Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response

Abstract

Human cytomegalovirus (HCMV) is a major cause of disability in congenitally infected infants and in the immunosuppressed. There is currently no licensed prophylactic HCMV vaccine. The HCMV envelope glycoprotein B (gB) is considered a major vaccine target antigen based on its critical role in mediating viral-host cell fusion and thus viral entry. The natural conformation of HCMV gB within the viral envelope is a trimer, but there has been no reported success in producing a recombinant trimeric gB suitable for vaccine use. Phase II clinical trials of a monomeric recombinant gB protein demonstrated 50% efficacy in preventing HCMV infection in seronegative women of reproductive age, and in reducing viremia in solid organ transplantation recipients. We now report the production of a uniformly trimeric recombinant HCMV gB protein in Chinese ovary cells, as demonstrated by Western blot analysis under modified non-reducing conditions and size exclusion chromatography with multiangle scattering. Immunization of mice with trimeric HCMV gB induced up to 11-fold higher serum titers of total gB-specific IgG relative to monomeric HCMV gB using Alum + CpG as adjuvants. Further, trimeric HCMV gB elicited 50-fold higher complement-independent and 20-fold higher complement-dependent HCMV neutralizing titers compared to monomeric HCMV gB using the fibroblast cell line, MRC-5, and up to 6-fold higher complement-independent and -dependent HCMV neutralizing titers using the epithelial cell line, ARPE-19. The markedly enhanced HCMV neutralizing activity in response to trimeric HCMV gB was also observed using an additional four distinct clinical HCMV isolates. These data support a role for trimeric HCMV gB as an important component for clinical testing of a prophylactic HCMV vaccine.

Development of novel vaccines against human cytomegalovirus

“…We have produced, within CHO cells, a trimeric HCMV gB by insertion of a flexible 15 amino acid (Gly 4 Ser) 3 linker at the furin cleavage site that allowed for terminal protein folding and efficient expression. 53 Trimeric HCMV gB induced 5to 11-fold higher serum titers of gB-specific IgG relative to monomeric HCMV gB similar to the Chiron gB that was previously used in phase II clinical trials and elicited 50-fold higher complement-independent HCMV neutralization activity, suggesting that conformational epitopes of the trimeric HCMV gB played an important role in eliciting neutralization activity. 53 Soluble monomeric HCMV gB as well as different post-fusion HCMV trimeric gBs elicited mainly complementdependent HCMV-neutralizing antibodies.…”

“…53 Trimeric HCMV gB induced 5to 11-fold higher serum titers of gB-specific IgG relative to monomeric HCMV gB similar to the Chiron gB that was previously used in phase II clinical trials and elicited 50-fold higher complement-independent HCMV neutralization activity, suggesting that conformational epitopes of the trimeric HCMV gB played an important role in eliciting neutralization activity. 53 Soluble monomeric HCMV gB as well as different post-fusion HCMV trimeric gBs elicited mainly complementdependent HCMV-neutralizing antibodies. [79][80][81] In contrast, the trimeric HCMV gB produced in our laboratory elicited markedly higher serum HCMV-neutralizing antibodies that exhibited both complement-independent and complementdependent activity.…”

“…78,79 The conformational epitopes expressed by the two subdomains of the trimeric gB might play key role in eliciting neutralizing anybody responses. 53,78,79 In addition, the trimeric gB made in our laboratory elicited markedly higher cross-strain neutralization activity against several clinical HCMV strains and an HCMV strain AD169 variant expressing a functional pentameric complex (AD169 wt131 ), compared to the monomeric gB that was similar to the gB protein made by Chiron. 53 In contrast, in phase II clinical trials, Chiron gB/MF59 vaccine elicited antibodies exhibited limited neutralization of the autologous virus and negligible neutralization of multiple heterologous strains.…”

“…Preclinical study Antibodies to gB, high-titer cross-strain neutralizing antibody [53] Pentameric complex…”

“…[79][80][81] In contrast, the trimeric HCMV gB produced in our laboratory elicited markedly higher serum HCMV-neutralizing antibodies that exhibited both complement-independent and complementdependent activity. 53 These results may be due to the trimeric HCMV gB having a 15 amino acid flexible linker inserted into the furin cleavage site that allowed the two subdomains of HCMV gB to fold into their native conformation. 78,79 The conformational epitopes expressed by the two subdomains of the trimeric gB might play key role in eliciting neutralizing anybody responses.…”