Enhancement of the immunogenicity of a Mycobacterium tuberculosis fusion protein using ISCOMATRIX and PLUSCOM nano-adjuvants as prophylactic vaccine after nasal administration in mice.

Objectives: Tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new TB vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through intranasal administration in a mice model. Materials and Methods: In the present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Three weeks after the final vaccination, IFN-γ, IL-4. IL-17, and TGF-ß concentrations in the supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined. Results: According to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induce IFN-γ and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens. Conclusion: The results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM, and MPLA after nasal administration in enhancing the immune response against M. tuberculosis antigens. Both nanoparticles were good adjuvants in order to promote the immunogenicity of TB-fused antigens. So, nasal immunization with these formulations, could induce immune responses and be considered a new TB vaccine or a BCG booster.

Microfluidic-assisted preparation of PLGA nanoparticles loaded with insulin: a comparison with double emulsion solvent evaporation method.

Poly lactic-co-glycolic acid (PLGA) is an ideal polymer for the delivery of small and macromolecule drugs. Conventional preparation methods of PLGA nanoparticles (NPs) result in poor control over NPs properties. In this research, a microfluidic mixer was designed to produce insulin-loaded PLGA NPs with tuned properties. Importantly; aggregation of the NPs through the mixer was diminished due to the coaxial mixing of the precursors. The micromixer allowed for the production of NPs with small size and narrow size distribution compared to the double emulsion solvent evaporation (DESE) method. Furthermore, encapsulation efficiency and loading capacity indicated a significant increase in optimized NPs produced through the microfluidic method in comparison to DESE method. NPs prepared by the microfluidic method were able to achieve a more reduction of trans-epithelial electrical resistance values in the Caco-2 cells compared to those developed by the DESE technique that leads to greater paracellular permeation. Compatibility and interaction between components were evaluated by differential scanning calorimetry and fourier transform infrared analysis. Also, the effect of NPs on cell toxicity was investigated using MTT test. Numerical simulations were conducted to analyze the effect of mixing patterns on the properties of the NPs. It was revealed that by decreasing flow rate ratio, i.e. flow rate of the organic phase to the flow rate of the aqueous phase, mixing of the two streams increases. As an alternative to the DESE method, high flexibility in modulating hydrodynamic conditions of the microfluidic mixer allowed for nanoassembly of NPs with superior insulin encapsulation at smaller particle sizes.

Mucosal and systemic immunization against tuberculosis by ISCOMATRIX nano adjuvant co-administered with alginate coated chitosan nanoparticles.

Objectives: BCG vaccine has no longer been appreciated to immunize against tuberculosis, worldwide, so novel appropriate adjuvants have been dedicated to improve immune responses. This study aimed to evaluate the immunomodulatory effects of ISCOMATRIX as an adjuvant to stimulate potent humoral and cellular immune responses of the PPE17 loaded alginate coated nanoparticles through subcutaneous and intranasal vaccination. Materials and Methods: Size, polydispersity index, and morphology of the resulting colloidal particles were explored by dynamic light scattering (DLS). The cellular and/or humoral immune stimulation properties of ISCOMATRIX adjuvant were measured by measuring the level of IFNγ, IL-4, IL-17, and TGFß in spleen cell cultures and IgG1 and IgG2a in serum and sIgA in nasal lavage of immunized mice, respectively. Results: The spherical cage-like particles of ISCOMATRIX adjuvant have optimal size of 59±6 nm appropriate for an immune adjuvant vaccine. ISCOMATRIX induced robust Th1 (IFN-γ) and IL-17 cytokine response also significant IgG2a and IgG1antibodies in both subcutaneous and intranasal routes and elicited mucosal sIgA response when administered intranasally. As a booster for BCG, ISCOMATRIX induced immune responses only in subcutaneous route. Conclusion: These findings indicate that ISCOMATRIX is a promising adjuvant with the potential for increasing cellular and humoral immunity both after subcutaneous and intranasal administration.

