Transient Expression of Biologically Active Anti-rabies Virus Monoclonal Antibody in Tobacco Leaves.

BACKGROUND: Rabies virus is a neurotropic virus that causes fatal, but, a preventable disease in mammals. Administration of rabies immunoglobulin (RIG) is essential for the post-exposure of the prophylaxis to prevent the disease. However, replacement of polyclonal RIGs with alternative monoclonal antibodies (MAbs) that are capable of neutralizing rabies virus has been recommended. OBJECTIVES: Here, we have investigated the transient expression of the full-size human MAb against rabies virus glycoprotein; the MAb SO57 in the tobacco plants using vacuum agro-infiltration. Previously, stably transformed plants expressing the MAb have been reported. MATERIALS AND METHODS: In this study three vectors carrying the codon-optimized genes for the heavy or light chain and p19 silencing-suppressor were constructed. These vectors were co-infiltrated into Nicotiana tabacum leaves and the transgenes were expressed. RESULTS: Dot blot, Western blotting, ELISA, and in vitro neutralization assays of the plant extracts showed that the human MAb could assemble in tobacco leaves and was able to neutralize rabies virus. CONCLUSIONS: This study is the first report of transient expression of human MAb SO57 gene in tobacco plant within a few days after vacuum agro-infiltration.

Neutralizing Antibody Response after Intramuscular Purified Vero Cell Rabies Vaccination (PVRV) in Iranian Patients with Specific Medical Conditions.

OBJECTIVE: Post exposure prophylaxis using one of the WHO-approved vaccines is the method of choice for preventing rabies. Abnormal immune function in patients with some specific medical conditions, such as pregnancy, chronic hepatitis B virus infection, different types of cancers like lymphoma, diabetes I and II, corticosteroid consumption by patients with rheumatoid arthritis and lupus erythematosus, could impair the immunologic response to various vaccines. The immune response to rabies vaccination has never been examined in patients with any of these described medical conditions. This study purposed to evaluate the neutralyzing antibody response after vaccination with purified Vero cell rabies vaccine (PVRV) according to the WHO-recommended Post-Exposure Prophylaxis (PEP) "ESSEN" regimen. METHODS: Thirty healthy volunteers and 50 volunteers with different medical conditions who were exposed to a suspected rabid animal in the 2nd or 3rd category of exposure received 5 doses of PVRV under the ESSEN protocol. Three blood samples were collected on days 0 (before the first dose), 14, and 35. The anti-rabies antibody titer was measured using the Rapid Fluorescent Foci Inhibition Test (RFFIT) and an ELISA Bio-Rad, Platelia, Rabies II kit. RESULTS: All subjects reached NAb titers above 0.5 IU/ml by day 14 after vaccination. On day 35 (1 week after receiving the last rabies vaccine), anti-rabies antibodies were in the protective level (>0.5 IU/ml) in both groups. There was no statistically significant difference in anti-rabies antibody response due to the type of exposure (category 2 or 3), and successful seroconversion was confirmed in both groups. CONCLUSION: In conclusion, the ESSEN protocol using the PVRV vaccine is sufficient for rabies prophylaxis in patients with specific medical conditions.

Cell culture extraction and purification of rabies virus nucleoprotein.

BACKGROUND: Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate. OBJECTIVES: The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate. MATERIALS AND METHODS: Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 10(6) cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus. CONCLUSIONS: The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.

Human rabies in Iran.

BACKGROUND: Like most Asian and African countries, Iran is highly endemic for rabies, which is a preventable disease with the timely utilisation of post-exposure prophylaxis (PEP). With the availability of affordable vaccination in Iran, there are still several rabies deaths which are assumed misdiagnosed or received ineffective PEP. METHODS: We reviewed the files of 16 human rabies deaths, consisting of two groups: 1, ineffective treatment; and 2, erroneous PEP. RESULTS: Most of the studied cases were male and were from rural areas. Stray dogs were found to be the common biting animal (68.75%). Of the patients, 10/16 (62.5%) who had injuries on their head and/or face demonstrated shorter incubation periods. The incubation period was longer in a 4-year-old boy who sustained injuries in his abdomen and back. All the patients in group 1 received four doses of vaccine and administration of human rabies immune globulin (HRIG), and death occurred with the mean of 49 days after the bite. This mean was 27 days in three patients in group 2, who received vaccine without administration of HRIG. CONCLUSION: In a total of 1,188,579 cases of PEP given in Iran during: 2002-2011, it is not known whether all PEPs were correctly administered by World Health Organization standards. Extending rabies awareness programmes and timely PEP education in the community in accordance with the implementation of rabies control measures might lead to a decrease in these unfortunate scenarios and heavy financial burden of vaccination required due to the prevalence of rabies.

Rabies antibody levels in pregnant women and their newborns after rabies post-exposure prophylaxis.

BACKGROUND: Rabies is a fatal infectious disease and rabies post-exposure prophylaxis is the method of choice for prevention of human rabies. CASE SERIES: We report rabies antibody levels in cord blood and also in serum of pregnant women who were bitten by suspected animals to rabies and were immunized by purified Vero cell rabies vaccine (PVRV) and Human Rabies immunoglobulin (HRIG) serum. During the years of 2007-2010, six pregnant women by the age range of 22-35 years were admitted in treatment and prevention of rabies center in Pasture institute of Iran, in Tehran. Among them two cases were at first trimester, one at second trimester and three at third trimester of conception. The interval between biting with delivery was 5-265 days (mean 121 days). CONCLUSION: Results of immunoglobulin illustrate that levels of rabies antibody in maternal sera with the fetus are not equal and uniform but it is proved that baby will find efficient immunity as well with minimum protective level of 0.5 IU/ml in all cases except a newborn whom had been born just 5 days after the mother's immunization and in a shorter time than the appropriate immunization of the mother who had received her second vaccination courses.