Immunogenic Effects Research Articles

Immunofilaments Are Well Tolerated after Local or Systemic Administration in Mice.

The invention of nanosized biomaterials has paved the way for novel therapeutics that can manipulate cells on a nanoscale. Nanosized immunofilaments (IFs) are synthetic filamentous polymers consisting out of polyisocyanopeptides, which have been recently established as a powerful platform to activate specific immune cells in vivo such that they raise an antitumor immune response. However, toxicological effects or immunogenicity toward the IFs have not yet been investigated. In this study, we evaluated potential toxic or immunogenic effects in C57BL/6 mice upon intravenous or subcutaneous injection of nonfunctionalized IFs or immunostimulatory IFs over 30 days. We here present a detailed analysis of the gross pathology, hematological parameters, blood biochemistry, histology, and antibody-response against the IF backbone. Our results demonstrate that IFs do not induce severe acute or chronic toxicity in mice. After 30 days, we only found elevated IgG-titers in intravenously injected but not subcutaneously injected mice. In summary, we demonstrate that IFs can be administered into a living organism without adverse side effects, thereby establishing the safety of IFs as a therapeutic intervention.

Abstract PO4-20-04: A Randomized Window of Opportunity Study of Preoperative Letrozole and Simvastatin Versus Letrozole Alone in Stage I-III Hormone Receptor Positive, HER2 Negative Breast Cancer

Abstract Hormone receptor positive (HR+), HER2 negative (HER2-) breast cancers are poor responders to immunotherapy. There is a need for innovative approaches to improve immune responses in these tumors. The combination of statins and aromatase inhibitors have demonstrated antiproliferative and immunomodulatory properties. However, this combination has never been studied in the preoperative setting; thus, our ongoing study represents an opportunity to investigate this approach. We designed a randomized two-arm presurgical “window of opportunity” trial to evaluate the effects of letrozole and simvastatin versus letrozole alone 14 days prior to surgery. Tissue and blood will be obtained at baseline and 14 days after completion of therapy at the time of surgery. Inflammatory markers in the blood and multiplex immunohistochemistry (IHC) in tissue will be assessed. To be eligible for the trial, participants must be postmenopausal women with histologically confirmed stage I-III HR+, HER2- invasive breast cancer with baseline ki-67 ≥10% that have not received prior chemotherapy, endocrine therapy, and/or immunotherapy within 3 months prior to trial enrollment. They should also not have received any cholesterol lowering medication within 3 months prior to trial enrollment. The primary objective is to determine if the addition of simvastatin to letrozole compared to letrozole alone will result in a decrease in geometric mean % change in ki-67 from pre-surgical baseline to 14 days following preoperative therapy. Ki-67 is a validated surrogate marker for disease-free survival in HR+, HER2- breast cancer. The main secondary objective is to determine if the addition of simvastatin to letrozole compared to letrozole alone will result in increased immune activation from pre- to post-treatment based on the evaluation of the immune subtype composition in tissue via multiplex immunofluorescence. We aim to enroll 16 patients in each arm to achieve 90% power and detect a minimum difference of 7.5% in ki-67 using a two-sided Mann-Whitney U or Wilcoxon Rank-Sum test. After accounting for a possible drop off rate of 20%, the study’s target accrual will be 40 participants. Statistical analyses will be performed using SAS 9.4. the significance level will be set at alpha = 0.1. Descriptive statistics will be applied to tissue and blood biomarkers of interest at 2 designated time points, prior to preoperative therapy and following completion of preoperative therapy. The absolute change or percentage change of those biomarkers will also be calculated and compared between the 2 arms using the nonparametric Mann-Whitney U test. The correlation among biomarkers will be described by Pearson correlation coefficient with 95% confidence interval. The p-value will be adjusted by Benjamini-Hochberg procedure to control the false discovery rate. All adverse events experienced data will be described by summary statistics and will be assessed according to CTCAE version 5.0. Currently, the study has accrued 2 participants. After completing accrual, we will assess whether the tumor-immune milieu has undergone modulation, resulting in a more immunogenic environment. If an immunogenic effect is demonstrated, this will provide rationale for a future multi-center trial assessing the combination of letrozole, simvastatin, and immunotherapy. Citation Format: Ruth Sacks, Elizabeth Haas, Jade Jones, Manali Bhave, Keerthi Gogineni, Jane Meisel, Demetria Smith-Graziani, Suchi Pakkala, Sarah Friend, Cletus Arciero, Lauren Postlewait, Clara Farley, Cathy Graham, Toncred Styblo, Monica Rizzo, Rhonda Pickett, Nicholas Crasta, Lori Brown, Xiaoxian Li, Sunil Badve, Madhav Dhodapkar, Kevin Kalinsky. A Randomized Window of Opportunity Study of Preoperative Letrozole and Simvastatin Versus Letrozole Alone in Stage I-III Hormone Receptor Positive, HER2 Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-04.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Immunogenicity of Current and Next-Generation Pneumococcal Conjugate Vaccines in Children: Current Challenges and Upcoming Opportunities.

Global use of pneumococcal conjugate vaccines (PCVs) with increasingly broader serotype coverage has helped to reduce the burden of pneumococcal disease in children and adults. In clinical studies comparing PCVs, higher-valency PCVs have met noninferiority criteria (based on immunoglobulin G geometric mean concentrations and response rates) for most shared serotypes. A numeric trend of declining immunogenicity against shared serotypes with higher-valency PCVs has also been observed; however, the clinical relevance is uncertain, warranting additional research to evaluate the effectiveness of new vaccines. Novel conjugation processes, carriers, adjuvants, and vaccine platforms are approaches that could help maintain or improve immunogenicity and subsequent vaccine effectiveness while achieving broader protection with increasing valency in pneumococcal vaccines.

Enhanced efficacy of a TLR3 agonist delivered by cowpea chlorotic mottle virus nanoparticles.

Intratumoral immunotherapies are those that are administered directly into a tumor to remodel the local tumor microenvironment and stimulate systemic anti-tumor immunity. Small molecule Toll-like receptor (TLR) agonists are undergoing development as intratumoral immunotherapies, and here we considered the TLR3 agonist poly(I:C). Because small molecule therapeutics often suffer rapid washout effects and ineffective immune cell uptake, we encapsulated poly(I:C) into nanoparticles derived from cowpea chlorotic mottle virus (CCMV). The particles (but not the separate components) stimulated the activity of macrophages in vitro and were able to reduce tumor growth and prolong survival in mouse models of colon cancer and melanoma. We also combined CCMV-poly(I:C) with oxaliplatin and found the combination therapy to be even more potent, strongly inhibiting tumor growth and increasing survival. The analysis of immune markers revealed that CCMV-poly(I:C) VLPs with oxaliplatin promoted the infiltration and activation of CD4+ and CD8+ cells and the production of IL-4 and IFN-γ, indicating a synergistic immunogenic effect. The combined treatment also enhanced the rate of apoptosis and immunogenic cell death (ICD). Our data support the development of combination therapies involving immunomodulatory plant virus nanoparticles and antineoplastic drugs to attack tumors directly and via the activation of innate and adaptive immune responses.

Efficacy and safety of human umbilical cord-derived mesenchymal stem cells in the treatment of refractory immune thrombocytopenia: a prospective, single arm, phase I trial

Patients with refractory immune thrombocytopenia (ITP) frequently encounter substantial bleeding risks and demonstrate limited responsiveness to existing therapies. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) present a promising alternative, capitalizing on their low immunogenicity and potent immunomodulatory effects for treating diverse autoimmune disorders. This prospective phase I trial enrolled eighteen eligible patients to explore the safety and efficacy of UC-MSCs in treating refractory ITP. The research design included administering UC-MSCs at escalating doses of 0.5 × 106 cells/kg, 1.0 × 106 cells/kg, and 2.0 × 106 cells/kg weekly for four consecutive weeks across three cohorts during the dose-escalation phase, followed by a dose of 2.0 × 106 cells/kg weekly for the dose-expansion phase. Adverse events, platelet counts, and changes in peripheral blood immunity were monitored and recorded throughout the administration and follow-up period. Ultimately, 12 (with an addition of three patients in the 2.0 × 106 cells/kg group due to dose-limiting toxicity) and six patients were enrolled in the dose-escalation and dose-expansion phase, respectively. Thirteen patients (13/18, 72.2%) experienced one or more treatment emergent adverse events. Serious adverse events occurred in four patients (4/18, 22.2%), including gastrointestinal hemorrhage (2/4), profuse menstruation (1/4), and acute myocardial infarction (1/4). The response rates were 41.7% in the dose-escalation phase (5/12, two received 1.0 × 106 cells/kg per week, and three received 2.0 × 106 cells/kg per week) and 50.0% (3/6) in the dose-expansion phase. The overall response rate was 44.4% (8/18) among all enrolled patients. To sum up, UC-MSCs are effective and well tolerated in treating refractory ITP (ClinicalTrials.gov ID: NCT04014166).

Immunogenic and toxic effects of graphene oxide nanoparticles in mouse skeletal muscles and human red blood cells

To investigate immunogenic and toxic effects of graphene oxide (GO) nanoparticles in mouse skeletal muscles and in human blood in vitro. GO nanoparticles prepared using a probe sonicator were supended in deionized H2O or PBS, and particle size and surface charge of the nanoparticles were measured with dynamic light scattering (DLS). Different concentrations (0.5, 1.0 and 2.0 mg/mL) of GO suspension or PBS were injected at multiple sites in the gastrocnemius muscle (GN) of C57BL/6 mice, and inflammatory response and immune cell infiltrations were detected with HE and immunofluorescence staining. We also examined the effects of GO nanoparticles on human red blood cell (RBC) morphology, hemolysis and blood coagulation using scanning electron microscope (SEM), spectrophotometry, and thromboelastography (TEG). GO nanoparticles suspended in PBS exhibited better colloidal dispersity, stability and surface charge effects than those in deionized H2O. In mouse GNs, injection of GO suspensions dose- and time-dependently resulted in sustained muscular inflammation and myofiber degeneration at the injection sites, which lasted till 8 weeks after the injection; immunofluorescence staining revealed obvious infiltration of monocytes, macrophages, dendritic cells and CD4+ T cells around the injection sites in mouse GNs. In human RBCs, incubation with GO suspensions at 0.2, 2.0 and 20 mg/mL, but not at 0.002 or 0.02 mg/mL, caused significant alterations of cell morphology and hemolysis. TEG analysis showed significant abnormalities of blood coagulation parameters following treatment with high concentrations of GO. GO nanoparticles can induce sustained inflammatory and immunological responses in mouse GNs and cause RBC hemolysis and blood coagulation impairment, suggesting its muscular toxicity and hematotoxicity at high concentrations.

Immunoinformatics Design and In Vivo Immunogenicity Evaluation of a Conserved CTL Multi-Epitope Vaccine Targeting HPV16 E5, E6, and E7 Proteins.

Human papillomavirus type 16 (HPV16) infection is responsible for more than 50% of global cervical cancer cases. The development of a vaccine based on cytotoxic T-lymphocyte (CTL) epitopes is a promising strategy for eliminating pre-existing HPV infections and treating patients with cervical cancer. In this study, an immunoinformatics approach was used to predict HLA-I-restricted CTL epitopes in HPV16 E5, E6, and E7 proteins, and a set of conserved CTL epitopes co-restricted by human/murine MHCs was screened and characterized, with the set containing three E5, four E6, and four E7 epitopes. Subsequently, the immunogenicity of the epitope combination was assessed in mice, and the anti-tumor effects of the multi-epitope peptide vaccine E5E6E7pep11 and the recombinant protein vaccine CTB-Epi11E567 were evaluated in the TC-1 mouse tumor model. The results demonstrated that mixed epitope peptides could induce antigen-specific IFN-γ secretion in mice. Prophylactic immunization with E5E6E7pep11 and CTB-Epi11E567 was found to provide 100% protection against tumor growth in mice. Moreover, both types of the multi-epitope vaccine significantly inhibited tumor growth and prolonged mouse survival. In conclusion, in this study, a multi-epitope vaccine targeting HPV16 E5, E6, and E7 proteins was successfully designed and evaluated, demonstrating potential immunogenicity and anti-tumor effects and providing a promising strategy for immunotherapy against HPV-associated tumors.

Preclinical evaluation of two phylogenetically distant arenavirus vectors for the development of novel immunotherapeutic combination strategies for cancer treatment

BackgroundEngineered arenavirus vectors have recently been developed to leverage the body’s immune system in the fight against chronic viral infections and cancer. Vectors based on Pichinde virus (artPICV) and lymphocytic...

Enhanced Immunogenicity and Protective Effects against SARS-CoV-2 Following Immunization with a Recombinant RBD-IgG Chimeric Protein.

The unprecedented global impact caused by SARS-CoV-2 imposed huge health and economic challenges, highlighting the urgent need for safe and effective vaccines. The receptor-binding domain (RBD) of SARS-CoV-2 is the major target for neutralizing antibodies and for vaccine formulations. Nonetheless, the low immunogenicity of the RBD requires the use of alternative strategies to enhance its immunological properties. Here, we evaluated the use of a subunit vaccine antigen generated after the genetic fusing of the RBD with a mouse IgG antibody. Subcutaneous administration of RBD-IgG led to the extended presence of the protein in the blood of immunized animals and enhanced RBD-specific IgG titers. Furthermore, RBD-IgG immunized mice elicited increased virus neutralizing antibody titers, measured both with pseudoviruses and with live original (Wuhan) SARS-CoV-2. Immunized K18-hACE2 mice were fully resistant to the lethal challenge of the Wuhan SARS-CoV-2, demonstrated by the control of body-weight loss and virus loads in their lungs and brains. Thus, we conclude that the genetic fusion of the RBD with an IgG molecule enhanced the immunogenicity of the antigen and the generation of virus-neutralizing antibodies, supporting the use of IgG chimeric antigens as an approach to improve the performance of SARS-CoV-2 subunit vaccines.

Outcomes of a mandatory non-medical switch of infliximab to a biosimilar for inflammatory bowel disease in British Columbia, Canada.

Despite infliximab biosimilars becoming widely used in inflammatory bowel disease (IBD) patients, real-world non-medical switching is sparse. A biosimilar non-medical switch was launched in British Columbia in 2019, the first Canadian province to do so, from Remicade to an approved biosimilar (CT-P13 or SB2). This study aims to obtain real-world evidence evaluating the clinical outcomes of non-medical switching from Remicade to the infliximab biosimilars. This is a retrospective observational study of stable IBD patients from the IBD Centre of BC who underwent the non-medical infliximab switch. The primary outcome is treatment continuation at 12 ± 2 months post-switch. Secondary outcomes include frequency of loss of response, adverse events, and immunogenicity within the first 12 months post-switch. A control group of patients maintained on the originator served as a comparison. Patients in the biosimilar switch group (n = 264) and originator group (n = 99), show similar demographics and disease characteristics. There was no difference in infliximab continuation between the biosimilar group (94.9%) and the originator group (90.1%) (P = 0.18). Reasons for discontinuation of infliximab included loss of response (4.04% vs 4.91%), immunogenicity (1.01% vs 0.75%), or adverse effect (1.01% vs 2.3%) in the infliximab originator vs biosimilar switch group, respectively. Similarly, no differences in safety or efficacy were observed between patients switched to CT-P13 or SB2. Non-medical biosimilar switch of infliximab demonstrates similar clinical outcomes compared to originator molecule continuation for therapy of IBD. These data support the safety and efficacy of non-medical infliximab switching in IBD patients.

Abstract 6749: A dendritic cell targeted nanosystem to overcome immune suppression and enhance the efficacy of the standard of care for pancreatic cancer

Abstract The development of novel therapies for pancreatic ductal adenocarcinoma (PDAC) remains an urgent unmet medical need, as it was proven to be refractory to most conventional anti-neoplastic therapies. PDAC is characterized by highly dense stroma, immunosuppressive tumor microenvironment (TME), high resistance to chemotherapy, and poor presentation of tumor-associated antigens (TAA) by dendritic cells (DC), which results in low immune cell infiltration to the tumor site. To that end, we designed a DC-targeted nanovaccine (NV) containing PDAC TAA and TLR agonists to modulate the PDAC immune-microenvironment. In recent years, the combination of immunotherapy with chemotherapy has been explored for PDAC therapy to improve efficacy. Therefore, we demonstrate the effect of our NV on the anti-tumor immune-mediated response in PDAC-bearing mice, alone and in combination with Gemcitabine and Nab-Paclitaxel (Gem/NabP). Our DC-targeted NV was formulated by the double emulsion method and loaded with PDAC’s TAA and immune potentiators. We conjugated the NV to a mannose moiety as a targeting moiety for the mannose receptor expressed on DC to increase the NV uptake, which resulted in the enhanced presentation of the encapsulated TAA. Followed by physicochemical characterization and evaluation of preclinical safety of the NV, the stimulation of T cell-specific response against PDAC tumor was assessed in vivo and ex vivo. The NV induced significant changes in PDAC’s TME, such as increased CD8+ T cells, decreased CD4+ T regulatory cells, and induced memory immunity in the tumor site. The combination of NV and Gem/NabP inhibited tumor growth and prolonged survival compared to mice treated with Gem/NabP alone. This could also be related to the immunogenic cell death effect caused by the chemotherapies. In addition to our mice model of PDAC, we also established a patient-derived model, in which we generate 3D-spheroids from the patient’s tumor and activate PBMCs with our NV from the same patient. This unique 3D model enables us to evaluate ex vivo the NV effect in a more clinically relevant manner. The NV proposed herein could induce a strong antigen-specific immune response against PDAC with restricted tumor growth while re-shaping PDAC immunosuppressive TME. Furthermore, our 3D patient-derived PDAC model allows us to assess the ability of our NV to generate specific immune responses in patients and to test other possible drug combinations predicting the clinical outcome. Citation Format: Ron Kleiner, Daniella Vaskovich-Koubi, Rita Acúrcio, Barbara Carreira, Helena Florindo, Ronit Satchi-Fainaro. A dendritic cell targeted nanosystem to overcome immune suppression and enhance the efficacy of the standard of care for pancreatic cancer [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 6749.

RNA is a pro-apoptotic target of cisplatin in cancer cell lines and C. elegans

Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20 ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.

Effect of GnRH Active Immunisation on Reproductive Performance of Male Sprague Dawley Rats.

To investigate the effect of active immunisation with gonadotropin-releasing hormone (GnRH) on the reproductive function in male Sprague Dawley (SD) rats, 24 42-day-old rats were randomly assigned to treatment with GnRH6-MAP, GnRH-OVA, a surgical castration group, and a blank control group. Each rat in the treatment groups was intramuscularly injected at 6, 8, and 10 weeks of age. The serum concentrations of testosterone (T), follicle-stimulating hormone (FSH), luteinising hormone (LH), and anti-GnRH antibodies were determined using enzyme-linked immunosorbent assays. The results showed that active immunisation with recombinant GnRH6-MBP and GnRH-OVA significantly increased the serum levels of anti-GnRH antibodies and reduced the serum concentrations of testosterone compared to the black control. Eight weeks after immunisation, the rats' testes were surgically removed for morphological evaluation, showing atrophy of the convoluted vasculature, relative emptying of the lumen, and insignificant differentiation of spermatogonial cells, which were increased in weight and volume compared with the blank control group. These findings indicated that active immunisation with GnRH can lead to testicular atrophy and reduce gonadal hormone concentrations, suggesting that GnRH is a highly effective immunogen.

Dendritic nanomedicine enhances chemo-immunotherapy by disturbing metabolism of cancer-associated fibroblasts for deep penetration and activating function of immune cells

Inefficient drug penetration hurdled by the stroma in the tumor tissue leads to a diminished therapeutic effect for drugs and a reduced infiltration level of immune cells. Herein, we constructed a PEGylated dendritic epirubicin (Epi) prodrug (Epi-P4D) to regulate the metabolism of cancer-associated fibroblasts (CAFs), thus enhancing Epi penetration into both multicellular tumor spheroids (MTSs) and tumor tissues in mouse colon cancer (CT26), mouse breast cancer (4T1) and human breast cancer (MDA-MB-231) models. Enhanced cytotoxicity against CT26 MTSs and remarkable antitumor efficacy of Epi-P4D were ascribed to reduced fibronectin, α-SMA, and collagen secretion. Besides, thinning of the tumor tissue stroma and efficient eradication of tumor cells promoted the immunogenic cell death effect for dendritic cell (DC) maturation and subsequent immune activation, including elevating the CD4+ T cell population, reducing CD4+ and CD8+ T cell hyperactivation and exhaustion, and amplifying the natural killer (NK) cell proportion and effectively activating them. As a result, this dendritic nanomedicine thinned the stroma of tumor tissues to enhance drug penetration and facilitate immune cell infiltration for elevated antitumor efficacy.

Preclinical toxicity analyses of lentiviral vectors expressing the HIV-1 LTR-specific designer-recombinase Brec1.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.

Nanoparticles integrated withmild photothermal therapy and oxaliplatin for tumor chemotherapy and immunotherapy.

Aims: Preparation and evaluation of nanoparticles for tumor chemotherapy and immunotherapy mild photothermal therapy and oxaliplatin. Methods: The double emulsion method was used for nanoparticle preparations. Polydopamine was deposited on the surface, which was further modified with folic acid. Cytotoxicity assays were carried out by cell counting kit-8. In vivo antitumor assays were carried out on 4T1 tumor-bearing mice. Results: The nanoparticles exhibited a 190nm-diameter pomegranate-like sphere, which could increase temperature to 43-46°C. In vivo distribution showed enhanced accumulation. The nanoparticles generated stronger immunogenic cell death effects. By stimulating the maturation of dendritic cells, mild photothermal therapy combined with oxaliplatin significantly increased the antitumor effect by a direct killing effect and activation of immunotherapy. Conclusion: This study provided a promising strategy of combination therapy for tumors.

Sterile Water Versus Glycine in Transurethral Resection of Bladder Tumors—Immunogenic and Clinical Implications

Background and objectiveWe compared the oncologic outcomes of patients with non-muscle invasive bladder cancer (NMIBC) who underwent transurethral resection of bladder tumor (TUBRT) using sterile water vs glycine irrigation. The tumoricidal and immunogenic effects of these solutions on urothelial cancer cell lines were investigated. MethodsThe medical records of 530 consecutive patients who underwent TURBT using sterile water or glycine irrigation for NMIBC were reviewed. Recurrence and progression rates were evaluated using time dependent analyses.Bladder cancer cell lines (RT4, T24 and 5637) were treated with glycine and sterile water. Cell viability was evaluated with the XTT assay. Cell membrane calreticulin levels were evaluated with flow cytometry. Extracellular high mobility group box 1 (HMGB1) and heat shock 70 (HSP70) protein levels were evaluated using western blots. Key findings and limitationsAfter propensity score matching each study arm comprised 161 patients. Median follow-up was 13.6 months (IQR 6.2, 24.5). The 2-year recurrence free survival was significantly lower in the sterile water vs glycine group (43% vs 71%, respectively, p<0.0001). Similarly, the 2-years progression free survival was significantly lower in the sterile water vs glycine group (85% vs 94%, respectively, p<0.014). Sterile water treatment resulted in the lowest number of viable cells. Early and late immunogenic cell death markers were markedly elevated in cells treated with glycine. Conclusions and clinical implicationsSterile water compared to glycine irrigation during TURBT for NMIBC was associated with higher recurrence and progression rates. Possible explanation for these findings is the diminished immune response associated with sterile water reflected in a comparatively lesser expression of immune response inducers. Patient summaryWe compared two irrigation fluids in non–muscle-invasive bladder cancer surgery: glycine and sterile water. Glycine outperformed sterile water in cancer recurrence, possibly boosting immunogenicity over sterile water.

A subunit-based influenza/SARS-CoV-2 Omicron combined vaccine induced potent protective immunity in BALB/c mice.

Infection with influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant risk to human life, health, and the global economy. Vaccination is one of the most effective strategies in the fight against infectious viruses. In this study, we, for the first time, have evaluated the immunogenicity and protective effect of an influenza/SARS-CoV-2 Omicron subunit combined vaccine adjuvanted with MF59 and administered to BALB/c mice. Results showed that the combined vaccine induced high levels of IgG, IgG1 , and IgG2a antibodies, as well as influenza A H1N1/California/2009 virus-specific hemagglutination-inhibiting antibodies in BALB/c mice. Moreover, this subunit combined vaccine induced high titers of neutralization antibodies against SARS-CoV-2 Omicron sublineage BA.5 pseudovirus and effectively reduced the viral load of authentic SARS-CoV-2 Omicron sublineage BA.5.2 in the cell culture supernatants. These results suggested that this subunit combined vaccine achieved protective effect against both H1N1 A/California/07/2009 strain and SARS-CoV-2 Omicron BA.5.2 variant. It is therefore expected that this study will establish the scientific foundation for the next-step development of combined vaccines against other strains or variants of IAV and SARS-CoV-2.

Engineering self-deliverable ribonucleoproteins for genome editing in the brain

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.