Published papers

Background Humoral immunity wanes following SARS-CoV-2 mRNA vaccination. However, frequent breakthrough infections following the emergence of the Omicron variant make it difficult to evaluate long-term antibody decline attributable solely to vaccination. This study addresses this gap by characterizing long-term antibody waning after booster vaccination in SARS-CoV-2-naïve individuals. Methods SARS-CoV-2 spike protein-specific IgG (S-IgG) titers in the Keio University Healthcare Worker Vaccination Cohort have been monitored since February 2021. Using serial measurements of stored serum samples, we identified SARS-CoV-2-naïve participants who had received at least three doses of mRNA vaccine and had not received any additional vaccination for ≥2 years. Absence of previous SARS-CoV-2 infection was confirmed by serial questionnaires and persistently negative nucleocapsid protein-specific IgG (N-IgG). Temporal waning of S-IgG after the final vaccination was modeled using one-compartment, two-compartment, and bi-exponential decay models. Predictive performance of the models was compared using the leave-one-out information criterion. Results 19 individuals (11 with three doses, 7 with four doses, and 1 with five doses) met the selection criteria. S-IgG waning after the final vaccination was best described by the two-compartment model, with an initial phase of relatively rapid decline followed by a second phase with a substantially smaller decay slope. Conclusions Long-term waning of S-IgG following mRNA booster vaccination is best characterized by a two-compartment model. These findings provide a framework for evaluating long-term binding antibody kinetics after booster vaccination and may facilitate the evaluation of current and future mRNA vaccines.

Background Clot waveform analysis (CWA) is performed to extend the interpretation of clotting reaction curves in activated partial thromboplastin time (APTT) or prothrombin time (PT) assays. The present study aims to investigate the relationship between CWA parameters and fibrinogen concentrations for elucidating how to use CWA for coagulation assessment. Methods Data regarding APTT-CWA, PT-CWA, and fibrinogen measurements were analyzed from 83 different individuals with prolonged APTT or PT and from 145 different individuals with APTT and PT within their reference ranges. CWA was adjusted to cancel out the influence of fibrinogen. Results The coefficient of variation (CV) was much lower in adjusted CWA parameters than in non-adjusted CWA parameters, concordant with canceling out the influence of fibrinogen concentrations. The CV in adjusted CWA parameters was higher in cases with prolonged APTT or PT compared to those with normal APTT and PT, likely due to the presence of various coagulopathies and anticoagulants. Regardless of APTT or PT data, non-adjusted CWA parameters and non-adjusted/adjusted CWA parameter ratios were positively correlated with fibrinogen concentrations. In APTT-CWA, the correlation coefficients of non-adjusted/adjusted CWA parameter ratios with fibrinogen concentrations were lower in cases of prolonged APTT or PT than in cases with normal APTT and PT, suggesting that other determinants than fibrinogen concentrations may alter turbidity changes and affect the CWA. Conclusion The present study demonstrated correlations between CWA parameters and fibrinogen concentrations in clinical samples. The results are expected to help broaden the interpretation of clotting reaction curves in APTT and PT assays.

