Recombinant outer capsid glycoprotein [VP7] of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits

Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection. Many efforts have been done to produce recombinant VP7 that maintain native characteristics. We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus [AcNPV] downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants. Injection of recombinant VP7 in rabbits elicited the production of serum antibodies, which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture. Recombinant outer capsid glycoprotein [VP7] of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine

Microtitration of rubella virus in monovalent vaccinal products

Potency test for control of rubella vaccine is a significant factor to qualify production line and vaccination program. For this reason, WHO recommends to use the microtitration method by both vaccine companies and control laboratories. Then the study was done to improve this test. Three rubella virus samples, including an in-house standard, a lot of vaccine and an in-process product, were tittered in cell culture tubes. Then micro titration steps were tested on 96-well microplate using cocultivation of standard rubella vaccine dilutions and RK-13 cell line. After 6-7 days, final reading was done and calculated the titer. Two other samples were assayed with the micromethod. Titer reduction less than 0.5 log was acquired for each sample during frequent tests and between two methods. The procedure was profitable and accurate for potency and identity tests of rubella virus vaccine, on the basis of WHO recommendations

[Vaccine potential of rotavirus outer capsid protein [VP4] cloned into the plasmid]

Rotaviruses are associated with endemic diarrhea in children under the age of 5, leading to approximately 800,000 deaths every year. Introduction of rotavirus vaccines into childhood immunization programs can contribute to substantial reduction in mortality from rotavirus gastroenteritis in developing countries. VP4 is outer capsid spike protein of rotavirus by which virus can bind to its receptor. VP4 protein can induce production of neutralizing antibodies. Regarding these concepts we cloned VP4 gene of rotavirus in plasmid vector for future expression. BSC-1 cells were cultured as a monolayer. Rotavirus CPE positive cell cultures were freeze-thawed three times. Rotavirus was partially purified by ultracentrifuge. Oligonucleotide primers, specific for gene segment 4 which encodes VP4, were synthesized. Rotavirus RNA extracted and used as a template for synthesis of cDNA by reverse transcriptase. Then they proliferated by PCR and PCR products were cloned into plasmid vector which was analyzed by restriction enzymes and sequencing. Sequencing result was analyzed with BLAST software that had a 100% homology with SA11 rotavirus genome segment 4 in NCBI Gene Bank. Sequencing result confirms highly that the nucleotide sequences of VP4 gene after long and continuous passage of SA11 rotavirus is conserved in our laboratory