IFN-γ ELISPOT Assay Research Articles

Decreased variability in the site-specific results during participation in the External Quality Assurance Program Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay

The NIAID DAIDS-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) manages an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) external proficiency program. The ELISpot program evaluates the accuracy and variability of results across laboratories. The variability in the program is quantified via the dispersion, which is the ratio of the variance over the mean of the background-corrected spot-forming cells (SFC) replicates obtained under stimulation with different peptide pools (CMV, CEF). This report includes the longitudinal analysis of the ELISpot program cohort composed of 22 laboratories from 2011 to 2022 to assess whether the within-lab variability has improved over time. Random intercept models of the dispersion over time showed a significant decrease in overall dispersion from an average of approximately 1.8 in 2011 to approximately 1.25 in 2022. Out of the 21 sites, 16 sites (4 being statistically significant) had a negative trend for dispersion over time. Our finding of a reduction of overall within-lab variability demonstrates the need for and benefit of proficiency testing programs.

Assays for Assessing Mycobacterium avium Immunity and Evaluating the Effects of Therapeutics.

In Europe and North America, the prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing. Most pulmonary NTM infections are caused by the Mycobacterium avium complex (MAC). Sadly, the treatment of pulmonary MAC is suboptimal with failure rates ranging from 37% to 58%. Therefore, there is a need to develop new therapeutics. Developing new immunotherapies and studying their interaction with standard or new drugs requires reliable assays. Four different assays including CFSE-based flow cytometry, in vitro protection assays, IFN-γ ELISPOT, and murine infection models were optimized using a reference strain of MAC (ATCC 700898) to help with the development of immunotherapies for MAC. Expansion of proliferating and IFN-γ producing human T cells is optimal after 7 days of stimulation with MAC at a multiplicity of infection (MOI) of 0.1, achieving a stimulation index of 26.5 ± 11.6 (mean ± SE). The in vitro protection assay for MAC works best by co-culturing T cells expanded for 7 days with MAC (MOI 1)-infected autologous macrophages. Aerosol MAC infection of mice allows measurement of the effects of the BCG vaccine and clarithromycin. IFN-γ ELISPOT assays with live MAC (MOI 3) stimulation of splenocytes from mice immunized with BCG help identify differences between unimmunized mice and mice immunized with BCG. In conclusion, multiple assays are available for use to identify MAC-specific effector T cells, which will help in the development of new therapeutics or vaccines against pulmonary MAC.

Timing is essential: Humoral and cellular responses to SARS-CoV-2 vaccination in a cohort of patients with auto-immune diseases treated with rituximab

Rituximab (RTX), an anti CD20 monoclonal antibody, is now a gold standard treatment for several auto-immune and chronic inflammatory diseases. Receiving RTX exposes patients to more severe infections as vaccinations become virtually inefficient in terms of B cell responses. During the COVID-19 crisis, RTX–exposed patients exhibited more severe forms of the disease, and in some cases, the introduction of RTX was delayed or avoided to protect patients as much as possible against SARS-CoV-2 infections. We retrospectively collected cellular and humoral responses from thirteen patients with dermatological and rheumatological autoimmune diseases who had been vaccinated after receiving RTX. Memory T cells subsets from patients that exposed to RTX showed few differences when compared to a cohort of healthy donors. The IFNᵧ ELISpot assay using SARS-CoV-Prot_S1 showed that eight patients exhibited a positive response that was neither correlated to the time between RTX infusion and the sampling nor to the time between RTX and the vaccination. Conversely, analysis of the SARS-CoV-2 serology showed a clearly lower binding antibody units per mL in case of recent RTX infusion. The safe threshold forconsistently positive serology was to vaccinate at least 300 days after RTX infusion (p = 0.02). Our data illustrate the difficulty in obtaining a satisfactory response to vaccination after RTX treatment within almost a year after the latest infusion, and emphasize the need to better evaluate the risk of relapses in auto-immune diseases before administering RTX in order to maintain RTX only in patients whose medical situation requires it.

Assessment of HBV variants and novel viral and immune biomarkers in chronic hepatitis B patients with metabolic dysfunction associated steatotic liver disease.