Preparation and Evaluation of the In situ Gel-forming Chitosan Hydrogels for Nasal Delivery of Morphine in a Single Unit dose in Rats to Enhance the Analgesic Responses.

INTRODUCTION: In this study, an in situ gel-forming chitosan hydrogel was prepared with the use of glutamate salt of chitosan (Ch-Ga), ß-glycerophosphate (Gp), and morphine (Mor). The paper is focused on in vitro physicochemical properties and in-vivo analgesic effects of the prepared chitosan hydrogel. METHOD: The thermosensitive properties of prepared chitosan hydrogel were evaluated during the different temperatures and times. The physicochemical properties of chitosan hydrogel were investigated by infrared (IR) spectroscopy and X-ray diffraction analysis (XRD). Also, its cell cytotoxicity effects were evaluated in murine NIH/3T3 normal cells. Subsequently, the distribution of chitosan hydrogel in the nasal cavity of rats and its analgesic responses were evaluated. The prepared chitosan hydrogel showed that it could be gelled at the temperature of 34 °C before leaving the nose in the shortest possible time of 30 s. RESULT: The analgesic responses of the intranasal (IN) injection of chitosan hydrogel (IN-chitosan hydrogel, 10 mg Mor/kg) in a single unit dose in rat relative to the placebo and intranasal or intraperitoneal (IP) injection of free morphine solution (IN-Free Mor or IP-Free Mor, 10 mg Mor/kg) via the hot plate test, reveal that the IN-chitosan hydrogel could induce fast analgesic effects of morphine with maximum possible effect (MPE) of 93% after 5 min compare to the IN-Free Mor and IP-Free Mor with MPE of 80% after 15 min and 66% after 30 min, respectively. Also, prolonged analgesic effects with MPE of 78 % after 6 h of injection were only seen in the IN-chitosan hydrogel injected group. The obtained fluorescent images of rat's brain injected with IN-chitosan hydrogel containing doxorubicine (Dox) as a fluorescent agent showed that the mucosal adhesive and absorption enhancer properties of IN-chitosan hydrogel resulting in longer presence of them in the nasal cavity of rats followed by more absorption of Dox from the blood vessels of olfactory bulbs with a 74% color intensity compared to the IN-Free Mor and IN-Free Dox with 15%. CONCLUSION: These data reveal that the IN-chitosan hydrogel could induce fast and prolonged analgesic effects of morphine compare to the IN/IP-Free Mor, which could be considered as an in situ gel-forming thermosensitive chitosan hydrogel for nasal delivery of wide ranges of therapeutic agents.

A novel nanomicelle composed from PEGylated TB di-peptide could be successfully used as a BCG booster.

Objectives: Tuberculosis affects one-third of the world's population and leads to a high rate of morbidity and mortality. Bacillus Chalmette-Guerin (BCG) as the only approved vaccine for the Mycobacterium tuberculosis (Mtb) does not show enough protection in the vaccinated population. Materials and Methods: The main aim of this study was to prepare a self-assembled nanomicelle composed from a di-block polymer in which, a di-fusion peptide was the hydrophobic block and polyethylene glycol (PEG) was the hydrophilic block. The micelles were characterized in vitro and in vivo as an antigen delivery system/adjuvant both with and without a prime BCG. Results: The micellar nanovaccine was able to elicit good dendritic cell maturation. Nanomicelles could efficiently induce systemic cytokines as well as nasal secretory predominant antibody titers (sIgA). The expression pattern of cytokines indicated the superiority of cellular immunity. Nasal administration of two doses of nanomicelles after a prime subcutaneous administration of BCG induced the highest mucosal and systemic immune responses. Conclusion: Based on our results PEG-HspX/EsxS self-assembled nanomicelle is highly immunogenic and can be considered a potential vaccine candidate against Mtb to boost BCG efficiency.

Immunological Assessment of Chitosan or Trimethyl Chitosan-Coated PLGA Nanospheres Containing Fusion Antigen as the Novel Vaccine Candidates Against Tuberculosis.

The crucial challenge in tuberculosis (TB) as a chronic infectious disease is to present a novel vaccine candidate that improves current vaccination and provides efficient protection in individuals. The present study aimed to evaluate the immune efficacy of multi-subunit vaccines containing chitosan (CHT)- or trimethyl chitosan (TMC)-coated PLGA nanospheres to stimulate cell-mediated and mucosal responses against Mycobacterium Tuberculosis (Mtb) in an animal model. The surface-modified PLGA nanoparticles (NPs) containing tri-fusion protein from three Mtb antigens were produced by the double emulsion technique. The subcutaneously or nasally administered PLGA vaccines in the absence or presence of BCG were assessed to compare the levels of mucosal IgA, IgG1, and IgG2a production as well as secretion of IFN-γ, IL-17, IL-4, and TGF-ß cytokines. According to the release profile, the tri-fusion encapsulated in modified PLGA NPs demonstrated a biphasic release profile including initial burst release on the first day and sustained release within 18 days. All designed PLGA vaccines induced a shift of Th1/Th2 balance toward Th1-dominant response. Although immunized mice through subcutaneous injection elicited higher cell-mediated responses relative to the nasal vaccination, the intranasally administered groups stimulated robust mucosal IgA immunity. The modified PLGA NPs using TMC cationic polymer were more efficient to elevate Th1 and mucosal responses in comparison with the CHT-coated PLGA nanospheres. Our findings highlighted that the tri-fusion loaded in TMC-PLGA NPs may represent an efficient prophylactic vaccine and can be considered as a novel candidate against TB.

Development of curcumin-loaded Prunus armeniaca gum nanoparticles: Synthesis, characterization, control release behavior, and evaluation of anticancer and antimicrobial properties.

The present work was conducted to develop a new polysaccharide-based encapsulation system via electrostatic interactions between Prunus armeniaca gum exudates (PAGE) and Ca2+ ions to enhance the biological activity and bioavailability of curcumin. The effects of different levels of pH (6, 7, and 8) and ion concentrations (1, 3, and 5) on the particle diameter and surface charge of the samples were examined. The encapsulation efficiency in the PAGE-based nanoparticles was realized to be 86.1%, indicating the encapsulation technique applied in this study was effective to entrap most of the curcumin within the PAGE matrix. The nanoparticles showed a smooth surface with spherical shape. Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (X-ray) studies confirmed the formation of polyelectrolyte complexation. The cumulative release of curcumin in simulated gastrointestinal tract was less than 75%, revealing a gradual release trend. Both pure curcumin and curcumin-loaded nanoparticles were toxic to the cancer cell lines.

The impact of nanocarriers in the induction of antigen-specific immunotolerance in autoimmune diseases.

Immunotolerance induction in an antigen-specific manner is the long-term goal of immunotherapy to treat autoimmune diseases. Nanocarriers (NCs) can be designed as a new generation of delivery systems to modulate the immune responses through targeted delivery of antigens and immunomodulators to antigen presenting cells (APCs). In this manuscript, several formulation factors in the preparation of NCs which affect their uptake using APCs and generation of tolerance have been reviewed. The physicochemical properties and composition of NCs have been shown to play essential roles in achieving the desired immunological outcome. Also, targeting of dendritic cells and macrophages as APCs and direct targeting of the autoreactive lymphocytes have been presented as two main ways for induction of antigen-specific tolerance by these tolerogenic nanocarriers (tNCs). These particles herald a promising approach to treat or even prevent unwanted immune reactions in humans specifically.

T helper type 1 biased immune responses by PPE17 loaded core-shell alginate-chitosan nanoparticles after subcutaneous and intranasal administration.

