Overcoming the age-dependent SARS-CoV-2 vaccine response through hybrid immunity: analysis of humoral and cellular immunity with mass cytometry profiling.

BACKGROUND: Age-dependent immune responses to coronavirus disease 2019 (COVID-19) vaccinations and breakthrough infections (BIs) in young and middle-aged individuals are unclear. METHODS: This nationwide multicenter prospective cohort study analyzed immune responses in participants of the ChAdOx1 (ChAd)-ChAd-mRNA vaccine group using cytometry by time-of-flight, anti-spike protein antibody (Sab) and anti-nucleocapsid antibody (Nab) titers, plaque reduction neutralization tests (PRNTs), and interferon-gamma (IFN-γ) release assays at various time points. RESULTS: We evaluated 347 participants with an average age of 38.9 ± 9.4 years (range: 21-63). There was a significant inverse correlation between age and Sab levels after the second dose (slope - 14.96, P = 0.032), and this was more pronounced after the third dose (slope - 208.9, P < 0.001). After BIs, older participants showed significantly higher Sab titers (slope 398.8, P = 0.001), reversing the age-related decline observed post-vaccination. This reversal was also observed in PRNTs against wild-type SARS-CoV-2 and the BA.1 and BA.5 variants. IFN-γ responses increased markedly after the third dose and Bis, but showed a weak positive correlation with age, without statistical significance. Immune cell profiling revealed an age-dependent decrease in the proportions of B-cell lineage cells. The proportions of naive CD4+ and CD8+ T cells were inversely correlated with age, whereas the proportions of mature T cell subsets with memory function, including memory CD4+ T, CD8+ TEM, CD8+ TEMRA, and TFH cells, increased with age. CONCLUSIONS: Age-dependent waning of the serologic response to COVID-19 vaccines occurred even in middle-aged individuals, but was reversed after BIs. IFN-γ responses were preserved, compensating for the decrease in naive T cell populations, with an increase in memory T cell populations.

Identification of the novel HLA-DRB3*02:209 allele by next-generation sequencing.

HLA-DRB3*02:209 is identical to HLA-DRB3*02:02:01:01 except for a single nucleotide substitution in exon 4.

QuantiFERON monitor predicts early cytomegalovirus infection and viral burden in allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: Cytomegalovirus (CMV) infection is a major cause of transplantation-related morbidity and mortality. This study assessed the utility of the QuantiFERON monitor (QFM; Qiagen) for the prediction of early CMV infection and viral burden. METHODS: QuantiFERON-CMV (QF-CMV; Qiagen) and QFM were measured at the post-allogeneic hematopoietic stem cell transplantation (HSCT) week 4. CMV DNA was measured at every visit until post-HSCT week 24. The QFM cutoff specific to CMV infection was established. RESULT: At the post-HSCT week 4, the QFM cutoff predicting CMV infection was 86.95 IU/mL. While QF-CMV results at the post-HSCT week 4 were associated with high-level CMV infection (CMV DNA ≥ 5,000 IU/mL) but not with CMV infection (CMV DNA ≥ 500 IU/mL), QFM was associated with both CMV infection and high-level CMV infection. Both indeterminate QF-CMV and nonreactive QFM were associated with increased peak CMV DNA. CONCLUSION: Low QFM is a risk factor for CMV infection and increased CMV viral loads. QFM at post-HSCT week 4 can be utilized as an assay to predict the risk and burden of early CMV infection in HSCT recipients, in conjunction with other risk factors.

Persistent differences in the immunogenicity of the two COVID-19 primary vaccines series, modulated by booster mRNA vaccination and breakthrough infection.

