Expression, Purification, and In Vivo Administration of a Promising Anti-Idiotypic HIV-1 Vaccine

To date only a few neutralizing antibodies against HIV-1 exist. Since these neutralizing antibodies are only rarely found in sera of HIV-1 infected individuals an active vaccine is required. We recently developed murine anti-idiotypic antibody Ab2/3H6 against monoclonal antibody (mAb) 2F5, which is one of the most prominent neutralizing antibodies. Anti-idiotypic antibody Ab2/3H6 has been partially humanized and expressed as whole immunoglobulin G in Chinese hamster ovary cells in order to minimize the human anti-mouse antibody response. Here we describe the expression, purification, and immunohistochemical characterization of the chimeric Ab2/3H6 Fab fragment, which was finally used beside the whole IgG1 as an antigen for immunization of guinea pigs. The crude sera were screened for specific antibodies against the epitope of mAb 2F5 ELDKWA as well as for reactivity against HIV-1 gp41.     

Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies

Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV(+) plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (相似文献   

Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.

The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.     

Physiological response ofCandida tropicalis grown on n-paraffin to mixing in a tubular closed loop fermenter

Summary A special tubular closed loop fermenter was used in order to simulate the particular mixing condition of a large scale recycle fermenter.Some mixing parameters of the system are characerized.During continuous cultivation ofCandida tropicalis on n-paraffin as a substrate the biomass yield with respect to carbon and oxygen increased, when a controlled oxygen limit was imposed on the culture.Mixing in the closed loop fermenter generates undamped short period oscillations in the respiration activity, in the dissolved oxygen tension and in the actual ATP content of the culture. These oscillations likely represent oscillations of allosteric feedback loops which manifest themselves by some synchronising action of the particular environmental transients in the closed loop fermenter.     

Influence of transfected SV40 early region on growth and differentiation of human endothelial cells.

Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells.     

Stability of von Willebrand Factor secretion in different human endothelial hybrid cell lines

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. Thein vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.     

Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.