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. 2017 Feb 28;91(6):e01857-16.
doi: 10.1128/JVI.01857-16. Print 2017 Mar 15.

Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus

Flavia Chiuppesi  1 , Teodora Kaltcheva  1 , Zhuo Meng  1 , Peter A Barry  2 , Don J Diamond  3 , Felix Wussow  3
Affiliations

Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus

Flavia Chiuppesi et al. J Virol. .

Abstract

As human cytomegalovirus (HCMV) is the most common infectious cause of fetal anomalies during pregnancy, development of a vaccine that prevents HCMV infection is considered a global health priority. Although HCMV immune correlates of protection are only poorly defined, neutralizing antibodies (NAb) targeting the envelope pentamer complex (PC) composed of the subunits gH, gL, UL128, UL130, and UL131A are thought to contribute to the prevention of HCMV infection. Here, we describe a continuous target sequence within UL128 that is recognized by a previously isolated potent PC-specific NAb termed 13B5. By using peptide-based scanning procedures, we identified a 13-amino-acid-long target sequence at the UL128 C terminus that binds the 13B5 antibody with an affinity similar to that of the purified PC. In addition, the 13B5 binding site is universally conserved in HCMV, contains a previously described UL128/gL interaction site, and interferes with the 13B5 neutralizing function, indicating that the 13B5 epitope sequence is located within the PC at a site of critical importance for HCMV neutralization. Vaccination of mice with peptides containing the 13B5 target sequence resulted in the robust stimulation of binding antibodies and, in a subset of immunized animals, in the induction of detectable NAb, supporting that the identified 13B5 target sequence constitutes a PC-specific neutralizing epitope. These findings provide evidence for the discovery of a continuous neutralizing epitope within the UL128 subunit of the PC that could be an important target of humoral immune responses that are involved in protection against congenital HCMV infection.IMPORTANCE Neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) envelope pentamer complex (PC) are thought to be important for preventing HCMV transmission from the mother to the fetus, thereby mitigating severe developmental disabilities in newborns. However, the epitope sequences within the PC that are recognized by these potentially protective antibody responses are only poorly defined. Here, we provide evidence for the existence of a highly conserved, continuous, PC-specific epitope sequence that appears to be located within the PC at a subunit interaction site of critical importance for HCMV neutralization. These discoveries provide insights into a continuous PC-specific neutralizing epitope, which could be an important target for a vaccine formulation to interfere with congenital HCMV infection.

Keywords: HCMV; UL128; epitope; pentamer complex; peptide.

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Figures

FIG 1
FIG 1
Recognition of denatured UL128 by 13B5 and Z9G11. Shown is immunoblot detection of UL128 expressed from Ad vectors either alone (128) or in combination with the other four PC subunits (PC) in infected ARPE-19 EC using nonneutralizing Ab Z9G11 (top) or PC-specific NAb 13B5 (bottom). Cells infected with Ad-tet only (tet) were analyzed as a control. Mass markers (in kilodaltons) are shown next to each panel.
FIG 2
FIG 2
UL128 peptide library binding of 13B5 and Z9G11. An ELISA was used to detect the binding of PC-specific NAb 13B5 and nonneutralizing Ab Z9G11 to a UL128 peptide library consisting of 41 overlapping 15-mers with an offset of 4. OD, optical density.
FIG 3
FIG 3
Binding of human serum antibodies to the UL128 peptide library. An ELISA was performed to determine the reactivity of the UL128 peptide library with commercial serum products from HCMV+ (S4360, S4234, and S4371; SeraCare) or HCMV− (SNeg; SeraCare) individuals or CMV-HIG. Human serum was diluted 1:100, and CMV-HIG was used at a concentration of 1 μg/ml.
FIG 4
FIG 4
Mapping of the NAb 13B5 minimal binding site. (A and B) C- and N-terminally truncated peptides based on library peptide 40 were used in an ELISA to identify the shortest amino acid sequence needed for binding of NAb 13B5. (C) ELISA to compare 13B5 binding to K13M, comprising the minimal 13B5 epitope sequence, and peptides based on K13M with one (H14M) or two (K15M) additional amino acid residues of UL128 added to the N terminus. (D) Alanine scanning based on peptide K13M to identify amino acid residues involved in 13B5 binding. Bars represent standard deviations of data from triplicate wells. Vertical axes represent the optical density at 450 nm.
FIG 5
FIG 5
13B5 neutralization interference by 13B5 binding peptides. Serial dilutions of peptides 37 to 41 of the UL128 peptide library (A) and peptides K14L to K11G of the C-terminal truncation library (B) were evaluated for interference with 13B5 neutralizing activity to block TB40/E entry into ARPE-19 EC. Peptides were preincubated with 150 ng/ml 13B5 or 50 ng/ml 54E11 as a control and then incubated for 2 h with 9,000 PFU of HCMV TB40/E before the mixture was transferred to the cells.
FIG 6
FIG 6
Induction of binding and neutralizing antibodies by peptides based on the 13B5 epitope sequence. (A) Peptides based on the 13B5 binding site (K15M, K14CS, and K16CS) and peptides containing only partial sequences of the 13B5 binding site (UL128 library peptide 38) were coupled to KLH and tested for binding to 13B5 antibody by an ELISA. KLH alone was used as a control. Bars represent standard deviations of data from triplicate wells. (B) Pooled sera from mice immunized with the peptide constructs KLH-K15M, KLH-K14CS, and KLH-K16CS were tested by Western blot analysis for the detection of UL128 expressed from Ad vectors in ARPE-19 EC. EC infected with Ad-tet (tet) were analyzed as a control. Mass markers (in kilodaltons) are shown at the right. (C and D) BALB/c mice (5 animals per group) were immunized three times 4 weeks apart with KLH-coupled peptides admixed in Freund's adjuvant. (C) Levels of peptide-specific binding antibodies in sera of immunized mice were measured via an ELISA 1 week before (−1wp1st) and 3 weeks after the first, second, and third immunizations (3wp1st, 3wp2nd, and 3wp3rd, respectively) by using the peptides (not conjugated to KLH) that were used for immunization as coating antigens. (D) Serum neutralizing antibody titers (NT50) from immunized mice against HCMV TB40/E were measured on ARPE-19 EC by using a standard microneutralization assay. Lines in panels C and D indicate the group means.
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