Expression of influenza A and B virus nucleoprotein antigens in baculovirus

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1 Journal of General Virology (1990), 71, , Printed in Great Britain 1545 Expression of influenza A and B virus nucleoprotein antigens in baculovirus Paul A. Rota,* Renee A. Black, Barun K. De, Maurice W. Harmon and Alan P. Kendal Influenza Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333, U.S.A. Full-length cdna clones of the nucleoprotein (NP) genes of influenza A/Ann Arbor/6/60 and B/Ann Arbor/1/86 viruses were constructed from virion RNA and subsequently expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus vector, Autographa californica nuclear polyhedrosis virus. Western blot analysis oflysates prepared from Sf9 cells infected with the recombinant viruses confirmed that the baculovirus-expressed NP antigens were reactive with monoclonal antibodies specific for either type A or B NP and with anti-np antibodies in human serum samples. Eleetrophoretic analysis indicated that the expressed NP antigens comigrated with NP purified from influenza A or B virions and that the recombinant NP antigens represented greater than 10 % of total protein in infected cells. Dilutions of clarified Sf9 cell lysates were used as antigens in a standard enzyme immunoassay to detect serum antibody specific for influenza A or/b viruses. The results from assays using the baculovirus-expressed NP antigens showed good correlation with the results obtained using bacterially expressed NP antigen as well as complement fixation. Therefore, baculovirus-expressed NP antigens have the potential to be used to develop reproducible and routine assays for the serodiagnosis of influenza virus infections as an alternative to the complement fixation or haemagglutination inhibition tests. Introduction The nucleoprotein (NP) is the major protein component of the ribonucleoprotein core of influenza virions and is a type-specific antigen defining influenza viruses as either type A or B. Recent studies have also identified the NP antigen as a major target of the cross-reactive cytotoxic T cell response against influenza viruses (Townsend & Skehel, 1984); therefore, cellular immunity to NP may contribute to recovery from influenza infection (Wraith et al., 1987). Because antigenic variation of NP occurs more slowly than does that of influenza virus surface glycoproteins (Van Wyke et al., 1980), this antigen is particularly useful for serodiagnostic tests. Antibodies to NP have traditionally been measured by a complement fixation (CF) test. However, the CF test requires that a large number of biological reagents be standardized. In addition, NP antigen preparations used in the CF test are likely to contain other virus-specific antigens. Since the NP antigen is antigenically conserved, it is an ideal candidate for expression by recombinant DNA methodology as part of the development of a new generation of diagnostic assays for influenza virus infections. We have previously reported the use of NP antigen produced in Escherichh~ coli in an experimental diagnostic assay for influenza A virus (Harmon et al., 1989). This assay was found to be superior to CF for influenza virus diagnosis. The recently developed eukaryotic expression system using recombinant baculovirus, derived from Autographa californica nuclear polyhedrosis virus (AcNPV), should be useful for producing antigens for immunoassays for the serological diagnosis of viral infections (Luckow & Summers, 1987). Insect cells (Spodoptera frugiperda) infected with recombinant baculoviruses are capable of producing large amounts of antigen (Possee, 1986; Matsuura et al., 1987). Another important advantage of the baculovirus system is that the viral antigens are produced in cells that do not contain antigens that cross-react with antibodies in most human sera. Therefore, purification of the antigen, a step required for proteins expressed in bacterial and yeast systems, may not be required with baculovirus-expressed antigens. AcNPV does not infect humans and can therefore be safely handled in large quantities. In this report, we describe the construction of recombinant AcNPV for the expression of the NP antigens from influenza A and B viruses in insect cells. We also report on the antigenic reactivity of the baculovirus-expressed antigens with human serum SGM

