title: Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells.
abstract: BACKGROUND
  Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients.
  
  
  RESULTS
  The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency approximately 20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated.
  
  
  CONCLUSIONS
  The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.
authors.affiliation: Department of Genetics, Biology and Biochemistry, University of Torino Medical School, Via Santena 19, 10126 Torino, Italy. ada.funaro@unito.it
authors.forename: Ada
authors.initials: A
authors.lastname: Funaro
authors.forename: Giorgio
authors.initials: G
authors.lastname: Gribaudo
authors.forename: Anna
authors.initials: A
authors.lastname: Luganini
authors.forename: Erika
authors.initials: E
authors.lastname: Ortolan
authors.forename: Nicola
authors.initials: N
authors.lastname: Lo Buono
authors.forename: Elisa
authors.initials: E
authors.lastname: Vicenzi
authors.forename: Luca
authors.initials: L
authors.lastname: Cassetta
authors.forename: Santo
authors.initials: S
authors.lastname: Landolfo
authors.forename: Richard
authors.initials: R
authors.lastname: Buick
authors.forename: Luca
authors.initials: L
authors.lastname: Falciola
authors.forename: Marianne
authors.initials: M
authors.lastname: Murphy
authors.forename: Gianni
authors.initials: G
authors.lastname: Garotta
authors.forename: Fabio
authors.initials: F
authors.lastname: Malavasi
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000276:immunology* / Q000627:therapeutic use; D001402:B-Lymphocytes / Q000276:immunology* / Q000821:virology; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology / Q000517:prevention & control / Q000628:therapy*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D017455:Receptors, IgE / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000627:therapeutic use; D015203:Reproducibility of Results; D051217:Toll-Like Receptor 9 / Q000819:agonists / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007376:Interleukin-2; D017455:Receptors, IgE; D011994:Recombinant Proteins; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; C071440:glycoprotein H, Cytomegalovirus