Low Antibody Titers Research Articles

Leveraging CRISPR-Cas9 for Accurate Detection of AAV-Neutralizing Antibodies: The AAV-HDR Method.

The effectiveness of adeno-associated virus (AAV)-based gene therapy is frequently constrained by the presence of AAV-neutralizing antibodies (NAbs). Existing detection techniques have shown inconsistencies across laboratories and cellular dependencies, challenging their universal applicability. Here, we redefine the NAb titer concept to represent the capability to neutralize a specific number of AAV virions per milliliter of serum. We present the AAV-homology-directed repair (HDR) assay, which harnesses the CRISPR-Cas9 system, offering a precise and sensitive means of detecting AAV NAbs. This assay employs a promoterless AAV HDR vector for integration into electroporated cells, facilitating the stable expression of a quantifiable fluorescent reporter and subsequent NAb titer assessment. Comparative evaluations indicated that the AAV-HDR method outperforms the traditional AAV overexpression (AAV-OE) assay regarding sensitivity and consistency. Crucially, it produced consistent outcomes across various cell lines, suggesting its potential as a universal standard for NAb titer measurement. We further confirmed the validity of the AAV-HDR titration approach by juxtaposing it with the established NT50 assay. Notably, the AAV-HDR method correlated robustly with both the AAV-OE assay and NT50 NAb titer values, and it exhibited heightened efficacy in identifying low-titer antibodies compared with the NT50 method. Given its ability to address AAV NAb detection challenges, the AAV-HDR assay holds promise for refining therapeutic strategies in gene therapy, particularly in tailoring AAV doses to neutralize preexisting NAbs.

Serologic Response to Vaccine for COVID-19 in Patients with Hematologic Malignancy: A Prospective Cohort Study

BackgroundPatients with hematological cancers have increased COVID-19 morbidity and mortality, and these patients show attenuated vaccine responses. This study aimed to characterize the longitudinal humoral immune responses to COVID-19 vaccination in patients with hematological malignancies. Patients and MethodsWe conducted a prospective cohort study, collecting samples from March 2021 to July 2022, from patients seen at a cancer treatment center in London, Ontario, Canada, who met the following eligibility criteria: age ≥18 years, diagnosed with a hematological malignancy, recipient of a COVID-19 vaccine during the study period, and able to provide informed consent. ResultsMedian anti-S titers (MST) were 0.0, 64.0, and 680.5 U/mL following first (V1), second (V2), and third (V3) vaccine doses, respectively. Patients with lymphoid malignancies’ response to vaccination was attenuated compared to myeloid malignancy patients after V2 and V3 (P < .001, P < .01). Active treatment was associated with lower antibody titers (MST 10) compared to treatment 12–24 months (MST 465, P = .04367) and >24 months (MST 1660.5, P = .0025) prior to vaccination. V3 significantly increased antibody titers compared to V2 for patients less than 3 months from treatment. Increasing age was associated with smaller antibody response following V2 (P < .05), but not following V3. Patients receiving anti-CD20 therapy did not demonstrate increased antibody titer levels after V3 (V2 MST 0, V3 MST 0; P > .05). ConclusionWe report an attenuated serologic response to COVID-19 vaccination in our study population of patients with hematological malignancy. The immune response to vaccination was affected by patient age, diagnosis, treatment, and timing of treatment exposure.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

Humoral and cellular immune responses after COVID-19 vaccination of lung transplant recipients and patients on the waiting list: a 6-month follow-up.

Data on cellular response and the decay of antibodies and T cells in time are scarce in lung transplant recipients (LTRs). Additionally, the development and durability of humoral and cellular immune responses have not been investigated in patients on the waitlist for lung transplantation (WLs). Here, we report our 6-month follow-up of humoral and cellular immune responses of LTRs and WLs, compared with controls. Humoral responses to two doses of the mRNA-1273 vaccination were assessed by determining spike (S)-specific IgG antibodies and neutralizing antibodies. Cellular responses were investigated by interferon gamma (IFN-γ) release assay (IGRA) and IFN-γ ELISpot assay at 28 days and 6 months after the second vaccination. In LTRs, the level of antibodies and T-cell responses was significantly lower at 28 days after the second vaccination. Also, WLs had lower antibody titers and lower T-cell responses compared with controls. Six months after the second vaccination, all groups showed a decrease in antibody titers and T-cell responses. In WLs, the rate of decline of neutralizing antibodies and T-cell responses was significantly higher than in controls. Our results show that humoral and cellular responses in LTRs, if they develop, decrease at rates comparable with controls. In contrast, the inferior cellular responses and the rapid decay of both humoral and cellular responses in the WL groups imply that WLs may not be protected adequately by two vaccinations and repeat boostering may be necessary to induce protection that lasts beyond the months immediately post-transplantation.

Characterization of antibody-dependent cellular phagocytosis in patients infected with hepatitis C virus with different clinical outcomes.

Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.

Evaluation of inflammatory biomarkers and their association with anti-SARS-CoV-2 antibody titers in healthcare workers vaccinated with BNT162B2.

Vaccine-induced immunity against COVID-19 generates antibody and lymphocyte responses. However, variability in antibody titers has been observed after vaccination, and the determinants of a better response should be studied. The main objective of this investigation was to analyze the inflammatory biomarker response induced in healthcare workers vaccinated with BNT162b2, and its association with anti-Spike (a SARS-CoV-2 antigen) antibodies measured throughout a 1-year follow-up. Anti-spike antibodies and 92 biomarkers were analyzed in serum, along with socio-demographic and clinical variables collected by interview or exploration. In our study, four biomarkers (ADA, IL-17C, CCL25 and CD8α) increased their expression after the first vaccine dose; and 8 others (uPA, IL-18R1, EN-RAGE, CASP-8, MCP-2, TNFβ, CD5 and CXCL10) decreased their expression. Age, body mass index (BMI), smoking, alcohol consumption, and prevalent diseases were associated with some of these biomarkers. Furthermore, higher baseline levels of T-cell surface glycoprotein CD6 and hepatocyte growth factor (HGF) were associated with lower mean antibody titers at follow-up, while levels of monocyte chemotactic protein 2 (MCP-2) had a positive association with antibody levels. Age and BMI were positively related to baseline levels of MCP-2 (β=0.02, 95%CI 0.00-0.04, p=0.036) and HGF (β=0.03, 95%CI 0.00-0.06, p=0.039), respectively. Our findings indicate that primary BNT162b2 vaccination had a positive effect on the levels of several biomarkers related to T cell function, and a negative one on some others related to cancer or inflammatory processes. In addition, a higher level of MCP-2 and lower levels of HGF and CD6 were found to be associated with higher anti-Spike antibody titer following vaccination.

Comparison of SARS-COV-2 humoral response between rheumatoid arthritis, psoriatic arthritis and spondyloarthritis patients and controls in two unvaccinated cohorts.

To compare the humoral response after a SARS-CoV-2 infection in an inflammatory rheumatic disease population with a healthy control population in a case-control study. Cases: between March and September 2021, all consecutive unvaccinated patients followed for rheumatoid arthritis (RA), spondyloarthritis (SpA) or psoriatic arthritis (PsA) in 16 hospitals in France were systematically screened with a SARS-CoV-2 serological test. Patients with a positive test were included in the COVID-RIC-2 cohort. between June and July 2020, healthcare professionals working in the Toulouse University Hospital were screened with a SARS-CoV-2 serological test. Those with a positive test were included in the COVID-BIOTOUL cohort and matched to those from COVID-RIC-2 by age, sex and time-sampling on infection date. total SARS-CoV-2 antibody titres were centrally measured and compared. 95 patients from COVID-RIC-2 (mean age 49 years, 76% females, median delay of COVID infection: 149 days) including 48 RA, 33 SpA and 14 PsA were compared to 95 matched controls. Globally, there was no significant difference of SARS-CoV-2 antibody titres between both populations: 155 Binding Antibody Units (BAU) (IQR:7-376) in COVID-RIC-2 vs. 120 BAU (IQR:35-320) in COVID-BIOTOUL. There was a trend towards higher antibody titres in patients from COVID-RIC-2 with severe COVID-19 symptoms. In COVID-RIC-2, there was no impact of age, sex, time-sampling or underlying disease on antibody titres and patients taking glucocorticoids, abatacept or rituximab trended toward having lower antibody titres after COVID-19 infection. This study provides reassuring data on humoral response after COVID-19 infection in patients treated with disease-modifying anti-rheumatic drugs.

Prenatal Exposure to Poly- and Perfluoroalkyl Substances (2009-2014) and Vaccine Antibody Titers of Measles, Mumps, Rubella, and Varicella in Children Four to Eight Years Old from the Healthy Start Cohort.

Prenatal exposures to certain poly- and perfluoroalkyl substances (PFAS) are associated with reduced humoral responses to some childhood immunizations. We estimated associations between prenatal PFAS exposure and child antibody titers for measles, mumps, rubella (MMR), and varicella after immunization. We measured serum antibody titers of 145 children (4-8 y old) enrolled in the Healthy Start cohort in Colorado, whose mothers had PFAS quantified mid-pregnancy (2009-2014). We used linear and logistic regression models to assess the relationship between five PFAS detected in of mothers and continuous or non-high-censored ("low") antibody titers and quantile g-computation to evaluate the overall effect of the PFAS mixture. Median concentrations of individual PFAS were at or below the median reported among females in the United States. After receiving two vaccine doses, seropositive levels of antibodies were detected among most (93%-100%) children. Each log-unit increase in perfluorononanoate was associated with 2.09 [95% confidence interval (CI): 1.13, 3.87] times higher odds of a low measles titer, and each log-unit increase in perfluorooctanoate was associated with 2.46 (95% CI: 1.28, 4.75) times higher odds of a low mumps titer. Odds ratios for all other PFAS were elevated, but CIs included the null. Each quartile increase in the PFAS mixture was associated with 1.35 (95% CI: 0.80, 2.26) times higher odds of a low measles titer and 1.44 (95% CI: 0.78, 2.64) times higher odds of a low mumps titer. No significant associations were observed between PFAS and varicella or rubella antibodies. In stratified analyses, associations were negative among female children, except for perfluorohexane sulfonate and varicella, whereas they were positive among males. Some prenatal PFAS were associated with lower antibody titers among fully immunized children. The potential for immunotoxic effects of PFAS requires further investigation in a larger study, because exposure is ubiquitous globally. https://doi.org/10.1289/EHP12863.

Humoral response after breakthrough SARS-CoV-2 infection in type 2 diabetes mellitus patients

Abstract Type 2 diabetes mellitus (T2DM) remains an important public health problem in both developing and developed countries. In addition, the recent COVID-19 pandemic has revealed furthel risks for diabetes patients in terms of symptoms and disease progression. Higher mortality and morbidity are related to the complexity of the pathology of this chronic underlying disease, which negatively affects the immune response to the SARSCoV-2 virus. The humoral response plays an important role in the eradication of the virus; thus, it was analyzed in vaccinated diabetics who underwent COVID-19, as well as in the control group. The aim of this study was to assess the prevalence and level of IgG antibodies raised against the nucleocapsid protein (NCP), S1 subunit receptor binding domain (RBD) and subunit Spike 2 (S2) subunit of the virus’s S protein using the Microblot Array test. The results demonstrated significantly lower prevalence and titers of anti-SARS antibodies in diabetic patients compared to the control group. In addition, antibody titers were negatively related to the duration of this chronic disease, body mass index (BMI), comorbidities and HbA1c concentration. Further research is needed to develop the best strategy for specific prevention of SARS-CoV-2 infection in diabetic patients.

Exploring Appropriate Strategies for Vaccination against Classical Swine Fever under a Dynamic Change in Antibody Titer in Sows after Starting Vaccination in a Japanese Farm Setting

