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Recent years have seen an unprecedented rise in the incidence of multidrug-resistant (MDR) Gram-negative bacteria (GNBs) such as Acinetobacter and Klebsiella species. In view of the shortage of novel drugs in the pipeline, alternative strategies to prevent, and treat infections by GNBs are urgently needed. Previously, we have reported that the Candida albicans hypha-regulated protein Hyr1 shares striking three-dimensional structural homology with cell surface proteins of Acinetobacter baumannii. Moreover, active vaccination with rHyr1p-N or passive immunization with anti-Hyr1p polyclonal antibody protects mice from Acinetobacter infection. In the present study, we use molecular modeling to guide design of monoclonal antibodies (mAbs) generated against Hyr1p and show them to bind to priority surface antigens of Acinetobacter and Klebsiella pneumoniae. The anti-Hyr1 mAbs block damage to primary endothelial cells induced by the bacteria and protect mice from lethal pulmonary infections mediated by A. baumannii or K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAbs as a cross-kingdom immunotherapeutic strategy against MDR GNBs.

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.