Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris.

Recombinant protein-based SARS-CoV-2 vaccines are needed to fill the vaccine equity gap. Because protein-subunit based vaccines are easier and cheaper to produce and do not require special storage/transportation conditions, they are suitable for low-/middle-income countries. Here, we report our vaccine development studies with the receptor binding domain of the SARS-CoV-2 Delta Plus strain (RBD-DP) which caused increased hospitalizations compared to other variants. First, we expressed RBD-DP in the Pichia pastoris yeast system and upscaled it to a 5-L fermenter for production. After three-step purification, we obtained RBD-DP with > 95% purity from a protein yield of > 1 g/L of supernatant. Several biophysical and biochemical characterizations were performed to confirm its identity, stability, and functionality. Then, it was formulated in different contents with Alum and CpG for mice immunization. After three doses of immunization, IgG titers from sera reached to > 106 and most importantly it showed high T-cell responses which are required for an effective vaccine to prevent severe COVID-19 disease. A live neutralization test was performed with both the Wuhan strain (B.1.1.7) and Delta strain (B.1.617.2) and it showed high neutralization antibody content for both strains. A challenge study with SARS-CoV-2 infected K18-hACE2 transgenic mice showed good immunoprotective activity with no viruses in the lungs and no lung inflammation for all immunized mice.

Targeting HIF1-alpha/miR-326/ITGA5 axis potentiates chemotherapy response in triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer that is frequently treated with chemotherapy. However, many patients exhibit either de novo chemoresistance or ultimately develop resistance to chemotherapy, leading to significantly high mortality rates. Therefore, increasing the efficacy of chemotherapy has potential to improve patient outcomes. METHODS: Here, we performed whole transcriptome sequencing (both RNA and small RNA-sequencing), coupled with network simulations and patient survival data analyses to build a novel miRNA-mRNA interaction network governing chemoresistance in TNBC. We performed cell proliferation assay, Western blotting, RNAi/miRNA mimic experiments, FN coating, 3D cultures, and ChIP assays to validate the interactions in the network, and their functional roles in chemoresistance. We developed xenograft models to test the therapeutic potential of the identified key miRNA/proteins in potentiating chemoresponse in vivo. We also analyzed several patient datasets to evaluate the clinical relevance of our findings. RESULTS: We identified fibronectin (FN1) as a central chemoresistance driver gene. Overexpressing miR-326 reversed FN1-driven chemoresistance by targeting FN1 receptor, ITGA5. miR-326 was downregulated by increased hypoxia/HIF1A and ECM stiffness in chemoresistant tumors, leading to upregulation of ITGA5 and activation of the downstream FAK/Src signaling pathways. Overexpression of miR-326 or inhibition of ITGA5 overcame FN1-driven chemotherapy resistance in vitro by inhibiting FAK/Src pathway and potentiated the efficacy of chemotherapy in vivo. Importantly, lower expression of miR-326 or higher levels of predicted miR-326 target genes was significantly associated with worse overall survival in chemotherapy-treated TNBC patients. CONCLUSION: FN1 is central in chemoresistance. In chemoresistant tumors, hypoxia and resulting ECM stiffness repress the expression of the tumor suppressor miRNA, miR-326. Hence, re-expression of miR-326 or inhibition of its target ITGA5 reverses FN1-driven chemoresistance making them attractive therapeutic approaches to enhance chemotherapy response in TNBCs.

Polymeric siRNA delivery targeting integrin-ß1 could reduce interactions of leukemic cells with bone marrow microenvironment.

Uncontrolled proliferation of the myeloid cells due to BCR-ABL fusion has been successfully treated with tyrosine kinase inhibitors (TKIs), which improved the survival rate of Chronic Myeloid Leukemia (CML) patients. However, due to interactions of CML cells with bone marrow microenvironment, sub-populations of CML cells could become resistant to TKI treatment. Since integrins are major cell surface molecules involved in such interactions, the potential of silencing integrin-ß1 on CML cell line K562 cells was explored using short interfering RNA (siRNA) delivered through lipid-modified polyethyleneimine (PEI) polymers. Reduction of integrin-ß1 in K562 cells decreased cell adhesion towards human bone marrow stromal cells and to fibronectin, a major extracellular matrix protein for which integrin-ß1 is a primary receptor. Interaction of K562 cells with fibronectin decreased the sensitivity of the cells to BCR-ABL siRNA treatment, but a combinational treatment with integrin-ß1 and BCR-ABL siRNAs significantly reduced colony forming ability of the cells. Moreover, integrin-ß1 silencing enhanced the detachment of K562 cells from hBMSC samples (2 out of 4 samples), which could make them more susceptible to TKIs. Therefore, the polymeric-siRNA delivery targeting integrin-ß1 could be beneficial to reduce interactions with bone marrow microenvironment, aiding in the response of CML cells to therapeutic treatment.