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

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

Neutralization capacity of SM5-1 and of partially germline-reverted SM5-1<i>germ</i>.

<p>Viruses were preincubated with the antibody for 1 h and fibroblasts were infected with the virus-antibody mixture. Medium was changed 4 h after infection. 24 h post infection luciferase activity was measured in relative light units (RLU) as described previously and percent neutralization was calculated <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004377#ppat.1004377-Potzsch1" target="_blank">[26]</a>. Shown is one data set from two independent experiments.</p

HCMV gB Dom-II recognition by antibody SM5-1.

<p>(A) Trimeric model of HCMV glycoprotein B (gB) derived from the crystal structure of the homologous HSV-1 gB protein (PDB ID 2gum, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004377#ppat.1004377-Heldwein1" target="_blank">[18]</a>). Of the different gB domains, Dom-II is highlighted in green. N- (Thr-112) and C-terminal (Ser-438) amino acids of the Dom-II expression construct are labelled. Dashed lines represent regions that were not resolved in the crystal structure of HSV-1 gB and are therefore excluded from the model. (B) Cartoon representation of the Dom-II-SM5-1 Fab complex determined at an atomic resolution of 2.1 Å. Dom-II is colored in green, SM5-1 light and heavy chains are colored in red and blue, respectively. The CDR loops of the heavy chain are shown in orange and those of the light chain in yellow. Two segments of gB (residues 112–132 and 344–438) were fused together by an artificial linker segment in order to obtain a sequence-contiguous Dom-II protein <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004377#ppat.1004377-Spindler1" target="_blank">[32]</a>. (C) Top view of the antigen-binding site with the structure of Dom-II omitted in the presentation. Colors are as in (B). CDR loops are labeled L1 to L3 and H1 to H3. (D) 2mFo-DFc map contoured at 1σ and displayed within 2.5 Å around any CDR H3 atoms. Contiguous electron density is observed for the entire H3 loop in the antigen-bound structure. (E) Surface representation of the antigen-binding site of SM5-1 oriented as in (C). Shown in white is the surface area of SM5-1 that becomes buried upon antigen binding.</p

Contacts between Dom-II and SM5-1 together with an alignment of the CDRs of related antibodies.

<p>Black lines indicate intermolecular distances smaller than 3.5 Å. The Dom-II sequence is reported in its entirety, while for SM5-1 only the amino acid sequences of the CDRs and of frame work residue Lys67 are displayed. Asterisks indicate mutations in engineered Dom-II in the complex. The CDR sequences of the highly related antibodies are provided and listed either above or below the SM5-1 sequence. Amino acids that differ in any of the sequences are highlighted in red. SM5-1 residues that were substituted <i>in silico</i> for the molecular dynamics simulations are underlined.</p

Conformational flexibility of SM5-1 and SM5-1*.

<p>(A) Plot of the root mean square fluctuations (RMSF) per residue indicating the enhanced flexibility of residues 102–112 (CDR H3) in SM5-1* (red line) compared to SM5-1 (black line). Sequence positions that differ between SM5-1 and SM5-1* are marked by a yellow asterisk. (B, C) Overlay of 6 structures collected every 20 ns over the simulation time for SM5-1 (B) and SM5-1* (C). Note that the CDR H3 loop in SM5-1* exhibits a higher flexibility and deviates further from the Dom-II-bound conformation which served as starting structure. The six residues that are different between SM5-1 and SM5-1* are shown in stick presentation and a pink arrow points towards the CDR H3 loop.</p

Crystal structures of unbound Dom-II and SM5-1.

<p>(A) Cartoon representation of unbound engineered Dom-II solved at 1.8 Å resolution. No electron density is observed for the artificially introduced linker amino acids (grey dashed line). (B) Cartoon representation of unbound Fab SM5-1 solved at 1.9 Å resolution. The heavy and light chains are colored in blue and red, respectively. The CDR loops are depicted either in orange (H1 to H3) or yellow (L1 to L3). In the free SM5-1 structure, the polypeptide chain between residues H3 107 and 111 as well as heavy chain residues 145 to 152 could not be traced. (C) Superposition of Dom-II in the unbound (in green) and Fab SM5-1-bound state (in black). (D) Superposition of Fab SM5-1 in the unbound state (in color) and in the Dom-II-bound state (in black). The variable domains were superimposed, and the superposition shows that the differences between both structures are limited to CDR H3 and changes in the elbow angle (indicated by an arrow). (E) Close-up showing residual positive mFo-DFc difference electron density in the unbound Dom-II crystal structure. The density is contoured at a 3σ level and extends across a crystallographic two-fold axis (black oval).</p