Antigenic relationships among human herpesvirus-6 isolates

Journal of Medical Virology 37247-254 (1992)

Antigenic Relationships Among Human Herpesvirus-6 Isolates Bala Chandran, Suranan Tirawatnapong, Brian Pfeiffer, and Dharam V. Ablashi Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center (B.C., S.T., B.P.), Kansas City, Kansas; Laboratory of Cellular and Molecular Biology, National Cancer Institute (D.V.A.), Bethesda, Maryland

Human herpesvirus 6 (HHV-6) prototype isolate G S is a newly identified lymphotropic herpesvirus and several subsequent herpes isolates were recoanized as HHV-6 bv their hvbridization to a

KEY WORDS: antigenic variations, reactivity of human sera, HHV-6

HHVr6(GS) DNA probe pZVH14.’DNA restriction analysis and in vitro tropism studies show that HHV-6 isolates can be divided into two groups, designated group A and group B. Antigenic relationships among 15 HHV-6 isolates belonging to these two groups were examined using rabbit antibodies against HHV-6(GS) infected cells, 11 monoclonal antibodies against three glycoproteins and four non-glycoproteins of HHV-G(GS), and sera from 136 healthy adults. More than 20 polypeptides from all these isolates were immunoprecipitated by rabbit polyclonal antibodies against HHV-G(GS) infected cells. Reactivities of monoclonal antibodies segregated these isolates into the same two groups. Group A contains HHV-G(GS), HHV-G(U1102) from a Ugandan acquired immunodeficiency syndrome (AIDS) patient, and nine other HHV-6 isolates from various disorders. HHV-6(Z-29) from a Zairian AIDS patient, HHV-G(SF) isolated from the saliva of a human immunodeficiency virus (HIVI-infected individual, HHV-G(OK) from a child with exanthem subitum, and HHV-G(DC) from a leukopenia patient are in group B. Eighty-one percent of the sera showed similar antibody titer in immunofluorescence assay with group A HHV-G(GS) and group B HHV-6(Z-29) infected cells and 19% of the sera showed two- to four-fold antibody titer differences. The mobilities of many of the polypeptides irnmunoprecipitated from group A HHV-G(GS) and group 6 HHV-6(Z-29) infected cells were different and sera showed differences in the quantities and nature of polypeptides immunoprecipitated. Together, our data show that HHV-6 related isolates segregate into two antigenically closely related yet distinct groups, ex- hibiting group common and group specific antigenic epitopes. The complex patterns of reactivities of human sera suggest that individuals may be infected with viruses from one group or both groups. o 1992 Wiley-Liss, Inc.

0 1992 WILEY-LISS, INC.

INTRODUCTION Human herpesvirus 6 (HHV-6) is a lymphotropic

herpesvirus, first isolated from the peripheral blood lymphocytes (PBL) of patients with lymphoproliferative disorders and acquired immunodeficiency syndrome (AIDS) [Salahuddin et al., 19861. Immunological and molecular analyses have demonstrated that the new herpesvirus is distinct from other human herpesviruses [Ablashi et al., 1988a; Josephs et al., 1988; Salahuddin et al., 19861. The original prototype isolate HHV-G(GS) replicated in phytohemagglutinin (PHA) stimulated human umbilical cord blood lymphocytes (CBL), adult PBL, and spleen and bone marrow mono- nuclear leukocytes. Since the infected cells were human B cells, the new virus was initially designated human B-lymphotropic virus (HBLV) [Salahuddin et al., 19861. Later it was shown to infect human T cells and has now been designated HHV-6 [Ablashi et al., 1988a,b; Lusso et al., 19881. Herpesviruses were subse- quently isolated from PBL of AIDS patients [Agut et al., 1988; Downing et al., 1987; Levy et al., 1990b; Lopez et al., 1988; Tedder et al., 19871 and children with exanthem subitum [Kikuta et al., 1989; Suga et al., 1989; Takahashi et al., 1989; Yamanishi et al., 19881. All these isolates were identified as HHV-6 by their hybridization to a 8.7 kilobase (kb) DNA probe (pZVH14) from HHV-G(GS). HHV-6 related viruses have been also isolated from PBL of healthy adults [Ablashi et al., 19911, renal and liver transplant patients [Asano et al., 1989; Ward et al., 1989; Wrzos et al., 19901, fatal fulminant hepatitis [Asano et al., 19901, and from the saliva of healthy individuals and HIV

Accepted for publication January 23, 1992. Address reprint requests to Bala Chandran, Department of Mi-

crobiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, KS 66103.

