Neutrophil-activating Protein Research Articles

Neutrophil-activating protein in Bacillus spores inhibits casein allergy via TLR2 signaling

BackgroundMilk allergy commonly occurs in children, mainly caused by bovine-derived casein (CAS) protein. Neutrophil-activating protein (NAP) of Helicobacter pylori plays an immunomodulatory role with potential to suppress Th2-type immune responses. Bacillus subtilis (B. subtilis) spores are commonly used as oral vectors for drug delivery.ObjectiveTo investigate whether recombinantly expressed NAP on B. subtilis spores could be an effective treatment for CAS allergy in mouse.MethodsAfter CAS sensitization, mice were orally administered B. subtilis spores expressing recombinant NAP for 6 weeks. Allergic symptoms and parameters were evaluated after CAS challenge oral gavage, including allergic inflammation, splenic cytokines, and serum-specific antibodies. Protein levels of Toll-like receptor 2 (TLR2) and c-JUN in the jejunum tissue were measured by western blot. Bone marrow-derived macrophages (BMDMs) were stimulated with inactivated NAP spores to measure the influence on cytokine profiles in vitro.ResultsNAP recombinant spore treatment significantly reduced allergic symptoms and intestinal inflammation. Interleukin-12 and interferon-gamma levels increased, whereas serum CAS-specific IgG1 and IgE levels decreased. TLR2 and c-JUN expression levels were elevated in the jejunal tissue. Inactivated NAP spores polarized BMDMs to the M1 phenotype and enhanced cytokine expression, which were inhibited by a TLR2 neutralizing antibody.ConclusionNAP offers a new strategy in the treatment of CAS allergy by inhibiting the Th2 response, while eliciting macrophages to promote Th1 immune responses.

Impact of neutrophil‐activating protein conservation on diagnostic tests and vaccine design

AbstractBACKGROUNDThe neutrophil activating protein (NAP) is a highly immunogenic and virulence factor of Helicobacter pylori, presenting inflammatory and immunomodulatory activity. Consequently, NAP has been explored as a diagnostic and therapeutic target. However, when evaluating a target protein to design diagnostic methods or vaccines, it is critical to determine the protein conservation among the bacterial population, as well the impact of alterations of amino acid residues on the protein antigenic profile.RESULTSIn the present work, NAP conservation and theoretical antigenicity were determined among 51 sequences from H. pylori isolated from patients worldwide. A high NAP conservation (83%) was observed, where 17 amino acid residues, among the 144 residues of the protein, were polymorphic. Alterations at these polymorphic sites had a theoretically low impact on predicted antigenicity, where only 5 NAPs out of 51 NAPs presented a slightly different antigenic profile in relation to the consensus sequence. According to that, it was possible to recognize in western blotting 93% of NAP from different bacteria (n = 15) using polyclonal antibodies developed against a specific NAP.CONCLUSIONSIt was predicted that when working with polyclonal antibodies or large NAP fragments for diagnostic and vaccine design, slight variation in protein sequence will have a minimal impact on NAP recognition. However, if a NAP monoclonal antibody or small NAP epitopes are considered, it is critical to select the most conserved and antigenic NAP regions, to maximize the coverage of NAP variants. © 2024 Society of Chemical Industry (SCI).

An intranasal influenza virus vector vaccine protects against Helicobacter pylori in mice.

Helicobacter pylori is a human pathogen that infects almost half of the population. Antibiotic resistance in H. pylori threatens health and increases the demand for prophylactic and therapeutic vaccines. Traditional oral vaccine research faces considerable challenges because of the epithelial barrier, potential enterotoxicity of adjuvants, and the challenging conditions of the gastric environment. We developed an intranasal influenza A virus (IAV) vector vaccine based on two live attenuated influenza viruses with modified acidic polymerase protein (PA) genes encoding the A subunit of H. pylori neutrophil-activating protein (NapA), named IAV-NapA, including influenza virus A/WSN/33 (WSN)-NapA and A/Puerto Rico/8/34 (PR8)-NapA. These recombinant influenza viruses were highly attenuated and exhibited strong immunogenicity in mice. Vaccination with IAV-NapA induced antigen-specific humoral and mucosal immune responses while stimulating robust Th1 and Th17 cell immune responses in mice. Our findings suggest that prophylactic and therapeutic vaccination with influenza virus vector vaccines significantly reduces colonization of H. pylori and inflammation in the stomach of mice.IMPORTANCEHelicobacter pylori is the most common cause of chronic gastritis and leads to severe gastroduodenal pathology in some patients. Many studies have shown that Th1 and Th17 cellular and gastric mucosal immune responses are critical in reducing H. pylori load. IAV vector vaccines can stimulate these immune responses while overcoming potential adjuvant toxicity and antigen dosing issues. To date, no studies have demonstrated the role of live attenuated IAV vector vaccines in preventing and treating H. pylori infection. Our work indicates that vaccination with IAV-NapA induces antigen-specific humoral, cellular, and mucosal immunity, producing a protective and therapeutic effect against H. pylori infection in BALB/c mice. This undescribed H. pylori vaccination approach may provide valuable information for developing vaccines against H. pylori infection.

Identification, Genome Sequencing, and Characterizations of Helicobacter pylori Sourced from Pakistan.

The stomach's colonization by Helicobacter pylori (H. pylori) results in gastritis, ulcers, and stomach cancer. Frequently, pain is treated with medication, but resistant H. pylori infections are not. Therefore, it is important to find pharmacological targets and improved treatments for resistant H. pylori strains. The aim of the current study was sampling, identification, drug susceptibility testing following genome sequencing and comparative genome-wide analysis of selected H. pylori strains from Pakistan with three representative strains for virulence and drug-resistant characteristics. Based on culture, biochemistry, and molecular biology, 84 strains of H. pylori were identified, which made up 47% of the enrolled cases. Among all H. pylori strains, the highest resistance was reported for metronidazole with 82 H. pylori strains (98%), followed by clarithromycin with 62 resistant strains (74%). Among metronidazole-resistant strains, 38 strains (46%) were also resistant to clarithromycin, contributing 61% of clarithromycin resistant cases. Two strains, HPA1 and HPA2, isolated from 'gastritis' and 'gastric ulcer' patients, respectively, were further processed for WGS. The draft genome sequences of H. pylori strains HPA1 and HPA2 encode 1.66 Mbp and 1.67 Mbp genome size, 24 and 4 contiguous DNA sequences, and 1650 and 1625 coding sequences, respectively. Both the genomes showed greater than 90% similarity with the reference strain H. pylori ATCC 43504/PMSS1. The antibiotic-resistant genes were identified among all the strains with overall similarity above 95%, with minor differences in the sequence similarity. Using the virulent gene data obtained from the Virulence Factor Database, 75 to 85 virulent genes were identified in the five genome assemblies with various key genes such as cytolethal distending toxin (cdt), type IV secretion system, cag PAI, plasticity region, cell-motility- and flagellar-associated genes, neutrophil-activating protein (HP-NAP), T4SS effector cytotoxin-associated gene A (cagA), and urease-associated genes ureA and ureB, etc. Sequence similarity between the virulence factors found in this study and reference genes was at least 90%. In summary, the results of our study showed the relationship between clinical results and specific H. pylori strains' (HPA1 and HPA2) genetics such as antibiotic resistance and specific virulence factors. These findings provide valued understanding of the epidemiology of H. pylori-associated diseases. Moreover, identification and genomics analysis have provided insights into the epidemiology, genetic diversity, pathogenicity, and potential drug resistance genes of H. pylori strains, offering a foundation for developing more targeted and effective medical interventions, including anti-virulent medications.

Immune response modulation in inflammatory bowel diseases by Helicobacter pylori infection.

Many studies point to an association between Helicobacter pylori (H. pylori) infection and inflammatory bowel diseases (IBD). Although controversial, this association indicates that the presence of the bacterium somehow affects the course of IBD. It appears that H. pylori infection influences IBD through changes in the diversity of the gut microbiota, and hence in local chemical characteristics, and alteration in the pattern of gut immune response. The gut immune response appears to be modulated by H. pylori infection towards a less aggressive inflammatory response and the establishment of a targeted response to tissue repair. Therefore, a T helper 2 (Th2)/macrophage M2 response is stimulated, while the Th1/macrophage M1 response is suppressed. The immunomodulation appears to be associated with intrinsic factors of the bacteria, such as virulence factors - such oncogenic protein cytotoxin-associated antigen A, proteins such H. pylori neutrophil-activating protein, but also with microenvironmental changes that favor permanence of H. pylori in the stomach. These changes include the increase of gastric mucosal pH by urease activity, and suppression of the stomach immune response promoted by evasion mechanisms of the bacterium. Furthermore, there is a causal relationship between H. pylori infection and components of the innate immunity such as the NLR family pyrin domain containing 3 inflammasome that directs IBD toward a better prognosis.

Study on anti-inflammatory effect of peptides-conjugated alumina nanoparticle on allergic rhinitis mice model.

Allergic rhinitis (AR) is a common atopic problem in which immune response to the environmental factors leads to clinical symptoms. Helicobacter pylori neutrophil-activating protein (HP-NAP) as a peptide attenuates Th2 response and stimulates Th1 activation and mucus adhesion promoting protein (MapA) as a cell-surface protein binds to mucus. This study evaluated the effect of HP-NAP and MapA conjugated with alumina nanoparticle on AR. HP-NAP and HP-NAP with MapA were conjugated to alumina nanoparticle and two separate nanoparticles were produced. The AR mice were treated with these and HP-NAP in peptide form. The AR symptoms, gene expression of mucus, levels of IL-33 and IL-4, and total and ovalbumin (OVA)-specific IgE levels were evaluated. Nasal rubbing, sneezing, gene expression of mucus, and IL-33 and IL-4 levels, and OVA-specific and total IgE were decreased in three treated groups compared to AR, and there was a significant decrease in the symptoms in AR-H-M-A group (P < 0.05) when compared to the other treated groups. HP-NAP has a controlling effect on AR, and in nanoparticle-conjugated form it can strongly attach to the airway's mucus via MapA. Therefore, cooperation of HP-NAP-alumina with MapA can produce an effective and applicable treatment for AR.

RMBP-NAP Suppresses OXA-induced Allergic Dermatitis by Regulating the Th1/Th2 Balance.

Allergic dermatitis (AD) is an inflammatory skin disease that arises from abnormal T lymphocyte activation. A recombinant fusion protein comprising Helicobacter pylori neutrophil-activating protein and maltose binding protein, rMBP-NAP, has been documented as a novel immunomodulatory TLR agonist. To explore the effect of the rMBP-NAP on the OXA-induced AD in a mouse model and clarify the possible action mechanism. The AD animal model was induced by repeated administration of oxazolone (OXA) in BALB/c mice. H&E staining was used to analyze the ear epidermis thickness and the number of infiltrating inflammatory cells. TB staining was used to detect mast cell infiltration in the ear tissue. ELISA was used to analyze the secretion of cytokines IL-4 and IFN-γ in peripheral blood. qRT-PCR was used to determine the expression levels of IL-4, IFN-γ, and IL-13 in ear tissue. OXA induced the establishment of an AD model. After the rMBP-NAP treatment, the thickness of the ear tissue and the number of mast cells infiltrated in AD mice reduced, and the serum and ear tissue levels of IL-4 and IFN-γ increased, but the ratio of IFN-γ (rMBP-NAP group)/IL-4 (rMBP-NAP group) was greater than the ratio of IFN-γ (sensitized group)/IL-4 (sensitized group). The rMBP-NAP improved the disease symptoms including skin lesions in AD, alleviated the inflammation in ear tissue, and restored the Th1/2 balance by inducing a shift from the Th2 to the Th1 response. The results of our work support the use of rMBP-NAP as an immunomodulator for AD treatment in future investigations.

The Role of Helicobacter pylori Neutrophil-Activating Protein in the Pathogenesis of H. pylori and Beyond: From a Virulence Factor to Therapeutic Targets and Therapeutic Agents.

