Induction Of Protective Responses Research Articles

Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model

ABSTRACT Genetic optimization of Nucleic Acid immunogens is important for potentially improving their immune potency. A COVID-19 DNA vaccine is in phase III clinical trial which is based on a promising highly developable technology platform. Here, we show optimization in mice generating a pGX-9501 DNA vaccine encoding full-length spike protein, which results in induction of potent humoral and cellular immune responses, including neutralizing antibodies, that block hACE2-RBD binding of live CoV2 virus in vitro. Optimization resulted in improved induction of cellular immunity by pGX-9501 as demonstrated by increased IFN-γ expression in both CD8+ and CD4 + T cells and this was associated with more robust antiviral CTL responses compared to unoptimized constructs. Vaccination with pGX-9501 induced subsequent protection against virus challenge in a rigorous hACE2 transgenic mouse model. Overall, pGX-9501 is a promising optimized COVID-19 DNA vaccine candidate inducing humoral and cellular immunity contributing to the vaccine’s protective effects.

Probiotic Effects Upon Resilience And Recovery

The presence of microbiota, functionally in the role of therapeutic probiotics, exerts fundamental actions upon the induction, shaping, training, and function of the host immune system, e.g. among humans. In exchange, the host immune system has largely evolved as a means to maintain and develop the symbiotic relationship of the host with these highly diverse and evolving microbes, such as yeast cells. When operating optimally, this immune system-microbiota alliance allows the induction of protective responses to inflammatory agents and pathogens and the maintenance of regulatory pathways involved in the preservation of tolerance to innocuous antigens thereby promoting both cellular and functional resilience. Resilience presents the process(s) through which individuals are enhanced in their adaptation to their resistance to adverse conditions and enabled in their capacity recover from them. Resilient individuals possess a different immunophenotype from those presenting stress susceptibility and disease-propensity.

2132-P: Single-Cell RNA-Sequencing of Mouse Islets after Inflammatory Cytokine Exposure

Type 1 diabetes is an autoimmune disease caused by destruction of pancreatic β-cells. During disease progression, β-cells are exposed to proinflammatory cytokines like interleukin-1β (IL-1β) and interferon-γ (IFN-γ), stimulating them to express inducible nitric oxide synthase (iNOS) and produce micromolar levels of nitric oxide. While some cytokine-mediated changes in islet gene expression are known, we do not have a global view of the genes induced by cytokines nor do we know how non-β-cells respond to cytokines. Following 6-hr treatment with IL-1β and IFN-γ, adult C57BL/6 mouse islets were subjected to single-cell RNA-sequencing using the 10x Genomics Chromium platform. iNOS mRNA (Nos2) was induced in 73% of β-cells in response to both cytokines. Unexpectedly, 72% of Δ-cells and 44% of α-cells expressed Nos2 at levels similar to that found in β-cells. This is surprising since β-cells are thought to be the primary islet source of nitric oxide. Of the 241 genes enriched in Nos2-expressing β-cells, 70% were also enriched in Nos2-expressing Δ- and α-cells, suggesting that these cells initially respond to cytokine exposure by activating similar pathways. Among the enriched genes were those indicative of anti-viral defenses, suggesting induction of protective responses. Nos2 expression was negatively correlated with expression of genes encoding heat shock proteins, such as Hsp70, consistent with previous reports that heat shock inhibits cytokine-induced iNOS expression. Finally, we observed IL-1β-induced repression of β-cell identity genes, including Mafa, Pdx1, Slc2a2, and Ucn3. Repression of these identity genes was negatively correlated with Hsp70 expression, suggesting that cellular stress prevents this dedifferentiation. While these studies are ongoing, they suggest that (1) Δ- and α-cells respond to cytokines by upregulating Nos2 mRNA, (2) cytokines repress β-cell identity genes, and (3) both of these outcomes can be inhibited by induction of cellular stress. Disclosure J. Stancill: None. A. Khatun: None. M. Kasmani: None. W. Cui: None. J.A. Corbett: None.

Expression and secretion of the Giardia duodenalis variant surface protein 9B10A by transfected trophozoites causes damage to epithelial cell monolayers mediated by protease activity

Giardia duodenalis is the protozoan parasite responsible for most cases of parasitic diarrhea worldwide. The pathogenic mechanisms of giardiasis have not yet been fully characterized. In this context parasite's excretory/secretory products have been related to the damage induced by the parasite on enterocytes. Among these is the Variable Surface Proteins (VSPs) family involved in antigenic variation and in the induction of protective response. In proteomic analyses carried out to identify the proteases with high molecular weight secreted by Giardia trophozoites during the initial phase of interaction with IEC-6 cell monolayers we identified the VSP9B10A protein. In silico bioinformatics analyses predicted a central region in residues 324–684 displaying the catalytic triad and the substrate binding pocket of cysteine proteases. The analysis of the effect of the VSP9B10A protein on epithelial cell monolayers using trophozoites that were transfected with a plasmid carrying the vsp9b10a gene sequence under the control of a constitutive promoter showed that transfected trophozoites expressing the VSP9B10A protein caused cytotoxic damages on IEC-6 and MDCK cell monolayers. This was characterized by loss of cell-cell contacts and cell detachment from the substrate while no damage was observed with trophozoites that did not express the VSP9B10A protein. The same cytotoxic effect was detected when IEC-6 cell monolayers were incubated only with supernatants from co-cultures of IEC-6 cell monolayers with VSP9B10A transfected trophozoites and this effect was not observed when transfected trophozoites were incubated with a monospecific polyclonal antibody anti-VSP9B10A previous to interaction with IEC-6 monolayers. These results demonstrate that the VSP9B10A protein secreted upon interaction with epithelial cells caused damage in these cells. Thus this protein might be considered as a conditional virulence factor candidate. To our knowledge this is the first report on the proteolytic activity from a Giardia VSP opening new research lines on these proteins.

Regulation of immunity during visceral Leishmania infection.

Unicellular eukaryotes of the genus Leishmania are collectively responsible for a heterogeneous group of diseases known as leishmaniasis. The visceral form of leishmaniasis, caused by L. donovani or L. infantum, is a devastating condition, claiming 20,000 to 40,000 lives annually, with particular incidence in some of the poorest regions of the world. Immunity to Leishmania depends on the development of protective type I immune responses capable of activating infected phagocytes to kill intracellular amastigotes. However, despite the induction of protective responses, disease progresses due to a multitude of factors that impede an optimal response. These include the action of suppressive cytokines, exhaustion of specific T cells, loss of lymphoid tissue architecture and a defective humoral response. We will review how these responses are orchestrated during the course of infection, including both early and chronic stages, focusing on the spleen and the liver, which are the main target organs of visceral Leishmania in the host. A comprehensive understanding of the immune events that occur during visceral Leishmania infection is crucial for the implementation of immunotherapeutic approaches that complement the current anti-Leishmania chemotherapy and the development of effective vaccines to prevent disease.

Influence of low-dose-rate red and near-infrared radiations on the level of reactive oxygen species, the genetic apparatus and the tumor growth in mice in vivo

The effect of low-dose-rate red and near-infrared radiations from the matrix of light emitted diode (650 nm and 850 nm) and a He-Ne laser (633 nm) on activation of the reserve of a natural defense system in the mice exposed to radiation in vivo was studied by the level of reactive oxygen species (ROS) production in blood cells, the induction of cytogenetic adaptive response in bone marrow cells, thymus and spleen, and the rate of Ehrlich ascites carcinoma growth in a solid form. As a positive control animals were irradiated with X-rays by the scheme of the radiation-induced adaptive response (0.1 Gy + 1.5 Gy). The levels of ROS production was assessed in whole blood by luminol-dependent chemiluminescence, of cytogenetic damage--by the "micronucleus test" in the bone marrow, the weight of the thymus and spleen--by index of organ, and the rate of tumor growth--according to its size for 30 days after inoculation. Adaptogenic and anticarcinogenic effects of studied radiations were revealed. The values of these effects were not different from those in animals pre-irradiated with the X-rays. The relationship between the level of ROS production and adaptive response induction in the mice under the influence of non-ionizing radiation was first ascertained. The experimental data obtained may indicate a similar mechanism of induction of protective responses to ionizing and non-ionizing radiations in mice in vivo.

Interferon-α as Antiviral and Antitumor Vaccine Adjuvants: Mechanisms of Action and Response Signature

Interferon-α (IFN-α) are cytokines endowed with multiple biologic effects, including activities on cells of the immune system, which are important for inducing protective antiviral and antitumor responses. Studies in mouse models have been instrumental for understanding the immune adjuvant activity of these cytokines and some of their mechanisms of action. In particular, recent studies conducted on both mouse and human models suggest that IFN-α act as effective immune adjuvants for inducing antiviral and antitumor immunity and that the effects of IFN on the differentiation and activation of dendritic cells (DC) play an important role in the induction of protective responses. In spite of the long record of IFN-α clinical use, a few clinical trials have attempted to evaluate the efficacy of these cytokines used as vaccine adjuvants. Recently, studies on the IFN-α signature in cells from patients treated with IFN-α under different modalities and various clinical settings have provided important insights for understanding the in vivo mechanisms of the IFN immune adjuvant activity in humans and may contribute to the identification of molecular markers with a clinical response. These studies further support the interest of evaluating the clinical efficacy of IFN-α when used as a vaccine adjuvant and also suggest that the DC generated in vitro from monocytes in the presence of this cytokine can exhibit a special advantage for the development of effective therapeutic vaccination strategies in cancer patients.

Adverse environmental conditions influence age-related innate immune responsiveness

Background-The innate immune system plays an important role in the recognition and induction of protective responses against infectious pathogens, whilst there is increasing evidence for a role in mediating chronic inflammatory diseases at older age. Despite indications that environmental conditions can influence the senescence process of the adaptive immune system, it is not known whether the same holds true for the innate immune system. Therefore we studied whether age-related innate immune responses are similar or differ between populations living under very diverse environmental conditions.Methods-We compared cross-sectional age-related changes in ex vivo innate cytokine responses in a population living under affluent conditions in the Netherlands (age 20–68 years old, n = 304) and a population living under adverse environmental conditions in Ghana (age 23–95 years old, n = 562).Results-We found a significant decrease in LPS-induced Interleukin (IL)-10 and Tumor Necrosis Factor (TNF) production with age in the Dutch population. In Ghana a similar age-related decline in IL-10 responses to LPS, as well as to zymosan, or LPS plus zymosan, was observed. TNF production, however, did not show an age-associated decline, but increased significantly with age in response to co-stimulation with LPS and zymosan.Conclusion-We conclude that the decline in innate cytokine responses is an intrinsic ageing phenomenon, while pathogen exposure and/or selective survival drive pro-inflammatory responses under adverse living conditions.

Role of Nitric Oxide Dependence on Nitric Oxide Synthase‐like Activity in the Water Stress Signaling of Maize Seedling

Nitric oxide (NO) has been known as an important signal in plant antioxidative defense but its production and roles in water stress are less known. The present study investigated whether NO dependence on a NO synthase-like (NOS) activity is involved in the signaling of drought-induced protective responses in maize seedlings.NOS activity, rate of NO release and drought responses were analyzed when NO donor sodium nitroprusside (SNP), NO scavenger c-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) were applied to both detached maize leaves and whole plants. Both NOS activity and the rate of NO release increased substantially under dehydration stress. The high NOS activity induced by c-PTIO as NO scavenger and NO accumulation inhibited by NOS inhibitor L-NAME in dehydration-treated maize seedlings indicated that most NO production under water deficit stress may be generated from NOS-like activity. After dehydration stress for 3 h, detached maize leaves pretreated with NO donor SNP maintained more water content than that of control leaves pretreated with water. This result was consistent with the decrease in the transpiration rate of SNP-treated leaves subjected to drought treatment for 3 h. Membrane permeability, a cell injury index, was lower in SNP-treated maize leaves under dehydration stress for 4 h when compared with the control leaves. Also, superoxide dismutase (SOD) activity of SNP combined drought treatment maize leaves was higher than that of drought treatment alone, indicating that exogenous NO treatment alleviated the water loss and oxidative damage of maize leaves under water deficit stress. When c-PTIO as a specific NO scavenger was applied, the effects of applied SNP were overridden. Treatment with L-NAME on leaves also led to higher membrane permeability, higher transpiration rate and lower SOD activities than those of control leaves, indicating that NOS-like activity was involved in the antioxidative defense under water stress. These results suggested that NO dependence on NOS-like activity serves as a signaling component in the induction of protective responses and is associated with drought tolerance in maize seedlings.

Mast cell production of IL-4 and TNF may be required for protective and pathological responses in gastrointestinal helminth infection

Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with Th2 responses and intestinal inflammation, which correlate with a marked mast cell (MC) response. To address the role of MC-derived cytokines in the induction of protective responses, WBB6F1-KitW/KitW-v (W/Wv) mice were reconstituted with wild-type, tumor necrosis factor (TNF)-α−/−, or interleukin (IL)-4−/− bone marrow (BM) prior to infection with T. spiralis. W/Wv mice reconstituted with TNF-α−/− or IL-4−/− BM expelled the parasite less efficiently and showed diminished enteropathy, whereas protective responses were normal in W/Wv mice reconstituted with wild-type BM and were accompanied by intestinal pathology. MC responses were reduced in W/Wv mice reconstituted with IL-4−/− BM and to a lesser extent when reconstituted with TNF-α−/−. These results suggest that MC-derived IL-4 and TNF may regulate the induction of protective Th2 responses and intestinal inflammation associated with the expulsion of T. spiralis. Significantly, these studies suggest a role for MC-derived cytokines as autocrine growth factors.

Suppression of LethalPlasmodium yoeliiMalaria following Protective Immunization Requires Antibody-, IL-4-, and IFN-γ-Dependent Responses Induced by Vaccination and/or Challenge Infection

Immunization with Plasmodium yoelii merozoite surface protein (PyMSP)-8 protects mice from lethal malaria but does not prevent infection. Using this merozoite surface protein-based vaccine model, we investigated vaccine- and infection-induced immune responses that contribute to protection. Analysis of prechallenge sera from rPyMSP-8-immunized C57BL/6 and BALB/c mice revealed high and comparable levels of Ag-specific IgG, but differences in isotype profile and specificity for conformational epitopes were noted. As both strains of mice were similarly protected against P. yoelii, we could not correlate vaccine-induced responses with protection. However, passive immunization studies suggested that protection resulted from differing immune responses. Studies with cytokine-deficient mice showed that protection was induced by immunization of C57BL/6 mice only when IL-4 and IFN-gamma were both present. In BALB/c mice, the absence of either IL-4 or IFN-gamma led to predictable shifts in the IgG isotype profile but did not reduce the magnitude of the Ab response induced by rPyMSP-8 immunization. Immunized IL-4-/- BALB/c mice were solidly protected against P. yoelii. To our surprise, immunized IFN-gamma-/- BALB/c mice initially controlled parasite growth but eventually succumbed to infection. Analysis of cytokine production revealed that P. yoelii infection induced two distinct peaks of IFN-gamma that correlated with periods of controlled parasite growth in intact, rPyMSP-8-immunized BALB/c mice. Maximal parasite growth occurred during a period of sustained TGF-beta production. Combined, the data indicate that induction of protective responses by merozoite surface protein-based vaccines depends on IL-4 and IFN-gamma-dependent pathways and that vaccine efficacy is significantly influenced by host responses elicited upon infection.

Candida albicans mannoprotein influences the biological function of dendritic cells

Cell wall components of fungi involved in induction of host immune response are predominantly proteins and glycoproteins, the latter being mainly mannoproteins (MP). In this study we analyse the interaction of the MP from Candida albicans (MP65) with dendritic cells (DC) and demonstrate that MP65 stimulates DC and induces the release of TNF-alpha, IL-6 and the activation of IL-12 gene, with maximal value 6 h post treatment. MP65 induces DC maturation by increasing costimulatory molecules and decreasing CD14 and FcgammaR molecule expression. The latter effect is partly mediated by toll-like receptor 2 (TLR2) and TLR4, and the MyD88-dependent pathway is involved in the process. MP65 enables DC to activate T cell response, its protein core is essential for induction of T cell activation, while its glycosylated portion primarily promotes cytokine production. The mechanisms involved in induction of protective response against C. albicans could be mediated by the MP65 antigen, suggesting that MP65 may be a suitable candidate vaccine.

Factors in xenograft rejection.

Important mechanisms underlying immediate xenograft loss by hyperacute rejection (HAR), in the pig-to-primate combination, have been recently delineated. There are now several proposed therapies that deal with the problem of complement activation and xenoreactive natural antibody (XNA) binding to the vasculature that have been shown to prevent HAR. However, vascularized xenografts are still lost, typically within days, by delayed xenograft rejection (DXR), alternatively known as acute vascular rejection (AVR). This process is characterized by endothelial cell (EC) perturbation, localization of XNA within the graft vasculature, host NK cell and monocyte activation with platelet sequestration and vascular thrombosis. Alternative immunosuppressive strategies, additive anti-complement therapies with the control of any resulting EC activation processes and induction of protective responses have been proposed to ameliorate this pathological process. In addition, several potentially important molecular incompatibilities between activated human coagulation factors and the natural anticoagulants expressed on porcine EC have been noted. Such incompatibilities may be analogous to cross-species alterations in the function of complement regulatory proteins important in HAR. Disordered thromboregulation is potentially relevant to the progression of inflammatory events in DXR and the disseminated intravascular coagulation seen in primate recipients of porcine renal xenografts. We have recently demonstrated the inability of porcine tissue factor pathway inhibitor (TFPI) to adequately neutralize human factor Xa (FXa), the aberrant activation of both human prothrombin and FXa by porcine EC and the failure of the porcine natural anticoagulant, thrombomodulin to bind human thrombin and hence activate human protein C. The enhanced potential of porcine von Willebrand factor to associate with human platelet GPIb has been demonstrated to be dependent upon the isolated A1 domain of von Willebrand factor. In addition, the loss of TFPI and vascular ATPDase/CD39 activity following EC activation responses would potentiate any procoagulant changes within the xenograft. These developments could exacerbate vascular damage from whatever cause and enhance the activation of platelets and coagulation pathways within xenografts resulting in graft infarction and loss. Analysis of these and the other putative factors underlying DXR should lead to the development and testing of genetic approaches that, in conjunction with selected pharmacological means, may further prolong xenograft survival to a clinically relevant extent.