Cytomegalovirus results in poor graft function via bone marrow-derived endothelial progenitor cells.

Introduction: Poor graft function (PGF), characterized by myelosuppression, represents a significant challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with human cytomegalovirus (HCMV) being established as a risk factor for PGF. However, the underlying mechanism remains unclear. Bone marrow endothelial progenitor cells (BM-EPCs) play an important role in supporting hematopoiesis and their dysfunction contributes to PGF development. We aim to explore the effects of CMV on BM-EPCs and its underlying mechanism. Methods: We investigated the compromised functionality of EPCs derived from individuals diagnosed with HCMV viremia accompanied by PGF, as well as after infected by HCMV AD 169 strain in vitro, characterized by decreased cell proliferation, tube formation, migration and hematopoietic support, and increased apoptosis and secretion of TGF-ß1. Results: We demonstrated that HCMV-induced TGF-ß1 secretion by BM-EPCs played a dominant role in hematopoiesis suppression in vitro experiment. Moreover, HCMV down-regulates Vitamin D receptor (VDR) and subsequently activates p38 MAPK pathway to promote TGF-ß1 secretion by BM-EPCs. Discussion: HCMV could infect BM-EPCs and lead to their dysfunction. The secretion of TGF-ß1 by BM-EPCs is enhanced by CMV through the activation of p38 MAPK via a VDR-dependent mechanism, ultimately leading to compromised support for hematopoietic progenitors by BM EPCs, which May significantly contribute to the pathogenesis of PGF following allo-HSCT and provide innovative therapeutic strategies targeting PGF.

Growth defect of domain III glycoprotein B mutants of human cytomegalovirus reverted by compensatory mutations co-localizing in post-fusion conformation.

Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell-cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion.IMPORTANCEHuman cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.

Human cytomegalovirus infection among febrile hematological cancer patients at the Uganda Cancer Institute.

Hematological cancers, including Leukemias and Lymphomas, and their associated chemotherapy and disease-specific factors, are linked to impaired granulocyte function and numbers, increasing the risk of opportunistic infections, often presenting as fever. Human cytomegalovirus (HCMV) is one of the significant opportunistic infections in these patients, but limited data exists on its seroprevalence and active infection burden among febrile hematological cancer patients in Uganda. We conducted a cross-sectional study from June to August 2017 at the Uganda Cancer Institute (UCI). Blood samples from 161 febrile hematological cancer patients were collected. HCMV exposure was assessed using indirect enzyme-linked immunosorbent assay for IgG and IgM antibodies, and active infection was confirmed with PCR testing and gel electrophoresis. IgG positivity indicated previous exposure, while positive IgM or PCR results indicated active infection. Overall, HCMV seroprevalence based on IgG and/or IgM positivity was 106/161 (66%). IgG alone, IgM alone, and combined IgG/IgM positivity prevalence rates were 57/161 (35.4%), 22/161 (13.6%), and 27/161 (16.7%), respectively. HCMV DNA PCR was positive in 5 of the 161 (3%) samples. Among PCR-positive patients, one (20%) was positive for IgG alone, two (40%) for IgM alone, and two (40%) for both IgG and IgM. Active infection based on positive IgM and HCMV DNA PCR was found in 23 of the 161 (14.3%) patients. Two-thirds of febrile patients with hematological malignancies in Uganda had been exposed to HCMV infection, with 14.3% showing active infection. Routine testing for active HCMV infection among febrile hematological cancer patients at the UCI is essential for timely and appropriate antiviral treatment. IMPORTANCE: In this paper, we demonstrated that over two-thirds of feverish patients with blood cancers such as leukemia at the Uganda Cancer Institute are already exposed to a type of virus infection called the human cytomegalovirus (HCMV), and 14% of the patients have active disease due to this virus. This was confirmed through finding blood samples testing positive for a type of protective antibody called IgM and also upon virus DNA detection in the blood of those patients. Routine testing for this virus is not usually done in the study settings. Our findings reveal and emphasize the importance of routinely testing blood samples for active infection with this virus among the feverish patients with blood cancers in the study settings, and prompt initiation of antiviral treatment of the actively infected patients.

The Autophagy Receptor SQSTM1/p62 Is a Restriction Factor of HCMV Infection.

