Development of broadly neutralizing antibodies targeting the cytomegalovirus subdominant antigen gH

Abstract

Human cytomegalovirus (HCMV) is a β-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.

The CD46 ectodomain participates in human cytomegalovirus infection of epithelial cells

“…As expected, the anti-gH mAb 15G11 had no impact on virus infection under these conditions due to its specificity for the gH viral envelope protein. However, AD169R pre-incubated with 15G11 reduced virus infection of both ARPE-19 and MRC5 cells [34] (). Interestingly, the anti-CD46 2E7 mAb preincubated with HCMV limited infection of epithelial cells at highest mAb concentrations ().…”

“…CD46 was demonstrated to participate in HCMV infection during the early stages of infection of epithelial cells and trophoblasts, and not fibroblasts [25]. To further evaluate whether targeting CD46 prior to infection can limit subsequent HCMV infection, we examined virus infectivity in ARPE-19 and MRC5 cells pre-incubated with anti-CD46 monoclonal antibody (mAb) 2E7 [25], anti-gH mAb 15G11 [34], or an irrelevant mAb raised against influenza hemagglutinin (PY102) (). MRC5 and ARPE-19 cells were incubated with increasing concentrations of 2E7, 15G11, and PY102 for 30 min at 4 °C, followed by a wash step to remove unbound antibody.…”

“…As expected, the anti-gH mAb 15G11 had no impact on virus infection under these conditions due to its specificity for the gH viral envelope protein. However, AD169R pre-incubated with 15G11 reduced virus infection of both ARPE-19 and MRC5 cells [34] (Fig. 1b).…”

“…HCMV infection of ARPE-19 or MRC5 cells (10 000 cells/well in a 96-well plate) was typically analysed at 24 h post-infection (hpi), beginning with fixation using 4 % paraformaldehyde for 30 min at room temperature [34]. Infected cells were then permeabilized with 0.3 % Triton X-100 in PBS, followed by incubation with 4 % normal goat serum (NGS) in 0.3 % Triton X-100 in PBS for 1 h at 4 °C.…”

“…The antibody PY102 (anti-IAV) (gift from Dr Thomas Moran, Icahn School of Medicine), anti-gH antibody 15G11 [34], and anti-CD46 antibody [25] were purified from hybridoma supernatants using an ÄKTA pure FPLC system on protein G affinity columns (HiTrap-1ml, GE /Cytiva, #17-0404-01). The antibodies were dialysed against PBS and quantitated by both BCA and OD at 280 nm.…”