Mol Immunol

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http://www.jimmunol.org/content/183/10/6600.full

Mol Immunol. 2010 Aug;47(14):2330-41. Epub 2010 Jun 2.

Fish virus-induced interferon exerts antiviral function through Stat1 pathway.

Yu FF, Zhang YB, Liu TK, Liu Y, Sun F, Jiang J, Gui JF.

Source

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology,

Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences,

Wuhan 430072, China.

Abstract

Virus-induced interferons (IFNs) have been identified in various fish species and displayantiviral activities similar to mammalian type I IFNs. However, apart from the mammalian

IFN system, the IFN signaling pathway remains largely unknown. Using transient

transfection and recombinant protein, we are reporting in this study that a crucian carp

(Carassius auratus L.) IFN exhibits strong antiviral activity against grass carp hemorrhagic

virus (GCHV) infection and also mediates Poly I:C-induced antiviral response, which

correlates with its ability to induce a set of IFN-stimulated genes (ISGs). Strikingly,

overexpression of wild-type Stat1 increases the effects of IFN on both the expression of ISGs

and the inhibition of virus infection, whereas a dominant negative mutant of Stat1 (Stat1-

Delta C), which lacks of the C-terminal transcriptional activation domain (TAD), inhibits the

antiviral activity of IFN and reduces the expression of ISGs, demonstrating that fish IFN

induces the expression of ISGs and host antiviral response through Stat1 pathway reminiscent

that of mammalian IFNs. Significantly, unlike mammalian type I IFNs, recombinant fish IFN

is able to upregulate IFN itself, which is enhanced by overexpression of Stat1 but impaired by

knockdown of Stat1, indicating a positive feedback loop in regulation of IFN itself. These

results provide strong evidence for the existence of an evolutionary conserved Stat1 pathway

between fish and mammals, which is indispensable for fish virus-induced IFN antiviral

response.

J Interferon Cytokine Res. 2006 Apr;26(4):256-9.

In vitro effects of recombinant zebrafish IFN on spring viremia of carp virus and

infectious hematopoietic necrosis virus.

Wang L, Wang L, Zhang HX, Zhang JH, Chen WH, Ruan XF, Xia C.

Source

Department of Microbiology and Immunology, College of Veterinary Medicine, China

Agricultural University, Beijing 100094, China.
http://www.jimmunol.org/content/183/10/6600.fullhttp://www.jimmunol.org/content/183/10/6600.fullhttp://www.ncbi.nlm.nih.gov/pubmed/20627394http://www.ncbi.nlm.nih.gov/pubmed?term=%22Yu%20FF%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhang%20YB%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Liu%20TK%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Liu%20Y%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sun%20F%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Jiang%20J%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Gui%20JF%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/16704302http://www.ncbi.nlm.nih.gov/pubmed?term=%22Wang%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhang%20HX%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhang%20JH%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Chen%20WH%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Ruan%20XF%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Xia%20C%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Xia%20C%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Ruan%20XF%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Chen%20WH%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhang%20JH%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhang%20HX%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Wang%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Wang%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/16704302http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gui%20JF%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Jiang%20J%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sun%20F%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Liu%20Y%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Liu%20TK%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhang%20YB%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yu%20FF%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/20627394http://www.jimmunol.org/content/183/10/6600.full


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Abstract

In order to evaluate the effects of fish recombinant interferon (rIFN) on fish pathogenic

rhabdoviruses, the zebrafish (Danio rerio) IFN (DreIFN) allele B gene was cloned and

expressed in Escherichia coli. In addition, the effects of recombinant DreIFN (rDreIFN) on

spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV), and

vesicular stomatitis virus (VSV) were surveyed in fish and chicken cells. The mature peptide

of DreIFN allele B gene encodes 163 amino acids. Residues 3 and 98 are a pair of cysteines

that likely form an intrachain disulfide bridge. rDreIFN protein was detected as a band at 21.6

kDa by SDS-PAGE. The purified rDreIFN has anti-SVCV and anti-IHNV activity of 3 x

10(4) U/mg-10(7) U/mg. The results indicate that rDreIFN has higher activity against SVCV

and IHNV on epithelioma papulosum cyprinid (EPC) than on grass carp (C. idellus) ovary

(CO) cell lines and no activity against VSV on chick embryo fibroblasts (CEF).

Developmental & Comparative Immunology

Volume 32, Issue 10, 2008, Pages 1211-1220

doi:10.1016/j.dci.2008.03.010 | How to Cite or Link Using DOI Cited By in Scopus (14)

Permissions & Reprints

Innate immunomodulation with recombinant interferon-enhances resistance of

rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis virus

Ei Lin Ooia, Noel Verjan

a, Ikumi Haraguchi

a, Takeo Oshima

a, Hidehiro Kondo

b,

Ikuo Hironob, Takashi Aoki

b, Hiroshi Kiyono

a, Yoshikazu Yuki

a, ,

Purchase

aDivision of Mucosal Immunology, Department of Microbiology and Immunology, The

Institute of Medical Science, The University of Tokyo, Shirokanedai 4-6-1, Minato, Tokyo

108-8639, Japan
http://www.sciencedirect.com/science/journal/0145305Xhttp://www.sciencedirect.com/science/journal/0145305Xhttp://www.sciencedirect.com/science?_ob=PublicationURL&_hubEid=1-s2.0-S0145305X08X00072&_cid=271272&_pubType=JL&view=c&_auth=y&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=ab15c5a2032a2bf9901698d428ba37abhttp://dx.doi.org/10.1016/j.dci.2008.03.010http://www.sciencedirect.com/science?_ob=HelpURL&_file=doi.htm&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=cb97ff0c0cd0cf5e9c46d4a05370ec58http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=656&_eid=1-s2.0-S0145305X08000785&_pii=S0145305X08000785&_origin=article&_zone=art_page&_targetURL=http%3A%2F%2Fwww.scopus.com%2Finward%2Fcitedby.url%3Feid%3D2-s2.0-46549087482%26partnerID%3D10%26rel%3DR3.0.0%26md5%3Dc46889598d04fd01e40ef240db2c8f50&_acct=C000228598&_version=1&_userid=10&md5=acb6870db30608779c9fe6e8d63f7228http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=656&_eid=1-s2.0-S0145305X08000785&_pii=S0145305X08000785&_origin=article&_zone=art_page&_targetURL=http%3A%2F%2Fwww.scopus.com%2Finward%2Fcitedby.url%3Feid%3D2-s2.0-46549087482%26partnerID%3D10%26rel%3DR3.0.0%26md5%3Dc46889598d04fd01e40ef240db2c8f50&_acct=C000228598&_version=1&_userid=10&md5=acb6870db30608779c9fe6e8d63f7228http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=936&_eid=1-s2.0-S0145305X08000785&_pii=S0145305X08000785&_origin=article&_zone=art_page&_targetURL=https%3A%2F%2Fs100.copyright.com%2FAppDispatchServlet%3FpublisherName%3DELS%26contentID%3DS0145305X08000785%26orderBeanReset%3Dtrue&_acct=C000228598&_version=1&_userid=10&md5=0a3f3c39fd20076db4b45b59cb7ab552http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff2http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff2http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff2http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff2http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff2http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff2http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff2http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science/article/pii/S0145305X08000785#aff1http://www.sciencedirect.com/science?_ob=ShoppingCartURL&_method=add&_eid=1-s2.0-S0145305X08000785&_acct=C000228598&_version=1&_userid=10&_ts=1316244182&md5=2b0c834982a39351a7c1f66419bb9439http://www.sciencedirect.com/science?_ob=ShoppingCartURL&_method=add&_eid=1-s2.0-S0145305X08000785&_acct=C000228598&_version=1&_userid=10&_ts=1316244182&md5=2b0c834982a39351a7c1f66419bb9439mailto:[email 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bLaboratory of Genome Science, Graduate School of Marine Science and Technology,

Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-

8477, Japan

Received 18 February 2008; revised 12 March 2008; Accepted 25 March 2008. Available

online 17 April 2008.

Summary

We examined the in vivo immunostimulatory effects of a recombinant Atlantic salmon

(Salmo salar) interferon-2 (rSasaIFN-2). The mature rSasaIFN-2, expressed and purified

fromEscherichia coli, was administered to rainbow trout (Oncorhynchus mykiss) via the oral,

immersion, or intraperitoneal (IP) injection route. Injection of rSasaIFN-2 at 0.1 g/g fish

gave significantly greater protection than a phosphate buffered saline (PBS) injection against

a lethal challenge of infectious hematopoietic necrosis virus (IHNV), with a relative percent

survival of 39%. Relative percent survival (RPS) increased significantly to 92% when the fish

were injected with rSasaIFN-2 at 1 g/g fish. Antiviral protection was evident for up to 7

days post-injection of rSasaIFN-2. Administration of rSasaIFN-2 by the oral or immersion

route was not protective, and the fish succumbed to virus infection. The level of systemic

IFN-induced expression of the Mx1 gene was significantly greater (p


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REFERENCES (51)
https://s100.copyright.com/AppDispatchServlet?publisherName=Springer&imprint=Springer+Berlin+%2f+Heidelberg&publication=0093-7711&title=Differential+expression+of+two+%3ci%3eCarassius+auratus+Mx%3c%2fi%3e+genes+in+cultured+CAB+cells+induced+by+grass+carp+hemorrhage+virus+and+interferon&publicationDate=04%2f01%2f2004&author=Yi-Bing+Zhang&authorAddress=State+Key+Laboratory+of+Freshwater+Ecology+and+Biotechnology%2c+Wuhan+Center+for+Developmental+Biology%2c+Institute+of+Hydrobiology+Chinese+Academy+of+Sciences+Wuhan+430072+China&contentID=10.1007%2fs00251-004-0658-1&volumeNum=56&issueNum=1&startPage=68&endPage=75&orderBeanReset=true&openAccess=falsehttps://s100.copyright.com/AppDispatchServlet?publisherName=Springer&imprint=Springer+Berlin+%2f+Heidelberg&publication=0093-7711&title=Differential+expression+of+two+%3ci%3eCarassius+auratus+Mx%3c%2fi%3e+genes+in+cultured+CAB+cells+induced+by+grass+carp+hemorrhage+virus+and+interferon&publicationDate=04%2f01%2f2004&author=Yi-Bing+Zhang&authorAddress=State+Key+Laboratory+of+Freshwater+Ecology+and+Biotechnology%2c+Wuhan+Center+for+Developmental+Biology%2c+Institute+of+Hydrobiology+Chinese+Academy+of+Sciences+Wuhan+430072+China&contentID=10.1007%2fs00251-004-0658-1&volumeNum=56&issueNum=1&startPage=68&endPage=75&orderBeanReset=true&openAccess=falsehttp://www.springerlink.com/content/jpbn4qnryu6attk4/#RelatedSectionhttp://www.springerlink.com/content/jpbn4qnryu6attk4/#ui-tabs-1http://www.springerlink.com/content/jpbn4qnryu6attk4/#ui-tabs-2http://www.springerlink.com/content/023295k221461l63/http://www.springerlink.com/content/023295k221461l63/http://www.springerlink.com/content/023295k221461l63/http://www.springerlink.com/content/023295k221461l63/http://www.springerlink.com/content/7b70kerqy6bjqwjv/http://www.springerlink.com/content/7b70kerqy6bjqwjv/http://www.springerlink.com/content/7b70kerqy6bjqwjv/http://www.springerlink.com/content/7b70kerqy6bjqwjv/http://www.springerlink.com/content/114232457533k5n6/http://www.springerlink.com/content/114232457533k5n6/http://www.springerlink.com/content/114232457533k5n6/http://www.springerlink.com/content/114232457533k5n6/http://www.springerlink.com/content/h8jg11868tx56106/http://www.springerlink.com/content/h8jg11868tx56106/http://www.springerlink.com/content/h8jg11868tx56106/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/q0102t07064862u4/http://www.springerlink.com/content/q0102t07064862u4/http://www.springerlink.com/content/q0102t07064862u4/http://www.springerlink.com/content/q0102t07064862u4/http://www.springerlink.com/content/q5t4372032452071/http://www.springerlink.com/content/q5t4372032452071/http://www.springerlink.com/content/q5t4372032452071/http://www.springerlink.com/content/d225481u5111x472/http://www.springerlink.com/content/d225481u5111x472/http://www.springerlink.com/content/d225481u5111x472/http://www.springerlink.com/content/p010666863m24231/http://www.springerlink.com/content/p010666863m24231/http://www.springerlink.com/content/p010666863m24231/http://www.springerlink.com/content/p010666863m24231/http://www.springerlink.com/content/jpbn4qnryu6attk4/references/http://www.springerlink.com/content/jpbn4qnryu6attk4/references/http://www.springerlink.com/content/jpbn4qnryu6attk4/references/http://www.springerlink.com/content/p010666863m24231/http://www.springerlink.com/content/p010666863m24231/http://www.springerlink.com/content/d225481u5111x472/http://www.springerlink.com/content/q5t4372032452071/http://www.springerlink.com/content/q0102t07064862u4/http://www.springerlink.com/content/q0102t07064862u4/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/87056t8u16504042/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/t190l0m65149m5tl/http://www.springerlink.com/content/h8jg11868tx56106/http://www.springerlink.com/content/114232457533k5n6/http://www.springerlink.com/content/114232457533k5n6/http://www.springerlink.com/content/7b70kerqy6bjqwjv/http://www.springerlink.com/content/7b70kerqy6bjqwjv/http://www.springerlink.com/content/023295k221461l63/http://www.springerlink.com/content/023295k221461l63/http://www.springerlink.com/content/jpbn4qnryu6attk4/#ui-tabs-2http://www.springerlink.com/content/jpbn4qnryu6attk4/#ui-tabs-1http://www.springerlink.com/content/jpbn4qnryu6attk4/#RelatedSection


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CITED BY (15) EXPORT CITATION ABOUT

Abstract

UV-inactivated GCHV (grass carp hemorrhage virus) is able to induce an antiviral state in

cultured CAB cells (crucian carp Carassius auratus blastulae embryonic cells) via the

production of interferon (IFN). In the current work, the full-length cDNAs of two Mx genes,

termed CaMx1 and CaMx2, have been cloned and sequenced from UV-inactivated GCHV-

infected and still IFN-producing CAB cells by suppression subtractive hybridization. Their

putative proteins show the characteristically structural features of mammalian IFN-induced

Mx proteins, including GTP-binding motif, dynamin family signature and leucine zipper

motif. CaMx1 exhibits 85% sequence identity to zebrafish MxA and 7274% to threeAtlantic salmon Mx proteins. CaMx2 is most similar to zebrafish MxE, with 80% identity,

and then rainbow trout Mx3, with 52%. Constitutive expression was detected by RT-PCR

for CaMx1, but not for CaMx2, in normal CAB cells, but their up-regulations could be

induced after treatment with active GCHV, UV-inactivated GCHV and CAB IFN. Distinct

kinetics of expression was observed for either CaMx1 or CaMx2 corresponding to the three

stimuli, and even between CaMx1 and CaMx2, corresponding to the same stimulus. Upon

virus infection, the transcriptional induction was strongly blocked for CaMx2 by

cycloheximide (CHX), whereas almost nothing was observed for CaMx1. By contrast,

following treatment with CAB IFN, CHX did not inhibit either gene transcription.

Collectively, these results suggest that there are very distinct mechanisms for modulating the

expression of both CaMx1 and CaMx2 in normal and GCHV-infected CAB cells.

Keywords Mx - Differential expression - Interferon - Antiviral-relevant genes - Innate

immunity

Nucleotide sequence data reported are available in the GenBank databases under the

following accession numbers: CaMx1 (AY303813), CaMx2 (AY303812)

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Fish & Shellfish Immunology

Volume 26, Issue 1, January 2009, Pages 91-108

doi:10.1016/j.fsi.2008.03.010 | How to Cite or Link Using DOI Cited By in Scopus (11)

http://www.sciencedirect.com/science/journal/10504648http://www.sciencedirect.com/science/journal/10504648http://www.sciencedirect.com/science?_ob=PublicationURL&_hubEid=1-s2.0-S1050464808X00129&_cid=272333&_pubType=JL&view=c&_auth=y&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=d8fffcae77782f5c1110312299b19469http://dx.doi.org/10.1016/j.fsi.2008.03.010http://www.sciencedirect.com/science?_ob=HelpURL&_file=doi.htm&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=cb97ff0c0cd0cf5e9c46d4a05370ec58http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=656&_eid=1-s2.0-S1050464808000806&_pii=S1050464808000806&_origin=article&_zone=art_page&_targetURL=http%3A%2F%2Fwww.scopus.com%2Finward%2Fcitedby.url%3Feid%3D2-s2.0-59349102675%26partnerID%3D10%26rel%3DR3.0.0%26md5%3Dff09adbb52fb1b6ac1e49fb81041169e&_acct=C000228598&_version=1&_userid=10&md5=e7cd41266002bee183b32f2b44ffb80chttp://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=656&_eid=1-s2.0-S1050464808000806&_pii=S1050464808000806&_origin=article&_zone=art_page&_targetURL=http%3A%2F%2Fwww.scopus.com%2Finward%2Fcitedby.url%3Feid%3D2-s2.0-59349102675%26partnerID%3D10%26rel%3DR3.0.0%26md5%3Dff09adbb52fb1b6ac1e49fb81041169e&_acct=C000228598&_version=1&_userid=10&md5=e7cd41266002bee183b32f2b44ffb80chttp://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=936&_eid=1-s2.0-S1050464808000806&_pii=S1050464808000806&_origin=article&_zone=art_page&_targetURL=https%3A%2F%2Fs100.copyright.com%2FAppDispatchServlet%3FpublisherName%3DELS%26contentID%3DS1050464808000806%26orderBeanReset%3Dtrue&_acct=C000228598&_version=1&_userid=10&md5=8e1018fad4f9edf16ea9d3efe303d105http://resources.metapress.com/pdf-preview.axd?code=jpbn4qnryu6attk4&size=largesthttp://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=936&_eid=1-s2.0-S1050464808000806&_pii=S1050464808000806&_origin=article&_zone=art_page&_targetURL=https%3A%2F%2Fs100.copyright.com%2FAppDispatchServlet%3FpublisherName%3DELS%26contentID%3DS1050464808000806%26orderBeanReset%3Dtrue&_acct=C000228598&_version=1&_userid=10&md5=8e1018fad4f9edf16ea9d3efe303d105http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=656&_eid=1-s2.0-S1050464808000806&_pii=S1050464808000806&_origin=article&_zone=art_page&_targetURL=http%3A%2F%2Fwww.scopus.com%2Finward%2Fcitedby.url%3Feid%3D2-s2.0-59349102675%26partnerID%3D10%26rel%3DR3.0.0%26md5%3Dff09adbb52fb1b6ac1e49fb81041169e&_acct=C000228598&_version=1&_userid=10&md5=e7cd41266002bee183b32f2b44ffb80chttp://www.sciencedirect.com/science?_ob=HelpURL&_file=doi.htm&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=cb97ff0c0cd0cf5e9c46d4a05370ec58http://dx.doi.org/10.1016/j.fsi.2008.03.010http://www.sciencedirect.com/science?_ob=PublicationURL&_hubEid=1-s2.0-S1050464808X00129&_cid=272333&_pubType=JL&view=c&_auth=y&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=d8fffcae77782f5c1110312299b19469http://www.sciencedirect.com/science/journal/10504648


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Immunological responses of turbot (Psetta maxima) to nodavirus infection or

polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags

(ESTs) analysis and cDNA microarrays

Kyoung C. Parka, ,

, Jane A. Osbornea, Ariana Montes

b, Sonia Dios

b, Audun

H. Nerlandc, Beatriz Novoab, Antonio Figuerasb, Laura L. Brownd, Stewart C. Johnsone

Purchase

a

Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street,Halifax, NS, B3H 3Z1, Canada

bInstituto Investigaciones Marinas CSIC, Eduardo Cabello, 6. 36208 Vigo, Spain

cInstitute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway

dBiodiversity Institute of Ontario, University of Guelph, 579 Gordon

Street, Guelph, Ontario, N1G 2W1, Canada

e The Atlantic Genome Centre, 1411 Oxford Street, Halifax, NS, B3H 3Z13, Canada

Received 14 December 2007; revised 11 March 2008; Accepted 16 March 2008. Available

online 26 March 2008.

