Strong Systemic Immune Responses Research Articles

An optically responsive cancer vaccine for inducing robust anti-tumor immunity by apoptotic body carrying nanoadjuvants

A cancer vaccine based on nano-adjuvant fusion represents an effective strategy for anti-tumor immunity. Compared with other cancer vaccine formulations, the ones based on apoptotic bodies not only possess higher load-bearing capacity and biosafety, but also present greater clinical translational potential. In this study, we developed an optically responsive cancer vaccine that induced robust anti-tumor immunity by encapsulating genipin-crosslinked nano-adjuvants in tumor cell apoptotic bodies. The vaccine may potentially be used for prophylactic or therapeutic purposes. As an antigen source and a cargo vehicle, apoptotic bodies can co-deliver antigens and adjuvants simultaneously to antigen-presenting cells to activate T cells. Nano-adjuvants promote the targeted delivery of adjuvants for effective immune responses. Genipin-crosslinked nano-adjuvants enable visual tracking of the tumor vaccine. In addition, the photothermal properties of genipin-crosslinked nano-adjuvants promote lysosomal escape of tumor antigens to enhance cross-presentation. These characteristics enable visualization and controllability of cancer vaccines, thereby avoiding the need for exogenous photosensitive reagents. Apoptotic body-based vaccines can be used to prevent or treat tumors by triggering a strong systemic immune response by promoting T-cell infiltration and improving the tumor immune microenvironment.

An Active Self-Mitochondria-Targeting Cyanine Immunomodulator for Near-Infrared II Fluorescence Imaging-Guided Synergistic Photodynamic Immunotherapy.

Photodynamic therapy targeting mitochondria represents a promising therapeutic strategy for fighting diverse types of cancers. However, the currently available photosensitizers (PSs) suffer from insufficient therapeutic potency, limited mitochondria delivery efficiency, and the inability to treat invisible metastatic distal cancers. Herein, an active self-mitochondria-targeting heptapeptide cyanine (HCy) immunomodulator (I2HCy-QAP) is reported for near-infrared II (NIR-II) fluorescence imaging-guided photodynamic immunotherapy of primary and distal metastatic cancers. The I2HCy-QAP is designed by introducing a quaternary ammonium salt with a phenethylamine skeleton (QAP) into the iodinated HCy photosensitizer. The I2HCy-QAP can precisely target mitochondria due to the lipophilic cationic QAP unit, present strong NIR-II fluorescence tail emission, and effectively generate singlet oxygen 1O2under NIR laser irradiation, thereby inducing mitochondria-targeted damages and eliciting strong systemic immunogenic cell death immune responses. The combination of the I2HCy-QAP-mediated photodynamic immunotherapy with anti-programmed death-1 antibody therapy achieves remarkable therapeutic efficacy against both primary and distal metastatic cancers with significant inhibition of lung metastasis in a triple-negative breast cancer model. This work provides a new concept for designing high-performance NIR emissive cyanine immunomodulators for NIR-II fluorescence-guided photodynamic immunotherapy.

Uplifting Antitumor Immunotherapy with Lymph-Node-Targeted and Ratio-Controlled Codelivery of Tumor Cell Lysate and Adjuvant.

Cancer vaccines provide a potential strategy to cure patients. Their clinical utilization and efficacy is, however, limited by incomplete coverage of tumor neoantigens and unspecific and restricted activation of dendritic cells (DCs). Tumor cell lysates (TCLs) containing a broad spectrum of neoantigens, while are considered ideal in formulating personalized vaccines, induce generally poor antigen presentation and transient antitumor immune response. Here, intelligent polymersomal nanovaccines (PNVs) that quantitatively coload, efficiently codeliver, and responsively corelease TCL and CpG adjuvant to lymph node (LN) DCs are developed to boost antigen presentation and to induce specific and robust antitumor immunity. PNVs carrying CpG and ovalbumin (OVA) markedly enhance the maturation, antigen presentation, and downstream T cell activation ability of bone-marrow-derived dendritic cells and induce strong systemic immune response after tail base injection. Remarkably, PNVs carrying CpG and TCL cure 85% of B16-F10 melanoma-bearing mice and generate long-lasting anticancer immune memory at a low dose, protecting all cured mice from tumor rechallenge. These LN-directed PNVs being highly versatile and straightforward opens a new door for personalized cancer vaccines.

Gas-generating polymersomes-based amplified photoimmunotherapy for abscopal effect and tumor metastasis inhibition

Due to the heterogeneity of tumors, single phototherapy cannot completely ablate tumors and inhibit tumor metastasis. To overcome these, we formulated targeted and multifunctional polymersomes ABC@ICG-IMQ-LHRH (AIRL) that encapsulated Toll-like receptor (TLR) 7/8 agonist imiquimod (IMQ) and photosensitizer indocyanine green (ICG) in the hydrophobic layer as well as bubble-generator NH4HCO3 in the hydrophilic cavity to inhibit the growth of primary and distant tumors, and prevent tumor metastasis through synergistic photoimmunotherapy. The AIRL polymersomes exhibited uniform and stable size, and high drug encapsulation efficiency, acid/reduction/laser responsiveness, excellent photothermal conversion efficiency, effective reactive oxygen species generation, high tumor accumulation. AIRL could be effectively internalized by dendritic cells (DCs), achieve lysosome escape and enhance DCs maturation. The synergistic photoimmunotherapy via AIRL polymersomes remarkably promoted the differentiation and activation of T cells, elevated strong systemic immune response to eradicate primary tumors and inhibit the growth of distant tumors. Simultaneously, the endurable immunological memory prevented tumor metastasis, which provided a promising nanoplatform for the combination therapy of cancer.

Assembling Au8 clusters on surfaces of bifunctional nanoimmunomodulators for synergistically enhanced low dose radiotherapy of metastatic tumor

BackgroundRadiotherapy is one of the mainstays of cancer therapy and has been used for treating 65–75% of patients with solid tumors. However, radiotherapy of tumors has two limitations: high-dose X-rays damage adjacent normal tissue and tumor metastases cannot be prevented.ResultsTherefore, to overcome the two limitations of radiotherapy, a multifunctional core–shell R837/BMS@Au8 nanoparticles as a novel radiosensitizer were fabricated by assembling Au8NCs on the surface of a bifunctional nanoimmunomodulator R837/BMS nanocore using nanoprecipitation followed by electrostatic assembly. Formed R837/BMS@Au8 NP composed of R837, BMS-1, and Au8 clusters. Au8NC can enhance X-ray absorption at the tumor site to reduce X-ray dose and releases a large number of tumor-associated antigens under X-ray irradiation. With the help of immune adjuvant R837, dendritic cells can effectively process and present tumor-associated antigens to activate effector T cells, meanwhile, a small-molecule PD-L1 inhibitor BMS-1 can block PD-1/PD-L1 pathway to reactivate cytotoxic T lymphocyte, resulting in a strong systemic antitumor immune response that is beneficial for limiting tumor metastasis. According to in vivo and in vitro experiments, radioimmunotherapy based on R837/BMS@Au8 nanoparticles can increase calreticulin expression on of cancer cells, reactive oxygen species generation, and DNA breakage and decrease colony formation. The results revealed that distant tumors were 78.2% inhibited depending on radioimmunotherapy of primary tumors. Therefore, the use of a novel radiosensitizer R837/BMS@Au8 NPs realizes low-dose radiotherapy combined with immunotherapy against advanced cancer.ConclusionIn conclusion, the multifunctional core–shell R837/BMS@Au8 nanoparticles as a novel radiosensitizer effectively limiting tumor metastasis and decrease X-ray dose to 1 Gy, providing an efective strategy for the construction of nanosystems with radiosensitizing function.

Immune cell receptor-specific nanoparticles as a potent adjuvant for nasal split influenza vaccine delivery

Mucosal delivery systems have gained much attention as effective way for antigen delivery that induces both systemic and mucosal immunity. However, mucosal vaccination faces the challenges of mucus barrier and effective antigen uptake and presentation. In particular, split, subunit and recombinant protein vaccines that do not have an intact pathogen structure lack the efficiency to stimulate mucosal immunity. In this study, poly (lactic acid-co-glycolic acid-polyethylene glycol) (PLGA-PEG) block copolymers were modified by mannose to form a PLGA-PEG-Man conjugate (mannose modified PLGA-PEG), which were characterized. The novel nanoparticles (NPs) prepared with this material had a particle size of about 150 nm and a zeta potential of −15 mV, and possessed ideal mucus permeability, immune cell targeting, stability and low toxicity. Finally, PLGA-PEG-Man nanoparticles (PLGA-PEG-Man NPs) were successfully applied for intranasal delivery of split influenza vaccine in rat for the first time, which triggered strong systemic and mucosal immune responses. These studies suggest that PLGA-PEG-Man NPs could function as competitive potential nano-adjuvants to address the challenge of inefficient mucosal delivery of non-allopathogenic antigens.

Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFβ or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

Ferritin-Based Nanocomposite Hydrogel Promotes Tumor Penetration and Enhances Cancer Chemoimmunotherapy.

Hydrogels are prevailing drug delivery depots to improve antitumor efficacy and reduce systemic toxicity. However, the application of conventional free drug-loaded hydrogel is hindered by poor drug penetration in solid tumors. Here, an injectable ferritin-based nanocomposite hydrogel is constructed to facilitate tumor penetration and improve cancer chemoimmunotherapy. Specifically, doxorubicin-loaded human ferritin (Dox@HFn) and oxidized dextran (Dex-CHO) are used to construct the injectable hydrogel (Dox@HFn Gel) through the formation of pH-sensitive Schiff-base bonds. After peritumoral injection, the Dox@HFn Gel is retained locally for up to three weeks, and released intact Dox@HFn gradually, which can not only facilitate tumor penetration through active transcytosis but also induce immunogenic cell death (ICD) to tumor cells to generate an antitumor immune response. Combining with anti-programmed death-1 antibody (αPD-1), Dox@HFn Gel induces remarkable regression of orthotopic 4T1 breast tumors, further elicits a strong systemic anti-tumor immune response to effectively suppress tumor recurrence and lung metastasis of 4T1 tumors after surgical resection. Besides, the combination of Dox@HFn GelL with anti-CD47 antibody (αCD47) inhibits postsurgical tumor recurrence of aggressive orthotopic glioblastoma tumor model and significantly extends mice survival. This work sheds light on the construction of local hydrogels to potentiate antitumor immune response for improved cancer therapy.

Mucosal Immunization Has Benefits over Traditional Subcutaneous Immunization with Group A Streptococcus Antigens in a Pilot Study in a Mouse Model.

Group A Streptococcus (GAS) is a major human pathogen for which there is no licensed vaccine. To protect against infection, a strong systemic and mucosal immune response is likely to be necessary to prevent initial colonization and any events that might lead to invasive disease. A broad immune response will be necessary to target the varied GAS serotypes and disease presentations. To this end, we designed a representative panel of recombinant proteins to cover the stages of GAS infection and investigated whether mucosal and systemic immunity could be stimulated by these protein antigens. We immunized mice sublingually, intranasally and subcutaneously, then measured IgG and IgA antibody levels and functional activity through in vitro assays. Our results show that both sublingual and intranasal immunization in the presence of adjuvant induced both systemic IgG and mucosal IgA. Meanwhile, subcutaneous immunization generated only a serum IgG response. The antibodies mediated binding and killing of GAS cells and blocked binding of GAS to HaCaT cells, particularly following intranasal and subcutaneous immunizations. Further, antigen-specific assays revealed that immune sera inhibited cleavage of IL-8 by SpyCEP and IgG by Mac/IdeS. These results demonstrate that mucosal immunization can induce effective systemic and mucosal antibody responses. This finding warrants further investigation and optimization of humoral and cellular responses as a viable alternative to subcutaneous immunization for urgently needed GAS vaccines.

Oral Immunization with rVSV Bivalent Vaccine Elicits Protective Immune Responses, Including ADCC, against Both SARS-CoV-2 and Influenza A Viruses.

COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.

Synergistic strategy based on mild phototherapy and deep tumor hypoxia reversal comprehensively remodels the tumor microenvironment for improved immunotherapy

Immunotherapy has shown remarkable therapeutic efficacy in triple-negative breast cancer (TNBC), but successful immune responses occur in only a subset of patients due to the hostile tumor microenvironment (TME). Specifically, low immunogenicity, dense extracellular matrix, and immunosuppressive environment in the TME are difficult to bypass for immunotherapy. The hypoxic environment of solid tumors not only contributes to an unfavorable TME but also functions in inhibiting the therapeutic effect of multiple antitumor methods, especially photodynamic therapy (PDT). In this study, we successfully developed a synergistic regulation nanosystem with albumin-mediated tumor targeted delivery of the photosensitizer IR780 and oxygen-generating nanoenzyme MnO2. As expected, MnO2 responsively catalyzed the decomposition of endogenous tumor H2O2 into O2, thereby enhancing the effect of PDT in triggering immunogenic cell death (ICD) and boosting tumor immunogenicity. Additionally, the enhanced PDT also functions in eliminating the tumor stromal barrier, facilitating the infiltration of effector T cells and therapeutics. The subsequent delivery of nanosystems containing MnO2 could reach deeper into the core of the tumor to alleviate severe hypoxia. Furthermore, relief of deep tumor hypoxia could significantly induce vascular normalization and reversal of tumor immunosuppression, decreasing the negative immunomodulatory cells such as Tregs and MDSCs. Resultantly, a positive feedback loop between mild phototherapy and deep hypoxia reversal was established using the synergistic strategy, which achieved comprehensive TME remodeling. Its combination with PD-L1 checkpoint blockades could lead to the regression of primary tumors and untreated metastasis tumors by inducing a strong systemic immune response, enhancing the immunotherapeutic efficacy for TNBC.

ISCOMs/MPLA-Adjuvanted SDAD Protein Nanoparticles Induce Improved Mucosal Immune Responses and Cross-Protection in Mice.

The epidemics caused by the influenza virus are a serious threat to public health and the economy. Adding appropriate adjuvants to improve immunogenicity and finding effective mucosal vaccines to combat respiratory infection at the portal of virus entry are important strategies to boost protection. In this study, a novel type of core/shell protein nanoparticle consisting of influenza nucleoprotein (NP) as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by SDAD hetero-bifunctional crosslinking is exploited. Immune-stimulating complexes (ISCOMs)/monophosphoryl lipid A (MPLA) adjuvants further boost the NP/NA-M2e SDAD protein nanoparticle-induced immune responses when administered intramuscularly. The ISCOMs/MPLA-adjuvanted protein nanoparticles are delivered through the intranasal route to validate the application as mucosal vaccines. ISCOMs/MPLA-adjuvanted nanoparticles induce significantly strengthened antigen-specific antibody responses, cytokine-secreting splenocytes in the systemic compartment, and higher levels of antigen-specific IgA and IgG in the local mucosa. Meanwhile, significantly expanded lung resident memory (RM) T and B cells (TRM /BRM ) and alveolar macrophages population are observed in ISCOMs/MPLA-adjuvanted nanoparticle-immunized mice with a 100% survival rate after homogeneous and heterogeneous H3N2 viral challenges. Taken together, ISCOMs/MPLA-adjuvanted protein nanoparticles could improve strong systemic and mucosal immune responses conferring protection in different immunization routes.

Cationic crosslinked carbon dots-adjuvanted intranasal vaccine induces protective immunity against Omicron-included SARS-CoV-2 variants

Mucosal immunity plays a significant role in the first-line defense against viruses transmitted and infected through the respiratory system, such as SARS-CoV-2. However, the lack of effective and safe adjuvants currently limits the development of COVID-19 mucosal vaccines. In the current study, we prepare an intranasal vaccine containing cationic crosslinked carbon dots (CCD) and a SARS-CoV-2 antigen, RBD-HR with spontaneous antigen particlization. Intranasal immunization with CCD/RBD-HR induces high levels of antibodies with broad-spectrum neutralization against authentic viruses/pseudoviruses of Omicron-included variants and protects immunized female BALB/c mice from Omicron infection. Despite strong systemic cellular immune response stimulation, the intranasal CCD/RBD-HR vaccine also induces potent mucosal immunity as determined by the generation of tissue-resident T cells in the lungs and airway. Moreover, CCD/RBD-HR not only activates professional antigen-presenting cells (APCs), dendritic cells, but also effectively targets nasal epithelial cells, promotes antigen binding via sialic acid, and surprisingly provokes the antigen-presenting of nasal epithelial cells. We demonstrate that CCD is a promising intranasal vaccine adjuvant for provoking strong mucosal immunity and might be a candidate adjuvant for intranasal vaccine development for many types of infectious diseases, including COVID-19.

Abstract 694: TG6050 an oncolytic vaccinia virus armed with interleukin 12 and anti-CTLA4 antibody induces TME remodeling and strong anti-tumoral responses

Abstract Background: By replicating specifically into tumor cells, oncolytic vaccinia viruses (VACV) can turn “cold” tumors “hot” while delivering therapeutic payloads into tumor. Thanks to its large genome capacity, multiple cloning loci, and availability of different promoters, VACV is an optimal vector for the design of advanced immunotherapies. Payloads with an established clinical efficacy but limited in terms of tolerability require local delivery. We report here the preclinical characterization of TG6050, a VACV encoding single chain interleukin 12 (IL12) and anti-CTLA4, targeting multiple cancer indications. Methods: TG6050 was generated by insertion of both the single chain IL-12p70, and an anti-CTLA-4 full-length (heavy and light chains independently) into the thymidine kinase (TK) and ribonucleotide reductase (RR) loci, of a triple deleted (ΔJ2R,ΔI4L,ΔM2L) VACV, Copenhagen strain. TG6050 was fully characterized in vitro, i.e. replication in tumor and normal cells, transgenes expression, functionality, and genetic stability. Its preclinical surrogate mTG6050, expressing murine payloads, was used to investigate the in vivo anti-tumoral activities in a broad range of immunocompetent murine models, as well as its mode of action in the reprograming of the tumor microenvironment (TME). Results: TG6050 displayed the same replicative, oncolytic activity, and genetic stability features as benchmark recombinant vaccinia viruses. IL-12 and anti-CTLA4 were both expressed at high levels, and as functional molecules by a broad panel of reference tumor cell lines. Local administration of mTG6050 induced accumulation of transgenes into tumor, with low systemic exposure. Noteworthy, expression of IL-12 did not accelerate the clearance of the viral vector. The combined effects of viral replication, IL-12 and anti-CTLA4 expression translated into impressive antitumoral activities in several syngeneic tumor models including immune-resistant ones such as B16F10 or LLC1. Transcriptomic analyses of the TG6050-treated tumors demonstrated a strong dynamic of infiltration by innate and adaptive immune cells. IFNy-ELISpot analysis on splenocytes confirmed induction of a strong systemic and specific anti-tumoral immune response. Moreover, the combination of TG6050 with ICI improved tumor regression in several challenging tumor models. Conclusion: TG6050 is a novel oncolytic VACV designed for multiple routes of administration, and several tumor indications. Its strong impact on the TME, by massive infiltration of innate and adaptive immune cells, translated into very remarkable therapeutic activities in resistant tumors. This anti-tumor activity was further enhanced by combination with anti-PD-1. Citation Format: Jean-Baptiste Marchand, Fadi Azar, Christelle Demeusoit, Patricia Kleinpeter, Jules Deforges, Fend Laetitia, Chantal Hoffmann, Huguette Schultz, Nathalie Silvestre, Eric Quéméneur. TG6050 an oncolytic vaccinia virus armed with interleukin 12 and anti-CTLA4 antibody induces TME remodeling and strong anti-tumoral responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 694.

Engineering nano-cellulose bio-composites to improve protein delivery for oral vaccination

Oral vaccine is a non-invasive, ideal way to protect communities from infectious diseases. Effective vaccine delivery systems are required to enhance vaccine absorption in the small intestine and its cellular uptake by immune cells. Here, we constructed alginate/chitosan-coated cellulose nanocrystal (Alg-Chi-CNC) and nanofibril (Alg-Chi-CNF) nanocomposites to enhance ovalbumin (OVA) delivery in the intestine. In vitro mucosal permeation and diffusion and cellular uptake demonstrated that Chi-CNC exhibited better cellular uptake in epithelial and antigen-presenting cells (APCs). In vivo results revealed that alginate/chitosan-coated nanocellulose nanocomposites generated strong systemic and mucosal immune responses. Though the features of functional nano-cellulose composites affected mucus permeation and APC uptakes, in vivo specific-OVA immune responses have not shown significant differences due to the complexity of the small intestine.

Alterations in gut immunological barrier in SARS-CoV-2 infection and their prognostic potential.

Although coronavirus disease 2019 (COVID-19) is primarily associated with mild respiratory symptoms, a subset of patients may develop more complicated disease with systemic complications and multiple organ injury. The gastrointestinal tract may be directly infected by SARS-CoV-2 or secondarily affected by viremia and the release of inflammatory mediators that cause viral entry from the respiratory epithelium. Impaired intestinal barrier function in SARS-CoV-2 infection is a key factor leading to excessive microbial and endotoxin translocation, which triggers a strong systemic immune response and leads to the development of viral sepsis syndrome with severe sequelae. Multiple components of the gut immune system are affected, resulting in a diminished or dysfunctional gut immunological barrier. Antiviral peptides, inflammatory mediators, immune cell chemotaxis, and secretory immunoglobulins are important parameters that are negatively affected in SARS-CoV-2 infection. Mucosal CD4+ and CD8+ T cells, Th17 cells, neutrophils, dendritic cells, and macrophages are activated, and the number of regulatory T cells decreases, promoting an overactivated immune response with increased expression of type I and III interferons and other proinflammatory cytokines. The changes in the immunologic barrier could be promoted in part by a dysbiotic gut microbiota, through commensal-derived signals and metabolites. On the other hand, the proinflammatory intestinal environment could further compromise the integrity of the intestinal epithelium by promoting enterocyte apoptosis and disruption of tight junctions. This review summarizes the changes in the gut immunological barrier during SARS-CoV-2 infection and their prognostic potential.

A Preliminary Study in Immune Response of BALB/c and C57BL/6 Mice with a Locally Allergic Rhinitis Model.

BALB/c and C57BL/6 mouse strains are commonly used in allergy research. The current study investigated the immunological differences between these two mouse strains with a locally allergic rhinitis model. Eighteen BALB/c and eighteen C57BL/6 mice received different doses of ovalbumin (OVA) intranasally for eight weeks (each mouse strain has three subgroups, 25 mg/mL group, 0.25 mg/mL group, and the PBS group). The allergic symptoms, OVA-specific serum antibody (IgE, IgG1, IgG2a), cytokines (IL-4, IFN-γ, IL-10) in the splenic culture supernatant, infiltrating eosinophils and goblet cells in local nasal mucosa were measured. RNA-seq technology was applied to detect differential gene expression in the local nasal mucosa. With the same dose of OVA stimulation, the exacerbation of allergic symptoms was more pronounced in C57BL/6 than in BALB/c. BALB/c serum IgE, IgG1, and IgG2a gradually increased, and C57BL/6 produced fewer serum antibodies IgE and IgG1, while IgG2a never increased. BALB/c spleen cell culture supernatant IL-4 and IL-10 increased with increasing dose, and IFN-γ increased significantly in the intermediate dose group, while IL-4, IL-10, and IFN-γ did not increase in C57BL/6. The infiltration of eosinophils and goblet cells in both mice was proportional to the dose, while C57BL/6 was elevated more than BALB/c. RNA-seq suggested that the innate immune response, immune system process function, Jun kinase (JNK) pathway, and MAPKK pathway were upregulated in C57BL/6 compared to BALB/c. The core genes responsible for the differential immune response in both mice with allergic rhinitis were Kng2, Kng1, Gnb3, Lpar3, Lpar1, Pik3r1, Pf4, Apob, Rps9, and Fbxo2. There are significant differences in the immunologic responses between BALB/c mice and C57BL/6 mice. BALB/c mice developed mild local allergic inflammatory reactions and strong systemic immune responses. In contrast, C57BL/6 mice had stronger local allergic inflammatory responses and relatively mild systemic immune responses. Different mice strains can be selected according to the research purpose.

A cooperative nano-CRISPR scaffold potentiates immunotherapy via activation of tumour-intrinsic pyroptosis

Efficient cancer immunotherapy depends on selective targeting of high bioactivity therapeutic agents to the tumours. However, delivering exogenous medication might prove difficult in clinical practice. Here we report a cooperative Nano-CRISPR scaffold (Nano-CD) that utilizes a specific sgRNA, selected from a functional screen for triggering endogenous GDSME expression, while releasing cisplatin to initiate immunologic cell death. Mechanistically, cascade-amplification of the antitumor immune response is prompted by the adjuvantic properties of the lytic intracellular content and enhanced by the heightened GDSME expression, resulting in pyroptosis and the release of tumor associated antigens. Neither of the single components provide efficient tumour control, while tumor growth is efficiently inhibited in primary and recurrent melanomas due to the combinatorial effect of cisplatin and self-supplied GSDME. Moreover, Nano-CD in combination with checkpoint blockade creates durable immune memory and strong systemic anti-tumor immune response, leading to disease relapse prevention, lung metastasis inhibition and increased survival in mouse melanomas. Taken together, our therapeutic approach utilizes CRISPR-technology to enable cell-intrinsic protein expression for immunotherapy, using GDSME as prototypic immune modulator. This nanoplatform thus can be applied to modulate further immunological processes for therapeutic benefit.

Mucosal immune response after the booster dose of the BNT162b2 COVID-19 vaccine.

To date, only a few studies reported data regarding the development of mucosal immune response after the BNT162b2-booster vaccination. Samples of both serum and saliva of 50 healthcare workers were collected at the day of the booster dose (T3) and after two weeks (T4). Anti-S1-protein IgG and IgA antibody titres and the neutralizing antibodies against the Wuhan wild-type Receptor-Binding Domain in both serum and saliva were measured by quantitative and competitive ELISA, respectively. Data were compared with those recorded after the primary vaccination cycle (T2). Neutralizing antibodies against the variants of concern were measured in those individuals with anti-Wuhan neutralizing antibodies in their saliva. After eight months from the second dose, IgG decreased in both serum (T2GMC: 23,838.5ng/ml; T3GMC: 1473.8ng/ml) and saliva (T2GMC: 12.9ng/ml; T3GMC: 0.3ng/ml). Consistently, serum IgA decreased (T2GMC: 48.6ng/ml; T3GMC: 6.4ng/ml); however, salivary IgA showed a different behaviour and increased (T2GMC: 0.06ng/ml; T3GMC: 0.41ng/ml), indicating a delayed activation of mucosal immunity. The booster elicited higher titres of both IgG and IgA when compared with the primary cycle, in both serum (IgG T4GMC: 98,493.9ng/ml; IgA T4GMC: 187.5ng/ml) and saliva (IgG T4GMC: 21.9ng/ml; IgA T4GMC: 0.65ng/ml). Moreover, the booster re-established the neutralizing activity in the serum of all individuals, not only against the Wuhan wild-type antigen (N=50; INH: 91.6%) but also against the variants (Delta INH: 91.3%; Delta Plus INH: 89.8%; Omicron BA.1 INH: 85.1%). By contrast, the salivary neutralizing activity was high against the Wuhan antigen in 72% of individuals (N=36, INH: 62.2%), but decreased against the variants, especially against the Omicron BA.1 variant (Delta N=27, INH: 43.1%; Delta Plus N=24, INH: 35.2%; Omicron BA.1N=4; INH: 4.7%). This was suggestive for a different behaviour of systemic immunity observed in serum with respect to mucosal immunity described in saliva (Wald chi-square test, 3 df of interaction between variants and sample type=308.2, p<0.0001). The BNT162b2-booster vaccination elicits a strong systemic immune response but fails in activating an effective mucosal immunity against the Omicron BA.1 variant. This work was funded by the Department of Medicine and Surgery, University of Insubria, and supported by Fondazione Umberto Veronesi (COVID-19 Insieme per la ricerca di tutti, 2020), Italy.

Systemic and Mucosal Humoral Immune Response Induced by Three Doses of the BNT162b2 SARS-CoV-2 mRNA Vaccines.

BNT162b2 (BioNTech/Pfizer) was the first SARS-CoV-2 mRNA vaccine approved by the European Medicines Agency. We monitored the long-term humoral responses of healthcare workers (HCWs) who received three vaccine doses. A total of 59 healthcare workers were studied: 47 were never SARS-CoV-2-infected (naïve-HCWs), and 12 (infected-HCWs) recovered from COVID-19 before the first vaccine. Serum and saliva were collected at baseline (before the first dose), just before the second dose, 1, 3, 6, and 9 months after the second dose, and 10 days after the third vaccine. SARS-CoV-2-specific IgG and IgA were evaluated in serum and saliva, respectively, and the presence of neutralizing antibodies (NAb) was analyzed in serum. SARS-CoV-2-specific IgG peaked one month after the second vaccine in naïve-HCWs but right before this timepoint in infected-HCWs. IgG titers significantly decreased during follow-up and at month 9 were still detectable in 50% of naïve-HCWs and 90% of infected-HCWs. NAb were significantly decreased 6 months after the second vaccine in naïve-HCWs and 9 months after this dose in infected-HCWs. Salivary SARS-CoV-2-specific IgA titers were significantly higher in infected-HCWs and were undetectable 9 months after the second vaccine in 43% of the naïve-HCWs alone. The third vaccine greatly increased humoral IgG and mucosal IgA in both groups. Two BNT162b2 doses induced strong systemic and humoral immune responses; to note, these responses weakened over time, although they are more prolonged in individuals who had recovered from COVID-19. The third vaccine dose quickly boosts systemic and mucosal humoral responses.