Human Cytomegalovirus Glycoprotein Research Articles

Clinical Characteristics of Different Human Cytomegalovirus Glycoprotein N Genotypes among Child Patients in China

Clinical Characteristics of Different Human Cytomegalovirus Glycoprotein N Genotypes among Child Patients in China

Human cytomegalovirus glycoprotein variants governing viral tropism and syncytium formation in epithelial cells and macrophages.

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.

Multivalent cytomegalovirus glycoprotein B nucleoside modified mRNA vaccines did not demonstrate a greater antibody breadth

Human cytomegalovirus (HCMV) remains the most common congenital infection and infectious complication in immunocompromised patients. The most successful HCMV vaccine to date, an HCMV glycoprotein B (gB) subunit vaccine adjuvanted with MF59, achieved 50% efficacy against primary HCMV infection. A previous study demonstrated that gB/MF59 vaccinees were less frequently infected with HCMV gB genotype strains most similar to the vaccine strain than strains encoding genetically distinct gB genotypes, suggesting strain-specific immunity accounted for the limited efficacy. To determine whether vaccination with multiple HCMV gB genotypes could increase the breadth of anti-HCMV gB humoral and cellular responses, we immunized 18 female rabbits with monovalent (gB-1), bivalent (gB-1+gB-3), or pentavalent (gB-1+gB-2+gB-3+gB-4+gB-5) gB lipid nanoparticle-encapsulated nucleoside-modified RNA (mRNA–LNP) vaccines. The multivalent vaccine groups did not demonstrate a higher magnitude or breadth of the IgG response to the gB ectodomain or cell-associated gB compared to that of the monovalent vaccine. Also, the multivalent vaccines did not show an increase in the breadth of neutralization activity and antibody-dependent cellular phagocytosis against HCMV strains encoding distinct gB genotypes. Interestingly, peripheral blood mononuclear cell-derived gB-2-specific T-cell responses elicited by multivalent vaccines were of a higher magnitude compared to that of monovalent vaccinated animals against a vaccine-mismatched gB genotype at peak immunogenicity. Yet, no statistical differences were observed in T cell response against gB-3 and gB-5 variable regions among the three vaccine groups. Our data suggests that the inclusion of multivalent gB antigens is not an effective strategy to increase the breadth of anti-HCMV gB antibody and T cell responses. Understanding how to increase the HCMV vaccine protection breadth will be essential to improve the vaccine efficacy.

Human cytomegalovirus glycoprotein B genotypic distributions and viral load in symptomatic infants.

HCMV infection is widespread in humans. This retrospective study aimed to explore the relationship between human cytomegalovirus (HCMV) glycoprotein B (gB) genotype distribution, viral load, and the demographic and clinical features of symptomatic infants. The detection rate of HCMV in blood and urine samples was also compared. Retrospective data from 265 infants who underwent urine HCMV DNA testing were analyzed. The viral load and gB genotype were detected in 91 HCMV positive infants by quantitative fluorescence polymerase chain reaction (PCR) and DNA sequencing, respectively. The positive rate of HCMV infection was 46.04% (122/265) in all infants, and increased rapidly with age. Among the 91 infants investigated, liver function abnormality was the most common diagnosis (34/91, 37.36%), followed by pneumonia (21/91, 23.07%). Sequence analysis of gB yielded two genetic subtypes: the most prevalent gB3 (47/91, 51.65%), followed by gB1 (44/91, 48.35%). The gB3 HCMV infection was more prevalent in infants aged 0-2 months than in infants aged 3-12 months (χ2 = 4.38, p = 0.0364). The data showed that ALT and AST levels were significantly higher in the anti-HCMV IgM+IgG- group than in the anti-HCMV IgM+IgG+ and IgM-IgG+ groups. In addition, this study showed that the detection rate of HCMV DNA in the blood was significantly lower than that in the urine (χ2 = 6.7131, p = 0.0096). This study presents the HCMV infection status of infants and its relationship with their demographic characteristics and clinical manifestations. In addition, this study suggests that urinary PCR is the most appropriate assay for detecting HCMV infections.

Correlates of postnatal human cytomegalovirus transmission in term babies in the first year.

Postnatal human cytomegalovirus (HCMV) infection in newborns is well characterized for preterm infants but less so for term infants. We sought to analyze the rates and routes of HCMV transmission in full-term infants during the first year of life. A cohort of 120 HCMV seropositive mothers and their 122 newborns were tested after delivery for HCMV-DNA shedding in different bodily fluids. Postnatal HCMV infection was defined as the detection of >2.5 × 102 HCMV-DNA copies/mL in infants' saliva swabs. Maternal neutralizing antibody serum titer, HCMV-specific T-cell response, and HCMV glycoprotein B immunoglobulin G on breastmilk were analyzed. HCMV shedding was detected in 67 of 120 mothers (55.8%), and 20 of 122 infants (16.4%) developed HCMV infection within the first 3 months of life. Six additional infants were infected during the first year, for a postnatal infection rate of 21.3%. Viral shedding was more frequent in breastmilk than saliva, urine, and vaginal secretions, and the mothers of infected infants showed higher levels of HCMV-DNA in milk. No association was found between the antibody levels in serum or milk and maternal viral shedding, whereas a slightly lower frequency of HCMV-specific CD4+ T-cells with long-term memory phenotype was observed in women with HCM-DNA-positive milk. About one out of five infants develop HCMV infection within the first year of life. Breastmilk appears the major route of transmission of the infection, maternal saliva has a minor role whereas the role of vaginal secretions is negligible.

A fully human neutralizing monoclonal antibody targeting a highly conserved epitope of the human cytomegalovirus glycoprotein B.

Human cytomegalovirus causes severe diseases in children (by congenital infection) and immunocompromised patients. Treatment with antiviral agents, such as ganciclovir, is limited by their toxicity. In this study, we investigated the effectiveness of a fully human neutralizing monoclonal antibody to inhibit human cytomegalovirus infection and viral cell-to-cell spread. We isolated a potent neutralizing antibody, EV2038 (IgG1 lambda), targeting human cytomegalovirus glycoprotein B using Epstein-Barr virus transformation. This antibody inhibited human cytomegalovirus infection by all four laboratory strains and 42 Japanese clinical isolates, including ganciclovir-resistant isolates, with a 50% inhibitory concentration (IC50) ranging from 0.013 to 0.105 μg/mL, and 90% inhibitory concentration (IC90) ranging from 0.208 to 1.026 μg/mL, in both human embryonic lung fibroblasts (MRC-5) and human retinal pigment epithelial (ARPE-19) cells. Additionally, EV2038 prevented cell-to-cell spread of eight clinical viral isolates, with IC50 values ranging from 1.0 to 3.1 μg/mL, and IC90 values ranging from 13 to 19 μg/mL, in ARPE-19 cells. EV2038 recognized three discontinuous sequences on antigenic domain 1 of glycoprotein B (amino acids 549-560, 569-576, and 625-632), which were highly conserved among 71 clinical isolates from Japan and the United States. Pharmacokinetics study in cynomolgus monkeys suggested the potential efficacy of EV2038 in vivo, the concentration of which in serum remained higher than the IC90 values of cell-to-cell spread until 28 days after intravenous injection of 10 mg/kg EV2038. Our data strongly support EV2038 as a promising candidate and novel alternative for the treatment of human cytomegalovirus infection.

A Virus Genetic System to Analyze the Fusogenicity of Human Cytomegalovirus Glycoprotein B Variants.

