Session Chairs: Erle Robertson & Elizabeth White
Sarcoma and Endemic Burkitt Lymphoma
Ethel Cesarman1, Andrea Gardner1, Ryan Snodgrass2, Julio Alvarez1, Varun Kopparthy2, David Erickson2
1Weill Cornell Medical College, 2Cornell University
Diagnosis of cancer in Subequatorial Africa (SSA) is hampered by difficulty accessing pathology support and ancillary testing such as immunohistochemistry. This leads to patients presenting with advanced disease that results in a poor clinical outcome for patients with Kaposi sarcoma (KS) and endemic Burkitt lymphoma (eBL), two common cancers in this region. We developed a low-cost portable device, called TINY (Tiny Isothermal Nucleic acid Amplification sYstem). This lightweight device performs loop- mediated isothermal amplification (LAMP) that runs on a variety of power sources, including solar. We proposed the molecular detection of KSHV in KS biopsies could be provide diagnostic information and be faster than standard pathology. We tested this hypothesis using qPCR and TINY. Biopsies were taken and DNA extraction was performed, followed by conventional quantitative PCR (qPCR) amplification and assessment by TINY. qPCR had a sensitivity of 93% and specificity of 915 (N=347) and TINY had a specificity and sensitivity of 91% (N=128). Testing conducted in SAA provided results within 3 hours, and the longest time went to DNA extraction from skin biopsies. A second disease that could be diagnosed by molecular viral detection is eBL, regularly associated with EBV infection in SSA. In contrast to KS, diagnosis of eBL can be achieved by using cytological specimens obtained by fine needle aspiration (FNA) tumor biopsies, and presence of EBV can distinguish these from other conditions such non-malignant lymphadenopathies. Therefore, we have developed a process-free assay that seamlessly integrates into TINY. The limit of detection was determined to be 90 copies per reaction with sensitivity of 99% at a threshold cutoff time of 30 minutes. To overcome the need for DNA extraction, a solution that allows extraction-free amplification was developed and tested with cell-based samples that mimic material obtained by FNA. This cells-to-LAMP solution produces amplification threshold values on the same order as DNA purified with a commercial kit. In combination with our established TINY system, a direct cells-to-LAMP system has been developed to rapidly detect EBV in lymphomas from FNA at the point-of- care.
Portable TINY kit to obtain liquid biopsy from potential KS patients
TINY: tiny isothermal nucleic acid quantification system
Inexpensive :
LAMP: isothermal nucleic acid amplification (loop mediated isothermal amplification
TINY connects to a laptop for control if unit, real time amplification monitoring and reporting of results
Portable sample processing
Biopsy to process less than 2.5h
EBERs to LAMP in TINY kit for endemic BL children
Detection of EBV in fine needle aspirated (FNA) biopsies from mouse xenografts of BL cell line
Conclusion
An extraction – free cell to LAMP DNA detection method has been validated in the lab and in TINY
Neck Cancer through the Antigen-Specific CD8+ T Cell
Response
Joseph A. Westrich1, Daniel W. Vermeer2, Alexa Silva1, Louis Cicchini1, John H. Lee2, William C. Spanos2, Dohun Pyeon1,3
1University of Colorado School of Medicine, 2Sanford Research, 3Michigan State University
While several proinflammatory chemokines are upregulated during -associated cancer progression, expression of human papillomavirus (HPV) the homeostatic chemokine CXCL14 is epigenetically downregulated by the HPV oncoprotein E7. We have previously shown that restoration of CXCL14 expression in HPV-positive head and neck cancer (HNC) cells dramatically suppresses tumor growth in immunocompetent syngeneic mice, but not in immunodeficient mice. CXCL14 expression induces natural killer (NK) and T cell chemotaxis in vitro and increases NK and T cell populations in the tumor draining lymph node in vivo. To determine the mechanism of CXCL14-mediated tumor suppression, we tested tumor growth in NK or CD8+ T cell deficient mice transplanted with CXCL14-expressing HNC cells. The results show that all CD8+ T cell deficient mice transplanted with CXCL14-expressing HNC cells grow tumor and have significantly reduced survival. In contrast, NK cell deficiency facilitated tumor growth, but did not significantly reduce survival with ~20% of mice still not growing tumor. To determine if the antigen-specificity of CD8+ T cells is important for
- Cxcl14 increases MHC-I expression on HPV+HNSCC cells
- B2M KO abrogates Cxcl14-mediated tumor suppression
- HPVE7 represses Cxcl14 expression to create the immunosuppressive micro environment (HPV E7 also represses MHC I)
- CXCL 14 mediated tumor suppression is reliant on antigen-specific CD8 T cells
- MHC I expression is resorted on HPV-positive HNSCC cells by Cxcl14 expression
- Question
- Cxcl14, low, HPV negative HNSCC will be the next one to investigate
- (查查 NP460EBV, 550EBV)
- Rooney asks PDL1, L2 change or not
of T-cells in Epstein-Barr Virus Type 2 Latency and
Lymphomagenesis
Carrie B. Coleman1, Julie Lang1, Nicholas A. Smith1, Zenggang Pan2, Bradley Haverkos3, Roberta Pelanda1, Rosemary Rochford1
1Department of Immunology and Microbiology, University of Colorado, Anschutz Medical Campus, 2Department of Pathology, University of Colorado, Anschutz Medical Campus, 3Department of Hematology and Oncology, University of Colorado, Anschutz Medical Campus
Epstein-Barr virus (EBV) has been classified into two major strains, EBV Type-1 (EBV-1) and EBV Type-2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B-cells in culture while EBV-2 is poorly transforming. The differing ability to immortalize B-cells in vitro suggest that in vivo these virus strains likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T-cells, not only infecting T-cells in culture, but also infecting T-cells of healthy Kenyan infants, strongly suggesting EBV-2 infection of T-cells is a natural part of the EBV-2 life-cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T-cells to establish latency and/or persist. Thus, we developed an EBV-2 humanized mouse model, utilized BALB/c-Rag2null IL2rγnull SIRPα (BRGS) mice engrafted with CD34+ hematopoietic stem cells. Infection of humanized BRGS mice with EBV-2 led to infection of both T-cells and B-cells, unlike infection with EBV-1, in which virus was only detected in B-cells. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B- and T-cells. Importantly, EBV-2 infected mice developed tumors resembling diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas had morphological features comparable to EBV-1 induced DLBCL and developed at similar rates with equivalent frequency as well as a latency III gene expression profile. Thus, despite the impaired ability of EBV-2 to immortalize B-cells in vitro, EBV-2 efficiently induces lymphoma development in BRGS mice. This new in vivo model can now be utilized to better understand the methods used by EBV-2 to persist and induce lymphomagenesis with specific emphasis on deciphering how EBV-2 infection of T-cells contribute to these processes.
McGeoch and Gatherer 2007 Virology
EBV type 2 also infects T cells
Coleman et al 2017, JID T cell fractions 12 months J Infect Dis. 2017 Sep 15;216(6):670-677. Epstein-Barr Virus Type 2 Infects T Cells in Healthy Kenyan Children. Coleman CB1, Daud II2, Ogolla SO1,3, Ritchie JA4, Smith NA1, Sumba PO3, Dent AE5, Rochford R1.
METHODS: Purified T-cell fractions isolated from children positive for EBV-1 or EBV-2 and their mothers were examined for the presence of EBV and for EBV type.
RESULTS: We detected EBV-2 in all T-cell samples obtained from EBV-2-infected children at 12 months of age, with some children retaining EBV-2-positive T cells through 24 months of age, suggesting that EBV-2 persists in T cells. We were unable to detect EBV-2 in T-cell samples from mothers but could detect EBV-2 in samples of their breast milk and saliva.
CONCLUSIONS: These data suggest that EBV-2 uses T cells as an additional latency reservoir but that, over time, the frequency of infected T cells may drop below detectable levels. Alternatively, EBV-2 may establish a prolonged transient infection in the T-cell compartment. Collectively, these novel findings demonstrate that EBV-2 infects T cells in vivo and suggest EBV-2 may use the T-cell compartment to establish latency. PMID: 28934430
EBV-2 Hu-mouse model
BRG5 mice-BALB/.. with signal regulatory protein a gene from NOD
I pin binding its ligand CD47, SIROa imitated don’t eat me
EBV 2 (LCL10 from Kenya child BL) vs EBV 1 (B958)
EBV type 2 establishes a latency III infection in T and B cells in huMice
Is EBV 2 pathogenic in huMice? Yes
Frequency, rate of tumor development, spleen, LN, kidney and or liver (9-12 month fro type 2)
EBV type 1 and 2 tumors are EBER+ with latency III gene expression program
EBV type 1 and 2 Timor’s showed morphology of diffuse llarge B-cell lymphoma with plasma last phenotype (DLBCL) latency III in both B and T cells
Cd20 downregulated when B cells differ intake tinto plasmablast
EBV 1 and 2 tumors showed remarkable similarities in in immunophenotype
Receptors for infecting T cells: — paper will be coming out soon!
