Journal of Microbiology

Journal Article

Recombinant baculovirus-based vaccine expressing M2 protein induces protective CD8+ T-cell immunity against respiratory syncytial virus infection: Jeong-Yoon Lee , Jun Chang; J. Microbiol. 2017;55(11):900-908. Published online October 27, 2017; DOI: https://doi.org/10.1007/s12275-017-7306-6

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Respiratory syncytial virus (RSV) is an important cause of acute lower respiratory tract disease in infants, young children, immunocompromised individuals, and the elderly. However, despite ongoing efforts to develop an RSV vaccine, there is still no authorized RSV vaccine for humans. Baculovirus has attracted attention as a vaccine vector because of its ability to induce a high level of humoral and cellular immunity, low cytotoxicity against various antigens, and biological safety for humans. In this study, we constructed a recombinant baculovirus- based vaccine expressing the M2 protein of RSV under the control of cytomegalovirus promoter (Bac_RSVM2) to induce CD8+ T-cell responses which play an important role in viral clearance, and investigated its protective efficacy against RSV infection. Immunization with Bac_RSVM2 via intranasal or intramuscular route effectively elicited the specific CD8+ T-cell responses. Most notably, immunization with Bac_RSVM2 vaccine almost completely protected mice from RSV challenge without vaccine-enhanced immunopathology. In conclusion, these results suggest that Bac_RSVM2 vaccine employing the baculovirus delivery platform has promising potential to be developed as a safe and novel RSV vaccine that provides protection against RSV infection.

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Cytokines and CD8 T cell immunity during respiratory syncytial virus infection
Megan E. Schmidt, Steven M. Varga
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Functional Characterization of Autographa californica Multiple Nucleopolyhedrovirus ORF43 and Phenotypic Changes of ORF43-Knockout Mutant: Xue Ying Tao , Jae Young Choi , Yong Wang , Jong Yul Roh , Joo Hyun Lee , Qin Liu , Jong Bin Park , Jae Su Kim , Woojin Kim , Yeon Ho Je; J. Microbiol. 2013;51(4):515-521. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3058-0

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Abstract: ORF43 (ac43) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene of unknown function. To investigate the role of ac43 in the baculovirus lifecycle, we constructed an ac43-deleted mutant AcMNPV, Ac43KO. After transfection into Spodoptera frugiperda cells, Ac43KO produced polyhedra much larger in size than those of wild-type AcMNPV. Interestingly, some of the nucleocapsids were singly enveloped in the polyhedrin matrix while the nucleocapsids of AcMNPV are known to be multiply enveloped. Furthermore, Ac43KO led to a defect in the transcription and expression of polyhedrin, which resulted in reduced occlusion body production. However, Ac43KO did not affect production of budded virus as there was no remarkable difference in budded virus titer. These results suggest that ac43 plays an important role in the expression of polyhedrin, the morphogenesis of occlusion body, and the assembly of virions occluded in occlusion bodies.

Identification and Characterization of Host Factors Interacting with Bombyx mori Nucleopolyhedrovirus ORF8: WonKyung Kang , Susumu Katsuma , Noriko Matsuda-Imai , Masaaki Kurihara , Toyoshi Yoshiga , Toru Shimada , Shogo Matsumoto; J. Microbiol. 2012;50(3):469-477. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2010-z

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Abstract: The orf8 gene (Bm8) in Bombyx mori nucleopolyhedrovirus (BmNPV) is one of 17 genes unique to group I NPVs and is expressed as an early gene. We have reported that Bm8 may play an important role during viral infection and that Bm8 protein co-localized with IE1 to specific nuclear foci throughout infection. It was also demonstrated that both IE1 and BmNPV hr facilitate this localization of Bm8. To investigate further, host proteins interacting with Bm8 were screened using a yeast two-hybrid system. We identified 6 host clones as Bm8-interacting partners from three cDNA libraries derived from BmN cells or B. mori larvae. Further assays showed that the N-terminal region of Bm8 is important for the interaction with most host clones and that two of the clones can associate with IE1. Cloning and sequencing of full-length cDNAs revealed that most of the clones potentially encode either membrane-bound proteins or secreted proteins. Quantitative RT-PCR analysis revealed that some of these host genes were slightly induced during the early stage of infection in BmN cells, and that the expression of all genes was markedly reduced during the late stage of infection. Generation of mutant BmNPVs over-expressing these host genes also identified a gene that potentially functions as a negative factor during BmNPV infection. These features of Bm8-interacting host proteins strongly support that Bm8 is a multifunctional protein involved in multiple signaling pathways in host cells.

