Immunostimulatory Sequences Research Articles

Quantification of influenza virus mini viral RNAs using Cas13.

Influenza A virus (IAV) RNA synthesis produces full-length and deletion-containing RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that DVG and mvRNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Measuring the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we explored if IAV mvRNA molecules can be detected and quantified using amplification-free, CRISPR-LbuCas13a-based detection. We show that CRISPR-LbuCas13a can be used to measure the copy numbers of specific mvRNAs in samples from infected tissue culture cells. However, to efficiently detect mvRNAs in other samples, promiscuous CRISPR guide RNAs are required that activate LbuCas13a in the presence of multiple mvRNA sequences. One crRNA was able to detect full-length IAV segment 5 without amplification, allowing it to be used for general IAV infection detection nasopharyngeal swabs. Using CRISPR-LbuCas13a, we confirm that mvRNAs are present in ferret upper and lower respiratory tract tissue, as well as clinical nasopharyngeal swab extracts of hospitalized patients. Overall, CRISPR-LbuCas13a-based RNA detection is a useful tool for studying deletion-containing viral RNAs and it complements existing amplification-based approaches.

Therapeutic efficacy of a novel self-assembled immunostimulatory siRNA combining apoptosis promotion with RIG-I activation in gliomas

BackgroundCurrent cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5’-C; antisense strand, 3’-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy.MethodsIsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5’-C; antisense strand, 3’-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-β and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection.ResultsIsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-β and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors.ConclusionsThis study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5’-C; antisense strand, 3’-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.

Improving combination cancer immunotherapy by manipulating dual immunomodulatory signals with enzyme-triggered, cell-penetrating peptide-mediated biomodulators.

Immunosuppressive tumor microenvironments challenge the effectiveness of protein-based biopharmaceuticals in cancer immunotherapy. Reestablishing tumor cell immunogenicity by enhancing calreticulin (CRT) exposure is expected to improve tumor immunotherapy. Given that CRT translocation is inherently modulated by phosphorylated eIF2α, the selective inhibition of protein phosphatase 1 (PP1) emerges as an effective strategy to augment tumor immunogenicity. To harness the PP1-disrupting potential of GADD34-derived motifs and address their limited intracellular delivery, we integrated these sequences into an enzyme-triggered, cell-penetrating peptide-mediated chimeric protein scaffold. This design not only facilitates efficient cytoplasmic delivery of these immunostimulatory motifs to induce "eat-me" signaling, but also provides a versatile platform for combination immunotherapy. Fabrication of biomodulators with cytotoxic BLF1 provides additional "eat-me" signaling through phosphatidylserine exposure or that with an immunomodulatory designed ankyrin repeat protein disables "don't-find-me" signaling by antagonizing PD-L1. Notably, these bifunctional biomodulators exhibit remarkable ability to induce macrophage phagocytosis, dendritic cell maturation, and CD8+ T activation, ultimately substantially inhibiting tumor growth. This study presents a modular genetic coding strategy for PP1-centered therapies that enables seamless integration of immunostimulatory sequences into protein-based anti-tumor cocktail therapies, thereby offering novel alternatives for improving antitumor efficacy.

Advancements in monkeypox vaccines development: a critical review of emerging technologies.

Monkeypox (mpox) is a zoonotic illness caused by the monkeypox virus (MPXV), with higher health concerns among people who are pregnant, children, and persons who are immunocompromised, including people with untreated and advanced HIV disease. Significant progress has been made in developing vaccines against mpox, yet critical challenges and limitations persist in ensuring their effectiveness, safety, and accessibility. The pertinence of this review is highlighted by the World Health Organization's declaration of a global health emergency on August 14, 2024, due to the recent mpox outbreak, underscoring the critical necessity for effective vaccine solutions in the face of a rapidly evolving virus. Here, we comprehensively analyze various vaccine platforms utilized in mpox prevention, including attenuated and non-replicating virus vaccines, viral vector-based vaccines, recombinant protein vaccines, and DNA and mRNA vaccines. We evaluate the advantages and limitations of each platform, highlighting the urgent need for ongoing research and innovation to enhance vaccine efficacy and safety. Recent advancements, such as incorporating immunostimulatory sequences, improved delivery systems, and developing polyvalent vaccines, are explored for their potential to offer broader protection against diverse orthopoxvirus strains. This work underscores the need to optimize currently available vaccines and investigate novel vaccination strategies to address future public health emergencies effectively. By focusing on these advanced methodologies, we aim to contribute to the development of robust and adaptable vaccine solutions for mpox and other related viral threats.

Efficacy of a two-dose hepatitis B vaccination with a novel immunostimulatory sequence adjuvant (Heplisav-B) on patients with chronic liver disease: a retrospective study.

Patients with chronic liver disease (CLD) are more likely to have severe morbidity and mortality due to superimposed acute or chronic hepatitis B virus (HBV) infection and should receive routine vaccination against the virus. Heplisav-B is a two-dose, inactivated, yeast-derived vaccine that uses a novel immunostimulatory adjuvant. Our primary objective was to determine the efficacy of hepatitis B vaccination with Heplisav-B in patients with CLD. This retrospective cohort analysis included patients ≥18 years old with CLD who received Heplisav-B from January 2018 to January 2021. All patients had anti-HBs <10 IU/L prior to vaccination and received two doses of Heplisav-B. Post-vaccination anti-HBs of ≥10 IU/L was considered successful vaccination. Basic demographic information, laboratory markers, and medical history were collected from the electronic health record. A total of 120 patients were included in analysis. The average age of patients was 59 years, 37% were female, and the most common etiology of liver disease was nonalcoholic fatty liver disease. Median days from 2nd vaccination to post-vaccination HBsAb levels was 121 days. 81/120 (67.5%) of patients had evidence of active immunity after receipt of Heplisav-B. On multivariable analysis, age >50 was associated with reduced odds of successful vaccination (OR =0.19, 95% CI: 0.03-0.76). In patients with CLD, Heplisav-B's overall efficacy (67.5%) is greater than reports of Engerix-B (33-45%), and thus is an effective hepatitis B vaccine in this patient population, particularly in cirrhotic patients. Further studies regarding this vaccine are needed in patients with CLD and after liver transplantation.

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

PurposeThe 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.MethodsA 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.ResultsWe 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.ConclusionWe 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.

Immunopotentiation by Lymph-Node Targeting of a Malaria Transmission-Blocking Nanovaccine.

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

Toll-Like Receptor 21 of Chicken and Duck Recognize a Broad Array of Immunostimulatory CpG-oligodeoxynucleotide Sequences.

CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the function of microbial CpG-dideoxynucleotides containing DNA (CpG-DNA) are potent immune stimuli. The immunostimulatory activity and the species-specific activities of a CpG-ODN depend on its nucleotide sequence properties, including CpG-hexamer motif types, spacing between motifs, nucleotide sequence, and length. Toll-like receptor (TLR) 9 is the cellular receptor for CpG-ODNs in mammalian species, while TLR21 is the receptor in avian species. Mammalian cells lack TLR21, and avian cells lack TLR9; however, both TLRs are expressed in fish cells. While nucleotide sequence properties required for a CpG-ODN to strongly activate mammalian TLR9 and its species-specific activities to different mammalian TLR9s are better studied, CpG-ODN activation of TLR21 is not yet well investigated. Here we characterized chicken and duck TLR21s and investigated their activation by CpG-ODNs. Chicken and duck TLR21s contain 972 and 976 amino acid residues, respectively, and differ from TLR9s as they do not have an undefined region in their ectodomain. Cell-based TLR21 activation assays were established to investigate TLR21 activation by different CpG-ODNs. Unlike grouper TLR21, which was preferentially activated by CpG-ODN with a GTCGTT hexamer motif, chicken and duck TLR21s do not distinguish among different CpG-hexamer motifs. Additionally, these two poultry TLR21s were activated by CpG-ODNs with lengths ranging from 15 to 31 nucleotides and with different spacing between CpG-hexamer motifs. These suggested that compared to mammalian TLR9 and grouper TLR21, chicken and duck TLR21s have a broad CpG-ODN sequence recognition profile. Thus, they could also recognize a wide array of DNA-associated molecular patterns from microbes. Moreover, CpG-ODNs are being investigated as antimicrobial agents and as vaccine adjuvants for different species. This study revealed that there are more optimized CpG-ODNs that can be used in poultry farming as anti-infection agents compared to CpG-ODN choices available for other species.

Overcoming Strategies for Non-responders in HBV Vaccination

Non-responder refers to an individual did not develop their anti HBs, even after administration of a 2 complete series of the HBV vaccine. Due to mutant variants, vaccine failure occurred in numerous reports but the incidence of these mutants were unknown. Primary HBV vaccine series failed for nearly 5% of immunocompetent people. There is no clear vision on nonresponse but certain individuals are at major risk, those with persistent diseases, immunosuppressant medication and genetic predisposition. CD-4 T-helper cells that was obtained from viral peptides played an important role in Human Leukocyte antigen (HLA) along with Major Histocompatibility Complex (MHC) and individuals who failed to respond had defect in T helper cells stimulation or antigen presentation. After administration of a course of vaccine, antibody will be produced against major hydrophilic domain of HBsAg in determinant epitope cluster. As upcoming approaches, antigen dose were increased, alternative vaccination, new adjuvants such as immunostimulatory DNA sequences and accelerating vaccination schedules are followed in practice. Increased dosage vaccines, upgraded immunogenicity, adjuvants, surrogate mechanism, combined vaccines and administration of intradermal vaccines are the relevant approaches for a non-responder. Further studies has to be conducted on HLA alleles that can overcome the obstacles in HBV therapeutics.

In Celiac Disease Patients the In Vivo Challenge with the Diploid Triticum monococcum Elicits a Reduced Immune Response Compared to Hexaploid Wheat.

Gluten from the diploid wheat Triticum monococcum (TM) has low content of immunostimulatory sequences and a high gastro-intestinal digestibility. Gluten-reactive T cells elicited by diploid and hexaploid (Triticum aestivum-TA) wheat in celiac disease (CD) patients upon a brief oral challenge are analyzed. Seventeen patients with CD (median age 13 years) consumed for 3 days sandwiches made with TM (cultivar Norberto-ID331, N=11), or TA (cultivar Sagittario, N=11) flours, corresponding to 12gr of gluten/die. Immunostimulatory properties are assessed in blood by measuring the IFN-γ-secreting T cells by EliSpot and the expression of inflammatory cytokines/receptors (IL-12A, IL-15, IL-18RAP, IFN-γ) by qPCR. TA mobilizes a remarkable number of gliadin-specific, IFN-γ-secreting T cells (p<0.05), while no significant cell mobilization is induced by TM (p=ns). Similar results are obtained in response to five immunogenic peptides from α-, ω-, and γ-gliadins, although with a large individual variability. An increased mRNA expression for IL-12A and IFN-γ is detected in the group eating TA compared to those consuming TM (p<0.05). Although T. monococcum is a cereal not suitable for the diet of celiacs, this diploid wheat elicits a reduced in vivo T-cell response compared to T. aestivum in celiac patients.

Hepatitis B Vaccine and Immunoglobulin: Key Concepts.

Hepatitis B virus (HBV) immunization is safe and has been accepted worldwide as a routine practice. The target of such vaccination is to induce the immune response in the host, resulting in the prevention of replication of HBV. There are several immunological and clinical factors which determine the clinical efficacy and safety of the HBV vaccine. In this article we have highlighted the response of the host immune system to HBV vaccination (immunogenicity), efficacy, and safety of the vaccine, issues with booster dosing, paths of development (preclinical and clinical) of the HBV vaccine, novel and upcoming strategies for improvement of HBV vaccination, and the concept of therapeutic HBV vaccination. The different aspects and regulatory recommendations pertaining to HBV vaccine development are also discussed. The new strategies for improvement of HBV vaccination include pre-S1 and pre-S2 portions of the HBV surface antigen, increasing the antigen dose, accelerated vaccination schedules, alternative vaccination route, use of adjuvants like immunostimulatory DNA sequences, etc. Therapeutic vaccination is being explored for initiation of a multifunctional and multispecific T cell response against the major HBV antigens and also effective activation of humoral immunity for viral control.

Adjuvants in Allergen-Specific Immunotherapy: Modulating and Enhancing the Immune Response.

Allergen-specific immunotherapy (AIT) is the only treatment that can affect the natural course of allergic diseases such as allergic asthma, allergic rhinitis, and IgE-mediated food allergy. Adjuvants are used to induce a quicker, more potent, and longer-lasting immune response. Only 4 compounds are used as adjuvants in currently marketed AIT products: aluminum hydroxide, calcium phosphate, microcrystalline tyrosine (MCT), and monophosphoryl lipid A (MPL). The first 3 adjuvants are delivery systems with a depot effect, although they may also have immunomodulatory properties. These first-generation adjuvants are still widely used, especially aluminum hydroxide. However, aluminum is subject to limitations. MCT is the depot formulation of L-tyrosine; it enhances IgG production without inducing a significant increase in IgE, is biodegradable, and has good local and systemic tolerability. In turn, MPL is an immunostimulatory agent that is the only second-generation adjuvant currently used for AIT. In addition, multiple adjuvants are currently being studied, including immunostimulatory sequences (ISSs), nanoparticles (liposomes, virus-like particles, and biodegradable polymers), and phosphatidylserine derivatives. In a murine model of allergic bronchial inflammation by sensitization to olive pollen, the specific IgE level was significantly higher in sensitized mice treated with olive pollen and aluminum hydroxide. However, specific IgE levels were significantly reduced and bronchial hyperreactivity significantly improved in sensitized mice treated with olive pollen and bacterial derivatives (MPL or ISSs).

Novel strategies in immunotherapy for allergic diseases.

Conventional immunotherapy (IT) for optimal control of respiratory and food allergies has been fraught with concerns of efficacy, safety, and tolerability. The development of adjuvants to conventional IT has potentially increased the effectiveness and safety of allergen IT, which may translate into improved clinical outcomes and sustained unresponsiveness even after cessation of therapy. Novel strategies incorporating the successful use of adjuvants such as allergoids, immunostimulatory DNA sequences, monoclonal antibodies, carriers, recombinant proteins, and probiotics have now been described in clinical and murine studies. Future approaches may include fungal compounds, parasitic molecules, vitamin D, and traditional Chinese herbs. More robust comparative clinical trials are needed to evaluate the safety, clinical efficacy, and cost effectiveness of various adjuvants in order to determine ideal candidates in disease-specific and allergen-specific models. Other suggested approaches to further optimize outcomes of IT include early introduction of IT during an optimal window period. Alternative routes of administration of IT to optimize delivery and yet minimize potential side effects require further evaluation for safety and efficacy before they can be recommended.

Delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination

DNA rich in unmethylated CG motifs (CpGs) engage Toll-Like Receptor 9 (TLR-9) in endosomes and are well described stimulators of the innate and adaptive immune system. CpGs therefore can efficiently improve vaccines' immunogenicity. Packaging CpGs into nanoparticles, in particular into virus-like particles (VLPs), improves the pharmacological characteristics of CpGs as the protein shell protects them from DNAse activity and delivers the oligomers to the endosomal compartments of professional antigen presenting cells (APCs). The current consensus in packaging and delivering CpGs in VLP-based vaccines is that both adjuvants and antigens should be kept in close proximity (i.e. physically linked) to ensure delivery of antigens and adjuvants to the same APCs. In the current study, we harness the draining properties of the lymphatic system and show that also non-linked VLPs are efficiently co-delivered to the same APCs in lymph nodes. Specifically, we have shown that CpGs can be packaged in one VLP and mixed with another VLP displaying the antigen prior to administration in vivo. Both VLPs efficiently reached the same draining lymph node where they were taken up and processed by the same APCs, namely dendritic cells and macrophages. This resulted in induction of specific CTLs producing cytokines and killing target cells in vivo at levels seen when using VLPs containing both CpGs and chemically conjugated antigen. Thus, delivery of antigens and adjuvants in separate nanoparticles eliminates the need of physical conjugation and thus can be beneficial when designing precision medicine VLP-based vaccines or help to re-formulate existing VLP vaccines not naturally carrying immunostimulatory sequences.

Tolerogenic mechanisms elicited by extensively hydrolysed casein formula with L. rhamnosus GG

We showed that extensively hydrolyzed casein formula containing the probiotic L. rhamnosus GG (EHCF + LGG) stimulates tolerance acquisition in children with cow milk allergy (CMA). We aimed to investigate tolerogenic immune and non-immune mechanisms elicited by LGG immunostimulatory DNA sequence (ODN ID35) and by the three most abundant β-casein-derived peptides contained in the formula.

Polypod-Shaped DNAs: Small-Angle X-ray Scattering and Immunostimulatory Activity.

We explored in detail the relationship between the structure in aqueous solution and immunostimulatory activity of polypod-shaped DNAs, called polypodnas. The polypodnas were constructed using 3-6 oligodeoxynucleotides (ODNs) to obtain tri-, tetra-, penta-, and hexapodna, each of which had 3, 4, 5, and 6 arms made of double-stranded DNA, respectively. A highly potent immunostimulatory CpG sequence was included into each of the polypodnas. Synchrotron X-ray scattering analysis showed that the double-stranded DNA arms of all of the polypodnas adopted a B-form DNA conformation. The analysis also suggested that some nucleotides in the central parts of pentapodna and hexapodna did not form base pairs, whereas those of tripodna and tetrapodna all formed base pairs. This difference would occur because of an increase in steric hindrance and electrical repulsion with increasing number of arms. The pentapodna and hexapodna induced a large amount of tumor necrosis factor α-release from macrophage-like cells compared with the tripodna and tetrapodna, suggesting that the partly loosened DNA in polypodna with many arms is advantageous for exposing the immunostimulatory sequences of the polypodna.

Immunogenicity and immunizing protection effect of GAMA gene DNA vaccine on Plasmodium berghei.

To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion. Gene fragments were obtained using PCR technique and eukaryotic expression vector (containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the IgG, IgG1 and IgG2a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-γ levels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate invitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels. In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific IgG in DNA vaccine immunity group significantly increased (P<0.01), and IgG1 and IgG2a all increased (P<0.01). IL-4, IFN-γ content in study group significantly increased, compared with control group (P<0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection (caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced (P<0.05), and antiserum ookinete numbers (cultured invitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group. GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocyte-stage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA (as multi-stage antigen vaccine and multi-stage combined vaccine components of the life-cycle of plasmodium) is necessary.

Bifunctional siRNA containing immunostimulatory motif enhances protection against pandemic H1N1 virus infection.

The first influenza pandemic of 21(st) century was attributed to a novel quadruple reassortant H1N1 virus that emerged in 2009. Currently available therapies for influenza have drugresistant. Therefore, there is a need to develop new generation immunotherapeutic antiviral strategy. This study described the efficacy of a novel bifunctional immunostimulatory siRNAs against H1N1pdm swine flu virus by targeting the Nucleocapsid (NP) gene. Small interfering RNAs (siRNA) targeting conserved region of NP were screened for antiviral efficacy in human lung epithelial cells (A549). Further, a bifunctional siRNA was synthesized by combining immunostimulatory sequence (5'-UGUGU-3') with NP specific siRNA. This immunostimulatory siRNA (NP-1-is) revealed strong antiviral effect through reduction in mRNA copies (99.58%), reduction in virus associated cell apoptosis and inhibition of nucleocapsid protein in western blot. This immunostimulatory siRNA was found more effective than nontagged siRNA. Further studies including dose dependent and time course kinetics revealed that the NP-1-is siRNA is more effective at 20-80 nM with significant protection upto 48 hpi. Besides, the qRT-PCR and western blot analysis confirmed higher antiviral response of immunostimulatory siRNA was due to upregulation of TLR-7 MyD88, IRF-7 and IFN-α. This study paves the way for broad-spectrum RNAi-based therapeutics using immunostimulatory motif towards improved antiviral effect. Hence this approach will be useful to confront the sudden emergence of pandemic strains.

Base modification strategies to modulate immune stimulation by an siRNA.

Immune stimulation triggered by siRNAs is one of the major challenges in the development of safe RNAi-based therapeutics. Within an immunostimulatory siRNA sequence, this hurdle is commonly addressed by using ribose modifications (e.g., 2'-OMe or 2'-F), which results in decreased cytokine production. However, as immune stimulation by siRNAs is a sequence-dependent phenomenon, recognition of the nucleobases by the trigger receptor(s) is also likely. Here, we use the recently published crystal structures of Toll-like receptor 8 (TLR8) bound to small-molecule agonists to generate computational models for ribonucleotide binding by this immune receptor. Our modeling suggested that modification of either the Watson-Crick or Hoogsteen face of adenosine would disrupt nucleotide/TLR8 interactions. We employed chemical synthesis to alter either the Watson-Crick or Hoogsteen face of adenosine and evaluated the effect of these modifications in an siRNA guide strand by measuring the immunostimulatory and RNA interference properties. For the siRNA guide strand tested, we found that modifying the Watson-Crick face is generally more effective at blocking TNFα production in human peripheral blood mononuclear cells (PBMCs) than modification at the Hoogsteen edge. We also observed that modifications near the 5'-end were more effective at blocking cytokine production than those placed at the 3'-end. This work advances our understanding of how chemical modifications can be used to optimize siRNA performance.