Exanthem subitum is a common childhood illness caused by primary infection with human herpesvirus 6B (HHV-6B). It is occasionally complicated by febrile seizures and even encephalitis. HHV-6B reactivation also causes encephalitis, especially after allogeneic hematopoietic stem cell transplantation. However, no adequate antiviral treatment for HHV-6B has yet been established. Mouse-derived monoclonal antibodies (MAbs) against the HHV-6B envelope glycoprotein complex gH/gL/gQ1/gQ2 have been shown to neutralize the viral infection. These antibodies have the potential to become antiviral agents against HHV-6B despite their inherent immunogenicity to the human immune system. Humanization of MAbs derived from other species is one of the proven solutions to such a dilemma. In this study, we constructed chimeric forms of two neutralizing MAbs against HHV-6B to make humanized antibodies. Both showed neutralizing activities equivalent to those of their original forms. This is the first report of humanized antibodies against HHV-6B and provides a basis for the further development of HHV-6B-specific antivirals. IMPORTANCE Human herpesvirus 6B (HHV-6B) establishes lifelong latent infection in most individuals after the primary infection. Encephalitis is the most severe complication caused by both the primary infection and the reactivation of HHV-6B and is the cause of considerable mortality in patients, without any established treatments to date. The humanization of the murine neutralizing antibodies described in this research provided a feasible way to reduce the inherent immunogenicity of the antibodies without changing their neutralizing activities. These newly designed chimeric antibodies against HHV-6B have the potential to be candidates for antivirals for future use. a IC 50 , half-maximal inhibitory concentration. FIG 7 Model of the possible mechanisms of neutralization by MAbs KH-1 and OHV-3. For HHV-6B entry, the gH/gL/gQ1/gQ2 glycoprotein complex, especially gQ1, binds directly to the cell receptor CD134. (A) KH-1, which interacts with gQ1, could directly inhibit receptor binding. (B) The interaction of gH with OHV-3 could affect gH function (fusion or association with gB) and thus inhibit viral entry. Humanization of Neutralizing MAbs against HHV-6B Journal of Virology May 2019 Volume 93 Issue 10 e02270-18 jvi.asm.org 7proteins by MAbs may affect their functions for entry. Further studies are required to answer these questions. MATERIALS AND METHODS Cells and viruses.Human embryonic kidney (HEK) 293T cells were cultured at 37°C under 5% CO 2 in Dulbecco's modified Eagle medium (DMEM) containing 8% fetal bovine serum, 4 mM L-glutamine, and 20 g/ml gentamicin. Serum-free CD 293 medium (Thermo Fisher Scientific) supplemented with 4 mM L-glutamine and 20 g/ml gentamicin was used after transfections. The human T-cell line MT4 was cultured in RPMI 1640 medium with 8% fetal bovine serum containing 20 g/ml gentamicin. HHV-6B strain HST was prepared as described previously (15). For virus propagation, umbilical cord blood m...