Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

Abstract

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen-the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains.IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen-the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

Keywords: antibodies; epitope mapping; human cytomegalovirus; neutralization; pentameric complex; strain coverage; vaccines.

FIG 1

Biochemical characterizations of MAbs specific for the pentameric complex. (A and B) To evaluate the reactivity of selected MAbs to virus particles measured by flow cytometry, V160 virus with restored expression of the pentameric gH complex (A) and AD169 virus (B) was mixed with each MAb as indicated and then stained with fluorescence-labeled secondary antibody. The control samples were incubated with polyclonal antibodies from a seronegative donor. Antibodies that bound to both viruses are indicated in red. The data shown are representative of two experiments. (C) Relative binding affinity was determined by quantitative ELISA and is expressed as EC₅₀, which is defined as the IgG concentration needed to achieve 50% maximal binding signal. EC₅₀s were determined by four-parameter curve fitting, and if there was poor fit, an arbitrary EC₅₀ of 100 μg/ml was assigned. Recombinant pentameric complex (PENTAMER) or gH/gL homodimer (DIMER) were made based on the viral sequence of the Towne strain. (D) Western blot analysis of MAbs to denatured and reduced AD169 virus antigens.

FIG 2

Negative-staining EM 2D class averages of recombinant pentamer and gH/gL homodimer and their complexes with various Fabs. Three domains of the pentamer are labeled with red numbers. The EBV gH/gL structure (PDB 3PHF) is shown as ribbons, with gL colored magenta and gH colored gray. The images within the dotted ovals represent the indicated Fab.

FIG 3

EM 3D reconstruction of pentamer bound by Fabs and a summary diagram of identified immunogenic sites. (A) Structures of the pentamer bound with Fab 2-15, Fab 1-85, or Fab 1-103 and their overlay, which was rotated 180° horizontally with respect to the individual structures. The random conical tilt (RCT) method was used to reconstruct the 3D structures. The surface rendering was generated using the Chimera visualization package. To aid the interpretation of the structure, the crystal structures of EBV gH/gL (PDB 3PHF) and anti-gB Fab (PDB 4OSU) were manually fitted into the EM 3D map by use of Chimera. Glycoprotein gH is colored gray, and gL is colored yellow. The numbers 1 to 3 correlate with the domains visible in the EM 2D class averages (Fig. 2). (B) A diagram showing the four immunogenic regions (IRs) and eight immunogenic sites of the HCMV pentamer targeted by 20 neutralizing antibodies. The arrows show the approximate positions of the immunogenic sites based on EM images.

FIG 4

Potencies of IR1 to IR4 antibodies in neutralizing HCMV in ARPE-19 and MRC-5 cells. Representative antibodies to each immunogenic site (and IR) were incubated in titration with HCMV. The mixtures were then applied to ARPE-19 cells (red circles) or MRC-5 cells (blue squares), viral entry events were documented by determining viral immediate early gene expression, and IC₅₀s, defined as the IgG concentration to achieve 50% viral entry inhibition, were calculated using four-parameter curve fitting. The data shown are representative of three experiments.

FIG 5

Potencies of antibodies in neutralizing 11 clinical isolates and 2 laboratory strains in ARPE-19 cells. Representative antibodies from each immunogenic site were incubated in titration with HCMV virus and then applied to ARPE-19 cells. The viral entry events were documented by immune staining of viral immediate early gene expression 24 h later. IC₅₀s shown on the y axis were calculated using four-parameter curve fitting. Antibody designations and their classification to IR regions are marked on the x axis. CMV-HIG was included as a reference. Strain information is given in Table 2. The data shown are representative of two experiments.

FIG 6

Evaluation of V160-induced antibody responses in rhesus macaques. (A) Neutralization titers of five rhesus macaques immunized with V160 HCMV vaccine. The vaccine was administered at week 0, 8, and 24 (red arrowheads), and sera collected at the indicated time points were evaluated for neutralizing activities in ARPE-19 cells. The neutralizing titers were determined by reciprocal serum dilutions to achieve 50% viral entry reduction. The data shown are representative of two experiments. (B) AbI₅₀ titers of rhesus immune sera in comparison with CMV-HIG. An arbitrary number of 1 was assigned to the serum if there was no detectable activity. The horizontal black bars represent the geometric mean AbI₅₀ titers to six sites. Sera from A10L106 at week 26 and A10R088 at week 36 were not available for testing. (C) Neutralization titers of four immune sera at week 36 against 11 clinical isolates and 2 laboratory strains in ARPE-19 cells. The legend symbols for HCMV isolates are as shown in Fig. 5. In panels B and C, CMV-HIG values are normalized to a starting concentration of 10 mg/ml to approximate the IgG concentration in serum.