Adjuvant Candidate Research Articles

2-Aminobenzothiazole based adjuvant of polymyxin E against Gram-negative bacteria

Antibiotic resistance has emerged as a pressing global health issue. Polymyxin E, commonly regarded as a clinically important antibiotic, faces limitations due to its dose-dependent toxicity. A crucial strategy to combat this is the development of an antibiotic adjuvant to enhance efficacy while reducing dosage. Screening from our extensive in-house compound library, KJ1071 emerged as a promising adjuvant candidate for polymyxin E. Subsequent structure–activity relationship studies led to the discovery of compound A35, which demonstrated superior synergistic activity. However, its limited aqueous solubility posed challenges for biological experiments. To address this, we carried out a structural derivatization aimed at enhancing its solubility. The amino acid derivative A59 notably improved aqueous solubility to 3.237 mg/mL, a substantial 3237-fold increase compared to compound A35. Moreover, A59 amplified the efficacy of polymyxin E in eradicating a variety of Gram-negative bacteria, including certain clinical strains that are resistant to polymyxin E. The mechanism studies indicated that A59 might target the bacterial membrane. These findings suggest that the discovery of this innovative scaffold could provide critical insights for new adjuvant therapies.

CNSC-02. ANTICANCER ABILITY OF THE BERBERINE TO PROMOTE THE APOPTOSIS EFFECT AND ATTENUATE THE ROS GENERATION CAPACITY ON DBTRG BRAIN CANCER CELLS

Abstract Abstract In this research, berberine (BB), and its effect on the biological performance of DBTRG brain cancer cells and normal endothelial cells (EC) was investigated. Firstly, the DBTRG cancer cell and EC cell were cultured with different concentrations of BB (0.5, 1, 5 and 10 μg/ml), and it was confirmed that BB significantly reduced the proliferation of DBTRG cancer cells. However, there is no significant toxic effect on EC cells. The results of cell cycle analysis confirmed that BB significantly induced DBTRG cancer cell undergo apoptotic effect in a dose dependent manner. The assessment of cell migration behavior showed that BB significantly inhibited cell migration in DBTRG cancer cell, but not in BAEC as well as attenuated the matrix metalloproteinase 2/9 (MMP2/9) in a dose dependent manner. Furthermore, the reactive oxygen species (ROS) generation capacity among DBTRG cancer cell, EC cell and macrophages (Raw 264.7). It was demonstrated that BB was prominently decreased the ROS generation of DBTRG instead of EC or Raw264.7 cell. Moreover, the pro-inflammatory cytokines of TNF-α, IL-1β and IL-6 were significantly inhibited by BB in a dose dependent manner of DBTRG brain cancer cell. As mentioned above, it was demonstrated that bioactive BB molecules is potential for anti-brain cancer drugs may via anti-immune response and anti-oxidative generation capacity for brain cancer therapy. BB biomolecules may target for DBTRG cell which cannot cause inevitable toxic damage to the normal EC cell. Altogether, the promising findings of BB biomolecule on DBTRG cancer cell suggest that BB may be a good candidate of anticancer drug or adjuvant for the treatment of human brain cancer. Key words: berberine, DBTRG brain cancer, anti-immune response, anti-oxidative generation

Protective effects of astaxanthin solid lipid nanoparticle as a promising candidate against acute kidney injury in rats.

Acute kidney injury (AKI) is a sudden onset of renal injury that occurs within a few hours or days. Ischemia-reperfusion (IR) is a major cause of AKI. There are multiple dysregulated mechanisms behind the pathogenesis of AKI and IR which urges the need for finding multi-targeting therapies. Natural products are multi-targeting agents with promising sources of anti-inflammation, antioxidant, and antiapoptosis. Among them, astaxanthin (AST) is a keto-carotenoid with a high antioxidant potential. Using solid lipid nanoparticles (SLNs) as a novel formulation of AST helps to increase its efficacy and reduce side effects against AKI. After SLN preparation and loading of AST, the physicochemical properties were evaluated, using scanning electron microscopy (SEM) and dynamic light scattering (DLS) tests. For the in vivo study, 28 rats were divided into four groups, including sham, ischemia/reperfusion (I/R), and groups receiving protective and daily doses of AST-SLN (5 and 10 mg/kg, i.p.) during all 5 days before ischemia. Exactly 24 h after ischemia, kidneys were isolated for histological studies, and also, serum levels of catalase (CAT), glutathione (GSH), nitrite, blood urea, and creatinine were measured. The results indicated that intraperitoneal administration of SLN-AST reduced oxidative stress by decreasing serum nitrite levels, while increasing CAT and GSH. SLN-AST also improved renal function by decreasing serum urea and creatinine and preventing tissue damage. Therefore, SLN-AST could be a hopeful adjuvant candidate to prevent AKI by modulating renal function, preventing tissue damage, and through antioxidant mechanisms.

NK cell membrane/MnO2 hybrid nanoparticle-adjuvanted intranasal vaccines synergistically boost protective immunity against H1N1 influenza infection

Intranasal vaccines hold a huge promise for preventing influenza infection, however, their development is compromised due to mucosal barriers and limited systemic and mucosal immunity. Herein, a novel biomimetic nano-adjuvant based on NK cell membrane (MNK)/MnO2 hybrid nanoparticles (NLipoMn) was developed for intranasal administration of H1N1 inactivated influenza vaccine (IIV). NK cell membrane, served as TLR4 agonist was fused with cationic liposomes encapsulating STING agonist MnO2 NPs (LipoMn) to yield NLipoMn, which inherited versatile characteristics to prolong the nasal retention of IIV. Encouragingly, CCL20 secretion was enhanced by NLipoMn-activated the TLR4/NF-κB pathway in mucosal endothelial cells, thereby promoting submucosal dendritic cells (DCs) recruitment for IIV uptake. Moreover, NLipoMn-IIV synergistically harnessed TLR4 and STING signaling pathway to augment STING activation though NLipoMn-activated the TLR4/NF-κB pathway, which demonstrated a superiority to elicit strong DCs maturation, CD8+ T cells activation, and high levels of antigen-specific IgG, antigen-specific IgA, cytokines secretion, resulting in a robust systemic and mucosal immune responses. Notably, intranasal immunization with NLipoMn-IIV elicited an effective immune protection against homologous and heterologous H1N1 influenza virus infection. This study provides a promising adjuvant candidate to develop needle-free intranasal vaccines that are active against influenza and other respiratory viral infection.

Antibody titer after anti-idiotype rabies vaccination with nano-chitosan adjuvant

Background.The rabies virus neutralizing antibodies titers is a public health problem in the world, including Indonesia. Rabies is zoonotic and causes death in humans with a case fatality rate of 100%. Anti-idiotype antibody (Ab2) from chicken immunoglobulin (IgY) can be a substitute antigen for the rabies virus. This research aims to study the potential of rabies vaccine based on anti-idiotype antibody (Ab2) with nano-chitosan as an adjuvant.Materials and methods.The production of Ab2 is derived from purified and characterized chicken immunoglobulins. Nano-chitosan is made from chitosan from shrimp shell waste. Vaccination tests were carried out on rats compared with commercial vaccines as a positive control and physiological solutions as a negative control. The characterization results of IgY Ab2 were IgY rabies with BM ~180 kDa, heavy chains (BM ~60 kDa), and light chains (BM ~30 kDa) of IgY. Nano-chitosan is less than 100 nm in size, can dissolve well, and does not cause side effects in experimental animals.Results.The vaccine formula uses a concentration of 0.5%, where the ratio of nano-chitosan and Ab2 is 1:1. The Ab2 concentration used was about 1000 units per mL of the Ab2 suspension.Conclusion.This study concludes that anti-idiotype antibodies dissolved in nano-chitosan adjuvant can induce the formation of antibodies with titers that are not statistically different from the antibody titers induced by commercial rabies vaccines. Nano-chitosan has a potential vaccine adjuvant candidate, safe to use, and can enhance the immunogenicity of an antigen applied subcutaneously. According to the results of this study, it is recommended that post-vaccination antibody titers be measured regularly: for example, every one week for six weeks after vaccination. This measurement determines the time of the peak of the antibody titer so that the time for revaccination can be determined.

Systematic Evaluation of Regiochemistry and Lipidation of Aryl Trehalose Mincle Agonists.

The Macrophage-Inducible C-type Lectin receptor (Mincle) plays a critical role in innate immune recognition and pathology, and therefore represents a promising target for vaccine adjuvants. Innovative trehalose-based Mincle agonists with improved pharmacology and potency may prove useful in the development of Th17-mediated adaptive immune responses. Herein, we report on in vitro and in silico investigations of specific Mincle ligand-receptor interactions required for the effective receptor engagement and activation of Th17-polarizing cytokines. Specifically, we employed a library of trehalose benzoate scaffolds, varying the degree of aryl lipidation and regiochemistry that produce inflammatory cytokines in a Mincle-dependent fashion. In vitro interleukin-6 (IL-6) cytokine production by human peripheral blood mononuclear cells (hPBMCs) indicated that the lipid regiochemistry is key to potency and maximum cytokine output, with the tri-substituted compounds inducing higher levels of IL-6 in hPBMCs than the di-substituted derivatives. Additionally, IL-6 production trended higher after stimulation with compounds that contained lipids ranging from five to eight carbons long, compared to shorter (below five) or longer (above eight) carbon chains, across all the substitution patterns. An analysis of the additional cytokines produced by hPBMCs revealed that compound 4d, tri-substituted and five carbons long, induced significantly greater levels of interleukin-1β (IL-1β), tumor necrosis factor- α (TNF-α), interleukin-23 (IL-23), and interferon- γ (IFN-γ) than the other compounds tested in this study. An in silico assessment of 4d highlighted the capability of this analogue to bind to the human Mincle carbohydrate recognition domain (CRD) efficiently. Together, these data highlight important structure-activity findings regarding Mincle-specific cytokine induction, generating a lead adjuvant candidate for future formulations and immunological evaluations.

The Role of Essential Oils on Sleep Quality and Other Sleep-related Issues: Evidence from Clinical Trials.

Essential oils (EOs) are a volatile mixture of bioactive compounds extracted from aromatic plants. The composition of EOs varies, which majorly depends on the extraction methods and plant parts. Aromatherapy using EOs has been reported for its several beneficial effects in humans. Aromatherapy is considered a complementary and/ or adjuvant therapeutic approach for treating several illnesses, especially to improve mental health and well-being. The incidence of sleep disorders, specifically insomnia, is nowadays increased, possibly due to urbanization and lifestyle. The studies showed that EOs-based treatments using lavender EO, bergamot EO, cinnamon EO, and rosemary EO (alone or in combinations) could improve sleep quality, duration, and deprivation in healthy subjects and patients, those who suffer from sleep-related issues. The current manuscript details the outcomes of EO-based treatments on the sleep quality of humans and the possible mechanisms associated with the health-promoting properties of EOs. Also, the toxicity and adverse effects of EOs have been discussed. The study indicated that EOs are potent adjuvant therapeutic candidates to manage mood-associated complications in humans. Moreover, the aromatherapeutic field requires detailed studies on toxicity and dose determination, which could provide safe and effective therapeutic results.

Retention of methicillin susceptibility in Staphylococcus aureus using natural adjuvant as an allosteric modifier of penicillin-binding protein 2a

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global public health challenge due to its resistance to conventional antibiotics, primarily mediated by the mutated penicillin-binding protein, PBP2a. This study aims to investigate the potential of phytochemicals derived from medicinal plants in the Indian subcontinent to serve as adjuvants, enhancing the efficacy of methicillin against MRSA through allosteric modification of PBP2a using molecular docking and molecular dynamics (MD) simulation. After comprehensive Absorption, Distribution, Metabolism, and Excretion (ADME) profiling, along with AMES and hepatotoxicity tests, 9 compounds were shortlisted as suitable adjuvant candidates. Among them, nimbolide, quercetin, emodin, daidzein, eriodictyol, luteolin, and apigenin exhibited strong binding affinity to the allosteric site of PBP2a, with docking scores ranging from −8.7 to −7.3 kcal/mol. These phytochemicals facilitated enhanced methicillin binding, as evidenced by improved docking scores ranging from −6.1 to −6.8 kcal/mol, compared to −5.6 kcal/mol for methicillin alone. Molecular dynamics simulations confirmed the stability and favorable conformations of phytochemical-PBP2a complexes. Quercetin and daidzein were identified as the most promising adjuvant candidates, forming stable and energetically favorable complexes with PBP2a. Experimental validation showed that quercetin, at 30 mg/mL, effectively retained methicillin's antibacterial efficacy against MRSA. This study underscores the potential of natural compounds in overcoming antibiotic resistance and suggests that phytochemical-antibiotic synergism could be a viable strategy to combat multidrug-resistant bacterial infections.

Repurposing cetylpyridinium chloride and domiphen bromide as phosphoethanolamine transferase inhibitor to combat colistin-resistant Enterobacterales

The emergence of plasmid-encoded colistin resistance mechanisms, MCR-1, a phosphoethanolamine transferase, rendered colistin ineffective as last resort antibiotic against severe infections caused by clinical Gram-negative bacterial pathogens. Through screening FDA-approved drug library, we identified two structurally similar compounds, namely cetylpyridinium chloride (CET) and domiphen bromide (DOM), which potentiated colistin activity in both colistin-resistant and susceptible Enterobacterales. These compounds were found to insert their long carbon chain to a hydrophobic pocket of bacterial phosphoethanolamine transferases including MCR-1, competitively blocking the binding of lipid A tail for substrate recognition and modification, resulting in the increase of bacterial sensitivity to colistin. In addition, these compounds were also found to dissipate bacterial membrane potential leading to the increase of bacterial sensitivity to colistin. Importantly, combinational use of DOM with colistin exhibited remarkable protection of test animals against infections by colistin-resistant bacteria in both mouse thigh infection and sepsis models. For mice infected by colistin-susceptible bacteria, the combinational use of DOM and colistin enable us to use lower dose of colistin to for efficient treatment. These properties render DOM excellent adjuvant candidates that help transform colistin into a highly potent antimicrobial agent for treatment of colistin-resistant Gram-negative bacterial infections and allowed us to use of a much lower dosage of colistin to reduce its toxicity against colistin-susceptible bacterial infection such as carbapenem-resistant Enterobacterales.

Knock down of transforming growth factor beta improves expressions of co-stimulatory molecules, type I interferon-regulated genes, and pro-inflammatory cytokine in PRRSV-inoculated monocyte-derived macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFβ1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFβ1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFβ1 mRNA expression and protein translation. Transfection of TGFβAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFβ1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNβ, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFβAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2’-5’-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFβAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFβ expression. These findings also suggest TGFβ as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.

Adjuvant use for the management of pesticide drift, leaching and runoff.

Adjuvants are included in many pesticide spray mixtures to enhance the performance of the applied chemical. Many adjuvants which modify the emulsion or extensional viscosity of the tank-mixture have been found to offer benefits in drift management, primarily by eliminating or reducing the 'Fine' droplets included in the spray with diameters <100-200 μm that can move off-target in unfavorable conditions during ground, airblast and aerial pesticide applications. Among wind tunnel and field studies conducted around the world, there is consensus that while some adjuvants are effective for drift management, the performance varies on a case-by-case basis, requiring verification for each adjuvant which could be achieved through a programme such as certification based on showing a reduction in Fine droplets and/or a reduction in airborne drift. These can be measured in wind tunnel studies according to international standards. This article provides a review of the current science in this subject area, from the approaches to data collection to a review of existing data and regulatory application for encouraging and rewarding the use of appropriate adjuvants that have been demonstrated to reduce airborne spray drift potential and therefore the size of no-spray buffer zones appropriate to protect nontarget sensitive areas from drift exposure. Some adjuvants can offer the same reduction in drift as offered by hooded sprayer retrofits. A drift reduction programme based on adjuvant use could include testing candidate adjuvants for their effect on droplet size and reduction in Fine droplets when sprayed through reference nozzles and compared against sprays without the adjuvant. Testing could also be based alternatively on measurements of drift potential on collectors such as monofilament line in wind tunnel or field studies. Once shown to be effective in reducing 'Fines' or spray drift, adjuvants could be certified and then referenced on pesticide labels and/or regulatory or best management practice schemes to encourage their use and offer reductions in use restrictions or no-spray buffer zone sizes based on drift management. Studies have shown that some adjuvants can reduce pesticide leaching into soils and contamination of groundwater, as well as runoff of active ingredients from plants into the environment. Performance depends on the adjuvant type, the pesticide with which it is used, the soil or plant type, the timing and mass of water input from rainfall and climatic factors. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Vitamin D in tuberous sclerosis complex-associated tumors