Molecular Characterization of ORFs 2 to 7 of Korean Porcine Reproductive and Respiratory Syndrome Virus (CA) and Its Protein Expression by Recombinant Baculoviruses: Hyun Na Koo , Jeong Mi Oh , Jae Kyung Lee , Jae Young Choi , Kwang Sik Lee , Jong Yul Roh , Yeon Ho Je , Byung Rae Jin , Sung Sik Yoo , Jae Su Kim , Young In Kim , In Joong Yoon , Soo Dong Woo; J. Microbiol. 2008;46(6):709-719. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0224-x

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Abstract: To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in insect cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

Molecular and Phylogenetic Characterization of Spodoptera litura Granulovirus: Yong Wang , Jae Young Choi , Jong Yul Roh , Soo Dong Woo , Byung Rae Jin , Yeon Ho Je; J. Microbiol. 2008;46(6):704-708. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0133-z

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Abstract: Baculovirus, Spodoptera litura granulovirus (SlGV) was isolated from the infected S. litura larvae, and was characterized. The granule of SlGV was ovoidal shape with an approximate size of 240~340 nm×140~180 nm. Each granule contained one single rod-shape virion with a mean size of 180~200 nm×20~40 nm. Restriction endonuclease fragment analysis estimated that the total genome size of SlGV is about 115 kb. Necleotide sequence analysis of the granulin gene showed that the gene encodes 249 amino acids with a predicted molecular mass of 29 kDa. When the phylogenic relationship was analyzed using the nucleotide sequence of the granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XcGV) which belong to Type I granulovirus.

Promoter Analysis of Bombyx mori Nucleopolyhedrovirus Ubiquitin Gene: Xu’ai Lin , Yin Chen , Yongzhu Yi , Jie Yan , Zhifang Zhang; J. Microbiol. 2008;46(4):429-435. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0163-y

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Abstract: The aim of this study was to analyze the characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) ubiquitin gene promoter and the effects of conserved motifs, such as TAAG, TATA, and CAAT, along with baculovirus enhancer homologous region 3 (hr3), on promoter activity. Ubiquitin gene of BmNPV was expressed during the late phase of virus infection. In the presence of viral factors, significant reduction of promoter activity was observed by deletion of -382 to -124 bp upstream of ATG. The fragment between -187 and -383 bp upstream of ATG, including distal TAAG, CAAT motif, and TATA box, could also drive expression of the reporter gene. The mutation of cis-elements TATA boxes and TAAG motifs significantly decreased the promoter’s activity, while CAAT mutations enhanced promoter activity by 2- or 3-fold, as compared with the native promoter. In the presence of BmNPV, hr3, both located downstream of the reporter gene of the same vector, and separate vector, could significantly enhance transcription activity of ubiquitin promoter as compared to the control. We concluded that BmNPV ubiquitin gene might be regulated by dual sets of promoter elements, where TAAG and TATA box may positively regulate the expression of ubiquitin, while CAAT motif functions as a negative regulator. Viral factor(s) play an important role in the co-activation of hr3 and promoter.

Expression of immunologically active porcine recombinant TGF-β1 precursor protein in baculovirus system: Lim, Hyun , Kim, Pyeung Hyeun , Chun, Gie Taek , Choi, Eui Yul , Yie, Se Won; J. Microbiol. 1997;35(4):341-346.

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Abstract: In order to express recombinant porcine TGF-β1 protein in a baculovirus expression system the entire TGF-β1 gene containing extra amino acids at the N terminus was cloned into pFBa and pFBb of the Bac-To-Bac^TM baculovirus expression system. One of the clones contained 106 extra amino acids and was designated pFBa-106 TGF-β1, and the other had 28 extra amino acids and was designated pFBb-28 TGF-β1. The orientation of the gene was identified with restriction enzyme mapping and PCR with internal TGF-β1 primers. Sf-9 cells were infected at a m.o.i. of 10 by the recombinant viruses generated from the two expected sizes of 55 kD and 46.4kD. these precursor forms of TGF-β1 with a polyclonal antibody against human TGF-β1. No mature form of TGF-β1 protein was detected on SDS gels and an immunoblot indicated that TGF-β1 precursor is not properlu processed in insect cells.

Isolation and Characterization of a Lymantria dispar Multinucleocapsid Nucleopolyhedrovirus Isolate in Korea: Hee Jin Shim , Jong Yul Roh , Jae Young Choi , Ming Shun Li , Soo Dong Woo , Hyun Woo Oh , Kyung Saeng Boo , Yeon Ho Je; J. Microbiol. 2003;41(4):306-311.

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Abstract: In Korea, a Lymantria dispar multinucleocapsid nucleopolyhedrovirus, LdMNPV-NM, was isolated and characterized from dead L. dispar larvae. The polyhedra of LdMNPV-NM were irregularly shaped with a diameter of 1.62?0.33 ?m. Numerous virions comprised of the multinucleocapsid were evident in the electron microscopic examination of the polyhedra cross sections. These polyhedra were composed of a major protein of 30 kDa. The restriction enzyme digestion patterns of LdMNPV-NM showed that this isolate had some different fragments from those of the Gypchek팜?LdMNPV isolate, although their overall profiles were similar. The deduced amino acid sequence of the enhancin gene of LdMNPV-NM showed differences when compared to previously reported enhancin genes of other LdMNPV strains. These results suggested that the LdMNPV-NM isolate from Korea was a new NPV strain and had a new enhancin gene.

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