A temperature-dependent virus-binding assay reveals the presence of neutralizing antibodies in human cytomegalovirus gB vaccine recipients' sera.

Human cytomegalovirus (HCMV) remains an important cause of mortality in immune-compromised transplant patients and following congenital infection. Such is the burden, an effective vaccine strategy is considered to be of the highest priority. The most successful vaccines to date have focused on generating immune responses against glycoprotein B (gB) - a protein essential for HCMV fusion and entry. We have previously reported that an important component of the humoral immune response elicited by gB/MF59 vaccination of patients awaiting transplant is the induction of non-neutralizing antibodies that target cell-associated virus with little evidence of concomitant classical neutralizing antibodies. Here we report that a modified neutralization assay that promotes prolonged binding of HCMV to the cell surface reveals the presence of neutralizing antibodies in sera taken from gB-vaccinated patients that cannot be detected using standard assays. We go on to show that this is not a general feature of gB-neutralizing antibodies, suggesting that specific antibody responses induced by vaccination could be important. Although we can find no evidence that these neutralizing antibody responses are a correlate of protection in vivo in transplant recipients their identification demonstrates the utility of the approach in identifying these responses. We hypothesize that further characterization has the potential to aid the identification of functions within gB that are important during the entry process and could potentially improve future vaccine strategies directed against gB if they prove to be effective against HCMV at higher concentrations.

Evolutionary remodelling of N-terminal domain loops fine-tunes SARS-CoV-2 spike.

The emergence of SARS-CoV-2 variants has exacerbated the COVID-19 global health crisis. Thus far, all variants carry mutations in the spike glycoprotein, which is a critical determinant of viral transmission being responsible for attachment, receptor engagement and membrane fusion, and an important target of immunity. Variants frequently bear truncations of flexible loops in the N-terminal domain (NTD) of spike; the functional importance of these modifications has remained poorly characterised. We demonstrate that NTD deletions are important for efficient entry by the Alpha and Omicron variants and that this correlates with spike stability. Phylogenetic analysis reveals extensive NTD loop length polymorphisms across the sarbecoviruses, setting an evolutionary precedent for loop remodelling. Guided by these analyses, we demonstrate that variations in NTD loop length, alone, are sufficient to modulate virus entry. We propose that variations in NTD loop length act to fine-tune spike; this may provide a mechanism for SARS-CoV-2 to navigate a complex selection landscape encompassing optimisation of essential functionality, immune-driven antigenic variation and ongoing adaptation to a new host.

HCMV carriage in the elderly diminishes anti-viral functionality of the adaptive immune response resulting in virus replication at peripheral sites.

Human cytomegalovirus (HCMV) infection and periodic reactivation is, generally, well controlled by adaptative immune responses in the healthy. In older people, overt HCMV disease is rarely seen despite the association of HCMV with increased risk of mortality; evidence from studies of unwell aged populations suggest that HCMV seropositivity is an important co-morbidity factor. HCMV genomes have been detected in urine from older donors, suggesting that the immune response prevents systemic disease but possibly immunomodulation due to lifelong viral carriage may alter its efficacy at peripheral tissue sites. Previously we have demonstrated that there were no age-related expansions of T cell responses to HCMV or increase in latent viral carriage with age and these T cells produced anti-viral cytokines and viremia was very rarely detected. To investigate the efficacy of anti-HCMV responses with increasing age, we used an in vitro Viral Dissemination Assay (VDA) using autologous dermal fibroblasts to determine the anti-viral effector capacity of total PBMC, as well as important subsets (T cells, NK cells). In parallel we assessed components of the humoral response (antibody neutralization) and combined this with qPCR detection of HCMV in blood, saliva and urine in a cohort of young and old donors. Consistent with previous studies, we again show HCMV specific cIL-10, IFNγ and TNFα T cell responses to peptides did not show an age-related defect. However, assessment of direct anti-viral cellular and antibody-mediated adaptive immune responses using the VDA shows that older donors are significantly less able to control viral dissemination in an in vitro assay compared to young donors. Corroborating this observation, we detected viral genomes in saliva samples only from older donors, these donors had a defect in cellular control of viral spread in our in vitro assay. Phenotyping of fibroblasts used in this study shows expression of a number of checkpoint inhibitor ligands which may contribute to the defects observed. The potential to therapeutically intervene in checkpoint inhibitor pathways to prevent HCMV reactivation in the unwell aged is an exciting avenue to explore.

Characterisation of B.1.1.7 and Pangolin coronavirus spike provides insights on the evolutionary trajectory of SARS-CoV-2.

