Kazuo Ohnishi

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

Abstract The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.

The Identification of a Nonclassical Cadherin Expressed during B Cell Development and Its Interaction with Surrogate Light Chain

A 130-kDa glycoprotein (p130) has been found to be associated with surrogate light chain on pro- and pre-B I cells. Using peptide sequences obtained from purified p130 we have cloned its gene. The gene encodes a typical cadherin type 1 membrane protein with six extracellular cadherin domains (one pseudo domain) but lacking the catenin-binding site in its cytoplasmic part. Even without this catenin-binding site, p130 mediates Ca2+-dependent homotypic adhesion of cells. The interaction of p130 with surrogate light chain is confirmed by co-transfection and co-immunoprecipitation experiments. The expression of p130 is biphasic during the B cell development. Reverse transcriptase-polymerase chain reaction and flow cytometric analyses revealed that it is expressed on B220+c-Kit+ pro-B and pre-B-I cells as well as on B220+CD25−IgM+ immature and mature B cells but not on B220+CD25+pre-B-II cells. It is also expressed in fetal liver, at low levels in myeloid cells, and strongly in intestinal epithelial cells. In the spleen, p130-expressing cells are mainly localized in the marginal zone. We call this B lineage-, intestine-, liver- and leukocyte-expressed gene BILL-cadherin. The possible functions of BILL-cadherin in B cell development are discussed.

Lymphocyte‐expressed BILL‐cadherin/cadherin‐17 contributes to the development of B cells at two stages

The gene encoding BILL‐cadherin/cadherin‐17, a nonclassical cadherin expressed on B lymphocytes in a stage‐and‐site‐specific manner, was inactivated by targeted disruption of its transmembrane/cytoplasmic portion‐encoding parts. BILL‐cadherin deficiency caused a threefold proB cell accumulation, as well as a reduction to half of the numbers of immature B cells in bone marrow. In spleen, CD21hiCD23lo marginal zone B cells were found reduced and the structure of the marginal zone was impaired. In addition, the size and number of germinal center as well as the number of PNA+ cells were significantly reduced in BILL‐cadherin‐deficient mice. In the peritoneal cavity of mutant mice IgM+Mac‐1+CD5+ B1a cell, that express high BILL‐cadherin in wild‐type mice, was also reduced in number. The IgG1 and IgG3 antibody response to the T‐independent antigen, TNP‐Ficoll, was impaired in the mutant mice. These results indicate that BILL‐cadherin participates in B lymphocyte development at least at two stages, first at the transition of pro/preB‐I cells to preB‐II cells possibly in association with surrogate light chain in bone marrow, and later at the point of development, accumulation and reactiveness of immature B cells in spleen.

Severe acute respiratory syndrome (SARS) coronavirus: application of monoclonal antibodies and development of an effective vaccine

SARS‐CoV is a new type of human coronavirus identified as a causative agent of severe acute respiratory syndrome (SARS). On the occasion of the SARS outbreak, various monoclonal antibodies (mAbs) against SARS‐CoV have been developed and applied for diagnosis, clinical management and basic research. In this review, we overview the biochemical and functional properties and applications of these SARS‐CoV mAbs. We also focus on a variety of vaccines currently under development and discuss the immune response elicited by these vaccines in animal models, hopefully to better understand what we need to do next to fight against newly emerging pathogens in the future. Copyright

The Non-Ig Parts of the VpreB and lambda 5 Proteins of the Surrogate Light Chain Play Opposite Roles in the Surface Representation of the Precursor B Cell Receptor

The VpreB and λ5 proteins, together with Igμ-H chains, form precursor BCRs (preBCRs). We established λ5−/−/VpreB1−/−/VpreB2−/− Abelson virus-transformed cell lines and reconstituted these cells with λ5 and VpreB in wild-type form or with a deleted non-Ig part. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, whereas deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65, or PLC-γ2, and of Ca2+ mobilization from intracellular stores, stimulated by μH chain crosslinking Ab were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH domains of IgH chains for later pairing with IgL chains, and its non-Ig part fixes the preBCRs on the surface. By contrast, the non-Ig part of λ5 crosslinks preBCRs for downregulation and stimulation.

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV.

Abstract In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.

Deciphering antigen-responding antibody repertoires by using next-generation sequencing and confirming them through antibody-gene synthesis

Vast diversity and high specificity of antigen recognition by antibodies are hallmarks of the acquired immune system. Although the molecular mechanisms that yield the extremely large antibody repertoires are precisely understood, comprehensive description of the global antibody repertoire generated in individual bodies has been hindered by the lack of powerful measures. To obtain holistic understanding of the antibody-repertoire space, we used next-generation sequencing (NGS) to analyze the deep profiles of naive and antigen-responding repertoires of the IgM, IgG1, and IgG2c classes formed in individual mice. The overall landscapes of naive IgM repertoires were almost the same for each mouse, whereas those of IgG1 and IgG2c differed considerably among naive individuals. Next, we immunized mice with a model antigen, nitrophenol (NP)-hapten linked to chicken γ-globulin (CGG) carrier, and compared the antigen-responding repertoires in individual mice. To extract the complete antigen response, we developed an intelligible method for detecting common components of antigen-responding repertoires. The major responding antibodies were IGHV1-72/IGHD1-1/IGHJ2 for NP-hapten and IGHV9-3/IGHD3-1/IGHJ2 for CGG-carrier protein. The antigen-binding specificities of the identified antibodies were confirmed through ELISA after antibody-gene synthesis and expression of the corresponding NGS reads. Thus, we deciphered antigen-responding antibody repertoires by inclusively analyzing the antibody-repertoire space generated in individual bodies by using NGS, which avoided inadvertent omission of key antibody repertoires.

Broad cross-reactive epitopes of the H5N1 influenza virus identified by murine antibodies against the A/Vietnam/1194/2004 hemagglutinin.

There is an urgent need for a rapid diagnostic system to detect the H5 subtype of the influenza A virus. We previously developed monoclonal antibodies (mAbs) against the H5 hemagglutinin (HA) for use in a rapid diagnostic kit. In this study, we determined the epitopes of the anti-H5 HA murine mAbs OM-b, AY-2C2, and YH-1A1. Binding assays of the mAbs to different strains of H5 HAs indicated that OM-b and AY-2C2 cross-reacted with HAs from clades 1, 2.1.3.2, 2.2, and 2.3.4, whereas YH-1A1 failed to bind to those of clades 2.1.3.2 and 2.3.4. HA chimeras revealed that the epitopes for each of the mAbs were in the HA1 region. Analysis of escape mutants revealed that OM-b and AY-2C2 mAbs interacted mainly with amino acid residues D43 and G46, and the YH-1A1 mAb interacted with G139 and K or R140 of H5 HA. Multiple alignments of H5 HA protein sequences showed that D43 and G46 were very conserved among H5N1 HAs, except those in clade 2.2.1 and clade 7 (88.7%). The epitope for YH-1A1 mAb was highly variable in the HAs of H5N1, although it was well conserved in those of H5N2-N9. The OM-b and AY-2C2 mAbs could bind to the HAs of clades 1.1 and 2.3.2.1 that are currently epidemic in Asia, and we conclude that these would be effective for the detection of H5N1 infections in this region.

BILL-cadherin/cadherin-17 contributes to the survival of memory B cells.

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) are responsible for immunological “memory”, which can last for many years. The long-term survival niche for LLPCs in the bone marrow is well characterized; however, the corresponding niche for MBCs is unclear. BILL-cadherin/cadherin-17 (CDH17) is the only member of the cadherin superfamily that is expressed on mouse B lymphocytes in a spatiotemporally regulated manner. Here, we show that half of all MBCs regain expression of CDH17 during the later stage of development. The maintenance of high affinity antigen-specific serum antibodies was impaired in CDH17-/- mice and the number of antigen-specific MBCs was reduced as compared to wild-type mice (WT). Also, specific responses to secondary antigens were ablated in CDH17-/- mice, whereas primary antibody responses were the same as those in WT mice. Cell cycle analysis revealed a decline in the proliferation of CDH17- MBCs as compared to CDH17+ MBCs. In addition, we identified a subpopulation of splenic stromal cells, MAdCAM-1+ blood endothelial cells (BEC), which was CDH17+. Taken together, these results suggest that CDH17 plays a role in the long-term survival of MBCs, presumably via an “MBC niche” comprising, at least in part, BEC in the spleen.

Reprint of: Environments of B cell development

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.