Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WBpositive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions,

Vaccines are beneficial and universal tools to prevent infectious disease. Thus, safety of vaccines is strictly evaluated in the preclinical phase of trials and every vaccine batch must be tested by the National Control Laboratories according to the guidelines published by each country. Despite many vaccine production platforms and methods, animal testing for safety evaluation is unchanged thus far. We recently developed a systems biological approach to vaccine safety evaluation where identification of specific biomarkers in a rat pre-clinical study evaluated the safety of vaccines for pandemic H5N1 influenza including Irf7, Lgals9, Lgalsbp3, Cxcl11, Timp1, Tap2, Psmb9, Psme1, Tapbp, C2, Csf1, Mx2, Zbp1, Ifrd1, Trafd1, Cxcl9, β2m, Npc1, Ngfr and Ifi47. The current study evaluated whether these 20 biomarkers could evaluate the safety, batch-to-batch and manufacturer-to-manufacturer consistency of seasonal trivalent influenza vaccine using a multiplex gene detection system. When we evaluated the influenza HA vaccine (HAv) from four different manufactures, the biomarker analysis correlated to findings from conventional animal use tests, such as abnormal toxicity test. In addition, sensitivity of toxicity detection and differences in HAvs were higher and more accurate than with conventional methods. Despite a slight decrease in body weight caused by HAv from manufacturer B that was not statistically significant, our results suggest that HAv from manufacturer B is significantly different than the other HAvs tested with regard to Lgals3bp, Tapbp, Lgals9, Irf7 and C2 gene expression in rat lungs. Using the biomarkers confirmed in this study, we predicted batch-to-batch consistency and safety of influenza vaccines within 2 days compared with the conventional safety test, which takes longer. These biomarkers will facilitate the future development of new influenza vaccines and provide an opportunity to develop in vitro methods of evaluating batch-to-batch consistency and vaccine safety as an alternative to animal testing.

We have shown previously that serum insulin-like growth factor binding protein-3 (IGFBP-3) levels have good predictive value for complete, but not partial, growth hormone deficiency (GHD). In this study, we compare IGFBP-3 levels in short children previously divided into groups on the basis of their post-stimulation GH levels. Complete GHD (N = 59) included those children with peak post-stimulation GH < 5 micrograms/l. The partial GHD group (N = 49) had post-stimulation GH peaks of > 5 micrograms/l but < 10 micrograms/l. The normal children with short stature (N = 103) had post-stimulation GH peaks > 10 micrograms/l. Partial GHD and normal children with short stature also were divided into either low IGF-I or normal IGF-I subgroups. The clinical sensitivity of IGFBP-3 for complete GHD was 92%, whereas its sensitivity for partial GHD was 39%. For partial GHD, among those with low IGF-I (N = 19) 68% were also low for IGFBP-3, while 80% of those with normal IGF-I (N = 30) were also normal for IGFBP-3. The clinical specificity of IGFBP-3 for normal children with short stature was 69%. For these groups, among those with low IGF-I (N = 22) 73% also were low for IGFBP-3, while 80% of those with normal IGF-I (N = 81) also were normal for IGFBP-3. In addition, we tested whether IGFBP-3 can predict the response to GH treatment in prepubertal children by comparing pretreatment IGFBP-3 with the height gain achieved by 1 year of GH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TLOm1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR. Human T-lymphotropic virus 1 (HTLV-1) was the first retrovirus to be found in humans (1, 2). HTLV-1 is a cause of adult T-cell leukemia (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and HTLV-1-associated uveitis (3). Areas where HTLV-1 is endemic are distributed across several different regions, including southern Japan, the Caribbean, South America, and tropical Africa (4, 5). A recent report has shown that the area affected by this infection has expanded from the southern part of Japan to the entire country, particularly the Tokyo metropolitan area (6). Diagnostic tests for HTLV-1 infection are performed mainly with serological assays, such as enzyme-linked immunoabsorbent assay, particle agglutination assay, and Western blotting. Recently, another diagnostic test has been developed. Quantitation of integrated proviral DNA in peripheral blood (proviral load [PVL]) can be performed by quantitative PCR (qPCR) as a risk assessment for ATL or HAM/TSP (7,8).A few studies reported that several samples were positive for viral DNA when tested by PCR even though those samples had been found seroindeterminate for HTLV-1 when tested by Western blotting (9, 10). Their results suggest that HTLV-1 qPCR could be used as an additional test to confirm infection in seroindeterminate samples.Although many laboratories have developed qPCR methods for HTLV-1 detection in Japan, a wide variety of testing methods are used. For example, the target region, primers and probes, and internal control (IC) genes vary among the laboratories (8,(11)(12)(13)(14)(15). These variations lead to significa...

We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.