Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells.
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.
Funaro
Ada
A
Department of Genetics, Biology and Biochemistry, University of Torino Medical School, Via Santena 19, 10126 Torino, Italy. ada.funaro@unito.it
Gribaudo
Giorgio
G
Luganini
Anna
A
Ortolan
Erika
E
Lo Buono
Nicola
N
Vicenzi
Elisa
E
Cassetta
Luca
L
Landolfo
Santo
S
Buick
Richard
R
Falciola
Luca
L
Murphy
Marianne
M
Garotta
Gianni
G
Malavasi
Fabio
F
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
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