HCMV trimer- and pentamer-specific antibodies synergize for virus neutralization but do not correlate with congenital transmission

Abstract

Human cytomegalovirus (HCMV) causes substantial disease in transplant patients and harms the development of the nervous system in babies infected in utero. Thus, there is a major focus on developing safe and effective HCMV vaccines. Evidence has been presented that a major target of neutralizing antibodies (NAbs) is the HCMV pentamer glycoprotein gH/gL/UL128-131. In some studies, most of the NAbs in animal or human sera were found to recognize the pentamer, which mediates HCMV entry into endothelial and epithelial cells. It was also reported that pentamer-specific antibodies correlate with protection against transmission from mothers to babies. One problem with the studies on pentamer-specific NAbs to date has been that the studies did not compare the pentamer to the other major form of gH/gL, the gH/gL/gO trimer, which is essential for entry into all cell types. Here, we demonstrate that both trimer and pentamer NAbs are frequently found in human transplant patients' and pregnant mothers' sera. Depletion of human sera with trimer caused reductions in NAbs similar to that observed following depletion with the pentamer. The trimer- and pentamer-specific antibodies acted in a synergistic fashion to neutralize HCMV and also to prevent virus cell-to-cell spread. Importantly, there was no correlation between the titers of trimer- and pentamer-specific NAbs and transmission of HCMV from mothers to babies. Therefore, both the trimer and pentamer are important targets of NAbs. Nevertheless, these antibodies do not protect against transmission of HCMV from mothers to babies.

Keywords: cell-to-cell spread; congenital; neutralizing antibodies; transplant; vaccines.

Fig. 1.

Neutralizing capacity of human serum from transplant patients following depletion with gH/gL, trimer, or pentamer. Human sera collected following heart transplant were diluted to titers that resulted in 100% neutralization of HCMV infection of APRE-19 cells (NT₁₀₀). The diluted sera were then incubated with either no protein or with 1 μg of soluble gH/gL, trimer, or pentamer and then tested for the ability to neutralize HCMV (BADrUL131) infection of APRE-19 epithelial cells. The relative infectivity of HCMV after incubation with sera was determined by counting GFP+ (infected) cells after 24 h of infection and compared with the number of infected cells following incubation of HCMV with a seronegative donor serum (ctl serum).

Fig. 2.

(A–C) Progression of NAbs over time in human transplant patients. Sera from three transplant patients (patient 1, patient 2, and patient 3) not shown in Fig. 1 were collected at various times following transplantation (with the indicated dates). The sera were diluted to NT₁₀₀, then depleted of NAbs with gH/gL, trimer, or pentamer as in Fig. 1. The depleted sera were then tested for neutralization of HCMV on ARPE-19 cells.

Fig. 3.

(A–C) Effects of depleting trimer- and pentamer-specific Abs from sera from pregnant mothers. Sera were compared based on mothers with similar ages and demographics, and each group is shown in separate panels that included one mother who transmitted HCMV to her baby and one or two mothers who did not transmit the virus. All of the sera shown here were collected during the first trimester. Sera were diluted to NT₁₀₀ values, then depleted using no protein or 1 μg of gH/gL, trimer, or pentamer. Neutralization of HCMV was determined using ARPE-19 epithelial cells. There were 14 individual mother donors, designated A through O. Data obtained with sera from donors A–I are represented here, and data obtained with sera from donors J–O are represented in SI Appendix, Fig. S4 A and B. Note that serum from donor H was not included due to poor neutralizing activity.

Fig. 4.

Synergistic effects of trimer- and pentamer-specific NAbs on HCMV neutralization. Sera from either transplant patients, sera 106 and 077 (A and B), or pregnant mothers, sera A1 and B1 (C and D), were diluted to NT₁₀₀ values and then depleted using the soluble trimer or pentamer or depleted with both the soluble trimer and pentamer. In contrast to Figs. 1–3, depletions were performed with 0.125 μg of each of the soluble proteins to reduce the efficiency of the single-protein depletions to measure differences in the codepletions. The depleted sera were then mixed with HCMV and infectivity tested using APRE-19 cells.

Fig. 5.

Trimer- and pentamer-specific Abs act together to reduce HCMV cell-to-cell spread. (A) ARPE-19 cells were infected with HCMV (BADrUL131) that expresses GFP, then trypsinized and plated with uninfected ARPE-19 cells in the presence of no Abs or with anti-gH MAb 14-4b. (B) Similarly, cells were incubated with sera from three different transplant patients (sera 117, 163, and 209) that had been depleted of Abs by incubation with no protein (no depletion) or with 1 μg of the trimer (T) or pentamer (P) or with both the trimer and pentamer (T+P). The virus was allowed to spread for 10 d, and GFP expression in cells was characterized by fluorescence microscopy. Images were acquired with 100× magnification.