Metabolic engineering of Escherichia coli to produce a monophosphoryl lipid A adjuvant.

Monophosphoryl lipid A (MPLA) species, including MPL (a trade name of GlaxoSmithKline) and GLA (a trade name of Immune Design, a subsidiary of Merck), are widely used as an adjuvant in vaccines, allergy drugs, and immunotherapy to boost the immune response. Even though MPLA is a derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, bacterial strains producing MPLA have not been found in nature nor engineered. In fact, MPLA generation involves expensive and laborious procedures based on synthetic routes or chemical transformation of precursors isolated from Gram-negative bacteria. Here, we report the engineering of an Escherichia coli strain for in situ production and accumulation of MPLA. Furthermore, we establish a succinct method for purifying MPLA from the engineered E. coli strain. We show that the purified MPLA (named EcML) stimulates the mouse immune system to generate antigen-specific IgG antibodies similarly to commercially available MPLA, but with a dramatically reduced manufacturing time and cost. Our system, employing the first engineered E. coli strain that directly produces the adjuvant EcML, could transform the current standard of industrial MPLA production.

Fusion of pseudorabies virus glycoproteins to IgG Fc enhances protective immunity against pseudorabies virus.

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.

Production of controlled molecular weight hyaluronic acid by glucostat strategy using recombinant Lactococcus lactis cultures.

Hyaluronic acid (HA) is a biopolymer with wide biomedical and cosmetic applications, wherein the molecular weight of HA (MWHA) is an important quality parameter that determines its suitability for the targeted application. To produce HA with desired molecular weight, it is important to identify parameters that offer tunability and control of MWHA at a desired value during fermentation. In this work, two tunable parameters, viz. glucose concentration and combination of HA biosynthetic genes expressed, were used to produce HA of different molecular weights. Three recombinant strains of Lactococcus lactis were constructed, using a combination of the has-operon genes from Streptococcus zooepidemicus (hasA, hasB, hasE) and the α-phosphoglucomutase gene (pgmA) from L. lactis. Batch fermentations of these recombinant strains at different initial glucose concentrations enabled production of HA with different molecular weights. Co-expression of hasABE was observed to be particularly effective in improving the MWHA. It was observed during batch fermentations of all these recombinant L. lactis cultures that the MWHA decreases steadily during the later part of the fermentation and the final value is 19-43% lower than the peak MWHA produced. Analysis of the fermentation data showed that the decrease in MWHA correlated strongly with the decrease in specific productivity of the culture. To overcome this decrease in MWHA, a glucostat strategy was successfully devised which could maintain a high value of specific productivity throughout the glucostat phase and result in constant-MW HA production. Glucostat processes were designed with the three recombinant L. lactis strains at two different glucose concentrations to produce constant molecular weight HA ranging from 0.4 to 1.4 MDa. This is the first report of its kind in literature that demonstrates production of controlled MW HA over a wide range by using a combination of tunable parameters and suitable process control strategies.

Elucidating Bottlenecks to the Efficient Preparation of AB5-Hexamer Mucosal Adjuvant Protein LTm by Genetic Engineering.

Escherichia coli heat-labile enterotoxin (LT) and its non-toxic mutant (LTm) are well-known powerful mucosal adjuvants and immunogens. However, the yields of these adjuvants from genetically engineered strains remain at extremely low levels, thereby hindering their extensive application in fundamental and clinical research. Therefore, efficient production of these adjuvant proteins from genetically engineered microbes is a huge challenge in the field of molecular biology. In order to explore the expression bottlenecks of LTm in E. coli, we constructed a series of recombinant plasmids based on various considerations and gene expression strategies. After comparing the protein expression among strains containing different recombinant plasmids, the signal sequence was found to be critical for the expression of LTm and its subunits. When the signal sequence was present, the strong hydrophobicity and instability of this amino acid sequence greatly restricted the generation of subunits. However, when the signal sequence was removed, abundantly expressed subunits formed inactive inclusion bodies that could not be assembled into the hexameric native form, although the inclusion body subunits could be refolded and the biological activity recovered in vitro. Therefore, the dilemma choice of signal sequence formed bottlenecks in the expression of LTm. These results reveal the expression bottlenecks of LTm, provide guidance for the preparation of LTm and its subunits, and certainly help to promote efficient preparation of this mucosal adjuvant protein.

Relation between hyaluronan synthesis and cell morphology in ovarian clear cell carcinomas.

Ovarian clear cell carcinomas often show a spherule-like mucoid stroma. In ascitic fluid, they form spheroids with a hollow acellular space. In spite of the absence of stromal cells, both the mucoid stroma and hollow spheroids contain abundant extracellular matrix, and one of the major components is hyaluronan. It has been suggested that tumor-derived hyaluronan plays a significant role in the formation of these structures. To clarify this, a hyaluronan inhibition assay was performed on HAC-2, a clear cell carcinoma cell line, in vitro. When hyaluronan synthesis was inhibited by 4-methylumbelliferone, HAC-2 failed to show the spherule-like accumulation of hyaluronan or hollow spheroids. Inhibition of hyaluronan synthesis was associated with the reduction of cell growth. Analysis of 28 archival ascites cytology specimens showed that clear cell carcinomas expressed hyaluronan more frequently than serous carcinomas (11 of 14 vs 3 of 14, respectively, P < 0.05). All of these facts indicate that tumor-derived hyaluronan is essential for the formation of the mucoid stroma or hollow spheroids, and that hyaluronan is also involved in the regulation of cell growth in ovarian clear cell carcinomas. The inhibition of hyaluronan synthesis could be a potential adjunctive therapy for refractory clear cell carcinomas outside the ovary.

Interaction between antimicrobial peptides and mycobacteria.

Mycobacteria can cause different severe health problems, including tuberculosis (TB). The treatment of TB with conventional antibiotics is successful, however, the number of multi-drug and extensively-drug resistant Mycobacterium tuberculosis strains increases. Moreover, many classical antimycobacterial antibiotics have severe side effects. Therefore, antimicrobial peptides (AMPs) seem to be good candidates for new therapeutic strategies. On the one hand AMPs can be used as a single drug or in combination with conventional antibiotics to directly kill mycobacteria, or on the other hand to act as immunstimulatory agents. This review summarizes the findings on the role of endogenous human AMPs being involved in TB, the antimycobacterial activity of various AMPs, and the molecular modes of action. Most active AMPs interact with the mycobacterial cell envelope and in particular with the mycomembrane and the plasma membrane. The mycomembrane is a very rigid membrane probably leading to a lower activity of the AMPs against mycobacteria as compared to other Gram-negative or Gram-positive bacteria. For some AMPs also other targets have been identified. Because of the complex environment of intracellular mycobacteria being trapped in the phagosome, within the macrophage, within the granuloma, within the lung, the external administration of AMPs in the latent phase of TB is a challenge. However, in the acute phase the AMPs can attack mycobacteria in a direct way. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Rescue and evaluation of a recombinant PRRSV expressing porcine Interleukin-4.

BACKGROUND: The current vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) have failed to provide broad protection against infection by various strains of PRRSV. Porcine Interleukin-4 (pIL-4) plays an important role in the regulation of the immune response and has been used previously as an immunological adjuvant. The objective of this study was to construct a recombinant PRRSV expressing pIL-4 and to evaluate the immune response of the recombinant virus in piglets. METHODS: The pIL-4 gene was inserted in the PRRSV (CH-1R strain) infectious clone by overlap PCR. Indirect immunofluorescence assay (IFA) and Western blotting were used to confirm the recombinant virus. The stability of the recombinant virus was assessed by DNA sequencing and IFA after 15 passages in vitro. Recombinant virus was injected into pigs and efficacy of immune protection was evaluated in comparison with the parental virus. RESULTS: The recombinant virus (CH-1R/pIL-4) was successfully rescued and shown to have similar growth kinetics as the parental virus. The recombinant virus was stable for 15 passages in cell culture. Pigs vaccinated with CH-1R/pIL-4 produced a similar humoral response to the response elicited by parental virus, but IL-4 level in the supernatant of PBMCs from pigs vaccinated with CH-1R/pIL-4 was significantly higher than the parent virus at 28 days post-immunization (DPI). Flow cytometric (FCM) analysis showed that the percentage of CD4(+)CD8(+) double positive T (DPT) cells in the CH-1R/pIL-4 vaccinated group was significantly higher than the parental virus at 3 and 7 Days Post-Challenge (DPC), and the IL-4 level in the blood significantly increased at 7 DPC. However, the viral load and histopathology did not show significant difference between the two groups. CONCLUSIONS: A recombinant PRRSV expressing porcine IL-4 was rescued and it remained genetically stable in vitro. The recombinant virus induced higher DPT ratios and IL-4 levels in the blood after HP-PRRSV challenge compared to the parental virus in piglets. However, it did not significantly improve protection efficacy of PRRSV vaccine.

Biosynthetically engineered lipopolysaccharide as vaccine adjuvant.

Lipopolysaccharide (LPS), a dominant component of the Gram-negative bacterial outer membrane, is a strong activator of the innate immune system, and thereby an important determinant in the adaptive immune response following bacterial infection. This adjuvant activity can be harnessed following immunization with bacteria-derived vaccines that naturally contain LPS, and when LPS or molecules derived from it are added to purified vaccine antigens. However, the downside of the strong biological activity of LPS is its ability to contribute to vaccine reactogenicity. Modification of the LPS structure allows triggering of a proper immune response needed in a vaccine against a particular pathogen while at the same time lowering its toxicity. Extensive modifications to the basic structure are possible by using our current knowledge of bacterial genes involved in LPS biosynthesis and modification. This review focuses on biosynthetic engineering of the structure of LPS and implications of these modifications for generation of safe adjuvants.

HIV-1 adenoviral vector vaccines expressing multi-trimeric BAFF and 4-1BBL enhance T cell mediated anti-viral immunity.

Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia-Gag challenge, but the protection was independent of standard immune markers. Soluble multi-trimeric SP-D-4-1BBL and SP-D-BAFF provide a novel technology to enhance adenoviral vector vaccines against HIV-1.

Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

Separation and characterization of the immunostimulatory components in unpolished rice black vinegar (kurozu).

Unpolished rice black vinegar (kurozu), a traditional Japanese vinegar, is considered to have beneficial health effects. Kurozu is produced via a static fermentation process involving the saccharification of rice by Aspergillus oryzae, alcohol fermentation by Saccharomyces cerevisiae, and the oxidation of ethanol to acetic acid by acetic acid bacteria such as Acetobacter pasteurianus. Since this process requires about 6 months' fermentation and then over a year of aging, most of these organisms die during the production process and so microbial components, which might stimulate the innate immune system, are expected to be present in the vinegar. In this study, we investigated whether microbial components are present in kurozu, and after confirming this we characterized their immunostimulatory activities. Lyophilized kurozu stimulated murine spleen cells to produce tumor necrosis factor (TNF)-α, at least in part, via Toll-like receptor (TLR) 2 and the Nod-like receptors NOD1 and 2. The active components associated with TLR2 activation were concentrated by Triton X-114-water phase partitioning and hydrophobic interaction chromatography on Octyl Sepharose. TLR4-activating components were also enriched by these methods. The concentrated preparation stimulated murine spleen cells to produce TNF-α and interferon (IFN)-γ. These results indicate that long-term fermented kurozu contains immunostimulatory components and that the TLR2 and TLR4-activating immunostimulatory components of kurozu are hydrophobic. These components might be responsible for the beneficial health effects of kurozu.

Production of recombinant immunotherapeutics for anticancer treatment: the role of bioengineering.

Cancer is one of the most important health problems because many cases are difficult to prevent. Cancer still has unknown mechanisms of pathogenesis, and its capacity to produce temporary or permanent damage, besides death, is very high. Although many anticancer therapies are available, finding a cure for cancer continues to be a difficult task. Thus, many efforts have been made to develop more effective treatments, such as immunotherapy based on a new class of tumor-specific products that are produced using recombinant DNA technology. These recombinant products are used with the main objectives of killing the tumor and stimulating immune cells to respond to the cancer cells. The principal recombinant products in anticancer therapy are immunostimulants, vaccines, antibodies, immunotoxins and fusion proteins. This review focuses on the general aspects of these genetically engineered products, their clinical performance, current advances and future prospects for this type of anticancer therapy.

Suppression of hyaluronan synthesis alleviates inflammatory responses in murine arthritis and in human rheumatoid synovial fibroblasts.

OBJECTIVE: To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis. METHODS: DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively. RESULTS: Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor α-stimulated FLS, in a dose-dependent manner. The 4-MU-induced decreases in the serum HA concentration in mice with CIA and in "medium" and "pericellular" HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression. CONCLUSION: Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.

Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity.

The development of safe live, attenuated Salmonella vaccines may be facilitated by detoxification of its LPS. Recent characterization of the lipid A 1-phosphatase, LpxE, from Francisella tularensis allowed us to construct recombinant, plasmid-free strains of Salmonella that produce predominantly 1-dephosphorylated lipid A, similar to the adjuvant approved for human use. Complete lipid A 1-dephosphorylation was also confirmed under low pH, low Mg(2+) culture conditions, which induce lipid A modifications. LpxE expression in Salmonella reduced its virulence in mice by five orders of magnitude. Moreover, mice inoculated with these detoxified strains were protected against wild-type challenge. Candidate Salmonella vaccine strains synthesizing pneumococcal surface protein A (PspA) were also confirmed to possess nearly complete lipid A 1-dephosphorylation. After inoculation by the LpxE/PspA strains, mice produced robust levels of anti-PspA Abs and showed significantly improved survival against challenge with wild-type Streptococcus pneumoniae WU2 compared with vector-only-immunized mice, validating Salmonella synthesizing 1-dephosphorylated lipid A as an Ag-delivery system.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

In planta production of plant-derived and non-plant-derived adjuvants.

Recombinant antigen production in plants is a safe and economically sound strategy for vaccine development, particularly for oral/mucosal vaccination, but subunit vaccines usually suffer from weak immunogenicity and require adjuvants that escort the antigens, target them to relevant sites and/or activate antigen-presenting cells for elicitation of protective immunity. Genetic fusions of antigens with bacterial adjuvants as the B subunit of the cholera toxin have been successful in inducing protective immunity of plant-made vaccines. In addition, several plant compounds, mainly plant defensive molecules as lectins and saponins, have shown strong adjuvant activities. The molecular diversity of the plant kingdom offers a vast source of non-bacterial compounds with adjuvant activity, which can be assayed in emerging plant manufacturing systems for the design of new plant vaccine formulations.

Plant-based anti-HIV-1 strategies: vaccine molecules and antiviral approaches.

The introduction of highly active antiretroviral therapy has drastically changed HIV infection from an acute, very deadly, to a chronic, long-lasting, mild disease. However, this requires continuous care management, which is difficult to implement worldwide, especially in developing countries. Sky-rocketing costs of HIV-positive subjects and the limited success of preventive recommendations mean that a vaccine is urgently needed, which could be the only effective strategy for the real control of the AIDS pandemic. To be effective, vaccination will need to be accessible, affordable and directed against multiple antigens. Plant-based vaccines, which are easy to produce and administer, and require no cold chain for their heat stability are, in principle, suited to such a strategy. More recently, it has been shown that even highly immunogenic, enveloped plant-based vaccines can be produced at a competitive and more efficient rate than conventional strategies. The high variability of HIV epitopes and the need to stimulate both humoral neutralizing antibodies and cellular immunity suggest the importance of using the plant system: it offers a wide range of possible strategies, from single-epitope to multicomponent vaccines, modulators of the immune response (adjuvants) and preventive molecules (microbicides), either alone or in association with plant-derived monoclonal antibodies, besides the potential use of the latter as therapeutic agents. Furthermore, plant-based anti-HIV strategies can be administered not only parenterally but also by the more convenient and safer oral route, which is a more suitable approach for possible mass vaccination.

Cerebral interleukin-15 shows upregulation and beneficial effects in experimental autoimmune encephalomyelitis.

Interleukin (IL)-15 can cross the blood-brain barrier to act on its specific brain receptor (IL15Ralpha) and co-receptors. The important roles of neuronal IL15 and IL15Ralpha in experimental autoimmune encephalomeylitis (EAE) are suggested by the upregulation of IL15Ralpha mRNA in different regions of the brain and spinal cord, and by double-labeling immunohistochemistry showing neuronal localization of IL15 and IL15Ralpha in different neurons. Contrary to expectations, IL15 treatment lessened EAE severity. IL15 knockout mice showed heightened susceptibility to EAE with significantly higher scores that were decreased by treatment with IL15. Thus, IL15 improves this CNS autoimmune disorder as a potential therapeutic agent.

Constitutive CD40L expression on B cells prematurely terminates germinal center response and leads to augmented plasma cell production in T cell areas.

CD40/CD40L engagement is essential to T cell-dependent B cell proliferation and differentiation. However, the precise role of CD40 signaling through cognate T-B interaction in the generation of germinal center and memory B cells is still incompletely understood. To address this issue, a B cell-specific CD40L transgene (CD40LBTg) was introduced into mice with B cell-restricted MHC class II deficiency. Using this mouse model, we show that constitutive CD40L expression on B cells alone could not induce germinal center differentiation of MHC class II-deficient B cells after immunization with T cell-dependent Ag. Thus, some other MHC class II-dependent T cell-derived signals are essential for the generation of germinal center B cells in response to T cell-dependent Ag. In fact, CD40LBTg mice generated a complex Ag-specific IgG1 response, which was greatly enhanced in early, but reduced in late, primary response compared with control mice. We also found that the frequency of Ag-specific germinal center B cells in CD40LBTg mice was abruptly reduced 1 wk after immunization. As a result, the numbers of Ag-specific IgG1 long-lived plasma cells and memory B cells were reduced. By histology, large numbers of Ag-specific plasma cells were found in T cell areas adjacent to Ag-specific germinal centers of CD40LBTg mice, temporarily during the second week of primary response. These results indicate that CD40L expression on B cells prematurely terminated their ongoing germinal center response and produced plasma cells. Our results support the notion that CD40 signaling is an active termination signal for germinal center reaction.