Distinct T cell responsiveness to different COVID-19 vaccines and cross-reactivity to SARS-CoV-2 variants with age and CMV status.

Introduction: Accumulating evidence indicates the importance of T cell immunity in vaccination-induced protection against severe COVID-19 disease, especially against SARS-CoV-2 Variants-of-Concern (VOCs) that more readily escape from recognition by neutralizing antibodies. However, there is limited knowledge on the T cell responses across different age groups and the impact of CMV status after primary and booster vaccination with different vaccine combinations. Moreover, it remains unclear whether age has an effect on the ability of T cells to cross-react against VOCs. Methods: Therefore, we interrogated the Spike-specific T cell responses in healthy adults of the Dutch population across different ages, whom received different vaccine types for the primary series and/or booster vaccination, using IFNÉ£ ELISpot. Cells were stimulated with overlapping peptide pools of the ancestral Spike protein and different VOCs. Results: Robust Spike-specific T cell responses were detected in the vast majority of participants upon the primary vaccination series, regardless of the vaccine type (i.e. BNT162b2, mRNA-1273, ChAdOx1 nCoV-19, or Ad26.COV2.S). Clearly, in the 70+ age group, responses were overall lower and showed more variation compared to younger age groups. Only in CMV-seropositive older adults (>70y) there was a significant inverse relation of age with T cell responses. Although T cell responses increased in all age groups after booster vaccination, Spike-specific T cell frequencies remained lower in the 70+ age group. Regardless of age or CMV status, primary mRNA-1273 vaccination followed by BNT162b2 booster vaccination showed limited booster effect compared to the BNT162b2/BNT162b2 or BNT162b2/mRNA-1273 primary-booster regimen. A modest reduction in cross-reactivity to the Alpha, Delta and Omicron BA.1, but not the Beta or Gamma variant, was observed after primary vaccination. Discussion: Together, this study shows that age, CMV status, but also the primary-booster vaccination regimen influence the height of the vaccination-induced Spike-specific T cell response, but did not impact the VOC cross-reactivity.

Validation of novel conditional ligands and large-scale detection of antigen-specific T cells for H-2Dd and H-2Kd.

The UV-mediated peptide exchange has enabled the generation of multiple different MHC multimer specificities in parallel, surpassing tedious individual refolding of MHC molecules with peptide ligands. Murine models are acknowledged as an effective tool for preclinical research to advance our understanding of immunological mechanisms, with the potential translatability of key learnings from mouse models to the clinic. The common inbred mouse strain BALB/c is frequently used in immunological research. However, for the BALB/c histocompatibility (H)-2 alleles availability of conditional ligand has been limited. To overcome this challenge, we design and experimentally validate conditional ligands restricted to murine MHC class I alleles H2Dd and H2Kd. In addition, we demonstrate the ability of the three H2d molecules and two additional C57BL/6 H2b molecules folded in-house with conditional ligands to generate fluorescently labeled peptide-H2 tetramers that allow staining of antigen-specific CD8+ T cells in splenocyte samples. Finally, we generate large peptide-H-2 multimer libraries with a DNA-barcode labeling system for high-throughput interrogation of CD8+ T cell specificity in murine splenocyte samples. Consequently, the described techniques will contribute to our understanding of the antigen-specific CD8+ T cell repertoire in murine preclinical models of various diseases.

Engineered E. coli Nissle 1917 for delivery of bioactive IL-2 for cancer immunotherapy.

In this study we performed a step-wise optimization of biologically active IL-2 for delivery using E. coli Nissle 1917. Engineering of the strain was coupled with an in vitro cell assay to measure the biological activity of microbially produced IL-2 (mi-IL2). Next, we assessed the immune modulatory potential of mi-IL2 using a 3D tumor spheroid model demonstrating a strong effect on immune cell activation. Finally, we evaluated the anticancer properties of the engineered strain in a murine CT26 tumor model. The engineered strain was injected intravenously and selectively colonized tumors. The treatment was well-tolerated, and tumors of treated mice showed a modest reduction in tumor growth rate, as well as significantly elevated levels of IL-2 in the tumor. This work demonstrates a workflow for researchers interested in engineering E. coli Nissle for a new class of microbial therapy against cancer.