Metabolic recycling of storage lipids promotes squalene biosynthesis in yeast.

BACKGROUND: Metabolic rewiring in microbes is an economical and sustainable strategy for synthesizing valuable natural terpenes. Terpenes are the largest class of nature-derived specialized metabolites, and many have valuable pharmaceutical or biological activity. Squalene, a medicinal terpene, is used as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines, and plays diverse biological roles as an antioxidant and anticancer agent. However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds including squalene, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products. RESULTS: In this study, focusing on the recycling of surplus metabolic energy stored in lipid droplets, we show that the metabolic recycling of the surplus energy to acetyl-CoA can increase squalene production in yeast, concomitant with minimizing the metabolic interferences in inherent pathways. Moreover, by integrating multiple copies of the rate-limiting enzyme and implementing N-degron-dependent protein degradation to downregulate the competing pathway, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L. CONCLUSIONS: Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation. This approach will also provide a universal strategy for the more stable and sustainable production of high-value terpenes.

In Silico Phytochemical Compounds Screening of Allium sativum Targeting the Mpro of SARS-CoV-2

The global pandemic of coronavirus disease is still widely spread across the world causing catastrophic effect in both human life and global economy. By the end of year 2021, it has caused a total of 5.437.636 deaths across the world. Indonesia has rich plant biodiversity including medicinal plants that may be used for combating the virus. One of the commonly used medicinal plants comes from Allium species and it has been proved to have antiviral activity. Conducting an in silico study, we screened bioactive compounds that came from Allium sativum to fight against coronavirus through the inhibition of 3CL-Pro, one of the major protease that have an active role for viral replication. Molecular docking of compounds from Allium sativum to 3CL-Pro resulting in the discovery of 5 compounds that have the best binding affinity to 3CL-Pro, which are squalene, 1,4-dihydro-2,3-benzoxathiin 3-oxide, 1,2,3-propanetriyl ester, trans-13-octadecenoic acid and methyl-11-hexadecenoate with binding affinity of -7, -6.5, -5.9, -5.7 and -5.6 kcal/mol, respectively. It is very likely that these compounds can be candidates for therapeutic agents and these candidates need to be studied further.

From Sharks to Yeasts: Squalene in the Development of Vaccine Adjuvants.

Squalene is a natural linear triterpene that can be found in high amounts in certain fish liver oils, especially from deep-sea sharks, and to a lesser extent in a wide variety of vegeTable oils. It is currently used for numerous vaccine and drug delivery emulsions due to its stability-enhancing properties and biocompatibility. Squalene-based vaccine adjuvants, such as MF59 (Novartis), AS03 (GlaxoSmithKline Biologicals), or AF03 (Sanofi) are included in seasonal vaccines against influenza viruses and are presently being considered for inclusion in several vaccines against SARS-CoV-2 and future pandemic threats. However, harvesting sharks for this purpose raises serious ecological concerns that the exceptional demand of the pandemic has exacerbated. In this line, the use of plants to obtain phytosqualene has been seen as a more sustainable alternative, yet the lower yields and the need for huge investments in infrastructures and equipment makes this solution economically ineffective. More recently, the enormous advances in the field of synthetic biology provided innovative approaches to make squalene production more sustainable, flexible, and cheaper by using genetically modified microbes to produce pharmaceutical-grade squalene. Here, we review the biological mechanisms by which squalene-based vaccine adjuvants boost the immune response, and further compare the existing sources of squalene and their environmental impact. We propose that genetically engineered microbes are a sustainable alternative to produce squalene at industrial scale, which are likely to become the sole source of pharmaceutical-grade squalene in the foreseeable future.

Hyaluronic Acid-Glycine-Cholesterol Conjugate-Based Nanoemulsion as a Potent Vaccine Adjuvant for T Cell-Mediated Immunity.

Clinical cases of allergic reaction that are due to excipients containing polyethylene glycol (PEG), a hydrophilic molecule commonly used in drug/vaccine formulations, has attracted much attention in recent years. In order to develop PEG-free adjuvants, we investigated the feasibility of natural ingredients in the human body such as hyaluronic acid in the form of hyaluronic acid-glycine cholesterol (HACH) conjugate as an excipient for vaccine formulation. Interestingly, HACH grafted with ~13 wt.% cholesterol has good water dispersity and can serve as an emulsifier to stabilize the squalene/water interfaces, yielding a milky white and isotropic emulsion (SQ@HACH) after being passed through a high-shear microfluidizer. Our results show that SQ@HACH particles possessed a unimodal average hydrodynamic diameter of approximately 190 nm measured by dynamic light scattering and exhibited good stability upon storage at 4 °C and 37 °C for over 20 weeks. The results of immunogenicity using a mouse model with ovalbumin (OVA) as the antigen revealed that SQ@HACH significantly enhanced antigen-specific immune responses, including the polarization of IgG antibodies, the cytokine secretions of T cells, and enhancement of cytotoxic T lymphocyte (CTL) activation. Moreover, SQ@HACH revealed lower local inflammation and rapidly absorbing properties compared with AlPO4 after intramuscular injection in vivo, indicating the potential functions of the HA-derived conjugate as an excipient in vaccine formulations for enhancement of T cell-mediated immunity.

Assessment of adjuvantation strategy of lipid squalene nanoparticles for enhancing the immunogenicity of a SARS-CoV-2 spike subunit protein against COVID-19.