Viruses can induce the fusion of infected and neighboring cells, leading to the formation of syncytia. Cell-cell fusion is mediated by viral fusion proteins on the plasma membrane of infected cells that interact with cellular receptors on neighboring cells. Viruses use this mechanism to spread rapidly to adjacent cells or escape host immunity. For some viruses, syncytium formation is a hallmark of infection and a known pathogenicity factor. For others, the role of syncytium formation in viral dissemination and pathogenicity remains poorly understood. Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. Clinical HCMV isolates have broad cell tropism but differ in their ability to induce cell-cell fusions, and little is known about the molecular determinants. We developed a system to analyze HCMV glycoprotein B (gB) variants in a defined genetic background. HCMV strains TB40/E and TR were used as vectors to compare the fusogenicity of six gB variants from congenitally infected fetuses with those from three laboratory strains. Five of them conferred the ability to induce the fusion of MRC-5 human embryonic lung fibroblasts to one or both backbone strains, as determined by a split GFP-luciferase reporter system. The same gB variants were not sufficient to induce syncytia in infected ARPE-19 epithelial cells, suggesting that additional factors are involved. The system described here allows a systematic comparison of the fusogenicity of viral envelope glycoproteins and may help to clarify whether fusion-promoting variants are associated with increased pathogenicity.

Cytotoxic CD4+ T cells eliminate senescent cells by targeting cytomegalovirus antigen

Senescent cell accumulation has been implicated in the pathogenesis of aging-associated diseases, including cancer. The mechanism that prevents the accumulation of senescent cells in aging human organs is unclear. Here, we demonstrate that a virus-immune axis controls the senescent fibroblast accumulation in the human skin. Senescent fibroblasts increased in old skin compared with young skin. However, they did not increase with advancing age in the elderly. Increased CXCL9 and cytotoxic CD4+ Tcells (CD4 CTLs) recruitment were significantly associated with reduced senescent fibroblasts in the old skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), becoming direct CD4 CTL targets. Skin-resident CD4 CTLs eliminated HCMV-gB+ senescent fibroblasts in an HLA-II-dependent manner, and HCMV-gB activated CD4 CTLs from the human skin. Collectively, our findings demonstrate HCMV reactivation in senescent cells, which CD4 CTLs can directly eliminate through the recognition of the HCMV-gB antigen.

Characterization of Plasma Immunoglobulin G Responses in Elite Neutralizers of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is the most common infectious complication of organ transplantation and cause of birth defects worldwide. There are limited therapeutic options and no licensed vaccine to prevent HCMV infection or disease. To inform development of HCMV antibody-based interventions, a previous study identified individuals with potent and broad plasma HCMV-neutralizing activity, termed elite neutralizers (ENs), from a cohort of HCMV-seropositive (SP) blood donors. However, the specificities and functions of plasma antibodies associated with EN status remained undefined. We sought to determine the plasma antibody specificities, breadth, and Fc-mediated antibody effector functions associated with the most potent HCMV-neutralizing responses in plasma from ENs (n = 25) relative to that from SP donors (n = 19). We measured antibody binding against various HCMV strains and glycoprotein targets and evaluated Fc-mediated effector functions, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). We demonstrate that ENs have elevated immunoglobulin G binding responses against multiple viral glycoproteins, relative to SP donors. Our study also revealed potent HCMV-specific antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis activity of plasma from ENs. We conclude that antibody responses against multiple glycoprotein specificities may be needed to achieve potent plasma neutralization and that potently HCMV elite-neutralizing plasma antibodies can also mediate polyfunctional responses.

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.

Sequencing of the Viral UL111a Gene Directly from Clinical Specimens Reveals Variants of HCMV-Encoded IL-10 That Are Associated with Altered Immune Responses to HCMV.

