Pp65 Antigen Research Articles

An optimized IFN-\u03b3 ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

BackgroundIn healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions.MethodsObjective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay.ResultsOptimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+ (Th), CD3+CD8+ (CTL), CD3−CD56+ (NK) and CD3+CD56+ (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive.ConclusionThe combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

Expression of human cytomegalovirus components in the braintissues of patients with Rasmussen\u2019s encephalitis

Rasmussen’s encephalitis (RE) is a rare and severe progressive epileptic syndromewith unknown etiology. Infection by viruses, including human cytomegalovirus (HCMV),has been speculated to be a potential trigger for RE. However, no viral antigenshave been detected in the brains of patients with RE; thus, a possible clinicallinkage between viral infections and RE has not been firmly established. In thisstudy, we evaluated the expression of HCMV pp65 antigen in brain sections from 26patients with RE and 20 non-RE patients by immunohistochemistry and in situ hybridization, and assessed the associationsbetween HCMV infection and clinical parameters. Elevated expression of HCMV pp65protein and DNA was observed in 88.5% (23/26) and 69.2% (18/26) of RE cases,respectively. In the non-RE group, HCMV pp65 antigen was detected only in two cases(10%), both of which were negative for DNA staining. Additionally, the intensity ofHCMV pp65 staining was correlated with a shorter duration of the prodromal stage,younger age of seizure onset, and more severe unilateral cortical atrophy. Elevatedexpression of HCMV pp65 was observed in RE brain tissue and was correlated with theclinical features of RE disease. In summary, our results suggested that HCMVinfection may be involved in the occurrence and progression of RE disease. Thus,further studies are needed to determine whether early treatment with anti-HCMVantibodies could modulate the course of RE.

Use of CMV gB/pp65 eVLPs to harness the immunogenicity of foreign viral antigens to target solid tumors.

165 Background: Human cytomegalovirus (CMV) is a ubiquitous, generally asymptomatic virus that is present in over 90% of GBM and breast cancer tumors which have comparatively low mutational burdens and predicted neoantigens relative to melanoma or lung cancer. Memory CD4+ and CD8+T cells are most frequently directed against the gB and pp65 antigens, respectively. Thus, CMV gB and pp65 represent highly immunogenic “foreign” antigen components of a vaccine against GBM and breast cancer. Methods: Enveloped virus-like particles (eVLPs) are produced after transfection of HEK 293 cells with a plasmid encoding murine leukemia virus Gag plasmid fused in-frame with CMV pp65 antigen, which gives rise to particles. Co-transfected CMV gB plasmid enables particles budding from the cell surface to incorporate the gB protein into the lipid bilayer. Surface expression of gB and internal expression of pp65 have been confirmed by CryoEM and immunogold labelling. Production and purification of multiple batches at a GMP-compliant manufacturer has demonstrated consistent purity and yields that meet regulatory requirements. Results: eVLPs restimulate IFN-g secreting CD4+ and CD8+ T cells in ex vivo PBMCs from healthy subjects (n=8) at mean frequencies of 0.27% and 1.28%, respectively. eVLP formulation with GM-CSF augments IFN-g and CCL3 secretion in stimulated PBMCs from healthy subjects (n=5), GBM patients (n=5), and breast cancer (n=1) patients to comparable levels. This vaccine candidate also induces de novo CD4+ and CD8+responses in mice. The vaccine induces high titer antibody responses against CMV gB, and the potential for ADCC activity against gB-expressing GBM tumor cells is currently being investigated. Conclusions: CMV gB/pp65 eVLPs address several shortcomings of past theraeputic vaccines, including: 1) the poor immunogenicity of "self" tumor-associated antigens, 2) failure to induce robust CD4+ responses that support CD8+ tumor-specific responses, 3) inclusion of only one or a few epitopes that rapidly encourage tumor cell immunoselection of variants that no longer express the target(s). An IND submission to FDA for a phase I/IIa trial in GBM patients is planned for H1 2017.

Early abnormal liver enzyme levels may increase the prevalence of human cytomegalovirus antigenaemia after hematopoietic stem cell transplantation.

ObjectiveHuman cytomegalovirus (HCMV) infection is common after bone marrow transplantation (BMT), and it increases morbidity and mortality for transplant recipients. HCMV infection may cause hepatitis and elevate the liver enzymes aspartate transferase (AST) and alanine transferase (ALT). This study aimed to analyse the associations between liver enzyme levels and infection with HCMV antigenaemia after BMT.MethodsData from 30 patients after BMT were collected at different time points (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, and 6.0 months post-transplantation). The patients were divided into the HCMV antigenaemia-positive and HCMV antigenaemia-negative groups according to a peripheral blood pp65 antigen assay. Immunohistochemistry was used to identify HCMV pp65 antigen and conventional methods were used to detect liver enzyme levels.ResultsTwelve patients were pp65 antigenaemia-positive and 10 patients were positive in the first 3 months post-transplant. Liver enzyme levels were increased after positivity for HCMV antigenaemia (p = 0.034 and p = 0.018 for ALT and AST, respectively). One month before antigenaemia, AST levels were higher in the HCMV antigenaemia-positive group compared with the negative group (p = 0.006).ConclusionHCMV antigenaemia mostly occurs in the early stage of post-BMT and early abnormal liver enzyme levels may increase the chance of HCMV antigenaemia after BMT.

