In induction of systemic and mucosal immunity, particulate antigens are more effective than soluble antigens possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization of tetanus toxoid TT and CPG-ODN encapsulated within PLGA nanospheres. The mean diameter of (TT) and TT+CPG nanospheres were 753 ±193 and 684±324 nm, respectively. Encapsulation efficiency of TT and CPG-ODN was determined as 52±7.8% and 30.2±7.4% respectively. The highest nasal lavage (sIgA) titers were observed in groups immunized with nanosphere formulations, while the IgG and antitoxin titers were suppressed by these formulations. CPG-ODN as an adjuvant could increase the serum IgG and antitoxin titers when co-administered with TT solution or co-encapsulated with TT in PLGA nanospheres, but failed to potentiate the 19 Atiters in nasal lavages. No hemolysis was occurred on incubation of PLGA nanospheres and human (RBCs). Also after nasal administration of plain nanospheres to human volunteers, no local irritation was seen. Intranasal administration of nanospheres encapsulated with vaccines showed to be an effective way for inducing mucosal sIgA immune responses, and CPG-ODN could increase the systemic immune responses.

Background and objectives: Augmentation of Th2 mediated responses such as elevation of serum IgE is the characteristic feature in atopy and asthma. CPG oligonucleotides (CPG ODN) have been used during the recent two decades as the potent immunomodulatory agents in allergic conditions. In this paper, we aim to report the anti-inflammatory effects of CPG ODN in murine model of asthma.Methods: In this study, BALB/c mice were sensitized with ch.a. allergen and then treated intranasally with CPG ODN. Following the challenge with allergen inhalation, mice were sacrificed and their splenocytes were cultured in the presence of antigen. After three days of culture, supernatants were examined for IFN- g levels by ELISA method, as an indication of Th1 response, and the results were compared with those in control mice without CPG therapy.Results: The results indicate that the mice in the test group, which received CPG ODN, showed higher levels of systemic INF- g, and lower levels of serum IgE compared with either the antigen or CPG negative ODN-treated groups.Conclusion: Based on the results of this study, which shows higher INF-g levels in mice received CPG ODN, it can be concluded that CPG motifs have immune response modulation potential apparently through the deviation of Th2 into Th1 responses, which lead to a decrease in IgE antibody level. Accordingly, we suggest that these components can have therapeutic effects in the asthma caused by ch.a. allergen.

Objective(s): Several antigens, adjuvants and delivery systems have been evaluated for induction of protective immune responses against Leishmaniasis, but most of them have been inefficient. In this study, PLGA nanospheres as antigen delivery system CPG-ODN as an immunoadjuvant for increasing the immune responses against Autoclaved Leishmania major (ALM) were prepared and characterized.Materials and Methods: PLGA nanospheres prepared by a double-emulsion (W/O/W) technique. The internal aqueous phase contained ALM and CPG-ODN, while the oily phase contained the solution of PLGA in dichloromethan and the external aqueous phase was PVA 7.5% (WIV) solution. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. The encapsulation efficiency was determined by Lowry method for ALM and UV spectroscopy at 260 nm for CPG-ODN. The release profiles of antigen and CPG-ODN from nanospheres evaluated for one week.Results: Nanospheres were spherical in shape, having smooth surfaces. Mean diameters for blank and ALM + CPGODN loaded nanospheres recorded as 302±129 and 333±128 nm respectively. Also, the encapsulation efficiencies of ALM and CPG-ODN were 71.6±8.8 and 49.1±2.4%, respectively. Evaluation of the release profiles of ALM and CPG-ODN from nanospheres showed that 44.8±0.8% of ALM and 29.5±0.2% of CPGODN released from nanospheres in one week.Conclusion: The prepared nanospheres with desirable size, encapsulation efficiency, and slow rate of release, had acceptable features for future in vivo studies.