2 1546 P. A. Rota and others samples in a standard enzyme immunoassay (EIA) protocol and demonstrate that results obtained from this assay correlated with results obtained using bacterially expressed NP antigen and with the results from CF assays. 3 A NP 1565 bp 5' B NP 1811 bp ] BSX Polyhedrin s t a r t ~ [... \ 1. A NP :Sail, ecori 1. Small t ',~ [ p~inv ]I. BNP:BamHI, Pvull 2. CIP [2' DAcYM1B1 Methods cdna cloning of influenza type A and B virus NP genes. Virion RNA was extracted from gradient-purified influenza B/Ann Arbor/l/86 and A/Ann Arbor/6/60 (wild-type) viruses by standard methods (Cox et al., 1983). cdna copies of total viral RNA were prepared by the method of Lapeyre & Amalric (1985) except that first-strand synthesis by reverse transcriptase was primed by using universal influenza type A and B virus primers complementary to the 3' untranslated region of virion RNA. The double-stranded cdna fragment corresponding to RNA segment 5 (influenza A virus, 1565 base pairs; influenza B virus, 1811 base pairs) was isolated from agarose gels, purified, and ligated into the Smal site of plasmid puc8 using standard methods (Maniatis et al., 1982). Bacterial colonies (Escherichia coli HB101) containing recombinant plasmids with NP inserts were identified by in situ hybridization (Maniatis et al., 1982) to 3:p-labelled oligonucleotide primers with sequences specific for influenza A or B virus NP genes. The size, sequence and orientation of the inserted NP genes were determined by a rapid plasmid-sequencing technique (Chen & Seeberg, 1985) using the M13 forward and reverse primers and by restriction endonuclease analysis. Plasmids containing full-length cdna copies of the influenza A and B virus NP genes were designated panp and pbnp, respectively (Fig. 1). Expression of influenza virus NP in insect cells. NP genes were excised from panp and pbnp by restriction endonuclease digestion, purified by agarose gel electrophoresis, made blunt-ended by treatment with mung bean nuclease, and ligated into the Sinai site of the transfer vector pacym1b1 (Fig. 1). pacym1b1, a derivative of pacym1 (Matsuura et al., 1987) contains a multiple cloning site inserted at the unique BamHI site of pacym1 (girl from Anthony Sanchez, Centers for Disease Control). Transformant colonies containing the inserted NP genes were identified by in situ hybridization as mentioned earlier. The proper orientation of the NP genes with respect to the AcNPV polyhedrin gene promoter was confirmed by sequencing purified plasmid DNA using oligonucleotide primers, B1 and B10 (also a gift from Anthony Sanchez), designed for sequencing of the 5' and 3' termini of DNA fragments inserted into the multiple cloning site of pacym1bi (Fig. 1). Insect cell culture and propagation of AcNPV were done by using previously described methods (Summers & Smith, 1987). Purified pym1anp and pym1bnp DNA were each cotransfected with purified AcNPV DNA into S.frugiperda (Sf9, ATCC CRL 1711) cells according to the protocol described by Summers & Smith (1987). After 5 to 6 days of incubation, serial dilutions (103 to 106) of the culture supernatants were prepared in Hink's medium, and 20 ~tl amounts of each dilution were inoculated into Sf9 cells that had been seeded into 96-well tissue culture plates. The supernatants from each well, harvested after 5 to 6 days, were transferred to a sterile 96-well plate for storage and the remaining cells were resuspended in 100 lal of phosphate-buffered saline (PBS), lysed by freezing and thawing, and spotted onto nitrocellulose filters. Cultures expressing NP were detected by incubating the filters with either influenza A or B virus NPspecific monoclonal antibodies (Walls et al., 1986). Hybridization and wash conditions were identical to those used for Western blot analysis (Tsang et al., 1983) except that bound antibody was visualized with ~zsi-labelled staphylococcal Protein A. Recombinant viruses express- Am I~'rg:ntj dm HBIOI Amp T 5" A NP 1565 bp 3' t B NP 1811 bp ] "'-. B X.