Veterinary Immunology and Immunopathology, 35 ( 1992 ) 133-141 Elsevier Science Publishers B.V., Amsterdam
133
A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay B. Mermer, P. Hillman, R. Harris, T. Krogmann, Q. Tonelli, W. Palin and P. Andersen IDEXX Laboratories, One IDEXX Way, Westbrook, ME 04092, USA
ABSTRACT Mermer, B., Hillman, P., Harris, R., Krogmann, T., Tonelli, Q., Palin, w. and Andersen, P., 1992. A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay. Vet. Immunol. Immunopathol., 35: 133-141. We have developed an antibody detection enzyme-linked immunosorbent assay (ELISA) for the identification of animals infected by feline immunodeficiency virus (FIV). The ELISA solid-phase antigen consists of recombinant FIV gag proteins expressed in bacteria. The proteins are purified from bacterial lysates as insoluble inclusion bodies. In the case of bacterially expressed p24~ , it is shown that all of the linear, sequential epitopes presented by viral p24 during infection are retained. Purified preparations can be substituted for solid-phase whole virus in the IDEXX PetChektm immunoassay. The antibody ELISA duplicates the sensitivity and specificity of the whole virus based PetChek plate assay. ABBREVIATIONS CrFK, Crandell feline kidney (cells); FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; PCR, polymerase chain reaction; TM, transmembrane (protein).
INTRODUCTION
Infection with a variety of lentiviruses is associated with immunodeficiency disease. Cats infected with feline immunodeficiency virus (FIV) (Pedersen et al., 1987 ), for example, show a number of pathogenic symptoms reminiscent of acquired immunodeficiency disease (AIDS) (Yamamoto et al., 1988; Ackley et al., 1990; Siebelink et al., 1990). FIV-associated feline AIDS is an important feline disease, with incidences as high as 15% in popuCorrespondence to: B. Mermer, IDEXX Laboratories, One IDEXX Way, Westbrook, ME 04092, USA.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
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B. MERMER ET AL.
lations of sick animals (O'Connor et al., 1989). The transient, low level viremia often seen in spite of the persistence of intracellular proviruses makes detection of the antibody response to infection the most reliable current assay for infection by FIV. As is the case with diagnostics designed to detect infection by human immunodeficiency virus (HIV), most antibody detection enzyme-linked immunosorbent assay (ELISA) formats utilize purified, inactivated FIV virions as solid-phase reagents. Cats infected with FIV elaborate antibodies directed against a number of the viral structural proteins. Antibodies recognizing epitopes on the p24-CA and p 15-NC products of the gag gene and the gp40-TM and gp 100-SU products ofenv have been detected by radioimmunoprecipitation analysis (RIPA) or immunoblot (Hosie and Jarrett, 1990; Steinman et al., 1990). The expression of these proteins in heterologous cells containing cloned genes is therefore a potential alternative source for the preparation of viral antigens for diagnostic applications. A variety of bacterially expressed HIV proteins, for example, have been evaluated as diagnostic reagents, including fragments derived from gag (Dowbenko et al., 1985 ), pol (Padberg et al., 1989 ), and env (Cabradilla et al., 1986). We have evaluated a recombinant-based antibody ELISA for FIV infection based on bacterially expressed antigens derived from the gag gene. We have found that this ELISA shows strong predictive value for the detection of FIV-infected cats. MATERIALS AND METHODS
Cells and viruses
The Crandall feline kidney (CrFK) cell line was used to propagate the Petaluma strain of FIV (O'Connor et al., 1989). Antisera
Rabbit antiserum directed against bacterial fl-galactosidase was purchased from 5 ' - 3 ' , 5603 Arapahoe Inc., Boulder, CO. Feline antisera from infected and uninfected animals were randomly selected from the collection at IDEXX Laboratories. Cloning and expression in Escherichia coli
The genes for p24 gag and pl 5gag were amplified by the polymerase chain reaction (PCR) from lysates of FIV Petaluma-infected CrFK cells. Primers were based on the published sequence (Talbott et al., 1989). PCR products were cloned in pUC 19, and inserts with verified DNA sequence were transferred to the appropriate pEX vector (Boehringer-Mannheim, Indianapolis, IN) to allow for synthesis of a fl-galactosidase-p24 gag or a fl-galactosidase-
RECOMBINANT-BASED
FIV ANTIBODY
13 5
ELISA
p 15g=g fusion protein in E. coli N4830-1 (Boehringer-Mannheim). Expres-
sion of the proteins was induced by incubation of 42 °C for 2 h. Purification of fusion proteins
fl-Galactosidase fusion proteins were purified from bacterial lysates as insoluble inclusion bodies by sonication and centrifugation as described previously (Hoppe et al., 1989; Wingender et al., 1989). Purity was assessed after Coomassie blue staining of SDS polyacrylamide gels by inspection or densitometry after PhastSystem (Pharmacia, Piccataway, N J) electrophoresis. Immunoblots
Immunoblot analysis and serum preadsorption was performed as previously described (Padberg et al., 1989). ELISA
ELISA analysis was performed by substitution of the recombinant antigens for the solid-phase whole virus antigen in the PetChek assay (IDEXX Laboratories).
