Caveats of chimpanzee ChAdOx1 adenovirus-vectored vaccines to boost anti-SARS-CoV-2 protective immunity in mice.

Several COVID-19 vaccines use adenovirus vectors to deliver the SARS-CoV-2 spike (S) protein. Immunization with these vaccines promotes immunity against the S protein, but against also the adenovirus itself. This could interfere with the entry of the vaccine into the cell, reducing its efficacy. Herein, we evaluate the efficiency of an adenovirus-vectored vaccine (chimpanzee ChAdOx1 adenovirus, AZD1222) in boosting the specific immunity compared to that induced by a recombinant receptor-binding domain (RBD)-based vaccine without viral vector. Mice immunized with the AZD1222 human vaccine were given a booster 6 months later, with either the homologous vaccine or a recombinant vaccine based on RBD of the delta variant, which was prevalent at the start of this study. A significant increase in anti-RBD antibody levels was observed in rRBD-boosted mice (31-61%) compared to those receiving two doses of AZD1222 (0%). Significantly higher rates of PepMix™- or RBD-elicited proliferation were also observed in IFNγ-producing CD4 and CD8 cells from mice boosted with one or two doses of RBD, respectively. The lower efficiency of the ChAdOx1-S vaccine in boosting specific immunity could be the result of a pre-existing anti-vector immunity, induced by increased levels of anti-adenovirus antibodies found both in mice and humans. Taken together, these results point to the importance of avoiding the recurrent use of the same adenovirus vector in individuals with immunity and memory against them. It also illustrates the disadvantages of ChAdOx1 adenovirus-vectored vaccine with respect to recombinant protein vaccines, which can be used without restriction in vaccine-booster programs. KEY POINTS: • ChAdOx1 adenovirus vaccine (AZD1222) may not be effective in boosting anti-SARS-CoV-2 immunity • A recombinant RBD protein vaccine is effective in boosting anti-SARS-CoV-2 immunity in mice • Antibodies elicited by the rRBD-delta vaccine persisted for up to 3 months in mice.

Transforming growth factor receptor III (Betaglycan) regulates the generation of pathogenic Th17 cells in EAE.

The transforming growth factor receptor III (TßRIII) is commonly recognized as a co-receptor that promotes the binding of TGFß family ligands to type I and type II receptors. Within the immune system, TßRIII regulates T cell development in the thymus and is differentially expressed through activation; however, its function in mature T cells is unclear. To begin addressing this question, we developed a conditional knock-out mouse with restricted TßRIII deletion in mature T cells, necessary because genomic deletion of TßRIII results in perinatal mortality. We determined that TßRIII null mice developed more severe autoimmune central nervous neuroinflammatory disease after immunization with myelin oligodendrocyte peptide (MOG35-55) than wild-type littermates. The increase in disease severity in TßRIII null mice was associated with expanded numbers of CNS infiltrating IFNγ+ CD4+ T cells and cells that co-express both IFNγ and IL-17 (IFNγ+/IL-17+), but not IL-17 alone expressing CD4 T cells compared to Tgfbr3fl/fl wild-type controls. This led us to speculate that TßRIII may be involved in regulating conversion of encephalitogenic Th17 to Th1. To directly address this, we generated encephalitogenic Th17 and Th1 cells from wild type and TßRIII null mice for passive transfer of EAE into naïve mice. Remarkably, Th17 encephalitogenic T cells from TßRIII null induced EAE of much greater severity and earlier in onset than those from wild-type mice. The severity of EAE induced by encephalitogenic wild-type and Tgfbr3fl/fl.dLcKCre Th1 cells were similar. Moreover, in vitro restimulation of in vivo primed Tgfbr3fl/fl.dLcKCre T cells, under Th17 but not Th1 polarizing conditions, resulted in a significant increase of IFNγ+ T cells. Altogether, our data indicate that TßRIII is a coreceptor that functions as a key checkpoint in controlling the pathogenicity of autoreactive T cells in neuroinflammation probably through regulating plasticity of Th17 T cells into pathogenic Th1 cells. Importantly, this is the first demonstration that TßRIII has an intrinsic role in T cells.

Application of Multiparametric Flow Cytometry Panels to Study Lymphocyte Subpopulations in Tuberculin-Positive Cattle.

Flow cytometry (FC) is widely used in microbiology, immunology, hematology, and oncology. In the veterinary field, FC enabled the study of the immune response in cattle infected with different pathogens, as well as vaccine testing. However, few fluorochrome-conjugated antibodies recognize bovine antigens, limiting the possible benefits of FC and the implementation of multiparametric analysis for more complex studies. Two cytometry panels with five colors each were designed and implemented for the study and identification of populations and subpopulations of T cells derived from the peripheral blood mononuclear cells of dairy heifers. Both panels detected differences in T cell subpopulations between heifers positively and negatively tested for tuberculin; they detected overexpression of CD25+ and CD45RO+ in tuberculin-positive heifers after stimulation with a culture filtrate protein extract (CFPE) from Mycobacterium bovis (M. bovis). We identified subpopulations of T cells from peripheral blood mononuclear cells using two multicolor panels. These panels could be used to analyze total bovine blood in immunopathogenic studies and vaccine development. The same strategy could be implemented in other species of veterinary interest.

