Browsing by Subject "Adjuvant"

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Open Access
Biotin functionalized self‐assembled peptide nanofiber as an adjuvant for immunomodulatory response
(Wiley-VCH Verlag, 2020-12) Demircan, Muhammed Burak; Tohumeken, Sehmus; Gündüz, Nuray; Khalily, Mohammad Aref; Tekinay, T.; Güler, M. O.; Tekinay, Ayşe B.
Biotinylated peptide amphiphile (Biotin‐PA) nanofibers, are designed as a noncovalent binding location for antigens, which are adjuvants to enhance, accelerate, and prolong the immune response triggered by antigens. Presenting antigens on synthetic Biotin‐PA nanofibers generated a higher immune response than the free antigens delivered with a cytosine‐phosphate‐guanine oligodeoxynucleotides (CpG ODN) (TLR9 agonist) adjuvant. Antigen attached Biotin‐PA nanofibers trigger splenocytes to produce high levels of cytokines (IFN‐γ, IL‐12, TNF‐α, and IL‐6) and to exhibit a superior cross‐presentation of the antigen. Both Biotin‐PA nanofibers and CpG ODN induce a Th‐1‐biased IgG subclass response; however, delivering the antigen with Biotin‐PA nanofibers induce significantly greater production of total IgG and subclasses of IgG compared to delivering the antigen with CpG ODN. Contrary to CpG ODN, Biotin‐PA nanofibers also enhance antigen‐specific splenocyte proliferation and increase the proportion of the antigen‐specific CD8(+) T cells. Given their biodegradability and biocompatibility, Biotin‐PA nanofibers have a significant potential in immunoengineering applications as a biomaterial for the delivery of a diverse set of antigens derived from intracellular pathogens, emerging viral diseases such as COVID‐19, or cancer cells to induce humoral and cellular immune responses against the antigens.
Open Access
Biotinylated peptide nanofibers for modulating the immune response
(2016-06) Tohumeken, Şehmus
Despite the fact that vaccination eradicates many diseases, a broad variety of disorders cannot be treated using current vaccine development methods. In addition, it is difficult to rapidly develop new vaccines following the sudden onset of a new pandemic, as the production and transport of vaccines to impoverished areas is still a major issue. The lack of sufficient vaccine production, for example, enabled the spread of swine flu in 2009, while HIV, Zika and malaria viruses currently lack effective vaccinations. In addition, while cancer vaccines represent a promising area of research, their clinical implementation is also limited by the absence of rapid and effective vaccine development methods. The development of new and effective vaccines is therefore quite vital. Moreover, recently used vaccines promote either humoral or cellular immune responses, while effective treatment requires the induction of both systems. Consequently, there is an urgent need for effective and easy-to-prepare vaccines that are capable of eliciting immune action through multiple channels. Peptide amphiphiles are small molecules that are able to self-assemble into nanoscale fibrous networks. These nanofibers are biodegradable, biocompatible and do not generate toxic byproducts, making them ideal for designing biomaterials. As such, nanofibers are a promising class of materials for inducing an effective immune response and overcoming some of the problems faced by current vaccine development methods. In this thesis, I detail the use of biotinylated peptide nanofiber systems as immunomodulatory materials that are capable of incorporating a broad variety of antigens in a modular manner. Briefly, biotinylated and non-biotinylated peptide amphiphiles (PA) were first synthesized, purified and characterized to determine their material properties. PAs were then induced to self-assemble in the presence of CpG oligonucleotide (ODN) adjuvants, and ovalbumin was conjugated to self-assembled biotinylated-PA (B-PA) nanofibers by streptavidin linkers. Splenocytes were isolated from the mouse spleen and treated with bioactive nanofibers to investigate the effect of bioactive nanofibers on the immune response. Following the confirmation of an effective combined immune response, live mice were exposed to the nanofiber adjuvants as a proof-of-concept demonstration of in vivo PA-vaccine efficiency. Both in vivo and in vitro studies demonstrated that B-PA nanofibers are able to effectively modulate the immune response. Given these observations, I suggest that the B-PA nanofiber can be used as an immunomodulatory material for promoting effective immune response against extracellular and intracellular pathogens, and especially for the vaccine-based treatment of cancer. As the antigen presented by the PA system can be changed in a modular manner, B-PA nanofibers can also be employed to rapidly develop new vaccines against sudden outbreaks of new viral strains.
