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Therapeutic fragmented antibodies show a poor pharmacokinetic profile that leads to frequent high-dose administration. In the current study, for the first time, a novel proline, alanine, serine (PAS) repeat sequence called PAS#208 was designed to extend the plasma half-life of a nanosized anti-vascular endothelial growth factor-A single-domain antibody. Polyacrylamide gel electrophoresis, circular dichroism, dynamic light scattering, and electrophoretic light scattering were used to assess the physicochemical properties of the newly designed PAS sequence. The effect of PAS#208 on the biologic activity of a single-domain antibody was studied using an in vitro proliferation assay. The pharmacokinetic parameters, including terminal half-life, the volume of distribution, elimination rate constant, and clearance, were determined in mice model and compared with the native protein and PAS#1(200) sequence. The novel PAS repeat sequence showed comparable physicochemical, biologic, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The PAS#208 increased the hydrodynamic radius and decreased significantly the electrophoretic mobility of the native protein without any change in zeta potential. Surprisingly, the fusion of PAS#208 to the single-domain antibody increased the binding potency. In addition, it did not alter the biologic activity and did not show any cytotoxicity on the normal cells. The PAS#208 sequence improved the terminal half-life (14-fold) as well as other pharmacokinetic parameters significantly. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of therapeutic fragmented antibodies. SIGNIFICANCE STATEMENT: In the current study, a new proline, alanine, serine (PAS) sequence was developed that showed comparable physicochemical, biological, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of recombinant small proteins.

For induction of an appropriate immune response, especially in the case of an inactivated vaccine, the use of an adjuvant is crucial. In this study, adjuvanticity effect of G2 dendrimer in veterinary rabies vaccine has been investigated. A nonlinear globular G2 dendrimer comprising citric acid and polyethylene glycol 600 (PEG-600) was synthesized and the toxicity was studied in vitro on the J774A.1 cell line. The adjuvanticity effect of the dendrimer was then investigated on rabies virus in NMRI mice as a model. Different concentrations of dendrimer were used to determine the best formulation for the survival of the mice after virus challenge. The rise of neutralizing antibody was also checked by rapid fluorescent focus inhibition test (RFFIT). The relative potency of the prepared formulation was finally calculated using standard NIH test and the results were compared (and discussed) with the commercially available rabies vaccine. The accuracy of dendrimer synthesis was confirmed using Fourier transform infrared (FT-IR), size, and zeta potential analysis. The in vitro toxicity assay revealed that no significant toxic effect is observed in cells when data are compared with the control group. The in vivo assay showed that a higher survival rate in the mice received a special formulation due to adjuvanticity effect of dendrimer, which is also confirmed by RFFIT. However, the relative potency of that formulation does not give expected results when compared with the alum-containing rabies vaccine. In the current investigation, the adjuvanticity effect of G2 dendrimer was demonstrated for the first time in rising of neutralizing antibodies against rabies virus. Our data confirm that nanoparticles can enhance immune responses in an appropriate manner. Moreover, engineered nanoparticles will enable us to develop novel potent multivalent adjuvants in vaccine technology.

Manufacturing new Hepatitis B virus vaccines, specifically by the use of nanoparticles, is of high global interest. In this paper, a new structure of nano-sized hepatitis B virus' surface antigen (HBsAg) was generated by conjugation with dendrimers. Methods: The physicochemical properties of the conjugate were characterized by zeta potential and size distribution analyses and FT-IR spectrometry. Results: The results confirmed the conjugation between HBsAg and the dendrimers. Immunological assays indicated that the immunogenicity of the conjugated HBsAg is more than HBsAg alone. Conclusion: In the current study, a cost- effective, biodegradable and biocompatible polymer was used to enhance the immunogenicity of HBsAg. Also, further investigations are required to explore the mechanisms of action of this nanocomplex vaccine candidate.

