Effect of mild-thiol reducing agents and alpha2,3-sialyltransferase expression on secretion and sialylation of recombinant EPO in CHO cells.

We have previously reported that N-acetylcysteine (NAC) not only delayed apoptosis but also enhanced the production of recombinant erythropoietin (EPO) in Chinese hamster ovary (CHO) cell culture. To investigate the production enhancement mechanism, the effects of similar thiolreducing agents were studied. Intriguingly, all mild reducing agents examined including mercaptoethanesulfonic acid (MESNA), thiolactic acid (TLA), and thioglycolate (TG) were shown to block apoptosis and increase EPO production. A pulse-chase study of EPO secretion revealed that all four thiol-reducing agents increased the EPO secretion rate; among them TLA showed the highest rate. In terms of product quality, the sialic acid content of the glycoprotein is one of the most important factors. It was reported that a number of glycoproteins produced by CHO cells often have incomplete sialylation, particularly under high-producing conditions. Human alpha2,3-sialyltransferase (alpha2,3-ST) was introduced into EPO-producing CHO cells in order to compensate for the reduced sialylation during supplementation with NAC. When alpha2,3-ST was expressed in the presence of NAC, reduced sialylation was restored and an even more sialylated EPO was produced. Thus, our study is significant in that it offers increased EPO production while still allowing the prevention of decreased sialylation of EPO.

Comparison of DNA vaccines producing HIV-1 Gag and LAMP/Gag chimera in rhesus macaques reveals antigen-specific T-cell responses with distinct phenotypes.

Optimized DNA expression vectors encoding the native HIV-1 Gag or a fusion of Gag with the lysosomal membrane associated protein 1 (LAMP) were compared for immunogenicity upon intramuscular DNA delivery in rhesus macaques. Both vaccines elicited CD4(+) T-cell responses, but with significant differences in the phenotype of the Gag-specific cells: the native Gag induced CD4(+) responses with a phenotype of central memory-like T cells (CD28(+) CD45RA(-)), whereas the LAMP/Gag chimera induced CD4(+) responses with effector memory phenotype (CD28(-) CD45RA(-)). Antigen-specific T cells producing both IFN-gamma and TNFalpha were found in the animals receiving the native Gag, whereas the LAMP/Gag chimera induced humoral responses faster. These results demonstrate that modification of intracellular Gag trafficking results in the induction of distinct immune responses. Combinations of DNA vectors encoding both forms of antigen may be more potent in eliciting anti-HIV-1 immunity.