Recombinant lipidated FLIPr effectively enhances mucosal and systemic immune responses for various vaccine types.

Formyl peptide receptor-like 1 inhibitor protein (FLIPr) is an immune evasion protein produced by Staphylococcus aureus, and FLIPr is a potential vaccine candidate for reducing Staphylococcus aureus virulence and biofilm formation. We produced recombinant lipidated FLIPr (rLF) to increase the immunogenicity of FLIPr and showed that rLF alone elicited potent anti-FLIPr antibody responses to overcome the FLIPr-mediated inhibition of phagocytosis. In addition, rLF has potent immunostimulatory properties. We demonstrated that rLF is an effective adjuvant. When an antigen is formulated with rLF, it can induce long-lasting antigen-specific immune responses and enhance mucosal and systemic antibody responses as well as broad-spectrum T-cell responses in mice. These findings support further exploration of rLF in the clinic as an adjuvant for various vaccine types with extra benefits to abolish FLIPr-mediated immunosuppressive effects.

Induction of high affinity monoclonal antibodies against SARS-CoV-2 variant infection using a DNA prime-protein boost strategy.

BACKGROUND: Calls for the coronavirus to be treated as an endemic illness, such as the flu, are increasing. After achieving high coverage of COVID-19 vaccination, therapeutic drugs have become important for future SARS-CoV-2 variant outbreaks. Although many monoclonal antibodies have been approved for emergency use as treatments for SARS-CoV-2 infection, some monoclonal antibodies are not authorized for variant treatment. Broad-spectrum monoclonal antibodies are unmet medical needs. METHODS: We used a DNA prime-protein boost approach to generate high-quality monoclonal antibodies. A standard ELISA was employed for the primary screen, and spike protein-human angiotensin-converting enzyme 2 blocking assays were used for the secondary screen. The top 5 blocking clones were selected for further characterization, including binding ability, neutralization potency, and epitope mapping. The therapeutic effects of the best monoclonal antibody against SARS-CoV-2 infection were evaluated in a hamster infection model. RESULTS: Several monoclonal antibodies were selected that neutralize different SARS-CoV-2 variants of concern (VOCs). These VOCs include Alpha, Beta, Gamma, Delta, Kappa and Lambda variants. The high neutralizing antibody titers against the Beta variant would be important to treat Beta-like variants. Among these monoclonal antibodies, mAb-S5 displays the best potency in terms of binding affinity and neutralizing capacity. Importantly, mAb-S5 protects animals from SARS-CoV-2 challenge, including the Wuhan strain, D614G, Alpha and Delta variants, although mAb-S5 exhibits decreased neutralization potency against the Delta variant. Furthermore, the identified neutralizing epitopes of monoclonal antibodies are all located in the receptor-binding domain (RBD) of the spike protein but in different regions. CONCLUSIONS: Our approach generates high-potency monoclonal antibodies against a broad spectrum of VOCs. Multiple monoclonal antibody combinations may be the best strategy to treat future SARS-CoV-2 variant outbreaks.

Substrate uptake and subcellular compartmentation of anoxic cholesterol catabolism in Sterolibacterium denitrificans.

Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. (13)C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a ß-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C27 sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C27 acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.

An oxygenase-independent cholesterol catabolic pathway operates under oxic conditions.

Cholesterol is one of the most ubiquitous compounds in nature. The 9,10-seco-pathway for the aerobic degradation of cholesterol was established thirty years ago. This pathway is characterized by the extensive use of oxygen and oxygenases for substrate activation and ring fission. The classical pathway was the only catabolic pathway adopted by all studies on cholesterol-degrading bacteria. Sterolibacterium denitrificans can degrade cholesterol regardless of the presence of oxygen. Here, we aerobically grew the model organism with (13)C-labeled cholesterol, and substrate consumption and intermediate production were monitored over time. Based on the detected (13)C-labeled intermediates, this study proposes an alternative cholesterol catabolic pathway. This alternative pathway differs from the classical 9,10-seco-pathway in numerous important aspects. First, substrate activation proceeds through anaerobic C-25 hydroxylation and subsequent isomerization to form 26-hydroxycholest-4-en-3-one. Second, after the side chain degradation, the resulting androgen intermediate is activated by adding water to the C-1/C-2 double bond. Third, the cleavage of the core ring structure starts at the A-ring via a hydrolytic mechanism. The (18)O-incorporation experiments confirmed that water is the sole oxygen donor in this catabolic pathway.