Virus-specific Cytotoxic T Lymphocytes Research Articles

Combined treatment with allogeneic Epstein–Barr- and human polyomavirus 1 specific T-cells in progressive multifocal leukoencephalopathy and EBV infection: a case report

Opportunistic viral infections in individuals with severe immunodeficiency can lead to fatal conditions such as progressive multifocal leukoencephalopathy (PML), for which treatment options are limited. These infections pose significant risks, especially when co-infections with other viruses occur. We describe a combined therapy approach using directly isolated allogeneic Human Polyomavirus 1 (also known as BKV) and Epstein–Barr virus (EBV) specific cytotoxic T-cells for the treatment of PML in conjunction with identified EBV in the cerebrospinal fluid (CSF) of a male patient infected with human immunodeficiency virus (HIV). A 53-year-old HIV-positive male, recently diagnosed with PML, presented with rapidly worsening symptoms, including ataxia, tetraparesis, dysarthria, and dysphagia, leading to respiratory failure. The patient developed PML even after commencing highly active antiretroviral therapy (HAART) 3 months prior. Brain magnetic resonance imaging (MRI) revealed multifocal demyelination lesions involving the posterior fossa and right thalamus suggestive of PML. In addition to the detection of human polyomavirus 2 (also known as JCV), analysis of CSF showed positive results for EBV deoxyribonucleic acid (DNA). His neurological condition markedly deteriorated over the following 2 months. Based on MRI, there was no evidence of Immune Reconstitution Inflammatory Syndrome contributing to this decline. The patient did not have endogenous virus-specific T-cells. We initiated an allogeneic, partially human leukocyte antigen-matched transfer of EBV and utilizing the cross-reactivity between BKV and JCV–BKV specific T-cells. This intervention led to notable neurological improvement and partial resolution of the MRI lesions within 6 weeks. Our case of a patient with acquired immune deficiency syndrome demonstrates that PML and concurrent EBV co-infection can still occur despite undergoing HAART treatment. This innovative experimental therapy, involving a combination of virus-specific T-cells, was demonstrated to be an effective treatment option in this patient.

Treatment of Refractory Adenovirus (ADV) Infections Using Adv-Specific Cytotoxic T-Lymphocytes (CTLs) in Children, Adolescents and Young Adults (CAYA) Post Allogeneic Hematopoietic Stem Cell Transplantation (AlloHSCT), Solid Organ Transplantation (SOT), or with Primary Immunodeficiency (PID)

Background: AlloHSCT induces long-term, cure in CAYA with malignant and non-malignant disorders, including those with primary immunodeficiencies (PID). However, post-transplant complications secondary to immunosuppression and slow or delayed immune reconstitution remains a cause of morbidity and mortality amongst AlloHSCT recipients (Satwani/Cairo, BBMT, 2009). Major complications include refractory viral infections, most importantly adenovirus (ADV), which is secondary to absent or decreased adenovirus specific T-cell immunity which occurs due to delayed immune reconstitution and/or continued immunosuppression (George/Cairo, BJH, 2012). The use of virus-specific (vs) CTLs in immunocompromised patients offer an opportunity to restore temporary virus specific immunity. From the Viral Cytotoxic T-Lymphocyte Consortium (VIRCTLC), we present safety and efficacy results on immune compromised, AlloHSCT, or SOT patients treated with familial ADV specific CTLs. Objective: Determine the safety and efficacy of familial ADV specific CTLs for the treatment of refractory systemic ADV infections. Design/Methods: Patient were deemed eligible if they had a history of AlloHSCT, SOT, or PID and had evidence of ADV infections refractory to anti-viral agents. Refractory ADV infection was defined by increasing serum RT-PCR DNA (by 1 log) after 7 days or persistent quantitative RT-PCR DNA copies after 14 days of appropriate anti-viral therapy, and/or known resistance/intolerance to anti-viral agents. Related donors were required to be matched ≥3 HLA (A, B, or DRB1) loci and have an adequate T-cell response to viral specific MACS ADV PepTivators®. We chose a direct selection method to facilitate more rapid generation of cellular products in a point of care setting (and with 14 hrs eligibility determination). Peripheral blood mononuclear cells were collected from eligible family donors using non-mobilized apheresis. vsCTLs were isolated using the CliniMACS ® Prodigy following stimulation with viral specific MACS® GMP PepTivator® AdV5 Hexon, generously provided by Miltenyi Biotec®. ADV specific CTLs were enriched using a Cytokine Capture System (CCS). CD4+ and CD8+ vsCTLs were quantified by flow cytometry (Feuchtinger et al, Blood, 2010). The target cell dose was 0.5x10 4 CD3+ cells/kg (recipient weight) for haploidentical related donors and 2.5x10 4 CD3+ cells/kg for matched related donors. Repeated doses were permitted every 2 weeks to a maximum of doses in the absence of a complete response (CR) and adverse events. The following were used to define response: CR - undetected ADV PCR, partial response (PR) - at least one log decrease from baseline, progressive disease (PD) - at least one log increase from baseline, and patients with stable disease. Results: Fifteen patients were enrolled: 10 females and 5 males; aged 1-19 years. All patients were post-AlloHSCT. All vsCTL donors were haploidentical: 8 were the original HSCT donors and 7 were third party donors (5 maternal, 2 paternal). The mean number of ADV CTL infusions was 3.7 (range: 1-16). One patient with underlying aplastic anemia had progressive disease complicated by increased adenoviral load despite 5 infusions of vsCTLs. Patient received FDA approval for additional CTLs, totaling 16 doses. Thirteen patients achieved CR as defined and 2 achieved PR. The overall response (OR) was 100% and the CR was 88%. The average time to OR was 33 days (range 6-112 days) and time to CR was 34 days (range 6-112 days). Day 100 and 365 overall survival (OS) post-HSCT was 86.1% (CI95: 49.4-95.7) and 70.5% (CI95: 38.9-87.8), respectively (Figure 1). For patients who achieved CR, the Day 100 OS post AlloHSCT was 83.9% (CI95: 55.0-94.3) and Day 365 post AlloHSCT was 74.6% (CI95: 39.8-91.1). Day 100 and 365 probability of ADV related mortality was 0% (Figure 2). One patient developed acute grade 2 skin GVHD possibly related to infusion, which resolved. Conclusion: Our preliminary data demonstrate that haploidentical ADV specific CTLs manufactured by direct selections using the CCS with the CliniMACS® Prodigy and ADV specific peptivators are safe, well-tolerated, and efficacious in patients with refractory/persistent ADV infections after AlloHSCT. Manufacturing is rapid, reproducible, and effective. Accrual is ongoing. This research is supported by FDA RO1006301A1.

424 - Safety and Efficacy of Virus-Specific Cytotoxic T-Lymphocytes Manufactured By the IFN-γ Cytokine Capture System for the Treatment of Refractory Adenovirus, Cytomegalovirus, Epstein Barr Virus, and BK Virus Infections in Children, Adolescents and Young Adults after Allogeneic Hematopoietic Stem Cell Transplantation, Solid Organ Transplantation, or with Primary Immunodeficiency (IND# 17449)

424 - Safety and Efficacy of Virus-Specific Cytotoxic T-Lymphocytes Manufactured By the IFN-γ Cytokine Capture System for the Treatment of Refractory Adenovirus, Cytomegalovirus, Epstein Barr Virus, and BK Virus Infections in Children, Adolescents and Young Adults after Allogeneic Hematopoietic Stem Cell Transplantation, Solid Organ Transplantation, or with Primary Immunodeficiency (IND# 17449)

Manufacture and Characterization of Good Manufacturing Practice-Compliant SARS-COV-2 Cytotoxic T Lymphocytes.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 virus-specific cytotoxic T-cell lymphocytes (vCTLs) could provide a promising modality in COVID-19 treatment. We aimed to screen, manufacture, and characterize SARS-CoV-2-vCTLs generated from convalescent COVID-19 donors using the CliniMACS Cytokine Capture System (CCS). Donor screening was done by stimulation of convalescent COVID-19 donor peripheral blood mononuclear cells with viral peptides and identification of interferonγ (IFN-γ)+ CD4 and CD8 T cells using flow cytometry. Clinical-grade SARS-CoV-2-vCTLs were manufactured using the CliniMACS CCS. The enriched SARS-CoV-2-vCTLs were characterized by T-cell receptor sequencing, mass cytometry, and transcriptome analysis. Of the convalescent donor blood samples, 93% passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that were 79% (standard error of the mean 21%) IFN-γ+ T cells. SARS-CoV-2-vCTLs displayed a highly diverse T-cell receptor repertoire with enhancement of both memory CD8 and CD4 T cells, especially in CD8 TEM, CD4 TCM, and CD4 TEMRA cell subsets. SARS-CoV-2-vCTLs were polyfunctional with increased gene expression in T-cell function, interleukin, pathogen defense, and tumor necrosis factor superfamily pathways. Highly functional SARS-CoV-2-vCTLs can be rapidly generated by direct cytokine enrichment (12 hours) from convalescent donors. NCT04896606.

Dysfunctional Sars-CoV-2-M protein-specific cytotoxic T lymphocytes in patients recovering from severe COVID-19

Although the importance of virus-specific cytotoxic T lymphocytes (CTL) in virus clearance is evident in COVID-19, the characteristics of virus-specific CTLs related to disease severity have not been fully explored. Here we show that the phenotype of virus-specific CTLs against immunoprevalent epitopes in COVID-19 convalescents might differ according to the course of the disease. We establish a cellular screening method that uses artificial antigen presenting cells, expressing HLA-A*24:02, the costimulatory molecule 4-1BBL, SARS-CoV-2 structural proteins S, M, and N and non-structural proteins ORF3a and nsp6/ORF1a. The screen implicates SARS-CoV-2 M protein as a frequent target of IFNγ secreting CD8+ T cells, and identifies M198–206 as an immunoprevalent epitope in our cohort of HLA-A*24:02 positive convalescent COVID-19 patients recovering from mild, moderate and severe disease. Further exploration of M198–206-specific CD8+ T cells with single cell RNA sequencing reveals public TCRs in virus-specific CD8+ T cells, and shows an exhausted phenotype with less differentiated status in cells from the severe group compared to cells from the moderate group. In summary, this study describes a method to identify T cell epitopes, indicate that dysfunction of virus-specific CTLs might be an important determinant of clinical outcomes.

Dynamics of an HTLV-I infection model with delayed CTLs immune response

This paper deals with a four dimensional mathematical model for Human T-cell leukemia virus type-I (HTLV-I) infection that includes delayed CD8+ cytotoxic T-cells (CTLs) immune response. The proposed system has three biologically feasible steady states, namely disease-free steady state, CTL-inactive steady state and an interior steady state. Our theoretical analysis demonstrates that local and global stability analysis are established by the two critical parameters R0 and R1, basic reproduction numbers due to viral infection and due to CTLs immune response, respectively. The disease-free steady state E0 is globally stable if R0≤1, and the HTLV-I infections are eliminated. The asymptotic-carrier steady state E1 is globally stable if R1≤1<R0, which indicates HTLV-I infection is chronic without persistence of CTLs immune response. The interior steady state E2 is globally asymptotic stable if R1>1, which implies that the HTLV-I infection is choric in persistence of CTLs immune response. Due to immune response delay, our proposed model undergoes a destabilization of the interior steady state leading to Hopf bifurcation and periodic oscillations. We estimate the length of time delay that preserve the stability of period-1 limit cycle. We also derived the direction and stability of Hopf bifurcation around the interior steady state by center manifold theory and normal form method. To determine the robustness of the model, we performed normalized forward sensitivity analysis with reference to R0 and R1. Our proposed model undergoes Hopf bifurcation with respect to the production rate of uninfected CD4+T cells h, removal rate of virus-specific CTLs d4, spontaneous infected CD4+T cell activation d2 and transmissibility coefficient β. Implications of our numerical illustrations to the pathogenesis of HTLV-I infection and the development of HTLV-I related HAM/TSP are explored.

Could Changing the DNA Methylation Landscape Promote the Destruction of Epstein-Barr Virus-Associated Cancers?

DNA methylation at CpG motifs provides an epigenetic route to regulate gene expression. In general, an inverse correlation between DNA hypermethylation at CpG motifs and gene expression is observed. Epstein Barr-virus (EBV) infects people and the EBV genome resides in the nucleus where either its replication cycle initiates or it enters a long-term latency state where the viral genome becomes hypermethylated at CpG motifs. Viral gene expression shows a largely inverse correlation with DNA hypermethylation. DNA methylation occurs through the action of DNA methyl transferase enzymes: writer DNA methyl transferases add methyl groups to specific regions of unmethylated DNA; maintenance DNA methyl transferases reproduce the pattern of DNA methylation during genome replication. The impact of DNA methylation is achieved through the association of various proteins specifically with methylated DNA and their influence on gene regulation. DNA methylation can be changed through altering DNA methyl transferase activity or through the action of enzymes that further modify methylated CpG motifs. Azacytidine prodrugs that are incorporated into CpG motifs during DNA replication are recognized by DNA methyl transferases and block their function resulting in hypomethylation of DNA. EBV-associated cancers have hypermethylated viral genomes and many carcinomas also have highly hypermethylated cellular genomes. Decitabine, a member of the azacytidine prodrug family, reactivates viral gene expression and promotes the recognition of lymphoma cells by virus-specific cytotoxic T-cells. For EBV-associated cancers, the impact of decitabine on the cellular genome and the prospect of combining decitabine with other therapeutic approaches is currently unknown but exciting.

Culture expansion of epstein-barr virus-specific cytotoxic T-lymphocytes (EBV-CTLs) using bioreactor with expandable culture area (BECA)

Culture expansion of epstein-barr virus-specific cytotoxic T-lymphocytes (EBV-CTLs) using bioreactor with expandable culture area (BECA)

Increasing Efficacy of Enveloped Whole-Virus Vaccines by In situ Immune-Complexing with the Natural Anti-Gal Antibody.

The appearance of variants of mutated virus in course of the Covid-19 pandemic raises concerns regarding the risk of possible formation of variants that can evade the protective immune response elicited by the single antigen S-protein gene-based vaccines. This risk may be avoided by inclusion of several antigens in vaccines, so that a variant that evades the immune response to the S-protein of SARS-CoV-2 virus will be destroyed by the protective immune response against other viral antigens. A simple way for preparing multi-antigenic enveloped-virus vaccines is using the inactivated whole-virus as vaccine. However, immunogenicity of such vaccines may be suboptimal because of poor uptake of the vaccine by antigen-presenting-cells (APC) due to electrostatic repulsion by the negative charges of sialic-acid on both the glycan-shield of the vaccinating virus and on the carbohydrate-chains (glycans) of APC. In addition, glycan-shield can mask many antigenic peptides. These effects of the glycan-shield can be reduced and immunogenicity of the vaccinating virus markedly increased by glycoengineering viral glycans for replacing sialic-acid units on glycans with α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Vaccination of humans with inactivated whole-virus presenting α-gal epitopes (virusα-gal) results in formation of immune-complexes with the abundant natural anti-Gal antibody that binds to viral α-gal epitopes at the vaccination site. These immune-complexes are targeted to APC for rigorous uptake due to binding of the Fc portion of immunecomplexed anti-Gal to Fcγ receptors on APC. The APC further transport the large amounts of internalized vaccinating virus to regional lymph nodes, process and present the virus antigenic peptides for the activation of many clones of virus specific helper and cytotoxic T-cells. This elicits a protective cellular and humoral immune response against multiple viral antigens and an effective immunological memory. The immune response to virusα-gal vaccine was studied in mice producing anti-Gal and immunized with inactivated influenza-virusα-gal. These mice demonstrated 100-fold increase in titer of the antibodies produced, a marked increase in T-cell response, and a near complete protection against challenge with a lethal dose of live influenza-virus, in comparison to a similar vaccine lacking α-gal epitopes. This glycoengineering can be achieved in vitro by enzymatic reaction with neuraminidase removing sialic-acid and with recombinant α1,3galactosyltransferase (α1,3GT) synthesizing α-gal epitopes, by engineering host-cells to contain several copies of the α1,3GT gene (GGTA1), or by transduction of this gene in a replication-defective adenovirus vector into host-cells. Theoretically, these methods for increased immunogenicity may be applicable to all enveloped viruses with N-glycans on their envelope.

Cellular Immune Responses in Rainbow Trout (Onchorhynchus mykiss) Following Vaccination and Challenge Against Salmonid Alphavirus (SAV).

Viral disease outbreaks remain a significant limiting factor for aquaculture. The majority of licensed vaccines used in the industry are administered as oil-adjuvanted formulations carrying inactivated whole pathogens. Cell-mediated immune responses, in particular those based on virus-specific cytotoxic T-cells (CTLs) to conventional inactivated oil-based vaccines, are largely unexplored. As vaccines cannot be optimized against viral pathogens if knowledge of host cellular immune mechanisms remains unknown, in this study we examined fundamental cell-mediated immune responses after vaccination of rainbow trout with an oil-adjuvanted inactivated vaccine against salmonid alphavirus (SAV) and after infection with SAV. A unique in vitro model system was developed to examine MHC class I restricted CTL responses in a clonal line of rainbow trout. The levels of cell-mediated cytotoxicity were compared to pathology, virus load, specific antibody response, changes in immune cell populations, and mRNA expression. Our results hint that different protective mechanisms are being triggered by infection compared to vaccination. While vaccination itself did not cause a strong cytotoxic or humoral response, subsequent challenge of vaccinated fish resulted in significantly stronger and faster specific cytotoxicity, alongside reduced viral titers and pathology. Hence, testing a vaccine on the capacity to induce cell-mediated cytotoxicity will still require a challenge test. Examination of cellular markers additionally indicates that the initial innate response induced by the vaccine could play an important role in steering adaptive mechanisms.