Co-existing chronic hepatitis B virus (CHB) infection and metabolic dysfunction associated steatotic liver disease (MASLD) can exert complex effects on hepatic metabolism, requiring mechanistic study. CHB participants were assessed for MASLD and the impact of hepatic steatosis/metabolic syndrome (MetS) on novel viral and immunological markers. In this prospective, cohort study, untreated CHB subjects were assessed for liver disease by non-invasive tests (i.e. FibroScan, controlled attenuation parameter, CAP). Subjects were tested for cytokines and IFN-γ ELISPOT assay to HBV Surface (S) and Core (C) proteins. Standard HBV serological, exploratory biomarkers and deep sequencing of HBV S and C genes were performed. In 53 subjects (median age 45 years [SD = 10.6], 35% F, 56% Asian, 20% Black, 3% White), 94% (50) HBeAg negative, 63% genotype B/C, mean HBV DNA 3.2 log10 IU/mL (SD = 1.8), quantitative HBsAg 2.9 log10 IU/mL (SD = 1.2) and HBV pgRNA 2.1 log10 copies/mL (SD = 1.3). In enrolled subjects, the mean ALT was 41.9 U/L (SD = 24.0), FibroScan was 5.7 kPa (SD = 1.9) and CAP was 306.4 dB/m (SD = 49.0). The mean BMI was 28.2 kg/m2 (SD = 4.2), 20% (11/53) had diabetes, 35% (19/53) dyslipidaemia and 24% (13/53) hypertension. Subjects with MetS and steatosis showed lower HBV markers (p < .01), higher HBV S diversity (p = .02) and greater frequency of HBV variants associated with host-anti-viral immune escape. Pro-inflammatory cytokine levels and HBV-specific cellular responses were higher in participants with hepatic steatosis. In CHB, MASLD/hepatic steatosis was associated with HBV variants and systemic immune responses potentially impacting liver disease progression despite low-level viraemia.

COVID-19 Vaccine in Lung and Liver Transplant Recipients Exceeds Expectations: An Italian Real-Life Experience on Immunogenicity and Clinical Efficacy of BNT162b2 Vaccine.

This study assessed humoral and T cell-mediated immune responses to the BNT162b2 vaccine in orthotopic liver transplant (OLT) and lung transplant (LUT) recipients who received three doses of the vaccine from March 2021 at our institution. Serum samples were collected 60days post-second and third dose to quantify antibodies against the spike region of SARS-CoV-2 while whole blood samples were collected to analyze the SARS-CoV-2-specific T-cell response using an IFN-γ ELISpot assay. We enrolled 244 OLT and 120 LUT recipients. The third dose increased antibody titres in OLT recipients (from a median value of 131 after the second dose to 5523 IU/mL, p < 0.001) and LUT recipients (from 14.8 to 1729 IU/mL, p < 0.001). T-cell response also increased in OLT recipients (from 8.5 to 23 IFN-γ SFU per 250,000 PBMC, p < 0.001) and LUT recipients (from 8 to 15 IFN-γ SFU per 250,000 PBMC, p < 0.001). A total of 128 breakthrough infections were observed: two (0.8%) OLT recipients were hospitalized due to COVID-19 and one died (0.4%); among LUT recipients, seven were hospitalized (5.8%) and two patients died (1.7%). In conclusion, the three-dose schedule of the BNT162b2 vaccine elicited both humoral and T cell-mediated responses in solid organ transplant recipients. The risk of severe COVID-19 post-vaccination was low in this population.

Peripheral Cellular Immune Responses Induced by Subretinal Adeno-Associated Virus Gene Transfer Can Be Restrained by the Subretinal-Associated Immune Inhibition Mechanism.

After more than two decades of basic research and preclinical studies, adeno-associated virus (AAV)-mediated gene transfer has been tested successfully in clinical trials to treat inherited retinal diseases. Despite the eye's immune-privileged status, some patients display inflammatory events requiring the use of corticoids as an adjunct treatment which led us to question the immune consequences of a subretinal AAV administration. We first characterized anti-transgene immune responses induced in the periphery by injecting increasing doses of AAV8 encoding reporter proteins fused with the HY male antigen into the subretinal space of female C57BL/6 and rd10 mice. Transgene expression was monitored over time with bioluminescence imaging, and T cell immune responses in the spleen were analyzed by IFNγ ELISpot and cytokine multiplex assays. Our data show that AAV8 injections cause pro-inflammatory T cell immune response against the transgene product correlated with the transgene expression level at 2.109 vg and above. In addition, co-injection of immunodominant peptides from the transgene product, along with AAV8, modulates the immune response at all AAV doses tested. Taken together, our data suggest that injection of AAV8 in the subretinal space induces pro-inflammatory peripheral T cell responses to the transgene product that can be modulated by the subretinal-associated immune inhibition mechanism.

