Publications by authors named "Leighton Clancy"

27 Publications

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Prophylactic antigen-specific T-cells targeting seven viral and fungal pathogens after allogeneic haemopoietic stem cell transplant.

Clin Transl Immunology 2021 15;10(3):e1249. Epub 2021 Mar 15.

Sydney Medical School University of Sydney Sydney NSW Australia.

Objectives: Adoptive immunotherapy using donor-derived antigen-specific T-cells can prevent and treat infection after allogeneic haemopoietic stem cell transplant (HSCT).

Methods: We treated 11 patients with a prophylactic infusion of 2 × 10 cells per square metre donor-derived T-cells targeting seven infections (six viral and one fungal) following HSCT. Targeted pathogens were cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, varicella zoster virus, influenza, BK virus (BKV) and .

Results: T-cell products were successfully generated in all patients with 10 products responsive to 6 or 7 infections. T-cell infusions were associated with increases in antigen-experienced activated CD8 T-cells by day 30. CMV, EBV and BKV reactivation occurred in the majority of patients and was well controlled except where glucocorticoids were administered soon after T-cell infusion. Three patients in that circumstance developed CMV tissue infection. No patient required treatment for invasive fungal infection. The most common CMV and EBV TCR clonotypes in the infusion product became the most common clonotypes seen at day 30 post-T-cell infusion. Donors and their recipients were recruited to the study prior to transplant. Grade III/IV graft-versus-host disease developed in four patients. At a median follow-up of 390 days post-transplant, six patients had died, 5 of relapse, and 1 of multi-organ failure. Infection did not contribute to death in any patient.

Conclusion: Rapid reconstitution of immunity to a broad range of viral and fungal infections can be achieved using a multi-pathogen-specific T-cell product. The development of GVHD after T-cell infusion suggests that infection-specific T-cell therapy after allogeneic stem cell transplant should be combined with other strategies to reduce graft-versus-host disease.
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http://dx.doi.org/10.1002/cti2.1249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960021PMC
March 2021

Successful treatment of CMV, EBV, and adenovirus tissue infection following HLA-mismatched allogeneic stem cell transplant using infusion of third-party T cells from multiple donors in addition to antivirals, rituximab, and surgery.

Transpl Infect Dis 2020 Nov 24:e13528. Epub 2020 Nov 24.

Department of Haematology, Westmead Hospital, Sydney, NSW, Australia.

Viral infections, principally cytomegalovirus, Epstein Barr virus (EBV) and adenovirus, are a leading cause of morbidity and mortality after allogeneic stem cell transplantation. The use of systemic antivirals is limited by limited efficacy and organ toxicities. Inability to clear infection is exacerbated by transplant-related immunosuppression and prophylaxis or treatment of acute graft versus host disease. We report the first patient to clear three serious viral infections after stem cell transplant using third-party donor partially human leukocyte antigen (HLA) matched virus-specific cytotoxic T cells. The patient, a 53 year old female with transplanted for relapsed leukemia, with severe graft versus host disease received five T cell infusions from three separate donors that ultimately cleared serious systemic infections with cytomegalovirus and adenovirus, and an EBV-driven lymphoma. Systemic antivirals had resulted in failed clinical responses. Use of repeated infusions of partially HLA matched virus-specific T cells from banks containing cryopreserved cells should be strongly considered in transplant recipients with single or multiple refractory viral infections.
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http://dx.doi.org/10.1111/tid.13528DOI Listing
November 2020

enrichment of PRAME antigen-specific T cells for adoptive immunotherapy using CD137 activation marker selection.

Clin Transl Immunology 2020 21;9(10):e1200. Epub 2020 Oct 21.

Westmead Institute for Medical Research Westmead NSW Australia.

Objective: Adoptive immunotherapy with expanded tumor-specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP-compliant manufacturing method for PRAME-specific T cells from healthy donors for adoptive immunotherapy.

Methods: Mononuclear cells were pulsed with PRAME 15-mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137 fraction in medium supplemented with interleukin (IL)-2, IL-7 and IL-15. Cultured T cells were restimulated with antigen-pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re-exposure were assessed with flow cytometry, enzyme-linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T-cell receptor (TCR) beta clonality studies were performed on selected cultures.

Results: PRAME-stimulated cultures ( = 10) had mean expansion of 2500-fold at day 18. Mean CD3 percentage was 96% with CD4:CD8 ratio of 4:1. Re-exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)-γ (mean: 12.2%) and TNF-α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3/CCR4/CCR6/Tbet, mean: 73%) and cytokine production including IL-2, IL-4, IL-8, IL-13 and GM-CSF (2%, 6%, 8%, 4% and 11%, respectively).

Conclusion: PRAME-specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines.
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http://dx.doi.org/10.1002/cti2.1200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577233PMC
October 2020

Rapidly expanded partially HLA DRB1-matched fungus-specific T cells mediate in vitro and in vivo antifungal activity.

Blood Adv 2020 07;4(14):3443-3456

Westmead Institute for Medical Research, Sydney, NSW, Australia.

Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice-compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen-matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.
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http://dx.doi.org/10.1182/bloodadvances.2020001565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391149PMC
July 2020

Mass cytometry reveals immune signatures associated with cytomegalovirus (CMV) control in recipients of allogeneic haemopoietic stem cell transplant and CMV-specific T cells.