PURPOSE: Tuberculosis, a cost and life threatening disease, was being subjected for improving vaccine strategies beyond BCG. Thus, a novel particulate delivery system using alginate-coated chitosan nanoparticles including PPE17 protein and CpG were administered through intranasal (IN) and subcutaneous (SC) routes. METHODS: The encapsulated nanoparticles were first characterized for size, surface charge, encapsulation efficiency and in vitro release of PPE17 antigen. The nanoparticles were then administered intranasal and subcutaneously to evaluate the induction of systemic and/or mucosal immune responses in mice. RESULTS: According to our result, the mean size of nanoparticles was measured about 427 nm, and exhibited a negative zeta potential of -37 mV. Following subcutaneous and intranasal administration, the results from cytokines assay showed that an increasing in the level of IFN-γ, and adversely a decrease in the level of IL-4 (presumptive Th1 biased immune response) was happened and also a notable elicitation in IL-17 cytokine was observed. CONCLUSION: In conclusion, our study demonstrated that alginate-coated chitosan nanoparticles showed to be an effective way to improve BCG efficiency as booster strategy for subcutaneous vaccine, and also can induce strong immune responses as prime strategy through intranasal vaccination.

pH-sensitive soluble soybean polysaccharide/SiO2 incorporated with curcumin for intelligent packaging applications.

In the present work, the effect of various concentrations of SiO2 nanoparticles (5, 10, and 15%) on physicochemical and antimicrobial properties of soluble soybean polysaccharide (SSPS)-based film was investigated. Then, the migration of SiO2 nanoparticles to ethanol as a food simulant was evaluated. Subsequently, curcumin was added to the nanocomposite formulation to sense the pH changes. Finally, the cytotoxicity of the developed packaging system was investigated. With increasing nanoparticle concentration, the film thickness, water solubility, and water vapor permeability decreased and mechanical performance of the films improved. SSPS/SiO2 nanocomposite did not show antibacterial activity. SEM analysis showed that SiO2 nanoparticles are uniformly distributed in the SSPS matrix; however, some outstanding spots can be observed in the matrix. A very homogeneous surface was observed for neat SSPS film with R a and R q values of 3.48 and 4.26, respectively. With the incorporation of SiO2 (15%) into SSPS film, R a and R q values increased to 5.67 and 5.98, respectively. Small amount of SiO2 nanoparticles was released in food simulant. The nanocomposite incorporated with curcumin showed good physical properties and antibacterial activity. A strong positive correlation was observed between TVBN content of shrimp and a* values of the films during storage time (Pearson's correlation = 0.985).

The dry powder formulation of mixed cross-linked dextran microspheres and tetanus toxoid-loaded trimethyl chitosan nanospheres as a potent adjuvant for nasal delivery system.

OBJECTIVES: The present study aimed to determine the immunoadjuvant efficacy of mixed cross-linked dextran microspheres (CDM) and tetanus toxoid (TT)-loaded trimethyl chitosan (TMC) nanospheres in dry powder form. MATERIALS AND METHODS: The TMC nanoparticles (NPs) containing TT were produced using the ionic gelation method. Co-administration of TT-loaded TMC NPs and CDM as an absorption enhancer was performed to improve immunity against the antigen. Dry powder formulations were delivered via the nasal route in a rabbit model. RESULTS: Among immunization groups, mixing of CDM with TT encapsulated in TMC NPs could elicit the highest titer of systemic IgG antibody. Furthermore, the addition of CDM to TT-loaded TMC enhanced the sIgA response relative to the TT solution. CONCLUSION: The TMC NPs had a considerable effect on mucosal and systemic immunity against the TT antigen. Therefore, the CDM excipient can be utilized for nasal immunization to elevate systemic and mucosal responses.

Immunogenicity of HspX/EsxS fusion protein of Mycobacterium tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants after subcutaneous administration in animal model.

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is one of the most common and dangerous infectious diseases in the world. Despite vaccination with BCG, it is still considered as a major health problem. Therefore, design and production of an effective novel vaccine against TB is necessary. Our aim was to evaluate immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX, PLUSCOM nano-adjuvants and MPLA through the subcutaneous route in mice model. METHODS: HspX/EsxS fused protein of M. tuberculosis was cloned, expressed and purified in the prokaryotic system. ISCOMATRIX and PLUSCOM nano-adjuvants were prepared by film hydration method. Subcutaneous immunization of BALB/c mice was performed by different formulations. IFN-γ, IL-4, IL-17 and TGF-ß cytokines levels as well as serum IgG1, IgG2a. RESULTS: Our results showed that subcutaneous administration of mice with HspX/EsxS along with three adjuvants, ISCOMATRIX, PLUSCOM and MPLA increased immunogenicity of multi-stage fusion protein of M. tuberculosis. Additionally, HspX/EsxS protein + ISCOMATRIX or + PLUSCOM nano-adjuvants induced stronger Th1, IgG2a and IgG1 immune responses compared to MPLA adjuvant. Totally, HspX/EsxS/ISCOMATRIX/MPLA, HspX/EsxS/PLUSCOM/MPLA and two BCG booster groups could significantly induce higher Th1 and IgG2a immune responses. CONCLUSION: With regard to ability of ISCOMATRIX, PLUSCOM and MPLA adjuvants to increase immunogenicity of HspX/EsxS protein through induction of IFN-γ and IgG2a immune responses, it seems that these adjuvants and especially ISCOMATRIX and PLUSCOM, could also improve BCG efficacy as a BCG booster.

Prunus armeniaca gum exudates: An overview on purification, structure, physicochemical properties, and applications.

Prunus armeniaca gum exudate (PAGE) is obtained from the trunk branches of apricot trees. PAGE is a high-molecular-weight polysaccharide with arabinogalactan structure. The physicochemical and rheological characteristics of this gum have been investigated in various researches. PAGE offers a good potential for use as an emulsifying, binding, and stabilizing agent in food and pharmaceutical industries. It also can be used as an organic additive in tissue culture media, synthesizing of metallic nanoparticles, binding potential in tablets, antioxidant agent, and corrosion inhibitor. For desirable emulsifying, stabilizing, shelf life-enhancing properties, and antioxidant activity of PAGE, it can be used as additive in many foods. We present here a comprehensive review on the existing literatures on characterization of this source of polysaccharide to explore its potential applications in various systems.

Helicobacter pylori infection and autoimmune diseases; Is there an association with systemic lupus erythematosus, rheumatoid arthritis, autoimmune atrophy gastritis and autoimmune pancreatitis? A systematic review and meta-analysis study.

Autoimmune diseases are considered as one of the most important disorders of the immune system, in which the prolonged and chronic processes eliminate self-tolerance to the auto-antigens. The prevalence of autoimmune diseases has been increasing worldwide in the recent years. According to the literature, biological processes such as the host genome, epigenetic events, environmental condition, drug consumption, and infectious agents are the most important risk factors that make the host susceptible to the development of autoimmune diseases. In the recent years, the role of Helicobacter pylori in the induction of autoimmune diseases has attracted extensive attention. Via molecular mimicry, epitope spreading, bystander activation, polyclonal activation, dysregulation in immune response, and highly immune-dominant virulence, such as cagA, H. pylori causes tissue damage, polarity, and proliferation of the host cells leading to the modulation of host immune responses. Moreover, given the large population worldwide infected with H. pylori, it seems likely that the bacterium may develop into autoimmune diseases through dysregulation of the immune response. The frequency and relationship between H. pylori infection and systemic lupus erythematosus, rheumatoid arthritis, autoimmune atrophy gastritis, and autoimmune pancreatitis were evaluated using the data from 43 studies involving 5052 patients. According to statistical analysis it is probable that infection with more virulent strains of H. pylori (such as H. pylori cagA positive) can increase the risk of autoimmune diseases. In addition, it was shown that infection with H. pylori can prevent the development of atrophic gastritis by stimulating inflammation in the gastric antrum. However, future studies should confirm the validity of this study.

A novel formulation of Mtb72F DNA vaccine for immunization against tuberculosis.

OBJECTIVES: Mycobacterium tuberculosis (M. tuberculosis), an intracellular pathogen, causes 1.5 million deaths globally. Bacilli Calmette-Guérin (BCG) is commonly administered to protect people against M. tuberculosis infection; however, there are some obstacles with this first-generation vaccine. DNA vaccines, the third generation vaccines, can induce cellular immune responses for tuberculosis (TB) protection. In this study, optimized DNA vaccine (pcDNA3.1-Mtb72F) entrapped in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) was used to achieve higher immunogenicity. MATERIALS AND METHODS: Plasmid Mtb72F was formulated in PLGA NPs using double emulsion method in the presence of TB10.4 and/or CpG as an adjuvant. Female BALB/c mice were immunized either with NP-encapsulated Mtb72F or naked Mtb72F with or without each adjuvant, using the BCG-prime DNA boost regimen. RESULTS: These NPs were approximately 250 nm in diameter and the nucleic acid and protein encapsulation efficiency were 80% and 25%, respectively. The NPs smaller than 200 nm are able to promote cellular rather than humoral responses. The immunization with the formulation consisting of Mtb72F DNA vaccine and TB10.4 entrapped in PLGA NPs showed significant immunogenicity and induced predominantly interferon-É£ (IFN-É£) production and higher INF-É£/interleukin-4 (IL-4) ratio in the cultured spleen cells supernatant. CONCLUSION: PLGA NPs loaded with Mtb72F DNA-based vaccine with TB10.4 could be considered as a promising candidate for vaccination against TB. These results represent an excellent initial step toward development of novel vaccine for TB protection.

Folate targeted PEGylated liposomes for the oral delivery of insulin: In vitro and in vivo studies.

In this study using phospholipids with high transition temperature and taking advantage of PEGylation, we designed liposomal formulation targeted with folic acid (FA) to improve the stability of liposomes with high penetration efficiency at the same time. The results of characterization demonstrated that liposomal formulations are in range of 150-210 nm size with negative surface charge. The results of cell uptake indicated that FA conjugation resulted in the more uptake of insulin. However, the results of transepithelial electrical resistance (TEER) showed no statistical differences among the formulations. The results of biodistribution also demonstrated that PEGylated liposome targeted with FA had more residence time in stomach and intestine along with higher amounts in blood and liver. The anti-diabetic effects of formulation in vivo indicated the efficacy of PEGylated liposome targeted with FA had promising results in decreasing blood glucose and increasing insulin levels. The results of this study indicated that using phospholipids with high Tm along with PEGylation and using targeting ligand could improve efficiency of oral delivery of liposomes which merit further investigation.

In-vitro Release Evaluation of Growth Hormone from an Injectable In-Situ Forming Gel Using PCL-PEG-PCL Thermosensitive Triblock.

OBJECTIVE: An injectable long acting In-Situ Forming Gel (ISFG) of human Growth Hormone (hGH) was prepared by using triblock PCL-PEG-PCL (Mw 1500-1500-1500). Ring-Opening Polymerization (ROP) of triblock using microwave was applied. METHODS: The BCA protein assay Kit was used to determine the concentration of hGH in the in-vitro release medium. Finally, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) tests and Circular Dichroism (CD) spectrum were done to approve the stability of released hGH. The result of ROP demonstrated that the proportion of PCL to PEG accorded with the initial molar ratio of the monomers. The cross-section of the Surface Electron Microscopy (SEM) indicated the porous framework of the hydrogel could load the drug into its tridimensional matrixes structure. There is the low initial burst release of hGH from the supramolecular hydrogel. RESULTS: The maximum in-vitro release of hGH was 71.2 % ± 1.5 that were due to hGH degrading after this time (21 days). The CD spectrum and SDS-PAGE results confirmed the stability of hGH during invitro release evaluation. CONCLUSION: The results suggest that the sustained-release formulation using PCL-PEG-PCL can be applied to control the release of hGH.

