Enhancement of therapeutic DNA vaccine potency by melatonin through inhibiting VEGF expression and induction of antitumor immunity mediated by CD8+ T cells.

To be effective, therapeutic cancer vaccines should stimulate both an effective cell-mediated and a robust cytotoxic CD8+ T-cell response against human papillomavirus (HPV)-infected cells to treat the pre-existing tumors and prevent potential future tumors. In this study, the therapeutic experiments were designed in order to evaluate antitumor effect against the syngeneic TC-1 tumor model. The anti-tumor efficacy of a HPV-16 E7 DNA vaccine adjuvanted with melatonin (MLT) was evaluated in a C57BL/6 mouse tumor model by measuring tumor growth post vaccination and the survival rate of tumor-bearing mice, analyzing the specific lymphocyte proliferation responses in control and vaccinated mice by MTT assay. The E7-specific cytotoxic T cells (CTL) were analyzed by lymphocyte proliferation and lactate dehydrogenates (LDH) release assays. IFN-γ, IL-4 and TNF-α secretion in splenocyte cultures as well as vascular endothelial growth factor (VEGF) and IL-10 in the tumor microenvironment were assayed by ELISA. Our results demonstrated that subcutaneous administration of C57BL/6 mice with a DNA vaccine adjuvanted with MLT dose-dependently and significantly induced strong HPV16 E7-specific CD8+ cytotoxicity and IFN-γ and TNF-α responses capable of reducing HPV-16 E7-expressing tumor volume. A significantly higher level of E7-specific T-cell proliferation was also found in the adjuvanted vaccine group. Furthermore, tumor growth was significantly inhibited when the DNA vaccine was combined with MLT and the survival time of TC-1 tumor bearing mice was also significantly prolonged. In vivo studies further demonstrated that MLT decreased the accumulation of IL-10 and VEGF in the tumor microenvironment of vaccinated mice. These data indicate that melatonin as an adjuvant augmented the cancer vaccine efficiency against HPV-associated tumors in a dose dependent manner.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

A novel chimeric influenza virosome containing Vesicular stomatitis G protein as a more efficient gene delivery system.

OBJECTIVES: To enhance the efficiency of influenza virosome-mediated gene delivery by engineering this virosome. RESULTS: A novel chimeric influenza virosome was constructed containing the glycoprotein of Vesicular stomatitis virus (VSV-G), along with its own hemagglutinin protein. To optimize the transfection efficiency of both chimeric and influenza cationic virosomes, HEK cells were transfected with plasmid DNA and virosomes and the transfection efficiency was assessed by FACS analysis. The chimeric virosome was significantly more efficient in mediating transfection for all amounts of DNA and virosomes compared to the influenza virosome. CONCLUSIONS: Chimeric influenza virosome, including VSV-G, is superior to the conventional influenza virosome for gene delivery.

Proteomics analysis of human brain tissue infected by street rabies virus.

In order to extend the knowledge of rabies pathogenesis, a two-dimensional electrophoresis/mass spectrometry based postmortem comparative proteomics analysis was carried out on human brain samples. Alteration in expression profile of several proteins was detected. Proteins related to cytoskeleton, metabolism, proteasome and immune regulatory systems showed the most changes in expression levels. Among these groups, the cytoskeleton related proteins (dynein light chain, ß-centractin, tubulin alpha-1C chain and destrin) and metabolism associated proteins (fatty acid-binding protein, macrophage migration inhibitory factor, glutamine synthetase and alpha enolase) were the main altered proteins. These alterations may be considered as an evidence of disturbances in neuronal key processes including axonal transport, synaptic activity, signaling and metabolic pathways in rabies virus infected human brain.

Hyalomma anatolicum as the Main Infesting Tick in an Important Livestock Rearing Region, Central Area of Iran.

BACKGROUND: This study was carried out to determine the infestation of domestic ruminants to ticks in an important livestock-rearing region, located in central part of Iran. METHODS: Ticks were collected from cattle, sheep, and goats and then were identified with appropriate identification keys to species level in two different ecological regions of plains and mountain in 4 seasons in 2015. RESULTS: Totally 492 ticks from cattle, sheep, and goats in 34 herds were collected. Totally, 18.53% of domestic animals were infected by ticks. All ticks were belonged to family Ixodidae and classified into three genera and six species comprising Hyalomma anatolicum (38.83%), Hy. Asiaticum (23.37%), Hy. marginatum (2.85%), Hy. sp. (3.45%), Rhipicephalus sanguineus (14.02%) and Haemaphysalis sulcata (10.98%). Sex ratio of the collected specimens showed 241 (48.99%) male, 219 (44.51%) female and 32 (6.5%) nymph. CONCLUSION: Studied area is important for production of livestock and dairy products. Annually, many livestock products are exported to other parts from this region; therefore, it is very important to identify the infection rate of tick-borne diseases as well as safety factors on livestock.

Phylogeny of tick-derived Crimean-Congo hemorrhagic fever virus strains in Iran.

The presence of Crimean-Congo hemorrhagic fever virus (CCHFV) in Iran was assessed by collecting ticks from Golpayegan, Isfahan Province. Real time RT-PCR was used to detect the CCHFV RNA in the tick population and the origins of the viral sequences were determined. The CCHFV RNA was detected in 5.2% of 492 ticks collected from livestock in different regions of Golpayegan. The tick species that tested positive for the presence of CCHFV RNA included Hyalomma, Rhipicephalus and Haemaphysalis species. Phylogenetic analysis using the partial S-segment indicated that eight sequences clustered in clade IV (Asia-1) and three other sequences aligned within clade VI (Europe) with other CCHFV strains from Kosovo (Kosova1917) and Russia (Kashmanov).

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.

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.

Six fatal cases of classical rabies virus without biting incidents, Iran 1990-2010.

BACKGROUND: Rabies is an endemic fatal zoonotic disease, commonly transmitted to humans through contact (bites and scratches) with infected animals. OBJECTIVES: During the years 1990-2010, six patients with the clinical symptoms of rabies (fever, tinnitus, buzzing, delirium and hydrophobia), with no history of a bite, were diagnosed by physicians in Iran. To obtain laboratory confirmation of rabies infection, different clinical specimens from each patient were sent to the World Health Organisation (WHO) Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran. The first case was a 39-year-old male veterinary technician who entered his uncovered scratched hand into the mouth of a rabid bovine and became infected. Two years later, a herd of sheep being tended by a shepherd and his two sons were attacked by a rabid wolf. All three individuals were infected when they applied burnt thorny wool to the sheep's wounds as a bandage. Their hands were scratched and then infected through contact with the remaining saliva of the rabid wolf on the sheep's wounds. In 1994, two other human cases occurred through corneal transplantation from the same donor who had died with the clinical signs of food poisoning (according to his hospital record), which probably was a misdiagnosis of rabies infection. STUDY DESIGN: This is a case series study that describes human rabies cases without biting incidents. According to the WHO recommendation, human rabies cases are notifiable, therefore, in Iran, a rabies surveillance system has been established to follow these cases. During the last decade, six patients with no 'history of a bite' were hospitalised with growing symptoms of rabies. The data were collected from each patient by the physicians and transferred to the Ministry of Health and Medical Education of Iran, and to the WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran as the only testing laboratory. Thus, they came to the attention of the surveillance system. Ante-mortem diagnosis was performed on saliva, cerebrospinal fluid and blood samples that were collected from the first patient by the physicians. Fresh brain specimens from all patients were kept in a mixture of 50% glycerol in phosphate-buffered saline and transported on ice to the WHO Collaborating Center for Reference and Research on Rabies. RESULTS: For the first patient, rabies virus was investigated in saliva using the rapid tissue cell inoculation test (RTCIT) and the mouse inoculation test (MIT). Anti-rabies antibodies in this patient's serum and cerebrospinal fluid (CSF) were examined using the mouse neutralisation test (MNT). Fresh brain specimens from all patients were examined using the fluorescence antibody test (FAT) as recommended by the WHO laboratory manual in rabies as the post-mortem diagnostic test for rabies. Rabies infection was confirmed in all of the deceased patients. Anti-rabies antibodies were identified only in one patient's serum specimen. Testing also showed that the rabies virus isolated was the classic rabies virus (serotype 1), which is widespread in Iran. CONCLUSIONS: Prevention and control of this fatal disease require a sensitive surveillance system to follow 'suspected' animal and human rabies cases thoroughly through the improved reporting system, which contains the history of exposure, clinical examinations, symptoms and laboratory results. This study describes some notable human rabies infections and their transmission modes to prevent occupational accidents.

Antibody persistence, 32 years after post-exposure prophylaxis with human diploid cell rabies vaccine (HDCV).

In 1975-1976 forty-five persons severely bitten by rabid wolves and dogs in Iran were treated successfully against rabies with HDCV. In this study contact was made with 26 of 45 above persons, 32 years after their initial treatment and all had rabies neutralizing antibody ranging from 0.3 to 2.69 IU/ml of serum. Of the 26 persons, 17 had received a booster dose of HDCV, 28 years ago and the remaining 9 persons, who had not received any booster since the initial treatment, were given one booster dose of HDCV. All 9 of these patients developed an anamnestic response after their booster inoculation. This study confirms the persistence of rabies neutralizing antibody in persons that received post-exposure vaccination with HDCV, 32 years previously. Furthermore, a single booster inoculation with HDCV resulted in anamnestic response in all individuals.