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I... .. . z... . . .. . .. . . hm. .Hmfi .. ....n. .24... , llllllllllll \lllllllll ll \8‘ ll \lllllll 31 293 300897 This is to certify that the thesis entitled Production and Characterization of Monoclonal Antibodies to Bovine Respiratory syncytial Virus presented by Wendy Jane Underwood has been accepted towards fulfillment of the requirements for Master degree in Science Department of Large Animal Clinical Sciences &£ 6. 6.4% 3% a/‘a 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University L PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE Mm) . “ that: l .Iu ‘k 5"" . MSU I: An Affirmative Action/Equal Opportunity Institution chS-DJ - PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO BOVINE RESPIRATORY SYNCYTIAL VIRUS By Wendy Jane Underwood A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Clinical Sciences 1993 ABSTRACT PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO BOVINE RESPIRATORY SYNCYTIAL VIRUS By Wendy Jane Underwood Monoclonal antibodies (Mabs) to the SmithKline Beecham Animal Health BRSVR vaccine strain (375) were produced and characterized by radioimmunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by virus neutralization, by inhibition of viral-induced fusion, and by isotype. Nineteen hybridomas produced antibodies that were reactive with the F0, F1, F2, N, or P viral proteins of bovine respiratory syncytial virus. Hybridoma 1E72C4 produced Mabs that immunoprecipitated the F0 glycoprotein, neutralized virus (1 :8) in the presence of complement, but did not inhibit fusion. Hybridoma 8321137 produced Mabs that immunoprecipitated the F0, F1, and F2 glycoproteins, neutralized virus (1:4) with and without complement, and inhibited fusion. Antibodies from eleven hybridomas immunoprecipitated the N and F1 viral proteins, one hybridoma immunoprecipitated the N and P protein, and five hybridomas immunoprecipitated the N protein. All Mabs were of the IgGZb subtype with either kappa or lambda light chains. It gives me great pleasure to dedieate this work to George and Suzanne Underwood, my father and my mother, for their unfailing love, support, and encouragement. iii ACKNOWLEDGEMENTS I would like to extend my sincere and heartfelt gratitude to Dr. John C. Baker, my major professor, for his assistance, guidance, and encouragement during this project. I would also like to extend a special thank you to Dr. Leland F. Velicer for generously making his laboratory and his scientific expertise available for this project. Dr. John M. Kruger and Dr. Robert E. Holland, other members of my committee, deserve thanks for their guidance, suggestions, and input. Our co—investigator, Dr. Maurice A. Mufson, also deserves recognition for his assistance in development of this project as well as characterization of monoclonal antibodies. Quentin M. McCallum deserves special recognition. Without his technical assistance, perseverance, and unfailing friendship, this project may not have been completed. In addition, I would like to thank Ruth A. Stringer for her technical assistance, encouragement, and support during this study. Funding for this project was provided by SmithKline Beecham Animal Health. iv TABLE OF CONTENTS LIST OF TABLES .................................... LIST OF FIGURES .................................... LIST OF ABBREVIATIONS .............................. INTRODUCTION ..................................... LITERATURE REVIEW ................................ Overview ...................................... Comparative Features ....... - ....................... Classification ............................... Molecular Biology ............................ I Epidemiology ............................... Clinical Signs ............................... Pathologic lesions ................ ' ............ Immune Response ............................ Antigenic Variation ........................... Cross neutralization ...................... Complement fixation (CF) .................. Tissue culture host range ................... Monoclonal antibodies ..................... Protein analysis by gel electrophoresis ........... Epitope mapping ........................ Nucleotide sequence analysis ................. Ribonuclease A mismatch cleavage analysis ........ cDN A nucleic acid hybridization assay ........... Restriction endonuclease mapping .............. Stability of Antigenic Variation ........................ Clinical Importance of Antigenic Variation ................. Early Investigations ........................... Epidemiology ............................... Immunity ................................. Summary of Antigenic Variation ....................... MATERIALS AND METHODS ............................ Virus and Cells .................................. Virus Purification ................................. Plaque Assay ................................... Immunization ................................... Production of Monoclonal Antibodies .................. ' . . Indirect Fluorescent Antibody Staining ([FA) ................ Enzyme-linked Immunoassay (EIA) . . ’ .................... Concentration of Hybridoma Media ...... ' ................ Preparation of Ascites Fluid .......................... Radioactive Labeling of BRSV Viral Proteins ................. Radioimmunoprecipitation Assay (RIPA) ................... Polyacrylamide Gel Electrophoresis (SDS -PAGE) .............. Virus Neutralization Assay ............................ Fusion Inhibition Assay ............................. Isotype ....................................... RESULTS .......................... Virus Purification ................................. Response to Immunization ........................... Results of Fusion and Screening ........................ Fusions .................................. Immunofluorescent Staining ...................... Enzyme-linked Immunoassay (EIA) ......... w ........ ‘ Characterization of Hybridomas ........................ RIPA and SDS-PAGE ......................... Virus Neutralization ........................... Virus-induced Fusion Inhibition .................... Enzyme-linked Immunoassay (EIA) .......... - ........ Isotype ............. ' ...................... DISCUSSION ............. ‘ .......................... Virus Purification ................................. Response to Immunization ........................... Screening of Hybridomas ............................ Characterization of Hybridomas ........................ CONCLUSIONS AND RECOMMENDATIONS .................. LIST OF REFERENCES ................................ vi Table LIST OF TABLES Page Genera and species of the Paramyxoviridae family. 5 Comparison of viral proteins in Human and Bovine Respiratory Syncytial Viruses. Characterization of hybridomas. 52 [3I-I]uridine activity in cpm in the PEG pellet, 30% sucrose 5 3 pellet, 35-45 % sucrose interface of discontinuous sucrose gradient, and in 25 fractions of the linear sucrose gradient of virions purified from BRSV-infected BT cells. vii Figure 3A LIST OF FIGURES Graph of [3H]uridine radioactivity in cpm in 25 fractions of the linear sucrose gradient. Media was harvested from BRSV-infected BT cells at 24, 36, 48, and 60 hrs PI. Results of immunization of mice. Immunoprecipitation and SDS- PAGE analysis of F’S]methionine-labeled BRSV-infected BT cells. labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV polyclonal serum. BRSV“ indicates mice immunized with reconstituted SBAH BRSVR vaccine, DISC indicates mice immunized with purified extracellular virions obtained from the 35-45 % sucrose interface of the discontinuous sucrose gradient, and VL indicates mice immunized with extracellular virions purified on the linear sucrose gradient. IBS indicates immune bovine serum to BRSV (C =Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls (data not shown). Results of immunization of mice. Immunoprecipitation and SDS- PAGE analysis of [3H]glucosamine-labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV polyclonal serum. Discontinuous Gradient indicates mice immunized with purified virions obtained from the 35-45 % interface of the discontinuous sucrose gradient, and BRSVll indicates mice immunized with reconstituted SBAH BRSVR vaccine. IBS indicates immune bovine serum to BRSV (C =Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls. viii 54 55 56 3B Results of immunization of mice. Immunoprecipitation and SDS- PAGE analysis of [’lflglucosamine-labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV polyclonal serum. BRSVn indicates mice immunized with reconstituted SBAH BRSVR vaccine, and Linear Gradient indicates mice immunized with virions purified on the linear sucrose gradient. IBS indicates immune bovine serum to BRSV (C=Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls. Photomicrograph of Group I type indirect immunofluorescent antibody staining of BRSV-infected BT cells (by hybridoma 1E72C4). The Group I type Mabs produced a granular immunofluorescent pattern that stained the outer portions of BRSV-infected BT cells. Photographs were taken at a magnification of x225. Photomicrograph of Group H type indirect immunofluorescent antibody staining of BRSV-infected BT cells (by hybridoma 9D4). The Group 11 type immunofluorescent pattern was less granular than Group I and stained intracytoplasmic areas. Photographs were taken at a magnification of x225 . Characterization of hybridoma lE72C4 by immunoprecipitation and SDS-PAGE analysis of [’lflglucosamine-labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV ascites fluid. Mock infected BT cells were used as negative controls (C =Control or mock infected BT cells, I=BRSV-infected BT cells). Characterization of hybridoma 8321137 by immunoprecipitation and SDS-PAGE analysis of [’lflglucosamine—labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV ascites fluid. Mock infected BT cells were used as negative controls (C =Control or mock infected BT cells, I=BRSV-infected BT cells). Characterization of hybridomas 6D4 and 6E62B8 by immunoprecipitation and SDS-PAGE analysis of [”S]methionine— labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV ascites fluid. IBS indicates immune bovine serum to BRSV (C=Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls 57 58 59 61 62 Characterization of hybridomas l3C8 and 1365 by immunoprecipitation and SDS-PAGE analysis of [”S] methionine- labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV ascites fluid. IBS indicates immune bovine serum to BRSV (C =Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls 63 BRSV BM BL BRSVR BT CF CPE DISC DMEM FCS 313(8) ' hr(s) LIST OF ABBREVIATIONS Bovine respiratory syncytial virus Lectin coated agarose beads incubated with media from BRSV-infected BT cells Lectin coated agarose beads incubated with BRSV-infected cell lysates SBAH BRSVR vaccine used for immunization of mice Bovine turbinate cells Complement fixation Cytopathic effect counts per minute 35-45 % sucrose interface of the discontinuous gradient Dulbecco’s minimum essential medium Enzyme-linked immunoassay Eagle’s minimum essential medium Fetal calf serum 3 g1ywproteins 3G71D4 N/F, — - - IgG2b/h 6D4 N - - - ND'“ 6E62B8 N - - - IgG2b/x Fusion #2 8B21E7 N/F 1:4 1:4 1:32 IgG2b/x 9E31E5 N - - - IgG2b/x 9D11 N/F, - - - ND“ 9Ell N - - - ND* 10631B2 N/F, - - - IgG2b/x l 1C7 N/F, - - - ND* 1 1G4 N/ F, - - - ND“ 12B21C6 N/F, - — - IgG2b/x 12D101F2 N/P - - - IgG2b/x 12D7 N/F, - 1:4 - ND“ 13C5 - - - - ND“ 13C8 N/F, - - - ND* 1365 N/F, - - — ND* Controls IBS > 1:2048 > 1:2048 - F Mab 1:4 1:4 1:32 IBS = Immune bovine serum to BRSV *ND = Isotype was determined only for hybridomas subcloned twice by limit dilution S/ + = Strong positive W/+ = Weak positive Negative 53 Table 4. [3H]uridine activity in cpm in the PEG pellet, 30% sucrose pellet, 35—45 % sucrose interface of discontinuous sucrose gradient, and in 25 fractions of the linear sucrose gradient of virions purified from BRSV-infected BT cells. SOURCE 54 600 +24 hrs PI *36 hrs PI ‘48 hrs Pl +60 hrs Pl 500 .p. O O Counts per Minute 00 O o N O O 100 l 0 2 4 6 8101214161820222426 Linear Gradient Fraction Number Figure 1. Graph of [’H]uridine radioactivity in cpm in 25 fractions of the linear sucrose gradient. Media was harvested from BRSV-infected BT cells at 24, 36, 48, and 60 hrs PI. 55 1lBRSVR VL [IBSII v1. DISC I213|4u1|20| 3|41 r‘ d C. 97.4— ‘0‘ 9! ‘-: 68“.”0’ ,. F0 ~ ’ ‘ 0‘- -.~ 4“- " ‘. .wp 29- Figure 2. Results of immunization of mice. Immunoprecipitation and SDS-PAGE analysis of [35$]methionine-labeled BRSV-infected BT cells. labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV polyclonal serum. BRSVR indicates mice immunized with reconstituted SBAH BRSVll vaccine, DISC indicates mice immunized with purified extracellular virions obtained from the 35- 45% sucrose interface of the discontinuous sucrose gradient, and VL indicates mice immunized with extracellular virions purified on the linear sucrose gradient. IBS indicates immune bovine serum to BRSV (C=Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls (data not shown). 56 Discontinuous Gradient IBS ensv“ 1 2 3 4 1 2 CIICIlc 1|c1 Cl C llCl : -q’ ' '1 . 200- . .. . n r ‘ I I I - .. 97.4- _ 68- I:0 43- , F1 29- F2 18.4- Figure 3A. Results of immunization of mice. Immunoprecipitation and SDS-PAGE analysis of nglucosamine-labeled BRSV-infected BT cells. labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV polyclonal serum. Discontinuous Gradient indicates mice immunized with purified virions obtained from the 35-45% interface of the discontinuous sucrose gradient, and BRSVR indicates mice immunized with reconstituted SBAH BRSVR vaccine. IBS indicates immune bovine serum to BRSV (C=Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls. 57 i ‘ 31:1sz4 IBS Linear Gradient i 3 ‘ C||C4| CIICHIICIIC4I 200- j 914-..: 68- 43- - o . O - F1 29- F2 18.4- Figure 38. Results of immunization of mice. Immunoprecipitation and SDS-PAGE analysis of [’H]glucosamine—labeled BRSV-infected BT cells. labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti—BRSV polyclonal serum. - BRSVR indicates mice immunized with reconstituted SBAH BRSVR vaccine, and Linear Gradient indicates mice immunized with virions purified on the linear sucrose gradient. IBS indicates immune bovine serum to BRSV (C=Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls. 58 Figure 4. Photomicrograph of Group I type indirect immunofluorescent antibody staining of BRSV-infected BT cells (by hybridoma 1E72C4). The Group ,I type Mabs produced a granular immunofluorescent pattern that stained the outer portions of BRSV-infected BT cells. Photographs were taken at a magnification of x225. 59 Figure 5. Photomicrograph of Group 11 type indirect immunofluorescent antibody staining of BRSV-infected BT cells (by hybridoma 9D4). The Group 11 type immunofluorescent pattern was less granular than Group I and stained intracytoplasmic areas. Photographs were taken at a magnification of x225. Q t: N , h I.“ C| I 200— 9734- 68" Fo 43—) 29- Figure 6. Characterization of hybridoma 1E72C4 by immunoprecipitation and SDS- PAGE analysis of [3H]glucosamine-labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV ascites fluid. Mock infected BT cells were used as negative controls (C =Control or mock infected BT cells, I=BRSV-infected BT cells). 61 h I.” 'or' a: co C l l zoo- .’ 97.4- i 1 68- Fo ! 43- F1 29- F2 18.4- Figure 7. Characterization of hybridoma 8B21E7 by immunoprecipitation and SDS- PAGE analysis of [‘mglucosamine-labeled BRSV-infected BT cells. Labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV ascites fluid. Mock infected BT cells were used as negative controls (C =Control or mock ' infected BT cells, I=BRSV-infected BT cells). 62 6E62 38 -68 37:». a. —43 -29 Figure 8. Characterization of hybridomas 6D4 and 6E62B8 by immunoprecipitation and SDS-PAGE analysis of [35S]methionine-labeled BRSV-infected BT cells. labeling was for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BRSV ascites fluid. IBS indicates immune bovine serum to BRSV (C=Control or mock infected BT cells, I=BRSV-infected BT cells). Mock infected BT cells were used as negative controls. 63 2"“ 29- Figure 9. Characterization of hybridomas 13C8 and 1365 by immunoprecipitation SDS-PAGE analysis of [35$]methionine-labeled BRSV-infected BT cells. labeling for 4 hrs at 36 hrs PI. Immunoprecipitation was performed using mouse anti-BI ascites fluid. IBS indicates immune bovine serum to BRSV (C=Control or m infected BT cells, I=BRSV—infected BT cells). Mock infected BT cells were user negative controls. DISCUSSION Virus Purification Because of the inherent lability and the difficulty in propagating RSV, there are few reports of HRSV purification and no reports of BRSV purification. Attempts to purify HRSV resulted in poor total yields with loss of infectivity because the virus tends to remain associated with the cellular membrane (Levine 77, Wunner 76). However, we were able to purify extracellular virions that retained infectivity from both BRSV—infected BT cells and from reconstituted SBAH BRSVR vaccine in yields that were sufficient to immunize mice. A [3H]uridine radiolabel was used to monitor the presence of extracellular virions during purification of BRSV-infected BT cells. Based on cpm of [’I-I]uridine radioactivity, more extracellular virions could be recovered from BRSV-infected BT cells at 60 hrs PI than at 24, 36, or 48 hrs PI. The higher cpm at 60 hrs PI may have been due to either virus budding or to excessive loss of lytic BRSV-infected BT cells into the media. Scintillation assays of the linear sucrose gradient fractions (final purification step) indicated the presence of extracellular virions in fractions #9 through #14 (25 total fractions). Since the principle of density-gradient centrifugation is the segregation of particles on the basis of their densities, material differing from the virus in density are 64 65 separated. The viral band in linear fractions #9-#14 formed in the 1.18-1.22 g/cm3 density region, in accordance with previously published reports of RSV density (Pringle 85). These results demonstrate that BRSV can be purified from BRSV-infected BT cells. The ability to purify extracellular virions from tissue culture media harvested from BRSV-infected BT cells is important for future studies designed to produce Mabs against viral proteins of other BRSV isolates. Extracellular virions were also purified from reconstituted SBAH BRSVR vaccine. In cell culture systems, RSV typically exhibits two features that correspond to pathogenic functions, infectivity and syncytia formation (Cote 81). Infectivity is the only reliable marker available to follow the virus during nonradiolabeled purification procedures (Cote 81). Based on plaque assays, purified virions retained infectivity which was highest in linear fractions #9 through #14 (25 total fractions) where infectivity titers ranged from 1X105 to 1x10“ PFU/ ml. Retention of infectivity indicates that the virion envelope is intact and that purified virions are capable of attachment and cell-to-cell fusion, functions of the G and F gps, respectively. The location of viral infectivity in linear sucrose gradients of virions purified from SBAH BRSV“ vaccine as determined by plaque assay correlated well with the location of virions in linear sucrose gradients of virions purified from BRSV-infected BT cells as determined by scintillation assays of [’H] radioactivity. These results indicate that not only can BRSV be purified from BRSV-infected BT cells or from reconstituted SBAH BRSVR vaccine, but also that purified BRSV can retain infectivity and that purified BRSV can be obtained in sufficient amounts (0.02ng/u1) to ' immunize mice for the production of Mabs. Purification of virions from reconstituted 66 SBAH BRSVll vaccine is less time consuming, less costly, and easier to accomplish than purifying virions from BRSV-infected BT cell media. To produce panels of Mabs to isolates other than the SBAH BRSV‘ vaccine strain, however, extracellular virions must be purified from tissue culture media harvested from BRSV-infected BT cells. The work reported here establishes that method for future use. Response to Immunization Several methods were developed to prepare BRSV antigen for the immunization . of mice. Mice were inoculated with: extracellular virions purified on the 35-45 % sucrose interface of the discontinuous sucrose gradient (DISC), extracellular virions purified on the linear sucrose gradient (VL), reconstituted SBAH BRSVR vaccine (BRSVR) administered directly to mice, glutaraldehyde fixed BRSV-infected whole BT cell cultures (W CC), and lentil lectin coated agarose beads incubated with either tissue culture media (BM) or lysates (BL) from BRSV-infected BT cells. Based on RIPA SDS- PAGE analysis of [”S]methionine— and [3I-I]glucosamine-labeled BRSV-infected BT cell lysate, a specific immune response to the G, F,, F,, N, and P proteins was produced in the BRSV“, DISC, and VL groups of mice. The WCC group produced a specific immune response to F,, N and P proteins (RIPA SDS-PAGE analysis using [3H]glucosamine-labeled BRSV-infected BT cells was not done for the WCC group). These results demonstrate that when mice are immunized to BRSV, a specific antibody response to at least four structural proteins, most importantly to the G gp, can be obtained. Obtaining a Mab to the BRSV G gp is essential to the identification of BRSV subgroups since the majority of antigenic variation in BRSV, like HRSV, may lie in the 67 G gp. Because purified virions reduce the antibody response of the mouse to nonviral antigens (Campbell 84), extracellular virions purified from the linear gradient were used for all fusions. Lectins are proteins that precipitate complex carbohydrates, especially diverse oligosaccharides of cell surface glycoproteins. Lentil lectin coated agarose beads should . bind viral glycoproteins, and, in the case of BRSV, only the F and G gps. Lentil lectin coated agarose beads incubated with tissue culture media harvested from BRSV-infected BT cells (BM) should selectively bind the soluble form of G (Hendricks 87). The BM group of mice produced antibodies to the F, and N protein, but not the G gp. The lectin coated beads may have bound glycoproteins on extracellular virions that were shed into the media from BRSV-infected BT cells; therefore, mice immunized by the BM method would respond to several viral proteins. Pelleting extracellular virions by ultracentrifugation of the media or by precipitation with PEG prior to incubation with lectin coated beads may improve results of the BM immunization method. Lentil lectin coated agarose beads incubated with lysates of BRSV-infected BT cells (BL) should selectively bind the F and G viral gps. The BL group responded to the F, N, and P proteins, but did not respond to the G gp. Mice immunized by the BL method produced antibodies to several viral proteins probably because the lectin coated beads bound glycoproteins on either intact virions or on vesicles containing viral proteins. Based on these results, using beads incubated with BRSV-infected BT cell lysates to select for viral gps was ineffective. Several other immunization protocols could be examined that might result in a 68 Mab specific to the G gp after a fusion. First, column chromatography using Concanavalin A (a lectin) could be utilized to affinity purify the BRSV G gp which could then be used as an immunogen (Bernstein 80, Lambert 83, Walsh 84). One report indicated that almost 50% of a fusion protein applied to an immunoadsorbent column could be recovered in an antigenically active form (Stott 84b). Second, the BRSV G gp' could be removed from RIPA SDS-PAGE gels, recovered by electroelution through a small membrane (10 kDa molecular weight cut off), and used as an immunogen. Alternatively, the purified G obtained from SDS-PAGE could be injected into rabbits to produce rabbit anti-G. The rabbit anti-G could then be used to purify BRSV G by immunoaffinity column chromatography. Third, since the complete nucleotide sequence of the BRSV G gene is known and since the BRSV G gene can be amplified using polymerase chain reaction technology and cloned into vectors (using vaccinia or adenovirus vectors or a bacculovirus or semiliki virus expression system), the BRSV G gp could be produced in large quantities and used as an immunogen. And fourth, immunization of mice with a viable Balb/c mouse cell line persistently infected with HRSV has proven successful in the production of monoclonals to the HRSV G gp (Fernie 81,82). Screening of Hybridomas Two methods were utilized to screen hybridomas for the production of Mabs specific to BRSV viral proteins, enzyme—linked immunoassay (EIA) and indirect .immunofluorescence (IFA). Although the EIA worked well in preliminary experiments to identify the F Mab used as a control, similar results were not obtained using 69 hybridoma or ascites fluid during screening. A variable amount of background absorbance occurred in both uninfected and infected wells despite blocking for nonspecific protein binding. Increased background absorbance may have been caused by nonspecific binding of proteins in the sample, from loss of antigen in test wells, or from the type of EIA plate used. Hybridoma fluid, especially when concentrated, can contain large amounts of protein contaminants. These contaminants are largely albumins, but may contain bovine immunoglobulins (if FCS is used during the fusion), proteases, and nucleases (Campbell 84). Ascitic fluid contaminants also include nonspecific immunoglobulins together with larger amounts of proteases and nucleases (Campbell 84). Loss of antigen from test wells increases the likelihood of nonspecific protein binding to the EIA plate. Test wells were plated with either uninfected or BRSV-infected BT cells, allowed to dry at room temperature, and stored at -20°C until used. Antigen could have been lost from test wells during drying by desiccation, during storage by freeze-thaw fracture, or during EIA assays by washing. One way to avoid antigen loss would be to fix antigen to test wells with either ethanol or acetone (Bolin 91). Fixing antigen as above and hand washing during the EIA procedure may prevent antigen loss during the EIA procedure. Alternatively, nonspecific protein binding may have been a result of the type of EIA plate or the type of tissue culture serum used. The EIA was developed using techniques similar to others who had successful results (Baker 92, Mufson 85). One difference, however, was the type of EIA plate utilized. Anecdotal reports abound 70 concerning the success of some types of EIA plates over others (Liddell 91). Another difference was the use of DMEM containing horse serum. To avoid problems in the future, the EIA should be tested prior to screening using a variety of plates and different lots of the tissue culture serum to be utilized during production of hybridomas. In contrast to the EIA, the IFA worked well for screening hybridomas for Mabs specific to BRSV viral proteins. Two distinct patterns of immunofluorescent staining were observed. Group I hybridomas produced an annular staining pattern with a distinctly granular appearance. The staining pattern of the Group H hybridomas was less granular and stained intracytoplasmic areas as well as annular areas. The two group division was developed by researchers studying Mabs to HRSV (Cote 81 , Femie 82, Gimenez 84, Klucas 88, Stott 84b, Walsh 83a). These researchers determined that the Group I staining pattern was consistent with specificity of Mabs for a protein expressed on the cell surface, while the Group II staining pattern was consistent with hybridomas producing Mabs to proteins located in intracellular virions. Because the IFA screening procedure was faster, less costly, and easier to perform than the EIA, screening was done by IFA only during the second and third fusion. One disadvantage of screening by one method only is that some monoclonals may fail to react in the screening method being used. Why some Mabs fail to react by IFA or EIA is unknown. Similar results have been reported by others working on HRSV (Anderson 85, Gimenez 86, Routledge 85). Anderson, et al (85) found that different epitopes showed varying sensitivities to IFA acetone fixation and suggested that some epitopes may be lost during IFA preparation. 71 Some Mabs to the F gp may have a low affinity for antigen resulting in low EIA absorbance readings (Routledge 85). In addition, since hybridomas secrete varying amounts of antibody, low EIA absorbance for some Mabs is possible. Only those hybridomas that were positive by EIA and IPA, and only those hybridomas that were positive to BRSV-infected BT cells and negative to uninfected BT cells by IFA, were subcloned and further characterized. Clones that were positive to both BRSV-infected and uninfected BT cells may contain a hybridoma producing Mabs specific to BRSV. In addition, those clones that were positive by EIA but negative by IFA may also contain hybridomas producing Mabs specific to BRSV. Characterization of Hybridomas The primary purpose of this project was to produce and characterize a panel of Mabs to the SBAH BRSVR vaccine isolate (375) of BRSV. Only two previous reports of Mabs specific to BRSV exist (Kennedy 88, Klucas 88). Klucas, er al (88) produced five Mabs to the 375 isolate of BRSV, of which two were specific to the F gp, and three were not characterized as to protein specificity. The second report describes the production of six interspecies hybridomas (heterohybridomas using bovine lymphocytes and mouse myeloma cells) secreting Mabs against F,,, F,, and F2 gps of the 127 isolate of BRSV (Kennedy 88). The current report describes the production and characterization of 19 hybridomas reactive with the F,,, F,, F,, N, and P viral proteins of the SBAH BRSVR vaccine strain of BRSV (375 isolate). All hybridomas were of the Ing,, subtype with either kappa or lambda light chains. One hybridoma (1E72C4) produced Mabs reactive with the F0 gp 72 based on [’Iflglucosamine-labeled BT cell lysates in RIPA SDS-PAGE analysis, but also immunoprecipitated the N protein based on [”S] methionine-labeled BRSV-infected BT cell lysates in RIPA SDS-PAGE analysis. One hybridoma (8B21E7) produced Mabs that reacted to the F,,, F,, and F, gp, but also immunoprecipitated the N protein. Eleven hybridomas immunoprecipitated N and F, proteins, but did not immunoprecipitate a viral envelope gp. One hybridoma immunoprecipitated the N and P protein, and five hybridomas immunoprecipitated the N protein. The RSV quion protein (F,,) is a 68 kDa inactive precursor protein that is proteolytically cleaved into two active subunits, F, and F,, The hybridoma producing Mabs to the F0 gp (1E72C4) neutralized virus only in the presence of complement and did not inhibit fusion. The hybridoma producing Mabs reactive with the F gp (8B21E7), however, neutralized virus with and without complement and inhibited fusion. Many reports exist of Mabs specific to the HRSV F gp that neutralize virus with and without complement and inhibit fusion (Fernie 82, Olmstead 86, Orvell 87 Samson 86, Walsh 83a). These reports also describe Mabs to the F gp that neither neutralize virus nor inhibit fusion. The presence of complement has been reported to increase the neutralization titer four to five-fold (Buynak 79, Femie 82, Tsutsumi 87) suggesting that neutralization is mediated by antibody dependent viral membrane lysis (Walsh 83a). Since the fusion protein is involved in the fusion of enveloped viruses to host cell membranes, neutralization of RSV by anti-F antibodies may be mediated by inhibition of virus-to—cell fusion. One Mab, BB21E7, gave an unusual pattern of reactivity when tested by 73 [3H]glucosamine-labeled BRSV—infected BT cell lysate in RIPA SDS-PAGE in that it immunoprecipitated the F,,, F,, and F, components of the F gp. Although a given Mab would be expected to react with a unique epitope within a protein, similar results of an HRSV anti-F mab reacting to both the F, and F, subunits have been reported (Garcia- Barreno 89, Mufson 85, Orvell 87, Samson 86, Trudel 86, Tsutsumi 87, Walsh 83a,86). Samson, er al (86) suggested that this unusual behavior may be due to: 1) biclonal hybridomas where one clone produces antibodies to F, while the other clone produces antibodies to F,, 2) a 21 kDa portion of F, that is cleaved and carries the same epitope (and therefore mimics F, binding), or 3) a common epitope within F, and F,. Since the 8B21E7 hybridoma was subcloned twice by limiting dilution, the biclonal theory seems unlikely. No reports of a 21 kDa fraction of F, exist, although further experiments may be needed to establish whether such a fragment could be derived from F,. Samson, er a1 (86) proposed the term "geminiepitopic" to refer to the situation when a Mab reacts with two distinct epitopes occurring on the same protein molecule. Many hybridomas immunoprecipitated the N protein either alone or with the P or F, protein. The RSV N protein is an abundant structural protein of the viral nucleocapsid that is tightly complexed with viral genomic RNA. There are several reports of HRSV Mabs to P coprecipitating with N (Gimenez 84, Mufson 85, Orvell 87 , Routledge 85, Stott 84b). In addition, several reports exist of an HRSV anti-F Mab immunoprecipitationg both the F and N proteins (Mufson 85, Orvell 87). Gimenez, et al (84) considered it unlikely that N and P share common epitopes. Routledge, et al (85) suggested that N and P form a macromolecule since the band associated with the N 74 protein was greatly reduced when immunoprecipitation was repeated after ultracentrifugation (100,000 x g for 45 minutes) or after addition of the nonionic detergent Empigen in the RIPA buffer (Routledge 85). Orvell, er al (87) determined that antibodies against the P protein coprecipitated the N protein, but that antibodies against the N did not coprecipitate the P protein. Since some of the hybridomas that coprecipitated the N with either P or F, were not subcloned, it is possible that two hybridomas exist, one specific to N and one specific to either P or F,. Despite the fact that N is an abundant protein, it is somewhat surprising that so many hybridomas secreted antibodies to N. There are no reports of an HRSV anti-N Mab that neutralizes or inhibits fusion. Although it is possible that an anti-F Mab (immunoprecipitating the F and N proteins) neutralized virus or inhibited fusion, it is also possible that the highly concentrated hybridoma fluid or impure ascites fluid may have interfered with virus neutralization and fusion inhibition tests. Many standard protocols exist for purifying Mabs from ascites or tissue culture hybridoma fluid including ammonium sulfate precipitation, diethylaminoethyl-cellulose column dialysis, and affinity chromatography (Campbell 84). Perhaps characterization of Mabs by ability to neutralize virus in the presence of complement and ability to inhibit viral-induced fusion should be repeated using purified Mabs. The RIPA SDS-PAGE analysis did not enable the specificity of all Mabs to be definitively identified. First, some EIA and [FA positive Mabs failed to react in RIPA SDS-PAGE. Similar results have been reported with Mabs to HRSV (Fernie 82, Storch 87 , Stott 84b). Femie, et al (82) suggested that failure to immunoprecipitate viral 75 proteins may be due to SDS-PAGE alteration of protein conformation and classi Mabs by radioimmunoassay (Fernie 82). Stott, et 01 (84b) used competitive bint assays to identify Mabs that failed to react by RIPA SDS-PAGE. Monoclonal antit specificity could be also examined by western immunoblot assay (Stott 84) . Howe Klucas, et al (88) found that not all BRSV Mabs reacted by Western immunol Alternatively, if viral proteins could be purified, then Mabs could be identified by I using the purified protein as the test antigen or by competitive blocking studies u SDS-PAGE. Second, analysis of RIPA SDS-PAGE autoradiographs indicated the presenc unidentified nonspecific polypeptides. The presence of nonspecific protein bindin. RIPA SDS-PAGE suggests either immunoprecipitation of nonspecific proteins or c: contamination of infected to control lanes. Addition of mouse serum (free of antibo to BRSV) during the preclear step of the immunoprecipitation did not elimi nonspecific protein binding. One problem may have been the use of impure ascites l or concentrated tissue culture hybridoma media. Similar nonspecific protein binding occurred with other researchers. Stott, er al (84) suggested that the "contamination” 1 be due to "the difficulty in disrupting membrane bound vesicles" or to the possibility some of the proteins precipitated may be precursors to or degradation products of 0 viral polypeptides. Walpita, et al (92) found that viral proteins were obscured numerous host-cell specific protein bands produced during one-dimensional SDS -PA separation of virus-infected whole-cell lysates. In addition, Mabs to viral proteins 1 cross-react with host proteins (Tsutsumi 87). One study suggested that nearly 3.59 76 all Mabs produced to other viruses cross-react with host proteins (Srinivasappa 86). Third, and most importantly, the [’Iflglucosamine—labeled RIPA SDS-PAGE never consistently allowed identification of viral surface membrane gps. Analysis of immunized mice sera by [3H]glucosamine-labeled BRSV-infected BT cell lysate RIPA SDS-PAGE indicated that all surface glycoproteins, including G, F,,, F,, and F,, could be identified on two and four day exposures of autoradiographs. However, immunoprecipitations using mouse anti-BRSV concentrated hybridoma or ascites fluid never reproduced those results even on 32 day exposures of autoradiographs. Many RIPA SDS-PAGE procedure variables were analyzed including the [3I-I]glucosamine radioisotope source (manufacturer), radiolabeling pulses, viral passage level, BT cell passage level, acrylamide source, type of gel box, etc. , but no improvement in results occurred. Subsequently, autoradiographs of [’mglucosamine-labeled BRSV-infected BT cell lysates in RIPA SDS-PAGE never adequately provided identification of the G gp. NoMabsproducedwere specifictotheGgp. SincetheGgpcanoftenbe identified by SDS-PAGE when [”S]methionine—labeled BRSV-infected BT cell lysates are immunoprecipitated with hyperimmune antisera specific to BRSV, an anti-G Mab might have been identified in [35$]methionine-labeled BRSV-infected BT cell lysates by RIPA SDS-PAGE. To date, there are no reports of a Mab to the G gp of BRSV. Perhaps the G gp is present in reduced quantities in purified virions (Orvell 87). Wunner, er a1 (76) suggested that partial purification was accompanied by the loss of high molecular weight glycoproteins. However, Tsutsumi, et al (87) were able to produce Mabs to I-lRSV G gp using purified HRSV virions, but Klucas, et al (88), using a BRSV-infected whole cell 77 lysate as an immunogen, did not obtain an anti-G Mab. In our study, immunized mice were able to recognize the G gp. However, it is poSsible that the process of purification and that the conditions used in the RIPA SDS-PAGE assay produced enough alterations in BRSV G gp epitopes to prevent proper identification of Mabs by RIPA SDS-PAGE analysis. It may be possible to identify the BRSV G Mab using alternative methods. For example, in Western blot analysis viral proteins are separated by SDS-PAGE, incubated with serum or ascites, washed, incubated with an anti-mouse antibody conjugated to an enzyme or radiolabel, washed again, and incubated with a substrate. Another possibility would be to utilize a [’Iflthreonine-labeling in the RIPA SDS-PAGE analysis. Since the BRSV G gene codes for many threonine residues, a [3H]threonine labeling may better identify a G Mab (Collins 86). Previous work with this radiolabel in this laboratory, however, was disappointing for labeling the G gp (unpublished data). Investigators have radiolabeled other sugars such as mannose but have had difficulty identifying the G gp (Dubovi 82). Although the identification of antigenic variation in RSV has historically been based on reaction panels of Mabs to several isolates of RSV, other methods exist for defining antigenic variation. A better appreciation of strain variation might be obtained by using Mabs to the biologically important