Active evolution of memory B-cells specific to viral gH/gL/pUL128/130/131 pentameric complex in healthy subjects with silent human cytomegalovirus infection

Abstract

Human cytomegalovirus (HCMV) can cause life-threatening infection in immunosuppressed patients, and in utero infection that may lead to birth defects. No vaccine is currently available. HCMV infection in healthy subjects is generally asymptomatic, and virus persists as latent infection for life. Host immunity is effective against reactivation and super-infection with another strain. Thus, vaccine candidates able to elicit immune responses similar to those of natural infection may confer protection. Since neutralization is essential for prophylactic vaccines, it is important to understand how antiviral antibodies are developed in natural infection. We hypothesized that the developmental path of antibodies in seropositive subjects could be unveiled by interrogating host B-cell repertoires using unique genetic signature sequences of mAbs. Towards this goal, we isolated 56 mAbs from three healthy donors with different neutralizing titers. Antibodies specific to the gH/gL/pUL128/130/131 pentameric complex were more potent in neutralization than those to gB. Using these mAbs as probes, patterns of extended lineage development for B-cells and evidence of active antibody maturation were revealed in two donors with higher neutralizing titers. Importantly, such patterns were limited to mAbs specific to the pentamer, but none to gB. Thus, memory B-cells with antiviral function such as neutralization were active during latent infection in the two donors, and this activity was responsible for their higher neutralizing titers. Our results indicated that memory B-cells of neutralizing capacity could be frequently mobilized in host, probably responding to silent viral episodes, further suggesting that neutralizing antibodies could play a role in control of recurrent infection.

Specificity and effector functions of non-neutralizing gB-specific monoclonal antibodies isolated from healthy individuals with human cytomegalovirus infection

“…Interestingly, none of the non-neutralizing gB-specific mAbs in this panel had appreciable binding to AD1 or Domain I. AD1 is targeted by both neutralizing and non-neutralizing mAbs [34, 36]. As such, the lack of Domain I and AD1 binding from this representative panel could be the result of sampling bias from only three individuals [23], by selection bias of excluding mAbs which bind to traditionally neutralizing domains, or non-optimal conformation of the protein used for B cell sorting to isolate these mAbs.…”

“…gB-specific monoclonal antibodies (mAbs) were isolated from healthy adult volunteers with previous natural HCMV infection. Subjects were consented for blood sampling in accordance with NIH guidelines [23]. B cell isolation and recombinant monoclonal antibody isolation and production using a human IgG1 backbone was performed as previously described [23].…”

“…Subjects were consented for blood sampling in accordance with NIH guidelines [23]. B cell isolation and recombinant monoclonal antibody isolation and production using a human IgG1 backbone was performed as previously described [23]. Briefly, total RNA from isolated memory B cells, which screened positive for binding activity against gB, was converted to cDNA using a reverse transcription kit (Invitrogen), and IgG genes were identified by PCR primers.…”

“…Because there are stark differences in the gB epitope binding profiles of naturally-infected individuals and gB/MF59 vaccinees [13, 22], closing the gap in our understanding of immune-protection offered by prior exposure to natural HCMV infection and vaccination will require investigation into how gB as an antigen induces potentially protective effector antibody responses. The aim of this study is to characterize the non-neutralizing gB-specific antibody responses in natural HCMV infection for a panel of gB-specific mAbs isolated by memory B cell cultures from three naturally infected individuals [23]. Towards this goal, we analyzed a panel of 24 mAbs by defining their target specificity for gB domains as well as genotype specificity, and analyzed effector functions for these mAbs in ADCC and ADCP assays.…”

Neutralizing Monoclonal Antibodies Reduce Human Cytomegalovirus Infection and Spread in Developing Placentas

“…Recently, a large panel of anti-HCMV human monoclonal antibodies (mAbs) from healthy seropositive donors was isolated and characterized for specificity, neutralizing capacity on epithelial cells, and binding affinities for whole virion and recombinant gB and pentameric complex [34]. Here we examined a select subset of these mAbs with high neutralizing activity targeting gB, gH/gL, and the pUL128-131 portion of the pentamer for their ability to neutralize infection in primary placental cells and explants of developing anchoring villi, as it is unknown which antibodies are most critical for preventing transplacental transmission.…”