Separation of the Epitopes in a Multi-Epitope Chimera: Helical or Flexible Linkers.

BACKGROUND: The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera. OBJECTIVE: The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1). METHODS: For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures. RESULTS: Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study. CONCLUSION: This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.

The roles of latency-associated antigens in tuberculosis vaccines.

Tuberculosis (TB) is a re-emerging disease and is caused by Mycobacterium tuberculosis (M. tuberculosis). TB is currently one of the leading causes of morbidity and mortality, worldwide. The only available vaccine against TB infection, Bacillus Calmette-Guérin (BCG), fails to adequately protect against reactivation of latent infections in adults. Furthermore, recently developed subunit vaccines, which are in various stages of clinical trials, are all prophylactic vaccines based on proteins expressed in replicating stage of M. tuberculosis and they are not preventive of reactivation of latent TB infection. Thus, an appropriate subunit post-exposure vaccine needs to be developed to control all forms of TB infection. To produce a multi-stage subunit vaccine, scientists should combine the early secreted M. tuberculosis antigens with latency antigens. For this purpose, some latency proteins are known which could be important antigens in the production of specific humoral and cellular immune responses in latent M. tuberculosis infected individuals. Several studies have evaluated the immunogenicity of these proteins in improving the TB vaccines. The present study is a comprehensive review of several studies on the role of the latency antigens in the development of TB vaccines. Overall, the studies indicate that the latency-associated antigens including the resuscitation-promoting factors, the Dormancy of survival regulon (DosR) proteins and the starvation stimulant proteins are potential candidates for the development of subunit vaccines against TB infection.

Enhancing immunogenicity of novel multistage subunit vaccine of Mycobacterium tuberculosis using PLGA:DDA hybrid nanoparticles and MPLA: Subcutaneous administration.

OBJECTIVES: A new strategy in recent studies is using effective tuberculosis (TB) subunit vaccines combined with appropriate carriers and adjuvants which have shown promising results in preclinical and clinical studies. The aim of the present study was to evaluate the PLGA:DDA hybrid nanoparticles (NPs) for subcutaneous delivery of a novel multistage subunit vaccine along with MPLA adjuvant against Mycobacterium tuberculosis (M. tuberculosis). MATERIALS AND METHODS: PLGA and PLGA:DDA NPs containing HspX/EsxS fusion protein and MPLA were prepared by double emulsion method (w/o/w). After characterization, these NPs were subcutaneously administered to BALB/c mice aged 6-8 weeks old. Immunogenicity of formulations were assessed by measuring the level of IFN-γ, IL-4, IL-17 and TGF-ß cytokines as well as IgG1, IgG2a and IgA antibodies using ELISA. RESULTS: Both particles had spherical shape and smooth surface with 316.7±12.7 nm in size, surface charge of -33±1.7 mV, and encapsulation efficiency of 92.2±2% for PLGA NPs and 249.7±16.7 nm in size, surface charge of 39±1.8 mV, and encapsulation efficiency of 35.7±1.4% for PLGA:DDA NPs. The highest IFN-γ response and also IgG2a and IgG1 antibodies titers were observed in groups immunized with PLGA:DDA/HspX/EsxS/MPLA and PLGA:DDA/HspX/EsxS/MPLA as booster as well as PLGA:DDA/HspX/EsxS and PLGA:DDA/HspX/EsxS as booster. CONCLUSION: With regard to effective induction of IFN-γ and IgG2a immune responses, PLGA:DDA hybrid NP along with MPLA adjuvant have good potentials for improving the immunogenicity of HspX/EsxS multistage subunit vaccine as well as promoting BCG efficacy as a BCG prime-boost.