Human Cytomegalovirus Antigens Research Articles

Molecular Detection of Epstein-Barr Virus and Human Cytomegalovirus Antigen Expression in Breast Cancer in Khartoum State, Sudan 2018

Molecular Detection of Epstein-Barr Virus and Human Cytomegalovirus Antigen Expression in Breast Cancer in Khartoum State, Sudan 2018

Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping.

T cell immunity is traditionally assessed through functional recall assays, which detect the consequences of the T cells' antigen encounter, or via fluorescently labeled multimers that selectively bind peptide-specific T cell receptors. Using either approach, if the wrong antigen or peptide of a complex antigenic system, such as a virus, is used for immune monitoring, either false negative data will be obtained, or the magnitude of the antigen-specific T cell compartment will go largely underestimated. In this work, we show how selection of the “right” antigen or antigenic peptides is critical for successful T cell immune monitoring against human cytomegalovirus (HCMV). Specifically, we demonstrate that individual HCMV antigens, along with previously reported epitopes, frequently failed to detect CD8+ T cell immunity in test subjects. Through systematic assessment of T cell reactivity against individual nonamer peptides derived from the HCMVpp65 protein, our data clearly establish that (i) systematic testing against all potential epitopes encoded by the genome of the antigen of interest is required to reliably detect CD8+ T cell immunity, and (ii) genome-wide, large scale systematic testing of peptides has become feasible through high-throughput ELISPOT-based “brute force” epitope mapping.

Multiantigenic Modified Vaccinia Virus Ankara Vaccine Vectors To Elicit Potent Humoral and Cellular Immune Reponses against Human Cytomegalovirus in Mice.

As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in protection against HCMV infection and disease. Here we describe a vaccine approach based on the well-characterized modified vaccinia virus Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show that in mice, multiantigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the individual antigens. In addition, we demonstrate that the PC/gB antigen combination of these multiantigenic MVA vectors can enhance the stimulation of humoral immune responses that mediate in vitro neutralization of different HCMV strains and antibody-dependent cellular cytotoxicity. These results support the use of MVA to develop a multiantigenic vaccine candidate for controlling HCMV infection and disease in different target populations, such as pregnant women and transplant recipients.IMPORTANCE The development of a human cytomegalovirus (HCMV) vaccine to prevent congenital disease and transplantation-related complications is an unmet medical need. While many HCMV vaccine candidates have been developed, partial success in preventing or controlling HCMV infection in women of childbearing age and transplant recipients has been observed with an approach based on envelope glycoprotein B (gB). We introduce a novel vaccine strategy based on the clinically deployable modified vaccinia virus Ankara (MVA) vaccine vector to elicit potent humoral and cellular immune responses by multiple immunodominant HCMV antigens, including gB, phosphoprotein 65, and all five subunits of the pentamer complex. These findings could contribute to development of a multiantigenic vaccine strategy that may afford more protection against HCMV infection and disease than a vaccine approach employing solely gB.

Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors.

Human cytomegalovirus (HCMV) represents a major cause of clinical complications during pregnancy as well as immunosuppression, and the licensing of a protective HCMV vaccine remains an unmet global need. Here, we designed and validated novel Sendai virus (SeV) vectors delivering the T cell immunogens IE-1 and pp65. To enhance vector safety, we used a replication-deficient strain (rdSeV) that infects target cells in a nonproductive manner while retaining viral gene expression. In this study, we explored the impact that transduction with rdSeV has on human dendritic cells (DCs) by comparing it to the parental, replication-competent Sendai virus strain (rcSeV) as well as the poxvirus strain modified vaccinia Ankara (MVA). We found that wild-type SeV is capable of replicating to high titers in DCs while rdSeV infects cells abortively. Due to the higher degree of attenuation, IE-1 and pp65 protein levels mediated by rdSeV after infection of DCs were markedly reduced compared to those of the parental Sendai virus recombinants, but antigen-specific restimulation of T cell clones was not negatively affected by this. Importantly, rdSeV showed reduced cytotoxic effects compared to rcSeV and MVA and was capable of mediating DC maturation as well as secretion of alpha interferon and interleukin-6. Finally, in a challenge model with a murine cytomegalovirus (MCMV) strain carrying an HCMV pp65 peptide, we found that viral replication was restricted if mice were previously vaccinated with rdSeV-pp65. Taken together, these data demonstrate that rdSeV has great potential as a vector system for the delivery of HCMV immunogens.IMPORTANCE HCMV is a highly prevalent betaherpesvirus that establishes lifelong latency after primary infection. Congenital HCMV infection is the most common viral complication in newborns, causing a number of late sequelae ranging from impaired hearing to mental retardation. At the same time, managing HCMV reactivation during immunosuppression remains a major hurdle in posttransplant care. Since options for the treatment of HCMV infection are still limited, the development of a vaccine to confine HCMV-related morbidities is urgently needed. We generated new vaccine candidates in which the main targets of T cell immunity during natural HCMV infection, IE-1 and pp65, are delivered by a replication-deficient, Sendai virus-based vector system. In addition to classical prophylactic vaccine concepts, these vectors could also be used for therapeutic applications, thereby expanding preexisting immunity in high-risk groups such as transplant recipients or for immunotherapy of glioblastomas expressing HCMV antigens.

HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition.

Immune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-EUL40 CD8 αβT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host’s HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vβ repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 CD8 T cells are reactive toward a broader set of nonapeptides varying in 1–3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and life-long lasting HLA-EUL40 CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance.

Evaluation of human cytomegalovirus antigen expression in invasive breast carcinoma in a population of Iranian patients

BackgroundThe role of human cytomegalovirus (HCMV) in the development of breast carcinoma is questionable. The aim of this study was to evaluate the expression of the immediate early antigen (IE) of HCMV in breast carcinoma and its association with some clinicopathologic factors in a population of Iranian patients.MethodsFormalin-fixed and paraffin-embedded tissue blocks from the pathology laboratories of the Azahra and Shahid Beheshti hospitals, Isfahan, Iran, from 2013 to 2016, were used in the study. We used immunohistochemistry and real-time PCR to detect the IE-antigen of HCMV in breast carcinoma, normal tissue adjacent to carcinoma, and normal tissue from mammoplasty specimens.ResultsA total of 96 samples were evaluated: 70 invasive breast carcinoma of different histologic subtypes and 26 mammoplasty normal breast tissues. All the samples were negative for IE-antigen expression. No relationship was seen between breast cancer and HCMV in this study.ConclusionsThe results of this study failed to show any relationship between HCMV and the development of breast carcinoma.

Functionally inactivated dominant viral antigens of human cytomegalovirus delivered in replication incompetent adenovirus type 6 vectors as vaccine candidates

ABSTRACTT cell immunity is critical in controlling human cytomegalovirus (HCMV) infection in transplant recipients, and T cells targeting viral immediate early proteins such as IE1, IE2 and pp65 have been speculated to be more effective against reactivation. Here we report efforts to construct replication incompetent adenovirus 6 vectors expressing these viral antigens as vaccine candidates. To reduce the potential liabilities of these viral proteins as vaccine antigens, we introduced mutations to inactivate their reported functions including their nuclear localization signals. The modifications greatly reduced their localization to the nuclei, thus limiting their interactions with cellular proteins important for cell cycle modulation and transactivation. The immunogenicity of modified pp65, IE1 and IE2 vaccines was comparable to their wild-type counterparts in mice and the immunogenicity of the modified antigens was demonstrated in non-human primates.

Human cytomegalovirus (HCMV) UL44 and UL57 specific antibody responses in anti-HCMV-positive patients with systemic sclerosis.

