Enzyme-linked Immunosorbent Spot Research Articles

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.

Characterization of Drug-Specific CD4+ T-Cells Reveals Possible Roles of HLA Class II in the Pathogenesis of Carbamazepine Hypersensitivity Reactions

Carbamazepine (CBZ) is an aromatic anticonvulsant known to cause drug hypersensitivity reactions, which range in severity from relatively mild maculopapular exanthema to potentially fatal Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS-TEN). These reactions are known to be associated with human leukocyte antigen (HLA) class I alleles, and CBZ interacts preferentially with the related HLA proteins to activate CD8+ T-cells. This study aimed to evaluate the contribution of HLA class II in the effector mechanism(s) of CBZ hypersensitivity. CBZ-specific T-cells clones were generated from two healthy donors and two hypersensitive patients with high-risk HLA class I markers. Phenotype, function, HLA allele restriction, response pathways, and cross-reactivity of CBZ-specific T-cells were assessed using flow cytometry, proliferation analysis, enzyme-linked immunosorbent spot, and enzyme-linked immunosorbent assay. The association between HLA class II allele restriction and CBZ hypersensitivity was reviewed using Allele Frequency Net Database. Forty-four polyclonal CD4+ CBZ-specific T-cell clones were generated and found to be restricted to HLA-DR, particularly HLA-DRB1*07:01. This CD4+-mediated response proceeded through a direct pharmacological interaction between CBZ and HLA-DR molecules. Similar to the CD8+ response, CBZ-stimulated CD4+ clones secreted granulysin, a key mediator of SJS-TEN. Our database review revealed an association between HLA-DRB1*07:01 and CBZ-induced SJS-TEN. These findings implicate HLA class II antigen presentation as an additional pathogenic factor for CBZ hypersensitivity reactions. Both HLA class II molecules and drug-responsive CD4+ T-cells should be evaluated further to gain better insights into the pathogenesis of drug hypersensitivity reactions.

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19.

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis.

HBV Core-specific CD4+ T cells correlate with sustained viral control upon off-treatment in HBeAg-positive chronic hepatitis B patients

Background & aimsTreatment with nucleos(t)ide analogue (NA) efficiently suppresses viral replication in patients with chronic HBV infection, yet HBV relapses frequently upon NA withdrawal; the detailed immunomodulatory compounds for sustained viral control of HBV upon NA interruption have yet to be fully clarified. This study aimed to elucidate the role of T cells specific for distinct HBV peptides in sustained response upon discontinuation of antiviral treatment. MethodsA total of 48 patients with HBeAg-positive chronic hepatitis B receiving NA treatment and withdrawal were included longitudinally in a retrospective and prospective cohort. Enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) assays were performed to detect IFN-γ producing HBV-specific T cells following stimulation with overlapping peptides covering the whole HBV genome after 10 days of in vitro expansion. ResultsICS assays revealed that T cells specific for HBV Core and Polymerase induced more robust IFN-γ responses compared to Envelope and HBx. Notably, at the time of NA discontinuation, the intensity and breadth of HBV Core peptides-induced responses, predominately targeted by CD4+ T cells but not CD8+ T cells, were associated with sustained viral control upon off-treatment. Further exploration of longitudinal features in patients with sustained viral control revealed that the breadth of HBV-specific T cell responses does not increase following treatment cessation. ConclusionThis report emphasizes the essential role of HBV Core-specific CD4+ T cells in sustained response after therapy withdrawal, indicating it is a potential candidate for immunotherapeutic approaches in chronic HBV patients.

Survivin (BIRC5) Peptide Vaccine in the 4T1 Murine Mammary Tumor Model: A Potential Neoadjuvant T Cell Immunotherapy for Triple Negative Breast Cancer: A Preliminary Study

A triple negative breast cancer model using the murine 4T1 tumor cell line was used to explore the efficacy of an adjuvanted survivin peptide microparticle vaccine using tumor growth as the outcome metric. We first performed tumor cell dose titration studies to determine a tumor cell dose that resulted in sufficient tumor takes but allowed multiple serial measurements of tumor volumes, yet with minimal morbidity/mortality within the study period. Later, in a second cohort of mice, the survivin peptide microparticle vaccine was administered via intraperitoneal injection at the study start with a second dose given 14 days later. An orthotopic injection of 4T1 cells into the mammary tissue was performed on the same day as the administration of the second vaccine dose. The mice were followed for up to 41 days with subcutaneous measurements of tumor volume made every 3-4 days. Vaccination with survivin peptides was associated with a peptide antigen-specific gamma interferon enzyme-linked immunosorbent spot response in the murine splenocyte population but was absent from the control microparticle group. At the end of the study, we found that vaccination with adjuvanted survivin peptide microparticles resulted in statistically significant slower primary tumor growth rates in BALB/c mice challenged with 4T1 cells relative to the control peptideless vaccination group. These studies suggest that T cell immunotherapy specifically targeting survivin might be an applicable neoadjuvant immunotherapy therapy for triple negative breast cancer. More preclinical studies and clinical trials are needed to explore this concept further.

Abstract OT2-10-02: Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC)

Abstract Background: Triple-negative breast cancer (TNBC) is the subtype of breast cancer with the worst prognosis and is the subtype most often associated with germline mutations of BRCA1 as well as other genes. Alpha-lactalbumin (aLA) is a milk protein expressed in lactating breasts but not at other times or in other normal tissues. Expression of aLA is found in approximately 70% of TNBC (Cancers PMID: 27322324) so is an attractive immunologic target for TNBC based on the “retired protein hypothesis” (Semin Immunol PMID: 31926646). Pre-clinical studies have shown that vaccination with aLA inhibits the growth of established breast tumors and provides potent protection from development of autochthonous tumors in transgenic murine models of breast cancer and against 4T1 transplantable breast cancer in BALB/c mice (Nat Med PMID: 20512124). Specific Aims: We are conducting a Phase I trial of vaccination with an aLA vaccine in patients with early stage TNBC to demonstrate the safety of this approach and to document the ability to produce a relevant immunologic response to aLA. Trial Design: Patients are being entered into a Phase I trial of alpha-lactalbumin with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive a total of 3 vaccinations administered once every 2 weeks. Toxic events of Common Terminology Criteria for Adverse Events (CTCAE) grade 2 or greater are considered dose-limiting. Dose levels being tested in the current protocol are alpha-lactalbumin/zymosan 0.01/0.01, 0.1/0.01, 0.5/0.01, 0.5/0.03, and 0.5 mg/0.06 mg. Patients are being monitored for toxicity until 84 days after the first vaccination or resolution of toxicity, whichever is later. Blood is being drawn prior to therapy and 14, 28, and 56 days after the first vaccination to assess cellular response using enzyme-linked immunosorbent spot (ELISpot) assays of interferon-gamma and interleukin-17 production in response to aLA. Humoral response to aLA vaccination is being assessed by enzyme-linked immunosorbent assay (ELISA). Eligibility Criteria: Patients with ER-negative, PR-negative, HER2-negative breast cancer of pathologic stage II-III or who had residual disease after standard pre-operative systemic therapy. Participants must be within 3 years of initiation of treatment and have no evidence of recurrence. Dose Selection: Doses are being escalated using a 3+3 trial design to determine the maximum tolerated dose, the lowest immunologic dose and the optimal immunologic dose Present Accrual and Target Accrual: Dependent on toxicity, 18-30 patients will be treated. Future Plans and Contact Information: After identification of the maximum tolerated dose and expansion of the dose levels associated with effective tumor immunity we plan to enroll 2 additional cohorts of patients: 1) patients who have completed pre-operative chemo-immunotherapy and are receiving pembrolizumab as standard of care therapy, and 2) patients without cancer planning to undergo prophylactic bilateral mastectomy. Interested parties may contact Dr. Budd at buddg@ccf.org. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Citation Format: George Budd, Justin M. Johnson, Emily Rhoades, Halle Moore, Megan L. Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Elena Haury, Vincent K. Tuohy. Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-10-02.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination.

While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools.

Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05-0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

Lipid-Based Poly(I:C) Adjuvants Strongly Enhance the Immunogenicity of SARS-CoV-2 Receptor-Binding Domain Vaccine

Abstract Background The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly threatened public health. Recent studies have revealed that the spike receptor-binding domain (RBD) of SARS-CoV-2 is a potent target for vaccine development. However, adjuvants are usually required to strengthen the immunogenicity of recombinant antigens. Different types of adjuvants can elicit different immune responses. Methods We developed an RBD recombinant protein vaccine with a polyriboinosinic acid–polyribocytidylic acid [poly(I:C)] adjuvant to evoke a strong immune response. The delivery of poly(I:C) was optimized in two steps. First, poly(I:C) was complexed with a cationic polymer, poly-l-lysine (PLL), to form poly(I:C)–PLL, a polyplex core. Thereafter, it was loaded into five different lipid shells (group II, III-1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC], III-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE], IV-DOPE, and IV-DSPC). We performed an enzyme-linked immunosorbent assay and enzyme-linked immunosorbent spot assay to compare the ability of the five lipopolyplex adjuvants to enhance the immunogenicity of the SARS-CoV-2 RBD protein, including humoral and cellular immune responses. Finally, the adjuvant with the highest immunogenicity was selected to verify the protective immunity of the vaccine through animal challenge experiments. Results Recombinant RBD protein has low immunogenicity. The different adjuvants we developed enhanced the immunogenicity of the RBD protein in different ways. Among the lipopolyplexes, those containing DOPE (III-DOPE and IV-DOPE) elicited RBD-specific immunoglobulin G antibody responses, and adjuvants with four components elicited better RBD-specific immunoglobulin G antibody responses than those containing three components (P &lt; 0.05). The IC50 and IC90 titers indicated that the IV-DOPE lipopolyplex had the greatest neutralization ability, with IC50 titers of 1/117,490. Furthermore, in the challenge study, IV-DOPE lipopolyplex protected mice from SARS-CoV-2 infection. On the fourth day after infection, the average animal body weights were reduced by 18.56% (24.164 ± 0.665 g vs. 19.678 ± 0.455 g) and 0.06% (24.249 ± 0.683 g vs. 24.235 ± 0.681 g) in the MOCK and vaccine groups, respectively. In addition, the relative expression of viral RNA in the vaccinated group was significantly lower than that in the MOCK group (P &lt; 0.05). Interstitial inflammatory cell infiltration was observed in the MOCK group, whereas no obvious damage was observed in the vaccinated group. Conclusions The IV-DOPE–adjuvanted SARS-CoV-2 recombinant RBD protein vaccine efficiently protected mice from SARS-CoV-2 in the animal challenge study. Therefore, IV-DOPE is considered an exceptional adjuvant for SARS-CoV-2 recombinant RBD protein-based vaccines and has the potential to be further developed into a SARS-CoV-2 recombinant RBD protein-based vaccine.

509. Role of cell-mediated immune monitoring using Interferon-γ Enzyme-linked Immunosorbent Spot Assay to predict CMV infection within six months after kidney transplantation

Abstract Background CMV infection can cause substantial morbidity and mortality in kidney transplant (KT) recipients due to an impairment in cell-mediated immunity (CMI) from immunosuppressive drugs. We investigated the role of CMI monitoring prior to and after transplant to predict CMV infection after KT. Methods A prospective study was performed between December 2020 and December 2021. All adult KT recipients underwent CMI measurement by investigating IFN-γ-producing T cells using enzyme-linked immunosorbent (ELISpot) assay before and one month post-transplant. The incidence of CMV infection within six months after transplant was reported, and predictors of CMV infection were analyzed using the Cox proportional hazard model. Results We included 93 KT recipients with a mean (SD) age of 44 (11) years; 59.1% were male, and 98.9% were CMV seropositivity. Twenty-two (23.7%) participants received anti-thymocyte globulin (ATG) for induction therapy. A median (IQR) of IFN-γ-producing T cells measured one month after transplant was significantly lower compared to before transplant (148 [54-389] vs. 763 [409-1,067] SFUs per 2.5 x 105 PBMCs, p &amp;lt; 0.001). Forty (42.9%) KT recipients who developed CMV infection appeared to have significantly less IFN-γ-producing T cells compared to those did not develop CMV infection (47.1%) (115 [33-237] vs. 238[76-492] SFUs/2.5x105 PBMCs, p=0.019). In univariate analysis, predictors for CMV infection included higher panel-reactive antibody (HR 1.02 [95%CI, 1.01-1.03], p&amp;lt; 0.001), receiving ATG for induction therapy (HR 3.45 [95%CI, 1.82-6.56], &amp;lt; 0.001) and lack of CMI one month after transplant defined as IFN-γ-producing T-cells of &amp;lt; 250 SFUs/2.5x105 PBMCs (HR 3.11 [95%CI, 1.36-7.10], p=0.007). In multivariate analysis, lack of CMI one month after transplant is the only predictor which remained independently associated with CMV infection (HR 3.1 [95%CI 1.2-7.80], p=0.019) (Table 1). Conclusion KT recipients with low IFN-γ-producing T cell responses are more likely to develop CMV infection post-transplant. Quantification of CMI using ELISpot assay could potentially predict those at risk of CMV infection after KT. Disclosures All Authors: No reported disclosures.

Cellular assays to evaluate B-cell function

Inborn errors of immunity (IEI) that present with recurrent infections are largely due to antibody (Ab) deficiencies. Therefore, assessment of the B-cell and Ab compartment is a major part of immunologic evaluation. Here we provide an overview about cellular assays used to study B-cell function and focus on lymphocyte proliferation assay (LPA), opsonophagocytic assay (OPA), and the Enzyme-linked Immunosorbent Spot Assay (ELISPOT) including clinical applications and limitations of these techniques. LPAs assess ex-vivo cell proliferation in response to various stimuli. Clinically available LPAs utilize peripheral blood mononuclear cells and mostly assess T-cell proliferation with pokeweed mitogen considered the most B-cell specific stimulus. In the research setting, isolating B cells or using more B-cell specific stimuli such as CD40L with IL-4/IL-21 or the TLR9 ligand CpG can more specifically capture the proliferative ability of B cells. OPAs are functional in-vitro killing assays used to evaluate the ability of IgG Ab to induce phagocytosis applied when assessing the potency of vaccine candidates or along with avidity assays to evaluate the quality of secreted IgG. The B-cell ELISPOT assesses Ab production at a cellular level and can characterize the Ab response of particular B-cell subtypes. It can be used in patients on IgG therapy by capturing specific Abs produced by individual B cells, which is not affected by exogenous IgG from plasma donors, and when assessing the vaccine response in patients on immunomodulatory drugs that can affect memory B-cell function. Emerging approaches that are only available in research settings are also briefly introduced.

Long-Term CD4+ T-Cell and Immunoglobulin G Immune Responses in Oncology Workers following COVID-19 Vaccination: An Interim Analysis of a Prospective Cohort Study.

We conducted a prospective study to evaluate immune responses to SARS-CoV-2 in oncology workers in which we collected blood and clinical data every 6 months. Spike-specific CD4+ T-cells and immunoglobulin G responses were measured using interferon-gamma enzyme-linked immunosorbent spot and enzyme-linked immunosorbent assay, respectively. Sixty (81%) vaccinated and 14 (19%) unvaccinated individuals were enrolled. CD4+ T-cell responses of those individuals currently naturally infected were comparable to those who were 6 months from receiving their last dose of the vaccine; both responses were significantly higher than among those who were unvaccinated. Unvaccinated participants who became vaccinated while in the study showed a significant increase in both types of spike-specific immune responses. Previously vaccinated individuals who received a third dose (booster) showed a similar response to the spike protein. However, this response decreases as soon as 3 months but does not dip below the established response following two doses. Response to variants of concern B.1.617.2 (Delta) and B.1.1.529 (Omicron) also increased, with the Omicron variant having a significantly lower response when compared to Delta and the wild type. We conclude that antibody and T-cell responses increase in oncology workers after serial vaccination but can wane over time.

RIG-I and TLR-7/8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine.

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)-PEG-Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant.Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centers during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.