- Polyhedrin s t a ~ Amp r Fig. 1. Scheme for construction of baculovirus transfer vectors. Solid black line indicates plasmid DNA; black line with dashed inner line indicates AcNPV DNA. Abbreviations are: S, Sinai; P, PvuII; B, BamHI ; Sa, SalI; E, EcoRI ; X, XbaI ; CIP, calf intestinal phosphatase; MBN, mung bean nuclease; Amp r, plasmid ampicillin resistance gene. Arrows indicate start site ofacnpv polyhedrin gene mrna and binding regions of the BI and BI0 sequencing primers. ing either the influenza A or B virus NP were purified from cultures that were strongly positive for antigen expression by three successive rounds of plaque isolation (Summers & Smith, 1987). Only plaques that were negative for polyhedron production by light microscopic examination were selected. Analysis of recombinant baculoviruses. DNA from the plaque-purified recombinant baculoviruses, AcANP and AcBNP, was extracted from the cytoplasm of infected Sf9 cells and analysed by restriction endonuclease digestion and Southern blot hybridization by standard procedures (Maniatis et al., 1982). Sf9 cells infected with the recombinant viruses were removed from the culture vessel, washed with Grace's medium, fixed onto microscope slides with ice-cold acetone and examined by fluorescent antibody assay using fluorescein isothiocyanate (FITC)-conjugated influenza A or B virus NP-specific monoclonal antibodies (Boots-Celltech) (Walls et al., 1986). Lysates prepared from infected cells were subjected to electrophoresis on 10~ polyacrylamide gels containing 4 M-urea (Lamb et al., 1978) followed by Western blot analysis (Tsang et al., 1983). To quantify the amount of NP contained in cell lysates, we stained gels containing electrophoretic separations with Coomassie blue and determined the amount of NP present by scanning densitometry. Antigenic reactivity of baculovirus-expressed NP antigens. Postinfection mouse serum was prepared by infecting groups of six A/J mice intranasally with 106 TCID50 of either influenza B/England/222/82 or A/Ann Arbor/6/60 virus. Serum samples were obtained from each group at 14 days post-infection and pooled. Adult human

3 Baculovirus expression of influenza virus NP 1547 serum samples used in this study were from the same set as was used in a previous study with bacterially expressed influenza A virus NP (Harmon et al., 1989). These serum samples had been obtained during the acute and convalescent stages of illness of patients from whom influenza A or B viruses were isolated. All of these serum pairs used had rises in antibody titre to either virus as detected by haemagglutination inhibition and CF (Table 1, adults 1 to 13). Serum pairs from infant children who had natural influenza infections as documented by virus isolation (Table 1, children 1 and 2) and from infant children receiving monovalent influenza A vaccine (Table 1, children 3 and 4) were also used. Paired serum samples from adults and infant children with rises in antibody titre to other respiratory viruses (Table 1, adults 14 and 15, children 5 and 6) as well as normal human infant serum (Table 1, children 7 and 8) and normal chimpanzee serum were used as negative controls. To prepare antigens for EIA, Sf9 cells were infected with the recombinant AcNPVs, and the cells were harvested when the eytopathic effect was advanced (usually 4 or 5 days). The cells were pelleted, washed with Hink's medium, and resuspended in PBS with 1 M-NaC1. The cells were disrupted by freezing and thawing followed by Dounce homogenization and centrifuged for 1 h at g to pellet cellular debris. Electrophoretic analysis of pellet and supernatant fractions revealed that most of the NP antigen was present in the supernatant. Lysates of cells infected with a recombinant baculovirus expressing the Lassa fever virus glycoprotein gene (gift from David Auperin and Kimberly Hummel, Centers for Disease Control) were prepared as described above and used as negative control antigens. For EIA, the supernatants were diluted in PBS containing 1% foetal bovine serum and coated onto 96-well polystyrene plates for 24 h at room temperature. Wash buffer was PBS plus 0.5% Tween 20. After washing, the plates were preincubated for 1 h with PBS, 0-5~ Tween plus 1 ~ foetal bovine serum. Dilutions of the serum specimens were then added and allowed to incubate for 2 h at room temperature. The plates were washed three times, and bound antibody was detected by using Protein A conjugated to horseradish peroxidase with o- phenylenediamine and hydrogen peroxide as substrate. Titres were expressed as the reciprocal of the highest serum dilution giving a reading of greater than in the EIA. Titres listed in Table 1 are the average of duplicate tests on each sample. Human serum samples (at 1:200 dilutions) were also analysed by Western blotting to detect antigen in electrophoretic separations of cell lysates of infected Sf9 cells (Tsang et al., 1983). Results Cloning influenza A and B virus NP genes Full-length edna clones of NP genes were constructed in puc8 as described in Methods. The correct size and the orientation of each clone were determined by restriction endonuclease digestion and by sequencing the 5' and 3' ends of the inserted edna. The complete nucleotide sequences of both NP genes have been published (Cox et al., 1988; Rota, 1989). The A NP edna clone was 1565 bases in length; the B NP was Both clones contained the entire NP coding sequence and 5' untranslated region, and both NP genes were oriented in the puc plasrnids with the 5" end of the gene closest to the BamHI site on the puc polylinker (Fig. 1). Fig. 2 Detection of baculovirus-expressed influenza virus A or B NP by fluorescent antibody. Sf9 cells were infected with AcANP (a), AcBNP (c) or wild-type AcNPV (b, d) fixed and stained with FITCconjugated monoclonal antibody to type A NP (a, b) or type B NP (c, d). Bar marker represents 25 ~m. Expression of influenza virus NP genes in insect cells After the transfer of the cloned NP genes into the transfer vector, pacym1b1, sequencing of the pym1bnp and pym1anp plasmids using the BI and B10 primers (Fig. 1) verified that the NP genes had been inserted in the correct orientation. The sequences also indicated that the translation start sites of the A and B NPs were, respectively, 50 and 58 bases downstream from the deleted translation start site on the AcNPV polyhedrin gene (Matsuura et al., 1987) and therefore 100 and 108 bases downstream of the transcription start site of the AcNPV polyhedrin gene mrna. Initial evaluation of the baculovirus recombinants obtained after transfection of Sf9 cells with the transfer plasmids and AcNPV DNA was done by fluorescent antibody assay to detect NP expression. This was repeated after plaque purification of the recombinant viruses, AcANP and AcBNP (Fig. 2). Cells infected with purified recombinant baculovirus showed intense, granular fluorescence when stained with FITC-conju-

4 1548 P. A. Rota and others gated monoclonal antibodies specific for either influenza A or B virus NP. In some cases (not shown) large, positively staining aggregates were visible in the cytoplasm of the infected cells. Sf9 cells infected with wildtype AcNPV gave little background fluorescence. DNA was purified from Sf9 cells infected with the recombinant baculoviruses and analysed by Southern blotting (Fig. 3). Restriction fragments that comigrated with NP genes excised from the puc plasmids and also hybridized to influenza A or B virus NP-specific 32p. labelled oligonucleotide probes were detected in blots of restriction digests of DNA from AcANP and AcBNP. Therefore the recombinant baculoviruses appeared to contain complete copies of the appropriate NP genes. Western blot analysis of total cell lysates prepared from Sf9 cells infected with either the A or the B NPproducing recombinant virus indicated that the NP antigens being expressed by the recombinant viruses were recognized by A or B NP-specific monoclonal antibodies and had electrophoretic mobilities that were identical to those of NPs contained in purified influenza A or B virions (Fig. 4). The monoclonal antibodies did not cross-react with any other antigens contained in Sf9 cell lysates or with the NP antigen of the heterologous subtype. In