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ~80% of adults worldwide. Acute infections are often asymptomatic in healthy individuals but generate diverse syndromes in neonates, renal transplant recipients (RTR), and people with HIV (PWH). The HCMV gene UL111a encodes a homolog of human interleukin-10 (IL-10) that interacts with the human IL-10 receptor. Deep sequencing technologies were used to sequence UL111a directly from 59 clinical samples from Indonesian PWH and Australian RTR, healthy adults, and neonates. Overall, 93% of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous variation. Carriage of these variants differed between neonates and adults, Australians and Indonesians, and between saliva and blood leukocytes. The variant alleles of N41D and S71Y occurred together in Australian RTR and were associated with higher T-cell responses to HCMV pp65. The variant P122S was associated with lower levels of antibodies reactive with a lysate of HCMV-infected fibroblasts. L174F was associated with increased levels of antibodies reactive with HCMV lysate, immediate-early 1 (IE-1), and glycoprotein B (gB) in Australian RTR and Indonesians PWH, suggesting a higher viral burden. We conclude that variants of UL111a are common in all populations and may influence systemic responses to HCMV.

Heterosubtypic, cross-reactive immunity to human Cytomegalovirus glycoprotein B.

Cytomegalovirus (CMV) genome is highly variable and heterosubtypic immunity should be considered in vaccine development since it can enhance protection in a cross-reactive manner. Here, we developed a protein array to evaluate heterosubtypic immunity to CMV glycoprotein B (gB) in natural infection and vaccination. DNA sequences of four antigenic domains (AD1, AD2, AD4/5, and AD5) of gB were amplified from six reference and 12 clinical CMV strains, and the most divergent genotypes were determined by phylogenetic analysis. Assigned genotypes were in vitro translated and immobilized on protein array. Then, we tested immune response of variable serum groups (primarily infected patients, reactivated CMV infections and healthy individuals with latent CMV infection, as well gB-vaccinated rabbits) with protein in situ array (PISA). Serum antibodies of all patient cohorts and gB-vaccinated rabbits recognized many genetic variants of ADs on protein array, including but not limited to the subtype of infecting strain. High-grade cross-reactivity was observed. In several patients, we observed none or neglectable immune response to AD1 and AD2, while the same patients showed high antibody response to AD4/5 and AD5. Among the primary infected patients, AD5 was the predominant AD, in antibody response. The most successful CMV vaccine to date contains gB and demonstrates only 50% efficacy. In this study, we showed that heterosubtypic and cross-reactive immunity to CMV gB is extensive. Therefore, the failure of CMV gB vaccines cannot be explained by a highly, strain-specific immunity. Our observations suggest that other CMV antigens should be addressed in vaccine design.

Genotyping of Human Cytomegalovirus Glycoprotein N in Iraqi Breast cancer Patients

Human Cytomegalovirus (HCMV) is an enveloped ubiquitous ds-DNA virus that has been implicated in several types of malignancies. The current work was conducted in the period extending from (November 2018 to the end of October 2019) and aimed to assess the frequency of glycoprotein N (gN) genotypes of HCMV. A total number of 91serum and plasma specimens were collected to fulfill this purpose from females (71 breast cancer patients, and a control group of 20 females) attending Al-Amal hospital for cancer management and Baghdad teaching hospital.The molecular part of this data was achieved through both PCR and Multiplex PCR for detection of HCMV gN (UL73) entire gene as well as for genotyping. gN was detected in 36/71 (50.7%) of breast cancer patients, while gN was detected in 7/20 (35%) of the control group. Furthermore, HCMV gN was successfully genotyped by multiplex PCR in 32/36 (88.88%) of breast cancer patients that were positive for gN and the distribution of each genotype was 46.87% (15/32) for gN-4a, 34.37% (11/32) for gN-1, 12.50% (4/32) for gN-2, 6.25% and 0.0% for gN-4b/c and gN-3b respectively.

Human Cytomegalovirus Seropositivity and Viral DNA in Breast Tumors Are Associated with Poor Patient Prognosis

Simple SummaryHuman cytomegalovirus (HCMV) infects 40–70% of adult populations in developed countries and this is thought to be involved in breast cancer progression; however, reports of detection of the viral genome in breast tumors ranges from 0–100%. We optimized a method that is both sensitive and specific to detect HCMV DNA in tissues from Canadian breast cancer patients. Only ~42% of HCMV-seropositive patients expressed viral DNA in their breast tumors. Viral transcription was not detected in any HCMV-infected breast tumors, indicating a latent infection; however, HCMV seropositivity and the presence of latent infections in breast tumors were independently, and in combination, associated with increased metastasis. HCMV DNA-positive tumors were also associated with lower relapse-free survival. Therefore, HCMV infection status should be accounted for during the monitoring and treatment of breast cancer patients. Prevention or reducing the effects of HCMV infection could decrease morbidity and mortality from metastatic disease.Human cytomegalovirus (HCMV) infects 40–70% of adults in developed countries. Detection of HCMV DNA and/or proteins in breast tumors varies considerably, ranging from 0–100%. In this study, nested PCR to detect HCMV glycoprotein B (gB) DNA in breast tumors was shown to be sensitive and specific in contrast to the detection of DNA for immediate early genes. HCMV gB DNA was detected in 18.4% of 136 breast tumors while 62.8% of 94 breast cancer patients were seropositive for HCMV. mRNA for the HCMV immediate early gene was not detected in any sample, suggesting viral latency in breast tumors. HCMV seropositivity was positively correlated with age, body mass index and menopause. Patients who were HCMV seropositive or had HCMV DNA in their tumors were 5.61 (CI 1.77–15.67, p = 0.003) or 5.27 (CI 1.09–28.75, p = 0.039) times more likely to develop Stage IV metastatic tumors, respectively. Patients with HCMV DNA in tumors experienced reduced relapse-free survival (p = 0.042). Being both seropositive with HCMV DNA-positive tumors was associated with vascular involvement and metastasis. We conclude that determining the seropositivity for HCMV and detection of HCMV gB DNA in the breast tumors could identify breast cancer patients more likely to develop metastatic cancer and warrant special treatment.

Distribution of Human Cytomegalovirus Glycoprotein H Genotypes in Different Samples from Chinese Children

Abstract Objective This study aimed to investigate characteristics of human cytomegalovirus (HCMV) glycoprotein H (gH) genotypes in urine, throat swab, and serum from children and breast milk from children's mothers. Methods Fresh urine samples, throat swabs, or serum samples from children and breast milk samples from children's mothers were collected for HCMV DNA detection. The positive samples of HCMV DNA were further detected by fluorescent quantitative polymerase chain reaction (PCR) with gH typing. Results Of 1,703 HCMV DNA-positive samples, the highest proportion (83.3%, 85/102) of children aged between 21 days and 3 months was detected positive in breast milk samples (p = 0.002), and the highest proportion (70.5%, 110/156) of children aged above 3 months was detected positive in throat swab samples (p = 0.002). HCMV in throat swab specimens is mainly high copy (p < 0.0001), and low-copy HCMV is prevalent in breast specimens (p < 0.0001). Among them, 1,059 samples were identified as gH1 genotype, 530 samples were gH2, and 114 samples were coinfection (gH1/2). There had the highest gH2 rates (32.3%) and lowest gH1 (61.0%) rates in urine samples (p = 0.041), whereas the highest gH1 rates (71.6%) and lowest gH2 rates (19.6%) were found in breast milk samples (p = 0.032). Concerning age groups, patients aged between 21 days and 3 months had the highest gH1 proportion (p = 0.017), while patients aged above 3 months had the highest gH1 and gH2 HCMV coinfection proportion (p = 0.002). Among 43 pairs of maternal and child samples corresponding to positive samples, gH genotype of 35 pairs of samples was consistent with a rate of 81.4%. Conclusion gH1 is the predominant genotype of HCMV in each kind of sample in China. However, the distribution of the HCMV gH genotype is different among different samples.

