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Intramuscular vaccines present limitations in eliciting robust mucosal immunity and preventing respiratory pathogens transmission. Sublingual vaccine administration offers promising advantages, including interconnected mucosal protection. Despite these advantages, only a few clinical trials have explored sublingual vaccines, underscoring the necessity of optimizing next-generation vaccine formulas. Critical research priorities include understanding vector behavior in the oral environment, understanding their interactions with mucosal immunity and developing formulations enabling sustained mucosal contact to facilitate efficient transduction. Consequently, tonsil organoids, as representative human mucosal models, could offer critical insights into sublingual immunization. Thus, a multi-disciplinary approach integrating pharmacological, immunological, and manufacturing considerations is pivotal for sublingual vaccines in targeting pathogen-aggravated prevalent respiratory diseases including asthma, COPD and lung cancer, as well as the antimicrobial resistance crisis. [ABSTRACT FROM AUTHOR]

Background: Adenoviral vectors are among the most frequently used vectors for gene therapy and cancer treatment. Most vectors are derived from human adenovirus (Ad) serotype 5 despite limited applicability caused by pre‐existing immunity and unfavorable liver tropism, whereas the other more than 100 known human serotypes remain largely unused. Here, we screened a library of human Ad types and identified Ad4 as a promising candidate vector. Methods: Reporter‐gene‐expressing viruses representative of the natural human Ad diversity were used to transduce an array of muscle cell lines and two‐ or three‐dimensional tumor cultures. The time‐course of transgene expression was monitored by fluorescence or luminescence measurements. To generate replication‐deficient Ad4 vector genomes, successive homologous recombination was applied. Results: Ad4, 17 and 50 transduced human cardiomyocytes more efficiently than Ad5, whereas Ad37 was found to be superior in rhabdomyocytes. Despite its moderate transduction efficiency, Ad4 showed efficient and long‐lasting gene expression in papillomavirus (HPV) positive tumor organoids. Therefore, we aimed to harness the potential of Ad4 for improved muscle transduction or oncolytic virotherapy of HPV‐positive tumors. We deleted the E1 and E3 transcription units to produce first generation Ad vectors for gene therapy. The E1‐ and E1/E3‐deleted vectors were replication‐competent in HEK293 cells stably expressing E1 but not in the other cell lines tested. Furthermore, we show that the Ad5 E1 transcription unit can complement the replication of E1‐deleted Ad4 vectors. Conclusions: Our Ad4‐based gene therapy vector platform contributes to the development of improved Ad vectors based on non‐canonical serotypes for a broad range of applications. [ABSTRACT FROM AUTHOR]

Directed evolution of viral vectors involves the generation of randomized libraries followed by artificial selection of improved variants. Directed evolution only yielded limited results in adenovirus (AdV) engineering until now, mainly due to insufficient complexities of randomized libraries. Meanwhile, clinical applications of AdVs as gene therapy or oncolytic vectors are still hampered by the predetermined tropism of natural types. To overcome this challenge, we hypothesized that randomized peptide insertions on the capsid surface can be incorporated into the AdV bioengineering toolbox for retargeting. Here we developed AdV-directed EVOlution protocols based on fiber knob peptide display. Human AdV-C5-derived libraries were constructed following three distinct protocols and selected on a panel of cancer cell lines, with the goal of identifying variants able to infect and lyse these tumor cells more efficiently. All protocols enabled the construction of high complexity libraries with up to 9.6 × 10 5 unique variants, an approximate 100-fold improvement compared with previously published AdV libraries. After selection, the most enriched variants, which were robustly selected in various cancer cell lines, did not display enhanced infectivity but rather more efficient replication and cell lysis. Selected inserts also conferred enhanced lysis ability to oncolytic AdVs restricted to telomerase-expressing cell lines., Competing Interests: J.F. and Z.R. are co-inventors in patent application EP20198944 by the Albert Ludwig University of Freiburg that describes the use of CRISPR/Cas-mediated rescue of recombinant AdVs from circular plasmids., (© 2024 The Author(s).)

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a Designed Ankyrin Repeat Protein (DARPin) adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus toward LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein. Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared with blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared with BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared with BA-bound virus. Mechanistically, we show that the LA increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Altogether, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.