The role of human cytomegalovirus (HCMV) has been postulated as a trigger of systemic sclerosis (SSc). The aim of the study was to assess the prevalence of antibodies against HCMV UL44 and UL57 antigens not tested in the past. Sixty SSc patients, 40 multiple sclerosis and 17 normal controls (NCs), all anti-HCMV positive, were tested by immunoblotting. Reactivity to HCMV antigens, expressed as arbitrary units (AUs), was assessed for correlation with clinical and immunological parameters, including types of SSc-related autoantibodies. Anti-UL44 and anti-UL57 HCMV antibodies were present in 3/60 (5%) and 58/60 (96.7%) SSc patients, respectively (p<0.001). Anti-UL57 antibodies were present in 35/40 (87.5%) MS patients and 16/17 (94.1%) NCs (SSc vs MS, MS vs NC, p=ns). Strong (50-75 AU) and very strong (75-100AU) anti-UL57 immunoreactivity was found in 24 (41.4%) and 22 (37.9%) SSc patients, respectively (p=ns). Dilution experiments showed anti-UL57 antibody persistence in up to 1/5000. Overall, there was no difference in the frequency or the magnitude of anti-UL57 immunoreactivity between diffuse cutaneous systemic sclerosis and limited cutaneous systemic sclerosis patients (96.67 vs 96.67%; 65.45±20.19 vs 64.31±21.11AU, p>0.05) but strong anti-UL57 reactivity were more frequent in SSc compared to NCs (p=0.007). Anti-UL57 reactivity was not inhibited by SSc-specific autoantigens. Anti-UL57 seropositivity did not correlate with demographic, clinical or immunological features of SSc. Anti-HCMV UL57 antibodies are universally present in anti-HCMV-positive patients with SSc, while those against UL44 are rarely seen. Because anti-UL57 lack disease specificity and are not involved in cross-reactive responses, their immunopathogenetic potential is to be questioned.

A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line.

Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in comparative analyses of different human herpesviruses.

Reconstitution of cytomegalovirus-specific T-cell response in allogeneic hematopoietic stem cell recipients: the contribution of six frequently recognized, virus-encoded ORFs.

The reactivation of human cytomegalovirus (HCMV) in immunosuppressed patients is associated with significant morbidity. Testing HCMV-specific T-cell responses can help determine which patients are at high risk of HCMV disease. We optimized selection of HCMV antigens for detection of T-cell response of patients after allogeneic hematopoietic stem cell transplantation (HSCT) with the aim of identifying patients with insufficient control of HCMV reactivation. T-cell immune response to HCMV was monitored in 30 patients during the first year after HSCT. The HSCT recipients were classified according to their anti-HCMV T-cell response and the presence of HCMV DNA in the blood. We observed an inverse relationship between the magnitude of HCMV-specific T-cell responses against CMV lysate, phosphoprotein (pp) 65, immediate early-1 (IE-1), UL36, and UL55, but not to US3 and US29 detected by interferon-gamma (IFNγ)- ELISPOT and the level of HCMV DNA in the blood of patients during the 30 days following sampling. The study has revealed that patients who received a graft from a seronegative donor have a lower T-cell response against HCMV and increased probability of HCMV reactivation in comparison to the patients who had received their graft from a seropositive donor. The individual peptide pools and native HCMV antigens were useful for monitoring the time course of the anti-HCMV response by IFNγ-ELISPOT, which proved to have a prognostic value. Besides widely employed peptide pools of pp65 and IE-1, the use of antigens UL36 and UL55, but not US3 or US29, increased sensitivity of the test.

Responses to fibroblast-adapted human cytomegalovirus (HCMV) vaccines conform to CD8 T cell recognition paradigms

Abstract Recently, a rhesus CMV-based SIV vaccine that clears SIV from half of vaccinated monkeys was shown to elicit unconventional CD8 T cell responses to both SIV and RhCMV (IE1) antigens in all immunized monkeys. These responses were characterized by i) recognition of an unusually large number of peptide epitopes within each antigen, and ii) MHCII restriction by ~2/3 of responding CD8 T cells (Hansen et al, Science, 2013). Unconventional responses were only elicited by fibroblast-adapted rhCMV strain 68-1 that lacks ability to infect cells other than fibroblasts due to loss of pentameric viral glycoprotein expression. To determine whether fibroblast-adapted human CMV (HCMV) vaccines also elicit unconventional CD8 T cell responses, we exploited an ongoing phase I clinical trial of live HCMV vaccines. These vaccines have similar fibroblast-restricted tropism to rhCMV68-1, although the precise genetic lesions differ. We analyzed CD8 T cell responses in 12 seroconverters by stimulating PBMC with overlapping peptide pools covering 20 HCMV antigens. Seven subjects in this low dose trial (100 or 1000 pfu) made consistent, measurable CD8 T cell responses. All 7 responded to IE1; 3 also responded to 1–2 additional antigens. We have mapped responses to individual 15mers in 3 subjects, and all revealed highly focused responses to 1–3 epitopes within IE1. In one HLA-B08 positive subject, the two responses mapped to known immunodominant B08-restricted IE1 epitopes. We have mapped 4 other epitopes to date; of these, 3 are 9mers and are likely conventional, but one may be a 13mer. Thus far, in contrast to RhCMV in monkeys, the CD8 T cell response to fibroblast-adapted HCMV vaccines in humans appears to be predominantly conventional. Supported by NIH R21 AI116107

Potential Crossreactivity of Human Immune Responses Against HCMV Glycoprotein B.

Antigens of infectious agents share amino acid sequences with human proteins. Such a peptide matching may lead to autoimmunity through crossreactivity phenomena following pathogen infection and/or immunotherapeutic approaches. In this framework, we analyzed as a model the primary sequence of human cytomegalovirus (HCMV) glycoprotein B (gB) protein and searched for viral peptide sequences shared with human proteins. We show that the HCMV antigen has a high peptide identity with a large number of human proteins at the penta-, hexa-, and heptapeptide level, with the viral versus human peptide overlap involving host proteins implicated in crucial processes such as embryonic development, spermatogenesis, spatial learning, and hippocampal plasticity, inter alia. This study might help understand the etiology of the pathologic sequela associated with HCMV (re)activation and, in addition, address scientific and clinical research toward the definition of antiviral therapeutics based on non-crossreactive viral sequences.

Diversified phenotype of antigen specific CD8+ T cells responding to the immunodominant epitopes of IE and pp65 antigens of human cytomegalovirus

To study the cytomegalovirus (CMV)-specific CD8+ T cells in individuals with HLA A∗1101, A∗0201 and A∗2402, our findings showed that peptide SK-10-2, KI-10 and KV-10 of CMV IE and pp65 antigens were immunodominant in 198 individuals with HLA A∗1101, A∗0201 and A∗2402, the most frequent genotypes in Chinese. Interestingly, SK-10-2 induced the strongest T cell response to produce IFN-γ whereas the others did not induce prominent IFN-γ production despite they all induced remarkable T cell proliferation. The peptides induced different phenotypes including IFN-γhighTNF-αlow and TNF-αlowFoxp3low. It suggests that only some of CMV-reactive CD8+ T cells are real protective IFN-γhigh cytotoxic T cells.

Disposable immunosensor for human cytomegalovirus glycoprotein B detection

Combining the advantages of the biosensor field with the problem of detecting Human Cytomegalovirus (HCMV) in human samples, an inexpensive, simple and disposable electrochemical immunosensor for glycoprotein B detection in urine is proposed. Glycoprotein B has been chosen once is the dominant antigen of HCMV. The approach is based on a sandwich-type immunoassay, with the HCMV glycoprotein B sandwiched between the Anti-HCMV antibody adsorbed onto the working electrode, and the Anti-HCMV labeled with gold nanoparticles. Glycoprotein B detection was carried out through electrochemical stripping analysis of silver nanoparticles quantitatively deposited on the immunosensor through catalysis by the nanogold labels. The influence of different steps in the immunosensor construction, namely the silver deposition time, silver enhancer concentration, antibody concentration, BSA concentration and glycoprotein B incubation time, were examined and optimized. The method showed a linear dependence between glycoprotein B concentration and the corresponding anodic stripping peak current, resulting in detection limits of 3.3±1.7ng/mL for samples prepared in buffer and 3.2±0.2ng/mL for urine samples, suggesting that the biological matrix does not interfere with the immunosensor detection capability. Given its mode of preparation, by physical adsorption of the capture antibody in the working electrode, the immunosensor also exhibited an acceptable reproducibility, with a residual standard deviation of 13.5% for samples prepared in buffer and 11.2% for urine samples, thereby presenting a promising development potential for clinical applications.

Detection of human cytomegalovirus in different histopathological types of glioma in Iraqi patients.

Human Cytomegalovirus (HCMV) is an endemic herpes virus that reemerges in cancer patients enhancing oncogenic potential. HCMV infection is associated with certain types of cancer morbidity such as glioblastomas. HCMV, like all other herpes viruses, has the ability to remain latent within the body of the host and can contribute in chronic inflammation. To determine the role of HCMV in glioma pathogenesis, paraffin-embedded blocks from glioma patients (n = 50) and from benign meningioma patients (n = 30) were obtained and evaluated by immunohistochemistry and polymerase chain reaction for the evidence of HCMV antigen expression and the presence of viral DNA. We detected HCMV antigen and DNA for IEI-72, pp65, and late antigen in 33/36, 28/36, and 26/36 in glioblastoma multiforme patients whereas 12/14, 10/14, and 9/14 in anaplastic astrocytoma patients, respectively. Furthermore, 84% of glioma patients were positive for immunoglobulin G (IgG) compared to 72.5% among control samples (P = 0.04). These data indicate the presence of the HCMV virus in a high percentage of glioma samples demonstrating distinct histopathological grades and support previous reports showing the presence of HCMV infection in glioma tissue. These studies demonstrate that detection of low-levels of latent viral infections may play an active role in glioma development and pathogenesis.

Human cytomegalovirus antigens in malignant gliomas as targets for adoptive cellular therapy.

Malignant gliomas are the most common primary brain tumor in adults, with over 12,000 new cases diagnosed in the United States each year. Over the last decade, investigators have reliably identified human cytomegalovirus (HCMV) proteins, nucleic acids, and virions in most high-grade gliomas, including glioblastoma (GBM). This discovery is significant because HCMV gene products can be targeted by immune-based therapies. In this review, we describe the current level of understanding regarding the presence and role in pathogenesis of HCMV in GBM. We describe our success detecting and expanding HCMV-specific cytotoxic T lymphocytes to kill GBM cells and explain how these cells can be used as a platform for enhanced cellular therapies. We discuss alternative approaches that capitalize on HCMV infection to treat patients with HCMV-positive tumors. Adoptive cellular therapy for HCMV-positive GBM has been tried in a small number of patients with some benefit, but we reason why, to date, these approaches generally fail to generate long-term remission or cure. We conjecture how cellular therapy for GBM can be improved and describe the barriers that must be overcome to cure these patients.

The oncogenic potential of human cytomegalovirus and breast cancer.

Breast cancer is the leading causes of cancer-related death among women. The vast majority of breast cancers are carcinomas that originate from cells lining the milk-forming ducts of the mammary gland. Numerous articles indicate that breast tumors exhibit diverse phenotypes depending on their distinct physiopathological signatures, clinical courses, and therapeutic possibilities. The human cytomegalovirus (HCMV) is a multifaceted highly host specific betaherpesvirus that is regarded as asymptomatic or mildly pathogenic virus in immunocompetent host. HCMV may cause serious in utero infections as well as acute and chronic complications in immunocompromised individual. The involvement of HCMV in late inflammatory complications underscores its possible role in inflammatory diseases and cancer. HCMV targets a variety of cell types in vivo, including macrophages, epithelial cells, endothelial cells, fibroblasts, stromal cells, neuronal cells, smooth muscle cells, and hepatocytes. HCMV can be detected in the milk after delivery and thereby HCMV could spread to adjacent mammary epithelial cells. HCMV also infects macrophages and induces an atypical M1/M2 phenotype, close to the tumor-associated macrophage phenotype, which is associated with the release of cytokines involved in cancer initiation or promotion and breast cancer of poor prognosis. HCMV antigens and DNA have been detected in tissue biopsies of breast cancers and elevation in serum HCMV IgG antibody levels has been reported to precede the development of breast cancer in some women. In this review, we will discuss the potential role of HCMV in the initiation and progression of breast cancer.

Human cytomegalovirus Fcγ binding proteins gp34 and gp68 antagonize Fcγ receptors I, II and III.

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.

Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice

Human cytomegalovirus (HCMV) is a member of the β-herpesvirus family that causes significant disease worldwide. Although evidence exists that neutralizing antibodies and cytotoxic T cell responses to HCMV antigens can prevent HCMV disease and/or infection, there are no approved vaccines to prevent HCMV disease. Over the past 10 years, multiple HCMV vaccines have been tested in man but only partial protection has been achieved in these studies. HCMV contains multiple surface-expressed glycoproteins that are critical to viral entry, including gB, the gM/gN complex, the gH/gL complex, and a pentameric gH/gL/UL128/UL130/UL131A complex. Recently we showed that viral replicon particles (VRPs) expressing the gH/gL complex elicited more potently neutralizing antibodies than VRPs expressing gB in mice. Here we compare the immunogenicity of VRPs encoding the HCMV gH/gL and pentameric complexes, as well as purified gH/gL and pentameric complexes administered in the presence or absence of the MF59 adjuvant. The results of these studies indicate that the pentameric complex elicits significantly higher levels of neutralizing antibodies than the gH/gL complex, and that MF59 significantly increases the potency of each complex. In addition, we show that animals immunized with pentamer encoding VRPs or the pentameric subunit produce antibodies that recognize a broad range of antigenic sites on the complex. Taken together, these studies support the utility of the pentameric complex in HCMV vaccine candidates.

Human cytomegalovirus induces upregulation of arginase II: possible implications for vasculopathies

Both human cytomegalovirus (HCMV) and arginase II (ARG II) have been implicated in the pathogenesis of cardiovascular diseases. The effects of HCMV on ARG II are unknown. The aim of this study was to investigate the effects of HCMV on ARG II expression in endothelial and vascular smooth muscle cells (SMC) both in vitro and ex vivo. Endothelial and SMC were infected with either HCMV or UV-irradiated HCMV. Expression of ARG II, endothelial or inducible nitric oxide synthase (eNOS and iNOS, respectively) and viral immediate early (IE) was quantified using quantitative PCR. Ganciclovir and short interfering RNA were used to determine the viral gene mediating the effects on ARG II. Detection of viral antigens and ARG II expression was performed by immunofluorescence or immunohistochemistry. HCMV infection increased both ARG II mRNA and protein levels in the examined cells; this effect was mediated by the HCMV IE2-p86 protein. The upregulation of ARG II was accompanied by a downregulation of eNOS but an induction of iNOS in HCMV-infected endothelial cells. Both eNOS and iNOS expressions were induced in HCMV-infected SMC. ARG II was abundantly expressed in endothelial cells, foam cells and SMC and was importantly significantly upregulated in HCMV-immunoreactive human carotid atherosclerotic plaques. HCMV IE2-p86 mediates ARG II upregulation in vitro and ARG II is co-expressed with HCMV antigens in human carotid atherosclerotic plaques. We speculate that HCMV may contribute to endothelial dysfunction via ARG II induction and reduced eNOS production.