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.

The Effect of Inhibition of Perisynaptic Astrocyte Glycogen Utilization on Depression-Like Behavior. / Sinaptik Enerji Kaynagi Glikojenin Kullaniminin Engellenmesinin Depresyon-benzeri Davranislara Etkisi.

OBJECTIVE: Under physiological conditions, astrocytes produce lactate to meet the increased synaptic energy demand due to neuronal activity. In the light of the findings showing that this process is disrupted in the pathophysiology of major depression, the aim of this study is to investigate the effect of pharmacological inhibition of perisynaptic astrocyte glycogen utilization on anxiety-like behavior and depression-like behavior in female and male mice. METHODS: In this study, DAB (1,4-dideoxy-1,4-imino-D-arabinitol), which is an inhibitor of glycogen breaking enzyme glycogen phosphorylase, was intrahippocampally administered to 15 female and 14 male Swiss albino mice, while 15 female and 12 male Swiss albino mice received intrahippocampal saline injections. Three and five days after the injections, the anxiety-like and depression-like behaviors of the mice were assessed by locomotor activity, open-field test, light-dark box test, tail suspension test and sucrose preference test. RESULTS: Three days after injection, neither depression-like nor anxietylike significant behavioral changes were detected in the male experimental group mice compared to the control group; but an increase in locomotor activity (p=0.05) and time spent in the open-field (p=0.01) were observed on the fifth day. In evaluations of the female experimental group mice on the third and fifth days, depression-like and anxiety-like behaviors were found similar to the control group, as seen in the male mice. The only significant difference in the experimental group female mice was found in the sucrose preference test, which revealed an increased tendency to prefer sucrose (p=0.003) compared to the control group. CONCLUSION: The inhibition of glycogen use in the hippocampus by DAB did not affect anxiety-like and depression-like behaviors 3 and 5 days after injection in both female and male mice. The increase in the time spent in the open-field by male experimental group mice was associated not with anxiety, but with increase in the locomotor activity. The fact that no significant difference was observed in the light-dark box test, which is another test used to evaluate anxiety, supported this opinion. The increase seen in the sucrose preference test in female experimental group mice was not interpreted as an increase in hedonic behavior because prevention of glycogen breakdown in the hypothalamus might have homeostatically increased sugar-craving and therefore resulted in an increase in sucrose preference. Different set of tests better targeting the energy and glucose metabolism and applied at farther time points than surgery are recommended for future studies.

Macrophage and dendritic cell subset composition can distinguish endotypes in adjuvant-induced asthma mouse models.

Asthma is a heterogeneous disease with neutrophilic and eosinophilic asthma as the main endotypes that are distinguished according to the cells recruited to the airways and the related pathology. Eosinophilic asthma is the treatment-responsive endotype, which is mainly associated with allergic asthma. Neutrophilic asthma is a treatment-resistant endotype, affecting 5-10% of asthmatics. Although eosinophilic asthma is well-studied, a clear understanding of the endotypes is essential to devise effective diagnosis and treatment approaches for neutrophilic asthma. To this end, we directly compared adjuvant-induced mouse models of neutrophilic (CFA/OVA) and eosinophilic (Alum/OVA) asthma side-by-side. The immune response in the inflamed lung was analyzed by multi-parametric flow cytometry and immunofluorescence. We found that eosinophilic asthma was characterized by a preferential recruitment of interstitial macrophages and myeloid dendritic cells, whereas in neutrophilic asthma plasmacytoid dendritic cells, exudate macrophages, and GL7+ activated B cells predominated. This differential distribution of macrophage and dendritic cell subsets reveals important aspects of the pathophysiology of asthma and holds the promise to be used as biomarkers to diagnose asthma endotypes.

The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation.

The clear and unequivocal identification of immune effector functions is essential to understand immune responses. The cytokine IL-10 is a critical immune regulator and was shown, for example, to limit pathology during various lung diseases. However, the clear identification of IL-10-producing cells is challenging and, therefore, reporter mouse lines were developed to facilitate their detection. Several such reporter lines utilize GFP, including the IL-10GFP (VeRT-X) reporter strain studied here. In line with previous reports, we found that this IL-10GFP line faithfully reports on the IL-10 production of lymphoid cells. However, we show that the IL-10GFP reporter is not suitable to analyse IL-10 production of myeloid cells during inflammation. During inflammation, the autofluorescence of myeloid cells increased to an extent that entirely masked the IL-10-specific GFP-signal. Our data illustrate a general and important technical caveat using GFP-reporter lines for the analysis of myeloid cells and suggest that previous reports on effector functions of myeloid cells using such GFP-based reporters might require re-evaluation.

Improved Detection of Cytokines Produced by Invariant NKT Cells.

Invariant Natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after in vivo activation, allowing for the direct detection of a number of cytokines directly ex vivo. However, for some cytokines this approach is suboptimal. Here, we report technical variations that allow the improved detection of IL-4, IL-10, IL-13 and IL-17A ex vivo. Furthermore, we describe an alternative approach for stimulation of iNKT cells in vitro that allows a significantly improved detection of cytokines produced by iNKT cells. Together, these protocols allow the detection of iNKT cell cytokines ex vivo and in vitro with increased sensitivity.

Label-free Raman imaging of the macrophage response to the malaria pigment hemozoin.

Hemozoin, the 'malaria pigment', is engulfed by phagocytic cells, such as macrophages, during malaria infection. This biocrystalline substance is difficult to degrade and often accumulates in phagocytes. The macrophage response to hemozoin relates to the severity of the disease and the potential for malaria-related disease complications. In this study we have used Raman spectroscopy as a label-free method to investigate the biochemical changes occurring in macrophages during the first few hours of hemozoin uptake. We found a number of distinct spectral groups, spectrally or spatially related to the presence of the hemozoin inside the cell. Intracellular hemozoin was spectrally identical to extracellular hemozoin, regardless of the location in the cell. A small proportion of hemozoin was found to be associated with lipid-based components, consistent with the uptake of hemozoin into vesicles such as phagosomes and lysosomes. The spatial distribution of the hemozoin was observed to be inhomogeneous, and its presence largely excluded that of proteins and lipids, demonstrating that cells were not able to break down the biocrystals on the time scales studied here. These results show that Raman imaging can be used to answer some of the open questions regarding the role of hemozoin in the immune response. How different combinations of hemozoin and other molecules are treated by macrophages, whether hemozoin can be broken down by the cell, and more importantly, which co-factors or products are involved in the subsequent cell reaction are the expected issues to be elucidated by this technique.

Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria.

Cerebral malaria is a complication of Plasmodium falciparum infection characterized by sudden coma, death, or neurodisability. Studies using a mouse model of experimental cerebral malaria (ECM) have indicated that blood-brain barrier disruption and CD8 T cell recruitment contribute to disease, but the spatiotemporal mechanisms are poorly understood. We show by ultra-high-field MRI and multiphoton microscopy that the olfactory bulb is physically and functionally damaged (loss of smell) by Plasmodium parasites during ECM. The trabecular small capillaries comprising the olfactory bulb show parasite accumulation and cell occlusion followed by microbleeding, events associated with high fever and cytokine storm. Specifically, the olfactory upregulates chemokine CCL21, and loss or functional blockade of its receptors CCR7 and CXCR3 results in decreased CD8 T cell activation and recruitment, respectively, as well as prolonged survival. Thus, early detection of olfaction loss and blockade of pathological cell recruitment may offer potential therapeutic strategies for ECM.

Zanthoxylum usambarense (Engl.) Kokwaro (Rutaceae) extracts inhibit the growth of the breast cancer cell lines MDA-MB-231 and MCF-7, but not the brain tumour cell line U251 in vitro.

Zanthoxylum usambarense (Engl.) Kokwaro has traditionally been used for the treatment of malaria, upper respiratory tract infections, cough, rheumatism, tooth decay and sore gums in Kenya and other African countries. Dried ground parts of Z. usambarense were extracted by maceration using methanol (MeOH) at room temperature, extract was dried and reconstituted in 70% aq. MeOH and partitioned against n-hexane and chloroform (CHCl3 ) to obtain MeOH, n-hexane and CHCl3 extracts. All extracts were assessed for cytotoxicity against two breast cancer cell lines, MDA-MB-231 and MCF-7, and the brain tumour cell line U251 by the MTT assay. The free-radical scavenging activity of the extracts was also determined by the 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. In the DPPH assay, the MeOH extract was found to be the most active free-radical scavenger with a RC50 value of 41.1 × 10(-3) mg/mL. It also displayed significant cytotoxicity against the MCF-7 cell line (IC50 42.9 µg/mL) and appeared to have induced cell death through apoptosis. None of the test extracts showed any activity against the U251 cell line at test concentrations. The present findings demonstrated that Z. usambarense could be a potential source for new cytotoxic compounds for possible anticancer drug development.