Immunoinformatics Vaccine Design for Zika Virus.

Zika virus (ZIKV) is an emerging virus from the Flaviviridae family and Flavivirus genus that has caused important outbreaks around the world. ZIKV infection is associated with severe neuropathology in newborns and adults. Until now, there is no licensed vaccine available for ZIKV infection. Therefore, the development of a safe and effective vaccine against ZIKV is an urgent need. Recently, we designed an in silico multi-epitope vaccine for ZIKV based on immunoinformatics tools. To construct this in silico ZIKV vaccine, we used a consensus sequence generated from ZIKV sequences available in databank. Then, we selected CD4+ and CD8+ T cell epitopes from all ZIKV proteins based on the binding prediction to class II and class I human leukocyte antigen (HLA) molecules, promiscuity, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the construct and B cell epitopes were identified. Adjuvants were associated to increase immunogenicity. Distinct linkers were used for connecting the CD4+ and CD8+ T cell epitopes, EDIII, and adjuvants. Several analyses, such as antigenicity, population coverage, allergenicity, autoimmunity, and secondary and tertiary structures of the vaccine, were evaluated using various immunoinformatics tools and online web servers. In this chapter, we present the protocols with the rationale and detailed steps needed for this in silico multi-epitope ZIKV vaccine design.

Endotoxin tolerance and low activation of TLR-4/NF-κB axis in monocytes of COVID-19 patients.

Higher endotoxin in the circulation may indicate a compromised state of host immune response against coinfections in severe COVID-19 patients. We evaluated the inflammatory response of monocytes from COVID-19 patients after lipopolysaccharide (LPS) challenge. Whole blood samples of healthy controls, patients with mild COVID-19, and patients with severe COVID-19 were incubated with LPS for 2 h. Severe COVID-19 patients presented higher LPS and sCD14 levels in the plasma than healthy controls and mild COVID-19 patients. In non-stimulated in vitro condition, severe COVID-19 patients presented higher inflammatory cytokines and PGE-2 levels and CD14 + HLA-DRlow monocytes frequency than controls. Moreover, severe COVID-19 patients presented higher NF-κB p65 phosphorylation in CD14 + HLA-DRlow, as well as higher expression of TLR-4 and NF-κB p65 phosphorylation in CD14 + HLA-DRhigh compared to controls. The stimulation of LPS in whole blood of severe COVID-19 patients leads to lower cytokine production but higher PGE-2 levels compared to controls. Endotoxin challenge with both concentrations reduced the frequency of CD14 + HLA-DRlow in severe COVID-19 patients, but the increases in TLR-4 expression and NF-κB p65 phosphorylation were more pronounced in both CD14 + monocytes of healthy controls and mild COVID-19 patients compared to severe COVID-19 group. We conclude that acute SARS-CoV-2 infection is associated with diminished endotoxin response in monocytes. KEY MESSAGES: Severe COVID-19 patients had higher levels of LPS and systemic IL-6 and TNF-α. Severe COVID-19 patients presented higher CD14+HLA-DRlow monocytes. Increased TLR-4/NF-κB axis was identified in monocytes of severe COVID-19. Blunted production of cytokines after whole blood LPS stimulation in severe COVID-19. Lower TLR-4/NF-κB activation in monocytes after LPS stimulation in severe COVID-19.

Local tumour nanoparticle thermal therapy: A promising immunomodulatory treatment for canine cancer.

Distinct thermal therapies have been used for cancer therapy. For hyperthermia (HT) treatment the tumour tissue is heated to temperatures between 39 and 45°C, while during ablation (AB) temperatures above 50°C are achieved. HT is commonly used in combination with different treatment modalities, such as radiotherapy and chemotherapy, for better clinical outcomes. In contrast, AB is usually used as a single modality for direct tumour cell killing. Both thermal therapies have been shown to result in cytotoxicity as well as immune response stimulation. Immunogenic responses encompass the innate and adaptive immune systems and involve the activation of macrophages, dendritic cells, natural killer cells and T cells. Several heat technologies are used, but great interest arises from nanotechnology-based thermal therapies. Spontaneous tumours in dogs can be a model for cancer immunotherapies with several advantages. In addition, veterinary oncology represents a growing market with an important demand for new therapies. In this review, we will focus on nanoparticle-mediated thermal-induced immunogenic effects, the beneficial potential of integrating thermal nanomedicine with immunotherapies and the results of published works with thermotherapies for cancer using dogs with spontaneous tumours, highlighting the works that evaluated the effect on the immune system in order to show dogs with spontaneous cancer as a good model for evaluated the immunomodulatory effect of nanoparticle-mediated thermal therapies.