The International Harmonisation Protocol (IHP) for Reticulocyte Percentage (%Retic) in CLSI-H44-A2 is a microscopic procedure performed on a new-methylene-blue-stained blood film (NMB-IHP). However, IHP-compliant Measurement Procedures (MPs) based on flow cytometry (FCM) are recommended for practical and accurately assigned reference values. Thiazole orange (TO) for nucleic acid staining and CD235a immunostaining for erythrocyte gating are currently widespread for MP using FCM (TO/CD235a-MP); however they have not been validated using NMB-IHP. We recently developed MP using FCM with anti-CD45/CD41a/CD61 antibodies, that are utilized for excluding platelets from the un-nucleated blood cell fraction to select erythrocytes (TO/CD41a/CD61-MP). The aim of this study is to validate TO/CD41a/CD61-MP for measurement of reticulocytes as an IHP-compliant MP. First, TO/CD235a-MP was validated as an IHP-compliant MP by comparing it with NMB-IHP. Then TO/CD41a/CD61-MP was compared with TO/CD235a-MP. The practical utility of TO/CD41a/CD61-MP was evaluated using XN-2000™(Sysmex) and Celltac G + ™(Nihon Kohden) hematology analyzers to assess the accuracy of different nucleic acid staining methods. We first confirmed that TO/CD235a-MP was consistent with NMB-IHP, then evaluated TO/CD41a/CD61-MP using TO/CD235a-MP. Regression analysis demonstrated consistency between TO/CD41a/CD61-MP and TO/CD235a-MP, thereby establishing TO/CD41a/CD61-MP as IHP-compliant MP. In the accuracy assessment, the correlation coefficients to TO/CD41a/CD61-MP were observed to be 0.97 for both hematology analyzers with %Retic ranging from 0.0 to 8.2. TO/CD41a/CD61-MP serves effectively as an IHP-compliant MP. This study provides the first empirical validation of FCM-based MPs.

Background: While previous studies have assessed the clinical values of clot waveform analysis (CWA) for detecting lupus anticoagulants (LA), there are no reports focusing on whether CWA predicts the LA sensitivity of activated partial thromboplastin time (APTT) reagents. The present study aimed to assess the usefulness of CWA in evaluating the LA sensitivity of APTT reagents. Methods: Commercially available pooled normal plasma (PNP) and LA-positive plasma control (LPC) were used. CWA findings were compared among nine APTT reagents. Results: Waveform abnormality appeared in assays with six APTT reagents for LPC but not for PNP. No waveform abnormality was evident in assays with three reagents for LPC or for PNP. In assays using reagents exhibiting waveform abnormality, APTT for LPC was longer and ratios of APTT for LPC/APTT for PNP were higher. Waveform abnormality was associated with indexes for evaluating mixing tests. Quantitative evaluation regarding the reduction of CWA parameters adjusted to compensate the influence of fibrinogen was likely applicable for predicting the LA sensitivity of APTT reagents. Conclusion: The present study suggested the usefulness of CWA for evaluating the LA sensitivity of APTT reagents. The results provide insights into use of APTT reagents for LA screening.

Background: Hemodialysis patients, due to impaired kidney function and compromised immune responses, face increased risks from SARS-CoV-2. Anti-nucleocapsid IgG (anti-IgG N) antibodies are a commonly used marker to assess prior infection in the general population; however, their efficacy for hemodialysis patients remains unclear. Methods: A retrospective study of 361 hemodialysis patients evaluated anti-IgG N antibodies for detecting prior SARS-CoV-2 infection. Antibody levels were measured using a chemiluminescence immunoassay (CLIA) over the four time points. Boxplots illustrated antibody distribution across sampling stages and infection status. Logistic regression and receiver operating characteristic (ROC) curve analysis determined diagnostic accuracy, sensitivity, specificity, and optimal cutoff values. Results: Among the 361 hemodialysis patients, 36 (10.0%) had SARS-CoV-2 infection. Sex distribution showed a trend toward significance (p = 0.05). Boxplot analysis showed that anti-IgG N levels remained low in non-infected patients but increased in infected patients, peaking at the third sampling. Anti-IgG N demonstrated high diagnostic accuracy (AUC: 0.973–0.865) but declined over time (p = 0.00525). The optimal cutoff at C1 was 0.01 AU/mL (sensitivity 1.00, specificity 0.94). Adjusted models had lower predictive value. Conclusions: Anti-IgG N antibodies showed high diagnostic accuracy for detecting prior SARS-CoV-2 infection in hemodialysis patients, though performance declined over time. These findings highlight the need for tailored diagnostic strategies in this vulnerable population.