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of H. pylori, plays a role in bacterial protection and host inflammation. HP-NAP activates a variety of innate immune cells, including neutrophils, monocytes, and mast cells, to induce their pro-oxidant and pro-inflammatory activities. This protein also induces T-helper type 1 (Th1) immune response and cytotoxic T lymphocyte (CTL) activity, supporting that HP-NAP is able to promote gastric inflammation by activation of adaptive immune responses. Thus, HP-NAP is a potential therapeutic target for the treatment of H. pylori-induced gastric inflammation. The inflammatory responses triggered by HP-NAP are mediated by a PTX-sensitive G protein-coupled receptor and Toll-like receptor 2. Drugs designed to block the interactions between HP-NAP and its receptors could alleviate the inflammation in gastric mucosa caused by H. pylori infection. In addition, HP-NAP acts as a promising therapeutic agent for vaccine development, allergy treatment, and cancer immunotherapy. The high antigenicity of HP-NAP makes this protein a component of vaccines against H. pylori infection. Due to its immunomodulatory activity to stimulate the Th1-inducing ability of dendritic cells, enhance Th1 immune response and CTL activity, and suppress Th2-mediated allergic responses, HP-NAP could also act as an adjuvant in vaccines, a drug candidate against allergic diseases, and an immunotherapeutic agent for cancer. This review highlights the role of HP-NAP in the pathogenesis of H. pylori and the potential for this protein to be a therapeutic target in the treatment of H. pylori infection and therapeutic agents against H. pylori-associated diseases, allergies, and cancer.

Anti-Inflammatory Effect of Korean Propolis on Helicobacter pylori-Infected Gastric Mucosal Injury Mice Model.

Propolis, a natural resinous substance obtained from a variety of buds and plants, has been reported to possess various biological functions. Several recent studies have demonstrated the inhibitory effects of propolis on the growth of Helicobacter&amp;nbsp;pylori (H. pylori) in vitro; however, current research efforts on Korean propolis (KP) remain insufficient especially in vivo. Our study aims to investigate the anti-inflammatory effect and molecular mechanism of KP on mouse gastric mucosa during H. pylori infection. We examined an in vivo H. pylori-induced gastric mucosal injury mice model. We found that KP inhibited the growth of H. pylori and attenuated the expression of H. pylori virulence factors such as cytotoxin-associated gene A, encoding urease A subunit, surface antigen gene and neutrophil-activating protein A. Moreover, KP reduced both gross lesions and pathological scores in H. pylori-challenged mice. In addition, KP markedly restrained the production of pro-inflammatory cytokines and nitric oxide levels compared with an untreated H. pylori-infected group. In particular, we found that KP repressed the phosphorylation of IκBα and NF-κB p65 subunit, and subsequently suppressed their downstream target genes. Taken together, these findings demonstrate the beneficial effects of KP on inflammation through the inhibition of NF-κB signaling as well as inhibition of H. pylori growth in a mouse model infected with H. pylori. This suggests the potential application of KP as a natural supplement for patient's suffering from gastric mucosal injury caused by H. pylori infection.

Preclinical safety assessment of MV-s-NAP, a novel oncolytic measles virus strain armed with an H. pylori immunostimulatory bacterial transgene

Despite recent therapeutic advances, metastatic breast cancer (MBC) remains incurable. Engineered measles virus (MV) constructs based on the attenuated MV Edmonston vaccine platform have demonstrated significant oncolytic activity against solid tumors. The Helicobacter pylori neutrophil-activating protein (NAP) is responsible for the robust inflammatory reaction in gastroduodenal mucosa during bacterial infection. NAP attracts and activates immune cells at the site of infection, inducing expression of pro-inflammatory mediators. We engineered an MV strain to express the secretory form of NAP (MV-s-NAP) and showed that it exhibits anti-tumor and immunostimulatory activity in human breast cancer xenograft models. In this study, we utilized a measles-infection-permissive mouse model (transgenic IFNAR KO-CD46Ge) to evaluate the biodistribution and safety of MV-s-NAP. The primary objective was to identify potential toxic side effects and confirm the safety of the proposed clinical doses of MV-s-NAP prior to a phase I clinical trial of intratumoral administration of MV-s-NAP in patients with MBC. Both subcutaneous delivery (corresponding to the clinical trial intratumoral administration route) and intravenous (worst case scenario) delivery of MV-s-NAP were well tolerated: no significant clinical, laboratory or histologic toxicity was observed. This outcome supports the safety of MV-s-NAP for oncolytic virotherapy of MBC. The first-in-human clinical trial of MV-s-NAP in patients with MBC (ClinicalTrials.gov: NCT04521764) was subsequently activated.

HP-NAP of Helicobacter pylori: The Power of the Immunomodulation.

The miniferritin HP-NAP of Helicobacter pylori was originally described as a neutrophil-activating protein because of the capacity to activate neutrophils to generate oxygen radicals and adhere to endothelia. Currently, the main feature for which HP-NAP is known is the ability to promote Th1 responses and revert the immune suppressive profile of macrophages. In this review, we discuss the immune modulating properties of the protein regarding the H. pylori infection and the evidence that support the potential clinical application of HP-NAP in allergy and cancer immunotherapy.

CAR T cells expressing a bacterial virulence factor trigger potent bystander antitumour responses in solid cancers

Chimeric antigen receptor T cells (CAR T cells) are effective against haematologic malignancies. However, in solid tumours, their potency is hampered by local immunosuppression and by the heterogeneous expression of the antigen that the CAR targets. Here we show that CAR T cells expressing a pluripotent pro-inflammatory neutrophil-activating protein (NAP) from Helicobacter pylori trigger endogenous bystander T-cell responses against solid cancers. In mice with subcutaneous murine pancreatic ductal adenocarcinomas, neuroblastomas or colon carcinomas, CAR(NAP) T cells led to slower tumour growth and higher survival rates than conventional mouse CAR T cells, regardless of target antigen, tumour type and host haplotype. In tumours with heterogeneous antigen expression, NAP secretion induced the formation of an immunologically ‘hot’ microenvironment that supported dendritic cell maturation and bystander responses, as indicated by epitope spreading and infiltration of cytotoxic CD8+ T cells targeting tumour-associated antigens other than the CAR-targeted antigen. CAR T cells armed with NAP neither increased off-tumour toxicity nor hampered the efficacy of CAR T cells, and hence may have advantageous translational potential.

Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells.

Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam3CSK4, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.

Helicobacter cyclurae sp. Nov., Isolated From Endangered Blue Iguanas (Cyclura lewisi)

Blue iguanas (Cyclura lewisi) are endangered reptiles found only on Grand Cayman. Previously, DNA for a novel Helicobacter species GCBI1 was detected in sick and dead iguanas. In the current study, fecal and cloacal swab samples were obtained from 25 iguanas. Through molecular and microbiological techniques, a novel Helicobacter species was cultured from feces and characterized, for whom we propose the name Helicobacter cyclurae. This novel helicobacter had a prevalence of 56% by PCR and 20% by culture in samples analyzed. The type strain MIT 16-1353 was catalase, oxidase, and gamma-glutamyl transpeptidase positive. By electron microscopy, H. cyclurae has a curved rod morphology and a single sheathed polar flagellum. Phylogenetic analysis using 16S rRNA, gyrB, and hsp60 indicated that these strains were most closely related to Helicobacter sp. 12502256-12 previously isolated from lizards. H. cyclurae has a 1.91-Mb genome with a GC content of 33.37%. There were 1,969 genes with four notable virulence genes: high temperature requirement-A protein-secreted serine protease, gamma-glutamyl transpeptidase, fibronectin/fibrinogen binding protein, and neutrophil-activating protein. Whole-genome phylogeny, average nucleotide identity, and digital DNA–DNA hybridization analysis confirmed that H. cyclurae is a novel species, and the first helicobacter cultured and characterized from blue iguanas.

The crosstalk between H. pylori virulence factors and the PD1:PD-L1 immune checkpoint inhibitors in progression to gastric cancer

BackgroundThe progression to gastric cancer has been linked to chronic infection with Helicobacter pylori (H. pylori). Immune checkpoint inhibitors (programmed cell death -1, PD-1; programmed cell death –ligand 1, PD-L1) have a role in cancer immune escape. The relationship between H. pylori virulence factors with PD-1, PD-L1 T helper 1 (Th1), T helper 17 (Th17), and regulatory T cell (Treg) response genes, has not been thoroughly investigated in the development of gastric cancer. Therefore, we evaluated how H. pylori virulence factors influence the expression levels of immune-related genes in the development of gastric immunopathology. MethodsA total of 92 gastric tissues of normal controls and patients with gastritis, gastric ulcer, and gastric cancer were examined for the expression of immune-checkpoint inhibitor genes (PD-1 PD-L1), Th1 (interferon- γ, IFN-γ), Th17 (interleukin- 17, IL-17, Retinoic-acid-receptor- related orphan nuclear receptor gamma t, RORγ-t), and Treg (Forkhead box P3, FOXP3) response genes with quantitative real-time PCR (qRT-PCR). Furthermore, correlation of H. pylori virulence factors’ (cytotoxin-associated gene A, cagA; vacuolating cytotoxin gene A, vacA (s1,s2,m1,m2); blood group antigen-binding adhesin gene A, babA, duodenal ulcer promoting gene A, dupA; the putative neuraminyllactose-binding hemagglutinin homolog, hpaA; neutrophil-activating protein A napA; outer inflammatory protein A, oipA; urease A, ureA; and urease B, ureB) genotypes with a degree of inflammation and density of H. pylori were investigated. Next, the relationship between H. pylori virulence factors and immune-checkpoint inhibitor genes, and T-cell response genes was evaluated. Eventually, a decision tree model was developed to determine the clinical outcome of patients using expression data. ResultsThe intensity of PD-1 and PD-L1 mRNA expression was increased significantly in gastric tissue of patients with gastric ulcer (PD-1: 2.3 fold, p=0.01; PD-L1: 2.1 fold, p=0.004), and gastric cancer (PD-1: 2 fold, p= 0.04; PD-L1: 1.8 fold, p=0.05) compared with control subjects. Also, PD-1: PD-L1 expression was significantly higher in patients with gastritis, who were infected with a marked density of H. pylori compared with its mildly infected counterparts. Furthermore, a novel negative correlation was found between PD-1 (r= -0.43) and PD-L1 (r= -0.42) with FOXP3 in patients with gastritis. CagA-positive H. pylori strain's negative association with PD-L1 expression (r=-0.34) was detected in patients with gastritis. Interestingly, PD-1 mRNA expression correlated positively with vacA s2/m2, in gastritis (r=0.43) and ulcer (r=0.43) patients. Furthermore, PD-1: PDL1 expression negatively correlated with vacA m1/m2 (r=-0.43 for PD-1; r=-0.38 for PD-L1) in gastritis patients. Moreover, an inverse correlation of PDL1 was present with vacA m1 (r=0.52) and vacA s1/m1 (r=0.46) versus vacA m2 (r=-0.44) and vacA m1 (r=0.52) and vacA s1/m2 (r=-0.14) in ulcer patients, respectively. Also, a correlation of vacA m2 (r=-0.47) and vacA s1/s2 (r= 0.45) with PD-1 was detected in ulcer patients. In addition, a novel negative correlation between FOXP3 mRNA levels and napA was shown in patients with gastritis and ulcer (r=-0.59). Finally, a computer-based model that was developed showed that knowing the expression levels of PD-L1, RORγ-t, and vacA s1/m2 would be useful to detect the clinical outcome of a patient. ConclusionOur results suggested that PD-1:PD-L1 immune checkpoint inhibitors were increased in gastric pre-cancerous lesions that progress to gastric cancer. Herein, we report the relationship between H. pylori virulence factors and expression of host immune checkpoint inhibitors for diagnostic prediction of gastric malignancies using computer-based models.

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.

Concurrent expression of HP-NAP enhances antitumor efficacy of oncolytic vaccinia virus but not for Semliki Forest virus

Oncolytic viruses (OVs) represent promising therapeutic agents for cancer therapy by selective oncolysis and induction of anti-tumor immunity. OVs can be engineered to express tumor-associated antigens and immune-modulating agents to provoke stronger antitumor immunity. Here, we engineered vaccinia virus (VV) and Semliki Forest virus (SFV) to express neuroblastoma-associated antigen disialoganglioside (GD2) and the immune modulator Helicobacter pylori neutrophil-activating protein (NAP) and compared their therapeutic potency. Oncolytic VV did not exhibit any antitumor benefits, whereas SFV was able to delay subcutaneous neuroblastoma (NXS2) tumor growth. Additional expression of the GD2 mimotope (GD2m) by VV-GD2m or SFV-GD2m did not improve their anti-tumor capacity compared to the parent viruses. Further arming these OVs with NAP resulted in contrasting anti-tumor efficacy. VV (VV-GD2m-NAP) significantly improved therapeutic efficacy compared to VV-GD2m, which was also associated with a significantly elevated anti-GD2 antibody, whereas there was no additive antitumor efficacy for SFV-GD2m-NAP compared to SFV-GD2m, nor was the anti-GD2 antibody response improved. Instead, NAP induced higher neutralizing antibodies against SFV. These observations suggest that distinct immune stimulation profiles are elicited when the same immunostimulatory factor is expressed by different OVs. Therefore, careful consideration and detailed characterization are needed when engineering OVs with immune-modulators.