Pre-existing immunity to cytomegalovirus in macaques influences human CMV vaccine responses in preclinical models

Development of a human cytomegalovirus (HCMV) vaccine is a Tier 1 priority by the National Institutes of Medicine, as HCMV is the most common congenital infection globally and most frequent infectious complication in transplant patients. Relevant preclinical non-human primate models used for testing HCMV vaccine immunogenicity are rhesus and cynomolgous monkeys. However, a complication in using these models is that species-specific CMV variants are endemic in non-human primate breeding colonies. We hypothesize that natural immunity to species-specific CMV in rhesus and cynomolgous monkeys impacts HCMV vaccine immunogenicity and may interfere with our ability to fully interpret vaccine immunogenicity. A modified mRNA vaccine encoding HCMV glycoprotein (gB) and the pentameric complex (PC) packaged in lipid nanoparticles (LNP) was delivered intramuscularly to groups of cynomolgous (n = 16, CyCMV-seropositive) and rhesus macaques (n = 24, RhCMV-seropositive). High pre-vaccination IgG binding responses to HCMV gB were present in both species, but pre-vaccination binding responses to PC were mostly present in rhesus macaques. Yet, at least a log increase in both PC and gB-specific plasma IgG levels was detected post-second HCMV mRNA vaccination in both species. Both species responded with high epithelial cell neutralizing antibody responses at 4 weeks post second HCMV mRNA vaccination, but limited fibroblast neutralizing antibodies. HCMV gB + PC mRNA/LNP vaccine also elicited IgG binding responses to cell-associated gB, an identified immune correlate of protection, in both species after the second vaccination, and there was a moderately strong direct correlation between this pre- and post-vaccination response in rhesus macaques. Based on the correlation between pre-existing and post-vaccine gB-specific binding responses in rhesus macaques, we conclude that species-specific CMV variant-specific antibody responses contribute to antibody responses to HCMV vaccination in primate models, indicating that pre-existing immunity must be taken into account in non-human primate preclinical models and will impact immunogenicity of HCMV vaccines seropositive human vaccinees.

Mutagenesis of Human Cytomegalovirus Glycoprotein L Disproportionately Disrupts gH/gL/gO over gH/gL/pUL128-131.

Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but how the gH/gL portions of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB-dependent cell-cell fusion but were still able to form gH/gL/pUL128-131 and induce receptor interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR, and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. The effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRα, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a "hyperactive" gH/gL/gO. Recently published crystallography and cryo-electron microscopy studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB. IMPORTANCE The endemic betaherpesvirus HCMV circulates in human populations as a complex mixture of genetically distinct variants, establishes lifelong persistent infections, and causes significant disease in neonates and immunocompromised adults. This study capitalizes on our recent characterizations of three genetically distinct HCMV BAC clones to discern the functions of the envelope glycoprotein complexes gH/gL/gO and gH/gL/pUL128-13, which are promising vaccine targets that share the herpesvirus core fusion apparatus component, gH/gL. Mutations in the shared gL subunit disproportionally affected gH/gL/gO, demonstrating mechanistic differences between the two complexes, and may provide a basis for more refined evaluations of neutralizing antibodies.

A slowly cleaved viral signal peptide acts as a protein-integral immune evasion domain

Stress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains.

Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion.

Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to fuse the viral envelope with a host cell membrane during entry. We stabilized prefusion gB on the virion with a fusion inhibitor and a chemical cross-linker, extracted and purified it, and then determined its structure to 3.6-Å resolution by electron cryomicroscopy. Our results revealed the structural rearrangements that mediate membrane fusion and details of the interactions among the fusion loops, the membrane-proximal region, transmembrane domain, and bound fusion inhibitor that stabilized gB in the prefusion state. The structure rationalizes known gB antigenic sites. By analogy to successful vaccine antigen engineering approaches for other viral pathogens, the high-resolution prefusion gB structure provides a basis to develop stabilized prefusion gB HCMV vaccine antigens.

Detection of Genotype-Specific Antibody Responses to Glycoproteins B and H in Primary and Non-Primary Human Cytomegalovirus Infections by Peptide-Based ELISA.

Background: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. Methods: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). Results: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. Conclusions: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB.