Citrulline as a novel adjuvant candidate for vaccines.

For a long time, many types of vaccines have been useful for the prophylaxis of many infectious diseases. Thus far, many adjuvants that enhance the effects of vaccines have been explored. However, very few adjuvants are being used for humans worldwide. In this study, we investigated the adjuvant activity of various substances, and found citrulline to have high potential as an adjuvant. Citrulline is a type of amino acid present in the body of many organisms. A number of biological activities of citrulline have been reported; however, no adjuvant activity has been reported thus far. Aluminum salts, which are commonly used as adjuvants are not water soluble; therefore, some difficulties are encountered while using them as vaccine adjuvants. Citrulline is easy to use because of its water solubility. In this study, we showed for the first time the adjuvant activity of citrulline by using viral antigens and amyloid ß peptide. Water-soluble citrulline, which is present in our body, is a potential adjuvant candidate.

Influenza virus M2e with additional cysteine residues shows enhanced immunogenicity and protection against lethal virus challenge.

The amino acid sequence of the extracellular domain of matrix protein 2 (M2e) is conserved among all subtypes of influenza A viruses. Therefore, the M2e peptide can be considered as a target antigen for the development of a universal influenza vaccine. We evaluated the effects of adding cysteine residues to a peptide of amino acids 2-24 of M2e. Mice immunized with some of these peptides containing one, two, three, four, or five extra cysteines displayed enhanced antibody titers to M2e. In addition, immunization with a peptide containing three extra cysteines, along with an aluminum adjuvant, protected mice more effectively against a lethal influenza virus challenge than the original M2e peptide. These results indicated that an M2e peptide containing additional cysteine residues could be a universal influenza vaccine candidate even without the addition of strong adjuvants.

Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E.

A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

Characterization of neutralizing mouse-human chimeric and shuffling antibodies against botulinum neurotoxin A.

Mouse-human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2-4, 2-5, 9-4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V(H)) and light chains (V(L)) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and mouse-human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V(H) or V(L) domains were constructed. A shuffling antibody (AC2494) that derived its V(H) and V(L) domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high-potency neutralizing chimeric antibodies by selecting and shuffling V(H) and V(L) domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.

Amyloid beta peptides with an additional cysteine residue can enhance immunogenicity and reduce the amyloid beta burden in an Alzheimer's disease mouse model.

For the development of a safe vaccine for Alzheimer's disease (AD), we studied the immunogenicity of amyloid beta (Abeta) peptides without adjuvant. Addition of a cysteine residue (Cys) to Abeta peptides enhanced immunogenicity in mice compared to those without Cys. Vaccination with the Abeta-Cys peptides reduced Abeta deposits in AD model mice. From these results, the Abeta-Cys peptides, administered without adjuvant, are considered candidates for vaccine therapy for AD.

High-yield production and characterization of biologically active recombinant aprotinin expressed in Saccharomyces cerevisiae.

Aprotinin is a polypeptide composed of 58 amino acid residues and has a molecular weight of 6512Da. The 58 amino acid residues are arranged in a single polypeptide chain, which is cross-linked by three disulfide bridges and folded to form a pear-shaped molecule. To express recombinant aprotinin in Saccharomyces cerevisiae, a synthetic gene encoding aprotinin was constructed and fused in frame with the pre-sequence of the S. cerevisiae MATalpha1 gene at the cleavage site of signal peptidase. The expression of aprotinin in S. cerevisiae was carried out using the PRB1 promoter. Aprotinin was secreted as a biologically active protein at a concentration of 426 mg/L into high cell density fermentation medium of 70.9 g/L cell dry weight. The purification process consisted of only three major steps and provided consistent yields of recombinant aprotinin using gel filtration high-pressure liquid chromatographic (HPLC) with a purity level higher than 99% and was free of non-aprotinin-related impurities. The recombinant aprotinin had the same characteristics as bovine aprotinin in a number of analytical methods, including alpha2-plasmin inhibition assay, amino acid composition, N-terminal amino acid sequence determination, and mass spectrum analysis. With further optimization of the purification process and culture conditions for high-yield production by S. cerevisiae, this source of recombinant aprotinin may be a promising approach for the commercial manufacture of aprotinin for pharmaceutical use instead of bovine aprotinin.

Hemostatic efficacy of a recombinant thrombin-coated polyglycolic acid sheet coupled with liquid fibrinogen, evaluated in a canine model of pulmonary arterial hemorrhage.

BACKGROUND: In thoracic surgery, although infrequent, we encounter unexpected damage to the pulmonary artery (PA). In the present study, we evaluated the hemostatic efficacy of a newly developed fibrin-based sheet material, thrombin sheet, coupled with liquid fibrinogen (TSF), in an experimental model of PA hemorrhage. METHODS: Female beagles (n = 8) were used for the study. Left thoracotomy was performed under general anesthesia. PA injury (approximately 4 x 2 mm) was created, and repaired by TSF (TSF group) or TachoComb (TC group). The animals were allowed to survive, and the repaired site was evaluated 4 weeks after the experiment. RESULTS: The number of sheet application and compression procedures required for hemostasis was increased in the TC group compared with in the TSF group (TC vs. TSF, 4 +/- 1 vs. 1 +/- 0.5, p = 0.01, unpaired t test). The time required to achieve hemostasis was increased in the TC group compared with in the TSF group (TC vs. TSF, 7 +/- 3 vs. 1 +/- 0.5 minutes, p = 0.01, unpaired t test). The amount of bleeding during the hemostasis procedure was increased in the TC group compared with in the TSF group (TC vs. TSF, 48 +/- 22 vs. 3 +/- 3 g, p = 0.01, unpaired t test). At 4 weeks, rethoracotomy revealed no apparent indication of delayed bleeding, such as intrathoracic hematoma formation or excessive adhesion formation in the vicinity of PA, in either group. Histologically, the vessel lumen was well sustained in both groups, with no apparent stenosis or thrombus formation. CONCLUSION: The hemostatic efficacy of TSF was superior to TC in this particular experiment. Single application of TSF was sufficient to achieve hemostasis in all but one animal. Compression time of approximately 1 minute was also very short albeit that the bleeding was from the PA and not an artery. These results were presumably because the adhesion was stronger, faster, and the sheet was more pliable in TSF compared with TC.

Effects of several virucidal agents on inactivation of influenza, Newcastle disease, and avian infectious bronchitis viruses in the allantoic fluid of chicken eggs.

General theories on the inactivation of viruses in the presence of a concentrated protein, such as the allantoic fluid of chicken eggs, are not useful. That is, although sodium hypochlorite and sodium hydroxide are generally known as strong virucidal agents, they do not sufficiently inactivate viruses in allantoic fluid. We found that benzalkonium chloride (BC) is an effective virucidal agent against influenza, Newcastle disease, and avian infectious bronchitis viruses even in the presence of a concentrated protein. BC is easily biodegradable by activated sludge and is not very harmful to humans. We strongly recommend BC as a useful virucidal agent, especially in the manufacture of vaccines for these viruses.

Analysis on the molecular species and concentration of circulating ADAMTS13 in Blood.

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.

ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage.

A severe lack of von Willebrand factor-cleaving protease (VWF-CP) activity can cause thrombotic thrombocytopenic purpura (TTP). This protease was recently identified as a member of the ADAMTS family, ADAMTS-13. It consists of a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, additional Tsp1 repeats, and CUB domains. To explore the structural and functional relationships of ADAMTS-13, we prepared here 13 sequential COOH-terminal truncated mutants and a single-point mutant (ArgGlyAsp [RGD] to ArgGlyGlu [RGE] in the cysteine-rich domain) and compared the activity of each mutant with that of the wild-type protein. The results revealed that the truncation of the cysteine-rich/spacer domains caused a remarkable reduction in VWF-CP activity. We also prepared immunoglobulin G (IgG) fractions containing inhibitory autoantibodies against ADAMTS-13 from plasma from 3 patients with acquired TTP, and we performed mapping of their epitopes using the aforementioned mutants. The major epitopes of these antibodies were found to reside within the cysteine-rich/spacer domains. These results suggest that the ADAMTS-13 cysteine-rich/spacer domains are essential for VWF-CP activity.

Identification of selenoprotein P fragments as a cell-death inhibitory factor.

Megakaryoblastoma (Dami cells) cultured in a serum-free medium containing albumin, proliferated for three days but died on the fourth day. This cell death was not observed when human plasma was added, suggesting that human plasma contains a cell-death inhibitory factor. In order to identify this factor, we purified it from human plasma. N-terminal amino acid sequence analysis revealed that this factor is a mixture of C-terminal fragments of selenoprotein P, a major selenocysteine-containing protein in plasma. The specific activity (unit per pmol of selenium) of selenoprotein P fragments protein was 15-fold and 1900-fold higher than that of the full-length SeP and sodium selenite, respectively.

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer.

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.

Production of human monoclonal and polyclonal antibodies in TransChromo animals.

We have developed TransChromo (TC) technology, which enables the introduction of megabase-sized segments of DNA into cells. We have used this approach to derive mice that carry megabases of human DNA by the use of a human chromosome fragment (HCF) as a vector. TC technology has been applied to the construction of the TC Mouse,trade mark which incorporates entire human immunoglobulin (hIg) loci. TC Mouse expresses a fully diverse repertoire of hIgs, including all the subclasses of IgGs (IgG1-G4). Immunization of the TC Mouse with various human antigens produced antibody responses comprised of human antibodies. Furthermore, it was possible to obtain hybridoma clones expressing fully human antibodies specific for the target human antigen. However, because of the instability of the Igkappa locus-bearing HCF2, the efficiency of hybridoma production was less than one-tenth of that observed in normal mice. An instant solution to this problem was to cross-breed the Kirin TC Mouse carrying the HCF14, which was stable in mouse cells, with the Medarex YAC-transgenic mouse carrying about 50% of the hIgVkappa gene segments as a region that is stably integrated into the mouse genome. The resulting mouse, dubbed the KM Mouse, performed as well as normal mice with regard to immune responsiveness and efficiency of hybridoma production. Another application of TC technology is the production of polyclonal antibodies in large animals such as chickens and cows. To test the efficacy of human polyclonal antibodies derived from TC animals, feasibility studies were performed using antisera and purified gamma-globulin from TC mice immunized with Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), or Japanese encephalitis virus (JEV). The TC mouse-derived antisera and gamma-globulin showed a much higher titer and efficacy in terms of the neutralizing activity of the pathogens in vitro and in vivo than either human serum or gamma-globulin prepared from human blood.