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 Ca2+ concentration impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages in vitro.

Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca2+ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional αß T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, γδ T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca2+ concentrations. This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. A similarly increased cytokine response due to elevated Ca2+ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca2+ concentrations. Furthermore, elevated Ca2+ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca2+ concentration. In summary, our data demonstrate that the Ca2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca2+ levels can boost cytokine production by both mouse and human lymphoid cells.

Effect of stimulation time on the expression of human macrophage polarization markers.

Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1ß, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGFß) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.

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.

The Role of TRAIL/DRs in the Modulation of Immune Cells and Responses.

Expression of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by immune cells can lead to the induction of apoptosis in tumor cells. However, it becomes increasingly clear that the interaction of TRAIL and its death receptors (DRs) can also directly impact immune cells and influence immune responses. Here, we review what is known about the role of TRAIL/DRs in immune cells and immune responses in general and in the tumor microenvironment in particular.

The interaction between invariant Natural Killer T cells and the mucosal microbiota.

The surface of mammalian bodies is colonized by a multitude of microbial organisms, which under normal conditions support the host and are considered beneficial commensals. This requires, however, that the composition of the commensal microbiota is tightly controlled and regulated. The host immune system plays an important role in the maintenance of this microbiota composition. Here we focus on the contribution of one particular immune cell type, invariant Natural Killer T (iNKT) cells, in this process. The iNKT cells are a unique subset of T cells characterized by two main features. First, they express an invariant T-cell receptor that recognizes glycolipid antigens presented by CD1d, a non-polymorphic major histocompatibility complex class I-like molecule. Second, iNKT cells develop as effector/memory cells and swiftly exert effector functions, like cytokine production and cytotoxicity, after activation. We outline the influence that the mucosal microbiota can have on iNKT cells, and how iNKT cells contribute to the maintenance of the microbiota composition.

Development of Asthma in Inner-City Children: Possible Roles of MAIT Cells and Variation in the Home Environment.

Humans have populations of innate-like T lymphocytes with an invariant TCR α-chain that recognize nonpeptide Ags, including invariant NKT (iNKT) cells and mucosal-associated invariant T (MAIT) cells. iNKT cell involvement in human asthma is controversial, whereas there has been little analysis of MAIT cells. Using peripheral blood cells from 110 participants from the Urban Environment and Childhood Asthma (URECA) birth cohort study, these cells were analyzed for number and function. We determined whether iNKT cell or MAIT cell frequency at 1 y is correlated with the cytokine polarization of mainstream CD4+ T cells and/or the development of asthma by age 7 y. Dust samples from 300 houses were tested for iNKT cell antigenic activity. Our results show that a higher MAIT cell frequency at 1 y of age was associated with a decreased risk of asthma by age 7 y. The frequency of MAIT cells was associated with increased production of IFN-γ by activated CD4+ T cells from the URECA cohort. iNKT cell antigenic activity in bedroom dust samples was associated with higher endotoxin concentration and also with reduced risk of asthma. In conclusion, MAIT cell frequency at 1 y may reflect the tendency of the immune system toward Th1 responses and is associated with protection from asthma. Additionally, iNKT cell antigenic activity may be a marker of houses with increased microbial exposures and therefore also with protection from asthma.

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.

From the Deep Sea to Everywhere: Environmental Antigens for iNKT Cells.

Invariant natural killer T (iNKT) cells are a unique subset of innate T cells that share features with innate NK cells and adaptive memory T cells. The first iNKT cell antigen described was found 1993 in a marine sponge and it took over 10 years for other, bacterial antigens to be described. Given the paucity of known bacterial iNKT cell antigens, it appeared as if iNKT cells play a very specialist role in the protection against few, rare and unusual pathogenic bacteria. However, in the last few years several publications painted a very different picture, suggesting that antigens for iNKT cells are found almost ubiquitous in the environment. These environmental iNKT cell antigens can shape the distribution, phenotype and function of iNKT cells. Here, these recent findings will be reviewed and their implications for the field will be outlined.

Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness.

A Novel Glycolipid Antigen for NKT Cells That Preferentially Induces IFN-γ Production.

In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response. This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), with the only change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared with αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10-producing iNKT cells, which could counteract the benefits of increased early IFN-γ production.

IL-10-producing NKT10 cells are a distinct regulatory invariant NKT cell subset.

Invariant natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after activation, thereby impacting a wide variety of different immune reactions. However, strong activation of iNKT cells with α-galactosylceramide (αGalCer) reportedly induces a hyporeactive state that resembles anergy. In contrast, we determined here that iNKT cells from mice pretreated with αGalCer retain cytotoxic activity and maintain the ability to respond to TCR-dependent as well as TCR-independent cytokine-mediated stimulation. Additionally, αGalCer-pretreated iNKT cells acquired characteristics of regulatory cells, including production and secretion of the immunomodulatory cytokine IL-10. Through the production of IL-10, αGalCer-pretreated iNKT cells impaired antitumor responses and reduced disease in experimental autoimmune encephalomyelitis, a mouse model of autoimmune disease. Furthermore, a subset of iNKT cells with a similar inhibitory phenotype and function were present in mice not exposed to αGalCer and were enriched in mouse adipose tissue and detectable in human PBMCs. These data demonstrate that IL-10-producing iNKT cells with regulatory potential (NKT10 cells) represent a distinct iNKT cell subset.

Invariant natural killer T cells are depleted in renal impairment and recover after kidney transplantation.

BACKGROUND: Altered immune function in patients with renal failure results in both susceptibility to infection and increased inflammatory response. Invariant natural killer T (iNKT) cells are a conserved, immunoregulatory T lymphocyte subset that responds to lipid antigens with near-immediate cytokine production and cytotoxicity. iNKT cells are required for the antibacterial host response. Whether renal failure and renal replacement therapy alter iNKT cell abundance or phenotype has not been investigated. METHODS: iNKT cells were studied by flow cytometry in the peripheral blood of patients with acute renal failure, chronic haemo- and peritoneal dialysis (PD), chronic kidney disease and after renal transplantation. RESULTS: A very marked reduction in iNKT lymphocytes was found in acute renal failure before the first haemodialysis (HD) session. iNKT cells were depleted in end-stage renal disease patients receiving either HD or PD. iNKT cell depletion was accentuated after an HD session. Lesser degrees were observed in patients with non-dialysis-dependent chronic kidney disease. CD56 and CD161 NK cell marker expression was decreased in renal impairment. CD56(+) and CD161(+) iNKT cells produced more interferon-γ than negative cells of the same donor. Within the first year after kidney transplantation, the decrease in iNKT cells and their NK cell markers was reverted. CONCLUSIONS: We describe for the first time that iNKT lymphocytes are reduced in end-stage renal disease and further depleted by HD. iNKT cells are important for early host response including activation of other immune cells and their depletion may contribute to immune dysfunction in renal disease.

Fms-like tyrosine kinase 3 receptor ligand (Flt3L)-based vaccination administered with an adenoviral vector prevents tumor growth of colorectal cancer in a BALB/c mouse model.

PURPOSE: Colorectal cancer is the third most frequent cancer in industrial nations. Therapeutic strategies to treat metastatic disease and prevent recurrence are needed. Anti-tumor immunity can be induced by dendritic cells. Dendritic cells can be expanded by the fms-like tyrosine kinase 3 ligand (Flt3L) in vivo. The aim of this study was to develop an adenoviral-based immune-gene therapy of colorectal cancer with Flt3L in a BALB/c mouse model. METHODS: A new Flt3L-encoding adenoviral vector (pAdFlt3L) was administered in two approaches in a CT26 colon cancer model in female BALB/c mice. In the therapeutic approach, pAdFlt3L was injected into the tail vein or directly into subcutaneous CT26 colon carcinoma tumors in BALB/c mice. In the vaccination protocol, mice were vaccinated with CT26 cell lysate and pAdFlt3L subcutaneous prior to subcutaneous application of vital CT26 cells. RESULTS: Application of pAdFlt3L led to high levels of Flt3L in vitro and in vivo. Significant expansion of dendritic cells after application of pAdFlt3L in vivo was confirmed by the use of CD11c and CD11b surface markers in immunohistochemistry and flow cytometry (p = 0.019). In the therapeutic approach, neither intravenous nor intratumoral treatments with pAdFlt3L lead to regression of CT26 tumors. In the vaccination protocol, vaccination completely prevented tumor growth and resulted in superior survival compared to control mice (p < 0.001). CONCLUSIONS: Our results demonstrate that immunostimulatory therapy with pAdFlt3L is effective to prevent tumor development through vaccination and may represent a therapeutic tool to prevent metastatic disease.

ATP-binding cassette transporter G1 intrinsically regulates invariant NKT cell development.

ATP-binding cassette transporter G1 (ABCG1) plays a role in the intracellular transport of cholesterol. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid Ags. In this study, we demonstrate that ABCG1 regulates iNKT cell development and functions in a cell-intrinsic manner. Abcg1(-/-) mice displayed reduced frequencies of iNKT cells in thymus and periphery. Thymic iNKT cells deficient in ABCG1 had reduced membrane lipid raft content, and showed impaired proliferation and defective maturation during the early stages of development. Moreover, we found that Abcg1(-/-) mice possess a higher frequency of Vß7(+) iNKT cells, suggesting alterations in iNKT cell thymic selection. Furthermore, in response to CD3ε/CD28 stimulation, Abcg1(-/-) thymic iNKT cells showed reduced production of IL-4 but increased production of IFN-γ. Our results demonstrate that changes in intracellular cholesterol homeostasis by ABCG1 profoundly impact iNKT cell development and function.

Intestinal microbes affect phenotypes and functions of invariant natural killer T cells in mice.

BACKGROUND & AIMS: Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells. METHODS: We analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free environments. RESULTS: Specific pathogen-free mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vß7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hyporesponsive to activation with the antigen α-galactosylceramide. Intragastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require Toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation. CONCLUSIONS: Intestinal microbes can affect iNKT cell phenotypes and functions in mice.