Immune correlates of protection of the four-segmented Rift Valley fever virus candidate vaccine in mice

ABSTRACT Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

Downregulation of HNRNPA1 induced neoantigen generation via regulating alternative splicing

BackgroundImmunotherapies effectively treat human malignancies, but the low response and resistance are major obstacles. Neoantigen is an emerging target for tumor immunotherapy that can enhance anti-tumor immunity and improve immunotherapy. Aberrant alternative splicing is an important source of neoantigens. HNRNPA1, an RNA splicing factor, was found to be upregulated in the majority of tumors and play an important role in the tumor immunosuppressive microenvironment.MethodsWhole transcriptome sequencing was performed on shHNRNPA1 SKOV3 cells and transcriptomic data of shHNRNPA1 HepG2, MCF-7M, K562, and B-LL cells were downloaded from the GEO database. Enrichment analysis was performed to elucidate the mechanisms underlying the activation of anti-tumor immunity induced by HNRNPA1 knockdown. mRNA alternative splicing was analyzed and neoantigens were predicted by JCAST v.0.3.5 and Immune epitope database. The immunogenicity of candidate neoantigens was calculated by Class I pMHC Immunogenicity and validated by the IFN-γ ELISpot assay. The effect of shHNRNPA1 on tumor growth and immune cells in vivo was evaluated by xenograft model combined with immunohistochemistry.ResultsHNRNPA1 was upregulated in a majority of malignancies and correlated with immunosuppressive status of the tumor immune microenvironment. Downregulation of HNRNPA1 could induce the activation of immune-related pathways and biological processes. Disruption of HNRNPA1 resulted in aberrant alternative splicing events and generation of immunogenic neoantigens. Downregulation of HNRNPA1 inhibited tumor growth and increased CD8+ T cell infiltration in vivo.ConclusionOur study demonstrated that targeting HNRNPA1 could produce immunogenic neoantigens that elicit anti-tumor immunity by inducing abnormal mRNA splicing. It suggests that HNRNPA1 may be a potential target for immunotherapy.

Immunogenicity of an AI-designed personalized neoantigen vaccine, EVX-01, in combination with anti-PD-1 therapy in patients with metastatic melanoma.

9561 Background: Personalized vaccines, targeting mutation-derived neoantigens (neoAg), represent a promising frontier in cancer immunotherapy. Here, we characterize the vaccine-induced immune response in seven melanoma patients treated with the AI-generated personalized cancer vaccine, EVX-01, in the ongoing single arm multicenter trial (NCT05309421). Data from an additional five patients will be available at the time of presentation. Methods: Tumor-specific neoAgs were identified and selected using the proprietary vaccine target discovery AI-Immunology platform based on tumor DNA- and RNA-sequencing data. The top-ranked neoAgs for each patient were manufactured as synthetic long peptides and formulated with an adjuvant, creating the personalized cancer vaccine, EVX-01, tailored to the individual tumor and immune system characteristics. Patients initiated anti-PD1 therapy (Pembrolizumab) 12 weeks prior to EVX-01 administration. Each patient received six priming doses of EVX-01 administered bi-weekly and will be followed by four booster immunizations at later timepoints. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected at different timepoints to evaluate the effect of anti-PD1 therapy, EVX-01 priming immunizations and EVX-01 booster immunizations on T-cell immunogenicity. NeoAg-specific T-cell responses were evaluated by IFN-γ ELISpot assay and intracellular cytokine staining after T cell in vitro expansion with vaccine neoAgs. Results: Immunogenicity analysis of PBMC samples from seven patients demonstrated that EVX-01 induced neoAg-specific immune responses in all patients after the priming phase. The effect of booster immunizations on neoAg-specific T-cell responses was analyzed in the patients who had reached these late visits of the study, and it demonstrated a clear trend towards increased immune responses after the first booster dose. The observed responses were mediated by both CD4+ and CD8+ neoAg-specific T-cells. Investigations of the response induced by the individual vaccine neoAgs revealed that the majority of the EVX-01 neoAgs triggered a specific T-cell response, affirming the ability of the AI-Immunology™ platform to precisely select effective vaccine targets. Data from an additional five patients will be available at the time of presentation. Importantly EVX-01 was safe and well-tolerated, with only grade 1 and 2 ADRs related to EVX-01 even after boosting. The combination of EVX-01 and anti-PD1 also appeared safe and well tolerated. Conclusions: EVX-01 induced neoAg-specific immune responses in all analyzed patients and a broad response against the neoAgs. These results further validate the precision and predictive power of our proprietary vaccine target discovery AI-Immunology platform. The combination of EVX-01 and anti-PD1 was safe and well tolerated. Clinical trial information: NCT05309421 .

