Enzyme-linked Immunosorbent Spot Research Articles

Clinical immunogenicity outcomes from GENEr8-1, a phase 3 study of valoctocogene roxaparvovec, an AAV5-vectored gene therapy for hemophilia A

Gene transfer therapies utilizing adeno-associated virus (AAV) vectors involve a complex drug design with multiple components that may impact immunogenicity. Valoctocogene roxaparvovec is an AAV serotype 5 (AAV5)-vectored gene therapy for treatment of hemophilia A that encodes a B-domain–deleted human factor VIII (FVIII) protein controlled by a hepatocyte-selective promoter. Following previous results from the first-in-human phase 1/2 clinical trial, we assessed AAV5 capsid– and transgene-derived FVIII-specific immune responses with 2 years of follow-up data from GENEr8-1, a phase 3, single-arm, open-label study in 134 adult men with severe hemophilia A. No FVIII inhibitors were detected following administration of valoctocogene roxaparvovec. Immune responses were predominantly directed toward the AAV5 capsid, with all participants developing durable anti-AAV5 antibodies. Cellular immune responses specific for the AAV5 capsid were detected in most participants by interferon-ᵧ enzyme-linked immunosorbent spot assay 2 weeks following dose administration and declined or reverted to negative over the first 52 weeks. These responses were weakly correlated with alanine aminotransferase elevations and showed no association with changes in FVIII activity. FVIII-specific cellular immune responses were less frequent and more sporadic compared with those specific for AAV5 and showed no association with safety or efficacy parameters.

A bispecific antibody targeting HLA-DQ2.5-gluten peptides potently blocks gluten-specific t cells induced by gluten ingestion in patients with celiac disease

The gluten-free diet for coeliac disease (CeD) is restrictive and often fails to induce complete symptom and/or mucosal disease remission. Central to CeD pathogenesis is the gluten-specific CD4+ T cell that is restricted by HLA-DQ2.5 in over 85% of CeD patients, making HLA-DQ2.5 an attractive target for suppressing gluten-dependent immunity. Recently, a novel anti-HLA-DQ2.5 antibody that specifically recognizes the complexes of HLA-DQ2.5 and multiple gluten epitopes was developed (DONQ52). ObjectiveTo assess the ability of DONQ52 to inhibit CeD patient-derived T-cell responses to the most immunogenic gluten peptides encompassing immunodominant T cell epitopes. MethodsWe employed an in vivo gluten challenge model in patients with CeD that affords a quantitative readout of disease-relevant gluten-specific T-cell responses. HLA-DQ2.5+ CeD patients consumed food containing wheat, barley, or rye for 3 days with collection of blood before (D1) and 6 days after (D6) commencing the challenge. Peripheral blood mononuclear cells were isolated and assessed in an interferon (IFN)-gamma enzyme-linked immunosorbent spot assay (ELISpot) testing responses to gluten peptides encompassing a series of immunodominant T cell epitopes. The inhibitory effect of DONQ52 (4 or 40 μg/mL) was assessed and compared to pan-HLA-DQ blockade (SPVL3 antibody). ResultsIn HLA-DQ2.5+ CeD patients, DONQ52 reduced T cell responses to all wheat gluten peptides to an equivalent or more effective degree than pan-HLA-DQ antibody blockade. It reduced T cell responses to a cocktail of the most immunodominant wheat epitopes by a median of 87.3% (IQR 72.4–92.4). Notably, DONQ52 also substantially reduced T-cell responses to dominant barley hordein and rye secalin derived peptides. DONQ52 had no effect on T-cell responses to non-gluten antigens. ConclusionDONQ52 can significantly block HLA-DQ2.5-restricted T cell responses to the most highly immunogenic gluten peptides in CeD. Our findings support in vitro data that DONQ52 displays selectivity and broad cross-reactivity against multiple gluten peptide:HLA-DQ2.5 complexes. This work provides proof-of-concept multi-specific antibody blockade has the potential to meaningfully inhibit pathogenic gluten-specific T-cell responses in CeD and supports ongoing therapeutic development.

