HCMV Infection Research Articles

Human cytomegalovirus preferentially infects early gestation placental macrophages and evades their antiviral immunogenicity through evasion of the type I IFN response

Abstract Human cytomegalovirus (HCMV) is the leading cause of congenital viral infections. When a pregnant woman develops primary HCMV infection in early gestation, the risk of transplacental infection is ~40%. Studies have documented the presence of HCMV in placental macrophages (Hofbauer cells [HCs]) as a consequence of maternal viremia. However, the interplay between HCMV and placental macrophages (HCs) is poorly defined. With written informed consent, placentae were collected from 12 HIV-1/Hep B/HCMV seronegative women (>18 years). HCs were exposed to human HCMV (TB40/E). qPCR, FACS, ELISA, and Western blot analysis determined the expression of activation markers, cytokines, antiviral genes, and host proteins. Here we found that HCs isolated from early gestation (15–23 weeks) placentae are more susceptible to HCMV infection than HCs isolated from term (>39 weeks) placentae. HCMV infection of early gestation HCs led to a significant upregulation of activation markers, along with elevated secretion of proinflammatory cytokines, compared to HCMV-infected term HCs. In early gestation and term HC, HCMV rapidly induces the type I IFN pathway, leading to secretion of IFNs and strong induction of ISGs. However, HCMV diminishes type I IFN-mediated phosphorylation of STAT2, with early gestation HCs exhibiting the greatest level of inhibition. Our findings show that HCMV can subvert type I IFN signaling to support viral growth at the placenta, particularly during early gestation. Studying mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections.

Association of Human Cytomegalovirus Infection with Different Forms of Diabetes

Different forms of virus induced infections are related to the enlarged risk of diabetogenic diseases in humans. Human cytomegalovirus infection has been advised by multiple studies to be one amongst the causative agents. HCMV infection has been linked to the development of both type 1 and type 2 diabetes as well as post-transplantation diabetes. The limited amount of available data indicates that active HCMV infection increases the risk of diabetes by either inhibiting the release of insulin from pancreatic cells or specifically destructing the pancreatic β cells. The mechanism is mainly attributed to the expression of pro-inflammatory cytokines that eventually results in programmed cell death or critical disturbances to the functioning of the β-cells.

Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release

ABSTRACT Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4BC74A). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4BC74A, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment. Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short

Modeling Human Cytomegalovirus in Humanized Mice for Vaccine Testing.

Human cytomegalovirus (HCMV or HHV-5) is a globally spread pathogen with strictly human tropism that establishes a life-long persistence. After primary infection, high levels of long-term T and B cell responses are elicited, but the virus is not cleared. HCMV persists mainly in hematopoietic reservoirs, whereby occasional viral reactivation and spread are well controlled in immunocompetent hosts. However, when the immune system cannot control viral infections or reactivations, such as with newborns, patients with immune deficiencies, or immune-compromised patients after transplantations, the lytic outbursts can be severely debilitating or lethal. The development of vaccines for immunization of immune-compromised hosts has been challenging. Several vaccine candidates did not reach the potency expected in clinical trials and were not approved. Before anti-HCMV vaccines can be tested pre-clinically in immune-compromised hosts, reliable in vivo models recapitulating HCMV infection might accelerate their clinical translation. Therefore, immune-deficient mouse strains implanted with human cells and tissues and developing a human immune system (HIS) are being explored to test anti-HCMV vaccines. HIS-mice resemble immune-compromised hosts as they are equipped with antiviral human T and B cells, but the immune reactivity is overall low. Several groups have independently shown that HCMV infections and reactivations can be mirrored in HIS mice. However, these models and the analyses employed varied widely. The path forward is to improve human immune reconstitution and standardize the analyses of adaptive responses so that HIS models can be forthrightly used for testing novel generations of anti-HCMV vaccines in the preclinical pipeline.

Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion.

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.

The power of human cytomegalovirus (HCMV) hijacked UL/b' functions lost in vitro.