Open Access
Development and preclinical characterization of meningococcal outer membrane vesicle vaccine
(2024-03) Özsürekci, Yasemin
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 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 assays (SBA) based protective coverage of OMV vaccine to 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 ELISA and SBA. 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.
Open Access
In vivo applications of liposomal vaccines encapsulating single or dual pathogenassociated molecular patterns
(2017-03) Bayyurt Kocabaş, Banu
Nucleic acid-based pattern recognition receptor (PRR) agonists are promising adjuvants and immunotherapeutic agents. Combination of PRR ligands potentiates immune response by providing synergistic immune activity via triggering different signaling pathways and may impact antigen dependent T-cell immune memory. However, the duration of short circulation due to nuclease attacks is hampering their clinical performance. Liposomes enable protein and nucleic acid based compounds to have high encapsulation efficiency. Herein, we aimed to develop liposomal carrier systems that co-encapsulating single TLR9 or combinations with TLR3 or STING ligands and assess their potential as adjuvants and immunostimulatory agents in in vivo applications. Liposomal dual nucleic acid formulations induced synergistic innate immune activation, enhanced cytokine production along with internalization capacity of ligands. In anti-cancer vaccine study, CpG ODN and poly(I:C) coencapsulation significantly increased OVA-specific Th1-biased immune even after eight months post-booster injection. Challenge with OVA-expressing tumor cell line, E.G7, demonstrated that mice immunized with liposomes co-encapsulating CpG ODN and poly(I:C) had significantly slower tumor progression dependent on OVAspecific cytotoxic memory T-cells. In our second in vivo application, liposomal CDN and TLR9 therapy led to 80% remission of established melanoma tumor. Increased IgG2c/IgG1 ratio in mice treated with liposomal formulations indicating the development of antigen specific Th1-biased immunity was observed. Furthermore, along with the treatment, IFN-dual ligands into liposomes enhanced the anti-tumor activity of single ligands. In the third part, immunization with CpG ODN loaded liposomal formulations together with antigens increased antigen-specific humoral response against FMDV and Helicobacter. In addition, the liposomal CpG ODN reduced bacterial gastric colonization by antigen-dependent Th1 and Th17 immune responses after helicobacter challenging.
Open Access
Investigation of the role of cGAMP in differentiation of T lymphocytes
(2016-10) Yıldız, Begüm
STING is the pivotal mediator for the recognition of host and pathogenic cytosolic dsDNA as well as cyclic di-nucleotides metabolites from microbes. STING can either recognize DNA itself or sense the presence of cGAMP, which is converted from ATP and GTP upon DNA binding to cGAS enzyme. Not only strategy against intracellular pathogens makes STING an ideal target, but also the recognition of DNA from host cells has a significant role in tumor immunity. Previous studies demonstrated that DNA released from cancerous cells are internalized by innate immune cells such as macrophages and dendritic cells in tumor microenvironment and trigger the production of IFN-β and other pro-inflammatory cytokines including IL-6, TNF-α, and IL-12 through STING triggered signaling pathway. These cytokines then enhance cytotoxic activity of CD8+ T cells by further increasing IFNγ production. Since enhanced T cell immunity is the hallmark of vaccine adjuvants, cyclic di-nucleotides such as cGAMP become an important and effective vaccine adjuvants against intracellular pathogens and malignant cells. Although STING activating cyclic di-nucleotides are envisioned as novel and potent vaccine adjuvants, more thorough research is needed to unearth the mechanism of action of STING on different immune cells. Therefore, it will pave the way for the initiation of successful human trials. The important criteria while developing vaccine adjuvant are the magnitude, and the quality of an immune response and its toxic side effects. To identify these, members of the both innate and adaptive immune system should be taken into account. However, previous studies merely focus on the function and effect of cGAMP in innate immune cells such as macrophages, monocytes and dendritic cells. However, to date there is no explicit study investigating the effect of STING signaling cascade on T-cells. In the light of these findings, we aimed to investigate the direct effect and function of cGAMP on T lymphocytes. Since there were not any preliminary data, we firstly stimulated Pan T cells with cGAMP alone or together with various TLR ligands and then, checked the cytokine profiles and the viability of cells. Surprisingly, 2.5µg/ml dose of cGAMP had a toxic effect on T cell but not on bone marrow derived dendritic cells and macrophages. While cGAMP triggered cell death, interestingly IL-17 secretion from both CD4+ and CD8+ T cells was dramatically increased. Beside, cGAMP stimulation drastically increased CD4+ /CD8+ T cells ratio of Pan T cells population. Next, we sought to identify the source of IL-17. The IL17 inductive capacity of cGAMP was investigated on purified CD4+ T cells from mice. Unexpectedly, data revealed that cGAMP elicited apoptosis of CD4+ T cells. Moreover, there was no significant induction of IL-17 secretion. Next, we aimed to find a condition that will reduce the toxic effect of cGAMP, while maintaining IL-17 secretion. When Pan T cells were stimulated with cGAMP and R848 (a TLR7 ligand), the toxic action of cGAMP decreased while IL-17 secretion was enhanced. Lastly, the potency of T cells stimulated with cGAMP was investigated. According to our results, macrophages were activated in the presence of conditioned medium obtained from T cells stimulated with cGAMP. When taken together our findings point out that STING dependent direct activation of T-cells via cGAMP and its subsequent effect on macrophages might be utilized as an immunotherapeutic approach where IL17 induction is important and could be harnessed as vaccine adjuvants against mucosal infections or against cancer.
Open Access
Involvement of sting-activating cyclic Di-nucleotides on T-cell differentiation and function: an unresolved issue
(Turkish Society of Immunology, 2016) Yıldız, B.; Gürsel, İ.
STING is the pivotal mediator for the recognition of host and pathogenic cytosolic dsDNA as well as cyclic di-nucleotide metabolites from microbes. Studies demonstrated that DNA released from cancerous cells are internalized by innate immune cells such as macrophages and dendritic cells in tumor microenvironment and trigger the production of interferon beta and other pro-inflammatory cytokines including interleukin 6, tumor necrosis factor alpha, and interleukin 12 through STING triggered signaling pathway. Later, these cytokines increase the cytotoxic activity of CD8+ T-cells by increasing the production of interferon gamma. This review discusses the importance of the involvement of STING during the establishment of immunity against intracellular pathogens and its direct effect on T-cells. © 2016 Turkish Journal of Immunology. All rights reserved.
Open Access
Large-scale manufacturing and characterization of a Sars Cov-2 virus-like particle vaccine adsorbed onto alhydrogel and adjuvanted with K3 CpG oligodeoxynucleotide for use in phase 1/2 clinical trials
(2022-04-28) Bülbül, Artun
Emergence of COVID-19 pandemic has been met by an exceptionally fast response from vaccine makers around the globe. Vaccines that elicit excellent immunological responses against SARS-CoV-2 are now widely utilized. Existing platforms include mRNA-lipid nanoparticle-based vaccines, adenovirus vectored vaccines, various inactivated virus vaccines and subunit vaccines. We have previously described a novel virus-like particle (VLP) platform expressing the hexaproline prefusion stabilized Spike protein along with the nucleocapsid, membrane and envelope structural proteins. In mice, ferrets and rats, VLPs adjuvanted with K3 CpG Oligodeoxynucleotide (ODN) and adsorbed onto 2% Aluminum Hydroxide (Alum), induced robust humoral and cellular immune response against Spike and Nucleocapsid proteins. Herein, we have expanded our work to manufacture the virus like particles in a GMP compliant facility intended for testing in phase I/II clinical trials. The technology transfer comprises i) VLP production from suspension adapted HEK293 cells, ii) purification with multimodal fast protein liquid chromatography (FPLC) and iii) concentration and diafiltration using tangential flow filtration (TFF). We have successfully scaled up our production from 50 mL of HEK293 cell culture to 5 L bioreactor, achieving yields reaching up to 40 mg VLPs per L of cell culture. Furthermore, several methods were developed to determine protein identity, purity, functionality, stability and immunopotency of VLP vaccine that was finally formulated with Alum + CpG ODN. Moreover, we investigated the immunogenicity of VLPs decorated either with Wuhan (Hu-1) or with Alpha (B.1.1.7) variant Spike against receptor binding domains (RBD) specific to other variants of concern (VoC). Although our vaccine platform, could further benefit from process optimization to improve VLP yield, this study presents the first pilot scale production and purification of variant specific hexaproline prefusion stabilized SARS-CoV-2 VLPs. VLP preparations complying with our quality control parameters were released for fill and finish and were used for subsequent Phase 1 (NCT04818281) and Phase 2 clinical trials (NCT04962893).
Open Access
Modulation of immune responses using adjuvants to facilitate therapeutic vaccination
(Wiley, 2020) Schijns, V.; Fernández‐Tejada, A.; Barjaktarović, Ž.; Bouzalas, I.; Brimnes, J.; Chernysh, S.; Gizurarson, S.; Gürsel, İhsan; Jakopin, Ž.; Lawrenz, M.; Nativi, C.; Paul, S.; Pedersen, G. K.; Rosano, C.; Ruiz‐de‐Angulo, A.; Slütter, B.; Thakur, A.; Christensen, D.; Lavelle, Ed. C.
Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non‐infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen‐specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies.
Open Access
Rational vaccine design in times of emerging diseases: The critical choices of immunological correlates of protection, vaccine antigen and immunomodulation
(MDPI AG, 2021-04-06) Schijns, V.; Majhen, D.; Ley, P.; Thakur, A.; Summerfield, A.; Berisio, R.; Nativi, C.; Fernández-Tejada, A.; Alvarez-Dominguez, C.; Gizurarson, S.; Zamyatina, A.; Molinaro, A.; Rosano, C.; Jakopin, Z.; McClean, S.; Gürsel, İhsan
Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases.
Open Access
A self assembled nanofibrous structure as a novel vaccine adjuvant
(2017-01) Demircan, Muhammed Burak
Vaccination is the most effective and cost-efficient way of protection against the major infectious diseases but ideal vaccine formulation has not been found. Recent vaccine systems are mainly composed of two major substitutes that are antigen and adjuvant. Recently it was demonstrated that widely used adjuvants exhibit some safety problems that affect the neural system such as neurotoxicity and autoimmune diseases. Therefore, there are increased concerns about side effects of the adjuvants and many researchers focus on developing new adjuvants that are effective and safe. Peptide amphiphiles are chemically defined molecules that are able to self-assemble into nanofibrous structures. The nanofibrous structures are biocompatible, biodegradable, and biosafe and thereby they are ideal for vaccine systems. Also, nanofibrous structures don’t contain any substance that are potentially dangerous for neural system such as metals. Thus, nanofibrous structures are promising candidates to be alternative novel vaccine adjuvants. In this thesis, I investigated the potential of a biotinylated nanofibrous structure as a novel vaccine adjuvant that is potentially safe. Briefly, biotinylated peptide amphiphiles were synthesized, purified and characterized to analyze the features of the novel material. The peptide amphiphiles were induced to form nanofibrous structures by self-assembly and antigens (ovalbumin) were bound to the biotinylated nanofibrous structures through streptavidin linkers. Splenocytes were treated with the nanofibrous structures to demonstrate the effects of the nanofibrous structures on the immune response. After the confirmation of efficient immune response that are induced by the nanofibrous structure in vitro, as enhancing release of stimulatory cytokines, inducing dendritic cell maturation and triggering the cross-presentation of the antigen, mice were immunized with the nanofibrous structure in the presence of antigen for further analysis of the nanofibrous structure efficiency as adjuvant in vivo. Both in vivo and in vitro results showed that the nanofibrous structure is able to effectively trigger the antigen specific immune response and thereby exhibit adjuvant properties. Overall, I suggest that the nanofibrous structure is able to be used as a new vaccine adjuvant that induces effective antigen specific adoptive immune response and thereby it could be a good alternative of recently used adjuvants that are suspected to contribute some impairments in neural system.