INTRODUCTION: The long-term impact of initial immunogenicity induced by different primary COVID-19 vaccine series remains unclear. METHODS: A prospective cohort study was conducted at 10 tertiary hospitals in Korea from March 2021 to September 2022. Immunogenicity assessments included anti-spike protein antibody (Sab), SARS-CoV-2-specific interferon-gamma releasing assay (IGRA), and multiplex cytokine assays for spike protein-stimulated plasma. Spike proteins derived from wild-type SARS-CoV-2 and alpha variant (Spike1) and beta and gamma variant (Spike2) were utilized. RESULTS: A total of 235 healthcare workers who had received a two-dose primary vaccine series of either ChAdOx1 or BNT162b2, followed by a third booster dose of BNT162b2 (166 in the ChAdOx1/ChAdOx1/BNT162b2 (CCB) group and 69 in the BNT162b2/BNT162b2/BNT162b2 (BBB) group, based on the vaccine series) were included. Following the primary vaccine series, the BBB group exhibited significantly higher increases in Sab levels, IGRA responses, and multiple cytokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß, interleukin (IL)-1ra, IFN-γ, IL-2, IL-4, and IL-10) compared to the CCB group (all P < 0.05). One month after the third BNT162b2 booster, the CCB group showed Sab levels comparable to those of the BBB group, and both groups exhibited lower levels after six months without breakthrough infections (BIs). However, among those who experienced BA.1/2 BIs after the third booster, Sab levels increased significantly more in the BBB group than in the CCB group (P < 0.001). IGRA responses to both Spike1 and Spike2 proteins were significantly stronger in the BBB group than the CCB group after the third booster, while only the Spike2 response were higher after BIs (P = 0.007). The BBB group exhibited stronger enhancement of T-cell cytokines (IL-2, IL-4, and IL-17A) after BIs than in the CCB group (P < 0.05). CONCLUSION: Differences in immunogenicity induced by the two primary vaccine series persisted, modulated by subsequent booster vaccinations and BIs.

Identification of the novel HLA-DPA1*02:07:04 allele by next-generation sequencing.

The novel HLA-DPA1*02:07:04 allele was detected during the HLA typing for kidney transplantation.

Does coronavirus disease 2019 history alone increase the risk of postoperative pulmonary complications after surgery? Prospective observational study using serology assessment.

BACKGROUND: Concern exists about the increasing risk of postoperative pulmonary complications in patients with a history of coronavirus disease 2019 (COVID-19). OBJECTIVE: We conducted a prospective observational study that compared the incidence of postoperative pulmonary complications in patients with and without a history of COVID-19. METHODS: From August 2022 to November 2022, 244 adult patients undergoing major non-cardiac surgery were enrolled and allocated either to history or no history of COVID-19 groups. For patients without a history of confirming COVID-19 diagnosis, we tested immunoglobulin G to nucleocapsid antigen of SARS-CoV-2 for serology assessment to identify undetected infection. We compared the incidence of postoperative pulmonary complications, defined as a composite of atelectasis, pleural effusion, pulmonary edema, pneumonia, aspiration pneumonitis, and the need for additional oxygen therapy according to a COVID-19 history. RESULTS: After excluding 44 patients without a COVID-19 history who were detected as seropositive, 200 patients were finally enrolled in this study, 100 in each group. All subjects with a COVID-19 history experienced no or mild symptoms during infection. The risk of postoperative pulmonary complications was not significantly different between the groups according to the history of COVID-19 (24.0% vs. 26.0%; odds ratio, 0.99; 95% confidence interval, 0.71-1.37; P-value, 0.92). The incidence of postoperative pulmonary complications was also similar (27.3%) in excluded patients owing to being seropositive. CONCLUSION: Our study showed patients with a history of no or mild symptomatic COVID-19 did not show an increased risk of PPCs compared to those without a COVID-19 history. Additional precautions may not be needed to prevent PPCs in those patients.

Identification of the novel HLA-DPB1*1591:01 allele by next-generation sequencing.

The novel HLA-DPB1*1591:01 allele was detected during the HLA typing for kidney transplantation.

Ruxolitinib Improves Immune-Dysregulation Features but not Epigenetic Abnormality in a Patient with STAT1 GOF.

PURPOSE: Patients with STAT1 gain-of-function (GOF) mutations often exhibit autoimmune features. The JAK1/2 inhibitor ruxolitinib can be administered to alleviate autoimmune symptoms; however, it is unclear how immune cells are molecularly changed by ruxolitinib treatment. Then, we aimed to investigate the trnscriptional and epigenetic status of immune cells before and after ruxolitinib treatment in a patient with STAT1 GOF. METHODS: A patient with a heterozygous STAT1 GOF variant (p.Ala267Val), exhibiting autoimmune features, was treated with ruxolitinib, and peripheral blood mononuclear cells (PBMCs) were longitudinally collected. PBMCs were transcriptionally analyzed by single-cell cellular indexing of the transcriptomes and epitopes by sequencing (CITE-seq), and epigenetically analyzed by assay of transposase-accessible chromatin sequencing (ATAC-seq). RESULTS: CITE-seq analysis revealed that before treatment, the patient's PBMCs exhibited aberrantly activated inflammatory features, especially IFN-related features. In particular, monocytes showed high expression levels of a subset of IFN-stimulated genes (ISGs). Ruxolitinib treatment substantially downregulated aberrantly overexpressed ISGs, and improved autoimmune features. However, epigenetic analysis demonstrated that genetic regions of ISGs-e.g., STAT1, IRF1, MX1, and OAS1-were highly accessible even after ruxolitinib treatment. When ruxolitinib was temporarily discontinued, the patient's autoimmune features were aggravated, which is in line with sustained epigenetic abnormality. CONCLUSIONS: In a patient with STAT1 GOF, ruxolitinib treatment improved autoimmune features and downregulated aberrantly overexpressed ISGs, but did not correct epigenetic abnormality of ISGs.

Identification of the novel HLA-B*13:191 allele by next-generation sequencing.

The novel HLA-B*13:191 allele was detected during the HLA typing for kidney transplantation.

Efficacy and safety of BVAC-C in HPV type 16- or 18-positive cervical carcinoma who failed 1st platinum-based chemotherapy: a phase I/IIa study.

Background: BVAC-C, a B cell- and monocyte-based immunotherapeutic vaccine transfected with recombinant HPV E6/E7, was well tolerated in HPV-positive recurrent cervical carcinoma patients in a phase I study. This phase IIa study investigates the antitumor activity of BVAC-C in patients with HPV 16- or 18-positive cervical cancer who had experienced recurrence after a platinum-based combination chemotherapy. Patients and methods: Patients were allocated to 3 arms; Arm 1, BVAC-C injection at 0, 4, 8 weeks; Arm 2, BVAC-C injection at 0, 4, 8, 12 weeks; Arm 3, BVAC-C injection at 0, 4, 8, 12 weeks with topotecan at 2, 6, 10, 14 weeks. Primary endpoints were safety and objective response rate (ORR) as assessed by an independent radiologist according to Response Evaluation Criteria in Solid Tumors version 1.1. Secondary endpoints included the disease control rate (DCR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Results: Of the 30 patients available for analysis, the ORR was 19.2% (Arm 1: 20.0% (3/15), Arm 2: 33.3% (2/6), Arm3: 0%) and the DCR was 53.8% (Arm 1: 57.1%, Arm 2: 28.6%, Arm3: 14.3%). The median DOR was 7.5 months (95% CI 7.1-not reported), the median PFS was 5.8 months (95% CI 4.2-10.3), and the median OS was 17.7 months (95% CI 12.0-not reported). All evaluated patients showed not only inflammatory cytokine responses (IFN-γ or TNF-α) but also potent E6/E7-specific T cell responses upon vaccinations. Immune responses of patients after vaccination were correlated with their clinical responses. Conclusion: BVAC-C represents a promising treatment option and a manageable safety profile in the second-line setting for this patient population. Further studies are needed to identify potential biomarkers of response. Clinical trial registration: ClinicalTrials.gov, identifier NCT02866006.

