Immunology 1991 72 10-14
ADONIS
001928059100003E
MHC class II-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavius IBV A. M. H. BOOTS,*t M. J. VAN LIEROPt J. G. KUSTERS,*t P. J. S. VAN KOOTENt B. A. M. VAN DER ZEIJSTt & E. J. HENSENt *Institute of Molecular Biology and Medical Biotechnology, and tDepartments of Immunology and Bacteriology, Institute of Infectious Diseases and Immunology, School of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands
Acceptedfor publication 7 September 1990
SUMMARY Mice were immunized with purified infectious bronchitis virus (IBV), strain M41. Spleen cells, expanded in vitro by stimulation with M41, were immortalized by fusion to obtain T-cell hybridomas, and two major histocompatability complex (MHC) class II (I-E)-restricted T-cell hybridomas were selected with specificity for IBV. Both hybridomas selectively recognized the internal nucleocapsid protein. The responses to 12 different strains of IBV varied markedly. This demonstrates antigenic variation of the nucleocapsid protein in addition to the known variation of the surface glycoprotein S.
protein of IBV in the context of major histocompatibility complex (MHC) class II. This is the first identification of a coronaviral antigen recognized by T cells.
INTRODUCTION Infectious bronchitis virus (IBV), the type species of the Coronaviridae, is the causative agent of an acute respiratory disease in chickens. The positive stranded RNA genome of IBV encodes three structural proteins, two N-glycosylated envelope proteins (S and M) and a non-glycosylated nucleocapsid protein (N). The S or spike proteins give the virus its typical corona-like appearance. The two structurally unrelated subunits, Sl and S2, with relative molecular masses of 90,000 and 84,000 MW, protrude from the viral envelope. 1,2 The N protein, a 56,000 MW phosphoprotein, is closely associated with the viral RNA.3 The M or matrix protein varies in molecular mass from 27,000 to 36,000 MW, due to heterogeneous glycosylation.' Many serotypes of IBV have been defined on the basis of cross-neutralization studies, and five different serotypes (A to E) of the Dutch IBV strains have been recognized (Table 1).4 There is a constant emergence of new serotypes, generated by various mechanisms, posing a serious problem in vaccination programmes.5'6'34 Changes in serotype are generally correlated with changes in the spike protein which contains the determinants recognized by virus neutralizing antibodies.78 Little is known about the role of the N and M proteins in the induction of protective immunity. For effective activation of the humoral immune response, determinants recognized by both B cells and T-helper cells are required.9 Therefore it might be crucial that a vaccine contains viral antigenic determinants recognized by both cell populations. We report here on T-cell hybridomas recognizing the N
MATERIALS AND METHODS Virus growth and purification All strains, unless otherwise mentioned, were obtained from the Poultry Health Institute in Doorn, The Netherlands. New Castle disease virus (NCDV) was used as a control. All virus stocks were obtained from egg-grown virus and gradientpurified as described elsewhere.'0 The M42 laboratory strain was grown on Vero cells (multiplicity of infection 0-1) and the supernatant of the infected cells was harvested after 36 hr. Gradient-purified mouse hepatitis virus (MHV) strain A59 was kindly donated by Dr W. Spaan, University of Utrecht, The Netherlands. Immunization and generation of IB V-specific T-cell hybridomas To obtain T-cell hybridomas, an IBV-specific T-cell line was generated and subsequently immortalized by fusion. Two 6-8week-old BALB/c mice were immunized intraperitoneally with a mixture of the Ribi Adjuvant System (RAS; Immunochemical Research, Inc. Hamilton, MT) and 100 ug of the gradientpurified, UV-inactivated virus of the M41 strain of IBV. " Three weeks later, the mice were boosted with the same dose and 6 weeks thereafter spleen cells of these mice were cultured for 3-4 days at a concentration of 2 x 106 cells/ml in Iscove's modification of Dulbecco's medium (Gibco, Breda, The Netherlands) supplemented with 10% foetal calf serum (Gibco), antibiotics and 2-mercaptoethanol (2 x 10-I M) in the presence of M41 antigen (4 pg/ml). Subsequently, the cells were washed and resuspended in 1% conditioned medium.'2 Seven days later
Correspondence: Dr E. J. Hensen, Dept. of Immunology, Institute of Infectious Diseases and Immunology, School of Veterinary Medicine, University of Utrecht, P.O. Box 80.165, TD Utrecht, The Netherlands.