Vaccination is regarded as the most effective intervention for controlling the coronavirus disease 2019 (COVID-19) pandemic. The objective of this study is to provide comprehensive information on lipid squalene nanoparticle (SQ@NP)-adjuvanted COVID-19 vaccines regarding modulating immune response and enhancing vaccine efficacy. After being adjuvanted with SQ@NP, the SARS-CoV-2 spike (S) subunit protein was intramuscularly (i.m.) administered to mice. Serum samples investigated by ELISA and virus neutralizing assay showed that a single-dose SQ@NP-adjuvanted S-protein vaccine can induce antigen-specific IgG and protective antibodies comparable with those induced by two doses of nonadjuvanted protein vaccine. When the mice received a boosting vaccine injection, anamnestic response was observed in the groups of adjuvanted vaccine. Furthermore, the secretion of cytokines in splenocytes, such as interferon (IFN)-γ, interleukin (IL)-5 and IL-10, was significantly enhanced after adjuvantation of S-protein vaccine with SQ@NP; however, this was not the case for the vaccine adjuvanted with conventional aluminum mineral salts. Histological examination of injection sites showed that the SQ@NP-adjuvanted vaccine was considerably well tolerated following i.m. injection in mice. These results pave the way for the performance tuning of optimal vaccine formulations against COVID-19.

Evaluating the efficacy of extracted squalene from seed oil in the form of microemulsion for the treatment of COVID-19: A clinical study.

This study investigates the effect of the nanostructure of squalene in the form of microemulsion on COVID-19 patients. In this blinded clinical trial, a comparison was made between the efficacy of squalene treatment and controls. A total of 30 COVID-19 patients admitted to the emergency department, and the infection ward was equally allocated to case (n = 15) and control (n = 15) groups according to their age and underlying diseases. The baseline characteristics of subjects, including age, gender, time of treatment onset, underlying condition, white blood cells count, and lymphocyte count were similar (p < 0.05). Baseline laboratory tests and computed tomography (CT) scans were performed for the study groups. The treatment group received 5 mg of intravenous squalene twice a day and standard treatment for 6 days, while controls received only standard treatment. After 6 days of treatment, clinical and CT scan changes were evaluated and compared in intervention and control groups. The need for oxygen therapy (p = 0.020), 2 days of no fever (p = 0.025), cough alleviation (p = 0.010), and lung high-resolution computed tomography improvement (p = 0.033) were significantly different between cases and controls within 7 days of admission. No adverse effects were observed in the treatment group. Our data suggest that squalene could be considered as a potential treatment for COVID-19, and further studies are required to confirm the results.

(Poly-cyanoacrylate) nanomedicines for cancer and beyond: Lessons learned.

This « Magnum Opus ¼ emphasizes that serendipity is a corner stone in research. The paths of discovery and innovation often result from the interdisciplinarity of scientific areas that are a priori disconnected from each other. In the 1970s, fundamental discoveries in cell biology led to unexpected advances in galenic pharmacy with the emergence of nanotechnologies for the intracellular delivery of non diffusing molecules. As well, fluorescein-loaded polyacrylamide nanocapsules were shown to deliver this fluorescent agent precisely into cellular lysosomes which represented a seminal observation. However, due to the lack of biodegradability of this carrier polymer, this approach was still far from therapeutic application. The use of cyanoacrylates as surgical glue inspired us to use this material in the design of the first biodegradable nanoparticles for human use. Capable of transporting compounds with anti-tumor activity, these polyalkylcyanoacrylate nanoparticles demonstrated the unexpected property of overcoming multi-drug resistance. This discovery led to the development of a nanomedicine that has completed phase III clinical trials for the treatment of resistant hepatocarcinoma. Going beyond the state-of-the art, a step ahead in the nanomedicine field was the drug « squalenoylation ¼ technology, which represents a shift from the « physical ¼ to the « chemical ¼ encapsulation paradigm. The bioconjugation of anticancer and other drugs to squalene, a natural and biocompatible lipid, enabled a dramatic increase in drug payload, and eliminated the so-called « burst release ¼ of drug: Two major drawbacks commonly associated with drug nanoencapsulation. The drug « squalenoylation ¼ approach resulted in a generic nanomedicine platform with broad pharmacological applications.

Adjuvanted SARS-CoV-2 spike protein elicits neutralizing antibodies and CD4 T cell responses after a single immunization in mice.

BACKGROUND: SARS-CoV-2 has caused a global pandemic, infecting millions of people. A safe, effective vaccine is urgently needed and remains a global health priority. Subunit vaccines are used successfully against other viruses when administered in the presence of an effective adjuvant. METHODS: We evaluated three different clinically tested adjuvant systems in combination with the SARS-CoV-2 pre-fusion stabilized (S-2P) spike protein using a one-dose regimen in mice. FINDINGS: Whilst spike protein alone was only weakly immunogenic, the addition of either Aluminum hydroxide, a squalene based oil-in-water emulsion system (SE) or a cationic liposome-based adjuvant significantly enhanced antibody responses against the spike receptor binding domain (RBD). Kinetics of antibody responses differed, with SE providing the most rapid response. Neutralizing antibodies developed after a single immunization in all adjuvanted groups with ID50 titers ranging from 86-4063. Spike-specific CD4 T helper responses were also elicited, comprising mainly of IFN-γ and IL-17 producing cells in the cationic liposome adjuvanted group, and more IL-5- and IL-10-secreting cells in the AH group. INTERPRETATION: These results demonstrate that adjuvanted spike protein subunit vaccine is a viable strategy for rapidly eliciting SARS-CoV-2 neutralizing antibodies and CD4 T cell responses of various qualities depending on the adjuvant used, which can be explored in further vaccine development against COVID-19. FUNDING: This work was supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653.