Viruses have evolved sophisticated strategies to subvert immunity to benefit overall viral fitness. Human cytomegalovirus (HCMV, β-herpesvirus) represents a paradigm of very effective hijacking of gene functions that imitate host encoded immunomodulatory proteins. This co-evolution with the host immune system allowed for establishment of lifelong persistence. The HCMV infection is largely asymptomatic in healthy persons; however, it can induce serious disease in immunocompromised individuals. For this reason, great attention is paid to the development of therapeutics based on HCMV immunomodulatory 'tricks' as well as to the search for active vaccine against HCMV. While comparing the HCMV clinical isolates with extensively passaged laboratory strains, the unique long (UL) b' locus was commonly found to be deleted in HCMV genome while adapted to replication in human fibroblasts in vitro. This missing region, called UL/b' region, encodes up to 22 canonical genes with different functions, as of targeting cellular tropism (e.g. UL133-UL138); viral entry and assembly (e.g. UL128, UL130, UL131A); regulation of immunological synapses (e.g. UL135); inhibition of NK and T cell function (e.g. UL141, UL142, UL148, UL144), ablating activity (e.g. UL146, UL147), but mainly aimed at manipulating the host immune response. Moreover, the presence of UL/b' genomic region dramatically correlates with adverse effects in vaccinated persons, indicating that viral genes in this region play a significant role in controlling virulence. Here, we review how HCMV shapes our immunity by hijacked genes originated from UL/b' locus, discuss their impact in immunomodulation mechanism and how this knowledge may translate to clinical applications. Keywords: immunomodulation; HCMV genes; UL/b' locus; NK cell function; HCMV vaccine; immunity; immunotherapeutics.

Human Cytomegalovirus Latency and Myelosuppression: A microRNA-Dependent Yin and Yang Regulatory Loop

In this issue of Cell Host & Microbe, Hancock et al., 2020 show that latent HCMV infection, and specifically miR-US5-2, induces TGF-β secretion, which inhibits myelopoiesis in uninfected HPCs. They also show that HCMV-infected cells become resistant to TGF-β signaling through targeting of SMAD3 by miR-UL22-A-3p and -5p.

Human cytomegalovirus IE2 protein regulates macrophage-mediated immune escape by upregulating GRB2 expression in UL122 genetically modified mice.

Although cytomegalovirus (HCMV) infection is asymptomatic in healthy individuals, the virus can remain latent for many years due to its ability to evade host immune surveillance. However, reactivation of HCMV can lead to life-threatening disease. Recent studies have shown that HCMV infection mediates immune escape by regulating macrophage activity, although the role of the HCMV-encoded IE2 protein is unclear. A ul122 transgenic mouse model was created to stably expresses the IE2 protein, and the proportion of M1 and M2 macrophage populations in their spleen and bone marrow was compared to that in wild-type controls. In addition, the phagocytic function of the macrophages was evaluated in terms of neutral red dye uptake. Spleen and bone marrow macrophages in IE2-expressing mice were mainly of the M2 phenotype and displayed enhanced phagocytic function compared to that in control mice. The relative levels of expression of macrophage-related GRB2 and of IL-4, IFN-γ, IL-13, and TNF-α were also analyzed in the spleen and bone marrow of the two groups. The IE2-expressing mice had increased expression of GRB2 and increased expression of the M2-related cytokines IL-4 and IL-13. Taken together, the current results suggest that HCMV IE2 polarizes the host macrophages to the M2 type via a GRB2/IL-4-related pathway, which enables long-term survival of the virus in the host.

A highly potent trimeric derivative of artesunate shows promising treatment profiles in experimental models for congenital HCMV infection in vitro and ex vivo

• TF27, a derivate of artesunate , was evaluated in vitro and in an ex vivo model of placental against cytomegalovirus strains. • TF27 showed efficacy against various HCMV strains in different cell types. • TF27 decreased HCMV load in 1st trimester floating villi explant culture without significant impact on villi viability. • Experimental drug TF27, showed promising effects for further antiviral drug development.

Phenotypic and Functional Analysis of Human NK Cell Subpopulations According to the Expression of FcεRIγ and NKG2C.