epitopes of the. fusion protein (Walsh 86). A similar approach has been used to evaluate strain variation of Newcastle disease virus (Iorio 84). The Rnase A mismatch cleavage technique has been shown to be very sensitive for detecting small changes in RNA sequences that are not detectable by Mabs, 78 including Single-base substitutions (Myers 85); however, it may prove to be too sensitive a technique to distinguish subgroup variation. The cDNA hybridization approach to RSV subgroup determination has the advantage of providing broad-based discrimination of RSV subgroups based on nucleic acid homology without interference from minor antigenic differences detected by Rnase A mismatch assays (Sullender 90). The restriction endonuclease mapping technique is simple, rapid, and more discriminating than analysis using Mabs. One reason to emphasize the Mab approach is that antigenic variation is defined on the immunological level, whereas other approaches define antigenic variation on the nucleic acid level. CONCLUSIONS AND RECOMMENDATIONS Conclusions that can be made from this study include: 1. Virus Purification: Extracellular virions of BRSV can be purified from BRSV-infected BT cell tissue culture media and from reconstituted SBAH BRSVR , vaccine. When using tissue culture media from BRSV-infected BT cells to purify extracellular virions, media should be harvested at 60 hrs P1 to avoid excessive loss of lytic BRSV-infected BT cells from the monolayer into the tissue culture media. Extracellular virions can be recovered from BRSV-infected BT cell tissue culture media and from reconstituted SBAH BRSVR vaccine in amounts sufficient to immunize mice. To produce panels of Mabs to isolates other than the SBAH BRSVR vaccine strain, however, extracellular virions must be purified from tissue culture media harvested from BRSV-infected BT cells. The work reported here establishes that method for future use. 2. Immunization: Balb/c mice can be immunized to produce antibodies specific to BRSV viral proteins. Mice immunized with lentil lectin coated beads incubated with either tissue culture media harvested from BRSV-infected BT cells or from BRSV- infected BT cell lysates did not produce antibodies specific only to BRSV viral gps. Pelleting extracellular virions by ultracentrifugation of the media or precipitation by PEG 79 80 prior to incubation with lectin coated beads may improve results of immunization by the BM method. Using lectin coated agarose beads incubated with BRSV-infected BT cell lysates to select for viral gps was ineffective. 3. Fusion: Hybridomas secreting antibodies specific to BRSV can be produced using NS-l myeloma cells or SP2/0 myeloma cells. Using of SP2/0 myeloma cells for the production of hybridomas is less costly. 4. Screening: Indirect immunofluorescence is faster, easier, and more efficient than EIA in screening hybridomas for the production of antibodies specific to BRSV. Hybridomas secreting antibodies specific to BRSV produced two patterns of immunofluorescence dependent upon which viral protein the Mab is directed against. 5. Characterization of Hybridomas: Nineteen Mabs reactive to one or more BRSV viral proteins were produced. No Mabs specific to the G or M protein were produced. The [3H]glucosamine-labeled RIPA SDS-PAGE never consistently allowed identification of viral surface membrane glycoproteins in our laboratory. Recommendations for further studies include: 1. Immunization: Extracellular virions from the tissue culture media of BRSV- infected BT cells should be pelleted (by ultracentrifugation) or precipitated (by PEG) prior to incubation with lentil lectin coated beads to decrease the probability of lectin coated beads binding viral gps on intact extracellular virions. Other methods to immunize mice for the production of antibodies specific to BRSV G gp should be examined (see discussion). 81 2. Fusion: All hybridomas that were positive to BRSV-infected BT cells and uninfected control cells by IFA should be subcloned and rescreened. All hybridomas that were positive to BRSV-infected BT cells by EIA and negative by IFA negative hybridomas should be rescreened and characterized by RIPA SDS-PAGE to determine if any clones produce antibodies specific to BRSV viral proteins. The SP2/0 line of myeloma cells should be used for future fusions. 3. Screening: To avoid loss of hybridomas producing antibodies specific to BRSV when screening by IFA only, both IFA and EIA should be utilized. A variety of EIA plates should be tested prior to fusion using the tissue culture medium to be utilized during fusion and cultivation of hybridomas. 4. Characterization of Mabs: All hybridomas that immunoprecipitated N with either F, or P should be subcloned twice by limiting dilution and retested by RIPA SDS- PAGE. If these hybridomas still immunoprecipitate more than one viral specific protein, then radiolabeled RIPA material should be ultracentrifuged (100,00 x g for 45 minutes) or the nonionic detergent Empigen should be added to the buffer prior to SDS-PAGE analysis. Western blot analysis might be used to confirm viral protein specificity of Mabs. Ascites fluid or concentrated tissue culture hybridoma fluid should be purified {using standard protocols such as ammonium sulfate precipitation, diethylaminoethyl— cellulbse column dialysis, or affinity chromatography (Campbell 84). Virus neutralization and fusion inhibition assays should be repeated using purified ascites or tissue culture hybridoma fluid. 82 5 . Future studies: Other methods to determine antigenic variation in BRSV should be examined. These methods might include Rnase A mismatch cleavage analysis, cDNA hybridization assays, or restriction endonuclease analysis. 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