10
T-cell hybridomas specific for avian coronavirus IBV Table 1. IL-2 response of MJBIOO and MJB1OI to 12 IBV strains belonging to various serotypes
I
Table 2. MHC class II-specific response of Tcell hybridomas to 5 pg/ml of IBV strain M41
antigen
Strain
Serotype
M41 M42 H52 H120 V1259 V1385 V1397 D207 D274 D212 D1466 D3128
A A A A
IL-2 response (SI) S2 S1 Genotype group group MJBIOO MJBO1I
IL-2 response (SI) L cell transfectants
A/C B B/E C C D
1 1 2 2 2 2 2 3 3 3 3 3
a a
a
b c c
I I
-
53 6 53 3 320 6-0 359 29-8 12-4
I I
ADONIS
001928059100003E
MHC class II-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavius IBV A. M. H. BOOTS,*t M. J. VAN LIEROPt J. G. KUSTERS,*t P. J. S. VAN KOOTENt B. A. M. VAN DER ZEIJSTt & E. J. HENSENt *Institute of Molecular Biology and Medical Biotechnology, and tDepartments of Immunology and Bacteriology, Institute of Infectious Diseases and Immunology, School of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands
Acceptedfor publication 7 September 1990
SUMMARY Mice were immunized with purified infectious bronchitis virus (IBV), strain M41. Spleen cells, expanded in vitro by stimulation with M41, were immortalized by fusion to obtain T-cell hybridomas, and two major histocompatability complex (MHC) class II (I-E)-restricted T-cell hybridomas were selected with specificity for IBV. Both hybridomas selectively recognized the internal nucleocapsid protein. The responses to 12 different strains of IBV varied markedly. This demonstrates antigenic variation of the nucleocapsid protein in addition to the known variation of the surface glycoprotein S.
protein of IBV in the context of major histocompatibility complex (MHC) class II. This is the first identification of a coronaviral antigen recognized by T cells.
INTRODUCTION Infectious bronchitis virus (IBV), the type species of the Coronaviridae, is the causative agent of an acute respiratory disease in chickens. The positive stranded RNA genome of IBV encodes three structural proteins, two N-glycosylated envelope proteins (S and M) and a non-glycosylated nucleocapsid protein (N). The S or spike proteins give the virus its typical corona-like appearance. The two structurally unrelated subunits, Sl and S2, with relative molecular masses of 90,000 and 84,000 MW, protrude from the viral envelope. 1,2 The N protein, a 56,000 MW phosphoprotein, is closely associated with the viral RNA.3 The M or matrix protein varies in molecular mass from 27,000 to 36,000 MW, due to heterogeneous glycosylation.' Many serotypes of IBV have been defined on the basis of cross-neutralization studies, and five different serotypes (A to E) of the Dutch IBV strains have been recognized (Table 1).4 There is a constant emergence of new serotypes, generated by various mechanisms, posing a serious problem in vaccination programmes.5'6'34 Changes in serotype are generally correlated with changes in the spike protein which contains the determinants recognized by virus neutralizing antibodies.78 Little is known about the role of the N and M proteins in the induction of protective immunity. For effective activation of the humoral immune response, determinants recognized by both B cells and T-helper cells are required.9 Therefore it might be crucial that a vaccine contains viral antigenic determinants recognized by both cell populations. We report here on T-cell hybridomas recognizing the N
MATERIALS AND METHODS Virus growth and purification All strains, unless otherwise mentioned, were obtained from the Poultry Health Institute in Doorn, The Netherlands. New Castle disease virus (NCDV) was used as a control. All virus stocks were obtained from egg-grown virus and gradientpurified as described elsewhere.'0 The M42 laboratory strain was grown on Vero cells (multiplicity of infection 0-1) and the supernatant of the infected cells was harvested after 36 hr. Gradient-purified mouse hepatitis virus (MHV) strain A59 was kindly donated by Dr W. Spaan, University of Utrecht, The Netherlands. Immunization and generation of IB V-specific T-cell hybridomas To obtain T-cell hybridomas, an IBV-specific T-cell line was generated and subsequently immortalized by fusion. Two 6-8week-old BALB/c mice were immunized intraperitoneally with a mixture of the Ribi Adjuvant System (RAS; Immunochemical Research, Inc. Hamilton, MT) and 100 ug of the gradientpurified, UV-inactivated virus of the M41 strain of IBV. " Three weeks later, the mice were boosted with the same dose and 6 weeks thereafter spleen cells of these mice were cultured for 3-4 days at a concentration of 2 x 106 cells/ml in Iscove's modification of Dulbecco's medium (Gibco, Breda, The Netherlands) supplemented with 10% foetal calf serum (Gibco), antibiotics and 2-mercaptoethanol (2 x 10-I M) in the presence of M41 antigen (4 pg/ml). Subsequently, the cells were washed and resuspended in 1% conditioned medium.'2 Seven days later
Correspondence: Dr E. J. Hensen, Dept. of Immunology, Institute of Infectious Diseases and Immunology, School of Veterinary Medicine, University of Utrecht, P.O. Box 80.165, TD Utrecht, The Netherlands.
10
T-cell hybridomas specific for avian coronavirus IBV Table 1. IL-2 response of MJBIOO and MJB1OI to 12 IBV strains belonging to various serotypes
I
Table 2. MHC class II-specific response of Tcell hybridomas to 5 pg/ml of IBV strain M41
antigen
Strain
Serotype
M41 M42 H52 H120 V1259 V1385 V1397 D207 D274 D212 D1466 D3128
A A A A
IL-2 response (SI) S2 S1 Genotype group group MJBIOO MJBO1I
IL-2 response (SI) L cell transfectants
A/C B B/E C C D
1 1 2 2 2 2 2 3 3 3 3 3
a a
a
b c c
I I
-
53 6 53 3 320 6-0 359 29-8 12-4
I I