Abstract

To investigate the immunological responses of turbot to nodavirus infection or pIC

stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-

infected fish and examined the differential gene expression within turbot kidney in response

to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot wereexperimentally infected with nodavirus and samples of each tissue were collected at selected

time points post-infection. Using equal amount of total RNA at each sampling time, we made

three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173

(98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568

(80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768

unique genes were identified.

The two largest groups resulting from the classification of ESTs according to function were

the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes).

Using these clones, a 1920 element cDNA microarray was constructed and used toinvestigate the differential gene expression within turbot in response to experimental
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nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-

infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of

the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed

genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those

stimulated with pIC. Within the nodavirus-infected fish we observed the highest number ofdifferentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have

important roles in immune responses to nodavirus infection and dsRNA stimulation.


Volume 26, Issue 1, January 2009, Pages 91-108



Immunological responses of turbot (Psetta maxima) to nodavirus infection or

polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags

(ESTs) analysis and cDNA microarrays

Kyoung C. Parka, ,

, Jane A. Osbornea, Ariana Montes

b, Sonia Dios

b, Audun

H. Nerlandc, Beatriz Novoa

b, Antonio Figueras

b, Laura L. Brown

d, Stewart C. Johnson

e

Purchase

aInstitute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street,

Halifax, NS, B3H 3Z1, Canada

bInstituto Investigaciones Marinas CSIC, Eduardo Cabello, 6. 36208 Vigo, Spain

cInstitute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway

dBiodiversity Institute of Ontario, University of Guelph, 579 Gordon

Street, Guelph, Ontario, N1G 2W1, Canada
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eThe Atlantic Genome Centre, 1411 Oxford Street, Halifax, NS, B3H 3Z13, Canada

Received 14 December 2007; revised 11 March 2008; Accepted 16 March 2008. Available

online 26 March 2008.

Abstract

To investigate the immunological responses of turbot to nodavirus infection or pIC

stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-

infected fish and examined the differential gene expression within turbot kidney in response

to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were

experimentally infected with nodavirus and samples of each tissue were collected at selected

time points post-infection. Using equal amount of total RNA at each sampling time, we made

three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173

(98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568

(80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768unique genes were identified.

The two largest groups resulting from the classification of ESTs according to function were

the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes).

Using these clones, a 1920 element cDNA microarray was constructed and used to

investigate the differential gene expression within turbot in response to experimental

nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-

infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of

the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed

genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those

stimulated with pIC. Within the nodavirus-infected fish we observed the highest number ofdifferentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have

important roles in immune responses to nodavirus infection and dsRNA stimulation.

Abstract- selected

Article

Figures/Tables

References


Volume 30, Issues 4-5, April-May 2011, Pages 1064-1071
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Anti-viral effects of interferon administration on sevenband grouper,Epinephelus

septemfasciatus

Takashi Ohtaa, b

, Yusuke Uedac, Katsutoshi Ito

c, Chiemi Miura

c, Hirofumi Yamashita

d,

Takeshi Miurac, Yuzuru Tozawa

a, b, c, ,

Purchase

aCell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho,

Matsuyama, Ehime 790-8577, Japan

bThe Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime

790-8577, Japan

cResearch Group for Reproductive Physiology, Southern Ehime Fisheries Research Center,

Ehime University, 1289-1 Funakoshi, Ainan, Ehime 798-4292, Japan

dEhime Prefectural Fisheries Experimental Station, Shitaba 5516, Uwajima, Ehime 798-

0104, Japan

Received 18 September 2010; revised 24 December 2010; Accepted 4 February 2011.

Available online 11 February 2011.

Abstract

Interferon (IFN) plays crucial roles in innate immune responses against viral infections. In the

present study, we report cloning and characterization of the IFN gene from the sevenband

grouper (Epinephelus septemfasciatus), and the anti-viral effects of its recombinant IFN

protein in vivo. The isolated cDNA from sevenband grouper IFN encoded a protein consisting

of 178 amino acids, and its first 22 amino acids represented a putative signal peptide. We

named the identified sevenband grouper IFN gene as SgIFNa1 based on the result from

phylogenetic analysis that categorized the deduced protein sequence into fish IFNa family.

The expression of SgIFNa1 mRNA in the head kidney cells was induced by synthetic

Poly(I:C), which is known as an inducer of IFN. It has also been confirmed that injection of

recombinant SgIFNa1 protein (rSgIFNa1) upregulates expression of the Mx gene, which is
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known as an IFN-responsive gene, in head kidney cells. Moreover, we observed that

preliminarily injection of rSgIFNa1 provided significant protection against a lethal challenge

of nervous necrosis virus (NNV), which is a serious disease of sevenband grouper. These

results demonstrate that SgIFNa1 has anti-viral activity and the administration of rSgIFNa1 to

sevenband grouper is effective in preventing severe symptom development after NNVinfection.

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United States Patent 6,171,591

Hall January 9, 2001

Recombinant nodavirus compositions and methods

Abstract

Recombinant nodavirus related compositions are disclosed. These compositions include

chimeric proteins in which a nodavirus capsid protein is present together with a heterologous

peptide segment. The heterologous peptide includes at least one cell-specific targeting

sequence, such as a B cell epitope, a T cell epitope, or a sequence specific for another cell

type, such as a hepatocyte. The chimeric proteins can be assembled to form chimeric virus-

like particles. The chimeric virus-like particles are useful in therapeutic applications, such as

vaccines and gene-delivery vectors, and in diagnostic applications, such as kits for the testing

of body tissue or fluid samples. Methods for the use of recombinant nodavirus related

compositions in therapeutic and diagnostic applications are also described.

Inventors: Hall; Stephen G. (San Diego, CA)

Assignee: Pentamer Pharmaceuticals, Inc. (Anaheim, CA)

The Scripps Research Institute (La Jolla, CA)

Appl. No.: 08/986,659

Filed: December 8, 1997

Current U.S. Class: 424/186.1 ; 424/192.1; 424/199.1; 424/224.1; 435/5;

530/350; 977/802; 977/882; 977/920

Current International Class: A61K 47/48 (20060101); C07K 14/08 (20060101);

C07K 14/005 (20060101); A61K 39/00 (20060101);

A61K 039/12 ()


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Field of Search: 530/350 424/186.1,192.1,199.1,224.1 435/5

References Cited

U.S. Patent Documents

4873192 October 1989 Kunkel

5149650 September 1992 Wertz et al.

5932426 August 1999 Baralle et al.

Foreign Patent Documents

WO 96/05293 Feb., 1996 WO

Other References

Kreis, Microinjected antibodies against the cytoplasmic domain of the vesicular

stomatitis virus glycoprotein block its transport to the cell surface. The EMBOJournal 5(5):931-941, 1986.*.

Primary Examiner: Wortman; Donna C.

Attorney, Agent or Firm: Olson & Hierl, Ltd.

Claims

I claim:

1. A chimeric protein which comprises a Flock House virus capsid protein free from

deletions, having a core structure constituted by anti-parallel beta barrels, and a heterologous

peptide segment in a loop formed by said capsid protein in the region between beta-E and

beta-F strands on one of said beta barrels.

2. An immunogenic composition comprising the chimeric protein of claim 1.
http://www.patents.com/us-4873192.htmlhttp://www.patents.com/us-5149650.htmlhttp://www.patents.com/us-5932426.htmlhttp://www.patents.com/us-5932426.htmlhttp://www.patents.com/us-5149650.htmlhttp://www.patents.com/us-4873192.html


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3. A diagnostic kit comprising the chimeric protein of claim 1.

4. A chimeric virus-like particle comprising a capsid including at least one chimeric protein

of claim 1.

5. An immunogenic composition comprising the chimeric virus-like particle of claim 4.

6. A method of inducing an immune response in an animal comprising the step of

administering an effective amount of a chimeric protein defined by claim 1 in a

pharmaceutically accetptable excipient.

7. A chimeric protein which comprises a Flock House virus capsid protein free from deletions

and including a heterologous peptide segment in the region between amino acid residues 205

and amino acid residue 209 from the amino terminus of said capsid protein, wherein said

chimeric protein can assemble into a virus-like particle.

8. The chimeric protein of claim 4 wherein the heterologous protein segment has an amino

acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID

NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ

ID NO:9.

9. The chimeric protein of claim 7 wherein the heterologous protein segment is represented

by SEQ ID NO:1.


by SEQ ID NO:2.


by SEQ ID NO:3.


by SEQ ID NO:4.


by SEQ ID NO:5.


by SEQ ID NO:6.


by SEQ ID NO:7.



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by SEQ ID NO:8.


by SEQ ID NO:9.

18. The chimeric protein of claim 7 wherein the heterologous peptide comprises at least one

cell-specific targeting sequence chosen from the group consisting of a B cell epitope, a T cell

epitope, a CTL epitope and a hepatocyte targeting sequence.

19. The chimeric protein of claim 7 wherein the heterologous peptide includes both a B cell

epitope and a T cell epitope.

20. An immunogenic composition comprising the chimeric protein of claim 7.

21. A diagnostic kit comprising the chimeric protein of claim 7.

22. A chimeric virus-like particle comprising a capsid including at least one chimeric protein

of claim 7.

23. An immunogenic composition comprising the chimeric virus-like particle of claim 22.

24. A method of inducing an immune response in an animal comprising the step of

administering an effective amount of a chimeric protein defined by claim 7 in a

pharmaceutically acceptable excipient.

Description

FIELD OF THE INVENTION

This invention relates to chimeric nodavirus related proteins such as antigenic peptides, and

uses thereof. This invention also relates to virus-like particles comprising chimeric nodavirus

related proteins and uses thereof.

BACKGROUND OF THE INVENTION

The immune system has several different mechanisms for dealing with pathogens in the

system (Parker, D. C. 1993. Annu. Rev. Immunol. 11:331-360; Clinical Immunology:

Principles and Practice. Vols. 1 and 2. eds (Fleisher et al. 1996. Mosby-Year Book, Inc. NewYork, N.Y.). The first step in the immune response is the activation of a special subclass of T


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lymphocytes called helper T cells. Macrophages present fragments of foreign proteins, or

antigens, on their surfaces. Recognition of these antigens by specialized receptors found on

helper T cells then initiates the two responses: a cell-mediated immune response and a

humoral immune response.

The cell-mediated response involves principally the stimulation of another subclass of T

lymphocytes called cytotoxic T lymphocytes (CTLs) that recognize and destroy infected

cells. HLA class-I restricted molecules bind to peptides that have been processed

intracellularly and enable CD8+T cells to eliminate intracellular viruses. CTLs are the second

line of immune defense since the humoral response generates antibodies that may be able to

neutralize virus and decrease the number of infected cells from the original infection. The

CD4+T (Th) cells often have a helper phenotype, which is measured by their ability to assist

the humoral response. The cells function to facilitate the effector function of other cells, and

while Th cells have limited antiviral activity, they play a major role in providing

intermolecular cooperation with antibody-producing B cells (Vitetta et al. 1989. Adv.

Immunol. 45:1; Hodes, R. J. p. 587 In: 1989. Fundamental immunology, eds Paul, W. E.).

The humoral response, on the other hand, involves the activation of the second major class of

lymphocytes, the B cells, to produce circulating antibodies. Antibodies recognize and

neutralize soluble antigens, and mark cells or viruses bearing antigens for destruction by

phagocytic cells. The HLA class-II molecules present the exogenous antigenic peptides to the

class-II restricted CD4+B cells. Antibodies serve as efficient means of reducing the viral

infectivity by several mechanisms. The antibody response to surface antigens on an invading

pathogen can be very strong, and the antibody may be able to decrease the titer of virusparticles significantly. In addition, antibodies can alter the structural integrity of the invading

pathogen through interaction with surface antigens, which can make the virus non-infectious.

Neutralization of the pathogen can also occur by preventing interaction with the cellular

receptor and/or preventing endocytosis into the cytoplasm (Ruggeri, F. M. and Greenberg, H.

B. J. Virol. 1991;2211-2219)Dimmock, N.J. In: Current Topics in microbiology and

immunology. 1993. Springer-Verlag: New York).

Development of effective vaccines has been one of the most decisive advances leading to the

dramatic downward trend in incidence of viral diseases. Vaccination induces a "primed" state

in the vaccinated subject so that, following exposure to an antigen, a rapid secondary immune

response is generated leading to accelerated elimination of the organism and protection from

clinical disease. Designing vaccines requires attention to the safety of the system as well as to

the antigenicity and efficacy of the prophylaxis.

