Plasmablast Response to Primary Rhesus Cytomegalovirus (CMV) Infection in a Monkey Model of Congenital CMV Transmission

Abstract

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and the leading infectious cause of neurologic deficits and hearing loss in newborns. Development of a maternal HCMV vaccine to prevent vertical virus transmission is a high priority, yet protective maternal immune responses following acute infection are poorly understood. To characterize the maternal humoral immune response to primary CMV infection, we investigated the plasmablast and early antibody repertoire using a nonhuman primate model with two acutely rhesus CMV (RhCMV)-infected animals-a CD4⁺ T cell-depleted dam that experienced fetal loss shortly after vertical RhCMV transmission and an immunocompetent dam that did not transmit RhCMV to her infant. Compared to the CD4⁺ T cell-depleted dam that experienced fetal loss, the immunocompetent, nontransmitting dam had a more rapid and robust plasmablast response that produced a high proportion of RhCMV-reactive antibodies, including the first identified monoclonal antibody specific for soluble and membrane-associated RhCMV envelope glycoprotein B (gB). Additionally, we noted that plasmablast RhCMV-specific antibodies had variable gene usage and maturation similar to those observed in a monkey chronically coinfected with simian immunodeficiency virus (SIV) and RhCMV. This study reveals characteristics of the early maternal RhCMV-specific humoral immune responses to primary RhCMV infection in rhesus monkeys and may contribute to a future understanding of what antibody responses should be targeted by a vaccine to eliminate congenital HCMV transmission. Furthermore, the identification of an RhCMV gB-specific monoclonal antibody underscores the possibility of modeling future HCMV vaccine strategies in this nonhuman primate model.

Keywords: congenital cytomegalovirus; nonhuman primates; vaccine.

FIG 1

Differences in the early RhCMV-specific antibody responses between an RhCMV-nontransmitting (NT/CD4⁺) pregnant monkey and an RhCMV-transmitting (T/CD4⁻) pregnant monkey following acute maternal RhCMV challenge. (A and B) Maternal plasma (●) and amniotic fluid (□) RhCMV DNA loads in an immunocompetent, RhCMV-nontransmitting dam (NT/CD4⁺, 251-05; in blue throughout) and a CD4⁺ T cell-depleted RhCMV-transmitting dam (T/CD4⁻, 369-09; in red throughout). An arrow indicates the time of fetal loss due to spontaneous abortion. (C and D) IgM and IgG responses measured against whole RhCMV virions. (E and F) RhCMV gB and RhPC glycoprotein-specific responses. The dotted lines indicate the limits of detection at week 0 + 2 SD. (G and H) Neutralizing titers assessed against RhCMV variants in fibroblasts and epithelial cells. The viral load and antibody kinetics data shown were adapted from reference with permission of the publisher.

FIG 2

Kinetics of plasmablast response in an RhCMV-nontransmitting, immunocompetent (NT/CD4⁺) dam (251-05) and an RhCMV-transmitting (T/CD4⁻) dam following acute maternal RhCMV challenge. (A) The plasmablast populations (defined as CD80⁺/HLADR⁺ of negatively selected lineage), outlined in black, following primary RhCMV infection in 251-05 (NT/CD4⁺, upper panel) and in 369-09 (T/CD4⁻, lower panel) are shown. The percentages of CD80⁺/HLADR⁺ populations out of the negatively selected lineage are indicated. wkpi, weeks postinfection. (B) Percent plasmablasts (calculated as CD80⁺/HLADR⁺ lymphocytes of total viable lymphocytes [AqVD⁻/CD14⁻]) (left) and absolute plasmablast counts (right) are shown for animal 251-05 (NT/CD4⁺; in blue) and animal 369-09 (T/CD4⁻; in red) acutely infected with RhCMV.

FIG 3

RhCMV reactivity of antibodies isolated from plasmablasts of dams acutely infected with RhCMV and from total memory B cells of a monkey chronically coinfected with RhCMV/SIV. Plasmablasts at peak response were sorted from animal 251-05 (NT/CD4⁺) at 2 weeks postinfection (wkpi) (A) and animal 369-09 (T/CD4⁻) at 3 wkpi (B) and screened for RhCMV reactivity and specificity. Total memory B cells were sorted from peripheral blood of 206-96, a rhesus monkey chronically coinfected with RhCMV and SIV for 1 year (C). Proportions of isolated monoclonal antibodies with RhCMV or SIV reactivity and antibodies that were nonreactive to RhCMV or SIV are shown.

FIG 4

Isotype, subclass, and genetic analysis of antibodies in acute and chronic RhCMV infection. Distribution of immunoglobulin isotype (A), V_H family (B), and V_H gene usage (C) from total monoclonal antibodies (upper pie charts) and RhCMV-reactive antibodies (lower pie charts) isolated from an NT/CD4⁺ dam (251-05; in blue), an T/CD4⁻ dam (369-09; in red) during acute infection, and a chronically RhCMV/SIV-coinfected monkey (206-96; in gray). V_H mutation frequencies (D) and HCDR3 lengths (E) of isolated RhCMV-reactive (●) and nonreactive (■) monoclonal antibodies were similar between each monkey. AA, amino acids.

FIG 5

Characterization of plasmablast MAb glycoprotein binding specificities. (A) Flow chart depicting the sorting/screening of MAbs from plasmablasts of two monkeys. (B) BHK-21 cells infected with MVA recombinants were analyzed by flow cytometry for intracellular staining of RhCMV UL128C (expressing gH/gL/UL128/UL130/UL131A), gB, or gB/pp65. Left panel, staining of MVA-infected cells was performed with DH669 as the primary antibody and Alexa Fluor 647-coupled secondary anti-rhesus IgG Ab. Right panel, GFP expression was analyzed for confirming MVA infection of BHK-21 cells since all the constructs contain a GFP expression cassette. Uninfected cells were used as a control. (C) Recognition of linear RhgB by DH669 (left panel) and anti-RhgB polyclonal serum (center panel). Immunoblots were performed by using a lysate from MVA-RhUL128C (PC)- or MVA-RhgB (gB)-infected BHK-21 cells. Uninfected cells (U) were used as a control. For a loading control, samples were analyzed with anti-MVA BR-5 Ab (right panel). (D) The binding of RhCMV Ig (whole IgG from RhCMV-seropositive monkeys) and DH669 (upper panels), plasmablast MAbs from 251-05 (NT/CD4⁺) (middle panels), and plasmablast MAbs from 369-09 (T/CD4⁻) (lower panels) to soluble RhgB and to membrane-associated proteins from uninfected cells (U), epithelial cell-tropic UCD52-infected cells (E), and fibroblast-tropic 180.92-infected cells (F) was assessed. Arrows indicate MAb binding to membrane-associated RhgB. Mass markers (in kilodaltons) are shown next to each panel.