Age-Related Differences in Neutralizing Antibody Responses against SARS-CoV-2 Delta and Omicron Variants in 151 SARS-CoV-2-Naïve Metropolitan Residents Boosted with BNT162b2.

BACKGROUND: Although age negatively correlates with vaccine-induced immune responses, whether the vaccine-induced neutralizing effect against variants of concern (VOCs) substantially differs across age remains relatively poorly explored. In addition, the utility of commercial binding assays developed with the wild-type SARS-CoV-2 for predicting the neutralizing effect against VOCs should be revalidated. METHODS: We analyzed 151 triple-vaccinated SARS-CoV-2-naïve individuals boosted with BNT162b2 (Pfizer-BioNTech). The study population was divided into young adults (age < 30), middle-aged adults (30 ≤ age < 60), and older adults (age ≥ 60). The plaque reduction neutralization test (PRNT) titers against Delta (B.1.617.2) and Omicron (B.1.1.529) variants were compared across age. Antibody titers measured with commercial binding assays were compared with PRNT titers. RESULTS: Age-related decline in neutralizing titers was observed for both Delta and Omicron variants. Neutralizing titers for Omicron were lower than those against Delta in all ages. The multiple linear regression model demonstrated that duration from third dose to sample collection and vaccine types were also significant factors affecting vaccine-induced immunity along with age. The correlation between commercial binding assays and PRNT was acceptable for all age groups with the Delta variant, but relatively poor for middle-aged and older adults with the Omicron variant due to low titers. CONCLUSIONS: This study provides insights into the age-related dynamics of vaccine-induced immunity against SARS-CoV-2 VOCs, corroborating the need for age-specific vaccination strategies in the endemic era where new variants continue to evolve. Moreover, commercial binding assays should be used cautiously when estimating neutralizing titers against VOCs, particularly Omicron.

Identification of the novel HLA-C*14:159 allele by next-generation sequencing.

The novel HLA-C*14:159 allele was detected during the routine HLA typing for kidney transplantation.

Realistic Estimation of COVID-19 Infection by Seroprevalence Surveillance of SARS-CoV-2 Antibodies: An Experience From Korea Metropolitan Area From January to May 2022.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, leading to the coronavirus disease 2019 (COVID-19) pandemic. Because a significant proportion of the COVID-19 confirmed cases were concentrated in the capital metropolitan area of South Korea, and a large proportion of the population in the area had been adequately vaccinated against COVID-19, we conducted a seroprevalence surveillance study focusing on the residents of the capital metropolitan area in South Korea. METHODS: We used a quota-sampling method to obtain blood samples from 1,000 individuals per round, equally stratified across seven age categories and sexes and regions, from five medical institutions located within the capital metropolitan area of South Korea. During five consecutive months (rounds) between January 2022 and May 2022, a total of 5,000 samples were analyzed for anti-spike (S) and anti-nucleocapsid (N) antibodies. RESULTS: High anti-S seropositivity was observed in all age groups, which corresponded to the vaccine coverage during the study period. Both the cumulative incidence based on polymerase chain reaction (PCR) and the estimated seroprevalence based on anti-N seropositivity increased in the fourth and fifth rounds, which corresponded to April 2022 and May 2022. Seroprevalence coincided with the cumulative incidence during the first three rounds, but exceeded from the fourth survey onwards when infection with omicron variants was increased rapidly in Korea. CONCLUSION: Seroprevalence confirmed the number of infection cases outside of PCR testing-based surveillance. Seroepidemiological surveillance can help us understand vaccine responses and detect hidden infections, thereby providing appropriate public health guidance for achieving population-level immunity.

No Significant Differences in Presepsin Levels According to the Causative Microorganism of Bloodstream Infection.

