Cytomegalovirus Phosphoprotein 150

Transcription

1 JOURNAL OF CLIICAL MICROBIOLOGY, OCt. 1992, p Vol. 30, No /92/ $02.00/0 Copyright 1992, American Society for Microbiology Antibody Responses to Synthetic Peptides from Cytomegalovirus Phosphoprotein 150 VIVI-ANNE SUNDQVIST, 12* WENMEI XU,2 AND BRIITA WAHREN2 Department of Medical Laboratory Technology, Stockholm College of Health and Caring Sciences, S Stockholm, 1 and Department of Virology, National Bacteriological Laboratory, S Stockholm, 2* Sweden Received 26 December 1991/Accepted 9 July 1992 We have identified antigenic regions within phosphoprotein 150 of human cytomegalovirus (CMV ppl50) to which seroreactivity appears in patients with active CMV infection or persists in seropositive persons. A range of 8.3 to 61.6% of healthy CMV-seropositive blood donors were immunoglobulin G positive for single peptides, while 91.6% reacted to a mixture of four peptides. All convalescent-phase serum samples from 26 patients with active CMV infection reacted with either of two peptides encompassing amino acids (aa) 594 to 623 and aa 614 to 643. Patients with a primary CMV infection had patterns of reactivity to single peptides different from those of patients with reactivated CMV infection. The immunoglobulin M antibodies reacted preferentially with the peptides encompassing aa 594 to 663 of CMV ppl50. Infection with human cytomegalovirus (CMV) is common. Although nonsymptomatic infection occurs in many cases, serious illness caused by CMV is a problem in immunocompromised patients, including those with AIDS (7, 14). Also, congenitally infected infants can be severely affected, since they develop a spectrum of clinical signs (23). By now, use of cultivation and isolation of virus in cell culture for the diagnosis of CMV infections is often replaced by more rapid methods. CMV early antigen can be detected by immunofluorescence after a short period of virus cultivation, before any cytopathic effect is evident (5). An assay based on the detection of CMV antigens in leukocytes has been adapted for diagnosis of CMV antigenemia (25). Polymerase chain reaction used for the detection of CMV DNA in different kinds of specimens seems to be a promising method for rapid and sensitive detection of CMV DNA (2, 21), although the clinical significance of the presence of CMV DNA in samples has to be carefully evaluated and correlated to detection of CMV by other methods. Serological methods are commonly used for diagnosis of CMV. In primary CMV infection, evidence of immunoglobulin G (IgG) seroconversion remains a reliable diagnostic criterion, as does the presence of CMV IgM antibodies, which are produced and can be detected prior to CMV IgG. However, reactivated CMV infections do not always induce IgM antibodies, and anti-cmv IgG levels can be unchanged during the course of reactivated infection, even though virus can be isolated. Blood and organ donors are serologically screened for previous exposure to CMV and therefore for the potential for transmitting CMV. Currently, most serological assays are based on antigens extracted from fibroblasts infected with the laboratory CMV strain AD169 or Towne. The CMV proteins included are not well-defined, and the remaining cellular antigens can cause nonspecific reactions. With immunoblotting, the antibody responses to the different polypeptides of CMV can be demonstrated (8, 11, 17). There is considerable heterogeneity among different individuals, but several studies have shown both CMV IgG and IgM antibodies against CMV phosphoprotein 150 (ppl50) to be present in a high percentage of CMV-infected * Corresponding author persons (9, 13, 20). Fusion proteins of CMV containing the amino acid (aa) residues 486 to 554 or 594 to 663 of CMV ppl50 used in immunoblotting were recognized by human sera known to be seropositive for CMV (13). In order to identify potent antigenic sites within the immunodominant ppl50, peptides were used to explore the IgG and IgM responses. Twelve peptides, 20 aa long and overlapping by 10 aa, were synthesized (6, 18) for the CMV ppl50 residues 486 to 555 and 594 to 663 (Table 1). In order to obtain several epitopes on one molecule, two additional peptides encompassing aa 594 to 623 (peptide 150:15) and aa 614 to 643 (peptide 150:16) were produced. Serum samples from 70 healthy blood donors were included in the study. A total of 60 samples were CMV IgG positive and 10 were CMV IgG negative as determined by CMV enzyme-linked immunosorbent assay (ELISA) (24). Acute-phase and convalescent-phase serum samples were collected from 26 patients with active CMV infection, with fever as the main clinical symptom. Three renal allograft recipients were included. All 26 patients had significant rises in CMV IgG titers. Fourteen patients, including two renal allograft recipients, with primary infections had low CMV IgG titers (<100 to 500) in the acute-phase serum samples. These patients all had specific CMV IgM (24). Twelve patients were classified as having a secondary CMV infection because of CMV IgG titers of >5,000 in the acute-phase serum samples, which were collected within 2 weeks after the onset of illness. Seven of them also had CMV IgM. The two groups were comparable concerning age, sex, and clinical symptoms. Peptide ELISA was performed as previously described (22). The ELISA wells were coated with peptides at a concentration of 10 Fg/ml. When mixtures of peptides were used for coating, the total amount was still 10 p,g/ml, which included equal amounts of each peptide. Serum samples were diluted 1:50. Alkaline phosphataseconjugated sheep anti-human IgG and anti-human IgM (Sigma, St. Louis, Mo.) were used. IgG reactivity to CMV ppl5o peptides. The IgG reactivities to the 12 peptides were initially studied with a panel of CMV IgG-positive sera and a panel of CMV IgG-negative sera. The 10 reactive peptides (150:C to F and 150:G to M), reactive with >5% of the CMV IgG-positive panel, were

2 2736 NOTES J. CLIN. MICROBIOL. TABLE 1. IgG reactivities to synthetic peptides from CMV pp150 in serum samples from 60 seropositive blood donors Peptide(s) Positions (aa) Sequence % Reactive samples 150:A QQQRHLAAFSLVSPQVTKASP < :B VSPQVTKASPGRVBRDSAWD < :C GRVRRDSAWDVRPLTETRGD :D VBPLTETRGDLFSGDEDSDS :E LFSGDEDSDSSDGYPPNRQD :F SDGYPPNRQDPRFTDTLVDI :G LTPTPVNPSTAPAPAPTPTF :H APAPAPTPTFAGTQTPVNGN :I AGTQTPVNGNSPWAPTAPLP :K SPWAPTAPLPGDMNPANWPR :L GDMNPANWPRERAWALKNPH :M ERAWALENPHLAYNPFRLPT :G, H, I :F, G, H, I evaluated further with serum samples from 70 blood donors, 10 of which were seronegative by our conventional CMV ELISA. The mean absorbance value for the 10 CMV IgGnegative serum samples + 2 standard deviations was calculated for each peptide or combination of peptides and was used as the cutoff value. A range of 8.3 to 61.6% of CMV-seropositive serum samples reacted with 10 peptides (Table 1). The most-reactive peptides encompass aa 536 to 555 (150:F) and aa 614 to 633 (150:J). Four of 60 serum samples did not react with any of the CMV ppl50 peptides, and 11 of 60 serum samples reacted with just one peptide. The median number of peptides to which each serum reacted was three, and the maximum number was eight. Strong reactivity to individual peptides could be seen, although the peptides overlapped with neighboring peptides by 10 aa. Since the sera had different reactivity patterns (Fig. 1), the o* Serum 517 S2I - M-pp7te2 CMV ppl50 poptidesa6 IgG reactivities to combinations of peptides were also measured. It was possible to coat each well with up to six peptides and still detect antibodies known to react with one of the included peptides (Fig. 1). In such cases, the absorbance value for the mixture of six peptides was lower than the value obtained with the single most reactive peptide. Eighty percent of the CMV IgG-positive serum samples reacted with a mixture of the peptides 150:G, 150:H, and 150:J. When peptide 150:F was added to that mixture, a response was detected in 91.6% of CMV antibody-containing serum samples. The responses of acute-phase and convalescent-phase serum samples from 26 patients with active CMV infections were studied (Table 2). A clear difference between sera from patients with primary infection and sera from patients with reactivated infection was seen. In primary infection, reac- LrM 2 - Serum 615 Serum 535 Serum 557 IA (1I o. s IA a I - Y ei E _ 2 CMV pp150 peptidesa 6 0 i!2 _ y, _ (D CIUV ppl 50 poptides ( FIG. 1. IgG reactivities to individual peptides and combinations of peptides encompassing aa 594 to 663 of CMV pplso in serum samples from four healthy seropositive blood donors. Abs., absorbance.

3 VOL. 30, 1992 NOTES 2737 TABLE 2. IgG reactivities to CMV pplso peptides in convalescent-phase sera from patients with active CMV infection Primary CMV infection (n = 14) Reactivated CMV infection (n = 12) Peptide Positions (aa) A4s5 A405 % Positive % Positive Mean Range Mean Range 150:C :D :E :F :G > :H > :J > :K > :L :M > : > : > tivity greater than 50% was seen only with peptides 150:G and 150:H. In reactivated infection, the most pronounced reactivity was to peptide 150:1, to which 100% of the serum samples reacted. Overall, more sera from patients with secondary infection were reactive with the peptides than sera from patients with primary infection. The sequence aa 594 to 643 thus seems to encompass major antigenic sites. Therefore, additional 30-aa-long peptides, 150:15 and 150:16, were studied. All convalescent-phase sera from patients with a primary CMV infection reacted with both of these peptides, although the highest mean absorbance value was seen for peptide 150:15. Of the serum samples from patients with reactivated infection, 100% were reactive with peptide 150: 16, which also gave the highest mean absorbance value. A total of 66.6% reacted with peptide 150:15. Three serum samples with low levels of reactivity to peptide 150:H failed to react with the 30-aa-long peptide 150:15. Ten of 14 acute-phase serum samples from patients with primary infection were CMV IgG negative (titer of < 100) by E our conventional CMV ELISA, but 5 of those 10 serum samples had detectable IgG to peptide 150:15. IgM reactivity to CMV ppl50 peptides. Acute-phase and convalescent-phase serum samples from 26 CMV-infected patients (same patients as for Table 2) were studied with the same peptides (Fig. 2). In 12 of 14 acute-phase serum samples from the patients with primary infection, IgM antibodies to peptides were found. In cases of reactivated infection, CMV IgM antibodies were detectable in 3 of 12 acute-phase serum samples. In comparison with the IgG reactivity, a more general peptide reactivity was seen with IgM antibodies. The appearance of IgM antibodies to the different peptides varied. In most cases, IgM reactivity to peptide 150:G occurred first. With time, all sera that were IgM positive according to our total CMV IgM assay became IgM reactive to one or several peptides. The IgM antibodies reacted preferentially with peptides 150:G to 150:M, encompassing aa 594 to 663. Nine CMV IgG-negative blood donor serum samples had low levels of reactivity with all of the 0 C D E F G H I K L M CMV pp150 peptides FIG. 2. IgM reactivities to individual peptides from CMV ppls0 encompassing aa 506 to 555 (peptides C to F) and aa 594 to 663 (peptides G to M). The mean absorbance (Abs.) values obtained for CMV IgG-negative (-) and CMV IgG-positive (U) sera from healthy blood donors and for acute-phase (U) and convalescent-phase (0) sera from patients with active CMV infection are presented.

4 2738 NOTES peptides (mean absorbance values of 0.18 to 0.38). Nine CMV IgG-positive blood donor serum samples had higher mean levels of IgM reactivity, since two of nine serum samples were IgM positive for peptides 150:G, 150:K, 150:L, and 150:M. That may be a sign of reactivated CMV infection with a corresponding IgM response to peptides of CMV pp150. The sequences of the peptides included in our study were chosen on the basis of their presence in fusion proteins known to be recognized by CMV antibodies. A mapping procedure based on the synthesis of peptides on chemically activated rods (4) has been applied to CMV ppl50 by Novak et al. (15). The two most-reactive peptides in that study encompassed aa 595 to 614 and aa 617 to 636 and were thus within the sequences that are described in this paper. Consequently, it is possible to conclude that aa 594 through 643 comprise major antibody binding sites for CMV ppl50. Landini et al. (10) synthesized peptides from CMV ppl50 according to sequences with a high content of hydrophilic amino acids or according to the presence of sequences in the Dl fusion protein (9), which is known to be reactive with CMV antibody-containing sera. IgG reactivity was obtained in 25% of CMV IgG-positive serum samples with one peptide encompassing aa 1011 to 1048, while IgM reactivity to the same peptide was seen in 83.3% of CMV IgM-containing serum samples. Peptides synthesized on the basis of a high content of hydrophilic amino acids showed almost no reactivity. Two of these peptides correspond to sequences in our peptides 150:A to 150:D, and in agreement with our findings, low levels of reactivity with these peptides were noted. Thus, synthesizing peptides according to sequences present in CMV antibody-binding fusion proteins seems to be a successful way of designing peptides. This applies particularly when dealing with viral proteins consisting of >1,000 aa, such as CMV ppls0. Peptides from CMV ppls0 seem to be particularly suitable for measuring antibodies present in active CMV infection. IgG reactivity was found in all convalescent-phase sera from patients with active CMV infection and was directed to either of two 30-aa-long peptides encompassing aa 594 to 643. Within that sequence, individual patterns of reactivity to separate peptides were seen. A common pattern according to the type of CMV infection was also seen. An antibody response to peptide 150:J (aa 614 to 633) was present in reactivated infection, while reactivity to peptides 150:G and 150:H (aa 594 to 623) instead was characteristic of primary infection. Further studies art in progress to evaluate whether reactivity to different CMV ppls0 peptides could be used for distinguishing primary infection from reactivated infection. One clinically important application for this differentiation would be in maternal CMV infection. Although cases of symptomatic congenital CMV infection following nonprimary maternal infection have been reported, the consensus is that fetal damage is found most often as a result of primary infection of the mother. The methods previously described for differentiating primary infection from reactivated infection include determination of antibodies produced against early and late antigens (16), measurement of antibody avidity (1), and the use of blocking tests (19). The ELISA is useful for measuring the antibody response to CMV infection, and different Ig classes and IgG subclasses can easily be determined (12, 24). The use of defined antigens can further increase the evaluation of the antibody response and even make it possible to identify anti-cmv antibodies correlated with an active infection. Selective loss of antibodies to CMV antigens may also have a prognostic J. CLIN. MICROBIOL. value (3). In the future, antibody tests based on peptide antigens may be valuable tools for such evaluations. Excellent technical assistance by Michel Silvestri, Fredrik Jfiderling, and Michael Levi in synthesizing the peptides and by Elisabet Wiklund in performing ELISAs is gratefully acknowledged. This work was supported by a grant from the Swedish Medical Research Council (project 09088). REFERENCES 1. Blackburn, N. K, T. G. Besselaar, B. D. Schoub, and K. F. O'Conell Differentiation of primary cytomegalovirus infection from reactivation using the urea denaturation test for measuring antibody avidity. J. Med. Virol. 33: Brytting, M., V.-A. Sundqvist, P. Stalhandske, A. Linde, and B. Wahren Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides. J. Virol. Methods 32: Converse, P. J., T. E. Fehniger, A. Ehrnst, 0. Strannegard, and S. Britton Immune response to fractionated cytomegalovirus (CMV) antigens after HIV infection. 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