In Vitro Analysis of the Effect of SARS-CoV-2 Non-VOC and four Variants of Concern on MHC-Class-I Expression on Calu-3 and Caco-2 Cells.

As the MHC-I-pathway is key to antigen presentation to cytotoxic T-cells and, therefore, recognition by the host adaptive immune system, we hypothesized that SARS-CoV-2 including its Variants of Concern (VOCs), influences MHC-I expression on epithelial cell surfaces as an immune evasion strategy. We conducted an in vitro time course experiment with the human airway epithelial cell line Calu-3 and the human colorectal adenocarcinoma cell line Caco-2. Cells were infected with SARS-CoV-2 strains non-VOC/B.1.1, Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, and Delta/B.1.617.2. At 2, 24, 48 and 72 h post-infection we performed RT-qPCR to track viral replication. Simultaneously, we performed intracellular staining with a serum of a double-vaccinated healthy adult containing a high amount of spike protein antibody. In flow cytometry experiments, we differentiated between infected (spike protein positive) and bystander (spike protein negative) cells. To compare their HLA expression levels, cells were stained extracellularly with anti-HLA-A-IgG and anti-HLA-B,C-IgG. While HLA-A expression was stable on infected Calu-3 cells for all variants, it increased to different degrees on bystander cells in samples infected with VOCs Beta, Gamma, Delta, or non-VOC over the time course analyzed. In contrast, HLA-A levels were stable in bystander Calu-3 cells in samples infected with the Alpha variant. The upregulation of MHC-I on spike protein negative bystander cells in Calu-3 cell cultures infected with Beta, Gamma, Delta, and partly non-VOC might suggest that infected cells are still capable of secreting inflammatory cytokines like type-I interferons stimulating the MHC-I expression on bystander cells. In comparison, there was no distinct effect on HLA expression level on Caco-2 cells of any of the VOCs or non-VOC. Further investigations of the full range of immune evasion strategies of SARS-CoV-2 variants are warranted.

Properties of Adenovirus Vectors with Increased Affinity to DSG2 and the Potential Benefits of Oncolytic Approaches and Gene Therapy.

Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction.