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months and became undetectable 5 to 6 months after transplantation. .... other hand, samples 3 and 4, obtained at late post-transplant stages, had no HHV-6.

Microbiol. Immunol. Vol. 36 (5), 495-506, 1992

IgM Neutralizing Antibody Responses to Human Herpesvirus-6 in Patients with Exanthem Subitum or Organ Transplantation Sadao Toshihiko


YOSHIKAWA,1 Takehiko

Seiji KOJIMA,2 Takaharu




and Shinichi 1






Departmentof Pediatrics, Fujita Health UniversitySchoolof Medicine, Toyoake,Aichi 470-11, Japan, 2Divisionof Hematology-Oncology, Children's Medical Center, the Japanese Red Cross NagoyaFirst Hospital, Nagoya, Aichi 453, Japan, 3Departmentof Urology, Komaki Shimin Hospital, Komaki, Aichi 485, Japan, and 4Departmentof Urology, ChukyoHospital, Nagoya, Aichi 457, Japan (Accepted for publication, February 15, 1992)

Abstract The assay for detecting IgM neutralizing (NT) antibody activity to human herpesvirus-6 (HHV-6) was developed by using pretreatment of blood sample with staphylococcal protein A. The activity was mostly present in IgM fractions of serum but not in IgA fractions separated by ultracentrifugation. The assay was used for seroepiderniological studies for HHV-6 infection. In primary HHV-6 infection, IgM NT antibodies appeared 5 to 7 days after onset of exanthem subitum, reached maximum titers at 2 to 3 weeks, and tended to decline to undetectable levels after 2 months. In contrast, reactivation of HHV-6 observed in organ transplants showed somewhat greater degree of IgM NT antibody responses that persisted for 2 to 3 months and became undetectable 5 to 6 months after transplantation. The level and persistence of NT antibody titers measured by the conventional method was generally greater than those of the IgM titers. The prevalence of the IgM NT antibodies was examined in healthy individuals. The antibody was first detected at 4 to 7 months of age (5%), reached maximum level at 8 to 11 months (40%), and was detectable by 4 to 6 years (17%). A few (4 to 5%) of adolescents and adults were positive for the antibody.

Human herpesvirus-6 (HHV-6) is a newly identified herpesvirus, initially isolated from peripheral blood mononuclear cells (MNCs) of patients with lymphoproliferative disorders or AIDS (26). It was strongly suggested that the primary infection with HHV-6 caused exanthem subitum (roseola infantum) (1, 33), a common febrile illness with rash in infancy, and confirmed that the virus produced wide varieties of clinical manifestations of the disease (2, 7, 17, 29, 37) including cases with a fatal outcome (4, 15). The virus is also linked with self-limiting disorders including mononucleosis-like illness and acute hepatitis in adults (11, 18, 23, 27, 28). 495




These findings were proved by isolation of HHV-6 or a fourfold or greater increase of the antibody titer or both. It is also important to detect HHV-6 specific IgM antibody for supporting the findings mentioned above, since presence of the IgM antibody suggests recent replication of HHV-6 in the patients and subsequent stimulation of their immune system by the virus antigen during the course of diseases. In the present study, we investigated the experimental condition for detection of neutralizing (NT) antibodies in IgM fraction of sera and then used the assay for seroepidemiological studies. MATERIALS



Neutralization test (NT). Details of the method for NT have been described elsewhere (30). Briefly, serial 2-fold serum dilutions prepared on disposable plastic trays










(100 ƒÊl)



preparation (FG-80 strain) containing 102.5tissue culture infective doses5o/0.1 ml of the virus. The cell-free virus was prepared from supernatant tissue culture fluid after centrifugation of the virus-infected cord blood MNCs cultures. RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum, 0.1 U/ml of recombinant human interleukin-2 (Takeda Chemical Industries, Osaka, Japan), 5 ƒÊg/ml





2 •~ 105

and cord



diluent MNCs




(25 ƒÊl)

tinued for 7 days. The antibody dilution of serum that completely

virus were


and added


sera. to


After each


1-hr and




incubation incubation

titer was determined as the reciprocal prevented large cell formation.



37 C,



of highest

Indirect immunofluorescence assay (IFA) . The representative strain of HHV-6 (FG-80 strain) was used throughout the study. Procedures of IFA were described elsewhere (34). The virus-infected cells prepared on slides were fixed in cold acetone and methanol mixture for 20 min. For IgG antibody, two-fold dilutions of sera were applied on the slide, incubated for 1 hr at 37 C, and the slide was washed in phosphate-buffered saline (PBS) 3 times. Then one drop of fluorescein isothiocyanate (FITC)-conjugated goat antibody to human IgG (Cappel Laboratories, Cochranville, Pa., U.S.A.) diluted to 1: 40 was added to fixed cells on the slide. After incubation for 1 hr at 37 C, the slide was washed with PBS 3 times and examined using a fluorescence microscope. For detection of IgM antibody, FITC-conjugated goat antibody to human IgM (Cappel) was incubated with sera for 3 hr. Sucrosedensitygradient centrifugation. Sucrose gradient centrifugation was carried out by standard methods. Briefly, sera were layered onto a preformed sucrose gradient









100,000 •~

R in

a Hitachi



Quantitation of immunoglobulins. Amounts of IgG, IgA, and IgM in sera after various treatments were determined by the standard latex agglutination method (Kyowa Medex Co., Ltd., Japan). Immunoglobulin isotypes concentrations in fractions obtained after ultracentrifugation were measured by ELISA using monoclonal antibodies to each isotype.







Absorptionof IgG. The removal of IgG from sera was carried out by using staphylococcal protein A, Cowan I strain (Absorb-G, Kaketsuken, Japan). Briefly, a portion of the 10% bacterial cell suspension was centrifuged and washed with PBS, and the sediment was used for the treatment of sera. The amount of bacterial cells was quantified by several wet weights (0.2, 0.1, 0.05, 0.025, 0.01g). Serum samples were diluted 1: 2 in the medium and 0.2 ml of them were added to the bacterial pellets. The mixture was incubated for 1 hr at room temperature with constant shaking. After centrifugation of the mixture, the supernatant was used for various analyses. Study populations. Subjects with primary HHV-6 infection consisted of 16 patients (ranged 5 to 18 months), who were confirmed as having exanthem subitum by isolation of the virus from blood and by a significant increase in NT antibody titers. Blood samples were obtained at various times after onset of the disease. Subjects for reactivation of HHV-6 consisted of 10 organ transplants : 5 recipients of bone marrow transplantation (ranged 6 years to 16 years) and 5 recipients of renal transplantation (ranged 13 to 47 years). All recipients were seropositive for HHV-6 just before transplantation, and the virus was isolated from peripheral blood 2 to 3 weeks after the transplantation. Blood samples were obtained just before transplantation and subsequently at various intervals for a six-month period. Sera were also obtained from 3 donors (2 bone marrow transplants and 1 renal transplant) of the 10 organ transplants. Additional 229 sera from healthy children and adults were used for seroepidemiological studies. The protocol was thoroughly explained to the patients and the guardians of infants and children. The sera were stored frozen at -20 C until assay. RESULTS

Effect of VariousConcentrationsof Protein A on Amounts of Immunoglobulinand Antibody Titers to HHV-6 in Sera Four representative blood samples obtained at various times after exanthem subitum or renal transplantation were treated at various concentrations of protein A (Table 1). The concentration of protein A, 0.05 g or greater, removed IgG completely, whereas IgA and IgM were present at these concentrations, but were low. Protein A, 0.05 g removed specific antibody activity to HHV-6 in one (sample 1) of the 4 samples but protein A, 0.1 g, removed it completely. Samples 1 and 2, obtained at early convalescent or early post-transplant stages, had NT and IgM (IFA) antibody activities to HHV-6 after treatment with 0.1 g, protein A. On the other hand, samples 3 and 4, obtained at late post-transplant stages, had no HHV-6 antibody activities after the same treatment. The concentration of protein A, 0.1 g, was chosen for the removal of IgG antibody to HHV-6 throughout the present study.


S. SUGA Table

1. amounts


of various

of immunoglobulin

concentrations isotypes

ET AL of staphylococcal and




A on


Detectionof Neutralizing AntibodyActivity in IgM and IgG and A Fractions Separated by SucroseGradientCentrifugation Four blood samples obtained at early convalescent stage of exanthem subitum or at 3 to 4 weeks after renal transplantation were separated into 14 fractions by sucrose density gradient centrifugation. Fractions were tested for NT antibody activities and amounts of immunoglobulin isotypes. Results of one representative sample obtained at day 12 of exanthem subitum is shown in Fig. 1. IgM fractions (fraction numbers 3 to 5) and IgG and IgA fractions (fraction numbers 7 to 12) had NT antibody titers of 1 : 4 and 1 : 4 to 1 : 16, respectively, before treatment by protein A. After the treatment, the antibody activities decreased less than 4 in the latter (IgA) fractions. The 3 other samples showed similar results. IgM

Neutralizing Antibody Responses to HHV-6 in Patients with Exanthem Subitum Serially collected 49 blood samples from 16 patients with exanthem subitum





Neutralizing in fractions

antibody separated





by sucrose

day 12 of exanthem subitum was "MATERIALS AND METHODS

used ." RI,




gradient before



centrifugation. or





of Blood



A treatment






were tested for the NT antibody to HHV-6 before and after the protein A treatment of the samples (Fig. 2). NT antibody activity measured by the conventional method was first detected at day 4 of the disease, and the positive rate and level of antibody titers increased on day 15 to 2 months after the disease. On the other hand, the antibody in IgM fraction appeared at day 5-7 of the disease, and the level of antibody titers reached the maximum level on day 8 to day 29 of the disease. Then they decreased and were at an undetectable level by 2 months after the disease. Table 2 shows NT antibody responses to HHV-6 in representative 4 patients among the 16. Antibody activity in IgM fraction appeared at day 9 to day 19 of the illness, and disappeared at day 30 to day 60 of the illness. IgM Neutralizing AntibodyResponsesto HHV-6 in Patients with Organ Transplantation Serially collected 60 blood samples from 10 recipients with organ transplantation were tested for the NT antibody to HHV-6 before and after protein A treatment of the samples (Fig. 3). Antibody activity to HHV-6 measured by the conventional method was present in all cases just before and after transplantation, and some recipients showed significant increases in the antibody titers by 1 month after organ transplantation. On the other hand, IgM fractions of all blood samples were seronegative to HHV-6 just before and after transplantation. The antibody activity was detected in some cases at 3 to 4 weeks after transplantation, and the maximum level was maintained for 2 to 3 months. All recipients became seronegative by




Fig. 2. HHV-6 neutralizing antibody responses after onset of exanthem samples were used for the assay before or after the protein A treatment.



5 months after transplantation-. Table 3 shows NT antibody titers to HHV-6 in representative 4 recipients and 3 donors. These 4 recipients and 3 donors were seropositive to HHV-6 at the time of transplantation. The 2 recipients of renal transplantation had a significant increase in the antibody titer; IgM antibody response was detected during 1 to 3 months after the transplantation. The 2 bone marrow transplants showed no or a low antibody response. Two of the 3 donors had HHV-6 antibody activity in IgM fraction.

Prevalenceof Neutralizing IgM Antibodiesto HHV-6 in Healthy Individuals A total of 229 blood samples from healthy children and adults were examined for the age-specific prevalence of the IgM antibody activities to HHV-6 (Table 4). The antibody activity was first detected at 4 to 7 months of age and the frequency




antibody with






HHV-6 used



of fever

neutralizing the


antibody before

or after



of exanthem

TO HHV-6 to HHV-6

501 in patients



responses the protein



A treatment.















Neutralizing antibody responses to HHV-6 received organ transplantation






of neutralizing

1: 4 or greater






in patients



to HHV-6


in healthy








reached 40% at 8 to 11 months. Then it decreased and became undetectable by 7 years of age. After 10 years of age, 4 out of 120 samples were positive for the antibody. DISCUSSION

The serological diagnosis of recent HHV-6 infection is based on a finding of a fourfold or greater increase in the virus antibody titer, which has been most clearly established in children with exanthem subitum (1, 33). The presence of IgM antibody to the virus also provides an additional evidence of recent infection with HHV-6. The most widely used serological procedures for HHV-6 are the IFA which had provided much seroepidemiological information on HHV-6 infection (9, 10, 24, 34). However, the method, especially in detecting IgM antibody, had problems such as strong nonspecific fluorescent staining of cord blood MNCs that had frequently made the IFA difficult to read. Therefore, we had developed a sensitive, specific, and practical NT antibody assay for HHV-6 (30), and used the assay for clinical and virological studies of HHV-6 infections (1-8, 27, 29, 31, 35-37). This NT method was comparable with the IFA and ELISA techniques for detecting antibodies to HHV-6 that did not cross-react with those of other human herpesviruses (5, 30). In the present study, NT antibody activity was found in IgM fractions of blood samples separated by sucrose density gradient centrifugation. After removal of IgG with staphylococcal protein A treatment, the activity was still present in IgM fractions, but undetectable in IgA fractions. Accordingly we assumed that the NT activity measured after the treatment of blood sample was present mostly in IgM fractions. However, the present results must be compared with those by pretreatment of sera with streptococcal cells which remove IgG and IgA simultaneously (19). In primary HHV-6 infection, IgM NT antibodies appeared 5 to 7 days after onset, reached maximum titers at 2 to 3 weeks, and then tended to decline to undetectable levels after about 2 months, which were comparable with those by the IFA (13, 21). However, NT antibodies measured by the conventional method preceded the appearance of IgM antibodies, suggesting considerable loss of IgM by the protein A treatment. In contrast, some cases in organ transplants showed somewhat greater degree of IgM NT antibody responses that persisted for 2 to 3 months after transplantation. Since all of the transplants were immune to HHV-6 and had viremia with HHV-6 after the transplantation, it is likely that the IgM antibody response is due to reactivation of the virus harbored somewhere in the recipient's body. It has been reported that several organs or tissues, such as lymph nodes (12), liver (32), kidney (3), and salivary glands (14) contain HHV-6. It is not known why the IgM antibody responses in the organ transplants persisted longer than in normal individuals with primary HHV-6 infection. However, these findings are consistent with those of cytomegalovirus (CMV) infection (25). CMV IgM antibody measured by radioimmunoassay has persisted for up to three to five months in nonimmunocompromised patients with CMV mononucleosis. In contrast, CMV-specific




IgM antibodies have been found in recipients of cardiac transplantation up to 12 months after the operation. Profound immune dysfunction in these patients may permit prolonged viral replication in hosts and prolonged antigenic stimulation to the host. The duration and the degree of viremia in organ transplants must be compared with those in primary HHV-6 infection (8).

In the present study, there were two peaks of the prevalence of IgM NT antibody to HHV-6 in healthy individuals. The first peak observed in infants and young children suggests clearly that HHV-6 infection occurs in early infancy and childhood and the age-clustering of many blood samples reflecting recent infection (34, 35). However, it is hard to evaluate why some of the donors, adolescents and adults had the IgM antibodies. It may suggest subclinical reactivation of the virus in healthy individuals. Alternatively, infection with CMV or other viruses may be associated with the presence of IgM HHV-6 antibody, as suggested elsewhere (16, 20, 22, 23). More information about the natural history of HHV-6 infection is needed. Finally, the results reported here may be an underestimate of IgM NT antibody prevalence. Development of more efficient procedures for removal of IgG and IgA without loss of IgM is required. This work was supported in part by the grant from the Aichi Kidney Foundation. Recombinant human interleukin-2 was kindly supplied by Takeda Chemical Industries, Ltd., Osaka, Japan.


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