Final outcomes from a phase 2 trial of posoleucel in allogeneic hematopoietic cell transplant recipients

AbstractAllogeneic hematopoietic cell transplantation (allo-HCT) recipients are susceptible to viral infections. We conducted a phase 2 trial evaluating the safety and rate of clinically significant infections (CSIs; viremia requiring treatment or end-organ disease) after infusion of posoleucel, a partially HLA-matched, allogeneic, off-the-shelf, multivirus-specific T-cell investigational product for preventing CSIs with adenovirus, BK virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus-6, or JC virus. This open-label trial enrolled allo-HCT recipients at high risk based on receiving grafts from umbilical cord blood, haploidentical, mismatched, or matched unrelated donors; post-HCT lymphocytes of <180/mm3; or use of T-cell depletion. Posoleucel dosing was initiated within 15 to 49 days of allo-HCT and subsequently every 14 days for up to 7 doses. The primary end point was the number of CSIs due to the 6 target viruses by week 14. Of the 26 patients enrolled, only 3 (12%) had a CSI by week 14, each with a single target virus. In vivo expansion of functional virus-specific T cells detected via interferon-γ enzyme-linked immunosorbent spot assay was associated with viral control. Persistence of posoleucel-derived T-cell clones for up to 14 weeks after the last infusion was confirmed by T-cell–receptor deep sequencing. Five patients (19%) had acute graft-versus-host disease grade 2 to 4. No patient experienced cytokine release syndrome. All 6 deaths were due to relapse or disease progression. allo-HCT recipients at high risk who received posoleucel had low rates of CSIs from 6 targeted viruses. Repeat posoleucel dosing was generally safe and well tolerated and associated with functional immune reconstitution. This trial was registered at www.ClinicalTrials.gov as #NCT04693637.

An investigation of the immune epitopes of adeno-associated virus capsid-derived peptides among hemophilia patients

Adeno associated virus (AAV) is an optimal gene vector for monogenic disorders. However, neutralizing antibodies (Nab) against AAV hinder its widespread application in gene therapy. In this study, we biosynthesized peptides recognized by the Binding Antibodies (Bab) from the sera containing high Nab titer against AAV2. We established four immunological methods to detect immune epitopes of the AAV2-derived peptides, including Bab assay, Nab assay, B-cell receptors (BCR) detecting assay and immunoglobin-producing B cells enzyme-linked immunosorbent spot (B-cell ELISpot) assay. Correlations among the epitopes determined by these four methods were analyzed using the serum samples and peripheral blood mononuclear cells (PBMC) from 89 hemophilia A/B patients. As decoys, the peptides' ability to block the Nab of AAV2 particles was assessed using AAV transduction models both in vitro and in vivo. Overall, we provide insights into AAV2 capsid-derived peptides immune epitopes, involving the Nab, Bab, BCR, and B-cell ELISpot, offering alternative immunological evaluation approaches and strategy to overcome Nab barriers in AAV-mediated gene therapy.

Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4+ and CD8+ effector T cells. T cell receptor β-chain (TCRβ) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV’s mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117.

Development of an Enzyme-Linked Immunosorbent Spot Assay for the Assessment of Adeno-Associated Virus Peptides to Examine Immune Safety.

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.

Utility of the Interferon-Gamma Enzyme-Linked Immunosorbent Spot Assay to Predict Risk of Cytomegalovirus Infection in Kidney Transplant Recipients.

