Delivery of antigenic peptides to antigen presenting cells (APCs) such as dendritic cells (DCs) using monoclonal antibodies (mAbs) is an attractive approach to evoke antigen-specific T cell activation and improve drug efficacy. Peptide linkage to mAbs has previously been achieved through genetic fusion, chemical conjugation, nano-engineered platforms and high affinity peptides. In this study, we have developed a flexible antibody-peptide linking technology using oppositely charged coiled coil domains to non-covalently link peptides to mAbs. The technology comprises (1) an anti-CD40 mAb connected with negatively charged E domains and (2) an immunogenic OVA peptide (SIINFEKL) from ovalbumin used as a model antigenic peptide fused with positively charged K domains. Combining these constructs leads to the formation of complexes that can be targeted to CD40 expressed on cells. Proof of concept antibody constructs connected with E domains generated from transient expressions exhibited good manufacturability, binding, and stability attributes comparable to a control mAb. Also, optimal repeat lengths for coiled-coil oligomerization domains were identified in these studies. Binding kinetics studies showed that connecting E domains to mAbs do not impede Fc gamma and neonatal receptor interactions. Additionally, formation of stable complexes capable of binding CD40 expressing cells was demonstrated in vitro. In vivo functionality evaluations showed that treatment of human CD40 transgenic mice with complexes elicited expansion of OVA peptide-specific CD8+ T cells and potent antitumor effects superior to peptide monotherapies. Overall, these findings demonstrate that the technology has great potential for application as an in vivo tool for antigenic peptide delivery.

Secretory leukocyte protease inhibitor (SLPI) is described as a potent regulator of inflammation and tissue homeostasis with pleiotropic functions. It has been shown to inhibit pro-inflammatory responses in myeloid cells. However, its expression patterns and specific functions in different monocyte and macrophage populations remain poorly understood. Therefore, we investigated its expression patterns in murine tissue macrophage populations by analysis of publicly available datasets and flow cytometry. Among various tissues, peritoneal macrophages were identified as a major source of SLPI, suggesting the highest impact of this inhibitor on their physiological and pathophysiological functions. To elucidate the role of SLPI in the inflammatory response, SLPI-deficient mice were used. First, the response to LPS was compared in resident and thioglycolate-recruited peritoneal macrophages. Moreover, we evaluated the role of SLPI in an in vivo mouse model of LPS-induced septic shock. Results demonstrated that while the lack of SLPI did not affect pro-inflammatory cytokine production in activated resident macrophages, it regulated the production of matrix metalloproteinase-9 (MMP-9). Similar results were observed in thioglycolate-elicited and LPS-activated peritoneal macrophage populations, further highlighting the link between SLPI and MMP-9. Furthermore, in vivo LPS-induced changes in SLPI expression were evident among various myeloid populations, including monocytes. Loss of SLPI also influenced the frequency of blood monocyte populations in this model. Overall, these findings highlight a specific role for SLPI in regulating MMP-9 in response to LPS both in vitro and in vivo and suggest that SLPI might play a role in tissue remodeling orchestrated by macrophages.
Copyright © 2025 Tyshchenko, Pocałuń, Kwiecińska, Cichy, Wilk and Oleszycka.

Immune memory is influenced by the frequency and type of antigenic challenges. Here, we performed a cross-sectional comparison of immune parameters following a BA.1 breakthrough infection in individuals with prior hybrid immunity (conferred by infection and vaccination) versus those solely vaccinated in a cohort of health care workers in Lyon, France. The results showed higher levels of serum anti-receptor binding domain (RBD) antibodies and neutralizing antibodies against BA.1 post-infection in the vaccine-only group. Individuals in this group also showed a decrease in memory B cells against the ancestral strain but an increase in those specific and cross-reactive to BA.1, suggesting a more limited immune imprinting. Conversely, hybrid immunity prevents the decrease in antibody dependent cellular cytotoxicity (ADCC) response, possibly by limiting IgG4 class-switching and enhanced anti-N responses post-infection. This highlights that BA.1 breakthrough infection induces different immune responses depending on prior history of vaccination and infection, which should be considered for further vaccination guidelines.
© 2025 The Authors.

ABSTRACT Short-lived, clade-specific immune responses with limited mucosal priming are limitations faced by current COVID-19 mRNA vaccines against sarbecoviruses. We have developed a nasal booster vaccine candidate that induced robust and sustained, cross-clade, systemic and mucosal protective immunity. Two recombinant Clec9A-specific monoclonal antibodies fused to the Receptor Binding Domain (RBD) from Omicron XBB.1.5 and SARS-CoV-1, respectively were generated. In Comirnaty mRNA-vaccinated mice, boosting with each individual Clec9A-RBD construct induced immune responses that either were limited in breadth or waned over time; while boosting with both constructs combined (Clec9A OMNI ) elicited robust cross-clade neutralizing antibodies (nAb) and T cell responses that were significantly more sustained compared to Bivalent Comirnaty (BC) mRNA vaccine booster. The persistence of RBD-specific follicular helper CD4 + T cells, germinal centre B cells, and long-lived plasma cells that facilitated affinity maturation in Clec9A OMNI -boosted mice, correlated with the detection of triple cross-reactive B cells that bind to ancestral SARS-CoV-2 ancestral, SARS-CoV-2 XBB.1.5 and SARS-CoV-1 RBD. Remarkably, intranasal boosting with Clec9A OMNI generated robust and sustained mucosal immune responses in the upper and lower respiratory compartments, including RBD-specific IgA, cross-clade nAb and cellular immunity together with functional tissue-resident memory T cells, without compromising the systemic immune responses. Correspondingly, Clec9A OMNI booster conferred superior protection against Omicron BA.1 compared to BC booster when challenge was performed at six months post-boost. Hence, Clec9A OMNI is a promising nasal booster vaccine candidate that has the potential to mitigate pandemic threats from emerging sarbecoviruses. One Sentence Summary Nasal booster immunization with dendritic cell-targeting vaccine candidate in mRNA-vaccinated mice induced cross-clade, sustained, systemic and mucosal protective immunity.

Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge for early diagnosis due to the lack of sensitive and specific biomarkers. This encouraged us to explore the diagnostic value of cancer-derived small extracellular vesicles (sEVs) as early detection biomarkers. We previously showed that the recombinant malaria protein VAR2CSA (rVAR2) selectively binds to oncofetal chondroitin sulfate (ofCS) on the surfaces of cancer cells, which might be useful for identifying cancer-derived sEVs. Indeed, flow cytometry revealed strong ofCS expression in PDAC cell-derived sEVs, as evidenced by the presence of mutant KRAS, a common genetic alteration in PDAC. Plasma from PDAC patients showed significantly higher ofCS+ sEV levels compared to healthy donors and patients with benign gastrointestinal diseases. ROC analysis for ofCS+ sEVs revealed an AUC of 0.9049 for the detection of all-stage and 0.9222 for early-stage PDAC. Notably, mutant KRAS was also detected in these patient-derived sEVs. Most intriguingly, combining ofCS+ sEVs and CA19-9 resulted in an AUC of 0.9707 for the detection of early PDAC. Our study demonstrates that rVAR2 is suitable for detecting ofCS+ cancer-derived sEVs in plasma, thereby providing high efficiency for identifying PDAC patients among a diverse population. These findings suggest that rVAR2-based sEV detection could serve as a powerful diagnostic tool to improve patient survival through early detection.
© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.