The recent emergence of SARS-CoV-2 variants with increased transmission, pathogenesis and immune resistance has jeopardised the global response to the COVID-19 pandemic. Determining the fundamental biology of viral variants and understanding their evolutionary trajectories will guide current mitigation measures, future genetic surveillance and vaccination strategies. Here we examine virus entry by the B.1.1.7 lineage, commonly referred to as the UK/Kent variant. Pseudovirus infection of model cell lines demonstrate that B.1.1.7 entry is enhanced relative to the Wuhan-Hu-1 reference strain, particularly under low expression of receptor ACE2. Moreover, the entry characteristics of B.1.1.7 were distinct from that of its predecessor strain containing the D614G mutation. These data suggest evolutionary tuning of spike protein function. Additionally, we found that amino acid deletions within the N-terminal domain (NTD) of spike were important for efficient entry by B.1.1.7. The NTD is a hotspot of diversity across sarbecoviruses, therefore, we further investigated this region by examining the entry of closely related CoVs. Surprisingly, Pangolin CoV spike entry was 50-100 fold enhanced relative to SARS-CoV-2; suggesting there may be evolutionary pathways by which SARSCoV-2 may further optimise entry. Swapping the NTD between Pangolin CoV and SARS-CoV-2 demonstrates that changes in this region alone have the capacity to enhance virus entry. Thus, the NTD plays a hitherto unrecognised role in modulating spike activity, warranting further investigation and surveillance of NTD mutations.

Latent Cytomegalovirus-Driven Recruitment of Activated CD4+ T Cells Promotes Virus Reactivation.

Human cytomegalovirus (HCMV) infection is not cleared by the initial immune response but persists for the lifetime of the host, in part due to its ability to establish a latent infection in cells of the myeloid lineage. HCMV has been shown to manipulate the secretion of cellular proteins during both lytic and latent infection; with changes caused by latent infection mainly investigated in CD34+ progenitor cells. Whilst CD34+ cells are generally bone marrow resident, their derivative CD14+ monocytes migrate to the periphery where they briefly circulate until extravasation into tissue sites. We have analyzed the effect of HCMV latent infection on the secretome of CD14+ monocytes, identifying an upregulation of both CCL8 and CXCL10 chemokines in the CD14+ latency-associated secretome. Unlike CD34+ cells, the CD14+ latency-associated secretome did not induce migration of resting immune cell subsets but did induce migration of activated NK and T cells expressing CXCR3 in a CXCL10 dependent manner. As reported in CD34+ latent infection, the CD14+ latency-associated secretome also suppressed the anti-viral activity of stimulated CD4+ T cells. Surprisingly, however, co-culture of activated autologous CD4+ T cells with latently infected monocytes resulted in reactivation of HCMV at levels comparable to those observed using M-CSF and IL-1ß cytokines. We propose that these events represent a potential strategy to enable HCMV reactivation and local dissemination of the virus at peripheral tissue sites.

Validation of a commercially available indirect assay for SARS-CoV-2 neutralising antibodies using a pseudotyped virus assay.

OBJECTIVES: To assess whether a commercially available CE-IVD, ELISA-based surrogate neutralisation assay (cPass, Genscript) provides a genuine measure of SARS-CoV-2 neutralisation by human sera, and further to establish whether measuring responses against the RBD of S was a diagnostically useful proxy for responses against the whole S protein. METHODS: Serum samples from 30 patients were assayed for anti-NP responses, for 'neutralisation' by the surrogate neutralisation assay and for neutralisation by SARS-CoV-2 S pseudotyped virus assays utilising two target cell lines. Correlation between assays was measured using linear regression. RESULTS: The responses observed within the surrogate neutralisation assay demonstrated an extremely strong, highly significant positive correlation with those observed in both pseudotyped virus assays. CONCLUSIONS: The tested ELISA-based surrogate assay provides an immunologically useful measure of functional immune responses in a much quicker and highly automatable fashion. It also reinforces that detection of anti-RBD neutralising antibodies alone is a powerful measure of the capacity to neutralise viral infection.