(1) Background: Intrinsic defense mechanisms are pivotal host strategies to restrict viruses already at early stages of their infection. Here, we addressed the question of how the autophagy receptor sequestome 1 (SQSTM1/p62, hereafter referred to as p62) interferes with human cytomegalovirus (HCMV) infection. (2) Methods: CRISPR/Cas9-mediated genome editing, mass spectrometry and the expression of p62 phosphovariants from recombinant HCMVs were used to address the role of p62 during infection. (3) Results: The knockout of p62 resulted in an increased release of HCMV progeny. Mass spectrometry revealed an interaction of p62 with cellular proteins required for nucleocytoplasmic transport. Phosphoproteomics further revealed that p62 is hyperphosphorylated at position S272 in HCMV-infected cells. Phosphorylated p62 showed enhanced nuclear retention, which is concordant with enhanced interaction with viral proteins relevant for genome replication and nuclear capsid egress. This modification led to reduced HCMV progeny release compared to a non-phosphorylated version of p62. (4) Conclusions: p62 is a restriction factor for HCMV replication. The activity of the receptor appears to be regulated by phosphorylation at position S272, leading to enhanced nuclear localization, viral protein degradation and impaired progeny production.

The Autonomous Fusion Activity of Human Cytomegalovirus Glycoprotein B Is Regulated by Its Carboxy-Terminal Domain.

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.

Different Antigen-Specific CD4+ and CD8+ T-Cell Response against HCMV Proteins in Pregnant Women with Primary Infection and in Control Subjects with Remote Infection.

Background/Objectives: Human cytomegalovirus (HCMV) is the most frequent cause of congenital infections. The HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection in pregnancy. In this study, the antigen-specific T-cell response against different HCMV proteins (IE-1, pp65, gB, gHgLpUL128L) was investigated in pregnant women with primary infection and in control subjects with remote infection to identify possible components of a vaccine. Methods: Blood samples from 35 pregnant women with HCMV primary infection and 30 HCMV-seropositive healthy adult subjects with remote infection were tested. The antigen-specific T-cell response was measured using cytokine intracellular staining after stimulation with IE-1, pp65, gB and gHgLpUL128L peptides pool. Results: The pp65-specific CD4+ T-cell response was higher in pregnant women with HCMV primary infection at the late time point and in control subjects with remote infection, while the pregnant women at the early time point showed a higher gB-specific CD8+ T-cell response. Regarding the CD4+ and CD8+ T-cell phenotypes, we observed that HCMV-specific CD4+ and CD8+ T cells expressing CD45RA+ remained constant in pregnant women with primary infection at the early and late time points and in subjects with remote infection, while HCMV-specific CD4+ and CD8+ T cells expressing IL-7R+ or producing IL-2 were higher in control subjects with remote infection than in pregnant women with HCMV primary infection. Conclusions: The T-cell response was higher against gB in the early phase of infection and against pp65 in the late phase. Therefore, these proteins should be taken into consideration as candidates for a vaccine.

Cyclin-Dependent Kinase 8 Represents a Positive Regulator of Cytomegalovirus Replication and a Novel Host Target for Antiviral Strategies.

Background. Cyclin-dependent kinase 8 (CDK8) is a multifaceted regulator and represents a catalytic component of the transcriptional Mediator complex. CDK8 activity, on the one hand, increases transcriptional elongation by the recruitment of Mediator/super elongation complexes, but, on the other hand, negatively regulates CDK7-controlled transcriptional initiation through inactivating cyclin H phosphorylation. Recently, these combined properties of CDK8 have also suggested its rate-limiting importance for herpesviral replication. Objectives. In this paper, we focused on human cytomegalovirus (HCMV) and addressed the question of whether the pharmacological inhibition or knock-down of CDK8 may affect viral replication efficiency in cell culture models. Methods. A number of human and animal herpesviruses, as well as non-herpesviruses, were used to analyze the importance of CDK8 for viral replication in cell culture models, and to assess the antiviral efficacy of CDK8 inhibitors. Results. Using clinically relevant CDK8 inhibitors (CCT-251921, MSC-2530818, and BI-1347), HCMV replication was found strongly reduced even at nanomolar drug concentrations. The EC50 values were consistent for three different HCMV strains (i.e., AD169, TB40, and Merlin) analyzed in two human cell types (i.e., primary fibroblasts and astrocytoma cells), and the drugs comprised a low level of cytotoxicity. The findings highlighted the following: (i) the pronounced in vitro SI values of anti-HCMV activity obtained with CDK8 inhibitors; (ii) a confirmation of the anti-HCMV efficacy by CDK8-siRNA knock-down; (iii) a CDK8-dependent reduction in viral immediate early, early, and late protein levels; (iv) a main importance of CDK8 for viral late-stage replication; (v) several mechanistic aspects, which point to a strong impact on viral progeny production and release, but a lack of CDK8 relevance for viral entry or nuclear egress; (vi) a significant anti-HCMV drug synergy for combinations of inhibitors against host CDK8 and the viral kinase vCDK/pUL97 (maribavir); (vii) finally, a broad-spectrum antiviral activity, as seen for the comparison of selected α-, ß-, γ-, and non-herpesviruses. Conclusions. In summary, these novel data provide evidence for the importance of CDK8 as a positive regulator of herpesviral replication efficiency, and moreover, suggest its exploitability as an antiviral target for novel strategies of host-directed drug development.

Immune Control of Human Cytomegalovirus (HCMV) Infection in HCMV-Seropositive Solid Organ Transplant Recipients: The Predictive Role of Different Immunological Assays.

Human cytomegalovirus (HCMV) infection remains a major complication for solid organ transplant recipients (SOTRs). The aim of this study was to evaluate the role of HCMV-specific T cell immunity measured at the time of the HCMV-DNA peak in predicting the spontaneous clearance of infection. The performance of cytokine flow cytometry using infected dendritic cells (CFC-iDC), infected cell lysate (CFC-iCL) and pp65 peptide pool (CFC-pp65 pool) as stimuli, as well as ELISPOT assays using infected cell lysate (ELISPOT-iCL) and the pp65 peptide pool (ELISPOT-pp65 pool), was analysed. Among the 40 SOTRs enrolled, 16 patients (40%) required antiviral treatment for an HCMV infection (Non-Controllers), while the others spontaneously cleared the infection (Controllers). At the HCMV-DNA peak, the number of HCMV-specific CD4+ T cells detected by the CFC-iDC, CFC-iCL and CFC-pp65 pool assays in Controllers was higher than that detected in Non-Controllers, while no difference was observed in terms of HCMV-specific CD8+ T cell response. The same trend was observed when the HCMV-specific T cell response was measured by ELISPOT-iCL and ELISPOT-pp65 pool. We observed that the CD4+ CFC-pp65 pool assay was the best predictor of self-resolving HCMV infection at the time of the HCVM-DNA peak. The CFC-pp65 pool assay is able to discriminate between CD4+ and CD8+ T cell responses and could be used in daily clinical practice.

Understanding the Cytomegalovirus Cyclin-Dependent Kinase Ortholog pUL97 as a Multifaceted Regulator and an Antiviral Drug Target.

Herpesviral protein kinases, such as the therapy-relevant pUL97 of human cytomegalovirus (HCMV), are important for viral replication efficiency as well as pathogenesis, and represent key antiviral drug targets. HCMV pUL97 is a viral cyclin-dependent kinase (CDK) ortholog, as it shares functional and structural properties with human CDKs. Recently, the formation of vCDK/pUL97-cyclin complexes and the phosphorylation of a variety of viral and cellular substrate proteins has been demonstrated. Genetic mapping and structural modeling approaches helped to define two pUL97 interfaces, IF1 and IF2, responsible for cyclin binding. In particular, the regulatory importance of interactions between vCDK/pUL97 and host cyclins as well as CDKs has been highlighted, both as determinants of virus replication and as a novel drug-targeting option. This aspect was substantiated by the finding that virus replication was impaired upon cyclin type H knock-down, and that such host-directed interference also affected viruses resistant to existing therapies. Beyond the formation of binary interactive complexes, a ternary pUL97-cyclin H-CDK7 complex has also been described, and in light of this, an experimental trans-stimulation of CDK7 activity by pUL97 appeared crucial for virus-host coregulation. In accordance with this understanding, several novel antiviral targeting options have emerged. These include kinase inhibitors directed to pUL97, to host CDKs, and to the pUL97-cyclin H interactive complexes. Importantly, a statistically significant drug synergy has recently been reported for antiviral treatment schemes using combinations of pharmacologically relevant CDK7 and vCDK/pUL97 inhibitors, including maribavir. Combined, such findings provide increased options for anti-HCMV control. This review focuses on regulatory interactions of vCDK/pUL97 with the host cyclin-CDK apparatus, and it addresses the functional relevance of these key effector complexes for viral replication and pathogenesis. On this basis, novel strategies of antiviral drug targeting are defined.