Non-specific interferon-gamma (IFN-γ) enzyme-linked immunosorbent (ELISpot) responses after solid organ transplant (SOT) and their relationship with cytomegalovirus (CMV) reactivation have hardly been investigated. Adult kidney transplant (KT) recipients underwent measurement of IFN-γ-producing T cells using the ELISpot assay before and 1 month after transplantation. Data for CMV infection episodes were collected. Risk factors for post-transplant CMV infection, based on IFN-γ responses, were analyzed using a Cox proportional hazards model. A total of 93 KT recipients were enrolled in the study and 84 evaluable participants remained at 1 month post KT. Thirty-three (39%) recipients developed subsequent CMV infection within 6 months post-transplant. At 1-month post-transplant, IFN-γ-producing T cells with <250 spot-forming units (SFUs)/2.5 × 105 peripheral blood mononuclear cells (PBMCs) were significantly associated with CMV infection (HR 3.1, 95% CI 1.4-7.1, p = 0.007). On multivariable analysis, posttransplant IFN-γ-producing T cells with <250 SFUs/2.5 × 105 PBMCs remained independently associated with CMV infection (HR 3.1, 95% CI 1.2-7.8, p = 0.019). Conclusions: KT recipients with low IFN-γ-producing T cells measured by the ELISpot assay are more likely to develop CMV infection after transplantation. Therefore, measurement of nonspecific cell-mediated immunity ELISpot responses could potentially stratify recipients at risk of CMV infection (Thai Clinical Trials Registry, TCTR20210216004).

Development and Evaluation of a Promising Biomarker for Diagnosis of Latent and Active Tuberculosis Infection

Abstract Background Diagnosing latent tuberculosis (TB) infection (LTBI) and active TB (ATB) is crucial for preventing disease progression and transmission. However, current diagnostic tests have limitations in terms of accuracy and sensitivity, making it challenging to diagnose these different infection states. Therefore, this study intends to develop a promising biomarker for LTBI and ATB diagnosis to overcome the limitations of the current diagnostic tests. Methods We developed a novel multiepitope-based diagnostic biomarker (MEBDB) from LTBI region of differentiation antigens using bioinformatics and immunoinformatics. Immune responses induced by MEBDM were detected using enzyme-linked immunosorbent spot and cytometric bead assays. This study was conducted from April 2022 to December 2022 in the Senior Department of Tuberculosis at the 8th Medical Center of PLA General Hospital, China. Blood samples were collected from participants with ATB, individuals with LTBI, and healthy controls (HCs). The diagnostic efficacy of MEBDB was evaluated using receiver operating characteristic curves. Results A novel MEBDB, designated as CP19128P, was generated. CP19128P comprises 19 helper T lymphocyte epitopes, 12 cytotoxic T lymphocyte epitopes, and 8 B-cell epitopes. In silico simulations demonstrated that CP19128P possesses strong affinity for Toll-like receptors and elicits robust innate and adaptive immune responses. CP19128P generated significantly higher levels of tumor necrosis factor (TNF-α), interleukin 4 (IL-4), and IL-10 in ATB patients (n = 7) and LTBI (n = 8) individuals compared with HCs (n = 62) (P &lt; 0.001). Moreover, CP19128P-induced specific cytokines could be used to discriminate LTBI and ATB from healthy subjects with high sensitivity and specificity. Combining IL-2 with IL-4 or TNF-α could differentiate LTBI from HCs (the area under the receiver operating characteristic curve [AUC], 0.976 [95% confidence interval [CI], 0.934–1.000] or 0.986 [0.956–1.000]), whereas combining IL-4 with IL-17A or TNF-α could differentiate ATB from HCs (AUC, 0.887 [0.782–0.993] or 0.984 [0.958–1.000]). Conclusions Our study revealed that CP19128P is a potential MEBDB for the diagnosis of LTBI and ATB. Our findings suggest a promising strategy for developing novel, accurate, and sensitive diagnostic biomarkers and identifying new targets for TB diagnosis and management.

Contribution of SARS-CoV-2 infection preceding COVID-19 mRNA vaccination to generation of cellular and humoral immune responses in children.

Primary COVID-19 vaccination for children, 5-17 years of age, was offered in the Netherlands at a time when a substantial part of this population had already experienced a SARS-CoV-2 infection. While vaccination has been shown effective, underlying immune responses have not been extensively studied. We studied immune responsiveness to one and/or two doses of primary BNT162b2 mRNA vaccination and compared the humoral and cellular immune response in children with and without a preceding infection. Antibodies targeting the original SARS-CoV-2 Spike or Omicron Spike were measured by multiplex immunoassay. B-cell and T-cell responses were investigated using enzyme-linked immunosorbent spot (ELISpot) assays. The activation of CD4+ and CD8+ T cells was studied by flowcytometry. Primary vaccination induced both a humoral and cellular adaptive response in naive children. These responses were stronger in those with a history of infection prior to vaccination. A second vaccine dose did not further boost antibody levels in those who previously experienced an infection. Infection-induced responsiveness prior to vaccination was mainly detected in CD8+ T cells, while vaccine-induced T-cell responses were mostly by CD4+ T cells. Thus, SARS-CoV-2 infection prior to vaccination enhances adaptive cellular and humoral immune responses to primary COVID-19 vaccination in children. As most children are now expected to contract infection before the age of five, the impact of infection-induced immunity in children is of high relevance. Therefore, considering natural infection as a priming immunogen that enhances subsequent vaccine-responsiveness may help decision-making on the number and timing of vaccine doses.

The DNA Sensor AIM2 Determines B Cell Fate after BCR Activation in Chronic Graft-Vs-Host Disease

Prevention and treatment of chronic graft-vs-host disease (cGVHD), a debilitating late immune toxicity in patients after allogeneic hematopoietic stem cell transplantation (allo-HCT), who are otherwise cured of hematological malignancies, remains a major challenge. T cells incite and promote cGVHD through coordinated T-B cell responses. Alloantibody production ( Srinivasan et al, Blood 2012, Jin et al, Blood 2016) and aberrant B Cell Receptor (BCR) signaling ( Allen et al, Blood 2014, Flynn et al, Blood 2015) play fundamental roles in the pathology of cGVHD. B cell-intrinsic pathways in cGVHD remain incompletely understood, limiting our therapeutic capacity for patients. In a single cell RNA sequencing study, we found that transcript levels of the DNA sensor Absent in Melanoma 2 (AIM2) were significantly increased in CD27+ BCR-activated B cell clusters in circulating as well as in tissue-resident cGVHD B cells ( Poe et al, JCI Insight 2023). After confirming AIM2 expression is restricted to the nucleus, we also previously showed that AIM2-expressing B cells were augmented after BCR and interferon-gamma (IFNγ) and/or Toll-like receptor (TLR)-7 or 9 stimulation ( Visentin J, Blood 2022;140 Supp 1), suggesting a context-dependent role for AIM2 after allo-HCT. Our new findings now afford the hypothesis that an AIM2-BCR signaling axis determines B cell fate after allo-HCT. To address this hypothesis, we first examined whether AIM2 modulates the promotion of antibody-secreting cells (ASCs) after BCR activation using a hapten-carrier immunization model. We compared numbers of hapten-specific ASCs from immunized WT and AIM2-KO mouse splenocytes using enzyme-linked immunosorbent spot (ELISPOT). We compared numbers of ASCs on day 3 post-immunization, when early germinal center (GC) formation is known to occur. We found that hapten-specific IgM ASCs were significantly increased in WT splenocytes compared to AIM2-KO (p=0.002). By contrast, when we looked at day 8, when mature GC formation and rapid ASC proliferation is known to occur, we found that AIM2-KO immunized mice had significantly lower numbers of hapten-specific IgM (p=0.005) and IgG (p=0.004) ASCs in AIM2-expressing mice (Figure 1a). Our results suggest a role for AIM2 in regulating the survival of short-lived plasmablast-like ASCs after high affinity antigen encounter and maturation of germinal centers. To further investigate whether AIM2 affects expansion of GC-derived (GL7+CD95+) ASCs, we stimulated B cells purified from wildtype (WT) or global AIM2-deficient (AIM2-KO) mice in vitro with lipopolysaccharide (LPS) and IL-4 +/- BCR stimulation. We found that AIM2-deficient B cells, especially GC-like cell populations, expanded significantly in an anti-IgM concentration-dependent manner up to 12-fold (p=0.03) after 72 hr (Figure 1b). By contrast, B cell expansion was constrained in AIM2-sufficient B cells. This expansion was unlikely to be due to differences in proliferation alone, as we did not detect differential Ki67 expression in stimulated B cells with or without AIM2. Together, these data suggest a role for AIM2 in modulating BCR signaling. Finally, we examined the role of AIM2 in BCR-mediated cell death after chronic antigen exposure using our previously described bone marrow transplant (BMT) model, where recipients of bone marrow and splenocytes (BM+Spl) exhibit cGVHD manifestations ( Poe et al, JCI Insight 2018, Jia et al, Blood 2021). We have previously generated C57BL/6J mice with conditional deficiency of AIM2 only in B cells (AIM2 fl/flMb1 Cre/+, CKO), and used these in our BMT model as donors. At day 58 post BMT, we performed ex vivo stimulation of splenic B cells from BMT recipients. While B cells from CKO BM+Spl recipients expanded ex vivo in an anti-IgM concentration-dependent manner, B cells from AIM2-sufficient BM+Spl recipients demonstrated limited responsiveness ex vivo to BCR stimulation. This extends our in vitro observations by suggesting a role for AIM2 in B cell tolerance after chronic antigen exposure. Ongoing experiments will determine if B cell-intrinsic AIM2 contributes to cGVHD pathogenesis. Thus far, our data reveal a novel role for AIM2 as a B cell survival checkpoint after BCR activation. Further delineation of context-dependent and synergistic AIM2-BCR signaling after allo-HCT will lead to strategies that block the development of cGVHD without adversely affecting anti-tumor activity.

ACE configurator for ELISpot: optimizing combinatorial design of pooled ELISpot assays with an epitope similarity model.

The enzyme-linked immunosorbent spot (ELISpot) assay is a powerful in vitro immunoassay that enables cost-effective quantification of antigen-specific T-cell reactivity. It is used widely in the context of cancer and infectious diseases to validate the immunogenicity of predicted epitopes. While technological advances have kept pace with the demand for increased throughput, efforts to increase scale are bottlenecked by current assay design and deconvolution methods, which have remained largely unchanged. Current methods for designing pooled ELISpot experiments offer limited flexibility of assay parameters, lack support for high-throughput scenarios and do not consider peptide identity during pool assignment. We introduce the ACE Configurator for ELISpot (ACE) to address these gaps. ACE generates optimized peptide-pool assignments from highly customizable user inputs and handles the deconvolution of positive peptides using assay readouts. In this study, we present a novel sequence-aware pooling strategy, powered by a fine-tuned ESM-2 model that groups immunologically similar peptides, reducing the number of false positives and subsequent confirmatory assays compared to existing combinatorial approaches. To validate ACE's performance on real-world datasets, we conducted a comprehensive benchmark study, contextualizing design choices with their impact on prediction quality. Our results demonstrate ACE's capacity to further increase precision of identified immunogenic peptides, directly optimizing experimental efficiency. ACE is freely available as an executable with a graphical user interface and command-line interfaces at https://github.com/pirl-unc/ace.

Convalescent Adaptive Immunity Is Highly Heterogenous after SARS-CoV-2 Infection.

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis

BackgroundPeritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this...

Cellular immune response of SARS-CoV-2 vaccination in kidney transplant recipients: a systematic review and meta-analysis.

Evidence has demonstrated inferior humoral immune responses after SARS-CoV-2 vaccination in kidney transplant recipients compared to the general population. However, data on cellular immune responses in this population have not been established. We searched the MEDLINE, Scopus, and Cochrane databases and included studies reporting cellular immune response rates in kidney transplant recipients after receiving SARS-CoV-2 vaccines. Studies that reported factors associated with cellular immune responders or non-responders were also included (PROSPERO: CRD42022375544). From a total of 1,494 articles searched, 53 articles were included in the meta-analysis. In all, 21 studies assessed cellular immune response by interferon-γ enzyme-linked immunosorbent spot (IFN-γ ELISPOT), 22 studies used interferon-γ release assay (IGRA), and 10 studies used flow cytometric analysis. The pooled response rate after two doses (standard regimen) and three doses of vaccination was 47.5% (95%CI 38.4-56.7%) and 69.1% (95%CI 56.3-80.6%) from IFN-γ ELISPOT, 25.8% (95%CI 19.7-32.4%) and 14.7% (95%CI 8.5-22.2%) from IGRA, and 73.7% (95%CI 55.2-88.8%) and 86.5% (95%CI 75.3-94.9%) from flow cytometry, respectively. Recipients with seroconversion were associated with a higher chance of having cellular immune response (OR 2.58; 95%CI 1.89-3.54). Cellular immune response in kidney transplant recipients was lower than in dialysis patients (OR 0.24; 95%CI 0.16-0.34) and the general population (OR 0.10; 95%CI 0.07-0.14). Age and immunosuppressants containing tacrolimus or corticosteroid were associated with inferior cellular immune response. Cellular immune response after SARS-CoV-2 vaccination in kidney transplant recipients was lower than in dialysis patients and the general population. Age, tacrolimus, and corticosteroid were associated with poor response. Cellular immune response should also be prioritized in vaccination studies. https://www.crd.york.ac.uk/prospero/, identifier CRD42022375544.

Clinical Trial on the Safety and Tolerability of Personalized Cancer Vaccines Using Human Platelet Lysate-Induced Antigen-Presenting Cells.

Research and development of personalized cancer vaccines as precision medicine are ongoing. We predicted human leukocyte antigen (HLA)-compatible cancer antigen candidate peptides based on patient-specific cancer genomic profiles and performed a Phase I clinical trial for the safety and tolerability of cancer vaccines with human platelet lysate-induced antigen-presenting cells (HPL-APCs) from peripheral monocytes. Among the five enrolled patients, two patients completed six doses per course (2-3 × 107 cells per dose), and an interim analysis was performed based on the immune response. An immune response was detected by enzyme-linked immunosorbent spot (ELISpot) assays to HLA-A*33:03-matched KRASWT, HLA-DRB1*09:01-compliant KRASWT or G12D, or HLA-A*31:01-matched SMAD4WT, and HLA-DRB1*04:01-matched SMAD4G365D peptides in two completed cases, respectively. Moreover, SMAD4WT-specific CD8+ effector memory T cells were amplified. However, an attenuation of the acquired immune response was observed 6 months after one course of cancer vaccination as the disease progressed. This study confirmed the safety and tolerability of HPL-APCs in advanced and recurrent cancers refractory to standard therapy and is the first clinical report to demonstrate the immunoinducibility of personalized cancer vaccines using HPL-APCs. Phase II clinical trials to determine immune responses with optimized adjuvant drugs and continued administration are expected to demonstrate efficacy.

Impact of delayed PBMC processing on functional and genomic assays

Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results.Whole blood samples from 20 healthy adults were collected and incubated at 20–25 °C for 2–48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2–4 h) and longer (8–48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs.PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased.PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.

#5264 CELLULAR IMMUNITY ACTIVATION IN RENAL TRANSPLANT RECIPIENTS AFTER VACCINATION WITH TOZINAMERAN: DIFFERENCES BETWEEN RESPONDERS AND NON-RESPONDERS

