Transcriptional signature of durable effector T cells elicited by a replication defective HCMV vaccine.

Human cytomegalovirus (HCMV) is a leading infectious cause of birth defects and the most common opportunistic infection that causes life-threatening diseases post-transplantation; however, an effective vaccine remains elusive. V160 is a live-attenuated replication defective HCMV vaccine that showed a 42.4% efficacy against primary HCMV infection among seronegative women in a phase 2b clinical trial. Here, we integrated the multicolor flow cytometry, longitudinal T cell receptor (TCR) sequencing, and single-cell RNA/TCR sequencing approaches to characterize the magnitude, phenotype, and functional quality of human T cell responses to V160. We demonstrated that V160 de novo induces IE-1 and pp65 specific durable polyfunctional effector CD8 T cells that are comparable to those induced by natural HCMV infection. We identified a variety of V160-responsive T cell clones which exhibit distinctive "transient" and "durable" expansion kinetics, and revealed a transcriptional signature that marks durable CD8 T cells post-vaccination. Our study enhances the understanding of human T-cell immune responses to V160 vaccination.

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Characterization of prefusion-F-specific antibodies elicited by natural infection with human metapneumovirus.

Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies; however, little is known about the humoral immune response generated from natural infection. Here, using prefusion-stabilized F proteins to interrogate memory B cells from two older adults, we obtain over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal response. Characterization of 136 monoclonal antibodies reveals broad recognition of the protein surface, with potently neutralizing antibodies targeting each antigenic site. Cryo-EM studies further reveal two non-canonical sites and the molecular basis for recognition of the apex of hMPV F by two prefusion-specific neutralizing antibodies. Collectively, these results provide insight into the humoral response to hMPV infection in older adults and will help guide vaccine development.

Polymorphic Forms of Human Cytomegalovirus Glycoprotein O Protect against Neutralization of Fibroblast Entry by Antibodies Targeting Epitopes Defined by Glycoproteins H and L.

Human cytomegalovirus (CMV) utilizes different glycoproteins to enter into fibroblast and epithelial cells. A trimer of glycoproteins H, L, and O (gH/gL/gO) is required for entry into all cells, whereas a pentamer of gH/gL/UL128/UL130/UL131A is selectively required for infection of epithelial, endothelial, and some myeloid-lineage cells, but not of fibroblasts. Both complexes are of considerable interest for vaccine and immunotherapeutic development but present a conundrum: gH/gL-specific antibodies have moderate potency yet neutralize CMV entry into all cell types, whereas pentamer-specific antibodies are more potent but do not block fibroblast infection. Which cell types and neutralizing activities are important for protective efficacy in vivo remain unclear. Here, we present evidence that certain CMV strains have evolved polymorphisms in gO to evade trimer-specific neutralizing antibodies. Using luciferase-tagged variants of strain TB40/E in which the native gO is replaced by gOs from other strains, we tested the effects of gO polymorphisms on neutralization by monoclonal antibodies (mAbs) targeting four independent epitopes in gH/gL that are common to both trimer and pentamer. Neutralization of fibroblast entry by three mAbs displayed a range of potencies that depended on the gO type, a fourth mAb failed to neutralize fibroblast entry regardless of the gO type, while neutralization of epithelial cell entry by all four mAbs was potent and independent of the gO type. Thus, specific polymorphisms in gO protect the virus from mAb neutralization in the context of fibroblast but not epithelial cell entry. No influence of gO type was observed for protection against CMV hyperimmune globulin or CMV-seropositive human sera, suggesting that antibodies targeting protected gH/gL epitopes represent a minority of the polyclonal neutralizing repertoire induced by natural infection.

Structural basis for HCMV Pentamer recognition by neuropilin 2 and neutralizing antibodies.

Human cytomegalovirus (HCMV) encodes multiple surface glycoprotein complexes to infect a variety of cell types. The HCMV Pentamer, composed of gH, gL, UL128, UL130, and UL131A, enhances entry into epithelial, endothelial, and myeloid cells by interacting with the cell surface receptor neuropilin 2 (NRP2). Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here, we describe the cryo-EM structure of NRP2 bound to Pentamer. The high-affinity interaction between these proteins is calcium dependent and differs from the canonical carboxyl-terminal arginine (CendR) binding that NRP2 typically uses. We also determine the structures of four neutralizing human antibodies bound to the HCMV Pentamer to define susceptible epitopes. Two of these antibodies compete with NRP2 binding, but the two most potent antibodies recognize a previously unidentified epitope that does not overlap the NRP2-binding site. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization.

Pan-SARS neutralizing responses after third boost vaccination in non-human primate immunogenicity model.

The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500ß, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).