This study is an improvement on the antibody binding test, known as ABT method, to develop a simple and fast method in comparison with NIH for determination of rabies vaccine potency. In the current study, several commercial human and veterinary vaccines were tested using both modified ABT and NIH methods. The ED50 was calculated using the probit method and the relative potency of each vaccine was measured based on the reference vaccine. The test was repeated four times to calculate the reproducibility of the method. Statistical analysis indicated that there was no significant difference between the result obtained from NIH and modified ABT method for either human or veterinary vaccines (p > 0.05). In addition, the linearity of the method (R2) was calculated as 0.94 by serial dilution of a test vaccine. Coefficient variances were determined as less than and more than 10% for the human and veterinary rabies vaccines, respectively. In conclusion, the findings suggest that the modified method could ...

Vahid Asgary,1,2 Alireza Shoari,1 Fahimeh Baghbani-Arani,3 Seyed Ataollah Sadat Shandiz,4 Mohammad Sadeq Khosravy,5 Alireza Janani,1 Razieh Bigdeli,6 Rouzbeh Bashar,1 Reza Ahangari Cohan1,7 1Virology Research Group, Department of Rabies, Pasteur Institute of Iran, 2Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, 3Department of Genetics and Biotechnology, School of Biological Science, Varamin-Pishva Branch, Islamic Azad University, Varamin, 4Young Researchers and Elite Club, East Tehran Branch, Islamic Azad University, 5Department of Laboratory of Animal Sciences, Pasteur Institute of Iran, 6Department of Genetic, Science and Research Branch, Islamic Azad University, 7New Technologies Research Group, Department of Pilot Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran Background: Green synthesis of nanoparticles by plant extracts plays a significant role in different applications. Recently, several studies were conducted on the use of nanoparticles as adjuvant. The main aim of this study was to evaluate green synthesized silver nanoparticles (AgNPs) as adjuvant in rabies veterinary vaccine and compare the results with the existing commercially available alum adjuvant.Materials and methods: In the current study, AgNPs were prepared by the reduction of aqueous silver nitrate by leaf extract of Eucalyptus procera. The formation of AgNPs was confirmed by ultraviolet (UV)–visible spectrophotometer, scanning electron microscopy, dynamic light scattering, and X-ray diffraction analysis. Then, different amounts of AgNPs (200µg, 400µg, 600µg, and 800µg) were added to 1mL of inactivated rabies virus. The loaded vaccines (0.5mL) were injected intraperitoneally into six Naval Medical Research Institute mice in each group on days 1 and 7. On the 15th day, the mice were intracerebrally challenged with 0.03mL of challenge rabies virus (challenge virus strain-11, 20 lethal dose [20 LD50]), and after the latency period of rabies disease in mice (5days), the mice were monitored for 21days. Neutralizing antibodies against rabies virus were also investigated using the rapid fluorescent focus inhibition test method. The National Institutes of Health test was performed to determine the potency of optimum concentration of AgNPs as adjuvant. In vitro toxicity of AgNPs was assessed in L929 cell line using MTT assay. In addition, in vivo toxicity of AgNPs and AgNPs-loaded vaccine was investigated according to the European Pharmacopeia 8.0.Results: AgNPs were successfully synthesized, and the identity was confirmed by UV–visible spectrophotometry and X-ray diffraction analysis. The prepared AgNPs were spherical in shape, with an average size of 60nm and a negative zeta potential of -14mV as determined by dynamic light scattering technique. The highest percentage of viability was observed at 15mg/kg and 20mg/kg of AgNPs-loaded vaccine concentrations after injecting into the mice. The calculated potencies for alum-containing vaccine and AgNPs-loaded vaccine (dose 15mg/kg) were 1.897 and 1.303, respectively. MTT assay demonstrated that alum at the concentration of 10mg/mL was toxic, but AgNPs were not toxic. The in vivo toxicity also elucidated the safety of AgNPs and AgNPs-loaded vaccine in mice and dogs, respectively.Conclusion: In the current study, for the first time, the adjuvanticity effect of green synthesized AgNPs on veterinary rabies vaccine potency with no in vivo toxicity was elucidated according to the European Pharmacopeia 8.0. Keywords: green synthesis, nanoparticles, rabies virus, adjuvant