INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Primary infection with human herpesvirus 7 (HHV-7) occurs in infancy and is occasionally associated with exanthem subitum or fever without rash (1, 6, 20, 22). More severe complications of primary HHV-7 infection include encephalitis and seizures due to invasion of the central nervous system (21). In healthy children and adults, the virus is excreted in saliva, which is the most likely route of transmission (2, 8, 12, 23). In the general population, HHV-7 seroprevalence reaches at least 80% (3, 6, 24). Until today, HHV-7 has generally been considered an orphan virus that is not usually pathogenic beyond the self-limiting childhood disease. However, more recently it has been found that HHV-7 infection or reactivation is associated with an increased risk of progression to cytomegalovirus (CMV) disease in renal transplant recipients positive for human CMV (HCMV) (15), with a reduced survival time, and with an acute graft-versus-host disease in bone marrow transplant recipients (7). Thus, HHV-7 alone or in combination with other -herpesviruses may be an important cofactor for the development of severe disease in immunosuppressed individuals.

A specific diagnosis of infection with HHV-7 is needed (i) for children presenting with complications of primary infection in order to distinguish rash caused by HHV-7 from rashes caused by human herpesvirus 6 (HHV-6), measles virus, and the virus that causes rubella or from an adverse reaction to antibiotic treatment (3); (ii) for immunocompromised adults, mainly transplant recipients, to assess the association between the virus and clinical manifestations and to monitor the effect of antiviral therapy; and (iii) for accurate seroprevalence studies. Serologic diagnosis of HHV-7 infection poses a major problem of specificity because HHV-7 shares the same overall genome organization with HHV-6, with homologies varying from 41 to 75% (11, 14, 17). Consequently, some polyclonal antibodies and monoclonal antibodies (MAbs) directed to one virus cross-react with the other virus. Cross-reacting HHV-7 and HHV-6 antibodies are also present in human sera. They can be removed by preabsorption with the heterologous HHV-6 antigens (4, 19). However, this is a troublesome procedure that is not readily reproducible and it is unavailable to the vast majority of diagnostic laboratories, because it requires routine growth of these viruses. In addition, preabsorption decreases the sensitivities of the assays.

In studies in which different assays were compared and in which the reactivity of human sera following preabsorption with heterologous HHV-6 antigen was analyzed, it was observed that immunoblotting is the most specific assay for detection of HHV-7 antibodies (4). Ninety percent of the sera reactive to HHV-7-infected cell lysates recognized a protein with apparent molecular mass of 89 kDa (this protein was estimated to be 85 kDa in a different laboratory; therefore, it is designated 85-89 kDa herein). Most importantly, reactivity with this protein was not affected by preabsorption with heterologous HHV-6 antigen (4, 10). These findings suggested that a protein of 85-89 kDa is a specific determinant and marker of HHV-7 infection (4, 10). It has not been ascertained whether the 85-89-kDa protein represents one or multiple peptides. Double bands were observed in some cases (4).

Two further findings are relevant to the present study. First, an HHV-7 85-kDa tegument phosphoprotein (pp85) has been shown to be encoded by the U14 gene (19). MAb 5E1 is directed to an HHV-7-specific epitope, which has not so far been mapped. Second, the immunodominant proteins p100 and pp150 of two other -herpesviruses, HHV-6 and HCMV, are encoded by homologous genes, U11 and UL37, respectively (13, 25). The homologous gene of HHV-7 (U11) encodes a protein of 755 residues, with a calculated molecular mass of 86 kDa (14). Thus, the mass and properties of the HHV-7 U14 and U11 gene products make both proteins likely candidates for the immunoreactive 85-89-kDa protein.

The objective of this study was to identify the HHV-7 gene(s) that encodes the 85-89-kDa immunodominant protein(s) of HHV-7 and to develop a prototypic diagnostic assay based on the recombinant protein(s). We report that the immunodominant protein in HHV-7 is the product of the U14 gene and that the recombinant pp85(U14) protein made in bacteria is a suitable reagent for a serologic immunoblotting assay.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cells and viruses. SupT1 cells (obtained from the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, Rockville, Md.) and Molt-3 cells were grown in RPMI 1640 medium (Gibco BRL, Life Technologies, Karlsruhe, Germany) supplemented with 10% fetal calf serum (Gibco). SupT1 cells were infected with HHV-7(AL) and Molt-3 cells were infected with HHV-6B(Z29) as described previously (9, 18). Infection (5) was monitored at different days postinfection by an indirect immunofluorescence assay (IFA) with MAb 5E1 to HHV-7 pp85 or with MAb 2D10 to HHV-6 glycoprotein B (gB). For this purpose, an aliquot of the culture was pelleted, and the cells were resuspended in a few microliters, deposited on a coverslip, and air dried. When approximately 60 to 70% of the cells in the culture were positive by IFA, the culture was harvested, washed with sterile phosphate-buffered saline (PBS), and used for IFA and immunoblotting assays.

Sera. Twenty human serum samples (8 cord blood serum samples and 12 adult serum samples) obtained from the Department of Obstetrics and Department of Pathology, University of Bologna, were screened by IFA, immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Sera from healthy adults were obtained with the consent of the personnel of the Department of Experimental Pathology, who were working in a separate section and building, on occasion of routine analyses. Infant serum samples 21 and 22 were the generous gifts of K. Yamanishi and P. Pellet, respectively.

IFA. HHV-6- and HHV-7-infected cells and uninfected cells were washed once with PBS and were acetone fixed for 10 min. Slides were incubated with human sera (diluted 1:40) for 30 min at 37°C. After two washes in PBS, infected cells were stained with fluorescein-isothiocyanate-conjugated anti-human immunoglobulin G (IgG), diluted 1:100 (Jackson ImmunoResearch laboratories, West Grove, Pa.) for 30 min at 37°C, and examined with a fluorescence microscope (Axioplan; Zeiss). MAb 2D10 and MAb 5E1 were routinely included as positive controls.

Immunoblotting assays with HHV-7-infected cell lysates. Lysates of HHV-7(AL)-infected cells and mock-infected cells were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to nitrocellulose sheets and were then incubated with human sera (diluted 1:40). After 3 h of incubation, the samples were washed with PBS and incubated with biotinylated anti-human IgG (Sigma, St. Louis, Mo.). A complex of avidin-biotinylated horseradish peroxidase (Vectastain ABC Kit, Vector Laboratories, Burlingame, Calif.) was added for 30 min. The reactivity was detected by the addition of diaminobenzidine and hydrogen peroxide as substrates. Reactivity with MAb 5E1 was routinely used as a positive control.

Production and purification of recombinant proteins. For bacterial expression of pp85(U14), two constructs were generated in the pTrHisB vector, which contains a six-residue His tag (Invitrogen Corporation, Carlsbad, Calif.). One construct contained the entire U14 open reading frame (ORF; 1,942 bp) encoding a protein of 648 amino acids (aa) and was designated pp85(U14)rec. The other contained the portion from positions +1309 to +1942 (663 bp) of the U14 ORF, corresponding to the C-terminal fragment of the U14 protein from positions 427 to 648, and was designated pp85(U14)rec. U14 sequences were amplified by PCR with the following primers sets: (i) a 5' full-length primer (TGAAC GCAGA CACAA TGGATC CG) coupled with a 3' primer (GTTGT GGTAC CATGA ATTAG C) for the pp85 (U14)rec protein and (ii) a 5' deletion primer (GAAAC TGAAT TGGAT CCTAC) together with the 3' primer (GTTGT GGTAC CATGA ATTAG C) for the deleted protein pp85(U14)rec. The 5' primers contained a BamHI restriction site, and the 3' primer contained a KpnI restriction site. After cloning, the DNA constructs were transfected into BL21 cells (protease deficient) for protein expression. Cells were grown in SOB medium (Invitrogen Corporation), and protein expression was induced by the addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG) for 2 h. The cells were harvested by centrifugation and were lysed in 8 M urea for 1 h. The recombinant proteins were purified by absorption to columns of Ni-NTA Superflow resin (Quiagen, Valencia, Calif.) and were eluted with a low-pH buffer (8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris-HCl [pH 4.5]). Fractions were analyzed by their reactivity with MAb 5E1 by denaturing PAGE with SDS.

Reactivity of human sera to recombinant pp85(U14) and p86(U11) proteins and synthetic peptides. For the immunoblotting assay, purified recombinant proteins were separated by SDS-PAGE (1 µg of protein per lane) and were transferred to nitrocellulose sheets. Nitrocellulose strips were incubated with the human sera (diluted 1:40), followed by incubation with biotinylated anti-human IgG (Sigma). Positive bands were visualized with the avidin-biotin amplification system (Vectastain). Reactivities with MAb 5E1 and MAb anti-GST were routinely used as positive controls for the pp85 and p86 recombinant proteins, respectively.

Mapping of the pp85 epitope reacting with HHV-7-specific MAb 5E1. In order to preliminarily map the pp85 epitope recognized by MAb 5E1, we constructed four plasmids carrying different 3'-terminus deletions. Deletions were generated by the following double restriction enzyme digestions of clone pBluescript 2C containing the entire U14 cDNA (21): (i) NcoI (which cuts at position 1755 of the gene) plus XhoI (which cuts at the stop codon), (ii) HincII (which cuts at position 1245) plus XhoI, and (iii) ClaI (which cuts at position 420) plus XhoI. Moreover, clone pBluescript 8A carrying a deletion of 153 bp at the 5' region of the gene (21) was digested with XhoI and EcoRI (which cuts at position 1407). The deleted DNAs were religated and transfected into BL-21 cells for expression of the truncated proteins. Recombinant deleted proteins were separated by SDS-PAGE, transferred to nitrocellulose sheets, and tested for reactivity with MAb 5E1. For the fine mapping of the HHV-7-specific epitope, 10 overlapping synthetic peptides (Biopolymer Core Facility, University of Maryland at Baltimore, Department of Microbiology and Immunology, Baltimore, Md.) spanning the region from 470 to 586 aa residues of pp85 (U14) (see Fig. 3) were serially diluted (from 5 µg/ml to 10 ng/ml) in carbonate-bicarbonate buffer (pH 9.4) in 96-well plates. Reactivity with increasing dilutions of MAb 5E1 (from 1:1,000 to 1:10,000) was detected by reaction with peroxidase-conjugated goat anti-mouse IgG (diluted 1:5,000; Dako, Glostrup, Denmark) and with o-phenylenediamine (Sigma) and hydrogen peroxide as substrates. The optical density was read at 490 nm in a Bio-Rad reader.

Reactivity of human sera with synthetic peptides. Microplates were coated at 4°C overnight with synthetic peptides diluted in carbonate-bicarbonate buffer (pH 9.4) at a final concentration of 5 µg/ml. The wells were washed three times with 0.05% Tween 20 in PBS. Aspecific binding sites were blocked with 5% nonfat dry milk-0.1% Tween 20 in PBS for 1 h at 37°C. Human sera were serially diluted (from 1:20 to 1:1,280) in the blocking buffer and were incubated for 2 h at 37°C in the coated plates. Positive binding was detected by reaction with peroxidase-conjugated goat anti-human IgG (diluted 1:5,000; Dako) and with o-phenylenediamine (Sigma) and hydrogen peroxide as substrates.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of pp85(U14) and p86(U11) recombinant proteins. In order to identify the genes encoding the 85-89-kDa immunodominant protein(s) of HHV-7, the pp85 and the p86 proteins encoded by the U14 and U11 ORFs, respectively, were selected for expression in recombinant form. For pp85(U14), two constructs were generated in the pTrHisB vector (Invitrogen Corporation) in fusion with a six-residue His tag. One construct, pp85(U14)rec, contained the entire U14 ORF (1942 bp). The other, pp85(U14)rec, contained the 3' terminal 663 bp (221 aa) corresponding to the divergent portion between the HHV-7 and HHV-6 U14 proteins (19). For p86(U11), two constructs which contained residues 277 to 496 and 490 to 753 (p86rec-1 and p86rec-2, respectively) cloned in the pGEX-3X vector, were generated as GST fusion proteins. pp85(U14) and p86(U11) recombinant proteins were produced in bacteria and were purified by Ni-NTA (for pp85) or GST (for p86) affinity chromatography.

Comparative reactivities of human sera to pp85(U14) and p86(U11) recombinant proteins. Twenty-two human serum samples (8 cord blood, 2 infant, and 12 adult serum samples) to be assayed for reactivity with the recombinant proteins were tested for reactivity with HHV-7-infected cells by IFA and immunoblotting. On the basis of the pattern of immunoblotting reactivity to HHV-7-infected cell lysates, they were divided into groups. Group A included 13 serum samples reactive with the 85-89-kDa protein(s). Group B included seven serum samples with no or very weak reactivity with the 85-89-kDa protein(s) but with reactivity with other HHV-7 proteins. Group A and B sera were positive for HHV-7 by IFA, with only two of them having very weak reactivity. Their pattern of reactivity with HHV-6B by IFA was variable and is reported in Table 1. Group C, representing the negative controls, included two serum samples from infants that were IFA and immunoblotting negative for HHV-7 and IFA positive for HHV-6B.

View larger version (82K): [in this window] [in a new window]   FIG. 1.   Immunoreactivity of human sera with recombinant proteins pp85(U14)rec, pp85(U14)rec, p86(U11)rec-1, and p86(U11)rec-2. The recombinant proteins were expressed in bacteria and were purified by affinity chromatography. The purified proteins were separated by denaturing PAGE, transferred to a nitrocellulose sheet, and then allowed to react with human sera (lanes are numbered according to the serum sample numbers in Table 1). Staining with MAb 5E1 and MAb anti-GST was carried out as a positive control for the pp85 and the p86 recombinant proteins, respectively. Reaction with the secondary antibody alone (lane II°) was carried out as a negative control. Numbers to the left of each gel are in kilodaltons.

Mapping of the pp85 epitope recognized by HHV-7-specific MAb 5E1. MAb 5E1 reacts specifically with HHV-7-infected cells by IFA and immunoblotting but fails to react with HHV-6B-infected cells (10, 19), and its reactivity is directed to pp85(U14) (19). In order to preliminarily map the region containing the HHV-7-specific epitope recognized by MAb 5E1, we constructed a series of plasmids carrying a deletion of 153 bp at the 5' region and deletions of different length at the 3' region of the U14 gene (as shown in Fig. 2). The proteins encoded by these deletion constructs were tested for their immunoblotting reactivities with MAb 5E1. Deletion of the N-terminal 51 aa residues (clone 8A) or deletion of the C-terminal 63 aa residues (clone C) did not abolish the reactivity with MAb 5E1, while deletion of the C-terminal 116 aa residues (clone G) abolished the reactivity (Fig. 2). These results allowed a preliminary mapping of the MAb 5E1-reactive epitope to the protein region of 116 aa encompassing residues 469 to 585. 

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Mapping of the region of pp85(U14) containing the epitope recognized by HHV-7-specific MAb 5E1. The inserts of the pBluescript constructs containing either the full-length U14 ORF or deletions of the U14 ORF are depicted schematically. Plasmid 2C contains the full-length U14 ORF; plasmids C, 4, and 6A carry progressive deletions at the 3' region of U14 ORF; plasmid 8A contains a deletion at the 5' region; plasmid G contains deletions at both the 5' and the 3' regions. The truncated proteins were expressed in bacteria and were tested for their reactivities with MAb 5E1 by immunoblotting (+ and  indicate the presence or lack of reactivity, respectively). The aa coordinates of the region containing the epitope are shown.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Mapping of the pp85 epitope recognized by HHV-7-specific MAb 5E1. Ten overlapping peptides spanning the region from 470 to 586 aa residues of the pp85 protein were synthesized and were tested for their reactivities with MAb 5E1 by ELISA. The coordinates of the peptides and the results of MAb 5E1 reactivity testing are reported in the inset.

Reactivity of human sera with the peptide carrying the HHV-7-specific epitope. Having ascertained that pp85(U14) is a major target of the human immune response to HHV-7, it was of interest to determine whether the reactivity of human sera was directed to the HHV-7-specific epitope recognized by MAb 5E1 (sequence of peptide 10 [Fig. 3]). Peptide 10 was used to coat microtiter wells and was then incubated with increasing dilutions of 11 group A serum samples. The results did not reveal a strong reactivity (data not shown), ruling out the possibility that the peptide carrying the HHV-7-specific epitope can be used to develop an HHV-7 specific ELISA.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The study described in this paper shows several things, as described below.

(i) The immunodominant protein in HHV-7 is the product of the U14 gene. Twelve of 13 serum samples (92%) that reacted by immunoblotting with the 85-89-kDa protein(s) in infected cells reacted with the pp85(U14) recombinant protein, whereas only 8 (61%) reacted with p86(U11)rec-1 and p86(U11)rec-2. As all except one of the serum samples that reacted with the pp85(U14) protein also reacted with the p86(U11) protein, the inclusion of p86(U11) in a recombinant pp85(U14)-based diagnostic assay appears to increase the sensitivity of the assay only to a small extent.

(ii) Recombinant diagnostic reagents that detect HHV-7 and that consist of proteins made in bacteria can be developed. The lack of posttranslational modifications of recombinant pp85(U14) and p86(U11) proteins does not adversely affect their reactivities with human sera. Recombinant reagents, especially those made in bacteria, have the obvious advantages that they can be produced and purified easily and can readily be standardized.

(iii) The reactivity with the recombinant pp85(U14) protein by immunoblotting had the same or even higher sensitivity than the reactivity to the 85-89-kDa protein(s) in infected cells by immunoblotting. Evidence for this rests on the observation that some of the group B sera, which were positive for HHV-7-infected cell proteins other than the 85-kDa species, reacted with pp85(U14)rec. The higher sensitivity of the recombinant protein-based assay most likely reflects the greater availability of the recombinant protein. This is not surprising since HHV-7 grows poorly in cell cultures, and lysates with a large and reproducible contents of viral proteins are not readily obtainable. The adaptation of HHV-7 to SupT1 cells (18) has greatly improved the ability to grow the virus in the laboratory; however, this remains limited to a few research laboratories and is not available to the vast majority of viral diagnostic laboratories.

(iv) U14 is present in both HHV-7 and HHV-6 genomes (11, 14). About two-thirds of the molecules at the N terminus display an identity of 59.5%. It is therefore surprising that the pp85(U14) recombinant protein may constitute an HHV-7-specific diagnostic reagent. The reason for this specificity rests on the fact that in patients with HHV-7 infection the immune response is directed predominantly to the U14 protein, while in patients with HHV-6 infection the response is directed predominantly to the U11 protein (13, 25). Therefore, antibodies to pp85(U14) are predominantly formed in response to HHV-7 infection. Given that HHV-7 and HHV-6 have overlapping genomes and that the immune response is predominantly directed toward the U14 and U11 gene products, it follows that, for intrinsic properties, diagnostic reagents with higher specificities and sensitivities than those of pp85(U14)rec probably cannot be derived.

(v) Finally, we have identified the HHV-7-specific epitope of pp85(U14) recognized by MAb 5E1. As might be expected from comparative analyses of HHV-6 and HHV-7 U14 sequences (19), the epitope is located in the C-terminal portion of the protein. This epitope represents a potential HHV-7-monospecific reagent. However, human sera displayed an overall low level of reactivity with a truncated form of pp85(U14) carrying the C-terminal portion of the molecule and, consistently, with the peptide carrying the HHV-7-specific epitope. This ruled out the practical use of this peptide as the basis for an HHV-7-monospecific ELISA.