Double-Stranded Structure of the Polyinosinic-Polycytidylic Acid Molecule to Elicit TLR3 Signaling and Adjuvant Activity in Murine Intranasal A(H1N1)pdm09 Influenza Vaccination.

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.

Impact of rearing temperature on the innate antiviral immune response of growth hormone transgenic female triploid Atlantic salmon (Salmo salar).

AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) are a faster-growing alternative to conventional farmed diploid Atlantic salmon. To investigate optimal rearing conditions for their commercial production, a laboratory study was conducted in a freshwater recirculating aquaculture system (RAS) to examine the effect of rearing temperature (10.5 °C, 13.5 °C, 16.5 °C) on their antiviral immune and stress responses. When each temperature treatment group reached an average weight of 800 g, a subset of fish were intraperitoneally injected with either polyriboinosinic polyribocytidylic acid (pIC, a viral mimic) or an equal volume of sterile phosphate-buffered saline (PBS). Blood and head kidney samples were collected before injection and 6, 24 and 48 h post-injection (hpi). Transcript abundance of 7 antiviral biomarker genes (tlr3, lgp2, stat1b, isg15a, rsad2, mxb, ifng) was measured by real-time quantitative polymerase chain reaction (qPCR) on head kidney RNA samples. Plasma cortisol levels from blood samples collected pre-injection and from pIC and PBS groups at 24 hpi were quantified by ELISA. While rearing temperature and treatment did not significantly affect circulating cortisol, all genes tested were significantly upregulated by pIC at all three temperatures (except for tlr3, which was only upregulated in the 10.5 °C treatment). Target gene activation was generally observed at 24 hpi, with most transcript levels decreasing by 48 hpi in pIC-injected fish. Although a high amount of biological variability in response to pIC was evident across all treatments, rearing temperature significantly influenced transcript abundance and/or fold-changes comparing time- and temperature-matched pIC- and PBS-injected fish for several genes (tlr3, lgp2, stat1b, isg15a, rsad2 and ifng) at 24 hpi. As an example, significantly higher fold-changes of rsad2, isg15a and ifng were found in fish reared at 10.5 °C when compared to 16.5 °C. Multivariate analysis confirmed that rearing temperature modulated antiviral immune response. The present experiment provides novel insight into the relationship between rearing temperature and innate antiviral immune response in AquAdvantage Salmon.

Combined Vaccination with NY-ESO-1 Protein, Poly-ICLC, and Montanide Improves Humoral and Cellular Immune Responses in Patients with High-Risk Melanoma.

Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1-specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.

Immunotherapeutic effects of intratumoral nanoplexed poly I:C.

Poly I:C is a powerful immune adjuvant as a result of its agonist activities on TLR-3, MDA5 and RIG-I. BO-112 is a nanoplexed formulation of Poly I:C complexed with polyethylenimine that causes tumor cell apoptosis showing immunogenic cell death features and which upon intratumoral release results in more prominent tumor infiltration by T lymphocytes. Intratumoral treatment with BO-112 of subcutaneous tumors derived from MC38, 4 T1 and B16-F10 leads to remarkable local disease control dependent on type-1 interferon and gamma-interferon. Some degree of control of non-injected tumor lesions following BO-112 intratumoral treatment was found in mice bearing bilateral B16-OVA melanomas, an activity which was enhanced with co-treatment with systemic anti-CD137 and anti-PD-L1 mAbs. More abundant CD8+ T lymphocytes were found in B16-OVA tumor-draining lymph nodes and in the tumor microenvironment following intratumoral BO-112 treatment, with enhanced numbers of tumor antigen-specific cytotoxic T lymphocytes. Genome-wide transcriptome analyses of injected tumor lesions were consistent with a marked upregulation of the type-I interferon pathway. Inspired by these data, intratumorally delivered BO-112 is being tested in cancer patients (NCT02828098).

Role of MDA5 and interferon-I in dendritic cells for T cell expansion by anti-tumor peptide vaccines in mice.

Cytotoxic T lymphocytes (CTLs) are effective components of the immune system capable of destroying tumor cells. Generation of CTLs using peptide vaccines is a practical approach to treat cancer. We have previously described a peptide vaccination strategy that generates vast numbers of endogenous tumor-reactive CTLs after two sequential immunizations (prime-boost) using poly-ICLC adjuvant, which stimulates endosomal toll-like receptor 3 (TLR3) and cytoplasmic melanoma differentiation antigen 5 (MDA5). Dendritic cells (DCs) play an important role not only in antigen presentation but are critical in generating costimulatory cytokines that promote CTL expansion. Poly-ICLC was shown to be more effective than poly-IC in generating type-I interferon (IFN-I) in various DC subsets, through its enhanced ability to escape the endosomal compartment and stimulate MDA5. In our system, IFN-I did not directly function as a T cell costimulatory cytokine, but enhanced CTL expansion through the induction of IL15. With palmitoylated peptide vaccines, CD8α+ DCs were essential for peptide crosspresentation. For vaccine boosts, non-professional antigen-presenting cells were able to present minimal epitope peptides, but DCs were still required for CTL expansions through the production of IFN-I mediated by poly-ICLC. Overall, these results clarify the roles of DCs, TLR3, MDA5, IFN-I and IL15 in the generation of vast and effective antitumor CTL responses using peptide and poly-IC vaccines.

A 12-Day Course of Imiquimod 5% for the Treatment of Actinic Keratosis: Effectiveness and Local Reactions. / Tratamiento con imiquimod al 5% durante 12 días para las queratosis actínicas: estudio de la eficacia y la reacción local.

INTRODUCTION AND OBJECTIVES: Imiquimod is an excellent option for patients with actinic keratosis, although its use may be limited by the long course of treatment required (4 weeks) and the likelihood of local skin reactions. The objectives of the present study were to demonstrate the effectiveness of a 12-day course of imiquimod 5% for the treatment of actinic keratosis and to examine the association between treatment effectiveness and severity of local reactions. PATIENTS AND METHODS: We included patients with at least 8 actinic keratoses treated with imiquimod 5% cream for 12 consecutive days. Local reactions were classified as mild, moderate, or severe. The statistical analysis of the association between local reactions and clinical response was based on the Pearson χ2 test and the Spearman rank correlation test. RESULTS: Sixty-five patients completed the study. Complete response was recorded in 52.3% and partial response in 75.4%. We found a statistically significant association between severity of the local reaction and response to treatment in both the Pearson χ2 test and the Spearman rank correlation test. CONCLUSIONS: A 12-day course of imiquimod 5% proved effective for the treatment of actinic keratosis. Severity of local reactions during treatment was correlated with clinical response.

NY-ESO-1 Vaccination in Combination with Decitabine Induces Antigen-Specific T-lymphocyte Responses in Patients with Myelodysplastic Syndrome.

Purpose: Treatment options are limited for patients with high-risk myelodysplastic syndrome (MDS). The azanucleosides, azacitidine and decitabine, are first-line therapy for MDS that induce promoter demethylation and gene expression of the highly immunogenic tumor antigen NY-ESO-1. We demonstrated that patients with acute myeloid leukemia (AML) receiving decitabine exhibit induction of NY-ESO-1 expression in circulating blasts. We hypothesized that vaccinating against NY-ESO-1 in patients with MDS receiving decitabine would capitalize upon induced NY-ESO-1 expression in malignant myeloid cells to provoke an NY-ESO-1-specific MDS-directed cytotoxic T-cell immune response.Experimental Design: In a phase I study, 9 patients with MDS received an HLA-unrestricted NY-ESO-1 vaccine (CDX-1401 + poly-ICLC) in a nonoverlapping schedule every four weeks with standard-dose decitabine.Results: Analysis of samples serially obtained from the 7 patients who reached the end of the study demonstrated induction of NY-ESO-1 expression in 7 of 7 patients and NY-ESO-1-specific CD4+ and CD8+ T-lymphocyte responses in 6 of 7 and 4 of 7 of the vaccinated patients, respectively. Myeloid cells expressing NY-ESO-1, isolated from a patient at different time points during decitabine therapy, were capable of activating a cytotoxic response from autologous NY-ESO-1-specific T lymphocytes. Vaccine responses were associated with a detectable population of CD141Hi conventional dendritic cells, which are critical for the uptake of NY-ESO-1 vaccine and have a recognized role in antitumor immune responses.Conclusions: These data indicate that vaccination against induced NY-ESO-1 expression can produce an antigen-specific immune response in a relatively nonimmunogenic myeloid cancer and highlight the potential for induced antigen-directed immunotherapy in a group of patients with limited options. Clin Cancer Res; 24(5); 1019-29. ©2017 AACRSee related commentary by Fuchs, p. 991.

