In spite of the progress of conventional vaccines, improvements are required due to concerns about the low immunogenicity of the toxicity, instability, and the need for multiple administrations of the vaccines. To overcome the mentioned problems, nanotechnology has recently been incorporated into vaccine development. Nanotechnology increasingly plays an important role in vaccine development nanocarrier-based delivery systems that offer an opportunity to increase the cellular and humoral immune responses. The use of nanoparticles in vaccine formulations allows not only enhanced immunogenicity and stability of antigen, but also targeted delivery and slow release. Over the past decade, nanoscale size materials such as virus-like particles, liposomes, ISCOMs, polymeric, inorganic nanoparticles and emulsions have gained attention as potential delivery vehicles for vaccine antigens, which can both stabilize vaccine antigens and act as adjuvants. This advantage is attributable to the nanoscale particle size, which facilitates uptake by Antigen- Presenting Cells (APCs), then leading to efficient antigen recognition and presentation. Modifying the surfaces of nanoparticles with different targeting moieties permits the delivery of antigens to specific receptors on the cell surface, thereby stimulating selective and specific immune responses. This review provides an overview of recent advances in nanovaccinology.

Hydrophilic nanoparticles have been widely investigated in recent years as delivery systems for therapeutic macromolecules such as antigens. In the present study Mesobuthus eupeus venom-loaded chitosan nanoparticles were prepared via ionic gelation of tripolyphosphate (TPP) and chitosan. The optimum encapsulation efficiency (91.1%) and loading capacity (76.3%) were obtained by a chitosan concentration of 2 mg/mL, chitosan-to-TPP mass ratio of 2 and M. eupeus venom concentration of 500 μg/mL. The average nanoparticle size at optimum conditions was determined by Zetasizer (Malvern Instruments, UK). The nanoparticle size was about 370 nm (polydispersity index: 0.429) while the zeta potential was positive. Transmission electron microscope (TEM) imaging showed a spherical, smooth and almost homogenous structure for nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles. The in vitro release of nanoparticles showed an initial burst release of approximately 60% in the first ten hours, followed by a slow and much reduced additional release for about 60 hours. It is suggested that the chitosan nanoparticles fabricated in our study may provide a suitable alternative to traditional adjuvant systems.

During last decades, diphtheria has remained as a serious disease that still outbreaks and can occur worldwide. Recently, new vaccine delivery systems have been developed by using the biodegradable and biocompatible polymers such as alginate. Alginate nanoparticles as a carrier with adjuvant and prolong release properties that enhance the immunogenicity of vaccines. In this study diphtheria toxoid loaded nanoparticles were prepared by ionic gelation technique and characterized with respect to size, zeta potential, morphology, encapsulation efficiency, release profile, and immunogenicity. Appropriate parameters (calcium chloride and sodium alginate concentration, homogenization rate and homogenization time) redounded to the formation of suitable nanoparticles with a mean diameter of 70±0.5 nm. The loading studies of the nanoparticles resulted in high loading capacities (>90%) and subsequent release studies showed prolong profile. The stability and antigenicity of toxoid were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ouchterlony test and proved that the encapsulation process did not affect the antigenic integrity and activity. Guinea pigs immunized with the diphtheria toxoid-loaded alginate nanoparticles showed highest humoral immune response than conventional vaccine. It is concluded that, with regard to the desirable properties of nanoparticles and high immunogenicity, alginate nanoparticles could be considered as a new promising vaccine delivery and adjuvant system.

Oral vaccination is the preferred route of immunization. However, the degradative condition of the gastrointestinal tract and the higher molecular size of peptides pose major challenges in developing an effective oral vaccination system. One of the most excellent methods used in the development of oral vaccine delivery system relies on the entrapment of the antigen in polymeric nanoparticles. In this work, trimethyl chitosan (TMC) nanoparticles were fabricated using ionic gelation teqnique by interaction hydroxypropyl methylcellulose phthalate (HPMCP), a pHsensitive polymer, with TMC and the utility of the particles in the oral delivery of hepatitis B surface antigen (HBsAg) was evaluated employing solutions that simulated gastric and intestinal conditions. The particle size, morphology, zeta potential, loading capacity, loading efficiency, in vitro release behavior, structure, and morphology of nanoparticles were evaluated, and the activity of the loaded antigen was assessed. Size of the optimized TMC/HPMCP nanoparticles and that of the antigen-loaded nanoparticles were 85 nm and 158 nm, respectively. Optimum loading capacity (76.75%) and loading efficiency (86.29%) were achieved at 300 mg/mL concentration of the antigen. SEM images revealed a spherical shape as well as a smooth and near-homogenous surface of nanoparticles. Results of the in vitro release studies showed that formulation with HPMCP improved the acid stability of the TMC nanoparticles as well as their capability to preserve the loaded HBsAg from gastric destruction. The antigen showed good activity both before and after loading. The results suggest that TMC/HPMCP nanoparticles could be used in the oral delivery of HBsAg vaccine.