-
-
Recombinant FIV Proteins
RV
Precursom:
prrr~
Genes
gag
viral proteins:
pl 5
Pr130 pol
IO24
env
su
EX
I~1
D
EX15 EYe.4
i
i
EXSU2
m
r---1
EXIDR
Fig. 1. Recombinant FIV proteins. Schematic diagram of the structures of the recombinant proteins used in this study. The FIV structural genes gag, pol and env are indicated. The polyprotein precursor products of the gag and pol genes, synthesized in infected feline cells, are depicted above the genes as Pr55 and Pr130, respectively. Below the gene designations are indicated the mature viral proteins p 15g=g, p24 gag, SU e"v and TM e"v, and diagrams of the EX recombinant proteins produced in E. coli.
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12345
Fig. 2. Purification of recombinant FIV proteins. PhastGel analysis of purified recombinant FIV proteins. Lane l, 0.1 #g purified EX24; Lane 2, 1 #g purified EX 15; Lane 3, 1/tg EX control; Lane 4, 1 Ftgtotal lysate containing EX24; Lane 5, molecular weight markers (0.1/lg per protein).
A 200
B -
9768-
43-
2
3
4
2
3
4
Fig. 3. Immunoblot analysis of recombinant FIV proteins. Immunoblot analysis was performed as described in Materials and Methods. Proteins were separated on 7.5% SDS polyacrylamide gels. Proteins were detected with serum from an infected cat (Panel A) or anti-fl-galactosidase serum (Panel B). Each lane contained about 0.1 #g of the following purified proteins: EX control (Lane 2); EX24 (Lane 3 ); EX 15 (Lane 4 ). Lane 1 contained prestained molecular weight markers (Bethesda Research Laboratories) of 200 000, 97 400, 68 000, 43 000, 29 000, and 18 400.
RECOMBINANT-BASEDFIVANTIBODYELISA
1
137
2
p27gag-->
Fig. 4. Serum preadsorption experiment. Western blot strips containing purified whole FIV vidons were treated with serum from an infected cat which detected primarily p24 and p 15 with (Lane 1 ) or without (Lane 2) preadsorption with purified EX24 protein. Antibody bound to the Western blot strips was then detected as in Fig. 3. RESULTS
Expression of recombinant FIV proteins in E. coli In order to evaluate the diagnostic potential of recombinant proteins, FIV
gag-derived polypeptides were expressed in E. coli (Materials and Methods). The genes for FIV p24gagand p 15gagwere cloned in the fl-galactosidase fusion vector pEX, allowing for regulated expression under the control of the ;t promoter and the heat-sensitive C 1857 repressor (Stanley and Luzio, 1984 ). This expression system resulted in the synthesis in bacteria of FIV proteins fused with the C-terminus of bacterial fl-galactosidase (Fig. 1).
Purification of recombinant FIV fusion proteins The regulated expression system described above allowed for the accumulation of recombinant FIV fusion proteins in induced cultures at levels equivalent to about 20% of total cellular protein. Purification of the recombinant
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-- PetChek
®F I V
vs. rFIV
3.5 mm
3
m
m mmm mm
m
2.5
mmm
mml
mm
m
m
mm
m
m
2 rHV
NIL
1.5
m
m
V m
1
mm
m mm
0.5
m
~
0
0.5
1
1.5 2 PetChek FIV
2.5
3
3.5
Fig. 5. Scatter diagram comparison of PetChek and recombinant-basedELISA. A panel of sera were analyzedby conventional PetChek (x-axis) or recombinant antigen-modifiedPetChek (yaxis). Values shown represent the absorbance at 650 nm. This analysis represented the data from 436 negative sera and 73 positive sera. proteins from bacterial lysates was monitored by gel electrophoresis. As control for these experiments, fl-galactosidase truncated at its C-terminus, the product of the pEX vector with no insert, was used. Figure 2 shows the degree of purification for the EX 24 product. Comparison of Lane 4, which contained 1/tg of total protein from the induced culture, and Lane 1, which contained 0.1/tg of purified protein, demonstrated a degree of purity which routinely approached 90% by densitometry analysis (data not shown). Similar enrichment was obtained with cultures expressing EX 15 (Fig. 2, Lane 2 ) and the EX control (Fig. 2, Lane 3 ).