Recent progress in development of monoclonal antibodies against human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause permanent childhood disabilities following in utero infection and life threatening diseases in immune-compromised individuals such as those post transplantation. Without an effective vaccine, small molecule antiviral drugs are routinely used in high-risk transplant recipients, but the effectiveness of which is limited by side effects and drug resistance. The potentials of antibody-based passive immune therapies alone or in combination with the small molecule antivirals to treat or prevent HCMV infection have been actively studied. In this review, we focus on the recent publications on identification and characterization of monoclonal antibodies that have the potential to be developed as anti-HCMV therapies. We review the progress in clinical evaluation of antibody-based therapies to prevent HCMV-associated diseases.

Development of multivalent mRNA vaccine candidates for seasonal or pandemic influenza.

Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice.

Novel adjuvants enhance immune responses elicited by a replication-defective human cytomegalovirus vaccine in nonhuman primates.

Adjuvants have long been explored to enhance vaccine efficacy. Current adjuvants approved for human vaccines are mostly studied for their ability to improve antibody responses. There remains a need for development of novel adjuvants, especially those able to enhance cell-mediated immunity (CMI). In this preclinical study we assessed the effect of two novel adjuvants, a delta inulin microparticle Advax formulated with or without a toll-like receptor 9 (TLR9) agonist CpG oligonucleotide, and a Merck & Co., Inc., Kenilworth, NJ, USA proprietary lipid nanoparticle (LNP), on immune responses elicited by V160, an experimental replication-defective human cytomegalovirus vaccine. Adult rhesus macaques were immunized with a low dose of V160 (10 units) either alone or in combination with the adjuvants as compared to those immunized with a high dose of V160 alone (100 units). While neither adjuvant conferred a significant benefit to vaccine-elicited humoral immune responses at the dose tested, both enhanced cellular immune responses to V160, where Advax promoted both CD4+ and CD8+ T cells and LNP predominantly impacted the CD4+ T cell response. Transcriptome analyses of peripheral blood samples demonstrated different modes of action for these adjuvants. One day post vaccination, LNP induced upregulation of a large number of genes involved in the innate immune response similar to those triggered by viral infection. In contrast, Advax did not activate any known inflammatory pathways and did not significantly impact gene expression pattern until day 7 post administration, suggesting a unique, non-inflammatory mechanism. These data warrant further exploration of Advax and LNP as adjuvants in clinical trials for vaccines desiring to elicit both humoral and T cell responses.

Comparison of detection strategies for screening and confirming congenital cytomegalovirus infection in newborns in a highly seroprevalent population: a mother-child cohort study.

BACKGROUND: Universal screening of congenital cytomegalovirus (cCMV) infection is important for monitoring and intervention during critical stages of speech and language development. This study aimed to explore the optimal detection strategy for cCMV infection screening. METHODS: Serum samples from pregnant women and saliva and urine samples from their newborns were collected for the anti-CMV IgG and CMV DNA PCR tests, respectively. The sensitivity, specificity, and predictive values as well as the likelihood ratios of 12 potential screening strategies for cCMV infection, based on tests for saliva, urine, and their combination, were evaluated. FINDINGS: A total of 6729 pregnant women were enrolled, and the seroprevalence was 98.1%. Among 6350 newborns that were followed up, 49 were defined as having cCMV infection. In the screening test, the CMV DNA positivity rate remained similar from day 0 to day 5, increased slowly from day 6 to day 13, and became high in newborns beyond 13 days of birth. In the confirmatory testing, the positive rates increased significantly beyond day 21. For the 49 newborns with cCMV infection, the proportion of agreement between saliva and urine testing was poor. Upon evaluating alternative screening strategies, using saliva and urine screening with saliva and urine confirmation as the reference strategy, saliva screening with saliva and urine confirmation showed good diagnostic accuracy and feasibility, with sensitivity, specificity, positive predictive and negative predictive values of 85.7%, 100.0%, 100.0% and 99.9%, respectively. INTERPRETATION: In populations with high seroprevalence, saliva screening with saliva and urine confirmation might be an alternative strategy for screening cCMV infections. The suggested timeframes for screening and confirmation are within 13 (ideally 5) and 21 (ideally 13) days of birth, respectively. FUNDING: National Natural Science Foundation of China, National Science and Technology Major Project of China and Merck & Co., Inc., Kenilworth, New Jersey, U.S.A.

Protective antibodies elicited by SARS-CoV-2 spike protein vaccination are boosted in the lung after challenge in nonhuman primates.

