Programming peptide-oligonucleotide nano-assembly for engineering of neoantigen vaccine with potent immunogenicity.

Background: Neoantigen nanovaccine has been recognized as a promising treatment modality for personalized cancer immunotherapy. However, most current nanovaccines are carrier-dependent and the manufacturing process is complicated, resulting in potential safety concerns and suboptimal codelivery of neoantigens and adjuvants to antigen-presenting cells (APCs). Methods: Here we report a facile and general methodology for nanoassembly of peptide and oligonucleotide by programming neoantigen peptide with a short cationic module at N-terminus to prepare nanovaccine. The programmed peptide can co-assemble with CpG oligonucleotide (TLR9 agonist) into monodispersed nanostructures without the introduction of artificial carrier. Results: We demonstrate that the engineered nanovaccine promoted the codelivery of neoantigen peptides and adjuvants to lymph node-residing APCs and instigated potent neoantigen-specific T-cell responses, eliciting neoantigen-specific antitumor immune responses with negligible systemic toxicity. Furthermore, the antitumor T-cell immunity is profoundly potentiated when combined with anti-PD-1 therapy, leading to significant inhibition or even complete regression of established melanoma and MC-38 colon tumors. Conclusions: Collectively, this work demonstrates the feasibility and effectiveness of personalized cancer nanovaccine preparation with high immunogenicity and good biosafety by programming neoantigen peptide for nanoassembly with oligonucleotides without the aid of artificial carrier.

Extracellular vesicles derived from plasmodium-infected red blood cells alleviate cerebral malaria in plasmodium berghei ANKA-infected C57BL/6J mice.

RTS,S is the first malaria vaccine recommended for implementation among young children at risk. However, vaccine efficacy is modest and short-lived. To mitigate the risk of cerebral malaria (CM) among children under the age of 5, it is imperative to develop new vaccines. EVs are potential vaccine candidates as they obtain the ability of brain-targeted delivery and transfer plasmodium antigens and immunomodulators during infections. This study extracted EVs from BALB/c mice infected with Plasmodium yoelii 17XNL (P.y17XNL). C57BL/6J mice were intravenously immunized with EVs (EV-I.V. + CM group) or subcutaneously vaccinated with the combination of EVs and CpG ODN-1826 (EV + CPG ODN-S.C. + CM group) on days 0 and 20, followed by infection with Plasmodium berghei ANKA (P.bANKA) on day 20 post-second immunization. We monitored Parasitemia and survival rate. The integrity of the Blood-brain barrier (BBB) was examined using Evans blue staining.The levels of cytokines and adhesion molecules were evaluated using Luminex, RT-qPCR, and WB. Brain pathology was evaluated by hematoxylin and eosin and immunohistochemical staining. The serum levels of IgG, IgG1, and IgG2a were analyzed by enzyme-linked immunosorbent assay. Compared with those in the P.bANKA-infected group, parasitemia increased slowly, death was delayed (day 10 post-infection), and the survival rate reached 75 %-83.3 % in the EV-I.V. + ECM and EV + CPG ODN-S.C. + ECM groups. Meanwhile, compared with the EV + CPG ODN-S.C. + ECM group, although parasitemia was almost the same, the survival rate increased in the EV-I.V. + ECM group.Additionally, EVs immunization markedly downregulated inflammatory responses in the spleen and brain and ameliorated brain pathological changes, including BBB disruption and infected red blood cell (iRBC) sequestration. Furthermore, the EVs immunization group exhibited enhanced antibody responses (upregulation of IgG1 and IgG2a production) compared to the normal control group. EV immunization exerted protective effects, improving the integrity of the BBB, downregulating inflammation response of brain tissue, result in reduces the incidence of CM. The protective effects were determined by immunological pathways and brain targets elicited by EVs. Intravenous immunization exhibited better performance than subcutaneous immunization, which perhaps correlated with EVs, which can naturally cross BBB to play a better role in brain protection.

Innovative gelatin-based micelles with AS1411 aptamer targeting and reduction responsiveness for doxorubicin delivery in tumor therapy.

Herein, we constructed innovative reduction-sensitive and targeted gelatin-based micelles for doxorubicin (DOX) delivery in tumor therapy. AS1411 aptamer-modified gelatin-ss-tocopherol succinate (AGSST) and the control GSST without AS1411 modification were synthesized and characterized. Antitumor drug DOX-containing AGSST (AGSST-D) and GSST-D nanoparticles were prepared, and their shapes were almost spherical. Reduction-responsive characteristics of DOX release in vitro were revealed in AGSST-D and GSST-D. Compared with non-targeted GSST-D, AGSST-D demonstrated better intracellular uptake and stronger cytotoxicity against nucleolin-overexpressed A549 cells. Importantly, AGSST-D micelles showed more effective killing activity in A549-bearing mice than GSST-D and DOX⋅HCl. It was revealed that AGSST-D micelles had no obvious systemic toxicity. Overall, AGSST micelles would have the potential to be an effective drug carrier for targeted tumor therapy.

Layer-by-layer nanoparticle encapsulating all-trans retinoic acid and CpG as a mucosal adjuvant targeting colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, demanding innovative therapeutic strategies. Immunotherapy, a promising avenue, employs cancer vaccines to activate the immune system against tumors. However, conventional approaches fall short of eliciting robust responses within the gastrointestinal (GI) tract, where CRC originates. Harnessing the potential of all-trans retinoic acid (ATRA) and cytosine-phosphorothioate-guanine (CpG), we developed layered nanoparticles using a layer-by-layer assembly method to co-deliver these agents. ATRA, crucial for gut immunity, was efficiently encapsulated alongside CpG within these nanoparticles. Administering these ATRA@CpG-NPs, combined with ovalbumin peptide (OVA), effectively inhibited orthotopic CRC growth in mice. Our approach leveraged the inherent benefits of ATRA and CpG, demonstrating superior efficacy in activating dendritic cells, imprinting T cells with gut-homing receptors, and inhibiting tumor growth. This mucosal adjuvant presents a promising strategy for CRC immunotherapy, showcasing the potential for targeting gut-associated immune responses in combating colorectal malignancies.

Emerging nanoparticle platforms for CpG oligonucleotide delivery.

Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), which were therapeutic DNA with high immunostimulatory activity, have been applied in widespread applications from basic research to clinics as therapeutic agents for cancer immunotherapy, viral infection, allergic diseases and asthma since their discovery in 1995. The major factors to consider for clinical translation using CpG motifs are the protection of CpG ODNs from DNase degradation and the delivery of CpG ODNs to the Toll-like receptor-9 expressed human B-cells and plasmacytoid dendritic cells. Therefore, great efforts have been devoted to the advances of efficient delivery systems for CpG ODNs. In this review, we outline new horizons and recent developments in this field, providing a comprehensive summary of the nanoparticle-based CpG delivery systems developed to improve the efficacy of CpG-mediated immune responses, including DNA nanostructures, inorganic nanoparticles, polymer nanoparticles, metal-organic-frameworks, lipid-based nanosystems, proteins and peptides, as well as exosomes and cell membrane nanoparticles. Moreover, future challenges in the establishment of CpG delivery systems for immunotherapeutic applications are discussed. We expect that the continuously growing interest in the development of CpG-based immunotherapy will certainly fuel the excitement and stimulation in medicine research.

Targeted Delivery of Immunostimulatory CpG Oligodeoxynucleotides to Antigen-Presenting Cells in Draining Lymph Nodes by Stearic Acid Modification and Nanostructurization.

Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.

A Nucleic Acid-Based Orthopoxvirus Vaccine Targeting the Vaccinia Virus L1, A27, B5, and A33 Proteins Protects Rabbits against Lethal Rabbitpox Virus Aerosol Challenge.

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

AS1411 Nucleolin-Specific Binding Aptamers Reduce Pathological Angiogenesis through Inhibition of Nucleolin Phosphorylation.

Proliferative retinopathies produces an irreversible type of blindness affecting working age and pediatric population of industrialized countries. Despite the good results of anti-VEGF therapy, intraocular and systemic complications are often associated after its intravitreal use, hence novel therapeutic approaches are needed. The aim of the present study is to test the effect of the AS1411, an antiangiogenic nucleolin-binding aptamer, using in vivo, ex vivo and in vitro models of angiogenesis and propose a mechanistic insight. Our results showed that AS1411 significantly inhibited retinal neovascularization in the oxygen induced retinopathy (OIR) in vivo model, as well as inhibited branch formation in the rat aortic ex vivo assay, and, significantly reduced proliferation, cell migration and tube formation in the HUVEC in vitro model. Importantly, phosphorylated NCL protein was significantly abolished in HUVEC in the presence of AS1411 without affecting NFκB phosphorylation and -21 and 221-angiomiRs, suggesting that the antiangiogenic properties of this molecule are partially mediated by a down regulation in NCL phosphorylation. In sum, this new research further supports the NCL role in the molecular etiology of pathological angiogenesis and identifies AS1411 as a novel anti-angiogenic treatment.

Aptamer-Aptamer Chimera for Targeted Delivery and ATP-Responsive Release of Doxorubicin into Cancer Cells.

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5'-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411-ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411-ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.

CpG Oligodeoxynucleotide Developed to Activate Primate Immune Responses Promotes Antitumoral Effects in Combination with a Neoantigen-Based mRNA Cancer Vaccine.

PURPOSE: The purpose of our research was to identify and evaluate synthetic phosphorothioate-modified CPG oligodeoxynucleotides (CPG-ODNs) activating innate and adaptive immune responses. Furthermore, combined treatment with CpG and an mRNA cancer vaccine was evaluated in melanoma models as a therapeutic approach. METHODS: A molecular assay was used to screen new CpG molecules; mouse modeling and pathological analysis were used to confirm the antitumor effect of CpG alone or in combination with an mRNA vaccine. Finally, safety was assessed by monitoring blood biochemistry. RESULTS: We first screened and identified a new CpG-B class ODN (CpG2018B) that effectively stimulated type II interferons in both mouse plasmacytoid dendritic cells (pDCs) and human peripheral blood mononuclear cells (PBMCs). In addition, CpG2018B promoted cytokine production mainly via toll-like receptor 9 (TLR9) pathways. We further demonstrated that intratumoral (IT) injection of CpG2018B inhibited melanoma growth in syngeneic models and could turn "cold" tumors into "hot" tumors. Then, CpG2018B and an mRNA-based neoantigen cancer vaccine were encapsulated in lipid nanoparticles (LNPs) and intratumorally injected into melanoma mouse models. Interestingly, vaccination with CpG or the mRNA vaccine alone could inhibit tumor growth, while combination of CpG with the mRNA vaccine enhanced the antitumor effect. Finally, we described the long-term safety and tolerability of CpG2018B and mRNA therapy in mice model. CONCLUSION: We identified a novel CpG-B class ODN to promote the immune response, and CpG combined with mRNA cancer vaccines is an attractive candidate approach for immunostimulatory sequence (ISS)-based therapeutic strategies.

Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components.

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

Nucleic acid vaccines and CpG oligodeoxynucleotides for allergen immunotherapy.

PURPOSE OF REVIEW: Molecular forms of allergen-specific immunotherapy (AIT) are continuously emerging to improve the efficacy of the treatment, to shorten the duration of protocols and to prevent any side effects. The present review covers the recent progress in the development of AIT based on nucleic acid encoding allergens or CpG oligodeoxynucleotides (CpG-ODN). RECENT FINDINGS: Therapeutic vaccinations with plasmid deoxyribonucleic acid (DNA) encoding major shrimp Met e 1 or insect For t 2 allergen were effective for the treatment of food or insect bite allergy in respective animal models. DNA expressing hypoallergenic shrimp tropomyosin activated Foxp3+ T regulatory (Treg) cells whereas DNA encoding For t 2 down-regulated the expression of pruritus-inducing IL-31. Co-administrations of major cat allergen Fel d 1 with high doses of CpG-ODN reduced Th2 airway inflammation through tolerance induction mediated by GATA3+ Foxp3hi Treg cells as well as early anti-inflammatory TNF/TNFR2 signaling cascade. Non-canonical CpG-ODN derived from Cryptococcus neoformans as well as methylated CpG sites present in the genomic DNA from Bifidobacterium infantis mediated Th1 or Treg cell differentiation respectively. SUMMARY: Recent studies on plasmid DNA encoding allergens evidenced their therapeutic potential for the treatment of food allergy and atopic dermatitis. Unmethylated or methylated CpG-ODNs were shown to activate dose-dependent Treg/Th1 responses. Large clinical trials need to be conducted to confirm these promising preclinical data. Moreover, tremendous success of messenger ribonucleic acid (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 must encourage as well the re-exploration of mRNA vaccine platform for innovative AIT.

TLR ligand loaded exosome mediated immunotherapy of established mammary Tumor in mice.

Tumor-derived exosomes (TEXs) could be harnessed as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. Engineering TEXs to harbor immunostimulatory molecules still remains a challenge. Previously, we demonstrated that nucleic acid ligand encapsulated liposomes could trigger synergistic strong humoral, and cell mediated immune responses and provokes tumor regression to that of their standalone counterparts. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). Engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic cancer model, administration of4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regress tumor growth in 4T1 tumor-bearing mice. Taken together this work implicated that an exosome-based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. This approach offers a personalized tumor therapy strategy that could be implemented in the clinic.

Evaluation of the AV7909 Anthrax Vaccine Toxicity in Sprague Dawley Rats Following Three Intramuscular Administrations.

AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.

Ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of liposomal antigen and CpG-ODN improved PD-1 blockade immunotherapy.

Lack of pre-existing tumor infiltrated T cells resulting in resistance to programmed cell death protein 1 (PD-1) blockade therapies can be solved by combining with anti-cancer vaccines and CpG-ODN in increasing T cell expansion and infiltration. Therefore, we prepared an ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of either liposomal or non-liposomal gp100 antigen (2.8 µg) plus CpG-ODN (800 ng) formulations and evaluated its anti-tumor activity in combination with anti-PD-1 therapy. Our results showed a combination of liposomal peptide plus CpG-ODN pulsed DC with anti-PD-1 antibody was more efficacious, as evidenced by a significant increase in Teff/Treg TILs with a marked fourfold elevation of IFN-γ expression level in the tumor site of treated mice which reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, this combination also led to a remarkable tumor remission and prolonged survival rate in melanoma-bearing mice compared to non-liposomal peptide plus CpG-ODN or single-treated liposomal peptide formulations. Our results provide essential insights to devise combining regimens to improve the efficacy of immune checkpoint blockers even by a low dose of peptide and CpG-ODN.

Neospora caninum truncated recombinant proteins formulated with liposomes and CpG-ODNs triggered a humoral immune response in cattle after immunisation and challenge.

Abortions caused by Neospora caninum are a serious problem in cattle production and require effective immunoprophylaxis. The objective of this work was to assess the humoral immune response to four recombinant (r) N. caninum antigens in cattle after immunisation and challenge. MIC1 and MIC3 proteins from the micronemes, SRS2 from the surface of tachyzoites, and GRA7 from the dense granules were expressed as truncated recombinant proteins in Escherichia coli. Cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODNs) were used as adjuvant. Steers were assigned to three groups of six steers each and were inoculated twice subcutaneously, 21 days apart. The rP + Lip + CpG-ODN group received the truncated recombinant proteins rMIC1, rMIC3, rSRS2 and rGRA7 formulated with the adjuvant; the Lip + CpG-ODN group received the adjuvant alone; and the PBS group received sterile phosphate-buffered saline. All steers were subcutaneously challenged with the NC-1 strain of N. caninum 35 days after the second dose of immunisation. Steers from the rP + Lip + CpG-ODN group developed specific IgG, IgG1 and IgG2 against the four recombinant proteins after immunisation. After challenge, IgG against rMIC1 and rMIC3 was detected in rP + Lip + CpG-ODN group and against rSRS2 and rGRA7 in all groups. IgG1 and IgG2 against the four recombinant proteins remained high after challenge in the rP + Lip + CpG-ODN group. Indirect ELISA detected anti-N. caninum antibodies after challenge in all groups, with the highest level of antibodies being detected in the rP + Lip + CpG-ODN group. The recombinant vaccine formulated with rMIC1, rMIC3, rSRS2 and rGRA7 using Lip + CpG-ODN as adjuvant was immunogenic in cattle and the humoral immune response after challenge was enhanced in vaccinated cattle.

IL-6 signaling in macrophages is required for immunotherapy-driven regression of tumors.

BACKGROUND: High serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory cytokine has pleiotropic effects, this treatment is not always effective. METHODS: IL-6 and IL-6R blockade was applied in an IL-6-mediated immunotherapy-resistant TC-1 tumor model (TC-1.IL-6) and immunotherapy-sensitive TC-1. CONTROL: Effects on therapeutic vaccination-induced tumor regression, recurrence and survival as well on T cells and myeloid cells in the tumor microenvironment were studied. The effects of IL-6 signaling in macrophages under therapy conditions were studied in Il6rafl/fl×LysMcre+ mice. RESULTS: Our therapeutic vaccination protocol elicits a strong tumor-specific CD8+ T-cell response, leading to enhanced intratumoral T-cell infiltration and recruitment of tumoricidal macrophages. Blockade of IL-6 signaling exacerbated tumor outgrowth, reflected by fewer complete regressions and more recurrences after therapeutic vaccination, especially in TC-1.IL-6 tumor-bearing mice. Early IL-6 signaling blockade partly inhibited the development of the vaccine-induced CD8+ T-cell response. However, the main mechanism was the malfunction of macrophages during therapy-induced tumor regression. Therapy efficacy was impaired in Il6rafl/fl×LysMcre+ but not cre-negative control mice, while no differences in the vaccine-induced CD8+ T-cell response were found between these mice. IL-6 signaling blockade resulted in decreased expression of suppressor of cytokine signaling 3, essential for effective M1-type function in macrophages, and increased expression of the phagocytic checkpoint molecule signal-regulatory protein alpha by macrophages. CONCLUSION: IL-6 signaling is critical for macrophage function under circumstances of immunotherapy-induced tumor tissue destruction, in line with the acute inflammatory functions of IL-6 signaling described in infections.

Decoy Technology as a Promising Therapeutic Tool for Atherosclerosis.

Cardiovascular diseases (CVDs) have been classified into several types of disease, of which atherosclerosis is the most prevalent. Atherosclerosis is characterized as an inflammatory chronic disease which is caused by the formation of lesions in the arterial wall. Subsequently, lesion progression and disruption ultimately lead to heart disease and stroke. The development of atherosclerosis is the underlying cause of approximately 50% of all deaths in westernized societies. Countless studies have aimed to improve therapeutic approaches for atherosclerosis treatment; however, it remains high on the global list of challenges toward healthy and long lives. Some patients with familial hypercholesterolemia could not get intended LDL-C goals even with high doses of traditional therapies such as statins, with many of them being unable to tolerate statins because of the harsh side effects. Furthermore, even in patients achieving target LDL-C levels, the residual risk of traditional therapies is still significant thus highlighting the necessity of ongoing research for more effective therapeutic approaches with minimal side effects. Decoy-based drug candidates represent an opportunity to inhibit regulatory pathways that promote atherosclerosis. In this review, the potential roles of decoys in the treatment of atherosclerosis were described based on the in vitro and in vivo findings.

CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge.

The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 µg or 5 µg of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 µg or 5 µg of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.