A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

The cryo-electron microscopy structure of huntingtin.

Huntingtin (HTT) is a large (348 kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein-protein interaction hub. Furthermore, Huntington's disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely α-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Human Monocytes Are Suitable Carriers for the Delivery of Oncolytic Herpes Simplex Virus Type 1 In Vitro and in a Chicken Embryo Chorioallantoic Membrane Model of Cancer.

Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models.

HAP40 protein levels are huntingtin-dependent and decrease in Huntington disease.

The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In complexes of these proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains rich in HEAT repeats, and a smaller bridge domain connecting the two. We show that HAP40 steady-state protein levels are directly dependent on HTT (both normal and mutant HTT) and that HAP40 is strongly stabilized by the interaction with HTT resulting in an at least 5-fold increase in HAP40's half-life when bound to HTT. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of Huntington Disease (HD) patients and in brain tissue of a full-length HTT mouse model of HD, concomitant with decreased soluble HTT levels in these cell types. This data and our previous demonstration of coevolution between HTT and HAP40 and evolutionary conservation of their interaction suggest that HAP40 is an obligate interaction partner of HTT. Our observation of reduced HAP40 levels in HD invites further studies, whether HAP40 loss-of-function contributes to the pathophysiology of HD.

The evolution of the huntingtin-associated protein 40 (HAP40) in conjunction with huntingtin.

BACKGROUND: The huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington's disease (HD). Therefore, we analysed the evolution of HAP40 and its interaction with HTT. RESULTS: We found that in amniotes HAP40 is encoded by a single-exon gene, whereas in all other organisms it is expressed from multi-exon genes. HAP40 co-occurs with HTT in unikonts, including filastereans such as Capsaspora owczarzaki and the amoebozoan Dictyostelium discoideum, but both proteins are absent from fungi. Outside unikonts, a few species, such as the free-living amoeboflagellate Naegleria gruberi, contain putative HTT and HAP40 orthologs. Biochemically we show that the interaction between HTT and HAP40 extends to fish, and bioinformatic analyses provide evidence for evolutionary conservation of this interaction. The closest homologue of HAP40 in current protein databases is the family of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs). CONCLUSION: Our results indicate that the transition from a multi-exon to a single-exon gene appears to have taken place by retroposition during the divergence of amphibians and amniotes, followed by the loss of the parental multi-exon gene. Furthermore, it appears that the two proteins probably originated at the root of eukaryotes. Conservation of the interaction between HAP40 and HTT and their likely coevolution strongly indicate functional importance of this interaction.

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids.

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.

Evaluation of life cycle defective adenovirus mutants for production of adeno-associated virus vectors.

BACKGROUND: Adeno-associated virus-based vectors are efficient and safe drug candidates for different in vivo gene therapy applications. With increasing numbers of clinical studies based on AAV2 vectors that include not only rare, but also common diseases as a therapeutic target, there is an increased demand for the development of improved production technologies. METHODS: In the present study, we compared two life cycle defective adenovirus mutants as helper viruses for AAV2 vector production. They had deletions either in the gene coding for the preterminal protein (pTP) that is expressed early in the viral life cycle and is essential for genome replication or in the gene coding for the 100K protein, a protein with many functions, one of which is involved in virus assembly. AAV2 vector production efficiencies were evaluated by analyzing genome-containing particles using a real-time polymerase chain reaction and functional units were investigated by transduction assays. RESULTS: Somewhat contrary to our expectations, the ∆100K mutant virus showed only a moderate efficiency as a helper virus for AAV2 vector production, whereas the replication-deficient ∆pTP mutant supported AAV2 production almost as efficiently as adenovirus wild-type. We also showed that a temperature shift to 32°C together with extended incubation times improved AAV2 vector productivity. CONCLUSIONS: The present study indicates the advantages of using a ∆pTP mutant adenovirus rather than adenovirus wild-type as a helper virus for AAV2 production and also indicates that temperature shifts to lower temperatures may improve AAV2 vector production rates.

Recent advances in gene-enhanced bone tissue engineering.

