Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Immunostimulatory bacterial antigen-armed oncolytic measles virotherapy significantly increases the potency of anti-PD1 checkpoint therapy.

Clinical immunotherapy approaches are lacking efficacy in the treatment of glioblastoma (GBM). In this study, we sought to reverse local and systemic GBM-induced immunosuppression using the Helicobacter pylori neutrophil-activating protein (NAP), a potent TLR2 agonist, as an immunostimulatory transgene expressed in an oncolytic measles virus (MV) platform, retargeted to allow viral entry through the urokinase-type plasminogen activator receptor (uPAR). While single-agent murine anti-PD1 treatment or repeat in situ immunization with MV-s-NAP-uPA provided modest survival benefit in MV-resistant syngeneic GBM models, the combination treatment led to synergy with a cure rate of 80% in mice bearing intracranial GL261 tumors and 72% in mice with CT-2A tumors. Combination NAP-immunovirotherapy induced massive influx of lymphoid cells in mouse brain, with CD8+ T cell predominance; therapeutic efficacy was CD8+ T cell dependent. Inhibition of the IFN response pathway using the JAK1/JAK2 inhibitor ruxolitinib decreased PD-L1 expression on myeloid-derived suppressor cells in the brain and further potentiated the therapeutic effect of MV-s-NAP-uPA and anti-PD1. Our findings support the notion that MV strains armed with bacterial immunostimulatory antigens represent an effective strategy to overcome the limited efficacy of immune checkpoint inhibitor-based therapies in GBM, creating a promising translational strategy for this lethal brain tumor.

Effects of trans-resveratrol on type 1 diabetes-induced up-regulation of apoptosis and mitogen-activated protein kinase signaling in retinal pigment epithelium of Dark Agouti rats.

Microvascular changes and retinal degeneration precede diabetic retinopathy. Oxidative stress alters several intracellular signaling pathways, which form the basis of diabetic retinopathy. Many antioxidants have been investigated as possible preventive and therapeutic remedies for diabetic retinopathy. The current study investigated the modulatory effects of trans-resveratrol on streptozotocin-induced type 1 diabetes mediated changes in the transcription and levels of apoptosis-related proteins and mitogen-activated protein kinases (MAPKs) in the retinal pigment epithelium (RPE) of adult male dark Agouti rats. In control rats, 5 mg/kg/d trans-resveratrol administration for 30 days increased gene expressions of tumor suppressor protein 53, Bcl2-associated X protein, B-cell lymphoma-2 (Bcl2), Caspase-3 (CASP3), CASP8 and CASP9, p38αMAPK, c-Jun N-terminal kinase-1 (JNK1), and extracellular signal-regulated kinase-1 (ERK1). On the other hand, diabetes decreased gene expressions of CASP3, CASP8, p38αMAPK, JNK, and ERK1. Trans-resveratrol reversed the inhibited gene expressions of CASP8, p38αMAPK, JNK, and ERK1 to normal control levels in diabetic rats. Trans-resveratrol normalized diabetes-induced upregulation of CASP3 and -9, cytochrome-c, Bcl-2, and ERK1 proteins. In conclusion, Trans-resveratrol-induced alterations in gene expressions do not seem to affect RPE functions as they do not reflect as altered protein functions. Trans-resveratrol imparts its protective effects by normalizing apoptosis-related proteins and ERK1 but does not affect JNK proteins. Trans-resveratrol causes cytostasis in RPE of normal rats by upregulating Bcl2 protein and apoptotic proteins.

Prophylactic efficacy against Mycobacterium tuberculosis using ID93 and lipid-based adjuvant formulations in the mouse model.

