IFN-gamma ELISPOT Assay Research Articles

Combination of E- and NS1-Derived DNA Vaccines: The Immune Response and Protection Elicited in Mice against DENV2.

The occurrence of dengue disease has increased radically in recent decades. Previously, we constructed the pE1D2 and pcTPANS1 DNA vaccines encoding the DENV2 envelope (E) and non-structural 1 (NS1) proteins, respectively. To decrease the number of plasmids in a tetravalent candidate vaccine, we constructed a bicistronic plasmid, pNS1/E/D2, encoding these two proteins simultaneously. We evaluated the protective immunity induced in mice vaccinated with the pNS1/E/D2 candidate and compared to the responses elicited by immunization with the former vaccines isolated or in combination. We transfected BHK-21 cells with the different plasmids and detected recombinant proteins by immunofluorescence and mass spectrometry assays to confirm antigen expression. BALB/c mice were inoculated with the DNA vaccines followed by a lethal DENV2 challenge. ELISA, PRNT50, and IFN-gamma ELISPOT assays were performed for the investigation of the humoral and cellular responses. We observed the concomitant expression of NS1 and E proteins in pNS1/E/D2-transfected cells. All E-based vaccines induced anti-E and neutralizing antibodies. However, anti-NS1 antibodies were only observed after immunization with the pcTPANS1 administered alone or combined with pE1D2. In contrast, splenocytes from pNS1/E/D2- or pcTPANS1 + pE1D2-vaccinated animals responded to NS1- and E-derived synthetic peptides. All the DNA vaccines conferred protection against DENV2.

Phase I/II trial of a long peptide vaccine (LPV7) plus toll-like receptor (TLR) agonists with or without incomplete Freund’s adjuvant (IFA) for resected high-risk melanoma

BackgroundWe performed a clinical trial to evaluate safety and immunogenicity of a novel long peptide vaccine administered in combinations of incomplete Freund’s adjuvant (IFA) and agonists for TLR3 (polyICLC) and...

Validation of an IFN-gamma ELISpot assay to measure cellular immune responses against viral antigens in non-human primates

Adeno-Associated Virus (AAV)-based gene therapy vectors are in development for many inherited human disorders. In nonclinical studies, cellular immune responses mediated by cytotoxic T cells may target vector-transduced cells, which could impact safety and efficacy. Here, we describe the bioanalytical validation of an interferon-gamma (IFN-γ)-based Enzyme-Linked Immunospot (ELISpot) assay for measuring T cell responses against viral antigens in cynomolgus monkeys. Since ELISpots performed with antigen-derived peptides offer a universal assay format, method performance characteristics were validated using widely available peripheral blood mononuclear cells (PBMCs) responsive to cytomegalovirus peptides. The limit of detection and confirmatory cut point were established using statistical methods; precision, specificity, and linearity were confirmed. Monkey PBMCs from an AAV5 gene therapy study were then analyzed, using peptide pools spanning the vector capsid and transgene product. AAV5-specific T cell responses were detected only in 2 of 18 monkeys at Day 28, but not at Day 13 and 56 after vector administration, with no correlation to liver enzyme elevations or transgene expression levels. No transgene product-specific T cell responses occurred. In conclusion, while viral peptide-specific IFN-γ ELISpots can be successfully validated for monkey PBMCs, monitoring peripheral T cell responses in non-clinical AAV5 gene therapy studies was of limited value to interpret safety or efficacy.

247-OR: ADA Presidents’ Select Abstract: Detection of T-Cells Reactive to Hybrid Insulin Peptides in Subjects at Risk for Type 1 Diabetes

Our lab recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors and more recently, in PBMC from T1D patients. Using an IFN-gamma ELISPOT assay to test T cell reactivity in the peripheral blood of T1D patients or control subjects to a panel of 16 HIPs, we demonstrated that 70% of the responding patients showed reactivity to multiple HIPs. To determine whether the presence of HIP-reactive T cells might serve as a biomarker of disease activity in humans, we are following longitudinally a cohort of subjects positive for multiple islet autoantibodies (Ab+), individuals that are at high risk for developing T1D. Using IFN-gamma ELISPOT and CFSE dilution assays, our data indicate that HIP-reactive T cells with an inflammatory phenotype can be detected before disease onset. In one Ab+ individual that showed HIP reactivity, we used single-cell RNA sequencing to study the phenotype and TCR usage of HIP-responding T cells in 3 longitudinal samples collected 6 months apart. We observed that T cells with a regulatory phenotype (FoxP3+, CTLA-4+, LAG3+) can be observed when a proliferative response - but not inflammatory - is detected. In this ongoing study, we are reporting for the first time that HIP-reactive T cells can be observed in the peripheral blood of subjects at risk for T1D, and our data may indicate that the balance of inflammatory vs. regulatory signals of HIP-reactive T cells could be potential indicators of disease activity. Disclosure R.L. Baker: None. T. Delong: None. M. Rewers: None. P. Gottlieb: Advisory Panel; Self; Janssen Scientific Affairs, LLC., Tolerion, Inc., Viacyte, Inc. Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Janssen Pharmaceuticals, Inc., JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Mercia/Nova Laboratories, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. K.M. Haskins: None. Funding American Diabetes Association (1-15-ACE-14 to T.D.); JDRF (2-SRA-2017-511-S-B); National Institutes of Health (R21AI133059-01A1)

Elispots and Ex Vivo Expansion of FVIII-Specific T-Cell Clones Confirm Specificity of CD4+ T-Cell Responses to Self-FVIII in Healthy Non-Hemophilic Blood Donors

The existence of natural antibodies recognizing endogenous factor VIII (FVIII) and of FVIII-specific CD4+ T-cell responses in some healthy, non-hemophilic blood donors has been appreciated for >20 years. The Conti-Fine group measured CD4+ T-cell proliferation following in vitro stimulation with FVIII protein or synthetic FVIII peptides. More recently, FVIII-specific CD4+ T-cell lines were expanded from PBMCs isolated from large blood volumes donated by healthy individuals, and estimates of specific precursor frequency (~2/million CD4+ T cells) were calculated on the basis of interferon (IFN)-gamma ELISPOT assays of FVIII-stimulated cells (Meuniere et al., Blood Advances 1(21): 1842-7). Escape of these self-reactive precursor cells from thymic editing via deletion or anergy and their subsequent persistence in the periphery may contribute to the rare but potentially severe autoimmune reactions to FVIII ("acquired hemophilia A") and to the unusual immunogenicity of therapeutic FVIII administered i.v. to hemophilia A patients. The present study sought to further characterize CD4+ T-cell responses to endogenous FVIII and to map epitopes recognized by these self-reactive cells. We were particularly interested to learn if these cells recognize multiple epitopes in FVIII or if they respond to only several immunodominant epitopes. Accordingly, IFN-gamma ELISPOT assays were carried out by stimulating CD4+ T cells with 15-mer FVIII peptides having 12-residue overlaps and spanning the FVIII A1, A2, A3, C1 and C2 domains. For efficient mapping, initial assays utilized large pools of peptides, and positive responses were then "decoded" by ELISPOTs using smaller peptide pools or individual peptides. Blood samples were obtained from healthy controls under approved IRB protocols. The ELISPOT assays utilized CD4+ T cells isolated by negative selection, with irradiated autologous PBMCs as antigen presenting cells. Anti-CD49d/CD28 monoclonal antibodies were added for co-stimulation to increase the sensitivity of the assay and cells were cultured with IL-7 to improve cell viability. As a result, this assay required smaller blood volumes, but it should be noted that lower-avidity T-cell responses were likely detected that might be missed in ELISPOT assays without these modifications. Relevance of such low-avidity self-reactive cells is provided by the clinical observation, consistent with basic immunological principles, that risk factors for autoimmune responses to FVIII include old age (pro-inflammatory), trauma, surgery and postpartum status, all of which up-regulate T-cell co-stimulatory factors. The first subject had HLA-DRB1*01:01 and HLA-DRB1*08:04 alleles. Stimulation with large peptide pools and rFVIII protein indicated recognition of epitopes in at least 3 FVIII domains. Additional ELISPOTs tested the immunogenicity of 15 peptides corresponding to FVIII peptides previously demonstrated to be presented on dendritic cells from 2 individuals with an HLA-DRB1*01:01 allele (van Haren et al., Mol Cell Proteomics. 2011;10(6)), ensuring that our assays included tests of naturally processed FVIII peptides. Two of these peptides, both from the FVIII A1 domain, produced ELISPOT readings above background levels. T cells were then stimulated with these peptides for 19 days, stained with peptide-loaded MHC Class II (HLA-DRB1*01:01) tetramers, sorted and expanded for another 14 days. Tetramer staining then confirmed isolation of CD4+ T-cell clones recognizing one of these peptides. T cells that recognize their cognate antigen with high avidity are significant drivers of allo- and autoimmune responses. Lower-avidity T cells, however, can play significant roles in pro-inflammatory settings. Tetramer staining validated our ELISPOT-based identification of specific epitopes in FVIII. We are now carrying out ELISPOT assays using pooled peptides followed by individual FVIII peptides as stimulants, to estimate the repertoire of FVIII-specific CD4+ T cells in healthy non-hemophilic individuals. Mapping of HLA-restricted T-cell epitopes will also enable future tetramer-based isolation and phenotypic characterization of these rare T cells without expanding them in culture. This will allow us to investigate the interesting question of what peripheral tolerance mechanisms prevent expansion of these self-reactive cells in vivo, except in rare cases of FVIII autoimmunity. . Disclosures Pratt: Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding.

Simultaneous formulation of influenza vaccine and chitosan nanoparticles within CpG oligodesoxi nucleotides leads to dose-sparing and protects against lethal challenge in the mouse model.

Lack of efficient delivery systems for transporting antigenic molecules to the cytosol of antigen-presenting cells presents a major obstacle for antigen uptake by immune cells. To this end, influenza whole inactivated virus vaccines were formulated with chitosan nanoparticles and CpG oligonucleotide as a biodegradable delivery system and a Th1-specific adjuvant, respectively. Intradermal injections of a single high dose and low dose of formulated candidate vaccines were carried out. Thirty days after injection, cell proliferation assay (MTT), IFN-gamma and IL-4 ELISpot assays were conducted. Sera samples were collected 21 days after immunization to measure IgG1 and IgG2a levels. In addition, the mice challenged with mouse-adopted virus were monitored for weight loss. The results show a significant stimulation of both humoral and cellular immunities; also, weight gain and a decrease in mortality in the mice receiving both dosages of inactivated influenza virus vaccines with CpG and Chitosan coating were observed. Based on the results, it can be concluded that formulation of inactivated influenza virus with CpG and its delivery by chitosan as low-dose can return the same results as with high-dose balanced between cellular and humeral immune responses. This formulation could potentially lead to a significant saving in vaccine production.

Abstract 1059: Enhanced anti-tumor efficacy of a checkpoint inhibitor in combination with the HDAC inhibitor belinostat in a murine hepato-cellular carcinoma preclinical model

Abstract Introduction Belinostat is an HDAC inhibitor currently marketed in the US for the treatment of PTCL. A new oral formulation is under development (positive PoC in preclinical PK study) and provides an increase potential to develop new indications of belinostat in combination with other drugs. It has been extensively demonstrated that some anti-tumor agents, besides their direct anti-tumor effect, may induce additional mechanisms involving activation of immune responses. Thus, combination of these drugs with other immunotherapeutic protocols may yield improved therapeutic benefits. The objective of the present study is the characterization of the therapeutic efficacy of a combination of checkpoint inhibitors (anti-CTLA-4 antibodies) with belinostat in a murine HCC model. In addition, immunoprofiling was performed in order to assess the associated immune response. Methods In vivo efficacy was performed in a Hepa 129 murine hepatocellular carcinoma model implanted subcutaneously in immune-competent C3H mice using anti-CTLA4 alone, belinostat alone or in combination. Treatments were optimized to be able to demonstrate positive effect of the combination. Tumor volume was the primary endpoint. Samples from the spleen were taken to analyze immune mechanism mediating the antitumor activity. Percent of CD4, CD8 T cells and regulatory T cells were determined by flow cytometry. Anti-tumor T cell response was measured by IFN-gamma ELISPOT assay. Results Belinostat improved anti-tumor therapeutic response induced by the anti-CTLA4 checkpoint inhibitor with a significant superior tumor growth inhibition compared to control groups. Treatment with the combination resulted in a complete cessation in tumor growth in all mice during the belinostat treatment period which continued for 1 week after the final dose. Mechanistic studies showed that the underlying immune response correlated with the observed therapeutic effect of the combination with enhancement of IFN-gamma production as antitumor T cell response and decrease in regulatory T cells in the spleens of treated animals. Conclusion These results provide a rational for using belinostat in combination with checkpoint inhibitors to reinforce therapeutic response. Currently only approx. 20% of patients respond to check point inhibitors alone. The oral formulation of belinostat will allow greater flexibility in dosing schedule and use in multiple clinical situations. Further studies are ongoing in order to fully characterize this finding and to facilitate its translation into patients. Citation Format: Diana Llopiz, Marta Ruiz, Perrine Pivette, Véronique Trochon-Joseph, Bérangère Vasseur, Caroline Lemarchand, Graham Dixon, Pablo Sarobe, Bruno Sangro. Enhanced anti-tumor efficacy of a checkpoint inhibitor in combination with the HDAC inhibitor belinostat in a murine hepato-cellular carcinoma preclinical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1059. doi:10.1158/1538-7445.AM2017-1059

Abstract 4559: Novel T-cell targets for HPV16-associated oropharyngeal cancer immune therapy

Abstract Background: Human papillomavirus subtype 16 (HPV16) is the primary cause of an increasing number of oropharyngeal cancers (HPVOPC) worldwide. Despite effective prophylactic vaccines, low vaccine uptake and high incidence of HPV16 infections provides a strong rationale for the development of novel targeted immune therapies against HPVOPC. We sought to 1) Identify novel CD8+ T-cell (CTL) epitopes from HPV16 antigens; 2) Understand mechanisms of immune resistance in HPVOPC to enhance T-cell based immune therapies. Methods: We developed a comprehensive bioinformatic strategy to predict potential CTL epitopes for 10 HLA class I alleles representing common HLA-supertypes (with >90% global coverage) against HPV16 antigens E2, E6 and E7. T-cell reactivity against candidate epitopes was assessed by short term ex vivo T-cell cultures from healthy donors (n=15) and HPVOPC patients (n=18). We data mined publicly available OPC transcriptomes (University of Michigan (UM-OPC) cohort, n=36) for gene expression analysis. Results: IFN-gamma Elispot assays using 58 candidate epitopes from HPV16 E2, E6 and E7 revealed that >70% of HPVOPC patients had either E2 or E6-specific CTL reactivity. E2-CTL reactivity was >2-fold higher in HPVOPC patients than in healthy controls (mean = 68.75 vs. 24.6 SFUs/106 cells, P = 0.027), while no difference in E6- CTL responses were observed between patients and controls. In contrast, E7-specific CTL responses were low to undetectable. We have further identified 7 novel E2 and 3 novel E6 CTL-epitopes, and confirmed immunogenicity of previously described E2, E6 and E7 epitopes. RNAseq analysis of the UM-OPC dataset indicated that 78% of HPV16+ OPCs expressed E2 (P<0.05), while E6 and E7 was expressed in all HPVOPCs (P<0.01) demonstrating tumor antigen expression. Unsupervised hierarchical clustering of immune transcriptomes revealed that HPVOPCs have higher expression of immune-regulatory ligands than HPV-negative OPC tumors, indicating high immune infiltration and possible immune-editing. We identified high expression of several key immune exhaustion genes such as IDO-1, TIM-3 and LAG-3, previously undescribed in HPVOPC. Conclusion: HPV16-E2 represents a novel antigen target for HPVOPC immune therapy, as demonstrated by T-cell reactivity and tumor antigen expression. Several novel and previously defined E2, E6 and E7 CTL have been confirmed for T-cell reactivity in HPVOPC patients. Transcriptomic analysis of OPCs indicate extensive immune dysfunction in HPVOPCs that are distinct from non-HPV OPCs. The T-cell epitopes identified in this study in combination with blockade of HPVOPC-specific checkpoints may be useful for targeted immunotherapy. Citation Format: Sri Krishna, Falguni Parikh, Peaches Ulrich, Shanshan Yang, Amelia Clark, Seunghee Kim-Schulze, Marshall Posner, Jin Park, Andrew Sikora, Karen S. Anderson. Novel T-cell targets for HPV16-associated oropharyngeal cancer immune therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2017-4559

Cellular immunity patterns of rDEN2Δ30 (Tonga/74) support its suitability as a human DENV challenge strain.

Abstract The need for human DENV challenge model to better evaluate DENV candidate vaccines has become of extreme importance in the past years after neutralizing antibody failure to be a good correlate of protection and the lack of valid alternatives. rDEN2Δ30, a variant of DENV2 Tonga/74 strain lacking 30 nucleotides from its 3′ untranslated region, has been established as DENV human challenge model. In this study, DENV cellular immune responses of rDEN2Δ30 strain have been compared with natural infection to derive correlates of protection for DENV vaccines based on cellular immunity. For this purpose, HLA class I and class II restricted peptides have been predicted and tested in an IFN-gamma ELISPOT assay, to assess CD8+ and CD4+ T cell responses in healthy donors infected with rDEN2Δ30. CD8 and CD4 responses were detected in approximately 80% and 90% of donors respectively. Strong CD8 responses are detected in rDEN2Δ30 recipients and when compared with natural infections similarity in terms of magnitude and numbers of recognized epitopes is observed. In particular, the immunodominance hierarchy of the DENV nonstructural proteins NS3, NS5 is maintained in both analyzed cohorts. On the contrary, CD4 responses were mainly focused on nonstructural proteins and less strong respect to natural infection. Large overlap is observed in the epitopes recognized by rDEN2Δ30 infection and natural infection both for CD8 (100%) and CD4 (85%) responses. Finally, when rDEN2Δ30 CD8 response is compared with the one induced by other attenuated DENV isolates, a stronger response is observed. In conclusion, T cell response of rDENV2D30 has very similar specificity to natural infection supporting its suitability as an appropriate human DENV challenge model.

Recognition of Highly Diverse Type-1 and -2 Porcine Reproductive and Respiratory Syndrome Viruses (PRRSVs) by T-Lymphocytes Induced in Pigs after Experimental Infection with a Type-2 PRRSV Strain.

Background/AimLive attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies, suggesting the contribution of cell-mediated immunity (CMI). However, PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end, the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates.MethodsAn IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983) and another strain (type-2 PRRSVVR2332) with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection.ResultsAt 72 days post infection, T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates, while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore, five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes, including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes, neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate.ConclusionThese results demonstrated that T-lymphocytes recognizing antigenically and genetically diverse isolates were induced by infection with a type 2 PRRSV strain (SD23983). If these reponses have cytotoxic or other protective functions, they may help overcome the suboptimal heterologous protection conferred by conventional vaccines.