Abstract Background and Aims Response to vaccination is a complex procedure, resulting from the activation of various immune cell subpopulations and comprising stimulation of several interactive pathways. The aim of our study was to assess the differences in cellular subpopulations between Renal Transplant Recipients (RTRs) classified as “responders” and “non-responders” to vaccination with Tozinameran, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Method Our research was a prospective observational study. Our sample consisted of 39 RTRs on stable immunosuppression, who had a negative history of corona virus disease 2019 (COVID-19) infection and had not achieved to develop protective humoral immunity (i.e., had a negative test for neutralizing antibodies -Nab-) three weeks after the 2nd vaccine dose (T0). We attempted to evaluate immune response 48 hours before (T1) and 3 weeks after (T2) the 3rd Tozinameran dose. Patients with serum Nab levels &amp;gt; 0.3AU/ml (calculated with chemiluminescence immunoassay -CLIA-) and/or enzyme-linked immunosorbent spot (ELISpot) values &amp;gt; 30SFC/5 × 105 PBMCs were regarded as responders. Results were subsequently correlated with variations in cellular subpopulation compositions. Concentrations of CD4+, activated CD4+, CD8+, activated CD8+ T-cells, CD3+CD16+CD56+ (Natural Killer T -NKT-), activated NKT cells, CD3-CD16+CD56+ (Natural Killer -NK-) cells, CD19+, CD19+CD27- (Naïve), CD19+IgD+CD27- (Marginal), CD19+CD24+CD38high (Transitional), CD19+CD27+CD38high (Plasmablasts) and CD19+CD27+ (Memory) B cells were measured with flow cytometry at T1 and T2. We compared the results in the “responders” and the “non-responders” group. Results The number of responders significantly increased from T1 [17/39, responders: Nab(-) - ELISpot(+)] to T2 [34/39, responders: Nab and/or ELISpot (+)] (x2 = 16.2, p &amp;lt; 0.0001). No difference was observed between responders and non-responders regarding age, renal function, calcineurin inhibitor levels, dialysis and transplantation vintage. Compared to non-responders, responders presented increased concentrations of: activated CD8+ [23.6(29) vs 9.49(10)/μL, p: 0.044], activated NKT [4.77(8) vs 1.73(2)/μL, p: 0.001] and NK cells [198.91(161) vs 104.45(103)/μL, p: 0.024] at T1 and elevated levels of: total B cells [87.63(101) vs 26.86(42)/μL, p: 0.01], marginal [10.03(13) vs 1.12(5.9)/μL, p: 0.045] and memory B-cells [28.31(42) vs 10.57(19)/μL, p: 0.048] at T2 (see table). A significant increase of activated NKT and naïve B-cells from T1 to T2 [310(174) vs 241(200)/μL, p = 0.02 and 56.2(67) vs 1082(834)/μL, p&amp;lt;0.0001, respectively] was evident only in the responders’ group. Conclusion As anticipated, responders to vaccination against SARS-CoV-2 exhibited a quite different immune cell activation profile compared to non-responders. Besides, the 3rd Tozinameran vaccine dose substantially improved RTRs’ immune response and induced a prominent stimulation of the B lymphocyte compartment.

Effect of extended treatment with IAP inhibitor xevinapant post radiotherapy (RT) on efficacy and the tumor microenvironment (TME) in preclinical models.

6027 Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis, induced by RT and chemotherapy, and enhance antitumor immunity. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved 18-month locoregional control and improved 3-year progression-free survival and 5-year overall survival vs placebo + CRT. A phase 3 confirmatory study (TrilynX NCT04459715) and a phase 3 study to evaluate xevinapant + RT in post-operative, high-risk, cisplatin-ineligible patients (XRay Vision NCT05386550) are ongoing. In these studies, we investigate 3 additional cycles of xevinapant or placebo monotherapy after completing combination treatment. Based on the role of IAPs in apoptosis, immunity, and stromal activation, continual dosing of xevinapant post RT may have additional therapeutic benefits. Methods: MC38 syngeneic tumor-bearing mice were treated with vehicle control, RT alone (3.6Gy once daily [QD], 5 days), or RT + xevinapant (100 mg/kg QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation, immune cells in tumors were evaluated by fluorescence-activated cell sorting (FACS)–based immunophenotyping, and tumor-specific T cell responses were investigated by enzyme-linked immunosorbent spot (ELISpot) assays. To understand the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation, CAF gene expression was profiled on cell lines and patient-derived primary cultures. Results: Xevinapant + RT increased antitumor efficacy compared with vehicle control or RT alone. Moreover, RT in combination with extended dosing of xevinapant for 4 weeks significantly improved therapeutic efficacy and prolonged survival compared with RT + shorter xevinapant dosing durations. FACS analysis demonstrated trends of increased numbers of CD8+ T cells, natural killer cells, and dendritic cells and decreased numbers of regulatory T cells in mice treated with xevinapant + RT. FACS and ELISpot further suggested an increase in antigen-specific T cell counts from baseline with combination therapy arm vs vehicle control or RT alone. Xevinapant may also suppress RT-mediated CAF activation, as indicated by downregulation of activation markers and CAF-derived secretory proteins. Conclusions: In preclinical models, extended dosing of xevinapant for 4 weeks post RT improved antitumor efficacy. Our data suggest that the effect may be, in part, modulated by TME responses, including enhanced antitumor immunity and suppressed pro-tumorigenic phenotype of CAFs. Based on these results, further evaluation is warranted to determine the mechanisms that underscore the therapeutic benefit offered by extended dosing of xevinapant.

A Novel Multicolor Enzyme-linked Immunosorbent Spot Assay for the Functional Assessment of CAR T Cell Products

Abstract Introduction: Chimeric antigen receptor (CAR) T cell therapies are powerful anti-tumor immunotherapies and CD19-targeted CAR T cells have revolutionized the treatment of B cell lymphomas. Unfortunately, recurrent disease after CAR T cell therapy remains a clinical problem with up to 50% early relapses after CD19-targeted CAR T cells. Therefore, there is a critical and unmet need to identify biomarkers associated with durable responses to guide the optimization of CAR T cell therapies. Methods: We developed a novel multicolor enzyme-linked immunosorbent spot (ELISpot) assay for the functional assessment of CAR T Cell products on a single-cell level combining the numerical assessment of CAR T cell products with their functional characterization. Results: We first used a standard single-cell interferon (IFN)γ ELISpot assay to measure CD19-targeted CAR T cell responses to CD19 protein-coated beads or to beads coated with an irrelevant target. Beads coated with anti-CD3/anti-CD28 antibodies were used as a positive control. We then developed a multicolor ELISpot assay simultaneously measuring the secretion of combinations of different cytokines on a single-CAR T cell level. We identified IFNγ/TNFα/Granzyme B as the most relevant combination of cytokines and we used our novel multicolor ELISpot assay to functionally and numerically characterize two clinical-grade CAR T cell products. Conclusions: We have developed a novel multicolor, single-cell ELISpot for the quantitative and functional assessment of CAR T cell products. The clinical value of our novel assay will be assessed in clinical studies correlating the pre-infusion assessment of CAR T cell products with the patients’ outcome. Supported by the Kehlert Foundation (to D. A) by the Maryland Department of Health's Cigarette Restitution Fund Program and by the National Cancer Institute - Cancer Support Grant (CCSG) P30CA134274.

Alloreactive T cells to Assess Acute Rejection Risk in Kidney Transplant Recipients.

Samples from 103 consecutive kidney transplant recipients (2018-2019) were obtained pretransplantation and at time of for-cause biopsy sampling within 6 mo of transplantation. The number of donor-reactive interferon gamma (IFN-γ) and interleukin (IL)-21-producing memory T cells was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay. Of the 63 patients who underwent a biopsy, 25 had a biopsy-proven acute rejection (BPAR; 22 aTCMR and 3 aAMR), 19 had a presumed rejection, and 19 had no rejection. Receiver operating characteristic analysis showed that the pretransplant IFN-γ ELISPOT assay distinguished between patients who later developed BPAR and patients who remained rejection-free (area under the curve [AUC] 0.73; sensitivity 96% and specificity 41%). Both the IFN-γ and IL-21 assays were able to discriminate BPAR from other causes of transplant dysfunction (AUC 0.81; sensitivity 87% and specificity 76% and AUC 0.81; sensitivity 93% and specificity 68%, respectively). This study validates that a high number of donor-reactive memory T cells before transplantation is associated with the development of AR after transplantation. Furthermore, it demonstrates that the IFN-γ and IL-21 ELISPOT assays are able to discriminate between patients with AR and patients without AR at the time of biopsy sampling.