Immunoreactivity o f recombinant F I V gag proteins Immunologic evaluation of the purified recombinant gag proteins was performed on immunoblots with an antiserum from an FIV-infected cat. This analysis (Fig. 3) indicated that antibodies elicited in cats by infection with FIV specifically recognized epitopes expressed in recombinant p24 gag (Lane 3 ) and p 15gag (Lane 4 ). To determine the extent to which epitopes presented during infection were retained in the bacterially expressed EX24 product, a serum preadsorption experiment was carried out (Fig. 4). Preadsorption of an infected cat serum with purified EX24 (Lane 1 ) specifically prevented recognition of authenthic viral P24 on an i m m u n o b l o t (Lane 2 ). Thus, EX24 displayed all of the linear, sequential, immunogenic epitopes of the cognate virion protein.
RECOMBINANT-BASED FlV ANTIBODY ELISA
139
A recombinant-based F I V antibody ELISA
The immunoreactive EX24 and EX 15 bacterial recombinant proteins were purified as described above in order to provide reagents for a recombinantbased immunoassay to detect antibodies specific for FIV gag. The sensitivity and specificity of the ELISA was compared with the USDA-licensed PetChek assay (Fig. 5 ). With this panel of feline sera, the agreement between the two assays was 100%.
DISCUSSION
We have developed an antibody detection ELISA based on the use of recombinant FIV proteins. As a potential diagnostic product, a recombinantbased assay for FIV infection offers a variety of useful features. The use of bacteria eliminates any variability caused by viral replication in tissue culture. The relatively high levels of expression attainable in a bacterial expression system should contribute to the purity of the final antigen preparation, with a concomitant improvement in the specificity of the immunoassay. The use of a prokaryotic expression system would also eliminate the potential for contamination by other feline antigens. A recombinant test like the one described here, for example, should be less likely to score autoimmune antibodies as a false positive. The choice of gene products incorporated into a recombinant-based immunoassay might influence the sensitivity of the test. The recombinant antigens should be highly antigenic and conserved within the FIV family, but should not contain epitopes shared with other feline retroviruses. We have modeled our recombinant-based assay on the sensitive whole virus based immunoassays, which contain purified virus as the solid-phase antigen. Based on the serology of HIV infection, it is likely that the product of gag, pol and env would be of potential diagnostic importance (Chang et al., 1985; Di Marzo Veronese et al., 1986 ). Sequence conservation is likely to be highest among the pol proteins of different isolates, but may extend to other retroviruses (Johnson et al., 1986 ). The primary env protein in purified virus is the transmembrane (TM) protein. The sequence variability typical of lentiviral env proteins might complicate the design of immunodiagnostics based on env (Coffin, 1986). Although we have identified immunodominant epitopes in the FIV TM protein (Mermer et al., 1992), we have not observed any improvement in sensitivity by inclusion of recombinant TM in the PetChek format (P. Hillman and B. Mermer, unpublished observations, 1991 ). Like other retroviruses, FIV is primarily composed of the gag proteins, with much lower
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B. MERMER ET AL.
q u a n t i t i e s o f the o t h e r s t r u c t u r a l gene p r o d u c t s ( D i c k s o n et al., 1 9 8 4 ) . By a n a l o g y with H I V , the F I V g a g p r o d u c t s are likely to be highly i m m u n o g e n i c ( D o w b e n k o et al., 1985) yet c o n s e r v e d a m o n g d i f f e r e n t isolates ( C o f f i n , 1986 ). B a s e d o n these c o n s i d e r a t i o n s a n d the d a t a p r e s e n t e d here, we believe t h a t r e c o m b i n a n t F I V g a g p r o t e i n s will p r o v i d e a reliable d i a g n o s t i c p r o d u c t o f strong p r e d i c t i v e v a l u e for the d e t e c t i o n o f F I V i n f e c t i o n in cats.