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike protein trimers (preS dTM) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHPs). Binding and functional neutralization assays and systems serology revealed that the vaccinated NHP developed AS03-dependent multifunctional humoral responses that targeted distinct domains of the spike protein and bound to a variety of Fc receptors mediating immune cell effector functions in vitro. The neutralizing 50% inhibitory concentration titers for pseudovirus and live SARS-CoV-2 were higher than titers for a panel of human convalescent serum samples. NHPs were challenged intranasally and intratracheally with a high dose (3 × 106 plaque forming units) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days after challenge, vaccinated NHPs showed rapid control of viral replication in both the upper and lower airways. Vaccinated NHPs also had increased spike protein-specific immunoglobulin G (IgG) antibody responses in the lung as early as 2 days after challenge. Moreover, passive transfer of vaccine-induced IgG to hamsters mediated protection from subsequent SARS-CoV-2 challenge. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine were sufficient to mediate protection against SARS-CoV-2 in NHPs and that rapid anamnestic antibody responses in the lung may be a key mechanism for protection.

A conditionally replication-defective cytomegalovirus vaccine elicits potent and diverse functional monoclonal antibodies in a phase I clinical trial.

A conditionally replication-defective human cytomegalovirus (HCMV) vaccine, V160, was shown to be safe and immunogenic in a two-part, double-blind, randomized, placebo-controlled phase I clinical trial (NCT01986010). However, the specificities and functional properties of V160-elicited antibodies remain undefined. Here, we characterized 272 monoclonal antibodies (mAbs) isolated from single memory B cells of six V160-vaccinated subjects. The mAbs bind to diverse HCMV antigens, including multiple components of the pentamer, gB, and tegument proteins. The most-potent neutralizing antibodies target the pentamer-UL subunits. The binding sites of the antibodies overlap with those of antibodies responding to natural HCMV infection. The majority of the neutralizing antibodies target the gHgL subunit. The non-neutralizing antibodies predominantly target the gB and pp65 proteins. Sequence analysis indicated that V160 induced a class of gHgL antibodies expressing the HV1-18/KV1-5 germline genes in multiple subjects. This study provides valuable insights into primary targets for anti-HCMV antibodies induced by V160 vaccination.

Presence of complete murine viral genome sequences in patient-derived xenografts.

Patient-derived xenografts are crucial for drug development but their use is challenged by issues such as murine viral infection. We evaluate the scope of viral infection and its impact on patient-derived xenografts by taking an unbiased data-driven approach to analyze unmapped RNA-Seq reads from 184 experiments. We find and experimentally validate the extensive presence of murine viral sequence reads covering entire viral genomes in patient-derived xenografts. The existence of viral sequences inside tumor cells is further confirmed by single cell sequencing data. Extensive chimeric reads containing both viral and human sequences are also observed. Furthermore, we find significantly changed expression levels of many cancer-, immune-, and drug metabolism-related genes in samples with high virus load. Our analyses indicate a need to carefully evaluate the impact of viral infection on patient-derived xenografts for drug development. They also point to a need for attention to quality control of patient-derived xenograft experiments.

Immunogenicity and efficacy of mRNA COVID-19 vaccine MRT5500 in preclinical animal models.

Emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of coronavirus disease 2019 (COVID-19). However, there remains a need for additional effective vaccines to meet the global demand and address the potential new viral variants. mRNA technologies offer an expeditious path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on the spike (S) glycoprotein of SARS-CoV-2. Several mRNA constructs of S-protein, including wild type, a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), as well as others, were tested in animal models for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques, and for efficacy in a Syrian golden hamster model. The selected 2P/GSAS vaccine formulation, designated MRT5500, elicited potent nAbs as measured in neutralization assays in all three preclinical models and more importantly, protected against SARS-CoV-2-induced weight loss and lung pathology in hamsters. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate (NHP), thus alleviating a hypothetical concern of potential vaccine-associated enhanced respiratory diseases known associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for entering clinical development.

Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates.

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FC receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3×106 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.

Potent Bispecific Neutralizing Antibody Targeting Glycoprotein B and the gH/gL/pUL128/130/131 Complex of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause developmental disorders following congenital infection and life-threatening complications among transplant patients. Potent neutralizing monoclonal antibodies (MAbs) are promising drug candidates against HCMV infection. HCMV can infect a broad range of cell types. Therefore, single neutralizing antibodies targeting one HCMV glycoprotein often lack either potency or broad cell-type coverage. We previously characterized two human-derived HCMV neutralizing MAbs. One was the broadly neutralizing MAb 3-25, which targets the antigenic domain 2 of glycoprotein B (gB). The other was the highly potent MAb 2-18, which specifically recognizes the gH/gL/pUL128/130/131 complex (pentamer). To combine the strengths of gB- and pentamer-targeting MAbs, we developed an IgG-single-chain variable fragment (scFv) bispecific antibody by fusing the 2-18 scFv to the heavy-chain C terminus of MAb 3-25. The resulting bispecific antibody showed high-affinity binding to both gB and pentamer. Functionally, the bispecific antibody demonstrated a combined neutralization breadth and potency of the parental MAbs in multiple cell lines and inhibited postinfection viral spreading. Furthermore, the bispecific antibody was easily produced in CHO cells at a yield above 1 g/liter and showed a single-dose pharmacokinetic profile comparable to that of parental MAb 3-25 in rhesus macaques. Importantly, the bispecific antibody retained broadly and potent neutralizing activity after 21 days in circulation. Taken together, our research provides a proof-of-concept study for developing bispecific neutralizing antibody therapies against HCMV infection.