Abstract B098: Discovering immunotherapeutic T-cell epitopes for HPV16-associated cancers

Abstract HPV subtype 16 (HPV16) causes a persistent viral infection resulting in the majority of cervical cancers and oropharyngeal cancers (HPVOPC). Despite effective prophylactic vaccines, the high incidence of persistent HPV infections provides a strong rationale for the development of novel targeted immunotherapies. Immune therapies such as PD-1 checkpoint inhibitors have the potential to enhance tumor infiltrating CD8+ T-cells (CTLs) in HPV-lesions targeting human leukocyte antigen (HLA) class I-restricted HPV16 epitopes. CTL-epitopes derived from HPV16 are ideal candidates for therapeutic vaccine development as well as immune monitoring of HPV16 associated-cancers. However, the CTL epitope landscape of HPV-16 during viral clearance and tumor progression is largely unknown. We sought to identify epitopes from HPV16 E2, E6, and E7 due to the sustained expression of these proteins in pre-cancerous and in cancerous lesions. To identify potential epitopes with >90% global population coverage, we performed a comprehensive bioinformatics analysis to predict potential HPV16 epitopes from 10 HLA-alleles representing common HLA class I-supertypes (A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801, B*2705, B*3501, B*5701) using 5 different HLA-binding and antigen processing prediction servers. Over 2000 possible candidate peptides from each HPV16 antigen spanning the 10 HLAs were evaluated. Incorporating stringent selection criteria to control for inter-algorithmic variations and normalization, we selected the 58 top-ranked candidate peptides (24 from E2, 20 from E6 and 14 from E7) as potential HPV16-CTL epitope candidates. We confirmed our prediction strategy with well-described HLA-A2 restricted E6 (KLPQLCTEL, TIHDIILECV, FAFRDLCIV) and E7 epitopes (LLMGTLGIV, YMLDLQPET). The immunogenicity of the 58 candidate HPV16 epitopes were evaluated by IFN-gamma Elispot assays using short term ex vivo T-cell lines stimulated in the presence of PD-1 blockade from healthy donors and patients with HPVOPC. HPV-16 E2 and E6-reactive T-cells have been detected at varying specificity and frequencies in PBMCs isolated from healthy blood donors (ranging from 45 to 175 SFUs/10^6 PBMCs) indicating the high prevalence of HPV in the general population. We have confirmed the immunogenicity of previously identified HLA-A2 restricted E2 epitopes TLQDVSLEV and YICEEASVTV. We have also identified novel HLA-A11 and B07-restricted-E6 epitopes (130 and 70 SFUs/10^6 PBMCs respectively) and novel HLA-A11 and B07 restricted-E2 epitopes (445 and 251 SFUs/10^6 PBMCs respectively) from healthy donors. The role of the identified epitopes in HPV clearance is currently unknown and is under investigation. E2 and E6-reactive T-cells were also found in HPVOPC patients indicating the possible presence of viral episomes in HPVOPC lesions. Additional CTL screens for epitope discovery are ongoing. The identified epitopes may be targets for globally relevant immunotherapies and monitoring of HPV related disease. Citation Format: Sri Krishna, Jacqueline Nelson, Falguni Parikh, Amelia Clark, Seunghee Kim-Schulze, Marshall Posner, Andrew Sikora, Karen S. Anderson. Discovering immunotherapeutic T-cell epitopes for HPV16-associated cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B098.

Our journey to successful gene therapy for hemophilia B.