Human memory-like NK cells are commonly defined by either a lack of FcεRIγ or gain of NKG2C expression. Here, we investigated the heterogeneity of human CD56dim NK cell subpopulations according to the expression of FcεRIγ and NKG2C in a large cohort (n = 127). Although the frequency of FcεRIγ− and NKG2C+ NK cells positively correlated, the FcεRIγ− and NKG2C+ NK cell populations did not exactly overlap. The FcεRIγ+NKG2C+, FcεRIγ−NKG2C+, and FcεRIγ−NKG2C− NK cell populations were only evident after HCMV infection, but each had distinct characteristics. Among the subpopulations, FcεRIγ−NKG2C+ NK cells exhibited the most restricted killer immunoglobulin-like receptor repertoire, suggesting clonal expansion. Moreover, FcεRIγ−NKG2C+ NK cells exhibited the lowest Ki-67 and highest Bcl-2 expression, indicating the long-lived quiescent memory-like property. Functionally, FcεRIγ−NKG2C+ NK cells had weak natural effector function against K562 but strong effector functions by CD16 engagement, whereas FcεRIγ+NKG2C+ NK cells had strong effector functions in both settings. Anatomically, the FcεRIγ+NKG2C+, FcεRIγ−NKG2C+, and FcεRIγ−NKG2C− NK cell populations were present in multiple human peripheral organs. In conclusion, we demonstrate the heterogeneity of memory-like NK cells stratified by FcεRIγ and NKG2C and suggest both markers be utilized to better define these cells.

Computational assembly of a human Cytomegalovirus vaccine upon experimental epitope legacy

BackgroundHuman Cytomegalovirus (HCMV) is a ubiquitous herpesvirus affecting approximately 90% of the world population. HCMV causes disease in immunologically naive and immunosuppressed patients. The prevention, diagnosis and therapy of HCMV infection are thus crucial to public health. The availability of effective prophylactic and therapeutic treatments remain a significant challenge and no vaccine is currently available. Here, we sought to define an epitope-based vaccine against HCMV, eliciting B and T cell responses, from experimentally defined HCMV-specific epitopes.ResultsWe selected 398 and 790 experimentally validated HCMV-specific B and T cell epitopes, respectively, from available epitope resources and apply a knowledge-based approach in combination with immunoinformatic predictions to ensemble a universal vaccine against HCMV. The T cell component consists of 6 CD8 and 6 CD4 T cell epitopes that are conserved among HCMV strains. All CD8 T cell epitopes were reported to induce cytotoxic activity, are derived from early expressed genes and are predicted to provide population protection coverage over 97%. The CD4 T cell epitopes are derived from HCMV structural proteins and provide a population protection coverage over 92%. The B cell component consists of just 3 B cell epitopes from the ectodomain of glycoproteins L and H that are highly flexible and exposed to the solvent.ConclusionsWe have defined a multiantigenic epitope vaccine ensemble against the HCMV that should elicit T and B cell responses in the entire population. Importantly, although we arrived to this epitope ensemble with the help of computational predictions, the actual epitopes are not predicted but are known to be immunogenic.

Dissecting the Role of DDX21 in Regulating Human Cytomegalovirus Replication.

DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484-493, 2014, https://doi.org/10.1016/j.chom.2014.03.002; Dong et al., Biophys Res Commun, 473:648-653, 2016, https://doi.org/10.1016/j.bbrc.2016.03.120; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, https://doi.org/10.1371/journal.ppat.1000654). The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.

Human cytomegalovirus seroprevalence and titres in solid organ transplant recipients and transplant donors in Seoul, South Korea

BackgroundHuman cytomegalovirus (HCMV) can cause poor outcomes in solid organ transplant (SOT) recipients; moreover, it is associated with cardiovascular diseases (CVD) in the general population. Accordingly, anti-HCMV immunoglobulin G (IgG) seroepidemiology may be useful in identifying the risk of post-SOT HCMV infection or disease as well as immunosenescence or CVD. However, HCMV seroprevalence and titre have not been fully evaluated with regard to age distribution or compared between SOT recipients and healthy individuals in South Korea.MethodsWe retrospectively retrieved all unduplicated anti-HCMV IgG results of individuals aged > 1 year evaluated between July 2006 and November 2017 at Severance Hospital in Seoul. The cohort, excluding haematopoietic stem cell transplant recipients and subjects with equivocal values, included 2184 SOT recipients and 3015 healthy transplant donors. All IgG results in the SOT recipients were measured during the pre-transplant period.ResultsThe overall IgG seroprevalence and titres were significantly higher among SOT recipients than among healthy donors (98.7% vs. 88.6%, p < 0.001, and 64.7 ± 44.3 vs. 49.8 ± 20.6 arbitrary units/mL, p < 0.001, respectively). The lowest seropositive rate in the SOT group was observed in recipients aged between 11 and 15 years (70.6%). The frequency of seropositivity among adults aged ≥41 years increased to ≥90% in SOT recipients and healthy donors. Age was independently associated with higher HCMV seroprevalence (41–60 years, OR, 76.4, 95% CI, 24.5–238.9, p < 0.001; ≥ 61 years, OR, 4.4, 95% CI, 1.3–14.9, p < 0.001, compared to ≤40 years). The healthy donor group had an independently low HCMV seropositive rate (OR, 0.1, 95% CI, 0.1–0.2, p < 0.001).ConclusionsHCMV seropositivity was the lowest among school-aged children and adolescents. IgG testing revealed an intermediate serostatus risk of post-transplant HCMV infection and disease for most adult SOT recipients in South Korea.

