JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1994, p. 358-3630095-1137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Construction of Polyepitope Fusion Antigens of HumanCytomegalovirus ppUL32: Reactivity with Human AntibodiesA. RIPALTI,* Q. RUAN, M. C. BOCCUNI, F. CAMPANINI, G. BERGAMINI, AND M. P. LANDINI

Department of Microbiology, School of Medicine, University of Bologna, Bologna, Italy

Received 8 July 1993/Returned for modification 9 September 1993/Accepted 25 October 1993

We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV)antigens, expressed as fusion proteins or synthesized as oligopeptides, can be valuable diagnostic material in theserology of HCMV infection (M. P. Landini, M. X. Guan, G. Jahn, W. Lindenmaier, M. Mach, A. Ripalti,A. Necker, T. Lazzarotto, and B. Plachter, J. Clin. Microbiol. 28:1375-1379, 1990; M. P. Landini, T.Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa, J. Clin. Microbiol. 27:2324-2327, 1989; A. Ripalti,M. P. Landini, E. S. Mocarski, and M. La Placa, J. Gen. Virol. 70:1247-1251, 1989). In this work weaddressed the question of whether the expression of more than one linear epitope on a single fusion proteincould increase the reactivity of genetically engineered antigenic material with human antibody. To answer thisquestion we fused sequences expressing two different epitopes contained in the basic phosphoprotein of 150 kDaencoded by UL32 (M. S. Chee, A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, T. Cerny, T. Hornsel, C. A.Hutchinson, T. Kouzarides, J. A. Martignetti, and B. G. Barrell, Curr. Top. Microbiol. Immunol.154:125-169, 1990; G. Jahn, T. Kouzarides, M. Mach, B.-C. Scholl, B. Plachter, B. Traupe, E. Preddie,S. C. Satchwell, B. Fleckenstein, and B. G. Barrell, J. Virol. 61:1358-1367, 1987), ppUL32, which wasrepeatedly shown to be the strongest immunogen present in the viral particle. We also made fusions withsequences expressing a single epitope repeated once, twice, or three times. The different fusion proteins weretested with HCMV-positive human sera. We found that fusion proteins expressing different epitopes togetherwere recognized by a larger number of serum specimens and with more intense reactions in Western blot(immunoblot) experiments. We also found evidence that expression on the same polypeptide of the two distinctepitopes produced a stronger antigen than the mere addition of two fusion proteins which each carried one copyof one of these epitopes. Furthermore, we found that while the same epitope expressed two or three times onthe same fusion protein was not better recognized by immunoglobulin G than the single epitope, immunoglob-ulin M reactivities to the double and triple epitopes were enhanced.

Human cytomegalovirus (HCMV) infection of immuno-competent hosts does not usually cause clinical symptoms,whereas infection of immunocompromised hosts often givesrise to severe and even fatal disease. Diagnosis of HCMVinfection can be obtained by direct demonstration of thevirus or virus components in pathological materials or indi-rectly through serology (for a review see reference 4).However, serological diagnoses vary greatly because ofantigen composition and lack of antigen standardization.Antigenic materials composed of single well-characterizedviral proteins, or portions of them, produced via molecularbiology or peptide chemistry have proved to be promisingtools in improving serological diagnosis (3, 5, 6, 8, 10-12,14).Analyses of the humoral immune response elicited during

natural infection have repeatedly shown that the basic phos-phoprotein of 150 kDa encoded by UL32 (ppUL32) andlocalized in the viral tegument is highly immunogenic and isrecognized by sera from nearly 100% of the HCMV-seropos-itive subjects tested (3, 7). At least two epitopes have beenidentified in this molecule and have been shown to reactefficiently with human immunoglobulins. In particular, theanalysis of several ppUL32 fusion proteins (FPs) showedthat a region localized in the last 25 amino acids (aa) of thecarboxy terminus of the molecule (aa 1024 to 1048) reacted

* Corresponding author. Mailing address: Istituto di Microbiolo-gia, Policlinico S. Orsola, via Massarenti, 9, I-40138 Bologna, Italy.Phone: 39.51.341652. Fax: 39.51.341632.

with more than 80% of immunoglobulin M (IgM)-positivesera tested (8). When chemically synthesized, another regionlocalized between aa 595 and 614 (the 595-614 epitope) gavea positive reaction with almost 100% of IgG-positive seratested (10).

In an attempt to improve the antigenicity of these tworelatively short epitopes of ppUL32, we cloned copies of thecoding sequences for these epitopes on a prokaryotic expres-sion vector in order to express FPs carrying differentepitopes, or the same epitope repeated two or three times,on a single polypeptide that can be expressed in largeamounts in bacterial cells. Their coding sequences weresubcloned in a prokaryotic expression vector. Five differentconstructs were produced each expressing either one copyof the 595-614 epitope; one, two, or three copies of the1024-1048 epitope, or both epitopes together. All these FPswere tested with human sera to determine their abilities tobind HCMV-specific IgG and IgM.

MATERIALS AND METHODS

Cloning. Standard cloning techniques were used for theproduction of all constructs. Plasmid pMB28 was obtainedby inserting a PCR-amplified and -modified DNA fragmentbetween the SalI and the HindlIl sites of the polycloningregion of the prokaryotic expression vector pROS (11). Thissequence is present on UL32 from nucleotide 1783 to 1842(the 1783-1842 region). pMB30, pMB31, and pMB32 wereobtained by inserting in the SmaI site of pROS and in thesame orientation one, two, and three copies, respectively, of

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HUMAN ANTIBODY REACTIVITY WITH HCMV FUSION ANTIGENS 359

a

StopE E

EcoRi

M lutagene,sis

EcoRIE E

Digestioniaa0414

aalO24 1 t4SE E E

| Ligation

1024 '1048

2 x 1(124 1(146

.x 1024 1'048 II 024 1(048

pM3

T'% G A T C

stop < A%,TA -AG

G-GA

A

G A T C /T

TT >-EcoRI

- AG.,

A_.. GBt41 G

B

a) Fill inb) Ligate to

pRos/Stnal

2 x 1(124 1048S

pM3

3, \ I 124I 048

p\13

b-9- 614 1024 10)48

KL-QEF\//

Stutl1it 2 114

pB2 E)+ E) )pMB3

FIG. 1. (a) A schematic representation of the modification and cloning of the epitope located at aa 1024 to 1048 on ppUL32 in theexpression vector pROS as one, two, or three copies (a) or in conjunction with the epitope at aa 595 to 614 (b). E, EcoRI; (E), EcoRI afterfilling in. (Inset) Autoradiography of the sequence corresponding to the carboxy terminus of the UL32 gene product before (A) and after (B)mutagenesis. See Materials and Methods for details.

a 78-bp sequence located at the 3070-3144 region of UL32(Fig. 1). The original sequence, obtained from a Xgtll clone(9, 12), was modified by mutagenesis (see below) to abolishthe stop codon and to introduce an EcoRI site at the sameplace (Fig. la). This resulted in the introduction of anadditional terminal amino acid (Phe) in the translated se-quence and conservation of the correct reading frame whentwo or more copies of the fragment were ligated together inthe same orientation. Insertion in pROS was preceded byfilling in of the inserts. pMB33 was obtained by inserting inpMB28, at the StuI site, one filled-in copy of the 78-bpfragment described above. The resulting FP includes a 5-aarm (KLQEF) between the two epitopes (Fig. lb).

Oligonucleotides. Oligonucleotides were synthesized withan oligonucleotide synthesizer, PCR Mate 391 (Applied

Biosystems, Foster City, Calif.). The oligonucleotides syn-thesized for PCR amplification of the 1783-1842 sequence ofUL32 were as follows: 5'CCG GTC GAC CAC GCC GACGCC TGT CAA TCC T3' (Al) and 5'TAT AAG ClTT CGCGAA GGT AGG TGT CGG GGC3' (CII). The oligonucleo-tide used for mutagenizing the carboxy-terminal codingsequence of UL32 was 5'ACA CGG AGG AAT tcT TAAGAA ACA CA3' (Dlm), where lowercase letters indicatemodified nucleotides substituting for AG in the native se-quence. For sequencing pROS inserts the following oligonu-cleotide was synthesized: 5'GAA GGT CGT AAA GGTGGT GG3'.PCR. The sequence from UL32 spanning nucleotide 1783

to 1842 was amplified by an Ampliwax GemlOO (PerkinElmer-Roche, Branchburg, N.J.) hot-start PCR protocol.

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360 RIPALTI ET AL.

The primers described above were used with a PCR reagentkit (Perkin Elmer) according to instructions from the manu-facturer. A plasmid subgenomic clone harboring the XbaI Afragment of the viral genome (Towne strain, obtainedthrough the courtesy of M. Stinski, University of Iowa,Ames) was linearized and used as a template (10 copies peramplification reaction).

Mutagenesis. An 800-bp EcoRI DNA fragment bearingcoding sequence for the 25 aa located at the carboxyterminus of ppUL32 was extracted from a Xgtll clone andmoved into the EcoRI site of a phagemid vector (pAlter)supplied with the kit for altered-site in vitro mutagenesis(Promega, Madison, Wis.) and bearing an inactivated formof the ampicillin resistance gene. Single-stranded DNA waspurified from the resulting clone (pMB20), after infection ofthe JM109 host with a helper phage, and annealed to themutagenic oligonucleotide (Dlm) described above. In addi-tion to Dlm, we used a control oligonucleotide supplied bythe manufacturer and designed to restore the correct se-quence for ampicillin resistance in pAlter. T4 DNA poly-merase synthesis of the second strand was followed by tworounds of transformation in different strains of Escherichiacoli (BMH 71-18 mut S and JM109) and selection in ampi-cillin-containing medium, ensuring proper segregation ofmutant and wild-type plasmids. One clone with the correctmutagenized sequence (Fig. lb) was called pMB21 and wasused to purify the 78-bp EcoRI fragment used to produce theexpression construct described above.

Sequencing. All clones were sequenced to verify thecorrect sequence and frame of expected constructs. Stan-dard double-stranded DNA dideoxysequencing protocolswere followed by using a Sequenase kit (U.S. Biochemical,Cleveland, Ohio) according to the manufacturer's recom-mendations.

Screening of expressive recombinants and induction of FPs.Transformation of electrocompetent E. coli strains withplasmid constructs was performed with a Bio-Rad (Rich-mond, Calif.) electroporator in 0.2-cm cuvettes, accordingto instructions supplied by the manufacturer. Screeningwith DNA probes was performed by standard techniques(13) with nitrocellulose filters carrying colonies of trans-formed bacteria. Probes were prepared by using subgenomicfragments excised from either Xgtll (9) or pACYC184(15) genomic libraries; labeling was done by randomly prim-ing the incorporation of [32P]dCTP by the Klenow fragmentof E. coli DNA polymerase I (reagents from Promega).Sequencing reactions (see above) were carried out withpositive colonies to check in-frame insertions or correctmutagenesis (Fig. 1). Immunoscreening of expressive re-combinants was performed as described by Sambrook et al.(13). Briefly, transformed cells were grown on nitrocellulosefilters, layered on selective medium, and then transferredonto fresh agar plates containing isopropyl-13-D-thiogalacto-pyranoside (IPTG) and incubated at 37°C for 3 h. The filterswere submerged in lysis buffer and incubated overnight atroom temperature. The filters were then washed and treatedfor antibody reactions in the same manner as Western blot(immunoblot) filters.

Immunoblotting. The procedure for immunoblotting hasbeen described elsewhere (7). Briefly, induced bacteriallysates were resuspended in loading buffer and proteins wereseparated by polyacrylamide gel electrophoresis (PAGE) in9 to 12% gels. Sometimes lysates containing two or threedifferent FPs with distinct molecular masses were run on thesame gel. Separated polypeptides were electrotransferred tonitrocellulose, and the immune reaction was carried out. In

some experiments a Miniblotter chamber (Immunetics,Cambridge, Mass.) was used to screen several serum sam-ples on preparative blots. Purified whole IgG (Endobulin;ImmunoAG, Vienna, Austria) was routinely used at a 1:100dilution; a pool of high-level-IgM-positive sera was used at a1:100 dilution. Individual human serum samples were dilutedto 1:80 for both anti-HCMV IgG and anti-HCMV IgMdetection. Either an anti--y- or an anti-,u-chain peroxidaseconjugate was used as a second antibody.Human sera. Two groups of sera were used. The first

group of sera consisted of 17 serum samples positive forspecific IgG, eight of which had high and nine of which hadmedium to low enzyme-linked immunosorbent assay(ELISA) antibody titers. All serum titers were confirmed byimmunoblotting.The second group of sera consisted of 19 serum samples

with CMV-specific IgM, 10 of which had high and 9 of whichhad medium to low titers as determined by ELISA andconfirmed by immunoblotting.ELISA. The evaluation of anti-HCMV IgG was carried out

with a CMV test kit (M. A. Bioproducts, Walkersville, Md.).Plates were read on a microELISA automatic reader(Dynatech Products, Alexandria, Va.). To perform linearregression analysis and to standardize the test run, everyplate included three serum calibrators. For IgM determina-tion, an antibody-capture kit (CMV IgM ELA; Technoge-netics, Hamburg, Federal Republic of Germany) was used.Both kits were used and the results were interpreted assuggested by the manufacturers. In order to exclude thepossibility of false-positive IgM reactions due to the pres-ence of rheumatoid factor, all sera were tested for thepresence of rheumatoid factor by latex agglutination(Rheuma-Wellco test; Wellcome, Dartford, England) andonly rheumatoid factor-negative sera were included in thepresent study.

RESULTS

Five different constructs were produced in order to ex-press one copy of the 595-614 epitope (MB28); one (MB30),two (MB31), or three (MB32) copies of the 1024-1048epitope; or both epitopes fused together (MB33).

Reactivity of FPs with pooled human anti-HCMV IgG.When Western blot reactivities of comparable amounts ofrecombinant proteins (Fig. 2a) with pooled human anti-HCMV IgG were compared (Fig. 2b), we found that thestrongest reaction was given by MB33 and the next strongestwas given by MB28; both FPs were much more reactive thanMB30 to MB32. No relevant difference in reactivities amongthe FPs expressing one, two, or three copies of the carboxy-terminal epitope of ppUL32 was observed (Fig. 2b).

Reactivity of FPs with pooled human anti-HCMV IgM.When Western blot reactivities of recombinant proteins withpooled anti-HCMV IgM were compared (Fig. 3a), we ob-served results very similar to reactivity with IgG, except thatMB32 was significantly more reactive than MB31 and MB31was more reactive than MB30.

Screening of human sera for IgG and IgM reactivities withFPs. Seventeen human serum specimens which were CMVpositive as determined by ELISA were tested in parallel byimmunoblotting for their IgG reactivities with the productsof the five expressive clones. When FPs carrying multipleepitopes were tested with human serum specimens andcompared with FPs carrying the corresponding epitopes as asingle copy, we found that the double epitope expressed bypMB33 showed the strongest reactivity and was recognized

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HUMAN ANTIBODY REACTIVITY WITH HCMV FUSION ANTIGENS 361

b2 3 5 4 a. MW

(Kd)

116.297.4

66.2

45.0

2 3 5 4

FIG. 2. (a) Coomassie blue staining of a PAGE separation of induced bacterial lysates expressing the five different FPs and a phage lysatefrom a clone expressing one copy of the carboxy-terminal epitope (residues 1024 to 1048). FPs are indicated by arrowheads. The amount ofeach FP was approximately 600 ng as determined by comparison with a bovine serum albumin standard. (b) Same as in panel a (except thateach FP was 200 ng) after Western blotting and immunostaining with purified anti-CMV IgG. (c) Immunoblot of a preparative polyacrylamidegel containing a mixture of two FP lysates (A) and a mixture of three FP lysates (B). All lanes were incubated with CMV-positive human sera,except the first lane in panel B, which was reacted with a CMV-negative serum specimen. 1, MB30; 2, MB31; 3, MB32; 4, MB33; 5, MB28.

by a larger number of serum specimcMB33 was recognized by all sera te,nized by 11 of 17 serum specimens,nized by 10 of 17 serum samples. INspecimens positive (by ELISA) for Ctested similarly: while only 6 of 19with MB30 and nine reacted with Iserum specimens reacted with ti(MB33). Furthermore, 10 of 19 serwith MB31 and MB32. These results -

statistical analysis of data confirm(reactivity stronger than those of the c

b MVka

MW 116.2

kd 97.4

116.2-

97.4-

66.2 -

66.2 -

-ns than the other FPs: Qualitative reactivities of human sera with FPs. An exam-

sted, MB28 was recog- ple of the reactivities obtained with individual serum sam-

and MB30 was recog- ples is shown in Fig. 2c. We found that four serum speci-{ineteen human serum mens unreactive to MB28 (see lanes 4 and 5 in Fig. 2c for an'MV-specific IgM were example) did react strongly with MB33, while two serumserum samples reacted specimens unreactive to MB30 to MB32 reacted with MB28MB28, as many as 13 (data not shown). Interestingly, five serum specimens (e.g.,he combined epitope lanes 2, 4, and 5 of Fig. 2c) either reacted weakly with MB30um specimens reacted to MB32 and MB28 or reacted with only one of these FPs,are shown in Table 1. A but they showed reactivities to MB33 that were alwaysed that MB33 gave a higher than the sum of the reactivities to single-epitope FPs.)ther FPs (P < 0.0001). The reactivities to MB31 and MB32 were almost identical to

the reactivity to MB30. An example of IgM reactivity to FPsis shown in Fig. 3b. Here, too, some sera did not react withone of the single-epitope FPs but did react with the com-

v A B bined-epitope FP. In order to determine whether the absencei of reactivity to one of the FPs was due to a complete lack of

antibody or to poor sensitivity of our assay, we repeated thetesting of the sera which had reacted clearly with only one ofthe epitopes, this time with three times the amount of FP perserum sample (-150 ng). The results obtained are reportedin Table 2. For IgG, we found that four serum specimens ofseven that reacted with MB33 did not have any detectableantibody to MB28 and that two of seven did not have any

4 , - 3 antibody to MB30 but reacted strongly with MB28 and with2 MB33. In five cases the reactivity to MB33 was much

1 stronger than the sum of the reactivities to the single3_21 45.0 -

45.0 -

FIG. 3. (a) Western blot of a PAGE separation of induced lysatesidentical to that shown in Fig. 2b. (b) Immunoblot of a preparativepolyacrylamide gel containing a mixture of two FP lysates (MB33 [4]and MB28 [5]) and a mixture of three FP lysates (MB30; [1], MB31;[2], and MB32 [3]). For each lane the amount of each FP isapproximately 50 ng.

TABLE 1. Number of serum specimens giving IgG and/or IgMreactivity with different FPs

No. of serum specimens witha:

FP IgG reactivity (n = 17) IgM reactivity (n = 19)

- + .2+ - + .2+MB30 7 8 2 13 5 1MB28 6 10 1 10 3 6MB33 0 10 7 6 4 9MB31 7 10 0 9 5 5MB32 7 10 0 9 5 5

-, no reaction; +, positive reaction; >2+, very strong reaction.

aMW(Kd)

116.297.4

66.2

45.0

CA B

_ 4 1 Is 32

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362 RIPALTI ET AL.

TABLE 2. Western blot reactivity of selected sera with excess amounts of FPs

Reactivity of indicated sample witha:

FP IgG IgM

383 396 405 435 418 490 536 23 78 1038 1299 26 559 560

MB30 +++ ++ + + - - ± + - + ++++ - - -MB28 - - - - + ++ + - - - - + ++++ ++++MB33 +++++ +++ ++ ++ + +++ + ++ + ++ ++++ + ++++ ++++

a Reactivity was measured on a scale of - (no reactivity) to + + + + + (highest level of reactivity). Each sample was given a numerical code.

epitopes. For IgM, we found that four serum specimens ofseven did not show any reactivity to MB30. Three of themdid react with MB28 and MB33, while one reacted only withthe combined-epitope FP. Furthermore, three serum speci-mens that reacted with MB30 did not react with MB28 butgave a strong reactivity to MB33.

DISCUSSION

Analysis of the humoral immune response elicited duringnatural HCMV infection has repeatedly shown that ppUL32is highly immunogenic and is recognized by sera from nearly100% of HCMV-seropositive subjects tested (3, 5, 7, 11, 14).At least two epitopes have been identified in this moleculeand have been shown to react efficiently with human anti-bodies. In particular, the analysis of several ppUL32 FPsshowed that a region localized in the last 24 aa of the carboxyterminus of the molecule (aa 1024 to 1048) reacted with morethan 80% of IgM-positive sera tested (8). Another region waslocalized between aa 595 and 614 by peptide walking and wasshown to give a positive reaction with almost 100% ofIgG-positive sera tested (11).

In this work we compared the reactivities of two linearepitopes expressed as FPs with IgG and IgM present inhuman sera. As expected, when the two distinct epitopeswere expressed fused on a single polypeptide, we found thatreactivity was stronger and related to a higher number ofserum samples than that obtained when the two epitopeswere expressed in separate FPs. No similar increase inantibody (IgG) binding ability was observed when only oneepitope was repeated two or three times on the samepolypeptide. On the other hand, FP containing the sameepitope repeated three times had higher IgM reactivity thanthat obtained with the FP containing the epitope expressedonly once. This is probably because of the need for a longeror more complex region for stable IgM binding.We also found that while some sera show a strong re-

sponse to one epitope, they may completely lack reactivityto a different epitope carried on the same viral protein. Thissuggests that a single viral epitope, even if well establishedas a generally strong immunogen, can stimulate variableimmune reactions in different subjects. To us, this alsomeans that a well-balanced cocktail of different epitopes willbe necessary to replace virion preparations as antigenicmaterial for diagnostic use.

Furthermore, the reactivity of a given serum specimen tothe double epitope was often much higher than the sum ofthe observed reactivities of the same serum specimen to thesingle epitopes expressed on distinct FPs. This was also truefor some sera that did not show significant reactivity to one(or both) of the two epitopes. This observation may beexplained as a simple cumulative effect, but the formation ofa "bridge" epitope cannot be ruled out. This epitope would

result from the alignment of two linear epitopes that mightmimic a conformational epitope on the native protein.

It is tempting to speculate that a new generation ofrecombinant antigens for antibody detection in human seracould be obtained by constructing complex polyepitoperecombinant proteins. These multireactive proteins wouldbear distinct epitopes present on the same or differentantigenic polypeptides from a given microorganism. Theadvantages associated with such engineered antigenic mate-rial would be (i) the availability of both a quantitative and aqualitative standard antigen; (ii) the presence of all epitopesof interest on a single polyprotein, avoiding the need for aninternal standard to balance quantities of distinct recombi-nant antigens expressed separately; and (iii) the possibility ofmimicking a class of conformational epitopes generated onnative proteins by the simple alignment of distant amino acidsequences in space.To our knowledge, this is the first report of the production

of polyepitope fusion antigens. Our results encourage theconstruction of more complex antigens made of numerousepitopes from different viral proteins. Construction of apolyepitope FP carrying a third antigenic region in additionto the two described in this paper is under way. We havechosen an epitope normally expressed on a nonstructuralviral protein (the product of UL44) which is known to beamong the strongest viral immunogens.We predict that a single polyepitopic FP might eventually

substitute for whole-virus preparations as antigenic materialin the serology of HCMV infection.

ACKNOWLEDGMENTS

We thank M. La Placa for his constant support and T. Lazzarottofor supplying selected human sera.

This work was partially supported by the Italian CNR (Biotech-nology Project), the Italian Ministry of Public Health (ISS AIDSProjects), and the Italian Ministry of Education (40 and 60%).

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