this gel system, the A NP migrated with an apparent Mr of approximately 53K; the B NP was slightly larger, approximately 58K (Fig. 5). Electrophoretic separations of lysates from infected Sf9 cells were also stained with Coomassie blue and analysed by scanning densitometry (Fig. 5). The protein bands that had been identified in the Western blot as either A or B NP protein were found to constitute approximately 10 to 20~ of the total protein in the lysate. The results of a time-course study (not shown) done by infecting cells in stationary culture at a multiplicity of infection of 5 p.f.u./cell and calculating the amount of NP contained in total cell lysates by Western blotting indicated that the level of expression for the B NP was greater than that of the A NP. In this experiment, peak production of both NPs occurred 4 to 5 days post-inoculation and reached a maximum of 200 to 300 ~tg NP/106 cells for the B NP and 50 to 100 pg NP/106 cells for the A NP. (a) ~,' i,', :~ ~ /i ~:, i~i ' ' l (b)! 2 3 B NP-- (1800) Fig. 3. Detection of influenza vxrus A or B NP genes in recombinant baculovirus. DNA from panp (a) or pbnp (b) (lanes 1), AcANP or AcBNP (lanes 2) and wild-type AcNPV (lanes 3) was digested with SalI and EcoRI (a) or BamHI and PvulI (b), separated by agarose gel electrophoresis, transferred to nitrocellulose and hybridized to 3zp. labelled primers specific for either A NP (a) or B NP (b). ANP-: (a) (b) B NP- / Antigenic reactivity of baculovirus-expressed NP antigens Crude lysates of Sf9 cells infected with the baculovirus recombinants expressing either the A or B NP antigen were prepared as described in Methods and used to coat polystyrene plates for EIA. The optimal concentration of the lysates used for coating the plates was a 1:1000 dilution of the B NP lysate and a 1 : 500 dilution of the A NP lysate containing, respectively 3 and 6 ~g of protein/ml (approximately 30 ng NP/well) and was determined in assays using the NP-specific monoclonal Fig. 4. Western blot analysis of baculovirus-expressed influenza virus A or B NP antigens. Samples were purified influenza A (a) or B (b) virions (lanes 1) or lysates of cells infected with wild-type AcNPV (lanes 4), AcANP (a, lane 2; b, lane 3) and AcBNP (a, lane 3; b, lane 2). Samples were electroblotted after electrophoresis and hybridized to monoclonal antibodies specific for A NP (a) or B NP (b).

5 Baeulovirus expression of influenza virus NP 1549 (a) (b) A NP B NP A --B NP --A NP -B NP --43K Fig. 5. (a) Coomassie blue-stained gel from Fig. 4. Lanes 1 and 2 represent lysates of Sf9 cells infected with AcANP (1) or AcBNP (2); lane 3 contains Mr markers. Bands identified as either A or B NP by immunological staining are marked. (b) Spectrodensitometric scans of the gel lanes from (a). antibodies to detect bound NP. Fig. 6 shows the reactivity of the antigens with serum samples from mice infected with either type A or B influenza virus. Normal mouse serum showed little background reactivity with either of the test antigens. Serum samples obtained from mice 14 days after infection with influenza virus reacted strongly with the NP antigen of the same virus type used for the infection but not with the NP antigen preparation from the heterologous virus type. These results indicated that the NP antigens were antigenically reactive when coated onto the EIA plates and that the remainder of the proteins present in the lysate were causing little background reactivity. The EIA was then tested with a series of acute (S 1) and convalescent phase ($2) adult human serum pairs that had demonstrated fourfold rises in titre against either influenza A or B virus as determined by both CF and by haemagglutination inhibition. Table 1 shows that the EIA using the baculovirus-expressed antigens detected serum antibody rises specific for either type A or B. The titres obtained using the baculovirus-expressed A NP antigen showed excellent correlation with titres obtained in assays using highly purified bacterially expressed A NP (Fig. 7 a). The titres obtained using the baculovirusexpressed A and B NP antigens showed a strong positive correlation with titres obtained using the traditional CF assay (Fig. 7b). All of the adult serum pairs that had a fourfold rise in antibody titre against influenza A virus as detected by CF (Table 1, adults 1 to 7) also had a fourfold rise in titre when tested with the baculovirus-expressed A NP by EIA. The EIA using the baculovirus-expressed B NP was slightly more sensitive than CF, and for three of the five serum pairs tested it detected a greater rise in antibody titre than did the CF assay (Table 1, adults 8 to 13). Also, it appeared that the EIA using the baculovirusexpressed antigens was more sensitive than the CF assay for detecting low levels of antibody in acute serum samples. Of the baculovirus-expressed antigens, the B NP was generally more reactive and gave higher titres than the A NP, even though equivalent amounts of the two antigens were seeded onto the EIA plates. Many of the acute serum samples from adults reacted with both A and B NP antigens as would be expected for human serum. Control serum pairs from adults who had antibody rises to other respiratory viruses (respiratory syncytial virus and parainfluenza virus) also showed some reactivity with the A NP antigen. However, eight acute phase or normal serum samples from infant children and one sample from a chimpanzee gave no background reactivity with the baculovirus antigen

6 1550 P. A. Rota and others ~ I (a) I.~ (b) I < I I I ~ I I I I I I L, [ I I I I I I I I I "I \ we subjected lysates of Sf9 cells infected with the recombinant baculoviruses to SDS-PAGE, blotted them to nitrocellulose, and hybridized them to several of the human acute and convalescent phase (S1 and $2) serum pairs from Table 1. The Western blot shown in Fig. 8 confirmed that all the acute serum samples from adults contained antibodies that reacted with NP antigens. In serum pairs that had a previously determined rise in antibody titre to influenza virus type A, the amount of antibody to the A NP in the convalescent phase ($2) serum increased visibly relative to the acute phase serum (S1); the amount of antibody that recognized the B NP remained constant for both serum samples. The reverse was true for the serum pair in which there was a rise in antibody titre to influenza B virus. The control samples also had detectable levels of NP antibody, but no increase in anti-np activity was apparent between the acute and the convalescent phase serum samples. Acute and control serum samples from infant children showed no reactivity with the baculovirus-expressed NP antigens in Western blot assays (Fig. 9). These results confirm that the background from the adult acute serum samples was due to pre-existing antibody and showed that human serum did not react with any of the other proteins contained in these crude baculovirus antigen preparations Serum dilution (reciprocal) Fig. 6. Reactivity of post-infection mouse serum with baculovirusexpressed influenza virus A (a) or B (b) NP antigens by EIA. [7, Normal mouse serum; i, serum from mice infected with type B influenza virus;, serum from mice infected with type A influenza virus, preparations (Table 1, children 1 to 8). In addition, EIA plates prepared using lysates of Sf9 cells infected with a polyhedrin-minus recombinant AcNPV (see Methods) showed little background reactivity with any of the human serum samples (Table 1). A polyhedrin-minus baculovirus recombinant was used for the control in these experiments rather than wild-type AcNPV because other investigators have shown that human serum binds non-specifically to the very abundant AcNPV polyhedron protein (unpublished observations). Therefore, the reactivity of the human acute serum samples from adults was most likely due to pre-existing antibody to influenza virus NP antigens from prior exposure to or vaccination against influenza rather than to non-specific reactivity of the human serum to baculovirus or Sf9 cell antigens. To investigate further the reactivity of human serum with the baculovirus-expressed influenza NP antigens, Discussion Viral antigens produced by recombinant DNA expression systems can provide an inexhaustible source of chemically defined material for use in serodiagnostic