Human Gene TherapyVol. 25, No. 11 Pioneer PerspectivesOur Journey to Successful Gene Therapy for Hemophilia BAmit C. Nathwani, Arthur W. Nienhuis, and Andrew M. DavidoffAmit C. NathwaniKatharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free NHS Foundation Trust, London NW3 2QG, United Kingdom.Department of Haematology, UCL Cancer Institute, London WC1E 6DD, United Kingdom.National Health Services Blood and Transplant, Hertfordshire WD24 4QN, United Kingdom.Search for more papers by this author, Arthur W. NienhuisDepartment of Hematology, St. Jude Children's Research Hospital, Memphis, TN 38105.Search for more papers by this author, and Andrew M. DavidoffDepartment of Surgery, St. Jude Children's Research Hospital, Memphis, TN 38105.Search for more papers by this authorPublished Online:14 Nov 2014https://doi.org/10.1089/hum.2014.2540AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byProgress, and prospects in the therapeutic armamentarium of persons with congenital hemophilia. Defining the place for liver-directed gene therapyBlood Reviews, Vol. 58Gene therapy and editing in the treatment of hereditary blood disorders: Medical and ethical aspects6 November 2021 | Clinical Ethics, Vol. 17, No. 3Challenges in development and authorisation of gene therapy products10 March 2022 | Biological Products. Prevention, Diagnosis, Treatment, Vol. 22, No. 1Validation of an IFN-gamma ELISpot assay to measure cellular immune responses against viral antigens in non-human primates11 January 2021 | Gene Therapy, Vol. 29, No. 1-2Current Clinical Applications of In Vivo Gene Therapy with AAVsMolecular Therapy, Vol. 29, No. 2Gene Therapy Leaves a Vicious Cycle24 April 2019 | Frontiers in Oncology, Vol. 9Serotype survey of AAV gene delivery via subconjunctival injection in mice2 August 2018 | Gene Therapy, Vol. 25, No. 6Gene Therapy for HemophiliaMolecular Therapy, Vol. 25, No. 5Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in DogsMolecular Therapy, Vol. 25, No. 4Progress and challenges in viral vector manufacturing30 October 2015 | Human Molecular Genetics, Vol. 25, No. R1Gene Therapy for Hemophilia Arthur W. Nienhuis, Amit C. Nathwani, and Andrew M. Davidoff28 March 2016 | Human Gene Therapy, Vol. 27, No. 4Treating Hemophilia by Gene TherapyPlasma and Plasma Protein Product Transfusion: A Canadian Blood Services Centre for Innovation SymposiumTransfusion Medicine Reviews, Vol. 29, No. 3 Volume 25Issue 11Nov 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Amit C. Nathwani, Arthur W. Nienhuis, and Andrew M. Davidoff.Our Journey to Successful Gene Therapy for Hemophilia B.Human Gene Therapy.Nov 2014.923-926.http://doi.org/10.1089/hum.2014.2540Published in Volume: 25 Issue 11: November 14, 2014PDF download

O6.06 * PERIPHERAL T CELL RESPONSES IN GLIOBLASTOMA PATIENTS ARE ASSOCIATED WITH AN IMPROVED SURVIVAL

BACKGROUND: Spontaneous anti-tumor T cell responses have been detected in several tumor types. However, not only in glioblastoma it is unclear if such immune responses influence patient outcome. MATERIAL AND METHODS: To detect such immune responses, occurence and frequencies of memory T-cells were determined in the peripheral blood of GBM patients applying the IFN gamma ELISPOT assay and using corresponding tumor lysates (n = 106) or a set of immunogenic polypeptides (n = 32). Extent of resection was assessed by either intraoperative or early postoperative MRI. In addition, intratumoral infiltration of different T-cell subpopulations at the time of first diagnosis was analyzed by immunohistochemistry (n = 20) and compared to the occurrence of spontaneous immune responses. RESULTS: T-cell responses against autologous tumor lysate were detected in 61/106 patients. Subsequent survival analysis concentrating on IDH1wt glioblastoma revealed a significant difference towards an improved survival for patients with a spontaneous immune response (p = 0.0251). Most interestingly, when stratifying for the extent of surgery those patients, who received a complete tumor resection and showed a spontaneous T cell response, survived significantly longer (p = 0.0002). In contrast, in subtotally resected patients the immune response did not improve survival. In line with these observations, occurrence of memory T cell responses against a set of known GBM-associated antigens (TTR, MAGE A3, S100A9) also trended to a better survival in those GBM patients who responded to all three antigens. Finally, increased frequency of intratumoral T cell infiltrates was also associated with the occurrence of spontaneous immune responses and an improved survival. CONCLUSIONS: The present analysis for the first time depicts on several levels a significant influence of the patient's immune system on the course of the disease in GBM patients. In this regard the most crucial prerequisite seems to be the MRI-based complete tumor resection. Most likely surgical removal of the immunosuppressive tumor environment allows for the beneficial reactivation of pre-existing immune responses.

SPONTANEOUS IMMUNE RESPONSES IN GBM PATIENTS AFTER COMPLETE TUMOR RESECTION ARE ASSOCIATED WITH AN IMPROVED SURVIVAL

BACKGROUND: Spontaneous anti-tumor immune responses have been detected in several tumor types. However, not only in glioblastoma it is unclear if such immune responses influence patient outcome. METHODS: To detect such immune responses, occurence and frequencies of memory T-cells were determined in the peripheral blood of GBM patients applying the IFN gamma ELISPOT assay and using corresponding tumor lysates (n = 106) or a set of immunogenic polypeptides (n = 32). Extent of resection was assessed by either intraoperative or early postoperative MRI. In addition, intratumoral infiltration of different T-cell subpopulations at the time of first diagnosis was analyzed by immunohistochemistry (n = 20) and compared to the occurrence of spontaneous immune responses. RESULTS: Altogether, T-cell responses against autologous tumor lysate were detected in 61/106 patients. Subsequent survival analysis concentrating on IDH1wt glioblastoma revealed a significant difference towards an improved survival for patients with a spontaneous immune response (p = 0.0251). Most interestingly, when stratifying for the extent of surgery those patients, who got a complete tumor resection and showed a spontaneous T cell response, survived significantly longer (p = 0.0002). In contrast, in subtotally resected patients the immune response did not improve survival. In line with these observations, occurrence of memory T cell responses against a set of known GBM-associated antigens (TTR, MAGE A3, S100A9) also trended to a better survival in those GBM patients who responded to all three antigens (p = 0.0663). Finally, increased frequency of intratumoral T cell infiltrates was also associated with the occurrence of spontaneous immune responses and an improved survival (p = 0.0113). CONCLUSIONS: The present analysis for the first time depicts on several levels a significant influence of the patient's immune system on the course of the disease in GBM patients. In this regard the most crucial prerequisite seems to be the MRI-based complete tumor resection. Most likely surgical removal of the immunosuppressive tumor environment allows for the beneficial reactivation of pre-existing immune responses. SECONDARY CATEGORY: Tumor Biology.

