The stability of immune responses to SARS-CoV-2 vaccines, especially concerning the cross-reactive recognition of the Omicron variant, remains incompletely characterized in multiple myeloma (MM) patients. This study evaluated humoral responses in 29 MM patients and cellular responses in a subset of 19 MM patients, specific to Wuhan and Omicron spike proteins, between 16 and 26 weeks following the third vaccine dose. After 26 weeks, we highlighted a significant reduction in the neutralizing antibodies to both spikes and the percentages of IFN-γ+CD107a+ spike-specific CD8+ T cells. On the other hand, patients who underwent an additional stimulation between the two time points, through either a fourth vaccine dose or breakthrough infection, showed a significant increase in neutralizing antibodies and stable levels of cytotoxic CD8+ T cells. Additionally, those with only three doses experienced a higher rate of breakthrough infections during the 32-week follow-up period. These findings underscore the waning of vaccine-induced immunity over time and may help benefit-risk evaluation in vaccination strategies in MM patients.
Copyright © 2025 Raimondi, Storti, Vescovini, Franceschi, Toscani, Notarfranchi, Dalla Palma, Iannozzi, Minesso, Scita, Lungu, Dessena, Donofrio and Giuliani.

During the COVID-19 pandemic we utilized an AI-driven T cell epitope prediction tool, the NEC Immune Profiler (NIP) to scrutinize and predict regions of T cell immunogenicity (hotspots) from the entire SARS-CoV-2 viral proteome. These immunogenic regions offer potential for the development of universally protective T cell vaccine candidates. Here, we validated and characterized T cell responses to a set of minimal epitopes from these AI-identified universal hotspots. Utilizing a flow cytometry-based T cell activation-induced marker (AIM) assay, we identified 59 validated screening hits, of which 56% (33 peptides) have not been previously reported. Notably, we found that most of these novel epitopes were derived from the non-spike regions of SARS-CoV-2 (Orf1ab, Orf3a, and E). In addition, ex vivo stimulation with NIP-predicted peptides from the spike protein elicited CD8+ T cell response in PBMC isolated from most vaccinated donors. Our data confirm the predictive accuracy of AI platforms modelling bona fide immunogenicity and provide a novel framework for the evaluation of vaccine-induced T cell responses.
Copyright © 2023 Federico, Malone, Tennøe, Chaban, Osen, Gainullin, Smorodina, Kared, Akbar, Greiff, Stratford, Clancy and Munthe.

Poor overall survival of hematopoietic stem cell transplantation (HSCT) recipients who developed COVID-19 underlies the importance of SARS-CoV-2 vaccination. Previous studies of vaccine efficacy have reported weak humoral responses but conflicting results on T cell immunity. Here, we have examined the relationship between humoral and T cell response in 48 HSCT recipients who received two doses of Moderna's mRNA-1273 or Pfizer/BioNTech's BNT162b2 vaccines. Nearly all HSCT patients had robust T cell immunity regardless of protective humoral responses, with 18/48 (37%, IQR 8.679-5601 BAU/mL) displaying protective IgG anti-receptor binding domain (RBD) levels (>2000 BAU/mL). Flow cytometry analysis of activation induced markers (AIMs) revealed that 90% and 74% of HSCT patients showed reactivity towards immunodominant spike peptides in CD8+ and CD4+ T cells, respectively. The response rate increased to 90% for CD4+ T cells as well when we challenged the cells with a complete set of overlapping peptides spanning the entire spike protein. T cell response was detectable as early as 3 months after transplant, but only CD4+ T cell reactivity correlated with IgG anti-RBD level and time after transplantation. Boosting increased seroconversion rate, while only one patient developed COVID-19 requiring hospitalization. Our data suggest that HSCT recipients with poor serological responses were protected from severe COVID-19 by vaccine-induced T cell responses.
Copyright © 2023 Federico, Tvedt, Gainullin, Osen, Chaban, Lund, Tietze, Tran, Lund-Johansen, Kared, Lind, Vaage, Stratford, Tennøe, Malone, Clancy, Myhre, Gedde-Dahl and Munthe.

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T‑cells. In order to overcome a lot‑to‑lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum‑free media [Prime‑XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T‑Cell Expansion Xeno‑Free Medium, Takara Bio™ (TB) and TCM GMP‑Prototype, CellGenix™ (CG)] to the standard CAR T‑cell medium containing FBS (RCF). After 12 days of CD19.CAR T‑cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T‑cells was evaluated using intracellular staining, a chromium release assay and a long‑term co‑culture assay. Expansion and viability did not differ between the CAR T‑cells generated in serum‑free media compared to the standard FBS‑containing medium. The CG CAR T‑cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF‑α+ CAR T‑cells than the CAR T‑cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T‑cells generated with serum‑free media exhibited a higher cytotoxicity than the CAR T‑cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T‑cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte‑activation gene 3 and T‑cell immunoglobulin and mucin‑domain containing‑3. The CAR T‑cells cultured in FF had a higher percentage of central memory CAR T‑cells (40.0 vs. 14.3%, P=0.0470) than the CAR T‑cells cultured with FBS, whereas the CAR T‑cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T‑cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum‑free media. Given the advantages of freedom from bovine material and consistent quality, serum‑free media hold promise for the future development of the field of GMP manufacturing of CAR T‑cells.

Arginase-1 (ARG1) and Programed death ligand-1 (PD-L1) play a vital role in immunosuppression in myeloproliferative neoplasms (MPNs) and directly inhibit T-cell activation and proliferation. We previously identified spontaneous T-cell responses towards PD-L1 and ARG1 derived peptide epitopes in patients with MPNs. In the present First-in-Man study we tested dual vaccinations of ARG1- derived and PD-L1-derived peptides, combined with Montanide ISA-51 as adjuvant, in patients with Janus Kinase 2 (JAK2) V617F-mutated MPN.
Safety and efficacy of vaccination with ARG1- derived and PD-L1-derived peptides with montanide as an adjuvant was tested in 9 patients with MPN The primary end point was safety and toxicity evaluation. The secondary end point was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT04051307).
The study included 9 patients with JAK2-mutant MPN of which 8 received all 24 planned vaccines within a 9-month treatment period. Patients reported only grade 1 and 2 vaccine related adverse events. No alterations in peripheral blood counts were identified, and serial measurements of the JAK2V617F allelic burden showed that none of the patients achieved a molecular response during the treatment period. The vaccines induced strong immune responses against both ARG1 and PD-L1- derived epitopes in the peripheral blood of all patients, and vaccine-specific skin-infiltrating lymphocytes from 5/6 patients could be expanded in vitro after a delayed-type hypersensitivity test. In two patients we also detected both ARG1- and PD-L1-specific T cells in bone marrow samples at the end of trial. Intracellular cytokine staining revealed IFNγ and TNFγ producing CD4+- and CD8+- T cells specific against both vaccine epitopes. Throughout the study, the peripheral CD8/CD4 ratio increased significantly, and the CD8+ TEMRA subpopulation was enlarged. We also identified a significant decrease in PD-L1 mRNA expression in CD14+ myeloid cells in the peripheral blood in all treated patients and a decrease in ARG1 mRNA expression in bone marrow of 6 out of 7 evaluated patients.
Overall, the ARG1- and PD-L1-derived vaccines were safe and tolerable and induced strong T-cell responses in all patients. These results warrant further studies of the vaccine in other settings or in combination with additional immune-activating treatments.
Copyright © 2023 Grauslund, Holmström, Martinenaite, Lisle, Glöckner, El Fassi, Klausen, Mortensen, Jørgensen, Kjær, Skov, Svane, Hasselbalch and Andersen.