Multi-centre ethics and research governance review can impede non-interventional clinical research.

BACKGROUND: The inter-jurisdictional National Mutual Acceptance (NMA) scheme for Human Research Ethics Committee (HREC) approvals of human research is designed to reduce the reported delays and costs of ethical review. Introduction of the NMA set forth an uncoupling of the ethics and governance review processes, permitting a single ethical review for multiple sites, while continuing separate governance review for each centre covering financial and operational aspects of the research project. AIM: To compare the time required to gain ethics and governance approvals in Australia for a non-interventional investigator-led study from December 2015 to approval times for an earlier pre-NMA study utilising a similar study design and study sites and evaluate the effect that the NMA has had on total approval time for non-interventional multi-centre projects. METHODS: We recorded the time taken to obtain ethics and governance approval at 16 sites for our nationwide low-risk non-interventional study looking at the prevalence and aetiology of non-tuberculous mycobacterial infection in people with cystic fibrosis in Australia. RESULTS: Applications were submitted to three hospitals and one university HREC to conduct our study at 16 hospital sites, HREC approval took from 16 to 79 days (median 28). Subsequent site-specific governance approval at 15 hospital sites took 23-225 days (median 83). The entire process of gaining ethical and governance approval to conduct the study at 16 sites took 24 months at an estimated cost of AU$56000 (US$ 42 000). CONCLUSION: Lengthy governance approval processes negate benefits gained from centralised ethics review under the NMA.

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.

Clinical assessment of anti-viral CD8+ T cell immune monitoring using QuantiFERON-CMV® assay to identify high risk allogeneic hematopoietic stem cell transplant patients with CMV infection complications.

The reconstitution of anti-viral cellular immunity following hematopoietic stem cell transplantation (HSCT) is crucial in preventing cytomegalovirus (CMV)-associated complications. Thus immunological monitoring has emerged as an important tool to better target pre-emptive anti-viral therapies. However, traditional laboratory-based assays are too cumbersome and complicated to implement in a clinical setting. Here we conducted a prospective study of a new whole blood assay (referred to as QuantiFERON-CMV®) to determine the clinical utility of measuring CMV-specific CD8+ T-cell responses as a prognostic tool. Forty-one evaluable allogeneic HSCT recipients underwent weekly immunological monitoring from day 21 post-transplant and of these 21 (51.2%) showed CMV reactivation and 29 (70.7%) developed acute graft-versus-host disease (GvHD). Patients with acute GvHD (grade ≥ 2) within 6 weeks of transplant showed delayed reconstitution of CMV-specific T-cell immunity (p = 0.013) and a higher risk of CMV viremia (p = 0.026). The median time to stable CMV-specific immune reconstitution was 59 days and the incidence of CMV reactivation was lower in patients who developed this than those who did not (27% versus 65%; p = 0.031). Furthermore, a failure to reconstitute CMV-specific immunity soon after the onset of CMV viraemia was associated with higher peak viral loads (5685 copies/ml versus 875 copies/ml; p = 0.002). Hence, QuantiFERON-CMV® testing in the week following CMV viremia can be useful in identifying HSCT recipients at risk of complicated reactivation.

Ex vivo functional analysis, expansion and adoptive transfer of cytomegalovirus-specific T-cells in patients with glioblastoma multiforme.

The frequent detection of human cytomegalovirus (CMV) antigens in glioblastoma multiforme (GBM) has raised the possibility of exploiting CMV-specific T-cell immunotherapy to control this disease in CMV--seropositive patients. Here, we have conducted a comprehensive ex vivo profiling of CMV-specific CD8(+) T-cell responses in a cohort of GBM patients. Of the patients analyzed, approximately half exhibited serological evidence of past infection with CMV. Although no CMV-specific CD8(+) T-cell responses could be detected in the serologically negative GBM patients, virus-specific CD8(+) T-cell responses were detected in all seropositive GBM patients. Using major histocompatibility complex-peptide multimers, the frequency of CMV-specific T-cells in the patients detected ranged from 0.1 to 22% of CD8(+) T-cells and a high proportion of these cells were positive for the human natural killer-1 glycoprotein CD57. Furthermore, ex vivo polychromatic functional analysis of the CMV-specific T-cells from GBM patients revealed that large proportions of these cells were unable to produce multiple cytokines (macrophage inflammatory protein (MIP)-1ß, tumor necrosis factor (TNF)α and interferon (IFN)γ) and displayed limited cytolytic function (CD107a mobilization) following stimulation with CMV peptide epitopes. However, in vitro stimulation with CMV peptide epitopes in the presence of γC cytokine dramatically reversed the polyfunctional profile of these antigen-specific T-cells with high levels of MIP-1ß, TNFα, IFNγ and CD107a mobilization. Most importantly, adoptive transfer of these in vitro-expanded T-cells in combination with temozolomide (TMZ) therapy into a patient with recurrent GBM was coincident with a long-term disease-free survival. These studies provide an important platform for a formal assessment of combination therapies based on CMV-specific T-cells and TMZ for recurrent GBM.