Nef-Specific CD8+ T Cell Responses Contribute to HIV-1 Immune Control

Recent studies in the SIV-macaque model of HIV infection suggest that Nef-specific CD8+ T-cell responses may mediate highly effective immune control of viraemia. In HIV infection Nef recognition dominates in acute infection, but in large cohort studies of chronically infected subjects, breadth of T cell responses to Nef has not been correlated with significant viraemic control. Improved disease outcomes have instead been associated with targeting Gag and, in some cases, Pol. However analyses of the breadth of Nef-specific T cell responses have been confounded by the extreme immunogenicity and multiple epitope overlap within the central regions of Nef, making discrimination of distinct responses impossible via IFN-gamma ELISPOT assays. Thus an alternative approach to assess Nef as an immune target is needed. Here, we show in a cohort of >700 individuals with chronic C-clade infection that >50% of HLA-B-selected polymorphisms within Nef are associated with a predicted fitness cost to the virus, and that HLA-B alleles that successfully drive selection within Nef are those linked with lower viral loads. Furthermore, the specific CD8+ T cell epitopes that are restricted by protective HLA Class I alleles correspond substantially to effective SIV-specific epitopes in Nef. Distinguishing such individual HIV-specific responses within Nef requires specific peptide-MHC I tetramers. Overall, these data suggest that CD8+ T cell targeting of certain specific Nef epitopes contributes to HIV suppression. These data suggest that a re-evaluation of the potential use of Nef in HIV T-cell vaccine candidates would be justified.

Impact of anti-CD25 monoclonal antibody on dendritic cell-tumor fusion vaccine efficacy in a murine melanoma model

BackgroundA promising cancer vaccine involves the fusion of tumor cells with dendritic cells (DCs). As such, a broad spectrum of both known and unidentified tumor antigens is presented to the immune system in the context of the potent immunostimulatory capacity of DCs. Murine studies have demonstrated the efficacy of fusion immunotherapy. However the clinical impact of DC/tumor fusion vaccines has been limited, suggesting that the immunosuppresive milieu found in patients with malignancies may blunt the efficacy of cancer vaccination. Thus, novel strategies to enhance fusion vaccine efficacy are needed. Regulatory T cells (Tregs) are known to suppress anti-tumor immunity, and depletion or functional inactivation of these cells improves immunotherapy in both animal models and clinical trials. In this study, we sought to investigate whether functional inactivation of CD4+CD25+FoxP3+ Treg with anti-CD25 monoclonal antibody (mAb) PC61 prior to DC/tumor vaccination would significantly improve immunotherapy in the murine B16 melanoma model.MethodsTreg blockade was achieved with systemic PC61 administration. This blockage was done in conjunction with DC/tumor fusion vaccine administration to treat established melanoma pulmonary metastases. Enumeration of these metastases was performed and compared between experimental groups using Wilcoxon Rank Sum Test. IFN-gamma ELISPOT assay was performed on splenocytes from treated mice.ResultsWe demonstrate that treatment of mice with established disease using mAb PC61 and DC/tumor fusion significantly reduced counts of pulmonary metastases compared to treatment with PC61 alone (p=0.002) or treatment with control antibody plus fusion vaccine (p=0.0397). Furthermore, IFN-gamma ELISPOT analyses reveal that the increase in cancer immunity was mediated by anti-tumor specific CD4+ T-helper cells, without concomitant induction of CD8+ cytotoxic T cells. Lastly, our data provide proof of principle that combination treatment with mAb PC61 and systemic IL-12 can lower the dose of IL-12 necessary to obtain maximal therapeutic efficacy.ConclusionsTo our knowledge, this is the first report investigating the effects of anti-CD25 mAb administration on DC/tumor-fusion vaccine efficacy in a murine melanoma model, and our results may aide the design of future clinical trials with enhanced therapeutic impact.

