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. 2011 Aug;7(8):e1002172.
doi: 10.1371/journal.ppat.1002172. Epub 2011 Aug 11.

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies

Sonja Pötzsch  1 , Nadja Spindler, Anna-Katharina Wiegers, Tanja Fisch, Pia Rücker, Heinrich Sticht, Nina Grieb, Tina Baroti, Florian Weisel, Thomas Stamminger, Luis Martin-Parras, Michael Mach, Thomas H Winkler
Affiliations

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies

Sonja Pötzsch et al. PLoS Pathog. 2011 Aug.

Abstract

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.

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Conflict of interest statement

SP, NS, A-KW, HS, LM-P, MM, and THW are inventors on a patent application on the antibodies described. THW and MM received consulting fees from 4-Antibody AG.

Figures

Figure 1
Figure 1. Neutralization capacity of Dom I- and Dom II-specific mabs.
The indicated B-cell supernatants (SN) and recombinant IgGs were tested for neutralization using different target cells. The AD-169 derived recombinant virus was used for fibroblasts as target cells. Human umbilical cord endothelial cells (HUVEC) and human retinal pigment epithelial cells (ARPE) were infected with the HCMV strain TB40E . Color code of the antibodies according to their target structure on gB as shown in Fig. 3. Every antibody was tested at least 3 times and representative results are shown.
Figure 2
Figure 2. Antibody recognition of gB deletion mutants.
(A) The parts of gB expressed by deletion mutants are represented by lines and the encompassed residues are given. Peptides starting with amino acid 1 of gB include the authentic signal sequence. Peptides starting at internal parts of gB contain a heterologous signal sequence and a HA-epitope tag at the amino terminus, indicated by the black circle. Reactivity of the respective gB peptides with different groups of monoclonal antibodies is indicated on the left of the figure and the minimal protein domains recognized by Group A and Group B mabs is indicated at the top. (B) Cos7 cells were transfected with the indicated plasmids and 48 h later processed for immunofluorescence using culture supernatants from Group A or Group B specific B-cell clones. Control stains for protein expression included a mab directed at gB-AD2 (aa 68-77), and an anti-HA mab as indicated at the right.
Figure 3
Figure 3. Domain architecture of HCMV gB.
(A) The regions representing individual domains are displayed in different colors in analogy to the HSV gB structure by Heldwein et al. and the numbers of the starting residues are given. Brackets indicate disulfide bonds. Signal: signal sequence, TM: transmembrane helix. (B) Ribbon diagram of a gB monomer (left), trimer with two protomers shown in grey (middle) and accessible surface representation of the trimeric gB (right). Coloring scheme according to (A).
Figure 4
Figure 4. Identification of gB Dom I and Dom II as antibody binding structures.
Plasmids expressing Dom I or Dom II were transfected into Cos7 cells and protein expression was analyzed 48 h later by indirect immunofluorescence using Group A or Group B mabs and a control stain (anti-HA antibody).
Figure 5
Figure 5. Mechanistic aspects of virus neutralization by Dom I and Dom II antibodies.
(A) Virus (m.o.i. 0.5) was incubated with the indicated antibodies (10 µg/ml for Dom I-, and Dom II-specific mabs, 5 µg/ml for the AD-2-specific mab C23, 2 µg/ml heparin) for 1 h at 37°C and cooled to 4°C. The virus/antibody mixture was added to HFF and incubated for 1 h at 4°C. Lysates were prepared and processed for quantitative real time PCR analysis. The virus only sample was set to 100% and used to calculate the remaining samples. (B) HFF were adsorbed with virus at a m.o.i. of 0.2 at 4°C for 1 h. Antibody at the indicated concentrations was added and the culture was shifted to 37°C. Extent of infection was analyzed 48 h later and calculated relative to the virus only control (100%). Color code of the antibodies according to their target structure on gB as shown in Fig. 3.
Figure 6
Figure 6. Influence of non-neutralizing antibodies on neutralizing capacity of antibodies.
(A) SM10 at a concentration of 0.3 µg/ml was mixed with the indicated non-neutralizing antibodies (AD-X  =  unknown binding site on gB) at various concentrations and incubated with virus for 1 h. The virus/antibody mixture was added to HFF and neutralization was assayed 48 h later. (B) Neutralization capacity of the serum pool, the affinity purified Dom II-specific antibodies (Dom II poly) and a Dom II-specific mab (SM5-1). Shown are representative results from at least 3 independent analyses.
Figure 7
Figure 7. Recognition of the antigenic domains of gB by human sera.
80 randomly selected sera from HCMV seropositive individuals were analyzed in an ELISA for reactivity against recombinant gB (gB) and the antigenic domains 1 (AD-1), 2 (AD-2) and the structural domain I and II (Dom I, Dom II), respectively. The horizontal line represents the cutoff for the individual antigens. The cutoff for each antigen was defined as mean +2 SD of 10 HCMV-negative sera.
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