VARIABILITY OF NEUTROPHIL-ACTIVATING PROTEIN AMONG HELICOBACTER PYLORI STRAINS

The Helicobacter pylori neutrophil activating protein (NAP) presents relevant inflammatory and immunomodulatory activity and has consequently been explored as a diagnosis and therapeutic target. In the present work, nap gene sequences, retrieved from H. pylori isolated world-wide, were analyzed, a high genetic diversity (with 88% of alleles) being observed in accordance with other virulence factors. The phylogenetic analysis did not reveal the separation of strains per geographical region according to a bacterial panmictic population. When compared to other genes of virulence factors of H. pylori, such as the vacuolating cytotoxin A (vacA), nap presents slightly lower genetic variability, concerning the number of alleles and polymorphic sites, pointing to a possible lower pressure of the host immune system. The nap genetic diversity is associated to a high proportion of synonymous substitutions in relation to non-synonymous substitutions, pointing to equilibrium between the need for antigenic diversity as a mechanism to escape the host immune system and the maintenance of the proteins function. All this information could be put to good use when planning the NAP application as a therapeutic or diagnostic target.

Recombination Lactococcus lactis expressing Helicobacter pylori neutrophil-activating protein A attenuates food allergy symptoms in mice.

Food allergy has been a significant public health issue with growing severity, prevalence and limited treatments. The neutrophil-activating protein A subunit (NapA) of Helicobacter pylori has been shown to have therapeutic potential in allergic diseases. The NapA expression efficiency of recombinant Lactococcus lactis(L.lactis) were determined. The effects of recombinant bacterium on food allergy in Balb/c mice were also investigated. NapA were delivered and expressed efficiently via L. lactis. The engineered bacterium ameliorated food allergy symptoms (acute diarrhea and intestinal inflammation) and decreased serum histamine levels. In addition, the secretion of OVA-specific IgG2a, IFN-γ was promoted and the level of IL-4, OVA-specific IgE was restrained. The recombinant strain may attenuate food allergy in mice through immune regulatory effect, which may be a promising approach for preventing or treating food allergy.

Abstract PO-061: The role of Cathepsin E expression in pancreatic ductal adenocarcinoma tumorigenesis

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of 10% due to a lack of early detection and effective treatments. Aspartic protease Cathepsin E (CTSE) expression is increased early in PDAC, but its role in tumorigenesis is unknown. We hypothesize that high CTSE expression promotes proliferation, invasion, inhibition of apoptosis, and the secretion of cancer-associated cytokines in PDAC. Methods: Human PDAC cell lines (Mpanc96, Panc-1, and Miapaca2) were engineered to express high levels of CTSE. Cell proliferation, invasion, and apoptosis were analyzed and compared to control cell lines expressing low levels of CTSE. Cells were treated with aspartic protease inhibitors (Pepstatin A and Ritonavir) and monitored for proliferation (24 - 72 hours) and invasion (48 hours). Apoptosis was measured using propidium iodide and analyzed via flow cytometry. Conditioned media from cells was collected (72 hours) after treatment to assess differential secretion of cancer-associated cytokines using a cytokine array. Results: Proliferation was not affected by the overexpression of CTSE; however, inhibition of CTSE slowed proliferation in the Panc-1 and MiaPaca2, but not Mpanc96 cells. CTSE inhibitors promoted invasion of Mpanc96 and Panc-1, but not Miapaca2 cells, regardless of CTSE levels. Apoptosis was not significantly different between the cell lines treated with the aspartic protease inhibitors compared to controls. Increased expression of CTSE induced the secretion of various cytokines such as epithelial neutrophil-activating protein 78 (ENA-78), a chemokine expressed in inflammatory pancreatic disorders. Treatment with the inhibitor Pepstatin A decreased ENA-78 secretion. Conclusions: CTSE levels does not appear to affect proliferation or apoptosis; however, inhibition of CTSE increased cell invasion and resulted in altered cytokine secretion. Further studies examining how CTSE influences invasiveness, and cytokine secretion may create new opportunities for developing effective therapies for PDAC. Citation Format: Ericka Velez-Bonet, Sabrina Kaul, Corbin Pontious, Marcus Hong, Kelly Dubai, Zobeida Cruz-Monserrate. The role of Cathepsin E expression in pancreatic ductal adenocarcinoma tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-061.