BACKGROUND: CD14 recognizes lipopolysaccharide (LPS), and presepsin is a fragment of soluble CD14. Still, it remains uncertain whether Gram-negative bacteria induce higher presepsin levels than other microorganisms. To address this question, this study aimed to analyze presepsin levels based on microorganisms isolated in blood cultures. MATERIALS AND METHODS: This study was a single-center study comprising suspected sepsis patients enrolled from July 2020 to September 2020. A total of 95 patients with a single isolate confirmed in blood culture were analyzed to evaluate if there are any differences in presepsin levels according to microbial isolates. Plasma presepsin level was measured using PATHFAST assay kit and analyzer (LSI Medience Corporation, Tokyo, Japan). RESULTS: There were 26 Gram-positive bacteremia, 65 Gram-negative bacteremia, and 3 fungemia patients with median presepsin levels of 869, 1,439, and 11,951 pg/mL, respectively. Besides, one case of algaemia demonstrated a presepsin level of 1,231 pg/mL. Our results showed no statistically significant difference in presepsin levels among patients with Gram-positive bacteremia, Gram-negative bacteremia, and fungemia. Furthermore, presepsin levels did not differ significantly among bloodstream infections caused by bacteria that were isolated from at least three different patients. In particular, Gram-positive bacteria such as Staphylococcus aureus and Enterococcus faecalis were able to induce presepsin levels comparable to those induced by Gram-negative bacteria. CONCLUSION: We demonstrated that there were no significant differences in plasma presepsin levels according to microbial isolates in blood culture. The major cause of the variability in presepsin levels during bloodstream infection might be the immunogenicity of each microorganism rather than the presence of LPS in the microorganism.

Characterization of novel HLA-A*24:608N allele discovered in Koreans.

A novel null HLA-A*24 allele, HLA-A*24:608N, was identified in five Korean subjects including three from a family and two separate individuals. This study was performed to discern its immunological function in transplantation settings. Because this null variant had deletions of approximately 12 k base pairs from intron 3 to 3' end of the HLA-A gene, low resolution HLA typing and amplicon-based next generation sequencing (NGS) typing methods had failed to assign it. Hybrid capture-based NGS method confirmed that this novel variant had a large deletion. T-lymphocyte crossmatching by complement-dependent lymphocytotoxicity and flow cytometry with a serum consisting anti-HLA-A24 antibody revealed negative results, implying that an individual with this allele would not carry a functioning A24 antigen. These findings highlight the importance of identifying a null HLA allele by employing appropriate molecular method and providing expected crossmatching outcomes in a real-world transplantation setting.

Identification of the novel HLA-DQA1*01:138 allele by next-generation sequencing.

HLA-DQA1*01:138 is identical to HLA-DQA1*01:03 except for a single nucleotide substitution in exon 3.

Current Status of Flow Cytometric Immunophenotyping of Hematolymphoid Neoplasms in Korea.

Background: Flow cytometric immunophenotyping of hematolymphoid neoplasms (FCI-HLN) is essential for diagnosis, classification, and minimal residual disease (MRD) monitoring. FCI-HLN is typically performed using in-house protocols, raising the need for standardization. Therefore, we surveyed the current status of FCI-HLN in Korea to obtain fundamental data for quality improvement and standardization. Methods: Eight university hospitals actively conducting FCI-HLN participated in our survey. We analyzed responses to a questionnaire that included inquiries regarding test items, reagent antibodies (RAs), fluorophores, sample amounts (SAs), reagent antibody amounts (RAAs), acquisition cell number (ACN), isotype control (IC) usage, positive/negative criteria, and reporting. Results: Most hospitals used acute HLN, chronic HLN, plasma cell neoplasm (PCN), and MRD panels. The numbers of RAs were heterogeneous, with a maximum of 32, 26, 12, 14, and 10 antibodies used for acute HLN, chronic HLN, PCN, ALL-MRD, and multiple myeloma-MRD, respectively. The number of fluorophores ranged from 4 to 10. RAs, SAs, RAAs, and ACN were diverse. Most hospitals used a positive criterion of 20%, whereas one used 10% for acute and chronic HLN panels. Five hospitals used ICs for the negative criterion. Positive/negative assignments, percentages, and general opinions were commonly reported. In MRD reporting, the limit of detection and lower limit of quantification were included. Conclusions: This is the first comprehensive study on the current status of FCI-HLN in Korea, confirming the high heterogeneity and complexity of FCI-HLN practices. Standardization of FCI-HLN is urgently needed. The findings provide a reference for establishing standard FCI-HLN guidelines.

Clinical utility of quantitative immunoassays and surrogate virus neutralization tests for predicting neutralizing activity against the SARS-CoV-2 Omicron BA.1 and BA.5 variants.

Developing new antibody assays for emerging SARS-CoV-2 variants is challenging. SARS-CoV-2 surrogate virus neutralization tests (sVNT) targeting Omicron BA.1 and BA.5 have been devised, but their performance needs to be validated in comparison with quantitative immunoassays. First, using 1749 PRNT-positive sera, we noticed that log-transformed optical density (OD) ratio of wild-type (WT) sVNT exhibited better titer-correlation with plaque reduction neutralization test (PRNT) than % inhibition value. Second, we tried 798 dilutional titration tests with 103 sera, but nonlinear correlation between OD ratio and antibody concentration limited titration of sVNT. Third, the titer-correlations of two sVNT kits for BA.1 and two quantitative immunoassays for WT were evaluated with BA.1 and BA.5 PRNT. All tested kits exhibited a linear correlation with PRNT titers, but the sVNT kits exhibited high false-negative rates (cPass-BA.1 kit, 45.4% for BA.1 and 44.2% for BA.5; STANDARD F-BA.1 kit, 1.9% for BA.1 and 2.2% for BA.5), while quantitative immunoassays showed 100% sensitivity. Linear mixed-effects model suggested superior titer-correlation with PRNT for quantitative immunoassays compared to sVNT kits. Taken together, the use of quantitative immunoassays for WT, rather than rapid development of new kits, would be practical for predicting neutralizing activities against emerging new variants.

False-positive Aspergillus galactomannan immunoassay in the glucose component of total parenteral nutrition products.

IMPORTANCE: This manuscript describes an occurrence of false-positive GM tests in patients receiving TPN products from a manufacturer who had recently changed the supplier of the glucose component. We describe the clinical presentation of nine false-positive cases and the results of serologic and microbiological investigations of the TPN products suspected of contamination with GM. Attempts to detect GM in parenteral nutrition products were made since the detection of GM in sodium gluconate-containing solutions in 2007, but none of them identified the source of elevated GM indexes in TPN products. However, the present study demonstrated that the glucose component of the TPN products contained a high level of GM antigen, which caused false-positive GM assay results. The source of GM was glucoamylase, which was derived from A. niger in the manufacturing process. Physicians and clinical microbiology laboratories should be aware of this issue to improve interpretation and patient care.

Clinical Utility of Sero-Immunological Responses Against SARS-CoV-2 Nucleocapsid Protein During Subsequent Prevalence of Wild-Type, Delta Variant, and Omicron Variant.

As nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 is immunogenic but not targeted in vaccines, it could be useful in distinguishing natural infection from vaccination. We aimed to investigate the clinical utility of sero-immunological responses against the nucleocapsid protein. Nucleocapsid antibody immunoassay study with 302 coronavirus disease 2019 (COVID-19) patients showed lower titers in immunocompromised patients (P < 0.001), higher titers in higher severity (P = 0.031), and different seroconversion rates and titers according to variants of concern. Longitudinal evaluation of nucleocapsid antibodies using 513 samples from 291 COVID-19 patients revealed that it could persist up to 556 days from symptom onset. Interferon gamma release assay against the nucleocapsid protein showed poor response, precluding the deduction of a cut-off for the nucleocapsid protein. In conclusion, nucleocapsid antibody provides instructive clues about the immunogenicity of nucleocapsid proteins by different seroconversion rates and titers according to the severity of infection, host immune status, and different variants of concern.