Comprehensive Analysis of Antibodies Induced by Vaccination with 4 Kinds of Avian Influenza H5N1 Pre-Pandemic Vaccines.

Four kinds of avian-derived H5N1 influenza virus, A/Vietnam/1194/2004 (Clade 1), A/Indonesia/5/2005 (Clade 2.1), A/Qinghai/1A/2005 (Clade 2.2), and A/Anhui/1/2005 (Clade 2.3), have been stocked in Japan for use as pre-pandemic vaccines. When a pandemic occurs, these viruses would be used as vaccines in the hope of inducing immunity against the pandemic virus. We analyzed the specificity of antibodies (Abs) produced by B lymphocytes present in the blood after immunization with these vaccines. Eighteen volunteers took part in this project. After libraries of Ab-encoding sequences were constructed using blood from subjects vaccinated with these viruses, a large number of clones that encoded Abs that bound to the virus particles used as vaccines were isolated. These clones were classified into two groups according to the hemagglutination inhibition (HI) activity of the encoded Abs. While two-thirds of the clones were HI positive, the encoded Abs exhibited only restricted strain specificity. On the other hand, half of the HI-negative clones encoded Abs that bound not only to the H5N1 virus but also to the H1N1 virus; with a few exceptions, these Abs appeared to be encoded by memory B cells present before vaccination. The HI-negative clones included those encoding broadly cross-reactive Abs, some of which were encoded by non-VH1-69 germline genes. However, although this work shows that various kinds of anti-H5N1 Abs are encoded by volunteers vaccinated with pre-pandemic vaccines, broad cross-reactivity was seen only in a minority of clones, raising concern regarding the utility of these H5N1 vaccine viruses for the prevention of H5N1 pandemics.

Development of varicella vaccine in Japan and future prospects.

In Japan, Dr. Michiaki Takahashi (1928-2013) successfully developed the first live attenuated varicella vaccine in the world. The virus used for this vaccine was varicella-zoster virus isolated from the vesicular fluid of a child with typical varicella and it was named the Oka strain after the family name of the child. In 1974, a patient with nephrosis developed varicella in the Pediatric Ward, and uninfected pediatric patients received varicella vaccine immediately. As a result, there were no cases of varicella in the other children and all of the vaccinated children acquired immunity to the disease. These results were published in the Lancet, demonstrating the safety and efficacy of varicella Oka strain vaccine for the first time. When clinical studies were conducted at the start of vaccine development, most of the subjects were pediatric patients with a high risk of contracting severe varicella. Therefore, the development process was different from that for other vaccines, since clinical studies are generally performed in healthy individuals. This vaccine was approved in Japan in 1986, and voluntary single-dose vaccination for children aged 1 year or older was started in 1987. However, the vaccination coverage rate remained low and the number of patients with varicella did not decrease significantly. Due to its voluntary status, the cost of vaccination was borne by the child's family and this was considered to be a reason for the low coverage rate. Moreover, although the vaccine achieved a good antibody response, the number of cases of breakthrough varicella (BV) was relatively high and showed an increasing trend that was also a concern. In order to increase the coverage rate and reduce BV, the Japanese government changed the varicella vaccination policy from voluntary to routine vaccination in October 2014. At the same time, a two-dose schedule was introduced that involved administration of the vaccine twice at an interval of at least 3 months up to the age of 3 years. At present, cases of varicella are only monitored at the pediatric sentinel clinics in Japan. Therefore, we need to establish a system to survey all patients, in order to demonstrate the efficacy of varicella vaccine based on detailed surveillance data. We also need to investigate the optimum timing of the second dose of the vaccine and the necessity for further booster vaccination. A combined live vaccine containing varicella vaccine has not yet been approved in Japan. Because of the greater convenience of combined vaccines, development and introduction of such a vaccine in the future would be desirable. Routine varicella vaccination is also expected to eventually reduce the occurrence of herpes zoster, although there are no supporting epidemiological data. The prevalence of herpes zoster has attracted attention, but it is necessary to develop a surveillance system for this disease. In March 2016, use of varicella vaccine to prevent herpes zoster in adults aged 50 years or older was approved in Japan, and the results of this policy change need to be assessed.

Virus specific cell-mediated immunity may play a role in controlling reactivated human herpesvirus 6B in patients under measles induced immunosuppression.

For a better understanding of the cellular immune responses to reactivated HHV 6B the lymphoproliferative response to human herpesvirus 6B (HHV 6B) antigen was measured in three consecutive specimens obtained biweekly from 22 young children and infants suffering from acute measles, and in 19 influenza patients and nine healthy control subjects. HHV 6B DNA in peripheral blood mononuclear cells (PBMCs) was detected in 18 of 22 subjects with measles, but not in the influenza patients or the healthy population. A novel reactivation profile of HHV 6B was found in patients with measles in the milder form of immunosuppression than in patients with organ transplantation. HHV 6B specific lymphoproliferation activities increased correspondingly with reactivation of HHV 6B assessed by detecting HHV 6B DNA in PBMCs in patients with measles, but no significant change in either the antibody response to HHV 6B or DNAemia occurred in serial specimens obtained either from patients with influenza or healthy subjects. This novel form of HHV 6B reactivation without antibody response was observed in patients with measles. The dynamic fluctuations in lymphoproliferative responses in measles may represent the balance between HHV 6B reactivation and its suppression by the host immune system.

Cycling probe technology to quantify and discriminate between wild-type varicella-zoster virus and Oka vaccine strains.

Rapid differentiation between wild-type varicella zoster virus (VZV) and Oka-vaccine (vOka) strains is important for monitoring side reactions of varicella vaccination. To develop a high-throughput molecular diagnostic method for the differentiation of wild-type VZV and vOka strains based on cycling probe technology. The primers were designed to amplify common sequences spanning a single nucleotide polymorphism (SNP) in gene 62 of VZV. DNA-RNA chimeric probes (cycling probes) were designed to detect the SNP at nucleotide 105705. The cycling probe real-time PCR assays for VZV wild-type and vOka strains specifically amplified plasmids containing target sequences that ranged between 10 and 1×10(6) copies per reaction. The inter- and intra-assay coefficients of variation were less than 5%. After initial validation studies, the clinical reliability of this method was evaluated using 38 swab samples that were collected from patients suspected of being zoster. Compared to the loop mediated isothermal amplification method, which is defined as the gold standard, cycling probe real-time PCR was highly sensitive and specific. The cycling probe real-time PCR technology is a reliable tool for differentiating between wild-type VZV and vOka strains in clinical samples.

Serum biomarker kinetics with three different courses of HHV-6B encephalitis.

Human herpesvirus-6B (HHV-6B) encephalitis can clinically manifest as hemorrhagic shock and encephalopathy syndrome (HSES), acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), and acute necrotizing encephalopathy (ANE). To compare the underlying pathophysiology, we measured several biomarkers of interest in patients with these three different courses. Based on their clinical course and neuroimaging analysis, Cases 1, 2 and 3 were diagnosed as HSES, AESD, and ANE, respectively. HHV-6B was isolated from peripheral blood obtained during the acute phase in all three patients, and was detected in the cerebrospinal fluid of Cases 2 and 3. In Case 1, a marked increase in levels of several serum cytokines (IL-1ß, IL-6, and IL-10) and chemokines (IL-8, MIG, MCP-1, and IP-10) was observed at disease onset. Subsequently, serum cytokine levels gradually became undetectable and chemokine levels stabilized by day 11 of illness. In Case 2, only two cytokines (IL-6 and IL-10) were slightly elevated at disease onset. In Case 3, the kinetics appeared to follow an up-and-down pattern. Additionally, in all three patients, TIMP-1 concentrations remained high during the observation period, and MMP-9 decreased quickly a few days after disease onset, and then returned to normal level.

Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts.

The monitoring of active human herpesvirus 6 (HHV-6) B infection is important for distinguishing between the reactivation and latent state of the virus. The aim of this present study is to develop a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay for diagnosis of active viral infection. Primers and probes for in house quantitative RT-PCR methods were designed to detect the three kinetic classes of HHV-6B mRNAs (U90, U12, U100). Stored PBMCs samples collected from 10 patients with exanthem subitum (primary HHV-6B infection) and 15 hematopoietic stem cell transplant recipients with HHV-6B reactivation were used to evaluate reliability for testing clinical samples. Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1-100 ng/reaction) and C(t) value of each gene transcript. The U90 and U12 gene transcripts were detected in all of the peripheral blood mononuclear cells (PBMCs) samples collected in acute period of primary HHV-6B infection. Only one convalescent PBMCs sample was positive for the U90 gene transcript. Additionally, the reliability of HHV-6B quantitative RT-PCRs for diagnosis of viral reactivation in hematopoietic transplant recipients was evaluated. Relative to virus culture, U90 quantitative RT-PCR demonstrated the highest assay sensitivity, specificity, positive predictive value, and negative predictive value. Thus, this method could be a rapid and lower cost alternative to virus culture, which is difficult to perform generally, for identifying active HHV-6B infection.

Rhabdomyolysis in an infant with primary human herpesvirus 6 infection.

We report here the first case of rhabdomyolysis at the time of primary human herpesvirus 6 infection. The patient was a previously healthy 1-year-old girl who developed rhabdomyolysis 4 days after the onset of the primary human herpesvirus 6 infection. No other etiologic agent that might cause rhabdomyolysis was identified.

Correlation between serum matrix metalloproteinase and antigenemia levels in patients infected with rotavirus.

Rotavirus (RV) antigenemia has been reported in patients with gastroenteritis; however, the exact mechanism remains unclear. In order to elucidate the mechanism of RV antigenemia, an association between RV antigenemia and matrix metalloproteinase (MMP) were analyzed. The object of this study was to elucidate the role of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the pathogenesis of RV antigenemia. Forty children admitted to hospital with RV gastroenteritis were enrolled in this study. Paired serum samples were collected at the time of admission and discharge. Enzyme-linked immunosorbent assays (ELISA) were used to detect serum concentrations of viral antigens, MMP-1, -2, -9, -13, TIMP -1, and -2. Cytokines were measured using flow cytometric beads array. RV antigens were significantly higher in serum collected at the time of admission than discharge (P < 0.001). MMP-9 concentrations were significantly higher in serum collected at the time of admission than discharge (P < 0.001). MMP-2 concentrations were significantly lower in serum collected at the time of admission than discharge (P < 0.001). A weak but a significantly positive association (P = 0.034) was observed between RV antigen and MMP-9 in serum collected at the time of admission, and inverse association was observed between RV antigen and MMP-2. In addition, a weak but significantly positive association (P = 0.002) was observed between IL-6 and MMP-9. These data suggest that MMPs may contribute to the pathogenesis of RV antigenemia.

Development of a human herpesvirus 6 species-specific immunoblotting assay.

In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.

Host factors associated with the kinetics of Epstein-Barr virus DNA load in patients with primary Epstein-Barr virus infection.

The aims of this study were to elucidate the kinetics of Epstein-Barr virus (EBV) DNA load in serially collected peripheral blood mononuclear cells of patients with primary EBV infection, and to determine the correlated host factors. Blood samples were collected from 24 patients with primary EBV infection. EBV DNA copy numbers were measured using real-time polymerase chain reaction. Based on the kinetics of EBV DNA load, the 24 patients were divided into two groups: rapid regression and slow regression. Eighteen of the 24 patients (75%) were included in the slow regression and 6 (25%) in the rapid regression group. No statistically significant differences were observed between the two groups in clinical features and laboratory findings. However, acute phase (3 to 10 days after the onset of the illness) serum samples from six children in the slow regression and four in the rapid regression group revealed significantly higher serum interleukin (IL)-1ß (P= 0.018), IL-12 (P= 0.009), tumor necrosis factor-α (P= 0.019), interferon-inducible protein 10, and monokine induced by interferon γ concentrations in the rapid regression than the slow regression group. On the other hand, sera from six children in the slow regression and four in the rapid regression group in the convalescent phase (14 to 21 days after the onset of the illness) showed no statistically significant differences between the two groups in these biomarker concentrations. Based on this, it was concluded that the kinetics of EBV DNA load can be divided to two different patterns after primary EBV infection, and immune response might be associated with viral clearance.

Naturally occurring antibodies in humans can neutralize a variety of influenza virus strains, including H3, H1, H2, and H5.

Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the V(H)1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use V(H)1-69. The possible epitope recognized by these clones is discussed.

Neutralizing anti-gH antibody of Varicella-zoster virus modulates distribution of gH and induces gene regulation, mimicking latency.

The anti-glycoprotein H (gH) monoclonal antibody (anti-gH-MAb) that neutralizes varicella-zoster virus (VZV) inhibited cell-to-cell infection, resulting in a single infected cell without apoptosis or necrosis, and the number of infectious cells in cultures treated with anti-gH-MAb declined to undetectable levels in 7 to 10 days. Anti-gH-MAb modulated the wide cytoplasmic distribution of gH colocalized with glycoprotein E (gE) to the cytoplasmic compartment with endoplasmic reticulum (ER) and Golgi markers near the nucleus, while gE retained its cytoplasmic distribution. Thus, the disintegrated distribution of gH and gE caused the loss of cellular infectivity. After 4 weeks of treatment with anti-gH-MAb, no infectious virus was recovered, even after cultivation without anti-gH-MAb for another 8 weeks or various other treatments. Cells were infected with Oka varicella vaccine expressing hepatitis B surface antigen (ROka) and treated with anti-gH-MAb for 4 weeks, and ROka was recovered from the quiescently infected cells by superinfection with the parent Oka vaccine. Among the genes 21, 29, 62, 63, and 66, transcripts of gene 63 were the most frequently detected, and products from the genes 63 and 62, but not gE, were detected mainly in the cytoplasm of quiescently infected cells, in contrast to their nuclear localization in lytically infected cells. The patterns of transcripts and products from the quiescently infected cells were similar to those of latent VZV in human ganglia. Thus, anti-gH-MAb treatment resulted in the antigenic modulation and dormancy of infectivity of VZV. Antigenic modulation by anti-gH-MAb illuminates a new aspect in pathogenesis in VZV infection and the gene regulation of VZV during latency in human ganglia.

[Varicella vaccine potency and stability during transport and delivery].

Oka varicella vaccine was developed to confer active immunity to varicella-zoster virus (VZV) in immunocompromized and immunocompetent children. It is now used to prevent varicella in about 20 million people worldwide. Although VZV infectivion is relatively unstable compared to other viruses, cell-free virus is stabilized and lyophilized vaccine has been developed. Virus titers were evaluated in vaccine distributed to six clinics in 5 years. Yearly mean virus titers at the vaccine producer were 42,000-67,000 plaque-forming units per dose, corresponding to Oka varicella vaccine (Zostavax) used to prevent zoster and postherpetic neuralgia by Oxman et al. Virus titer was found to be stable during delivery to clinics. Virus titers of varicella vaccine were equivalent to Zostavax and vaccine delivered to clinics had enough virus titer to confer active immunity to VZV in this study.

Different characteristics of human herpesvirus 6 encephalitis between primary infection and viral reactivation.

BACKGROUND: Pathogenesis of human herpesvirus 6 (HHV-6) encephalitis, in particular difference between HHV-6 encephalitis at the time of primary infection and reactivation remains unclear. OBJECTIVES: To elucidate the mechanism of HHV-6 encephalitis at the time of primary infection and reactivation. STUDY DESIGN: Twenty-two HHV-6 encephalitis patients at the time of primary infection, 6 febrile convulsion (FC) patients caused by HHV-6 infection, and 14 FC patients without HHV-6 infection (non HHV-6 FC) were enrolled. Additionally, 7 stem cell transplant recipients with HHV-6 encephalitis and eight adult controls were also enrolled in this study. Cerebrospinal fluid (CSF) HHV-6 DNA copy numbers and biomarkers levels were compared. RESULTS: Low copy number of CSF HHV-6 DNA was detected in 7 of the 22 patients with HHV-6 encephalitis in primary infection, whereas all seven CSF samples collected from post-transplant HHV-6 encephalitis patients contained high viral DNA copy numbers (P<0.001). CSF concentrations of IL-6 (P=0.032), IL-8 (P=0.014), MMP-9 (P=0.004), and TIMP-1 (P=0.002) were significantly higher in patients with HHV-6 encephalitis in primary infection than non-HHV-6 FC. CSF IL-6 (P=0.008), IL-8 (P=0.015), and IL-10 (P=0.019) concentrations were significantly higher in patients with post-transplant HHV-6 encephalitis than adult controls. CONCLUSION: The present study suggests that the characteristics of HHV-6 encephalitis are different between HHV-6 encephalitis at the time of primary infection and reactivation in transplant recipients.

A case of erythema multiforme associated with primary Epstein-Barr virus infection.

We present a case of primary Epstein-Barr virus (EBV) infection with erythema multiforme. A 1-year-old Japanese boy presented with skin eruptions, including typical target lesions and a low-grade fever. Just before the skin biopsy, 95 copies/µg DNA of EBV genome was detected in peripheral blood mononuclear cells, which subsequently increased to 6,834 copies/µg DNA. Skin tissue collected from the skin lesion showed the typical pathologic findings of erythema multiforme. EBV-encoded small nuclear RNA signals were not detected in the skin tissue by in situ hybridization.

Evaluation of reverse transcription loop-mediated isothermal amplification assays for rapid diagnosis of pandemic influenza A/H1N1 2009 virus.

Two genetic diagnosis systems using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology were evaluated: one for detecting the HA gene of the pandemic influenza A/H1N1 2009 virus (H1pdm RT-LAMP) and the other for detecting the matrix gene of the influenza A virus (TypeA RT-LAMP). The competence of these two RT-LAMP assay kits for the diagnosis of the pandemic influenza A/H1N1 2009 virus was compared using real-time RT-PCR assays developed recently on viruses isolated and clinical specimens collected from patients with suspected infection. TypeA RT-LAMP and H1pdm RT-LAMP showed almost the same sensitivity as real-time RT-PCR for viruses isolated. The sensitivity and specificity of TypeA RT-LAMP and H1pdm RT-LAMP were 96.3% and 88.9%, respectively, for clinical specimens. Considering that the ability of the two RT-LAMP assay kits for detection of the pandemic influenza A/H1N1 2009 virus was comparable to that of the real-time RT-PCR assays, and that the assays were completed within 1 hr and did not require any expensive equipment, these two RT-LAMP assays are promising rapid diagnostic tests for the pandemic influenza A/H1N1 2009 virus at the hospital bedside.

Localization of epitopes recognized by monoclonal antibodies that neutralized the H3N2 influenza viruses in man.

Through extensive isolation of neutralizing mAbs against H3N2 influenza viruses representing the in vivo repertoire in a human donor, we examined the relationships between antigenic drift of influenza virus and protective antibodies generated in an infected individual. The majority of mAbs isolated from a donor born in 1960 were divided into three major groups with distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003. In the present study, we developed a new method that allowed us to comprehensively determine the location of epitopes recognized by many mAbs. Original haemagglutinins (HAs) of several strains and chimaeric variants, in which one of the seven sites (A, B1, B2, C1, C2, D or E) was replaced by some other strain-derived sequence, were artificially expressed on the cell surface. The binding activity of mAbs to the HAs was examined by flow cytometry. By using this method, we determined the location of epitopes recognized by 98 different mAbs. Clones that neutralize the 1968-1973 strains bind to site B2/D, A or A/B1. While sites C, E and B were recognized by clones that neutralized the 1977-1993 strains, the majority of these clones bind to site C. Clones that neutralize the 1997-2003 strains bind to site B, A/B1, A/B2 or E/C2.