REFERENCES Ackley, C.D., Yamamoto, J.K., Levy, N.B., Pedersen, N.C. and Cooper, M.D., 1990. Immunologic abnormalities in pathogen free cats experimentally infected with feline immunodeficiency virus. J. Virol., 64: 5652-5655. Cabradilla, C.D., Groopman, J.E., Lanigan, J., Renz, M., Lasky, L. and Capon, D.J., 1986. Serodiagnosis of antibodies to the human AIDS retrovirus with a bacterially synthesized env polypeptide. Bioteehnology, 4:128-133. Chang, N.T., Chanda, P.D., Barone, A.D., McKinney, S., Rhodes, D.P., Tam, S.H., Sherman, C.W., Huang, J., Chang, T.W., Gallo, R.C. and Wong-Staal, F.W., 1985. Expression in Escherichia coli of open reading frame gene segments of HTLV-III. Science, 228: 93-96. Coffin, J.M., 1986. Genetic variation in AIDS viruses. Cell, 46: 1-4. Dickson, C., Eisenman, R., Fan, H., Hunter, E. and Teich, N., 1984. Protein biosynthesis and assembly. In: R. Weiss, N. Teich, H. Varmus and J. Coffin (Editors), RNA Tumor Viruses. Cold Spring Harbor Laboratory, pp. 513-648. Di Marzo Veronese, F., Copeland, T.D., DeVico, A.L., Rahman, R., Oroszlan, S., Gallo, R.C. and Sangadharan, M.G., 1986. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science, 231: 1289-1291. Dowbenko, D.J., Bell, J.R., Benton, C.V., Groopman, J.E., Nguyen, H., Betterlein, D., Capon, D.J. and Lasky, L.A., 1985. Bacterial expression of acquired immunodeficiency syndrome retrovirus p24 gag protein and its use as a diagnostic reagent. Proc. Natl. Acad. Sci. USA, 82: 7748-7752. Hoppe, J., Weich, H.A. and Eichner, W., 1989. Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli. Biochemistry, 28: 2956-2960. Hosie, M.J. and Jarrett, O., 1990. Serological response of cats to feline immunodeficiencyvirus. AIDS, 4: 215-220. Johnson, M.S., Mcclure, M.A., Feng, D.F., Gray, J. and Doolittle, R.G., 1986. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc. Natl. Acad. Sci. USA, 83: 7648-7652. Mermer, B., Harris, R., Seymour, C,, Krogmann, T., Lawrence, K., Hamblen, M., Lemieux, L., Jackson, T. and Andersen, P., 1992. Monoclonal antibodies directed against an immunodominant epitope in the transmembrane protein of Feline Immunodeficiency Virus. In preparation. O'Connor, T.P., Tangnay, S., Steinman, R., Smith, R., Barr, M.C., Yamamoto, J.K., Pedersen, N.C., Andersen, P.R. and Tonelli, Q.J., 1989. Development and evaluation ofimmunoassay for detection of antibodies to the Feline T-Lymphotropic Lentivirus (Feline Immunodeficiency Virus). J. Clin. Microbiol., 27: 474-479. Padberg, C., Nowlan, S. and Mermer, B., 1989. Recombinant polypeptides from the Human
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Immunodeficiency Virus reverse transcriptase define three epitopes recognized by antibodies in sera from patients with acquired immunodeficiency syndrome. AIDS Res. Hum. Retroviruses, 5:61-71. Pedersen, N.C., Ho, E.W., Brown, M.L. and Yamamoto, J.K., 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science, 235: 790793. Siebelink, K.H.J., Chu, I., Rimmelzwaan, G.F., Weijer, K., van Herwijnen, R., Knell, P., Egberink, H.F., Bosch, M.L. and Osterhaus, A.D.M.E., 1990. Feline immunodeficiency virus (FIV) infection in the cat as a model for HIV infection in man: FIV-induced impairment of immune function. AIDS Res. Hum. Retroviruses, 6: 1373-1378. Stanley, K.K. and Luzio, J.P., 1984. Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J., 3: 1429-1434. Steinman, R., Dombrowski, J., O'Connor, T., Montelaro, R.C., ToneUi, Q., Lawrence, K., Seymour, C., Goodness, J., Pedersen, N.C. and Andersen, P.R., 1990. Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus. J. Gen. Virol., 71: 701-706. Talbott, R.L., Sparker, E., Lovelace, K.M., Fitch, W.M., Pedersen, N.C., Luciw, P.A. and Elder, J.H., 1989. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc. Natl. Acad. Sci. USA, 86: 5743-5747. Wingender, E., Bercz, G., Blocker, H., Frank, R. and Mayer, H., 1989. Expression of human parathyroid hormone in Escherichia coli. J. Biol. Chem., 264: 4367-4373. Yamamoto, J.K., Sparger, E., Eo, E.W., Andersen, P.R., O'Connor, T.P., Mandell, C.P., Lowenstine, L. and Pedersen, N.C., 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res., 49: 1246-1258.
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A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay B. Mermer, P. Hillman, R. Harris, T. Krogmann, Q. Tonelli, W. Palin and P. Andersen IDEXX Laboratories, One IDEXX Way, Westbrook, ME 04092, USA
ABSTRACT Mermer, B., Hillman, P., Harris, R., Krogmann, T., Tonelli, Q., Palin, w. and Andersen, P., 1992. A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay. Vet. Immunol. Immunopathol., 35: 133-141. We have developed an antibody detection enzyme-linked immunosorbent assay (ELISA) for the identification of animals infected by feline immunodeficiency virus (FIV). The ELISA solid-phase antigen consists of recombinant FIV gag proteins expressed in bacteria. The proteins are purified from bacterial lysates as insoluble inclusion bodies. In the case of bacterially expressed p24~ , it is shown that all of the linear, sequential epitopes presented by viral p24 during infection are retained. Purified preparations can be substituted for solid-phase whole virus in the IDEXX PetChektm immunoassay. The antibody ELISA duplicates the sensitivity and specificity of the whole virus based PetChek plate assay. ABBREVIATIONS CrFK, Crandell feline kidney (cells); FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; PCR, polymerase chain reaction; TM, transmembrane (protein).
INTRODUCTION
Infection with a variety of lentiviruses is associated with immunodeficiency disease. Cats infected with feline immunodeficiency virus (FIV) (Pedersen et al., 1987 ), for example, show a number of pathogenic symptoms reminiscent of acquired immunodeficiency disease (AIDS) (Yamamoto et al., 1988; Ackley et al., 1990; Siebelink et al., 1990). FIV-associated feline AIDS is an important feline disease, with incidences as high as 15% in popuCorrespondence to: B. Mermer, IDEXX Laboratories, One IDEXX Way, Westbrook, ME 04092, USA.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
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lations of sick animals (O'Connor et al., 1989). The transient, low level viremia often seen in spite of the persistence of intracellular proviruses makes detection of the antibody response to infection the most reliable current assay for infection by FIV. As is the case with diagnostics designed to detect infection by human immunodeficiency virus (HIV), most antibody detection enzyme-linked immunosorbent assay (ELISA) formats utilize purified, inactivated FIV virions as solid-phase reagents. Cats infected with FIV elaborate antibodies directed against a number of the viral structural proteins. Antibodies recognizing epitopes on the p24-CA and p 15-NC products of the gag gene and the gp40-TM and gp 100-SU products ofenv have been detected by radioimmunoprecipitation analysis (RIPA) or immunoblot (Hosie and Jarrett, 1990; Steinman et al., 1990). The expression of these proteins in heterologous cells containing cloned genes is therefore a potential alternative source for the preparation of viral antigens for diagnostic applications. A variety of bacterially expressed HIV proteins, for example, have been evaluated as diagnostic reagents, including fragments derived from gag (Dowbenko et al., 1985 ), pol (Padberg et al., 1989 ), and env (Cabradilla et al., 1986). We have evaluated a recombinant-based antibody ELISA for FIV infection based on bacterially expressed antigens derived from the gag gene. We have found that this ELISA shows strong predictive value for the detection of FIV-infected cats. MATERIALS AND METHODS
Cells and viruses
The Crandall feline kidney (CrFK) cell line was used to propagate the Petaluma strain of FIV (O'Connor et al., 1989). Antisera
Rabbit antiserum directed against bacterial fl-galactosidase was purchased from 5 ' - 3 ' , 5603 Arapahoe Inc., Boulder, CO. Feline antisera from infected and uninfected animals were randomly selected from the collection at IDEXX Laboratories. Cloning and expression in Escherichia coli
The genes for p24 gag and pl 5gag were amplified by the polymerase chain reaction (PCR) from lysates of FIV Petaluma-infected CrFK cells. Primers were based on the published sequence (Talbott et al., 1989). PCR products were cloned in pUC 19, and inserts with verified DNA sequence were transferred to the appropriate pEX vector (Boehringer-Mannheim, Indianapolis, IN) to allow for synthesis of a fl-galactosidase-p24 gag or a fl-galactosidase-
RECOMBINANT-BASED
FIV ANTIBODY
13 5
ELISA
p 15g=g fusion protein in E. coli N4830-1 (Boehringer-Mannheim). Expres-
sion of the proteins was induced by incubation of 42 °C for 2 h. Purification of fusion proteins
fl-Galactosidase fusion proteins were purified from bacterial lysates as insoluble inclusion bodies by sonication and centrifugation as described previously (Hoppe et al., 1989; Wingender et al., 1989). Purity was assessed after Coomassie blue staining of SDS polyacrylamide gels by inspection or densitometry after PhastSystem (Pharmacia, Piccataway, N J) electrophoresis. Immunoblots
Immunoblot analysis and serum preadsorption was performed as previously described (Padberg et al., 1989). ELISA
ELISA analysis was performed by substitution of the recombinant antigens for the solid-phase whole virus antigen in the PetChek assay (IDEXX Laboratories).
-
-
Recombinant FIV Proteins
RV
Precursom:
prrr~
Genes
gag
viral proteins:
pl 5
Pr130 pol
IO24
env
su
EX
I~1
D
EX15 EYe.4
i
i
EXSU2
m
r---1
EXIDR
Fig. 1. Recombinant FIV proteins. Schematic diagram of the structures of the recombinant proteins used in this study. The FIV structural genes gag, pol and env are indicated. The polyprotein precursor products of the gag and pol genes, synthesized in infected feline cells, are depicted above the genes as Pr55 and Pr130, respectively. Below the gene designations are indicated the mature viral proteins p 15g=g, p24 gag, SU e"v and TM e"v, and diagrams of the EX recombinant proteins produced in E. coli.
136
B. MERMERETAL.
12345
Fig. 2. Purification of recombinant FIV proteins. PhastGel analysis of purified recombinant FIV proteins. Lane l, 0.1 #g purified EX24; Lane 2, 1 #g purified EX 15; Lane 3, 1/tg EX control; Lane 4, 1 Ftgtotal lysate containing EX24; Lane 5, molecular weight markers (0.1/lg per protein).
A 200
B -
9768-
43-
2
3
4
2
3
4
Fig. 3. Immunoblot analysis of recombinant FIV proteins. Immunoblot analysis was performed as described in Materials and Methods. Proteins were separated on 7.5% SDS polyacrylamide gels. Proteins were detected with serum from an infected cat (Panel A) or anti-fl-galactosidase serum (Panel B). Each lane contained about 0.1 #g of the following purified proteins: EX control (Lane 2); EX24 (Lane 3 ); EX 15 (Lane 4 ). Lane 1 contained prestained molecular weight markers (Bethesda Research Laboratories) of 200 000, 97 400, 68 000, 43 000, 29 000, and 18 400.
RECOMBINANT-BASEDFIVANTIBODYELISA
1
137
2
p27gag-->
Fig. 4. Serum preadsorption experiment. Western blot strips containing purified whole FIV vidons were treated with serum from an infected cat which detected primarily p24 and p 15 with (Lane 1 ) or without (Lane 2) preadsorption with purified EX24 protein. Antibody bound to the Western blot strips was then detected as in Fig. 3. RESULTS
Expression of recombinant FIV proteins in E. coli In order to evaluate the diagnostic potential of recombinant proteins, FIV
gag-derived polypeptides were expressed in E. coli (Materials and Methods). The genes for FIV p24gagand p 15gagwere cloned in the fl-galactosidase fusion vector pEX, allowing for regulated expression under the control of the ;t promoter and the heat-sensitive C 1857 repressor (Stanley and Luzio, 1984 ). This expression system resulted in the synthesis in bacteria of FIV proteins fused with the C-terminus of bacterial fl-galactosidase (Fig. 1).
Purification of recombinant FIV fusion proteins The regulated expression system described above allowed for the accumulation of recombinant FIV fusion proteins in induced cultures at levels equivalent to about 20% of total cellular protein. Purification of the recombinant
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-- PetChek
®F I V
vs. rFIV
3.5 mm
3
m
m mmm mm
m
2.5
mmm
mml
mm
m
m
mm
m
m
2 rHV
NIL
1.5
m
m
V m
1
mm
m mm
0.5
m
~
0
0.5
1
1.5 2 PetChek FIV
2.5
3
3.5
Fig. 5. Scatter diagram comparison of PetChek and recombinant-basedELISA. A panel of sera were analyzedby conventional PetChek (x-axis) or recombinant antigen-modifiedPetChek (yaxis). Values shown represent the absorbance at 650 nm. This analysis represented the data from 436 negative sera and 73 positive sera. proteins from bacterial lysates was monitored by gel electrophoresis. As control for these experiments, fl-galactosidase truncated at its C-terminus, the product of the pEX vector with no insert, was used. Figure 2 shows the degree of purification for the EX 24 product. Comparison of Lane 4, which contained 1/tg of total protein from the induced culture, and Lane 1, which contained 0.1/tg of purified protein, demonstrated a degree of purity which routinely approached 90% by densitometry analysis (data not shown). Similar enrichment was obtained with cultures expressing EX 15 (Fig. 2, Lane 2 ) and the EX control (Fig. 2, Lane 3 ).
Immunoreactivity o f recombinant F I V gag proteins Immunologic evaluation of the purified recombinant gag proteins was performed on immunoblots with an antiserum from an FIV-infected cat. This analysis (Fig. 3) indicated that antibodies elicited in cats by infection with FIV specifically recognized epitopes expressed in recombinant p24 gag (Lane 3 ) and p 15gag (Lane 4 ). To determine the extent to which epitopes presented during infection were retained in the bacterially expressed EX24 product, a serum preadsorption experiment was carried out (Fig. 4). Preadsorption of an infected cat serum with purified EX24 (Lane 1 ) specifically prevented recognition of authenthic viral P24 on an i m m u n o b l o t (Lane 2 ). Thus, EX24 displayed all of the linear, sequential, immunogenic epitopes of the cognate virion protein.
RECOMBINANT-BASED FlV ANTIBODY ELISA
139
A recombinant-based F I V antibody ELISA
The immunoreactive EX24 and EX 15 bacterial recombinant proteins were purified as described above in order to provide reagents for a recombinantbased immunoassay to detect antibodies specific for FIV gag. The sensitivity and specificity of the ELISA was compared with the USDA-licensed PetChek assay (Fig. 5 ). With this panel of feline sera, the agreement between the two assays was 100%.
DISCUSSION
We have developed an antibody detection ELISA based on the use of recombinant FIV proteins. As a potential diagnostic product, a recombinantbased assay for FIV infection offers a variety of useful features. The use of bacteria eliminates any variability caused by viral replication in tissue culture. The relatively high levels of expression attainable in a bacterial expression system should contribute to the purity of the final antigen preparation, with a concomitant improvement in the specificity of the immunoassay. The use of a prokaryotic expression system would also eliminate the potential for contamination by other feline antigens. A recombinant test like the one described here, for example, should be less likely to score autoimmune antibodies as a false positive. The choice of gene products incorporated into a recombinant-based immunoassay might influence the sensitivity of the test. The recombinant antigens should be highly antigenic and conserved within the FIV family, but should not contain epitopes shared with other feline retroviruses. We have modeled our recombinant-based assay on the sensitive whole virus based immunoassays, which contain purified virus as the solid-phase antigen. Based on the serology of HIV infection, it is likely that the product of gag, pol and env would be of potential diagnostic importance (Chang et al., 1985; Di Marzo Veronese et al., 1986 ). Sequence conservation is likely to be highest among the pol proteins of different isolates, but may extend to other retroviruses (Johnson et al., 1986 ). The primary env protein in purified virus is the transmembrane (TM) protein. The sequence variability typical of lentiviral env proteins might complicate the design of immunodiagnostics based on env (Coffin, 1986). Although we have identified immunodominant epitopes in the FIV TM protein (Mermer et al., 1992), we have not observed any improvement in sensitivity by inclusion of recombinant TM in the PetChek format (P. Hillman and B. Mermer, unpublished observations, 1991 ). Like other retroviruses, FIV is primarily composed of the gag proteins, with much lower
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q u a n t i t i e s o f the o t h e r s t r u c t u r a l gene p r o d u c t s ( D i c k s o n et al., 1 9 8 4 ) . By a n a l o g y with H I V , the F I V g a g p r o d u c t s are likely to be highly i m m u n o g e n i c ( D o w b e n k o et al., 1985) yet c o n s e r v e d a m o n g d i f f e r e n t isolates ( C o f f i n , 1986 ). B a s e d o n these c o n s i d e r a t i o n s a n d the d a t a p r e s e n t e d here, we believe t h a t r e c o m b i n a n t F I V g a g p r o t e i n s will p r o v i d e a reliable d i a g n o s t i c p r o d u c t o f strong p r e d i c t i v e v a l u e for the d e t e c t i o n o f F I V i n f e c t i o n in cats.
REFERENCES Ackley, C.D., Yamamoto, J.K., Levy, N.B., Pedersen, N.C. and Cooper, M.D., 1990. Immunologic abnormalities in pathogen free cats experimentally infected with feline immunodeficiency virus. J. Virol., 64: 5652-5655. Cabradilla, C.D., Groopman, J.E., Lanigan, J., Renz, M., Lasky, L. and Capon, D.J., 1986. Serodiagnosis of antibodies to the human AIDS retrovirus with a bacterially synthesized env polypeptide. Bioteehnology, 4:128-133. Chang, N.T., Chanda, P.D., Barone, A.D., McKinney, S., Rhodes, D.P., Tam, S.H., Sherman, C.W., Huang, J., Chang, T.W., Gallo, R.C. and Wong-Staal, F.W., 1985. Expression in Escherichia coli of open reading frame gene segments of HTLV-III. Science, 228: 93-96. Coffin, J.M., 1986. Genetic variation in AIDS viruses. Cell, 46: 1-4. Dickson, C., Eisenman, R., Fan, H., Hunter, E. and Teich, N., 1984. Protein biosynthesis and assembly. In: R. Weiss, N. Teich, H. Varmus and J. Coffin (Editors), RNA Tumor Viruses. Cold Spring Harbor Laboratory, pp. 513-648. Di Marzo Veronese, F., Copeland, T.D., DeVico, A.L., Rahman, R., Oroszlan, S., Gallo, R.C. and Sangadharan, M.G., 1986. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science, 231: 1289-1291. Dowbenko, D.J., Bell, J.R., Benton, C.V., Groopman, J.E., Nguyen, H., Betterlein, D., Capon, D.J. and Lasky, L.A., 1985. Bacterial expression of acquired immunodeficiency syndrome retrovirus p24 gag protein and its use as a diagnostic reagent. Proc. Natl. Acad. Sci. USA, 82: 7748-7752. Hoppe, J., Weich, H.A. and Eichner, W., 1989. Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli. Biochemistry, 28: 2956-2960. Hosie, M.J. and Jarrett, O., 1990. Serological response of cats to feline immunodeficiencyvirus. AIDS, 4: 215-220. Johnson, M.S., Mcclure, M.A., Feng, D.F., Gray, J. and Doolittle, R.G., 1986. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc. Natl. Acad. Sci. USA, 83: 7648-7652. Mermer, B., Harris, R., Seymour, C,, Krogmann, T., Lawrence, K., Hamblen, M., Lemieux, L., Jackson, T. and Andersen, P., 1992. Monoclonal antibodies directed against an immunodominant epitope in the transmembrane protein of Feline Immunodeficiency Virus. In preparation. O'Connor, T.P., Tangnay, S., Steinman, R., Smith, R., Barr, M.C., Yamamoto, J.K., Pedersen, N.C., Andersen, P.R. and Tonelli, Q.J., 1989. Development and evaluation ofimmunoassay for detection of antibodies to the Feline T-Lymphotropic Lentivirus (Feline Immunodeficiency Virus). J. Clin. Microbiol., 27: 474-479. Padberg, C., Nowlan, S. and Mermer, B., 1989. Recombinant polypeptides from the Human
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Immunodeficiency Virus reverse transcriptase define three epitopes recognized by antibodies in sera from patients with acquired immunodeficiency syndrome. AIDS Res. Hum. Retroviruses, 5:61-71. Pedersen, N.C., Ho, E.W., Brown, M.L. and Yamamoto, J.K., 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science, 235: 790793. Siebelink, K.H.J., Chu, I., Rimmelzwaan, G.F., Weijer, K., van Herwijnen, R., Knell, P., Egberink, H.F., Bosch, M.L. and Osterhaus, A.D.M.E., 1990. Feline immunodeficiency virus (FIV) infection in the cat as a model for HIV infection in man: FIV-induced impairment of immune function. AIDS Res. Hum. Retroviruses, 6: 1373-1378. Stanley, K.K. and Luzio, J.P., 1984. Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J., 3: 1429-1434. Steinman, R., Dombrowski, J., O'Connor, T., Montelaro, R.C., ToneUi, Q., Lawrence, K., Seymour, C., Goodness, J., Pedersen, N.C. and Andersen, P.R., 1990. Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus. J. Gen. Virol., 71: 701-706. Talbott, R.L., Sparker, E., Lovelace, K.M., Fitch, W.M., Pedersen, N.C., Luciw, P.A. and Elder, J.H., 1989. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc. Natl. Acad. Sci. USA, 86: 5743-5747. Wingender, E., Bercz, G., Blocker, H., Frank, R. and Mayer, H., 1989. Expression of human parathyroid hormone in Escherichia coli. J. Biol. Chem., 264: 4367-4373. Yamamoto, J.K., Sparger, E., Eo, E.W., Andersen, P.R., O'Connor, T.P., Mandell, C.P., Lowenstine, L. and Pedersen, N.C., 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res., 49: 1246-1258.
