Pore-forming alpha-hemolysin efficiently improves the immunogenicity and protective efficacy of protein antigens

Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.

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Vaccination against Anthrax with Attenuated Recombinant Strains of Bacillus anthracis That Produce Protective Antigen

Infection and Immunity ◽

10.1128/iai.67.2.562-567.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 562-567 ◽

Cited By ~ 53

Author(s):

John P. Barnard ◽

Arthur M. Friedlander

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Plasmid Stability ◽

Protective Efficacy ◽

Live Vaccines ◽

Antibody Titers ◽

Anthrax Protective Antigen ◽

Anthrax Vaccines

ABSTRACT The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming ΔANR and ΔSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro.

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Salmonella Vaccine Vectors Displaying Delayed Antigen Synthesis InVivo To Enhance Immunogenicity

Infection and Immunity ◽

10.1128/iai.00444-10 ◽

2010 ◽

Vol 78 (9) ◽

pp. 3969-3980 ◽

Cited By ~ 47

Author(s):

Shifeng Wang ◽

Yuhua Li ◽

Giorgio Scarpellini ◽

Wei Kong ◽

HuoYing Shi ◽

...

Keyword(s):

Immune Responses ◽

Fluorescent Protein ◽

Protective Efficacy ◽

Start Codon ◽

Antibody Titers ◽

Iga Antibody ◽

Green Fluorescent ◽

High Level

ABSTRACTWe have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuatedSalmonellavaccine (RASV) strains to enhance immune responses by reducing the adverse effects of high-level antigen synthesis. This system includes a chromosomal repressor gene,lacI, expressed from the arabinose-regulatedaraCPBADpromoter. LacI serves to regulate expression from a plasmid promoter, Ptrc, that directs antigen synthesis. In the presence of arabinose LacI is produced, which binds to Ptrc, blocking antigen synthesis.In vivo, an arabinose-poor environment, the concentration of LacI decreases with each cell division, allowing increased antigen synthesis. To optimize the system and for comparison, we altered thelacIribosome-binding site, start codon, and/or codon content to construct RDAS strains χ9095, χ9959, and χ9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain χ9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initialin vitroassessment and theStreptococcus pneumoniaePspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression oflacIdid not interfere with the capacity to induce an immune response. Strain χ9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain χ9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain χ9241 also induced significantly greater protective efficacy against challenge with virulentS. pneumoniae. These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.

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Development of combination adjuvant for efficient T cell and antibody response induction against protein antigen

PLoS ONE ◽

10.1371/journal.pone.0254628 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0254628

Author(s):

Yasunari Haseda ◽

Lisa Munakata ◽

Chiyo Kimura ◽

Yumi Kinugasa-Katayama ◽

Yasuko Mori ◽

...

Keyword(s):

T Cell ◽

Neutralizing Antibodies ◽

Aluminum Salt ◽

Protein Antigen ◽

New Combination ◽

Vaccine Adjuvants ◽

Protein Antigens ◽

Antigen Uptake

Most current clinical vaccines work primarily by inducing the production of neutralizing antibodies against pathogens. Vaccine adjuvants that efficiently induce T cell responses to protein antigens need to be developed. In this study, we developed a new combination adjuvant consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), D35, and an aluminum salt. Among the various combinations tested, the DOTAP/D35/aluminum salt adjuvant induced strong T cell and antibody responses against the model protein antigen with a single immunization. Adjuvant component and model antigen interaction studies in vitro also revealed that the strong mutual interactions among protein antigens and other components were one of the important factors for this efficient immune induction by the novel combination adjuvant. In addition, in vivo imaging of the antigen distribution suggested that the DOTAP component in the combination adjuvant formulation elicited transient antigen accumulation at the draining lymph nodes, possibly by antigen uptake DC migration. These results indicate the potential of the new combination adjuvant as a promising vaccine adjuvant candidate to treat infectious diseases and cancers.

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β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

Cellular & Molecular Biology Letters ◽

10.1186/s11658-021-00261-0 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Guoying Zhang ◽

Cheng Xue ◽

Yiming Zeng

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Model ◽

Protective Efficacy ◽

Rabbit Model ◽

Akt Pathway ◽

Loss Of Function ◽

Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

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Peyer's Patch Dendritic Cells Process Viral Antigen from Apoptotic Epithelial Cells in the Intestine of Reovirus-infected Mice

Journal of Experimental Medicine ◽

10.1084/jem.20041132 ◽

2004 ◽

Vol 200 (2) ◽

pp. 235-245 ◽

Cited By ~ 106

Author(s):

Marina N. Fleeton ◽

Nikhat Contractor ◽

Francisco Leon ◽

J. Denise Wetzel ◽

Terence S. Dermody ◽

...

Keyword(s):

T Cells ◽

Epithelial Cells ◽

Cd4 T Cells ◽

Cell Protein ◽

Peyer’S Patch ◽

Peyer's Patch ◽

Antigen Uptake ◽

Cross Presentation

We explored the role of Peyer's patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (σ1) and nonstructural (σNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both σ1 and σNS, indicating productive viral replication. In contrast, σ1, but not σNS, was detected in the subepithelial dome (SED) in association with CD11c+/CD8α−/CD11blo DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained σ1, but not σNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, σ1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4+ T cells in vitro. These studies show that CD8α−/CD11blo DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4+ T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.

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Cell-Penetrating Peptide-Mediated Nanovaccine Delivery

Current Drug Targets ◽

10.2174/1389450122666210203193225 ◽

2021 ◽

Vol 22 ◽

Author(s):

Jizong Jiang

Keyword(s):

Immune System ◽

Immune Responses ◽

Cell Penetrating Peptide ◽

Antigen Delivery ◽

Efficient Manner ◽

Antigen Uptake ◽

Cell Penetrating ◽

The Stability ◽

Antigen Presenting

Abstract: Vaccination with small antigens, such as proteins, peptides, or nucleic acids, is used to activate the immune system and trigger the protective immune responses against a pathogen. Currently, nanovaccines are undergoing development instead of conventional vaccines. The size of nanovaccines is in the range of 10–500 nm, which enables them to be readily taken up by cells and exhibit improved safety profiles. However, low-level immune responses, as the removal of redundant pathogens, trigger counter-effective activation of the immune system invalidly and present a challenging obstacle to antigen recognition and its uptake via antigen-presenting cells (APCs). In addition, toxicity can be substantial. To overcome these problems, a variety of cell-penetrating peptide (CPP)-mediated vaccine delivery systems based on nanotechnology have been proposed, most of which are designed to improve the stability of antigens in vivo and their delivery into immune cells. CPPs are particularly attractive components of antigen delivery. Thus, the unique translocation property of CPPs ensures that they remain an attractive carrier with the capacity to deliver cargo in an efficient manner for the application of drugs, gene transfer, protein, and DNA/RNA vaccination delivery. CPP-mediated nanovaccines can enhance antigen uptake, processing, and presentation by APCs, which are the fundamental steps in initiating an immune response. This review describes the different types of CPP-based nanovaccines delivery strategies.

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323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.323 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A348-A348

Author(s):

Jessie Wang ◽

Kaixia Lian ◽

Jia Zheng ◽

Chenpan Nie ◽

Annie An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Immunity ◽

Immune Evasion ◽

Syngeneic Mouse ◽

Ifn Γ ◽

Draining Lymph Nodes ◽

Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

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Kinetics of Targeted Phage Rescue in a Mouse Model of SystemicEscherichia coliK1

BioMed Research International ◽

10.1155/2018/7569645 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

György Schneider ◽

Nikolett Szentes ◽

Marianna Horváth ◽

Ágnes Dorn ◽

Alysia Cox ◽

...

Keyword(s):

Capsular Polysaccharide ◽

Protective Efficacy ◽

Lytic Bacteriophage ◽

E Coli ◽

Causative Agents ◽

High Level ◽

Systemic Infections ◽

Kinetics Of

Escherichia (E.) coliK1 strains remain common causative agents of neonatal sepsis and meningitis. We have isolated a lytic bacteriophage (ΦIK1) againstE. colistrain IHE3034 and tested its specificityin vitro, as well as distribution and protective efficacyin vivo. The phage was shown to be specific to the K1 capsular polysaccharide. In the lethal murine model, a high level of protection was afforded by the phage with strict kinetics. A single dose of 1 x 108phage particles administered 10 and 60 minutes following the bacterial challenge elicited 100 % and 95 % survival, respectively. No mice could be rescued if phage administration occurred 3 hours postinfection. Tissue distribution surveys in the surviving mice revealed that the spleen was the primary organ in which accumulation of active ΦIK1 phages could be detected two weeks after phage administration. These results suggest that bacteriophages have potential as therapeutic agents in the control of systemic infections.

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The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc

Infection and Immunity ◽

10.1128/iai.00651-19 ◽

2019 ◽

Vol 87 (12) ◽

Author(s):

Yongli Bi ◽

Qingan Xu ◽

Lingkai Su ◽

Jiantao Xu ◽

Zhongfang Liu ◽

...

Keyword(s):

Lipid A ◽

Vaccine Development ◽

Protection Effect ◽

Content Type ◽

Monophosphoryl Lipid A ◽

Monophosphoryl Lipid ◽

Tooth Surfaces ◽

Antibody Titers

ABSTRACT We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro. PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.

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Indigenous enteric eosinophils control DCs to initiate a primary Th2 immune response in vivo

Journal of Experimental Medicine ◽

10.1084/jem.20131800 ◽

2014 ◽

Vol 211 (8) ◽

pp. 1657-1672 ◽

Cited By ~ 74

Author(s):

Derek K. Chu ◽

Rodrigo Jimenez-Saiz ◽

Christopher P. Verschoor ◽

Tina D. Walker ◽

Susanna Goncharova ◽

...

Keyword(s):

Lymph Nodes ◽

Eosinophil Peroxidase ◽

Th2 Immune Response ◽

Intestinal Immune System ◽

Eosinophil Activation ◽

And Migration ◽

Deficient Mice ◽

Draining Lymph Nodes

Eosinophils natively inhabit the small intestine, but a functional role for them there has remained elusive. Here, we show that eosinophil-deficient mice were protected from induction of Th2-mediated peanut food allergy and anaphylaxis, and Th2 priming was restored by reconstitution with il4+/+ or il4−/− eosinophils. Eosinophils controlled CD103+ dendritic cell (DC) activation and migration from the intestine to draining lymph nodes, events necessary for Th2 priming. Eosinophil activation in vitro and in vivo led to degranulation of eosinophil peroxidase, a granule protein whose enzymatic activity promoted DC activation in mice and humans in vitro, and intestinal and extraintestinal mouse DC activation and mobilization to lymph nodes in vivo. Further, eosinophil peroxidase enhanced responses to ovalbumin seen after immunization. Thus, eosinophils can be critical contributors to the intestinal immune system, and granule-mediated shaping of DC responses can promote both intestinal and extraintestinal adaptive immunity.

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