assays, experimental vaccines and fundamental research. These techniques also eliminate the costs and potential hazards in the large-scale cultivation of pathogenic viruses. For example, the use of baculovirusexpressed Hantaan virus nucleoprotein as a diagnostic antigen has been reported recently (Schmaljohn et al., 1988). In this report, we describe the construction of recombinant AcNPVs that express the NP antigens of influenza type A and B virus at levels similar to those obtained for other non-glycosylated viral antigens using the same transfer vector (Matsuura et al., 1987). The level of expression of the type A NP was lower than that of the type B NP, even though both genes were inserted into the transfer vector in similar orientations. The influenza virus-specific sequences immediately upstream from the NP initiation codons are different in type A and B viruses and could have different effects on expression levels. Optimum levels of expression have been obtained when the intervening sequences were removed and the initiation codon of the expressed

7 Baculovirus expression of influenza virus NP 1551 Table 1. Comparison of the reactivity of human serum samples with baculovirus-expressed influenza NP antigens with standard diagnostic tests EIA titre when tested with Complement fixation titre Baculovirus-expressed Bacterially expressed Influenza A Influenza B Serum A NP B NP Control A NP* virus virus Rises to influenza A virus Adult 1 S < 16 $ Adult 2 S <8 $ Adult 3 S $ Adult 4 S $ Adult 5 S $ < 32 Adult 6 S < 16 <8 $ < 16 Adult 7 S <8 $ I < 8 Child 1 S1 200 < 100 < 100 $ < 100 < 100 Child 2 SI < 100 < 100 < 100 $2 800 < 100 < 100 Child 3 t $1 200 < 100 < 100 $ < 100 < 100 Child 4 t SI < 100 < 100 < 100 $2 400 < 100 < 100 Rises to influenza B virus Adult 8 S < 8 < 8 $ < Adult 9 S <8 <8 $ < Adult 10 S <8 <8 $ < Adult 12 S <8 $ Adult 13 S < 16 < 16 $ < Rises to other respiratory viruses Adult 14 S $ Adult 15 S $ Child 5 SI < $2 < Child 6 S1 < 100 < 100 < 100 $2 < 100 < 100 < 100 Normal serum Child 7 < 100 < 100 < 100 Child 8 < 100 < 100 < 100 Chimpanzee < 100 < 100 < 100 * Titres from Harmon et al. (1989). t Monovalent influenza vaccine recipients. antigen was placed as close as possible to the deleted polyhedrin protein initiation codon on the transfer vector (Matsuura et al., 1987). A previous report has demonstrated that bacterially expressed NP antigen could be used in an EIA for the serological diagnosis of influenza virus infection, and the

8 1552 P. A. Rota and others i I I I I / (a) / /o ~1) ~ SI $2 Sl $2. *** / ooo 640 o o I I I I I I I I / 512 (b) '064 ~ ~ ~ I I I l I I I I I I I 1 I 1 I I! 512 (c) a Titre Fig. 7. Comparison of titres obtained in EIAs using baculovirusexpressed NPs compared to titres obtained using bacterially expressed A NP or CF. (a) Comparison of EIA titres using bacterial (y axis) or baculovirus-expressed (x axis) A NP for 24 adult serum samples (Table 1, adults 1 to 7 and 8 to 13). (b, c) Comparison of EIA titres (x axis) using baculovirus-expressed A NP (b, 24 points; Table 1, adults 1 to 7 and 8 to 13) or B NP (c, 18 points; Table 1, adults 4 to 7 and 8 to 13) antigens with titres obtained in CF assays (y axis). Coefficient of correlation (r) = 0.88 (a), 0-58 (b), 0.83 (c). Level of significance was 0.01 for all panels. (6) ~) S1 $2 S1 $2 Fig. 8. Western blot reactivity of human serum with baculovirusexpressed influenza virus NP antigens. Lysates of Sf9 cells infected with AcANP (left lanes) or AcBNP (right lanes) were subjected to electrophoresis and transferred to nitrocellulose and hybridized to pairs of acute phase (S1) and convalescent phase ($2) human serum specimens that had increased antibody reactivity specifically to either influenza A virus (al : Table 1, adult 1 ; a2: Table 1, adult 5) or influenza B virus (b: Table 1, adult 8) or to respiratory syncytial virus (c: Table 1, adult 14). results were superior to those obtained using the standard CF test (Harmon et al., 1989). The results of the analyses we present here indicated that the reactivities of the crude baculovirus-expressed antigens and highly purified bacterially expressed NP antigens were equivalent and that the titres obtained using the baculovirusexpressed antigens in a standard EIA gave titres that correlated with titres obtained with the CF assay. The EIA using the baculovirus-expressed B NP appeared to be more sensitive than that using the A NP for detecting increases in antibody titre. Since type A influenza viruses circulate more frequently than type B viruses, acute serum samples often contained higher levels of preexisting antibody to A NP than to B NP. These higher acute serum titres could account for the inability of the