The loss of bone tissue represents a critical clinical condition that is frequently faced by surgeons. Substantial progress has been made in the area of bone research, providing insight into the biology of bone under physiological and pathological conditions, as well as tools for the stimulation of bone regeneration. The present review discusses recent advances in the field of gene-enhanced bone tissue engineering. Gene transfer strategies have emerged as highly effective tissue engineering approaches for supporting the repair of the musculoskeletal system. By contrast to treatment with recombinant proteins, genetically engineered cells can release growth factors at the site of injury over extended periods of time. Of particular interest are the expedited technologies that can be applied during a single surgical procedure in a cost-effective manner, allowing translation from bench to bedside. Several promising methods based on the intra-operative genetic manipulation of autologous cells or tissue fragments have been developed in preclinical studies. Moreover, gene therapy for bone regeneration has entered the clinical stage with clinical trials for the repair of alveolar bone. Current trends in gene-enhanced bone engineering are also discussed with respect to the movement of the field towards expedited, translational approaches. It is possible that gene-enhanced bone tissue engineering will become a clinical reality within the next few years.

Alpha-Ketoglutarate Curbs Differentiation and Induces Cell Death in Mesenchymal Stromal Precursors with Mitochondrial Dysfunction.

Increased concentrations of reactive oxygen species (ROS) originating from dysfunctional mitochondria contribute to diverse aging-related degenerative disorders. But so far little is known about the impact of distinct ROS on metabolism and fate of stromal precursor cells. Here, we demonstrate that an increase in superoxide anion radicals due to superoxide dismutase 2 (Sod2) deficiency in stromal precursor cells suppress osteogenic and adipogenic differentiation through fundamental changes in the global metabolite landscape. Our data identify impairment of the pyruvate and l-glutamine metabolism causing toxic accumulation of alpha-ketoglutarate in the Sod2-deficient and intrinsically aged stromal precursor cells as a major cause for their reduced lineage differentiation. Alpha-ketoglutarate accumulation led to enhanced nucleocytoplasmic vacuolation and chromatin condensation-mediated cell death in Sod2-deficient stromal precursor cells as a consequence of DNA damage, Hif-1α instability, and reduced histone H3 (Lys27) acetylation. These findings hold promise for prevention and treatment of mitochondrial disorders commonly associated with aged individuals. Stem Cells 2017;35:1704-1718.

CD169+ macrophages are sufficient for priming of CTLs with specificities left out by cross-priming dendritic cells.

Dendritic cells (DCs) are considered the most potent antigen-presenting cells (APCs), which directly prime or cross-prime MHC I-restricted cytotoxic T cells (CTLs). However, recent evidence suggests the existence of other, as-yet unidentified APCs also able to prime T cells. To identify those APCs, we used adenoviral (rAd) vectors, which do not infect DCs but selectively accumulate in CD169(+) macrophages (MPs). In mice that lack DCs, infection of CD169(+) MPs was sufficient to prime CTLs specific for all epitopes tested. In contrast, CTL responses relying exclusively on cross-presenting DCs were biased to selected strong MHC I-binding peptides only. When both DCs and MPs were absent, no CTL responses could be elicited. Therefore, CD169(+) MPs can be considered APCs that significantly contribute to CTL responses.

Temperature-sensitive miR-483 is a conserved regulator of recombinant protein and viral vector production in mammalian cells.

Cell engineering and bioprocess optimizations such as low temperature cultivation represent powerful tools to improve cellular performance and product yields of mammalian production cells. Besides monoclonal antibodies (mABs), novel biotherapeutic formats such as viral vectors will gain increasing importance. Here, we demonstrate that similar to Chinese hamster ovary (CHO) cells, product yields of recombinant adeno-associated virus (rAAV) producing HeLa cells can be markedly increased by low temperature cultivation. MicroRNAs (miRNAs) are small non-coding RNAs that critically regulate cell phenotypes. We thus investigated differential miRNA expression in response to mild hypothermia in CHO and HeLa production cells. We discovered miR-483 to be substantially up-regulated upon temperature down-shift in both cell types. Functional validation experiments revealed that introduction of miR-483 mimics led to a significant increase in both rAAV and mAB production in HeLa and CHO cells, respectively. Furthermore, inhibition of miR-483 up-regulation during mild hypothermia significantly decreased product yields, suggesting that miR-483 is a key regulator of cellular productivity in mammalian cells. In addition, miRNA target gene identification indicated that miR-483 might regulate genes directly involved in cellular survival and protein expression. Our results highlight that miR-483 is a valuable tool for product-independent engineering of mammalian production cells.

The transcription factor serum response factor stimulates axon regeneration through cytoplasmic localization and cofilin interaction.

Axonal injury generates growth inert retraction bulbs with dynamic cytoskeletal properties that are severely compromised. Conversion of "frozen" retraction bulbs into actively progressing growth cones is a major aim in axon regeneration. Here we report that murine serum response factor (SRF), a gene regulator linked to the actin cytoskeleton, modulates growth cone actin dynamics during axon regeneration. In regeneration-competent facial motoneurons, Srf deletion inhibited axonal regeneration. In wild-type mice after nerve injury, SRF translocated from the nucleus to the cytoplasm, suggesting a cytoplasmic SRF function in axonal regeneration. Indeed, adenoviral overexpression of cytoplasmic SRF (SRF-ΔNLS-GFP) stimulated axonal sprouting and facial nerve regeneration in vivo. In primary central and peripheral neurons, SRF-ΔNLS-GFP stimulated neurite outgrowth, branch formation, and growth cone morphology. Furthermore, we uncovered a link between SRF and the actin-severing factor cofilin during axonal regeneration in vivo. Facial nerve axotomy increased the total cofilin abundance and also nuclear localization of phosphorylated cofilin in a subpopulation of lesioned motoneurons. This cytoplasmic-to-nucleus translocation of P-cofilin upon axotomy was reduced in motoneurons expressing SRF-ΔNLS-GFP. Finally, we demonstrate that cytoplasmic SRF and cofilin formed a reciprocal regulatory unit. Overexpression of cytoplasmic SRF reduced cofilin phosphorylation and vice versa: overexpression of cofilin inhibited SRF phosphorylation. Therefore, a regulatory loop consisting of SRF and cofilin might take part in reactivating actin dynamics in growth-inert retraction bulbs and facilitating axon regeneration.

ProteinCoLoc streamlines Bayesian analysis of colocalization in microscopic images.

Colocalization, the spatial overlap of molecular entities, is often key to support their involvement in common functions. Existing colocalization tools, however, face limitations, particularly because of their basic statistical analysis and their low-throughput manual entry processes making them unsuitable for automation and potentially introducing bias. These shortcomings underscore the need for user-friendly tools streamlining colocalization assessments and enabling their robust and automated quantitative analyses. We have developed ProteinCoLoc, an innovative software designed for automated high-throughput colocalization analyses and incorporating advanced statistical features such as Bayesian modelling, automatic background detection and localised correlation analysis. ProteinCoLoc rationalises colocalization assessments without manual input, comes with a user-friendly graphical user interface and provides various analytics allowing to study and locally quantify colocalization. This easy-to-use application presents numerous advantages, including a direct comparison with controls employing a Bayesian model and the analysis of local correlation patterns, while reducing hands-on time through automatic background detection. The software was validated while studying the colocalization pattern of two proteins forming a stable complex: the huntingtin protein (HTT) and its partner huntingtin-associated protein 40 (HAP40). Our results showcase the software's capacity to quantitatively assess colocalizations. ProteinCoLoc is available both as a Julia package and as a compiled software ( https://github.com/ma-seefelder/ProteinCoLoc ).

Foamy virus-adenovirus hybrid vectors for gene therapy of the arthritides.

BACKGROUND: Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints. METHODS: We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats. RESULTS: FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10-35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3-5 days after injection of FAD vectors to the knee joints. CONCLUSIONS: Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides.

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation.

UNLABELLED: Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. CONCLUSION: Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases.

CAP, a new human suspension cell line for influenza virus production.

Forced by major drawbacks of egg-based influenza virus production, several studies focused on the establishment and optimization of cell-based production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high virus titers. From primary virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza virus production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log(10) HA units/100 µL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log(10) HA units/100 µL and maximum cell-specific virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza virus vaccine production.

Affilin-based retargeting of adenoviral vectors to the epidermal growth factor receptor.

INTRODUCTION: Treatment of head and neck squamous cell carcinomas (HNSCC) by oncolytic adenoviral vectors holds promise as an efficient anti-cancer therapy. The epidermal growth factor receptor (EGFR) represents an attractive target receptor since it is frequently overexpressed in many types of HNSCC. METHODS: To achieve EGFR-specific targeting by human adenovirus type 5 (HAdV-5) based vectors, the EGFR affinity ligand Affilin was covalently attached in a position specific manner either to the fiber or the hexon protein of the vector capsid. In vitro and in vivo studies investigated EGFR-specific cancer cell transduction, susceptibility to natural sequestration mechanisms, pharmacokinetics and biodistribution profiles of Affilin-decorated vectors. RESULTS: Affilin-decorated vectors showed strongly enhanced and EGFR-specific cancer cell transduction in vitro and less susceptibility to known sequestration mechanisms of HAdV-5 particles. However, in vivo neither systemic nor intratumoral vector administration resulted in an improved transduction of EGFR-positive tumors. Comprehensive analyses indicated hampered EGFR-targeting by Affilin-decorated vectors was caused by rapid vector particle consumption due to binding to the murine EGFR, insufficient tumor vascularization and poor target accessibility for Affilin in the solid tumor caused by a pronounced tumor stroma. CONCLUSION: In vitro studies yielded proof-of-concept results demonstrating that covalent attachment of a receptor-specific Affilin to the adenoviral capsid provides an effective and versatile tool to address cancer-specific target receptors by adenoviral vectors. Regarding EGFR as the vector target, off-target tissue transduction and low receptor accessibility within the tumor tissue prevented efficient tumor transduction by Affilin-decorated vectors, rendering EGFR a difficult-to-target receptor for adenoviral vectors.