2651. Protection Against Human Cytomegalovirus Acquisition Is Associated with IgG Binding to Cell-Associated CMV glycoprotein B in Two Historical gB/MF59 Vaccine Cohorts

BackgroundHuman cytomegalovirus (CMV) is the most common congenital infection worldwide. A CMV glycoprotein B (gB) subunit vaccine with MF59 adjuvant achieved ~50% protection in phase II clinical trials in postpartum and adolescent women. Interestingly, postpartum vaccinees showed poor virus neutralization but robust antibody-dependent cellular phagocytosis (ADCP). In this study, we performed a combined humoral immune correlate of risk analysis in vaccinees to define vaccine-elicited immune responses associated with protection and targets for vaccine candidate immunogenicity.MethodsgB/MF59 vaccinees who became infected and those who remained uninfected were 2:1 matched on race and number of vaccine doses. This study included 42 women from the adolescent (14 infected, 28 uninfected) and 33 from the postpartum cohorts (11 infected, 22 uninfected). IgG binding to whole gB, gB-neutralizing epitopes, FCRs, and whole virions were assessed by standard or multiplex ELISA. IgG binding to gB mRNA-transfected HEK293Ts was measured by flow cytometry. Neutralization of Towne, TB40/E, and AD169-repaired-GFP strains were measured in MRC-5, BJ5Ta, and/or ARPE-19 cells. Phagocytosis was assessed by THP-1 uptake of fluorescently conjugated TB40/E and AD169-repaired-GFP virions. Multiple linear regression controlling for cohort was performed for the combined log-transformed group data (apriori significance cut-off of P < 0.05, Benjamin–Hochberg FDR < 0.2).ResultsVaccine-elicited antibodies in adolescent and postpartum cohorts exhibited similar magnitude IgG binding to soluble HCMV gB protein, gB-neutralizing domains, and gB-transfected cells. Autologous Towne strain neutralization was observed in both cohorts, but heterologous strain neutralization was observed only in adolescent vaccinees (P = 0.001). Both cohorts exhibited robust phagocytosis of HCMV virions. Regression analyses revealed that risk of HCMV acquisition in vaccinees was associated with magnitude IgG binding to gB-transfected cells (P = 0.006, FDR = 0.15), not neutralization or phagocytosis responses.ConclusionProtection against primary HCMV infection was significantly associated with vaccine-elicited IgG binding to gB-transfected cells, suggesting the importance of a native, cell-associated gB conformation in future vaccine candidates.Disclosures All authors: No reported disclosures.

Cell Line Models for Human Cytomegalovirus Latency Faithfully Mimic Viral Entry by Macropinocytosis and Endocytosis.