An estimated 10 million people developed tuberculosis (TB) disease in 2019 which underscores the need for a vaccine that prevents disease and reduces transmission. The aim of our current studies is to characterize and test a prophylactic tuberculosis vaccine comprised of ID93, a polyprotein fusion antigen, and a liposomal formulation [including a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant, GLA) and QS-21] in a preclinical mouse model of TB disease. Comparisons of the ID93+GLA-LSQ vaccines are also made to the highly characterized ID93+GLA-SE oil-in-water emulsion adjuvant, which are also included these studies. The recent success of vaccine candidate M72 combined with adjuvant AS01E (GlaxoSmithKline Biologicals) in reducing progression to active disease is promising and has renewed excitement for experimental vaccines currently in the TB vaccine pipeline. The AS01E adjuvant contains monophosphoryl lipid A (MPL) and QS-21 (a saponin) in a liposomal formulation. While AS01E has demonstrated potent adjuvant activity as a component of both approved and experimental vaccines, developing alternatives to this adjuvant system will become important to fill the high demand envisioned for future vaccine needs. Furthermore, replacement sources of potent adjuvants will help to supply the demand of a TB vaccine [almost one-quarter of the world's population are estimated to have latent Mycobacterium tuberculosis (Mtb) according to the WHO 2019 global TB report], addressing (a) cost of goods, (b) supply of goods, and (c) improved efficacy of subunit vaccines against Mtb. We show that both ID93+GLA-SE (containing an emulsion adjuvant) and ID93+GLA-LSQ (containing a liposomal adjuvant) induce ID93-specific TH1 cellular immunity including CD4+CD44+ T cells expressing IFNγ, TNF, and IL-2 (using flow cytometry and intracellular cytokine staining) and vaccine-specific IgG2 antibody responses (using an ELISA). In addition, both ID93+GLA-SE and ID93+GLA-LSQ effectively decrease the bacterial load within the lungs of mice infected with Mtb. Formulations based on this liposomal adjuvant formulation may provide an alternative to AS01 adjuvant systems.

Inhibitory Effects of a Reengineered Anthrax Toxin on Canine Oral Mucosal Melanomas.

Canine oral mucosal melanomas (OMM) are the most common oral malignancy in dogs and few treatments are available. Thus, new treatment modalities are needed for this disease. Bacillus anthracis (anthrax) toxin has been reengineered to target tumor cells that express urokinase plasminogen activator (uPA) and metalloproteinases (MMP-2), and has shown antineoplastic effects both, in vitro and in vivo. This study aimed to evaluate the effects of a reengineered anthrax toxin on canine OMM. Five dogs bearing OMM without lung metastasis were included in the clinical study. Tumor tissue was analyzed by immunohistochemistry for expression of uPA, uPA receptor, MMP-2, MT1-MMP and TIMP-2. Animals received either three or six intratumoral injections of the reengineered anthrax toxin prior to surgical tumor excision. OMM samples from the five dogs were positive for all antibodies. After intratumoral treatment, all dogs showed stable disease according to the canine Response Evaluation Criteria in Solid Tumors (cRECIST), and tumors had decreased bleeding. Histopathology has shown necrosis of tumor cells and blood vessel walls after treatment. No significant systemic side effects were noted. In conclusion, the reengineered anthrax toxin exerted inhibitory effects when administered intratumorally, and systemic administration of this toxin is a promising therapy for canine OMM.

AMD3100 Augments the Efficacy of Mesothelin-Targeted, Immune-Activating VIC-008 in Mesothelioma by Modulating Intratumoral Immunosuppression.

AMD3100 (plerixafor), a CXCR4 antagonist, has been demonstrated to suppress tumor growth and modulate intratumoral T-cell trafficking. However, the effect of AMD3100 on immunomodulation remains elusive. Here, we explored immunomodulation and antitumor efficacy of AMD3100 in combination with a previously developed mesothelin-targeted, immune-activating fusion protein, VIC-008, in two syngeneic, orthotopic models of malignant mesothelioma in immunocompetent mice. We showed that combination therapy significantly suppressed tumor growth and prolonged animal survival in two mouse models. Tumor control and survival benefit were associated with enhanced antitumor immunity. VIC-008 augmented mesothelin-specific CD8+ T-cell responses in the spleen and lymph nodes and facilitated intratumoral lymphocytic infiltration. However, VIC-008 treatment was associated with increased programmed cell death protein-1 (PD-1) expression on intratumoral CD8+ T cells, likely due to high CXCL12 in the tumor microenvironment. AMD3100 alone and in combination with VIC-008 modulated immunosuppression in tumors and the immune system through suppression of PD-1 expression on CD8+ T cells and conversion of regulatory T cells (Tregs) into CD4+CD25-Foxp3+IL2+CD40L+ helper-like cells. In mechanistic studies, we demonstrated that AMD3100-driven Treg reprogramming required T cell receptor (TCR) activation and was associated with loss of PTEN due to oxidative inactivation. The combination of VIC-008 augmentation of tumor-specific CD8+ T-cell responses with AMD3100 abrogation of immunosuppression conferred significant benefits for tumor control and animal survival. These data provide new mechanistic insight into AMD3100-mediated immunomodulation and highlight the enhanced antitumor effect of AMD3100 in combination with a tumor antigen-targeted therapy in mouse malignant mesothelioma, which could be clinically relevant to patients with this difficult-to-treat disease. Cancer Immunol Res; 6(5); 539-51. ©2018 AACR.

Immunization with an adenovirus-vectored TB vaccine containing Ag85A-Mtb32 effectively alleviates allergic asthma.

