CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1995, p. 325–329 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 2, No. 3
Human Antibody Reactivity against the Lower Matrix Protein (pp65) Produced by Cytomegalovirus MATS OHLIN,1* BODO PLACHTER,2 VIVI-ANNE SUNDQVIST,3 PETER G. A. STEENBAKKERS,4 JAAP M. MIDDELDORP,5 AND CARL A. K. BORREBAECK1 Department of Immunotechnology, Lund University, S-220 07 Lund,1 and Department of Medical Laboratory Technology, Stockholm College of Health and Caring Sciences, S-112 96 Stockholm,3 Sweden; Institut fu ¨r Klinische und Molekulare Virologie, Friedrich-Alexander-Universita ¨t Erlangen-Nu ¨rnberg, D-91054 Erlangen, Germany2; and Organon International B.V., NL-5340 BH Oss,4 and Organon Teknika B.V., NL-5281 RM Boxtel,5 The Netherlands Received 7 November 1994/Returned for modification 15 December 1994/Accepted 1 February 1995
The lower matrix protein (pp65) is a major product of many laboratory strains of cytomegalovirus (CMV). It is thus an integral part of many CMV serological assays based on native antigen. Recombinant fragments of pp65 have previously been investigated for their usefulness in more-defined assays. The latter antigens have, however, failed to develop a positive response with serum samples derived from a substantial number of infected individuals. Here we show that the human humoral immune response to CMV pp65 is highly diverse and recognizes at least seven distinct but in some cases partly overlapping epitopes. Most of these epitopes could not be mimicked by any of the investigated recombinant or synthetic antigens. Furthermore, when we investigated the ability of human CMV-seropositive serum samples to block the reactivity of pp65-specific antibodies recognizing five different epitopes within pp65, it was evident that several sera did not contain significant levels of antibodies against any of these or overlapping structures. It was thus concluded that the antibody response against CMV pp65 is weak in some CMV-infected individuals, making this antigen unsuitable for use alone in serological screening systems for CMV infection. as pp150, have been suggested to function better in serological assays (15). On the other hand, studies using radioimmunoprecipitation assays have suggested that sera frequently recognize an immunodominant protein with a molecular mass of approximately 65 kDa in CMV-infected cells (3, 23). The possible occurrence of overlapping protein bands in radioimmunoprecipitation assays may, however, obscure the nature of the precipitated antigen, making the evaluation of the results difficult. However, the frequent occurrence of pp65-specific human-antibody-producing cell lines in experiments designed to obtain CMV-specific hybridomas from fusions with human lymphocytes (20) may suggest that a substantial portion of the CMV-specific B cells are specific for pp65. In this study we investigated the human humoral immune response to native pp65 antigen at the level of individual epitopes expressed by this structural protein, in order to further characterize seroreactivity against this antigen.
Cytomegalovirus (CMV) is a virus frequently associated with disease in immunocompromised individuals, including patients undergoing organ transplantation. It has also been identified as a major cause of mental retardation following prenatal infection. As a consequence of the impact of CMV on the health of the population, development of both diagnostic and therapeutic strategies has been extensive. Diagnosis of ongoing disease has recently been improved by the use of PCR-based detection of the viral genome in clinical specimens. Similarly, assays allowing immunological detection of the presence of CMVencoded antigens in blood leukocytes have been extensively utilized to identify active disease. Originally, the latter assays were believed to be detecting immediate-early antigens expressed in lymphocytes infected with CMV. Recent evaluation of the specificity of the antibodies used to detect CMV antigenemia has, however, shown that they recognize the viral phosphoprotein pp65 (lower matrix protein) (2, 16). This antigen is frequently heavily overexpressed by cells infected with standard laboratory strains of CMV, such as Ad-169 (4), and it is thus an important component of viral antigen preparations which form the basis of many serological assays used to detect CMV-specific antibodies. In addition, the conserved nature of pp65 (13, 17) makes it a good candidate antigen for serological analysis. The usefulness of pp65 in general serological screening has, however, been questioned. It has been claimed to be poorly immunogenic, possibly as a consequence of its low level of expression by wild-type viral strains (5). Furthermore, serological assays in which prokaryotically expressed fragments of pp65 are used have frequently failed to detect specific antibodies in many CMV-seropositive sera (6, 8). Other antigens, such
MATERIALS AND METHODS Antibodies, serum samples, and antigens. Human and murine antibodies against CMV pp65 which were developed independently in several different laboratories (Table 1) were used to investigate epitopes of CMV pp65. In addition, antibody 28-103 (provided by W. J. Britt, Birmingham, Ala.) was used as a positive control in immunoblot experiments. Antibodies were included in this investigation on the basis of their ability to recognize recombinant pp65 (2) or fragments thereof. Some antibodies which efficiently competed with antibodies known to recognize pp65 or which functioned in sandwich assays with such pp65-specific antibodies were also investigated. Serum samples known to be seropositive were obtained from healthy blood donors (n 5 23) or patients with active CMV disease (n 5 8). Nuclear and membrane antigen preparations of CMV-infected fibroblasts were prepared as previously described (9). Prokaryotically derived fragments of pp65 (Fig. 1), including HE68-1 (residues 184 to 379), SK-5 (residues 401 to 426), SEML68-2 (residues 401 to 470), pp65/1 (residues 2 to 161), and pp65/3 (residues 372 to 546), were produced as described previously (14, 22). The recombinant proteins C35 (residues 303 to 467) (8), C47 (residues 388 to 561), and C74 (residues 297 to 460) (6) were kindly provided by W. Lindenmaier (GBF, Braunschweig, Germany). Analytical procedures. An antigen-specific enzyme-linked immunosorbent as-
* Corresponding author. Mailing address: Department of Immunotechnology, Lund University, P.O. Box 7031, S-220 07 Lund, Sweden. Phone: 46-46 222 43 22. Fax: 46-46 222 42 00. Electronic mail address:
[email protected]. 325
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TABLE 1. Monoclonal antibodies against pp65 used in this investigation Antibody
Source
Reference
A4B4 A6B3 CMVHU.03A CMVHU.07A CMVHU.12 CMVHU.15 MO53 MO58 MO61 MO81a CF5II MINA8II 1-5-12 (CMV-C10) 1-5-17 (CMV-C11) 28-77
Human Human Human Human Human Human Human Human Human Human Mouse Mouse Mouse Mouse Mouse
1 1 19 19 18a 18a 12 12 12 10a 19a 19a 10 10 2
a This clone has a specificity identical to that of the previously described clone MO79 (12).
say (ELISA) (11) was performed by coating polystyrene microtiter plates (Costar, Cambridge, Mass.) with viral or recombinant antigen (0.1 to 0.5 mg per well) diluted in phosphate-buffered saline (pH 7.4) and letting the plates stand at room temperature overnight. All washing steps were performed in a 0.9% NaCl solution containing 0.05% Tween 20. Samples were diluted in sample dilution buffer (10 mM sodium phosphate, 500 mM NaCl, 0.1% Tween 20 [pH 8.0]) supplemented with 20% tissue culture medium (RPMI 1640) containing 10% fetal calf serum and incubated on the coated and washed plates for 1 h at 378C. Following incubation, antibody binding was detected with peroxidase-labelled affinity-purified antibodies against human immunoglobulin G (Zymed Laboratories, Inc., San Francisco, Calif.) diluted in sample dilution buffer. Blocking of epitopes with monoclonal antibodies or sera was performed by preincubating CMV nuclear antigen-coated microtiter plates with the diluted blocking sample for 2 h at 378C prior to the addition of the monoclonal antibody being studied. The antibody was biotinylated, if necessary. Binding of the tested antibody was detected with peroxidase-labelled streptavidin, rabbit anti-mouse immunoglobulin (Dakopatts A/S, Glostrup, Denmark), or goat anti-human immunoglobulin (Zymed Laboratories, Inc.). Complementation of antibody 1-5-12 was determined following coating of microtiter plates with this antibody (700 ng per well) and incubation with nuclear antigen. The ability of human antibodies to recognize captured antigen was subsequently detected by peroxidase-labelled goat anti-human immunoglobulin (Zymed Laboratories, Inc.). Reactivity was, in all cases, visualized with o-phenylenediamine as the chromogen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were carried out as previously described (22) with the enhanced chemiluminescence detection system (Amersham Buchler GmbH, Braunschweig, Germany).
RESULTS AND DISCUSSION The selection of an antigen like CMV pp65, or fragments thereof, for serological analysis requires knowledge of the Bcell epitopes expressed by the protein and their relative immunogenicities in vivo. In order to establish a map of immunogenic B-cell epitopes of native pp65, several human and mouse antibodies were tested for the capacity to block the reactivity of five pp65-specific antibodies exhibiting different fine specificities. From this investigation, it was clear that at least eight distinct but in some cases overlapping epitopes could be detected, seven of which were recognized by human immunoglobulin G antibodies (Table 2). None of the 10 human antibodies bound the epitope recognized by mouse antibody 28-77, as shown by blocking experiments (Table 2). Therefore, this particular epitope, which is highly immunogenic in mice (14), does not appear to be a major component of the human humoral immune response against pp65. This finding explains the poor recognition of this sequence, as expressed by recombinant proteins, by a majority of human sera (14). The differences in major epitopes detected by the murine and human antibody responses might reflect differences in the form in which or the
FIG. 1. Locations of recombinant fragments of pp65 within the linear sequence of the intact antigen. The positions of the epitopes recognized by MO58 and 28-77 are indicated by arrows.
route by which the antigen is originally presented to the immune systems (active immunization and infection). In order to confirm the differences in fine specificity, some of the antibodies in this study were evaluated for the ability to recognize other recombinant fragments of pp65. As suggested by the epitope mapping described above, none of the tested antibodies (MO53, MO58, MO61, or MO81) recognized the recombinant protein SK-5 mimicking the epitope recognized by 28-77 (data not shown). However, in agreement with previously published data on the recognition of synthetic peptides (12), MO58 specifically bound the HE68-1 protein (amino acids 184 to 379 of pp65), as determined by immunoblot analysis (Fig. 2). When the same analysis was used, neither MO53, MO61, nor MO81 showed any reactivity above the background level against the HE68-1, pp65/1, pp65/3 (Fig. 2), or SEML68-2 recombinant protein. Further investigations of the recognition of the recombinant fragments of pp65 by ELISA proved that none of eight human antibodies (MO53, MO58, MO61, MO81, CMVHU.03A, CMVHU.07A, CMVHU.12, and CM VHU.15, representing six of the epitopes defined in Table 2) or two murine antibodies (1-5-12 and 1-5-17) recognized fragment C35, C47, or C74, which together cover the C-terminal half of pp65 (data not shown). It thus appears that the major immunogenic epitopes of pp65 are not expressed by these proteins. This finding agrees with the relatively low frequency of antibody reactivity with C35 and C74 in most CMV-seropositive sera (6, 8). In addition to the possibility of improper folding in a nonnatural host cell environment or the lack of certain residues making up a conformational epitope, the recombinant forms of pp65 may have aberrant posttranslational modifications. Taken together, these factors may result in a conformation which is altered relative to that of viral pp65, eliminating antibody reactivity. The extensive complexity of the antibody response against pp65 is well illustrated by the fact that the behavior of murine antibody 1-5-12 is substantially different from the behavior of antibody 28-77 in epitope blocking studies (Table 2). However, murine antibody 1-5-12 recognizes residues (amino acids 410 to 427, as determined by peptide mapping [9a]), overlapping the epitope (amino acids 401 to 426) recognized by 28-77. In these investigations, almost the entire length of pp65 has been studied with many overlapping fragments. Only a short segment (residues 162 to 183) has not been studied in these experiments. There is thus strong evidence that most human antibodies against pp65 recognize conformation-dependent epitopes and that this recognition, in some cases (e.g., MO61), is dependent on the intact native protein structure (Fig. 2). The established array of pp65-specific antibodies provides
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TABLE 2. Summary of epitopes detected by an array of human and murine monoclonal antibodies specific for CMV pp65 Ability to blocka: Epitope
Antibody MO53
MO58
MO61
MO81
28-77
1-5-12
Complementation of 1-5-12 in sandwich ELISA
I
MO53 CMVHU.03A CMVHU.12
1 1 1
2 2 2
2 2 2
1 1 1
2 2 2
2 2 2
NDb ND ND
II
MO81 MINA8II
1 1
2 2
1 1
1 1
2 ND
1 ND
ND ND
III
A6B3
1
1
2
2
2
ND
1
IV
MO58 A4B4 CF5II
2 2 2
1 1 1
2 2 2
2 2 2
2 2 ND
2 ND ND
1 1 ND
V
CMVHU.15
2
2
1
2
2
1
ND
VI
MO61 1-5-12 1-5-17
2 2 2
2 2 2
1 1 1
1 1 1
2 ND ND
1 ND ND
2 ND ND
VII
28-77
2
2
2
2
ND
ND
ND
VIII
CMVHU.07A
2
2
2
2
2
2
1
a b
Positive reactivity was established by an inhibitory activity of at least 25% at a concentration of 1 mg of immunoglobulin G per ml. ND, not determined.
an efficient tool for characterizing the repertoire of human polyclonal antibodies against this antigen. In order to evaluate the ability of human polyclonal antibodies to recognize pp65, we investigated the ability of sera to block the reactivity of the monoclonal antibodies. This approach also allowed detection of conformation-dependent epitopes not detected by Western blotting (immunoblotting). Furthermore, the results are not obscured by reactivities to proteins with molecular weights similar to that of pp65, which may pose a problem in radioimmunoprecipitation assays. From these blocking experiments, it appeared that the reactivities of all of the investigated antibodies (MO53, MO58, MO61, MO81, and 28-77) could be blocked by some human CMV-seropositive sera (Fig. 3 and 4). Thus, structures overlapping (but not necessarily identical to) the epitope recognized by antibody 28-77 bound human antibodies. This occurs despite the fact that the short recombinant protein which is recognized by 28-77 (14) frequently is not recognized well by human sera (see above). It furthermore appeared that the immune response against pp65 was highly diverse in all individuals who actually responded by producing
FIG. 2. Western blot analysis of the reactivity of MO53 (A), MO58 (B), MO61 (C), MO81 (D), and mouse antibody 28-103 (E) with (from left to right in each panel) virion-derived pp65, pp65/1 (residues 2 to 161), HE68-1 (residues 184 to 379), and pp65/3 (residues 372 to 546).
antibody against pp65. This diversity was evident because most investigated epitopes expressed by pp65 were recognized by each pp65-seropositive serum sample. As would be expected, all sera which were strongly seropositive for CMV, as determined by ELISA, were also positive for pp65-specific antibodies as determined by this assay (Fig. 3). Similar results were obtained irrespective of whether serum samples were obtained from healthy individuals or from patients with an active CMV infection. However, several epitopes were not detected by lowtiter sera, and thus they may contain a more restricted set of specificities. Such nonreactive samples were seen among healthy seropositive individuals (Fig. 3) as well as among patients with active disease (Fig. 4). In fact, the inability of most of the samples investigated in this study that were clearly but weakly CMV seropositive (as judged by both nuclear and membrane-derived antigen preparations) to block the binding of any of the investigated pp65-specific antibodies suggests that the inability to recognize pp65-related structures was not restricted to individual epitopes. This may, to a larger extent, reflect a quantitative aspect of the poor immune response against this antigen rather than a qualitative difference in the ability to mount an antibody response against particular structures. However, the assay is quite sensitive, requiring, e.g., only the equivalent of approximately 15 ng of MO58 per ml of diluted sample for 50% blocking of the binding of biotinylated MO58. This strongly suggests that the level of antibody specific for pp65 is indeed very low in many CMV-seropositive samples. Immunoblot analysis has shown that a 66-kDa antigen (probably pp65) is not as frequently detected among healthy seropositive individuals as it is among patients suffering from an ongoing CMV infection, while reactivity towards other antigens remains (21). Whether a shift towards a low level of reactivity against pp65 develops following recovery from acute disease as a consequence of, e.g., B-lymphocyte tolerance or inefficient antigen processing and/or presentation in some in-
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FIG. 4. Blocking of the binding of biotin-labelled human pp65-specific monoclonal antibodies MO53 (A) and MO58 (B) to native antigen by sera obtained from normal CMV-seropositive individuals (h) and individuals with active CMV infections (■). The abilities of monoclonal antibodies MO58 (E) and MO81 (F) to block the binding of the biotinylated detection antibody are also illustrated.
FIG. 3. Blocking of the epitopes recognized by biotin-labelled human monoclonal antibodies MO53 (A), MO58 (B) and MO61 (C) by human sera obtained from healthy individuals. Sera were obtained from strongly CMV-seropositive donors (Ç), weakly CMV-seropositive individuals (as determined with both nuclear and membrane CMV antigen preparations) (å), and CMV-seronegative donors (E). The blocking activities of unlabelled monoclonal antibodies MO58 (F) and MO81 (■) are also shown.
dividuals (although pp65 is a major antigen recognized by T lymphocytes) is unknown, but the matter merits further investigation. This subject is of particular interest in view of the recent finding that low levels of a humoral pp65-specific response correlate with the excretion of virus among seropositive children (18). Regardless of the mechanism behind the waning of the anti-pp65 antibody responses, it seems that this antigen is unsuitable for use alone in serodiagnostic procedures to detect CMV seroreactivity, in agreement with previous reports (6, 8). These results also suggest that previous failures to detect pp65-specific antibodies in some CMV-positive sera with recombinant proteins (6, 8) and by immunoblot analysis (7) were
not always due to an inability of these systems to mimic the structure of this major structural antigen but in some cases reflected a low level of humoral immune response of this protein in vivo. Consequently, other targets, such as pp150 (7, 15, 22), pp28, and gp58, should be investigated for diagnostic use. It may also be proposed that antigen derived from CMVinfected cells, which frequently is dominated by pp65, may not be ideal as a source of antigen in diagnostic systems. In view of the sensitivity of certain epitopes of pp65 to denaturation (data not shown), highly variable assay results may be obtained by using pp65 as a major target antigen in serological analysis of CMV infection. In conclusion, it has been shown that several human serum samples obtained from CMV-seropositive individuals did not contain significant levels of antibodies reacting with any of the investigated epitopes expressed by pp65. As it was shown that individual sera may contain low or undetectable levels of antibodies to epitopes expressed by pp65, this protein by itself is not sufficient for use in diagnostic screening systems for CMV seroreactivity. ACKNOWLEDGMENTS We are grateful to W. Lindenmaier for providing recombinant fragments of pp65, W. J. Britt for providing mouse monoclonal antibodies,
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and H. Alexander for providing human monoclonal antibody preparations. We acknowledge the skillful technical assistance of Ann-Charlott Olsson. This investigation was supported by grants from NUTEK (C.A.K.B.) and in part by the Deutsches Bundesministerium fu ¨r Forschung und Technologie (B.P.) and the Swedish Medical Research Council (grant 09088) (V.-A.S.). REFERENCES 1. Alexander, H., J. Harpprecht, H.-G. Podzuweit, P. Rautenberg, and W. Mu ¨ller-Ruchholtz. 1994. Human monoclonal antibodies recognize early and late viral proteins of human cytomegalovirus. Hum. Antib. Hybrid. 5:81–90. 2. Grefte, J. M. M., B. T. F. van der Gun, S. Schmolke, M. van der Giessen, W. J. van Son, B. Plachter, G. Jahn, and T. H. The. 1992. The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocytes during an active cytomegalovirus infection. J. Gen. Virol. 73:2923– 2932. 3. Hayes, K., C. Alford, and W. Britt. 1987. Antibody response to virus-encoded proteins after cytomegalovirus mononucleosis. J. Infect. Dis. 156:615–621. 4. Irmiere, A., and W. Gibson. 1983. Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology 130:118–133. 5. Jahn, G., B.-C. Scholl, B. Traupe, and B. Fleckenstein. 1987. The two major structural phosphoproteins (pp65 and pp150) of human cytomegalovirus and their antigenic properties. J. Gen. Virol. 68:1327–1337. 6. Landini, M. P., M. X. Guan, G. Jahn, W. Lindenmaier, M. Mach, A. Ripalti, A. Necker, T. Lazzarotto, and B. Plachter. 1990. Large-scale screening of human sera with cytomegalovirus recombinant antigens. J. Clin. Microbiol. 28:1375–1379. 7. Landini, M. P., M. C. Re, G. Mirolo, B. Baldassarri, and M. La Placa. 1985. Human immune response to cytomegalovirus structural polypeptides studied by immunoblotting. J. Med. Virol. 17:303–311. 8. Lindenmaier, W., A. Necker, S. Krause, R. Bonewald, and J. Collins. 1990. Cloning and characterization of major antigenic determinants of human cytomegalovirus Ad169 seen by the human immune system. Arch. Virol. 113:1–16. 9. Ljungman, P., B. Wahren, and V.-A. Sundqvist. 1985. Lymphocyte proliferation and IgG production with herpesvirus antigens in solid phase. J. Virol. Methods 12:199–208. 9a.Middeldorp, J. M. Unpublished data. 10. Middeldorp, J. M., J. Jongsma, and T. H. The. 1986. Immunofluorescence for detection of antibodies against human cytomegalovirus-induced membrane antigens. J. Clin. Microbiol. 24:405–413. 10a.Ohlin, M., et al. Unpublished data. 11. Ohlin, M., and C. A. K. Borrebaeck. 1993. Production of human monoclonal antibodies, p. 298–325. In R. F. Masseyeff, W. H. Albert, and N. A. Staines (ed.), Methods of immunological analysis, vol. 2. VCV Verlagsgesellschaft mbH, Weinheim, Germany.
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