IgA binds to the AD-2 epitope of glycoprotein B and neutralizes human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that is potentially pathogenic in immunosuppressed individuals and pregnant females during primary infection. The HCMV envelope glycoprotein B (gB) facilitates viral entry into all cell types and induces a potent immune response. AD-2 epitope is a highly conserved linear neutralizing epitope of gB and a critical target for antibodies; however, only 50% of sero-positive individuals make IgG antibodies to this site and IgA responses have not been fully investigated. This study aimed to compare IgG and IgA responses against gB and the AD-2 epitope in naturally exposed individuals and those receiving a recombinant gB/MF59 adjuvant vaccine. Thus, vaccination of sero-positive individuals improved pre-existing gB-specific IgA and IgG levels and induced de novo gB-specific IgA and IgG responses in sero-negative recipients. Pre-existing AD-2 IgG and IgA responses were boosted with vaccination, but de novo AD-2 responses were not detected. Naturally exposed individuals had dominant IgG responses towards gB and AD-2 compared with weaker and variable IgA responses, although a significant IgA binding response to AD-2 was observed within human breastmilk samples. All antibodies binding AD-2 contained kappa light chains, whereas balanced kappa/lambda light chain usage was found for those binding to gB. V region-matched AD-2-specific recombinant IgG and IgA bound both to gB and to AD-2 and neutralized HCMV infection in vitro. Overall, these results indicate that although human IgG responses dominate, IgA class antibodies against AD-2 are a significant component of human milk, which may function to protect neonates from HCMV.

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells.

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) - the promoter that regulates immediate early (IE) gene expression - is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

Cell signaling and cytomegalovirus reactivation: what do Src family kinases have to do with it?

Primary infection with human cytomegalovirus (HCMV) is usually asymptomatic and leads to the establishment of lifelong latent infection. A major site of latency are the CD34+ hematopoietic progenitor cells. Importantly, normal cellular differentiation of CD34+ cells to a macrophage or dendritic cell phenotype is concomitant with viral reactivation. Molecular studies of HCMV latency have shown that the latent viral genome is associated with histone proteins and that specific post-translational modifications of these histones correlates with the transcriptional activity of the genome arguing that expression of key viral genes that dictate latency and reactivation are subject to the rules of the histone code hypothesis postulated for the regulation of eukaryotic gene expression. Finally, many studies now point to a key role for multiple signaling pathways to provide the cue for HCMV reactivation. The challenge now is to understand the complex interplay between cell identity, transcriptional regulation and cell signaling that occurs to promote reactivation and, additionally, how HCMV may further manipulate these events to support reactivation. Understanding how HCMV utilizes these pathways to drive HCMV reactivation will provide new insight into the mechanisms that govern viral and host gene expression and, potentially, illuminate new, host-directed, therapeutic opportunities to support our attempts to control this important medical pathogen of immune-compromised individuals.

Immune Correlates of Protection Against Human Cytomegalovirus Acquisition, Replication, and Disease.

Human cytomegalovirus (HCMV) is the most common infectious cause of infant birth defects and an etiology of significant morbidity and mortality in solid organ and hematopoietic stem cell transplant recipients. There is tremendous interest in developing a vaccine or immunotherapeutic to reduce the burden of HCMV-associated disease, yet after nearly a half-century of research and development in this field we remain without such an intervention. Defining immune correlates of protection is a process that enables targeted vaccine/immunotherapeutic discovery and informed evaluation of clinical performance. Outcomes in the HCMV field have previously been measured against a variety of clinical end points, including virus acquisition, systemic replication, and progression to disease. Herein we review immune correlates of protection against each of these end points in turn, showing that control of HCMV likely depends on a combination of innate immune factors, antibodies, and T-cell responses. Furthermore, protective immune responses are heterogeneous, with no single immune parameter predicting protection against all clinical outcomes and stages of HCMV infection. A detailed understanding of protective immune responses for a given clinical end point will inform immunogen selection and guide preclinical and clinical evaluation of vaccines or immunotherapeutics to prevent HCMV-mediated congenital and transplant disease.

Seronegative patients vaccinated with cytomegalovirus gB-MF59 vaccine have evidence of neutralising antibody responses against gB early post-transplantation.

BACKGROUND: Human cytomegalovirus (HCMV) causes a ubiquitous infection which can pose a significant threat for immunocompromised individuals, such as those undergoing solid organ transplant (SOT). Arguably, the most successful vaccine studied to date is the recombinant glycoprotein-B (gB) with MF59 adjuvant which, in 3 Phase II trials, demonstrated 43-50% efficacy in preventing HCMV acquisition in seronegative healthy women or adolescents and reduction in virological parameters after SOT. However, the mechanism of vaccine protection in seronegative recipients remains undefined. METHODS: We evaluated samples from the cohort of seronegative SOT patients enroled in the Phase II glycoprotein-B/MF59 vaccine trial who received organs from seropositive donors. Samples after SOT (0-90 days) were tested by real-time quantitative PCR for HCMV DNA. Anti-gB antibody levels were measured by ELISA. Neutralization was measured as a decrease in infectivity for fibroblast cell cultures revealed by expression of immediate-early antigens. FINDINGS: Serological analyses revealed a more rapid increase in the humoral response against gB post transplant in vaccine recipients than in those randomised to receive placebo. Importantly, a number of patient sera displayed HCMV neutralising responses - neutralisation which was abrogated by pre-absorbing the sera with recombinant gB. INTERPRETATION: We hypothesise that the vaccine primed the immune system of seronegative recipients which, when further challenged with virus at time of transplant, allowed the host to mount rapid immunological humoral responses even under conditions of T cell immune suppression during transplantation.

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.

Src family kinase activity drives cytomegalovirus reactivation by recruiting MOZ histone acetyltransferase activity to the viral promoter.

Human cytomegalovirus (HCMV) latency and reactivation rely on a complex interplay between cellular differentiation, cell signaling pathways, and viral gene functions. HCMV reactivation in dendritic cells (DCs) is triggered by IL-6 and extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase signaling. However, activation of the same pathway fails to reactivate HCMV in other myeloid cell types, despite this signaling axis being active in those cells. We hypothesized that IL-6-induced ERK activation initiates the changes in chromatin structure required for viral reactivation but that a concomitant signal is necessary to complete the changes in chromatin structure required for gene expression to occur. Using a differential phosphoproteomics approach in cells that do or do not support IL-6-induced viral reactivation, we identified the concomitant activation of an Src family kinase (SFK), hematopoietic cell kinase (HCK), specifically in DCs in response to IL-6. Pharmacological and genetic inhibition of HCK activity indicated that HCK is required for HCMV reactivation. Furthermore, the HCK/SFK activity was linked to recruitment of the monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase to the viral promoter, which promoted histone acetylation after ERK-mediated histone phosphorylation. Importantly, pharmacological and genetic inhibition of MOZ activity prevented reactivation. These results provide an explanation for the selective activation of viral gene expression in DCs by IL-6, dependent on concomitant SFK and ERK signaling. They also reveal a previously unreported role for SFK activity in the regulation of chromatin structure at promoters in eukaryotic cells via MOZ histone acetyltransferase activity.

The core promoter controls basal and inducible expression of duck retinoic acid inducible gene-I (RIG-I).

Retinoic acid inducible gene-I (RIG-I) is a cytoplasmic RNA sensor for detecting a variety of RNA viruses including influenza A viruses. Detection ultimately produces Type I interferon (IFN), which stimulates expression of interferon stimulated genes (ISGs), including RIG-I itself in a positive feedback loop. The structure and function of RIG-I is conserved across phylogeny, despite significant protein sequence divergence, however, the promoter sequences do not show the expected phylogenetic relationships and it is not known whether they are similarly regulated. We previously cloned duck RIG-I and showed it is highly induced during influenza A infection consistent with induction by the interferon produced. Here, we identified the Pekin duck RIG-I promoter and constructed promoter reporter vectors, which we transfected into duck embryonic fibroblasts or chicken DF-1 cells and tested in dual luciferase assays. We showed that activation of the Mitochondrial Antiviral Signalling (MAVS) pathway using the constitutively active N-terminal region of RIG-I or polyinosinic-polycytidylic acid (poly I:C) led to stimulation of duck RIG-I promoter activity. Using deletion constructs we showed the core promoter lies in the proximal 250 basepairs, and we identified essential cis-regulatory elements, a GC-box and an interferon-sensitive response element (ISRE), responsible for basal and inducible expression, respectively. Using mCherry-tagged interferon regulatory factors (IRFs) cloned from chickens and ducks, we show overexpression of chIRF7 induced the duck RIG-I promoter, and this required the ISRE site. Finally, we also demonstrated that overexpressed chIRF7 translocated to the nucleus, which was augmented by MAVS activation using RIG-I 2CARD. Our findings demonstrate that RIG-I expression is induced by chIRF7, in a positive regulatory loop. These studies show that the duck RIG-I promoter is appropriately regulated in chicken cells, necessary for the potential generation of transgenic chickens expressing RIG-I.

A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency.

A subset of viral genes is required for the long-term latent infection of hematopoietic cells by human cytomegalovirus (HCMV). Here, we show that a latency-associated gene product (LUNA) promotes the disruption of cellular PML bodies during latency. Mutation and inhibitor studies reveal that LUNA encodes a deSUMOylase activity responsible for this disruption. Specifically, LUNA encodes a conserved Asp-Cys-Gly motif common to all deSUMOylases. Importantly, mutation of the putative catalytic cysteine is sufficient to reverse LUNA-mediated PML dispersal and markedly reduces the efficiency of viral reactivation. The depletion of PML from cells is sufficient to rescue the reactivation of the LUNA-deficient viruses, arguing that targeting PML is an important biological role of LUNA. Finally, we demonstrate that reactivation of naturally latent HCMV is blocked by deSUMOylase inhibitors. Thus, latent HCMV primes the cellular environment for efficient reactivation via the activity of a virally encoded deSUMOylase.

