Mutational mapping of pUL131A of human cytomegalovirus emphasizes its central role for endothelial cell tropism.

The UL131A protein is part of a pentameric variant of the gcIII complex in the virion envelope of human cytomegalovirus (HCMV), which has been found essential for efficient entry into endothelial cells (ECs). Using a systematic mutational scanning approach, we aimed to define peptide motifs within the UL131A protein that contribute to EC infection. Mutant viruses were generated in which charged amino acids within frames of 2 to 6 amino acids were replaced with alanines. The resulting viruses were evaluated with regard to their potential to infect EC cultures. Four clusters of charged amino acids essential for EC infection were identified (amino acids 22 to 27, 32 to 35, 64 to 69, and 116 to 121). Mutations of individual charge clusters within amino acids 72 to 104 caused minor reductions of EC tropism, but these effects were additive in a combined mutation, showing that this region also contributes to EC tropism. Only charge clusters within amino acids 46 to 58 were found irrelevant for EC infection. In conclusion, the unusual sensitivity to mutations, together with the remarkable conservation of the UL131A protein, emphasizes its particular role for EC tropism of HCMV.

West Nile virus noncoding subgenomic RNA contributes to viral evasion of the type I interferon-mediated antiviral response.

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.

Mutational mapping of UL130 of human cytomegalovirus defines peptide motifs within the C-terminal third as essential for endothelial cell infection.

The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.

Charge cluster-to-alanine scanning of UL128 for fine tuning of the endothelial cell tropism of human cytomegalovirus.

The viral genes UL128, UL130, and UL131A have been identified as major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV), with deletion of either gene causing a null phenotype. We hypothesized that a functional scanning of these genes by minor genetic modifications would allow for the generation of mutants with an intermediate phenotype. By combining charge cluster-to-alanine (CCTA) mutagenesis with markerless mutagenesis of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain, we analyzed UL128 in order to identify functional sites and hence enable targeted modulation of the EC tropism of HCMV. A total of nine mutations in eight charge clusters were tested. Three of the CCTA mutations severely reduced EC tropism, three were irrelevant, two had a weak effect on cell tropism, and one mutation in the most C-terminal cluster caused an intermediate phenotype. All of the highly effective mutations were located in a core region (amino acids 72 to 106) which appears to be particularly crucial for EC tropism. The intermediate effect of mutations in the C-terminal cluster could be modulated by varying the number of amino acids replaced with alanine. This study provides a rational approach for targeted modulation of HCMV cell tropism, which may aid in the development of HCMV strains with a desired degree of attenuation.

Pre-emptive and therapeutic adoptive immunotherapy for nasopharyngeal carcinoma: Phenotype and effector function of T cells impact on clinical response.

Adoptive T cell therapy has emerged as a powerful strategy to treat human cancers especially haematological malignancies. Extension of these therapies to solid cancers remains a significant challenge especially in the context of defining immunological correlates of clinical responses. Here we describe results from a clinical study investigating autologous Epstein-Barr virus (EBV)-specific T cells generated using a novel AdE1-LMPpoly vector to treat patients with nasopharyngeal carcinoma (NPC) either pre-emptively in at-risk patients with no or minimal residual disease (N/MRD) or therapeutically in patients with active recurrent/metastatic disease (ARMD). Tolerability, safety and efficacy, including progression-free survival (PFS) and overall survival (OS), were evaluated following adoptive T-cell immunotherapy. Twenty-nine patients, including 20 with ARMD and nine with N/MRD, successfully completed T-cell therapy. After a median follow-up of 18.5 months, the median PFS was 5.5 months (95% CI 2.1 to 9.0 months) and the median OS was 38.1 months (95% CI 17.2 months to not reached). Post-immunotherapy analyses revealed that disease stabilization in ARMD patients was significantly associated with the functional and phenotypic composition of in vitro-expanded T cell immunotherapy. These included a higher proportion of effector CD8+ T-cells and an increased number of EBV-specific T-cells with broader antigen specificity. These observations indicate that adoptive immunotherapy with AdE1-LMPpoly-expanded T cells stabilizes relapsed, refractory NPC without significant toxicity. Promising clinical outcomes in N/MRD patients further suggest a potential role for this approach as a consolidation treatment following first-line chemotherapy.

Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients.

Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as "off-the-shelf" therapeutics as well as autologous T-cell therapies for transplant patients.

Cytomegalovirus as a novel target for immunotherapy of glioblastoma multiforme.

Progress in the treatment of glioblastoma multiforme (GBM) over the last few decades has remained marginal and GBM is still universally fatal with short survival times after initial diagnosis. Much research is focused on finding new therapeutics for GBM and immune-based approaches have shown great promise. The detection of cytomegalovirus (CMV) antigens in malignant cells has suggested that treatment strategies based on immunological intervention, such as adoptive transfer of antiviral T cells or vaccination with viral epitopes, could be exploited as cancer therapy. Here, we review the rationale for using CMV as a therapeutic target and discuss the first clinical evidence for safety and efficacy of CMV-specific cellular immunotherapy for GBM.

Cellular immunotherapy directed against human cytomegalovirus as a novel approach for glioblastoma treatment.

Glioblastoma multiforme (GBM) has a very poor prognosis, despite multimodal therapy including surgery, radiation and chemotherapy. A novel adoptive immunotherapy that exploits the presence of cytomegalovirus antigens in malignant brain cancer cells has been shown to be safe and elicit potential clinical benefit for the treatment of recurrent GBM.

Induction of innate immune signatures following polyepitope protein-glycoprotein B-TLR4&9 agonist immunization generates multifunctional CMV-specific cellular and humoral immunity.

Recent studies have suggested that a successful subunit human cytomegalovirus (CMV) vaccine requires improved formulation to generate broad-based anti-viral immunity following immunization. Here we report the development of a non-live protein-based vaccine strategy for CMV based on a polyepitope protein and CMV glycoprotein B (gB) adjuvanted with TLR4 and/or TLR9 agonists. The polyepitope protein includes contiguous multiple MHC class I-restricted epitopes with an aim to induce CD8(+) T cell immunity, while gB is an important target for CD4(+) T cell immunity and neutralizing antibodies. Optimal immunogenicity of this bivalent non-live protein vaccine formulation was dependent upon the co-administration of both the TLR4 and TLR9 agonist, which was associated with the activation of innate immune signatures and the influx of different DC subsets including plasmacytoid DCs and migratory CD8-DEC205+CD103-CD326- langerin-negative dermal DCs into the draining lymph nodes. Furthermore these professional antigen presenting cells also expressed IL-6, IL-12p70, TNFα, and IFNα which play a crucial role in the activation of adaptive immunity. In summary, this study provides a novel platform technology in which broad-based anti-CMV immune responses upon vaccination can be maximised by co-delivery of viral antigens and TLR4 and 9 agonists which induce activation of innate immune signatures and promote potent antigen acquisition and cross-presentation by multiple DC subsets.

Autologous T-cell therapy for cytomegalovirus as a consolidative treatment for recurrent glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive human brain malignancies. Even with optimal treatment, median survival is less than 6 months for patients with recurrent GBM. Immune-based therapies have the potential to improve patient outcome by supplementing standard treatment. Expression of human cytomegalovirus (CMV) antigens in GBM tissues provides the unique opportunity to target viral antigens for GBM therapy. Here, we report findings of a formal clinical assessment of safety and potential clinical efficacy of autologous CMV-specific T-cell therapy as a consolidative treatment for recurrent GBM. From a total of 19 patients with recurrent GBM, CMV-specific T cells were successfully expanded from 13 patients (68.4%), 11 of whom received up to four T-cell infusions. Combination therapy based on T-cell infusion and chemotherapy was well tolerated, and we detected only minor adverse events. The overall survival of these patients since first recurrence ranged from 133 to 2,428 days, with a median overall survival of 403 days. Most importantly, 4 of 10 patients that completed the treatment remained progression free during the study period. Furthermore, molecular profiling of CMV-specific T-cell therapy from these patients revealed distinct gene expression signatures, which correlated with their clinical response. Our study suggests that a combination therapy with autologous CMV-specific T cells and chemotherapy is a safe novel treatment option and may offer clinical benefit for patients with recurrent GBM.

Improvement of a quantitative cell tropism assay for rapid and reliable characterization of human cytomegalovirus mutants.

Endothelial cell tropism is an inherent property of isolates of human cytomegalovirus (HCMV), and has been proposed as a pathogenicity factor of HCMV. Mutational approaches can be applied for analysis of the molecular determinants of endothelial cell tropism, but the evaluation of mutants is limited by the low reliability of the widely employed cell tropism assay. The aim of this study was to improve the assay conditions in order to allow for accurate discrimination of phenotypic differences between HCMV mutants. Target cell density and input virus titres were identified to account for most of the variation in the apparent endothelial cell tropism of a given cytomegalovirus strain. Coating of culture dishes, cell attachment at ambient temperature, and standardization of virus input titres together with automated counting of immunofluorescence signals enabled the discrimination of differences of as little as 25% in endothelial cell tropism of HCMV strains. This improvement will facilitate rapid and reliable quantitation of cell tropism of HCMV, and is particularly suitable for the analysis of large numbers of mutants with only minor changes in tropism.