Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B

Abstract

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...

Structural basis for the interaction of human herpesvirus 6B tetrameric glycoprotein complex with the cellular receptor, human CD134

“…The interpretation of the 2D images was difficult compared to that of the tetramer/anti-gQ1 Mab KH-1 complex because the Stalk, Palm and the Fab density could not be distinguished from each other in the 2D images showing multiple knobs. However, the 3D reconstruction with 81,721 particles at 16.8 Å resolution revealed that the Fab density bound to the Stalk part, i.e., the gH/gL area of the tetramer (Fig 5E) which is consistent with the report that OHV-3 recognizes gH [28,29,32].…”

“…The anti-gQ1 Mab KH-1 and anti-gH Mab OHV-3 have neutralizing activity against HHV-6B [27,29], and we recently reported a humanization trial of these murine Mabs as candidate anti-HHV-6B antiviral agents [28]. The present study's SPR analysis revealed the considerable affinities of these Mabs for the soluble tetramer, and the affinities were comparable to those of hCD134.…”

“…The immunization of mice with inactivated HHV-6B elicited neutralizing antibodies, and interestingly, most of the monoclonal antibodies (Mabs) with neutralizing potency target the tetramer [27]. An HHV-6B-specific neutralizing Mab for gQ1 named KH-1 was analyzed in detail [27,28]. The neutralizing potency against HHV-6B infection has also been shown for a Mab for gH, OHV-3 [28,29].…”

“…An HHV-6B-specific neutralizing Mab for gQ1 named KH-1 was analyzed in detail [27,28]. The neutralizing potency against HHV-6B infection has also been shown for a Mab for gH, OHV-3 [28,29]. One possible scenario regarding the mechanism of Mabs for the neutralization of gQ1 is as follows: the access of the gQ1 in the tetramer to the host receptor hCD134 is blocked and the gQ1 itself cannot bind to hCD134.…”

“…We determined the sequences of the complementarity-determining regions (CDRs) of anti-gQ1 Mab KH-1 and anti-gH Mab OHV-3 in our previous study [28], but their spatial arrangements remained to be elucidated. Herein, the Fab domains of the anti-gQ1 Mab KH-1 and those of the anti-gH Mab OHV-3 were prepared by papain digestion, and the crystal structures were determined.…”