Clin Transl Immunology 2020 2;9(7):e1149. Epub 2020 Jul 2.

Faculty of Medicine and Health The University of Sydney Camperdown NSW Australia.

Objectives: Cytomegalovirus (CMV) is known to have a significant impact on immune recovery post-allogeneic haemopoietic stem cell transplant (HSCT). Adoptive therapy with donor-derived or third-party virus-specific T cells (VST) can restore CMV immunity leading to clinical benefit in prevention and treatment of post-HSCT infection. We developed a mass cytometry approach to study natural immune recovery post-HSCT and assess the mechanisms underlying the clinical benefits observed in recipients of VST.

Methods: A mass cytometry panel of 38 antibodies was utilised for global immune assessment (72 canonical innate and adaptive immune subsets) in HSCT recipients undergoing natural post-HSCT recovery ( = 13) and HSCT recipients who received third-party donor-derived CMV-VST as salvage for unresponsive CMV reactivation ( = 8).

Results: Mass cytometry identified distinct immune signatures associated with CMV characterised by a predominance of innate cells (monocytes and NK) seen early and an adaptive signature with activated CD8 T cells seen later. All CMV-VST recipients had failed standard antiviral pharmacotherapy as a criterion for trial involvement; 5/8 had failed to develop the adaptive immune signature by study enrolment despite significant CMV antigen exposure. Of these, VST administration resulted in development of the adaptive signature in association with CMV control in three patients. Failure to respond to CMV-VST in one patient was associated with persistent absence of the adaptive immune signature.

Conclusion: The clinical benefit of CMV-VST may be mediated by the recovery of an adaptive immune signature characterised by activated CD8 T cells.
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http://dx.doi.org/10.1002/cti2.1149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332355PMC
July 2020

Pathogen-Specific T Cells Beyond CMV, EBV and Adenovirus.

Curr Hematol Malig Rep 2019 08;14(4):247-260

Faculty of Medicine and Health, The University of Sydney, Camperdown, Australia.

Purpose Of Review: Infectious diseases contribute significantly to morbidity and mortality in recipients of allogeneic haematopoietic stem cell transplantation (aHSCT), particularly in the era of highly immunosuppressive transplant regimens and alternate donor transplants. Delayed cellular immune recovery is a major mechanism for the increased risk in these patients. Adoptive cell therapy with ex vivo manipulated pathogen-specific T cells (PSTs) is increasingly taking its place as a treatment strategy using donor-derived or third party-banked cells.

Recent Findings: The majority of clinical trial data in the form of early-phase studies has been in the prophylaxis or treatment of cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV). Advancements in methods to select and enrich PSTs offer the opportunity to target the less common viral pathogens as well as fungi with this technology. Early clinical studies of PSTs targeting polyomaviruses (BK virus and JC virus), human herpesvirus 6 (HHV6), varicella zoster virus (VZV) and Aspergillus spp. have shown promising results in small numbers of patients. Other potential targets include herpes simplex virus (HSV), respiratory viruses and other invasive fungal species. In this review, we describe the burden of disease of this wider spectrum of pathogens, the progress in the development of manufacturing capability, early clinical results and the opportunities and challenges for implementation in the clinic.
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http://dx.doi.org/10.1007/s11899-019-00521-zDOI Listing
August 2019

Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program.

Biol Blood Marrow Transplant 2018 12 29;24(12):2433-2442. Epub 2018 Aug 29.

Westmead Institute for Medical Research, University of Sydney, Australia; Blood and Bone Marrow Transplant Unit, Westmead Hospital, Sydney, Australia; Department of Haematology, Westmead Hospital, Sydney, Australia; Sydney Cellular Therapies Laboratory, Westmead Hospital, Sydney, Australia; Sydney Medical School, University of Sydney, Sydney, Australia. Electronic address:

Hematopoietic stem cell transplantation (HSCT) donor-generated virus-specific T cells (VSTs) can provide effective treatment for viral infection post-HSCT but are not readily accessible to all patients. Off-the-shelf cryopreserved VSTs suitable for treatment of multiple patients are an attractive alternative. We generated a bank of 17 cytomegalovirus (CMV)-, 14 Epstein-Barr virus (EBV)-, and 15 adenovirus (AdV)-specific T cell products from 30 third-party donors. Donors were selected for expression of 6 core HLA antigens expressed at high frequency in the local transplant population. T cells were generated by co-culturing venous blood or mobilized hematopoietic stem cell (HSC)-derived mononuclear cells with monocyte-derived dendritic cells pulsed with overlapping peptides covering CMV pp65, AdV5 hexon, or EBV BZLF1/LMP2A/EBNA1 proteins. Addition of a CD14 selection step instead of plate adherence to isolate monocytes before culture initiation significantly improved expansion in cultures from HSC material. Phenotyping showed the CD8 subset to have significantly higher numbers of terminal effector T cells (CD45RA62L) and lower numbers of effector memory T cells (CD45RA62L) when compared with the CD4 subset. Increased expression of the immunoinhibitory markers PD-1 and TIM-3 was noted on CD4 but not CD8 cells when compared with the control group. VST showed antiviral activity restricted through a variety of common HLAs, and modelling suggested a suitably HLA-matched product would be available for >90% of HSCT patients. Only a small number of carefully selected third-party donors are required to generate a VST bank of broad coverage, indicating the feasibility of local banking integrated into existing allogeneic HSCT programs.
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http://dx.doi.org/10.1016/j.bbmt.2018.08.024DOI Listing
December 2018

Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies.

Biol Blood Marrow Transplant 2018 05 2;24(5):1069-1078. Epub 2018 Jan 2.

Westmead Institute of Medical Research, University of Sydney, Westmead, Australia; Sydney Medical School, University of Sydney, Camperdown, Australia; Sydney Cellular Therapies Laboratory, Westmead, Australia; Blood and Marrow Transplant Unit, Department of Haematology, Westmead Hospital, Westmead, Australia. Electronic address:

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300 ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies.
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http://dx.doi.org/10.1016/j.bbmt.2017.12.802DOI Listing
May 2018

Long-term control of recurrent or refractory viral infections after allogeneic HSCT with third-party virus-specific T cells.

Blood Adv 2017 Nov 2;1(24):2193-2205. Epub 2017 Nov 2.

Westmead Institute for Medical Research, University of Sydney, Sydney, NSW, Australia.

Donor-derived adoptive T-cell therapy is a safe and effective treatment of viral infection posttransplant, but it is limited by donor serostatus and availability and by its personalized nature. Off-the-shelf, third-party virus-specific T cells (VSTs) appear promising, but the long-term safety and durability of responses have yet to be established. We conducted a prospective study of 30 allogeneic hemopoietic stem cell transplant (HSCT) patients with persistent or recurrent cytomegalovirus (CMV) (n = 28), Epstein-Barr virus (n = 1), or adenovirus (n = 1) after standard therapy. Patients were treated with infusions of partially HLA-matched, third-party, ex vivo-expanded VSTs (total = 50 infusions) at a median of 75 days post-HSCT (range, 37 to 349 days). Safety, viral dynamics, and immune recovery were monitored for 12 months. Infusions were safe and well tolerated. Acute graft versus host disease occurred in 2 patients, despite a median HLA match between VSTs and the recipient of 2 of 6 antigens. At 12 months, the cumulative incidence of overall response was 93%. Virological control was durable in the majority of patients; the reintroduction of antiviral therapy after the final infusion occurred in 5 patients. CMV-specific T-cell immunity rose significantly and coincided with a rise in CD8 terminal effector cells. PD-1 expression was elevated on CD8 lymphocytes before the administration of third-party T cells and remained elevated at the time of viral control. Third-party VSTs show prolonged benefit, with virological control achieved in association with the recovery of CD8 effector T cells possibly facilitated by VST infusion. This trial was registered at www.clinicaltrials.gov as #NCT02779439 and www.anzctr.org.au as #ACTRN12613000603718.
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http://dx.doi.org/10.1182/bloodadvances.2017010223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737128PMC
November 2017

Prospects for adoptive T-cell therapy for invasive fungal disease.

Curr Opin Infect Dis 2017 Dec;30(6):518-527

aWestmead Institute for Medical Research at the University of Sydney bDepartment of Haematology, Westmead Hospital, Westmead cUniversity of Sydney, Camperdown, New South Wales, Australia.

Purpose Of Review: Invasive fungal disease (IFD) is a cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. As more potent broad-spectrum antifungal agents are used in prophylaxis, drug resistance and less common fungal species have increased in frequency. Here we review current treatments available for IFD and examine the potential for adoptive T-cell treatment to enhance current therapeutic choices in IFD.

Recent Findings: There is growing evidence supporting the role of T cells as well as phagocytes in antifungal immunity. T cells recognizing specific antigens expressed on fungal morphotypes have been identified and the role of T-cell transfer has been explored in animal models. The clinical efficacy of adoptive transfer of antigen-specific T cells for prophylaxis and treatment of viral infections post-HSCT has raised interest in developing good manufacturing practice (GMP)-compliant methods for manufacturing and testing fungus-specific T cells after HSCT.

Summary: As the outcomes of IFD post-HSCT are poor, reconstitution of antifungal immunity offers a way to correct the underlying deficiency that has caused the infection rather than simply pharmacologically suppress fungal growth. The clinical development of fungus specific T cells is in its early stages and clinical trials are needed in order to evaluate safety and efficacy.
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http://dx.doi.org/10.1097/QCO.0000000000000403DOI Listing
December 2017

Adjuvant Peptide Pulsed Dendritic Cell Vaccination in Addition to T Cell Adoptive Immunotherapy for Cytomegalovirus Infection in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Biol Blood Marrow Transplant 2018 01 31;24(1):71-77. Epub 2017 Aug 31.

The Westmead Institute for Medical Research, The University of Sydney, Sydney, Australia; Blood and Marrow Transplant Unit, Westmead Hospital, Sydney, Australia; Sydney Cell and Gene Therapy Laboratory, Westmead Hospital, Sydney, Australia. Electronic address:

Adoptive cellular immunotherapy has been shown to be effective in the management of cytomegalovirus (CMV) reactivation and disease. Whether adjuvant dendritic cell (DC) vaccination will provide additional benefit in prophylaxis or treatment of CMV in hematoietic cell transplantation (HSCT) recipients is unknown. In this study, we administered prophylactic CMV-peptide specific T cell infusions, followed by 2 doses of intradermal CMV peptide-pulsed DC vaccine, to 4 HSCT recipients. There were no immediate adverse events associated with T cell infusion or DC vaccinations. One of the 4 patients developed grade III acute gut graft-versus-host disease. Immune reconstitution against CMV was detected in all 4 patients. Patients receiving CMV peptide-specific T cells and DC vaccination had peak immune reconstitution at least 10 days after the second DC vaccination. In summary, combining DC vaccine with T cell infusion appears feasible, although further study is required to ascertain its safety and efficacy in augmenting the effects of infusing donor-derived CMV-specific T cells.
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http://dx.doi.org/10.1016/j.bbmt.2017.08.028DOI Listing
January 2018

Herpes simplex virus type 1 (HSV-1) specific T-cell generation from HLA-A1- and HLA-A2-positive donors for adoptive immunotherapy.

Cytotherapy 2017 01 25;19(1):107-118. Epub 2016 Oct 25.

The Westmead Institute for Medical Research, Australia; Blood and Marrow Transplant Unit, Australia; Sydney Cell and Gene Therapy Laboratory, Westmead Hospital, The University of Sydney, Sydney, Australia. Electronic address:

Background Aims: Herpes simplex virus (HSV) reactivation and infection is common in patients undergoing hematopoietic stem cell transplant (HSCT) and requires routine antiviral prophylaxis. Drug-resistant strains are increasingly common, and effective alternative therapy is currently unavailable. We generated and characterized HSV-1-specific T cells for use in adoptive cellular immunotherapy following allogeneic stem cell transplantation.

Methods: Peripheral blood mononuclear cells from HLA-A1 and HLA-A2 HSV-seropositive hereditary hemochromatosis donors were used as the antigen source. Three HLA-A1 and four HLA-A2 specific epitopes were used for stimulation of T cells. Cells were stimulated with antigen-pulsed dendritic cells and cultured for 21 days in medium with interleukin (IL)-2. Cultured cells were phenotyped and tested for cytokine production, proliferation and cytotoxicity.

Results: There was a 5.3-fold expansion in total cell numbers over 21 days of culture, with 35% of T cells being CD8 positive. Thirty-five percent, 21% and 5% of CD8 cells secreted interferon-γ, tumor necrosis factor-α and IL-2 upon HSV antigen re-stimulation. More than 50% of antigen-specific T cells secreted multiple cytokines. Cultured T cells proliferated upon antigen re-stimulation and lysed HSV-1 peptide and virus-infected targets.

Conclusions: It is feasible to generate functional HSV-1 specific T cells from the blood of HLA-A1 and HLA-A2 HSV-seropositive donors using specific peptides. The utility of these cells in preventing and treating HSV-1 reactivation in allogeneic HSCT will need to be tested clinically.
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http://dx.doi.org/10.1016/j.jcyt.2016.09.013DOI Listing
January 2017

CMV-specific immune reconstitution following allogeneic stem cell transplantation.

Virulence 2016 11 9;7(8):967-980. Epub 2016 Aug 9.

a Westmead Institute for Medical Research at the University of Sydney , Westmead , Sydney , Australia.

Cytomegalovirus (CMV) remains a major contributor to morbidity and mortality following allogeneic haemopoietic stem cell transplant (HSCT) despite widespread use of viraemia monitoring and pre-emptive antiviral therapy. Uncontrolled viral replication occurs primarily in the first 100 d post transplant but this high risk period can extend to many months if immune recovery is delayed. The re-establishment of a functional population of cellular effectors is essential for control of virus replication and depends on recipient and donor serostatus, the stem cell source, degree of HLA matching and post-transplant factors such as CMV antigen exposure, presence of GVHD and ongoing use of immune suppression. A number of immune monitoring assays exist but have not yet become widely accessible for routine clinical use. Vaccination, adoptive transfer of CMV specific T cells and a number of graft engineering processes are being evaluated to enhance of CMV specific immune recovery post HSCT.
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http://dx.doi.org/10.1080/21505594.2016.1221022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5160403PMC
November 2016

Stimulation with lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae maximizes cross-reactivity of anti-fungal T cells.

Cytotherapy 2016 Jan 6;18(1):65-79. Epub 2015 Nov 6.

Centre for Cancer Research, Westmead Millennium Institute for Medical Research, Sydney, Australia; Sydney Medical School, University of Sydney, Australia; Sydney Cellular Therapies Laboratory, Westmead Hospital, Sydney, Australia; Blood and Marrow Transplant Unit, Department of Haematology, Westmead Hospital, Sydney, Australia.

Background Aims: Invasive fungal diseases caused by filamentous fungi and yeasts are significant causes of morbidity and mortality in immunosuppressed hematology patients. We previously published a method to expand Aspergillus fumigatus-specific T cells for clinical cell therapy. In the present study, we investigated expansion of T cells specific for other fungal pathogens and creation of a broadly reactive panfungal T-cell product.