Suranan Tirawatnapong’s current address is the Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

248 Chandran et al.

seropositive individuals [Fox et al., 1990; Harnett et al., 1990; Levy et al., 1990a,bl. HHV-6 DNA and antigens have been detected in the salivary glands and bronchial glands from healthy individuals and patients with various disorders [Krueger. et al., 19901

HHV-6 isolates are tropic for CD4+ human T cells and T-lymphocyte derived cell lines and studies from several laboratories show differences in the in vitro tropism of HHVB isolates [Ablashi et al., 1988a, 1991; Black et al., 1989; Downing et al., 1987; Levy et al., 1990b; Lopez et al., 1988; Takahashi e t al., 1989; Wyatt et al., 19901. HHV-G(GS) infected phenotypically imma- ture CD4+T cells [Ablashi et al., 1988a,b; Lusso et al., 19881 and HHVB related isolates from PBL of children with exanthem subitum infected CD4+ CD8- T cells of mature phenotype. HHV-6(Z-29) isolated from a Zair- ian AIDS patient infected T lymphocytes of CBL and PBL and require prior mitogenic stimulation [Black et al., 1989; Wyatt et al., 19901. This isolate did not show efficient replication in several T-cell lines tested [Black et al., 1989; Lopez et al., 1988; Wyatt et al., 19901. In contrast, HHV-6 (U1102) from Ugandan AIDS patient infected unstimulated PBL as well as T-cell lines, HSB-2 and J-JHAN [Wyatt et al., 19901. HHV-G(SF) isolated from the saliva of an HIV-infected individual replicated substantially better in adult PBL than in CBL [Levy et al., 1990bI. HHV-6 isolates also exhibit DNA restriction site polymorphism [Ablashi et al., 1991; Aubin et al., 1991; Chang and Balachan- dran, 1991; Jarrett et al., 1989; Kikuta et al., 1989; Levy et a]., 1990b; Schirmer et al., 19911 and DNA restriction studies show that HHV-6 isolates could be separated into two groups [Ablashi e t al., 1991; Aubin et al., 1991; Schirmer et al., 19911. Group A contains HHV-G(GS) and HHV-6(U1102) and group B contains HHV-6(Z-29) and HHV-6 isolates from exanthem subitum. Antigenic differences between these HHV-6 isolates have been detected in an immunofluorescence assay using a limited number of our monoclonal antibodies (MAbs) against HHV-G(GS) [Ablashi et al., 1991; Schirmer et al., 19911. In this study, we used rabbit antibodies against HHV-G(GS) infected cells, a panel of 11 MAbs against three glycoproteins and four non-glycoproteins of HHV-G(GS), as well as sera from healthy adults to examine the antigenic relationships among 15 different isolates in the two groups of HHV-6. Our studies suggest that these two groups of HHV-6 are an antigenically closely related, yet distinct group of viruses and exhibit group common and group specific epitopes.

MATERIALS AND METHODS Cells

Suspension cultures of human T-cell lines, HSB-2 (ATCC CCL 120.1. CCRF-HSB-2) and M o b 3 (ATCC CRL 1552), and PHA stimulated CBL and adult PBL were used for virus propagation. Cells were grown in RPMI 1640 medium (Sigma, St. Louis, MO) supple- mented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics.

Viruses Fifteen HHV-6 isolates were used in this study.

HHV-G(GS) [Salahuddin et al., 19861 was a gift from Dr. R.C. Gallo, National Cancer Institute, Washington, D.C. HHV-6(Z-29) [Lopez et al., 19881 was a gift from Dr. P. Pellett, Center for Disease Control, Atlanta, GA. HHV-6(U1102) [Downing et al., 19871 was a gift from the late Dr. R.W. Honess, National Institute for Medi- cal Research, Mill Hill, London, England. HHV-G(SF) isolated from the saliva of an HIV-infected individual [Levy et al., 1990bl was a gift from Dr. J. Levy, San Francisco. HHV-G(DA) was isolated from a patient with chronic fatigue syndrome [Ablashi et al., 1988a1. HHV- 6(OK) from a child with exanthem subitum was a gift from Dr. H. Kikuta, Haikido University, Saporro, Ja- pan [Kikuta et al., 19891. HHV-G(DE) from an AIDS patient was a gift from Dr. M. Kaplan, Northshore Uni- versity Hospital, Long Island, NY. HHV-G(DC) was isolated from a leukopenic patient and was a gift from Dr. R. Carrigan, University of Wisconsin, Milwaukee, WI. HHV-6 isolates Col to C08 were from the laboratory of Dr. G.R. Krueger, University of Cologne, West Ger- many and were isolated from PBL of patients with atypical lymphoproliferation with rheumatoid arthritis (Col), unclassified collagen vascular disease ( C O ~ ) , sys- temic lupus erythematosus (co3, co4, and C O ~ ) , chronic fatigue syndrome ( C O ~ ) , rheumatoid arthritis ( C O ~ ) , and from PBL of a healthy adult (co5) [Krueger et al., 19911.

Infection and Assay Procedures HSB-2 cells were used for the routine propagation of

HHV-6 isolates GS, DE, DA, U1102, and Col to C08. For some experiments, these isolates were also grown in PHA stimulated CBL and adult PBL. HHV-6 isolates 2-29, OK, and DC were propagated in PHA stimulated PBL and CBL. HHV-6(Z-29) was also grown in M o b 3 cells. PHA was used at a concentration of 10 pg/ml two days prior to infection and 5 pg/ml after infection IWyatt et al., 19901. Infection was carried out by mixing lo6 uninfected cells with infected cells a t a ratio of 5:1 [Balachandran et al., 1989, 19911. Growth of HHV-6 was monitored by observing cytopathic effect (CPE) and by testing viral antigens by indirect immunofluorescence assay (IFA) as described below. Infected cells were suspended in medium containing 20% FBS and 5% dimethylsulfoxide and stored in liquid nitrogen.

Antibodies The production and characterization of polyclonal

rabbit antibodies (R anti-HHV-6) and MAbs against HHV-G(GS) infected cells have been described before [Balachandran et al., 19891. High-titer ascitic fluids of MAbs were raised by injecting hybridoma clones in- traperitoneally into pristane-primed BALB/c mice. The characterization of these MAbs has been described else- where [Balachandran., 1991; Balachandran et al., 1989,1991; Chang and Balachandran, 19911. The reactivities of MAbs with HHV-G(GS) infected cells were

Antigenic Relationships Among HHV-6 Isolates

tested by IFA with acetone fixed cells, IFA with unfixed live cells, immune electron microscopy (IEM) with infected cells using goat anti-mouse antibodies conjugated with gold particles, Western blot with infected cell extracts, and radioimmunoprecipitation of cells labeled with [35Sl methionine, 13H] glucosamine, and [12511. MAbs were tested with acetone fixed cells infected with different HHV-6 isolates in IFA. Human sera from healthy adults (ages 18-50) collected from the Kansas City area were inactivated a t 56°C for 30 minutes before use.

IFA MAbs and human sera were tested for their reactivi-

ties with acetone fixed uninfected and HHV-6 infected cells by IFA. Cells were collected, washed in phosphate buffered saline (PBS, pH 7.4), air dried on slides (5 mm inner diameter, 10 circles per slide. Roboz Surgical In- strument Co., Washington, D.C.), and fixed in cold acetone for 10 minutes. Fixed cells were incubated for 30 minutes at 37°C with two-fold dilutions of MAbs beginning a t 1:lO. After incubation, slides were washed with PBS and incubated for 30 minutes at 37°C with pre- standardized dilution of fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG (HyClone Labo- ratories, Logan, UT). After washing, slides were mounted with 50% (vol/vol) glycerol in PBS. Human sera were tested for their reactivity by incubating two- fold dilutions of sera beginning a t 1:lO. After reacting with FITC labeled goat anti-human IgG (HyClone), slides were counterstained with 1:20,000 dilution of Evansblue (Sigma) for 5 minutes at room temperature before mounting. Slides were examined under an Olympus fluorescence microscope.

Radiolabeling, Radioimmunoprecipitation, and Sodium Dodecyl Sulfate-Polyacrylamide

Gel Electrophoresis Uninfected and HHV-6 infected cells (day 6-12

postinfection) were washed once with PBS and lo7 cells were labeled for 20 hours with 250 &i of [35S] methionine (specific activity, 1,072 Ci/mmol; NEN DuPont, Wilmington, DE). Radioimmunoprecipitation (RIP) was carried out essentially as described previously [Balachandran et al., 1989,1991; Chang and Balachan- dran, 19911. Briefly, cells were solubilized with lysing buffer (0.05 M Tris hydrochloride, 0.15 M NaC1, 0,1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1% Triton X-100, 100 U of aprotinidml, 0.1 mM phe- nylmethylsulfonyl fluoride), sonicated, and centrifuged a t 100,OOOg for 1 hour. Equal amounts of trichloroacetic acid precipitable radioactivity (5 x lo5 cpm) from control and virus-infected cell lysates were mixed with 10 pl of antibodies and 100 ~1 of Protein-A-agarose beads (Genzyme Corp., Boston, MA) and were kept rocking at 4°C for 2 hours. The precipitates were washed, dissoci- ated by boiling in sample buffer, and analyzed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE) in 9% acrylamide cross-linked with 0.28% N, N'-diallyltar- tardiamide (DATD). Molecular weight markers

249

Fig. 1. SDS-PAGE analysis of polypeptides from cells infected with HHV-6 isolates immunoprecipitated by rabbit polyclonal antibodies against HHV-G(GS). Cells were labeled with [%I methionine. Lane 1: uninfected HSB-2 cells (C). Lanes 2-10: different HHV-6 isolates as indicated at the bottom of the Figure. Isolates HHV-G(GS), HHV- 6(DA), HHV-6(U1102), HHV-G(Col), HHV-G(Co21, HHV-G(Co5), and HHV-fXCo6) were grown in HSB-2 cells. HHV-6(Z-29) was grown in Molt-3 cells and HHV-G(DC) was grown in CBL. Rabbit anti-HHV- 6(GS) antibodies were absorbed with uninfected cells before use. Im- munoprecipitated samples were analyzed on 9% acrylamide cross- linked with N, N'-DATD and standard molecular weight markers were included in parallel lanes. Numbers indicate approximate molecular weights (kDa) of the prominent polypeptides identified as specific for HHV-6 infected cells.

(Sigma) were electrophoresed in parallel channels. Gels were stained, destained, infused with 2,5-diphenylox- azole, dried on filter paper, and placed in contact with XAR-5 film at -70°C for fluorography.

RESULTS

Reactivities of Rabbit Antibodies Against HHV-G(GS) With Groups A and B HHV-6 Isolates

To determine the antigenic relationships between the two groups of HHV-6 isolates, cells infected with different isolates were labeled with [35S] methionine and the lysates were immunoprecipitated with polyclonal rabbit antibodies against HHV-G(GS) infected cells. The results with HHV-6 isolates GS, 2-29, DA, U1102, DC, Col, C02, co5, and C06 are shown in Figure 1. No reactivity was seen with pre-immune serum and immune serum showed very minimal cross- reactivity with cytomegalovirus (CMV)(AD169) infected cells (data not shown). To remove the reactivities against host cell polypeptides, rabbit antiserum was absorbed with uninfected cells [Balachandran et al., 19891. However, a prominent polypeptide of about 38k as well as less intense polypeptide bands of about 80k and 66k were detected from uninfected HSB-2 cells and from infected cells (Fig.1, lanes 2-10), suggesting that the absorption was not complete. Nevertheless, this did not interfere in the reactivity of the rabbit antibodies

250 Chandran et al.

TABLE I. Immunofluorescence Reactivities of MAbs With Various HHVB Isolates*

DC and

MAb reactinn Drotein GS DE U1102 CO1-CO8 DA 2-29 OK SF

HHV-6 isolates

9A5 D12 + + + + + + + + 12B3 G4 + + + + + + + + 4A6 P180 + + + + + - - - 4B2 P70 + + + + f - - - 7A2 gp102 + + + + + + + + 6A5gp116/gp64/gp54 + + + + + + + + 2D6 gp82-gp105 + + + + f - - - 2D4 gp82-gp105 + + + + f - - - 13D6 gp82-gp105 + + + + + - - - 3B5 gp82-gp105 + + + + + - - - 2G3 gp82-gp105 + + + + + - - - *MAbs (ascitic fluids) against HHV-6(GS) [Balachandran, 1991; Balachandran et al., 1989, 1991; Chang and Balachandran, 19911 were tested a t dilutions of 1 : lO to 1:2,560 with actone fixed cells infected with different HHV-6 isolates. + = reactivity; - = non-reactivity a t 1 : l O dilution.

with polypeptides specific for cells infected with different HHV-6 isolates (Fig.1, lanes 2-10).

More than 20 polypeptides specific for cells infected by different HHV-6 isolates were immunoprecipitated by the rabbit antibodies against HHV-6(GS). Promi- nent polypeptides with approximate molecular weights of 135k, 116k, 105k, 82k, 78k, 74k, 64k, 58k, 54k, 41k, 33k, 31k, and less intense polypeptides of about 225k and 185k were immunoprecipitated from cells infected with HHV-6 isolates GS, DA, U1102, Col, C02, co5, and C06. The polypeptides with the approximate molecular weights of 135k, 78k, 74k, 58k, 41k, 33k, and 31k were also immunoprecipitated from cells infected with HHV-6(Z-29) (Fig.1, lane 31, HHV-G(DC) (Fig.1, lane 6), and HHV-G(OK and SF) (data not shown). In addi- tion, polypeptides of about 110k, 102k, 92k and 52k were predominantly seen in cells infected with group B HHV-6 isolates (Fig.1, lanes 3 and 6). Similar autorad- iographic profiles were also seen in CBL and/or PBL infected with all these isolates (data not shown), indicating that the observed differences in the mobilities of polypeptides were not due to cell type differences. Im- munoprecipitation of several polypeptides from different HHV-6 isolates by rabbit antibodies against HHV- 6(GS) clearly demonstrates the presence of shared antigenic epitopes and suggests that HHV-6 isolates examined here are antigenically closely related to the prototype HHV-G(GS).

HHV-G(GS) MAbs Detect Antigenic Differences Between the Two Groups of HHV-6

Isolates in IFA We have previously detected antigenic differences

between these HHV-6 isolates in an immunofluorescence assay using a limited number of our MAbs against HHV-G(GS) [Ablashi et al., 1991; Schirmer et al., 1991; Wyatt et al., 19901. To determine further the antigenic relationships between the two groups of HHV-6 isolates, in this study we used 11 independently derived MAbs against four non-glycoproteins and three

glycoproteins of HHV-G(GS) (Table I). The characterization of these MAbs has been described previously [Balachandran, 1991; Balachandran et al., 1989, 1991; Chang and Balachandran, 19911. HHV-6 isolates infected cells were collected a t different times postinfection, fixed with acetone, and were tested with MAbs in IFA. The results are shown in Table I. MAbs against HHV-G(GS) non-glycoproteins P41 and P135 and against glycoproteins gp116igp64/gp54 and gp102 reacted with all 16 isolates tested, and the titers of these four MAbs were identical with all these isolates (data not shown). MAbs 4A6 and 4B2 against non-glycoproteins P180 and P72 and five MAbs against gp82-gp105 reacted only with group A HHV-6 isolates GS, DE, U1102, Col to COB, and DA, and not with group B HHV-6 isolates 2-29, SF, OK, and DC even at a dilution of 1:lO. Similar reactive patterns were also seen with viruses grown in PHA stimulated PBL and CBL (data not shown). The reactivities of MAbs segregated these isolates into the same two groups as grouped by their growth properties and DNA restriction analysis [Ablashi et al., 1991; Schirmer et al., 19911. These data further demonstrate the differences in the antigenic make up of these two groups of HHV-6 isolates and suggest that HHV-6 isolates possess group common and group specific antigenic epitopes.

Human Sera Detect Antigenic Differences Between the Two Groups of H H V S Isolates

To examine whether human sera could detect antigenic differences among HHV-6 isolates, we used 136 sera from healthy adults (ages 18-50) residing in the Kansas City area. We choose HHV-G(GS) and HHV- 6(DA) as representative examples for group A and HHV-6(Z-29) for group B. Sera were tested in IFA with acetone fixed infected cells. The IF IgG antibody titer of sera ranged from 1:lO to 1:1,280 and the geometric mean titers (GMT) were 128 for GS, 120 for DA, and 110 for 2-29. Majority of the sera (110/136,81%) showed similar antibody titer with all three HHV-6 isolates.

Antigenic Relationships Among HHV-6 Isolates 251

Fig. 2. SDS-PAGE analysis of c3%1 methionine labeled HHV-6 isolates GS, DA, and 2-29 infected cell extracts immunoprecipitated by sera from healthy adults. Isolates HHV-G(GS) and HHV-G(DA) were grown in HSB-2 cells. HHV-6(Z-29) was grown in Molt-3. Lanes 1,2: reactivities of serum no. 2 with uninfected HSB-2 and Molt-3 cells. Lanes 3-5: reactivities of serum no. 2 with HHV-6 isolates GS (lane 3), DA (lane 4), and 2-29 (lane 5). Lanes 6-8: reactivities of serum no. 16 with HHV-6 isolates GS (lane 61, DA (lane 71, and 2-29 (lane 8). Numbers indicate approximate molecular weights (kDa) of prominent HHV-6 polypeptides immunoprecipitated. IF titer indicates HHV-6 antibody (IgG) titers of sera as measured in an immunofluorescence assay with acetone fixed cells infected with the three isolates.

However, 19% of the sera (26/136) showed differences in their reactivities and two- to four-fold higher IF titers against one of the isolates were observed. These differences could not be related to age or sex. To identify the reacting HHV-6 polypeptides and to determine the antigenic relationships among these isolates, human sera were tested in immunoprecipitation reactions with [35S] methionine labeled uninfected and HHV-6 isolates infected cells. Single batch labeled antigens were used in these reactions and lysates with equal amounts of trichloroacetic acid precipitable radioactive counts (5 x lo5 cpm) were used. Equal counts of immunoprecipitated samples were analyzed and the autoradiographs were developed at 5,10, and 15 days. Results of 10-day exposures are shown here.

Sera did not show any reactivities with uninfected cells (Fig.2, lanes 1 and 2). Complex patterns of reactivities with infected cells were observed and the representative examples are shown in Figures 2-4. The following observations were made from these autoradiographs. a) Individual sera with IF titers of 5 1:20 recognized more than 20 HHV-6 specific polypeptides from all three isolates and exhibited variations in the quantities of polypeptides immunoprecipitated from individual isolate. b) The mobilities of many of the HHV-6(Z-29) polypeptides could be distinguished from HHV-G(GS) polypeptides. c) Except for minor differences, the approximate molecular weights of poly-

Fig. 3. SDS-PAGE analysis of t3%1 methionine labeled HHV-6 isolates GS, DA, and 2-29 infected cell extracts immunoprecipitated by sera from healthy adults. Lanes 1 3 : reactivities of serum no. 18 with HHV-6 isolates GS (lane l), DA (lane 21, and 2-24 (lane 3). Lanes Q-6: reactivities of serum no. 128 with HHV-6 isolates GS (lane 4), DA (lane 5), and 2-29 (lane 6). Lanes 7-9 reactivities of serum no. 8 with HHV-6 isolates GS (lane 71, DA (lane 81, and 2-29 (lane 9). Numbers indicate approximate molecular weights (kDa) of prominent HHV-6 polypeptides immunoprecipitated. IF titer indicates HHV-6 antibody (IgG) titers of sera as measured in an immunofluorescence assay with acetone fixed cells infected with the three isolates.

peptides immunoprecipitated from HHV-6 isolates GS and DA were identical and individual sera immunoprecipitated identical number of polypeptides from these isolates (Fig.2, lanes 3,4,6, and 7; Fig.3, lanes 1,2,4,5, 7, and 8; Fig.4, lanes 1 ,2 ,4 ,5 , 7, and 8). d) Some of the polypeptides with approximate molecular weights of 250k, 135k, 78k, 41k, 36k, 34k, and 31k were immunoprecipitated from all three isolates (Figs. 2-4). e) The polypeptides with the approximate molecular weights of 210k, 185k, 150k, 110k, 105k, 92k, 72k, 70k, and 52k were immunoprecipitated from HHV-6(Z-29) infected cells and not from HHV-6 GS and DA infected cells (Fig.2, lanes 5 and 8; Fig.3, lanes 3,6, and 9; Fig. 4, lanes 3,6, and 9). 0 Even though some sera had similar IF titer with all three isolates, they showed variations in the quantities of polypeptides immunoprecipitated from these isolates. Some sera immunoprecipitated considerable quantities of polypeptides in the 135k- 52k region from all three isolates (Fig.2, lanes 3-5). However, only low quantities of HHV-6(Z-29) polypeptides with the approximate molecular weights of 210k and 185k were immunoprecipitated by these sera and an example is shown in Figure 2, lane 5. g) In contrast, some sera with low 2-29 IF titer (1:40 to 1:80) readily immunoprecipitated the HHV-6(Z-29) 210k and 185k polypeptides (Fig. 2, lane 8; Fig. 3, lane 3; Fig.4, lanes 3, 6, and 9), although they did not immunoprecipitate higher quantities of polypeptides in the 135k-52k region as seen with sera shown in Figure 2, lane 5. h) Some sera had four-fold higher 2-29 IF antibody titer

252 Chandran et al.

Fig. 4. SDS-PAGE analysis of ["S] methionine labeled HHV-6 isolates GS, DA, and 2-29 infected cell extracts immunoprecipitated by sera from healthy adults. Lanes 1-3: reactivities of serum no. 97 with HHV-6 isolates GS (lane l), DA (lane 2), and 2-29 (lane 3). Lanes 4-6: reactivities of serum no. 56 with HHV-6 isolates GS (lane 4), DA (lane 5). and 2-29 (lane 6). Lanes 7-9: reactivities of serum no. 72 with HHV-6 isolates GS (lane 7), DA (lane 81, and 2-29 (lane 9). Numbers indicate approximate molecular weights (kDa) of prominent HHV-6 polypeptides immunoprecipitated. IF titer indicates HHV-G antibody (IgG) titers of sera as measured in an immunofluorescence assay with acetone fixed cells infected with the three isolates.

and immunoprecipitated considerable quantities of the 185k and 210k polypeptides from HHV-6(Z-29) infected cell lysate (Fig.3, lane 6), and not from HHV-6 GS and DA infected cell lysates (Fig.3, lanes 4 and 5). i) Differ- ent patterns of reactivities were also exhibited by some sera with high IF titer against all three isolates. In contrast to the serum described in Figure 2, lanes 1-5, these sera immunoprecipitated considerable amounts of 210k and 185k polypeptides from HHV-6(Z-29) infected cell lysate, as well as 180k and 225k polypeptides from GS and DA infected cells (Fig.3, lanes 7-9).

The observed differences in the mobilities and quantities of polypeptides immunoprecipitated from these three isolates are not just due to cell type differences, as similar polypeptide profiles were also seen with infected CBL and PBL (Fig. 5). We have also tested a limited number of these sera with other HHV-6 isolates and the results with a serum is shown in Figure 6. This serum showed an IF titer of 1:640 with all these isolates. The approximate molecular weights of polypeptides immunoprecipitated from group A isolates U1102, Col, C02, Co5, and C06 were similar to HHV-G(GS) polypeptides (Fig.6, lanes 2,5,7-10). The molecular weights of polypeptides immunoprecipitated from group B isolates HHV-G(DC) (Fig.6, lane 6) and HHV- 6(OK and SFXdata not shown) were identical to the molecular weights of HHV-6(Z-29) polypeptides (Fig.6, lane 3) and the 210k and 185k polypeptides were readily immunoprecipitated only from HHV-6 2-29 and

Fig. 5. SDS-PAGE analysis of ["'Sl methionine labeled HHV-6 isolates GS, DA, and 2-29 infected PBL and CBL extracts immunoprecipitated by sera from healthy adults. Lanes 1-4: reactivities of serum no. 56 with uninfected cells (lane l), HHV-6 isolates GS (lane 21, DA (lane 31, and 2-29 (lane 4) grown in CBL. Lanes 5-8: reactivities of serum no. 56 with uninfected cells (lane 5), HHV-G isolates GS (lane 6) , DA (lane 71, and 2-29 (lane 8 ) grown in PBL. Lanes 9-11: reactivities of serum no. 72 with HHV-6 isolates GS (lane 9), DA (lane lo), and 2-29 (lane 11) grown in PBL. Numbers indicate approximate molecular weights (kDa) of prominent HHV-6 polypeptides immunoprecipitated.

DC infected cells. The above reactivities of human sera further demonstrate the antigenic differences between the two groups of HHV-6 isolates.

DISCUSSION Studies presented here represent the early stages in

sorting out the antigenic properties of subtypes of the new human herpesvirus pathogen. Even these limited studies suggest that these two groups of HHV-6 isolates differ in their proteins and antigenic epitopes, as evi- denced by the reactivities of MAbs and human sera. Even though non-reactivity with a MAb is not unex- pected, non-reactivities only with group B isolates infected cells by seven MAbs against three different proteins of HHV-G(GS) are unlikely due to random occurrence. Non-reactivities of seven MAbs with group B isolates in IFA suggest the absence or modified epitopes or the absence of corresponding proteins. Test- ing these possibilities require monospecific polyclonal rabbit antibodies against these proteins and such studies are in progress.

Immunoprecipitation by polyclonal rabbit antibodies against HHV-G(GS) demonstrated close antigenic re- latedness between the two groups of HHV-6 isolates. The reactivities of human sera from healthy adults also show the existence of antigenic differences between the two groups of HHV-6 related isolates. The majority of the sera could not distinguish the antigenic differences between the two groups of HHV-6 isolates by IFA

Antigenic Relationships Among HHV-6 Isolates 253

Fig. 6. SDS-PAGE analysis of r3%] methionine labeled HHV-6 isolates infected cell extracts immunoprecipitated by serum no 8. Lane 1: uninfected cells (C). Lanes 2-10 cells infected with different HHV-6 isolates as indicated. Isolates HHV-G(GS), HHV-G(DA), HHV- 6(U1102), HHV-G(Col), HHV-G(CoZ), HHV-G(Co5), and HHV-G(Co6) were grown in HSB-2 cells. HHV-6(Z-29) was grown in M o b 3 cells and HHV-G(DC) was grown in CBL. Numbers indicate approximate molecular weights (kDa) of the prominent polypeptides identified as specific for HHV-6 infected cells.

alone. A two-fold difference in the IF titer against an isolate could not be considered significant due to the inherent subjective nature of the IF assay. However, sera with similar or two-fold difference in IF titer against the isolates from the two groups could be distinguished by their reactivities in RIP assay, especially by their reactivities with the HHV-6(Z-29) 185k and 210k polypeptides. The failure to immunoprecipitate the corresponding molecular weight polypeptides from HHV-6 GS and DA infected cells by majority of the sera could be due to low solubility of group A polypeptides under the conditions used, or these polypeptides may be less abundant or less immunogenic or may be due to low methionine content. Nevertheless, these also indicate the differences among the proteins of these two groups of viruses. Immunoprecipitation of 185k and 210k polypeptides only from HHV-6(Z-29) infected cells by sera shown in Figure 2, lanes 6-8, Figure 3, lanes 1-3,4-6, and Figure 4, lanes 1-3 and P 6 , suggests that these polypeptides may contain predominantly group B specific epitopes. Further work is in progress to determine whether these polypeptides could be used to serologi- cally distinguish infection by these two groups.

The complex pattern of reactivities seen with human sera suggests that individuals may be infected with viruses from one or both groups. Differences in the IF titer together with the reactivities with the 185k and 210k polypeptides of HHV-6(2-29) by the sera shown in

Figure 2, lanes 6-8, Figure 3, lanes 1-3, 4-6, and Figure 4, lanes 1-3 and 4-6 could be interpreted as suggestive of infection by a group B isolate related andlor similar to 2-29 isolate. Reactivities shown in Figure 2, lanes 3-5 could be interpreted as suggestive of an infection by a group A isolate related to the GS isolate as well as by a 2-29 related isolate. Stronger reactivities of this serum with many polypeptides in the 135k-52k region of HHV-6 isolates GS and 2-29 and not with HHV-6(Z-29) 185k and 210k polypeptides could be suggestive of cross-reactive epitopes in the 135k-52k polypeptide region, reactivation of GS related isolate in this individual, and/or stronger re- sponse to cross-reactive epitopes. Reactivities shown in Figure 3, lanes 7-9 could be interpreted as suggestive of infection by HHV-6 related to GS as well as 2-29 related isolates. However, for a meaningful interpreta- tion of these data, larger samples from different age groups need to be analyzed and such studies are in progress.

Among the MAbs described here, only MAbs 9A5D12 and 12B3G4 show weak cross-reactivity with HHV-7 [Wyatt et al., 19911, indicating that the other nine MAbs are ideal reagents for the identification of HHV-6 related isolates as well as to group the newer isolates. Segregation by growth properties, DNA restriction analysis, and reactivities of MAbs, together with the diverse reactivities of human sera indicate the existence of potential relationships between these segrega- tions. Further in depth studies such as comparison of DNA restriction maps and DNA sequences of open reading frames, identification of type specific proteins and their genes, cross-neutralization, and cross-absorption studies are essential to fully understand the biolog- ical significance of the observed differences between the two groups of HHV-6 isolates, their relationship to different tropism, the number of groups in the population, and to determine the human antibody responses. The specific disease association and time of acquisition of infection by these two groups of HHVB need to be determined. Variations among HHV-6 isolates demonstrated here also indicate a need for a unified nomen- clature of these two distinct groups.

ACKNOWLEDGMENTS This study was supported in part by the United

States Public Health Service grants A1 24224, AI30355, A1 32109, and BRSG-507-RR0573.

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Antigenic relationships among human herpesvirus-6 isolates

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