Functional Evaluation and Genetic Evolution of Human T-Cell Responses After Vaccination With a Conditionally Replication-Defective Cytomegalovirus Vaccine.

BACKGROUND: Cytomegalovirus (CMV) can cause congenital infection and is the leading cause of nongenetic newborn disabilities. V160, a conditionally replication-defective virus, is an investigational vaccine under evaluation for prevention of congenital CMV. The vaccine was well tolerated and induced both humoral and cellular immunity in CMV-seronegative trial participants. T-cell-mediated immunity is important for immune control of CMV. Here we describe efforts to understand the quality attributes of the T-cell responses induced by vaccination. METHODS: Using multicolor flow cytometry, we analyzed vaccine-induced T cells for memory phenotype, antigen specificity, cytokine profiles, and cytolytic potential. Moreover, antigen-specific T cells were sorted from 4 participants, and next-generation sequencing was used to trace clonal lineage development during the course of vaccination using T-cell receptor ß-chain sequences as identifiers. RESULTS: The results demonstrated that vaccination elicited polyfunctional CD4 and CD8 T cells to 2 dominant antigens, pp65 and IE1, with a predominantly effector phenotype. Analysis of T-cell receptor repertoires showed polyclonal expansion of pp65- and IE1-specific T cells after vaccination. CONCLUSION: V160 induced a genetically diverse and polyfunctional T-cell response and the data support further clinical development of V160 for prevention of CMV infection and congenital transmission. CLINICAL TRIALS REGISTRATION: NCT01986010.

Antibody binding to native cytomegalovirus glycoprotein B predicts efficacy of the gB/MF59 vaccine in humans.

Human cytomegalovirus (CMV) is the most common infectious cause of infant brain damage and posttransplant complications worldwide. Despite the high global burden of disease, vaccine development to prevent infection remains hampered by challenges in generating protective immunity. The most efficacious CMV vaccine candidate tested to date is a soluble glycoprotein B (gB) subunit vaccine with MF59 adjuvant (gB/MF59), which achieved 50% protection in multiple historical phase 2 clinical trials. The vaccine-elicited immune responses that conferred this protection have remained unclear. We investigated the humoral immune correlates of protection from CMV acquisition in populations of CMV-seronegative adolescent and postpartum women who received the gB/MF59 vaccine. We found that gB/MF59 immunization elicited distinct CMV-specific immunoglobulin G (IgG)-binding profiles and IgG-mediated functional responses in adolescent and postpartum vaccinees, with heterologous CMV strain neutralization observed primarily in adolescent vaccinees. Using penalized multiple logistic regression analysis, we determined that protection against primary CMV infection in both cohorts was associated with serum IgG binding to gB present on a cell surface but not binding to the soluble vaccine antigen, suggesting that IgG binding to cell-associated gB is an immune correlate of vaccine efficacy. Supporting this, we identified gB-specific monoclonal antibodies that differentially recognized soluble or cell-associated gB, revealing that there are structural differences in cell-associated and soluble gB are relevant to the generation of protective immunity. Our results highlight the importance of the native, cell-associated gB conformation in future CMV vaccine design.

Specificity and effector functions of non-neutralizing gB-specific monoclonal antibodies isolated from healthy individuals with human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common congenital infection. A glycoprotein B (gB) subunit vaccine (gB/MF59) is the most efficacious clinically tested to date, having achieved 50% protection against primary infection of HCMV-seronegative women. We previously identified that gB/MF59 vaccination primarily elicits non-neutralizing antibody responses, with variable binding to gB genotypes, and protection associated with binding to membrane-associated gB. We hypothesized that gB-specific non-neutralizing antibody binding breadth and function are dependent on epitope and genotype specificity, and ability to interact with membrane-associated gB. We mapped twenty-four gB-specific monoclonal antibodies (mAbs) from naturally HCMV-infected individuals for gB domain specificity, genotype preference, and ability to mediate phagocytosis or NK cell activation. gB-specific mAbs were primarily specific for Domain II and demonstrated variable binding to gB genotypes. Two mAbs facilitated phagocytosis with binding specificities of Domain II and AD2. This investigation provides novel understanding on the relationship between gB domain specificity and antigenic variability on gB-specific antibody effector functions.

Recognition of a highly conserved glycoprotein B epitope by a bivalent antibody neutralizing HCMV at a post-attachment step.

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.