Epstein-Barr Virus (EBV) Transformation:

B cell immortalization by Epstein-Barr vius (EBV) is an established method for antibody production. EBV infects B cells via their CD21 receptor and subsequently transforms them into continually dividing, lymphoblastoid cell lines that produce antibodies representing the humoral immune response in vivo. The major advantage of B cell immortalization when compared to other antibody producing techniques is the generation of fully human antibodies that truly reflect both the specificity and diversity of the human imune response. Fraussen (J. of Autoimmunity 35 (2010) 130-134)

EBV in combination with CpG oligonucleotides

Esslinger (WO2008/001372) discloses isolation of memory B cells from human peripheral blood lymphocytes by a two step selection prcedure. The B cell marker CD22 was used for positive selection of B cells using the MACS technology. PBL were labeled using MACS conjugated anti human CD22 mAbs, phycoerythrin conjugated mAbs aanti human IgD and APC conjugated antibodies anti human IgM, IgA, CD3, CD8, CD56. Pan B cells were isolated by positive selecting CD22 positive cell using a midi MACS device followed by selection of phycoerythrin and APC negative cells using a MoFlo cell soter. CD22 positive IgM-, IgD-, IgA- B cells where then incubated with EBV containing supernatant obtained from B95-8 cells in the presence of CpG2006.

Fraussen (J. of Autoimmunity 35 (2010) 130-134) discloses a B cell immortalization method using simultaneous B cell stimulation by CpG2006 and B cell infection by EBV, followed by an additional CpG2006 and Il-2 stimulus. 

Funaro (WO2007/068758) discloses a method for immortalizing a population of cells that secrete antibodies which includes selecting the antibody producing cells by using B cell markers such as CD22, stimulating them with a stimulating agent such as an activator of TLRs expressed on B cells (activators include CpG2006 and IL-2), eliminating the stimulating agent from the cell culture, selecting the population of stimulated cells that express antibodies of specific isotypes, exposing the population of selected and stimulated cells to an immortalizing agent, elimaiting the immortalizing agent from the cell culture.

Hallybone (WO2004/076677) discloses a method for producing a clone of an immortalised human B memory lymphocyte using the steps of transforming human B memory lymphocytes using EBV in the presence of a polyclonal B cell activator such as CpG2006.

Simmons (PLoS Med 2007, 4(5): e178) discloses using epstein-Barr virus to immotalize memory B cells from aduls who had recovered from infections with highy pathogenic avian influenza (HPAI) H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cells secreting neutrlizing antibodies were cloned and mAbs purified.

Traggiai (Nature Medicine, 10(8) 2004, pp. 871-) discloses an improved method for Epstein-Barr virus transformation of human B cells which was used to analyze memory B cells from a patient who recovered from severe acute respiratory syndrome coronavirus (SARS0CoV) infection. According to the method, peripheral blood was collected from a patient who had recovered from SARC. Memory B cells were isolated by binding to CD22 microbeads followed by depletion of cells carrying IgM, IgD and IgA by cell sorting. Memory B cells were seeded in plates with medium containing CpG 2006 in the presence of EBV. After 2 weeks, the culture supernatants were screened for specific antibodies.

EBV Stimulation but not Immortilization:

Hayday (US 14/368,749) discloses a method of producing human antibody by providing a B cell culture from peripheral blood mononuclear cells (PBMC) which are subjected to a polyclonal B cell activator such as EBV, under conditions which are sufficient for stimulating/activating proliferation and immunoglobulin secretion but wihout relying on the immortalizing properties of EBV.  In contrast to EBV based methods where the B lymphocytes are immortalized, the cells are only simulated/activated with EBV by incubating the cells with EBV for a limited time and then either diluting the EBV out such as by seeding in a different medium with a second polyclonal B cell activator such as CpG2006. 

Enhancing telomerase activity in B lymphocytes:

Esslinger (US12/991422 and WO2010/003529) discloses lentivector-mediated gene transfer of human Telomerase-Reverse-Transcriptase (hTert), the catalytic protein subunit of human telomerase, into blood dervied membory B cells to immortalize or prolong the life span of the human memory B cells.