Background: The long-term immune implications of administering more than four doses of COVID-19 vaccine and the impact of breakthrough infections are not fully understood. Research Design and Methods: We conducted a follow-up cohort study on Japanese healthcare workers who received more than three doses of the BNT162b2 vaccine. We assessed both the anti-SARS-CoV-2 antibody titer and cellular immunity in 429 participants and investigated the numbers, types, and brands of COVID-19 vaccines administered, as well as the episodes of COVID-19 infections after the third dose. Results: Individuals who received three total doses of vaccines with BTI episodes demonstrated higher antibody titers than those who received four total doses of vaccines with no BTIs. The cellular immune responses between these two groups were comparable. Conclusions: These findings suggest that BTIs occurring after the primary series of COVID-19 vaccinations (first to third dose) induced humoral immunity to the spike protein that is greater than that induced by booster doses (fourth or fifth dose) and elicit cellular immunity to the spike protein comparable to that of booster doses.

Introduction: Accurate and precise platelet (PLT) count is critical for the appropriate management of patients with thrombocytopenia. This study evaluated the performance of PLT counting with the Abbott Alinity hq hematology analyzer, which utilizes multi-dimensional optical technology. Methods: Imprecision, linearity, and accuracy were assessed per CLSI guidelines. Alinity hq PLT results were compared to the international flow cytometry reference method (IRM) in the concentration range of 6.3 to 103.0 × 109/L. Additional comparisons were made with Sysmex XN-3000 PLT counts: impedance (PLT-I), optical (PLT-O), and optical fluorescent (PLT-F) methods. Results: The average within-run %CV was 4.7% on patient samples with PLT concentrations ranging from 13.1 to 41.7 × 109/L, and the within-laboratory %CV was 3.6% at the level of 68.2 × 109/L. Linearity evaluation indicated a maximum deviation of 3.1% from the linear fit in the range of 0.1 to 316.8 × 109/L. Comparison between Alinity hq and the IRM PLT counts yielded a correlation coefficient of 0.99 and predicted bias of 0.0 and −0.5 × 109/L at 10.0 and 20.0 × 109/L transfusion thresholds, respectively. Alinity hq PLT counts also correlated well with Sysmex PLT counts, with strongest correlation obtained with PLT-F and PLT-O (r =.99) methods. Conclusion: This study demonstrated excellent analytical performance of Alinity hq PLT counting in thrombocytopenic samples, equivalency with the IRM and strong agreement with Sysmex PLT-F and PLT-O methods. The Alinity hq multi-dimensional optical PLT count is available with every CBC without additional reagents and may help promote efficiency in clinical laboratories.

Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60-80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.

Tools that can be used to estimate antibody waning following COVID-19 vaccinations can facilitate an understanding of the current immune status of the population. In this study, a two-compartment-based mathematical model is formulated to describe the dynamics of the anti-SARS-CoV-2 antibody in healthy adults using serially measured waning antibody concentration data obtained in a prospective cohort study of 673 healthcare providers vaccinated with two doses of BNT162b2 vaccine. The datasets of 165 healthcare providers and 292 elderly patients with or without hemodialysis were used for external validation. Internal validation of the model demonstrated 97.0% accuracy, and external validation of the datasets of healthcare workers, hemodialysis patients, and nondialysis patients demonstrated 98.2%, 83.3%, and 83.8% accuracy, respectively. The internal and external validations demonstrated that this model also fits the data of various populations with or without underlying illnesses. Furthermore, using this model, we developed a smart device application that can rapidly calculate the timing of negative seroconversion.

Triple-negative breast cancer (TNBC) chemoresistance hampers the ability to effectively treat patients. Identification of mechanisms driving chemoresistance can lead to strategies to improve treatment. Here, we revealed that protein arginine methyltransferase-1 (PRMT1) simultaneously methylates D-3-phosphoglycerate dehydrogenase (PHGDH), a critical enzyme in serine synthesis, and the glycolytic enzymes PFKFB3 and PKM2 in TNBC cells. 13C metabolic flux analyses showed that PRMT1-dependent methylation of these three enzymes diverts glucose toward intermediates in the serine-synthesizing and serine/glycine cleavage pathways, thereby accelerating the production of methyl donors in TNBC cells. Mechanistically, PRMT1-dependent methylation of PHGDH at R54 or R20 activated its enzymatic activity by stabilizing 3-phos-phoglycerate binding and suppressing polyubiquitination. PRMT1-mediated PHGDH methylation drove chemoresistance independently of glutathione synthesis. Rather, activation of the serine synthesis pathway supplied a-ketoglutarate and citrate to increase palmitate levels through activation of fatty acid synthase (FASN). Increased palmitate induced protein S-palmitoylation of PHGDH and FASN to further enhance fatty acid synthesis in a PRMT1-dependent manner. Loss of PRMT1 or pharmacologic inhibition of FASN or protein S-palmitoyltransferase reversed chemoresistance in TNBC. Furthermore, IHC coupled with imaging MS in clinical TNBC specimens substantiated that PRMT1-mediated methylation of PHGDH, PFKFB3, and PKM2 correlates with chemoresistance and that metabolites required for methylation and fatty acid synthesis are enriched in TNBC. Together, these results suggest that enhanced de novo fatty acid synthesis mediated by coordinated protein arginine methylation and protein S-palmitoylation is a therapeutic target for overcoming chemoresistance in TNBC. Significance: PRMT1 promotes chemoresistance in TNBC by methylating metabolic enzymes PFKFB3, PKM2, and PHGDH to augment de novo fatty acid synthesis, indicating that targeting this axis is a potential treatment strategy.

As we learned during the COVID-19 pandemic, vaccines are one of the most important tools in infectious disease control. To date, an unprecedentedly large volume of high-quality data on COVID-19 vaccinations have been accumulated. For preparedness in future pandemics beyond COVID-19, these valuable datasets should be analyzed to best shape an effective vaccination strategy. We are collecting longitudinal data from a community-based cohort in Fukushima, Japan, that consists of 2,407 individuals who underwent serum sampling two or three times after a two-dose vaccination with either BNT162b2 or mRNA-1273. Using the individually reconstructed time courses of the vaccine-elicited antibody response based on mathematical modeling, we first identified basic demographic and health information that contributed to the main features of the antibody dynamics, i.e., the peak, the duration, and the area under the curve. We showed that these three features of antibody dynamics were partially explained by underlying medical conditions, adverse reactions to vaccinations, and medications, consistent with the findings of previous studies. We then applied to these factors a recently proposed computational method to optimally fit an “antibody score”, which resulted in an integer-based score that can be used as a basis for identifying individuals with higher or lower antibody titers from basic demographic and health information. The score can be easily calculated by individuals themselves or by medical practitioners. Although the sensitivity of this score is currently not very high, in the future, as more data become available, it has the potential to identify vulnerable populations and encourage them to get booster vaccinations. Our mathematical model can be extended to any kind of vaccination and therefore can form a basis for policy decisions regarding the distribution of booster vaccines to strengthen immunity in future pandemics.

Background: Understanding the waning of immunity after booster vaccinations is important to identify which immune-low populations should be prioritized. Methods: We investigated longitudinal cellular and humoral immunity after the third vaccine dose in both high- and low-cellular and humoral immunity groups at the peak immunity phase after the booster vaccination in a large community-based cohort. Blood samples were collected from 1045 participants at peak (T1: median 54 days post-third dose) and decay (T2: median 145 days post-third dose) phases to assess IgG(S), neutralizing activity, and ELISpot responses. Participants were categorized into high/low ELISpot/IgG(S) groups at T1. Cellular and humoral responses were tracked for approximately five months after the third vaccination. Results: In total, 983 participants were included in the cohort. IgG(S) geometric mean fold change between timepoints revealed greater waning in the >79 years age group (T2/T1 fold change: 0.27) and higher IgG(S) fold change in the low-ELISpot group at T1 (T2/T1 fold change: 0.32–0.33) than in the other groups, although ELISpot geometric mean remained stable. Conclusions: Antibody level of those who did not respond well to third dose vaccination waned rapidly than those who responded well. Evidence-based vaccine strategies are essential in preventing potential health issues caused by vaccines, including side-effects.