9 Baculovirus expression of influenza virus NP ) (b) ~) S1 $2 $1 $2 SI $2 Fig. 9. Western blot reactivity of serum from infant children with baculovirus-expressed influenza virus A and B NP antigens. Acute and convalescent serum pairs (S1 and $2) were from individuals receiving monovalent influenza type A vaccine (a: Table 1, child 3), having a natural influenza type A infection (b: Table 1, child 1) or from a child infected with parainfluenza virus (c: Table 1, child 5). Lysate containing baculovirus-expressed A NP was run in right lane, B NP was run in left lane as in Fig. 8. EIA using the A NP to detect greater than fourfold rises in antibody titre. The results presented here suggest that it may be more advantageous to use baculovirus-expressed antigens in future assays because higher levels of expression can be achieved with the recombinant baculoviruses and because purification of the NP antigens from the infected ceils or supernatant medium is not necessary. None of the serum specimens from humans, mice, ferrets, rabbits or chimpanzees that we have tested in Western blot assays reacted with antigens other than the NPs in baculovirus-infected cells. In addition, with the standard EIA format, specialized reagents such as those required for CF do not need to be prepared and titrated. Studies directed at optimizing the EIA protocol with baculovirus-expressed NP antigens using larger sets of human serum samples are in progress. The reason for the decreased reactivity of the influenza A virus NP relative to that of the type B NP in the EIA tests is not clear. The A NP antigen may have formed aggregates in which antigenic sites on the molecule were partially blocked. We tried various treatments without success, including the use of detergents and heat to denature the A NP before using it as an EIA antigen. Other investigators have expressed influenza virus NP genes in bacteria (Kingsbury et al., 1987) or transient expression systems (Ryan et al., 1986) to study the biophysical properties of NP. Although the biological activity of the baculovirus-expressed NP antigen has not yet been examined, the results from the expression of other viral proteins suggest that insect cells were able to modify these expressed proteins correctly after translation and that biological activity will be maintained (Lanford, 1988; Murphy et al., 1988; Lanford et al., 1989; Overton et al., 1989; Schmaljohn et al., 1989). These recombinant viruses should provide a supply of influenza virus NP for future structure-function and immunological studies. The authors are grateful to Wendy Keitel, Baylor College of Medicine, Houston, Tx. and Peter F. Wright, Vanderbilt University, Nashville, Tenn., U.S.A. for the human serum samples, to Dean Erdman, David Auperin, Kimberly Hummel, Anthony Sanchez and James Ebert of the Division of Viral and Rickettsial Diseases of the Centers for Disease Control (CDC) for contributing materials used in this study, and to Drew Abernathy for technical assistance. The authors thank George Carlone of the CDC for help with scanning densitometry and David Bishop for initially providing the baculovirus transfer vector to CDC. References CHEN, E. Y. & SEEBERG, P. H. (1985). Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, Cox, N. J., BAI, Z. S. & KENDAL, A. P. (1983). Laboratory-based surveillance of influenza A (H1N1) and (H3N2) viruses in : antigenic and genomic analyses. Bulletin of the World Health Organization 61, COX, N. J., KITAME, E., KENDAL, A. P., MAASSAB, H. F. & NAEVE, C. (1988). 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