Multi-drug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. Due to its environmental persistence, virulence, and limited treatment options, this organism causes both increased patient mortality and incurred healthcare costs. Thus, prophylactic vaccination could be ideal for intervention against MDR Acinetobacter infection in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B- and T-cell epitopes from proteins associated with A. baumannii pathogenesis. A novel Acinetobacter Multi-Epitope Vaccine (AMEV2) was constructed using an A. baumannii thioredoxin A (TrxA) leading protein sequence followed by five identified peptide antigens. Antisera from A. baumannii infected mice demonstrated reactivity to rAMEV2, and subcutaneous immunization of mice with rAMEV2 produced high antibody titer against the construct as well as peptide components. Immunization results in increased frequency of IL-4-secreting splenocytes indicative of a Th2 response. AMEV2-immunized mice were protected against intranasal challenge with a hypervirulent strain of A. baumannii and demonstrated reduced bacterial burden at 48 h. In contrast, all mock vaccinated mice succumbed to infection within 3 days. Results presented here provide insight into the effectiveness of immunoinformatic-based vaccine design and its potential as an effective strategy to combat the rise of MDR pathogens.

Background/Objectives: Acinetobacter baumannii is an opportunistic nosocomial pathogen characterized by its multidrug-resistant (MDR) phenotype, increasing patient mortality and healthcare costs as a result. Previously, we constructed an immunoinformatics-based Acinetobacter Multi-Epitope Vaccine (AMEV2) candidate and demonstrated robust protection against this MDR pathogen. In this study, we delineate the mechanisms of AMEV2-mediated protective immunity. Methods: In vivo passive immunization with AMEV2 antisera and in vitro opsonophagocytic killing assays (OPKAs) were used to assess the critical role of antibody-mediated protection induced by AMEV2 vaccination. Results: The passive transfer of AMEV2 immune sera to naïve mice afforded 67% protection in a pulmonary challenge mouse model. Although AMEV2 sera reacts with bacterial antigens, it is not bactericidal on its own and does not enhance the complement-mediated direct killing of A. baumannii. However, OPKAs demonstrate AMEV2 sera enhancement of the killing of A. baumannii in the presence of primary bone marrow-derived macrophages. This killing occurs via complement and Fc gamma receptor-mediated phagocytosis. A highly immunogenic AMEV2 component peptide, pTonB, elicits pTonB-specific antibodies and protection in vivo. The depletion of pTonB antibodies from AMEV2 immune sera by pTonB absorption significantly reduced the opsonophagocytic killing of A. baumannii in vitro. Conclusions: The data presented here demonstrate the importance of humoral immunity and its protective mechanisms against A. baumannii. These findings further expand the in vivo evaluation of in silico-designed vaccines as a viable alternative to combat the current global MDR pathogen health crisis.

Platelet activation is a hallmark of antiphospholipid syndrome (APS). However, the mechanisms through which antiphospholipid antibodies activate platelets and the optimal way to inhibit this pathophysiology are still not fully understood. Extracellular adenosine, produced by the ectoenzyme CD73, activates cell-surface receptors to increase intracellular cyclic adenosine monophosphate (cAMP), raising the threshold for platelet activation. Here, we found that platelets from patients with APS exhibited lower CD73 activity and cAMP content than healthy controls. Platelet activation, as measured by the surface expression of CD62P and activated αIIbβ3, was negatively correlated with CD73 activity and cAMP content. Exposing healthy platelets to APS patient immunoglobulin G (IgG) in vitro significantly reduced CD73 activity and intracellular cAMP while simultaneously increasing activation markers. We identified critical roles for FcγRIIa, phospholipase C, and the Akt signaling pathway in platelet activation by APS IgG. We also tested whether various agents that boost intracellular cAMP could blunt APS IgG–induced platelet activation. Agonism of the adenosine 2A receptor (A2AR) and inhibition of phosphodiesterase 3 (PDE3) were both highly effective in this regard. In summary, a dysregulated adenosinergic axis appears to potentiate APS platelets for prothrombotic activation by antiphospholipid antibodies. A2AR agonists and PDE3 inhibitors may be useful strategies for restoring platelet homeostasis in APS.