Oligodeoxynucleotides (ODN) having a CPG motif (CPG-ODN) are regarded as a new class of potent immunoadjuvants. It has been shown that concomitant delivery of an antigen with CPG-ODN to the same APC will be able to increase the immune responses against the relevant antigen. The main objective of the present study was co-encapsulation of tetanus toxoid (TT) and CPG-ODN in PLGA microspheres and evaluation of immune responses gained by the concomitant delivery of TT and CPG-ODN via the same delivery system. Both TT alone and [TT+CPG-ODN] were encapsulated in PLGA microspheres. The following groups of mice were immunized twice 2 Lf TT in the presence or absence of 4µg CPG-ODN via subcutaneous route: 1) encapsulated [TT + CPG-ODN]; 2) encapsulated TT alone; 3) encapsulated TT with CPG-ODN solution; 4) solution of [TT+ CPG-ODN]; 5) alum adsorbed TT (routine vaccine). Mice immunized with microspheres encapsulated with [TT+CPG-ODN] showed a very high T cell proliferation (stimulation index=45 in T cell proliferation assay and very high levels of INF-γ (2694.7±41.1 pg/ml) which was significantly higher than those of other groups. Anti-sera obtained from this group also showed very high total IgG titers (end point titers 2560000) which was 16 times that of the group immunized with the solution of [TT+CPG-ODN]. Also the titers of IgGl, IgG3 and IgG2b obtained from group 1 were considerably higher than those from other groups. Interestingly however the IgG titers induced in group 5 were far less than that in group 1 (total IgG, end point titers 160000). This study clearly showed that [TT+CPG-ODN] encapsulated in PLGA microspheres was more efficient than the relevant solution in terms of inducing immune responses. Also the results indicated that the co-delivery of TT and CPG-ODN in PLGA microspheres elicits a stronger Th1 (i.e. IgG2b, IgG3, INF- γ ) as well as Th2 (i.e. IgG1) type responses.        

Background: This study aims to prepare high stability chitosan nanoparticles (CNP) and examine the ability of CNP in CPG-ODN delivery when treating allergic mice model. Methods: Preparation and characterization of CNP were performed by ionic gelation, dynamic light scattering, and zeta sizer. The CNP cytotoxicity and activation ability of CPG ODN delivered with CNP were tested using a cell counting kit-8 and Quanti blue method. Allergic mice were injected intraperitoneal with 10 ug ovalbumin on day 0 and 7, and then treated with intranasal CPG ODN/CPG ODN, delivered with CNP/CNP, on the third week three times per week for three weeks. The ELISA method measured cytokine and IgE profiles in the allergic mice’, s plasma and spleen. Results: CNP results have sizes 27. 73 nm±, 3. 67 dan 188. 23 nm±, 53. 47, spherical in shape and nontoxic, and did not alter the NF-κ, B activation of CPG ODN in RAW-blue cells. The application of CPG ODN delivered by chitosan nanoparticles shows no statistical difference between groups of IFN-γ, , IL-10, and IL-13 in Balb/c mice’, s plasma and spleen, in contrast with IgE level. Conclusions: The results showed that using chitosan nanoparticles as a delivery system for CPG ODN has the potency to safely CPG ODN efficacy.

Objective: The goal of this study was to prepare and characterize alginate microspheres as an antigen delivery system and adjuvant for immunization against leishmaniasis. Materials and Methods: Microspheres were prepared by an emulsification technique and characterized for size, encapsulation efficiency, and release profile of encapsulates. Selection of appropriate parameters (viscosity of alginate, emulsifier, and sonication times) enabled the preparation of alginate microspheres with a mean diameter of 1.8±1.0mm, as determined by Scanning Electron Microscopy and Particle Size Analyzer. Results: The encapsulation efficiency was about 34.2±6.7% for autoclaved leishmania major and 63.5±6.9% for CPG-ODN, as determined by spectrophotometric assays. In vitro release profile showed a slow release rate for encapsulated ALM, while higher release rate was observed for CPG-ODN. The molecular weight was evaluated by SDS-PAGE and showed that the process of encapsulation did not affect the molecular weight of the entrapped antigen. Conclusion: With regard to the optimum diameter (less than 5 mm), slow release rate and preservation of antigen molecules, alginate microspheres could be considered as a promising antigen delivery system for ALM.

Background: Historically, leishmanization is the most effective protective measure against Cutaneous Leishmaniasis (CL), CL lesion induced by leishmanization sometimes takes a long time to heal. Manipulation of leishmanization inoculums needed to induce a mild and acceptable CL lesion. The aim of this study was to explore if liposomal form of CPG ODN (Cytosin phosphate Guanin Oligodeoxynucleotides) mixed with Leishmania major would induce a milder lesion size in Balb/c mice.Methods: This study was performed in Biotechnology Research Center, Mashhad, and Center for Research and Training in Skin Diseases and Leprosy, Tehran, Iran during 2008-2009. Different groups of BALB/c mice were subcutaneously (SC) inoculated with L. major mixed with liposomal form of CPG ODN, or L. major plus free CPG ODN, or L. major mixed with empty liposomes or L. major in PBS. The lesion onset and the size of lesion were recorded; the death rate was also monitored.Result: Footpad thickness was significantly (P<0.01) smaller, death rate was also significantly (P<0.05) lower in the mice received L. major mixed with liposomal CPG ODN or free CPG ODN than control groups received L. major in PBS or L. major plus liposomes, also mice which received L. major mixed with CPG ODN in soluble form showed a significantly (P<0.001) smaller lesion size than control groups.Conclusion: CPG ODN seems to be an appropriate immunopotentiator mixed with Leishmania stabilate in leishmanization.

Objective(s): Program death 1 (PD-1)/ program death-ligand 1 (PD-L1) pathways, as the main inhibitory checkpoints, induce immunosuppression in the tumor microenvironment (TME). Despite the importance of inhibitor checkpoint receptor (ICR) blockers, their outcomes have been limited by the low immune response rate and induced acquired resistance. Pre-existing tumor-specific T cells is related to the improvement of their therapeutic efficacy. In the present study, we show that the combination of liposomal gp100 nanovaccine with anti PD-1 monoclonal antibody (mAb) potentiates the therapeutic effect in the melanoma model. Materials and Methods: In this study, we first decorate the cationic liposome with gp10025-33 selfantigen and then characterize it. Mice bearing B16F10 melanoma tumors were vaccinated with different formulations of gp100 peptide (free or liposomal form) with or without CPG ODN adjuvant in combination with anti PD-1 mAb. Results: Therapeutic combination of liposomal nanovaccine and CPG with anti PD-1 mAb, demonstrated the increased number of tumor infiltrated lymphocytes (TILs) in TME with the highest IFN-γ production and cytotoxic activity, which led to remarkable tumor regression. Conclusion: Our results demonstrated the synergism between Lip-peptide+CPG nanovaccine and anti PD-1 regime, which improved the therapeutic efficacy of PD-1 checkpoint blocker in melanoma mice models.

ENGLISH: Un-methylated cytosine-phosphate-guanosine oligodeoxynucleotides (CPG-ODN) has been considered as a powerful vaccine adjuvant and recognition of CPG-ODN by chicken leukocytes promotes their ability to fight against infections. In our study, efficacy of different routes of CPG-ODN application as an adjuvant on immune responses (antibody titer together with leukogram) following vaccination against Newcastle disease (ND) has been evaluated in broiler chickens (Ross-308). The results indicated that routes of CPG-ODN administration influence immune responses and comparison effectiveness of CPG-OND delivery routes showed that group vaccinated by eye-drop application had the highest antibody titer than that of the group injected intramuscularly (im) and the difference was significant (p = 0. 04) on day 35 of age. Antibody titer of the group treated with Clone 30 plus CPG-ODN via eye-drop route was higher than that of the group vaccinated with clone 30 alone on days 28 and 35 of age and the difference was significant (p = 0. 04). Co-administration of both vaccine and CPG improved outcome of leukogram of the chickens on days 21 to 42 of age and among the treated groups, WBC of the group received both vaccine and CPG by eye-drop route significantly (p < 0. 05) differed from that of the group vaccinated with clone 30 alone on days 28 and 35 but not on day 42 of age. Average final body weight of the control group did not significantly differ from those of the treated groups at end of the experiment. In conclusion, co-administration of ND vaccine plus CPG-ODN via eye-drop route improves immune responses. FRENCH: Ré sumé : Les oligodé soxynuclé otides non mé thylé s de cytosine-phosphate-guanosine (ODN-CPG) ont é té considé ré s comme un puissant adjuvant vaccinal et la reconnaissance de l'ODN-CPG par les leucocytes du poulet favorise leur capacité à lutter contre les infections. Dans notre é tude, l'efficacité des diffé rentes voies d'application de l'ODN-CPG en tant qu'adjuvant des ré ponses immunitaires (é valuation du titre d'anticorps ainsi que du leucogramme) à la suite de la vaccination contre la maladie de Newcastle (ND) a é té é tudié e chez des poulets de chair (Ross-308). Les ré sultats montraient que les voies d'administration de l'ODN-CPG influent sur les ré ponses immunitaires. Le groupe vacciné par l'application de gouttes oculaires avait un titre d’ anticorps plus é levé que celui traité pat injection intramusculaire (im) et la diffé rence é tait significative (p = 0, 04) au trentecinquiè me jour. Le titre en anticorps du groupe traité par les gouttes oculaires comprenant le clone 30 combiné à l'ODN-CPG é tait supé rieur aux jours 28 et 35 à celui du groupe vacciné uniquement avec le clone 30 (p = 0, 04). La co-administration du vaccin et du CPG a amé lioré les ré sultats du leucogramme des poulets du 21è me au 42è me jour. Parmi les groupes traité s, le leucogramme du groupe ayant reç u à la fois le vaccin et le CPG par voie oculaire montrait des diffé rences significatives (p <0, 05) de celui du groupe vacciné uniquement avec le clone 30 aux 28è me et 35è me jours, mais au 42è me jour. Le poids corporel final moyen du groupe té moin ne diffé rait pas significativement de celui des groupes traité s à la fin de l'expé rience. En conclusion, la co-administration du vaccin ND et de l'ODN-CPG amé liore la ré ponse immunitaire aprè s vaccination.