HIV-derived lentiviral particles promote T-cell independent activation and differentiation of naïve cognate conventional B2-cells in vitro

Divergence of Primary Cognate B- and T-Cell Proliferative Responses to Subcutaneous and Intravenous Immunization with Virus-Like Particles

“…The plasmids, Hgpsyn (a codon-optimized HIV GagPol expression plasmid) [11], pConBgp140GCD (a codon-optimized HIV-Env clade B consensus sequence) [10] and pC-HEL-TM (encoding the membrane-anchored form of HEL) [12], have been described. The HIV-Gag expression plasmid was constructed from Hgpsyn by deleting the coding sequences for the amino acid sequences downstream of those encoding GNDPSSQ.…”

“…The p24 content of the VLP preparations was determined with an anti-p24CA-ELISA, and western blot analyses for Gag were performed, as reported elsewhere [13]. The anti-HEL ELISA was described previously [12]. The endotoxin levels were measured by the QCL-1000 ® Chromogenic LAL Endpoint Assay (Lonza, Walkersville, MD, USA).…”

“…On the same day, recipient mice were immunized with HEL-VLPs by intravenous or by subcutaneous injection in a final concentration of 100 ng of HEL per mouse. Mice having received CFSE-labeled B-cells were sacrificed three days after the VLP injections; spleen and draining LNs were removed and stained with HEL-Alexa647 and with anti-B220 APC-eFlour (eBioscience, San Diego, CA, USA) antibody for HEL-transgenic B-cell receptor (BCR) [12] and total B-cells, respectively. CFSE dilution in HEL + B220 + CFSE + cells was analyzed with flow cytometry.…”

Application of surface plasmon resonance imaging technique for the detection of single spherical biological submicrometer particles

“…HIV-VLPs were produced in HEK293T cells human cell line as described previously [12]. Briefly, to produce lentiviral particles HEK293T cells were transiently transfected using polyethylenimine with a codon-optimized HIV GagPol expression plasmid (Hgpsyn) [13].…”

“…To produce control VLP, the cells were cotransfected with Carrier DNA (Invitrogen, Karlsruhe, Germany) and the resulting VLP carried all producer-cell-derived surface proteins, but lacked OVA. The VLP were purified from the cell-culture medium and concentrated by ultracentrifugation through a 20% sucrose cushion [12]. According to previous observations normally prepared HIV-VLPs are round-shaped nanoobjects with size around 100-140 nm [15,16] The concentrations of OVA and HIV-Gag (p24) proteins in the different VLP preparations were determined by ELISA as described elsewhere [14,17].…”