Towards the development of an epitope-focused vaccine for SARS-CoV-2.

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions. Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

Lupin γ-conglutin protects against cell death induced by oxidative stress and lipotoxicity, but transiently inhibits in vitro insulin secretion by increasing KATP channel currents.

Lupin γ-conglutin beneficially modulates glycemia, but whether it protects against oxidative and lipotoxic damage remains unknown. Here, we studied the effects of γ-conglutin on cell death provoked by hydrogen peroxide and palmitate in HepG2 hepatocytes and insulin-producing MIN6 cells, and if a modulation of mitochondrial potential and reactive oxygen species (ROS) levels was involved. We also investigated how γ-conglutin influences insulin secretion and electrical activity of ß-cells. The increased apoptosis of HepG2 cells exposed to hydrogen peroxide was prevented by γ-conglutin, and the viability and ROS content in γ-conglutin-treated cells was similar to that of non-exposed cells. Additionally, γ-conglutin partially protected MIN6 cells against hydrogen peroxide-induced death. This was associated with a marked reduction in ROS. No significant changes were found in the mitochondrial potential of γ-conglutin-treated cells. Besides, we observed a partial protection against lipotoxicity only in hepatocytes. Unexpectedly, we found a transient inhibition of insulin secretion, plasma membrane hyperpolarization, and higher KATP channel currents in ß-cells treated with γ-conglutin. Our data show that γ-conglutin protects against cell death induced by oxidative stress or lipotoxicity by decreasing ROS and might also indicate that γ-conglutin promotes a ß-cell rest, which could be useful for preventing ß-cell exhaustion in chronic hyperglycemia.

Analysis of Tumor-Derived Exosomes by Nanoscale Flow Cytometry.

The study of tumor exosomes has gained relevance in the last decades due to their potential use for therapeutic and diagnostic application. Although there is extensive knowledge of exosome biology, some biological samples like tumor-derived exosomes have been difficult to characterize due to their complexity and heterogeneity. This distinctive feature makes difficult the identification of specific exosome subpopulations with a shared molecular signature that could allow for targeting of exosomes with therapeutic and diagnostic potential use in cancer patients. Nanoscale flow cytometry has lately emerged as an alternative tool that can be adapted to the study of nanoparticles, such as exosomes. However, the physicochemical properties of these particles are an important issue to consider as nanoparticles need the application of specific settings which differ from those used in conventional flow cytometry of cells. Therefore, in the last few years, one of the main aims has been the optimization of technical and experimental protocols to improve exosome analysis. In this chapter, we discuss several aspects of cytometric systems with a special emphasis in technical considerations of samples and equipment.

Glucose modulates proliferation in root apical meristems via TOR in maize during germination.

The Glucose-Target of Rapamycin (Glc-TOR) pathway has been studied in different biological systems, but scarcely during early seed germination. This work examines its importance for cell proliferation, expression of cell cycle key genes, their protein levels, besides morphology and cellularization of the root apical meristem of maize (Zea mays) embryo axes during germination under the influence of two simple sugars, glucose and sucrose, and a specific inhibitor of TOR activity, AZD 8055. The two sugars promote germination similarly and to an extent, independently of TOR activity. However, the Glc-TOR pathway increases the number of cells committed to proliferation, increasing the expression of a cell cycle gene, ZmCycD4;2, a putative G1/S regulator. Also, Glc-TOR may have influence on the protein stability of another G1/S cyclin, ZmCycD3, but had no influence on ZmCDKA;1 or ZmKRP3 or their proteins. Results suggest that the Glc-TOR pathway participates in the regulation of proliferation through different mechanisms that, in the end, modify the timing of seed germination.

CD5 on dendritic cells regulates CD4+ and CD8+ T cell activation and induction of immune responses.

The role of CD5 as a regulator of T cell signaling and tolerance is well recognized. Recent data show expression of CD5 on different subtypes of human dendritic cells, however its functional relevance in modulating DC mediated responses remains poorly understood. In this study, we show CD5 is expressed on CD11c+ DC from murine thymus, lymph node, spleen, skin and lung. Although the development of DC subpopulations in CD5-/- mice was normal, CD5-deficient DC produced significantly higher levels of IL-12 than wild type DC in response to LPS. CD5-/- DC, in comparison to CD5+/+ DC, enhanced the activation of CD4+ and CD8+ T cells in vitro and in vivo and induced significantly higher production of IL-2 and IFN-gamma by T cells. Consequently, CD5-/- DC were significantly more potent than wild type DC in the induction of anti-tumor immunity and contact hypersensitivity responses in mice. Restoration of CD5 expression in CD5-/- DC reduced IL-12 production and inhibited their capacity to stimulate T cells. Collectively, these data demonstrate that the specific expression of CD5 on DC inhibits the production of inflammatory cytokines and has a regulatory effect on their activity to stimulate T cells and induce immune responses. This study reveals a previously unrecognized regulatory role for CD5 on DC and provides novel insights into mechanisms for DC biology in immune responses.

Inhibins regulate peripheral regulatory T cell induction through modulation of dendritic cell function.

We have previously reported that the absence of inhibins results in impaired dendritic cell (DC) maturation and function, leading to decreased T cell activation and diminished delayed-type hypersensitivity responses. Here, we investigated the role of inhibins in peripheral regulatory T cell (Treg) induction in vitro and in vivo. Inhibin deficient (Inhα-/-) mice showed an increased percentage of peripherally induced Tregs in colonic lamina propria and mesenteric lymph nodes, compared to Inhα+/+ mice, which correlated with increased expression of PD-L1 in CD103+ and CD8α+ DCs. Lipopolysaccharide-stimulated bone marrow-derived and ex vivo spleen- and lymph node-purified CD11c+ Inhα-/- DCs induced higher Tregs in vitro. Moreover, in vivo anti-DEC205-ovalbumin (OVA) DC targeting of mice with adoptively transferred OVA-specific T cells showed enhanced induced peripheral Treg conversion in Inhα-/- mice. These data identify inhibins as key regulators of peripheral T cell tolerance.

Tilmicosin modulates the innate immune response and preserves casein production in bovine mammary alveolar cells during Staphylococcus aureus infection.

Tilmicosin is an antimicrobial agent used to treat intramammary infections against Staphylococcus aureus and has clinical anti-inflammatory effects. However, the mechanism by which it modulates the inflammatory process in the mammary gland is unknown. We evaluated the effect of tilmicosin treatment on the modulation of the mammary innate immune response after S. aureus infection and its effect on casein production in mammary epithelial cells. To achieve this goal, we used immortalized mammary epithelial cells (MAC-T), pretreated for 12 h or treated with tilmicosin after infection with S. aureus (ATCC 27543). Our data showed that tilmicosin decreases intracellular infection (P < 0.01) and had a protective effect on MAC-T reducing apoptosis after infection by 80% (P < 0.01). Furthermore, tilmicosin reduced reactive oxygen species (ROS) (P < 0.01), IL-1ß (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.05) production. In an attempt to investigate the signaling pathways involved in the immunomodulatory effect of tilmicosin, mitogen-activated protein kinase (MAPK) phosphorylation was measured by fluorescent-activated cell sorting. Pretreatment with tilmicosin increased ERK1/2 (P < 0.05) but decreased P38 phosphorylation (P < 0.01). In addition, the anti-inflammatory effect of tilmicosin helped to preserve casein synthesis in mammary epithelial cells (P < 0.01). This result indicates that tilmicosin could be an effective modulator inflammation in the mammary gland. Through regulation of MAPK phosphorylation, ROS production and pro-inflammatory cytokine secretion tilmicosin can provide protection from cellular damage due to S. aureus infection and help to maintain normal physiological functions of the bovine mammary epithelial cell.

TßRIII is induced by TCR signaling and downregulated in FoxP3+ regulatory T cells.

TGF-ß type III receptor (TßRIII) is a co-receptor for TGFß family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-ßs, Bone Morphogenetic Proteins (BMP2/4) and Inhibins/Activins regulate different checkpoints during T cell differentiation. We have previously reported that TßRIII modulates T cell development by protecting developing thymocytes from apoptosis, however the role of this co-receptor in peripheral lymphocytes still remains elusive. Here we describe a detailed characterization of TßRIII expression in murine and human lymphocyte subpopulations demonstrating that this co-receptor is significantly expressed in T but not B lymphocytes and among them, preferentially expressed on naïve and central memory T cells. TßRIII was upregulated after TCR stimulation, in parallel to other early activation markers. In contrast, natural and induced Tregs downregulated TßRIII in association with FoxP3 upregulation. Finally, anti-TßRIII blocking experiments demonstrated that TßRIII promotes TGFß-dependent iTreg conversion in vitro, and suggest that this co-receptor may be involved in modulating peripheral T cell tolerance and could be considered as a potential target to boost T cell immune responses.

A Key Role for Inhibins in Dendritic Cell Maturation and Function.

Inhibins are members of the TGFß superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The "semi-mature" phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance.