JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1375-1379
Vol. 28, No. 6
0095-1137/90/061375-05$02.00/0
Large-Scale Screening of Human Sera with Cytomegalovirus Recombinant Antigens M. P. LANDINIJ* M. X. GUAN,' G. JAHN,2 W. LINDENMAIER,3 M. MACH,2 A. RIPALTI,' A. NECKER,3 T. LAZZAROTTO,' AND B. PLACHTER2 Institute of Microbiology, University of Bologna, Bologna, Italy,' and Institute fur Klinische und Molekulare Virologie, University of Erlangen-Nurnberg, Erlangenm and Gesellschaftfur Biotechnologische Forschung, Braunschweig, Federal Republic of Germany Received 18 January 1990/Accepted 23 March 1990
One of the major problems regarding cytomegalovirus (CMV) serodiagnosis is the use of poorly defined viral antigens. Individual CMV proteins expressed via recombinant DNA procedures are a promising approach to solving this problem. In this work, 10 different fusion proteins containing antigenic epitopes of the major CMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons and of the nonstructural protein of 52 kilodaltons were subjected to Western (immuno-) blotting to assay their reactivities with immunoglobulin G and M in 395 CMV-seropositive and 100 CMV-seronegative unselected human serum samples. As a whole, the results obtained indicate that CMV can be replaced by recombinant viral proteins in the serological evaluation of anti-CMV antibodies.
Human cytomegalovirus (HCMV) is a ubiquitous member of the family Herpesviridae which mainly causes subclinical infections in humans. However, HCMV can cause a wide spectrum of disease in patients with impaired immune functions, such as transplant recipients, patients receiving antineoplastic therapy, and patients with acquired immunodeficiency syndrome. Viral diagnosis is best accomplished either by the direct detection of the virus by its isolation in tissue culture or by demonstration of the viral genome or viral antigens with DNA probes or immunological reagents, respectively. Other procedures for the indirect demonstration of the virus are based on serological determinations, but none of them has ever been proved to be completely straightforward. One of the major problems hampering HCMV serodiagnosis is the use of poorly defined viral antigens. Individual HCMV proteins expressed in bacterial cells via recombinant DNA procedures are a promising approach to solving this problem. HCMV codes for 7 to 10 immediate early proteins, approximately 25 early proteins, and some 50 late proteins (8), several of which are recognized by the host immune system during viral infection (5, 8, 9). Although the antigenic potential of the major CMV antigens is undisputed, the question still remains as to whether one or a few antigens alone, and in particular bacterially synthesized parts of these polypeptides, will meet all the necessary requirements for replacing the virus in a reliable standard diagnostic assay. Within the last 5 years, several fusion proteins containing significant epitopes of the major HCMV immunogenic polypeptides have been expressed in procaryotic cells (4, 10-12, 14, 17; W. Lindenmaier, A. Necker, S. Krauss, R. Bonewald, and J. Collins, unpublished data), and their use in HCMV serodiagnosis has been proposed (7, 11, 17; Lindenmaier et al., unpublished). However, no study has been carried out on a large number of human serum samples to evaluate the effectiveness of these epitopes as antigenic material for serological procedures. In this work, 10 different fusion proteins containing anti*
genic epitopes of the major HCMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons (kDa) and of the nonstructural protein of 52 kDa were used in Western (immuno-) blotting to assay their reactivity with immunoglobulin G (IgG) and IgM in 395 HCMV-seropositive and 100 HCMVseronegative unselected human serum samples. MATERIALS AND METHODS Conventional serological procedures. Enzyme immunoassay (EIA) for both IgM and IgG detection was performed with the following commercial kits: (i) an indirect HCMV EIA for IgG detection (from M.A. Bioproducts, Walkersville, Md.) and (ii) an antibody capture EIA for IgM detection (from Technogenetics, Hamburg, Federal Republic of Germany). As antigenic material, both kits contained a partially purified preparation of HCMV particles of the AD169 strain. The kits were used and the results were interpreted as suggested by the manufacturers. Serum samples. In this work, a total of 495 serum samples (395 HCMV-seropositive and 100 HCMV-seronegative serum samples) from unselected adults were used. The sera were sent to the diagnostic laboratory of the Institute of Microbiology, University of Bologna, Italy, for serological monitoring of HCMV infection in groups of individuals at risk for HCMV infection, such as pregnant women, renal transplant recipients, acquired immunodeficiency syndrome patients, or patients suffering from non-A, non-B hepatitis. The samples were stored at -20°C until tested. Sera were divided into four groups with respect to their anti-HCMV IgG titer and the presence or absence of HCMV-specific IgM (Table 1). Immunoblotting. The procedure for immunoblotting has been described in detail elsewhere (9). Briefly, bacterial lysates were denatured in the presence of sodium dodecyl sulfate and P-mercaptoethanol, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 7% acrylamide gels. Two different fusion proteins with different Mws were run on the same gel. Separated polypeptides were electrotransferred to nitrocellulose, and the immune reaction was performed in a Miniblotter chamber (Immunetics, Cambridge, Mass.). Sera were routinely
Corresponding author. 1375
1376
LANDINI ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Overall IgG and IgM reactivities to one or more HCMV recombinant proteins % of reactivity Serum group no. (no. of serum samples)
1 2 3 4 5
(65) (120) (120)
(90) (100)
EIA results for':
IgG (OD)
IgM
>0.28 >1.15 0.46-1.14 0.28-0.45 ci c v0.0 '~~~~~~~~~c > ? x -
0
C4
~CL
w
o.
cy*
0v U
Recombinant
~~~~proteins
2 ' '~~~~~~inCMV C CL.L o. CL.L C
proteins i
FIG. 1. Percentages of reactivity of individual HCMV recombinant proteins with antibodies present in various groups of human sera. (A) IgM reactivities of sera strongly positive for IgG and positive for IgM (group 1); (Bi) IgG reactivities of group 1. (B2) IgG reactivities of sera with a high IgG level but without IgM (group 2). (B3) IgG reactivities of sera with a medium IgG level and with no IgM (group 3). (B4) IgG reactivities of sera with a low IgG level to CMV and with no IgM (group 4).
96% in the serum samples with a high IgG titer but with no IgM (group 2) and decreased to 89 and 87% in groups 3 and 4, respectively (serum samples with medium or low EIA titer for IgG and with no IgM). However, when the 12 serum samples of group 4 that did not react with any recombinant protein at a 1:100 dilution were retested at a dilution of 1:20, even when the background staining was high, 8 gave a positive reaction with at least one recombinant protein (data not shown). The IgG reactivities to each individual protein are shown in Fig. 1B, where the results are divided with respect to the anti-HCMV IgG titer as detected by EIA. A high percentage of reactivity was found in group 1 of serum samples for several clones, especially G2 (71%), XP1 (65%), and C74 (54%). The reactivity to all the proteins progressively decreased in the serum samples of groups 2, 3, and 4, with XP1 always being the most reactive. Analysis of the antibody cross-reactivity to recombinant CMV proteins showed that the percentage of positive reac-
1378
LANDINI ET AL.
tivity described in Table 2 can be obtained by using clones Dl, XP1, C74, G2, BiB, and p271 together. In fact, none of the other recombinant proteins was recognized by sera that could not recognize one or more of the aforementioned recombinant clones (data not shown). Nonspecific reactivity. When 100 HCMV-negative human serum samples were tested with the 10 recombinant proteins, only 3 gave some IgG positivity and only 1 gave some IgM reactivity (Table 2). The positivity was against the great majority of the fusion proteins, suggesting the presence of antibodies to E. coli ,-galactosidase. To assess the percentage of human serum samples with anti-p-galactosidase antibodies, 100 serum samples with positive reactions to one or more recombinant proteins were tested with E. coli -galactosidase produced by infecting E. coli with nonrecombinant Xgtll. Three serum samples gave a pale positive reaction (data not shown). This result indicates that even when anti-E. coli antibodies are largely present in the population, ,-galactosidase is not one of the most reactive antigens. However, several other bacterial polypeptides with masses ranging from 60 to 30 kDa were often recognized by human antibodies (data not shown). DISCUSSION To evaluate whether one or more HCMV recombinant proteins could be used to detect anti-HCMV antibodies in serological procedures, 10 different recombinant proteins containing important antigenic epitopes of five structural (pplS0, pp7l, pp65, p38, and pp28) and one nonstructural (p52) HCMV antigens were used in Western blotting to study the specific IgG and IgM reactivities of 395 HCMV-seropositive and 100 HCMV-seronegative human serum samples. As a whole, the results obtained indicated that HCMV can be replaced by recombinant viral proteins in the serological evaluation of anti-HCMV antibodies. In particular, 95% of the serum samples considered IgM positive for HCMV by EIA were also positive for one or more of the following HCMV recombinant proteins: XP1, C74, G2, and BiB. These clones contain significant portions of piS0, p65, p52, and p38 antigens and might represent an antigenic complex specific enough to detect HCMV IgM. This result is in agreement with other data already present in the literature regarding the specificity of IgM reactivity to some HCMV polypeptides (8, 9). Furthermore, a correct evaluation of IgG to HCMV is best accomplished by using a higher number of recombinant proteins. In fact, the satisfying percentage of positivity detected in this work, ranging from a minimum of 87% (in the group of sera with a low level of HCMV-specific IgG as detected by EIA) to a maximum of 96% (in the group of sera with a high IgG level to HCMV), was obtained with six clones: Di, XP1, C74, G2, BiB, and p271. Therefore, in addition to the clones necessary for IgM detection, recombinant proteins Di, containing a different portion of ppiS0, and p271, containing a large fragment of p28, seem to be necessary to obtain an antigenic complex sensitive enough to detect even low levels of antibodies due to a past infection or to a very early phase of a primary HCMV infection. In view of the results obtained in this study, we think it is possible to introduce the use of recombinant HCMV proteins produced in bacteria in the serodiagnosis of HCMV infection, because they would make it easier to standardize antigenic material to be used in serological tests such as EIA or latex agglutination. A drawback to the large-scale use of bacterially produced
J. CLIN. MICROBIOL.
HCMV antigens in serological tests could arise from the presence of antibodies against E. coli proteins in human sera that could give rise to a high number of false-positive reactions. In this respect, there are two major problems: (i) several human sera showed the presence of antibodies to different and variable bacterial proteins, and (ii) a low percentage (3%) of serum samples showed the presence of antibody to 3-galactosidase. To solve the first problem, it could be advantageous to purify the fusion proteins from the whole-cell lysates in order to avoid the presence of other bacterial proteins in the antigenic material to be used. The purified antigen preparations could then be easily used in EIA or other serological procedures. The second point could be overcome by expressing useful epitopes as nonfusion proteins. This would possibly increase the specificity from 97 to 100% in an immunoblotting test. Another problem that has to be faced before using the HCMV recombinant proteins for mass screening is the 11 to 13% rate of false-negative results found in sera with mediumto-low antibody titers to HCMV. In this respect, the availability of purified fusion proteins and/or nonfused proteins would allow the use of sera at lower dilutions, and this might very likely decrease the percentage of false-negative results. This is supported by the finding that most serum samples giving a negative result at 1:100 dilution were judged positive at a 1:20 dilution. Additionally, the percentage of false-negative reactions might be reduced by the use of recombinant proteins in a serological test (EIA, latex agglutination, etc.) which allows the detection of antibodies directed against at least some conformational epitopes that could not be detected in the present study because of the method used. Alternative prospects would be the determination of the most important antigenic epitopes present in the recombinant proteins and the chemical synthesis of them for the subsequent use in a conventional serological test. However, according to our (limited) experience with using small subclones and synthetic peptides, the reactivities of individual serum samples to parts of the cloned determinants can be rather low and heterogeneous (S. Krause and W. Lindenmaier, unpublished results). Therefore, the number of peptide epitopes necessary to achieve the same percentage of positivity could be considerably larger than the number of fusion proteins. ACKNOWLEDGMENTS We thank M. La Placa for critical reading of the manuscript and P. Dal Monte, M. Mugnaini, L. Bertacchi, and P. Guglielmi for technical assistance. This work was partially supported by the Italian National Research Council, the Italian Ministry of Education, and Regione Emilia Romagna. LITERATURE CITED 1. Bernhard, H. U., and D. R. Helsinki. 1980. Bacterial plasmid cloning vehicles, p. 133-168. In A. Setlow and J. Hollander (ed.), Genetic engineering principles and methods. Plenum Publishing Corp., New York. 2. Fleckenstein, B., I. Muller, and J. Collins. 1982. Cloning of the complete human cytomegalovirus genome in cosmids. Gene 18:39-46. 3. Hattori, M., and Y. Sakaki. 1986. Dideoxy sequencing method using denatured templates. Anal. Biochem. 152:232-238. 4. Jahn, G., T. Kouzarides, M. Mach, B. C. Scholl, B. Plachter, R. Predy, S. C. Satchwell, B. Traupe, B. Fleekenstein, and B. G. Barrel. 1987. Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovi-
VOL. 28, 1990
SERUM SCREENING WITH RECOMBINANT CMV ANTIGENS
rus. J. Virol. 61:1358-1367. 5. Jahn, G., V. C. SchoIl, B. Traupe, and B. Fleekenstein. 1988. The two major structural phosphoproteins (pp65 and ppl5O) of human cytomegalovirus and their antigenic properties. J. Gen. Virol. 68:1327-1337. 6. Klages, S., B. Ruger, and G. Jahn. 1989. Multiplicity dependent expression of the predominant phosphoprotein pp65 of human cytomegalovirus. Virus Res. 12:159-168. 7. Landini, M. P., T. Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa. 1989. Antibody response to recombinant lambda gtll fusion proteins in cytomegalovirus infection. J. Clin. Microbiol. 27:2324-2327. 8. Landini, M. P., and S. Michelson. 1988. Human cytomegalovirus proteins. Prog. Med. Virol. 35:152-185. 9. 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. 10. Mach, M., U. Utz, and B. Fleckenstein. 1986. Mapping of the major glycoprotein gene of human cytomegalovirus. J. Gen. Virol. 67:1461-1467. 11. Meyer, H., A. T. Bankier, M. P. Landini, C. M. Brown, B. G. Barrel, B. Ruger, and M. Mach. 1988. Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus. J. Virol. 62:2243-2250. 12. Mocarski, E. S., L. Pereira, and N. Michael. 1985. Precise localization of genes on large animal virus genomes: use of lambda gtll and monoclonal antibodies to map the gene for a
13.
14.
15. 16.
17.
18.
19.
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cytomegalovirus protein family. Proc. Natl. Acad. Sci. USA 82:1266-1270. Re, M. C., M. P. Landini, P. Coppolecchia, G. Furlini, and M. La Placa. 1985. A 28000 d human cytomegalovirus structural polypeptide studied by means of a specific monoclonal antibody. J. Gen. Virol. 66:2507-2511. Ripalti, A., M. P. Landini, E. S. Mocarski, and M. La Placa. 1989. Identification and preliminary use of recombinant lambda gtll fusion proteins in human cytomegalovirus diagnosis. J. Gen. Virol. 70:1247-1251. Robson, L., and W. Gibson. 1989. Primate cytomegalovirus assembly protein: genome location and nucleotide sequence. J. Virol. 63:669-676. Ruger, B., S. Klages, B. Walla, J. Albrecht, B. Fleckenstein, P. Tomlinson, and B. Barrell. 1987. Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp7l of human cytomegalovirus. J. Virol. 61:446-453. School, B. C., J. Von Hintzenstern, B. Borisch, B. Traupe, M. Broker, and G. Jahn. 1988. Prokaryotic expression of immunogenic polypeptides of the large phosphoprotein (pplSO) of human cytomegalovirus. J. Gen. Virol. 69:1195-1204. Stanley, K. K., and J. P. Luzio. 1984. Construction of a new family of high efficiency bacterial expression vectors: identification of c-DNA clones coding for human liver proteins. EMBO J. 3:1429-1434. Wingender, B., G. Bercz, H. Blocker, R. Frank, and H. Mayer. 1989. Expression of human parathyroid hormone in Escherichia coli. J. Biol. Chem. 264:4367-4373.
Vol. 28, No. 6
0095-1137/90/061375-05$02.00/0
Large-Scale Screening of Human Sera with Cytomegalovirus Recombinant Antigens M. P. LANDINIJ* M. X. GUAN,' G. JAHN,2 W. LINDENMAIER,3 M. MACH,2 A. RIPALTI,' A. NECKER,3 T. LAZZAROTTO,' AND B. PLACHTER2 Institute of Microbiology, University of Bologna, Bologna, Italy,' and Institute fur Klinische und Molekulare Virologie, University of Erlangen-Nurnberg, Erlangenm and Gesellschaftfur Biotechnologische Forschung, Braunschweig, Federal Republic of Germany Received 18 January 1990/Accepted 23 March 1990
One of the major problems regarding cytomegalovirus (CMV) serodiagnosis is the use of poorly defined viral antigens. Individual CMV proteins expressed via recombinant DNA procedures are a promising approach to solving this problem. In this work, 10 different fusion proteins containing antigenic epitopes of the major CMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons and of the nonstructural protein of 52 kilodaltons were subjected to Western (immuno-) blotting to assay their reactivities with immunoglobulin G and M in 395 CMV-seropositive and 100 CMV-seronegative unselected human serum samples. As a whole, the results obtained indicate that CMV can be replaced by recombinant viral proteins in the serological evaluation of anti-CMV antibodies.
Human cytomegalovirus (HCMV) is a ubiquitous member of the family Herpesviridae which mainly causes subclinical infections in humans. However, HCMV can cause a wide spectrum of disease in patients with impaired immune functions, such as transplant recipients, patients receiving antineoplastic therapy, and patients with acquired immunodeficiency syndrome. Viral diagnosis is best accomplished either by the direct detection of the virus by its isolation in tissue culture or by demonstration of the viral genome or viral antigens with DNA probes or immunological reagents, respectively. Other procedures for the indirect demonstration of the virus are based on serological determinations, but none of them has ever been proved to be completely straightforward. One of the major problems hampering HCMV serodiagnosis is the use of poorly defined viral antigens. Individual HCMV proteins expressed in bacterial cells via recombinant DNA procedures are a promising approach to solving this problem. HCMV codes for 7 to 10 immediate early proteins, approximately 25 early proteins, and some 50 late proteins (8), several of which are recognized by the host immune system during viral infection (5, 8, 9). Although the antigenic potential of the major CMV antigens is undisputed, the question still remains as to whether one or a few antigens alone, and in particular bacterially synthesized parts of these polypeptides, will meet all the necessary requirements for replacing the virus in a reliable standard diagnostic assay. Within the last 5 years, several fusion proteins containing significant epitopes of the major HCMV immunogenic polypeptides have been expressed in procaryotic cells (4, 10-12, 14, 17; W. Lindenmaier, A. Necker, S. Krauss, R. Bonewald, and J. Collins, unpublished data), and their use in HCMV serodiagnosis has been proposed (7, 11, 17; Lindenmaier et al., unpublished). However, no study has been carried out on a large number of human serum samples to evaluate the effectiveness of these epitopes as antigenic material for serological procedures. In this work, 10 different fusion proteins containing anti*
genic epitopes of the major HCMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons (kDa) and of the nonstructural protein of 52 kDa were used in Western (immuno-) blotting to assay their reactivity with immunoglobulin G (IgG) and IgM in 395 HCMV-seropositive and 100 HCMVseronegative unselected human serum samples. MATERIALS AND METHODS Conventional serological procedures. Enzyme immunoassay (EIA) for both IgM and IgG detection was performed with the following commercial kits: (i) an indirect HCMV EIA for IgG detection (from M.A. Bioproducts, Walkersville, Md.) and (ii) an antibody capture EIA for IgM detection (from Technogenetics, Hamburg, Federal Republic of Germany). As antigenic material, both kits contained a partially purified preparation of HCMV particles of the AD169 strain. The kits were used and the results were interpreted as suggested by the manufacturers. Serum samples. In this work, a total of 495 serum samples (395 HCMV-seropositive and 100 HCMV-seronegative serum samples) from unselected adults were used. The sera were sent to the diagnostic laboratory of the Institute of Microbiology, University of Bologna, Italy, for serological monitoring of HCMV infection in groups of individuals at risk for HCMV infection, such as pregnant women, renal transplant recipients, acquired immunodeficiency syndrome patients, or patients suffering from non-A, non-B hepatitis. The samples were stored at -20°C until tested. Sera were divided into four groups with respect to their anti-HCMV IgG titer and the presence or absence of HCMV-specific IgM (Table 1). Immunoblotting. The procedure for immunoblotting has been described in detail elsewhere (9). Briefly, bacterial lysates were denatured in the presence of sodium dodecyl sulfate and P-mercaptoethanol, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 7% acrylamide gels. Two different fusion proteins with different Mws were run on the same gel. Separated polypeptides were electrotransferred to nitrocellulose, and the immune reaction was performed in a Miniblotter chamber (Immunetics, Cambridge, Mass.). Sera were routinely
Corresponding author. 1375
1376
LANDINI ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Overall IgG and IgM reactivities to one or more HCMV recombinant proteins % of reactivity Serum group no. (no. of serum samples)
1 2 3 4 5
(65) (120) (120)
(90) (100)
EIA results for':
IgG (OD)
IgM
>0.28 >1.15 0.46-1.14 0.28-0.45 ci c v0.0 '~~~~~~~~~c > ? x -
0
C4
~CL
w
o.
cy*
0v U
Recombinant
~~~~proteins
2 ' '~~~~~~inCMV C CL.L o. CL.L C
proteins i
FIG. 1. Percentages of reactivity of individual HCMV recombinant proteins with antibodies present in various groups of human sera. (A) IgM reactivities of sera strongly positive for IgG and positive for IgM (group 1); (Bi) IgG reactivities of group 1. (B2) IgG reactivities of sera with a high IgG level but without IgM (group 2). (B3) IgG reactivities of sera with a medium IgG level and with no IgM (group 3). (B4) IgG reactivities of sera with a low IgG level to CMV and with no IgM (group 4).
96% in the serum samples with a high IgG titer but with no IgM (group 2) and decreased to 89 and 87% in groups 3 and 4, respectively (serum samples with medium or low EIA titer for IgG and with no IgM). However, when the 12 serum samples of group 4 that did not react with any recombinant protein at a 1:100 dilution were retested at a dilution of 1:20, even when the background staining was high, 8 gave a positive reaction with at least one recombinant protein (data not shown). The IgG reactivities to each individual protein are shown in Fig. 1B, where the results are divided with respect to the anti-HCMV IgG titer as detected by EIA. A high percentage of reactivity was found in group 1 of serum samples for several clones, especially G2 (71%), XP1 (65%), and C74 (54%). The reactivity to all the proteins progressively decreased in the serum samples of groups 2, 3, and 4, with XP1 always being the most reactive. Analysis of the antibody cross-reactivity to recombinant CMV proteins showed that the percentage of positive reac-
1378
LANDINI ET AL.
tivity described in Table 2 can be obtained by using clones Dl, XP1, C74, G2, BiB, and p271 together. In fact, none of the other recombinant proteins was recognized by sera that could not recognize one or more of the aforementioned recombinant clones (data not shown). Nonspecific reactivity. When 100 HCMV-negative human serum samples were tested with the 10 recombinant proteins, only 3 gave some IgG positivity and only 1 gave some IgM reactivity (Table 2). The positivity was against the great majority of the fusion proteins, suggesting the presence of antibodies to E. coli ,-galactosidase. To assess the percentage of human serum samples with anti-p-galactosidase antibodies, 100 serum samples with positive reactions to one or more recombinant proteins were tested with E. coli -galactosidase produced by infecting E. coli with nonrecombinant Xgtll. Three serum samples gave a pale positive reaction (data not shown). This result indicates that even when anti-E. coli antibodies are largely present in the population, ,-galactosidase is not one of the most reactive antigens. However, several other bacterial polypeptides with masses ranging from 60 to 30 kDa were often recognized by human antibodies (data not shown). DISCUSSION To evaluate whether one or more HCMV recombinant proteins could be used to detect anti-HCMV antibodies in serological procedures, 10 different recombinant proteins containing important antigenic epitopes of five structural (pplS0, pp7l, pp65, p38, and pp28) and one nonstructural (p52) HCMV antigens were used in Western blotting to study the specific IgG and IgM reactivities of 395 HCMV-seropositive and 100 HCMV-seronegative human serum samples. As a whole, the results obtained indicated that HCMV can be replaced by recombinant viral proteins in the serological evaluation of anti-HCMV antibodies. In particular, 95% of the serum samples considered IgM positive for HCMV by EIA were also positive for one or more of the following HCMV recombinant proteins: XP1, C74, G2, and BiB. These clones contain significant portions of piS0, p65, p52, and p38 antigens and might represent an antigenic complex specific enough to detect HCMV IgM. This result is in agreement with other data already present in the literature regarding the specificity of IgM reactivity to some HCMV polypeptides (8, 9). Furthermore, a correct evaluation of IgG to HCMV is best accomplished by using a higher number of recombinant proteins. In fact, the satisfying percentage of positivity detected in this work, ranging from a minimum of 87% (in the group of sera with a low level of HCMV-specific IgG as detected by EIA) to a maximum of 96% (in the group of sera with a high IgG level to HCMV), was obtained with six clones: Di, XP1, C74, G2, BiB, and p271. Therefore, in addition to the clones necessary for IgM detection, recombinant proteins Di, containing a different portion of ppiS0, and p271, containing a large fragment of p28, seem to be necessary to obtain an antigenic complex sensitive enough to detect even low levels of antibodies due to a past infection or to a very early phase of a primary HCMV infection. In view of the results obtained in this study, we think it is possible to introduce the use of recombinant HCMV proteins produced in bacteria in the serodiagnosis of HCMV infection, because they would make it easier to standardize antigenic material to be used in serological tests such as EIA or latex agglutination. A drawback to the large-scale use of bacterially produced
J. CLIN. MICROBIOL.
HCMV antigens in serological tests could arise from the presence of antibodies against E. coli proteins in human sera that could give rise to a high number of false-positive reactions. In this respect, there are two major problems: (i) several human sera showed the presence of antibodies to different and variable bacterial proteins, and (ii) a low percentage (3%) of serum samples showed the presence of antibody to 3-galactosidase. To solve the first problem, it could be advantageous to purify the fusion proteins from the whole-cell lysates in order to avoid the presence of other bacterial proteins in the antigenic material to be used. The purified antigen preparations could then be easily used in EIA or other serological procedures. The second point could be overcome by expressing useful epitopes as nonfusion proteins. This would possibly increase the specificity from 97 to 100% in an immunoblotting test. Another problem that has to be faced before using the HCMV recombinant proteins for mass screening is the 11 to 13% rate of false-negative results found in sera with mediumto-low antibody titers to HCMV. In this respect, the availability of purified fusion proteins and/or nonfused proteins would allow the use of sera at lower dilutions, and this might very likely decrease the percentage of false-negative results. This is supported by the finding that most serum samples giving a negative result at 1:100 dilution were judged positive at a 1:20 dilution. Additionally, the percentage of false-negative reactions might be reduced by the use of recombinant proteins in a serological test (EIA, latex agglutination, etc.) which allows the detection of antibodies directed against at least some conformational epitopes that could not be detected in the present study because of the method used. Alternative prospects would be the determination of the most important antigenic epitopes present in the recombinant proteins and the chemical synthesis of them for the subsequent use in a conventional serological test. However, according to our (limited) experience with using small subclones and synthetic peptides, the reactivities of individual serum samples to parts of the cloned determinants can be rather low and heterogeneous (S. Krause and W. Lindenmaier, unpublished results). Therefore, the number of peptide epitopes necessary to achieve the same percentage of positivity could be considerably larger than the number of fusion proteins. ACKNOWLEDGMENTS We thank M. La Placa for critical reading of the manuscript and P. Dal Monte, M. Mugnaini, L. Bertacchi, and P. Guglielmi for technical assistance. This work was partially supported by the Italian National Research Council, the Italian Ministry of Education, and Regione Emilia Romagna. LITERATURE CITED 1. Bernhard, H. U., and D. R. Helsinki. 1980. Bacterial plasmid cloning vehicles, p. 133-168. In A. Setlow and J. Hollander (ed.), Genetic engineering principles and methods. Plenum Publishing Corp., New York. 2. Fleckenstein, B., I. Muller, and J. Collins. 1982. Cloning of the complete human cytomegalovirus genome in cosmids. Gene 18:39-46. 3. Hattori, M., and Y. Sakaki. 1986. Dideoxy sequencing method using denatured templates. Anal. Biochem. 152:232-238. 4. Jahn, G., T. Kouzarides, M. Mach, B. C. Scholl, B. Plachter, R. Predy, S. C. Satchwell, B. Traupe, B. Fleekenstein, and B. G. Barrel. 1987. Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovi-
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rus. J. Virol. 61:1358-1367. 5. Jahn, G., V. C. SchoIl, B. Traupe, and B. Fleekenstein. 1988. The two major structural phosphoproteins (pp65 and ppl5O) of human cytomegalovirus and their antigenic properties. J. Gen. Virol. 68:1327-1337. 6. Klages, S., B. Ruger, and G. Jahn. 1989. Multiplicity dependent expression of the predominant phosphoprotein pp65 of human cytomegalovirus. Virus Res. 12:159-168. 7. Landini, M. P., T. Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa. 1989. Antibody response to recombinant lambda gtll fusion proteins in cytomegalovirus infection. J. Clin. Microbiol. 27:2324-2327. 8. Landini, M. P., and S. Michelson. 1988. Human cytomegalovirus proteins. Prog. Med. Virol. 35:152-185. 9. 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. 10. Mach, M., U. Utz, and B. Fleckenstein. 1986. Mapping of the major glycoprotein gene of human cytomegalovirus. J. Gen. Virol. 67:1461-1467. 11. Meyer, H., A. T. Bankier, M. P. Landini, C. M. Brown, B. G. Barrel, B. Ruger, and M. Mach. 1988. Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus. J. Virol. 62:2243-2250. 12. Mocarski, E. S., L. Pereira, and N. Michael. 1985. Precise localization of genes on large animal virus genomes: use of lambda gtll and monoclonal antibodies to map the gene for a
13.
14.
15. 16.
17.
18.
19.
1379
cytomegalovirus protein family. Proc. Natl. Acad. Sci. USA 82:1266-1270. Re, M. C., M. P. Landini, P. Coppolecchia, G. Furlini, and M. La Placa. 1985. A 28000 d human cytomegalovirus structural polypeptide studied by means of a specific monoclonal antibody. J. Gen. Virol. 66:2507-2511. Ripalti, A., M. P. Landini, E. S. Mocarski, and M. La Placa. 1989. Identification and preliminary use of recombinant lambda gtll fusion proteins in human cytomegalovirus diagnosis. J. Gen. Virol. 70:1247-1251. Robson, L., and W. Gibson. 1989. Primate cytomegalovirus assembly protein: genome location and nucleotide sequence. J. Virol. 63:669-676. Ruger, B., S. Klages, B. Walla, J. Albrecht, B. Fleckenstein, P. Tomlinson, and B. Barrell. 1987. Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp7l of human cytomegalovirus. J. Virol. 61:446-453. School, B. C., J. Von Hintzenstern, B. Borisch, B. Traupe, M. Broker, and G. Jahn. 1988. Prokaryotic expression of immunogenic polypeptides of the large phosphoprotein (pplSO) of human cytomegalovirus. J. Gen. Virol. 69:1195-1204. Stanley, K. K., and J. P. Luzio. 1984. Construction of a new family of high efficiency bacterial expression vectors: identification of c-DNA clones coding for human liver proteins. EMBO J. 3:1429-1434. Wingender, B., G. Bercz, H. Blocker, R. Frank, and H. Mayer. 1989. Expression of human parathyroid hormone in Escherichia coli. J. Biol. Chem. 264:4367-4373.