Human cytomegalovirus (HCMV) enters primary CD34+ hematopoietic progenitor cells by macropinocytosis, where it establishes latency in part because its tegument-transactivating protein, pp71, remains associated with endosomes and is therefore unable to initiate productive, lytic replication. Here we show that multiple HCMV strains also enter cell line models used to study latency by macropinocytosis and endocytosis. In all latency models tested, tegument-delivered pp71 was found to be colocalized with endosomal markers and was not associated with the seven other cytoplasmic localization markers tested. Like the capsid-associated pp150 tegument protein, we initially detected capsid proteins in association with endosomes but later detected them in the nucleus. Inhibitors of macropinocytosis and endocytosis reduced latent viral gene expression and precluded reactivation. Importantly, we utilized electron microscopy to observe entry by macropinocytosis and endocytosis, providing additional visual corroboration of the findings of our functional studies. Our demonstration that HCMV enters cell line models for latency in a manner indistinguishable from that of its entry into primary cells illustrates the utility of these cell lines for probing the mechanisms, host genetics, and small-molecule-mediated inhibition of HCMV entry into the cell types where it establishes latency.IMPORTANCE Primary cells cultured in vitro currently provide the highest available relevance for examining molecular and genetic requirements for the establishment, maintenance, and reactivation of HCMV latency. However, their expense, heterogeneity, and intransigence to both long-term culture and molecular or genetic modification create rigor and reproducibility challenges for HCMV latency studies. There are several cell line models for latency not obstructed by deficiencies inherent in primary cells. However, many researchers view cell line studies of latency to be physiologically irrelevant because of the perception that these models display numerous and significant differences from primary cells. Here, we show that the very first step in a latent HCMV infection, entry of the virus into cells, occurs in cell line models in a manner indistinguishable from that in which it occurs in primary CD34+ hematopoietic progenitor cells. Our data argue that experimental HCMV latency is much more similar than it is different in cell lines and primary cells.

Blocking cellular N-glycosylation suppresses human cytomegalovirus entry in human fibroblasts

N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of host cell N-glycosylation in human cytomegalovirus (hCMV) infection remains to be elucidated. In this study, we found that blocking or removal of cellular N-glycosylation by tunicamycin, peptide-N-glycosidase F (PNGase F) treatment, or N-acetylglucosaminyltransferase I (MGAT1) knockdown resulted in suppression of hCMV infection in human fibroblasts. This suppression was reversed following N-glycosylation restoration. Immunofluorescence and flow cytometry analysis showed that blockade of cellular N-glycosylation interfered with hCMV entry rather than binding. Removal of N-glycosylation on epidermal growth factor (EGFR) and integrin β3, two proposed hCMV receptors, blocked their interaction with hCMV glycoproteins B and H. It also suppressed activation of these receptors and downstream integrin β3/Src signaling. Taken together, these results suggest that N-glycosylation of host cell glycoproteins including two proposed hCMV receptors is critical for hCMV entry rather than attachment. They provide novel insights into the biological process important for the early stage of hCMV infection with potential therapeutic implications.

MicroRNA-221 restricts human cytomegalovirus replication via promoting type I IFN production by targeting SOCS1/NF-κB pathway

ABSTRACT HCMV is a common pathogen for human with relatively high prevalence, which could be life-threatened in immunodeficient patients and lead to significant birth defects in newborns. In this study, we firstly report that HCMV infection significantly enhances the expression of microRNA-221 (miR-221) in Neural Precursor Cells (NPCs). We found that miR-221 directly targets at the 3ʹ-UTR of suppressor of cytokine signaling 1 (SOCS1) and suppresses SOCS1 expression at the both mRNA and protein levels. MiR-221 overexpression restrained HCMV replication by promoting type I interferon (IFN) and interferon stimulating genes (ISGs) production, whereas reintroduction of SOCS1 abrogated the miR-221-induced effects on HCMV replication. Importantly, miR-221 positively regulated the phosphorylation and activation of NF-κB by suppressing SOCS1. What’s more, miR-221 agomir alleviated MCMV-induced tissue injury by promoting type I IFN antiviral activities in vivo. Thus, miR-221 modulates the infection and replication of HCMV as an intrinsic antiviral factor, and could be developed as a treatment target for anti-HCMV treatment.

Simian Immunodeficiency Virus Infection Modulates CD94+ (KLRD1+) NK Cells in Rhesus Macaques.