[Clinical efficacy of the immunomodulatory agent cycloferon (tablets) in viral respiratory infections: Results of a systematic review and meta-analysis]. / Klinicheskaia éffektivnost' immunomoduliatora tsikloferona (tabletki) pri virusnykh infektsiiakh organov dykhaniia: rezul'taty sistematicheskogo obzora i metaanaliza.

The authors carried out a systematic review and subsequent meta-analysis of randomized clinical trials evaluating the efficacy of the immunomodulator agent cycloferon as tablets in adults and children with viral respiratory diseases. A total estimate of its clinical efficacy was obtained in terms of compared heterogeneous groups and response variables. The data published in 16 articles were used to calculate the formal parameters of the clinical efficacy of cycloferon (increased absolute and relative benefits, odds ratio (OR); the number of patients needed to be additionally treated with cycloferon to achieve a favorable outcome or to prevent a poor outcome in one patient, etc.). High heterogeneity hampered the unequivocal interpretation of results; however, combining the compared homogeneous groups in the meta-analysis (with adjustments for fixed and random effects) increased the statistical power of the investigation. In children aged 6 to 18 years, the OR for the positive effect of the drug (no new cases after its preventive administration) was 5.3 (95% confidence interval (CI), 4.8-5.9), heterogeneity test, χ2 = 249.5; p=0.000...; I2 = 94.8% (95% CI, 92.7-96.3%). This suggested the heterogeneity of clinical trial data and extrapolated this estimate to medical practice. The use of cycloferon in adults to treat acute respiratory viral infection enhanced their chances of enduring the disease in a mild form and avoiding serious complications: the OR for positive outcomes was 9.7 (95% CI, 7.0-13.0), while the effect was more homogeneous than in children (heterogeneity test, χ2 = 7.4; p=0.061...; I2 = 59.4% (95% CI, 0-86.5). Thus, the use of cycloferon to treat and prevent acute viral respiratory infections showed a more than 5-fold increase in the probability of avoiding the disease or enduring the latter in a mild form.

[The role of local immunotherapy in the treatment of the patients presenting with virus-associated allergic rhinitis]. / Lokal'naia immunoterapiia v lechenii patsientov s virus-assotsiirovannym allergicheskim rinitom.

The objective of the present study was to evaluate the clinical effectiveness of intranasal immunotherapy with the use of сycloferon in the patients presenting with seasonal virus-associated allergic rhinitis during the period of pollination. The results of the treatment of 50 patients with virus-associated allergic rhinitis (i.e. those allergic to the sagebrush pollen) were analyzed. The patients were divided into two groups depending on the therapeutic modality being used. Those comprising the main group (21 patients) received immunotherapy with сycloferon in addition to allergen-specific immunotherapy (by sublingual administration). A cycloferon solution in the physiological saline (2:1) at a concentration of 125 mg/ml was applied to the intranasal mucous membrane with the use of a nebulizer. The inhalation was performed every other day as a course of 10 procedures at a total cycloferon dose of 1250 mg. The control group was composed of 29 patients given sublingual allergen-specific immunotherapy alone. The study included the evaluation of the patients' quality of life in accordance with the adapted and modified RQLQ questionnaire. In addition, the demand for other relevant medications following the course of intranasal immunotherapy was determined by analyzing the data extracted from the self-monitoring diaries and questionnaires. The study has shown that the inclusion of сycloferon therapy in the scheme for the management of the patients presenting with virus-associated allergic rhinitis during the period of pollination enhanced the effectiveness of the treatment as assessed from the improvement of the patients' quality of life that correlated with the reduction in the severity of rhino-conjunctival manifestations of the disease (r= -0.4, p<0.05). It is concluded that the intranasal administration of the immunomodulator сycloferon has resulted in a significant decrease of requirements for chromones (p<0.001) and antihistamines (p<0.05) among the patients suffering from allergic rhinitis.

Prophylactic and therapeutic intranasal administration with an immunomodulator, Hiltonol® (Poly IC:LC), in a lethal SARS-CoV-infected BALB/c mouse model.

Hiltonol®, (Poly IC:LC), a potent immunomodulator, is a synthetic, double-stranded polyriboinosinic-polyribocytidylic acid (poly IC) stabilized with Poly-L-lysine and carboxymethyl cellulose (LC). Hiltonol® was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. Hiltonol® at 5, 1, 0.5 or 0.25 mg/kg/day by intranasal (i.n.) route resulted in significant survival benefit when administered at selected times 24 h prior to challenge with a lethal dose of mouse-adapted severe acute respiratory syndrome coronavirus (SARS-CoV). The infected BALB/c mice receiving the Hiltonol® treatments were also significantly effective in protecting mice against weight loss due to infection (p < 0.001). Groups of 20 mice were dosed with Hiltonol® at 2.5 or 0.75 mg/kg by intranasal instillation 7, 14, and 21 days before virus exposure and a second dose was given 24 h later, prophylactic Hiltonol® treatments (2.5 mg/kg/day) were completely protective in preventing death, and in causing significant reduction in lung hemorrhage scores, lung weights and lung virus titers. Hiltonol® was also effective as a therapeutic when give up to 8 h post virus exposure; 100% of the-infected mice were protected against death when Hiltonol® was administered at 5 mg/kg/day 8 h after infection. Our data suggest that Hiltonol® treatment of SARS-CoV infection in mice leads to substantial prophylactic and therapeutic effects and could be used for treatment of other virus disease such as those caused by MERS-CoV a related coronavirus. These properties might be therapeutically advantageous if Hiltonol® is considered for possible clinical use.

Self-administered interventions for anogenital warts in immunocompetent patients: a systematic review and meta-analysis.

BACKGROUND: Anogenital warts (AGWs, condylomata acuminata) are among the most common STIs and may severely impact quality of life (QoL). Available treatment options are characterised by a high proportion of non-responders and recurrences. OBJECTIVE: To systematically review and meta-analyse the available evidence from randomised controlled trials (RCTs) on topical treatments for AGWs considering short-term and long-term efficacy, effects on QoL and adverse events (AE). METHODS: A comprehensive literature search was performed in Cochrane Central Register of Controlled Trials, Embase and MEDLINE. Included studies were evaluated with the Cochrane Collaboration's risk of bias tool. The confidence in the pooled effect estimates was evaluated according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach and categorised as 'very low', 'low', 'moderate' or 'high'. RESULTS: Eighteen RCTs met the inclusion criteria. Regarding complete clearance (CC), imiquimod 3.75% and 5% cream, podophyllotoxin 0.5% solution and gel and polyphenon E 10% and 15% ointment were superior to placebo. Although more local AE and pain occurred in the actively treated groups, differences regarding dropouts due to AE were not statistically significant. For podophyllotoxin 0.15% cream, no placebo-controlled trials were available; however, in an active-controlled trial, it was inferior to podophyllotoxin 0.5% solution with respect to CC. No significant differences were detected between imiquimod 5% cream and podophyllotoxin 0.5% solution and between polyphenon E 10% and 15% ointment. No data on the influence on health-related QoL were available. CONCLUSION: Our confidence in the pooled estimates (GRADE quality of the evidence) ranged from very low to high. Apart from the given results, other aspects such as availability, costs or patient preference have to be considered when making a treatment choice. Due to the limited number of direct comparisons, conclusions on the relative efficacy of the different treatment options are restricted.

Fractional Laser-Assisted Topical Imiquimod 5% Cream Treatment for Recalcitrant Common Warts in Children: A Pilot Study.

BACKGROUND: Conventional treatments for warts like cryotherapy are limited by the pain during procedures, especially in pediatric patients. Imiquimod is a topical immune response modifier, but the thick stratum corneum of common warts prevents drug permeation through skin. OBJECTIVE: To evaluate the efficacy and safety of fractional laser/topical 5% imiquimod cream for the treatment of warts in children. METHODS: Eleven pediatric patients with multiple recalcitrant common warts were included. Lesions were treated using an ablative fractional 2,940-nm Er:YAG laser at 1- or 2-week interval. After each laser treatment session, imiquimod 5% cream was self-applied once daily 5 days a week. Response and adverse effects were assessed 2 weekly until complete clearance or up to maximum of 48 weeks. Pain during fractional laser was assessed using a visual analogue scale (0-10). RESULTS: Eight of the 11 (72.7%) children experienced complete clearance. Mean duration was 29.7 (16-48) weeks, and the mean number of fractional laser was 17.5 (8-37). No significant adverse effect was observed. Pain visual analogue scale during fractional laser was 2.4 (1-4) compared to 6.2 (5-8) during cryotherapy. CONCLUSION: This pilot study indicates that fractional laser-assisted topical imiquimod may provide benefit for recalcitrant warts in children.

Immune responses and outcome after vaccination with glioma-associated antigen peptides and poly-ICLC in a pilot study for pediatric recurrent low-grade gliomas.

BACKGROUND: Low-grade gliomas (LGGs) are the most common brain tumors of childhood. Although surgical resection is curative for well-circumscribed superficial lesions, tumors that are infiltrative or arise from deep structures are therapeutically challenging, and new treatment approaches are needed. Having identified a panel of glioma-associated antigens (GAAs) overexpressed in these tumors, we initiated a pilot trial of vaccinations with peptides for GAA epitopes in human leukocyte antigen-A2+ children with recurrent LGG that had progressed after at least 2 prior regimens. METHODS: Peptide epitopes for 3 GAAs (EphA2, IL-13Rα2, and survivin) were emulsified in Montanide-ISA-51 and administered subcutaneously adjacent to intramuscular injections of polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose every 3 weeks for 8 courses, followed by booster vaccines every 6 weeks. Primary endpoints were safety and T-lymphocyte responses against GAA epitopes. Treatment response was evaluated clinically and by MRI. RESULTS: Fourteen children were enrolled. Other than grade 3 urticaria in one child, no regimen-limiting toxicity was encountered. Vaccination induced immunoreactivity to at least one vaccine-targeted GAA in all 12 evaluable patients: to IL-13Rα2 in 3, EphA2 in 11, and survivin in 3. One child with a metastatic LGG had asymptomatic pseudoprogression noted 6 weeks after starting vaccination, followed by dramatic disease regression with >75% shrinkage of primary tumor and regression of metastatic disease, persisting >57 months. Three other children had sustained partial responses, lasting >10, >31, and >45 months, and one had a transient response. CONCLUSIONS: GAA peptide vaccination in children with recurrent LGGs is generally well tolerated, with preliminary evidence of immunological and clinical activity.

[USING INTERFERON INDUCTORS IN THERAPY OF PRE-SCHOOL-AGE PATIENTS WITH REACTIVATED EPSTEIN - BARR VIRUS INFECTION.]

The efficacy of including interferon inductor - meglumine acridone acetate (cycloferon) - into a complex therapy of pre-school-age patients with reactivated Epstein - Barr virus infection in the form of mononucleosis has been assessed. It is established that the use of cycloferon leads to regression of the main clinical-hematological symptoms of disorder during the progression period (with a decrease in the clinical treatment period on the average by 4.39 bed days) and favors smoother course of the restoration period. The maximum effect was achieved using prolonged cycloferon treatment schedule, which led to more pronounced relief of residual phenomena, decreased recurrence frequency, and number of acute respiratory complications as comparted to a short-term treatment and control groups.

[EFFICACY OF CYCLOFERON LINIMENT IN THE COMBINED TREATMENT OF CHRONIC GINGIVITIS IN PATIENTS WITH CHRONIC INFECTIOUS DISEASES].

In order to study the clinical-pathogenetic efficacy of using cycloferon liniment in the combined therapy of patients with gingivitis on the background of chronic infectious diseases (HIV infection, hepatitis C, brucellosis), medical examination and treatment of 42 patients with this diagnosis has been carried out. It is established, that the use of cycloferon liniment in the combined therapy decreases the infection load in periodontal recess and manifestation of local inflammation, normalizes the immunity indices, and reduces the level of endogenous intoxication. All these factors provide acceleration of the recuperation processes and decrease the frequency of recidivating.

[ASSESSMENT OF INDICATORS FOR INTERFERON SYSTEM IN PATIENTS WITH COMMON FORMS OF PULMONARY TUBERCULOSIS RECEIVING COMPLEX THERAPY WITH CYCLOFERON].

We have evaluated the efficacy of cycloferon inclusion in the complex therapy of newly diagnosed patients with common forms of pulmonary tuberculosis, based on monitoring of the number of monocytes with receptors to interferon-gamma (flow cytometry) and the concentration of interferon-gamma in the serum (ELISA). For this purpose, a group of 36 patients (18 patients received standard chemotherapy, and 18 additionally received 600 mg cycloferon tablets 3 times per week) was examined for 3 months. Control group consisted of 18 apparently healthy patients. The study did not include patients with multiple or extensively drug-resistant M. tuberculosis strains. The analysis of results showed a statistically significant positive dynamics of the level of monocyte receptors to interferon-gamma in patients receiving cycloferon as manifested by an increase in their number in the first 2 months of therapy (period of clinical manifestations of the disease), followed by a decrease in the 3rd months of treatment, which corresponds to clinical improvement, in contrast to patients treated with standard chemotherapy alone.

Molecular cloning and expression analysis of interferon stimulated gene 15 (ISG15) in turbot, Scophthalmus maximus.

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by double-stranded RNA (polyinosinic: polycytidylic acid, poly I:C) and viral infection. In this study, we described the nucleotide, mRNA tissue distribution and regulation of an ISG15 gene from turbot, Scophthalmus maximus (SmISG15). SmISG15 gene is 862 bp in length, composed of two exons and one intron, and encodes 158 amino acids. The deduced protein exhibits the highest homology (44.7-71.2% identity) with ISG15s from other fishes and possesses two conserved tandem ubiquitin-like (UBL) domains and a C-terminal RLRGG conjugating motif known to be important for the functions of ISG15s in vertebrates. Phylogenetic analysis grouped SmISG15 into fish ISG15. SmISG15 mRNA was constitutively expressed in all tissues examined, with higher levels observed in immune organs. Gene expression analysis was performed for SmISG15 in the spleen, head kidney, gills and muscle of turbots challenged with poly I:C or turbot reddish body iridovirus (TRBIV) over a 7-day time course. The result showed that SmISG15 was upregulated by both stimuli in all four tissues, with induction by poly I:C apparently stronger and initiated more quickly. A two-wave induced expression of SmISG15 was seen in the spleen, head kidney and gills, suggesting an induction of SmISG15 either by IFN-dependent or -independent pathway. These results provide insights into the roles of fish ISG15 in antiviral immunity.

Prior chronic stress induces persistent polyI:C-induced allodynia and depressive-like behavior in rats: Possible involvement of glucocorticoids and microglia.

When animals suffer from viral infections, they develop a set of symptoms known as the "sickness response." Recent studies suggest that psychological stress can modulate the sickness response. However, it remains uncertain whether acute and chronic psychosocial stresses have the same effect on viral infection-induced sickness responses. To address this question, we compared changes in polyI:C-induced sickness responses, such as fever, change of body weight and food intake, mechanical allodynia, and depressive-like behavior, in rats that had been pre-exposed to single and repeated social defeat stresses. Intraperitoneal injection of polyI:C induced a maximal fever of 38.0°C 3h after injection. Rats exposed to prior social defeat stress exhibited blunted febrile responses, which were more pronounced in the repeated stress group. Furthermore, only the repeated stress group showed late-onset and prolonged mechanical allodynia lasting until 8days after injection in the von Frey test and prolonged immobility time in the forced swim test 9days post-injection. To assess the role of glucocorticoids and microglia in the delayed and persistent development of these sickness responses in rats exposed to repeated stress, we investigated the effect of pretreatment with RU486, a glucocorticoid receptor antagonist, and minocycline, an inhibitor of microglial activation, on polyI:C-induced allodynia and depressive-like behavior. Pretreatment with either drug inhibited both the delayed allodynia and depressive-like behavior. The present study demonstrates that repeated, but not single, social defeat stress followed by systemic polyI:C administration induced prolonged allodynia and depressive-like behavior in rats. Our results show that even though a single-event psychosocial stress does not have any effect by itself, animals may develop persistent allodynia and depressive-like behavior when they suffer from an infectious disease if they are pre-exposed to repeated or chronic psychosocial stress. Furthermore, this study suggests that stress-induced corticosterone and microglial activation play a pivotal role in this phenomenon.

Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation.

The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In preclinical and clinical studies adjuvants based on mineral oils (such as incomplete Freund's adjuvant (IFA) and Montanide) are used to create a sustained release depot at the injection site. While the depot effect of mineral oils is important for induction of robust immune responses, their administration is accompanied with severe adverse and long lasting side effects. In order to develop an alternative for IFA family of adjuvants, polymeric nanoparticles (NPs) based on hydrophilic polyester (poly(d,l lactic-co-hydroxymethyl glycolic acid) (pLHMGA)) were prepared. These NPs were loaded with a synthetic long peptide (SLP) derived from HPV16 E7 oncoprotein and a toll like receptor 3 (TLR3) ligand (poly IC) by double emulsion solvent evaporation technique. The therapeutic efficacy of the nanoparticulate formulations was compared to that of HPV SLP+poly IC formulated in IFA. Encapsulation of HPV SLP antigen in NPs substantially enhanced the population of HPV-specific CD8+ T cells when combined with poly IC either co-encapsulated with the antigen or in its soluble form. The therapeutic efficacy of NPs containing poly IC in tumor eradication was equivalent to that of the IFA formulation. Importantly, administration of pLHMGA nanoparticles was not associated with adverse effects and therefore these biodegradable nanoparticles are excellent substitutes for IFA in cancer vaccines.