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Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients., Human cytomegalovirus (HCMV) can evade immune responses in latent reservoirs, which is a specific problem during transplantation. Here the authors develop a bivalent nanobody that is specific for US28, a viral receptor required by latent HCMV, and show it can partially reactivate the virus and make it susceptible to killing by T cells.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. In healthy people, primary infection is generally asymptomatic, and the virus can go on to establish lifelong latency in cells of the myeloid lineage. However, HCMV often causes severe disease in the immunosuppressed: transplant recipients and people living with AIDS, and also in the immunonaive foetus. At present, there are several antiviral drugs licensed to control HCMV disease. However, these are all faced with problems of poor bioavailability, toxicity and rapidly emerging viral resistance. Furthermore, none of them are capable of fully clearing the virus from the host, as they do not target latent infection. Consequently, reactivation from latency is a significant source of disease, and there remains an unmet need for treatments that also target latent infection. This review briefly summarises the most common HCMV antivirals used in clinic at present and discusses current research into targeting the latent HCMV reservoir.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe disease following congenital infection and in immunocompromised individuals. No vaccines are licensed, and there are limited treatment options. We now show that the addition of anti-HCMV antibodies (Abs) can activate NK cells prior to the production of new virions, through Ab-dependent cellular cytotoxicity (ADCC), overcoming viral immune evasins. Quantitative proteomics defined the most abundant HCMV proteins on the cell surface, and we screened these targets to identify the viral antigens responsible for activating ADCC. Surprisingly, these were not structural glycoproteins; instead, the immune evasins US28, RL11, UL5, UL141, and UL16 each individually primed ADCC. We isolated human monoclonal Abs (mAbs) specific for UL16 or UL141 from a seropositive donor and optimized them for ADCC. Cloned Abs targeting a single antigen (UL141) were sufficient to mediate ADCC against HCMV-infected cells, even at low concentrations. Collectively, these findings validated an unbiased methodological approach to the identification of immunodominant viral antigens, providing a pathway toward an immunotherapeutic strategy against HCMV and potentially other pathogens.

The extensive tropism of human cytomegalovirus (HCMV) results in the productive infection of multiple cell types within the human host. However, infection of other cell types, such as undifferentiated cells of the myeloid lineage, give rise to nonpermissive infections. This aspect has been used experimentally to model latent infection, which is known to be established in the pluripotent CD34+ hematopoietic progenitor cell population resident in the bone marrow in vivo. The absence of a tractable animal model for studies of HCMV has resulted in a number of laboratories employing experimental infection of cells in vitro to simulate both HCMV lytic and latent infection. Herein, we will focus on the techniques used in our laboratory for the isolation and use of primary cells to study aspects of HCMV latency, reactivation, and lytic infection.

While CD8+ T cells specific for human cytomegalovirus (HCMV) have been extensively studied in both healthy HCMV seropositive carriers and patients undergoing immunosuppression, studies on the CD4+ T cell response to HCMV had lagged behind. However, over the last few years there has been a significant advance in our understanding of the importance and contribution that CMV-specific CD4+ T cells make, not only to anti-viral immunity but also in the potential maintenance of latently infected cells. During primary infection with HCMV in adults, CD4+ T cells are important for the resolution of symptomatic disease, while persistent shedding of HCMV into urine and saliva is associated with a lack of HCMV specific CD4+ T cell response in young children. In immunosuppressed solid organ transplant recipients, a delayed appearance of HCMV-specific CD4+ T cells is associated with prolonged viremia and more severe clinical disease, while in haematopoietic stem cell transplant recipients, it has been suggested that HCMV-specific CD4+ T cells are required for HCMV-specific CD8+ T cells to exert their anti-viral effects. In addition, adoptive T-cell immunotherapy in transplant patients has shown that the presence of HCMV-specific CD4+ T cells is required for the maintenance of HCMV-specific CD8+ T cells. HCMV is a paradigm for immune evasion. The presence of viral genes that down-regulate MHC class II molecules and the expression of viral IL-10 both limit antigen presentation to CD4+ T cells, underlining the important role that this T cell subset has in antiviral immunity. This review will discuss the antigen specificity, effector function, phenotype and direct anti-viral properties of HCMV specific CD4+ T cells, as well as reviewing our understanding of the importance of this T cell subset in primary infection and long-term carriage in healthy individuals. In addition, their role and importance in congenital HCMV infection and during immunosuppression in both solid organ and haemopoietic stem cell transplantation is considered.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/β/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection., Author summary Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects, can trigger disease in immune-compromised patients, and plays roles in cancer development. Cytotoxic lymphocytes kill HCMV-infected cells via releasing a set of five cytotoxic serine proteases called granzymes. However, the killing capacity of cytotoxic cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we show that cytotoxic lymphocytes can also use granzymes to inhibit HCMV replication in absence of cell death. All five granzymes cleave and inactivate both viral immediate-early (IE1/2) proteins, which are essential players for initiating HCMV infection. Our data support the model that cytotoxic cells employ granzymes to dampen HCMV replication prior to accumulation of sufficient hits to kill the infected cell.

