Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Dengue viruses induce cell proliferation and morphological changes of endothelial cells.

Replication of dengue viruses (type 1, 2, 3 and 4) in vitro in endothelial cells from human umbilical cord vein was demonstrated by virus titers and immunofluorescent antibody studies. Both showed highest peak at Day 6 after inoculation and declined to origin at Day 14. Some of the cultured endothelial cells detached from the culture well. Most of these floating cells were rarely viable as shown by failure in trypan blue exclusion whereas the adhering cells are mostly viable. More frequent and higher intensity of immunofluorescent positive cells were found in the detached cells as compared to adhering cells. The virus titers in the supernatant and in the adhering cell population were comparable, although floating cells were maximally 26.2% of the total cultured endothelial cells. Many floating cells and occasional adhering cells had numerous blebs on their surface. Endothelial cell proliferation was markedly increased after virus inoculation as compared with the control. Increased number of mitotic cells was also observed in the dengue virus-endothelial cell culture. Comparing among the four types, dengue type 4 induced highest peaks of cell proliferation and cell mitosis at Day 10 after inoculation. Dengue type 2 had the highest virus titers both in adhering cells and in supernatant at Day 6 as compared with other types.

T cell responses in Hodgkin's disease: frequency distribution of IL-2 producing cells and quantitation of IL-2 produced per cell.

T cell dysfunction in Hodgkin's disease (HD) is well documented. Since interleukin-2 (IL-2) plays a pivotal role in T cell proliferation, we have investigated frequency distribution of IL-2 producing phytohaemagglutinin (PHA)-stimulated lymphocytes from HD patients compared to that of healthy donors using two limiting dilution (LD) culture systems in which autologous peripheral blood lymphocytes (PBL) and Epstein Barr Virus transformed allogeneic B lymphoblastoid cell lines (EBV-LCL) have been used as feeders. The latter provided better conditions for IL-2 production by single cells, as evident from the enhanced frequencies obtained (For healthy donors: 1/67 +/- 1545.5 using EBV-LCL and 1/1123 +/- 1.7438 using autologous PBL as feeders). The data showed significantly reduced frequency of IL-2 producing cells as well as reduced quantity of IL-2 produced per cell in HD even after using/EBV-LCL as feeders, the amount of IL-2 produced per activated responder cell in HD patients being 0.825-1.3 pg/well (p < 0.001) as compared to 1.48-2.43 pg/well in healthy donors. Thus, the EBV-LCL feeders did provide better culture conditions for estimating frequencies of functional T cells. However these cell lines were unable to restore in vitro the abnormalities in functional properties of T cells in HD.