Recently, we and others have shown that natural killer (NK) cells exhibit memory-like recall responses against cytomegalovirus (CMV) and human immunodeficiency/virus simian immunodeficiency virus (HIV/SIV) infections. Although the mechanism(s) have not been fully delineated, several groups have shown that the activating receptor NKG2C is elevated on NK cells in the context of rhesus CMV (rhCMV) or human CMV (hCMV) infections. CD94, which heterodimerizes with NKG2C is also linked to adaptive NK cell responses. Because nonhuman primates (NHP) play a crucial role in modeling HIV (SIV) infections, it is crucial to be able to assess and characterize the NKG2 family in NHP. Unfortunately, it is not possible to detect CD94 using commercially available antibodies in NHP. Our work, a first for NHP, has focused on developing RNA flow cytometry using mRNA transcripts as proxies distinguishing NKG2C from NKG2A. We have expanded the application of this technology and here we show the first characterization of CD94+ (KLRD1+) NK cells in NHP using multiparametric RNA flow cytometry. Peripheral blood mononuclear cells from naive and matched acutely (n = 4) or chronically (n = 12) SIV-infected rhesus macaques were analyzed by flow cytometry using commercially available antibodies, determining expression of transcripts for NKG2A, NKG2C, and CD94 (KLRC1, KLRC2, and KLRD1, respectively) on NK cells using RNA flow cytometry. Our data show that KLRC1+/- KLRC2+ KLRD1+ NK cells decrease following chronic, but not acute, infection with SIV. This approach will allow us to investigate the kinetics of infection and NK memory formation and will further improve our understanding of basic NK cell biology, especially in the context of SIV infection.IMPORTANCE Nonhuman primates play a crucial role in approximating human biology and many diseases that are difficult, if not impossible, to achieve in other animal models, notably HIV. Current advances in adaptive NK cell research positions us to address fundamental deficiencies in our fight against infection and disease at the earliest moments after infection or substantially earlier in disease progression. We show here that we can identify specific NK cell subpopulations that are modulated following chronic, but not acute, SIV infection. The ability to identify these subsets more precisely will inform therapeutic and vaccine strategies targeting an optimized NK cell response.

Human Cytomegalovirus Enters the Primary CD34+ Hematopoietic Progenitor Cells Where It Establishes Latency by Macropinocytosis.

Viral entry is targeted by immunological and pharmacological measures to inhibit viral infection. Human cytomegalovirus (HCMV) entry into cells where it initiates productive infection has been well studied, but its entry into cell types where it establishes latency has not. Therefore, we examined the entry of HCMV into CD34+ hematopoietic progenitor cells where the virus establishes latency. We determined that HCMV enters into the primary CD34+ hematopoietic progenitor cells in which it establishes latency by macropinocytosis. The capsid-associated tegument protein pp150 is released from maturing endosomes and migrates to the nucleus, whereas other tegument proteins, including pp71, remain endosome associated in the cytoplasm. The inhibition of macropinocytosis impairs entry, thereby diminishing latency-associated transcription and reducing viral reactivation. We conclude that HCMV virions enter CD34+ cells by macropinocytosis but fail to fully uncoat or disassemble their tegument layers, leading to the establishment of latency.IMPORTANCE Virion entry is targeted by antivirals and natural immunity to prevent infection. Natural preexisting immunity is ineffective at clearing an HCMV infection, and an incomplete understanding of the viral glycoproteins and cellular receptors that mediate entry has hampered inhibitor development. Nevertheless, HCMV entry remains a viable drug target. Our characterization here of HCMV entry into primary CD34+ hematopoietic progenitor cells through macropinocytosis and our comparison to viral entry into fibroblast cells highlight virion uncoating and tegument disassembly as a divergence point between productive and latent infections. Further definition of tegument disassembly may permit the development of interventions to inhibit this process to block productive infection or to trigger it in incompletely differentiated cells to prevent the seeding of the latent reservoirs that make HCMV infections incurable.

The Humoral Immune Response Against the gB Vaccine: Lessons Learnt from Protection in Solid Organ Transplantation.

Human cytomegalovirus (hCMV) is considered to be the highest priority for vaccine development. This view is underscored by the significant morbidity associated with congenital hCMV infection and viraemia in transplant patients. Although a number of vaccines have been trialed, none have been licensed. The hCMV vaccine candidate that has performed best in clinical trials to date is the recombinant glycoprotein B (gB) vaccine that has demonstrated protection, ranging from a 43% to 50% efficacy in three independent phase II trials. In this review, we focus on data from the phase II trial performed in solid organ transplant patients and the outcomes of follow-up studies attempting to identify immunological and mechanistic correlates of protection associated with this vaccine strategy. We relate this to other vaccine studies of gB as well as other vaccine strategies to determine areas of commonality and divergence. Finally, through the review, we discuss the unique challenges and opportunities presented with vaccine studies in transplant populations with recommendations that could empower subsequent trials.