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. 2013 Dec 17;110(51):E4997-5005.
doi: 10.1073/pnas.1316517110. Epub 2013 Dec 2.

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine

Daniel C Freed  1 , Qi Tang, Aimin Tang, Fengsheng Li, Xi He, Zhao Huang, Weixu Meng, Lin Xia, Adam C Finnefrock, Eberhard Durr, Amy S Espeseth, Danilo R Casimiro, Ningyan Zhang, John W Shiver, Dai Wang, Zhiqiang An, Tong-Ming Fu
Affiliations

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine

Daniel C Freed et al. Proc Natl Acad Sci U S A. .

Abstract

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.

Keywords: antiviral antibody; monoclonal antibody.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
A vaccine virus with the pentameric gH complex restored can elicit high neutralizing titers in rabbits. Groups of rabbits (n = 4) immunized with the vaccine virus at indicated dose at week 0, 3, and 8. The immune sera were collected at week 11 and evaluated along with prevaccination sera in viral neutralization assay. Prevaccination sera showed no neutralizing activity. A serum panel from HCMV-seropositive donor (n = 30) was included in the experiments as a comparison. NT50 titers represent the reciprocal serum dilutions to block 50% viral entry. The geometric means with 95% confidence intervals are marked in the plot for each group.
Fig. 2.
Fig. 2.
Correlation analysis of neutralizing and binding properties of rabbit mAbs. The neutralizing and binding functions of each antibody were analyzed in viral neutralization and binding assays, respectively. Human CMV hyperimmune IgG (HCMV-HIG) (e.g., CytoGam) is provided as an example and also as a reference for comparison. HCMV-HIG is analyzed for neutralization in ARPE-19 cells (A) and binding to virions (B). EC50 neutralizing and EC50 binding, defined as the IgG concentration required to block 50% of viral infection (A) or reach 50% maximal binding signal (B), respectively, were calculated by four-parameter curve fitting. For mAbs with weak neutralizing or binding capacity, the EC50 value was arbitrarily assigned as 100 μg/mL (Materials and Methods). Each mAb was plotted for its EC50 neutralizing (y axis) and EC50 binding (x axis) (C). The solid square symbol in the center represents HCMV-HIG (e.g., CytoGam); the dashed horizontal line represents the EC50 neutralizing of HCMV-HIG, which is used as the threshold to differentiate neutralizing mAbs (triangles above the line) and nonneutralizing mAbs (circles below the line). The 11 elite neutralizing antibodies are identified by solid triangles. mAbs 57.4 and 276.10 are marked with the clone identification. The 11 elite binders are identified by filled circles.
Fig. 3.
Fig. 3.
Neutralizing properties of mAbs in ARPE-19 cells do not predict their activity in MRC-5 cells. The EC50 neutralizing values were calculated for each antibody in ARPE-19 epithelial cells and MRC-5 fibroblasts (Table S1). Three distinct groups are marked by circles for the mAbs only neutralizing in ARPE-19 cells (group A), the mAbs neutralizing in both ARPE-19 and MRC-5 cells (group B), and the mAbs nonneutralizing in either cell line (group C), respectively.
Fig. 4.
Fig. 4.
Preferential binding of mAbs to the vaccine virus is associated with their neutralizing activity. AD169 virus and the vaccine virus were immobilized in titration on ELISA plates. Reactivity of rabbit mAb to AD169 versus the vaccine virions was tested at a fixed concentration of 2 μg/mL. The representative curves illustrate three patterns of binding to the antigen pair in A (mAb 57.4), B (mAb 58.5), and C (mAb 295.5), respectively. In D, the elite neutralizing mAbs (open circles) and elite binding mAbs (closed triangles) are plotted based on their binding patterns (x axis) and EC50 neutralizing value in ARPE-19 cells (y axis).
Fig. 5.
Fig. 5.
Eight of the 11 elite neutralizing mAbs recognize pentameric gH complex in ELISA. Recombinant gB antigen or pentameric gH complex antigen was immobilized at 2 μg/mL on ELISA plates and tested for reactivity with elite neutralizing mAbs and elite binding mAbs in titration (Fig. S1). Fluorescent signals to either recombinant pentameric gH complex (recombinant 5′gH, open circles) or recombinant gB (solid dots) are plotted for each mAb at a concentration of ∼1 μg/mL.
Fig. 6.
Fig. 6.
Phylogenetic analysis of 45 rabbit mAbs and their lineages in correlation to their binding and neutralizing properties. Phylogenetic trees were constructed based on the entire heavy-chain variable-region amino acid sequence, and lineage groups were classified based on similarities in the heavy-chain CDR3. Lineage groups containing two or more mAbs are highlighted with different background colors. The solid dots identify neutralizing mAbs, with those of three dots indicating the elite neutralizing mAbs, whereas the open circles identify nonneutralizing mAbs, with those of three circles indicating the elite binding mAbs.
Fig. 7.
Fig. 7.
Neutralizing function for an antibody is associated with long CDR3 for heavy or light chain. Heavy- and light-chain CDR3 lengths are plotted for the 11 elite neutralizing antibodies (circles), the 11 elite binding antibodies without mAb 57.4 (inverted triangles), all neutralizing antibodies (n = 25; triangles), and all nonneutralizing antibodies (n = 20; diamonds). The solid and open symbols represent heavy and light chains, respectively. Average CDR3 length is indicated by the horizontal line. Unpaired two-tailed t test was performed for statistical comparisons of indicated groups with P values marked.
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