mechanisms(s) by which these viruses cause disease has not been established. ...... We thank R. Lerner, S. Alexander, T. Shinnick, H. Alexander, R. Arlinghaus, P. Wright, and T. ... from Johnson and Johnson Biotechnology, Inc. LITERATURE ...
Vol. 61, No. 1
JOURNAL OF VIROLOGY, Jan. 1987, p. 8-15
0022-538X/87/010008-08$02.00/0 Copyright ©D 1987, American Society for Microbiology
Localization of Neutralizing Regions of the Envelope Gene of Feline Leukemia Virus by Using Anti-Synthetic Peptide Antibodiest JOHN H.
ELDER,'* JULI S.
McGEE,' MELINDA MUNSON,' RICHARD A. HOUGHTEN,' WILLIAM KLOETZER,2 JAMES L. BITTLE,' AND CHRIS K. GRANT3
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 920371; Johnson and Johnson Biotechnology Center, Inc., San Diego, California 921212; and Pacific Northwest Research Foundation, Seattle, Washington 981043 Received 26 June 1986/Accepted 10 September 1986
We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp7O and pl5E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp7O and three to pl5E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure. The feline leukemia viruses (FeLVs) are a group of horizontally transmitted retroviruses that are found associated with malignant and degenerative diseases of hematopoietic origin in domestic cats (19, 20, 26, 31, 45). Although leukemia can result from FeLV infection, general immune deficiency brought on by viremia is a major cause of death in the cat population (25). As in other retrovirus systems, including the murine and primate retroviruses, the mechanisms(s) by which these viruses cause disease has not been established. As more work is done, an increasing amount of structural variability has been detected. However, FeLVs may basically be divided into three subgroups, defined by type-specific neutralization and interference assays (47, 48). These include FeLV-A, which is the most frequently isolated subgroup; FeLV-B, which to date has been found only in association with FeLV-A; and FeLV-C, which has been isolated rarely but has been found along with FeLV-A and FeLV-B in cats with polycythemia. These virus subtypes have an overall homology of approximately 85% by nucleic acid hybridization (35). More variability has been detected by T, oligonucleotide analyses (46). The major changes detected between the various FeLV isolates reside within the envelope gene encoding gp7O, similar to the murine retroviruses (4, 8, 14, 29, 34, 50).
The extent of involvement of the envelope gene in the overall biology and pathogenesis of the retroviruses is unresolved. Within the murine retroviruses, recombination with endogenous virus-related sequences occurs with high frequency in association with transformation (7, 13, 27), and the recombinant viruses so formed are candidates for the actual transforming agents in virus-induced leukemia. Changes in the U3 enhancer region (5, 6, 10) may also be important to the transforming potential of the retroviruses. Rearrangements of the c-myc oncogene have been observed in association with transformation by FeLV (36, 42, 43), offering an additional explanation for the transforming ability of these and other retroviruses. Regardless of the ultimate mechanism of transformation, the envelope gene controls two important aspects of the retrovirus life cycle which have direct bearing on the ability of these viruses to infect and transform the host cell. The first is the phenomenon of recognition of the host cell and penetration, which is directly related to the structure of gp7O. Secondly, gp7O is the primary target for neutralizing antibody and thus directly influences the perception of the virion by the host during viral invasion. Therefore, structural (antigenic) variability within gp70 plays a pivotal role in the success of viral infection, regardless of the ultimate mechanism of transformation of the host cell. This may be particularly true of the FeLVs, which are primarily spread through exogenous infection and thus are confronted with the full force of the host immune surveillance system.
* Corresponding author. t Manuscript no. 4294-MB of the Molecular Biology Department of Scripps Clinic and Research Foundation.
8
VOL. 61, 1987
We wished to determine which regions of the FeLV envelope gene were involved in neutralization of the virus. We chose the synthetic peptide approach, which has been used with varied degrees of success in a number of viral systems, including foot-and-mouth disease virus (3), rabies virus (39, 40), hepatitis B virus (11, 22), herpesvirus (12), and influenza virus (2, 24, 49). We prepared antibodies to synthetic peptides corresponding to approximately 80% of the envelope gene of Gardner-Arnstein FeLV and tested the ability of these antibodies to neutralize several FeLV subtypes in vitro. The results indicate that several discrete regions of both gp7O and pi5E can serve as targets for a neutralizing response. The data, however, also indicate that changes distal to certain of these targeted epitopes can influence their availability in certain subtypes of FeLV. Thus, some subtypes may be resistant to neutralization at epitopes which are identical in susceptible subtypes. MATERIALS AND METHODS Virus strains and cell lines. All viruses were propagated on the dog thymus-derived CF2th cell line (ATCC CRL 1430). The viruses used include a molecularly cloned transfected isolate of Gardner-Arnstein FeLV-B (17), a molecularly cloned transfected type B isolate from Rickard virusinfected cells (16), and three prototype isolates representing subgroups A, B, and C which were obtained from Oswald Jarrett and subsequently molecularly cloned in our laboratory. The latter viruses were transfected into CF2th cells, and the resultant virus progeny were used for subsequent studies. Peptide synthesis and antisera production. Peptides derived from the sequence of the Gardner-Arnstein virus envelope gene (17) were prepared as described previously (30, 41). The peptides were then conjugated to keyhole limpet hemocyanin (KLH) for preparation of antisera in rabbits as described previously (32, 37). The resultant antisera were subsequently tested for titer and specificity by an enzymelinked immunosorbent assay (ELISA) against homologous and heterologous peptides in the absence of carrier protein
(32, 37). Virus infectivity and neutralization. In vitro virus infectivity and titers were determined by a modified immunoblotting procedure as previously described (18, 33), except that virus-infected cells were detected with a rabbit anti-p27 antiserum and peroxidase-conjugated goat anti-rabbit immunoglobulin instead of anti-gp70 antibody and "251-labeled Staphylococcus aureus protein A. The presence of peroxidase conjugate was detected with 4-chloro-1-naphthol dye (28). The antisera prepared above were tested along with the corresponding prebleed sera for ability to neutralize FeLV in vitro by preincubating virus supernatants for 30 min at 37°C prior to dilution and addition to cell monolayers. Immunoblotting procedures. Peptides were bound to nitro-
cellulose in the presence of carrier bovine serum albumin (0.1%) by fixation in 0.8% glutaraldehyde in phosphatebuffered saline. The blots were washed extensively with deionized water and then blocked and reacted with antibody essentially as described for Western blotting with nonfat dry milk (BLOTTO [18]) as a diluent. Immune reactivity was detected with either peroxidase conjugated anti-immunoglobulin or l25l-protein A. Antibody competition assays. Titers of antipeptide antisera and monoclonal antibodies reactive with the same peptide were determined against FL74 virus (provided by the Resources branch of the National Cancer Institute) in antigen
NEUTRALIZING ANTIPEPTIDE ANTIBODIES TO FeLV
9
excess by ELISA (21) with modifications. Dilutions which gave 50% maximal reactivity were chosen for each antibody and subsequently tested against dilutions of virus to define limiting antigen concentrations. The amount of viral antigen which gave 25% maximal reactivity at these antibody dilutions was then used in subsequent assays. Experiments were performed in which the mouse monoclonal antibody concentration was held constant and competed with serial dilutions of antipeptide antibody as well as experiments in which antipeptide antibody was held constant and competed with serial dilutions of monoclonal antibody. Antipeptide antibodies bound to virus-coated plates were specifically detected with a peroxidase-conjugated goat anti-rabbit immunoglobulin antibody. Monoclonal antibody binding was quantitated with a peroxidase-conjugated goat anti-mouse
immunoglobulin antibody. RESULTS Reactivity and neutralization by antipeptide antibodies to Gardner-Arnstein FeLV envelope gene. Based on the predicted amino acid sequence of the envelope gene of GardnerArnstein FeLV (17), we prepared 27 synthetic peptides corresponding to approximately 80% of the sequence encoding gp7O and pi5E (Fig. 1). The peptides were conjugated to KLH via cysteine residues (either naturally occurring or artificially added to the N or C terminus), and antisera were prepared in rabbits. These antisera were then analyzed for relative titer and for specificity. The titers of the antipeptide antisera varied considerably, but the response in each case was specific for the appropriate peptide (data not shown). Of the 27 antipeptide antisera, all reacted to some degree with nondisrupted FeLV in ELISA plate assays (Table 1). However, the majority had very weak antivirus titers. In vitro neutralization assays were performed to determine which of these antisera would inhibit virus infection (Table 1, Fig. 2). Eight of the antipeptide antisera were found to neutralize Gardner-Arnstein FeLV. Five of these sera were directed to gp7O, and three reacted with pi5E (Fig. 2). One of the neutralizing regions of gp7O (the 1-26 peptide, Fig. 2) corresponded to the region reported previously by Nunberg and colleagues (44) as a site of neutralization of FeLV. The neutralizing titers of all the antipeptide antisera were low compared with those of heterosera prepared in goats or rabbits against whole virions. Titers for 50% neutralization ranged from 1:20 to 1:60 with antipeptide sera versus 1:500 to 1:1,500 for the heterosera. However, the neutralizing titers of the antipeptide sera were considerably higher than normally found in regressing FeLV infection, which have titers in the range of 1:8 to 1:10 in comparable assays (data not shown). Neutralization of FeLV subtypes. We next assayed the neutralizing antisera against representative FeLV isolates from the A, B, and C subgroups (Table 2). FeLV-A was obtained from Oswald Jarrett and was subsequently molecularly cloned and transfected in CF2th dog thymus cells for our assay. Gardner-Arnstein virus, representative of FeLVB, was obtained from James Mullins and was the virus from which the sequence of our peptides was derived (17). Another B-type virus, cloned by Dr. Mullins from Rickard FeLV-infected cells (16) was also used in these studies. An additional B-type virus was obtained from Dr. Jarrett which has restriction characteristics similar to Snyder-Theilen FeLV (cloned isolate termed XB1). The FeLV-C subgroup is represented by the Sarma isolate (47). All the antipeptide antibodies which neutralized Gardner-Arnstein virus also
10
J. VIROL.
ELDER ET AL. 1 813 gp 70 IlI ( M4ESPTHPKPSKDKTLSWNLVFLVGILFT ID I GMANPSPIIQVYtNVTWT ITJLVTGTKAtNATStiLGTLTDAFPTMYFDL CD IIGJTWtNPSDQEPFPGYGCDQ
¢,Leader
C IB
C2B
121 1;
c5I
C4B
1RRWQQRnP YVCPGHA RKQCGGPQDGFCAVWGCETTGETYWRPTSS KY
C6B
C7B
1-26B
QGIYQCSGGGWCGPCYDKAVHSSTTGASEGGRCt!PL ILQ
C8
F TQKGRA1> WDGPKSWGL RL YRSGYDP I ALF SV SiVtlIT I TPPQAt-lGP tIL VL P,,QKPPSRQSQ I E SRVT P,1111SQG11GGTPG I TL VIIAS I APL ST PVT PAS
r1iILI R ri on r1R k rl LAh 1) 'L CI1OB C9B3 PK RI GTGDR,L I NLVQGTYL AL NATDPNRTKDC,WL$LVSRPPYYEG IA.ILG6NYSNQT tJPPPSCL S I PQIIKL T I SE VSGQGL,,C I GTVPKTIIQALC IE TQQGH
C15D
1 C16B
127B
1313
15E
_,GAHYLAAPNGTYWA\CNTGLTPCIS1 IAVLNWTTSDFCVLIELWPRVTYHQPEYVYTHFAKAARFRRtPISLTV0L ISLGGLTVGGIAAGVGGTKAL IETAQ C18B QA EES SL ESLTS FRQ QA1tH,D
14B
128B
15B
16B
17B
FtALRRLKRQFSQQGVIF EGFIK SEVVQNRRRGL D ILFL QEGGLCiAAL KEECXC YDGL VRD~k
IMGPLLILLLILLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDPDRP FIG. 1. Synthetic peptides synthesized for antibody preparation, as predicted from the envelope gene sequence of Gardner-Arnstein FeLV (17). Cysteine residues were added to peptides lacking endogenous cysteines to facilitate coupling to KLH (37). Peptides were synthesized as described previously (30, 41). PWFT
neutralized the other representatives of FeLV-B subgroup. Antibodies to the I-26 and C-8 peptides neutralized all five virus isolates to about the same degree, whereas antibodies to the 1-6 peptide of pl5E neutralized all the isolates but was considerably less effective against FeLV-A and FeLV-C (40% neutralization of the latter two isolates at concentrations which neutralized the B-type viruses by >80%). AntiC-18 partially neutralized FeLV-A but failed to neutralize FeLV-C. Anti-I-7 partially neutralized FeLV-C but failed to neutralize FeLV-A. Antibodies to peptides 1-10, C-9, and C-14 did not neutralize appreciably either FeLV-A or FeLVC isolates. Nucleotide sequence analyses of the FeLV-A isolate used in these studies as well as the B-type AB1 isolate are still in progress, so we cannot yet compare their reactivity with their primary amino acid sequences. However, comparison of the neutralizing regions to the envelope gene sequence of Sarma FeLV-C (38) yielded interesting results (Fig. 3). The I-10 peptide region, which failed to neutralize the FeLV-C isolate, had a totally different amino acid sequence in that virus, explaining the failure of the anti-I-10 antibody to neutralize it. We have recently prepared an antiserum to the FeLV-C equivalent of 1-10 which specifically neutralizes FeLV-C (data not shown). The I-26 and C-8 regions are homologous between Gardner-Arnstein and Sarma isolates, and thus cross-neutralization is compatible with the the sequence homology. The C-9 peptide resides in a hypervariable region and only served as a neutralizing target in the subtype B viruses, which are homologous in this
region. However, discrepancies arose in comparisons of the other four regions, all of which elicited neutralization of B-type viruses but failed to neutralize or only weakly neutralized FeLV-C. The C-14 region varied by four amino acids between these two isolates, but the changes were identical to differences between the Gardner-Arnstein and Rickard isolates, both of which were neutralized by anti-C-14 antibody. The C-18, 1-6, and 1-7 regions were identical between the Gardner-Arnstein and Sarma isolates. Together, the data indicate that the availability of these epitopes in the Sarma subtype C isolate is such that antibodies to these regions cannot neutralize the virus. Identification of epitopes required for monoclonal and antipeptide antibody binding. We carried out an extensive analysis of the region corresponding to the 1-26 and C-8 peptides to determine the amino acids critical for neutralization. These studies were facilitated by a monoclonal antibody (C11D8 [23]) made to FL74 virus which reacts with the I-26 peptide. C11D8 reacts with the same epitope as another rhonoclonal antibody previously used to identify this neutralizing region (44; data not shown). By preparing a "nest" of overlapping peptides within this region, we are able to define the minimal epitope size for reactivity with these antibodies and compare the results with those for the antisynthetic peptide antibodies (Fig. 4). The monoclonal antibody reacted against 1-26, I-3b, and 13-Bl, which were reductive syntheses from the N-terminal end of I-26 (Fig. 4, lane A). However, once the methionine residue was removed from the N terminus (peptide 13-B2), the monoclonal
NEUTRALIZING ANTIPEPTIDE ANTIBODIES TO FeLV
VOL. 61, 1987
11
TABLE 1. Comparison of the relative antipeptide, antivirus, and neutralization titers of antisera to peptides corresponding to the Gardner-Arnstein FeLV envelope gene Antiserum
1-8 I-10 C-i C-2 1-21 C-4 C-S C-6 C-7 1-26 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 1-27 C-16 1-3 C-18 1-4 1-28 I-5 1-6 1-7
Titer
Amino acid sequencea
VTWTITNLVTGTKA(C) CDIIGNTWNPSDQEPFPGYG CDQPMRRWQQRNTPF CPGHANRKQCGGPQDGFC CETTGETYWRPTSSWD YITVKKGVTQGIYQC CYDKAVHSSTTGASEGGR CNPLILQFTQKGRQTS
RLYRSGYDPIALFSVSR(C) QVMTITPPQAMGPNLVLP(C) DQKPPSRQSQIESRVTP(C) LSTPVTPASPKRIGTGDR(C)
LNATDPNRTKDC CLVSRPPYYEGIA GNYSNQTNPPPSC CLSIPQHKLTISEVSG CIGTVPKTHQALCNETQQGHT CNTGLTPCISMAVLNWTSDF CVLIELWPRVTYHQPEY FAKAARFRREPISLTVA(C) GTGTKALIETAQFR(C)
TDIQALEESISALEKSLTSLSE(C) RRGLDILFLQEGGLC QEGGLCAALKEEC CFYADHTGLVRDN AKLRERLKQRQQLF(C) DSQQGWFEGWFNKSPWFTTLISS(C)
Antipeptideb
Antivirusc
>10 > 160 >320 >320 1,280 >1,280 >640 >1,280 >10 >640 >320 >10 320
20 20 320 >80 >320 >40
>1,280
>640 20
>40
>20 >20
Based on the sequence of the Gardner-Arnstein FeLV envelope gene (17). (C), Cysteine artificially added to facilitate coupling of peptides to carrier protein. Titers of antibodies raised against peptides conjugated to KLH (37). Titers were determined by ELISA plate assay with 10 pmol of peptide per well in 96-well tissue culture plates. Values shown are the reciprocal of the antibody solution which yielded 50% maximal reactivity for each antibody against homologous peptide. The prebleed sera from the injected animals were tested and found to be negative at all dilutions. High-stringency conditions (33) were used in these assays, which yield lower absolute values but more accurate relative values. c Titers of antipeptide antibodies-against virus affixed to tissue culture plates at pH 8.5 and assayed by ELISA under high-stringency conditions (33). Absolute titers are not directly comparable to antipeptide titers, since the amount of peptide equivalents was not equal. d Neutralization assays were performed as described previously (18). Values are the reciprocal of the antisera dilution which yield 50% neutralization of Gardner-Arnstein FeLV relative to prebleed serum from each injected animal. Neutralization tests were repeated more than 20 times and were totally reproducible. -, No neutralization detected at the lowest dilution (1:5). a
b
antibody failed
to react. Reductive syntheses from the C terminus of 1-26 (peptides 1-85 to 1-87) indicated that the C-terminal residue critical for reactivity was the leucine residue of this sequence, yielding a five-amino-acid epitope (MGPNL) which was required for the reactivity of both of these monoclonal antibodies. We next tested three antipeptide polyclonal antisera to this region to compare the reactivity obtained with the monoclonal antibodies. The first (Fig. 4, lane B) was a neutralizing antiserum made to the 1-26 peptide. The second serum (lane C) was also made to 1-26, but failed to neutralize; and the third (lane D) was a neutralizing antiserum made against the C-8 peptide, which immediately adjoins the 1-26 peptide on the C-terminal side (see Fig. 1). These analyses demonstrated several points. (i) The neutralizing anti-I-26 serum reacted with all the peptides which were positive with the monoclonal antibodies (compare lanes A and B) and also reacted to a degree with two peptides (13-B2 and 1-86) not recognized by the monoclonals. The nonneutralizing anti-I26 serum (lane C) reacted only to peptides of the N-terminal reductive series (1-26, I-3B, 13-Bl, and 13-B2) and not to the C-terminal reductive peptides (I-85 to I-87). The reason for the different response to the I-26 peptide in these two sera is unclear at present. However, the immune reactivity of the nonneutralizing anti-I-26 antiserum was biased toward the C terminus. Additionally, the minimal epitope size for the
nonneutralizing sera was larger than for the neutralizing sera. As shown in Fig. 4, lane D, the epitope reactive with the C-8 peptide was also discrete, although more peptides need to be synthesized to determine the precise immunoreactive region. Reactivity was observed with the homologous peptide (C-8) and with peptide 15B, which lacked the C-terminal three amino acids of C-8. The anti-C-8 antibody, however, failed to react with the 14B peptide, which lacked an additional four amino acids from the C-terminal end of C-8. Comparison of binding and neutralizing properties of monoclonal and anti-synthetic peptide antisera. The neutralizing anti-synthetic peptide antibody to the 1-26 peptide was compared with the C1iD8 monoclonal antibody for antipeptide, antivirus, and neutralization titers (Table 3). Both of these antisera had equivalent titers against the peptide by ELISA plate assay. However, the monoclonal antibody had a fourfold-higher titer against intact virus. Additionally, the neutralizing titer of the monoclonal antibody was approximately 10-fold greater than the titer of the anti-synthetic peptide antibody. The nonneutralizing anti-I26 antiserum had antipeptide and antivirus titers equivalent to those of the neutralizing anti-I-26 antiserum, indicating that failure to neutralize with the former serum was not simply due to failure of the antibody to bind to the virus. We performed competition experiments between the neutraliz-
J. VIROL.
ELDER ET AL.
12
I f3l O1 I r gp7O ME SPTHIPKPSKDKTL SWtILVFLVG ILFT ID I GMANP SPIIQVYtIVIWT ITt.LVTGTKANJATSftLGTLTDAFPT14YFDLrD II GtI JIWPSUQEPF PGY DQ
F* L.eade
1211;
C2B3
ClI
C5l1
C4n
P11RRWQQRNfTPFYVCPGHAtIRKQCGGPQDGFCAVWGCETTGETYWRPTSSLIDY I TVKKGVTQGIYQCSGGGWCGPCYDKAVHSSTTGASEGGRCt!PL ILQ
CEl
CR
1-2613
C77n
VL tQKPPSRQSQ I ESRVT ItiSQGIJGGTPGI TLVYIAS IAF STPVTPAS I FTQKGRATSWDGPKSWGLRLYRSGYDP IALFSVSRtV-TIPPQA1GPtJL
C913
C. 1II
ri ?R
r1 In
%,I
ClOB
ID
U
r II I LR
II
PK RIGTlGDIL INLVQGTYLALT ATDPtRTKDCWLCLVSRPPYYEGIAILtNYSNJQT1JPPPSCLS IPQIIKLT ISEVSGQGL, IGTVPKTIIQALCINETQQGII
C16B 54. lSE
127B
C15D
1313
J_GAHYLAAPNGTYWACNTGLTPC ISIlAVLtJWTSDFCVL IELWPRVTYHQPEYVYTHiFAKAARFRREP)I SLTVAL1LGGLTVGGIAAGVGTGTKAL I ETAQ 14B
C18B
1281
1 513
1611
171
FRQLQMAMI1D IQALEESISALEKSLTSLSE VLQNRRGLDILFLQEGGLCALKEECCFYADIITGLVRDtO AKLRERLKQRQQL DSQQGFEGWFtIKS
PVJFTTLISSPMGPLLLLLILLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDPDRP FIG. 2. Location of peptides which elicited neutralizing antibody responses to Gardner-Arnstein FeLV. Peptides eliciting neutralization are boxed.
ing anti-I-26 antibody and the C11D8 monoclonal antibody for intact virus affixed to tissue culture plates (Fig. 5). Assays were performed with constant anti-synthetic peptide antibody or monoclonal antibody against a dilution series of the other. The monoclonal antibody competed more successfully with the anti-synthetic peptide antibody for virus binding than the latter did against the former (Fig. 5). The data suggest that the affinity of the monoclonal antibody for virus is higher than that of the anti-synthetic peptide antiTABLE 2. Neutralizationa of FeLV isolates by anti-synthetic
peptide antisera Neutralization of FeLVs Rickard ABl GardnerArnstein
Antiserum
Subtype A
1-10 1-26 C-8 C-9 C-14 C-18 1-6 1-7
+ +
+ + +
+ + +
+ + +
+ +
-
+
+
+
_
+ + -
+ + + +
+ + + +
+ + + +
_
Subtype C
± ±
aNeutralization assays were performed as described previously (18). Symbols: -, no neutralization; +, neutralization equivalent in magnitude to neutralization of Gardner-Arnstein FeLV (see Table 1); +, virus isolate neutralized to approximately 10 to 40% of the level of neutralization of Gardner-Arnstein FeLV (see Table 1).
body, which may explain the lower degree of neutralization obtained with the latter antibody. DISCUSSION The results of this study indicate that neutralization of FeLV can occur through antibody reaction at several distinct sites throughout the envelope gene. Neutralization can be facilitated by reaction to both gp7O and pl5E. However, the results further show that certain epitopes homologous between the A, B, and C subtypes of FeLV (including all three pl5E epitopes) preferentially neutralize the subtype B viruses. These results suggest that exposure of these peptide regions within the FeLV subtypes can be influenced by structural changes which are distal in the linear molecule. The most notable sequence changes in the envelope genes of FeLV-A (Glasgow-I [38, 52]) and Sarma subtype C virus (38) relative to the B-type viruses occur in the N-terminal portion of gp7O. These changes include both primary amino acid changes and substitution of additional sequences relative to the subtype B viruses. Homologous epitopes which neutralize subtype B but not subtypes A or C reside in the C-terminal portion of the envelope gene. Since the three-dimensional structure of gp7O is unknown, it is not possible to structurally predict such a masking phenomenon. However, if the overall structure of the gp70-p15 complex is similar to that of the complex formed between influenza virus hemagglutinins I and 11 (53), one could envision an influence of the N terminus of the molecule on the availability of epitopes on
NEUTRALIZING ANTIPEPTIDE ANTIBODIES TO FeLV
VOL. 61, 1987 PEPTIDE REGION
SEQUENCE
VIRUS
A
Rickard Sarma
1-26/C-e
Gardner-Arnstein
CDI IGNTWNPSDQEP--------- FPGYG LV D E IAPD TNVRSWARYSSSTH
QVMTITPPQAMGPNLVLPDQKPPSRQSQIESRVTP
Rickard Sarma C-9
C-14
Gardner-Arnstein
CIGTVPKTHQALCNETQQGHT
Rickard Sarma C-18
KK
K
K
KK
K
K
TDIQALEESISALEKSLTSLSE
Rickard Sarma 1-6
Gardner-Arnstein
Rickard Sarma
I-7
Gardner-Arnstein
1382
GPNLVLPI DaKPPSR aS0
1383
PNLVLPE3OKPPSROSQ
AKLRERLKQRQQLF K
DSQQGWFEGWFNKSPWFTTL ISS
Rickard Sarma
FIG. 3. Protein sequence comparisons of Gardner-Arnstein, Rickard, and Sarma isolates of FeLV within the peptide regions of the envelope gene of Gardner-Arnstein FeLV which elicited neutralizing antibody responses. Only amino acid changes are shown for Rickard and Sarma isolates. Dashed lines represent relative deletions. Gardner-Arnstein (17) and Rickard (16) isolates are subtype B. The Sarma (38) isolate is subtype C.
the C terminus. Another possible explanation of the differential susceptibility of the FeLV subtypes at common epitopes could be through altered glycosylation. The FeLV gp70s thus far sequenced all contain unique glycosylation sites which may influence the availability of certain distal epitopes. Recent studies in our laboratory (1, 15) have shown that although carbohydrates are not the targets of neutralizing antibodies, they do in fact have a marked influence on the perception of the virus by the host as an antigen. Others (51) have also reported that glycosylation changes have occurred in variants of influenza virus which have become resistant to neutralization at discrete sites. From the standpoint of relating neutralizing epitopes to the actual epitopes involved in cell-specific binding, this study suggests that at the least, a direct relationship is not mandatory. It seems unlikely that the majority of the neutralizing sites are directly involved in cell binding, especially if one invokes primary amino acid changes to explain the observed host range differences between the FeLV subtypes (47, 48). Of the eight neutralizing epitopes, only the 1-10 region and the C-9 region contain substantial amino acid changes between subtypes A, B, and C. Whether these regions of the gp7O molecule are involved in specific recognition of the respective host cells of the FeLV subtypes is currently under investigation. Detailed studies of one of the neutralizing regions, encompassed by peptides 1-26 and C-8, yielded interesting data about the epitopes recognized by the antibodies used in these studies. Both of these peptides contain discrete regions which are critical for reactivity. The neutralizing epitope of the 1-26 peptide, as assessed by reacting nested peptides from this region with a neutralizing monoclonal antibody (prepared against intact virions, but reactive with the pep-
_
NLVLPI D)KPPSR(S0
148
LPN D a KP P S R a S Q I E S R ra aKPPSRaSQI E SR VT P
1-85
MTITPPQAMGPNL
1-86
VMTITPPQAMGPN TITPPQA MG PN LV
187
f -
m
_
MGPNLVLPCDQKPPSR
CS
LSTPVTPASPKR IGTGDR N R VASATMG
Gardner-Arnstein
1381
15B
Rickard Sarma
Gardner-Arnstein
D
__
PA MGPNLVLPEDQK PPS
38 Gardner-Arnstein
C
UV MTI TPPaAMGPNLVLP
1-26
1-10
B
13
_~ _
_
...
FIG. 4. Slot blot analyses with monoclonal and anti-synthetic peptide antisera to react against a nest of synthetic peptides. Synthetic peptides were prepared as described previously (30, 41), using the derived amino acid sequence of the Gardner-Arnstein FeLV envelope gene (see Fig. 1). Peptides 1-26 and C-8 represent the continuous sequence through this region; the other peptides represent reductive synthesis from either the N- or C-terminal end of the I-26/C-8 region. Immunoblot analyses were performed against these peptides affixed to nitrocellulose membranes, essentially as described for Western blotting (see Materials and Methods section). Lanes: A, reactivity of the neutralizing monoclonal antibody (C11D8) made against FL74 virus but reactive with the 1-26 peptide; B, reactivity of neutralizing anti-synthetic peptide antibody made against peptide 1-26; C, reactivity of nonneutralizing anti-synthetic peptide antibody against 1-26; D, reactivity of neutralizing antibody against the C-8 peptide. Critical amino acids involved in the I-26 epitope, as judged by reactivity requirements for the monoclonal antibodies, are underlined in I-87.
tides), involved the five-amino-acid sequence methionineglycine-proline-asparagine-leucine. Whether substitutions within this sequence would be tolerated is unknown, but these studies indicate that shifting of the peptide sequence to either the right or the left of these five amino acids abrogated all reactivity. A search of protein sequences stored in Genbank and Dayhouf atlases indicated that the sequence Met-Gly-Pro-Asn-Leu could only be found in FeLV gp7O. Although the protein sequence repertoire is admittedly biased in these sources, the results are supportive of the frequency of occurrence one would expect from an immunologically specific epitope. This epitope is also interesting in that it contains three amino acids in a row (Gly-Pro-Asn) which are found in high frequency in reverse turns, as judged by the predictions of Chou and Fasman (9). The C-12 peptide, which elicited the second highest antivirus reTABLE 3. Comparison of the relative antipeptide, antivirus, and neutralization titers of monoclonal and anti-synthetic peptide antisera reactive with peptide 1-26 Titerb Antibodya
Anti-I-26 1 Anti-I-26 2 C11D8 a
Antipeptide
Antivirus
Neutralization
>640 >640 >640
>5,120 >5,120 >20,480
>60 >500
Anti-I-26 1 and 2 are two antisera prepared against different preparations
of I-26 peptide. Both antisera had high titers against both virus and peptide, but only anti-1-26 1 elicited neutralization. C11D8 is a monoclonal antibody prepared against whole FL74 virus (23) which reacts with the 1-26 peptide. Immunoglobulin G was prepared from ascites fluid and adjusted to 1 mg/ml for use as antibody in tests with C11D8. b Titers are expressed as the reciprocal of the antibody dilution which elicited 50% maximal reactivity or neutralization. See also Table 1, footnotes b, c, and d.
J. VIROL.
ELDER ET AL.
14
protein is critical for optimal binding. The appropriate conformation represents only a portion of the conformations assumed by peptide bound to KLH. Experiments are in progress to optimize the presentation of peptide antigen. From the standpoint of a vaccine, the significance of lower neutralizing titers is unclear, since it is unknown what constitutes a response sufficient for in vivo protection. The neutralizing titers of the antipeptide antisera were higher than observed in regressing cat sera. Challenge studies are in progress to test these peptides for protection in cats.
100
80
rl-.
-j
0
z 601 0
z
40[
w
06
201
10
1puu
100
/Ab
FIG. 5. Comparison of the ability of a monoclonal antibody made against whole virions and an anti-synthetic peptide antibody to compete for virus binding. Experiments were performed with FL74 virus affixed to tissue culture plates as detailed in Materials and Methods. Both the monoclonal antibody (C11D8 [23]) and the anti-I-26 antipeptide antibody bound to an epitope on the 1-26 peptide and elicited neutralization of FeLV. Competitions were performed by holding the monoclonal antibody concentration constant and competing with a dilution series of antipeptide antibody (x) or by holding the antipeptide antibody concentration constant and competing with a dilution series of the monoclonal antibody (0). 1/Ab, Reciprocal of the antibody dilution plotted on a logarithmic scale; percent control, percentage of binding obtained in the absence of competing antibody.
also had four amino acids in a row (asparagineproline-proline-proline) which frequently occur in reverse turns. Such regions may thus be prime targets for antibody responses. No correlation may, however, be drawn to neutralization, since anti-C-12 failed to neutralize. The antipeptide antiserum to the I-26 region which elicited neutralization contained antibodies reactive with and dependent on the Met-Gly-Pro-Asn-Leu epitope. Other minor reactivities are also observed, in keeping with the polyclonal nature of these preparations. One antiserum to the 1-26 peptide which failed to elicit neutralization also required a larger epitope size for reactivity. The predominant reactivity, as judged by reaction with the nested peptides, was shifted one amino acid to the right of the methionine residue. The 1-87 peptide, which contains the Met-Gly-Pro-Asn-Leu epitope as well as Val on the C terminus, was not immunoreactive with this antiserum, indicating that at least six amino acids were required for reactivity, and possibly as many as seven. However, the antivirus titers of the neutralizing and nonneutralizing antipeptide sera were equivalent, indicating that failure of this latter antibody to neutralize could not be explained simply by failure to bind to the virus. The neutralizing titers of all the anti-synthetic peptide antisera were low relative to those of heterosera made against whole virions or neutralizing monoclonal antibodies. The results of competition experiments and comparison of reactivity with virus particles indicated that antibodies to the synthetic peptides did not react as well as the monoclonal antibody to the epitope in the native virus under conditions in which titers against the peptide were equivalent. It seems probable that the conformation of the epitope within the viral sponse,
ACKNOWLEDGMENTS We thank R. Lerner, S. Alexander, T. Shinnick, H. Alexander, R. Arlinghaus, P. Wright, and T. Fieser for helpful discussions and comments regarding this work. The technical assistance of P. MacIsaac, L. A. Smith, and P. A. Van Hook is also acknowledged. We also thank S. Null for preparation of this manuscript. This work was supported by departmental funds and by a grant from Johnson and Johnson Biotechnology, Inc. LITERATURE CITED 1. Alexander, S., and J. H. Elder. 1984. Carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens. Science 226:1328-1330. 2. Atassi, M. Z., and R. G. Webster. 1980. Localization, synthesis, 3.
4.
5.
6.
7.
8. 9. 10. 11.
12.
13.
14.
and activity of an antigenic site on influenza virus hemagglutinin. Proc. Natl. Acad. Sci. USA 80:840-844. Bittle, J. L., R. A. Houghton, H. Alexander, T. M. Shinnick, J. G. Sutcliffe, R. A. Lerner, D. J. Rowlands, and F. Brown. 1982. Protection against foot-and-mouth disease by immunization with a chemically synthesised peptide predicted from the viral nucleotide sequence. Nature (London) 298:30-33. Bosselman, R. A., F. Van Straaten, C. Van Beveren, I. M. Verma, and M. Vogt. 1982. Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA. J. Virol. 44:19-31. Celander, D., and W. A. Haseltine. 1984. Tissue-specific transcription preference as a determinant of cell tropism and leukaemogenic potential of murine retroviruses. Nature (London) 312:159-162. Chatis, P. A., C. A. Holland, J. W. Hartley, W. P. Rowe, and N. Hopkins. 1983. Role for the 3' end of the genome in determining disease specificity of Friend and Moloney murine leukemia viruses. Proc. Natl. Acad. Sci. USA 80:4408-4411. Chattopadhyay, S. K., M. W. Cloyd, D. L. Linemeyer, M. R. Lander, E. Rands, and D. R. Lowy. 1982. Cellular origin and role of mink cell focus-forming viruses in murine thymic lymphomas. Nature (London) 295:25-31. Chen, R. 1982. Complete amino acid sequence and glycosylation sites of glycoprotein gp71A of Friend murine leukemia virus. Proc. Natl. Acad. Sci. USA 79:5788-5792. Chou, P. Y., and G. D. Fasman. 1974. Prediction of protein conformation. Biochemistry 13:222-224. DesGroseillers, L., E. Rassart, and P. Jolicoeur. 1983. Thymotropism of murine leukemia virus is conferred by its long terminal repeat. Proc. Natl. Acad. Sci. USA 80:4203-4207. Dreesman, G. R., Y. Sanchez, I. Ionescu-Matiu, J. T. Sparrow, H. R. Six, D. L. Peterson, F. B. Hollinger, and J. L. Melnick. 1982. Antibody to hepatitis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides. Nature (London) 295:158-160. Eisenberg, R. J., C. P. Cerini, C. J. Heilman, A. D. Joseph, B. Dietzschold, E. Golub, D. Long, M. P. deLeon, and G. H. Cohen. 1985. Synthetic glycoprotein D-related peptides protect mice against herpes simplex virus challenge. J. Virol. 56:1014-1017. Elder, J. H. 1984. On the role of recombinant retroviruses in murine leukemia, p. 86-93. In A. L. Notkins and M. B. A. Oldstone (ed.), Concepts in viral pathogenesis. Springer-Verlag, New York. Elder, J. H., F. C. Jensen, M. L. Bryant, and R. A. Lerner. 1977. Polymorphism of the major envelope glycoprotein (gp7O) of
VOL. 61, 1987
murine C-type viruses: virion associated and differentiation antigens encoded by a multi-gene family. Nature (London) 267:23-28. 15. Elder, J. H., J. S. McGee, and S. Alexander. 1986. Carbohydrate side chains of Rauscher leukemia virus envelope glycoproteins are not required to elicit a neutralizing antibody response. J. Virol. 57:340-342. 16. Elder, J. H., and J. I. Muilins. 1985. Feline leukemia virus (FeLV), strains B and B/Glasgow-7, env genes, p. 1005-1011. R. Weiss, N. Teich, H. Varmus, and J. Coffin (ed.), RNA tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, 17.
18. 19. 20.
21.
22.
23.
24. 25.
26.
N.Y. Elder, J. H., and J. I. Mullins. 1983. Nucleotide sequence of the envelope gene of Gardner-Arnstein feline leukemia virus B reveals unique sequence homologies with a murine mink cell focus-forming virus. J. Virol. 46:871-880. Elder, J. H., and M. Munson. 1984. Modification of Western blotting technique for detection and quantitation of infectious virus. Biotechniques 2:170-172. Essex, M. 1975. Horizontally and vertically transmitted oncornaviruses of cats. Adv. Cancer Res. 21:175-248. Essex, M., S. M. Cotter, A. H. Sliski, W. D. Hardy, J. R. Stephenson, S. A. Aaronson, and 0. Jarrett. 1977. Horizontal transmission of feline leukemia virus under natural conditions in a feline leukemia cluster household. Int. J. Cancer 19:90-96. Friguet, B., A. F. Chaffotte, L. Djavadi-Ohaniance, and M. E. Goldberg. 1985. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods 77:305-319. Gerin, J. L., H. Alexander, J. W. K. Shih, R. H. Purcell, G. Dapolito, R. Engle, N. Green, J. G. Sutcliffe, T. M. Shinnick, and R. A. Lerner. 1983. Chemically synthesized peptides of hepatitis B surface antigen duplicate the dly specificities and induce subtype-specific antibodies in chimpanzees. Proc. Nati. Acad. Sci. USA 80:2365-2369. Grant, C. K., B. J. Ernisse, 0. Jarrett, and F. R. Jones. 1983. Feline leukemia virus envelope gp7O of subgroups B and C defined by monoclonal antibodies with cytotoxic and neutralizing functions. J. Immunol. 131:3042-3048. Green, N., H. Alexander, A. Olsen, S. Alexander, T. M. Shinnick, J. G. Sutcliffe, and R. A. Lerner. Immunogenic structure of the influenza virus hemagglutinin. Cell 28:477-487. Hardy, W. D. 1980. Feline leukemia virus diseases, p. 3-31. In W. D. Hardy, M. Essex, and A. J. McClelland (ed.), Feline leukemia viruses. Elsevier Biomedical Press, Amsterdam. Hardy, W. D., L. J. Old, P. W. Hess, M. Essex, and S. Cotter. 1973. Horizontal transmission of feline leukemia virus. Nature
(London) 144:266-269. 27. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe. 1977. A new class of murine leukemia virus associated with development of spontaneous lymphomas. Proc. Natl. Acad. Sci. USA 74:789-792. 28. Hawkes, R., E. Niday, and J. Gordon. 1982. A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119:142-147. 29. Hino, S., J. R. Stephenson, and S. A. Aaronson. 1976. Radioimmunoassays for the 70,000-molecular weight glycoproteins of endogenous mouse type C viruses: viral antigen expression in normal mouse tissues and sera. J. Virol. 18:933-941. 30. Houghten, R. A. 1985. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigenantibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. 31. Jarrett, O., W. D. Hardy, M. C. Golden, and D. Hay. 1978. The frequency of occurrence of feline leukemia virus subgroups in cats. Int. J. Cancer 21:334-337. 32. Johnson, D. A., and J. H. Elder. 1983. Antibody directed to determinants of a Moloney virus derived MCF gp7O recognizes antigens on normal immature thymus cells, brain, testes, and murine leukemia cells. J. Exp. Med. 159:1751-1756. 33. Johnson, D. A., J. W. Gautsch, R. Sportsman, and J. H. Elder. 1983. Improved technique utilizing non-fat dry milk for analysis
NEUTRALIZING ANTIPEPTIDE ANTIBODIES TO FeLV
34. 35. 36. 37.
38.
39.
40.
41.
42. 43.
44.
45. 46. 47.
48. 49.
50. 51.
52.
53.
15
of protein and nucleic acids transferred to nitrocellulose. Gene Anal. Tech. 1:3-7. Lenz, J., R. Crowther, A. Straceski, and W. A. Haseltine. 1982. Nucleotide sequence of the AKV env gene. J. Virol. 42:519-529. Levin, R., S. K. Ruscetti, W. P. Parks, and E. M. Scolnick. 1976. Expression of feline type-C virus in normal and tumor tissues of the domestic cat. Int. J. Cancer 18:661-671. Levy, L. S., M. B. Gardner, and J. W. Casey. 1984. Isolation of a feline leukaemia provirus containing the oncogene myc from a feline lymphosarcoma. Nature (London) 308:853-856. Liu, F. T., M. Zinnecker, T. Hamaska, and D. H. Katz. 1979. New procedures for preparation and isolation of conjugates and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry 18:690-697. Luciw, P., D. Parkes, S. Potter, and R. Najerian. 1985. Feline leukemia virus (FeLV), strains A/Glasgow-1 and c, env genes, p. 1000-1004. R. Weiss, N. Teich, H. Varmus, and J. Coffin (ed.), RNA tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. MacFarlan, R. I., B. Dietzschold, H. Koprowski, M. Kiel, R. Houghton, R. A. Lerner, and J. G. Sutcliffe. 1984. Localization of the immunodominant domains of rabies virus glycoprotein, p. 139-143. In R. M. Chanock and R. A. Lerner (ed.), Modern approaches to vaccines. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. MacFarlan, R. I., B. Dietzschold, T. J. Wiktor, M. Kiel, R. Houghton, R. A. Lerner, J. G. Sutcliffe, and H. Koprowski. 1984. T-cell responses to cleaved rabies virus glycoprotein and to synthetic peptides. J. Immunol. 133:2748-2752. Merrifield, R. B. 1963. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-2154. Mullins, J. I., D. S. Brody, R. C. Binari, Jr., and S. M. Cotter. 1984. Viral transduction of c-myc gene in naturally occurring feline leukaemias. Nature (London) 308:856-858. Neil, J. C., D. Hughes, R. McFarlane, N. M. Wilkie, D. E. Onions, G. Lees, and 0. Jarrett. 1984. Transduction and rearrangement of the myc gene by feline leukaemia virus in naturally occurring T-cell leukaemias. Nature (London) 308:814-820. Nunberg, H., G. Rodgers, J. H. Gilbert, and R. M. Snead. 1984. Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp7O. Proc. Natl. Acad. Sci. USA 81:3675-3679. Rickard, C. G., J. E. Post, F. Noronka, and L. M. Barr. 1969. A transmissible virus-induced lymphocytic leukemia of the cat. J. Natl. Cancer Inst. 42:987-1013. Rosenberg, Z. F., F. S. Pedersen, and W. A. Haseltine. 1980. Comparative analysis of the genomes of feline leukemia viruses. J. Virol. 35:542-546. Sarma, P., and T. Log. 1973. Subgroup classification of feline leukemia and sarcoma viruses by viral interference and neutralization tests. Virology 54:160-169. Sarma, P. S., and T. Log. 1971. Viral interference in feline leukemia-sarcoma complex. Virology 44:352-358. Shapira, M., M. Jibson, G. Muller, and R. Arnon. 1984. Immunity and protection against influenza virus by synthetic peptide corresponding to antigenic sites of hemagglutinin. Proc. Natl. Acad. Sci. USA 81:2461-2465. Shinnick, T. M., R. A. Lerner, and J. G. Sutcliffe. 1981. Nucleotide sequence of Moloney murine leukaemia virus. Nature (London) 293:543-548. Skehel, J. J., D. J. Stevens, R. S. Daniels, A. R. Douglas, M. Knossow, I. A. Wilson, and D. C. Wiley. 1984. A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc. Natl. Acad. Sci. USA 81:1779-1783. Stewart, M. A., M. Warnock, A. Wheeler, N. Wilkie, J. I. Mullins, D. E. Onions, and J. C. Neil. 1986. Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses. J. Virol. 58:825-834. Wilson, I. A., J. J. Skehel, and D. C. Wiley. 1981. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 289:366-373.
