Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.

An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice.

The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity.

Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo.

Rabies is an acute fatal encephalitis disease that affects many warm-blooded mammals. The causative agent of the disease is Rabies virus (RABV). Currently, no approved therapy is available once the clinical signs have appeared. Aptamers, oligonucleotide ligands capable of binding a variety of molecular targets with high affinity and specificity, have recently emerged as promising therapeutic agents. In this study, sixteen high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Viral titer assays revealed aptamers could specifically inhibit the replication of RABV in cells but did not inhibit the replication of canine distemper virus or canine parvovirus. In addition, the FO21 and FO24 aptamers, with and without PEGylation, were found to effectively protect mice against lethal RABV challenge. When mice were inoculated with aptamers for 24h prior to inoculation with CVS-11, approximately 87.5% of the mice survived. Here, we report aptamers that could significantly protect the mice from a lethal dose of RABV in vitro and in vivo, as demonstrated by the results for survival rate, weight loss and viral titers. These results indicate that FO21 and FO24 aptamers are a promising agent for specific antiviral against RABV infections.

PIKA provides an adjuvant effect to induce strong mucosal and systemic humoral immunity against SARS-CoV.

Severe Acute Respiratory Syndrome (SARS) is a deadly infectious disease caused by SARS Coronavirus (SARS-CoV). Inactivated SARS-CoV has been explored as a vaccine against SARS-CoV. However, safe and potent adjuvants, especially with more efficient and economical needle-free vaccination are always needed more urgently in a pandemic. The development of a safe and effective mucosal adjuvant and vaccine for prevention of emergent infectious diseases such as SARS will be an important advancement. PIKA, a stabilized derivative of Poly (I:C), was previously reported to be safe and potent as adjuvant in mouse models. In the present study, we demonstrated that the intraperitoneal and intranasal co-administration of inactivated SARS-CoV vaccine together with this improved Poly (I:C) derivative induced strong anti-SARS-CoV mucosal and systemic humoral immune responses with neutralizing activity against pseudotyped virus. Although intraperitoneal immunization of inactivated SARS-CoV vaccine alone could induce a certain level of neutralizing activity in serum as well as in mucosal sites, co-administration of inactivated SARS-CoV vaccine with PIKA as adjuvant could induce a much higher neutralizing activity. When intranasal immunization was used, PIKA was obligatorily for inducing neutralizing activity in serum as well as in mucosal sites and was correlated with both mucosal IgA and mucosal IgG response. Overall, PIKA could be a good mucosal adjuvant candidate for inactivated SARS-CoV vaccine for use in possible future pandemic.