Acinetobacter baumannii ( Ab ) is one of the most treacherous pathogens among those causing hospital-acquired pneumonia (HAP). A. baumannii possesses an adaptable physiology, seen not only in its antibiotic resistance and virulence phenotypes but also in its metabolic versatility. In this study, we observed that A. baumannii undergoes global transcriptional changes in response to human pleural fluid (PF), a key host-derived environmental signal. Differential gene expression analyses combined with experimental approaches revealed changes in A. baumannii metabolism, affecting cytotoxicity, persistence, bacterial killing, and chemotaxis. Over 1,220 genes representing 55% of the differentially expressed transcriptomic data corresponded to metabolic processes, including the upregulation of glutamate, short chain fatty acid, and styrene metabolism. We observed an upregulation by 1.83- and 2.61-fold of the pyruvate dehydrogenase complex subunits E3 and E2, respectively. We also found that pyruvate (PYR), in conjunction with PF, triggers an A. baumannii pathogenic behavior that adversely impacts human epithelial cell viability. Interestingly, PF also amplified A. baumannii cytotoxicity against murine macrophages, suggesting an immune evasion strategy implemented by A. baumannii . Moreover, we uncovered opposing metabolic strategies dependent on the degree of pathogenicity of the strains, where less pathogenic strains demonstrated greater utilization of PYR to promote persister formation in the presence of PF. Additionally, our transcriptomic analysis and growth studies of A. baumannii suggest the existence of an alternative phenylalanine (PA) catabolic route independent of the phenylacetic acid pathway, which converts PA to phenylpyruvate (PP) and shuttles intermediates into styrene metabolism. This alternative route promoted a neutrophil-evasive state, as PF-induced degradation of PP significantly reduced overall human neutrophil chemotaxis in ex vivo chemotactic assays. Taken together, these data highlight A. baumannii pathoadaptabililty in response to host signals and provide further insight into the role of bacterial metabolism in virulence traits, antibiotic persistence strategies, and host innate immune evasion.

The production of reactive oxygen species (ROS) is a prominent response to infection among innate immune cells such as macrophages and neutrophils. To better understand the relationship between antimicrobial and regulatory functions of blood cell ROS, we have characterized the ROS response to infection in Drosophila hemocytes. Using fluorescent probes, we find a biphasic hemocyte ROS response to bacterial infection. In the first hour, virtually all hemocytes generate a transient ROS signal, with nonphagocytic cells including prohemocytes and crystal cells displaying exceptionally strong responses. A distinct, and more delayed ROS response starting at 90 minutes is primarily within cells that have engulfed bacteria, and is sustained for several hours. The early response has a clear regulatory function, as dampening or intensifying the intracellular ROS level has profound effects on plasmatocyte activation. In addition, ROS are necessary and sufficient to activate JNK signaling in crystal cells, and to promote JNK-dependent crystal cell rupture. These findings indicate that Drosophila will be a promising model in which to dissect the mechanisms of ROS stimulation of immune activation.

To address antigen escape and loss of T-cell functionality, we report a phase-1 clinical trial (NCT04007029) evaluating autologous naive and memory T (TN/MEM) cells engineered to express a bispecific anti-CD19/CD20 CAR (CART19/20) for patients with relapsed/refractory NHL, with safety as the primary end point. Ten patients were treated with 36–165 x 106 CART19/20 cells. No patient experienced neurotoxicity of any grade, or over grade-1 cytokine release syndrome. One case of dose-limiting toxicity (persistent cytopenia) was observed. Nine of ten patients achieved objective response (90% ORR), with seven achieving complete remission (70% CR rate). One patient relapsed after 18 months in CR, but returned to CR after receiving a second dose of CART19/20 cells. Median progression-free survival and overall survival were not reached with a 17-month median follow-up. In conclusion, CART19/20 TN/MEM cells are safe and effective in patients with relapsed/refractory NHL, with durable responses achieved at low dosage levels.

2543 Background: Although chimeric antigen receptor (CAR)-T cells produce impressive outcomes in B-cell malignancies, a substantial fraction of patients with relapsed/refractory B-cell leukemia and lymphoma treated with anti-CD19 CAR-T cell therapy (CART19) either do not respond to treatment or relapse, with poor CAR-T cell persistence or CD19 antigen escape being two key factors that limit durability of response. In order to address these factors, we initiated a clinical trial with naïve/memory T (TN/MEM) cells engineered to express bispecific anti-CD19/CD20 CARs (CART19/20) (NCT04007029). Methods: This trial is a Phase 1, first-in-human, dose-escalation trial enrolling patients with relapsed or refractory follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Following lymphodepletion chemotherapy with fludarabine and cyclophosphamide, patients received CART19/20 cell doses ranging from 50 x 106 to 200 x 106 CAR-positive cells. The primary endpoint was to evaluate the safety of CART19/20 as measured by adverse events and dose limiting toxicities. Secondary endpoints were efficacy as assessed by disease response, progression-free survival (PFS), overall survival (OS), and CAR transgene persistence. Results: As of February 7, 2022, dose-escalation has been completed with 9 patients enrolled and 8 patients infused (3 FL, 4 DLBCL including 2 transformed follicular and 1 primary mediastinal B cell, and 1 MCL). with CART19/20 cells on this study. The median age at the time of CART19/20 infusion was 59 and median prior lines of therapy was 3.5. All patients had stage IV disease and 7 of 9 patients required bridging therapy. Grade-1 cytokine release syndrome (CRS) occurred in 6 of 8 patients, and no patient experienced immune effector cell-associated neurotoxicity syndrome ( ICANS). Among all patients, only one dose of tocilizumab was administered to one subject, and no steroids were given. With a median follow-up of 12 months from time of CART19/20 infusion (range: 4+ to 24+ months), 7 of 8 of patients remain in a complete remission. Median PFS and OS were not reached, and all patients with a complete remission demonstrate ongoing B-cell aplasia. Conclusions: This study demonstrates that CART19/20 cells are safe and effective in patients with relapsed/refractory NHL and potentially obviates the challenges of the commonest causes of relapse after CAR-T cell therapy by means of modifying TN/MEM cells and dual-antigen targeting, respectively. Given the strong safety and response observed, dose escalation was completed with the second dosing level (DL2) of 200 x 106 CAR-positive cells, and DL1 of 50 x 106 CAR-positive cells was chosen as the therapeutic dose for future trial expansion. Clinical trial information: NCT04007029.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which can readily mutate to escape acquired immunity. Other regions in the spike S2 subunit, such as the fusion peptide and the stem helix, are highly conserved across sarbecoviruses and recognized by broadly reactive antibodies, providing hope that targeting these epitopes by vaccination could offer protection against both current and emergent viruses. Here we employed computational modeling to design epitope scaffolds that display the fusion peptide and the stem helix epitopes. The engineered proteins bound both mature and germline versions of multiple broad and protective human antibodies with high affinity. Binding specificity was confirmed both biochemically and via high resolution crystal structures. Finally, the epitope scaffolds showed potent engagement of antibodies and memory B-cells from subjects previously exposed to SARS-CoV2, illustrating their potential to elicit antibodies against the fusion peptide and the stem helix by vaccination.