RESUMEN
Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.
RESUMEN
BACKGROUND: Despite effective antiretroviral therapy, patients with human immunodeficiency virus type-1 (HIV) suffer from a high frequency of malignancies, but related risk factors remain elusive. Here, we focused on blood-circulating viral protein R (Vpr) of HIV, which induces proinflammatory cytokine production and genotoxicity by exogenous functions. METHODS AND FINDINGS: A total 404 blood samples of HIV patients comprising of 126 patients with malignancies (tumor group) and 278 patients without malignancies (non-tumor group), each of 96 samples was first selected by one-to-one propensity score matching. By a detergent-free enzyme-linked immunosorbent assays (detection limit, 3.9 ng/mL), we detected Vpr at a higher frequency in the matched tumor group (56.3%) than in the matched non-tumor group (39.6%) (P = 0.030), although there was no different distribution of Vpr levels (P = 0.372). We also detected anti-Vpr immunoglobulin (IgG), less frequently in the tumor group compared with the tumor group (22.9% for tumor group vs. 44.8% for non-tumor group, P = 0.002), and the proportion of patients positive for Vpr but negative of anti-Vpr IgG was significantly higher in the tumor group than in the non-tumor group (38.6% vs. 15.6%, respectively, P < 0.001). Additionally, Interleukin-6 (IL-6), the levels of which were high in HIV-1 infected patients (P < 0.001) compared to non-HIV-infected individuals, was significantly higher in advanced cases of tumors (P < 0.001), and IL-6 level was correlated with Vpr in the non-tumor group (P = 0.010). Finally, multivariate logistic regression analysis suggested a positive link of Vpr with tumor occurrence in HIV patients (P = 0.002). CONCLUSION: Vpr and IL-6 could be risk factors of HIV-1 associated malignancies, and it would be importance to monitor these molecules for well managing people living with HIV-1.