The clinical implication of HAV viral load and HAV proteins-specific IFN-gamma production in patients with acute hepatitis A (P6120)

Abstract The relationship between immune responses to hepatitis A virus (HAV) and the clinical manifestation of HAV infection have been obscured. We investigated the clinical manifestation of acute hepatitis A (AHA) and the relationship between HAV-specific, IFN-gamma-producing T cell response and clinical outcomes in patients with AHA. We analyzed 770 patients diagnosed with AHA for their clinical manifestation. Among them, 128 patients with a prothrombin time (PT) of less than 40% of normal were classified as “severe acute hepatitis (s-AH)”. Others were defined as having “mild acute hepatitis (m-AH)”. In 110 patients, direct ex vivo IFN-gamma ELISpot assays were performed with peripheral blood mononuclear cells using HAV proteins. Mean values of serum HAV RNA at admission were 4.79 log10 copies/ml in s-AH, and 3.30 log10 copies/ml in m-AH (P = 0.003). Viral load showed a significant correlation with ALT (r = 0.417, P = 0.0032) and PT levels (r = -0.453, P = 0.0012). In addition, the mean frequencies of IFN-gamma-producing T cells against VP1-2A (aa 669-782), and 2C(aa 1121-1234) proteins were significantly lower in patients with s-AH, compared to patients with m-AH (3.3 vs. 17.7, P < 0.001; 2.1 vs. 13.3, P < 0.001). In view of viral factors, the load of HAV is closely correlated with liver damage and disease severity in patients with AHA. As a host factor, HAV-specific, IFN-gamma-producing T cells might induce effective viral eradication and result in mild hepatitis

Multiple therapeutic peptide vaccines consisting of combined novel cancer testis antigens and anti-angiogenic peptides for patients with non-small cell lung cancer

BackgroundVaccine treatment using multiple peptides derived from multiple proteins is considered to be a promising option for cancer immune therapy, but scientific evidence supporting the therapeutic efficacy of multiple peptides is limited.MethodsWe conducted phase I trials using a mixture of multiple therapeutic peptide vaccines to evaluate their safety, immunogenicity and clinical response in patients with advanced/recurrent NSCLC. We administered two different combinations of four HLA-A24-restricted peptides. Two were peptides derived from vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2), and the third was a peptide derived from up-regulated lung cancer 10 (URLC10, which is also called lymphocyte antigen 6 complex locus K [LY6K]). The fourth peptide used was derived from TTK protein kinase (TTK) or cell division associated 1 (CDCA1). Vaccines were administered weekly by subcutaneous injection into the axillary region of patients with montanide ISA-51 incomplete Freund’s adjuvant, until the disease was judged to have progressed or patients requested to be withdrawn from the trial. Immunological responses were primarily evaluated using an IFN-gamma ELiSPOT assay.ResultsVaccinations were well tolerated with no severe treatment-associated adverse events except for the reactions that occurred at the injection sites. Peptide-specific T cell responses against at least one peptide were observed in 13 of the 15 patients enrolled. Although no patient exhibited complete or partial responses, seven patients (47%) had stable disease for at least 2 months. The median overall survival time was 398 days, and the 1- and 2-year survival rates were 58.3% and 32.8%, respectively.ConclusionPeptide vaccine therapy using a mixture of four novel peptides was found to be safe, and is expected to induce strong specific T cell responses.Trial registrationThese studies were registered with ClinicalTrials.gov NCT00633724 and NCT00874588.

A single dose of SAAVI MVA-C reboosts rhesus macaques after more than 3 years post DNA-MVA prime-boost vaccination

Background We have previously reported induction of robust immune responses in rhesus macaques following a prime boost immunization with candidate HIV-1 vaccines, SAAVI DNA-C (DNA) and SAAVI MVA-C (MVA). These vaccines are already in clinical evaluation. In the current study, we investigated whether re-boosting these animals with as ingle MVA inoculation after more than 3y ears was sufficient to restore previous magnitudes of HIVspecific immune responses. Methods Seven rhesus macaques which had been vaccinated with three doses of DNA vaccine (4mg DNA/dose) and two doses of MVA (10 9 pfu MVA/dose) in a past study, >3 years previously, were re-boosted with a single dose of MVA. HIV-1-specific responses were quantified in the peripheral blood using an IFN-gamma ELISPOT assay. Results A peak magnitude of response (1146±240 sfu/10 6 PBMC) was reached 1 week after vaccination with the first dose of MVA. The second MVA inoculation did not increase these responses which declined to undetectable levels by 1 year post vaccination. After re-boosting with MVA after 3.5 years post the second MVA, all animals responded, with a peak response (1824±672 sfu/10 6 PBMC) being reached 1 week after vaccination. Although the mean magnitude of the second peak was not significantly higher than the one seen in the first peak, boosting of responses in 3 of 7 animals with an apparent broadening of the breadth of responses was observed. Conclusion These preliminary data suggest a long-term preservation of vaccine memory following a prime-boost vaccination regimen with SAAVI DNA-C and SAAVI MVA-C vaccines.

Use of ESAT-6-CFP-10 fusion protein in the bovine interferon-gamma ELISPOT assay for diagnosis of Mycobacterium bovis infection in cattle

Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay.