatidina contra vários gêneros de tripanossomatídeos de insetos e plantas e Trypanosoma cruzi

Glycoalkaloids are important secondary metabolites accumulated by plants as protection against pathogens. One of them, α-tomatine, is found in high concentrations in green tomato fruits, while in the ripe fruits, its aglycone form, tomatidine, does not present a protective effect, and it is usual to find parasites of tomatoes like Phytomonas serpens in these ripe fruits. To investigate the sensitivity of trypanosomatids to the action of α-tomatine, we used logarithmic growth phase culture of 20 trypanosomatids from insects and plants and Trypanosoma cruzi. The lethal dose 50% (LD50) was determined by mixing 107 cells of the different isolates with α-tomatine at concentrations ranging from 10-3 to 10-8 M for 30 min at room temperature. The same tests performed with the tomatidine as a control showed no detectable toxicity against the same trypanosomatid cultures. The tests involved determination of the percentage (%) survival of the protozoan cultures in a Neubauer chamber using optical microscopy. The LD50 values varied from 10-4 to 10-6 M α-tomatine. Slight differences were detected among the LD50 values of the analyzed samples, and none of them showed evidence of resistance to the action of tomatinase, as shown by some pathogenic fungi.

Os glicoalcaloides são metabólitos secundários importantes produzidos pelas plantas e estão envolvidos em sua proteção contra agentes patogênicos. Um deles, α-tomatina, é encontrado em altas concentrações em frutos de tomate verde, enquanto que, nos frutos maduros, sua forma aglicona, tomatidina, não apresenta um efeito protetor, sendo comum encontrar parasitas de tomates como Phytomonas serpens nesses frutos maduros. Para investigar a sensibilidade dos tripanossomatídeos à ação da α-tomatina, utilizamos formas de cultura em fase logarítmica de 20 tripanossomatídeos de plantas e insetos e Trypanosoma cruzi. A dose letal 50% (DL50) foi determinada, misturando 107 células das formas de cultura com concentrações de 10-3 a 10-8 M de α-tomatina durante trinta minutos a temperatura ambiente. Testes realizados com a tomatidina como controle não mostraram toxicidade detectável contra os mesmos tripanossomatídeos. Os testes foram avaliados pela porcentagem (%) de sobrevivência das formas de cultura dos protozoários observados por microscopia óptica em câmara de Neubauer. Os resultados da determinação de DL50 mostraram que esta variou entre 10-4 a 10-6 M de α-tomatina. Pequenas diferenças foram observadas entre os valores de DL50 das amostras analisadas, e nenhuma delas mostrou evidência de resistência pela ação da tomatinidase, como demonstrado em alguns fungos patogênicos.

Behavior of alfa-tomatine and tomatidine against several genera of trypanosomatids from insects and plants and Trypanosoma cruzi

Glycoalkaloids are important secondary metabolites accumulated by plants as protection against pathogens. One of them, a-tomatine, is found in high concentrations in green tomato fruits, while in the ripe fruits, its aglycone form, tomatidine, does not present a protective effect, and it is usual to find parasites of tomatoes like Phytomonasserpens in these ripe fruits. To investigate the sensitivity of trypanosomatids to the action of a-tomatine, we used logarithmic growth phase culture of 20 trypanosomatids from insects and plants and Trypanosoma cruzi. The lethal dose 50% (LD50) was determined by mixing 107 cells of the different isolates with a-tomatine at concentrations ranging from 10-3 to 10-8 M for 30 min at room temperature. The same tests performed with the tomatidine as a control showed no detectable toxicity against the same trypanosomatid cultures. The tests involved determination of the percentage (%) survival of the protozoan cultures in a Neubauer chamber using optical microscopy. The LD50 values varied from 10-4 to 10-6 M a-tomatine. Slight differences were detected among the LD50 values of the analyzed samples, and none of them showed evidence of resistance to the action of tomatinase, as shown by some pathogenic fungi.

Behavior of alfa-tomatine and tomatidine against several genera of trypanosomatids from insects and plants and Trypanosoma cruzi

Glycoalkaloids are important secondary metabolites accumulated by plants as protection against pathogens. One of them, a-tomatine, is found in high concentrations in green tomato fruits, while in the ripe fruits, its aglycone form, tomatidine, does not present a protective effect, and it is usual to find parasites of tomatoes like Phytomonasserpens in these ripe fruits. To investigate the sensitivity of trypanosomatids to the action of a-tomatine, we used logarithmic growth phase culture of 20 trypanosomatids from insects and plants and Trypanosoma cruzi. The lethal dose 50% (LD50) was determined by mixing 107 cells of the different isolates with a-tomatine at concentrations ranging from 10-3 to 10-8 M for 30 min at room temperature. The same tests performed with the tomatidine as a control showed no detectable toxicity against the same trypanosomatid cultures. The tests involved determination of the percentage (%) survival of the protozoan cultures in a Neubauer chamber using optical microscopy. The LD50 values varied from 10-4 to 10-6 M a-tomatine. Slight differences were detected among the LD50 values of the analyzed samples, and none of them showed evidence of resistance to the action of tomatinase, as shown by some pathogenic fungi.