Vaccine immunogenicity is influenced by the vaccinee's genetic background. Here, we perform a genome-wide association study of vaccine-induced SARS-CoV-2-specific immunoglobulin G (IgG) antibody titers and T cell immune responses in 1,559 mRNA-1273 and 537 BNT162b2 vaccinees of Japanese ancestry. SARS-CoV-2-specific antibody titers are associated with the immunoglobulin heavy chain (IGH) and major histocompatibility complex (MHC) locus, and T cell responses are associated with MHC. The lead variants at IGH contain a population-specific missense variant (rs1043109-C; p.Leu192Val) in the immunoglobulin heavy constant gamma 1 gene (IGHG1), with a strong decreasing effect (β = −0.54). Antibody-titer-associated variants modulate circulating immune regulatory proteins (e.g., LILRB4 and FCRL6). Age-related hematopoietic expanded mosaic chromosomal alterations (mCAs) affecting MHC and IGH also impair antibody production. MHC-/IGH-affecting mCAs confer infectious and immune disease risk, including sepsis and Graves’ disease. Impacts of expanded mosaic loss of chromosomes X/Y on these phenotypes were examined. Altogether, both germline and somatic mutations contribute to adaptive immunity functions.

© The Author(s) 2021. Background: The usability of laboratory tests related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critically important for the world undergoing the COVID-19 pandemic. The present study aimed to assess the diagnostic usability of rapid tests for the detection of antibody against SARS-CoV-2 through comparison of their results with the results of reverse transcription polymerase chain reaction (RT-PCR) test for the detection of SARS-CoV-2 genomic RNA and with the results of a quantitative test for antibody detection. Methods: Serum samples were collected from 18 patients undergoing RT-PCR testing for SARS-CoV-2. Twelve patients were RT-PCR positive while six were negative. A quantitative test based on chemiluminescent immunoassay and three rapid tests based on immunochromatography were performed to detect anti-SARS-CoV-2 IgG and IgM. Results: All the antibody tests exhibited poor sensitivity at the timing of initial RT-PCR diagnosis. IgG responses occurring prior to or simultaneously with IgM responses were observed through not only the quantitative test but also the three rapid tests. Based on concordance with the quantitative test results, the large variance among the three rapid tests was revealed. Conclusions: All antibody tests were unsatisfactory to replace RT-PCR for the early diagnosis of COVID-19. Rapid antibody tests as well as a quantitative antibody test were useful in the assessment of immune responses in COVID-19. The obvious variance among the three rapid tests suggested limited accuracy and difficult standardization. Diagnostic usability of rapid antibody tests for COVID-19 should be investigated rigorously.

Background: Quantitative antibody tests are expected to be useful in diagnostics of COVID-19 and investigation of herd immunity against SARS-CoV-2. To make it proper to perform them, understanding of the immunological aspects is critically important. The present study aimed to assess humoral responses in COVID-19 using various quantitative antibody tests. Methods: Four quantitative antibody tests that are different in targeted antigens, detectable immunoglobulin classes and avidity were used. Diagnosis was confirmed by RT-PCR for SARS-CoV-2 detection. Antibody titres of 117 samples collected from 24 COVID-19 patients and 23 non-COVID-19 patients were measured to evaluate correlations between different tests. For 24 COVID-19 patients, antibody titres measured at various time points after the onset or the RT-PCR diagnosis were subjected to assessment of humoral responses. Results: Correlations between tests were observed to some degree, although there were discrepancies putatively due to differences in measurement principle. Seronegative COVID-19 was diagnosed for some patients, in whom antibody titres were less than the cut-off value in each test throughout the time courses. IgG seroconversion without prior IgM seroconversion most frequently occurred, while predominance of IgM responses over IgG responses was observed in some severe cases. Viral burdens estimated according to threshold cycle values at the RT-PCR seemed to impact antibody responses. Conclusions: The results provide insights into the nature of humoral responses to SARS-CoV-2 and diagnostic performance of antibody tests.