ASRM: American Society of Reproductive Medicine; HHCY: hyperhomocysteinemia; MTHFR: methylenetetrahydrofolate reductase; PCR: polymerase chain reaction; PAGE: poly-acrylamide gel electrophoresis; RPL: recurrent pregnancy loss.

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 (200 µg, 400 µg, 600 µg, and 800 µg) were added to 1 mL of inactivated rabies virus. The loaded vaccines (0.5 mL) 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 0.03 mL of challenge rabies virus (challenge virus strain-11, 20 lethal dose [20 LD 50 ]), and after the latency period of rabies disease in mice (5 days), the mice were monitored for 21 days. 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 60 nm and a negative zeta potential of −14 mV as determined by dynamic light scattering technique. The highest percentage of viability was observed at 15 mg/kg and 20 mg/kg of AgNPs-loaded vaccine concentrations after injecting into the mice. The calculated potencies for alum-containing vaccine and AgNPs-loaded vaccine (dose 15 mg/kg) were 1.897 and 1.303, respectively. MTT assay demonstrated that alum at the concentration of 10 mg/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.

To date, selective blockade of Toll-like receptor (TLR) signalling has been developed as a new approach for treatment for many inflammatory diseases. As β-D-mannuronic acid (M2000) has been known as an anti-inflammatory molecule in several experimental models, we investigated the antagonistic effects of M2000 on TLR2 and TLR4 downstream signalling transduction pathway in human embryonic kidney (HEK) 293 cell lines overexpressing TLR2/CD14 and the TLR4/MD2/CD14 complex, respectively. M2000 effectively inhibited mRNA expression of MyD88 and p65, major subunit of nuclear factor-κB, in HEK293 cells stimulated by lipoteichoic acid (LTA, a TLR2 agonist) and lipopolysaccharide (LPS, a TLR4 agonist) with no evidence of cytotoxicity. In addition, M2000 also suppressed LTA and LPS-induced production of TNF-α and IL-6 inflammatory cytokines in these cells. Furthermore, the results revealed that M2000 had no significant effect on Tollip mRNA expression as a negative regulator of TLR signalling in aforesaid cells. Overall, these data point to M2000 inhibitory effect on Toll-like receptor (TLR) 2, 4 signalling in HEK293 cells. This information might provide new insights into the possible roles of this small drug in order to introduce it as a TLR signalling pathway inhibitor. However, more studies are needed to confirm β-D-mannuronic acid antagonistic effects including the effects of M2000 on peritoneal isolated macrophages and also on blood cells in patients with inflammatory diseases such as ankylosing spondylitis.

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.