Open Access
TLR agonists on autoimmunity, cancer and M1/M2 macrophage polarization
(2019-07) Horuluoğlu, Begüm Han
Macrophages play an important role in the initiation of immune responses and the maintenance of immune homeostasis. Alterations in their phenotype, function and activation state have been implicated in the pathogenesis of autoimmune and inflammatory diseases. An increased M1:M2 ratio is associated with the development of several autoimmune diseases including Systematic Lupus Erythematosus (SLE), vasculitis and myositis. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This work seeks to investigate the ability of PAM3 to induce M2 macrophage differentiation from patient monocytes and evaluate the therapeutic efficacy of PAM3 in a murine model of lupus. Our findings revealed that patients with indicated autoimmune diseases have a significant increase in monocytes of the inflammatory subtypes coupled with a decrease in non-inflammatory classical monocytes compared to healthy controls. Additionally, in the absence of stimulant patient monocytes differentiated into more M1-like macrophages. Nevertheless, phenotypic analysis of in vitro generated macrophages revealed that, PAM3 stimulation induced M2-like macrophage differentiation without any difference from patient and healthy monocytes. Phenotypic analysis was further supported by the high endocytic abilities and secretion of anti-inflammatory cytokines instead of pro-inflammatory cytokines by PAM3 generated macrophages. Lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Upon PAM3 treatment the increased M1:M2 ratio, which was observed in PBS treated group, was decreased to normal levels. The increase in M2 macrophages was accompanied by decreased autoantibody and inflammatory cytokines along with an increase in anti-inflammatory cytokine production. Moreover, kidney damage was significantly suppressed and M2 macrophages were detected in the kidneys of PAM3 treated group. PAM3 treatment prolonged to survival of NZB/W significantly, at 45 weeks of age %60 of mice were still alive whereas in PBS group only %5 were. Our results indicate that, PAM3 induces immunosuppressive macrophages and thus could represent a novel approach to the therapy of autoimmune diseases. The second part of this thesis focused on enhancing the immunomodulatory effects of TLR9 ligands, CpG ODN upon encapsulation within liposomes as cancer vaccine adjuvants. Although both D and K ODN are strictly dependent on TLR9, K ODN trigger pDCs to mature and secrete TNFα while D ODN stimulate pDC to produce IFNα. When cells are incubated with a mixture of K and D ODN, K masks the activity of D. The use of both K and D ODN would be of benefit when preparing vaccine adjuvants and for immunotherapy. Our data indicate that simultaneous delivery of D ODN loaded into neutral liposomes plus K ODN loaded into cationic liposomes improved rather than masked IFNα production while continuing to support TNFα by PBMCs. Liposomal encapsulation did not alter the subcellular localization of either class of ODN, but internalization studies revealed that cationic liposome encapsulation slows and reduces the uptake of K ODN whereas neutral encapsulation of D increases their uptake by pDCs. The efficiency of K plus D liposome combinations was examined in a murine tumor vaccine model. The liposome combinations induced pronounced Th1-biased anti-OVA immunity and led to a significant reduction of B16-OVA tumors following inoculation. Our findings, demonstrate that the beneficial features of D and K ODN could be obtained simultaneously by appropriate liposomal formulation, further extending the breadth of CpG ODN-dependent immunotherapy.