HCMV infection, reinfection or reactivation occurs in 60% of untreated solid organ transplant (SOT) recipients. Current clinical approaches to HCMV management include pre-emptive and prophylactic antiviral treatment strategies. The introduction of immune monitoring to better stratify patients at risk of viraemia and HCMV mediated disease could improve clinical management. Current approaches quantify T cell IFNγ responses specific for predominantly IE and pp65 proteins ex vivo, as a proxy for functional control of HCMV in vivo. However, these approaches have only a limited predictive ability. We measured the IFNγ T cell responses to an expanded panel of overlapping peptide pools specific for immunodominant HCMV proteins IE1/2, pp65, pp71, gB, UL144, and US3 in a cohort of D+R- kidney transplant recipients in a longitudinal analysis. Even with this increased antigen diversity, the results show that while all patients had detectable T cell responses, this did not correlate with control of HCMV replication in some. We wished to develop an assay that could directly measure anti-HCMV cell-mediated immunity. We evaluated three approaches, stimulation of PBMC with (i) whole HCMV lysate or (ii) a defined panel of immunodominant HCMV peptides, or (iii) fully autologous infected cells co-cultured with PBMC or isolated CD8+ T cells or NK cells. Stimulation with HCMV lysate often generated non-specific antiviral responses while stimulation with immunodominant HCMV peptide pools produced responses which were not necessarily antiviral despite strong IFNγ production. We demonstrated that IFNγ was only a minor component of secreted antiviral activity. Finally, we used an antiviral assay system to measure the effect of whole PBMC, and isolated CD8+ T cells and NK cells to control HCMV in infected autologous dermal fibroblasts. The results show that both PBMC and especially CD8+ T cells from HCMV seropositive donors have highly specific antiviral activity against HCMV. In addition, we were able to show that NK cells were also antiviral, but the level of this control was highly variable between donors and not dependant on HCMV seropositivity. Using this approach, we show that non-viraemic D+R+ SOT recipients had significant and specific antiviral activity against HCMV.

The complexity of host-associated microbial ecosystems requires host-specific reference catalogs to survey the functions and diversity of these communities. We generate a comprehensive resource, the integrated mouse gut metagenome catalog (iMGMC), comprising 4.6 million unique genes and 660 metagenome-assembled genomes (MAGs), many (485 MAGs, 73%) of which are linked to reconstructed full-length 16S rRNA gene sequences. iMGMC enables unprecedented coverage and taxonomic resolution of the mouse gut microbiota; i.e., more than 92% of MAGs lack species-level representatives in public repositories (

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50 to 100% of humans worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals but is a serious cause of mortality and morbidity in the immunocompromised and neonates. In particular, reactivation of HCMV in the transplant setting is a major cause of transplant failure and related disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide insights into potential ways to target the latent viral reservoir in at-risk patient populations., Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected cell to optimize latent carriage and reactivation. This is achieved, in part, through the expression of viral genes, including the G-protein-coupled receptor US28. Here, we use an unbiased proteomic screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes are downregulated by US28. We validate that major histocompatibility complex (MHC) class II and two pyrin and HIN domain (PYHIN) proteins, myeloid cell nuclear differentiation antigen (MNDA) and IFI16, are downregulated during experimental latency in primary human CD14+ monocytes. We find that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner but only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early gene expression during latency via NF-κB, a function which explains why downregulation of IFI16 during latency is advantageous for the virus.

Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials.

Herpesviruses undergo life-long latent infection which can be life-threatening in the immunocompromised. Models of latency and reactivation of human cytomegalovirus (HCMV) include primary myeloid cells, cells known to be important for HCMV latent carriage and reactivation in vivo. However, primary cells are limited in availability, and difficult to culture and to genetically modify; all of which have hampered our ability to fully understand virus/host interactions of this persistent human pathogen. We have now used iPSCs to develop a model cell system to study HCMV latency and reactivation in different cell types after their differentiation down the myeloid lineage. Our results show that iPSCs can effectively mimic HCMV latency/reactivation in primary myeloid cells, allowing molecular interrogations of the viral latent/lytic switch. This model may also be suitable for analysis of other viruses, such as HIV and Zika, which also infect cells of the myeloid lineage.