It has been known for some time that humoral and cell-mediated responses to antigens can be

quite different. In general, B cell epitopes of antigens are longer, and are known as

conformational epitopes. Conformational epitopes require the proper 3-dimensional structure

for efficient recognition by antibodies (Elner et al. 1977. J. Immunol. 118:2053). In contrast,

T cells usually recognize small linear epitopes based on sequential information. Since

effective resistance to viral or bacterial infection requires the activities of both humoral and


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cellular components, it is important to optimize the presentation of antigens to both B and T

cells.

Despite recent advances in epitope presentation systems, there is still a need in the art for a

system that is genetically capable of expressing of B and T cell epitopes concurrently, as wellas presenting these epitopes to the immune system in the proper context.

There is evidence that simultaneous expression of T and B cell epitopes on the same carrier

system can enhance the antibody response through a cooperative mechanism between B and

T cells. For example, enhanced B cell vaccines to Hepatitis B are possible by stimulating

hepatitis B virus (HBV) envelope protein-specific B cells though a carrier system that mimics

HBV (Chisari, F. V. 1995. pp. 29-60 In: Annu. Rev. Immunol). If this carrier, or mimic,

carries Th cell epitopes, the subsequent processing of these Th epitopes can induce the

proliferation of B cells. For example, the HBV clearance depends on a vigorous and

polyclonal B cell and T cell response to the envelope, nucleocapsid, and polymerase antigens.

While the antibody responses to the HBV nucleocapsid and polymerase antigens are not well

understood, it is quite clear that the antibody response to HBV envelope antigen requires T-

cell cooperation. Despite the need for the vigorous immune response necessary to clear HBV,

the class-II restricted envelope-specific response is quite low in acute and chronic hepatitis.

The processing of Th epitopes can induce the proliferation of B cells and anti-envelope

antibodies. An efficient means of enhancing the cooperation between B and Th cells due to

increasing the exposure of B and Th cell epitopes via a carrier system would likely result in

disease remission in patients. There is evidence that enhanced B cell vaccines to Hepatitis B

are possible by stimulating HBV envelope-specific B cells though a carrier system thatmimics HBV by properly presenting HBV determinants to antigen presenting cells (APCs).

While there is a substantial response of Class I-restricted CTL in acute cases, the CTL

response to HBV is not easily detected in chronically infected patients who have been unable

to clear virus. The ability to induce vigorous CTL expansion along with B and Th cell

cooperation could have profound significance for the treatment of HBV infection as well as

many other infectious diseases and cancer.

There have been remarkable advances made in vaccination strategies recently, yet there

remains a need for improvement on existing strategies. Recombinant pathogenic viruses have

often induced the strongest humoral and cell-mediated responses, but the issues of safety and

potential interference with pre-existing immunity to the vectors have not made them an

attractive system for continued use. Peptide vaccines have been shown to be relatively safe

with a strong monofunctional immune response. Despite these advantages, they tend to be

poorly immunogenic and have a limited ability to bind selectively to different MHC

determinants found in genetically distinct populations of individuals. While it may be

possible to produce numerous peptides for formulation into a single vaccine, such an

undertaking presents a formidable task. Genetic immunization, or DNA vaccines, has shown

promise, but has not shown the ability to target APCs and produce a broad polyclonal

response. Despite these advances, the current technologies have not provided a system that


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was competent in the simultaneous expression of B and T cell epitopes that effectively prime

B and T cells of the immune system.

In these non-limiting examples, genetically-engineered Nodaviral or specifically the Flock

House Virus (FHV) chimeric coat protein constructs were made to include well-definedligands within the insertion region as well as encapsidation of therapeutic genes or antisense.

Gene delivery or gene therapy can be defined as the delivery of a functional gene (for

expression of a protein) or an antisense molecule (for blocking translation of a protein) to

somatic cells. See, for example, U.S. Pat. No. 5,589,466 to Felgner et al. and U.S. Pat. No.

5,676,954 to Brigham. For reviews see Mitani, K, and Caskey, C. T. (1993) TIBTECH

11:162-166, Findeis, M. A. et al. (1993) TIBTECH 11:202-205; Friedmann, T. (1994) TIG:

10:210-214, Smith, C. (1994) TIG: 10:139-144; Karpati et al. (1996) TIBS 19:49-54; Calos,

M. P. (1996) TIG: 12:463-466. Several gene delivery technologies that are being used to treat

a variety of diseases and acquired and genetic disorders are summarized in Table 1.

TABLE 1 Comparison of Gene Delivery Technologies Trans- duction Insert effi- Vector Size

Integration ciency Advantages Disadvantages Retrovirus 8 Yes High Stable Infects only kB

transfection rapidly divid- of dividing ing cells. Can cells. be oncogenic. Adenovirus 7.5 No

High Transfects Transient kB nearly all expression cell types triggers im- dividing or mune

response, nondividing. common human virus Adeno 4 Yes. High Stable Small insert

Associated kB (chr. 19) transfection. size, integra- Virus (AAV) tion poorly understood.

Helper virus required. Herpes 20 No Low Large insert Transient Simplex kB size. Neuron

expression, Virus (HSV) specific. potential to generate infectious HSV in humans Vaccinia25 No High Infects a Limited to non- kB variety of smallpox cells vaccinated or effectively.

immuno- compromised individuals. Liposomes 20 No N/A Large insert Transient kB size.

expression is disadvantage combined with variable delivery. Ballistic 20 No N/A Large insert

Requires ex- ("biollistic") kB size. posed tissue. Injection Plasmid 20 No N/A Large insert

Poor delivery. DNA kB size. Only sustained Injection expression in muscle.

Recombinant Flock House virus (FHV) proteins displaying viral antigens have been

described (Tisminetzky, S. G. et al., FEBS Lett. 353:1-4 (1994); Scodeller, E. A., et al.

Vaccine, 13:1233-1239 (1995); Buratti, E., et al., J. Immunol. Methods, 197:7-18 (1996);

Schiappacassi, M., et al., J. Virol. Methods, 63:121-127 (1997); Buratti, E., et al., Clin.

Diagn. Lab. Immunol., 4:117-12 (1997). See also Baralle, F. E. et al., PCT Published

Application WO 96/05293 (1996). These previous attempts suffer from difficulties in

forming a virus-like particle due to the deletion of amino acid residues in one or more regions

of the capsid protein, however.

What is needed are recombinant nodavirus related proteins that can incorporate heterologous

peptides as long as 100 amino acid residues or larger yet still be capable of self-assembly into

chimeric virus-like particles.

SUMMARY OF THE INVENTION


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The present invention provides a chimeric protein which comprises a nodavirus capsid (or

coat) protein free from any deletion, having a core structure constituted by anti-parallel beta

barrels, and a heterologous peptide segment situated between a pair of strands of a beta

barrel. A preferred chimeric protein is a Flock House virus capsid protein together with aheterologous peptide segment present in the capsid protein at a location between amino acid

residue 205 and amino acid residue 209 from the amino terminus of the capsid protein. All

amino acid residues normally present in the nodavirus capsid protein are retained. The amino

acid sequence of the heterologous peptide segment is chosen from cell-specific targeting

sequences such as B cell epitopes, T cell epitopes, and sequences targeting other cell types.

