Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, frequently causing hearing loss and brain damage in afflicted infants. A vaccine to prevent maternal acquisition of HCMV during pregnancy is necessary to reduce the incidence of infant disease. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine platform is the most successful HCMV vaccine tested to date, demonstrating ∼50% efficacy in preventing HCMV acquisition in multiple phase 2 trials. However, the mechanism of vaccine protection remains unknown. Plasma from 33 postpartum women gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and nonneutralizing anti-HCMV effector functions and compared with an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and domain I. Vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this nonneutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, although were poor mediators of NK cell activation. Altogether, these data suggest that nonneutralizing antibody functions, including virion phagocytosis, likely played a role in the observed 50% vaccine-mediated protection against HCMV acquisition.

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4 + T-cell depleted at the time of inoculation. Animals that received the CD4 +

Risk of congenital cytomegalovirus (cCMV) transmission is highly dependent on the presence of preexisting maternal immunity, with the lowest rates observed in CMV-seroimmune populations. Among infants of CMV-seroimmune women, those who are exposed to human immunodeficiency virus (HIV) have an increased risk of acquiring cCMV infection as compared to HIV-unexposed infants. To better understand the risk factors of nonprimary cCMV transmission in HIV-infected women, we performed a casecontrol study in which CMV-specific plasma antibody responses from 19 CMV-transmitting and 57 CMV-nontransmitting women with chronic CMV/HIV coinfection were evaluated for the ability to predict the risk of cCMV infection. Primary multivariable conditional logistic regression analysis revealed an association between epithelial-tropic CMV neutralizing titers and a reduced risk of cCMV transmission (odds ratio [OR], 0.18; 95% confidence interval [CI], .03-.93; P = .04), although this effect was not significant following correction for multiple comparisons (false-discovery rate, 0.12). Exploratory analysis of the CMV specificity of plasma antibodies revealed that immunoglobulin G (IgG) responses against the glycoprotein B (gB) neutralizing epitope AD-2 had a borderline association with low risk of transmission (OR, 0.72; 95% CI, .51-1.00; P = .05), although this was not confirmed in a post hoc plasma anti-AD-2 IgG blocking assay. Our data suggest that maternal neutralizing antibody responses may play a role in protection against cCMV in HIV/CMV-coinfected populations.

word count: 242 (250 max) 24Significance word count: 120 (120 max) 25Manuscript character count: 48,559 (49,000 max) 26 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/246884 doi: bioRxiv preprint first posted online 2 Abstract: 27Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, frequently 28 causing hearing loss and brain damage in afflicted infants. A vaccine to prevent maternal 29 acquisition of HCMV during pregnancy is necessary to reduce the incidence of infant disease. 30The glycoprotein B (gB) + MF59 adjuvant subunit vaccine platform is the most successful 31HCMV vaccine tested to-date, demonstrating approximately 50% efficacy in preventing HCMV 32 acquisition in phase II trials. However, the mechanism of vaccine protection remains unknown. 33Plasma from 33 gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope

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