Updated results on multiple antigens stimulating cellular therapy (MASCT-I) in metastatic urothelial carcinoma: A randomized, open-label, phase I trial.

2545 Background: The appropriate treatment for mUC remains unmet medical needs. MASCT-I, an adoptive transfer of dendritic cells (DCs) presenting 15 types of tumor-associated antigens (TAAs), showed valuable treatment in solid tumors. We previously reported the efficacy and safety of MASCT-I alone, or plus camrelizumab or chemotherapy in mUC (NCT03034304). The biomarker analysis and long-term follow-up to for this promising approach was unknown. Methods: All enrolled patients were divided into in five groups (G) as previous reported. Patients in G2 receiving maintenance therapy of MASCT-I after first-line platinum based-chemotherapy were allowed to enter enrolled in G3 and receiving MASCT-I plus camrelizumab when disease progressed. We updated the exploratory endpoint of the IFN-γ ELISPOT assay, which was used to measure activation of the cytotoxic T Lymphocyte (CTL) response in eligible patients. Long-term follow-up results for safety, progression-free survival (PFS) and overall survival (OS) were reported. Results: 39 patients were enrolled. By January 31st, 2024, median follow-up time was 31.1 months. No new safety signals were updated. As previously reported, the most common adverse events (AEs) associated with MASCT-I included grade 1 and 2 flushing, pruritus, rash, muscle cramp, fever, and arthralgia. No MASCT-I-related death occurred. Median PFS and OS for all patients were 2.3 months, and 16.9 months, respectively. Patients in G2 presented superior OS of 41.2 months and duration of response (DOR) of 6.4 months. Nine patients treated with MASCT-I and camrelizumab after progression on MASCT-I in G2 presented a 24-month PFS rate of 55.6% and a 48-month DOR rate of 71.8%. Five patients had prolonged PFS for more than 20 months while two of them had PFS of more than 43 months. To identify the potentially beneficial population, IFN-γ ELISPOT data analysis revealed that patients who had positive TAAs ELISPOT response had significant prolonged PFS and OS compared to the non-responders (median PFS: 10.1 months vs 4.8 months, HR=0.30, 95% CI: 0.14-0.61, P&lt;0.005; median OS: not reached vs 13.6 months, HR=0.16, 95% CI: 0.05-0.57, P&lt;0.005). The Cmax value of INF-γ-positive T cells after cell infusion was also higher in patients with better PFS. In addition, we observed a significant increase in INF-γ-positive T cell activities in patients who progressed from G2 and were subsequently treated with a combination of MASCT-I and camrelizumab (Cmax mean (SD): 72 spots per million bulk PBMCs at G2 to 186 spots per million bulk PBMCs after transfer to G3, P=0.002). Conclusions: MASCT-I alone or in combination with immunotherapy or chemotherapy are safe and well tolerated, with inspiring survival benefit. The combination of PD-1 inhibitors may enhance the antitumor activity of MASCT-I. IFN-γ ELISPOT might be able to predict potentially beneficial populations. Clinical trial information: NCT03034304 .