After 26 years of absence in Japan, a classical swine fever (CSF) outbreak occurred at a domestic pig farm in 2018. Vaccination against the CSF virus with a live attenuated vaccine at pig farms was restarted in October 2019, which was 13 years after the 2006 ban on vaccination. An individual-based simulation model for CSF antibody dynamics was developed to determine an effective CSF vaccination strategy for pig populations. In creating a simulated pig herd, the optimal vaccination age of piglets and the effect of vaccinating piglets twice were evaluated. Additionally, the herd immunity was monitored every 6 months for 4 years after the start of vaccination, and the effects of intensive sow replacement policies were assessed. The simulation results indicated that the vaccination age should be delayed relative to the age used before the 2006 ban on vaccination and shifted earlier, from 8 weeks to 6 weeks, as time elapses. The simulations indicated a tradeoff in protection between the weaning period (i.e., maternally derived antibodies) and the fattening period (i.e., by vaccine-induced antibodies). Mixing sows with high and low antibody titers, particularly sows that received the first vaccination and those born after the start of vaccination, resulted in a high variation in antibody titer among pigs on the farm. This study also clarified the positive effect of intensive sow replacement strategies on shortening the period in which sows show diverse titers. Differences in sow replacement rates among farms and/or the time lag in starting vaccination in different prefectures result in heterogeneity in herd immunity in Japan; thus, herd immunity status should be examined at every farm using this simulation model.

SARS-CoV-2 mRNA Vaccines Induce Greater Complement Activation and Decreased Viremia and Nef Antibodies in Men With HIV-1.

Immune dysregulation in people with human immunodeficiency virus-1 (PWH) persists despite potent antiretroviral therapy and, consequently, PWH tend to have lower immune responses to licensed vaccines. However, limited information is available about the impact of mRNA vaccines in PWH. This study details the immunologic responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in PWH and their impact on HIV-1. We quantified anti-S immunoglobulin G (IgG) binding and neutralization of 3 SARS-CoV-2 variants of concern and complement activation in blood from virally suppressed men with HIV-1 (MWH) and men without HIV-1 (MWOH), and the characteristics that may impact the vaccine immune responses. We also studied antibody levels against HIV-1 proteins and HIV-1 plasma RNA. MWH had lower anti-S IgG binding and neutralizing antibodies against the 3 variants compared to MWOH. MWH also produced anti-S1 antibodies with a 10-fold greater ability to activate complement and exhibited higher C3a blood levels than MWOH. MWH had decreased residual HIV-1 plasma viremia and anti-Nef IgG approximately 100 days after immunization. MWH respond to SARS-CoV-2 mRNA vaccines with lower antibody titers and with greater activation of complement, while exhibiting a decrease in HIV-1 viremia and anti-Nef antibodies. These results suggest an important role of complement activation mediating protection in MWH.

Vaccination Recommendations and Clinical Characteristics of Children after Allogeneic Hematopoietic Stem Cell Transplantation

To investigate the clinical characteristics, the risk factors related to antibody-negative conversion, and the effectiveness and safety of revaccination in children who underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT). The clinical characteristics of 516 children after Allo-HSCT who were eligible for revaccination in the Vaccination Evaluation Clinic of Shanghai Children's Medical Center from March 2017 to March 2023 were analyzed. Of the 516 patients enrolled, 464 (89.9%) had started revaccination, with a median time of 2.1 (IQR, 1.8-2.6) years after transplantation and 1.1 (IQR, 0.7-1.5) years after cessation of immunosuppressive therapy. There were 418 cases of hepatitis B surface antibody (HBsAb) seropositive before transplantation, and 339 cases (81.1%) became negative at the time of evaluation after transplantation. The visit time for children with persistently HBsAb seropositive was 1.9 (IQR, 1.3-2.3) years after transplantation, which was shorter than the time for children with HBsAb seronegative (2.2 [IQR, 1.9-2.8] years, P&amp;lt;0.001). HBsAb negative conversion was related to the HBsAb titers pre-transplantation, with a lower proportion of high titers in the negative conversion group (4.7% vs. 17.7%, P&amp;lt;0.001). Both univariate and multivariate analyses suggested that HBsAb seroconversion was statistically related to uncompleted three doses of the hepatitis B vaccine before transplantation, low pre-transplantation antibody titers, and ≥2 years after transplantation. Among 40 patients with HBsAb seronegative who received hepatitis B vaccination, 39 (97.5%) had HBsAb titers converted to seropositive. The immune response rates of measles, mumps, and rubella (MMR) were 96.6% (28/29), 69.0% (20/29,) and 88.5% (23/26), respectively. There were no serious adverse side effects after revaccination.After Allo-HSCT, standard evaluation procedures for revaccination are highly recommended. The immune response rates after hepatitis B and MMR revaccination were highly recommended. No severe adverse side effects were observed.

Efficacy and Safety of Concizumab Prophylaxis in Patients with Hemophilia A or B without Inhibitors: 56-Week Cut-Off Results of the Phase 3 explorer8 Study

Introduction Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody in development as a once-daily subcutaneous prophylactic treatment for hemophilia of all subtypes. Results from the 56-week cut-off of the prospective, multicenter, open-label explorer8 study (NCT04082429; phase 3) in patients with hemophilia A or B without inhibitors (HA/HB) are presented, further to the 32-week confirmatory analysis cut-off (CACO) results previously reported (Chowdary P et al. ISTH 2023, OC 59.1). Aim To assess the longer-term efficacy and safety of daily concizumab prophylaxis in patients with HA/HB. Methods Male patients (aged ≥12 years) with HA/HB were randomized 1:2 to no prophylaxis (arm 1; 24 weeks) or concizumab prophylaxis (arm 2; ≥32 weeks) or assigned to non-randomized concizumab prophylaxis (arms 3 and 4; ≥32 weeks). After the main part of the trial (24 weeks for arm 1), patients in arm 1 could switch to concizumab prophylaxis. After treatment restart following the treatment pause due to thromboembolic events, patients received a 1.0 mg/kg concizumab loading dose on Day 1, followed by an initial 0.20 mg/kg daily dose starting on Day 2, with potential adjustment to 0.15 or 0.25 mg/kg based on measured plasma concizumab concentration after week 4. Assessments at the 56-week cut-off included efficacy, safety, pharmacokinetic and pharmacodynamic measurements. Informed consent/ethics committee approval were obtained. Results The study included 148 patients (HA, n=82; HB, n=66), of which 21 patients were randomized to no prophylaxis (arm 1: HA, n=9; HB, n=12), 42 patients to concizumab prophylaxis (arm 2: HA, n=18; HB, n=24) and the remaining 85 patients were assigned to concizumab prophylaxis (non-randomized arms 3 and 4). After the main part of the trial, 17 patients from arm 1 switched to concizumab prophylaxis, receiving the same dosing regimen as patients in arms 2‒4 when first starting on concizumab. A total of 132 patients (HA, n=73; HB, n=59) completed the 56-week cut-off. The overall median (interquartile range) annualized bleeding rate (ABR) on concizumab prophylaxis in arms 1‒4 was 1.7 (0.0‒4.5) for HA and 2.8 (0.0‒6.4) for HB (Table 1); these results were also supported by the supportive secondary efficacy assessments. After treatment restart, no thromboembolic events were reported (Table 2). A total of 30 serious adverse events (AEs) were reported in 20 patients at the 56-week cut-off. As previously reported at CACO, one patient experienced a serious AE with fatal outcome (intra-abdominal hemorrhage) and 6 patients experienced AEs leading to withdrawal of concizumab treatment. The rate of injection site reactions was comparable to CACO. No hypersensitivity type reactions were reported in the study and low-titer anti-concizumab antibodies were detected in a total of 21 patients at the 56-week cut-off, with no apparent impact on bleeds or AEs. Concizumab plasma concentration remained stable over time. Free TFPI and thrombin peak levels continued to be stable over time. Conclusions Concizumab prophylaxis showed longer-term (≥1 year) efficacy in adult and adolescent patients with HA/HB at the 56-week cut-off, which was consistent with the results observed at CACO. Concizumab prophylaxis was considered safe and well tolerated in patients with HA/HB.

Large-Scale Characterization and Functional Assessment of Adaptive Immunity in Patients with MGUS and SMM Reveals Significant Defects

Introduction Patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Multiple Myeloma (SMM) exhibit signs of immune dysregulation despite their early stage of oncogenesis. The SARS-CoV-2 pandemic and subsequent broad vaccination of the population presented an opportunity to compare the real-life immune response of patients and healthy individuals following exposure to a common antigen. Here, we have used SARS-CoV-2 vaccination as a model to study the adaptive immune response and have thus comprehensively assessed the adaptive immune response of patients with MGUS/SMM to SARS-CoV-2 vaccination compared to healthy donors (HD). Methods We performed (i) paired 5' single-cell RNA and T cell receptor (TCR) sequencing on 224 peripheral blood mononuclear cell (PBMC) samples drawn serially before and after two doses of SARS-CoV-2 vaccination (HD, n=26; MGUS, n=20; SMM, n=48; Multiple Myeloma (MM), n=24), (ii) ELISA for anti-Spike IgG antibodies post-vaccination (post-vx), (HD, n=237; MGUS, n=550; SMM, n=947) and MMR antibodies at baseline (HD, n=20; SMM, n=20), (iii) bulk TCR sequencing pre- and post-vx (HD, n=2; SMM, n=2) (Adaptive Biotechnologies), and (iv) IFN gamma ELISpot assay following in vitro stimulation with Spike peptide (HD, n=20; SMM, n=20). All post-vx samples were drawn after 2 doses of vaccination. Results Patients and HD showed comparable anti-Spike antibody titers within 14-50 days post-vx, however, we observed faster waning in patients with MGUS/SMM, who had significantly lower titers 50-100 days post-vx (p=0.014 &amp; p=0.007, respectively), suggesting a potential defect in long-lived plasma cell maintenance. Indeed, patients with SMM showed significantly lower titers of antibodies against Mumps and Rubella, antigens encountered in childhood, compared to age-matched HD (p=0.002 &amp; p=0.03, respectively), showing that this defect may also lead to loss of plasma cell memory. Next, we performed single-cell RNA/TCR-seq to assess the patients' T cell response to vaccination. Patients with SMM showed a significant increase in TCR repertoire diversity post-vx (paired t-test, p=0.046), suggesting at least partial preservation of T cell responses with recruitment of rare clones. Indeed, using bulk TCR-seq, we observed Spike-specific T cells in patients with MGUS/SMM. Interestingly, however, a significant enrichment in Spike-specific clones by single-cell TCR-seq was only observed in HD post-vx (paired t-test, p=0.01), suggesting patient T cell responses may be suboptimal. Functional assessment of T cell activation following stimulation with the Spike peptide showed significantly lower activation in patients with SMM compared to HD (t-test, p=0.002), confirming the suspected defect. Notably, a larger proportion of Spike-specific clones in patients was of the Treg phenotype compared to HD (empirical p=0.002), which may partly explain the observed defect. Conclusions This study demonstrates that despite the absence of symptoms, patients with MGUS/SMM may demonstrate defective humoral and cellular immune responses, including loss of plasma cell memory. Specific interventions, such as changes in the vaccination schedule, may be warranted for this population, and the morbidity associated with these defects should be studied further.

Effects of dietary inclusion of Moringa oleifera methanolic extract on productive performance, humoral immunity and nutrient digestibility in Japanese quails

ABSTRACT A total of 180 unsexed, 1-day-old quail chicks were divided into four treatments (three replicates). The treatment groups were designated as MOLE-0, MOLE-1, MOLE-2 and MOLE-3. In the MOLE-0 group, quails received a basal diet without supplementation. For MOLE-1, quails were provided with 100 mg/L of MOLE in their water, while MOLE-2 received 200 mg/L of water with MOLE supplementation. The fourth treatment, MOLE-3, received a concentration of 300 mg/L of MOLE in their drinking water. The findings revealed that MOLE-3 exhibited significantly higher weight gain and dressing percentage, along with a lower feed conversion ratio (P < 0.05), while the control group (MOLE-0) exhibited the least growth performance. MOLE-3 showed statistically higher antibody titres and immune organ weights, followed by MOLE-2 and MOLE-1. Conversely, the control group (MOLE-0) had a lower antibody titre against ND. Additionally, nutrient digestibility analysis indicated that MOLE-3 had higher digestibility of dry matter and crude protein (P < 0.05), while group MOLE-0 exhibited lower digestibility (P < 0.05). We concluded that supplementing Moringa oleifera leaf extract at a rate of 300 mg/L resulted in improved growth performance, immunity and nutrient digestibility in Japanese quails.

