Naoko Nakagawa

B Cell Growth and Differentiation Factors and Mechanism of B Cell Activation

B cells, one of the best understood eukariotic cells, originate from pluripotent hematopoietic stem cells and differentiate into immunoglobulin {Ig)-secreting cells through multistep developmental stages, such as pre-pre-B cells, pre-B cells, immature B cells, mature B cells, activated B cells and Ig-secreting cells. An understanding of the stimuli which cause the activation, proliferation and differentiation of B cells is critical to the delineation of the normal regulation of the immune responses as well as proliferation and differentiation of eukariotic cells. Since the discovery of T-B collaboration in the antibody response, extensive studies on the regulatory molecules involved in B cell activation have been done. More than a decade ago, Dutton and his colleagues (1971) suggested the involvement of soluble helper factors in the T cell-dependent activation of B cells into Ig-secreting cells. Since then, hundreds of factors, antigen specific or nonspecific, have been reported and those results have supported the notion that helper or suppressor function of T cells in B cell activation can be replaced by the soluble products released from T cells. Kishimoto and Ishizaka (1975) and Kishimoto and his colleagues (1975) have shown Ig-induction in rabbit B cells by anti-Ig and T cell-derived helper factors, suggesting that 2 signals, crosslinkage of Ig-receptors and helper factors, could induce activation of B cells into Igproducing cells. This finding was confirmed by several investigators in murine or human B cells (Parker etal. 1979, Isakson etal. 1981, Yoshizaki et al. 1982)and also supported the notion that helper function of T cells was mediated by T cellderived helper factors.

Reactivation of human herpesvirus 6 by infection of human herpesvirus 7.

We have attempted to reactivate human herpesvirus 6 (HHV‐6) by infection with HHV‐7 using childhood exanthem subitum patients in vitro. Peripheral blood mononuclear cells (PBMCs) were collected from children who had a history of exanthem subitum(ES) by HHV‐6 and were infected by human herpesvirus 7 (HHV‐7) in vitro. The antigen positive rate to HHV‐6 started to increase 7 days after the infection and reached a maximum by Day 15 using an immunofluorescence antibody test. The copy number of HHV‐6 DNA also increased in the samples in 10 days after infection in vitro. No antigen or increase in DNA was detected in PBMCs, that were mock‐infected or infected with supernatant of stock virus after ultracentrifugation, suggesting that an infection by HHV‐7 is necessary to reactivate HHV‐6. In the paired sera samples during the acute and the convalescent phases of ES, seven to ten bands, that were specific for HHV‐6, were recognized in samples from the acute phase, and at least 5 dominant polypeptides were found more intensively after HHV‐7 infection. J. Med. Virol. 60:284–289, 2000.

Antigenic variants with amino acid deletions clarify a neutralizing epitope specific for influenza B virus Victoria group strains

To study the neutralizing epitopes of influenza B virus Victoria group strains, two monoclonal antibodies (MAbs) were used to select antigenic variants of the virus. MAbs 10B8 and 8E6 were found to react with B/Victoria group strains in three tests, peroxidase-antiperoxidase staining, haemagglutination inhibition and neutralization tests; no reactivity with B/Yamagata group strains was observed. Analysis of the deduced amino acid sequences of 10B8-induced variants identified a single amino acid deletion at residue 165 or 170, as well as a single amino acid substitution at residues 164 (Asp-->Tyr), 165 (Asn-->Ser or Thr) or 203 (Lys-->Thr or Asn). A single amino acid substitution at residue 241 (Pro-->Ser) was observed in 8E6-induced variants. Three-dimensional analysis showed that the epitopes for both MAbs were situated in close proximity to each other. Since B/Yamagata group strains are characterized by amino acid deletions at residues 164-166, the epitope for MAb 10B8 is strictly specific for B/Victoria group strains.

Monoclonal antibodies in man that neutralized H3N2 influenza viruses were classified into three groups with distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

We tried to reveal the strain specificity of neutralizing mAbs against H3N2 influenza viruses in individuals. A large number of B lymphocytes of a pediatrician were collected by apheresis and two Ab libraries were constructed at 2004 and 2007 by using the phage-display technology. The libraries were screened against 12 different H3 strains of flu isolated between 1968 and 2004. Large numbers of clones that bound to the Ags were isolated and mAbs that specifically bound to H3 strain viruses were selected. Their binding activity to the 12 strains and neutralizing activity were studied by ELISA and focus reduction test, respectively. Furthermore, the binding activity to hemagglutinin (HA) was examined by Western blot. The majority of clones showing the neutralizing activity turned out to be anti-HA mAbs and could be divided into three major groups showing distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

Sensitivity of rapid immunoassay for influenza A and B in the early phase of the disease.

Background:  Immunochromatography (IC) tests are often used for the rapid diagnosis of influenza. Once influenza is diagnosed, an anti‐influenza drug can be administered. Physicians claim, however, that they are not sufficiently sensitive, especially in the early phase of the disease. The aim of the present study was therefore to analyze the sensitivity of the IC test from the standpoint of virology.

Emergence of an Influenza B Virus with Antigenic Change

ABSTRACT Influenza B virus Yamagata group strains, isolated in the 2000 to 2001 influenza epidemic season, reacted poorly to the polyclonal ferret sera prepared against strains isolated earlier. The results of genetic analysis clarified that a point mutation of the nucleotide at position 126 in the HA1 region and the corresponding one-amino-acid substitution altered viral antigenicity.

Antigenic analysis of human herpesvirus 7 (HHV-7) and HHV-6 using immune sera and monoclonal antibodies against HHV-7

Using polyclonal and monoclonal (MAbs) antibodies to human herpesvirus 7 (HHV-7), we have studied HHV-7-specific polypeptides. Human sera were obtained during the convalescent phase from patients with exanthem subitum due to HHV-7, and at least 16 HHV-7-specific polypeptides with apparent molecular masses of 26-210 kDa were immuno-precipitated. Sera prepared in mice also precipitated at least 17 HHV-7-specific polypeptides with molecular masses of 26-210 kDa. Among them, the most commonly observed antigenic protein had an apparent molecular mass of 52 kDa. Forty-two clones secreting MAbs against HHV-7-specific proteins, as determined by immunofluorescence assays, were established from BALB/c mice immunized with HHV-7-infected cell extracts. Seven MAbs which immunoprecipitated HHV-7-specific polypeptides were further characterized. Two of these, MAbs 5E12 and 5F12, reacted predominantly with glyco-proteins of 78 kDa and 85 kDa, respectively, and possessed neutralizing activity. This suggests that there are at least two neutralization-inducing proteins in HHV-7. MAb 16B4 reacted with the major immunogenic protein of 52 kDa. Five of the 42 MAbs also reacted in immunofluorescence assays with HHV-6 antigens to the same degree as to HHV-7. Two other MAbs, 7C10 and 10F1, recognized an HHV-7 protein of 40 kDa, and only 7C10 cross-reacted with an HHV-6 protein of 45 kDa.

Variation of the conserved neutralizing epitope in influenza B virus victoria group isolates in Japan.

ABSTRACT For almost 20 years, the neutralizing-epitope site specific for influenza B virus Victoria group isolates was conserved at the “tip” of the hemagglutinin molecule; however, it was not detected in half of the isolates from the 2002-2003 epidemic in Japan. Amino acid substitutions (D164E or N165K) were observed at the “tip,” and the epitope was altered. The viral antigenicities were affected, and human antibodies did not substantially inhibit the hemagglutination in the hemagglutination inhibition tests. It is suspected that such variants will be important in future epidemics.

Detection of antigenic variants of the influenza B virus by melting curve analysis with LCGreen

Automated, high-throughput detection methods for single-nucleotide polymorphisms have been applied to the routine genotyping of genetic polymorphisms influencing drug metabolism. Melting curve analysis with LCGreen was introduced recently as one such technique which can be performed rapidly and easily. This technique was used to detect antigenic variants of the influenza B virus. The antigenic variants and vaccine-type strains of the influenza B virus are isolated from clinical specimens of one epidemic season, and they usually differ in one nucleotide in the HA1 gene, corresponding to one amino-acid substitution. By means of melting curve analysis with LCGreen, an antigenic variant clone and a vaccine-type clone were clearly distinguished. In addition, the proportions of the antigenic variants in the mixture-type isolates were estimated. The clinical isolates were detected as the vaccine-type strains, antigenic variants, or a mixture of both. It became clear that humans were infected with a mixture of the vaccine-type strains and the antigenic variants for a certain period after which the viral antigenicities vary. This technique will contribute to the analysis of antigenic shifts in influenza B virus.

Discovery of the Neutralizing Epitope Common to Influenza B Virus Victoria Group Isolates in Japan

ABSTRACT Monoclonal antibody 9B2 possesses hemagglutination inhibition activity against all the 2002/2003 influenza B virus Victoria group isolates in Kobe, Japan, as well as representative strains isolated between 1987 and 1997. The 9B2 epitope localizes three-dimensionally in the vicinity of antigenic site A of the hemagglutinin molecule, and amino acid substitutions in this region affected the binding of 9B2.