BackgroundHuman cytomegalovirus (HCMV) is a threat to immunologically weak patients. HCMV cannot yet be eliminated with a vaccine, despite recent advances.Sources of dataSources of data are recently published research papers and reviews about HCMV treatments.Areas of agreementCurrent antivirals target the UL54 DNA polymerase and are limited by nephrotoxicity and viral resistance. Promisingly, letermovir targets the HCMV terminase complex and has been recently approved by the FDA and EMA.Areas of controversyShould we screen newborns for HCMV, and use antivirals to treat sensorineural hearing loss after congenital HCMV infection?Growing pointsGrowing points are developing drugs against latently infected cells. In addition to small molecule inhibitors, a chemokine-based fusion toxin protein, F49A-FTP, has shown promise in killing both lytically and latently infected cells.Areas timely for developing researchWe need to understand what immune responses are required to control HCMV, and how best to raise these immune responses with a vaccine.

Human cytomegalovirus (HCMV) is a major cause of viral disease in the young and the immune-suppressed. At sites of infection, HCMV recruits the neutrophil, a cell with a key role in orchestrating the initial immune response. Herein, we report a profound survival response in human neutrophils exposed to the clinical HCMV isolate Merlin, but not evident with the attenuated strain AD169, through suppression of apoptosis. The initial survival event, which is independent of viral gene expression and involves activation of the ERK/MAPK and NF-κB pathways, is augmented by HCMV-stimulated release of a secretory cytokine profile that further prolongs neutrophil lifespan. As aberrant neutrophil survival contributes to tissue damage, we predict that this may be relevant to the immune pathology of HCMV, and the presence of this effect in clinical HCMV strains and its absence in attenuated strains implies a beneficial effect to the virus in pathogenesis and/or dissemination. In addition, we show that HCMV-exposed neutrophils release factors that enhance monocyte recruitment and drive monocyte differentiation to a HCMV-permissive phenotype in an IL-6-dependent manner, thus providing an ideal vehicle for viral dissemination. This study increases understanding of HCMV–neutrophil interactions, highlighting the potential role of neutrophil recruitment as a virulence mechanism to promote HCMV pathology in the host and influence the dissemination of HCMV infection. Targeting these mechanisms may lead to new antiviral strategies aimed at limiting host damage and inhibiting viral spread.

Human cytomegalovirus (HCMV) primary infection and periodic reactivation of latent virus is generally well controlled by T-cell responses in healthy people. In older donors, overt HCMV disease is not generally seen despite the association of HCMV infection with increased risk of mortality. However, increases in HCMV DNA in urine of older people suggest that, although the immune response retains functionality, immunomodulation of the immune response due to lifelong viral carriage may alter its efficacy. Viral transcription is limited during latency to a handful of viral genes and there is both an IFNγ and cellular IL-10 CD4+ T-cell response to HCMV latency-associated proteins. Production of cIL-10 by HCMV-specific CD4+ T-cells is a candidate for aging-related immunomodulation. To address whether long-term carriage of HCMV changes the balance of cIL-10 and IFNγ-secreting T-cell populations, we recruited a large donor cohort aged 23–78 years and correlated T-cell responses to 11 HCMV proteins with age, HCMV IgG levels, latent HCMV load in CD14+ monocytes, and T-cell numbers in the blood. IFNγ responses by CD4+ and CD8+ T-cells to all HCMV proteins were detected, with no age-related increase in this cohort. IL-10-secreting CD4+ T cell responses were predominant to latency-associated proteins but did not increase with age. Quantification of HCMV genomes in CD14+ monocytes, a known site of latent HCMV carriage, did not reveal any increase in viral genome copies in older donors. Importantly, there was a significant positive correlation between the latent viral genome copy number and the breadth and magnitude of the IFNγ T-cell response to HCMV proteins. This study suggests in healthy aged donors that HCMV-specific changes in the T cell compartment were not affected by age and were effective, as viremia was a very rare event. Evidence from studies of unwell aged has shown HCMV to be an important comorbidity factor, surveillance of latent HCMV load and low-level viremia in blood and body fluids, alongside typical immunological measures and assessment of the antiviral capacity of the HCMV-specific immune cell function would be informative in determining if antiviral treatment of HCMV replication in the old maybe beneficial.

Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8 + T cell response to HCMV has been extensively studied, the HCMV-specific CD4 + T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-γ) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4 + T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-γ response was still dominant. CD4 + T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1β secretion) effector responses. Importantly, when we measured the CD4 + T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro , we observed that the CD4 + T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4 + T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4 + T cell responses, even those from elderly individuals, are highly functional and are directly antiviral. IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated during the host's lifetime. The dysfunction of immune cells associated with the long-term carriage of HCMV has been linked with poor responses to new pathogens and vaccines when people are older. In this study, we investigated the response of a subset of immune cells (CD4 + T cells) to HCMV proteins in healthy donors of all ages, and we demonstrate that the functionality of CD4 + T cells is maintained. We also show that CD4 + T cells produce effector functions in response to HCMV-infected cells and can prevent virus spread. Our work demonstrates that these HCMV-specific immune cells retain many important functions and help to prevent deleterious HCMV disease in healthy older people.

Since Looney at al. published their seminal paper a decade ago [1] it has become clear that many of the differences in T cell immunological parameters observed between young and old people are related to the age-associated increasing prevalence of infection with the persistent β-herpesvirus HHV-5 (Cytomegalovirus). Ten years later, studies suggest that hallmark age-associated changes in peripheral blood T cell subset distribution may not occur at all in people who are not infected with this virus [[2]; Derhovanessian et al., in press]. Whether the observed changes are actually caused by CMV is an open question, but very similar, rapid changes observed in uninfected patients receiving CMV-infected kidney grafts are consistent with a causative role [3]. This meeting intensively discussed these and other questions related to the impact of CMV on human immune status and its relevance for immune function in later life.

Several decades of research on human cytomegalovirus (HCMV) and the principal mammalian cytomegaloviruses which to varying degrees act as models of HCMV infection, particularly murine, guinea pig and rhesus CMV, have led to the recognition of the CMVs as interesting models of persistent infection with a large and complex DNA virus, which have been highly informative of the immunology and molecular pathogenesis of the virus–host relationship in the normal host. However, it is appropriate to ask how this relative wealth of knowledge has influenced the understanding and management of clinical disease due to HCMV. This article considers the immunology of cytomegalovirus in the normal human host, and the interrelated issue of the sites of HCMV latency and mechanisms of reactivation in the myeloid cell lineage, and in related in vitro model systems. The way in which this site of latency conditions the immune response, and emerging information on the special features of the adaptive immune response to HCMV during latency are also considered. Examples of HCMV disease associated with acquired immunosuppression, principally in the context of transplantation, but also as a consequence of HIV/AIDS and immune reconstitution inflammatory syndrome, are then discussed, with a particular emphasis on how understanding the immunology of persistent infection may contribute to managing CMV disease now and in future.

Cytotoxic T-lymphocytes (CTL) play an important role in the control of human immunodeficiency virus (HIV) and of human cytomegalovirus (HCMV) infection. Following highly active antiretroviral therapy (HAART), most studies have demonstrated a decline in the frequency of HIV-specific CTL. We analysed the effect of HAART on the size, phenotype and function of individual HIV- and HCMV-specific CTL clones, using clonotypic oligonucleotide probing specific for the T-cell receptor (TCR) beta-chain hypervariable sequence of defined immunodominant CTL clones specific for peptides of HIV or HCMV, and quantified the limiting dilution analysis frequencies of CTL precursors (CTLp) specific for the same viral peptides. We found that the clonal composition of CD8+ T cells specific for HIV gag and env epitopes was highly focused and did not change after HAART. Following HAART, there was progressive contraction of HIV-specific CD8+ clones, especially in the CD28- CD27- subpopulation--the remaining cells of contracting HIV-specific clones were predominantly CD28- CD27+ CD45RO(hi). We observed maintenance of strong functional HIV-specific CD8+ T-cell responses in limiting dilution analysis following HAART, indicating preferential loss of HIV-specific cells that have reduced cloning efficiency in vitro. Following HAART, we also observed selective expansion of HCMV-specific CD8+ clones. Most HCMV-specific CD8+ clones were predominantly CD28- CD27+/- CD45RA(hi) following HAART. In one subject, a Vbeta6.4+ clone specific for HCMV pp65 selectively expanded following HAART, without expansion of two other Vbeta6.4+ clones, indicating that individual clonotypes specific for the same peptide can show different kinetics and phenotypes in response to antiretroviral therapy.