Ming-Han: NPC EBV is type 1
Amplification
Kavi Mehta1, Laimonis Laimins1
1Northwestern University
High-risk human papillomaviruses (HPV) activate the ataxia telangiectasia mutated- dependent (ATM) DNA damage response as well as the ataxia telangiectasia mutated- dependent DNA related (ATR) pathway in the absence of external DNA damaging agents for differentiation-dependent genome amplification. Through the use of comet assays and pulsed-gel electrophoresis our studies show that these pathways are activated in response to DNA breaks induced by the viral proteins E6 and E7 alone and independent of viral replication. The majority of these virally induced DNA breaks are present in cellular DNAs and only minimally in HPV episomes. Treatment of HPV positive cells with inhibitors of both ATM and ATR leads to the fragmentation of viral episomes indicating that DNA breaks are introduced into HPV genomes but are rapidly repaired. This preferential repair occurs as a result of the preferential recruitment of homologous recombination repair enzymes, such as RAD51 and BRCA1, to viral genomes at the expense of cellular DNAs. When HPV positive cells are treated with hydroxyurea, this recruitment of RAD51 and BRCA1 to viral genomes is greatly enhanced with little recruitment to damaged cellular DNAs. Despite substantial damage to cellular DNAs, the ability of viral genomes to amplify is maintained. Overall our studies demonstrate that human papillomaviruses induce breaks into cellular and viral DNAs and that the preferential repair of these lesions in viral episomes leads to genome amplification.
ATM and ATR DNA repair pathways
How does HPV activate DDR? Does HPV induce breaks into DNA?
V Didenko 2002 (T4 DNA algae’s assay on tissue slide)
Neutral Comet assay detects DNBs
HPV induces dsDNA breaks
E6 and E7 alone induce DNA breaks by Comet assay)
DNA breakage detection FISH on comet assay (pretty!)
Breaks are induced in HPV DNAs bit are rapidly repaired
Comet-EdU (label for newly replicating DNA also show minimal breaks in viral DNAs
DNA damage factors such as RAD51 and BRCA1 bind preferentially to HPVC genomes
Comet with 5mm hydrxyurea (Tail lengths increase upon HU)
RAD51 is preferentially recruited to the HPV genome during stress) SMC-1 RAD51
HPV amplifies normally in the presence of ydroxyurea (inducing a great number of DNA breaks)
Potently Inhibit EBV Glycoprotein-Mediated B Cell and
Epithelial Cell Membrane Fusion and Neutralize Virus Infection
Wei Bu1, Gordon Joyce2, Hanh Nguyen1, Yusuke Tsujimura3, Ulrich Baxa4, Yasuhiro Yasutomi3, Gary Nabel2, Masaru Kanekiyo2, Jeffrey Cohen1
1Laboratory of Infectious Diseases, NIH, 2Vaccine Research Center, NIH, 3Laboratory of Immunoregulation and Vaccine Research, National Institutes of Biomedical Innovation, Health, and Nutrition, 4Cancer Research Technology Program, LEIDOS Biomedical Research
Epstein-Barr virus (EBV) infects both B cells and oropharyngeal epithelial cells and is associated with two distinct types of malignancies – B cell lymphoma and epithelial cell cancers. An effective prophylactic vaccine should be able to elicit neutralizing antibodies to prevent EBV infection of both B cells and epithelial cells. Since glycoproteins gH/gL are part of the viral fusion machinery and required for EBV to fuse with host cell membranes, we reasoned that antibodies specifically targeting these viral glycoproteins should block virus infection in both B cells and epithelial cells. We determined that gH/gL-specific antibodies in human plasma are the predominant antibodies that neutralize EBV infection of epithelial cells, and these antibodies, along with those to gp42, also contribute to B cell neutralization. We developed self-assembling nanoparticles that display gH/gL or gH/gL/gp42 and studied their immunogenicity in animals. Mice and nonhuman primates immunized with gH/gL or gH/gL/gp42 nanoparticles developed high levels of neutralizing antibodies that potently neutralize B cell and epithelial cell infection and exceeded titers observed in naturally infected humans. In addition to neutralizing activity, immune serum from nonhuman primates potently inhibited EBV glycoprotein-mediated fusion of epithelial cells and B cells. Sera from animals immunized twice with gH/gL nanoparticles inhibited epithelial cell fusion by 80%, while sera from animals immunized with gH/gL/gp42 nanoparticles inhibited B cell fusion by > 90%. Thus, by targeting the EBV fusion machinery with gH/gL and gH/gL/gp42 nanoparticles, both neutralizing and fusion-inhibitory antibody responses could be generated in mice as well as in nonhuman primates. Eliciting these immune response is likely to be a robust strategy that can be applied to develop vaccines to other herpesviruses.
EBV vaccine should induce antibodies to neutralise infection of both B cells and epithelial cells
EBV vaccine should include gH/gL +- gp42 each are compinets of the EBV fusion machinery
EBV gH/gL+- gp42-ferritin nanoparticles
ferritin 24-mer for repetitive array of antigen （transfect HEK293 with gH-ferritin + gL
B cell neutralizeing antibody titers remain hight titers 》 100 days after lat vaccination in mice and in Cynomologus Monkeys
Epithelial cell neutralizing antibody titers remain higher 》 100 days after last vaccination in Cynomologys Monkeys
gH/gL/gp42-ferritin induces higher EBV B cell and epithelial cell neutralizing antibodies in monkeys htan serum from EBV + humans
Epithelial and B cell fusion assays
sera from immunised monkeys inhibit B cell and epithelial cell fusion