Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus

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.

Multiantigenic Modified Vaccinia Virus Ankara Vaccine Vectors To Elicit Potent Humoral and Cellular Immune Reponses against Human Cytomegalovirus in Mice

“…Peptide libraries of gH, gL, UL130, and UL131A were composed of 15-mers with a 9-aa overlap and purchased from Genscript. The UL128 peptide library was composed of 15-mers with an 11-aa overlap and produced at the City of Hope Core Facility (99). Equal amounts of peptides of the individual PC subunits were used to create the PC peptide library.…”

Human Cytomegalovirus Particles Treated with Specific Antibodies Induce Intrinsic and Adaptive but Not Innate Immune Responses

“…This gives HCMV an advantage in transmission by shielding the virus from neutralizing antibodies. Current HCMV therapeutic approaches are focused on the generation of neutralizing antibodies (3,(34)(35)(36)(37), some of which are potent for neutralizing cell-free virus in vitro but may have no effect on cell-associated transmission. Our observation offers a new approach to developing therapeutic interventions by using viral particles and neutralizing antibodies to trigger antiviral responses without introducing infections.…”

Comparison of homologous and heterologous prime-boost vaccine approaches using Modified Vaccinia Ankara and soluble protein to induce neutralizing antibodies by the human cytomegalovirus pentamer complex in mice

“…The construction and generation of MVA-PC was described previously [ 27 ]. HCMV strain TB40/E and MVA-PC virus stocks were prepared as described previously following virus propagation of ARPE-19 or BHK-21 cells, respectively [ 21 , 27 , 28 ].…”

“…For the inhibition of HCMV entry study, a neutralization assay was performed as previously described [ 21 , 25 , 28 ]. Briefly, ARPE-19 cells were seeded at 1.5x10 4 cells/well in a clear-bottom 96-well plate (Corning).…”