Cytomegalovirus Protein Research Articles

Regulation of the cell surface expression of classical and non-classical MHC proteins by the human cytomegalovirus UL40 and rhesus cytomegalovirus Rh67 proteins.

The protective immune response induced by a rhesus cytomegalovirus (RhCMV)-vectored simian immunodeficiency virus (SIV) vaccine in rhesus macaques depends on the presence of the viral Rh67 gene in the vaccine. The Rh67 protein contains a peptide that allows the RhCMV-infected cells to maintain expression of major histocompatibility complex (MHC) antigen E at the cell surface. We show that production of this peptide, referred to as "VL9," mirrors that of the equivalent peptide present in the human cytomegalovirus (CMV) protein UL40, despite the little sequence similarity between the two CMV proteins. We also show that the mature UL40 and Rh67 proteins, which have no previously described function, also contribute to CMV immune evasion by reducing cell surface expression of MHC proteins important for the immune system to detect infected cells. Despite these similarities, our work also reveals possible differences between Rh67 and UL40, and these may have implications for the use of human CMV as the vector for a potential HIV-1 vaccine.

IGH Complementarity Determining Region-3-Cytomegalovirus Protein Chemical Complementarity Linked to Better Overall Survival Probabilities for Glioblastoma.

Cytomegalovirus (CMV) has long been thought to have an association with glioblastoma multiforme (GBM), although the exact role of CMV and any subsequent implications for treatment have yet to be fully understood. This study addressed whether IGH complementarity determining region-3 (CDR3)-CMV protein chemical complementarity, with IGH CDR3s representing both tumor resident and blood-sourced IGH recombinations, was associated with overall survival (OS) distinctions. IGH recombination sequencing reads were obtained from (a) the Clinical Proteomic Tumor Analysis Consortium, tumor RNAseq files; and (b) the cancer genome atlas, blood exome-derived files. The Adaptive Match web tool was used to calculate chemical complementarity scores (CSs) based on hydrophobic interactions, and those scores were used to group GBM cases and assess survival probabilities. We found a higher OS probability for cases whose hydrophobic IGH CDR3-CMV protein chemical complementarity scores (Hydro CSs) were in the upper 50th percentile for several CMV proteins, including UL99 and UL123, as well as for CSs based on known B cell epitopes representing these proteins. We also identified multiple immune signature genes, including CD79A and TNFRSF17, for which higher RNA expression was associated with higher Hydro CSs. Results were consistent with the idea that stronger immunoglobulin responses to CMV are associated with better OS probabilities for GBM.

In Vitro Amplification and Selection of Engineered RNase P Ribozyme for Gene Targeting Applications.

Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications.

‘Synthesis, antiviral activity, molecular docking, and molecular dynamics studies of ethoxy phthalimide pyrazole derivatives against Cytomegalovirus and Varicella-Zoster virus: potential consequences and strategies for developing new antiviral treatments’

Substituted ethoxy phthalimide pyrazole derivatives (6a–e) have been produced using a one-pot synthesis technique. Spectral analysis was used to establish the molecular structure of the synthesized compounds, and they were examined in silico and in vitro for their ability to bind to and inhibit replication of the AD-169 strain, the Davis strain of CMV, the OKA strain and the 07/1 strain of Varicella-Zoster virus (VZV). Molecular Docking was used to estimate the binding mechanism and energy of compounds 4, 6a–e to their respective target proteins, thymidine kinase (TK), Varicella-Zoster protease (VZP) of VZV and tegument protein pp71 (TPpp71) of Cytomegalovirus (CMV). The MIC50 and EC50 were utilized to evaluate the antiviral and cytotoxic activities of test compounds in human embryonic lung (HEL) cells against the two reference medicines, Ganciclovir and Acyclovir. The chemicals studied showed a high affinity for binding sites and near binding sites of target proteins by generating H-bonds, carbon-hydrogen bonds, π-anion, π-sulfur, π-sigma, alkyl and π-alkyl interactions. All of the test compounds (6a–e) had higher binding energy than the standard medications. The ADME/T data suggests that these potential inhibitors are less toxic. Drug-protein complexes are structurally compact and demonstrate minimal conformational change in molecular dynamics (MDs) simulations, indicating stability and stiffness. MM-PBSA and post-simulation analysis can predict lead compound active cavity binding stability. By inhibiting multitargeted proteins, these synthetic compounds may improve antiviral therapy. Our research suggests that these unique synthesized chemicals may be useful and accessible adjuvant antiviral therapy for Varicella Zoster and CMV. Highlights Two components synthesis of substituted ethoxy phthalimide pyrazole derivatives (6a–e). Tested compounds (6a–e) have antiviral and cytotoxicity activity against CMV and Varicella-Zoster virus (VZV) in HEL cells. Compounds bind to TK, Varicella-Zoster protease (VZP) of VZV, and modeled TPpp71 of Cytomegalovirus (CMV). In comparison to reference drugs, compounds have strong binding free energy and interactions with VZV and CMV protein complexes. The RMSD, RMSF, Rg, residual correlative motion (RCM), No. of hydrogen bonds, protein secondary structure content, per-residue protein secondary structure and MM/PBSA energy calculated for the selected compound with thymidine kinase (TK), VZP of VZV, and modeled tegument protein pp71 (TPpp71) of CMV through MD simulation studies for 50 ns. In comparison to the two reference drugs, ligands/compounds were found to meet the Lipinski rule of five and to have strong biological activity. Communicated by Ramaswamy H. Sarma

IgG-antibodies to individual cytomegalovirus proteins and the peripheral blood lymphocyte subset profile in patients with anterior uveitis of varying severity

Background. Cytomegalovirus (CMV), a human beta-herpesvirus, persists latently lifelong after infection. CMV reactivation that occurs periodically can cause disease in target organs including the eye, not only in immunosuppressed, but also in immunocompetent people. Due to its immunosuppressive properties, persistent CMV is involved in the pathogenesis of diverse diseases. The aim was to study the features of antibody formation against six individual CMV proteins and to conduct a correlation analysis with parameters of peripheral blood lymphocyte profile in patients with anterior uveitis of varying severity (without hypopion-mild course, with hypopion-more severe course). Materials and methods. 36 patients with anterior uveitis were examined. Group I consisted of 20 patients without hypopion, group II 16 people with hypopion. No characteristic features of CMV anterior uveitis (elevated intraocular pressure, stromal iris atrophy) were found. Blood serum anti-CMV IgG antibodies (markers of chronic infection) were measured by ELISA, antibodies against individual recombinant CMV antigens were assessed by Line-Immunoassay specific to the main non-structural immediate early protein (IE), DNA-binding phosphoprotein p52, phosphoproteins of the tegument p150, p65, p28, GB-AD envelope glycoprotein. Lymphocyte subset composition was studied by flow cytometry: T-lymphocytes (CD3+), T-helper cells (CD3+CD4+CD8), T-cytotoxic (CD3+CD4CD8+), T-double-positive (CD3+CD4+CD8+), natural killers (CD16+CD56+), B-lymphocytes (CD19+). Statistical analysis was performed using StatTech v. 3.0.2 program (Stattech LLC, Russia). Results. In mild anterior uveitis, there was observed a significantly increased percentage of CD16+CD56+ and a decreased absolute number of CD3+CD8+ compared with anterior uveitis of a more severe course. Only in the group of patients with hypopion (more severe course) there was a direct significant correlation between level of antibodies against IE, p65, p28 antigens and percentage of double-positive cells (CD3+CD4+CD8+) as well as an negative significant relationship between the level of antibodies against IE, p65 and p52 antigens and CD19+ lymphocytes. Conclusion. The data obtained are consistent with few reports on the role of double-positive lymphocytes in the pathogenesis of severe forms of some viral diseases. Further studies are needed to assess an effect of individual viral antigens on CD3+CD4+CD8+ level in patients with chronic CMV infection to confirm the role of double-positive lymphocytes in the pathogenesis of a more severe course of anterior uveitis.

Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses

Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host’s immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts.

Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selectively eliminate carcinoma cells

ABSTRACT Typically, anticancer CD8pos T cells occur at low frequencies and become increasingly impaired in the tumor micro environment. In contrast, antiviral CD8pos T cells display a much higher polyclonality, frequency, and functionality. In particular, cytomegalovirus (CMV) infection induces high numbers of ‘inflationary’ CD8pos T cells that remain lifelong abundantly present in CMV-seropositive subjects. Importantly, these so-called inflationary anti-CMV T cells increase with age, maintain a ready-to-go state, populate tumors, and do not become exhausted or senescent. Given these favorable attributes, we devised a novel series of recombinant Fab-peptide-HLA-I fusion proteins and coined them ‘ReTARGs’. A ReTARG fusion protein consists of a high-affinity Fab antibody fragment directed to carcinoma-associated cell surface antigen EpCAM (or EGFR), fused in tandem with soluble HLA-I molecule/β2-microglobulin, genetically equipped with an immunodominant peptide derived from CMV proteins pp65 (or IE-1). Decoration with EpCAM-ReTARGpp65 rendered EpCAM-expressing primary patient-derived carcinoma cells highly sensitive to selective elimination by cognate anti-CMV CD8pos T cells. Importantly, this treatment did not induce excessive levels of proinflammatory T cell-secreted IFNγ. In contrast, analogous treatment with equimolar amounts of EpCAM/CD3-directed bispecific T-cell engager solitomab resulted in a massive release of IFNγ, a feature commonly associated with adverse cytokine-release syndrome. Combinatorial treatment with EpCAM-ReTARGpp65 and EGFR-ReTARGIE-1 strongly potentiated selective cancer cell elimination owing to the concerted action of the corresponding cognate anti-CMV CD8pos T cell clones. In conclusion, ReTARG fusion proteins may be useful as an alternative or complementary form of targeted cancer immunotherapy for ‘cold’ solid cancers.

Human Cytomegalovirus Protein Expression Is Correlated with Shorter Overall Survival in Breast Cancer Patients: A Cohort Study.

Background: Human cytomegalovirus (HCMV) is increasingly suggested to be involved in human carcinogenesis and onco-modulation due to its ability to contribute to all hallmarks of cancer. Growing evidence demonstrates a link between HCMV infection and various malignancies, including breast cancer, which incidence and mortality are still on the rise. The etiology of breast cancer remains mostly unclear, leaving 80% of breast cancer cases considered to be sporadic. Identifying novel risk- and prognostic factors for improved breast cancer treatment and increased survival rates, were the objectives of this study. Methods: Automated immunohistochemical staining results for HCMV proteins in 109 breast tumors and lymph node metastasis were correlated with clinical follow-up data (>10 years). Statistical analyses for median Overall Survival (OS) were performed. Results: Survival analyses revealed shorter median OS for patients with HCMV-IE positive tumors of 118.4 months compared to 202.4 months for HCMV-IE negative tumors. A higher number of HCMV-LA positive cells in the tumors was also associated with a shorter OS in patients (146.2 months vs. 151.5 months). Conclusions: Our findings suggest a link between HCMV-infections and breast cancer prognosis, which paves the way for potential novel clinical intervention and targeted therapy that may prolong the overall survival of selected patients with breast cancer.

Targeted Protein-Specific Multi-Epitope-Based Vaccine Designing against Human Cytomegalovirus by Using Immunoinformatics Approaches.

Cytomegaloviruses are emerging pathogenic agents known to cause congenital disorders in humans. In this study, immune epitopes (CTL, B cell and HTL) were screened for highly antigenic target proteins of the Human Cytomegalovirus. These shortlisted epitopes were then joined together through suitable linkers to construct multi epitope-based vaccine constructs (MEVCs). The functionality of each vaccine construct was evaluated through tertiary vaccine structure modelling and validations. Furthermore, physio-chemical properties including allergenicity, antigenicity molecular weight and many others were also predicted. The vaccine designs were also docked with the human TLR-4 receptor to demonstrate the receptor specific affinity and formed interactions. The vaccine peptides sequences were also subjected to codon optimization to confirm the potential vaccines expression in E. coli hosts. Additionally, all the MEVCs were also evaluated for immune response (IgG and IgM) induction. However, further in vivo tests are needed to ensure the efficacy of these vaccine designs.

Immunohistochemistry for hepatitis E virus capsid protein cross-reacts with cytomegalovirus-infected cells: a potential diagnostic pitfall.

Immunohistochemistry for hepatitis E virus (HEV) ORF2 (capsid) protein is a powerful tool for tissue-based diagnosis of hepatitis E, particularly useful in evaluating abnormal liver values in immunocompromised patients. We report here a previously unobserved reactivity of the HEV ORF2 antibody to human cytomegalovirus (CMV) proteins and contrast the staining patterns encountered in HEV and CMV infection, respectively. As part of a routine diagnostic work-up, the liver biopsy of an immunocompromised patient with elevated liver values was examined histologically for infection with viruses including CMV and HEV. Cytopathic changes were found, suggestive of CMV infection, which was confirmed by immunohistochemistry. Surprisingly, reactivity of a portion of CMV-infected cells with a mouse monoclonal antibody (clone 1E6) against HEV ORF2 protein was also detected. This observation prompted a screening of 22 further specimens (including liver, gastrointestinal, lung, brain and placental biopsies) with confirmed CMV infection/reactivation. Immunoreactivity of CMV-infected cells with HEV ORF2 antibody was observed in 18 of 23 specimens. While the HEV ORF2 antibody showed cytoplasmic, nuclear and canalicular positivity in hepatitis E cases, positivity in CMV-infected cells was limited to the nucleus. In conclusion, the HEV ORF2 antibody (clone 1E6) shows unexpected immunoreactivity against CMV proteins. In contrast to the hepatitis E staining pattern with cytoplasmic, nuclear and occasional canalicular positivity, reactivity in CMV-infected cells is restricted to the nucleus. Awareness of this cross-reactivity and knowledge of the differences in staining patterns will prevent pathologists from misinterpreting positive HEV ORF2 immunohistochemistry in liver specimens.

Mechanisms of Survival of Cytomegalovirus-Infected Tumor Cells.

Human cytomegalovirus (HCMV) DNA and proteins are often detected in malignant tumors, warranting studies of the role that HCMV plays in carcinogenesis and tumor progression. HCMV proteins were shown to regulate the key processes involved in tumorigenesis. While HCMV as an oncogenic factor just came into focus, its ability to promote tumor progression is generally recognized. The review discusses the viral factors and cell molecular pathways that affect the resistance of cancer cells to therapy. CMV inhibits apoptosis of tumor cells, that not only promotes tumor progression, but also reduces the sensitivity of cells to antitumor therapy. Autophagy was found to facilitate either cell survival or cell death in different tumor cells. In leukemia cells, HCMV induces a “protective” autophagy that suppresses apoptosis. Viral factors that mediate drug resistance and their interactions with key cell death pathways are necessary to further investigate in order to develop agents that can restore the tumor sensitivity to anticancer drugs.

Human Cytomegalovirus UL24 and UL43 Cooperate to Modulate the Expression of Immunoregulatory UL16 Binding Protein 1.

The human cytomegalovirus (HCMV) UL24 and UL43 are tegument proteins that have recently been shown to interact with each other in a yeast two-hybrid system. By their overexpression in MRC5 cells, we demonstrate that these viral proteins interact with several important host proteins, especially Dicer and trans-activation response RNA binding protein. As these hots proteins are involved in regulating the production of cellular micro-RNAs, the cytomegalovirus (CMV) proteins could interfere with their actions to favor viral replication directly or through an immune escape mechanism. Double knockout of UL24 and UL43 does not show a remarkable effect on CMV entry or replication, but it significantly downregulates the expression of CMV-encoded miR-UL59, which is thought to regulate the expression of a downstream target UL16 binding protein 1 (ULBP1). Interestingly, the double knockout increases the expression of the ULBP1 recognized by the NKG2D activating receptor of natural killer cells. This study investigates the potential role of several proteins encoded by HCMV in regulating the host cellular environment to favor escape from immunity, and it also provides some basis for the future development of RNA-targeted small molecules to control HCMV infection.

Insight into Viral Hijacking of CRL4 Ubiquitin Ligase through Structural Analysis of the pUL145-DDB1 Complex.

Viruses evolve mechanisms to exploit cellular pathways that increase viral fitness, e.g., enhance viral replication or evade the host cell immune response. The ubiquitin-proteosome system, a fundamental pathway-regulating protein fate in eukaryotes, is hijacked by all seven classes of viruses. Members of the Cullin-RING family of ubiquitin (Ub) ligases are frequently co-opted by divergent viruses because they can target a broad array of substrates by forming multisubunit assemblies comprised of a variety of adapters and substrate receptors. For example, the linker subunit DDB1 in the cullin 4-RING (CRL4)-DDB1 Ub ligase (CRL4DDB1) interacts with an H-box motif found in several unrelated viral proteins, including the V protein of simian virus 5 (SV5-V), the HBx protein of hepatitis B virus (HBV), and the recently identified pUL145 protein of human cytomegalovirus (HCMV). In HCMV-infected cells, pUL145 repurposes CRL4DDB1 to target STAT2, a protein vital to the antiviral immune response. However, the details of how these divergent viral sequences hijack DDB1 is not well understood. Here, we use a combination of binding assays, X-ray crystallography, alanine scanning, cell-based assays, and computational analysis to reveal that viral H-box motifs appear to bind to DDB1 with a higher affinity than the H-box motifs from host proteins DCAF1 and DDB2. This analysis reveals that viruses maintain native hot-spot residues in the H-box motif of host DCAFs and also acquire favorable interactions at neighboring residues within the H-box. Overall, these studies reveal how viruses evolve strategies to produce high-affinity binding and quality interactions with DDB1 to repurpose its Ub ligase machinery. IMPORTANCE Many different viruses modulate the protein machinery required for ubiquitination to enhance viral fitness. Specifically, several viruses hijack the cullin-RING ligase CRL4DDB1 to degrade host resistance factors. Human cytomegalovirus (HCMV) encodes pUL145 that redirects CRL4DDB1 to evade the immune system through the targeted degradation of the antiviral immune response protein STAT2. However, it is unclear why several viruses bind specific surfaces on ubiquitin ligases to repurpose their activity. We demonstrate that viruses have optimized H-box motifs that bind DDB1 with higher affinity than the H-box of native binders. For viral H-boxes, native interactions are maintained, but additional interactions that are absent in host cell H-boxes are formed, indicating that rewiring CRL4DDB1 creates a selective advantage for the virus. The DDB1-pUL145 peptide structure reveals that water-mediated interactions are critical to the higher affinity. Together, our data present an interesting example of how viral evolution can exploit a weakness in the ubiquitination machinery.

New mechanistic insights of the pathogenicity of high-risk cytomegalovirus (CMV) strains derived from breast cancer: Hope for new cancer therapy options.

Breast cancer is the most common cancer type among women, and concerningly its prevalence is steadily increasing. Some breast cancers are hereditary, but most are sporadic with an unknown cause. Accumulated evidence over the past two decades implies a very high prevalence of cytomegalovirus (CMV) in breast cancer. Detection of CMV proteins, which signifies an ongoing infection, has been found in 90%-100% of breast, colon, prostate and ovarian cancer, in sarcomas and in neural derived cancers such as glioblastoma, neuroblastoma and medulloblastoma.

Reproducibility of outcomes in sequential trials using CMV-targeted dendritic cell vaccination for glioblastoma.

2005 Background: Vaccination with dendritic cells (DCs) fares poorly in primary and recurrent glioblastoma (GBM). Moreover, GBM vaccine trials are often underpowered due to limited sample size. Methods: To address these limitations, we conducted three sequential clinical trials utilizing Cytomegalovirus (CMV)-specific DC vaccines in patients with newly diagnosed GBM eligible to receive standard of care resection and adjuvant radiation therapy and temozolomide chemotherapy. Autologous DCs were generated and electroporated with mRNA encoding for the CMV protein pp65. Serial vaccination was given throughout adjuvant temozolomide cycles, and 111Indium radiolabeling was implemented to assess migration efficiency of DC vaccines. Patients were followed for median overall survival (mOS) and OS. Results: Our initial study was the phase II ATTAC study (NCT00639639; total n = 12) with 6 patients randomized to vaccine site preconditioning with tetanus-diphtheria (Td) toxoid. This led to an expanded cohort trial (ATTAC-GM; NCT00639639) of 11 patients receiving CMV DC vaccines containing granulocyte-macrophage colony-stimulating factor (GM-CSF). Follow-up data from ATTAC and ATTAC-GM revealed 5-year OS rates of 33.3% (mOS 38.3 months; CI95 17.5-undefined) and 36.4% (mOS 37.7 months; CI95 18.2-109.1), respectively. ATTAC additionally revealed a significant increase in DC migration to draining lymph nodes following Td preconditioning ( P = 0.049). Increased DC migration was associated with OS (Cox proportional hazards model, HR = 0.820, P = 0.023). Td-mediated increased migration has been recapitulated in our larger confirmatory trial ELEVATE (NCT02366728) of 43 patients randomized to preconditioning (Wilcoxon rank sum, Td n = 24, unpulsed DC n = 19; 24h, P = 0.031 and 48h, P = 0.0195). In ELEVATE, median follow-up of 42.2 months revealed significantly longer OS in patients randomized to Td ( P = 0.026). The 3-year OS for Td-treated patients in ELEVATE was 34% (CI95 19-63%) compared to 6% given unpulsed DCs (CI95 1-42%). Conclusions: We report reproducibility of our findings across three sequential clinical trials using CMV pp65 DCs. Despite their small numbers, these successive trials demonstrate consistent survival outcomes, thus supporting the efficacy of CMV DC vaccine therapy in GBM. Clinical trial information: NCT00639639, NCT02366728.

Cytomegalovirus proteins, maternal pregnancy cytokines, and their impact on neonatal immune cytokine profiles and acute lymphoblastic leukemogenesis in children.

Not available.

Dual inhibition of innate immunity and apoptosis by human cytomegalovirus protein UL37x1 enables efficient virus replication.

Immune evasion and inhibition of apoptosis are required for successful virus infection. However, inhibition of apoptosis can increase antiviral immune responses, which can then clear viral infections. Here we show that human cytomegalovirus (HCMV)-encoded UL37 exon-1 protein (UL37x1) not only inhibits apoptosis but also suppresses the cGAS-STING immune pathway. Using co-immunoprecipitation assays, we found that UL37x1 binds to TBK1 to abrogate the TBK1-STING-IRF3 interaction. Although the anti-apoptosis function of UL37x1 increases immune signalling, the immunosuppressive role of UL37x1 counteracts this undesirable side-effect. Furthermore, we used mutational analyses to show that the loss of either immunosuppressive or anti-apoptotic function of UL37x1 significantly reduced HCMV replication in human primary foreskin fibroblasts and humanized mice by over twofold. Finally, loss of both functions resulted in over fourfold reduction of HCMV replication in the same cell type and mouse model, showing that both UL37x1 functions are crucial for HCMV infection. We conclude that this sophisticated mechanism enables HCMV to control innate immunity and apoptosis to ensure efficient infection.

Characteristics of antibody production to individual cytomegalovirus proteins in children with keratitis and uveitis

The clinical diagnosis of cytomegalovirus (CMV) eye pathology in children needs laboratory confirmation. The disadvantages of determining antibodies to CMV in serum in enzyme-linked immunosorbent assay (ELISA) include the presence of false positive and false negative results.
 AIM: To determine the features of the synthesis of antibodies to the proteins of the tegument pp65, pp150, pp28 and the DNA-binding protein pp52 of cytomegalovirus, as well as to compare the diagnostic effectiveness of two laboratory methods of serodiagnostics of CMVI in children with uveitis and keratitis of different genesis: enzyme immunoassay and linear immunolysis.
 MATERIAL AND METHODS: A total of 30 children (age 516 years) with uveitis (n=14) and keratitis (n=16) were included in the study. The IgM and IgG antibodies to the immediate early (IE) and late antigens of the virus (serological markers of primary, chronic, and reactivation of chronic infection) were determined in the ELISA. The IgG antibodies to individual recombinant CMV antigens containing only immunodominant protein fragments of viral antigens were studied in the line immunoassay (LIA): the main nonstructural IE protein, DNA-binding phosphoprotein p52, and phosphoproteins of the tegument p150, p65, and p28. The results of LIA were evaluated visually.
 RESULTS: CMV infection (CMVI) in children with uveitis in ELISA was almost 2 times higher than that with keratitis (10/14%71% vs. 6/16%37.5%, p 0.05). Of the four positive results for detection of antibodies to IE antigen in ELISA, one was confirmed in LIA. In general, the discrepancy between the results of the determination of IgG antibodies to IE antigen in ELISA and LIA was noted in 13% of serum samples. LIA showed the increased frequency (p 0.05) and intensity (p 0.05) of antibody formation to viral antigens p65 and p52 in uveitis compared with keratitis, which confirmed the important role of CMVI in the pathogenesis of uveitis.
 CONCLUSION: Both methods revealed a higher level of CMVI in children with uveitis than those with keratitis. IgG antibodies to IE-antigen serological markers of the reactivation of chronic CMVI have clinical importance because they serve as the basis for the appointment of antiviral therapy. Discrepancies between the results of ELISA and LIA in the detection of antibodies to IE antigen indicate the expediency of confirming the results of ELISA in LIA.
 Thus, laboratory examination of sera in ELISA with subsequent analysis of antibodies to individual recombinant CMV antigens in LIA is an effective, highly sensitive, and highly specific method to verify CMVI and determine its activity. The most informative action is the determination of IgG antibodies to recombinant CMV antigens: IE antigens, p65, and p52.

Development and Characterization of an HCMV Multi-Antigen Therapeutic Vaccine for Glioblastoma Using the UNITE Platform.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with a median survival of 15 months that has remained unchanged despite advances in the standard of care. GBM cells express human cytomegalovirus (HCMV) proteins, providing a unique opportunity for targeted therapy. We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins pp65, gB, and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated membrane protein 1 (LAMP1) with target ntigens. We demonstrate evidence of increased antigen presentation by both MHC-I and -II, delivering a robust antigen-specific CD4 and CD8 T-cell response in addition to a strong humoral response. Using a syngeneic orthotopic GBM mouse model, therapeutic treatment with the ITI-1001 vaccine resulted in ~56% survival of tumor-bearing mice. Investigation of the tumor microenvironment showed significant CD4 infiltration as well as enhanced Th1 and cytotoxic CD8 T activation. Regulatory T cells were also upregulated after ITI-1001 vaccination. In addition, tumor burden negatively correlated with activated interferon (IFN)γ+ CD4 T cells, reiterating the importance of CD4 activation in ITI-1001 efficacy and in identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+, and CD8+ T cells in responders compared to non-responders. Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant antitumor activity, leading to enhanced survival in a mouse model of GBM.

Mechanisms of Survival of Cytomegalovirus-Infected Tumor Cells

Human cytomegalovirus (HCMV) DNA and proteins are often detected in malignant tumors, warranting studies of the role that HCMV plays in carcinogenesis and tumor progression. HCMV proteins were shown to regulate the key processes involved in tumorigenesis. While HCMV as an oncogenic factor just came into focus, its ability to promote tumor progression is generally recognized. The review discusses the viral factors and cell molecular pathways that affect the resistance of cancer cells to therapy. CMV inhibits apoptosis of tumor cells, that not only promotes tumor progression, but also reduces the sensitivity of cells to antitumor therapy. Autophagy was found to facilitate either cell survival or cell death in different tumor cells. In leukemia cells, HCMV induces a "protective" autophagy that suppresses apoptosis. Viral factors that mediate drug resistance and their interactions with key cell death pathways are necessary to further investigate in order to develop agents that can restore the tumor sensitivity to anticancer drugs.