Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection.

BACKGROUND: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Allogeneic bone marrow mesenchymal stem cell-derived exosomes alleviate human hypoxic AKI-on-a-Chip within a tight treatment window.

BACKGROUND: Acute hypoxic proximal tubule (PT) injury and subsequent maladaptive repair present high mortality and increased risk of acute kidney injury (AKI) - chronic kidney disease (CKD) transition. Human bone marrow mesenchymal stem cell-derived exosomes (hBMMSC-Exos) as potential cell therapeutics can be translated into clinics if drawbacks on safety and efficacy are clarified. Here, we determined the real-time effective dose and treatment window of allogeneic hBMMSC-Exos, evaluated their performance on the structural and functional integrity of 3D microfluidic acute hypoxic PT injury platform. METHODS: hBMMSC-Exos were isolated and characterized. Real-time impedance-based cell proliferation analysis (RTCA) determined the effective dose and treatment window for acute hypoxic PT injury. A 2-lane 3D gravity-driven microfluidic platform was set to mimic PT in vitro. ZO-1, acetylated α-tubulin immunolabelling, and permeability index assessed structural; cell proliferation by WST-1 measured functional integrity of PT. RESULTS: hBMMSC-Exos induced PT proliferation with ED50 of 172,582 µg/ml at the 26th hour. Hypoxia significantly decreased ZO-1, increased permeability index, and decreased cell proliferation rate on 24-48 h in the microfluidic platform. hBMMSC-Exos reinforced polarity by a 1.72-fold increase in ZO-1, restored permeability by 20/45-fold against 20/155 kDa dextran and increased epithelial proliferation 3-fold compared to control. CONCLUSIONS: The real-time potency assay and 3D gravity-driven microfluidic acute hypoxic PT injury platform precisely demonstrated the therapeutic performance window of allogeneic hBMMSC-Exos on ischemic AKI based on structural and functional cellular data. The novel standardized, non-invasive two-step system validates the cell-based personalized theragnostic tool in a real-time physiological microenvironment prior to safe and efficient clinical usage in nephrology.

Comparison of total anastomosis time between four different combinations of suturing and knot tying techniques in microsurgical anastomosis.

BACKGROUND: Various techniques have been described for performing microsurgical anastomosis with providing high patency rates. Although the total anastomotic time may not be an issue when dealing with a single set of anastomoses, using a faster technique may save significant amount of time in cases of transferring flaps with shorter critical ischemia time or where multiple anastomoses are required. This study compares the total anastomosis time between four different combinations of commonly used suturing and knot tying techniques. METHODS: Twenty-four rats were divided into 4 groups. Simple interrupted suture with conventional knot tying technique (SIS-CT) was used in group I, continuous suture technique with conventional knot tying (CST) was used in group II, simple interrupted suture with airborne knot tying technique(SIS-AT) was used in group III, and continuous-interrupted suture with airborne knot tying technique(CIS-AT) was used in group IV for microsurgical anastomosis. Total anastomosis time and patency rates with each technique and samples from anastomotic sites were analyzed. RESULTS: The mean time required for microvascular anastomosis of the femoral artery was 1075 s in group I, 799 s in group II, 844 s in group III, and 973 s in group IV. The difference between four groups was statistically significant. The anastomoses in group II and group III were completed in the shortest period of time. Intergroup comparison revealed that the difference between group II and group III was not statistically significant, however, total anastomosis time for completion of the anastomosis was significantly longer for group I, followed by group IV. Thrombosis rates and histological analysis revealed no significant differences among four groups. CONCLUSION: CST and SIS-AT techniques can significantly reduce microsurgical anastomosis time and provide high patency rates. Also, the time needed to complete an anastomosis was significantly shorter for CIS-AT when compared to SIS-CT.