Immunogenicity and protective capacity of a CpG ODN adjuvanted alum adsorbed bivalent meningococcal outer membrane vesicle vaccine.

Invasive meningococcal disease (IMD) is caused by Neisseria meningitidis, with the main serogroups responsible for the disease being A, B, C, W, X, and Y. To date, several vaccines targeting N. meningitidis have been developed albeit with a short-lived protection. Given that MenW and MenB are the most common causes of IMD in Europe, Turkey, and the Middle East, we aimed to develop an outer membrane vesicle (OMV) based bivalent vaccine as the heterologous antigen source. Herein, we compared the immunogenicity, and breadth of serum bactericidal activity (SBA) assay-based protective coverage of OMV vaccine to the X serotype with existing commercial meningococcal conjugate and polysaccharide (PS) vaccines in a murine model. BALB/c mice were immunized with preclinical batches of the W + B OMV vaccine, either adjuvanted with Alum, CpG ODN, or their combinations, and compared with a MenACYW conjugate vaccine (NimenrixTM, Pfizer), and a MenB OMV-based vaccine (Bexsero®, GSK), The immune responses were assessed through enzyme-linked immunosorbent assay (ELISA) and SBA assay. Antibody responses and SBA titers were significantly higher in the W + B OMV vaccine when adjuvanted with Alum or CpG ODN, as compared to the control groups. Moreover, the SBA titers were not only significantly higher than those achieved with available conjugated ACYW vaccines but also on par with the 4CMenB vaccines. In conclusion, the W + B OMV vaccine demonstrated the capacity to elicit robust antibody responses, surpassing or matching the levels induced by licensed meningococcal vaccines. Consequently, the W + B OMV vaccine could potentially serve as a viable alternative or supplement to existing meningococcal vaccines.

Immunization with the glutathione S-transferase Sj26GST with Chi-CpG NP against Schistosoma japonicum in mice.

Schistosomiasis caused by Schistosoma japonicum (S. japonicum) is a major public health problem in the Philippines, China and Indonesia. In this study, the immunopotentiator CpG-ODN was encapsulated within chitosan nanoparticles (Chi NPs) to create a combination adjuvant (Chi-CpG NP). This approach was employed to enhance the immunogenicity of 26 kDa glutathione S-transferase (Sj26GST) from S. japonicum through intranasal immunization. The results demonstrated higher levels of specific anti-Sj26GST antibodies and Sj26GST-specific splenocyte proliferation compared to mice that were immunized with Sj26GST + Chi-CpG NP. Cytokine analysis of splenocytes revealed that the Sj26GST + Chi-CpG NP induced a slight Th1-biased immune response, with increased production of IFN-γ by CD4+ T-cells in the spleen. Subsequently, mice were intradermally inoculated with 1 × 107 organisms in the Coeliac cavity. The bacterial organ burden detected in the liver of immunized mice suggested that Sj26GST + Chi-CpG NP enhances protective immunity to inhibit S. japonicum colonization. Therefore, Sj26GST + Chi-CpG NP vaccination enhances Sj26GST-specific immunogenicity and provides protection against S. japonicum.

Tumor-Targeted Codelivery of CpG and siRNA by a Dual-Ligand-Functionalized Curdlan Nanoparticle.

The simultaneous delivery of CpG oligonucleotide along with short interfering RNA (siRNA) has the potential to significantly boost the anticancer impact of siRNA medications. Our previous research demonstrated that Curdlan nanoparticles functionalized with adenosine are capable of selectively delivering therapeutic siRNA to cancerous cells through endocytosis mediated by adenosine receptors. Herein, we synthesized a dual-ligand-functionalized Curdlan polymer (denoted by CuMAN) to simultaneously target tumor cells and tumor-associated macrophages (TAMs). CuMAN nanoparticles containing CpG and siRNA demonstrated enhanced uptake by B16F10 tumor cells and bone marrow-derived macrophages, which are facilitated by AR on tumor cells and mannose receptor on macrophages. This led to increased release of pro-inflammatory cytokines in both in vitro and in vivo settings. The synergistic effect of CpG on TAMs and RNAi on tumor cells mediated by the CuMAN nanoparticle not only suppressed the tumor growth but also strongly inhibited the lung metastasis. Our findings indicate that the CuMAN nanoparticle has potential as an effective dual-targeting delivery system for nucleic acid therapeutics.

An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer.

OBJECTIVES: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer. METHODS: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response. RESULTS: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment. CONCLUSIONS: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.

Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.

Characterization of the impact of dietary immunostimulant CpG on the expression of mRNA biomarkers involved in the immune responses in Atlantic salmon (Salmo salar).

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.

NF-κB Decoy Oligodeoxynucleotide-Loaded Poly Lactic-co-glycolic Acid Nanospheres Facilitate Socket Healing in Orthodontic Tooth Movement.

Orthodontic space closure following tooth extraction is often hindered by alveolar bone deficiency. This study investigates the therapeutic use of nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides loaded with polylactic-co-glycolic acid nanospheres (PLGA-NfDs) to mitigate alveolar bone loss during orthodontic tooth movement (OTM) following the bilateral extraction of maxillary first molars in a controlled experiment involving forty rats of OTM model with ethics approved. The decreased tendency of the OTM distance and inclination angle with increased bone volume and improved trabecular bone structure indicated minimized alveolar bone destruction. Reverse transcription-quantitative polymerase chain reaction and histomorphometric analysis demonstrated the suppression of inflammation and bone resorption by downregulating the expression of tartrate-resistant acid phosphatase, tumor necrosis factor-α, interleukin-1ß, cathepsin K, NF-κB p65, and receptor activator of NF-κB ligand while provoking periodontal regeneration by upregulating the expression of alkaline phosphatase, transforming growth factor-ß1, osteopontin, and fibroblast growth factor-2. Importantly, relative gene expression over the maxillary second molar compression side in proximity to the alveolus highlighted the pharmacological effect of intra-socket PLGA-NfD administration, as evidenced by elevated osteocalcin expression, indicative of enhanced osteocytogenesis. These findings emphasize that locally administered PLGA-NfD serves as an effective inflammatory suppressor and yields periodontal regenerative responses following tooth extraction.

Antimetastatic effect of intratumoral Treg antagonists in mice with renal cancer.

The interplay of regulatory T cells (Tregs) within the tumour microenvironment presents a significant challenge in anticancer immunotherapy. This study investigates the potential of Treg blockade to enhance the efficiency of effector T cells. Two distinct treatment cocktails were examined: 3p-hpRNA (5' triphosphate hairpin RNA) combined with unmethylated CpG oligonucleotide (CpG); CpG in combination with OX40 receptor-specific monoclonal antibody (anti-OX40). Treatment efficacy was assessed using a murine model of kidney adenocarcinoma.Renca cells (renal cortical cells with adenocarcinoma) were subcutaneously engrafted in 30 BALB/c mice, then animals were allocated into three treatment groups: Group 1: CpG+anti-OX40, Group 2: CpG+3p-hpRNA, Group 3: untreated control. Treatment efficacy was evaluated based on tumour growth, the occurrence of metastases and overall survival.On day 28 post-implantation, experiments had to be terminated due to tumour progression. Although comparisons of survival times and primary tumour sizes thus became inconsequential, histological examinations provided valuable insights. We observed distinct variations in primary tumour characteristics among the different groups: Groups 1 and 2 displayed demarcations, while Group 3 exhibited diffuse tumours with necrosis. Lung metastases were evident in 70% of untreated mice, whereas none were observed in either of the treated groups.Our findings instil confidence in the potential efficacy of the treatments, thereby laying a solid foundation for future investigations.

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.

Efficient Gene Suppression by DNA/DNA Double-Stranded Oligonucleotide In Vivo.

We recently reported the antisense properties of a DNA/RNA heteroduplex oligonucleotide consisting of a phosphorothioate DNA-gapmer antisense oligonucleotide (ASO) strand and its complementary phosphodiester RNA/phosphorothioate 2'-O-methyl RNA strand. When α-tocopherol was conjugated with the complementary strand, the heteroduplex oligonucleotide silenced the target RNA more efficiently in vivo than did the parent single-stranded ASO. In this study, we designed a new type of the heteroduplex oligonucleotide, in which the RNA portion of the complementary strand was replaced with phosphodiester DNA, yielding an ASO/DNA double-stranded structure. The ASO/DNA heteroduplex oligonucleotide showed similar activity and liver accumulation as did the original ASO/RNA design. Structure-activity relationship studies of the complementary DNA showed that optimal increases in the potency and the accumulation were seen when the flanks of the phosphodiester DNA complement were protected using 2'-O-methyl RNA and phosphorothioate modifications. Furthermore, evaluation of the degradation kinetics of the complementary strands revealed that the DNA-complementary strand as well as the RNA strand were completely cleaved in vivo. Our results expand the repertoire of chemical modifications that can be used with the heteroduplex oligonucleotide technology, providing greater design flexibility for future therapeutic applications.

Targeted In Vivo Delivery of NF-κB Decoy Inhibitor Augments Sensitivity of B Cell Lymphoma to Therapy.

Despite recent advances, non-Hodgkin's B cell lymphoma patients often relapse or remain refractory to therapy. Therapeutic resistance is often associated with survival signaling via nuclear factor κB (NF-κB) transcription factor, an attractive but undruggable molecular target. In this study, we describe a bipartite inhibitor comprising a NF-κB-specific decoy DNA tethered to a CpG oligodeoxynucleotide (ODN) targeting Toll-like receptor-9-expressing B cell lymphoma cells. The Bc-NFκBdODN showed efficient uptake by human diffuse large B cell (U2932, OCI-Ly3), Burkitt (RaJi), and mantle cell (Jeko1) lymphomas, respectively. We confirmed that Bc-NFκBdODN inhibited NF-κB nuclear translocation and DNA binding, resulting in CCND2 and MYC downregulation. Bc-NFκBdODN enhanced radiosensitivity of lymphoma cells in vitro. In xenotransplanted human lymphoma, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors. When combined with a local 3-Gy dose of radiation, Bc-NFκBdODN effectively arrested OCI-Ly3 lymphoma progression. In immunocompetent mice, intratumoral injections of Bc-NFκBdODN suppressed growth of directly treated and distant A20 lymphomas, as a result of systemic CD8 T cell-dependent immune responses. Finally, systemic administration of Bc-NFκBdODN to mice bearing disseminated A20 lymphoma induced complete regression and extended survival of most of the treated mice. Our results underscore clinical relevance of this strategy as monotherapy and in support of radiation therapy to benefit patients with resistant or relapsed B cell lymphoma.

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.

Oligodeoxynucleotides containing unmethylated cytosine-guanine motifs are effective immunostimulants against pneumococcal meningitis in the immunocompetent and neutropenic host.

BACKGROUND: Bacterial meningitis is a fatal disease with a mortality up to 30% and neurological sequelae in one fourth of survivors. Available vaccines do not fully protect against this lethal disease. Here, we report the protective effect of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG ODN) against the most frequent form of bacterial meningitis caused by Streptococcus pneumoniae. METHODS: Three days prior to the induction of meningitis by intracerebral injection of S. pneumoniae D39, wild-type and Toll-like receptor (TLR9)-/- mice received an intraperitoneal injection of 100 µg CpG ODN or vehicle. To render mice neutropenic, anti-Ly-6G monoclonal antibody was daily administrated starting 4 days before infection with a total of 7 injections. Kaplan-Meier survival analyses and bacteriological studies, in which mice were sacrificed 24 h and 36 h after infection, were performed. RESULTS: Pre-treatment with 100 µg CpG ODN prolonged survival of immunocompetent and neutropenic wild-type mice but not of TLR9-/- mice. There was a trend towards lower mortality in CpG ODN-treated immunocompetent and neutropenic wild-type mice. CpG ODN caused an increase of IL-12/IL-23p40 levels in the spleen and serum in uninfected animals. The effects of CpG ODN on bacterial concentrations and development of clinical symptoms were associated with an increased number of microglia in the CNS during the early phase of infection. Elevated concentrations of IL-12/IL-23p40 and MIP-1α correlated with lower bacterial concentrations in the blood and spleen during infection. CONCLUSIONS: Pre-conditioning with CpG ODN strengthened the resistance of neutropenic and immunocompetent mice against S. pneumoniae meningitis in the presence of TLR9. Administration of CpG ODN decreased bacterial burden in the cerebellum and reduced the degree of bacteremia. Systemic administration of CpG ODN may help to prevent or slow the progression to sepsis of bacterial CNS infections in healthy and immunocompromised individuals even after direct inoculation of bacteria into the intracranial compartments, which can occur after sinusitis, mastoiditis, open head trauma, and surgery, including placement of an external ventricular drain.

Murine Cross-Reactive Nonneutralizing Polyclonal IgG1 Antibodies Induced by Influenza Vaccine Inhibit the Cross-Protective Effect of IgG2 against Heterologous Virus in Mice.

Annual vaccination against influenza viruses is the most reliable and efficient way to prevent and control annual epidemics and protect from severe influenza disease. However, current split influenza vaccines are generally not effective against antigenically mismatched (heterologous) strains. To broaden the protective spectrum of influenza vaccines, adjuvants that can induce cross-reactive antibodies with cross-protection via Fc-mediated effector functions are urgently sought. Although IgG2 antibodies are generally more efficient than IgG1 antibodies in Fc-mediated effector functions, it is not yet clear which IgG isotypes show superior cross-protection against heterologous strains. It also remains unclear whether these IgG isotypes interfere with each other's protective effects. Here, we found that influenza split vaccine adjuvanted with aluminum salts, which predominantly induce cross-reactive IgG1, did not confer cross-protection against heterologous virus challenge in mice. In contrast, split vaccine adjuvanted with CpG oligodeoxynucleotides, which predominantly induce cross-reactive IgG2, showed cross-protection through the interaction of cross-reactive nonneutralizing IgG2 and alveolar macrophages, indicating the importance of cross-reactive nonneutralizing IgG2 for cross-protection. Furthermore, by using serum samples from immunized mice and isolated polyclonal antibodies, we show that vaccine-induced cross-reactive nonneutralizing IgG1 suppress the cross-protective effects of IgG2 by competitively inhibiting the binding of IgG2 to virus. Thus, we demonstrate the new concept that cross-reactive IgG1 may interfere with the potential for cross-protection of influenza vaccine. We propose that adjuvants that selectively induce virus-specific IgG2 in mice, such as CpG oligodeoxynucleotides, are optimal for heterologous protection.IMPORTANCE Current influenza vaccines are generally effective against highly similar virus strains by inducing neutralizing antibodies. However, these antibodies fail to neutralize antigenically mismatched (heterologous) strains and therefore provide limited protection against them. Efforts are being made to develop vaccines with cross-protective ability that would protect broadly against heterologous strains, because the mismatch between predicted and epidemic strains cannot always be avoided, resulting in low vaccine efficacy. Here, we show that nonneutralizing IgG2 antibodies induced by an optimal adjuvant play a crucial role in cross-protection against heterologous virus challenge in mice. Furthermore, nonneutralizing polyclonal IgG1 suppressed the cross-protective effects of nonneutralizing polyclonal IgG2 by competitively blocking the binding of IgG2 to its antigen. These data shed new light on the importance of IgG isotypes and the selection of appropriate adjuvants for the development of universal influenza vaccines. Furthermore, our findings are applicable to the rational design of vaccines against other pathogens.

Protective immunity induced by CpG ODN-adjuvanted virus-like particles containing Toxoplasma gondii proteins.

AIMS: To date, a Toxoplasma gondii vaccine for clinical use remains unavailable, though multiple vaccine candidates have been suggested. In our previous studies, unadjuvanted virus-like particles (VLPs) vaccines expressing multiple T. gondii antigens were confirmed to be protective against T. gondii challenge infection. Yet, the protective efficacy of adjuvanted T. gondii VLP in comparison with the unadjuvanted counterpart requires elucidation. METHODS AND RESULTS: In the present study, mice were immunized with the multi-antigenic VLP vaccines (TG146 VLP) with or without CpG adjuvants and their protective efficacies were compared. CpG-adjuvanted TG146 VLP vaccine elicited enhanced T gondii-specific IgG and IgA antibody responses in the sera, mucosal tissue and the brain compared to unadjuvanted VLPs vaccine. Inclusion of CpG adjuvant in vaccines also induced greater CD4+ and CD8+ T-cell responses, as well as B cell and germinal centre B cell responses from splenocytes and mesenteric lymph nodes. Pro-inflammatory cytokine response and cyst counts in the brain were drastically diminished in mice immunized with CpG-adjuvanted VLP vaccines. CONCLUSION: Our results demonstrated that CpG-adjuvanted T. gondii VLPs can significantly enhance the protective efficacy of vaccines against T. gondii infection.

Therapeutic efficacy and cardioprotection of nucleolin-targeted doxorubicin-loaded ultrasound nanobubbles in treating triple-negative breast cancer.

Targeted lipid nanobubbles as theranostic ultrasound molecular probes with both targeted contrast-enhanced ultrasound molecular imaging and synergistic treatment capabilities are expected to overcome severe challenges in the diagnosis and treatment of refractory triple-negative breast cancer (TNBC). In this study, AS1411 aptamer-functionalised nucleolin-targeted doxorubicin-loaded lipid nanobubbles (AS1411-DOX-NBs) were constructed, and their physicochemical properties as well as anti-tumour and cardioprotective efficacies were systematically tested and evaluated. The results showed that AS1411-DOX-NBs can carry and maintain the physicochemical and pharmacodynamic properties of doxorubicin (DOX) and show stronger tumour cell-killing abilityin vitroby increasing the active uptake of drugs. AS1411-DOX-NBs also significantly inhibited the growth of TNBC xenografts while maintaining the weight and health of the mice. Echocardiography and pathological examination further confirmed that AS1411-DOX-NBs effectively caused tumour tissue apoptosis and necrosis while reducing DOX-induced cardiotoxicity. The AS1411-DOX-NBs constructed in this study enable both targeted contrast-enhanced ultrasound molecular imaging and synergistic therapeutic efficacy and can be used as safe and efficient theranostic ultrasound molecular probes for the diagnosis and treatment of TNBC.

Topical Adjuvant Application during Subcutaneous Vaccination Promotes Resident Memory T Cell Generation.

Skin tissue resident memory T cells (TRM) provide superior protection to a second infection. In this study, we evaluated the use of topical CpG oligodeoxynucleotide (ODN) as adjuvant to generate skin TRM in mice. Topical or s.c. CpG ODN adjuvant administration at the time of a s.c. Ag injection led to an accumulation of CD103- CD8 T cells in the epidermis. However, only mice with CpG ODN administered topically had significant numbers of CD103+ Ag-specific CD8 T cells persisting in the local epidermal skin, enhanced circulating memory cells in the blood, and showed protection from intradermal challenge with melanoma cells. Generation of Ag-specific CD8 T cells was dependent on TLR9 expression on hematopoietic cells and partially dependent on receptor expression on stromal cells. Topical challenge of immunized mice at a distal site led to significant expansion of Ag-specific T cells in the blood and accumulation in the challenged skin. We demonstrate that local and systemic T cell memory can be generated with topical CpG ODN at the time of s.c. immunization, suggesting a new method of enhancing current vaccine formulations to generate tissue TRM.

CpG preconditioning reduces accumulation of lysophosphatidylcholine in ischemic brain tissue after middle cerebral artery occlusion.

Ischemic stroke is one of the major causes of death and permanent disability in the world. However, the molecular mechanisms surrounding tissue damage are complex and further studies are needed to gain insights necessary for development of treatment. Prophylactic treatment by administration of cytosine-guanine (CpG) oligodeoxynucleotides has been shown to provide neuroprotection against anticipated ischemic injury. CpG binds to Toll-like receptor 9 (TLR9) causing initialization of an inflammatory response that limits visible ischemic damages upon subsequent stroke. Here, we use nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) to characterize molecular effects of CpG preconditioning prior to middle cerebral artery occlusion (MCAO) and reperfusion. By doping the nano-DESI solvent with appropriate internal standards, we can study and compare distributions of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the ischemic hemisphere of the brain despite the large changes in alkali metal abundances. Our results show that CpG preconditioning not only reduces the infarct size but it also decreases the degradation of PC and accumulation of LPC species, which indicates reduced cell membrane breakdown and overall ischemic damage. Our findings show that molecular mechanisms of PC degradation are intact despite CpG preconditioning but that these are limited due to the initialized inflammatory response.

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.

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.

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.