Abstract PO4-16-05: Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors

Abstract The adoptive transfer of genetically engineered T-cell receptors (TCR-T) has emerged as a promising therapeutic strategy for the treatment of solid tumors. Recent clinical trials have demonstrated the safety and efficacy of TCR-T cell therapies against certain types of metastatic solid cancers. In our study, we aimed to investigate the potential of TCR-T cell therapies targeting neoantigens, which are exclusively expressed in cancer cells, with a focus on metastatic breast cancer (MBC). Specifically, we identified four driver missense mutations (PIK3CA E542K, E545K, H1047L, and H1047R) frequently observed in MBC patients and utilized computational tools to predict the generation of neoantigens. Utilizing NetMHCpan V.4.1, we performed screening of peptides spanning the mutation region and predicted a neoantigen epitope, PIK3CA_H1047L, to bind with HLA-A*11:01. To validate the presentation and stability of the neoantigen epitope, we employed TAP1-deficient K562 cells and observed stable expression of the mutant peptide-major histocompatibility complex (MHC) complex on the cell membrane. Subsequently, we isolated T cells from healthy donors possessing the HLA-A11:01 genotype, and subjected them to successive stimulations with neopeptide-pulsed dendritic cells. T cells stimulated with neopeptide were examined for neoantigen specificity using an IFN-γ ELISpot assay. Employing pMHC tetramer staining and single-cell sorting via flow cytometry, we successfully isolated five HLA-A11:01-restricted PIK3CA_H1047L-specific TCRs. Through lentivirus transduction of these TCRs into T cells, we achieved the generation of TCR-T cells that exhibited potent and specific activity exclusively against the PIK3CA_H1047L neoantigen. The functionality of the TCR-T cells was confirmed through assessments of CD137 expression, IFN-γ secretion, and cytotoxicity assays. In conclusion, our study demonstrates T cell responses towards neoantigens derived from PIK3CA, a commonly observed driver mutation in MBC. Furthermore, we successfully isolated five distinct neoantigen-specific TCRs that specifically recognized the PIK3CA_H1047L mutation and were restricted to the prevalent HLA-A*11:01 allele. The transfer of these TCRs into T cells resulted in the generation of TCR-T products exhibiting remarkable activity against the PIK3CA_H1047L neoantigen. These findings strongly encourage further investigation into TCRs targeting driver mutations in MBC, with the aim of advancing neoantigen-targeted TCR-T therapies into clinical trials. Additionally, the PIK3CA_H1047L neoantigen holds great promise as a potential candidate for the development of effective cancer vaccines. Citation Format: Meiying Shen, Xiaojian Han, Siyin Chen, Aishun Jin, Shengchun Liu. Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-16-05.

Dose escalation study of a personalized peptide-based neoantigen vaccine (EVX-01) in patients with metastatic melanoma

BackgroundNeoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated...

Stealth transgenes enable CAR-T cells to evade host immune responses

BackgroundAdoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based...

A universal recombinant adenovirus type 5 vector-based COVID-19 vaccine.

A universal recombinant adenovirus type-5 (Ad5) vaccine against COVID19 (Ad-US) was constructed, and immunogenicity and broad-spectrum of Ad5-US were evaluated with both intranasal and intramuscular immunization routes. The humoral immune response of Ad5-US in serum and bronchoalveolar lavage fluid were evaluated by the enzyme-linked immunosorbent assay (ELISA), recombinant vesicular stomatitis virus based pseudovirus neutralization assay, and angiotensin-converting enzyme-2 (ACE2) -binding inhibition assay. The cellular immune response and Th1/Th2 biased immune response of Ad5-US were evaluated by the IFN-γ ELISpot assay, intracellular cytokine staining, and Meso Scale Discovery (MSD) profiling of Th1/Th2 cytokines. Intramuscular priming followed by an intranasal booster with Ad5-US elicited the broad-spectrum and high levels of IgG, IgA, pseudovirus neutralizing antibody (PNAb), and Th1-skewing of the T-cell response. Overall, the adenovirus type-5 vectored universal SARS-CoV-2 vaccine Ad5-US was successfully constructed, and Ad5-US was highly immunogenic and broad spectrum. Intramuscular priming followed by an intranasal booster with Ad5-US induced the high and broad spectrum systemic immune responses and local mucosal immune responses.

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

Integrating Multisector Molecular Characterization into Personalized Peptide Vaccine Design for Patients with Newly Diagnosed Glioblastoma.

