RESUMEN
Subunit vaccines feature critical advantages over other vaccine platforms such as stability, price, and minimal adverse effects. To maximize immunological protection of subunit vaccines, adjuvants are considered as main components that are formulated within the subunit vaccine. They can modulate adverse effects and enhance immune outcomes. However, the most suitable formulation providing the best immunological outcomes and safety are still under investigation. In this report, we combined recombinant RBD with human IgG1 Fc to create an RBD dimer. This fusion protein was expressed in CHO and formulated with alternative adjuvants with different immune activation including Montanide ISA51, Poly (I:C), and MPLA/Quil-A® as potential vaccine candidate formulations. Using the murine model, a potent induction of anti-RBD IgG antibodies in immunized mice sera were observed. IgG subclass analyses (IgG1/IgG2a) illustrated that all adjuvanted formulations could stimulate both Th1 and Th2-type immune responses in particular Poly (I:C) and MPLA/Quil-A®, eliciting greater balance. In addition, Montanide ISA51-formulated RBD-Fc vaccination provided a promising level of neutralizing antibodies against live wild-type SARS-CoV-2 in vitro followed by Poly (I:C) and MPLA/Quil-A®, respectively. Also, mice sera from adjuvanted formulations could strongly inhibit RBD:ACE2 interaction. This study offers immunogenicity profiles, forecasted safety based on Vaccine-associated enhanced disease (VAED) caused by Th1-skewed immunity, and neutralizing antibody analysis of candidates of RBD-Fc-based subunit vaccine formulations to obtain an alternative subunit vaccine formulation against SARS-CoV-2.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Animales , Ratones , COVID-19/prevención & control , Adyuvantes Inmunológicos , Anticuerpos Neutralizantes , Vacunas de Subunidad , Adyuvantes Farmacéuticos , Inmunoglobulina G , Inmunidad , Anticuerpos Antivirales , Glicoproteína de la Espiga del CoronavirusRESUMEN
Protein-based drugs have increasingly become an important segment of cancer treatment. In comparison with chemotherapy, they offer high efficacy and fewer side effects due to specifically targeting only cancer cells. Monoclonal antibodies are currently the main protein-based drugs in the market but their complexity and limitations in tumour penetration led to the development of alternative protein therapeutics such as pore-forming toxins. Colicin N (ColN), a pore-forming protein produced by E. coli, was previously found to exhibit cytotoxicity and selectivity in human lung cancer cells with promising potential for further development. Here we aimed to screen for the cytotoxicity of ColN in breast (MCF-7 and MDA-MB-231), lung (A549) and colon cancer cells (HT-29 and HCT-116) by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay with various concentrations for 72 h and to investigate the cytotoxic effect of ColN domains on cancer cells. It showed that ColN mildly mediated the decrease in cell viability except for MCF-7. The highest effect was seen in A549 and HCT-116 cells which showed 31.9% and 31.5% decrease in cell viability, respectively. The mild inhibition or promotion of cancer cell proliferation by ColN tend to be based on the cell types. Furthermore, to search for the functional domain of ColN used for cytotoxicity, full-length ColN and truncated ColN with deletion of translocating, receptor binding and pore-forming domains were also tested on HCT-116 colon cancer cells. The findings indicated that HCT-116 cells were not significantly sensitive to ColN but full length ColN caused slight decrease in cancer cell viability. The data in this study will benefit the further development of ColN for alternative protein-based cancer therapy.
RESUMEN
Colicin N (ColN) is a bacteriocin secreted by Escherichia coli (E. coli) to kill other Gram-negative bacteria by forcefully generating ion channels in the inner membrane. In addition to its bactericidal activity, ColN have been reported to selectively induce apoptosis in human lung cancer cells via the suppression of integrin modulated survival pathway. However, ColN showed mild toxicity against human lung cancer cells which could be improved for further applications. The protein resurfacing strategy was chosen to engineer ColN by extensive mutagenesis at solvent--exposed residues on ColN. The highly accessible Asp and Glu on wild-type ColN (ColNWT) were replaced by Lys to create polycationic ColN (ColN+12). Previous studies have shown that increase of positive charges on proteins leads to the enhancement of mammalian cell penetration as well as increased interaction with negatively charged surface of cancer cells. Those solvent--exposed residues of ColN were identified by Rosetta and AvNAPSA (Average number of Neighboring Atoms Per Side-chain Atom) approaches. The findings revealed that the structural features and stability of ColN+12 determined by circular dichroism were similar to ColNWT. Furthermore, the toxicity of ColN+12 was cancer -selective. Human lung cancer cells, H460 and H23, were sensitive to ColN but human dermal papilla cells were not. ColN+12 also showed more potent toxicity than ColNWT in cancer cells. This confirmed that polycationic resurfacing method has enabled us to improve the anticancer activity of ColN towards human lung cancer cells.
RESUMEN
The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5-15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10-15 µM) for 12 h. Moreover, 5-15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin ß1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.