Anti-varicella-zoster virus antibody titers and seroprotection status from before the first dose of varicella vaccination to before entering elementary school in one region in Japan.

OBJECTIVE: We aimed to examine changes in anti-varicella-zoster virus (VZV) antibody titers and seroprotection status from before the first dose of vaccination to before 7 years old entering elementary school in children who received the routine two-dose varicella vaccination. METHODS: Participants were 37 healthy children who received the routine two-dose varicella vaccination at our hospital. A total of five serum samples per child were collected immediately before and 4-6 weeks after each dose of the vaccination and in the year before entry to elementary school. We measured anti-VZV antibody titers by immune adherence hemagglutination (IAHA) method and glycoprotein-based enzyme-linked immunosorbent assay (gpELISA). A positive antibody titer and the seroprotection level were set as ≥2-fold and ≥16-fold, respectively, for IAHA antibody and as ≥50 units and ≥105 units, respectively, for gpELISA-IgG antibody. RESULTS: The rates of IAHA antibody positivity in the five samples (in order of collection) were 0%, 65%, 38%, 100%, and 59%, and the rates of seroprotection were 0%, 43%, 8%, 100%, and 43%. The rates of gpELISA-IgG antibody positivity were 8%, 81%, 89%, 100%, and 100%, and the rates of seroprotection were 5%, 54%, 70%, 100%, and 89%. The mean IAHA antibody titer and mean gpELISA-IgG antibody titer before entering elementary school were both lower than the respective titers obtained after the second vaccination (both p < 0.01). CONCLUSIONS: Routine two-dose varicella vaccination leads to good antibody production, but titers of acquired antibodies decrease before children enter elementary school.

An Investigation of Japanese Neonatal and Maternal Antibody Status against Pertussis.

To clarify the pertussis immune status of the Japanese population, we investigated levels of serum pertussis toxin (PT)-specific immunoglobulin G (IgG) antibody in infants and mothers between April 2016 and March 2018. A total of 206 infants (n = 22, < 32 weeks of gestational age [wGA]; n = 70, 32-36 wGA; n = 114, ≥ 37 wGA) and 170 mothers were enrolled. The maternal seroprevalence and antibody geometric mean titer (GMT) were 52.4% and 10.7 EU/mL, respectively. The antibody GMT, seroprevalence, and mean ratio of infant to maternal antibody titers of infants at < 32 wGA were 3.2 EU/mL, 13.6%, and 42.5%, respectively, and were significantly lower than those of infants at 32-36 wGA (9.7 EU/mL, 54.3%, and 110.2%) and infants at ≥ 37 wGA (12.1 EU/mL, 57.9%, and 112.6%). Of the 21 infants who underwent a second examination, five were positive in the first examination. Of those five, the GMT for PT had decreased by an average of 52.6% at 4.3- week intervals. In the second examination, two infants were seropositive. Approximately half of the mothers and infants were negative for anti-PT antibody. Thus, new vaccination strategies, such as the vaccination of pregnant women, are needed to prevent pertussis infection in early infancy.

[Results of Booster Vaccination in Children with Primary Vaccine Failure after Initial Varicella Vaccination].

In October 2014, the varicella vaccination policy in Japan was changed from a single voluntary inoculation to two routine inoculations. This paper reports the results of booster vaccination in children who did not show seroconversion after initial vaccination (i.e., primary vaccine failure : PVF) over a 7-year period prior to the introduction of routine varicella vaccination. Between November 2007 and May 2014, 273 healthy children aged between 1.1 and 14.5 years (median : 1.7 years) underwent varicella vaccination. Before and 4 to 6 weeks after vaccination, the antibody titers were measured using an immune adherence hemagglutination (IAHA) assay and a glycoprotein-based enzyme-linked immunosorbent assay (gpELISA). In addition, side reactions were examined during the four-week period after vaccination. Children who did not show IAHA seroconversion (PVF) were recommended to receive a booster vaccination, and the measurement of antibody titers and an assessment of side reactions were performed after the booster dose. In May 2015, a questionnaire was mailed to each of the 273 participants to investigate whether they had developed varicella and/or herpes zoster after vaccination. After initial vaccination, the IAHA seroconversion rate was 75% and the mean antibody titer (Log2) with seroconversion was 4.7, while the gpELISA seroconversion rate was 84% and the mean antibody titer (Log10) with seroconversion was 2.4. Among children with PVF, 54 received booster vaccination within 81 to 714 days (median : 139 days) after the initial vaccination. After booster vaccination, the IAHA seroconversion rate was 98% and the mean antibody titer (Log2) with seroconversion was 5.8. Both the seroconversion rate and the antibody titer were higher compared with the values after the initial vaccination (p < 0.01). After booster vaccination, the gpELISA seropositive rate was 100% and the mean positive antibody titer (Log 10) was 3.6 ; similar results were obtained for the IAHA assay, with a significantly higher, antibody response than that after the initial vaccination (p < 0.01). Side reactions were generally minor, including fever (≥ 37.5 degrees C), rash at the injection site, and rash at other sites. There were no significant differences in the incidences of side reactions between the initial and booster vaccinations. A total of 185 participants responded to the questionnaire (response rate : 68%), and the period between receiving the initial vaccination and their response to the questionnaire ranged from 1.0 to 7.5 years (median : 4.0 years). The prevalence of breakthrough varicella after the initial vaccination was 17% among seroconverters who did not receive booster vaccination and 14% among non-seroconverters who received booster vaccination, showing no significant difference between the two groups. In conclusion, there are no safety issues regarding the administration of a booster vaccination to children with PVF after an initial varicella vaccination, and,a good antibody response can be expected.

A Novel IgM-capture enzyme-linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection.

An ELISA that measures anti-PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti-Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.

Role of matrix metalloproteinases in the pathogenesis of childhood gastroenteritis.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the pathogenesis of gastrointestinal diseases, such as rotavirus gastroenteritis (GE). Kinetics of these biomarkers were examined in paired serum samples collected from bacterial enteritis patients with Campylobacter (n = 2) and Salmonella (n = 4) and viral GE patients with rotavirus (n = 27), norovirus (n = 25), and adenovirus (n = 11). At the time of hospital admission, all viral GE patients demonstrated increased MMP-9 and decreased MMP-2 and TIMP-2 serum levels. In contrast to viral GE patients, serum MMP-9 levels were not elevated at the time of hospital admission but elevated at the time of discharge; serum MMP-2 and TIMP-2 levels were decreased both at the time of admission and discharge in bacterial enteritis patients. Interestingly, the kinetics of serum MMP-2, MMP-9, and TIMP-2 levels were similar among the viral GE patients but distinct from bacterial enteritis patients. Thus, the involvement of MMPs and TIMPs in the pathophysiology of gastrointestinal symptoms likely varies depending on the etiological agent. Further studies are required to verify whether the extent of the bacterial enteritis or age of the patients influences these serum biomarkers. J. Med. Virol. 88:1341-1346, 2016. © 2016 Wiley Periodicals, Inc.

Time-Series Analysis Comparing the Prevalence of Antibodies against Nine Viral Species Found in Umbilical Cord Blood in Japan.

In this study, we investigated the prevalence of antibodies against 9 viral species found in umbilical cord blood from 561 neonates in 2013. Serum IgG antibodies against the following viruses were measured: herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), measles virus (MV), rubella virus (RV), mumps virus (MuV), and human parvovirus B19 (HPV B19). A survey questionnaire regarding past medical history and maternal immunization status for the vaccine-preventable diseases of varicella, measles, rubella, and mumps was simultaneously administered. The results were compared with previous data collected in 2001-2002 from 378 umbilical cord blood samples. Viral seroprevalence data were: HSV, 54%; VZV, 96%; EBV, 96%; CMV, 67%; HHV-6, 100%; MV, 95%; RV, 94%; MuV, 64%; and HPV B19, 55%. The seroprevalence of CMV, MV, and MuV were significantly lower in 2013 than in 2001-2002 (CMV, 76%; MV, 98%; MuV, 93%). Compared with the 2001-2002 data, the mean IgG antibody values of the 4 vaccine-preventable diseases were significantly lower, and vaccination coverage for those diseases among mothers was significantly higher. Thus, attention should be paid to antibody levels in women of childbearing age in the future.

Evaluation of varicella zoster virus-specific cell-mediated immunity by using an interferon-γ enzyme-linked immunosorbent assay.

BACKGROUND: Administration of the varicella vaccine induces both varicella-zoster virus (VZV)-specific humoral and cell-mediated immunity (CMI). OBJECTIVE: To assess VZV-CMI, we developed an interferon γ enzyme-linked immunosorbent assay (IFN-γ ELISA) that measures the quantity of total IFN-γ in culture supernatants of human peripheral blood mononuclear cells. STUDY DESIGN: We evaluated this method by comparing the pre- and post-vaccination immune response in peripheral blood mononuclear cells of 30 healthy children who were administered an initial varicella vaccination at Konan Kosei hospital. RESULTS: IFN-γ ELISA showed well-validated results; CMI was not detectable pre-immunization but became detectable post-immunization. Seroconversion was detected in 92.6% of subjects by the immune adherence hemagglutination test; however, half of the subjects did not display an increase in CMI levels. We also compared the incidence of breakthrough varicella and herpes zoster development between CMI post-positive and post-negative vaccinees at 1-2years after the last VZV vaccination. Eight subjects had a history of varicella or herpes zoster exposure post-VZV vaccination. Two of them with post-negative CMI contracted breakthrough varicella 15-16months after the last vaccination, even though they had sufficient VZV-specific antibody levels to be considered seropositive and seroprotected. Conversely, the others with post-positive CMI did not contract breakthrough varicella, despite experiencing extensive VZV exposure through casual contact with playmates and family. CONCLUSIONS: The CMI data generated by this IFN-γ ELISA may accurately reflect real-world immune status, and CMI may be closely related to immunoprotection against breakthrough varicella development.

Clinical manifestations of children with microbiologically confirmed pertussis infection and antimicrobial susceptibility of isolated strains in a regional hospital in Japan, 2008-2012.

We conducted a retrospective study in 57 children (median age, 3.5 years; range, 1 month-14.5 years) with microbiologically confirmed pertussis infection over a recent 4-year period in a regional hospital in Japan. We obtained nasal swabs from all patients for Bordetella pertussis isolation as well as performed B. pertussis DNA detection using loop-mediated isothermal amplification (LAMP). Of the 57 cases, 34 (60%) were culture-positive and 57 (100%) were LAMP-positive. The frequency of each symptom was as follows: typical paroxysmal cough for over 14 days, 96% (55/57); paroxysms, 86% (49/57); posttussive vomiting, 33% (19/57); inspiratory whoop, 25% (14/57); and apnea, 12% (7/57). Hospitalization was required in 14 cases (25%), 93% (13/14) of which were aged <1 year. The proportion of patients previously immunized against diphtheria-tetanus-acellular pertussis vaccine (DTaP) was 19% (4/21) in children aged <1 year and 92% (11/12) in children aged ≥ 10 years. Minimum inhibitory concentrations for 6 antimicrobials (erythromycin, clarithromycin, azithromycin, minocycline, amoxicillin, and sulfamethoxazole/trimethoprim) were measured for 30 isolated strains, and all strains were susceptible to all aforementioned antimicrobials. Thus, an additional pertussis vaccination in older children is necessary, and the current macrolides-based treatment strategy is considered reasonable.

Assessment of the loop-mediated isothermal amplification assay for rapid diagnosis of Mycoplasma pneumoniae in pediatric community-acquired pneumonia.

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for timely treatment with effective antibiotics; however, PCR-based methods are often too expensive and technologically intensive for general use in clinical practice. In this study, the efficacy of the loop-mediated isothermal amplification (LAMP) assay for diagnosis of M. pneumoniae pneumonia in clinical practice was prospectively evaluated. From July 2011 to March 2012, 531 children hospitalized for community-acquired pneumonia were enrolled. In all patients, throat swabs were obtained on admission for the detection of M. pneumoniae DNA, and paired serum samples were obtained to assay M. pneumoniae particle agglutination (PA) antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or an increase of 4-fold or greater in the PA titer. Overall, 271 children (51.0% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia. Among these, 258 (95.2%) and 248 (91.5%) were identified by the LAMP assay and serological tests, respectively. When the results of serological tests were considered as standard, the sensitivity, specificity, and positive and negative predictive values of the LAMP assay were 94.8%, 91.9%, and 91.1% and 95.2%, respectively. The median duration of pharyngeal carriage, as measured by the LAMP assay, was 9.5 days. Thus, the LAMP assay is useful in the rapid diagnosis of M. pneumoniae pneumonia.

[Immunogenicity of additional varicella vaccination 3-5 years after the initial vaccination].

Additional varicella vaccination was carried out targeting 16 subjects who had immune adherence hemagglutination (IAHA) seroconversion following the initial varicella vaccination and did not contract breakthrough varicella after the initial vaccination. The median ages at the initial and additional vaccination were 2.1 (1.1-6.9) years old and 6.1 (4.4-10.5) years old, respectively. The mean interval between the initial and additional vaccination was 4.0 (3.2-5.2) years. IAHA and glycoprotein-based enzyme-linked immunosorbent assay (gpELISA) antibody titers were measured just before and 4-6 weeks after the additional vaccination. Side reaction was surveyed at four weeks after the additional vaccination, and compared with the results at the initial vaccination. IAHA and gpELISA seroconversion rates at the initial vaccination were 100% and 88% respectively. Prior to the additional vaccination, IAHA antibody titers significantly decreased in 50% of the subjects, and became negative in 38% of the subjects. On the other hand, a significant increase in IAHA antibody titers was observed in 25% of the subjects, and this is assumed to be the result of a subclinical infection after the initial vaccination. The positive rate of both antibodies after the additional vaccination was 100%, the mean IAHA antibody titer (Log2) after the initial/additional vaccination in seropositive subjects was 4.6/6.5, and the mean gpELISA antibody titer (Log10) was 2.3/4.0. The mean IAHA and gpELISA antibody titers were higher after the additional vaccination than after the initial vaccination (p < 0.01, p < 0.01). This is considered to be the booster effect due to the additional vaccination. At 0-2 days after the additional vaccination, a rash at the injection site was observed in 56% of the subjects, higher than the incidence after the initial vaccination (13%) (p < 0.05), but no severe systemic side reactions were observed at either the initial or the additional vaccination. In conclusion, an additional varicella vaccination 3-5 years after the initial vaccination is thought to have greater immunogenicity and is considered effective.

Plasma viral microRNA profiles reveal potential biomarkers for chronic active Epstein-Barr virus infection.

BACKGROUND: Chronic active Epstein-Barr virus (CAEBV) infection has high mortality and morbidity, and biomarkers for disease severity and prognosis are required. MicroRNAs (miRNAs) are small noncoding RNAs, and EBV encodes multiple miRNAs. Because plasma contains sufficiently stable miRNAs, circulating EBV-associated miRNA profiles were investigated as novel biomarkers in CAEBV infection. METHODS: Plasma miRNA expression was assessed for 12 miRNAs encoded within 2 EBV open reading frames (BART and BHRF). Expression levels were investigated in 19 patients with CAEBV infection, 14 patients with infectious mononucleosis, and 11 healthy controls. Relative expression levels of plasma miRNAs were determined by TaqMan probe-based quantitative assay. RESULTS: Plasma miR-BART1-5p, 2-5p, 5, and 22 levels in patients with CAEBV infection were significantly greater than those in patients with infectious mononucleosis and in controls. Plasma miR-BART2-5p, 4, 7, 13, 15, and 22 levels were significantly elevated in patients with CAEBV infection with systemic symptoms, compared with levels in patients with no systemic symptoms. The levels of miR-BART2-5p, 13, and 15 showed clinical cutoff values associated with specific clinical conditions, in contrast to plasma EBV loads. CONCLUSIONS: Levels of specific plasma EBV miRNAs were elevated differentially in patients with CAEBV infection. Several EBV miRNAs, particularly miR-BART2-5p, 13, and 15, are potentially biomarkers of disease severity or prognosis.

Application of flow cytometric in situ hybridization assay to Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.

Epstein-Barr virus (EBV) infects various types of lymphocytes and is associated with not only B cell-origin lymphoma, but also T or natural killer cell lymphoproliferative diseases (T/NK LPD). Recently, we established a novel assay to identify EBV-infected cells using FISH. Using this assay, dual staining with antibodies to both surface antigens and an EBV-encoded small RNA (EBER) probe can be performed. In the present study, we applied this recently developed FISH assay to EBV-associated T/NK LPD to confirm its diagnostic utility. Using FISH, we prospectively analyzed peripheral blood from patients with suspected EBV-associated T/NK LPD. The results were compared with those obtained using immunobead sorting followed by quantitative PCR. In all, 26 patients were included study. Using FISH, 0.15-67.0% of peripheral blood lymphocytes were found to be positive for EBER. Dual staining was used to determine EBER-positive cell phenotypes in 23 of 26 subjects (88.5%). In five of seven patients with hydroa vacciniforme-like lymphoma (an EBV-positive cutaneous T cell lymphoma), EBER-positive cells were identified as CD3(+) CD4(-) CD8(-) TCRγδ(+) T cells. Furthermore, in a 25-year-old male patient with systemic EBV-positive T cell LPD, two lymphocyte lineages were positive for EBER: CD4(+) CD8(-) and CD4(-) CD8(+) T cells. Thus, we confirmed that our newly developed assay is useful for quantifying and characterizing EBV-infected lymphocytes in EBV-associated T/NK LPD and that it can be used not only to complement the pathological diagnosis, but also to clarify the pathogenesis and to expand the spectrum of EBV-associated diseases.

Detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification (LAMP) assay and serology in pediatric community-acquired pneumonia.

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1-14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia.

EBV-associated T/NK-cell lymphoproliferative diseases in nonimmunocompromised hosts: prospective analysis of 108 cases.

EBV-associated T/NK-cell lymphoproliferative disease (T/NK-LPD) is defined as a systemic illness characterized by clonal proliferation of EBV-infected T or NK cells. We prospectively enrolled 108 nonimmunocompromised patients with this disease (50 men and 58 women; median onset age, 8 years; age range, 1-50 years) evidenced by expansion of EBV(+) T/NK cells in the peripheral blood; these were of the T-cell type in 64 cases and of the NK-cell type in 44, and were clinically categorized into 4 groups: 80 cases of chronic active EBV disease, 15 of EBV-associated hemophagocytic lymphohistiocytosis, 9 of severe mosquito bite allergy, and 4 of hydroa vacciniforme. These clinical profiles were closely linked with the EBV(+) cell immunophenotypes. In a median follow-up period of 46 months, 47 patients (44%) died of severe organ complications. During the follow-up, 13 patients developed overt lymphoma or leukemia characterized by extranodal NK/T-cell lymphoma and aggressive NK-cell leukemia. Fifty-nine received hematopoietic stem cell transplantation, 66% of whom survived. Age at onset of disease (≥ 8 years) and liver dysfunction were risk factors for mortality, whereas patients who received transplantation had a better prognosis. These data depict clinical characteristics of systemic EBV(+) T/NK-LPD and provide insight into the diagnostic and therapeutic approaches for distinct disease.

Antitumor activities of valproic acid on Epstein-Barr virus-associated T and natural killer lymphoma cells.

Epstein-Barr virus (EBV), which infects B cells, T cells, and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, histone deacetylase (HDAC) inhibitors have been reported to have anticancer effects against various tumor cells. In the present study, we evaluated the killing effect of valproic acid (VPA), which acts as an HDAC inhibitor, on EBV-positive and -negative T and NK lymphoma cells. Treatment of multiple T and NK cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3 and KHYG1) with 0.1-5 mM of VPA inhibited HDAC, increased acetylated histone levels and reduced cell viability. No significant differences were seen between EBV-positive and -negative cell lines. Although VPA induced apoptosis in some T and NK cell lines (SNT16, Jurkat and KHYG1) and cell cycle arrest, it did not induce lytic infection in EBV-positive T or NK cell lines. Because the killing effect of VPA was modest (1 mM VPA reduced cell viability by between 22% and 56%), we tested the effects of the combination of 1 mM of VPA and 0.01 µM of the proteasome inhibitor bortezomib. The combined treated of cells with VPA and bortezomib had an additive killing effect. Finally, we administered VPA to peripheral blood mononuclear cells from three patients with EBV-associated T or NK lymphoproliferative diseases. In these studies, VPA had a greater killing effect against EBV-infected cells than uninfected cells, and the effect was increased when VPA was combined with bortezomib. These results indicate that VPA has antitumor effects on T and NK lymphoma cells and that VPA and bortezomib may have synergistic effects, irrespective of the presence of EBV.

[A study for the necessity of virus titer of varicella vaccine presently used].

Recently available varicella vaccine titers are several dozen times higher than the formulation standard in accompanying information, i.e., > or = 1,000 PFU/dose. We investigated changes in immunogenicity associated with vaccination using a reduced dose whose virus titer was close to that used when the vaccine was developed, and examined the need for the virus titer presently used. In a non blinded study of 43 children with no history of varicella infection, we administered 0.1 mL of varicella vaccine (1/5 of the normal dose) to 20 children 1 year 0 month to 4 years 5 months old (median: 1 year 5 months) and the standard 0.5mL dose to 23 children 1 year 2 months to 3 years 7 months old (median: 1 year 9 months). We measured IAHA and gpELISA antibody titer before vaccination and 4 to 6 weeks after vaccination. We defined "positive" as > or = 2 fold of IAHA titer and > or = 50 U of gpELISA antibody titer. We administered an additional 0.5mL of varicella vaccine to children whose IAHA titer failed to show seroconversion and remeasured antibody titer 4 to 6 weeks after revaccination. IAHA seroconversion was 25.0% (5/20) and gpELISA seroconversion 55.0% (11/20) in the 0.1 mL vaccination group, which was lower than that of 76.2% (16/21) and 87.0% (20/23), IAHA p < 0.01, gpELISA p < 0.05, in the 0.5 mL vaccination group. We administered an additional vaccination to 19 children--15 in the 0.1 mL vaccination group and 4 in the 0.5 mL vaccination group-with 100% seroconversion for both methods. Mean antibody titer after revaccination in the 0.1 mL vaccination group (IAHA 2 (6.0), gpELISA 10 (3.7)) was higher than those in the 0.5mL vaccination group who seroconverted following initial vaccination (IAHA 2(4.5), gpELISA 10(2.6)) (p < 0.01). We also measured virus titer in the remaining vaccine following vaccination of 0.1 mL (n = 20), and estimated virus titer administered to the 0.1 mL vaccination group to be 2,600-6,400 PFU/ dose. Varicella vaccine immunogenicity decreased if dosage was reduced to 1/5 of the standard dose, indicating that the present virus titer is necessary to maintain adequate immunogenicity. An additional administration of the standard dose to children who failed to seroconvert after initial 0.1 mL administration produced high antibody titers thought to constitute a booster effect.

Replication of Epstein-Barr virus primary infection in human tonsil tissue explants.

Epstein-Barr virus (EBV) may cause a variety of virus-associated diseases, but no antiviral agents have yet been developed against this virus. Animal models are thus indispensable for the pathological analysis of EBV-related infections and the elucidation of therapeutic methods. To establish a model system for the study of EBV infection, we tested the ability of B95-8 virus and recombinant EBV expressing enhanced green fluorescent protein (EGFP) to replicate in human lymphoid tissue. Human tonsil tissues that had been surgically removed during routine tonsillectomy were sectioned into small blocks and placed on top of collagen sponge gels in culture medium at the air-interface, then a cell-free viral suspension was directly applied to the top of each tissue block. Increasing levels of EBV DNA in culture medium were observed after 12-15 days through 24 days post-infection in tissue models infected with B95-8 and EGFP-EBV. Expression levels of eight EBV-associated genes in cells collected from culture medium were increased during culture. EBV-encoded small RNA-positive cells were detected in the interfollicular areas in paraffin-embedded sections. Flow cytometric analyses revealed that most EGFP(+) cells were CD3(-) CD56(-) CD19(+) HLA-DR(+), and represented both naïve (immunoglobulin D(+)) and memory (CD27(+)) B cells. Moreover, EBV replication in this model was suppressed by acyclovir treatment in a dose-dependent manner. These data suggest that this model has potential for use in the pathological analysis of local tissues at the time of primary infection, as well as for screening novel antiviral agents.