Radiotherapy: An immune response modifier for immuno-oncology.

The ability of radiotherapy to enhance antigenicity and adjuvanticity of an irradiated tumor has stimulated the interest for its combination with immuno-oncology agents. However, radiotherapy often generates multiple layers of host responses which likely depends on the tumor biology, the immune cell infiltration and the induction of immunosuppressive signals post radiotherapy. Consequently, translation of preclinical findings to the clinic is more convoluted than anticipated which underscore the need to decipher molecular and cellular mechanisms elicited by radiotherapy. Here we review pro-inflammatory and immunosuppressive mechanisms triggered by radiotherapy that impact the outcome of antigen specific T cell killing and discuss how radiation-induced immunostimulatory mechanisms can be exploited to reactivate the host's immune system, especially in the context of immunotherapy.

Design, synthesis and biological evaluation of novel lipophilic 2, 5-disubstituted tetrazole analogues of muramyl dipeptide as NOD2 agonists.

A focused library of six new 2, 5-disubstituted tetrazole (2, 5-DST) analogues of N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) as potential immunomodulators were synthesized by the bioisosteric replacement of α-amide of d-isoglutamine with 5-substituted tetrazole (5-ST). Another parameter 'lipophilicity' was also considered to improve the pharmacological properties of MDP through the alkylation of 5-substituted tetrazole during synthesis. In total, six 2, 5-DST analogues of MDP were synthesized and bio-evaluated for the study of human NOD2 stimulation activity in the innate immune response. Interestingly, among the varied lengths of the alkyl chain in 2, 5-disubstituted tetrazole derivatives, the tetrazole analogues 12b bearing the -Butyl (C4) and 12c having -Octyl (C8) chain showed the best NOD2 stimulation potency equivalent with reference compound MDP. These analogues were evaluated for their adjuvanticity against dengue antigen and analogues 12b and 12c have elicited a potent humoral and cell mediated response.

Different serotypes of Escherichia coli flagellin exert identical adjuvant effects.

Bacterial flagellin is a potent powerful adjuvant, which exerts its adjuvant activity by activating the Toll-like receptor 5 (TLR5) signaling pathway to induce host pro-inflammatory responses. Flagellin of Salmonella typhimurium (S. typhimurium) has shown strong adjuvant effects for a variety of vaccine candidates, however, the adjuvanticity of different serotypes of Escherichia coli (E. coli) flagellin (FliC) is unclear. To explore the adjuvant activity of different serotypes of E. coli flagellin, FliCH1, FliCH7, and FliCH19 recombinant flagellins were prokaryotically-expressed and purified. The adjuvanticity of three recombinant flagellins was evaluated by analyzing their abilities to induce the IL-8 production in human colorectal adenocarcinoma (Caco-2) cells and the immune responses to co-administrated FaeG antigen in mice. Sequence analysis showed that the N-and C-terminal regions are highly conserved, whereas the central region is hypervariable. The TLR5 recognized site is identical among these three serotypes of flagellins. Coomassie blue staining SDS-PAGE showed the molecular mass of FliCH1, FliCH7, and FliCH19 recombinant flagellin are 66 kDa, 64 kDa, and 68 kDa, which can be recognized by anti-FliCH1, FliCH7, and FliCH19 serum, respectively. Moreover, the flagellin serotypes induced similar levels of IL-8 and TNF-α production in Caco-2 cells, anti-FaeG specific IgG antibodies in mice, and IL-4 production in mice spleen cells. Our results indicated that E. coli flagellins can be an adjuvant for vaccine candidates and that different serotypes of E. coli flagellins possess identical adjuvant effects.

Immunoadjuvant and Humoral Immune Responses of Garlic (Allium sativum L.) Lectins upon Systemic and Mucosal Administration in BALB/c Mice.

Dietary food components have the ability to affect immune function; following absorption, specifically orally ingested dietary food containing lectins can systemically modulate the immune cells and affect the response to self- and co-administered food antigens. The mannose-binding lectins from garlic (Allium sativum agglutinins; ASAs) were identified as immunodulatory proteins in vitro. The objective of the present study was to assess the immunogenicity and adjuvanticity of garlic agglutinins and to evaluate whether they have adjuvant properties in vivo for a weak antigen ovalbumin (OVA). Garlic lectins (ASA I and ASA II) were administered by intranasal (50 days duration) and intradermal (14 days duration) routes, and the anti-lectin and anti-OVA immune (IgG) responses in the control and test groups of the BALB/c mice were assessed for humoral immunogenicity. Lectins, co-administered with OVA, were examined for lectin-induced anti-OVA IgG response to assess their adjuvant properties. The splenic and thymic indices were evaluated as a measure of immunomodulatory functions. Intradermal administration of ASA I and ASA II had showed a four-fold and two-fold increase in anti-lectin IgG response, respectively, vs. the control on day 14. In the intranasal route, the increases were 3-fold and 2.4-fold for ASA I and ASA II, respectively, on day 50. No decrease in the body weights of animals was noticed; the increases in the spleen and thymus weights, as well as their indices, were significant in the lectin groups. In the adjuvanticity study by intranasal administration, ASA I co-administered with ovalbumin (OVA) induced a remarkable increase in anti-OVA IgG response (~six-fold; p < 0.001) compared to the control, and ASA II induced a four-fold increase vs. the control on day 50. The results indicated that ASA was a potent immunogen which induced mucosal immunogenicity to the antigens that were administered intranasally in BALB/c mice. The observations made of the in vivo study indicate that ASA I has the potential use as an oral and mucosal adjuvant to deliver candidate weak antigens. Further clinical studies in humans are required to confirm its applicability.

Targeted Delivery of IL-12 Adjuvants Immunotherapy by Oncolytic Viruses.

The great hopes raised by the discovery of the immunoregulatory cytokine interleukin 12 (IL-12) as an anticancer agent were marred during early clinical experimentation because of severe adverse effects, which prompted a search for alternative formulations and routes of administration. Onco-immunotherapeutic viruses (OIVs) are wild-type or genetically engineered viruses that exert antitumor activity by causing death of the tumor cells they infect and by overcoming a variety of immunosuppressive mechanisms put in place by the tumors. OIVs have renewed the interest in IL-12, as they offer the opportunity to encode the cytokine transgenically from the viral genome and to produce it at high concentrations in the tumor bed. A large body of evidence indicates that IL-12 serves as a potent adjuvant for the immunotherapeutic response elicited by OIVs in murine tumor models. The list of OIVs includes onco-immunotherapeutic herpes simplex, adeno, measles, Newcastle disease, and Maraba viruses, among others. The large increase in IL-12-mediated adjuvanticity was invariably observed for all the OIVs analyzed. Indirect evidence suggests that locally delivered IL-12 may also increase tumor antigenicity. Importantly, the OIV/IL-12 treatment was not accompanied by adverse effects and elicited a long-lasting immune response capable of halting the growth of distant tumors. Thus, OIVs provide an avenue for reducing the clinical toxicity associated with systemic IL-12 therapy, by concentrating the cytokine at the site of disease. The changes to the tumor microenvironment induced by the IL-12-armed OIVs primed the tumors to an improved response to the checkpoint blockade therapy, suggesting that the triple combination is worth pursuing in the future. The highly encouraging results in preclinical models have prompted translation to the clinic. How well the IL-12-OIV-checkpoint inhibitors' combination will perform in humans remains to be fully investigated.

Hydrophobicity and the Physico-Chemical Basis of Immunotolerance.

This study analyzes the primary electrostatic interaction between the membrane-bound B-cell receptor and antigen peptide determinants, and identifies peptide frequency and hydrophobicity as the main factors that govern and shape immunotolerance versus immunoreactivity.

Adjuvanticity Regulation by Biodegradable Polymeric Nano/microparticle Size.

Polymeric nano/microparticles as vaccine adjuvants have been researched in experimental and clinical studies. A more profound understanding of how the physicochemical properties regulate specific immune responses has become a vital requirement. Here we prepared poly(d,l-lactic-co-glycolic acid) (PLGA) nano/microparticles with uniform sizes (500 nm, 900 nm, 2.1 µm, and 4.9 µm), and the size effects on particle uptake, activation of macrophages, and antigen internalization were evaluated. Particle uptake kinetic studies demonstrated that 900 nm particles were the easiest to accumulate in cells. Moreover, they could induce macrophages to secrete NO and IL-1ß and facilitate antigen internalization. Furthermore, 900 nm particles, mixed with antigen, could exhibit superior adjuvanticity in both humoral and cellular immune responses in vivo, including offering the highest antibody protection, promoting the maximum secretion levels of IFN-γ and IL-4 than particles with other sizes. Overall, 900 nm might be the optimum choice for PLGA particle-based vaccine adjuvants especially for recombinant antigens. Understanding the effect of particle size on the adjuvanticity based immune responses might have important enlightenments for rational vaccine design and applications.

Application of phosphorylcholine derivative as mucosal adjuvant enhancing mucosal immune responses in the upper respiratory tract.

OBJECTIVE: A phosphorylcholine (PC)-derivative with high binding ability (PCDB) was intranasally administered to mice with ovalbumin (OVA), and immune responses were investigated to determine whether PCDB has antigenicity and adjuvanticity. METHODS: BALB/c mice were intranasally immunized with PCDB coupled with OVA, unbound PCDB plus OVA, cholera toxin (CT) plus OVA, OVA alone, and PCDB alone. Then, the production of OVA- and PC-specific antibodies in external secretions and serum, and the secretion of cytokines such as IL-4 and IFN-γ from splenic mononuclear cells by stimulation with PCDB and OVA were examined. Furthermore, the secretion of IL-12p40 from CD11c+ cells following stimulation with PCDB was observed to clarify the adjuvant effect of PCDB through TLR4. RESULTS: Intranasal immunization with PCDB plus OVA increased OVA- and PC-specific IgA in external secretions and OVA- and PC-specific antibodies in the serum. The analysis of IgG subclasses specific to OVA and PC showed a higher production of IgG1 than IgG2, and the secretion of both IL-4 and IFN-γ was enhanced. However, IL-12p40 secretion from CD11c+ cells was increased and OVA-specific IgE production was not promoted by PCDB stimulation. CONCLUSION: Intranasal administration of the protein antigen with PCDB enhanced immune responses specific to the mixed antigen and PC. Although PCDB acted to bias the immune response toward the Th2-type, antigen-specific IgE production did not increase. These findings suggest that PCDB has the potential to be a mucosal vaccine with both adjuvanticity and antigenicity without causing side effects due to type I allergy.

Sequence-Optimized mRNA Vaccines Against Infectious Disease.

Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA vaccines (RNActive® technology) with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by formulation into lipid nanoparticles. Methods described here include the synthesis, purification, and formulation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.

mRNA Vaccination: An Outlook on Innate Sensing and Adaptive Immune Responses.

Vaccination has led to significant dismantling of infectious diseases worldwide. Since the dawn of the SARS-CoV-2 pandemic, there has been increased popularity in the usage and study of the mRNA vaccine platform. Here, we highlight fundamental knowledge on mRNA vaccine pharmacology, followed by the immunity conferred by innate sensing and adaptive responses resulting from exposure to the mRNA vaccine construct and encapsulation materials. A better understanding of these immune mechanisms will shed light on further improvements in mRNA vaccine design, aiming to improve efficiency and optimize immune responses upon inoculation.

Preconditioning with immunogenic cell death-inducing treatments for subsequent immunotherapy.

Since the dawn of anticancer immunotherapy, the clinical use of immune checkpoint inhibitors (ICI) has increased exponentially. Monoclonal antibodies targeting CTLA-4 and the PD-1/PD-L1 interaction were first introduced for the treatment of patients with unresectable melanoma. In melanoma, ICI lead to durable regression in a significant number of patients and have thus been clinically approved as a first-line treatment of advanced disease. Over the past years an increasing number of regulatory approvals have been granted for the use of ICI in patients affected by a large range of distinct carcinomas. In retrospect surprisingly, it has been discovered that particularly successful chemotherapeutic treatments are able to trigger anticancer immune responses because they induce immunogenic cell death (ICD), hence killing cancer cells in a way that they elicit an immune response against tumor-associated antigens. Logically, preclinical studies as well as clinical trials are currently exploring the possibility to combine ICD inducers with ICI to obtain optimal therapeutic effects. Here, we provide a broad overview of current strategies for the implementation of combinatorial approaches involving ICD induction followed by ICI in anticancer therapy.

Vaccination-Route-Dependent Adjuvanticity of Antigen-Carrying Nanoparticles for Enhanced Vaccine Efficacy.

Vaccination-route-dependent adjuvanticity was identified as being associated with the specific features of antigen-carrying nanoparticles (NPs) in the present work. Here, we demonstrated that the mechanical properties and the decomposability of NP adjuvants play key roles in determining the antigen accessibility and thus the overall vaccine efficacy in the immune system when different vaccination routes were employed. We showed that soft nano-vaccines were associated with more efficient antigen uptake when administering subcutaneous (S.C.) vaccination, while the slow decomposition of hard nano-vaccines promoted antigen uptake when intravenous (I.V.) vaccination was employed. In comparison to the clinically used aluminum (Alum) adjuvant, the NP adjuvants were found to stimulate both humoral and cellular immune responses efficiently, irrespective of the vaccination route. For vaccination via S.C. and I.V. alike, the NP-based vaccines show excellent protection for mice from Staphylococcus aureus (S. aureus) infection, and their survival rates are 100% after lethal challenge, being much superior to the clinically used Alum adjuvant.

Ym1 protein crystals promote type 2 immunity.

Spontaneous protein crystallization is a rare event, yet protein crystals are frequently found in eosinophil-rich inflammation. In humans, Charcot-Leyden crystals (CLCs) are made from galectin-10 (Gal10) protein, an abundant protein in eosinophils. Although mice do not encode Gal10 in their genome, they do form pseudo-CLCs, made from the chitinase-like proteins Ym1 and/or Ym2, encoded by Chil3 and Chil4 and made by myeloid and epithelial cells respectively. Here, we investigated the biological effects of pseudo-CLCs since their function is currently unknown. We produced recombinant Ym1 crystals which were shown to have identical crystal packing and structure by X-ray crystallography as in vivo native crystals derived from murine lung. When administered to the airways of mice, crystalline but not soluble Ym1 stimulated innate and adaptive immunity and acted as a type 2 immune adjuvant for eosinophilic inflammation via triggering of dendritic cells (DCs). Murine Ym1 protein crystals found at sites of eosinophilic inflammation reinforce type 2 immunity and could serve as a surrogate model for studying the biology of human CLCs.

Optimizing high-intensity focused ultrasound-induced immunogenic cell-death using passive cavitation mapping as a monitoring tool.

Over the past decade, ultrasound (US) has gathered significant attention and research focus in the realm of medical treatments, particularly within the domain of anti-cancer therapies. This growing interest can be attributed to its non-invasive nature, precision in delivery, availability, and safety. While the conventional objective of US-based treatments to treat breast, prostate, and liver cancer is the ablation of target tissues, the introduction of the concept of immunogenic cell death (ICD) has made clear that inducing cell death can take different non-binary pathways through the activation of the patient's anti-tumor immunity. Here, we investigate high-intensity focused ultrasound (HIFU) to induce ICD by unraveling the underlying physical phenomena and resulting biological effects associated with HIFU therapy using an automated and fully controlled experimental setup. Our in-vitro approach enables the treatment of adherent cancer cells (B16F10 and CT26), analysis for ICD hallmarks and allows to monitor and characterize in real time the US-induced cavitation activity through passive cavitation detection (PCD). We demonstrate HIFU-induced cell death, CRT exposure, HMGB1 secretion and antigen release. This approach holds great promise in advancing our understanding of the therapeutic potential of HIFU for anti-cancer strategies.

Immunogenicity of ferroptosis in cancer: a matter of context?

Ferroptosis is a variant of regulated cell death (RCD) elicited by an imbalance of cellular redox homeostasis that culminates with extensive lipid peroxidation and rapid plasma membrane breakdown. Since other necrotic forms of RCD, such as necroptosis, are highly immunogenic, ferroptosis inducers have attracted considerable attention as potential tools to selectively kill malignant cells while eliciting therapeutically relevant tumor-targeting immune responses. However, rather than being consistently immunogenic, ferroptosis mediates context-dependent effects on anticancer immunity. The inability of ferroptotic cancer cells to elicit adaptive immune responses may arise from contextual deficiencies in intrinsic aspects of the process, such as adjuvanticity and antigenicity, or from microenvironmental defects imposed by ferroptotic cancer cells themselves or elicited by the induction of ferroptosis in immune cells.

Mucosal adjuvanticity and mucosal booster effect of colibactin-depleted probiotic Escherichia coli membrane vesicles.

There is a growing interest in development of novel vaccines against respiratory tract infections, due to COVID-19 pandemic. Here, we examined mucosal adjuvanticity and the mucosal booster effect of membrane vesicles (MVs) of a novel probiotic E. coli derivative lacking both flagella and potentially carcinogenic colibactin (ΔflhDΔclbP). ΔflhDΔclbP-derived MVs showed rather strong mucosal adjuvanticity as compared to those of a single flagellar mutant strain (ΔflhD-MVs). In addition, glycoengineered ΔflhDΔclbP-MVs displaying serotype-14 pneumococcal capsular polysaccharide (CPS14+MVs) were well-characterized based on biological and physicochemical parameters. Subcutaneous (SC) and intranasal (IN) booster effects of CPS14+MVs on systemic and mucosal immunity were evaluated in mice that have already been subcutaneously prime-immunized with the same MVs. With a two-dose regimen, an IN boost (SC-IN) elicited stronger IgA responses than homologous prime-boost immunization (SC-SC). With a three-dose regimen, serum IgG levels were comparable among all tested regimens. Homologous immunization (SC-SC-SC) elicited the highest IgM responses among all regimens tested, whereas SC-SC-SC failed to elicit IgA responses in blood and saliva. Furthermore, serum IgA and salivary SIgA levels were increased with an increased number of IN doses administrated. Notably, SC-IN-IN induced not only robust IgG response, but also the highest IgA response in both serum and saliva among the groups. The present findings suggest the potential of a heterologous three-dose administration for building both systemic and mucosal immunity, e.g. an SC-IN-IN vaccine regimen could be beneficial. Another important observation was abundant packaging of colibactin in MVs, suggesting increased applicability of ΔflhDΔclbP-MVs in the context of vaccine safety.

Intrinsic features of the cancer cell as drivers of immune checkpoint blockade response and refractoriness.

Immune checkpoint blockade represents the latest revolution in cancer treatment by substantially increasing patients' lifetime and quality of life in multiple neoplastic pathologies. However, this new avenue of cancer management appeared extremely beneficial in a minority of cancer types and the sub-population of patients that would benefit from such therapies remain difficult to predict. In this review of the literature, we have summarized important knowledge linking cancer cell characteristics with the response to immunotherapy. Mostly focused on lung cancer, our objective was to illustrate how cancer cell diversity inside a well-defined pathology might explain sensitivity and refractoriness to immunotherapies. We first discuss how genomic instability, epigenetics and innate immune signaling could explain differences in the response to immune checkpoint blockers. Then, in a second part we detailed important notions suggesting that altered cancer cell metabolism, specific oncogenic signaling, tumor suppressor loss as well as tight control of the cGAS/STING pathway in the cancer cells can be associated with resistance to immune checkpoint blockade. At the end, we discussed recent evidences that could suggest that immune checkpoint blockade as first line therapy might shape the cancer cell clones diversity and give rise to the appearance of novel resistance mechanisms.

A Large Cluster of New Onset Autoimmune Myositis in the Yorkshire Region Following SARS-CoV-2 Vaccination.

BACKGROUND: The novel SARS-CoV-2 vaccines partially exploit intrinsic DNA or RNA adjuvanticity, with dysregulation in the metabolism of both these nucleic acids independently linked to triggering experimental autoimmune diseases, including lupus and myositis. METHODS: Herein, we present 15 new onset autoimmune myositis temporally associated with SARS-CoV-2 RNA or DNA-based vaccines that occurred between February 2021 and April 2022. Musculoskeletal, pulmonary, cutaneous and cardiac manifestations, laboratory and imaging data were collected. RESULTS: In total, 15 cases of new onset myositis (11 polymyositis/necrotizing/overlap myositis; 4 dermatomyositis) were identified in the Yorkshire region of approximately 5.6 million people, between February 2021 and April 2022 (10 females/5 men; mean age was 66.1 years; range 37-83). New onset disease occurred after first vaccination (5 cases), second vaccination (7 cases) or after the third dose (3 cases), which was often a different vaccine. Of the cases, 6 had systemic complications including skin (3 cases), lung (3 cases), heart (2 cases) and 10/15 had myositis associated autoantibodies. All but 1 case had good therapy responses. Adverse event following immunization (AEFI) could not be explained based on the underlying disease/co-morbidities. CONCLUSION: Compared with our usual regional Rheumatology clinical experience, a surprisingly large number of new onset myositis cases presented during the period of observation. Given that antigen release inevitably follows muscle injury and given the role of nucleic acid adjuvanticity in autoimmunity and muscle disease, further longitudinal studies are required to explore potential links between novel coronavirus vaccines and myositis in comparison with more traditional vaccine methods.

Effects of N-acylation on the immune adjuvanticity of analogs of the Quillaja saponins derivative GPI-0100.

Modification of the 3-glucuronic acid (GlcA) residue from the Quillaja saponin (QS) adjuvants by N-acylation, yields derivatives with linear alkylamides that show structural and functional changes. Structural, since the relatively unreactive added hydrophobic alkyl chains may modify these glycosides' conformation and micellar structure. Functional, because altering the availability of proposed pharmacophores, like fucose (Fucp) and aldehyde groups, to interact with their cellular receptors, may change these glycosides' adjuvanticity. While deacylated QS (DS-QS) adjuvants bias the response toward a sole anti-inflammatory Th2 immunity against an antigen, their N-alkylated derivatives carrying octyl to dodecylamide residues, modify that response to a pro-inflammatory Th1 immunity. As shown by their IgG2a/IgG1 titer ratios, which are higher than those for Th2 immunity. A result of the fact that in mice, the IgG2a levels are dependent on the direct influence of secreted interferon-γ (IFN-γ), a crucial Th1 cytokine. But addition of the longer and more lipophilic tetradecylamide group, yields derivatives that like DS-QS induce Th2 immunity, as shown by their low IgG2a/IgG1 ratio. Results that imply that changes in these analogs' conformation and micellar structure, would affect the immunomodulatory properties or adjuvanticity of N-acylated DS-QS. Physical changes that may alter the availability of groups like Fucp, to bind to its presumed dendritic cells' lectin receptor DC-SIGN; an essential step in the stimulation of Th2 immunity. Structural properties that in an aqueous environment, would depend on these glycosides' balance of their hydrophilic and lipophilic moieties (HLB), and the interactions of the newly introduced alkyl chain with the native QS' lipophilic triterpene aglycone and hydrophilic oligosaccharide chains. A situation that would explain these new derivatives' qualitative and quantitative changes in adjuvanticity.

Nanoclays: Promising Materials for Vaccinology.

Clay materials and nanoclays have gained recent popularity in the vaccinology field, with biocompatibility, simple functionalization, low toxicity, and low-cost as their main attributes. As elements of nanovaccines, halloysite nanotubes (natural), layered double hydroxides and hectorite (synthetic) are the nanoclays that have advanced into the vaccinology field. Until now, only physisorption has been used to modify the surface of nanoclays with antigens, adjuvants, and/or ligands to create nanovaccines. Protocols to covalently attach these molecules have not been developed with nanoclays, only procedures to develop adsorbents based on nanoclays that could be extended to develop nanovaccine conjugates. In this review, we describe the approaches evaluated on different nanovaccine candidates reported in articles, the immunological results obtained with them and the most advanced approaches in the preclinical field, while describing the nanomaterial itself. In addition, complex systems that use nanoclays were included and described. The safety of nanoclays as carriers is an important key fact to determine their true potential as nanovaccine candidates in humans. Here, we present the evaluations reported in this field. Finally, we point out the perspectives in the development of vaccine prototypes using nanoclays as antigen carriers.