Fluorescent-protein co-expression to select CHO cells expressing high quantities of vaccine antigens.

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen.

Multiple risk factor analysis of recurrent respiratory tract infection after Mycoplasma pneumoniae pneumonia in children / 临床儿科杂志

Objective To investigate the occurrence trend and risk factors of recurrent respiratory tract infection during the ifrst year after Mycoplasma pneumoniae pneumonia (MPP) in children. Methods The clinical data of 133 children complete-ly recoved from MPP and one year follow-up after MPP were included in this study, MPP IgM IgG double antibody titer were measured in different time slots (3 month, 6 months, 9 months, 12 months) after discharge. Information on frequency of recurrent respiratory tract infections, respiratory tract infection site, and drug use within one year were collected. Possible factors affecting the occurrence of recurrent respiratory tract infection were analyzed by means of single factor and multi factor analyses. Results In 133 patients, the recurrent rate was 31.58%in the ifrst year;aged 3-6 years old (OR=2.29,95%CI:1.13~4.64), or continuous positive or negative to positive antibodies (OR=4.47,95%CI:1.47~13.65), or low CD4/CD8 (OR=10.26,95%CI:3.30~31.90), or low IgA (OR=1.90,95%CI:1.06~3.40) is independent risk factor of recurrent respiratory tract after MPP;immune enhancer therapy is an independent protective factor (OR=0.29,95%CI:0.11~0.78). Conclusions Immune function disorders in the ifrst year after MPP were independent risk factors of recurrent respiratory tract infection. MP antibody positive without clinical symptoms and sustained antibody positive can not prevent the recurrence of respiratory tract infection without use of antibiotics. Immune enhancer was advocate to adjust immune function and reduce the incidence of repeated respiratory tract infection.