The heterologous peptide segment can have a size of up to about 100 amino acid residues.

An embodiment of the present invention is a nodavirus system for delivery of a biologically

active moiety, which system includes a chimeric capsid protein. The biologically active

moiety can be a direct immunostimulant, an indirect immunostimulant, a gene encoding a

direct immunostimulant, or a gene encoding an indirect immunostimulant. Chimeric proteins,

as well as nodavirus particles, conjugated to an antigenic protein can also serve as effective

immunostimulants or immunomodulators, and are useful for diagnostic as well as

immunizing purposes.

In accordance with the present invention, the need for nonpathogenic vectors as described

above has been satisfied by a novel viral-like based system that provides therapeutic

compositions including vaccines, as well as diagnostic embodiments, such as diagnostic kits.

Flock House virus (FHV) is one such nodavirus that can be used to genetically engineervirus-like particles carrying antigenic peptides on their surface. The basis for this system is

centered on the remarkable functional versatility of the FHV capsid protein. Extensive

computational chemical analysis of the high resolution atomic structure of FHV has led to the

identification of a region in the capsid protein that is amenable to insertion of heterologous

peptide segments without affecting assembly of the viral coat or capsid. The predictions from

the structural and computational studies have been used to genetically construct FHV-like

chimeric virus-like particles that contain well-defined antigenic determinants as inserted

heterologous peptides. These inserted heterologous peptides include B cell epitopes that are

presented on the surface of the carrier in the proper conformation to generate a humoral

response. Other heterologous peptide segments, such as those containing T cell epitopes,

have been expressed on the surface of such chimeric virus-like particles, producing a

structure that generates a proliferative and CTL response. Heterologous peptide segments

comprising contiguous B cell epitopes and T cell epitopes inserted to form chimeric proteins

have demonstrated enhanced immunogenicity.

BRIEF DESCRIPTION OF THE DRAWINGS

In the Drawings,

FIG. 1 shows the tertiary structure of the FHV coat protein subunit.


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FIG. 2 shows predicted the secondary structure that represent the encapsidation signal in

RNA 2 of several nodaviruses.

FIG. 3 shows the grafting of FHV encapsidation signal to the end of a gene of therapeuticinterest, human interferon-.gamma..

FIG. 4 shows the p2BS(+)-wt sequence immediately flanking the RNA2 insert, determined

by sequencing the ends of p2BS(+)-wt, p2BS(+)-RNA2:VSV-G #1, p2BS(+)-RNA2:BRSV

#1.

FIG. 5 shows the sequence of p2BS(+)-RNA2:VSV-G constructs.

FIG. 6 shows the sequences of the RNA2:VSV-G mutagenic primer and the hybridization

probe.

FIG. 7 shows the sequence of p2BS(+)-RNA2:BRSV constructs.

FIG. 8 shows the sequence of the RNA2:BRSV mutagenic primer.

FIG. 9 shows the scheme of the construction of the pVL1392-RNA2, pVL1392-RNA2:VSV-

G & pVL1392-RNA2:BRSV baculovirus expression vectors.

FIG. 10 shows the sequences of primers For subcloning RNA2 constructs into pVL1392.

FIG. 11 shows the sequences of p2BS(+)-RNA2:HBV constructs.

FIG. 12 shows the scheme of the construction of the functional RNA2:CSP fusion construct.

FIG. 13 shows the sequences of RNA2 and other sequencing primers.

FIG. 14 shows an electron micrograph of chimeric FHV particles with an VSV epitope

inserted into the coat protein. An epitope was derived from Vesicular Stomatitis Virus (VSV)

to generate a FHV:VSV chimeric particle. The epitope

TyrThrAspIleGluMetAsnArgLeuGlyLys (SEQ ID NO:1) was inserted into the FHV coat

protein gene and introduced into the baculovirus expression system. Panel A shows chimeric

virus-like particles that were isolated from baculovirus-infected S. frugiperda cells and

subjected to electron microscopy using standard negative staining conditions

(Magnification.times.39,000). Note the icosahedral shape of the stable chimeric virions.

Panel B shows chimeric FHV:VSV chimeric particles were isolated from baculovirus-

infected S. frugiperda cells and subjected to immuno-electron microscopy

(Magnification.times.39,000). The FHV:VSV chimeric particles are decorated with the

monoclonal antibody P5D4 (MAb P5D4) against the aforementioned 11-residue VSV epitope

(SEQ ID NO:1) which was inserted into the FHV coat protein. MAb P5D4 is of subtype


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IgG.sub.1 k and can be seen binding to the VSV epitope, which gives a dark halo-like

appearance to the chimeric particles. The antibody also induces mild aggregation due to the

presence of IgG.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention uses members of the Nodaviridae virus family, commonly known as

nodaviruses, such those listed in Table 2, below, to produce chimeric proteins capable of self-

assembly into chimeric virus-like particles.

TABLE 2 Nodaviruses Propagation in Member Cell Culture Protein Expression Systems

Nodamura virus (NV) BHK-21, Baculovirus and E. coli. mosquito cells expression systems

Flock House virus (FHV) Drosophila Baculovirus and E. coli. cells, Black expression

systems Black Beetle virus (BBV) Beetle cells, Boolarra virus (BoV) protoplasts Gypsy moth

virus (GMV) Manawatu virus (MwV)

Suitable nodaviruses include Nodamura virus (NV), Flock House virus (FHV), Black Beetle

virus (BBV), Boolarra virus (BoV), Gypsy moth virus (GMV), and Manawatu virus (MwV).

A preferred nodavirus is the Flock House virus (FHV).

The structure of the Flock House virus coat protein, also known as the capsid protein, is

shown in FIG. 1. The amino acid residue sequence, left-to-right in the direction from the

amino or N-terminus to the carboxy or C-terminus of this protein is as follows:

(SEQ ID NO:10) Met Val Asn Asn Asn Arg Pro Arg Arg Glu Arg Ala Glu Arg Val Val Val

Thr Thr Thr Glu Thr Ala Pro Val Pro Glu Glu Asn Val Pro Arg Asn Gly Arg Arg Arg Arg

Asn Arg Thr Arg Arg Asn Arg Arg Arg Val Arg Gly Met Asn Met Ala Ala Leu Thr Arg Leu

Ser Gln Pro Gly Leu Ala Phe Leu Lys Cys Ala Phe Ala Pro Pro Asp Phe Asn Thr Asp Pro

Gly Lys Gly Ile Pro Asp Arg Phe Glu Gly Lys Val Val Ser Arg Lys Asp Val Leu Asn Gln

Ser Ile Ser Phe Thr Ala Gly Gln Asp Thr Phe Ile Leu Ile Ala Pro Thr Pro Gly Val Ala Tyr

Trp Ser Ala Ser Val Pro Arg Gly Thr Phe Pro Thr Ser Ala Thr Thr Phe Asn Pro Val Asn Tyr

Pro Gly Phe Thr Ser Met Phe Gly Thr Thr Ser Thr Ser Arg Ser Asp Gln Val Ser Ser Phe Arg

Tyr Ala Ser Met Asn Val Gly Ile Tyr Pro Thr Ser Asn Leu Met Gln Phe Ala Gly Ser Ile Thr

Val Trp Lys Cys Pro Val Lys Leu Ser Thr Val Gln Phe Pro Val Ala Thr Asp Pro Ala Thr Ser

Ser Leu Val His Thr Leu Val Gly Leu Asp Gly Val Leu Ala Val Gly Pro Asp Asn Phe Ser

Glu Ser Phe Ile Lys Gly Val Phe Ser Gln Ser Ala Cys Asn Glu Pro Asp Phe Glu Phe Asn Asp

Ile Leu Glu Gly Ile Gln Thr Leu Pro Pro Ala Asn Val Ser Leu Gly Ser Thr Gly Gln Pro Phe

Thr Met Asp Ser Gly Ala Glu Ala Thr Ser Gly Val Val Gly Trp Gly Asn Met Asp Thr Ile Val

Ile Arg Val Ser Ala Pro Glu Gly Ala Val Asn Ser Ala Ile Leu Lys Ala Trp Ser Cys Ile Glu

Tyr Arg Pro Asn Pro Asn Ala Met Leu Tyr Gln Phe Gly His Asp Ser Pro Pro Leu Asp Glu

Val Ala Leu Gln Glu Tyr Arg Thr Val Ala Arg Ser Leu Pro Val Ala Val Ile Ala Ala Gln Asn

Ala Ser Met Trp Glu Arg Val Lys Ser Ile Ile Lys Ser Ser Leu Ala Ala Ala Ser Asn Ile Pro

Gly Pro Ile Gly Val Ala Ala Ser Gly Ile Ser Gly Leu Ser Ala Leu Phe Glu Gly Phe Gly Phe


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The nucleotide sequence for the foregoing amino acid residue sequence is known, and is

described by Dasgupta, R. et al., Nucleic Acids Res. 17(18):7525-7526 (1989).

The core of the structure is made up of eight stranded anti-parallel beta-barrels, as seen in

many other viral capsid proteins. The helical domain is made up of 3 alpha-helices, formedby the polypeptide chain located sequentially, `N` and `C` terminal to the beta-barrel. The

helix is the gamma-peptide, one of the cleavage products. A region between amino acids 205-

209 from the "N" terminus forms a loop that is exposed on the exterior surface of the

assembled capsid. The formed loop is between the .beta.E-.beta.F strands of a beta barrel.

The insertion itself forms a pair of .beta.-strands, .beta.E'-.beta.", with a short loop

therebetween, as can be seen in FIG. 1.

The FHV genome contained within the capsid includes two messenger-sense RNA

molecules, RNA 1 and RNA 2 (Schneemann, A., et al. 1993. In W. Doerfler and P. Bhm

(eds.), Viral Strategies, Verlag Chemie, Weinheim, Germany. p. 167-176). RNA 1 (3.1 kb)

directs synthesis of protein A (102 kDa) the putative RNA-dependent RNA polymerase

(Fisher, A. J. and J. E. Johnson. 1993. Nature (London) 361:176-179). RNA 2 (1.4 kb)

encodes protein alpha (43 kDa), the precursor of the coat protein (Gallagher, T. M. and R. R.

Rueckert. 1988. J. Virol. 62:3399-3406). In addition to the genomic RNAS, infected cells

also contain a subgenomic RNA 3 (0.4 kb) derived from the 3' end of RNA 1. It encodes

protein B (10 kDa), whose function is to modulate replication (Ball, L. A. (1994) PNAS

91:12443-12447; Ball, L. A. (1995) J. Virol. 69:720-727).

A specific region of FHV RNA 2 (bases 186-217) has a predicted stem-loop structure (FIG.2), which serves as the packaging signal for in vivo encapsidation of RNA 2 (Zhong, W.,

Dasgupta, R., and Rueckert, R. 1992. Proc. Natl. Acad. Sci. USA 89:11146-11150). Similar

regions of the other Nodaviral RNA 2 sequences are also shown in FIG. 2 and serve an

identical function. The initial step is believed to involve the formation of a nucleating

complex, in which a coat protein substructure interacts with this encapsidation signal on the

viral RNA. The initiation complex may then be propagated into a spherical particle by

addition of coat protein subunits, which are guided into the growing shell by binding to the

viral RNA.

Flock House virus (FHV) can be grown to high titers in Drosophila cell culture with synthesis

of proteins A and B peaking at about 5 hours and 8 hours, respectively (Schneemann, A., et

al. 1993. In W. Doerfler and P. Bhm (eds.), Viral Strategies, Verlag Chemie, Weinheim,

Germany. p. 167-176). In contrast, synthesis of protein alpha remains low during the first 12

hours but rises rapidly thereafter with peak production at about 15 hours. The newly

synthesized alpha chains are assembled within minutes into labile precursor particles, called

provirions. Provirions contain 180 alpha subunits, arranged with icosahedral symmetry, as

well as a copy of each of the genomic RNA molecules. The assembly process triggers an

autoproteolytic reaction in the 407 amino acid alpha chain which results in cleavage between

asparagine 363 and alanine 364 (Hosur, M. V. et al. 1987. Proteins: Struc. Funct. Genet.

2:167-176; Fisher, A. J. and J. E. Johnson. 1993. Nature (London) 361:176-179).


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The newly formed polypeptides, beta (38 kDa, 363 amino acids) and gamma (5 kDa, 44

amino acids), remain associated with the mature virion.

Following injection of wild-type FHV, antibody formation occurs without symptoms ordisease in pigs, adult mice, rabbits, guinea pigs, Syrian hamsters, and chickens. In addition,

there is no cytopathology in mammalian cell culture lines including primate kidney and

human amino cells (Hendry, D. A. 1991. In Viruses of Invertebrates (ed. E. Kurstak) Marcel

Dekker, Inc.: New York).

The structure of FHV was solved to atomic resolution and shows that the viral capsid is

composed of 60 triangular units, which consist of three identical polypeptides related by

icosahedral symmetry (Hosur, M. V. et al., 1987. Struc. Funct. Genet. 2:167-176). All three

subunits, designated A, B and