Is There a Difference between the Preoperative and Postoperative Serum Levels of Interleukin-6 and Tumor Necrosis Factor-α in Children Submitted to Adenotonsillectomy?

Abstract Introduction Palatine and pharyngeal tonsils are the first line of defense against pathogens. Clinically, two alterations may require surgical removal of the tonsils: hypertrophy and recurrent tonsillitis. The two conditions probably result from a dysfunction of the immune system. Objective To evaluate possible differences in the plasma levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in patients submitted to adenotonsillectomy. Methods Prospective, longitudinal study with 25 children undergoing adenotonsillectomy separated into 3 different groups: recurrent tonsillitis (RT), composed of 7 patients; recurrent hypertrophy tonsillitis (RTTH), with 8 patients; and the tonsillar hypertrophy (TH) group, with 10 patients. Ten healthy control children (SD) were also included in the study. Peripheral blood was collected, and plasma was separated to measure the levels of TNF-α, IL-6, and IL-10. The Mann-Whitney test was used for statistical analysis. Results The plasma level of IL-6 was higher in the RT (p= 0.0394) and TH (p= 0.0009) groups, compared with the control group. The TH group also had higher levels of IL-6 than the RT group (p= 0.039). The IL-6/IL-10 ratio was higher in the RT (p= 0.029) and TH (p= 0.0005) groups compared with the control group. Between the RT and RTTH groups, the IL-6/IL-10 ratio was higher in the RT group, with a statistically significant difference (p= 0.0091). Conclusion Patients with a history of chronic tonsillitis had higher levels of IL-6, compared with the control group.

Is There a Difference between the Preoperative and Postoperative Serum Levels of Interleukin-6 and Tumor Necrosis Factor-α in Children Submitted to Adenotonsillectomy?

Introduction Palatine and pharyngeal tonsils are the first line of defense against pathogens. Clinically, two alterations may require surgical removal of the tonsils: hypertrophy and recurrent tonsillitis. The two conditions probably result from a dysfunction of the immune system. Objective To evaluate possible differences in the plasma levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in patients submitted to adenotonsillectomy. Methods Prospective, longitudinal study with 25 children undergoing adenotonsillectomy separated into 3 different groups: recurrent tonsillitis (RT), composed of 7 patients; recurrent hypertrophy tonsillitis (RTTH), with 8 patients; and the tonsillar hypertrophy (TH) group, with 10 patients. Ten healthy control children (SD) were also included in the study. Peripheral blood was collected, and plasma was separated to measure the levels of TNF-α, IL-6, and IL-10. The Mann-Whitney test was used for statistical analysis. Results The plasma level of IL-6 was higher in the RT ( p = 0.0394) and TH ( p = 0.0009) groups, compared with the control group. The TH group also had higher levels of IL-6 than the RT group ( p = 0.039). The IL-6/IL-10 ratio was higher in the RT ( p = 0.029) and TH ( p = 0.0005) groups compared with the control group. Between the RT and RTTH groups, the IL-6/IL-10 ratio was higher in the RT group, with a statistically significant difference ( p = 0.0091). Conclusion Patients with a history of chronic tonsillitis had higher levels of IL-6, compared with the control group.

In silico construction of a multiepitope Zika virus vaccine using immunoinformatics tools.

Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine's average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation.

Correlation between intestinal BMP2, IFNγ, and neural death in experimental infection with Trypanosoma cruzi.

Megacolon is one of the main late complications of Chagas disease, affecting approximately 10% of symptomatic patients. However, studies are needed to understand the mechanisms involved in the progression of this condition. During infection by Trypanosoma cruzi (T. cruzi), an inflammatory profile sets in that is involved in neural death, and this destruction is known to be essential for megacolon progression. One of the proteins related to the maintenance of intestinal neurons is the type 2 bone morphogenetic protein (BMP2). Intestinal BMP2 homeostasis is directly involved in the maintenance of organ function. Thus, the aim of this study was to correlate the production of intestinal BMP2 with immunopathological changes in C57Bl/6 mice infected with the T. cruzi Y strain in the acute and chronic phases. The mice were infected with 1000 blood trypomastigote forms. After euthanasia, the colon was collected, divided into two fragments, and a half was used for histological analysis and the other half for BMP2, IFNγ, TNF-α, and IL-10 quantification. The infection induced increased intestinal IFNγ and BMP2 production during the acute phase as well as an increase in the inflammatory infiltrate. In contrast, a decreased number of neurons in the myenteric plexus were observed during this phase. Collagen deposition increased gradually throughout the infection, as demonstrated in the chronic phase. Additionally, a BMP2 increase during the acute phase was positively correlated with intestinal IFNγ. In the same analyzed period, BMP2 and IFNγ showed negative correlations with the number of neurons in the myenteric plexus. As the first report of BMP2 alteration after infection by T. cruzi, we suggest that this imbalance is not only related to neuronal damage but may also represent a new route for maintaining the intestinal proinflammatory profile during the acute phase.

Immunoinformatic construction of an adenovirus-based modular vaccine platform and its application in the design of a SARS-CoV-2 vaccine.

The current SARS-CoV-2 pandemic has imposed new challenges and demands for health systems, especially in the development of new vaccine strategies. Vaccines for many pathogens were developed based on the display of foreign epitopes in the variable regions of the human adenovirus (HAdV) major capsid proteins (hexon, penton and fiber). The humoral immune response against the HAdV major capsid proteins was demonstrated to play a role in the development of an immune response against the epitopes in display. Through the immunoinformatic profiling of the major capsid proteins of HAdVs from different species, we developed a modular concept that can be used in the development of vaccines based on HAdV vectors. Our data suggests that different immunomodulatory potentials can be observed in the conserved regions, present in the hexon and penton proteins, from different species. Using this modular approach, we developed a HAdV-5 based vaccine strategy for SARS-CoV-2, constructed through the display of SARS-CoV-2 epitopes indicated by our prediction analysis as immunologically relevant. The sequences of the HAdV vector major capsid proteins were also edited to enhance the IFN-gamma induction and antigen presenting cells activation. This is the first study proposing a modular HAdV platform developed to aid the design of new vaccines by inducing an immune response more suited for the epitopes in display.

Distinct inflammatory profiles in HIV-infected individuals under antiretroviral therapy using cannabis, cocaine or cannabis plus cocaine.

OBJECTIVES: To evaluate the effects of cannabis and/or cocaine use on inflammatory, oxidative stress status and circulating monocyte subsets in HIV-infected individuals under antiretroviral therapy. DESIGN: Soluble CD14 (sCD14), intestinal fatty acid-binding protein (IFABP), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, IL-10, C-reactive protein (CRP) and oxidative stress markers were examined. The monocyte subsets and their activation and cytokine production by peripheral blood mononuclear cells (PBMCs) of HIV-1 infected individuals upon lipopolysaccharide (LPS)-stimulation were also investigated. METHODS: sCD14, IFABP, TNF-α, IL-6, IL-8 and IL-10 levels were evaluated using ELISA, CRP by turbidimetry; lipid peroxidation (TBARS) spectrofluometrically and total thiol levels by using 5-5'-dithio-bis (2-nitrobenzoic acid) reagent. Monocyte subsets and activation were assessed by flow cytometry. RESULTS: All HIV-infected drug user groups showed higher sCD14 levels compared with HIV+ nondrug users. IFABP was increased in HIV+ drug-users in relation to healthy individuals. Cannabis use lowered the percentages of inflammatory, nonclassical, activated-classic and activated-inflammatory monocytes. Cocaine users showed increased plasmatic TNF-α and TBARS levels, decreased thiols content and lower activated-classic and inflammatory-monocyte percentages. Cannabis-plus-cocaine use increased CRP, IL-8 and IL-6/IL-10 ratio, but decreased thiol content, and inflammatory and activated-classic monocyte percentages. PBMCs of cannabis and cannabis-plus-cocaine users showed low-potential cytokine production either spontaneously or under LPS-stimulation. CONCLUSION: In HIV infection, the use of cannabis induces predominantly an anti-inflammatory profile. The use of cocaine and cannabis-plus-cocaine showed a mixed pro-inflammatory and anti-inflammatory profile, with predominance of inflammatory status. Further studies are required to better understand the action of these drugs in HIV infection.

Cardiorespiratory fitness modulates the proportions of monocytes and T helper subsets in lean and obese men.

This study investigated the peripheral frequency of monocytes, CD4 + T cell subsets and the systemic levels of cytokines in lean and obese men with different levels of cardiorespiratory fitness (CRF). Mononuclear cells were obtained from 45 lean and 45 obese men who were assigned into six groups according to their body mass index and CRF (low, moderate, or high VO2Peak ) to analyze the frequency of monocyte subsets and subpopulations of CD4 + T cells (Treg cells, CD4 + CD25high CD127low ; mTeff, CD4 + CD25-CD39+; mTreg, CD4 + CD25+CD39+). The systemic levels of interleukin (IL)-6, IL-10, IL-17a, IL-33, leptin, and tumor necrosis factor-alpha (TNF-α) were also evaluated. Seven sedentary obese men performed one week of high-intensity interval training (HIIT, 3 sessions/week), and blood samples were collected before and 24 hours after the last session for phenotypic analysis of T cells and monocytes. Obese individuals presented an inflammatory profile characterized by lower frequencies of Treg and mTreg cells and higher proportions of proinflammatory monocytes. However, higher CRF status increased the frequencies of Treg cells and mTreg cells and decreased the percentage of CD4 + mTeff cells and intermediate and non-classical monocytes in the peripheral blood from lean and obese men. Systemic lower levels of proinflammatory (IL-6 and TNF-) cytokines and higher concentrations of IL-10 and IL-33 were observed in moderate and higher CRF in all subjects. HIIT increased the proportions of circulating mTreg and Treg cells in sedentary obese individuals. The immunoregulatory role of CRF contributes to the maintenance of low levels of inflammatory mediators.

Phloroglucinol derivatives from Hypericum species trigger mitochondrial dysfunction in Leishmania amazonensis.

Bioactive molecules isolated from plants are promising sources for the development of new therapies against leishmaniasis. We investigated the leishmanicidal activity of cariphenone A (1), isouliginosin B (2) and uliginosin B (3) isolated from Hypericum species. Promastigotes and amastigotes of Leishmania amazonensis were incubated with compounds 1-3 at concentrations 1-100 µm for 48 h. The anti-promastigote effect of compounds was also tested in combinations. The cytotoxicity against macrophages and human erythrocytes were determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method and hemolysis assay, respectively. The compounds 1-3 showed high leishmanicidal activity against promastigotes, IC50 values of 10.5, 17.5 and 11.3 µm, respectively. Synergistic interactions were found to the associations of compounds 1 and 2 [Σ fractional inhibitory concentration (FIC) = 0.41], and 2 and 3 (ΣFIC = 0.28) on promastigotes. All Hypericum compounds induced mitochondrial hyperpolarization and reactive oxygen species production in promastigotes. The compounds showed low cytotoxicity toward mammalian cells, high selectivity index and killed intracellular amastigotes probably mediated by oxidative stress. These results indicate that these compounds are promising candidates for the development of drugs against leishmaniasis.

Use of Bidens pilosa L. (Asteraceae) and Curcuma longa L. (Zingiberaceae) to treat intestinal mucositis in mice: Toxico-pharmacological evaluations.

INTRODUCTION: Several studies towards the development of an effective treatment for intestinal mucositis have been reported, since this condition represents a major problem in clinical oncology practice due to cytotoxic effects of chemotherapy. However standardized protocols and universally accepted treatment options are yet to be established. OBJECTIVES: Given above, this study evaluated the protective effects of a mucoadhesive formulation containing both Bidens pilosa L. (Asteraceae) (BP) and curcuminoids from Curcuma longa L. (Zingiberaceae) (CL) on intestinal mucositis induced by 5-fluoruoacil (5-FU) in mice. RESULTS: As expected, animals only treated with 5-FU (200 mg/kg) showed a significant reduction of 60.3 and 42.4% in villi and crypts size, respectively, when compared to control. On the other hand, the proposed therapeutic/prophylactic treatment with mucoadhesive formulations managed to reduce histopathologic changes in mice bearing mucositis, especially at 125 mg/kg BP + 15 mg/kg CL dose. The formulation promoted an increase of 275.5% and 148.7% for villi and crypts size, respectively. Moreover, chemotherapy-related weight loss was reduced by 7.4% following the treatment. In addition, an increase of 10 and 30.5% in red and white blood cells was observed when compared to 5-FU group. Furthermore, treatments with the mucoadhesive formulation containing BP/CL up modulated Ki-67 and Bcl-2 expression while reduced pro-apoptotic regulator Bax. The formulation also modulated inflammatory response triggered by 5-FU through reduction of 68% of myeloperoxidase activity and a 4-fold increase in anti-inflammatory IL-10 levels. In parallel, the oxidative stress via lipid peroxidation was reduced as indicated by decrease of 63% of malondialdehyde concentrations. Additionally, the new formulation presented low acute oral systemic toxicity, being classified in the category 5 (2000 mg/kg < LD50 < 5000 mg/kg) of the Globally Harmonized Classification System. CONCLUSIONS: This study showed an interesting potential of the mucoadhesive formulation of BP/CL for the treatment of 5-FU-induced intestinal mucositis. Given the perspectives for the development of a new medicine, clinical studies are in progress to better understand the protective effects of this innovative formulation in treating mucositis.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.