Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

doi:10.1016/S1671-2927(11)60178-8

摘要/Abstract

摘要： Classical swine fever (CSF) and porcine reproduction and respiratory syndrome (PRRS) are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus (PRRSV) and the E2 protein of classical swine fever virus (CSFV). Foot-and-mouth disease virus (FMDV) 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1:128 and a PRRSV-neutralizing antibody titer of 1:16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

关键词: porcine reproductive and respiratory syndrome virus, classical swine fever virus, recombinant adenovirus, immunogenicity

Abstract: Classical swine fever (CSF) and porcine reproduction and respiratory syndrome (PRRS) are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus (PRRSV) and the E2 protein of classical swine fever virus (CSFV). Foot-and-mouth disease virus (FMDV) 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1:128 and a PRRSV-neutralizing antibody titer of 1:16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

Key words: porcine reproductive and respiratory syndrome virus, classical swine fever virus, recombinant adenovirus, immunogenicity

LI Hong-yu, SUN Yuan, ZHANG Xing-juan, CHANG Tian-ming, WANG Xiang-peng, HE Fan, HUANG Junhua , QIU Hua-ji . Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus [J]. Journal of Integrative Agriculture, 2011, 10(11): 1781-1791.

参考文献

[1]Ambriovic A, Adam M, Monteil M, Paulin D, Eloit M. 1997. Efficacy of replication-defective adenovirus-vectored vaccines: protection following intramuscular injection is linked to promoter efficiency in muscle representative cells. Virology, 238, 327-335.

[2]Bastos R G, Dellagostin O A, Barletta R J, Doster A R, Nelson E, Osorio F A. 2002. Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive and respiratory syndrome virus. Vaccine, 21, 21-29.

[3]Bilodeau R, Dea S, Martineau G P, Sauvageau R. 1991. Porcine reproductive and respiratory syndrome in Quebec. Veterinary Record, 129, 102-103.

[4]Chiuchiolo M J, Boyer J L, Krause A, Senina S, Hackett N R, Crystal R G. 2006. Protective immunity against respiratory tract challenge with Yersinia pestis in mice immunized with an adenovirus-based vaccine vector expressing V antigen. Journal of Infectious Disease, 194, 1249-1257.

[5]Conzelmann K K, Visser N, van Woensel P, Thiel H J. 1993. Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology, 193, 329-339.

[6]Dea S, Gagnon C A, Mardassi H, Pirzadeh B, Rogan D. 2000. Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Achieves of Virology, 145, 659-688.

[7]Donnelly M L L, Luke G, Mehrotra A, Li X J, Hughes L E, Gani D, Ryan M D. 2001. Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. Journal of General Virology, 82, 1013-1025.

[8]Dong X N, Chen Y H. 2007. Marker vaccine strategies and candidate CSFV marker vaccines. Vaccine, 25, 205-230.

[9]EU. 2002. EU Diagnostic Manual for Classical Swine Fever (CSF) Diagnosis: Technical Part (Second Draft March 2002). [2002-9-2]. http://viro08.tiho-hannover.de/eg/TechnicalPart4. pdf pp. 28-35.

[10]Fauquet C M, Mayo M A, Maniloff J, Desselberger U, Ball L A. 2005. Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press. de Felipe P, Hughes L E, Ryan M D, Brown J D. 2003. Cotranslational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide. Journal of Biological Chemistry, 278, 11441-11448.

[11]Graham B S, Koup R A, Roederer M, Bailer R T, Enama M E, Moodie Z, Martin J E, McCluskey M M, Chakrabarti B K, Lamoreaux L, et al. 2006. Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector. Journal of Infectious Disease, 194, 1638-1649.

[12]Graham F L, Smiley J, Russell W C, Nairn R. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus 5. Journal of General Virology, 36, 59-74.

[13]Greiser-Wilke I, Blome S, Moennig V. 2007. Diagnostic methods for detection of classical swine fever virus-status quo and new developments. Vaccine, 25, 5524-5530.

[14]Han J, Wang Y, Faaberg K S. 2006. Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus. Virus Research, 122, 175-182.

[15]Hou Q, Peng W P, Sun Y, Qiu H J. 2008. Expression of the truncated E2 protein of classical swine fever virus in Escherichia coli and preparation of a monoclonal antibody against the E2 protein. Veterinary Science in China, 38, 6-10. (in Chinese)

[16]Jiang W M, Jiang P, Li Y F, Tang J Y, Wang X W, Ma S. 2006. Recombinant adenovirus expressing GP5 and M fusion proteins of porcine reproductive and respiratory syndrome virus induce both humoral and cell-mediated immune responses in mice. Veterinary Immunology and Immunopathology, 113, 169-180.

[17]Kim S J, Sung H W, Han J H, Jackwood D, Kwon H M. 2004. Protection against very virulent infectious bursal disease virus in chickens immunized with DNA vaccines. Veterinary Microbiology, 101, 39-51.

[18]Lengler J, Holzmüller H, Salmons B, Günzburg W H, Renner M. 2005. FMDV-2A sequence and protein arrangement contribute to functionality of CYP2B1-reporter fusion protein.

[19]Analatical Biochemistry, 343, 116-124.

[20]Li Y F, Jiang P, Jiang W M, Tang J Y. 2006. Construction and immunogenecity of GP5 recombinant adenovirus of porcine reproductive and respiratory syndrome virus. Virologica Sinica, 21, 364-367. (in Chinese)

[21]Li Y F, Wang X L, Bo K T, Wang X W, Tang B, Yang B S, Jiang W M, Jiang P. 2007. Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China. Veterinary Journal, 174, 577-584.

[22]Mattion N M, Harnish E C, Crowley J C, Reilly P A. 1996. Foot-and-mouth disease virus 2A protease mediates cleavage in attenuated Sabin 3 poliovirus vectors engineered for delivery of foreign antigens.Journal Virology, 70, 8124-8127.

[23]Meulenberg J J M, de Meijer E J, Moonen R J. 1993. Subgenomic RNAs of Lelystad virus contain a conserved leader-body junction sequence. Journal of General Virology, 74, 1697-1701.

[24]Meulenberg J J M, Petersen-Den B A, de Kluyvier E P, Moormann R J, Schaaper W M, Wensvoort G. 1995. Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology, 206, 155-163.

[25]Meyers G, Thiel H J. 1996. Molecular characterization of pestiviruses. Advances in Virus Research, 47, 53-118.

[26]Miyamoto T, Min W, Lillehoj H S. 2002. Lymphocyte proliferation response during Eimeria tenella infection assessed by new, reliable nonradioactive colorimetric assay. Avian Diseases, 46, 10-16.

[27]Moennig V. 2000. Introduction to classical swine fever virus, disease and control policy. Veterinary Microbiology, 73, 93-102.

[28]van Oirschot J T. 2003. Vaccinology of classical swine fever: from lab to field. Veterinary Microbiology, 96, 367-384.

[29]Pacheco J M, Brum M C S, Moraes M P, Golde W T, Grubman M J. 2005. Rapid protection of cattle from direct challenge with foot-and-mouth disease virus (FMDV) by a single inoculation with an adenovirus-vectored FMDV subunit vaccine. Virology, 337, 205-209.

[30]Peng W P. 2007. Epitope identification of classical swine fever virus E2 protein by phage display. MSc thesis, Chinese Academy of Agricultural Sciences. (in Chinese) Qiu H J, Tian Z J, Tong G Z, Zhou Y J, Ni J Q, Luo Y Z, Cai X H. 2005. Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets. Veterinary Immunology and Immunopathology, 106, 309-319.

[31]Ryan M D, Donnelly M, Lewis A, Mehrotra A P, Wilkie J, Gani D. 1999. A model for nonstoichiometric, cotranslational protein scission in eukaryotic ribosomes. Bioorganic Chemistry, 27, 55-79. (in Chinese)

[32]Santosuosso M, McCormick S, Xing Z. 2005. Adenoviral vectors for mucosal vaccination against infectious diseases. Viralal Immunology, 18, 283-291.

[33]Sullivan N J, Geisbert T W, Geisbert J B, Xu L, Yang Z Y, Roederer M, Koup R A, Jahrling P B, Nabel G J. 2003. Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates. Nature, 424, 681-684.

[34]Sun Y, Liu D F, Wang Y F, Liang B B, Cheng D, Li N, Qi Q F, Zhu Q H, Qiu H J. 2010. Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus. Research in Veterinary Science, 88, 77-82.

[35]Sun Y, Xia Z H, Liang B B, Peng W P, Qiu H J. 2008. Development of an indirect ELISA for detecting antibodies against classical swine fever virus based on the baculovirus expressed E2 protein. Veterinary Science in China, 38, 315-319. (in Chinese)

[36]Szymczak A L, Workman C J, Wang Y, Vignali K M, Dilioglou S, Vanin E F, Vignali D A A. 2004. Correction of multi-gene defciency in vivo using a single ‘self-cleaving’ 2A peptidebased retroviral vector. Nature Biotechnology, 22, 589-594.

[37]Tan Y, Hackett N R, Boyer J L, Crystal R G. 2003. Protective immunity evoked against anthrax lethal toxin after a single intramuscular administration of an adenovirus-based vaccine encoding humanized protective antigen. Human Genetic Therapy, 14, 1673-1682.

[38]Tatsis N, Ertl H C. 2004. Adenoviruses as vaccine vectors. Molecular Therapy, 10, 616-629.

[39]Tong G Z, Zhou Y J, Hao, X F, Tian Z J, An T Q, Qiu H J. 2007. Highly pathogenic porcine reproductive and respiratory syndrome, China. Emerging Infectious Diseases, 13, 1434-1436.

[40]de Vries A A, Post S M, Raamsman M J, Horzinek M C. Rottier P J. 1995. The two major envelope proteins of equine arteritis virus associate into disulfide-linked heterodimers. Journal of Virology, 69, 4668-4674.

[41]Wensvoort G, de Kluyver E P, Pol J M, Wagenaar F, Moormann R J, Hulst M M, Bloemraad R, den Besten A, Zetstra T, Terpstra C. 1992. Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at Lelystad. Veterinary Microbiology, 33, 185-193.

[42]Yun T, Ni Z, Yu B, Chen L, Hua J G, Wang G R, Liu G Q. 2009. Construction and immunogenicity of recombinant adenovirus co-expressing the GP5 and M protein of porcine reproduction and respiratory syndrome virus in mice. Chinese Journal of Biotechnology, 25, 488-495. (in Chinese)

[43]Zhao H P, Sun J F, Li N, Sun Y, Xia Z H, Wang Y, Qiu H J. 2009. Assessment of the cell-mediated immunity induced by alphavirus replicon-vectored DNA vaccines against classical swine fever in a mouse model. Veterinary Immunology and Immunopathology, 129, 57-65.

[44]Zhao J J, Cheng D, Li N, Sun Y, Shi Z X, Zhu Q H, Tu C C, Tong G Z, Qiu H J. 2008. Evaluation of a multiplex real-time RTPCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of classical swine fever virus. Veterinary Microbiology, 126, 1-10.

[45]Zhou Y J, An T Q, Xue Q, Qiu H J, Tong G Z, Liu J X, Tian Z J, Wang Y F, Peng J M. 2005. Expression of GP5 gene of PRRSV CH-1a strain and development of monoclonal antibodies against GP5. Veterinary Science in China, 11, 859-864. (in Chinese)