Author Correction: Viral programming of progenitor cell commitment.

In the version of this News & Views originally published, ref. 6 was incorrectly cited instead of ref. 5 at the end of the sentence shown below. This has now been corrected."Indeed, a proof of concept study has shown that latently expressed US28 can be targeted using an immunotoxin-based approach to eliminate infected cells in vitro5."

Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies.

Human cytomegalovirus (HCMV) is an important pathogen in transplant patients and in congenital infection. Previously, we demonstrated that vaccination with a recombinant viral glycoprotein B (gB)/MF59 adjuvant formulation before solid organ transplant reduced viral load parameters post transplant. Reduced posttransplant viremia was directly correlated with antibody titers against gB consistent with a humoral response against gB being important. Here we show that sera from the vaccinated seronegative patients displayed little evidence of a neutralizing antibody response against cell-free HCMV in vitro. Additionally, sera from seronegative vaccine recipients had minimal effect on the replication of a strain of HCMV engineered to be cell-associated in a viral spread assay. Furthermore, although natural infection can induce antibody-dependent cellular cytotoxicity (ADCC) responses, serological analysis of seronegative vaccinees again presented no evidence of a substantial ADCC-promoting antibody response being generated de novo. Finally, analyses for responses against major antigenic domains of gB following vaccination were variable, and their pattern was distinct compared with natural infection. Taken together, these data argue that the protective effect elicited by the gB vaccine is via a mechanism of action in seronegative vaccinees that cannot be explained by neutralization or the induction of ADCC. More generally, these data, which are derived from a human challenge model that demonstrated that the gB vaccine is protective, highlight the need for more sophisticated analyses of new HCMV vaccines over and above the quantification of an ability to induce potent neutralizing antibody responses in vitro.

Navigating the Host Cell Response during Entry into Sites of Latent Cytomegalovirus Infection.

The host cell represents a hostile environment that viruses must counter in order to establish infection. Human cytomegalovirus (HCMV) is no different and encodes a multitude of functions aimed at disabling, re-directing or hijacking cellular functions to promulgate infection. However, during the very early stages of infection the virus relies on the outcome of interactions between virion components, cell surface receptors and host signalling pathways to promote an environment that supports infection. In the context of latent infection-where the virus establishes an infection in an absence of many gene products specific for lytic infection-these initial interactions are crucial events. In this review, we will discuss key host responses triggered by viral infection and how, in turn, the virus ameliorates the impact on the establishment of non-lytic infections of cells. We will focus on strategies to evade intrinsic antiviral and innate immune responses and consider their impact on viral infection. Finally, we will consider the hypothesis that the very early events upon viral infection are important for dictating the outcome of infection and consider the possibility that events that occur during entry into non-permissive cells are unique and thus contribute to the establishment of latency.

Epitope-Specific Humoral Responses to Human Cytomegalovirus Glycoprotein-B Vaccine With MF59: Anti-AD2 Levels Correlate With Protection From Viremia.

The human cytomegalovirus (HCMV) virion envelope protein glycoprotein B (gB) is essential for viral entry and represents a major target for humoral responses following infection. Previously, a phase 2 placebo-controlled clinical trial conducted in solid organ transplant candidates demonstrated that vaccination with gB plus MF59 adjuvant significantly increased gB enzyme-linked immunosorbent assay (ELISA) antibody levels whose titer correlated directly with protection against posttransplant viremia. The aim of the current study was to investigate in more detail this protective humoral response in vaccinated seropositive transplant recipients. We focused on 4 key antigenic domains (AD) of gB (AD1, AD2, AD4, and AD5), measuring antibody levels in patient sera and correlating these with posttransplant HCMV viremia. Vaccination of seropositive patients significantly boosted preexisting antibody levels against the immunodominant region AD1 as well as against AD2, AD4, and AD5. A decreased incidence of viremia correlated with higher antibody levels against AD2 but not with antibody levels against the other 3 ADs. Overall, these data support the hypothesis that antibodies against AD2 are a major component of the immune protection of seropositives seen following vaccination with gB/MF59 vaccine and identify a correlate of protective immunity in allograft patients.