Outcomes for patients with glioblastoma (GBM) remain poor despite multimodality treatment with surgery, radiation, and chemotherapy. There are few immunotherapy options due to the lack of tumor immunogenicity. Several clinical trials have reported promising results with cancer vaccines. To date, studies have used data from a single tumor site to identify targetable antigens, but this approach limits the antigen pool and is antithetical to the heterogeneity of GBM. We have implemented multisector sequencing to increase the pool of neoantigens across the GBM genomic landscape that can be incorporated into personalized peptide vaccines called NeoVax. In this study, we report the findings of four patients enrolled onto the NeoVax clinical trial (NCT0342209). Immune reactivity to NeoVax neoantigens was assessed in peripheral blood mononuclear cells pre- and post-NeoVax for patients 1 to 3 using IFNγ-ELISPOT assay. A statistically significant increase in IFNγ producing T cells at the post-NeoVax time point for several neoantigens was observed. Furthermore, a post-NeoVax tumor biopsy was obtained from patient 3 and, upon evaluation, revealed evidence of infiltrating, clonally expanded T cells. Collectively, our findings suggest that NeoVax stimulated the expansion of neoantigen-specific effector T cells and provide encouraging results to aid in the development of future neoantigen vaccine-based clinical trials in patients with GBM. Herein, we demonstrate the feasibility of incorporating multisector sampling in cancer vaccine design and provide information on the clinical applicability of clonality, distribution, and immunogenicity of the neoantigen landscape in patients with GBM.

Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

Prevalence Study of Cellular Capsid-Specific Immune Responses to AAV2, 4, 5, 8, 9, and rh10 in Healthy Donors.

Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for invivo gene transfer as illustrated by the approvals of 7 drugs across Europe and the United States. Nevertheless, preexisting immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas a preexisting humoral response to AAV capsid is well documented, the prevalence of preexisting capsid-specific T cell responses still needs to be studied and characterized. In this study, we investigated the prevalence of AAV-specific circulating T cells toward AAV2, 4, 5, 8, 9, and rh10 in a large cohort of healthy donors using the standard IFNγ ELISpot assay. We observed the highest prevalence of preexisting cellular immunity to AAV9 serotype followed by AAV8, AAV4, AAV2, AAVrh10, and AAV5 independently of the donors' serological status. An in-depth analysis of T cell responses toward the 2 most prevalent serotypes 8 and 9 shows that IFNγ secretion is mainly mediated by CD8 T cells for both serotypes. A polyfunctional analysis reveals different cytokine profiles between AAV8 and AAV9. Surprisingly, no IL-2 secretion was mediated by anti-AAV9 immune cells suggesting that these cells may rather be exhausted or terminally differentiated than cytotoxic T cells. Altogether, these results suggest that preexisting immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anticapsid cellular immunity and foresee its impact in rAAV-mediated clinical trials.

Abstract 4106: A novel synthetic long peptide vaccine composition targeting multiple PD-L1 T-cell epitopes exhibits anti-tumor efficacy in a syngeneic mouse colorectal cancer model

Abstract Background Cancer immunotherapy targeting immune checkpoints has shown promising clinical anti-tumor efficacy over the past decades. Monoclonal antibodies against PD-1 or PD-L1 have demonstrated clinical efficacy in a wide spectrum of cancer types. Despite the clinical success of therapies using immune checkpoint inhibitors (ICI), only a subset of patients shows long-lasting tumor remission, while resistance and toxicities to these agents remain to be addressed. This supports the need to explore alternative immunotherapeutic approaches or additional therapies for synergistic combinations to augment the anti-tumor efficacy for cancer patients receiving ICI therapies. Methods As an active immunotherapy approach, we developed a blend of four novel synthetic peptide vaccines each containing one PD-L1 T-cell epitope with individual covalent conjugation of the carrier protein, keyhole limpet haemocyanin (KLH), which is to break immune tolerance towards self-molecules. The four PD-L1 epitopes range from 30 to 40 amino acids in length, which target different domains of mouse PD-L1 protein. The PD-L1 peptides were synthesized using the technique of solid-phase peptide synthesis and purified by high performance liquid chromatography. All naked peptides were conjugated to KLH protein by thiol-maleimide reaction, followed by dialysis for purification. The peptide vaccines were formulated with a Toll-like receptor 9 agonist CpG ODN 1826 and aluminum hydroxide gel for animal immunization. Results These synthetic long PD-L1 peptide vaccines are highly immunogenic in a syngeneic BALB/c mouse model for a cell-mediated immune response when admixed and formulated with adjuvants. The vaccine composition is well tolerated by the mice with no evidence of toxicity and autoimmunity. In vivo administration of the vaccine formulation can elicit a positive PD-L1-specific T cell response in splenocytes with an IFN-γ ELISPOT assay. Moreover, in vivo vaccination of these PD-L1 peptide vaccines in a CT26 syngeneic mouse preventive colorectal cancer model exhibits a potent anti-tumor activity, with a 54% tumor growth inhibition (p&amp;lt;0.001) in tumor volume compared to that of the adjuvant-control group at the end of the study. Conclusions Taken together, our data demonstrate a proof-of-concept that T cell-based PD-L1 peptide vaccines are a promising therapeutic strategy for inducing anti-tumor immunity to combat cancers. Citation Format: Kenneth Nansheng Lin, Melvin Toh. A novel synthetic long peptide vaccine composition targeting multiple PD-L1 T-cell epitopes exhibits anti-tumor efficacy in a syngeneic mouse colorectal cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4106.