Narrowing of T-cell receptor beta variable repertoire during symptomatic herpesvirus infection in transplant patients.

Primary infection or recrudescence of latent virus infection in transplant recipients can be manifested either as asymptomatic or symptomatic disease. Here we show that symptomatic human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV) infection or recrudescence following solid organ transplantation (SOT) was coincident with a dramatic skewing of T-cell receptor beta variable (TRBV) repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no viral infection or recrudescence showed minimal or no skewing of the T-cell receptor repertoire to maintain peripheral blood repertoire diversity. More importantly, we show that large monoclonal/oligoclonal repertoire expansions are associated with the loss of HCMV-specific T-cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, whereas SOT recipients who maintain peripheral blood TRBV repertoire diversity and functional antigen-specific T-cell responses can resist clinical symptomatic disease in spite of high levels of viral load.

Discerning regulation of cis- and trans-presentation of CD8+ T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation.

Activation of the nuclear factor-kappaB pathway by Epstein-Barr virus-encoded latent membrane protein-1 (LMP-1) leads to an up-regulation of the major histocompatibility complex class I antigen-processing pathway. Paradoxically, LMP-1 itself induces a subdominant CD8+ T-cell response and appears to have evolved to avoid immune recognition. Here we show that, although expression of LMP-1 in human cells dramatically enhanced the trans-presentation of CD8+ T-cell epitopes, cis-presentation of LMP-1-derived epitopes was severely impaired. Testing of a series of LMP-1 mutants revealed that deletion of the first transmembrane domain of LMP-1, which prevented self-aggregation, significantly enhanced cis-presentation of T-cell epitopes from this protein, whereas it lost its ability to up-regulate trans-presentation. Interestingly, we also found that cis-presentation of LMP-1 epitopes was rescued by blocking the proteasome function. Taken together, these results delineate a novel mechanism of immune evasion, which renders a virally encoded oncogene inaccessible to the conventional major histocompatibility complex class I pathway limiting its cis-presentation to effector cells.

Immunobiology of human cytomegalovirus: from bench to bedside.

SUMMARY: Following primary infection, human cytomegalovirus (HCMV) establishes lifelong latency and periodically reactivates without causing symptoms in healthy individuals. In the absence of an adequate host-derived immune response, this fine balance of permitting viral reactivation without causing pathogenesis is disrupted, and HCMV can subsequently cause invasive disease and an array of damaging indirect immunological effects. Over the last decade, our knowledge of the immune response to HCMV infection in healthy virus carriers and diseased individuals has allowed us to translate these findings to develop better diagnostic tools and therapeutic strategies. The application of these emerging technologies in the clinical setting is likely to provide opportunities for better management of patients with HCMV-associated diseases.

Symptomatic and asymptomatic viral recrudescence in solid-organ transplant recipients and its relationship with the antigen-specific CD8(+) T-cell response.

Using ex vivo antigen-specific T-cell analysis, we found that symptomatic cytomegalovirus recrudescence in transplant recipients was coincident with reduced expression of gamma interferon (IFN-gamma) by virus-specific CD8(+) T cells and an up-regulation of CD38 expression on these T cells, although there was no significant change in the absolute number of virus-specific cells (as assessed by major histocompatibility complex-peptide multimers). In contrast, HLA class I-matched transplant patients with asymptomatic viral recrudescence showed increased expansion of antigen-specific T cells and highly stable IFN-gamma expression by epitope-specific T cells. These studies suggest that a strong functional T-cell response plays a crucial role in defining the clinical outcome of acute viral recrudescence.

Ex vivo expansion of human cytomegalovirus-specific cytotoxic T cells by recombinant polyepitope: implications for HCMV immunotherapy.

Stem cell transplantation (SCT) remains the most effective curative therapy for the majority of hematopoietic malignancies. Unfortunately, SCT is limited by its toxicity and infectious complications that result from profound immunosuppression. In particular, acquisition of exogenous or reactivation of endogenous human cytomegalovirus (HCMV) is common after SCT. More recently, reconstitution of host immunity through augmentation of anti-HCMV T cell responses has been proposed as an exciting candidate therapy to avoid the requirement for antiviral drug use. Here we have developed a novel antigen presentation system based on a replication-deficient adenovirus that encodes multiple HLA class I-restricted epitopes from eight different antigens of HCMV as a polyepitope (referred to as AdCMVpoly). Ex vivo stimulation of peripheral blood mononuclear cells with AdCMVpoly consistently showed rapid stimulation and expansion of multiple epitope-specific T cells that recognized endogenously processed epitopes presented on virus-infected cells. Interestingly, the AdCMVpoly expression system is capable of expanding antigen-specific T cells even in the absence of CD4(+) T cells. These studies show the effectiveness of a polyepitope antigen presentation system for reproducible expansion of antigen-specific T cells from immunocompetent and immunocompromised settings.

Contemporaneous fluctuations in T cell responses to persistent herpes virus infections.

The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.

Granulocyte colony-stimulating factor modulates alpha-galactosylceramide-responsive human Valpha24+Vbeta11+NKT cells.

Despite more than a 10-fold increase in T cell numbers in G-CSF-mobilized peripheral blood stem cell (PBSC) grafts, incidence and severity of acute graft-vs-host disease (GVHD) are comparable to bone marrow transplantation. As CD1d-restricted, Valpha24+Vbeta11+ NKT cells have pivotal immune regulatory functions and may influence GVHD, we aimed to determine whether G-CSF has any effects on human NKT cells. In this study, we examined the frequency and absolute numbers of peripheral blood NKT cells in healthy stem cell donors (n = 8) before and following G-CSF (filgrastim) treatment. Effects of in vivo and in vitro G-CSF on NKT cell cytokine expression profiles and on responsiveness of NKT cell subpopulations to specific stimulation by alpha-galactosylceramide (alpha-GalCer) were assessed. Contrary to the effects on conventional T cells, the absolute number of peripheral blood NKT cells was unaffected by G-CSF administration. Furthermore, responsiveness of NKT cells to alpha-GalCer stimulation was significantly decreased (p < 0.05) following exposure to G-CSF in vivo. This hyporesponsiveness was predominantly due to a direct effect on NKT cells, with a lesser contribution from G-CSF-mediated changes in APC. G-CSF administration resulted in polarization of NKT cells toward a Th2, IL-4-secreting phenotype following alpha-GalCer stimulation and preferential expansion of the CD4+ NKT cell subset. We conclude that G-CSF has previously unrecognized differential effects in vivo on NKT cells and conventional MHC-restricted T cells, and effects on NKT cells may contribute to the lower than expected incidence of GVHD following allogeneic peripheral blood stem cell transplantation.

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H.

Although the importance of CD4+ T cell responses to human cytomegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4+ CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HCMV isolates revealed that the gB and gH epitope sequences recognized by human CD4+ T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4+ CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse peptide-sensitized Patr-DR7+ cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class II lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.

Loss of PKR activity in chronic lymphocytic leukemia.

There are a number of observations that suggest the dsRNA-activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full-length PKR is present but not active in 21 of 28 patient samples from B-cell chronic lymphocytic leukemia (B-CLL). PKR from these patients was unable to auto-activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58(IPK). We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B-CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL.

Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers.

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.

Donor-derived b2a2-specific T cells for immunotherapy of patients with chronic myeloid leukemia.

The BCR-ABL fusion proteins, b2a2 and b3a2, are potential targets for a beneficial graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation for chronic myeloid leukemia (CML). This study demonstrates that CD4+ T cells specific to the b2a2 peptide can be generated from a normal allogeneic stem cell transplant donor after stimulation with monocyte-derived dendritic cells (Mo-DC) using culture conditions applicable to clinical use. Stimulation of donor T-cell enriched mononuclear cells (MNC) with b2a2-pulsed Mo-DC produced approximately 3 x 10(9) b2a2-specific CD4+ T cells. The CD4+ T cells were HLA-DR7 restricted. These results confirm that the generation of donor derived b2a2-specific T cells for clinical use is feasible and warrants clinical testing after stem cell transplantation.