Safety and Efficacy of Virus-Specific Cytotoxic T-Lymphocytes Manufactured By the IFN-g Cytokine Capture System for the Treatment of Refractory Adenovirus, Cytomegalovirus, Epstein Barr Virus, and BK Virus Infections in Children, Adolescents and Young Adults after Allogeneic Hematopoietic Stem Cell Transplantation, Solid Organ Transplantation, or with Primary Immunodeficiency (IND# 17449)

Background: Viral infection remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Bollard/Heslop Blood 2016). Anti-viral agents for treatment of viral infection in immunocompromised patients are limited in efficacy and are associated with significant toxicities (Gerdemann BBMT 2004; Sili Cytother 2012). The use of virus-specific cytotoxic T-lymphocytes (VST) for immunocompromised patients with viral infections has been associated with therapeutic benefit and improved OS (Bollard/Heslop Blood 2016; Sutrave Cytother 2017). Methods of VST production include ex-vivo expansion and direct selection (Gottlieb Cytother 2017). Ex-vivo expansion requires prolonged manufacturing time, is associated with T-cell exhaustion, and results in a limited donor pool. Direct selection is rapid (12-24 hours), can be done locally, allows for expanded HLA matching, permits a low degree of HLA match to the recipient, and can be adapted for many viruses. A multicenter consortium, the Viral Cytotoxic T-Lymphocyte Consortium (VIRCTLC) was created to investigate the safety and efficacy of VST manufactured by direct selection using the IFN-g Cytokine Capture System process automated on the CliniMACS® Prodigy device (Miltenyi Biotec) for immunocompromised patients with viral infection (Figure 1). Objective: Determine the safety and efficacy of VST for the treatment of immunocompromised child, adolescent and young adult (CAYA) patients with refractory, systemic viral infection and/or viral infection and intolerance to appropriate anti-viral medical therapy. Design/Methods: CAYA patients after allo-HSCT, solid organ transplantation (SOT), or with primary immunodeficiency (PID) with refractory adenovirus (ADV), cytomegalovirus (CMV), Epstein Barr virus (EBV) or BK virus (BKV) infections as evidenced by increasing serum RT-PCR DNA (by 1 log) after 7 days or persistent quantitative RT-PCR DNA copies after 14 days of appropriate anti-viral therapy, and/or known resistance to anti-viral agents, and/or intolerance to anti-viral agents were eligible. Related donors with ≥1 HLA A, B, or DR match to recipient and with an adequate T-cell response to virus specific MACS® PepTivators were eligible. Donors were screened with viral specific antigen (PepTivator®) to predict successful VST manufacturing. Peripheral blood mononuclear cells (PBMC) were collected from eligible related donors using non-mobilized apheresis. VST were isolated using the CliniMACS® Prodigy following stimulation of PBMC with specific viral MACS PepTivator® pools, generously provided by Miltenyi Biotec. Production of CD4+ and CD8+ VST was performed as previously described (Feuchtinger Blood 2010). The target cell dose was 0.5x104 CD3+/kg for HLA mismatched haploidentical related donors and 2.5x104 CD3+/kg for matched related donors. Based on response and safety, VST were given every 2 weeks for a maximum of 5 infusions. Results: Eleven patients have been enrolled to date. Seven patients were treated for ADV, 2 for BKV, 1 for CMV, and 1 for EBV. There were 8 males and 3 females enrolled, aged 1-38 years. There were 10 patients post allo-HSCT and 1 patient post SOT. There were 8 haploidentical, related, original allo-HSCT donors and 3 haploidentical, related, third party donors. There have been no matched related donors enrolled to date. The mean±SEM %CD4+ IFN-g+ of total CD4+, %CD8+ IFN-g+ of total CD8+, and %CD3 cells recovered in the final product were 21.5±4.8, 25.0±7.0, and 50.4.2±7.2, respectively. The median number of VST infusions was 2 (1-5). The mean±SEM CD3+ cell dose was 0.49±0.001x104. Ten patients achieved complete response (PCR negative) and 1 patient achieved partial response (PCR≥1 log decrease). The overall response and complete response rates were 100% and 90.9%, respectively. The median time to maximal response was 34 days (7-141) (Table 1). No patient developed aGVHD, cGVHD, infusion reaction or CRS associated with VST. Conclusion: Preliminary results of this pilot study demonstrate that VST are safe, well tolerated and efficacious in CAYA with refractory viral infections after allo-HSCT, SOT or with PID. Manufacturing utilizing the CliniMACS® Prodigy device is rapid, reproducible and effective. Accrual is ongoing. This research is supported by FDA RO10063-01A1. Disclosures Flower: Lentigen Technology Inc/Miltenyi Biotec: Research Funding. O'Donnell:Kiadis Pharma: Other: Licensing of intellectual property. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Johnson:Cell Vault: Research Funding; Miltenyi Biotec: Research Funding. Cairo:Technology Inc/Miltenyi Biotec: Research Funding; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Miltenyi: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Outcomes and Challenges of Manufacturing Virus-Specific Cytotoxic T-lymphocytes Using IFN-gamma Cytokine Capture System

Background & Aim Background Viral infection/reactivation after allogeneic hematopoietic stem cell (HSCT) or solid organ transplant (SOT) is a significant cause of morbidity and mortality in drug-refractory patients. Adoptive transfer of allogeneic virus-specific cytotoxic T lymphocytes (vCTLs) can rapidly restore antiviral T cell immunity. The IFN-gamma Cytokine Capture System (CCS, Miltenyi) provides a fast and automated method of isolating vCTLs by stimulating them with virus-specific peptides. Aim To evaluate the safety and efficacy of vCTLs manufactured using IFN-gamma CCS on CliniMACS Prodigy® in children and young adults (CAYA) and highlight technical aspects of vCTL manufacturing by this method. Methods, Results & Conclusion Methods CAYA HSCT or SOT recipients with refractory viral infections (CMV, ADV, EBV, BK) received adoptive transfer of vCTLs from haploidentical relatives or matched donors who screened positive for the relevant vCTL. Following non-mobilized leukocytapheresis, virus-specific CD4+ and CD8+ T cells were isolated using IFN-gamma CCS on CliniMACS Prodigy®. Results Fourteen patients have been treated to date (Table 1). Of patients treated for CMV, EBV, ADV, or their combination, 100% (9) achieved complete remission (CR). 2 out of 4 patients treated for BK achieved CR, 2 had partial remission (PR), and one patient treated for CMV/BK had resolution of CMV and no response (NR) to BK. There were no episodes of acute Grade II-IV GVHD, chronic GVHD, CRS, or infusion reactions. We noted occasional discordance between donor viral serology and their response to the stimulation by the viral Peptivator; for example, we successfully manufactured anti-ADV vCTLs from a donor who was serologically negative to adenovirus. Flow cytometry analysis of rare events in a limited sample required sequential gating strategy to maximally reduce background noise. Products thus manufactured have unique properties, such as low cell dose and high debris content, which may face additional regulatory scrutiny. Conclusion Despite its unique challenges, vCTL manufacturing is rapid, reproducible and effective, and vCTLs are safe and well tolerated in CAYA with HSCT and SOT.

Immunization of Pigs with Recombinant Plasmids Containing Genes of Ubiquitinated p30, p54 and CD2v Proteins of African Swine Fever Virus

Abstract Three recombinant plasmid constructs, expressing chimeric proteins containing human ubiquitin fused to an ectodomain of one of the potentially protective proteins (p30, p54 and CD2v) of the attenuated MK-200 strain of African swine fever virus (ASFV), were created as potential inductors of specific antiviral cellular immunity. Three-time immunization of pigs with the mixture of these plasmids led to the formation of virus-specific cytotoxic T-lymphocytes (CTL), but did not induce production of virus-specific antibodies. After challenge with the homologous parental virulent ASFV strain M-78 at a dose of 103 HAD50, all five animals (four immunized pigs and one naïve) fell between the 4th and 7th days post infection. The obtained results demonstrated that induction of CTL did not protect pigs against challenge with the virulent ASFV. Balanced activation of CTL and antibody-mediated cellular mechanisms should be investigated.

The Safety and Efficacy of Targeted Virus Specific Cytotoxic T-Lymphocytes (VST) Manufactured By the IFN-g Cytokine Capture System (CCS) for the Treatment of Refractory Adenovirus (ADV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV) and BK Virus (BKV) in Children, Adolescents and Young Adults (CAYA) after Allogenic Hematopoietic Stem Cell Transplantation (Allo-HSCT), Solid Organ Transplantation (SOT), or with Primary Immunodeficiency (PID) (IND#

Viral infection remains a major cause of morbidity and mortality after allo-HSCT (Bollard/Heslop <i>Blood</i> 2016). Antibiotics for treatment of viral infection in immunocompromised patients are limited in efficacy and associated with toxicity (Gerdemann <i>BBMT</i> 2004; Sili <i>Cytother</i> 2012). Increasing evidence suggests that use of VST for immunocompromised patients with viral infections can provide therapeutic benefit and improve OS (Bollard/Heslop <i>Blood</i> 2016; Sutrave <i>Cytother</i> 2017). Methods of VST production include ex-vivo expansion and direct selection (Gottlieb <i>Cytother</i> 2017). Ex-vivo expansion requires prolonged manufacturing time, is associated with exhaustion, and provides a limited donor pool. Direct selection results in rapid production and allows for expanded HLA matching. A multicenter consortium (VIRCTLC) was created to investigate the safety and efficacy of VST (Figure 1) manufactured by the IFN- CCS process automated on the CliniMACS® Prodigy device (Miltenyi Biotec) for immunocompromised patients with viral infection. To determine the safety and efficacy of VST therapy for immunocompromised CAYA patients with viral infection. CAYA patients after allo-HSCT, SOT or with PID with refractory ADV, CMV, EBV or BKV infections despite 2 weeks of appropriate anti-viral therapy, and/or resistance to antibiotics and/or intolerance to antibiotics were eligible. Donors were screened with viral specific antigen (PepTivator®) to predict successful VST manufacturing. Eligible donor PBMC were collected with non-mobilized apheresis. VST were isolated using the CliniMACS® Prodigy following specific stimulation of PBMC with specific viral MACS PepTivator® pools, generously provided by Miltenyi Biotec. Production of CD4+ and CD8+ VST was performed as previously described (Feucthinger <i>Blood</i> 2010). Target cell dose was 0.5 × 10⁴ CD3⁺/kg for HLA mismatched related donors and 2.5 × 10⁴ CD3⁺/kg for matched related donors. Based on response and safety, additional doses of VST were given every 2 weeks, for a maximum of 5 infusions. Thirteen patients were treated under IND 17449 and other single INDs. Median number of VST infusions was 2 (1-5). Mean±SEM CD3⁺ cell dose infused was 0.48±0.03 × 10⁴. There were 10 CR (PCR negative), 2 PR (PCR≥1 log decrease), and 1 too early to assess (ORR 100%, CRR 83%). Median time to maximal response was 24 days (range 3-111). No patient developed aGVHD, extensive cGVHD, infusion reaction or CRS associated with VST infusions. One patient experienced recurrence of aGVHD, possibly related to VST. (Table 1) Preliminary results of this pilot study demonstrate that VST are safe and efficacious in CAYA with refractory viral infections after alloHSCT, SOT or with PID. VST manufacturing utilizing the CliniMACS® Prodigy device is rapid (24 hours), reproducible and effective. Accrual is ongoing.

A unique variant of lymphocytic choriomeningitis virus that induces pheromone binding protein MUP: Critical role for CTL

Lymphocytic choriomeningitis virus (LCMV) WE variant 2.2 (v2.2) generated a high level of the major mouse urinary protein: MUP. Mice infected with LCMV WE v54, which differed from v2.2 by a single amino acid in the viral glycoprotein, failed to generate MUP above baseline levels found in uninfected controls. Variant 54 bound at 2.5 logs higher affinity to the LCMV receptor α-dystroglycan (α-DG) than v2.2 and entered α-DG-expressing but not α-DG-null cells. Variant 2.2 infected both α-DG-null or -expressing cells. Variant 54 infected more dendritic cells, generated a negligible CD8 T cell response, and caused a persistent infection, while v2.2 generated cytotoxic T lymphocytes (CTLs) and cleared virus within 10 days. By 20 days postinfection and through the 80-day observation period, significantly higher amounts of MUP were found in v2.2-infected mice. Production of MUP was dependent on virus-specific CTL as deletion of such cells aborted MUP production. Furthermore, MUP production was not elevated in v2.2 persistently infected mice unless virus was cleared following transfer of virus-specific CTL.

Long-term eradication of extranodal natural killer/T-cell lymphoma, nasal type, by induced pluripotent stem cell-derived Epstein-Barr virus-specific rejuvenated T cells in vivo.

Functionally rejuvenated induced pluripotent stem cell (iPSC)-derived antigen-specific cytotoxic T lymphocytes (CTL) are expected to be a potent immunotherapy for tumors. When L-asparaginase-containing standard chemotherapy fails in extranodal natural killer/T-cell lymphoma, nasal type (ENKL), no effective salvage therapy exists. The clinical course then is miserable. We demonstrate prolonged and robust eradication of ENKL in vivo by Epstein-Barr virus-specific iPSC-derived antigen-specific CTL, with iPSC-derived antigen-specific CTL persisting as central memory T cells in the mouse spleen for at least six months. The anti-tumor response is so strong that any concomitant effect of the programmed cell death 1 (PD-1) blockade is unclear. These results suggest that long-term persistent Epstein-Barr virus-specific iPSC-derived antigen-specific CTL contribute to a continuous anti-tumor effect and offer an effective salvage therapy for relapsed and refractory ENKL.

Rapid Manufacture and Isolation of Highly Viable Interferon-γ Expressing Virus-Specific T Cells: Validation Results for the Virus-Specific Cytotoxic T-Lymphocyte Consortium for Refractory EBV, CMV, and Adenovirus Infections

Background Children and young adults who develop treatment-refractory infection with EBV, CMV, and/or adenovirus (AdV) after allogeneic hematopoietic stem cell transplant (allo-HSCT) have a dismal prognosis (≥90% mortality). Adoptive transfer of ex-vivo expanded virus-specific cytotoxic T lymphocytes (CTLs) is a treatment option for these patients, but the cell manufacturing process is labor-intensive and typically takes several weeks of culturing to complete. More recently, a culture-free method has been developed to directly isolate virus-specific T cells from peripheral blood for adoptive transfer. The process is fully automated using the CliniMACS Prodigy device (Miltenyi Biotec), a cGMP-compliant, temperature-controlled, closed system. Importantly, virus-specific T cells ready for infusion can be isolated with Methods Peripheral blood mononuclear cells from volunteer donors were stimulated with PepTivators (CMV, EBV, and/or AdV), followed by isolation of the virus-specific T cells using the interferon-γ (IFNγ) cytokine capture system (CCS) on the CliniMACS Prodigy device (n=11). Expression of lineage and T-cell subset markers, IFNγ, and cell viability were determined by flow cytometry. Results The viability of recovered T cells was remarkably and consistently high (92.9% ± 7.4%, Table 1). The number of recovered viable T cells was 5.03 × 106 ± 5.09 × 106. The purity of IFNγ+ cells was 69.0% ± 23.8% for CD4 T cells and 82.9% ± 15.3% for CD8 T cells. At a dose of 2.5 × 104 T cells/kg, the mean T cell collection for a 70 kg recipient would suffice for 3 separate infusions. Conclusion The results presented here show that manufacture of virus-specific CTLs by the CCS method reproducibly produced sufficient quantities of viable T cells for patient treatment. These results thus support the feasibility of the multicenter VIRCTLC trial. The trial is currently enrolling patients.

Generation of virus-specific CD8+ T cells by vaccination with inactivated virus in the intestine of ginbuna crucian carp

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8+ cytotoxic T cells (CTLs) in teleost fish are generated by vaccination with exogenous antigens. In the present study, we investigated whether antigen-specific CD8+ CTLs in ginbuna crucian carp can be elicited by intestinal immunization with an exogenous antigen without any adjuvant. The IFNγ-1 and T-bet mRNA expressions were up-regulated in intestinal leukocytes following the administration of formalin-inactivated crucian hematopoietic necrosis virus (FI-CHNV), whereas the down-regulation of these genes was observed in kidney leukocytes. Furthermore, an increase in the percentage of proliferating CD8+ cells was detected in the posterior portion of the hindgut, suggesting that the virus-specific CTLs are locally generated in this site. In addition, cell-mediated cytotoxicity against CHNV-infected syngeneic cells and the in vivo inhibition of viral replication were induced by immunization with FI-CHNV. Unexpectedly, intraperitoneal immunization with FI-CHNV induced a type I helper T cell (Th1)-response in the intestine, but not in the kidney; however, its effect was slightly lower than that reported after intestinal immunization. These findings suggest that the posterior portion of the intestine is an important site for generating virus-specific CTLs by vaccination with the inactivated vaccine.

Successful Treatment Using Ebstein Barr Virus-Specific Cytotoxic T-Lymphocytes for Post-Transplant Lymphoproliferative Disorder in Pediatric Small Bowel Transplantation

Successful Treatment Using Ebstein Barr Virus-Specific Cytotoxic T-Lymphocytes for Post-Transplant Lymphoproliferative Disorder in Pediatric Small Bowel Transplantation

Granzyme K-deficient mice show no evidence of impaired antiviral immunity.

The biological role of granzyme K, a serine protease of cytotoxic T lymphocytes (CTL), is controversial. It has been reported to induce perforin-mediated cell death in vitro, but is also reported to be non-cytotoxic and to operate in inflammatory processes. To elucidate the biological role of this protease, we have deleted the granzyme K gene in mice (mutant allele: Gzmktm1.1Pib; MGI:5636646). Gzmk -/- mice are healthy, anatomically normal, fecund and show normal hematopoietic development. Gzmk -/- mice readily recover from lymphocytic choriomeningitis virus and mouse pox Ectromelia virus infection. Ex vivo, virus-specific granzyme K-deficient CTL are indistinguishable from those of wild-type mice in apoptosis induction of target cells. These data suggest that granzyme K does not play an essential role in viral immunity or cytotoxicity. Our granzyme K knockout line completes the collection of mouse models for the human granzymes, and will further our understanding of their biological roles and relationships.