Abstract 4094: Immune modulatory cancer vaccines against IDO1 and PD-L1 trigger distinct pathways and cooperatively reduce tumor growth in preclinical models

Abstract While immune checkpoint inhibitors have shown clinical efficacy in many cancers, drug and immune resistance remain challenging. Indoleamine 2,3-dehydrogenase (IDO1) and Programmed Death Ligand 1 (PD-L1) both contribute to immunosuppression, leading to immune escape and cancer progression. In this context, vaccination to promote T-cell immunity against IDO1+ and PD-L1+ cells is an attractive strategy that demonstrated encouraging clinical results in melanoma (Kjeldsen et al. Nat Med 2021). Our study aims to further evaluate the efficacy and mode of action of combined IDO1 and PD-L1 peptide vaccines. Mice injected with tumor cells and treated with IDO1 or PD-L1 peptides were monitored for tumor growth. Vaccine activity was determined by IFNγ Elispot assay. Gene expression analysis was performed on tumors using Nanostring nCounter PanCancer IO360 Panel. IDO1 and PD-L1 expression were also examined via multiplex immunofluorescence in a human tumor tissue microarray (TMA) panel. Human IDO1 and PD-L1 specific CD4+ T cell clones were isolated and expanded from melanoma patients treated with IDO1/IO102 or PD-L1/IO103 peptide vaccines in combination with aPD1 therapy. Immunofluorescence analysis of murine tumor sections showed expression of IDO1 and PD-L1 by different cell populations. Vaccination against each target led to expansion of specific T cells in splenocytes and reduced tumor growth in MC38 and CT26 models. Combining IDO1 and PD-L1 treatments further enhanced the anti-tumor response. Gene expression analysis of tumors revealed distinct signatures following monotherapy and combination treatments. While PD-L1 vaccination predominantly enhanced the cytotoxic effector T cell function in the tumor microenvironment (TME) and concomitantly increased gene signatures associated with enhanced activation of T effector cells, IDO1 vaccine primarily exerted its effect via reduction in the immune suppression loop. TMA analysis of IDO1+ and PD-L1+ cells in human tumors also revealed that they represent largely distinct cell populations. Lastly, IDO1 and PDL1-specific CD4+ T cell clones from melanoma patients treated with IO102-IO103 vaccine could selectively target cells differentially expressing IDO1 or PDL1. Our data collectively show that cells expressing IDO1 and PD-L1 represent distinct populations in the TME thus targetable by the IDO1-PD-L1 vaccination approach. The vaccines targeting IDO1 and PD-L1 cooperatively reduce tumor outgrowth, and each contributes to the anti-tumor effect through distinct molecular programs regulating immunosuppression in the TME and T cell activation respectively. These data are supported by ex vivo functional assays using target specific T cells from vaccinated patients. Altogether, our data support the use of a dual antigen approach to reduce the immunosuppression and enhance anti-tumor effect. Citation Format: Marion Chapellier, Evelina Martinenaite, Preeyam Patel, Lea Svendsen, Shamaila Munir Ahmad, Hannah Jorinde Glöckner, Marco Carretta, Erika Sutanto-Ward, James DuHadaway, Souvik Dey, Shih-Chun Shen, Dema Ghaban, Marcos Iglesias, Megan Biller, Inés Lecoq, Alexander J. Muller, Mads Hald Andersen, Ayako Wakatsuki Pedersen. Immune modulatory cancer vaccines against IDO1 and PD-L1 trigger distinct pathways and cooperatively reduce tumor growth in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4094.