Evaluation of Human Mesenchymal Stromal Cells as Carriers for the Delivery of Oncolytic HAdV-5 to Head and Neck Squamous Cell Carcinomas.

Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest due to their ability to deliver oncolytic adenoviruses to tumors. This approach is also investigated for targeting head and neck squamous cell carcinomas (HNSCCs). HAdV-5-HexPos3, a recently reported capsid-modified vector based on human adenovirus type 5 (HAdV-5), showed strongly improved infection of both hMSCs and the HNSCC cell line UM-SCC-11B. Given that, we generated life cycle-unmodified and -modified replication-competent HAdV-5-HexPos3 vector variants and analyzed their replication within bone marrow- and adipose tissue-derived hMSCs. Efficient replication was detected for both life cycle-unmodified and -modified vectors. Moreover, we analyzed the migration of vector-carrying hMSCs toward different HNSCCs. Although migration of hMSCs to HNSCC cell lines was confirmed in vitro, no homing of hMSCs to HNSCC xenografts was observed in vivo in mice and in ovo in a chorioallantoic membrane model. Taken together, our data suggest that HAdV-5-HexPos3 is a potent candidate for hMSC-based oncolytic therapy of HNSCCs. However, it also emphasizes the importance of generating optimized in vivo models for the evaluation of hMSC as carrier cells.

Heterologous DNA-prime/protein-boost immunization with a monomeric SARS-CoV-2 spike antigen redundantizes the trimeric receptor-binding domain structure to induce neutralizing antibodies in old mice.

A multitude of alterations in the old immune system impair its functional integrity. Closely related, older individuals show, for example, a reduced responsiveness to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. However, systematic strategies to specifically improve the efficacy of vaccines in the old are missing or limited to simple approaches like increasing the antigen concentration or injection frequencies. We here asked whether the intrinsic, trimeric structure of the SARS-CoV-2 spike (S) antigen and/or a DNA- or protein-based antigen delivery platform affects priming of functional antibody responses particularly in old mice. The used S-antigens were primarily defined by the presence/absence of the membrane-anchoring TM domain and the closely interlinked formation/non-formation of a trimeric structure of the receptor binding domain (S-RBD). Among others, we generated vectors expressing prefusion-stabilized, cell-associated (TM+) trimeric "S2-P" or secreted (TM-) monomeric "S6-PΔTM" antigens. These proteins were produced from vector-transfected HEK-293T cells under mild conditions by Strep-tag purification, revealing that cell-associated but not secreted S proteins tightly bound Hsp73 and Grp78 chaperones. We showed that both, TM-deficient S6-PΔTM and full-length S2-P antigens elicited very similar S-RBD-specific antibody titers and pseudovirus neutralization activities in young (2-3 months) mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. The trimeric S2-P antigen induced high S-RBD-specific antibody responses in old (23-24 months) mice through DNA-prime/DNA-boost vaccination. Unexpectedly, the monomeric S6-PΔTM antigen induced very low S-RBD-specific antibody titers in old mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. However, old mice efficiently elicited an S-RBD-specific antibody response after heterologous DNA-prime/protein-boost immunization with the S6-PΔTM antigen, and antibody titers even reached similar levels and neutralizing activities as in young mice and also cross-reacted with different S-variants of concern. The old immune system thus distinguished between trimeric and monomeric S protein conformations: it remained antigen responsive to the trimeric S2-P antigen, and a simple change in the vaccine delivery regimen was sufficient to unleash its reactivity to the monomeric S6-PΔTM antigen. This clearly shows that both the antigen structure and the delivery platform are crucial to efficiently prime humoral immune responses in old mice and might be relevant for designing "age-adapted" vaccine strategies.