Current treatments for allergic asthma primarily ameliorate symptoms rather than inhibit disease progression. Regulating the excessive T helper type 2 (Th2) responses may prevent asthma exacerbation. In this study, we investigated the protective effects of Ad5-gsgAM, an adenovirus vector carrying two mycobacterial antigens Ag85A and Mtb32, against allergic asthma. Using an ovalbumin (OVA)-induced asthmatic mouse model, we found that Ad5-gsgAM elicited much more Th1-biased CD4+T and CD8+T cells than bacillus Calmette-Guérin (BCG). After OVA challenge, Ad5-gsgAM-immunized mice showed significantly lowered airway inflammation in comparison with mice immunized with or without BCG. Total serum immunoglobulin E and pulmonary inducible-nitric-oxide-synthase were efficiently reduced. The cytokine profiles in bronchial-alveolar-lavage-fluids (BALFs) were also modulated, as evidenced by the increased level of interferon-γ (IFN-γ) and the decreased level of interleukin (IL)-4, IL-5, and IL-13. Anti-inflammatory cytokine IL-10 was sharply increased, whereas pro-inflammatory cytokine IL-33 was significantly decreased. Importantly, exogenous IL-33 abrogated the protective effects of Ad5-gsgAM, revealing that the suppression of IL-33/ST2 axis substantially contributed to protection against allergic inflammation. Moreover, regulatory T cells were essential for regulating aberrant Th2 responses as well as IL-33/ST2 axis. These results suggested that modulating the IL-33/ST2 axis via adenovirus-vectored mycobacterial antigen vaccination may provide clinical benefits in allergic inflammatory airways disease. KEY MESSAGES: •Ad5-gsgAM elicits Th1 responses and suppresses Th2-mediated allergic asthma in mice. •Ad5-gsgAM inhibits IL-33/ST2 axis by reducing IL-33 secretion but not ILC2 recruiting. •Treg is essential for modulating Th2 responses and IL-33/ST2 axis by Ad5-gsgAM.

A Multistage Subunit Vaccine Effectively Protects Mice Against Primary Progressive Tuberculosis, Latency and Reactivation.

Adult tuberculosis (TB) is the main cause of TB epidemic and death. The infection results mainly by endogenous reactivation of latent TB infection and secondarily transmitted by exogenous infection. There is no vaccine for adult TB. To this end, we first chose antigens from a potential antigenic reservoir. The antigens strongly recognized T cells from latent and active TB infections that responded to antigens expressed by Mycobacterium tuberculosis cultured under different metabolic states. Fusions of single-stage polyprotein CTT3H, two-stage polyprotein A1D4, and multistage CMFO were constructed. C57BL/6 mice vaccinated with DMT adjuvant ed CMFO (CMFO-DMT) were protected more significantly than by CTT3H-DMT, and efficacy was similar to that of the only licensed vaccine, Bacillus Calmette-Guérin (BCG) and A1D4-DMT in the M. tuberculosis primary infection model. In the setting of BCG priming and latent TB infection, M. tuberculosis in the lung and spleen was eliminated more effectively in mice boosted with CMFO-DMT rather than with BCG, A1D4-DMT, or CTT3H-DMT. In particular, sterile immunity was only conferred by CMFO-DMT, which was associated with expedited homing of interferon-gamma+CD4+ TEM and interleukin-2+ TCM cells from the spleen to the infected lung. CMFO-DMT represents a promising candidate to prevent the occurrence of adult TB through both prophylactic and therapeutic methods, and warrants assessment in preclinical and clinical trials.

Solid tumor therapy by selectively targeting stromal endothelial cells.

Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors.

[THE CORRECTION OF THE LOCAL IMMUNE RESPONSE AT THE PATIENTS WITH HELICOBACTER PYLORI INFECTION].

INTRODUCTION: Helicobacter pylori infection is due to the high prevalence in population attracts the clinical interest of researchers in the whole World. It is well known that this microorganism not only resides in the mucosa of the gastrointestinal tract, but is also defined in the periodontal pocket of the oral cavity. THE AIM OF INVESTIGATION: to evaluate Helicobacter pylori diagnostics in the mouth and prove a method of relief of the inflammatory process by applying immunomodulator Imudon. RESULTS. On the basis of obtained results it was found that the inclusion of topical immunomodulator Imudon in the complex therapy of Helicobacter pylori-associated diseases leads to reduction of inflammatory potential through the decrease of the TNFα, IL-6 activity in saliva and to increase the protective properties of saliva as a result of increased levels of mucin, significantly reduces the frequency of relapses in the one year after therapy. CONCLUSION: It is practically important to determine the effectiveness of eradication therapy by the study of the contents of the tooth-gingival pocket for the detection of genetic material of Helicobacter pylori, as well as to include in the complex therapy of Helicobacter pylori-associated diseases of the immune modulator Imudon.

A commercially available preparation of Staphylococcus aureus bio-products potently inhibits tumour growth in a murine model of mesothelioma.

BACKGROUND AND OBJECTIVE: Mesothelioma is an incurable cancer with a rising global incidence. Intrapleural delivery of a commercially available compound made up of proteins produced by Staphylococcus aureus has been used clinically to induce pleurodesis. We investigate if this bacterial compound has anti-tumoural activities against pleural malignancies, in addition to its pleurodesing effect. METHODS: The effects of the treatment on mesothelioma cells were evaluated in vitro and further tested in two validated murine models. RESULTS: This S. aureus bio-product mixture effectively kills mesothelioma cells and induces the release of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and vascular endothelial growth factor from primary human mesothelial cells but not malignant pleural mesothelioma cells in vitro. Intratumoural delivery of the treatment in BALB/c mice induced tumour necrosis and local activation of T cells. Tumour growth was significantly inhibited in the treatment group during and after the treatment period (size of tumour 58.8 ± 10.3 mm(2) vs 118.3 ± 6.7 mm(2) from saline controls at day 23, n = 9-12 per group), P < 0.001. Tumour growth resumed on cessation of treatment, confirming the inhibition was treatment related. Treatment benefits were further validated in an orthotopic peritoneal model of mesothelioma and the compound significantly reduced the mesothelioma load (P < 0.05 vs saline controls). Mice in the treatment group had a significant increase in the percentage of activated CD4(+) and CD8(+) T cells in tumour-draining lymph nodes. No histological side-effects were observed with the treatment. CONCLUSIONS: This proof-of-principle study demonstrates promising antitumoural activity of a commercially available compound of S. aureus bio-products against mesothelioma.

Stimulation and analysis of the immune response in calves from vaccinated pregnant cows.

The effect of vaccinating pregnant cows with an inactivated vaccine against Mannheimia haemolytica, BRSV and PI3V infections on selected immune responses in their offspring was examined. Blood samples were collected weekly for 12 weeks from six newborn calves from each of vaccinated (experimental) and unvaccinated (control) dams. Specific antibodies to M. haemolytica, BRSV and PI3V and mean values of IgA, IgG concentrations were significantly higher in the experimental calves compared with the controls. However, specific antibody titres to adenovirus type 3, BHV1 and BVDV in the experimental calves had constant levels while the control group levels changed. The IgM, Hp and SAA concentrations generally increased until week 8 in the experimental group, but the control group titres became higher after week 9. This study demonstrates that specific immunisation of cows pre-partum significantly stimulated parameters associated with immunity and it also controlled the acute phase response intensity in their offspring. Therefore the vaccination of dams may provide additional antibody protection against infection to their offspring.

[Effect of Immunovac-VP-4 and Kagocel immunotherapy on cytokine levels in patients with erythema nodosum].

Fixed erythema--a kind of clinical and histopathologic reaction, fixed drug eruption. The purpose of the study--the study of characteristics of the cytokine profile in patients with erythema and the dynamics of the basal levels of proinflammatory and antiinflammatory cytokines during immunotherapy. All 41 patients with fixed erythema at baseline and after treatment was carried out determination of levels of pro-, anti-inflammatory and regulatory cytokines in the serum by ELISA using test systems "Biosource" (Austria). In patients with fixed erythema Immunovac treatment increased serum IFN-gamma (p < 0.05), IL-1beta (p > 0.05), IL-6. While Kagocel led to an increase in IFN-gamma (p < 0.05), IL-1beta, IL-6 and reduction of TGF-beta (p < 0.05). At the same time in patients with fixed erythema basic therapy contributed to the significant increase in TGF-â and decrease in IL-10. Immunovac-VP-4 had the highest activity for the induction of IFN-gamma. Inclusion in the range of therapeutic and prophylactic measures in patients with fixed erythema immunomodulators promotes activation links innate and adaptive immunity triggers mechanisms, thus increasing the antiviral response in patients with erythema.

Receptor-directed chimeric toxins created by sortase-mediated protein fusion.

Chimeric protein toxins that act selectively on cells expressing a designated receptor may serve as investigational probes and/or antitumor agents. Here, we report use of the enzyme sortase A (SrtA) to create four chimeric toxins designed to selectively kill cells bearing the tumor marker HER2. We first expressed and purified: (i) a receptor recognition-deficient form of diphtheria toxin that lacks its receptor-binding domain and (ii) a mutated, receptor-binding-deficient form of anthrax-protective antigen. Both proteins carried at the C terminus the sortase recognition sequence LPETGG and a H6 affinity tag. Each toxin protein was mixed with SrtA plus either of two HER2-recognition proteins--a single-chain antibody fragment or an Affibody--both carrying an N-terminal G5 tag. With wild-type SrtA, the fusion reaction between the toxin and receptor-recognition proteins approached completion only after several hours, whereas with an evolved form of the enzyme, SrtA*, the reaction was virtually complete within 5 minutes. The four fusion toxins were purified and shown to kill HER2-positive cells in culture with high specificity. Sortase-mediated ligation of binary combinations of diverse natively folded proteins offers a facile way to produce large sets of chimeric proteins for research and medicine.

Prevention of recurrent urinary tract infections in women.

Around 40-50% of women experience at least one urinary tract infection (UTI) during their lifetime and 20-30% of these have a recurrence within 3-4 months of the initial infection.¹ Recurrent UTI (usually defined as three episodes in the last 12 months or two episodes in the last 6 months) can have a considerable impact on a woman's quality of life. Each episode of acute UTI in young women is typically associated with about 6 days of symptoms, 2.4 days of restricted activities and 0.4 days of bed rest.¹ Antibacterial prophylaxis is effective in preventing recurrent episodes, but at the expense of unwanted effects and a risk of promoting bacterial resistance. Here we assess the efficacy of different antibacterial regimens and non-antibacterial alternatives (cranberry, probiotics, oestrogens, immunostimulation, hyaluronic acid and chondroitin, acupuncture and herbs) in the prevention of recurrent uncomplicated UTIs in women.

Administration of mycobacterial Ag85A and IL-17A fusion protein attenuates airway inflammation in a murine model of asthma.

Interleukin (IL)-17A contributes to the development of asthma, especially in severe asthma which has characteristic neutrophil infiltration in airways. However, IL-17A-blocking antibody could escalate T helper (Th) 2 cytokines, such as IL-13, IL-4 in murine models. We aimed at determining the effect of mycobacterial Ag85A and IL-17A fusion protein­Ag85A-IL-17A on airway inflammation in a murine model of asthma. IL-17A recombinant protein fused mycobacterial immunodominant antigen Ag85A was constructed, expressed and purified. The fusion protein was then administrated into BALB/c mice and its anti-inflammatory effects in the infiltration of inflammatory cells, Th2/Th17 cytokines in BALF, histopathological changes of lung tissues as well as chemokines in lung tissues were evaluated in the murine model of asthma. We found that administration of mycobacterial Ag85A and IL-17A fusion protein induced IL-17A specific immunoglobulin (Ig)G in sera and significantly decreased IL-17A and IL-6 levels in bronchoalveolar lavage fluid (BALF). Ag85A-IL-17A vaccinated mice also showed marked reduction in the infiltration of inflammatory cells in peribronchiolar region and significant decrease in total cells, eosinophil cells and neutrophil cells in BALF. The increased levels of IL-13 and IL-4 in BALF of ovalbumin-sensitized mice were significantly reduced by the administration of Ag85A-IL-17A. Furthermore, CD3+CD4+IL-13+ splenocytes stimulated with OVA and CXCL1 mRNA, CCL2 mRNA and GATA-3 mRNA expressed in lung tissues were decreased markedly in Ag85A-IL-17A vaccinated group. Our results demonstrate remarkable antiallergic effects of Ag85A-IL-17A in a murine model of asthma and it may have protective effects on allergic asthma.

Efficacy of a topical suspension of bacterial antigens for the management of chronic suppurative otitis media.

The aim of this study was to compare the efficacy and safety of a topical suspension of bacterial antigens for the management of chronic suppurative otitis media (CSOM). Two-hundred ten patients with CSOM were enrolled in this study. The patients were divided into two numerically equal groups and randomized to receive a topical suspension of bacterial antigens, Lantigen B (group A), or placebo (group B), according to the following protocol: 5 drops thrice a day for 12 consecutive weeks in the external acoustic duct. The topical suspension of bacterial antigens and placebo were administered by identical, anonymous dispensers in order to maintain double-blind conditions. Before, at the end and 3 months, and after the end of the treatment, all patients who entered in the study underwent pure tone audiometry, microscopic examination of ears, microbiological examination of the ear swab, aural suctioning and subjective assessment of symptoms by visual analog scale. In group A, we saw a statistically significant improvement in the different items measured. A topical suspension of bacterial antigens was significantly more effective than placebo in the treatment of CSOM.