Methods: Fungal strains selected were those frequently observed in the clinical hematology setting and included Aspergillus, Candida, Fusarium, Rhizopus and Lomentospora/Scedosporium. Four T-cell cultures specific to each fungus were established. We selected lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae to expand panfungal T cells. Allelic restriction of anti-fungal activity was determined through the use of specific major histocompatibility complex class II-blocking antibodies.

Results: Individual T-cell cultures specific to each fungus could be expanded in vitro, generating predominantly CD4(+) T cells of which 8% to 20% were fungus-specific. We successfully expanded panfungal T cells from the peripheral blood (n = 8) and granulocyte-colony-stimulating factor-primed stem cell products (n = 3) of normal donors by using a combination of lysates from Aspergillus terreus, Candida krusei and Rhizopus oryzae. Anti-fungal activity was mediated through human leukocyte antigen (HLA)-DR alleles and was maintained when antigen-presenting cells from partially HLA-DRB1-matched donors were used to stimulate T cells.

Conclusions: We demonstrate a method to manufacture panfungal T-cell products with specificity against a range of clinical fungal pathogens by use of the blood and stem cells of healthy donors as the starting material. The safety and efficacy of these products will need to be tested clinically.
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http://dx.doi.org/10.1016/j.jcyt.2015.09.013DOI Listing
January 2016

Addition of varicella zoster virus-specific T cells to cytomegalovirus, Epstein-Barr virus and adenovirus tri-specific T cells as adoptive immunotherapy in patients undergoing allogeneic hematopoietic stem cell transplantation.

Cytotherapy 2015 Oct;17(10):1406-20

Faculty of Medicine, University of Sydney, Sydney, Australia; Westmead Millennium Institute, Centre for Cancer Research, Sydney, Australia; Blood and Marrow Transplant Unit, Department of Haematology, Westmead Hospital, Sydney, Australia; Sydney Cell and Gene Therapy Laboratory, Westmead Hospital, Sydney, Australia. Electronic address:

Background Aims: Virus-specific T-cell immunotherapy is emerging as a promising management strategy for virus infections in patients after hematopoietic stem cell transplant (HSCT). Here we present outcomes of 10 adult patients who received multi-virus-specific T cells prophylactically after HSCT.

Methods: Donor-derived cytomegalovirus (CMV)-, Epstein-Barr virus (EBV)-, adenoviral- and varicella zoster virus (VZV)-specific T cells were generated in a single culture and administered to HSCT patients at a dose of 2 × 10(7)/m(2) virus-specific T cells at a median of 63 days post-transplant. Patients were monitored for 12 months for evidence of viral reactivation and graft-versus-host disease.

Results: There was no acute infusion-related toxicity. Six patients developed CMV reactivation after T-cell infusion with a median peak CMV DNA titer of 600 copies per milliliter, and 1 received CMV-specific pharmacotherapy post-infusion. No EBV, adenoviral or VZV reactivation or disease was reported. Using interferon-γ Elispot analysis on post-infusion samples, we identified anti-viral immunity against all viruses including VZV. Three patients (30%) developed grade II-IV acute graft-versus-host disease.

Conclusions: This is the first description of the use of a multi-virus-specific T-cell product containing cells specific for VZV after allogeneic HSCT. The T-cell product appears safe in the setting of HSCT and confirms our previous findings regarding CMV control and treatment. A larger study with longer follow-up is required to determine the efficacy of VZV-specific T cells given prophylactically in controlling episodes of herpes zoster and disseminated varicella infection after cessation of prophylactic anti-viral treatment.
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http://dx.doi.org/10.1016/j.jcyt.2015.07.005DOI Listing
October 2015

Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials.

Cytotherapy 2015 Sep 23;17(9):1251-67. Epub 2015 Jul 23.

The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia. Electronic address:

Background Aims: Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification.

Methods: We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period.

Results: Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer.

Discussion: We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies.
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http://dx.doi.org/10.1016/j.jcyt.2015.05.013DOI Listing
September 2015

Human cytomegalovirus interleukin-10 polarizes monocytes toward a deactivated M2c phenotype to repress host immune responses.

J Virol 2013 Sep 17;87(18):10273-82. Epub 2013 Jul 17.

Centre for Virus Research, Westmead Millennium Institute, Westmead, New South Wales, Australia.

Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14(+) monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1β, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4(+) T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4(+) T cell responses at sites of infection.
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http://dx.doi.org/10.1128/JVI.00912-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754025PMC
September 2013

Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation.

Blood 2013 May 22;121(18):3745-58. Epub 2013 Feb 22.

Westmead Millennium Institute, University of Sydney, Sydney, Australia.

We investigated the use of adoptively transferred donor-derived cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) as immune reconstitution postallogeneic transplant in a phase 2 study. Fifty patients were infused with a single dose of 2 × 10(7)cells/m(2) after day 28 post-transplant. Twenty-six patients reactivated CMV posttransplant (only 5 post-CTL infusion) and 9 required therapy with ganciclovir or foscarnet (only 1 post-CTL infusion). There was 1 case of fatal CMV disease, attributable to high levels of antithymocyte globulin at the time of T cell infusion. We compared the patients in the phase 2 study with a group of contemporaneous controls also treated at the trial centers. There was no increase in acute or chronic graft-versus-host disease attributable to CTL infusion; overall and progression-free survival were similar in both groups. There was a reduction in the percentage of patients who required CMV directed antiviral therapy (17% vs 36%, P = .01) and in the total number of treatment days in the cohort receiving CTL (3.4 days vs 8.9 days, P = .03) without a reduction in CMV reactivation rates. We postulate that adoptively transferred cells are able to expand in response to viral antigen, limit viral replication, and prevent progression to tissue infection. This study was registered on the Australian Clinical Trial Registry as #ACTRN12605000213640 and #ACTRN12607000224426.
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http://dx.doi.org/10.1182/blood-2012-08-448977DOI Listing
May 2013

Cytomegalovirus-specific cytotoxic T lymphocytes can be efficiently expanded from granulocyte colony-stimulating factor-mobilized hemopoietic progenitor cell products ex vivo and safely transferred to stem cell transplantation recipients to facilitate immune reconstitution.

Biol Blood Marrow Transplant 2013 May 1;19(5):725-34. Epub 2013 Feb 1.

Westmead Millennium Institute, Westmead Institute for Cancer Research, University of Sydney, Westmead, Australia.

Uncontrolled cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation causes significant morbidity and mortality. Adoptive transfer of CMV-specific cytotoxic T lymphocytes (CTLs) is a promising therapy to treat reactivation and prevent viral disease. In this article, we describe the generation of clinical-grade CMV-specific CTLs directly from granulocyte colony-stimulating factor-mobilized hemopoietic progenitor cell (G-HPC) products collected for transplantation. This method requires less than 2.5% of a typical G-HPC product to reproducibly expand CMV-specific CTLs ex vivo. Comparison of 11 CMV CTL lines generated from G-HPC products with 52 CMV CTL lines generated from nonmobilized peripheral blood revealed similar expansion kinetics and phenotype. G-HPC-derived CTLs produced IFN-γ after reexposure to CMVpp65 antigen and exhibited CMV-directed cytotoxicity but no alloreactivity against transplantation recipient-derived cells. Seven patients received CMV-specific CTL lines expanded from G-HPC products in a prophylactic adoptive immunotherapy phase I/II clinical trial. Use of G-HPC products will facilitate integration of CTL generation into established quality systems of transplantation centers and more rapid inclusion of T cell therapies into routine clinical care.
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http://dx.doi.org/10.1016/j.bbmt.2013.01.021DOI Listing
May 2013

Robust polyfunctional T-helper 1 responses to multiple fungal antigens from a cell population generated using an environmental strain of Aspergillus fumigatus.

Cytotherapy 2012 Oct 7;14(9):1119-30. Epub 2012 Aug 7.

Westmead Institute for Cancer Research, Westmead Millennium Institute and Faculty of Medicine, The University of Sydney, Westmead, New South Wales, Australia.

Background And Aims: Aspergillus fumigatus infections are the leading cause of invasive fungal infection-related deaths in stem cell transplant patients, and may be amenable to correction with adoptive immunotherapy providing T lymphocytes specific for A. fumigatus. However, a clinically usable source of antigen and a reliable procedure for the generation of large numbers of Aspergillus-specific T lymphocytes to clinical-grade standards is not available.

Methods: An environmental strain of A. fumigatus (WMAfES) was isolated and cultured using materials and reagents suitable for clinical manufacture. Water-soluble lysate from germinated conidia of WMAfES was used as the antigen source. Peripheral blood mononuclear cells were stimulated with antigen-pulsed autologous dendritic cells on days 0 and 7. Cells were expanded with a cocktail of interleukin (IL)-2, IL-7 and IL-15 from days 7 to 21.

Results: We obtained a mean 32.8-fold increase in cell numbers over 21 days of culture (n = 8). Resultant cultures were predominantly effector and central memory CD4(+) T cells, which produced T-helper (h)1 and Th17 cytokines when restimulated with A. fumigatus antigen derived from environmental or clinically isolated A. fumigatus. Cultured cells exhibited a high level of specific expansion and chemokine production when restimulated. Moreover, cultured cells cross-reacted with antigens from other fungi, including Penicillium, Candida albicans and other non-fumigatus Aspergillus species.

Conclusions: We describe a simple, robust, reproducible and clinically applicable procedure using a clinically appropriate antigen preparation for the expansion of polyfunctional A. fumigatus-specific T cells from normal donors of varying HLA types.
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http://dx.doi.org/10.3109/14653249.2012.704013DOI Listing
October 2012

Clinical-grade varicella zoster virus-specific T cells produced for adoptive immunotherapy in hemopoietic stem cell transplant recipients.

Cytotherapy 2012 Jul 12;14(6):724-32. Epub 2012 Mar 12.

Westmead Millennium Institute, University of Sydney, Westmead, Australia.

Background Aims: Varicella zoster virus (VZV) causes life-long latent infection in healthy individuals, which reactivates in 10-68% of stem cell transplant patients. Reconstituting immunity through adoptive transfer of T cells specific for VZV may aid in the prophylaxis and treatment of VZV infections. The potential for generating T cells specific for VZV using a clinically approved VZV vaccine strain was investigated.

Methods: The Varivax® vaccine was used to stimulate peripheral blood mononuclear cells from healthy donors. Only reagents approved for clinical manufacture were used. Monocyte-derived dendritic cells pulsed with Varivax (R) were used to stimulate autologous mononuclear cells at a responder to stimulator ratio of 10:1. On day 7, a second stimulation was performed; 20 U/mL interleukin (IL)-2 were added from day 7 and 50 U/mL IL-2 from day 14 onwards. Cell phenotype and functionality were assessed after 21 days of culture.

Results: A mean increase of 11-fold in cell number was observed (n= 18). Cultures were mainly T cells (mean CD3 (+) 89.7%, CD4 (+) 54.2%, CD8 (+) 28.7%) with effector and central memory phenotypes. Cells produced one or more T helper (Th)1 cytokine (interferon-γ, tumor necrosis factor-α and IL-2), and CD4 (+) (but not CD8 (+) ) cells expressed the cytoxicity marker CD107 when restimulated with VZV antigens.

Conclusions: We have demonstrated a clinically applicable method that yields high numbers of highly reactive T cells specific for VZV. We propose that reconstructing host immunity through adoptive transfer of VZV-specific T cells will reduce the frequency of clinical VZV infection in the period of severe immune suppression that follows allogeneic stem cell transplantation.
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http://dx.doi.org/10.3109/14653249.2012.663486DOI Listing
July 2012

BK virus-specific T cells for use in cellular therapy show specificity to multiple antigens and polyfunctional cytokine responses.

Transplantation 2011 Nov;92(10):1077-84

Westmead Institute of Cancer Research, Westmead Millennium Institute, University of Sydney, Westmead, Australia.

Background: BK virus (BKV) infection causes hemorrhagic cystitis posthemopoietic stem-cell transplant and graft loss in renal transplant recipients. Reactivation occurs in up to 60% of patients in both groups. Treatment-related cellular immunosuppression is a major contributor to the development of BKV-related disease, but the targets of the immune response are not well characterized. Immunotherapy by adoptive transfer of cellular effectors has been shown to be effective in controlling and preventing some virus-related diseases in transplant recipients, particularly Epstein-Barr virus and cytomegalovirus. Infusion of BKV-specific T cells may potentially reconstitute functional BKV immunity and reduce clinical complications of BKV infection.

Methods: BKV-specific T cells for clinical use in adoptive immunotherapy were generated using monocyte-derived dendritic cells pulsed with overlapping peptide mixes spanning the five BKV proteins VP1, VP2, VP3, large T antigen, and small T antigen. Phenotypic and functional characteristics of the cells were investigated as well as their antigen specificity.

Results: Expanded CD4(+) and CD8(+) cells responded to restimulation with BKV peptides principally from VP1, large T, or small T antigens; produced multiple cytokines; and showed cytotoxic activity against antigen-coated targets.

Conclusions: Possible clinical uses for BKV-specific T cells generated using this method include immune reconstitution posthemopoietic stem-cell transplantation or prophylaxis and treatment of immune deficiency in renal transplant recipients, fulfilling the need for effective therapy for BKV-related hemorrhagic cystitis and renal dysfunction.
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http://dx.doi.org/10.1097/TP.0b013e31823328c0DOI Listing
November 2011

In vitro generation of influenza-specific polyfunctional CD4+ T cells suitable for adoptive immunotherapy.

Cytotherapy 2012 Feb 28;14(2):182-93. Epub 2011 Sep 28.

Westmead Institute for Cancer Research, Westmead Millennium Institute and Faculty of Medicine, The University of Sydney, NSW, Australia.

Background Aims: Influenza viruses cause potentially fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived host adaptive immunity to influenza viruses, and the potential for generating such cells for clinical use was investigated.

Methods: The inactivated influenza vaccine (Fluvax) approved for human use was used as the antigen source. Monocyte-derived dendritic cells pulsed with Fluvax were used to stimulate autologous peripheral blood mononuclear cells (PBMC) on days 0 and 7. Cells were expanded with interleukin (IL)-2 from day 7 onwards. Cell numbers and phenotype were assessed on day 21. The presence of influenza virus-specific cells was assessed by cytokine production and proliferative responses following restimulation with influenza antigens.

Results: Over 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n = 7). Cultures were predominantly effector and central memory CD4+ cells, and expressed a phenotype characteristic of activated antigen-specific cells capable of B-cell helper function. Cytotoxic CD4+ and CD8+ cells specific for influenza and a high percentage of CD4+ cells specific for each of three influenza viruses targeted by Fluvax (H1N1, H3N2 and Brisbane viruses) were generated. In addition, T cells expanded when restimulated with antigens derived from influenza viruses.

Conclusions: We have demonstrated a clinically usable method for producing influenza virus-specific T cells that yield high numbers of highly reactive CD4+ cells suitable for adoptive immunotherapy. We propose that reconstructing host immunity through adoptive transfer of influenza virus-specific T cells will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows stem cell transplantation.
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http://dx.doi.org/10.3109/14653249.2011.613932DOI Listing
February 2012

Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation.

Blood 2008 Nov 3;112(10):3974-81. Epub 2008 Sep 3.

Westmead Millennium Institute, University of Sydney at Westmead Hospital, Australia.

Cytomegalovirus (CMV) and its therapy continue to contribute to morbidity and mortality in hemopoietic stem cell transplantation (HSCT). Many studies have demonstrated the feasibility of in vitro generation of CMV-specific T cells for adoptive immunotherapy of CMV. Few clinical trials have been performed showing the safety and efficacy of this approach in vivo. In this study, donor-derived, CMV-specific T cells were generated for 12 adult HSCT patients by stimulation with dendritic cells transduced with an adenoviral vector encoding the CMV-pp65 protein. Patients received a prophylactic infusion of T cells after day 28 after HSCT. There were no infusion related adverse events. CMV DNAemia was detected in 4 patients after infusion but was of low level. No patient required CMV-specific pharmacotherapy. Immune reconstitution to CMV was demonstrated by enzyme linked immunospot assay in all recipients with rapid increases in predominantly CMV-pp65 directed immunity in 5. Rates of graft-versus-host disease, infection, and death were not increased compared with expected. These results add to the growing evidence of the safety and efficacy of immunotherapy of CMV in HSCT, supporting its more widespread use. This study was registered at www.anzctr.org.au as #ACTRN12605000213640.
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http://dx.doi.org/10.1182/blood-2008-06-161695DOI Listing
November 2008

High-affinity aptamers to subtype 3a hepatitis C virus polymerase display genotypic specificity.

Antimicrob Agents Chemother 2006 Sep;50(9):3019-27

Department of Microbiology, Prince Wales Hospital, Randwick, Sydney, NSW 2031, Australia.

Research into antiviral agents directed at hepatitis C virus (HCV) proteins is commonly based and tested on a single genotype, namely, genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than genotype 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach used in this study that allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNA-dependent RNA polymerase (RdRp) (nonstructural protein 5B [NS5B]) of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 as the partitioning system. Two aptamers, r10/43 and r10/47, were chosen for further studies on the basis of their abilities to bind the HCV RdRp and inhibit polymerase activity. The affinities (equilibrium dissociation constants) of these aptamers for the HCV subtype 3a polymerase were estimated to be 1.3 +/- 0.3 nM (r10/43) and 23.5 +/- 6.7 nM (r10/47). The inhibition constants of r10/43 and r10/47 were estimated to be 1.4 +/- 2.4 nM and 6.0 +/- 2.3 nM, respectively. Inhibition of HCV 3a polymerase was specific for r10/47, while r10/43 also demonstrated some inhibitory effect on norovirus and phi6 polymerase activity. Neither r10/43 nor r10/47 was able to inhibit the RdRp activity of HCV genotype 1a and 1b polymerases. This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase.
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http://dx.doi.org/10.1128/AAC.01603-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563542PMC
September 2006

Norovirus recombination in ORF1/ORF2 overlap.

Emerg Infect Dis 2005 Jul;11(7):1079-85

School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, New South Wales, Australia.

Norovirus (NoV) genogroups I and II (GI and GII) are now recognized as the predominant worldwide cause of outbreaks of acute gastroenteritis in humans. Three recombinant NoV GII isolates were identified and characterized, 2 of which are unrelated to any previously published recombinant NoV. Using data from the current study, published sequences, database searches, and molecular techniques, we identified 23 recombinant NoV GII and 1 recombinant NoV GI isolates. Analysis of the genetic relationships among the recombinant NoV GII isolates identified 9 independent recombinant sequences; the other 14 strains were close relatives. Two of the 9 independent recombinant NoV were closely related to other recombinants only in the polymerase region, and in a similar fashion 1 recombinant NoV was closely related to another only in the capsid region. Breakpoint analysis of recombinant NoV showed that recombination occurred in the open reading frame (ORF)1/ORF2 overlap. We provide evidence to support the theory of the role of subgenomic RNA promoters as recombination hotspots and describe a simple mechanism of how recombination might occur in NoV.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3371806PMC
http://dx.doi.org/10.3201/eid1107.041273DOI Listing
July 2005

Human enterovirus isolates from an outbreak typed using heteroduplex mobility analysis.

J Med Virol 2005 Jun;76(2):215-22

Virology Division, Department of Microbiology, SEALS, The Prince of Wales Hospital, Randwick, Sydney, NSW, Australia.

Genotyping and serotyping of enteroviruses is important for epidemiological, prognostic, and therapeutic reasons. In this study clinical isolates of enterovirus 71 during an outbreak of childhood meningoencephalitis in Sydney, Australia were identified using heteroduplex mobility analysis (HMA) of products from RT-PCR amplification of the 5' untranslated region. Five enterovirus 71 isolates shared identical heteroduplex patterns and nucleotide sequences in the 5' untranslated region. A sixth isolate exhibited minor differences in heteroduplex pattern and sequencing confirmed the isolate varied by 1% at the nucleotide level. The use of multiple reference strains and the analysis of heteroduplex patterns increased the confidence of isolate identification, and allowed identification of strain variation which could be subsequently further analyzed using sequencing. HMA can be used to accurately distinguish identical and variant isolates derived from sporadic cases and clustered infections with enteroviruses, including those causing serious infections.
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http://dx.doi.org/10.1002/jmv.20344DOI Listing
June 2005