Engineered Nanovesicles Expressing Bispecific Single Chain Variable Fragments to Protect against SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in high morbidity and mortality rates worldwide. Although the epidemic has been controlled in many areas and numerous patients have been successfully treated, the risk of reinfection persists due to the low neutralizing antibody titers and weak immune response. To provide long-term immune protection for infected patients, novel bispecific CB6/dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (SIGN) nanovesicles (NVs) were constructed to target both the SARS-CoV-2 spike protein (S) and the DC receptors for virus neutralization and immune activation. Herein, we designed NVs expressing both CB6 and DC-SIGN single chain variable fragments (scFvs) on the surface to block SARS-CoV-2 invasion and activate DC function. Monophosphoryl lipid A (MPLA) was loaded into the CB6/DC-SIGN NVs as an adjuvant to promote this process. The CB6/DC-SIGN NVs prevented a pseudovirus expressing the S protein from infecting the target cells expressing high levels of angiotensin-converting enzyme 2 in vitro. Additionally, CB6/DC-SIGN NVs admixed with S-expressing pseudoviruses activated the DCs, which was promoted by the adjuvant MPLA loaded in the NVs. Using a mouse model, we also confirmed that the CB6/DC-SIGN NVs effectively improved the neutralizing antibody titer and inhibited the growth of tumors expressing the S protein after 3 weeks of treatment. This potential NV-based treatment not only exerts a blocking effect by binding the S protein in the short term but may also provide patients with long-term protection against secondary infections.

Impact of Nutritional Status on Antibody Titer After Booster mRNA COVID-19 Vaccine Among Elderly Adults in Japan.

Published studies on mRNA coronavirus disease 2019 (COVID-19) vaccine effects focus on younger individuals, comprising the majority of the workforce. Studies in elderly adults are sparse. In total, 107 subjects were recruited (median age 78; interquartile range [IQR], 58.5-90.5; range, 35-105 years). Factors associated with antibody titer after the third mRNA COVID-19 vaccination were compared between 49 elderly (age ≥80; median, 94; IQR, 86-97; range, 80-105 years) and 58 younger (age ≤79; median, 61; IQR, 46-71; range, 35-79 years) adults. Among body mass index (BMI) categories, the group of underweight elderly adults had a lower antibody titer compared to those with normal weight (P < .01 after 1, 3, and 5 months). Elderly adults were less likely to maintain effective antibody titer (≥4160 AU/mL) compared to younger adults: 76% versus 98%, P < .001 after 1 month, and 45% versus 78%, P < .001 after 3 months. Elderly adults who maintained effective antibody titer for 5 months had a higher BMI (22.9 kg/m2 vs 20.1 kg/m2, P = .02), and were less likely to have underweight BMI (0% vs 31%, P = .02) compared to the subjects who failed to maintain effective antibody titer. These results highlight the impact of nutritional status and the deleterious effect of underweight BMI on antibody titer and its maintenance among elderly adults following booster mRNA COVID-19 vaccination.

Serum antibody levels to SARS-CoV-2 receptor-binding domain (RBD) in convalescent patients and vaccinated individuals of northern Nevada.

Antibodies reactive with the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein are associated with viral neutralization, however low antibody titers, specifically against SARS-CoV-2 variants, may result in reduced viral immunity post naturally acquired infection. A cohort study comprised of 121 convalescent individuals from northern Nevada was conducted looking at anti-RBD antibody levels by enzyme-linked immunosorbent assay. Serum was collected from volunteers by staff at the University of Nevada, Reno School of Medicine Clinical Research Center and assessed for antibodies reactive to various SARS-CoV-2 RBD domains relevant to the time of the study (2020-2021). A nonpaired group of vaccinated individuals were assessed in parallel. The goal of the study was to identify antibody levels against the RBD subunit in convalescent and vaccinated individuals from northern Nevada.

Acquired Hemophilia a: A Single-Center Study of 165 Patients

Introduction and aim: Acquired hemophilia A (AHA) is a rare hemorrhagic disorder caused by the existence of factor VIII (FVIII) inhibitors with an estimated annual incidence of 1.5 per million people. AHA patients are characterized by exceeding bleeding of varying severity, while approximately 10% of AHA patients have no bleeding symptoms. It's a challenge to diagnose and manage AHA due to its rarity and heterogeneity. We conducted a retrospective study to report the characteristics and outcomes of AHA and enhance its recognition and management. Methods: Patients diagnosed and treated with AHA at the Blood Disease Hospital, Chinese Academy of Medical Sciences between July 1997 to December 2021 were retrospectively enrolled. Demographics, clinical characteristics, treatments, and outcomes were collected. All Patients were followed immediately from admission to our center and ended in March 2022. Bleeding severity was assessed retrospectively in March 2022 by bleeding score (BS), a summary score aggregated by the International Society on Thrombosis and Hemostasis/ Scientific and Standardization Committee (ISTH/SSC) bleeding assessment tool (BAT) (2010 edition). Results: A total of 165 AHA patients admitted to our center were enrolled. The median age at diagnosis was 45 (10-90) years. Overall, a female preponderance (106/165, 64.2%) was observed in this cohort. Notable, the proportion of male patients increased with age. Most patients (126/165, 76.4%) were idiopathic with no underlying diseases discovered. Of the 39 (23.6%) patients with associated diseases, 12 patients had autoimmune diseases, 16 patients had infections, eight patients had malignancies (all solid tumors) and three patients were secondary to pregnancy. All patients presented at least one bleeding episode. Subcutaneous bleeding was most common affecting 84.8% (140/165) of patients, while hemarthrosis was infrequent (n=11, 6.7%). The median time from bleeding to be diagnosed as AHA was delayed (30, 2-5650 days). Anti-FVIII antibody titer and FVIII level showed no correlation. A significant difference in FVIII activity was observed between patients with anti-FVIII antibody titer &amp;lt; 20 BU/ml and ≥ 20 BU/ml. The BS varied among all patients ranging from 2 to 12 with a median of 4. A significant difference in BS was observed among the mild (FVIII: C &amp;gt; 5 IU/dl), moderate (1 IU/dl &amp;lt; FVIII: C ≤ 5 IU/dl), and severe (FVIII: C ≤ 1 IU/dl) AHA patients (p &amp;lt; 0.05). The same conclusion was also drawn between patients with low anti-FVIII antibody titer (&amp;lt; 20 BU/ml) and high antibody titer (≥ 20 BU/ml). BS was not correlated with APTT (r=0.-0.04, 95% CI (-0.19-0.12), p &amp;gt; 0.05). Hemostatic treatment was applied to 129 (78.2%) AHA patients. Of the 163 patients who received immunosuppressive therapy (IST), 22 (13.5%) received steroids alone, with an 82.4% complete remission (CR) rate, 50 (30.7%) received steroids plus cyclophosphamide (CTX), with an 85.0% CR rate, 80 (49.1%) received rituximab-based therapy, which includes rituximab plus steroids, rituximab plus steroids and CTX, and rituximab plus steroids and other immunosuppressants, with a 93.3% CR rate (Figure 1A). There were seven deaths. Causes of death were bleeding (n=3), underlying disease (n=1), pulmonary infection (m=1), and unknown (n=2). FVIII inhibitor titer ≥ 20 BU/ml was the only significant poor prognostic factor for CR (HR 0.56; 95% CI, 0.37-0.85; p &amp;lt; 0.01). In addition, patients with FVIII inhibitor titers ≥ 20 BU/ml needed more time to achieve CR (130 days vs. 35 days; p &amp;lt; 0.01) and showed a lower CR rate (77.6% vs. 94.9%, p &amp;lt; 0.01) than those with FVIII inhibitor titers &amp;lt; 20 BU/ml (Figure 1B). Conclusions: Delayed diagnosis of AHA patients is a severe issue. The rituximab-based regimen is more likely to achieve a CR in AHA patients. The only poor predictor of CR for AHA patients was FVIII inhibitor titer ≥ 20 BU/ml.

Regulatory T cell-like response to SARS-CoV-2 in Jamaican fruit bats (Artibeus jamaicensis) transduced with human ACE2.

Insectivorous Old World horseshoe bats (Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats (Rousettus aegyptiacus) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats (Eptesicus fuscus) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats (Artibeus jamaicensis) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyer's patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4+ helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-β, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptible to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease.