Antigenic signature analysis reflects differences among plant virus isolates

Journal of Virological Methods, 42 0 1993 Elsevier Science Publishers

281

(1993) 28 l-292 B.V. / All rights reserved / 01660934/93/$06.00

VIRMET 01479

Antigenic signature analysis reflects differences among plant virus isolates Rong Di, John H. Hill and Richard A. Van Deusen ~~~urt~e~t

of Plant Pathff~#gy, Iowa State University, Ames, IA (USA]

(Accepted 25 November

1992)

Summary Antigenic differences among cowpea severe mosaic virus (CPSMV) isolates were clearly reflected in signature analysis employing a panel of seven wellcharacterized, monoclonal antibodies. Separate binding curves were generated by reacting serial dilutions of extracts from infected plant tissue containing each antigen simultaneously with each antibody in the panel. An iterative procedure was used to align unknown CPSMV antigen concentrations from different antigen preparations to allow comparison of binding profiles from different assays. Signature analysis was shown to be highly useful for the elucidation of subtle antigenic differences among viral agents because it requires neither purified virus nor knowledge of virus coucentration in sap from infected plants. Cowpea severe mosaic virus; Comovirus; Diagnosis

Introduction Several methods have been used to demonstrate serological relationships among plant viruses and/or virus strains. Historically, these have employed polyclonal antibodies in classical techniques such as microprecipitin tests, serologically specific electron microscopy, agar diffusion and intragel absorption. By comparison with newer technologies, these methods are laborious, difficult to interpret, and/or less sensitive. Viruses and many other plant pathogens are currently more easily detected and identi~ed by Correspondence to:

J. Hill, Department of Plant Pathology, Iowa State University, Ames, IA 50011, USA.

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immunosorbent assays than by other procedures. Further, discrimination may be greatly improved when monoclonal antibodies (MAbs) are used in such assays. Antigenic differences between two virus strains may result from changes as slight as a single amino acid substitution or as great as a major change in the amino acid sequence of a portion of the coat protein. Such variation may result in observable differences in the way MAbs bind with a virus. When differences are sufficient to result in at least one unique epitope in each strain of the virus, identification of strains is trivial, provided one MAb specific to one unique epitope in each virus strain is available. This approach necessarily leads to large panels of MAbs, since differentiation of each variant is based upon the pattern of positive and negative reactions with a panel of MAbs. Further, data interpretation is often ambiguous because concentration of antigen in sap varies with respect to strain, plant and experimental replication (D’Arcy et al., 1989). Significant difficulties in data management and interpretation occur, especially when changes in selected epitopes modify virus-host/vector interactions greatly but antibody-epitope affinity only slightly. These difficulties can sometimes be addressed only by using purified antigen preparations to allow adjustment of antigen concentrations to equivalent values (Hill et al., 1989; Jordan and Hammond, 1991). Rigid adherence to a one antibody-one strain philosophy ignores one of the most essential attributes of MAbs; that is, the ability to bind with different affinity depending on differences in the epito e to which each is directed. Thus, a single MAb with a binding affinity of 10 k M-r for the epitope which the immune system ori inally recognized upon immunization may show affinities ranging down to 10$ M- ’ when tested against a group of strains which express structural differences in the particular epitope. A second significant attribute of MAbs is realized when the differences among strains are not structural, but rather are in epitope frequency; thus, not binding affinity. but binding frequency will differ among the strains. In theory then, a single MAb can be selected which might elucidate differences among a large group of strains of a single virus. Wands et al. (1984) demonstrated the usefulness of these features in studying geographical variants of Hepatitis B virus strains. Their ‘signature analysis’ method is based on the simultaneous testing of serially diluted virus with a panel of six to eight antibodies to different epitopes of Hepatitis B virus. Subsequently, signature analysis showed that antigenic signatures of Dengue-2 virus correlated with geographic distribution (Monath et al., 1986). Several investigations with plant viruses have used large MAb panels to accurately classify isolates (e.g., Jordan and Hammond, 1991). Antibodies to common epitopes, which are often produced and discarded in the process of choosing panel members, may effect differentiation more efficiently than those which do not cross-react. Furthermore, the cross-reacting MAbs may provide useful information regarding interactive relationships of epitopes if their attributes are effectively utilized. The goal of this work was to apply the philosophy inherent in signature analysis to plant viruses. The sensitivity of the

283

immunosorbent assay has been coupled with the discrimination of MAbs to rapidly differentiate plant viruses in infected leaf samples. To accomplish this, nine isolates of cowpea severe mosaic virus (CPSMV) and seven MAbs were chosen for development of a model.

Materials and Methods Source of virus isolates Isolates representing CPSMV serotypes I-IV have been described (Lin et al., 1984). Other isolates, designated V (CR7), VI (S-l), VII (11 I), VIII (DG) and IX (ATCC PV 273) were obtained from the collection of J. P. Fulton (University of Arkansas; designation in parentheses) and have been maintained in dried tissue. Isolates were maintained and propagated in Vigna unguiculata (L.) Walp. CPSMV I-III were purified as previously described (Lin et al., 1984). Antibody preparation Balb/c mice were injected intraperitoneally with 50 ,ug purified CPSMV I, II or III emulsified in Freund’s complete adjuvant. One month later, mice were injected intravenously with 50 pg of virus in 0.05 M phosphate buffer, pH 7.0. Three days later, mice were exsanguinated, and spleen cells were fused with Sp2/0 myeloma cells (Van Deusen, 1984). Antibody-secreting hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) (Voller et al., 1976) and cloned by limiting dilution (Mernaugh et al., 1987). Antibody isotype of the MAb preparations was determined by ELISA using each of the following class- and subclass-specific rabbit anti-mouse immunoglobulins: rabbit anti-mouse IgA, IgGl, IgG2a, IgG2b, IgG3, IgM, kappa light chain and lambda light chain (Zymed Laboratories, Burlingame, CA). Ascites fluids were produced as described (Van Deusen, 1984). When necessary, antibodies were isolated from ascites fluid as described by Jones et al. (1988) or by affinity chromatography on anti-mouse IgM (p-chain specific) agarose (Sigma Chemical Co.). Antibody was bound to the column at pH 7.4. After washing the column with PBS (0.01 M sodium phosphate containing 0.15 M NaCl), pH 7.4 and, subsequently, pH 4.0, bound antibody was eluted with PBS (pH 3.0). Antibody-containing fractions were adjusted to pH 7.0 by addition of 0.01 M NaOH. Protein concentration of MAb was estimated spectrophotometrically using &so = 1.4. Purified antibody was biotin-labelled according to procedures similar to those described previously for IgG (Disco et al., 1986). However, because IgM molecules are five times larger than IgG, IgM was labelled in a 1:50 (v/v) and 2O:l (mol/mol) ratio of biotin to IgM in this study. Antibody selection Antibodies were selected for the signature analysis panel on the basis of competition curves obtained from simultaneous and consecutive assays

284 TABLE I Characteristics of monoclonal virus (CPSMV) demonstrated Un~~belIed MAb

(35: 4-5B 4-6F 5B5 4DS 5C6

immunogen

CPSMV CPSMV CPSMV CPSMV CPSMV CPSMV CPSMV

I I II II HI III III

antibodies (MAb) used for signature analysis ofcowpea severe mosaic from simultaneous and consecutive competition assays

Coating antigen and competing labelled MAb: Comaetition i shane of curves CPSMV I + MAb 5B

CPSMV It + MAb 4-SE

CPSMV III + MAb SBS

i-b/l i-j2 -“I2 --iI -/l +/l c/2

-i-/2. +/I +/2 +-I1 --/I

‘j2 -+-I2 +j2 + I2 +/I -t/l +/2

‘Shape of competition curves is designated as (f) monophasic if both simultaneous and consecutive curves are monophasic; or as (2) if the s~mu~taneo~ and/or consecutive curve is diphasrc. Significance of these curves has been previously discussed (Kubanek et al., 1991). ‘-t indicates that the unlabelled MAb competed with biotin-iabeifed MAb for antigenic sites on the antigen in at least one competition (simultaneous or consecutive) assay c - Indicates that the unlabelled MAb did not compete with biotin-labelled MAb in both simultaneous and consecutive competition assays.

(Kubanek et al., 1991). For the competition assay, Immulo~ I microtiter plates were coated with optimal concentrations (as determined by calculation of the maximum P/N (Hill et al., 1981)) of CPSMV I, II or III at 75, 75 or 50 ng/well, respectively. MAbs chosen as the labelled (biotin-labelled) antibody were 5B, 4SB, and 5B5 (Table 1) used at concentrations of 25 ng/well in assays employing CPSMV I, II and III, respectively. Serial dilutions of competing antibodies, in culture medium, were generally in the range up to 1:1024. However, dilutions of some antibodies were extended to 1:lOOOOif it was suggested in initial assays that competition still occurred at dilutions as high as 1: 1024. Reciprocal assays, using CPSMV I, II and III, were also performed in pairs with the seven MAbs chosen for the panel to identify competition that may have been due to steric hindrance rather than true competition for epitopes. The criteria used for selection of the panel were (i) the ability to bind CPSMV I, II and III, and (ii) to recognize distinct epitopes as demonstrated by lack of competition with the biotin-labelled antibody. The CPSMV group-reactive MAb 4D8, which is able to capture all CPSMV isolates, was bound to wells (50 $/well, 1:4000 dilution of ascites fluid) in Immulon I Removaweli strips (Dynatech Laboratories) in PBS (pH 7.4). After incubation overnight at 22”C, wells were washed with PBS (pH 7.4) containing 0.05% Tween-20 and blocked with BLOTTO (Johnson et al., 1984; 300 $/well) for 1 h at 37°C. After washing, two-fold serial dilutions (i.e., 1:2 to 1:2048) of freshly prepared extracts of CPSMV-infected cowpea leaf tissue (1 g tissue ground in 1.0 ml 0.05 M sodium potassium phosphate (pH 7.0) and strained through cheesecloth) were added to wells (SO~l~well~which were incubated for

285

1 h at 37°C. Extracts from noninoculated cowpea leaves prepared in the same way were added to wells as negative controls. After washing, the seven biotinlabelled MAbs chosen for the signature panel were added to separate wells (2.50 ng/well) so that each antigen dilution was tested simultaneously against all antibodies in the panel independently. After incubation for 1 h at 37°C and washing, 200000 cpmjwell of 12SI-labelled avidin (20-40 $Zi/pg, Amersham Corp.) was added. Wells, after incubation for 1 h at 22°C were mechanically washed (Pentawash, Abbott Laboratories) and bound radioactivity was measured. Each experiment consisted of three trials. Antigenic signatures were generated from three independent replications using different preparations of virus antigen. Data analysis

After counting bound radioactivity, the In of the ratio of mean cpm bound in the antigen sample to the mean cpm bound in the healthy control sample (at the dilution equivalent to the antigen sample) (ln S/N) was plotted against log2 of the antigen dilution for each CPSMV isolate with each of the MAbs used. The separate curves generated for each MAb, with which the antigen had been simultaneously tested, were arranged as in Fig. 1 to construct a signature for

Log Dilution Factor

Fig, 1. Antigenic signatures for (A) aligned binding values of CPSMV I against seven MAbs. Symbols o, + and x represent data points for three different replications, respectively. Note that the data points are not aligned vertically because the antigen concentration in plant sap varies for each replication. Data points are aligned with an ‘index sample’ by an iterative maximum likelihood procedure which determines the equivalent antigen concentration by simultaneously comparing data from all seven antibodies and generates an x value correction which is applied to a11data points for that sample. (B) Logistic regression curves generated to fit bindmg values of (A) which constitutes the an&genie signature of CPSMV I.

286

each virus isolate. Because the initial virus concentration in each antigen sample was unknown, an iterative procedure (logistic regression with estimation of relative concentrations) adopted from that of Ben-Porath et al. (1985), was used to estimate the multiple response functions between dilution and In S/N. A generalized computer program for performing the procedure was developed, using Microsoft QuickBasic, for use on a Macintosh SE computer (J. Robison-Cox, Department of Statistics, Iowa State University; unpublished). The QuickBasic programming language will permit use of the program in either Macintosh or IBM-compatible personal desktop computers.

Results and Discussion Selection of monoclonal antibodies Twenty-three MAbs were generated against CPSMV I, II or III and analyzed for potential application in the signature analysis of CPSMV isolates. All antibodies were of the IgM class. Radioimmunoassay, rather than enzymelinked immunoassay, was used in the signature analysis to avoid introduction of potential spurious effects of enz me kinetics upon antigenic signatures. Y Efforts to label IgM directly with ’ 5I resulted in inactive antibody or low specific activity. Therefore, biotin-labelled antibody and “‘1-avidin was used. Antibodies selected for the panel were 5B, 6A and 5C6, chosen for their ability to bind CPSMV I, II and III, and 4-5B, 4-6F, 5B5 and 4D8, shown to recognize distinct epitopes on at least one isolate as demonstrated by lack of competition with a biotin-labelled antibody (Kubanek et al., 1991; Table 1). Antigenic signature analysis of CPSMV isolates The alignment of binding values of seven MAbs with CPSMV I is shown in Fig. 1. The antigen concentration obtained from plants at different sample times varied. However, simultaneously shifting each set of seven curves for each antigen preparation along the X-axis revealed that the experimental values for all seven antibodies did align to form continuous binding curves which collectively form the antigenic signature for this virus isolate. Signatures generated for all 9 isolates of CPSMV are shown in Figs. l-4. Although no antibodies were generated against CPSMV IV-IX, the signatures demonstrate that these isolates possessed epitopes which were reactive with each MAb in the panel. The signatures reflect binding to the native configuration of viral coat protein. Signatures for some CPSMV isolates changed when purified virus was used rather than sap from infected plants (data not shown). We attribute this to epitopic modification during virus purification. Comparison of signatures for CPSMV II and CPSMV IV (Fig. 2) showed distinct differences in relative reactivity of several epitopes. Antibodies 5B, 45B and 5B5 appear to recognize a determinant that is identical in composition and frequency on both isolates. More of MAb 4-6F bound than MAbs 5B, 4-

287

0

12 0

3 --w_

58: -w-

12

- ---,__6A:

z .---WV__

. . . . . . . ...* ::. 12 G 120 -w ww 408‘ ---,N5C6 .w_ * -_

4-58

: - -%?-6F \

.-

: 585 -“W -*__

-1 Is.--: 3

-1 P..... 0

12 0

. .. . ..d 12

0

12 0

12

120

12

0

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Log Dilution Factor Fig. 2. Antigemc signatures for (A) CPSMV II and (B) CPSMV IV. (C) Binding curves from (A) and (B) are overlayed for comparison of signatures. All binding curves differ except those usmg MAbs 5B, 4-SB and 585.

5B, 5B5,4D8 and 5C6 when tested with CPSMV IV (Fig. 2B). This could be due to differences in affinity, relative epitope frequency or relative epitope availability. Qualitative differences, as demonstrated by shifts of the binding profiles of MAbs 6A, 4-6F, 4D8 and SC6 to the left with respect to CPSMV II, as compared to CPSMV IV, demonstrate epitopic heterogeneity between CPSMV II and CPSMV IV (Fig. 2C). By contrast, all of the curves could be exactly aligned when signatures of CPSMV III and CPSMV V were compared (Fig. 3C). This suggests that the epitopes defined by these antibodies were highly conserved on these virus isolates. In a similar fashion, the relative reactions of the MAbs in the panel varied when tested with the other virus isolates (Fig. 4). Comparison of curves by overlaying in all possible combinations (data not shown) demonstrated that signatures of 8 of the 9 isolates could be readily differentiated. As shown in Fig. 3, only CPSMV III and CPSMV V had

288

-1 3 4-6F

.U

&_-g

-_ 1

31

-\

406 -NW

_ :’ _

.-

......

...’

;,

585

_ ---_

2.;_

5C6 --N_

-_

Log Dilution Factor Fig. 3. Antlgemc signatures for (A) CPSMV III and (B) CPSMV V and (C) comparison of signatures for CPSMV III versus CPSMV V. These two isolates are identical with respect to the seven epitopes under observation.

identical antigenic signatures. Although the signature of CPSMV VIII (Fig. 4A) showed the greatest amount of similarity to signatures of the other isolates, it was still discernibly different. Signatures of CPSMV III (Fig. 3A) and CPSMV V (Fig. 3B), followed by CPSMV VII (Fig. 4B), revealed the greatest differences relative to the other isolates. Caution must be exercised, however, regarding claims of antigenic homogeneity or heterogeneity of the viral coat protein using this analysis. Signatures reflect only the relative antibody binding ability of the specific epitopes recognized by the MAbs selected for the panel. It is probable that these antibodies do not reflect all epitopic diversity in these isolates. In general, epitopes defined by antibodies 4-6F and 5C6 were highly conserved. Epitopic differences among isolates were revealed most frequently by binding curves of MAbs 4-5B, 5B and 6A. MAb 4D8 was the least discriminating of the antibodies in the panel. This could be predicted because, in this example, the capture antibody was also used

289

3 I

1

0 3

12 0

12

5B: -1

_

4-5B+.@F~

5B5

11 YAL_-L ......t.......*r.mi-....>...*.. ........_ 120

0

12 0

12

12 log

Fig. 4. Antigem

12 0

Dilution Factor

signatures for (A) CPSMV VI, (B) CPSMV VII, (C) CPSMV VIII and (D) CPSMV IX.

as a labelled antibody. However, binding curves employing the same MAb as capture and labelled antibody should not be expected to overlap precisely when antigenic signatures of all virus isolates are compared. Differences in the slope of the binding curve reflect relative epitopic affinity/frequency differences among isolates for a MAb used as illustrated by MAb 4D8. If affinity is high, virus particles are tightly bound by the capture antibody with a relatively low dissociation rate of the labelled antibody. This yields a binding curve which is gradual and is primarily frequency dependent. Conversely, if affmity is lower,

virus particles are not held as tightly and the dissociation rate of the labelled antibody is relatively greater. The resulting binding curve, with a relatively steeper slope, reflects affinity more than frequency. While the above is true, differences in the affinity of the capture antibody for the various virus isolates will have no effect on the reproducibility of each isolate’s signature because it affects all the curves in the antigenic signature in the same manner. investigations of virus epidemiology would be enhanced if a specific virus isolate could be readily monitored through the course of an epidemic. Although it is common to monitor the temporal and spatial progression of specific isolates of plant pathogenic bacteria by specific sensitivity to antibiotics, it has not been possible to differentiate specific virus isolates easily from isolates endemic in a population. A previous report suggested MAb-defined epitopes may be useful for monitoring progression of plant virus epidemics (Hill et al.. 1989). The methods employed in their study, however, were not readily suitabte for field application. This report now provides a solution to that problem. A small, well-selected panel of MAbs, used in combination with a statistical method to compensate for initially unknown virus concentrations in plant sap, has allowed comparison of binding curves to detect both subtle and major antigenic differences. This procedure will allow rapid classification of single virus isolates from the field. The classification is based upon antigenic differences whose correlation with the phenotypic response of a host to infection by diverse strains is unclear. Although there is no a priori reason to suggest that alterations in antigenic specificity of virus coat protein should be such a correlated with host response, there are examples demonstrating correlation (e.g., Rochow and Carmichael, 1979). The methods we report should be applicable to differentiation of other plant pathogens, as well as viruses, taken directly from host material. Also. the recent interest in isolation of plant pathogenicity genes (e.g., Yoder and Turgeon, 1985) provides the opportunity for correlating alterations in epitopic domains of virulence dete~inants with modi~cations in pathogenicity. Variations in antigenic signatures, reflecting altered pathogenicity domains, will permit studies in which such changes can be correlated with alterations in hostpathogen interaction.

Acknowledgements This work was supported in part by the Iowa Agricultural Biotechnology Program. We thank Helen Benner for helpful discussions. Journal paper J14852 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project 2700.

291

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