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1、Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein A.M.H. Boots*, B.J. Benaissa-Trouw+, W. Hesselink:, E. Rijke:, C. Schrierf and E.J. Hensen*O Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein wer
2、e studied using a recombinant-DNA expression product. In mice, a lymphocyte proltferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a prot
3、ective immune response to IBV in chickens. Chickens were primed with nucleocapsidprotein and subsequently boosted with inactivated IBV, strain M41. Proltferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finall
4、y, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsidprotein is a relevant target for immune recognition in both the mouse and the chicken. Keywords: Infectious bronchitis virus; mice; chickens; nucleoca
5、psid; recombinant DNA INTRODUCTION Infectious bronchitis virus (IBV) is the prototype of the Coronaviridae. The virus consists of a lipid-containing membrane, a single-stranded RNA genome and three structural proteins. Apart from the internally localized nucleocapsid protein (N), the virus consists
6、of two glycoproteins anchored in the lipid membrane,2. The integral matrix protein (M ) protrudes only slightly from the membrane in contrast to the spike protein (S) exposed at the surface which gives the virus its coronaviral image. The virus can cause an acute respiratory disease in young chicken
7、s and a reduction in egg production in laying hens. Attention has been focused primarily on responses to the S protein. This was based on the observation that neutralizing antibody showed specificity for the S protein3. By generating antigenic variants of the S protein the virus is capable of avoidi
8、ng elimination by virus neutralizing antibody4. These distinct antigenic variations pose a problem in IBV vaccine design5. To circumvent the problem of the observed antigenic variability of the S protein we directed our attention to the IBV N protein which is more conserved among IBV strainsv6. Obse
9、rvations in other *Department of Immunology, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, PO Box 80.165, 3508 TD Utrecht, The Netherlands, +Section of Experimental Virology, Eijkman-Winkler Labora- tory, Utrecht, The Netherlands. llntervet I
10、nternational BV, PO Box 31, 5830 AA Boxmeer, The Netherlands. “To whom correspondence should be addressed. (Received 29 May 1991; revised 23 August 1991; accepted 17 September 1991) 0264410X/92/0201 1 SO6 c) 1992 Butterworth-Heinemann Ltd pathogenic virus systems indicate that internal viral antigen
11、s can contribute significantly to the induction of protective immunity7-9. Protection is not only induced by generating cytotoxic T cells (36), but also by generating T helper cell responses that augment the activity of B cells in production of virus-neutralizing antibody . l-l 3 Recently, we have s
12、hown that two murine CD4-positive T cell hybridomas generated from an IBV-specific T cell line were responsive to N proteins of several IBV strains14. Now the immunogenicity of recombinant-DNA encoded N protein in relation to the cellular immune response to IBV was studied. First it was shown that d
13、elayed-type hypersensitivity (DTH) and lymphocyte proliferative responses to IBV were induced upon immunization of mice with the expressed N protein. The purpose of the work described in this paper was secondly, to assess the role of the N protein in induction of cellular immune responses to IBV in
14、the chicken, thirdly to test whether the N protein can accelerate the induction of virus-neutralizing (VN) and haemagglutin- ation-inhibition (HI) antibodies, and finally to ascertain whether priming of chickens with the N protein results in increased tracheal protection against challenge with IBV.
15、MATERIALS AND METHODS Antigens IBV strain M41 was obtained from egg-grown virus and gradient purified as described”. The M42 strain, an IBV laboratory strain, was grown in Vero cells Vaccine, Vol. 10, Issue 2, 1992 119 Immunization with recombinant-DNA encoded infectious bronchitis (multiplicity of
16、infection 0.1 )16. Supernatant of infected cells was harvested after 36 h and stored at -80C. Expression of the nucleocapsid fusion protein The IBV nucleocapsid pEX clone was constructed as described by Kusters 17. Briefly, the DNA encoding the N protein was isolated from the IBV M41 cDNA library and thereafter cut with restriction enzymes and cloned in the expression vector pEX TM. The recombinant plasmid was expressed in Escherichia coll. In this system heterologous expressio
