Report for the Eighth Asian National Control Laboratory Network meeting in 2023: Self-sufficiency strategy of plasma-derived medicinal products and regulatory harmonisation.

The Eighth Asian National Control Laboratory (NCL) Network meeting, entitled "Biological Products Quality Control and Self-Sufficiency Strategy focusing on plasma-derived medicinal products (PDMPs)" was held in Seoul on 31 August 2023. The participants were NCL experts from Indonesia, Japan, Malaysia, the Philippines, Vietnam, and the Republic of Korea. Special lectures included the PDMPs self-sufficiency strategies of the World Health Organization (WHO) and Indonesian Food and Drug Authority, and a case study on Global Benchmarking Tool (GBT) assessment for vaccines by the Korea Ministry of Food and Drug Safety. The NCL delegates shared their current experiences with national lot releases and biological standardisation. The meeting contributed to a mutual understanding of the progress of the PDMPs self-sufficiency among Asian countries, the WHO's support strategies, and the NCL's plan for the preparation of the WHO GBT assessment. In the panel discussion, all participants agreed that building capacity in blood safety in the Asian region and harmonisation of relevant international regulatory requirements will support appropriate emergency preparedness, particularly source materials in the region, and will build the foundation for resolving the PDMPs supply insecurity that has worsened after the COVID-19 pandemic in some countries.

Report on the seventh meeting of national control laboratories for vaccines and biologicals of the WHO Western Pacific and South-East Asia member states.

The Biregional Network of National Control Laboratories (NCLs) of the WHO Western Pacific and South-East Asia Regions has been meeting annually since 2018 to enhance NCLs' voluntary participation capacity. Its seventh meeting was hosted by the Korea National Institute of Food and Drug Safety Evaluation (NIFDS) of the Ministry of Food and Drug Safety (MFDS), in conjunction with the Global Bio Conference, in Seoul on September 6, 2022. Over 60 participants from seven countries, (India, Indonesia, Japan, Korea, Malaysia, the Philippines, and Vietnam) attended the meeting on-site and online. The theme of this meeting was 'Quality Control Issues and International Trends for Biologicals including Vaccines and Plasma-Derived Medicinal Products.' Three special speeches were presented on sharing the quality control system for biologicals, including NCLs' considerations in preparing the WHO Listed Authorities and sharing MFDS experiences. Furthermore, the participating NCLs shared country-specific issues related to national lot releases during the COVID-19 pandemic and acknowledged the meeting's crucial role in response preparedness for pandemic emergencies and enhancing regulatory capacity through coalitions and information exchange among NCLs. The NIFDS will cooperate closely with other Asian NCLs to enhance biological product quality control, aiming to establish regional standards and standardize test methods through collaboration.

Anthocyanin regulatory and structural genes associated with violet flower color of Matthiola incana.

MAIN CONCLUSION: MiMYB1 and MibHLH2 play key roles in anthocyanin biosynthesis in Matthiola incana flowers. We established a transient expression system using Turnip mosaic virus vector in M. incana. Garden stock (Matthiola incana (L.) R. Br.) is a popular flowering plant observed from winter to spring in Japan. Here we observed that anthocyanin accumulation in 'Vintage Lavender' increased with flower development, whereas flavonol accumulation remained constant throughout flower development. We obtained five transcription factor genes, MiMYB1, MibHLH1, MibHLH2, MiWDR1, and MiWDR2, from M. incana floral cDNA contigs. Yeast two-hybrid analyses revealed that MiMYB1 interacted with MibHLH1, MibHLH2, and MiWDR1, but MiWDR2 did not interact with any transcription factor. Expression levels of MiMYB1 and MibHLH2 increased in petals during floral bud development. Their expression profiles correlated well with the temporal profiles of MiF3'H, MiDFR, MiANS, and Mi3GT transcripts and anthocyanin accumulation profile. On the other hand, MibHLH1 was expressed weakly in all organs of 'Vintage Lavender'. However, high expression levels of MibHLH1 were detected in petals of other cultivars with higher levels of anthocyanin accumulation than 'Vintage Lavender'. MiWDR1 and MiWDR2 maintained constant expression levels in petals during flower development and vegetative organs. Transient MiMYB1 expression in 1-month-old M. incana seedlings using a Turnip mosaic virus vector activated transcription of the endogenous anthocyanin biosynthetic genes MiF3'H, MiDFR, and MiANS and induced ectopic anthocyanin accumulation in leaves. Therefore, MiMYB1 possibly interacts with MibHLH2 and MiWDR1, and this trimeric protein complex activates the transcription of anthocyanin biosynthetic genes in M. incana flowers. Moreover, MibHLH1 acts as an enhancer of anthocyanin biosynthesis with the MiMYB1-MibHLH2-MiWDR1 complex. This study revealed the molecular mechanism involved in the regulation of anthocyanin accumulation levels in M. incana flowers.

Establishment of a reference material for standardization of the anti-complementary activity test in intravenous immunoglobulin products used in Japan: A collaborative study.

Aggregates of human plasma-derived intravenous immunoglobulins (IVIGs) carries a risk of severe adverse events after nonspecific complement activation induced in humans administrated. Therefore, the anti-complementary activity (ACA) test is legally required in every batch of IVIGs in Japan. However, due to the intrinsic nature of this bioassay, there might be large differences in the results of ACA tests from laboratories, even when the same batch of IVIGs was measured. Our six laboratories evaluated whether there were such differences and argued for establishment of a reference material (RM) for standardization of the ACA test. Our results revealed inter-laboratory differences in ACA values, indicating a need to establish an RM. Therefore, after ACA values in candidate RMs were measured collaboratively, one RM was selected from two candidates and unit value-assigned. The RM in fact normalized the ACA test values for samples measured in parallel at almost all the laboratories, when the values were calculated relative to the assigned unit value of the RM. Thus, we established a first RM to standardize the ACA test in Japan, which enabled each laboratory to normalize ACA values constantly for IVIGs. This indicates that the establishment of an RM can contribute to quality control of IVIGs.

Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study.

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.

A need for careful evaluation of endotoxin contents in acellular pertussis-based combination vaccines.

Two batches each of diphtheria-tetanus-acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from foreign markets were tested by mouse body weight decreasing (BWD) toxicity test and Limulus amaebocyte lysate (LAL) test. Three out of the four imported vaccine batches showed the levels of BWD toxicity even comparable to that of DT-whole cell pertussis vaccine. BWD toxicity test is based on endotoxin dose-dependent weight loss of mice and has been used for controlling endotoxin in DTaP. Although of the strong BWD toxicity of the imported vaccines, there was no marked difference in LAL test results between the imported vaccines and Japanese DTaP. However, one imported DTaP batch showed very strong interference with LAL activity of spiked lipopolysaccharide (LPS). The batch interfered not only with LAL activity but also with pyrogenicity and prostaglandin E(2) induction activity. However, the pyrogenicity of the spiked LPS could be recovered from the precipitated fraction of the batch by treating with phosphate buffer to suggest the possibility of recovering in vivo toxicity. As an adequate in vitro test method could not be identified for controlling the safety of the interfering batch, an appropriate in vivo test would be required for testing such vaccines.

Anaphylaxis after vaccination for cats in Japan.

Severe adverse reactions in cats after vaccination were examined from 316 cases reported to the Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan during 15-year period from April 2004 to March 2019. We found that 130 (41%) showed anaphylaxis, and 99 (76%) of the 130 cases of anaphylaxis resulted in death. Veterinarians should be well prepared to deal with vaccine-associated anaphylaxis in cats. Bovine serum albumin (BSA) as indicator of purification was detected at high levels in commercially available feline vaccines. BSA might derive from fetal calf serum in culture media. This study provides useful information about anaphylaxis including critical details of the potential clinical signs associated with adverse events to feline vaccination.

Characteristics and incidence of vaccine adverse events after Bacille Calmette-Guérin vaccination: A national surveillance study in Japan from 2013 to 2017.

BACKGROUND: Japan amended the recommended age for the Bacille Calmette-Guérin (BCG) vaccination to less than 6 months after 2005, but subsequently amended the recommended age to 5-8 months (latest amendment, <1 year) in April 2013 due to the increasing incidence of BCG-associated osteitis/osteomyelitis since 2005. METHODS: We collected data on BCG-associated vaccine adverse events (VAEs) in the population aged <1 year between April 2013 and March 2017. The incidence of BCG-associated VAE was analyzed using census and vaccine coverage data from the government website. We compared the incidence of VAEs in patients vaccinated at less than 6 months with those vaccinated at 6 months or older. RESULTS: Among the 581 BCG-associated VAEs recorded during the study period, 354 (61%) were male, and the average age at vaccination was 5.7 months. The incidence of VAEs per million population aged <1 year at vaccination was highest for suppurative lymphadenitis (63.7), followed by skin lesions (38.4), and BCG-associated osteitis/osteomyelitis (3.1). Disseminated BCG and anaphylaxis were rare (1.1 and 1.6%, respectively). The incidence of VAEs in the population vaccinated at <6 months of age was higher for BCG-associated osteitis/osteomyelitis (3.8) and disseminated BCG (1.3) than in the population vaccinated at ≥6 months. CONCLUSIONS: The population vaccinated at <6 months of age was more likely to develop BCG-associated osteitis/osteomyelitis than the population vaccinated at ≥6 months of age, indicating that the change in the recommended vaccination age in 2013 might have contributed to the subsequent decrease in the incidence of BCG-associated osteitis/osteomyelitis.

Vasovagal reactions after COVID-19 vaccination in Japan.

Coronavirus disease 2019 (COVID-19) vaccine administration started in February 2021 in Japan. As of December 2021, approximately 75% of the population aged ≥12 years had received two doses of vaccine. We conducted a study to investigate vasovagal reactions (VVR) after COVID-19 vaccination using data on adverse events following immunization. The crude reporting rate of VVR (cases/1,000,000 doses) after vaccination was 9.6 in all age groups combined, and was more frequent in the younger age groups: 28.6 and 37.2 in individuals aged 10-19 years and 20-29 years, respectively. In individuals aged 10-29 years, the rate was similar in males and females (33.0 and 34.2, respectively, p = 0.53); but was higher after dose 1 than after dose 2 (57.4 and 8.8, respectively, p < 0.001). Based on these results, caution needs to be exercised when vaccinating adolescents and young adults, especially with dose 1 of COVID-19 vaccines.

Anaphylaxis after rabies vaccination for dogs in Japan.

Severe adverse reactions after rabies vaccination in dogs were examined from 317 cases reported to the Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan during 15-year period from April 2004 to March 2019. We found that 109 of the 317 dogs showed anaphylaxis (0.15/100,000 vaccinated dogs), and 71 of the 109 cases of anaphylaxis resulted in death (0.10/100,000 vaccinated dogs). We measured bovine serum albumin (BSA) in four commercially available rabies vaccines and found the levels ranged from 0.1 to 16.6 µg/dose. Our survey showed that the rate of anaphylaxis to rabies vaccines in dogs is rare, although some cases of anaphylaxis resulted in death. Veterinarians should be well prepared to deal with vaccine-associated anaphylaxis.

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.

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)-ß-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.

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.

Study on the Procedure for Lot Release of Vaccines in Japan.

Biological products, such as vaccines, blood products, antitoxins, and antivenoms, are released into the market following a lot release conducted by National Regulatory Authorities or National Control Laboratories, even if their manufacturing and marketing have been authorized. Independent lot release by regulatory authorities is not a procedure unique to Japan, but is performed worldwide. Previously, Japan carried out lot release mainly by laboratory tests, and the manufacturers' in-house test records were used as a reference, not involved in the decision of lot release. Conversely, the international standard procedure promoted by the World Health Organization (WHO) includes a document review of the manufacturers' summary protocols, and laboratory tests are listed as an optional procedure. To harmonize with the WHO recommended international method, Japan modified the procedure and introduced a document review in addition to laboratory tests for vaccines in 2012. Since then, substantial knowledge regarding vaccine quality has been obtained during the process of summary protocol reviewing. Here, we outline the current status of the lot release procedure in Japan. We shed light on its history and show recent research based on the knowledge obtained from the protocol review to improve efficiency of laboratory testing and international harmonization.

A Comparative Study on the Lot Release Systems for Vaccines as of 2016.

Many countries have already established their own vaccine lot release system that is designed for each country's situation: while the World Health Organization promotes for the convergence of these regulatory systems so that vaccines of assured quality are provided globally. We conducted a questionnaire-based investigation of the lot release systems for vaccines in 7 countries and 2 regions. We found that a review of the summary protocol by the National Regulatory Authorities was commonly applied for the independent lot release of vaccines, however, we also noted some diversity between countries, especially in regard to the testing policy. Some countries and regions, including Japan, regularly tested every lot of vaccines, whereas the frequency of these tests was reduced in other countries and regions as determined based on the risk assessment of these products. Test items selected for the lot release varied among the countries or regions investigated, although there was a tendency to prioritize the potency tests. An understanding of the lot release policy may contribute to improving and harmonizing the lot release system globally in the future.

Endotoxin content in Haemophilus influenzae type b vaccine.

Haemophilus influenzae type b (Hib) is a major cause of bacterial meningitis among children. Hib conjugate vaccines have effectively prevented Hib infection, and routine immunization with Hib conjugate vaccine has diminished the incidence of the disease in the United States and European countries. Introduction of Hib conjugate vaccines is also required in Japan. However, endotoxin that can carry over from Gram-negative H. influenzae with a purified component may contribute to adverse events following Hib vaccination. In the present study, we examined the endotoxin content in Hib conjugate vaccines. The Hib conjugate vaccine batches, which were produced by a European vaccine manufacturer, were shown to have considerably high endotoxin activity and to vary from 13.9 to 173.7 endotoxin units/dose. These results suggest that it is necessary to monitor the endotoxin content of the vaccine batches to ensure the quality and safety of the vaccines.

A cell line assay system for predicting the response of human blood to endotoxin.

Some parenteral drugs augment the in vivo action of endotoxin. It is necessary to regulate the overall toxic action of contaminating endotoxin by developing a clinically relevant test method for the safety control of such drugs. Although the responses of human peripheral blood cells (hPBC) to endotoxins to produce tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-1beta showed considerable variation depending on the endotoxin and also on the individuals used as sources of blood, the responses to each of the endotoxins evaluated relative to that to the Japanese reference standard endotoxin were found to be highly reproducible irrespective of the sources of hPBC. The evaluation procedure based on the relative responsiveness to various endotoxins was shown to be highly effective to detect differences in responsiveness among the endotoxin test, the pyrogen test, and the cytokine induction in hPBC. When eight human monocytoid cell lines were examined, only THP-1 and 28SC cells showed a significant dose-dependent IL-6 production. However, THP-1 failed to show consistency with hPBC in responses to the panel of endotoxins. 28SC cells showed appropriate consistency with hPBC not only in terms of respective responses to the endotoxins but also with regard to detection of the effect of human interferons to augment endotoxin to induce IL-6.

Investigation in a murine model of possible mechanisms of enhanced local reactions to post-primary diphtheria-tetanus toxoid boosters in recipients of acellular pertussis-diphtheria-tetanus vaccine.

In recipients primed with acellular pertussis diphtheria-tetanus combined vaccine (DTaP) an increased incidence of severe local reactions with extensive redness/swelling has been reported for each subsequent dose of diphtheria-tetanus based combination vaccine given as a booster. This has been attributed to residual active pertussis toxin (PT) in the primary vaccine. In this study, we investigated the possible contribution of the A-subunit enzymatic activity and the B-oligomer carbohydrate binding activity of residual PT in DTaP to local reactions in a murine model using Japanese DTaP batches produced before and after the introduction of a test for reversion of pertussis toxoid to toxin. Residual PT activity was correlated with the B-oligomer carbohydrate binding activity. The in vivo mouse footpad swelling model assay indicated that the B-oligomer carbohydrate binding activity and possibly other factors were associated with intensified sensitization to local reaction following diphtheria toxoid booster.

A national reference for inactivated polio vaccine derived from Sabin strains in Japan.

As one aspect of its campaign to eradicate poliomyelitis, the World Health Organization (WHO) has encouraged development of the inactivated polio vaccine (IPV) derived from the Sabin strains (sIPV) as an option for an affordable polio vaccine, especially in low-income countries. The Japan Poliomyelitis Research Institute (JPRI) inactivated three serotypes of the Sabin strains and made sIPV preparations, including serotypes 1, 2 and 3 D-antigens in the ratio of 3:100:100. The National Institute of Infectious Diseases, Japan, assessed the immunogenic stability of these sIPV preparations in a rat potency test, according to an evaluation method recommended by the WHO. The immunogenicity of the three serotypes was maintained for at least 4 years when properly stored under -70°C. Based on these data, the sIPV preparations made by JPRI have been approved as national reference vaccines by the Japanese national control authority and used for the quality control of the tetracomponent sIPV-containing diphtheria-tetanus-acellular pertussis combination vaccines that were licensed for a routine polio immunization in Japan.

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.