The herpesvirus Human Cytomegalovirus (HCMV) is an important opportunistic infection in recipients of allogeneic haemopoietic stem cell transplants, in whom HCMV-specific CD8+ and CD4+ T-cell responses are impaired. The nature of the HCMV-specific T-cell response in healthy virus carriers has been characterised in detail. High frequencies of circulating CD8+ T-cells that recognise defined viral peptides are maintained for years, and include individual CD8+ clones that have undergone extensive clonal expansion and phenotypic diversification in vivo. Following stem cell transplantation, the kinetics of HCMV-specific CD8+ T-cell reconstitution in the recipient are related to the presence or absence of antigen-experienced CD8+ T-cells transferred via the allograft, and to the presence of the virus in the recipient. We discuss recent progress in our understanding of HCMV-specific immunity in healthy virus carriers and in recipients after alloSCT.

CD8 + T cells specific for pp65, IE1, and IE2 are present at high frequencies in human cytomegalovirus (HCMV)-seropositive individuals, and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described, but little is known about their phenotypes and functions. Consequently, in this study, we chose to determine the diversity of HCMV-specific CD8 + T cell responses to the products of 11 HCMV open reading frames (ORFs) in a cohort of donors aged 20 to 80 years old as well as the ability of the T cells to secrete gamma interferon (IFN-γ). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8 + T cell responses by IFN-γ enzyme-linked immunospot (ELISPOT) assays to all 11 of these HCMV proteins, and across the cohort, individuals displayed a range of responses, from tightly focused to highly diverse, which were stable over time. CD8 + T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA + , CD57 + , and CD28 − , across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex vivo isolation. Taken together, our data argue that HCMV-specific CD8 + T cells have effective antiviral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion. IMPORTANCE Human cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population; however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells, and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease, the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The study presented in this paper examined immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the virus' lytic cycle immune evasion mechanisms.

Alone among herpesviruses, persistent Cytomegalovirus (CMV) markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates “immunosenescence”. This contention remains controversial and was the primary subject of the Third International Workshop on CMV & Immunosenescence which was held in Cordoba, Spain, 15-16th March, 2012. Discussions focused on several main themes including the effects of CMV on adaptive immunity and immunosenescence, characterization of CMV-specific T cells, impact of CMV infection and ageing on innate immunity, and finally, most important, the clinical implications of immunosenescence and CMV infection. Here we summarize the major findings of this workshop. The Workshop was partially supported by the University of Cordoba and the Spanish Ministry of Science (SAF2011-16169-E)

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-γ secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR β-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3–1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28−CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.

Cytomegalovirus (CMV) infection continues to be a problem in selected populations following hematopoietic stem cell transplantation (SCT). Although there have been no new antiviral agents for management of this infection in recent years, the methods for using the existing agents have improved with newer assays for detection of virus. In addition, our understanding of immunity to CMV has undergone considerable expansion. This paper will address these new aspects relating to CMV infection in the setting of SCT. In Section I Dr. Zaia reviews the pathogenesis of CMV and the current epidemiology of CMV disease following marrow or blood allo-SCT with emphasis on late-onset disease. The current lab tests available for preemptive management are summarized including the role for conventional shell vial cultures, and a comparison of the CMV antigenemia assay with the new nucleic acid-based assays, including the hybrid capture assay, the NASBA assay, and “real-time” PCR assays. Use of antiviral agents with these tests in the preemptive management of CMV infection is discussed. Ultimately, what is necessary is restoration of adequate CMV immunity, and that requires understanding the basics of the CMV-specific immune response. In Section II, Dr. Sissons traces the evolution of the CTL response from primary infection into memory and reviews recent advances in the understanding of cytotoxic T cell based immunity to CMV, based on the use of T cell clonotypic analysis and markers of T cell memory and activation, with conventional CTL functional assays. In Section III Dr. Riddell presents approaches to correction of the problem of CMV pathogenesis, namely direct restoration of the CMV-specific cellular immune deficiency. Attempts at passive therapies will be reviewed with the focus on current problems and approaches to these problems. In Section IV, Dr. Diamond presents work on the identification of multiple HLA-allele specific cytotoxic T cell epitopes specific for CMV-pp65 and - pp150. Specific epitopes are recognized by CMV-seropositive individuals including healthy donors, SCT recipients, and AIDS patients, indicating their potential usefulness as vaccines. One of these epitopes is recognized by most individuals who express the HLA A*0201 Class I allele. Pre-clinical evaluation in HLA2.1 transgenic mice of vaccine structures utilizing this epitope, and alternative delivery systems are described. Possible methods for vaccination of donor and/or recipient of a SCT as well as their limitations, utilizing synthetic or viral vaccines, are discusseed.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.