Masaki Ochiai

Antigen-loaded dissolving microneedle array as a novel tool for percutaneous vaccination.

Antigen-loaded dissolving microneedle array (dMNA) patches were investigated as novel systems for vaccine delivery into the skin, where immuno-competent dendritic cells are densely distributed. We fabricated micron-scale needles arrayed on patches, using chondroitin sulfate mixed with a model antigen, ovalbumin. Insertion of dMNA effectively delivered substantial amounts of ovalbumin into the skin within 3 min and induced robust antigen-specific antibody responses in the sera of mice. The antibody dose-response relationship showed that the efficiency of dMNA patch immunization was comparable to that of conventional intradermal injections. Thus, Antigen-loaded dMNA patches are a promising antigen-delivery system for percutaneous vaccination.

An in vitro assay system as a potential replacement for the histamine sensitisation test for acellular pertussis based combination vaccines

The histamine sensitisation test (HIST) for pertussis toxin is currently an official batch release test for acellular pertussis containing combination vaccines in Europe and North America. However, HIST, being a lethal endpoint assay, often leads to repeated tests due to large variations in test performance. Although a more precise HIST test based on measurement of temperature reduction after the histamine challenge is used in Asian countries, this test still uses animals. An in vitro test system based on a combination of enzyme coupled-HPLC and carbohydrate-binding assays with results analysed by a mathematical formula showed a good agreement with the in vivo HIST results based on measurement of temperature reduction after histamine challenge. The new in vitro test system was shown to be a potential alternative to the current in vivo HIST.

Lipopolysaccaride-binding peptides obtained by phage display method

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. It has strong toxicity and might cause sepsis or septic shock. Thus early detection of LPS and neutralization of LPS toxicity are required. We obtained several new LPS-binding peptides using a phage display method. We synthesized 3 of these peptides and analyzed their binding affinity and capacity to LPS. One of these peptides, named Li5-001, showed high binding affinity to LPS and lipid A; the K(d) values were 10 and 1 nM, respectively. Li5-001 showed a high binding capacity to LPS, and was estimated to bind 130 ng LPS/mg, which is higher than that of polymyxin B (80 ng LPS/mg); however, its LPS-neutralizing activity was low. Li5-001 coupled with beads will be useful for eliminating endotoxin contamination from pharmaceuticals. Its low LPS-neutralizing activity allows to be used in the Limulus amebocyte lysate test without eluting LPS from the Li5-001 coupled beads.

Enhanced sensitisation of mice with diphtheria tetanus acellular pertussis vaccine to local swelling reaction to the booster immunisation

Severe local swelling has been regarded as a serious safety problem for the booster immunisations of diphtheria tetanus acellular pertussis combined (DTaP) vaccine and DT combined toxoids (DT-td). We attempted to search for the factor of DTaP vaccines possibly contributing to the enhanced local reaction by using the mouse hind paw swelling reaction. Mice were immunised intramuscularly with DTaP vaccine twice at 1-month interval and were challenged their hind paw with one of the antigens of DTaP vaccine 2 weeks later. D-td was shown to elicit the strongest swelling among the vaccine antigens. No causal relationship was found between the swelling and the level of immunoglobulin G (IgG) or IgE in mice. Residual pertussis toxin (PT) activity of DTaP vaccines for immunisation was shown to play a role in the enhanced sensitisation of mice to the D-td-related hind paw swelling.

Transferability of dermal temperature histamine sensitization test for estimation of pertussis toxin activity in vaccines

All current acellular pertussis vaccines (ACVs) contain detoxified pertussis toxin (PT) as a major component. An essential part of the safety evaluation of these vaccines, required by regulatory authorities, is to monitor their active PT content and to check for reversion to toxicity of the detoxified PT. Although various in vitro tests are under investigation, the only practicable means for detecting active PT at present is the histamine sensitization test. The methods given in the European Pharmacopoeia and in the US Pharmacopoeia are based on recording a binary response to histamine challenge (using a lethal end point). A more sensitive method based on measurement of rectal temperature is given in the Japanese Minimum Requirements for Biological Products. More recently, a refinement of this method based on dermal temperature measurement has been developed for ACVs in combination with diphtheria and tetanus vaccines (DTaP). We show that this method also can be used for more complex combination vaccines and is readily transferable. Furthermore use of dermal temperature provides a more precise quantitative estimate of toxin activity than the binary response, leading to an increase in information from a specified number of animals, or allowing a reduction in the number of animals required. We suggest that, pending the development of an alternative in vitro replacement method, the temperature based method may serve as an intermediate solution to the estimation of PT activity giving a precise estimate with reduction in animal numbers.

A New Method for the Evaluation of Vaccine Safety Based on Comprehensive Gene Expression Analysis

For the past 50 years, quality control and safety tests have been used to evaluate vaccine safety. However, conventional animal safety tests need to be improved in several aspects. For example, the number of test animals used needs to be reduced and the test period shortened. It is, therefore, necessary to develop a new vaccine evaluation system. In this review, we show that gene expression patterns are well correlated to biological responses in vaccinated rats. Our findings and methods using experimental biology and genome science provide an important means of assessment for vaccine toxicity.

A Limulus Amoebocyte Lysate Activating Activity (LAL Activity) That Lacks Biological Activities of Endotoxin Found in Biological Products

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test. Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF‐α) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS‐binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IHA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.

Standardization of Quantitative PCR for Human T-cell Leukemia Virus Type 1 in Japan: A Collaborative Study

ABSTRACT Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1-3)-β-D-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.

Comparison of acellular pertussis-based combination vaccines by Japanese control tests for toxicities and laboratory models for local reaction

Two batches each of diphtheria -- tetanus -- acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from the U.S.A., European and Asian markets were compared with Japanese DTaPs by Japanese control tests for DTaP and laboratory models for local reaction. All the imported vaccines met Japanese criteria for toxicities of acellular pertussis vaccine except for the toxicity to mouse weight gain (body weight decreasing (BWD) toxicity). When injecting into mouse footpad, rabbit back skin and mouse quadriceps muscle, the imported vaccines induced much severer inflammation and tissue injury comparing to Japanese DTaPs irrespective of animal species, injection site and injection volume suggesting that these vaccines may induce stronger local reactogenicity.