Modified vaccinia Ankara (MVA) is a promising vaccine vector due to its highly attenuated phenotype and good immunogenicity. However, obtaining a new recombinant MVA remains a tedious and laborious procedure involving many rounds of plaque purification. Recombinant MVA generation can be greatly improved and facilitated by different selection techniques. Here, we describe a comparison between techniques based on K1L, F13L and D4R genes.

The CARD11-BCL10-MALT1 (CBM) complex drives NF-κB signaling and MALT1 protease activation after T cell receptor (TCR) stimulation, forming a central signaling hub in adaptive immunity. Both linear ubiquitin chain assembly complex (LUBAC), consisting of HOIP, HOIL-1 and SHARPIN, and TRAF6 interact with the CBM complex. Still, the coordinated activity of these E3 ligases in controlling CBM activity remains elusive. Here we demonstrate that LUBAC, unlike TRAF6, is largely dispensable for TCR-induced NF-κB activation in human CD4 ⁺ T cells. However, HOIP contributes to NF-κB target gene expression and, with TRAF6, modulates MALT1 substrate recognition, influencing T cell responses. Further, LUBAC-mediated conjugation of Met1-linked ubiquitin chains to BCL10 strictly depends on TRAF6, but putative Met1-ubiquitin acceptor lysines in BCL10 serve essential structural roles that limit accessibility within BCL10-MALT1 filaments. Thus, LUBAC acts downstream of TRAF6 to modulate MALT1 substrate recognition and to catalyze BCL10 ubiquitination, which is incompatible with BCL10-MALT1 filament formation.