Mammalian target of rapamycin inhibitors (mTORi) have been used to treat pediatric tuberous sclerosis complex (TSC)-associated tumors, particularly in cases with contraindications to surgery or difficulties in complete tumor resection. However, some patients experience side effects and tumor regression after discontinuation of the treatment. Therefore, there is an urgent need to develop drugs that can be used in combination with mTORi to increase their efficacy and minimize their side effects. 1,25-Dihydroxyvitamin D3 (1,25-D), which has anticancer properties, may be a promising candidate for adjuvant or alternative therapy because TSC and cancer cells share common mechanisms, including angiogenesis, cell growth, and proliferation. Vitamin D receptor-mediated signaling can be epigenetically modified and plays an important role in susceptibility to 1,25-D. Therefore, vitamin D signaling may be a promising drug target, and in vitro studies are required to evaluate the efficacy of 1,25-D in TSC-associated tumors, brain development, and core symptoms of psychiatric disorders.

High production of IL-12 by human dendritic cells stimulated with combinations of pattern-recognition receptor agonists

The cytokine IL-12p70 is crucial for T helper 1 (Th1) polarization and the generation of type 1 immunity required to fight cancer and pathogens. Therefore, strategies to optimize the production of IL-12p70 by human dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by monocyte-derived DCs. We used ten well-characterized agonists and stimulated DCs in vitro with either single agonists or 27 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and MDA5, and LPS which stimulates TLR4, were the only agonists that could elicit notable IL-12p70 production when used as single ligands. We identified six different combinations of PRR agonists, all containing either the TLR3/MDA5 agonist poly(I:C) or the TLR7/8 agonist R848, that could synergize to elicit high production of IL-12p70 by human DCs. Five of the six combinations also triggered high production of the antiviral and antitumor cytokine IFNβ. Overall, the tested PRR ligands could be divided into three groups depending on whether they triggered production of both IL-12p70 and IFNβ, only one of the two, or neither. Thus, combinations of PRR agonists were found to increase the production of IL-12p70 by human DCs in a synergistic manner, and we identified six PRR agonist combinations that may represent strong adjuvant candidates, in particular for therapeutic cancer vaccines.

Abstract 6743: Identifying new cellular adjuvants for cancer vaccines and immunotherapies

Abstract Background: Cancer vaccines represent a promising strategy for antitumor immunotherapy. Despite considerable research efforts and progress, the effectiveness of cancer vaccines remains a challenge. This is primarily due to relatively low immune responses against tumors, stemming from issues of poor immunogenicity and the immunosuppressive microenvironment commonly found in many solid tumors. Use of an optimal adjuvant can augment immune responses against tumor antigens and guide them towards a specific adaptive immune enhancement. Dendritic cells (DCs) play a primary role in initiating immunes response to vaccines, often through activation of toll-like receptors (TLRs). Identifying novel immunomodulatory proteins that regulate DCs or TLR activation can identify novel adjuvant candidates with applications for cancer immunotherapy. Methods: We constructed a Membrane Proteome Array (MPA) encompassing ~6,000 structurally intact human membrane proteins in their native conformation (94% of the entire membrane proteome) and used it to screen for cellular proteins that can modulate response to vaccination through DCs and TLRs. To identify proteins that interact with TLRs, the library was expressed in live TLR reporter cells, and these cells were tested for response to known activators. In addition, cells expressing the MPA were presented to DCs to identify membrane proteins that modulate DC activity. TLR-expressing cell activation was measuring using an NF-kB-activated luciferase reporter, while DC assays used upregulation of cell surface markers (CD80, CD83, and MHCII) to measure the activation of immature monocyte-derived dendritic cells. Results: Screening of TLR2, TLR3, and TLR5 reporter cell lines identified both known and novel regulators of TLR activation. Activators common to several TLRs were identified as well as activators unique to individual TLRs. Screening of primary human DCs likewise identified known regulators of activation (for example CD40L) and several novel targets. Putative novel regulators of DC activation are being further investigated to determine their biological significance. Conclusion: Discovering novel immunomodulatory targets and proteins that regulate DCs and TLR activation can identify novel adjuvant candidates with targeted immune outcomes, such as immune stimulating and anti-cancer responses. This study identified novel regulators of immune activation which are being further investigated as adjuvant candidates for cancer vaccines. Citation Format: Joseph Rucker, Nnenna Nwogu, Nicholas M. Molino, Ami Snyder, Jonathan T. Sullivan, Benjamin J. Doranz. Identifying new cellular adjuvants for cancer vaccines and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6743.

Abstract 5138: Proteomic analyses of the urothelial cancer landscape reveal highly distinct prognostic and predictive subtypes

Abstract Introduction: Urothelial cancer (UC) is a challenging disease with a wide tumor-biological spectrum. Most molecular classifications cover only muscle-invasive bladder cancer and are transcriptome-based, relating only indirectly to the therapeutically relevant and more stable protein level. Also, the recent introduction of antibody-drug conjugates (ADCs) requires quantitative data about the tumor specificity of their target proteins. We turned to the proteome to address these questions. Methods: We performed deep proteomic profiling of a comprehensive cohort by optimized tandem mass tag-labelled liquid chromatography-coupled tandem mass spectrometry. Data acquisition was validated internally with immunoblotting and externally by bioinformatic reclassification within existing, filtered transcriptomic data. After bioinformatics, the top cluster defining proteins were quantified immunohistochemically under real-world conditions. Protein profiles were individualized and separately evaluated with drug repurposing libraries. Cell viability assays were performed for a panel of twelve UC cell lines to validate these predictions in vitro. Results: We analyzed 434 samples with 242 tumors and 192 paired normal mucosae, covering all stages of UC. 9542 proteins were quantified and revealed five distinct proteomic subtypes. These were validated internally and externally, showing relevant survival stratification also in the TCGA dataset. The proteomic subtypes were independent from pathological groups with relevant stratification of progression- free and overall survival (low vs. high-risk: median 103 vs. 27 months). Tumor specificity of all proteins was highly heterogeneous across stages and subtypes. As an example, the ADC target NECTIN4 was generally overexpressed mainly in non-muscle-invasive UC. Drug repurposing revealed several new candidate drugs, each specific to different proteomic subtypes. In vitro data showed increased sensitivity by subtype in line with four out of seven representative predictions. Conclusions: Proteomic subtypes add independent prognostic information and carry predictive value for several newly identified adjuvant drug candidates. The actual tumor specificity of biomarkers and ADC targets is highly dependent on stage and subtype and calls for individualized patient-specific predictive testing. Citation Format: Franz Friedrich Dressler, Falk Diedrichs, Deema Sabtan, Sofie Hinrichs, Paulina Mackedanz, Mareile Schlotfeldt, Christoph Krisp, Martin Hennig, Hartmut Schlueter, Ulrich Wetterauer, Roman Zubarev, David Horst, Sven Perner, Philipp Wolf, Ákos Végvári. Proteomic analyses of the urothelial cancer landscape reveal highly distinct prognostic and predictive subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5138.

Site-Specific Differences of Eligibility for Adjuvant Immunotherapy Among Urothelial Carcinoma Patients Treated With Radical Surgery: Results From a Multicenter Cohort Study

BackgroundThe CheckMate274 trial has reported enhanced disease-free survival rates in patients with stage pT3–4/ypT2–4 or pN+ urothelial carcinoma (UC) undergoing adjuvant nivolumab therapy. This study compares prognostic differences between urothelial carcinoma of the bladder (UCB) and upper tract urothelial carcinoma (UTUC). MethodsWe retrospectively analyzed data from 719 patients with UC who underwent radical surgery, stratifying to patients at stage pT3–4 and/or pN+ without neoadjuvant chemotherapy (NAC) or at ypT2–4 and/or ypN+ with NAC (potential candidates for adjuvant immunotherapy), and to those who were not candidates for adjuvant immunotherapy. We used Kaplan–Meier curves to assess oncological outcomes, particularly nonurothelial tract recurrence-free survival (NUTRFS), cancer-specific survival (CSS), and overall survival (OS). Risk factors were identified by Cox regression analysis. ResultsKaplan–Meier curves showed significantly lower NUTRFS, CSS, and OS for potential adjuvant immunotherapy candidates than for noncandidates in each UCB and UTUC group. NUTRFS, CSS, and OS did not differ significantly between adjuvant immunotherapy candidates with UBC or UTUC. Trends were similar among patients ineligible for adjuvant immunotherapy. Pathological T stage (pT3–4 or ypT2–4), pathological N stage, and lymphovascular invasion (LVI) were independent predictors of oncological outcomes on multivariate analysis. ConclusionThe criteria for adjuvant immunotherapy candidates from the CheckMate 274 trial can also effectively stratify UC patients after radical surgery. Substantial clinical significance is attached to LVI status as well as to pathological T and N status, suggesting that LVI status should be considered when selecting suitable candidates for adjuvant immunotherapy.

Adjuvant effects of β-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus)

β-defensin of flounder plays an important role in immunomodulation by recruiting immune cells and has a potential vaccine adjuvant effect in addition to its bactericidal activity. In this study, adjuvant effects of β-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus) were investigated. The bicistronic eukaryotic expression plasmid pBudCE4.1 plasmid vector with two independent coding regions was selected to construct DNA vaccine of p-OmpC which express only the gene for the outer membrane protein of Edwardsiella tarda and the vaccine of p-OmpC-βdefensin which express both the outer membrane protein of the bacterium and β-defensin of flounder. In vitro and in vivo studies have shown that the constructed plasmids can be expressed in flounder embryonic cell lines and injection sites of muscles. After vaccination by intramuscular injection, both p-OmpC and p-OmpC-βdefensin groups showed significant upregulation of immune-response. Compared to the pBbudCE4.1 and the p-OmpC vaccinated groups, the p-OmpC-βdefensin vaccinated group showed significantly more cell aggregation at the injection site and intense immune response. The proportion of sIgM+ cells, as well as the CD4-1+ and CD4-2+ cells in both spleen and kidney was significantly higher in the p-OmpC-βdefensin vaccinated group at peak time point than in the control groups. The relative survival rate of the p-OmpC-βdefensin vaccine was 74.17%, which was significantly higher than that of the p-OmpC vaccinated group 48.33%. The results in this study determined that β-defensin enhances the responses in cellular and humoral immunity and evokes a high degree of protection against E. tarda, which is a promising candidate for vaccine adjuvant.

PGM-CSF as an adjuvant in DNA vaccination against SARS-CoV-2

The emergence of SARS-CoV-2 presents a significant global public health dilemma. Vaccination has long been recognized as the most effective means of preventing the spread of infectious diseases. DNA vaccines have attracted attention due to their safety profile, cost-effectiveness, and ease of production. This study aims to assess the efficacy of plasmid-encoding GM-CSF (pGM-CSF) as an adjuvant to augment the specific humoral and cellular immune response elicited by DNA vaccines based on the receptor-binding domain (RBD) antigen. Compared to the use of plasmid-encoded RBD (pRBD) alone, mice that were immunized with a combination of pRBD and pGM-CSF exhibited significantly elevated levels of RBD-specific antibody titers in serum, BALF, and nasal wash. Furthermore, these mice generated more potent neutralization antibodies against both the wild-type and Omicron pseudovirus, as well as the ancestral virus. In addition, pGM-CSF enhanced pRBD-induced CD4+ and CD8+ T cell responses and promoted central memory T cells storage in the spleen. At the same time, tissue-resident memory T (Trm) cells in the lung also increased significantly, and higher levels of specific responses were maintained 60 days post the final immunization. pGM-CSF may play an adjuvant role by promoting antigen expression, immune cells recruitment and GC B cell responses. In conclusion, pGM-CSF may be an effective adjuvant candidate for the DNA vaccines against SARS-CoV-2.

Type I and II interferons, transcription factors and major histocompatibility complexes were enhanced by knocking down the PRRSV-induced transforming growth factor beta in monocytes co-cultured with peripheral blood lymphocytes.

The innate and adaptive immune responses elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection are known to be poor. This study investigates the impact of PRRSV-induced transforming growth factor beta 1 (TGFβ1) on the expressions of type I and II interferons (IFNs), transcription factors, major histocompatibility complexes (MHC), anti-inflammatory and pro-inflammatory cytokines in PRRSV-infected co-cultures of monocytes and peripheral blood lymphocytes (PBL). Phosphorothioate-modified antisense oligodeoxynucleotide (AS ODN) specific to the AUG region of porcine TGFβ1 mRNA was synthesized and successfully knocked down TGFβ1 mRNA expression and protein translation. Monocytes transfected with TGFβAS1 ODN, then simultaneously co-cultured with PBL and inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) showed a significant reduction in TGFβ1 mRNA expression and a significant increase in the mRNA expressions of IFNα, IFNγ, MHC-I, MHC-II, signal transducer and activator of transcription 1 (STAT1), and STAT2. Additionally, transfection of TGFβAS1 ODN in the monocyte and PBL co-culture inoculated with cPRRSV-2 significantly increased the mRNA expression of interleukin-12p40 (IL-12p40). PRRSV-2 RNA copy numbers were significantly reduced in monocytes and PBL co-culture transfected with TGFβAS1 ODN compared to the untransfected control. The yields of PRRSV-2 RNA copy numbers in PRRSV-2-inoculated monocytes and PBL co-culture were sustained and reduced by porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα), respectively. These findings highlight the strategy employed by PRRSV to suppress the innate immune response through the induction of TGFβ expression. The inclusion of TGFβ as a parameter for future PRRSV vaccine and vaccine adjuvant candidates is recommended.

Suppressing the Substance P-NK1R Signalling Protects Mice against Sepsis-Associated Acute Inflammatory Injury and Ferroptosis in the Liver and Lungs.

Substance P (SP), encoded by the TAC1/Tac1 gene, acts as a significant mediator in dysregulated systemic inflammatory response and associated organ injury in sepsis by activating the neurokinin-1 receptor (NK1R). This study investigated the impact of SP-NK1R signaling on ferroptosis in the liver and lungs of mice with sepsis. Sepsis was induced by caecal ligation puncture (CLP) surgery in mice. The SP-NK1R signaling was suppressed by Tac1 gene deletion, NK1R blockade, and a combination of these two approaches. The physiological conditions of mice were recorded. The profile of the SP-NK1R cascade, inflammatory response, ferroptosis, and tissue histology were investigated in the liver and lungs. Several manifestations of sepsis occurred in Tac1+/+ mice during the development of sepsis. Notably, hypothermia became significant four hours after the induction of sepsis. In the liver and lungs of mice subjected to CLP surgery, the concentrations of SP and NK1R were upregulated. Additionally, the concentrations of pro-inflammatory mediators, including cytokines (IL-1β, IL-6, and TNF-α) and chemokines (MCP-1 and MIP-2), were increased. Moreover, ferroptosis was elevated, as evidenced by increased concentrations of iron and MDA and reduced concentrations of GSH, Nrf2, and Gpx4. Suppressing the SP-NK1R cascade significantly mitigated CLP-surgery-induced alterations in mice. Importantly, these three approaches used to suppress SP-NK1R signaling showed similar effects on protecting mice against sepsis. In conclusion, increased SP-mediated acute inflammatory response and injury in the liver and lungs in mice with CLP-surgery-induced sepsis was associated with elevated ferroptosis. The detrimental effect of SP on sepsis was predominantly mediated by NK1R. Therefore, the suppression of increased SP-NK1R signaling and ferroptosis may be a promising adjuvant therapeutic candidate for sepsis and associated acute liver and lung injury.