Real-time PCR and pp65-antigen test for monitoring human cytomegalovirus infection in kidney transplant patients

Background: Human cytomegalovirus (HCMV) is a common virus that infects people of all ages. HCMV infection is an important and common complication in immunocompromised patients, especially transplant recipients. Antigenemia test and real-time PCR are one of the most common assays for diagnosis and monitoring of CMV infections. The aims of this study were to compare two common detection methods in order to identify clinically useful CMV infection in kidney transplant patients. Material and methods : One hundred and fifty peripheral blood samples from kidney transplant patients, including 78 men and 72 women aged from 4 to 73 years; with mean age of 36 years, collected during March 2016 to June 2016. Then samples were investigated for pp65-antigen on polymorphonuclear cells and HCMV DNA viral load on plasma and whole blood. Results: Out of 150 samples analyzed, HCMV DNA was detected in 47(31.33%) cases; with 26 (55.32%) and 21(44.68%) cases in men and women, respectively. The pp65 antigen was detected in 42(28%) casas; with 23 (54.76%) and 19 (45.24%) cases in men and women, respectively. Of the 150 samples, 42 (28%) were positive for both assays and 108 (72%)were negative. Conclusion: Our findings showed both tests were significantly correlated and can be useful for monitoring of CMV infection. Hence, higher viral loads have been found to be associated with increase of disease complication, Real- time PCR is more suitable. The findings merit more investigations involving larger numbers of samples

THE RESULTS OF LONG-TERM STUDY OF HERPESVIRUS INFECTION AT THE DEPARTMENT OF INFECTIOUS DISEASES IN CHILDREN OF RUSSIAN NATIONAL RESEARCH MEDICAL UNIVERSITY

As a result of many years of research, the role of Herpesviridae IV, V and VI types in the structure of various pathologies in children (with infectious mononucleosis (MI), CNS damage, long-term subfebrile syndrome, vasculitis syndrome, hepatitis, etc.) has been proven. MI is a polyethological disease associated with the Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpesvirus type 6 (HHV6), occurring in mono(58%) and mixed infections in various combinations of herpesviruses (42%). Clinical manifestations of MI are typical and do not depend on etiology. With CMV and HHV6 IM, the response to heterophilic antibodies is always negative. Markers of herpesvirus infections were detected in 87% of children observed with neurological pathology. Children with diagnoses such as convulsive syndrome, epilepsy, neuropathy need a comprehensive examination (ELISA, PCR, Indirect immunofluorescence reaction) to identify markers of active replication of herpes viruses, with an emphasis on the detection of HHV6. In 44% of cases, the role of active herpesvirus infection in the etiology of prolonged subfebrile fever in children was proven, with mixed forms (78%) prevailing over monoinfection (22%). To identify the active forms of herpesvirus infection, it is important to detect the markers of viral replication. For EBV, this is the DNA of the virus in the blood, a smear from the tonsils and saliva, antigens of the complete assembly of EBV in blood lymphocytes, antibodies to the capsid antigen (VCA) of IgM and IgG classes, and IgG to the early (EA) antigen. With CMV infection, complete virus assembly antigens (pp65 and pp72) in blood lymphocytes and/or DNA of the virus in the blood, virus DNA in saliva and urine, antibodies to the IgM class virus and high IgG class titer in the blood. With HHV6 infection, the DNA of the virus in the blood, a smear from tonsils and saliva, complete antigens of HHV6 in blood lymphocytes, specific IgM and/or IgG antibodies are 2—4 times higher than the diagnostic values.

Cytomegalovirus Infection in Pediatric Haploidentical Hematopoietic Stem Cell Transplantation at a Single Center

Introduction : Clinical aspects and risk factors of Cytomegalovirus (CMV) reactivation and diseases are an emerging interest in recent investigation fields of hematopoietic stem cell transplantation (HSCT). Several risk factors and clinical manifestations of CMV infection have been reported in numbers of previous reports. Monitoring of viral replication and timely given antiviral agents have resulted in a decreased incidence of CMV-related death recently. However, CMV in still a major cause of infectious complications after HSCT. Specially in haploidentical HSCT setting, clinical manifestations and associated risk factors have been rarely reported. This study is conducted to identify risk factors and clinical aspects of CMV reactivation and disease in pediatric haploidentical HSCT.Methods : We retrospectively reviewed the medical records of 92 pediatric patients who underwent haploidentical HSCT using ex vivo T cell-depleted grafts at Asan Medical Center between December, 2004 and June, 2016. After haploidentical HSCT, all recipients were monitored for CMV infection by CMV pp65 antigen and/or CMV-PCR. Both recipient and donor were screened for CMV serology before the HSCT. During the study period, methodologic changes in ex vivo T cell depletion and CMV prophylaxis policy have been modulated with the course of time. During the 1st study period (Group I, n=19), CD3+ T cell was depleted without CMV prophylaxis while ganciclovir for CMV prophylaxis was given with CD3+ T cell depletion during the 2nd period (Group II, n=16). The T cells expressing alpha-beta T cell receptor (TCRαβ) was depleted during the 3rd period (Group III, n=57) with ganciclovir prophylaxis.Results : Of the 92 patients, 49 (53.3%) developed CMV reactivation after haploidentical HSCT. CMV reactivation significantly reduced in group II and III compared to group I (84±8.4% vs 46±5.9%; p<0.001). Patients with chronic graft-versus-host-disease (GVHD) also experienced more reactivation (p=0.041). In the multivariate analysis, difference of study group was the only variable predicted the occurrence of CMV reactivation (Group I vs Group III; hazard ratio, 3.728; 95% confidence interval (CI), 1.910-7.278). CMV disease was developed in 16/92 (17.4%) patients during the study period (16/49, 32.7% among CMV reactivation patients). The peak CMV pp65 antigen level, peak CMV DNA-PCR level, and duration of CMV antigenemia were significantly higher in patients with CMV disease (p=0.024, p=0.009, p=0.046, respectively). In univariate analysis, the peak CMV pp65 antigen level (p=0.020), and duration of CMV antigenemia (p=0.05) were associated with increased occurrence of CMV disease. In multivariate analysis, CMV disease significantly increased in patients with high CMV pp65 antigenemia greater than 40/200,000 cells (hazard ratio, 3.568; 95% CI 1.062-12.594). CMV diseases included retinitis in 12 (75%), pneumonitis in 3 (25%), gastritis, colitis, and encephalitis in 1 (8.3%) each, respectively. Of the 16 patients, 3 patients (2 pneumonitis, 1 encephalitis) died of CMV disease.Conclusions : Our study demonstrated that ganciclovir prophylaxis has effectively reduced the occurrence of CMV reactivation in ex vivo T cell depleted haploidentical HSCT. In addition, close monitoring of CMV pp65 antigen and timely preemptive management is important to prevent CMV related death. DisclosuresNo relevant conflicts of interest to declare.

Safety and Preliminary Efficacy of "Ready to Administer" Cytomegalovirus (CMV)-Specific T Cells for the Treatment of Patients with Refractory CMV Infection

Despite advances in antiviral drugs, Cytomegalovirus (CMV) infections remain a significant cause of morbidity and mortality in immunocompromised individuals. We have recently demonstrated in hematopoietic stem cell transplant (HSCT) recipients that adoptively-transferred virus-specific T cells, generated from healthy 3rd party donors and administered as an "ready to administer" product, can be curative, even in patients with drug-refractory CMV infections. However, broader implementation has been hindered by the postulated need for extensive panels of T cell lines representing a diverse HLA profile, as well as the complexities of large scale manufacturing for widespread clinical application. To address these potential issues, we have developed a decision tool that identified a short list of donors who provide HLA coverage for >90% of the stem cell transplant population. Furthermore, to generate banks of CMV-specific T cells from these donors, we have created a simple, robust, and linearly scalable manufacturing process. To determine whether these advances would enable the widespread application of "ready to administer" T cells, we generated CMV cell banks (Viralym-C™) from 9 healthy donors selected by our decision tool, and initiated a fixed-dose (2x107 cells/m2) Phase I clinical trial for the treatment of drug-refractory CMV infections in pediatric and adult HSCT recipients.To generate the Viralym-C™ banks, we stimulated donor peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries spanning the immunodominant CMV antigens pp65 and IE1. Cells were subsequently expanded in a G-Rex device, resulting in a mean fold expansion of 103±12. The lines were polyclonal, comprising both CD4+ (21.3±6.7%) and CD8+ (74.8±6.9%) T cells, and expressed central CD45RO+/CD62L+ (58.5±4.2%) and effector memory markers CD45RO+/CD62L- (35.3±12.2%). Furthermore, the lines generated were specific for the target antigens (IE1: 419±100; pp65 1070±31 SFC/2x105, n=9).To date, we have screened 12 patients for study participation, and from our bank of just 9 lines we have successfully identified a suitable line for all patients within 24 hours. Of these, 6 patients have been infused; 5 received a single infusion and 1 patient required 2 infusions for sustained benefit. There were no immediate infusion-related toxicities; and despite the HLA disparity between the Viralym-C lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (GvHD). One patient developed a transient fever a few hours post-infusion, which spontaneously resolved. Based on viral load, measured by quantitative PCR, or symptom resolution (in patients with disease), Viralym-C™ cells controlled active infections in all 5 evaluable patients; 4 patients had complete responses, and 1 patient had a partial response within 4 weeks of cell infusion. One patient with CMV retinitis had complete resolution of symptoms following Viralym-C™ infusion.In conclusion, our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" Viralym-C™ cells that have been generated from a small panel of healthy, eligible CMV seropositive donors identified by our decision support tool. These data suggest that cost-effective, broadly applicable T cell anti-viral therapy may be feasible for patients following HSCT and potentially other conditions. DisclosuresTzannou:ViraCyte LLC: Consultancy. Leen:ViraCyte LLC: Equity Ownership, Patents & Royalties. Kakarla:ViraCyte LLC: Employment.

Heterogeneity of Specific CD4+ and CD8+ T Cells Stimulated by CMV pp65 and IE1 Antigens.

Characterization of human cytomegalovirus-specific T cells (CMV-T) is of critical importance for their potential use in adoptive immunotherapy after allogeneic hematopoietic stem cell transplantation. Background frequencies of CMV-T in peripheral blood mononuclear cells (PBMCs) of CMV-seropositive healthy subjects are usually very low, hence the requirement for prolonged culture time and multiple stimulations to expand them. The evaluation of the end-culture specificity and composition has sometimes been neglected or difficult to assess in these settings. We explored the identity and the functionality of pp65-specific and IE1-specific T cells, enriched in short-term cultures from PBMCs. Antigen-specific T cells were further isolated by IFN-γ capture system and/or CD154 microbeads. Frequency of IE1-specific cytotoxic T cells in PBMCs secreting IFN-γ was higher compared with the pp65-specific one, whereas the latter cell types showed a higher median CD107a degranulation. Cell viability, rate of CMV-T increase, and multicytokine secretion profile after epitope-specific short-term cultures were heterogenous. T cells were mainly of late effector stages but they significantly dropped off upon CMV rechallenge with peptide pools. In parallel, CMV-T expansion was accompanied by a significant increase of cytotoxic naive/memory stem cells (CTLs), whereas the CD4 counterpart significantly increased only upon stimulation with IE1. Outcome was variable and showed donor and epitope dependency. Differences in human leukocyte antigen and epitope dominance and variability in the relative number of CD3 effector cells and IFN-γ/CD154 expression among healthy donors could reflect the observed individual CMV-specific cellular immunity. This heterogeneity raises points to be considered when approaching adoptive immunotherapy.

Abstract B028: Blockade of the CD47 “Do not eat” signal by TTI-621 (SIRPαFc) leads to enhanced antitumor CD8+ T cell responses in vitro

Abstract High expression of the CD47 “do-not-eat” signal is a mechanism commonly used by tumor cells to escape phagocytosis by macrophages. CD47 transmits an inhibitory signal upon binding to its receptor signal-regulatory protein α (SIRPα) on the surface of macrophages. We have previously shown that TTI-621, a soluble SIRPαFc fusion protein, neutralizes the suppressive effects of CD47 and triggers macrophage-mediated phagocytosis of tumor cells in vitro, and effectively controls tumor growth in vivo. Using a human culture system, we investigated whether this increase in macrophage-mediated phagocytosis results in augmented T cell responses. To evaluate the T cell response to a model tumor antigen, Jurkat, a human leukemia cell line, was stably transfected with a construct containing the human cytomegalovirus phosphoprotein pp65 (CMV-Jurkat). Consistent with our previous studies, blockade of CD47 on CMV-Jurkat cells using TTI-621 led to a dramatic increase in tumor cell phagocytosis by primary macrophages derived from peripheral blood monocytes of healthy, HLA-A2+ donors. Using a high-affinity soluble TCR multimer that specifically recognizes an immunodominant epitope from pp65 complexed with HLA-A2, we found that TTI-621-mediated phagocytosis of CMV-Jurkat resulted in increased pp65 antigen presentation on the surface of macrophages. In cultures treated with a control Fc fragment, where macrophages only exhibited a low level of phagocytosis, no presentation of pp65 peptide could be detected. Moreover, mock-transfected tumor cells were efficiently phagocytosed in the presence of TTI-621, yet did not result in presentation of CMV pp65 peptide. To assess whether this increase in antigen presentation results in augmented T cell responses, macrophages from the phagocytosis assay were co-cultured with autologous CD8+ T cells for five days. We observed robust proliferation of CMV pp65-specific CD8+ T cells following co-culture with macrophages that had phagocytosed CMV-Jurkat in the presence of TTI-621 compared to control Fc treatment. In contrast, no proliferation of CMV pp65-specific CD8+ T cells occurred when macrophages had phagocytosed mock-transfected tumor cells, suggesting that the proliferation of CMV-specific CD8+ T cells is a tumor antigen-specific response. In addition, preliminary data indicate that primed CMV-specific CD8+ T cells are fully functional and are capable of exhibiting cytotoxicity against CMV-Jurkat. Collectively, our study demonstrates for the first time that in a human culture system, blockade of the CD47 “do not eat” signal results in increased phagocytosis, augmented tumor antigen presentation and enhanced anti-tumor CD8+ T cell responses. These results provide further support for the concept that CD47 lies at the intersection of the innate and adaptive immune systems. TTI-621, which is currently in a Phase I trial in patients with advanced hematological malignancies (NCT02663518), may thus be able to harness the anti-tumor activity of both macrophages and T cells. Citation Format: Natasja Nielsen Viller, Tran Truong, Emma Linderoth, Lisa D. Johnson, Stephane Viau, Gloria H. Y. Lin, Mark Wong, Xinli Pang, Penka S. Petrova, Robert A. Uger. Blockade of the CD47 “Do not eat” signal by TTI-621 (SIRPαFc) leads to enhanced antitumor CD8+ T cell responses in vitro [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B028.

Relationship between human cytomegalovirus infection and glioma

Objective To study the relationship between human cytomegalovirus (HCMV) infection and glioma. Methods HCMV DNA in glioma samples (n=35) and control samples (n=17) was detected by nested polymerase chain reaction (PCR). The expression of HCMV pp65 protein was detected by immunohistochemistry (IHC). Results HCMV DNA was positive in 51.4% (18/35) of glioma samples and was detectable in normal brain samples (P<0.05). The positive rate of HCMV pp65 antigen was 60% (21/35) in glioma samples and 0 in the normal brain controls (P<0.05). Conclusion Based on the results of nested PCR and IHC there was significant difference in HCMV infection between glioma and normal brain tissue. HCMV infection may play a role in the pathagenesis and progression of glioma. Key words: Human cytomegalovirus; Glioma; Nested polymerase chain reaction; Immunohistochemistry

469. Improved Protocol and Use of Mitochondrial DNA as Reference for qPCR Quantification of Integrase-Defective Lentiviral Vectors (IDLVs)

Integrase defective lentiviral vectors (IDLVs) are attractive tools for genetic manipulation and are increasingly being tested as recombinant viral vaccines against infectious pathogens and cancer. IDLVs remain mostly as extrachromosomal/episomal DNA circles in the infected cells, hence with reduced risk of genotoxicity mediated by insertional mutagenesis. We have demonstrated in our previous work the production and characterization of a tricistronic IDLV (packaged with the D64V mutation in the integrase and co-expressing GM-CSF, IFN-alpha and the CMV pp65 antigen) and IDLV-mediated monocyte transduction achieved under Good Manufacturing Process (GMP) compliant conditions (Sundarasetty et al., JTM 2015). One of the main criteria for batch release of the cell vaccine is to confirm and quantify IDLV copies in the thawed cell product. Currently used DNA extraction methods are not efficient in isolating small molecular weight episomal DNA from cells. In addition, genomic DNA loci used as reference controls are not informative regarding quantification of episomal DNAs. Mitochondrial DNA (mtDNA) is an episomal small molecular weight circular DNA (16.5 kb) that can serve as a reference for IDLV quantification. In order to maximize recovery of episomal DNAs, we explored the total DNA (tDNA) extraction described for mtDNA isolation (Badralmaa et al., J Vir. Meth. 2013), based on dehydration and precipitation of proteins and subsequent tDNA precipitation from the supernatants by isopropanol. As a reference for the qPCR quantification, we constructed and validated a plasmid containing a sequence homologous to a regulatory region of transfer vector (wPRE), a sequence for a genomic house-keeping gene (PTBP2) and a sequence for a mitochondrial house-keeping gene (Cytochrome B). The amplification of three target regions was validated by generating a standard curve with the reference plasmid ranging from 5 x10 to 5×105 copies (n=3). As a reference, we used an in-house generated 293T cell line (B5) containing three LV genomic copies. Total DNA extracted form B5 was serially diluted in non-transduced 293T DNA in order to result into 0.25, 0.5, 1, 2, 3 LV copies and the assay linearity was assessed in three independent runs. The reliable detection limit of the qPCR assay was 0.5 LV copies/cell and 0.9 copies/ng of genomic DNA. Having assessed the linearity, we assessed if this method could be used to quantify IDLV in transduced monocytes. The tricistronic IDLV was used to transduce monocytes of different donors in triplicates at increasing multiplicities of infection (MOI: 1, 2.5, 5 and 10). After thawing each cryopreserved batch, tDNA was extracted and analyzed. Our results showed a direct correlation between the IDLV copies per cell and ng of DNA used for the assay and MOI, whereas detection of the genomic and mtDNA references remained constant. Thus, this simple methodological adaptation provided an internal mtDNA control to better quantify episomal vector copies, which can be also used for qPCR quantification of other types of non-integrating vectors.

476. No GVHD, but Human Inflammatory “Cytokine Storm” and Mouse Macrophage Activation Upon Accelerated Development of Human CD4+ Effector Memory T Cells in Long-Term Humanized Mice

Non clinical assessment of immunotoxicity of human cellular products in vivo is limited by the lack of availability of pharm-tox models enabling a full range of human innate and adaptive responses. We previously demonstrated improved development of human adaptive T and B cell responses in mice receiving human hematopoietic stem cell transplantation (HSCT) in combination with prime/boost immunization with induced dendritic cells (iDCs), i.e. monocytes derived the stem cell donor reprogramed with a lentiviral vector for co-expression of GM-CSF, IFN-alpha and the CMV antigen pp65 that self differentiate into DCs in vivo (Salguero et al, 2014 J. Immunology; Daenthanasanmak et al, 2015 Mol. Ther. Meth.). For these previous studies, mice were kept for 6-10 weeks after iDC administration, and we did not observe signs of tumorigenicity or graft-versus-host disease (GVHD). After consultation with the German regulatory authorities, we were requested to perform pharm-tox analyses 26 weeks after iDC immunization. We report here a pilot-feasibility study with humanized mice showing long-term (more than 33-36 weeks after HSCT) robust immune reconstitution with human T cells (control mice, n=2 Vs. iDC-immunized mice, n=6). Longitudinal analyses of human immune reconstitution in blood samples confirmed accelerated development of CD4+ effector memory T cells 5-10 weeks after iDC immunization. No weight loss, GVHD or malignancies were observed after iDC immunization. 3/6 immunized mice developed skin erythema and inflammation around 23 weeks after iDC immunization and were sacrificed earlier for analyses. Analyses of all mice were performed at autopsy by macroscopic observations, H&E histopathology, immunohistochemistry and flow cytometry of target organs. Positivity for anti-human nuclei antibody, anti-human HLA-DR and anti-human CD3 leucocytes were detected in several tissues (spleen, bone marrow, brain, lungs, skin, eyelids, liver, and kidneys), confirming the long-term persistency of human T cells in the humanized mice. In inflamed skin, abundant human CD4+ T cells were localized in the basal layers of the epidermis and hair follicles in the dermis. Human macrophages (CD68+) could be detected in several tissues, but were rare in inflamed skin. However, in the inflamed dermis areas of ulceration, mouse macrophages (F4/80+) were observed frequently with strongly positive cytoplasm. Mice with ulcerations had significantly higher levels of several human inflammatory cytokines in plasma (IFN-gamma, IL-6, GM-CSF and IL-8) than immunized mice with no skin inflammation or control mice. In summary, a long-term pilot study exploring a hybrid human-mouse model system was feasible and iDCs accelerated mature human T cell reconstitution with no signs of tumorigenicity or GVHD. However, accumulation of human CD4+ memory T cells and activated mouse macrophages in inflamed skin was associated with elevated levels of human inflammatory cytokines resembling a “cytokine storm”.

Risk Factors and Clinical Features of Cytomegalovirus Disease in Children Receiving Anticancer Chemotherapy.

This study was conducted to identify risk factors for cytomegalovirus (CMV) infection and demonstrate the spectrum of CMV disease in children receiving anticancer chemotherapy without hematopoietic stem cell transplantation (HSCT). A total of 289 children who received chemotherapy and were tested for CMV infection were included in the study. CMV antigenemia and DNAemia were determined by identifying the pp65 antigen in leukocytes and performing real-time PCR. CMV disease was diagnosed by tissue biopsy, culture, or ophthalmic examination. Of the 289 children, CMV infection was demonstrated in 46 patients (15.9%). Young age at cancer diagnosis was the risk factor for CMV infection by multivariate analysis (7 mo vs. 7 y, P<0.001). Among 46 children with CMV infection, 10 (21.7%) were diagnosed with CMV disease; hepatitis (n=4), retinitis (n=3), hepatitis and pneumonia (n=2), and hepatitis and retinitis (n=1). The age of the patients with CMV disease was significantly younger than those without (3 vs. 16 mo, P=0.023). Retinoblastoma and neuroblastoma were the 2 most common underlying malignancies. There were 2 fatal cases associated with CMV disease, including 1 who died of CMV pneumonia. The findings of this study demonstrated significant morbidity of CMV infection and disease in young children during the course of chemotherapy without HSCT.

Impact of Persistent Cytomegalovirus Infection on Dynamic Changes in Human Immune System Profile.

Human cytomegalovirus (HCMV) imprints the immune system after primary infection, however its effect during chronic infection still needs to be deciphered. In this study we report the variation of blood cell count along with anti-HCMV IgG and T cell responses to pp-65 and IE-1 antigens, that occurred after an interval of five years in a cohort of 25 seropositive healthy adults. We found increased anti-viral IgG antibody responses and intracellular interferon-gamma secreting CD8+ T cell responses to pp-65: a result consistent with memory inflation. With the only exception of shortage in naive CD8+ T cells most memory T cell subsets as well as total CD8+ T cells, T cells, lymphocytes, monocytes and leukocytes had increased. By contrast, none of the cell types tested were found to have increased in 14 subjects stably seronegative. Rather, in addition to a shortage in naive CD8+ T cells, also memory T cell subsets and most other cell types decreased, either in a statistically significant or non-significant manner. The trend of T cell pool representation with regard to CD4/CD8 ratio was in the opposing directions depending on HCMV serology. Globally, this study demonstrates different dynamic changes of most blood cell types depending on presence or absence of HCMV infection. Therefore, HCMV plays a continual role in modulating homeostasis of blood T cells and a broader expanding effect on other cell populations of lymphoid and myeloid origin.

A Single Center Experience: Short Term CD4 Count Monitoring and Rate of Opportunistic Infections in Human Immunodeficiency Virus (HIV) Infected Patients

Background/Objectives: CD4 cell count is a key measure of Human immunodeficiency virus (HIV) disease progression and for the risk of developing a life-threatening opportunistic infections (OIs). We designed this study to assess and monitor CD4 count at baseline and response to antiretroviral therapy (ART) at 6 and 12 months and to know the prevalence of OIs among all adult with HIV-infection in a tertiary care hospital in Saudi Arabia (SA). Material/methods: Retrospective study was conducted with a total of 61 HIV seropositive patients attended at King Abdulaziz Medical City-Riyadh from January 2005 to November 2015, data was collected and recorded form patients’ charts, electronic health record system and HIV database for age, gender, nationality, CD4 count at baseline for all patients and at 6 months and 12 months for patients receiving ART and OIs. Flow cytometry was used for absolute CD4 count measurement. Identification of all causative microorganisms of OIs was performed by the standard microbiologic methods with clinical correlation. Results: Majority, 46/61(75.4%) of study participants were male patients. The mean age of participants was 44.39 with std of +/- 13.375 years. Mean baseline CD4 count for all study participants was 285.28 with std of +/- 306.333 cells/μL. Distribution of baseline CD4 count for all patients was, DC4 count less than 200 cells/μL were 34/61(55.7%), CD4 count from 200-500 cells/μL were 13/61(21.3%) and CD4 count more than 500 cells/μL were 14/61(23.0%). A total of 30/61 patients (49.2%) were not receiving ART and 31/61 patients (51.8%) were receiving ART. A total of 22/61 (36.1%) OIs were observed from the study participants. Commonly observed OIs were Cytomegalovirus (CMV) 14/61 (23.0%) (Positive IgG and IgM antibodies, pp65 antigen and PCR without clinical correlation), Tuberculosis 4/61(6.6%), pneumocystis pneumonia 2/61(3.3%) Toxoplasmosis 2/61(3.3%), Syphilis 2/61(3.3), varicella zoster virus (VZV) 1/61(1.6%), herpes simplex virus (HSV) 1/61 (1.6%). Conclusions: Half of HIV-infected patients 55.7% in this study were severely immunocompromised at the time of HIV diagnosis. Half of patients receiving ART were nonadherence to medications and 21% of patients had a poor immunologic response after one year of adherence to ART. One-third of our HIV-positive patients were infected with one or more OIs.

Electrochemical Immunoassay for Cytomegalovirus Antigen Detection with Multiple Signal Amplification Using HRP and Pt‐Pd Nanoparticles Functionalized Single‐walled Carbon Nanohorns

AbstractCytomegalovirus is typically associated with immunocompromised hosts, pregnant women and transplant patients, who require a timely diagnosis. In this work, a sensitive and highly specific electrochemical amplification immunosensor was established for detecting Cytomegalovirus pp65 antigen based on Pt‐PdNPs@SWCNHs with horseradish peroxidase (HRP) as a signal enhancer and thionine as a signal probe. First, Pt nanoparticle (PtNP) and Pd nanoparticle (PdNP) functionalized single‐walled carbon nanohorn (SWCNH) nanocomposites, i.e. Pt‐PdNPs@SWCNHs, was used as a carrier for immobilization of antibody through the Pt‐N bond and the Pd‐N bond. Next, HRP was used to block the rest of the binding‐sites. Signal amplification was obtained by the cooperative catalytic activities of Pt‐PdNPs and HRP to H2O2. SWCNHs loaded with a large amount of Pt‐PdNPs further amplified the signal due to the excellent surface area. The fabricated immunosensor was used to detect different concentrations of Cytomegalovirus pp65 antigen under optimized conditions. The tests showed a linear range from 0.1 to 80 ng mL−1 with a low detection limit of 30 pg mL−1, and exhibited excellent selectivity, stability and reproducibility. Therefore, this project presented a potential approach for the early diagnosis of Cytomegalovirus infection in clinical trials.

Evaluation of JC and Cytomegalo Viruses in Glioblastoma Tissue

Glioblastoma multiforme (GBM) is the most aggressive of the gliomas, a collection of tumors arising from glia in the central nervous system. Possible associations between the human cytomegalovirus (HCMV) and the JC virus with GBM are now attracting interest. Our present aim was to investigate the prevalence of the two viruses in Iranian patients from Kerman’s cities in the south of Iran. In addition, the expression rates of pp65, large T antigen and p53 proteins were assessed and their relation with GBM evaluated using reverse transcription real time PCR (rReal Time PCR). A total of 199 patients with GBM cancer were enrolled, with mean±SD ages of 50.0±19.5 and 50.7±19.6 years for males and females, respectively. The P53 rate was dramatically low suggesting an aetiological role,. Large T antigen expression was found in JC positive samples, while the PP65 antigen was observed in patients positive for CMV and JC. HCMV products and JC virus with oncogenic potential may induce the development of various tumors including glioblastomas. The JC virus produces an early gene product, T-antigen, which has the ability to associate with and functionally inactivate well-studied tumor suppressor proteins including p53 and pRB.

NIK- and IKKβ-binding protein promotes colon cancer metastasis by activating the classical NF-κB pathway and MMPs.

The nuclear factor (NF)-κB pathway has been implicated in colorectal cancer (CRC) tumorigenesis. Here, we investigated the role of a novel NIK- and IKKβ-binding protein (NIBP) in CRC metastasis through activation of the canonical NF-κB pathway. NIBP, p-p65, and matrix metalloproteases (MMPs) were assessed by immunohistochemistry in 114 CRC tissues, and the time to metastasis was recorded after surgery. Furthermore, the activity of the NF-κB pathway, MMP expression, and the metastatic potential of HT-29 cells overexpressing NIBP after treatment with the NF-κB inhibitor pyrrolidinecarbodithioic acid (PDTC) were examined in vitro and in vivo. NIBP-positive CRC exhibited a higher rate of metastasis, and the time to metastasis of NIBP-positive patients was shorter in the early tumor, lymph node, metastasis (TNM) stages (I and II), while NIBP and p-p65 expression was higher in later TNM stages (III and IV). However, there was no difference in terms of the positive rate of NIBP, p-p65, MMP-2, and serum carcinoembryonic antigen (CEA) level was no difference in the pathological type, gender, tumor location, or size. The NF-κB pathway, MMP-2 and MMP-9 activity, and cell motility and invasion were increased in NIBP-overexpressing cells, even after PDTC treatment. Moreover, these cells exhibited high metastasis in mice, and p-p65, MMP-2, and MMP-9 expression levels were elevated in the primary tumor and liver metastases. In conclusion, NIBP overexpression increases the CRC metastatic potential through activation of the NF-κB pathway and increasing MMP-2 and MMP-9 expression. In addition, NIBP overexpression, at least in part, may reduce inhibition of the canonical NF-κB pathway and MMPs caused by PDTC treatment.

Is CMV a target in pediatric glioblastoma? Expression of CMV proteins, pp65 and IE1-72 and CMV nucleic acids in a cohort of pediatric glioblastoma patients.

While the 5-year overall survival is better in pediatric than in adult patients diagnosed with glioblastoma (GBM), outcomes in children remain very poor. Understanding the mechanisms of tumorigenesis and tumor propagation can identify therapeutic targets to improve these outcomes. Human cytomegalovirus (CMV) proteins and nucleic acids are present in the majority of adult GBM. Indeed, CMV is emerging as a potential glioma-associated target for anti-CMV agents and cellular therapeutics. Furthermore, CMV appears to contribute to GBM’s malignant phenotype, although its role in tumorigenesis is less certain. In this cohort of 25 serially diagnosed pediatric GBMs, the largest described cohort to date, we used immunohistochemical staining and in situ hybridization to show the presence of CMV antigens pp65 and IE1-72 as well as CMV nucleic acids, respectively. Our cohort indicated either CMV antigen pp65 or IE1-72 was present in approximately 67 % of pediatric GBM samples. The majority of samples stained positive for either CMV antigen showing a cytoplasmic pattern in 25-50 % of cells within the sample at a moderate intensity, while a few samples showed nuclear staining and higher grade/intensity. Of 16 samples where in situ hybridization was performed, 13 (81 %) showed specific staining using a CMV genome specific probe cocktail. ISH positive samples showed high concordance with being pp65 or IE1-72 positive. These findings, paired with the association of CMV expression with poor prognosis and overall survival, indicate the need to further investigate how these antigens are promoting tumor growth and preventing cell death. Also, the expression of these antigens in a majority of tumor tissues should be considered for immunotherapeutic targets in cases of pediatric GBM.