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IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins
Journal of Virological Methods ELSEVIER
Journal of Virological Methods 60 (1996) 73-80
IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins Walter Hinderer”, Hans-H. Sonneborn”, Gregor Beinb, Lukas Rolf Vornhagen”, Matterc, T. Hauw Thed, Gisela Ender9, Gerhard Jahnf, Bodo Plachterg,* “Biotesr AG, Research Department, 63303 Dreieich, Germany bInstitut fur Immunologie und Transfusionsmedi;i~~, Universitat Liibeck, 23562 Liibeck, Germany ‘Institut fir Medizinische Mikrobiologie, Uniuersitiit Bern, 3010 Bern, Switzerland ‘Deptartment of Clinical Immunology and Renal Transplant Unit, Unioersity of Groningen, Groningen, Netherlands ‘Institut Jiir Virologie, Infektiologie und Epidemiologie e. V., Medizinisch-diagnostisches Gemeinschaftslabor, 70193 Stuttgart, Germany fAbteilung fur Virologie, Hygiene Institut, Uniaersitiit Tiibingen, 72076 Tiibingen, Germany “Institiit ,fiir Virologie, Unirersitiit Maim, 55101 Maim, Germany
Accepted 26 February 1996
Abstract Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (~52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (~~150, aa 994- 1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELBA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection. Keylt’ords:
Cytomegalovirus;
Recombinant
antigens;
ELISA;
* Corresponding author. Tel.: + 49 6131 173652; fax: + 49 6131 395604; email: [email protected] 0166-0934,‘96/$15.00 @ 1996 Elsevier Science B.V. All rights reserved PII SOl66-0934(96)02047-2
IgM
1. Introduction Human cytomegalovirus (HCMV) infection usually remains asymptomatic in immunocompetent individuals but causes major clinical problems in patients whose immune functions are impaired or not yet fully developed. Prenatal transmission of HCMV is considered the most frequent cause of congenital sequelae related to virus infection in the Western world (Britt, 1994). In addition, primary and secondary infection in transplant recipients and in AIDS patients may lead to various organ manifestations and even life threatening conditions (for review, see Ho, 1991). To ensure timely and efficient antiviral treatment, diagnostic methods have been requested that allow close monitoring of patients endangered by HCMV infection (Balfour, 1990). Determination of specific antibodies against viral antigens is a diagnostic procedure which is used commonly for the identification of acute and past HCMV-infection. However, currently available serological assays, such as enzyme linked immunosorbent assays (ELISAs), use antigen preparations characterized poorly derived from infected cell culture. Yet, only a few of the over 200 putative proteins of HCMV serve as dominant targets for the humoral immune response. The over-representation of irrelculture derived antigen proteins in evant preparations, especially for IgM specific test systems, may explain the limited performance of available assays in relation to either sensitivity or specificity. This leads to discordant results when assays from different suppliers are compared (Lazzarotto et al., 1992). Careful selection of viral antigens for the design of improved tests therefore appears essential. Recombinant technology offers a strategy to identify relevant polypeptides for serological assays and to provide pure preparations of such antigens in large quantities. In previous investigations recombinant antigen fragments were identified which permit sensitive and specific diagnosis of acute and past HCMV al., 1995). infection (Vornhagen et 1994, Polypeptide fragments representing the C-termi-
nal portions of pUL44 (~~52, aa 297-433) and pUL32 (~~150, aa 99441048) as well as a 57 amino acid fragment from the central part of the major DNA-binding protein pUL57 (aa 545-601) appeared to be promising candidates for improving of IgM-specific serodiagnosis of acute HCMV-infection. In the present study, the immunodominant antigen fragments from pUL32, pUL44 and pUL57 of HCMV were combined to form different autologous fusion proteins. Using these polypeptides as antigens in ELISA experiments, the following questions were addressed: (i) whether autologous fusion of different highly reactive antigens would result in a better performance of the test systems compared to assays using culture derived antigens and (ii) which combination of the recombinant antigens would provide optimal test results concerning sensitivity and specificity for the detection of acute HCMV infection.
2. Material
and methods
2.1. Cloning and eqwession
of’ antigens
The construction of plasmids expressing autologous fusion proteins was carried out with DNA-fragments which had been generated by PCR-technology. The general strategy has been described in detail previously (Vornhagen et al., 1992). Amplification was undertaken with pairs recognition of PCR-primers containing sequences for restriction endonucleases Burn HI and EcoRI, 5’ and 3’ of the original priming sequence respectively. The 3’-primers contained an additional recognition sequence for BglIl 5’ to the EcoRI-site which was used for in-frame insertion of other PCR-derived BamHI/EcoRIfragments. The autologous fusion proteins CMl, CM2, CM3 and the reference antigens 5213 and 150/7 (Fig. 1) were expressed in E. co/i using vector pET5c which permits expression with an N-terminal leader sequence of 11 amino acids (Stud1986). The antigens were ier and Moffatt, purified to homogeneity using differential cen-
R. Vornhagen et al. 1 Journal qf Virological Methods 60 (1996) 73-80
pCC150/7/2 *w%
pcc57/3 %*a
pCC52/3 % *>
229kbp
HCMV -
-
c-
CM1 150/l/2 5713
52/3
CM2 52/3
57/3
CM3
52/3
150/l/2
52/3
Fig. 1. Schematic representation of the HCMV genome (central part) with the open reading frames UL32, UL44 and UL57. The relative localization of the DNA-fragments encoding the portions of the proteins expressed in E. coli are shown on top. Numbers indicate the amino acids of the respective proteins contained in the recombinant polypeptides. The composition of the autologous fusion proteins CMl, CM2 and CM3 evaluated in this study is given at the bottom.
and washing steps with subsequent ion-exchange and gel chromatography. The final purity was demonstrated by SDS-polyacrylamide gel electrophoresis and Western blot analysis. All three antigen preparations contained less than 1% residual E. coli protein contamination. trifugation
2.2. Evaluation experiments
of recombinant
antigens
in ELISA
The autologous fusion proteins were purified to homogeneity and evaluated by ELISA. Microtiter plates (Nunc, Maxisorb) were coated with 100 ng of purified antigens solubilized in 100 ~1 of 0.01 M carbonate (Na,CO,, NaHCO,) buffer, pH 9.5. ELISA plates were incubated with human sera diluted 1:21 for 1 h at 40°C in a water bath. After washing, bound IgM-antibodies were detected using a specific mouse monoclonal antibody (Jansen, Beerse, Belgium) conjugated with horeseradish peroxidase (30 min, 40°C). The conjugate was diluted 1: 1000 in a phosphate buffer containing 10% fetal calf serum. Color was developed for 30 min at room temperature using a 3,3’, 5,5’-tetramethylbenzidine (TMB) solution (Sigma, Munchen, Germany). Optical density was measured at 450 nm.
2.3. Serum samples In this study the recombinant HCMV-antigens were evaluated with human sera from different sources. Sera from healthy blood donors were obtained from a local transfusion center (Suhl, Germany) and HCMV-specific seroprevalence was determined by Biotest anti-CMV IgG ELISA. Patients receiving a renal allograft at the University Hospital Groningen, Netherlands, and at University Hospital, Liibeck, Germany, were monitored by the pp65specific antigenemia assay. Sera obtained from immunocompetent individuals with acute HCMV infection were taken from persons with primary infection proven by IgG seroconversion and/or from patients with HCMV excretion revealing typical clinical symptoms. In order to identify false positive results caused by rheumatoid factors selected sera were retested after treatment with RF absorbent (Behring, Marburg, Germany). 2.4. Reference
ELISAs
Recombinant HCMV-antigens together with two commercially
were evaluated available HCMV-
76 Table I IgM-specific
ELISA
results
of recombinant
viral antigens
52:‘3, 150/7 and autologous
Samples
II
fusion
protein
Number
CM3
of samples
with positive
result
52,!3
I so/7
CM3
36 17
27 IO
33 14
33 13
54 54
I 0
I9 0
0
Acute HCM V inf2ction.s Immunocompetent Renal transplant Randomly selected seropositive
individuals recipients with acute
HCMV
primary
infection”
hertlth_v blood donors
seronegative “Sera from individuals symptoms. %era were taken 2-3
with HCMV
primary
infection
proven
by seroconversion
and/or
viral excretion
together
I
with typical
clinical
weeks after onset of antigenemia
specific ELISAs which utilize cell culture derived antigens. While reference ELISA 1 was an indirect ELISA which uses control antigen, reference ELBA 2 was a p-capture assay, in which bound IgM-molecules were detected with enzyme labelled HCMV-antigens. With reference ELBA 1 a preincubation step was performed in order to remove rheumatoid factors.
3. Results 3.1. Construction md expression uutologous fusion proteins
oJ’ recombinant
In preceding evaluations we could show that recombinant antigen fragments representing the C-terminal portions of pp150 (150/7) and ~52 (52/3), and a small segment from the central part of the major DNA binding protein (UL57/3) are major target antigens of the IgM-specific antibody response during acute HCMV infection (Vornhagen et al., 1994, 1995). IgM antibodies against 15Oj7 (UL32, aa862-1048) however, were also detected in a substantial number of serum samples from healthy blood donors without any signs of acute infection (Vornhagen et al., 1995). Additional experiments were carried out with a recombinant fragment which consists only of the last 54 amino acids of ~~150. This portion (150/7/2), which harbours a potential IgM-epitope (Landini et al., 1991) was expressed in fusion with fragment
5213 to form the recombinant autologous fusion protein CM3. After purification CM3 was tested by ELISA together with the recombinant antigens 52/3 and 150/7. In this first series of experiments CM3 proved to be more reactive than 52/3 when tested with samples from acutely infected individuals but has only low reactivity with sera from healthy blood donors (Table 1). Considering these results two additional autologous fusion proteins were constructed (Fig. 1). While CM1 combines the three immunodominant fragments 52/3, UL57/3 and 150/7/2, CM2 consists of the central and highly reactive portion of pUL57 (UL57/3) with one copy of 5213 on either side. This combination appeared necessary, since a C-terminal fusion protein of 5213 and UL57/3 showed substantial proteolytic degradation during expression and purification; CM2 was found to be very stable. 3.2. ELISA evaluution ,fiaion proteins
of recombinant
autologous
Purified recombinant antigens CMl, CM2 and CM3 were evaluated further by ELISA experiments in comparison with an indirect assay and a /L-capture ELBA which both use cell culture derived antigens. These two reference ELISAs represent the two different assay formats of the currently available HCMV-specific IgM-ELISAs. The sensitivity of the recombinant antigens was determined with sera from immunocompetent in-
R. Vornhagen et al. 1 Journal of Virological Table 2 IgM-specific IgM-ELISAs
ELBA
results
of recombinant
autologous
n
Samples
Acute HCMV injkctions Immuncompetent individuals” Renal transplant recipients with acute primary infectioni Randomly selected healthy blood donors Total Seropositive Seronegative
HCMV
fusion
proteins
Number
Methods
in comparison
of samples
17
60 (1996) 73-80
with two commercially
with positive
CM2
CM3
Reference
47 13
41 12
41 13
44 12
39 10
46 11
175 121 54
I 7 0
3 3 0
6 6 0
1 1 0
14 12 2
dividuals with acute HCMV-infection and from renal transplant recipients with acute HCMV-primary infection. The latter samples were drawn 223 weeks after onset of antigenemia. The results are shown in Table 2 and Fig. 2. While recombinant antigens CM1 and CM2 revealed high IgM specific prevalence, CM3 was less reactive. CM2 was the only recombinant antigen which was positive with all samples tested. The two reference ELISAs showed highly discordant results. While reference ELISA 2 revealed a lower but still comparable sensitivity as CM1 and CM2, reference ELISA 2 was much less reactive and gave negative results with 17% of the samples. These results indicated IgM assays using CM1 and CM2 as antigens show comparable or better sensitivity than the commercially available assays. Secondly, the time course of the IgM response against the recombinant antigens was tested with follow-up sera from renal transplant recipients (Fig. 2). HCMV infection in these patients had been monitored by pp65-specific antigenemia assay. While Fig. 2 A-C show the data obtained with sera from patients with primary infection, Fig. 2 D represents a follow-up from a patient with acute secondary infection. The results demonstrate that IgM antibodies against all recombinant autologous fusion proteins were detectable very early during the acute phase of infection. In all
by seroconversion
and/or
reference
result
CM1
“Indirect ELISA with control antigen. “p-capture ELISA. ‘Sera from individuals with HCMV primary infection proven symptoms. “Sera were taken 223 weeks after onset of antigenemia.
available
ELISA
viral excretion
I”
Reference
together
ELISA
with typical
2’
clinical
three patients with acute primary infection, IgM antibodies against the recombinant antigens were detectable within a time period of two weeks after onset of antigenemia, while in the patient suffering from acute secondary infection recombinant ELISAs became positive concomitantly (CM2) or even 1 week before (CMl, CM3) the antigenemia assay. Similar results were obtained with two additional follow-ups from renal transplant recipients with acute secondary infection (data not shown). Both reference ELISAs were less sensitive with the follow-up samples from transplant recipients. Reference ELISA 2 revealed a positive result only in one patient (C) as early as the recombinant ELISAs but became positive in all other patients l-2 weeks after the recombinant assays. Reference ELISA 1 was even less sensitive and revealed a positive result l-5 weeks after the recombinant ELISAs. While the three recombinant antigens revealed comparable results during seroconversion, differences in reactivity were more pronounced after the acute phase of infection. Although IgM antibodies against CM2 were still detectable in all four patients at the end of the observation period, IgM specific reactivity in three patients already declined several weeks after the acute phase of infection. In contrast, high levels of IgM antibodies against both autologous fusion proteins which
R. Vornhagen et al. i Journal c$ Virological
Methods
60 (1996) 73-80
30
0
20 weeks
10
20 weeks
post TX
post TX
10
.g
d s5
D
0
0
0
10
20
30 weeks
0
20
IO weeks
post TX
post TX
Fig. 2. IgM-specific reactivities of recombinant autologous fusion proteins CMI, CM2 and CM3 in ELISA in comparison with two reference ELlSAs with follow ups from renal transplant recipients with acute HCMV-primary infection (A + C) and acute HCMV-secondary infection (D). HCMV infection was monitored using the pp65-specific antigenemia assay. Sample/cut OK ratios for CMI, n ; CM2, A; CM3, a; reference ELISA 1, *; reference ELISA 2, x ; and number of antigen positive ceils per 100000 cells, 0.
contain the 54 C-terminal amino acids of pp150 (CMl, CM3) were present even several months after the acute stage of infection. To evaluate the specificity of tests using recombinant antigens, sera from healthy blood donors (Table 2, panel II) were analysed. Here, CM2 revealed the lowest IgM-reactivity of all recombinant antigens. All three positive samples were taken from seropositive individuals. One of these sera was highly reactive (OD > 2.0) with all three recombinant antigens and was also positive with reference ELISA 1. The two other reactive samples showed optical density values just above cutoff. CM1 and CM3 revealed substantially higher IgM specific reactivity than CM2. With sera from healthy donors both reference ELISAs again revealed highly discordant results. While reference ELISA 1 revealed a positive result with a single
donor, reference samples.
ELBA
2 was positive
with
14
4. Discussion Conventional assays for the detection of IgM antibodies against HCMV, which use cell culture derived antigens suffer from either limited sensitivity or specificity (Lazzarotto et al., 1992). Several attempts have been made to identify recombinant viral proteins which can serve as antigens for the detection of IgM antibodies during acute HCMV infection (Lindenmaier et al., 1990; Landini et al., 1990; Vornhagen et al., 1995). These studies led to the conclusion that only a combination of carefully selected components will enable IgM specific serodiagnosis with improved specificity and sensitivity.
R. Vornhagen et al. /Journal
qf Virological Methods 60 (1996) 73-80
In a previous study, it was shown that antigen fragments from p52 (52/3) and pp150 (150/l and 150/7) of HCMV enable highly sensitive detection of IgM antibodies in patient sera (Vornhagen et al., 1994). However, especially with fragments from the tegument protein ~~150, IgM antibodies were also detected in sera from patients without acute infection. These data suggested that antigen composition used in these experiments was not optimal to indicate clinically relevant infection. In recent analyses we identified a major IgM antigen of HCMV consisting of aa 545-601 of pUL57 (UL57/3; Vornhagen et al., 1995). ELISAs using this polypeptide as antigen indicated acute HCMV infection with high sensitivity and specificity. The aim of this study was to evaluate, whether a combination of UL57/3 and 5213 together with a truncated version of 150/7 (150/7/2) would combine highly sensitive and specific detection of acute HCMV infection using IgM ELISA and whether such a recombinant test would be superior to conventional IgM ELISAs. The use of severai individual recombinant proteins in a single assay requires complicated procedures in order to guarantee the presence of standardized quantities of the different antigens. In addition, high level expression of very small heterologous proteins in E. coli may not always be possible. We therefore chose to express the different immunodominant HCMV antigen fragments as autologous fusion proteins. This strategy allows the synthesis of high amounts of different antigens in one molecule, thus providing a defined composition of the material. A previous study in which single as well as multiple copies of immunodominant portions of pp150 (pUL32) were expressed in E. coli as so-called polypeptide fusion antigens has already demonstrated the general usefulness of this approach (Ripalti et al., 1994). We generated constructs expressing three different autologous fusion proteins (Fig. 1). The fusion protein CM2, which combines 5213 with UL57/3, but lacks portions of ~~150, turned out to be the most reliable recombinant IgM antigen. Using this polypeptide, IgM antibodies were detected in all sera from patients undergoing acute HCMV infection. Accordingly, the sensitivity of the assay for the identification of acute infection
19
was superior to all other assays with different antigen composition including both commercial tests. In sera from blood donors without symptoms of HCMV infection, the CM2-ELISA detected IgM antibodies in three cases. One of these was also positive with all other recombinant ELISAs and with one commercial ELISA; this indicates that this particular donor might have undergone acute infection at the time the sample was obtained. Two other samples were positive just above cut-off, probably reflecting recent infection. ELISAs using the two other recombinant proteins, containing the C-terminal portion of ~~150, showed comparable sensitivity to tests using CM2, but where less specific in the detection of acute infection. In addition, high levels of IgM antibodies against CM1 and CM3 could be detected in follow-up sera from transplant recipients even serveral months after acute infection, while IgM-reactivity against CM2 declined in most patients within several weeks. These results confirm previous data, which demonstrated that although pp150 represents a very sensitive antigen, detection of IgM antibodies against this component is not reliably correlated with acute HCMV-infection (Vornhagen et al., 1994, 1995). In a recent study, the IgM-reactivity of different recombinant fusion proteins including a construct which contains the C-terminal half of p52 and two small portions of pp150 has been evaluated (Landini et al., 1995). The authors concluded that only a combination of this triple antigen fusion protein with additional recombinant viral proteins will enable sensitive IgM-serodiagnosis. These results are in agreement with our findings, which demonstrate that a combination of ~52 and pp150 (CM3) is not sufficient for the generation of an optimal recombinant IgM-assay and emphasize the essential role of the antigenic fragment UL57/3 present in CM1 and CM2 to achieve optimal sensitivity. The reactivity of our recombinant proteins was evaluated in comparison with two commercially available IgM ELISAs. These tests represent two different assay formats of the currently available HCMV specific IgM ELISAs. Although this comparative analysis was carried out with a limited number of carefully selected sera, the results indi-
80
R. Vornhagen et al. )/)Journal qf Virological
cate that using CM2 as an antigen will enable the design of a recombinant ELBA that combines, in contrast to the assays available currently, high sensitivity with high specificity for the detection of acute HCMV infection.
Acknowledgements The authors wish to thank Christine Rhode, Andrea Heim, Erika Seufert and Marianne Nashir-Heyer for their excellent technical assistance.
References Balfour, H.H.J. (1990) Management of cytomegalovirus disease with anti-viral drugs. Rev. Infect. Dis. 12. Suppl. 7, 8499860. Britt, W.J. (1994) Infections associated with human cytomegalovirus. In: R. Glaser and J.F. Jones (Ed%), Herpesvirus Infections. Marcel Dekker. NY. pp. 59-l 16. Ho. M. (1991) Cytomegalovirus Biology and Infection. Plenum Medical Book Co., NY. Landini. M.P., Guan, M.X., Jahn, G., Lindenmeier, W., Mach, M.. Ripalti, A., Necker, A., Lazzarotto. T. and Plachter, B. (1990) Large-scale screening of human sera with cytomegalovirus recombinant antigens. J. Clin. Microbiol. 28, 1375- 1379. Landini, M.P.. Ripalti, A., Sra. K. and Pouletty, P. (1991) Human cytomegalovirus structural proteins: immune reaction against ppl50 synthetic peptides. J. Clin. Microbial. 29, 1868 1872.
Methods
60 (1996) 73-80
Landini, M.P., Lazzarotto, T., Maine, G.T., Ripalti, A. and Flanders. R. (1995) Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay. J. Clin. Microbial. 33. 2535 2542. Lazzarotto. T., Dalla-Casa, B., Campisi, B. and Landini, M.P. (1992) Enzyme-linked immunoadsorbent assay for the detection of cytomegalovirus-IgM: comparison between eight commercial kits, immunofluorescence. and immunoblotting. J. Clin. Lab. Anal. 6, 216-218. Lindenmaier. W.. Necker, A., Krause, S., Bonewald, R. and Collins, J. (1990) Cloning and characterization of major antigenic determinants of human cytomegalovirus AD169 seen by the human immune system. Arch. Viral. 113,I 16. Ripalti, A., Ruan, Q.. Boccuni, M.C.. Campanini, F., Bergamini, G. and Landini, M.P. (1994) Construction of a polyepitope fusion antigen of human cytomegalovirus ppUL32: reactivity with human antibodies. J. Clin. Microbiol. 32, 3588363. Studier. F.W. and Motfatt, B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 1133130. Vornhagen, R., Plachter. B.. Hinderer, W.. Sonneborn. H.-H. and Jahn G. (1992) Application of PCR-technology for the generation of DNA-fragments coding for viral antigens. In: G. Kahl, H. Appelhans, J. Koempf and A.J. Driesel (Eds.), Bio Tech Forum IO, Hiithig, Heidelberg, pp. 2799288. Vornhagen, R., Plachter, B., Hinderer, W., The, T.H., van Zanten, J., Matter. L.. Schmidt, C.A., Sonneborn. H.-H. and Jahn. G. (1994) Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens. J. Clin. Microbial. 32, 981-986. Vornhagen, R., Hinderer. W., Sonneborn, H.-H.. Bein, G.. Matter. L., The, T.H.. Jahn, G. and Plachter, B. (1995) The DNA-binding protein pUL57 of human cytomegalovirus is a major target antigen for the immunoglobulin M antibody response during acute infection. J. Clin. Microbial. 33. 1927 1930.
Journal of Virological Methods 60 (1996) 73-80
IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins Walter Hinderer”, Hans-H. Sonneborn”, Gregor Beinb, Lukas Rolf Vornhagen”, Matterc, T. Hauw Thed, Gisela Ender9, Gerhard Jahnf, Bodo Plachterg,* “Biotesr AG, Research Department, 63303 Dreieich, Germany bInstitut fur Immunologie und Transfusionsmedi;i~~, Universitat Liibeck, 23562 Liibeck, Germany ‘Institut fir Medizinische Mikrobiologie, Uniuersitiit Bern, 3010 Bern, Switzerland ‘Deptartment of Clinical Immunology and Renal Transplant Unit, Unioersity of Groningen, Groningen, Netherlands ‘Institut Jiir Virologie, Infektiologie und Epidemiologie e. V., Medizinisch-diagnostisches Gemeinschaftslabor, 70193 Stuttgart, Germany fAbteilung fur Virologie, Hygiene Institut, Uniaersitiit Tiibingen, 72076 Tiibingen, Germany “Institiit ,fiir Virologie, Unirersitiit Maim, 55101 Maim, Germany
Accepted 26 February 1996
Abstract Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (~52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (~~150, aa 994- 1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELBA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection. Keylt’ords:
Cytomegalovirus;
Recombinant
antigens;
ELISA;
* Corresponding author. Tel.: + 49 6131 173652; fax: + 49 6131 395604; email: [email protected] 0166-0934,‘96/$15.00 @ 1996 Elsevier Science B.V. All rights reserved PII SOl66-0934(96)02047-2
IgM
1. Introduction Human cytomegalovirus (HCMV) infection usually remains asymptomatic in immunocompetent individuals but causes major clinical problems in patients whose immune functions are impaired or not yet fully developed. Prenatal transmission of HCMV is considered the most frequent cause of congenital sequelae related to virus infection in the Western world (Britt, 1994). In addition, primary and secondary infection in transplant recipients and in AIDS patients may lead to various organ manifestations and even life threatening conditions (for review, see Ho, 1991). To ensure timely and efficient antiviral treatment, diagnostic methods have been requested that allow close monitoring of patients endangered by HCMV infection (Balfour, 1990). Determination of specific antibodies against viral antigens is a diagnostic procedure which is used commonly for the identification of acute and past HCMV-infection. However, currently available serological assays, such as enzyme linked immunosorbent assays (ELISAs), use antigen preparations characterized poorly derived from infected cell culture. Yet, only a few of the over 200 putative proteins of HCMV serve as dominant targets for the humoral immune response. The over-representation of irrelculture derived antigen proteins in evant preparations, especially for IgM specific test systems, may explain the limited performance of available assays in relation to either sensitivity or specificity. This leads to discordant results when assays from different suppliers are compared (Lazzarotto et al., 1992). Careful selection of viral antigens for the design of improved tests therefore appears essential. Recombinant technology offers a strategy to identify relevant polypeptides for serological assays and to provide pure preparations of such antigens in large quantities. In previous investigations recombinant antigen fragments were identified which permit sensitive and specific diagnosis of acute and past HCMV al., 1995). infection (Vornhagen et 1994, Polypeptide fragments representing the C-termi-
nal portions of pUL44 (~~52, aa 297-433) and pUL32 (~~150, aa 99441048) as well as a 57 amino acid fragment from the central part of the major DNA-binding protein pUL57 (aa 545-601) appeared to be promising candidates for improving of IgM-specific serodiagnosis of acute HCMV-infection. In the present study, the immunodominant antigen fragments from pUL32, pUL44 and pUL57 of HCMV were combined to form different autologous fusion proteins. Using these polypeptides as antigens in ELISA experiments, the following questions were addressed: (i) whether autologous fusion of different highly reactive antigens would result in a better performance of the test systems compared to assays using culture derived antigens and (ii) which combination of the recombinant antigens would provide optimal test results concerning sensitivity and specificity for the detection of acute HCMV infection.
2. Material
and methods
2.1. Cloning and eqwession
of’ antigens
The construction of plasmids expressing autologous fusion proteins was carried out with DNA-fragments which had been generated by PCR-technology. The general strategy has been described in detail previously (Vornhagen et al., 1992). Amplification was undertaken with pairs recognition of PCR-primers containing sequences for restriction endonucleases Burn HI and EcoRI, 5’ and 3’ of the original priming sequence respectively. The 3’-primers contained an additional recognition sequence for BglIl 5’ to the EcoRI-site which was used for in-frame insertion of other PCR-derived BamHI/EcoRIfragments. The autologous fusion proteins CMl, CM2, CM3 and the reference antigens 5213 and 150/7 (Fig. 1) were expressed in E. co/i using vector pET5c which permits expression with an N-terminal leader sequence of 11 amino acids (Stud1986). The antigens were ier and Moffatt, purified to homogeneity using differential cen-
R. Vornhagen et al. 1 Journal qf Virological Methods 60 (1996) 73-80
pCC150/7/2 *w%
pcc57/3 %*a
pCC52/3 % *>
229kbp
HCMV -
-
c-
CM1 150/l/2 5713
52/3
CM2 52/3
57/3
CM3
52/3
150/l/2
52/3
Fig. 1. Schematic representation of the HCMV genome (central part) with the open reading frames UL32, UL44 and UL57. The relative localization of the DNA-fragments encoding the portions of the proteins expressed in E. coli are shown on top. Numbers indicate the amino acids of the respective proteins contained in the recombinant polypeptides. The composition of the autologous fusion proteins CMl, CM2 and CM3 evaluated in this study is given at the bottom.
and washing steps with subsequent ion-exchange and gel chromatography. The final purity was demonstrated by SDS-polyacrylamide gel electrophoresis and Western blot analysis. All three antigen preparations contained less than 1% residual E. coli protein contamination. trifugation
2.2. Evaluation experiments
of recombinant
antigens
in ELISA
The autologous fusion proteins were purified to homogeneity and evaluated by ELISA. Microtiter plates (Nunc, Maxisorb) were coated with 100 ng of purified antigens solubilized in 100 ~1 of 0.01 M carbonate (Na,CO,, NaHCO,) buffer, pH 9.5. ELISA plates were incubated with human sera diluted 1:21 for 1 h at 40°C in a water bath. After washing, bound IgM-antibodies were detected using a specific mouse monoclonal antibody (Jansen, Beerse, Belgium) conjugated with horeseradish peroxidase (30 min, 40°C). The conjugate was diluted 1: 1000 in a phosphate buffer containing 10% fetal calf serum. Color was developed for 30 min at room temperature using a 3,3’, 5,5’-tetramethylbenzidine (TMB) solution (Sigma, Munchen, Germany). Optical density was measured at 450 nm.
2.3. Serum samples In this study the recombinant HCMV-antigens were evaluated with human sera from different sources. Sera from healthy blood donors were obtained from a local transfusion center (Suhl, Germany) and HCMV-specific seroprevalence was determined by Biotest anti-CMV IgG ELISA. Patients receiving a renal allograft at the University Hospital Groningen, Netherlands, and at University Hospital, Liibeck, Germany, were monitored by the pp65specific antigenemia assay. Sera obtained from immunocompetent individuals with acute HCMV infection were taken from persons with primary infection proven by IgG seroconversion and/or from patients with HCMV excretion revealing typical clinical symptoms. In order to identify false positive results caused by rheumatoid factors selected sera were retested after treatment with RF absorbent (Behring, Marburg, Germany). 2.4. Reference
ELISAs
Recombinant HCMV-antigens together with two commercially
were evaluated available HCMV-
76 Table I IgM-specific
ELISA
results
of recombinant
viral antigens
52:‘3, 150/7 and autologous
Samples
II
fusion
protein
Number
CM3
of samples
with positive
result
52,!3
I so/7
CM3
36 17
27 IO
33 14
33 13
54 54
I 0
I9 0
0
Acute HCM V inf2ction.s Immunocompetent Renal transplant Randomly selected seropositive
individuals recipients with acute
HCMV
primary
infection”
hertlth_v blood donors
seronegative “Sera from individuals symptoms. %era were taken 2-3
with HCMV
primary
infection
proven
by seroconversion
and/or
viral excretion
together
I
with typical
clinical
weeks after onset of antigenemia
specific ELISAs which utilize cell culture derived antigens. While reference ELISA 1 was an indirect ELISA which uses control antigen, reference ELBA 2 was a p-capture assay, in which bound IgM-molecules were detected with enzyme labelled HCMV-antigens. With reference ELBA 1 a preincubation step was performed in order to remove rheumatoid factors.
3. Results 3.1. Construction md expression uutologous fusion proteins
oJ’ recombinant
In preceding evaluations we could show that recombinant antigen fragments representing the C-terminal portions of pp150 (150/7) and ~52 (52/3), and a small segment from the central part of the major DNA binding protein (UL57/3) are major target antigens of the IgM-specific antibody response during acute HCMV infection (Vornhagen et al., 1994, 1995). IgM antibodies against 15Oj7 (UL32, aa862-1048) however, were also detected in a substantial number of serum samples from healthy blood donors without any signs of acute infection (Vornhagen et al., 1995). Additional experiments were carried out with a recombinant fragment which consists only of the last 54 amino acids of ~~150. This portion (150/7/2), which harbours a potential IgM-epitope (Landini et al., 1991) was expressed in fusion with fragment
5213 to form the recombinant autologous fusion protein CM3. After purification CM3 was tested by ELISA together with the recombinant antigens 52/3 and 150/7. In this first series of experiments CM3 proved to be more reactive than 52/3 when tested with samples from acutely infected individuals but has only low reactivity with sera from healthy blood donors (Table 1). Considering these results two additional autologous fusion proteins were constructed (Fig. 1). While CM1 combines the three immunodominant fragments 52/3, UL57/3 and 150/7/2, CM2 consists of the central and highly reactive portion of pUL57 (UL57/3) with one copy of 5213 on either side. This combination appeared necessary, since a C-terminal fusion protein of 5213 and UL57/3 showed substantial proteolytic degradation during expression and purification; CM2 was found to be very stable. 3.2. ELISA evaluution ,fiaion proteins
of recombinant
autologous
Purified recombinant antigens CMl, CM2 and CM3 were evaluated further by ELISA experiments in comparison with an indirect assay and a /L-capture ELBA which both use cell culture derived antigens. These two reference ELISAs represent the two different assay formats of the currently available HCMV-specific IgM-ELISAs. The sensitivity of the recombinant antigens was determined with sera from immunocompetent in-
R. Vornhagen et al. 1 Journal of Virological Table 2 IgM-specific IgM-ELISAs
ELBA
results
of recombinant
autologous
n
Samples
Acute HCMV injkctions Immuncompetent individuals” Renal transplant recipients with acute primary infectioni Randomly selected healthy blood donors Total Seropositive Seronegative
HCMV
fusion
proteins
Number
Methods
in comparison
of samples
17
60 (1996) 73-80
with two commercially
with positive
CM2
CM3
Reference
47 13
41 12
41 13
44 12
39 10
46 11
175 121 54
I 7 0
3 3 0
6 6 0
1 1 0
14 12 2
dividuals with acute HCMV-infection and from renal transplant recipients with acute HCMV-primary infection. The latter samples were drawn 223 weeks after onset of antigenemia. The results are shown in Table 2 and Fig. 2. While recombinant antigens CM1 and CM2 revealed high IgM specific prevalence, CM3 was less reactive. CM2 was the only recombinant antigen which was positive with all samples tested. The two reference ELISAs showed highly discordant results. While reference ELISA 2 revealed a lower but still comparable sensitivity as CM1 and CM2, reference ELISA 2 was much less reactive and gave negative results with 17% of the samples. These results indicated IgM assays using CM1 and CM2 as antigens show comparable or better sensitivity than the commercially available assays. Secondly, the time course of the IgM response against the recombinant antigens was tested with follow-up sera from renal transplant recipients (Fig. 2). HCMV infection in these patients had been monitored by pp65-specific antigenemia assay. While Fig. 2 A-C show the data obtained with sera from patients with primary infection, Fig. 2 D represents a follow-up from a patient with acute secondary infection. The results demonstrate that IgM antibodies against all recombinant autologous fusion proteins were detectable very early during the acute phase of infection. In all
by seroconversion
and/or
reference
result
CM1
“Indirect ELISA with control antigen. “p-capture ELISA. ‘Sera from individuals with HCMV primary infection proven symptoms. “Sera were taken 223 weeks after onset of antigenemia.
available
ELISA
viral excretion
I”
Reference
together
ELISA
with typical
2’
clinical
three patients with acute primary infection, IgM antibodies against the recombinant antigens were detectable within a time period of two weeks after onset of antigenemia, while in the patient suffering from acute secondary infection recombinant ELISAs became positive concomitantly (CM2) or even 1 week before (CMl, CM3) the antigenemia assay. Similar results were obtained with two additional follow-ups from renal transplant recipients with acute secondary infection (data not shown). Both reference ELISAs were less sensitive with the follow-up samples from transplant recipients. Reference ELISA 2 revealed a positive result only in one patient (C) as early as the recombinant ELISAs but became positive in all other patients l-2 weeks after the recombinant assays. Reference ELISA 1 was even less sensitive and revealed a positive result l-5 weeks after the recombinant ELISAs. While the three recombinant antigens revealed comparable results during seroconversion, differences in reactivity were more pronounced after the acute phase of infection. Although IgM antibodies against CM2 were still detectable in all four patients at the end of the observation period, IgM specific reactivity in three patients already declined several weeks after the acute phase of infection. In contrast, high levels of IgM antibodies against both autologous fusion proteins which
R. Vornhagen et al. i Journal c$ Virological
Methods
60 (1996) 73-80
30
0
20 weeks
10
20 weeks
post TX
post TX
10
.g
d s5
D
0
0
0
10
20
30 weeks
0
20
IO weeks
post TX
post TX
Fig. 2. IgM-specific reactivities of recombinant autologous fusion proteins CMI, CM2 and CM3 in ELISA in comparison with two reference ELlSAs with follow ups from renal transplant recipients with acute HCMV-primary infection (A + C) and acute HCMV-secondary infection (D). HCMV infection was monitored using the pp65-specific antigenemia assay. Sample/cut OK ratios for CMI, n ; CM2, A; CM3, a; reference ELISA 1, *; reference ELISA 2, x ; and number of antigen positive ceils per 100000 cells, 0.
contain the 54 C-terminal amino acids of pp150 (CMl, CM3) were present even several months after the acute stage of infection. To evaluate the specificity of tests using recombinant antigens, sera from healthy blood donors (Table 2, panel II) were analysed. Here, CM2 revealed the lowest IgM-reactivity of all recombinant antigens. All three positive samples were taken from seropositive individuals. One of these sera was highly reactive (OD > 2.0) with all three recombinant antigens and was also positive with reference ELISA 1. The two other reactive samples showed optical density values just above cutoff. CM1 and CM3 revealed substantially higher IgM specific reactivity than CM2. With sera from healthy donors both reference ELISAs again revealed highly discordant results. While reference ELISA 1 revealed a positive result with a single
donor, reference samples.
ELBA
2 was positive
with
14
4. Discussion Conventional assays for the detection of IgM antibodies against HCMV, which use cell culture derived antigens suffer from either limited sensitivity or specificity (Lazzarotto et al., 1992). Several attempts have been made to identify recombinant viral proteins which can serve as antigens for the detection of IgM antibodies during acute HCMV infection (Lindenmaier et al., 1990; Landini et al., 1990; Vornhagen et al., 1995). These studies led to the conclusion that only a combination of carefully selected components will enable IgM specific serodiagnosis with improved specificity and sensitivity.
R. Vornhagen et al. /Journal
qf Virological Methods 60 (1996) 73-80
In a previous study, it was shown that antigen fragments from p52 (52/3) and pp150 (150/l and 150/7) of HCMV enable highly sensitive detection of IgM antibodies in patient sera (Vornhagen et al., 1994). However, especially with fragments from the tegument protein ~~150, IgM antibodies were also detected in sera from patients without acute infection. These data suggested that antigen composition used in these experiments was not optimal to indicate clinically relevant infection. In recent analyses we identified a major IgM antigen of HCMV consisting of aa 545-601 of pUL57 (UL57/3; Vornhagen et al., 1995). ELISAs using this polypeptide as antigen indicated acute HCMV infection with high sensitivity and specificity. The aim of this study was to evaluate, whether a combination of UL57/3 and 5213 together with a truncated version of 150/7 (150/7/2) would combine highly sensitive and specific detection of acute HCMV infection using IgM ELISA and whether such a recombinant test would be superior to conventional IgM ELISAs. The use of severai individual recombinant proteins in a single assay requires complicated procedures in order to guarantee the presence of standardized quantities of the different antigens. In addition, high level expression of very small heterologous proteins in E. coli may not always be possible. We therefore chose to express the different immunodominant HCMV antigen fragments as autologous fusion proteins. This strategy allows the synthesis of high amounts of different antigens in one molecule, thus providing a defined composition of the material. A previous study in which single as well as multiple copies of immunodominant portions of pp150 (pUL32) were expressed in E. coli as so-called polypeptide fusion antigens has already demonstrated the general usefulness of this approach (Ripalti et al., 1994). We generated constructs expressing three different autologous fusion proteins (Fig. 1). The fusion protein CM2, which combines 5213 with UL57/3, but lacks portions of ~~150, turned out to be the most reliable recombinant IgM antigen. Using this polypeptide, IgM antibodies were detected in all sera from patients undergoing acute HCMV infection. Accordingly, the sensitivity of the assay for the identification of acute infection
19
was superior to all other assays with different antigen composition including both commercial tests. In sera from blood donors without symptoms of HCMV infection, the CM2-ELISA detected IgM antibodies in three cases. One of these was also positive with all other recombinant ELISAs and with one commercial ELISA; this indicates that this particular donor might have undergone acute infection at the time the sample was obtained. Two other samples were positive just above cut-off, probably reflecting recent infection. ELISAs using the two other recombinant proteins, containing the C-terminal portion of ~~150, showed comparable sensitivity to tests using CM2, but where less specific in the detection of acute infection. In addition, high levels of IgM antibodies against CM1 and CM3 could be detected in follow-up sera from transplant recipients even serveral months after acute infection, while IgM-reactivity against CM2 declined in most patients within several weeks. These results confirm previous data, which demonstrated that although pp150 represents a very sensitive antigen, detection of IgM antibodies against this component is not reliably correlated with acute HCMV-infection (Vornhagen et al., 1994, 1995). In a recent study, the IgM-reactivity of different recombinant fusion proteins including a construct which contains the C-terminal half of p52 and two small portions of pp150 has been evaluated (Landini et al., 1995). The authors concluded that only a combination of this triple antigen fusion protein with additional recombinant viral proteins will enable sensitive IgM-serodiagnosis. These results are in agreement with our findings, which demonstrate that a combination of ~52 and pp150 (CM3) is not sufficient for the generation of an optimal recombinant IgM-assay and emphasize the essential role of the antigenic fragment UL57/3 present in CM1 and CM2 to achieve optimal sensitivity. The reactivity of our recombinant proteins was evaluated in comparison with two commercially available IgM ELISAs. These tests represent two different assay formats of the currently available HCMV specific IgM ELISAs. Although this comparative analysis was carried out with a limited number of carefully selected sera, the results indi-
80
R. Vornhagen et al. )/)Journal qf Virological
cate that using CM2 as an antigen will enable the design of a recombinant ELBA that combines, in contrast to the assays available currently, high sensitivity with high specificity for the detection of acute HCMV infection.
Acknowledgements The authors wish to thank Christine Rhode, Andrea Heim, Erika Seufert and Marianne Nashir-Heyer for their excellent technical assistance.
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Landini, M.P., Lazzarotto, T., Maine, G.T., Ripalti, A. and Flanders. R. (1995) Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay. J. Clin. Microbial. 33. 2535 2542. Lazzarotto. T., Dalla-Casa, B., Campisi, B. and Landini, M.P. (1992) Enzyme-linked immunoadsorbent assay for the detection of cytomegalovirus-IgM: comparison between eight commercial kits, immunofluorescence. and immunoblotting. J. Clin. Lab. Anal. 6, 216-218. Lindenmaier. W.. Necker, A., Krause, S., Bonewald, R. and Collins, J. (1990) Cloning and characterization of major antigenic determinants of human cytomegalovirus AD169 seen by the human immune system. Arch. Viral. 113,I 16. Ripalti, A., Ruan, Q.. Boccuni, M.C.. Campanini, F., Bergamini, G. and Landini, M.P. (1994) Construction of a polyepitope fusion antigen of human cytomegalovirus ppUL32: reactivity with human antibodies. J. Clin. Microbiol. 32, 3588363. Studier. F.W. and Motfatt, B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 1133130. Vornhagen, R., Plachter. B.. Hinderer, W.. Sonneborn. H.-H. and Jahn G. (1992) Application of PCR-technology for the generation of DNA-fragments coding for viral antigens. In: G. Kahl, H. Appelhans, J. Koempf and A.J. Driesel (Eds.), Bio Tech Forum IO, Hiithig, Heidelberg, pp. 2799288. Vornhagen, R., Plachter, B., Hinderer, W., The, T.H., van Zanten, J., Matter. L.. Schmidt, C.A., Sonneborn. H.-H. and Jahn. G. (1994) Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens. J. Clin. Microbial. 32, 981-986. Vornhagen, R., Hinderer. W., Sonneborn, H.-H.. Bein, G.. Matter. L., The, T.H.. Jahn, G. and Plachter, B. (1995) The DNA-binding protein pUL57 of human cytomegalovirus is a major target antigen for the immunoglobulin M antibody response during acute infection. J. Clin. Microbial. 33. 1927 1930.