Evaluation of the Potency of the Anti-Idiotypic Antibody Ab2/3H6 Mimicking gp41 as an HIV-1 Vaccine in a Rabbit Prime/Boost Study

The HIV-1 envelope protein harbors several conserved epitopes that are recognized by broadly neutralizing antibodies. One of these neutralizing sites, the MPER region of gp41, is targeted by one of the most potent and broadly neutralizing monoclonal antibody, 2F5. Different vaccination strategies and a lot of efforts have been undertaken to induce MPER neutralizing antibodies but little success has been achieved so far. We tried to consider the alternative anti-idiotypic vaccination approach for induction of 2F5-like antibodies. The previously developed and characterized anti-idiotypic antibody Ab2/3H6 was expressed as antibody fragment fusion protein with C-terminally attached immune-modulators and used for immunization of rabbits to induce antibodies specific for HIV-1. Only those rabbits immunized with immunogens fused with the immune-modulators developed HIV-1 specific antibodies. Anti-anti-idiotypic antibodies were affinity purified using a two-step affinity purification protocol which revealed that only little amount of the total rabbit IgG fraction contained HIV-1 specific antibodies. The characterization of the induced anti-anti-idiotypic antibodies showed specificity for the linear epitope of 2F5 GGGELDKWASL and the HIV-1 envelope protein gp140. Despite specificity for the linear epitope and the truncated HIV-1 envelope protein these antibodies were not able to exhibit virus neutralization activities. These results suggest that Ab2/3H6 alone might not be suitable as a vaccine

Experimental design and small-scale model for high-performing perfusion media and processes scalable to 50 L bioreactors

Cell culture perfusion processes are considered optimal for a truly integrated continuous biomanufacturing pipeline. The nutrient-rich but balanced media should be designed to support very low cell-specific perfusion rates (CSPR) that minimize media consumption but maximize viable cell days and productivities. Optimized processes at low CSPR drastically reduce equipment costs, lab space, and product dilution. Finally, operating at very low CSPR enables running mammalian cell bioprocesses as true chemostat cultures in the future. We demonstrate a general workflow to develop high-performing perfusion media using small-scale models and transferred the process to 50 L scale at CSPR of 20 pL/c/d. Recombinant CHO cells were evaluated at small scale in shaking tubes. Cells were grown in HyClone™ ActiPro or CDM4NS0 basal media, and optimal spike concentrations of HyClone Cell Boost™ supplements were determined using a DoE-supported workflow. The identified high-performing perfusion medium was applied to ReadyToProcess WAVE™ 25 and XDR-50 bioreactor runs. Different strategies were tested to find the critical minimum CSPR and maximum supported viable cell density (VCD). The obtained product profile was compared between scales, as determined by glycan-, charge-, and size-variant distribution. Scale-down models were leveraged to define high-performing media and applied to bioreactor runs at constant volumetric perfusion rate, VCD, or CSPR. CSPR values as low as 10 pL/c/d at 2 × 108 c/mL were achieved. These results make high-density perfusion processes suitable for inoculum preparation (N-1) or high cell density cryopreservation. The developed perfusion processes supported steady-state production at constant 5 × 107 c/mL by applying a continuous cell bleed and were scaled to 50 L

Anti-idiotypic antibody Ab2/3H6 mimicking gp41: a potential HIV-1 vaccine?

Meeting abstract from 22nd European Society for Animal Cell Technology(ESACT) Meeting on Cell Based Technologies Vienna, Austria. 15-18 May 2011(VLID)90658

Genotype of CHO host cell line has higher impact on mAb production and quality than process strategy or cell culture medium

Chinese hamster ovary (CHO) cells comprise a variety of lineages, including CHO-DXB11, CHO-K1, CHO-DG44 and CHO-S. Despite the fact that CHO cell lines share a common ancestor, extensive mutagenesis and clonal selection have resulted in substantial genetic heterogeneity among them. Data from sequencing shows that different genes are lacking from individual CHO cell lines and that each cell line harbors a unique set of mutations that are relevant to the bioprocess. However, literature outlining how the observed genetic differences affect CHO cell performance during bioprocess operations remains scarce. In this study, we examined host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format in all cell lines by using bacterial artificial chromosomes (BACs) as transfer vector. Cell-specific growth, product formation and heavy and light chain mRNA levels were studied in batch, fed-batch and perfusion cultures. Furthermore, two different cell culture media were investigated. Product quality was studied through glycoprofiling, and the thermal denaturation was analyzed using differential scanning calorimetry (DSC). We found CHO cell line-specific preferences for mAb production or biomass synthesis that were determined by the host cell line rather than product-specific mRNA levels. Additionally, quality attributes of the expressed mAb were influenced by the host cell line and medium used

Kırk bin frank cihaz

Émile Richebourg'ün Saadet'te yayımlanan Kırk Bin Frank Cihaz adlı romanının ilk ve son tefrikalarıTefrikanın devamına rastlanmamış, tefrika yarım kalmıştır

Stress-Induced PARP Activation Mediates Recruitment of Drosophila Mi-2 to Promote Heat Shock Gene Expression

Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-[ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. We show here that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP–dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. We have identified several dMi-2 regions which bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. We suggest that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response