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The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised concerns regarding vaccine effectiveness. We investigated humoral and cellular immune responses against SARS-CoV-2 in healthcare workers before and after a third (booster) dose of the BNT162b2 messenger RNA vaccine. It significantly enhanced both humoral and cellular immunity in previously uninfected individuals. However, cellular immunity was not enhanced in previously infected persons, suggesting that 3 antigenic stimuli by vaccination or natural infection reached a plateau of cellular immunity. Even with reinforced immunity to SARS-CoV-2, we confirmed several postbooster breakthrough cases caused by the Omicron variant.

To evaluate vaccine-induced humoral and cell-mediated immunity at 6 months after completion of two doses of BNT162b2 vaccination, immunoglobulin G against SARS-CoV-2 spike protein (SP IgG), 50% neutralizing antibody (NT

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised concerns regarding vaccine effectiveness. We investigated humoral and cellular immune responses against SARS-CoV-2 in healthcare workers before and after a third (booster) dose of the BNT162b2 mRNA vaccine. It significantly enhanced both humoral and cellular immunity in previously uninfected individuals. However, cellular immunity was not enhanced in previously infected persons, suggesting that three antigenic stimuli by vaccination or natural infection reached a plateau of cellular immunity. Even with reinforced immunity to SARS-CoV-2, we confirmed several post-booster breakthrough cases caused by the Omicron variant.

BackgroundLittle is known about the population prevalence of antibodies against emerging immune escape variants of SARS-CoV-2.MethodsA population-based prevalence study was conducted in Yokohama City, the most populous municipality of Japan. Quantitative measurements of immunoglobulin G against SARS-CoV-2 spike protein (SP-IgG) and qualitative measurements of neutralization antibodies against the Omicron BA.1 and BA.2 variants were performed.ResultsOf 6,000 randomly selected residents aged 20–74, 1,277 participated in the study during a period from January 30 to February 28, 2022. Of them, 3% had prior diagnosis of COVID-19, 96% received at least two-doses of SARS-CoV-2 vaccines, and 94% were positive for SP-IgG. The positive rates of neutralizing antibodies were 28% to Omicron BA.1 and BA.2 variants in a random sample of 10% of participants (n=123) and 100% to BA.1 and BA.2 among participants who received the third vaccination at least 7 days before (n=66).ConclusionsIn this population-based prevalence study in Japan, most had SP-IgG antibodies but the overall neutralizing antibody positive rate was 28% against the Omicron BA.1 and BA.2 variants. The population-level insufficient humoral immunity against the Omicron variants may explain the outbreak of COVID-19 during this period in Japan.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection elicits varying degrees of protective immunity conferred by neutralizing antibodies (nAbs). In this study, we report the persistence of nAb responses over 12 months after infection despite their decreasing trend noticed from 6 months. Methods The study included sera from 497 individuals who had been infected with SARS-CoV-2 between January and August 2020. Samples were collected at 6 and 12 months after onset. The titers of immunoglobulin (Ig)G to the viral nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were measured by chemiluminescence enzyme immunoassay. The nAb titer was determined using lentivirus-based pseudovirus or authentic virus. Results Antibody titers of NP-IgG, RBD-IgG, and nAbs were higher in severe and moderate cases than in mild cases at 12 months after onset. Although the nAb levels were likely to confer adequate protection against wild-type viral infection, the neutralization activity to recently circulating variants in some of the mild cases (~30%) was undermined, implying the susceptibility to reinfection with the variants of concerns (VOCs). Conclusions Coronavirus disease 2019 convalescent individuals have robust humoral immunity even at 12 months after infection albeit that the medical history and background of patients could affect the function and dynamics of antibody response to the VOCs.

BackgroundLevels of 50% neutralizing titer (NT50) reflect a vaccine-induced humoral immunity after the vaccination against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Measurements of NT50 are difficult to implement in large quantities. A high-throughput laboratory test is expected for determining the level of herd immunity against SARS-CoV-2.MethodsWe analyzed samples from 168 Japanese healthcare workers who had completed two doses of the BNT162b2 vaccine. We analyzed immunoglobulin G (IgG) index values against spike protein (SP) using automated chemiluminescent enzyme immunoassay system AIA-CL and analyzed the background factors affecting antibody titer. SP IgG index was compared with 50% neutralization titers.ResultsThe median SP IgG index values of the subjects (mean age = 43 years; 75% female) were 0.1, 1.35, 60.80, and 97.35 before and at 2, 4, and 6 weeks after the first dose, respectively. At 4 and 6 weeks after the first dose, SP IgG titers were found to have positive correlation with NT50 titer (r=0.7535 in 4 weeks; r=0.4376 in 6 weeks). Proportions of the SP IgG index values against the Alpha, Beta, Gamma, and Delta variants compared with the original strain were 2.029, 0.544, 1.017, and 0.6096 respectively. Older age was associated with lower SP IgG titer index 6 weeks after the first dose.ConclusionsSP IgG index values were raised at 3 weeks after two doses of BNT162b2 vaccination and have positive correlation with NT50. SP IgG index values were lower in the older individuals and against Beta and Delta strain.

To evaluate vaccine-induced humoral and cell-mediated immunity at 6 months post BNT162b2 vaccination, immunoglobulin G against SARS-CoV-2 spike protein (SP IgG), 50% neutralizing antibody (NT50), and spot-forming cell (SFC) counts were evaluated by interferon-γ releasing ELISpot assay of 98 healthy subjects (median age, 43 years). The geometric mean titers of SP IgG and NT50 decreased from 95.2 (95% confidence interval (CI) 79.8–113.4) to 5.7 (95% CI 4.9–6.7) and from 680.4 (588.0–787.2) to 130.4 (95% CI 104.2–163.1), respectively, at 3 weeks and 6 months after the vaccination. SP IgG titer was negatively correlated with age and alcohol consumption. Spot-forming cell counts at 6 months did not correlate with age, gender, and other parameters of the patients. SP IgG, NT50, and SFC titers were elevated in the breakthrough infected subjects. Although the levels of vaccine-induced antibodies dramatically declined at 6 months after vaccination, a certain degree of cellular immunity was observed irrespective of the age.

SARS-CoV-2 infection elicits varying degrees of protective immunity conferred by neutralizing antibodies (nAbs). Here we report the persistence of nAb responses over 12 months after infection despite its decreasing trend noticed from 6 months. The study included sera from 358 individuals who had been infected with SARS-CoV-2 between January and May 2020. Samples were collected at 6 and 12 months after onset. The titers of IgG to the viral nucleocapsid protein (NP) and receptor-binding domain of the spike protein (RBD) were measured by CLEIA. The nAb titer was determined using lentivirus-based pseudovirus or authentic virus. Antibody titers of NP-IgG, RBD-IgG, and nAbs were higher in severe and moderate cases than in mild cases at 12 months after onset. While the nAb levels were likely to confer adequate protection against wild-type viral infection, the neutralization activity to recently circulating variants in some of the mild cases (∼30%) was undermined, implying the susceptibility of reinfection to the variants of concerns (VOCs). COVID-19 convalescent individuals have robust humoral immunity even at 12 months after infection albeit that the medical history and background of patients could affect the function and dynamics of antibody response to the VOCs.

ObjectiveSerological tests for COVID-19 have been instrumental in studying the epidemiology of the disease. However, the performance of the currently available tests is plagued by the problem of variability. We have developed a high-throughput serological test capable of simultaneously detecting total immunoglobulins (Ig) and immunoglobulin G (IgG) against two of the most immunologically relevant SARS-CoV-2 antigens, nucleocapsid protein (NP) and spike protein (SP) and report its performance in detecting COVID-19 in clinical samples.MethodsWe designed and prepared reagents for measuring NP-IgG, NP-Total Ig, SP-IgG, and SP-Total Ig (using N-terminally truncated NP (ΔN-NP) or receptor-binding domain (RBD) antigen) on the advanced chemiluminescence enzyme immunoassay system TOSOH AIA-CL. After determining the basal thresholds based on 17 sera obtained from confirmed COVID-19 patients and 600 negative sera. Subsequently, the clinical validity of the assay was evaluated using independent 202 positive samples and 1,000 negative samples from healthy donors.ResultsAll of the four test parameters showed 100% specificity individually (1,000/1,000; 95%CI, 99.63-100). The sensitivity of the assay increased proportionally to the elapsed time from symptoms onset, and all the tests achieved 100% sensitivity (153/153; 95%CI, 97.63-100) after 13 days from symptoms onset. NP-Total Ig was the earliest to attain maximal sensitivity among the other antibodies tested.ConclusionOur newly developed serological testing exhibited 100% sensitivity and specificity after 13 days from symptoms onset. Hence, it could be used as a reliable method for accurate detection of COVID-19 patients and to evaluate seroprevalence and possibly for surrogate assessment of herd immunity.

Tissue factor pathway inhibitor‑2 (TFPI‑2) is a promising candidate as a serum biomarker of ovarian clear cell carcinoma (OCCC), a lethal histological subtype of epithelial ovarian cancer (EOC). TFPI‑2 is a secreted serine protease inhibitor that suppresses cancer progression through the inhibition of matrix protease activities. Previous studies have also identified TFPI‑2 in the nucleus, and a possible function of nuclear TFPI‑2 as a transcriptional repressor of matrix metalloproteinase‑2 (MMP‑2) was recently demonstrated. We are currently establishing TFPI‑2 as a serum biomarker for OCCC patients; however, TFPI‑2 expression in OCCC tissues has not been previously investigated. In the present study, we examined TFPI‑2 expression and its localization in 11 OCCC cell lines by western blotting and enzyme‑linked immune assay. Four cell lines expressed TFPI‑2 in the nucleus, cytoplasm and culture plate-attached extracellular fraction, while four other cell lines expressed TFPI‑2 only in the extracellular fraction. In the remaining three cell lines, TFPI‑2 was not identified in any fraction. The amount of secreted soluble TFPI‑2 showed similar trends to that of the plate‑attached fraction. We next investigated the expression levels and distribution of TFPI‑2 in surgically resected EOC tissues by immunohistochemistry. In 52 of the 77 (67.5%) OCCC tumors, TFPI‑2 expression was detected in at least one of the nuclear, cytoplasmic and extracellular matrix fractions. In contrast, we did not identify TFPI‑2 in the other EOC subtypes (n=65). TFPI‑2‑positive expression distinguished CCC from the other EOC tissues with a sensitivity of 67.5% and specificity of 100%. Although the inherent tumor suppressor function, statistical analyses failed to demonstrate correlations between TFPI‑2 expression and clinical parameters, including 5‑year overall survival, except for the patient age. In conclusion, we identified TFPI‑2 expression in the nucleus, cytoplasm and extracellular matrix in OCCC tissues. The high specificity of TFPI‑2 may support its use for diagnosis of OCCC in combination with existing markers.

SARS-CoV-2 neutralizing antibodies confer protective immunity against reinfection. We have developed a rapid test for screening SARS-CoV-2 neutralization antibodies using genome-free virus-like particles incorporated with a small luciferase peptide, HiBiT. Their entry into LgBiT-expressing target cells reconstitutes NanoLuc luciferase readily detected by a luminometer. This newly developed HiBiT-tagged Virus-like particle-based Neutralization Test (hiVNT) can readily quantify SARS-CoV-2 neutralizing antibodies within three hours with a high-throughput in a low biosafety setting. Moreover, the neutralizing activity obtained from hiVNT was highly consistent with that measured by the conventional neutralization test using authentic SARS-CoV-2. Furthermore, antibody responses to both viral spike and nucleocapsid proteins correlated with the neutralization activity assessed by hiVNT. Our newly-developed hiVNT could be instrumental to survey individuals for the presence of functional neutralizing antibody against SARS-CoV-2.

Background There is currently no reliable serum biomarker for ovarian clear cell carcinoma (CCC), a highly lethal histological subtype of epithelial ovarian cancer (EOC). Previously, using a proteome-based approach, we identified tissue factor pathway inhibitor 2 (TFPI2) as a candidate serum biomarker for CCC. In this study, we sought to evaluate the clinical diagnostic performance of TFPI2 in preoperative prediction of CCC. Methods Serum TFPI2 levels were measured in serum samples from a retrospective training set consisting of patients with benign and borderline ovarian tumors, EOC subtypes, and uterine diseases. Via receiver operating characteristic (ROC) analyses, we compared the diagnostic performance of TFPI2 with that of CA125 in discrimination of patients with ovarian CCC from other patient groups. The observed diagnostic performances were examined in a prospective validation set. Results The 268-patient training set included 29 patients with ovarian CCC. Unlike CA125, which was also elevated in patients with endometriosis and several EOC subtypes, serum TFPI2 levels were specifically elevated only in ovarian CCC patients, consistent with the mRNA expression pattern in tumor tissues. The area under the ROC curve (AUC) of serum TFPI2 was obviously higher than that of CA125 for discrimination of CCC from other ovarian diseases (AUC = 0.891 versus 0.595). Applying a cut-off value of 280 pg/mL, TFPI2 could distinguish early-stage (FIGO I and II) CCC from endometriosis with 72.2% sensitivity, 93.3% specificity, and 88.8% accuracy. Similar results were confirmed in an independent 156-patient prospective validation set. Conclusions TFPI2 is a useful serum biomarker for preoperative clinical diagnosis of CCC.

Prostate specific antigen (PSA) is useful for screening prostate cancer, while it has no reliable correlation with pathological malignancy of prostate cancer and is not a predictor for castration resistant prostate cancer (CRPC). In order to explore novel biomarkers to predict the development of CRPC, we performed proteomics from secreted proteins from human prostate cancer cells and identified the candidates of biomarker for CRPC. Materials and Methods: The proteins secreted from 6 prostate cancers in culture medium were analyzed and compared with 8 other cancer cells including renal and urothelial cancers using LTQ Orbitrap mass spectrometer. With the focus on high tissue specificity, the candidate biomarker proteins were then identified through analysis of gene expressions in proteins common to human prostate cancers by real time qPCR. Next, a system to measure the identified mouse monoclonal antibodies against the focused proteins was established. Finally, serum levels of these proteins from 33 patients with benign prostate hyperplasia (BPH), 31 with untreated prostate cancer (PCa) and 35 with CRPC, were measured. Results: The proteome analysis identified 12 candidates of secreted cell membrane proteins as new biomarkers. The proteome analysis indicated that not only matured GDF15, but pro-peptide as well as fragments (GDPP) are released from prostate cancer cells. Patients’ serum was analyzed for matured and pro-peptide GDF15 using ELISA and immunoprecipitation-MRM mass spectrometry. The results showed that the serum level of GDDP-1, one of the processing forms of GDPP, was significantly higher in CRPC than those in BPH and untreated PCa (P Conclusion: GDPP-1 shows potential as a novel biomarker for CRPC. Citation Format: Hiroji Uemura, Noriaki Arakawa, Yusuke Itoh, Takashi Kawahara, Yasuhide Miyoshi, Masahiro Yao, Shouhei Akiba, Norihisa Ohtake. Identification of a novel biomarker to predict castration-resistant prostate cancer (CRPC) by the proteome analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 228. doi:10.1158/1538-7445.AM2017-228

Of all of the epithelial ovarian cancers (EOC), clear cell adenocarcinoma (CCA) has the worst clinical prognosis. Furthermore, the conventional EOC biomarker CA125 is more often negative in CCA than in other subtypes of EOC. This study sought to discover a new diagnostic biomarker that would allow more reliable detection of CCA. Using mass spectrometry, we compared proteins in conditioned media from cell lines derived from CCA and other types of EOC. We identified 30 extracellular or released proteins specifically present in CCA-derived cell lines. Bioinformatics analyses identified a serine protease inhibitor, tissue factor pathway inhibitor 2 (TFPI2), as a potential biomarker for CCA. Real time RT-PCR and Western blot analyses revealed that TFPI2 was exclusively expressed in CCA-derived cell lines and tissues. For clinical validation, we measured levels of TFPI2 and CA125 in a set of sera from 30 healthy women, 30 patients with endometriosis, and 50 patients with CCA, using an automated enzyme-linked immunosorbent assay systems. Serum levels of TFPI2 were significantly elevated in CCA patients, even those with normal CA125 levels. In terms of area under the receiver operating characteristic curve (AUC), TFPI2 was superior to CA125 in discriminating CCA patients from healthy women (AUC 0.97 for TFPI2 versus AUC 0.80 for CA125), or from patients with endometriosis (AUC 0.93 for TFPI2 versus 0.80 for CA125). This is the first evidence for TFPI2 as a serum biomarker of CCA. We propose that this biomarker may be useful for detection of CCA and for monitoring the transformation from endometriosis into CCA.

247 Background: It is well known that prostate specific antigen (PSA) level has no reliable correlation with pathological malignancy of prostate cancer and is not a predictor for the development of castration resistant prostate cancer (CRPC). The aim of this study is to explore novel biomarkers to predict the development of CRPC by using proteomics from secreted proteins from human prostate cancer cells. Methods: The proteins secreted from 6 prostate cancers in culture medium were analyzed and compared with 8 other cancer cells including renal and urothelial cancers using LTQ Orbitrap mass spectrometer. With the focus on high tissue specificity, the candidate biomarker proteins were then identified through analysis of gene expressions in proteins common to human prostate cancers by real time qPCR. Next, a system to measure the identified mouse monoclonal antibodies against the focused proteins was established. Finally, serum levels of these proteins from 33 patients with benign prostate hyperplasia (BPH), 31 with untreated prostate cancer (PCa) and 35 with CRPC, were measured. Results: The proteome analysis identified 12 candidates of secreted cell membrane proteins as new biomarkers. The proteome analysis indicated that not only matured GDF15, but pro-peptide as well as fragments (GDDP) are released from prostate cancer cells. Patients’ serum was analyzed for matured and pro-peptide GDF15 using ELISA and immunoprecipitation-MRM mass spectrometry. The results showed that the serum level of GDDP-1, one of the processing forms of GDDP, was significantly higher in CRPC than those in BPH and untreated PCa (P < 0.01). ROC analysis also showed that the AUC of GDDP-1(0.86) was higher than that of matured GDF15 (0.76). When the cutoff value of GDDP-1 was set at 4.0 ng/mL, there was a significant difference of overall survival (OS) in CRPC patients between those with more than 4.0 ng/mL compared to less than 4.0 ng/mL of GDDP-1, whereas there was no significant difference of OS measurable by PSA in CRPC patients. These data suggest that GDDP-1 may be a novel biomarker for CRPC. Conclusions: GDDP-1 shows potential as a novel biomarker for CRPC.