Single-cell RNA-sequencing reveals a profound immune cell response in human cytomegalovirus-infected humanized mice.

Human cytomegalovirus (HCMV) is a common herpesvirus that persistently infects a large portion of the world's population. Despite the robust host immune response, HCMV is able to replicate, evade host defenses, and establish latency throughout the lifespan by developing multiple immunomodulatory strategies, making the studies on the interaction between HCMV infection and host response particularly important. HCMV has a strict host specificity that specifically infects humans. Therefore, most of the in vivo researches of HCMV rely on clinical samples. Fortunately, the establishment of humanized mouse models allows for convenient in-lab animal experiments involving HCMV infection. Single-cell RNA sequencing enables the study of the relationship between viral and host gene expressions at the single-cell level within host cells. In this study, we assessed the gene expression alterations of PBMCs at the single-cell level within HCMV-infected humanized mice, which sheds light onto the virus-host interactions in the context of HCMV infection of humanized mice and provides a valuable dataset for the related researches.

Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy.

Human cytomegalovirus (HCMV) represents a highly medically important pathogen which has constantly been the subject of both molecular and clinical investigations. HCMV infections, especially those in high-risk patients, still raise many unanswered questions, so current investigations are focused on viral pathogenesis, vaccine development, and options for antiviral drug targeting. To this end, the use of suitable viral strains as well as recombinant reporter constructs in cultured cells and model systems has specific significance. We previously reported on the application of various herpesviruses that express green, red, or related fluorescent proteins, especially in the fields of virus-host interaction and antiviral research. Here, we characterized a recombinant version of the clinically relevant and cell type-adaptable HCMV strain TB40, which expresses firefly luciferase as a quantitative reporter of viral replication (TB40-FLuc). The data provide evidence for five main conclusions. First, HCMV TB40-FLuc is employable in multiple settings in primary human cells. Second, viral reporter signals are easily quantifiable, even at early time points within viral replication. Third, the FLuc reporter reflects the kinetics of viral intracellular replication, cascade-like viral IE-E-L protein production, and progeny release. Fourth, as relates to specific applications of the TB40-FLuc system, we demonstrated the reliability of quantitative antiviral compound determination in multi-well formats and its independence from fluorescence-based measurements in the case of autofluorescent inhibitors. Finally, we illustrated increased reporter sensitivity in comparison to other recombinant HCMVs. In essence, recombinant HCMV TB40-FLuc combines several molecular properties that are considered beneficial in studies on viral host tropism, replication efficiency, and antiviral drug assessment.

Polyploid Giant Cancer Cells: A Distinctive Feature in the Transformation of Epithelial Cells by High-Risk Oncogenic HCMV Strains.

Human cytomegalovirus (HCMV) infection is common in tumor tissues across different types of cancer. While HCMV has not been recognized as a cancer-causing virus, numerous studies hint at its potential role in cancer development where its presence in various cancers corresponds with the hallmarks of cancer. Herein, we discuss and demonstrate that high-risk HCMV-DB and BL strains have the potential to trigger transformation in epithelial cells, including human mammary epithelial cells (HMECs), ovarian epithelial cells (OECs), and prostate epithelial cells (PECs), through the generation of polyploid giant cancer cells (PGCCs). A discussion is provided on how HCMV infection creates a cellular environment that promotes oncogenesis, supporting the continuous growth of CMV-transformed cells. The aforementioned transformed cells, named CTH, CTO, and CTP cells, underwent giant cell cycling with PGCC generation parallel to dedifferentiation, displaying stem-like characteristics and an epithelial-mesenchymal transition (EMT) phenotype. Furthermore, we propose that giant cell cycling through PGCCs, increased EZH2 expression, EMT, and the acquisition of malignant traits represent a deleterious response to the cellular stress induced by high-risk oncogenic HCMV strains, the latter being the origin of the transformation process in epithelial cells upon HCMV infection and leading to adenocarcinoma of poor prognosis.

The effect of early life cytomegalovirus infection on the immune profile of children.

Cytomegalovirus (CMV) infection has a life-long impact on the immune system, particularly on memory T cells. However, the effect of early life CMV infection on the phenotype and functionality of T cells in infants and especially longitudinal changes occurring during childhood have not been explored in detail. The phenotype and functionality of peripheral blood CD8+ and CD4+ T cells from children infected with CMV in early life (< 6 months of age) was analyzed using high-dimensional flow cytometry. Samples from CMV IgG-seropositive (CMV+) children were collected at 6 months and 6 years of age and compared to samples from CMV-seronegative (CMV-) children. Early life CMV infection caused multiple alterations within T cells. These include downregulation of CD28 expression and upregulation of CD57 expression within both CD27+ early and CD27- late effector memory CD8+ and CD4+ T-cells at 6 months of age. Of these changes, only alterations within the highly differentiated late effector memory compartment persisted at the age of 6 years. Early life CMV-infection has a distinct impact on developing CD8+ and CD4+ memory T cell compartments. It appears to induce both temporary as well as longer-lasting alterations, which may affect the functionality of the immune system throughout life.

Designing multi-epitope vaccine against human cytomegalovirus integrating pan-genome and reverse vaccinology pipelines.

Human cytomegalovirus (HCMV) is accountable for high morbidity in neonates and immunosuppressed individuals. Due to the high genetic variability of HCMV, current prophylactic measures are insufficient. In this study, we employed a pan-genome and reverse vaccinology approach to screen the target for efficient vaccine candidates. Four proteins, envelope glycoprotein M, UL41A, US23, and US28, were shortlisted based on cellular localization, high solubility, antigenicity, and immunogenicity. A total of 29 B-cell and 44 T-cell highly immunogenic and antigenic epitopes with high global population coverage were finalized using immunoinformatics tools and algorithms. Further, the epitopes that were overlapping among the finalized B-cell and T-cell epitopes were linked with suitable linkers to form various combinations of multi-epitopic vaccine constructs. Among 16 vaccine constructs, Vc12 was selected based on physicochemical and structural properties. The docking and molecular simulations of VC12 were performed, which showed its high binding affinity (-23.35 kcal/mol) towards TLR4 due to intermolecular hydrogen bonds, salt bridges, and hydrophobic interactions, and there were only minimal fluctuations. Furthermore, Vc12 eliciting a good response was checked for its expression in Escherichia coli through in silico cloning and codon optimization, suggesting it to be a potent vaccine candidate.

Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis.

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Evaluation of the In Vitro Capacity of Anti-Human Cytomegalovirus Antibodies to Initiate Antibody-Dependent Cell Cytotoxicity.

In the setting of infectious diseases, antibodies show different functions beyond neutralizing activity. In this study, we investigated the activation of NK cells in vitro in the presence of human cytomegalovirus (HCMV)-specific antibodies and their potential role in the control of HCMV infection through antibody-dependent cell cytotoxicity (ADCC). Retinal pigmented epithelial cells (ARPE-19) infected with the HCMV strain VR1814 were co-cultured with cytokine-activated peripheral blood mononuclear cells (PBMCs) in the presence of sera collected from 23 HCMV-seropositive and 9 HCMV-seronegative donors. Moreover, 13 pregnant women sampled 3 and 6 months after HCMV primary infection and 13 pregnant women with pre-conception immunity were tested and compared. We determined the percentage of activated NK cells via the analysis of CD107a expression as a marker of degranulation. Significantly higher levels of NK-cell activation were observed using 1/100 and 1/10 dilutions of sera from HCMV-seropositive individuals, and when cells were infected for 96 and 120 h, suggesting that NK cells are activated by antibodies directed against late antigens. In the absence of serum NK cells, activation was negligible. In seropositive subjects, the median percentages of CD107a-positive NK cells in the presence of autologous serum and pooled HCMV-positive serum were similar (14.03% [range 0.00-33.56] and 12.42% [range 1.01-46.00], respectively), while NK-cell activation was negligible using an HCMV-negative serum pool. In HCMV-seronegative subjects, the median percentage of activated NK cells was 0.90% [range 0.00-3.92] with autologous serum and 2.07% [0.00-5.76] in the presence of the HCMV-negative serum pool, while it was 8.97% [0.00-26.49] with the pool of HCMV-positive sera. NK-cell activation using hyperimmune globulin is comparable to what is obtained using autologous serum. Sera from subjects at 3 and 6 months post primary infection showed a lower capacity of NK-cell activation than sera from subjects with past infection (p < 0.001). NK activation against HCMV-infected epithelial cells is dependent on the presence of HCMV-specific antibodies. This serum activity increases with time after the onset of HCMV infection. The protective role of NK-cell activation by HCMV-specific serum antibodies should be verified in clinical settings.

Bidirectional transfer of human cytomegalovirus strains in donor and recipient seropositive lung transplant patients.

Donor and recipient human cytomegalovirus (HCMV) seropositive (D+R+) lung transplant recipients (LTRs) often harbor multiple strains of HCMV, likely due to transmitted donor (D) strains and reactivated recipient (R) strains. To date, the extent and timely occurrence of each likely source in shaping the post-transplantation (post-Tx) strain population is unknown. Here, we deciphered the D and R origin of the post-Tx HCMV strain composition in blood, bronchoalveolar lavage (BAL), and CD45+ BAL cell subsets. We investigated either D and/or R formalin-fixed paraffin-embedded blocks or fresh D lung tissue from four D+R+ LTRs obtained before transplantation. HCMV strains were characterized by short amplicon deep sequencing. In two LTRs, we show that the transplanted lung is reseeded by R strains within the first 6 months after transplantation, likely by infiltrating CD14+ CD163+/- alveolar macrophages. In three LTRs, we demonstrate both rapid D-strain dissemination and persistence in the transplanted lung for >1 year post-Tx. Broad inter-host diversity contrasts with intra-host genotype sequence stability upon transmission, during follow-up and across compartments. In D+R+ LTRs, HCMV strains of both, D and R origin can emerge first and dominate long-term in subsequent episodes of infection, indicating replication of both sources despite pre-existing immunity.

Adaptive natural killer cell expression in response to cytomegalovirus infection in blood and solid cancer.

Natural Killer (NK) cells are conventionally thought to be an indefinite part of innate immunity. However, in a specific subset of NK cells, recent data signify an extension of their "duties" in immune surveillance and response, having characteristics of adaptive immunity, in terms of persistence and cytotoxicity. These cells are known as the adaptive or memory-like NK cells, where human cytomegalovirus (HCMV) infection has been shown to drive the expansion of adaptive NKG2C+ NK cells. HCMV is a ubiquitous pathogen whose prevalence differs worldwide with respect to the socioeconomic status of countries. The adaptive NK cell subpopulation is often characterized by the upregulated expression of NKG2C, CD16, and CD2, and restricted expression of NKG2A, FCεRγ and killer immunoglobulin-like receptors (KIR), although these phenotypes may differ in different disease groups. The reconfiguration of these receptor distributions has been linked to epigenetic factors. Hence, this review attempts to appraise literature reporting markers associated with adaptive or memory-like NK cells post-HCMV infection, in relation to solid cancers and hematological malignancies. Adaptive NK cells, isolated and subjected to ex vivo modifications, have the potential to enhance anti-tumor response which can be a promising strategy for adoptive immunotherapy.

Immunomodulatory soluble HLA-G and HLA-E are associated with rapidly deteriorating CLAD and HCMV viremia after lung transplantation.

Plasma-soluble (s)HLA-G and sHLA-E are immunoregulatory proteins that balance the activation of NKG2A+ immune cells. In lung-transplant recipients (LTRs), dysregulated NKG2A+ natural killer cell responses may result in high-level human cytomegalovirus (HCMV) replication as well as chronic lung allograft dysfunction (CLAD), and especially the development of rapidly deteriorating CLAD is associated with high mortality. We thus analyzed the kinetics and function of sHLA-G and sHLA-E in follow-up samples of N = 76 LTRs to evaluate whether these immunoregulatory proteins are associated with the risk for CLAD and high-level HCMV replication. Here, we demonstrate that rapidly deteriorating CLAD LTRs are hallmarked by continually low (<107 ng/ml) sHLA-G levels. In contrast, high sHLA-E levels were associated with the following development of high-level (>1,000 copies/ml) HCMV episodes. Thus, sHLA-G and sHLA-E may serve as novel biomarkers for the development of rapidly deteriorating CLAD and high-level HCMV replication in LTRs.