In this study chitosan nanoparticles (CS NPs) and mannosylated chitosan nanoparticles (MCH NPs) loaded with recombinant hepatitis B surface antigen (rHBsAg) was synthesized as a vaccine delivery system and assessed toxically and immunologically. The physicochemical properties of the nanoparticles (NPs) were determined by methods including scanning electron microscope (SEM) and dynamic light scattering (DLS). The morphology of the NPs was semi spherical and the average diameter of the loaded CS and MCH NPs was found to be 189 and 239 nm, respectively. The release studies showed that after the initial burst, both of the loaded NPs provided a continuous and slow release of the antigens. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed concentration and time dependent toxicity profile for both formulations, but rHBsAg loaded CS nanoparticle showed higher toxicity due to smaller particle size and larger zeta potential. Abnormal toxicity test (ATT) results showed no signs of toxicity in mice and guinea-pigs treated with loaded MCHNPs. Stability test for six months showed acceptable changes in size, surface charge, and antigenicity for loaded MCH nanoparticles. Finally, in vivo immunogenicity study revealed greater adjuvant capability of MCH nanoparticles than others formulations. Our results showed MCH NPs can be used as a controlled and targeted vaccine delivery system.

Chicken egg yolk antibodies against Vipera lebetina venom were evaluated for their antivenom potential. White leghorn hens were immunized with detoxified V. lebetina venom (g-irradiated venom). The detoxified venom (200 mg) was mixed with an equal volume of complete Freund's adjuvant and was injected intramuscularly into the hens. The antibodies showed high activity (1.6 LD 50 /mL) in egg yolks after 12 d of venom injection. The eggs were collected after 12 days, and the egg yolks were removed and washed with purified water to remove any contamination with egg whites. The purification was performed using a method described by Maya Devi et al., followed by gel filtration (Sephadex G-50). The purity and molecular weight of antivenom antibodies (IgY) were determined using electrophoresis, and the molecular weight was found to be approximately 185 kDa. The potency of IgY was 6 LD 50 /mL (mice), i.e., 1 mL of IgY could neutralize 43.8 mg of standard V. lebetina venom). Our results showed that chicken egg yolk antibodies were effective in neutralizing the lethality and several pharmacological effects of V. lebetina venom and could be used for developing effective antivenom.

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There is no doubt about the whole cell pertussis vaccine efficacy, but it is necessary to improve the vaccine quality specially to decrease its toxicity by obtaining good immunogenicity with low bacterial content. In this work, under optimum condition inactivated B. pertussis bacteria cells entrapped with alginate microparticles were fabricated and in vivo immunogenicity and ptency of new microparticle based vaccine were evaluated in mice. Microspheres loaded with inactive B. pertussis bacterium cells were prepared via an emulsification method and analyzed for morphology, size, polydispersity index, loading efficiency, loading capacity, release profile and in vivo potency. The inactivated bacterial suspension mixture prepared in this work was nontoxic and showed potent ED50 (1:333 of human dose) and preserved agglutinins 1, 2, 3. The optimum conditions for the preparation of microparticles were achieved at alginate concentration 3.8% (w/v), CaCl2 8% (w/v), PLL 0.1% (w/v), lipophilic surfactant 0.22 (%w/v), hydrophilic surfactant 3.6 (%w/v), cross linking time 3min, homogenization rate 600 rpm, and alginate to CaCl2 solution ratio 4. Both empty and B. pertussis loaded microparticles exhibited smooth surface texture and relatively spherical shape. The B. pertussis encapsulated microspheres fabricated under optimized conditions showed mean particle size 151.1 μm, polydispersity index 0.43, loading efficiency 89.6%, loading capacity 36.3%, and relatively constant release rate lasted to 15 days. In vivo immunogenicity and protection study against wild type challenge showed strongly higher potency (approximately 2.5 fold) of encapsulated B. pertussis organisms than non-encapsulated conventional aluminum hydroxide adsorbed vaccine. It can be concluded that microencapsulation of inactive B. pertussis cells appears to be a suitable approach for improving the wP vaccine quality, specially by obtaining good immunogenicity with low bacterial content.