Serum Titers Research Articles

Seroprevalence of Francisella tularensis in patients with neck mass complaints

Aim: Tularemia is a zoonotic disease endemic in the northern hemisphere. The causative agent of the disease is Francisella tularensis. F.tularensis is endemic in Turkey, predominantly in the Marmara and Black Sea regions, and causes small outbreaks. This study aimed to investigate the seroprevalence of F.tularensis in patients admitted to the Otorhinolaryngology outpatient clinic with the complaint of neck mass by using two different methods.Methods: Serum samples were collected from patients who were admitted to the Otorhinolaryngology outpatient clinic of Dicle University Faculty of Medicine Hospitals between January 2021 and December 2021 with the complaint of neck mass. A commercially available immunochromatographic lateral flow test (ICT) and a single-assay chemiluminescence test (CHT) were used to detect F.tularensis antibodies. Rose-Bengal test was performed on all sera to determine cross-reactions with antibodies produced in brucellosis. Brucella immunocapture agglutination tests (BCT) were performed on the sera of patients with positive screening tests.Results: The ages of patients diagnosed with neck mass ranged between 14–70 years, with a mean age of 44.5±12.1 years. Sixty two (62%) of the patients were male, and 38 (38%) were female. When the test results were evaluated, two sera were positive for F.tularensis by both ICT and CHT methods. The first serum tests were negative for Brucella. The titer of the second serum in the chemiluminescence test was low positive.Conclusion: Tularemia should be considered in the differential diagnosis of patients presenting with neck mass complaints, especially in patients living in rural areas, and specific diagnostic tests should be performed. In addition, a more comprehensive seroprevalence study supported by molecular testing techniques to be conducted in Diyarbakır will provide clearer data on the extent to which tularemia affects our region and which subspecies is the causative agent.

Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology.

Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was observed in different organs of vaccinated fish whereas minor deteriorative changes were found in fish vaccinated with higher concentrations of the vaccine.

Impact of xenogenic mesenchimal stem cells secretome on a humoral component of the immune system

Objective. To determine such parameters of humoral immunity as the number of antibody-forming cells and the titer of antibodies in the blood under the action of the whole fraction of the secretome of xenogenic mesenchymal stem cells in mice with a normal immune status and mice with secondary immunodeficiency caused by the hydrocortisone acetate. Methods. The following methods were used in the study: isolation of mesenchymal stem cells from the bone marrow of cattle; culturing of isolated cells and obtaining a conditioned medium containing exometabolites with subsequent purification; modeling of secondary immunodeficiency in mice; setting up a test on mice to determine the number of antibody-forming cells in the spleen and hemagglutinin titers in blood serum; statistical data analysis. Results. A study of the impact of the secretome of xenogenic mesenchymal stem cells (MSCs) on the humoral arm of immunity in mice with a normal immune status showed a significant increase in the antibody-forming cells count compared with the control by 3.4-8.8 times when administered intramuscularly and by 4.2-5.4 times when administered subcutaneously (at the same time, in the group of the reference drug, the increase was four times), as well as an increase in the titer of hemagglutinins concerning the control by 25-95% when administered intramuscularly and 32-52% when administered subcutaneously (the reference drug Thymalin was at the control level). With intramuscular administration, a clear relationship was observed between the concentration of MSC secretome and the biological effect (high significant positive correlation: ρ = 0.99, p ≤ 0.05 for calculating AFC; ρ = 0.97, p ≤ 0.05 for HA titer). Lethality in the group of animals what administered only hydrocortisone acetate (HCA, positive control) was 100%. The use of the mesenchymal stem cells secretome increased the survival of animals by 50% by stimulating the formation of the required number of antibody-forming cells and antibody titer, except for the subcutaneous route of administration (at the level of immunized control, animals with a normal immune status). The reference drug showed a result at a level significantly lower than the immunized control. The antibody titer with the subcutaneous route of administration of secretion of MSCs was significantly lower than the immunized control but significantly higher concerning the reference drug. Conclusion. The administration of the secretome of xenogenic mesenchymal stem cells stimulated the humoral arm of immunity as same in mice with normal immune status as in mice with secondary immunodeficiency. The data obtained supplement the information on the introduction of live mesenchymal stem cells. Live allogeneic MSCs have a suppressive effect on B cells, while xenogenic MSCs cause a response on themselves. The secretome of xenogenic MSCs does not contain surface immunogenic molecules that are carried by living cells, but nevertheless increases the activity of the humoral component of immunity. The mechanisms of this effect require further study.

Pseudotyped Viruses for Influenza.

We have developed an influenza hemagglutinin (HA) pseudotype (PV) library encompassing all influenza A (IAV) subtypes from HA1-HA18, influenza B (IBV) subtypes (both lineages), representative influenza C (ICV), and influenza D (IDV) viruses. These influenza HA (or hemagglutinin-esterase fusion (HEF) for ICV and IDV) pseudotypes have been used in a pseudotype microneutralization assay(pMN), an optimized luciferase reporter assay, that is highly sensitive and specific for detecting neutralizing antibodies against influenza viruses. This has been an invaluable tool in detecting the humoral immune response against specific hemagglutinin or hemagglutinin-esterase fusion proteins for IAV to IDV in serum samples and for screening antibodies for their neutralizing abilities. Additionally, we have also produced influenza neuraminidase (NA) pseudotypes for IAV N1-N9 subtypes and IBV lineages. We have utilized these NA-PV as surrogate antigens in in vitro assays to assess vaccine immunogenicity. These NAPV have been employed as the source of neuraminidase enzyme activity in a pseudotype enzyme-linked lectin assay (pELLA) that is able to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies, and postvaccination sera. Here we show the production of influenza HA, HEF, and NAPV and their employment as substitutes for wild-type viruses in influenza serological and neutralization assays. We also introduce AutoPlate, an easily accessible web app that can analyze data from pMN and pELLA quickly and efficiently, plotting inhibition curves and calculating half-maximal concentration (IC50) neutralizing antibody titers. These serological techniques coupled with user-friendly analysis tools are faster, safer, inexpensive alternatives to classical influenza assays while also offering the reliability and reproducibility to advance influenza research and make it more accessible to laboratories around the world.

Viabilidade do Toxoplasma gondii em sêmen de bovino criopreservado em diferentes concentrações de crioprotetores

ABSTRACT: Toxoplasma gondii can be eliminated in bovine semen. Cryopreserved semen is often used due to the fact that artificial insemination in dairy and beef cattle provides benefits in terms of production. However, little is known regarding the viability and infectivity of T. gondii tachyzoites in cryopreserved bovine semen. In the present study, cattle semen negative for T. gondii were contaminated with 1 x 106 tachyzoites (RH strain) and cryopreserved with and without different cryoprotectants, such as DMSO (concentrations of 2.5%, 5.0%, 7.5%, 8.0% and 10.0%) and glycerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10.0%), followed by freezing in liquid nitrogen (-196°C). After 24 hours, the samples were thawed and inoculated in 10 mice per cryoprotectant concentration. The mice were evaluated for clinical signs of toxoplasmosis (rough coat, diarrhea, hypoactivity and sudden death) as well as serum titers of IgM and IgG and the presence of tachyzoites in the peritoneal lavage. The results revealed that T. gondii remained infective in all samples. Clinical signs of toxoplasmosis were observed in the mice beginning with the 6th day post-inoculation (DPI) and 100% lethality was found between the 7th and 9th DPI. Viable tachyzoites were recovered from peritoneal exudate of dead mice (except for the control group), with higher mean of tachyzoite counts in the intraperitoneal lavage for 5% DMSO (±3.32 x 106), 8% DMSO (±3.53 x 106), 3% glycerol (±4.75 x 106), 7.5% glycerol (±6.26 x 106) and the absence of cryoprotectant (±3.11 x 106). Seroconversion occurred in the treated groups, with titers of IgG from 1:16 to 1:128 and IgM from 1:16 to 1:512. T. gondii viability and infectivity were maintained in cattle semen during 24 hours of cryopreservation at -196°C with and without cryoprotectant. However, further studies are necessary to determine whether cryopreserved semen contributes to the spread of toxoplasmosis through artificial insemination.

Reproducibility of double agar gel immunodiffusion test using stored serum and plasma from paracoccidioidomycosis patients.

Serological evaluation performed by double agar gel immunodiffusion test (DID) is used for diagnosis, evaluation of severity, management of paracoccidioidomycosis patients, and development of new clinical studies. For these reasons, the Botucatu Medical School of UNESP maintains a serum bank at the Experimental Research Unit with patient clinical data. This study aimed to evaluate the influence of the freeze-thaw cycle and different blood matrices on the titration of circulating antibodies. The study included 207 patients with confirmed (etiology-demonstrated) or probable (serology-demonstrated) paracoccidioidomycosis, and DID was performed with culture filtrate from Paracoccidioides brasiliensis B339 as antigen. First experiment: the antibody levels were determined in serum samples from 160 patients with the chronic form and 20 with the acute/subacute form, stored at -80oC for more than six months. Second experiment: titers of 81 samples of serum and plasma with ethylenediaminetetraacetic acid (EDTA) or heparin, from 27 patients, were compared according to matrix and effect of storage at -20oC for up to six months. Differences of titers higher than one dilution were considered discordant. First experiment: test and retest presented concordant results in serum stored for up to three years, and discordant titers in low incidence in storage for four to six years but high incidence when stored for more than six years, including conversion from reagent test to non-reagent retest. Second experiment: serum, plasma-EDTA and plasma-heparin samples showed concordant titers, presenting direct correlation, with no interference of storage for up to six months. Storage at -80oC for up to six years has no or little influence on the serum titers determined by DID, permitting its safe use in studies depending on this parameter. The concordant titrations in different blood matrices demonstrated that the plasma can be used for immunodiffusion test in paracoccidioidomycosis, with stability for at least six months after storage at -20oC.

Serum titer of neutralizing antibodies after COVID-19 vaccination in Japanese patients with inflammatory bowel disease.

Vaccination is an important strategy to reduce the infection rate and adverse events of coronavirus disease 2019 (COVID-19). However, the effect of COVID-19 vaccination for Japanese patients with inflammatory bowel disease (IBD) has not been fully elucidated. In the present study, we investigated the serum titer of neutralizing antibodies after COVID-19 vaccination in patients with IBD, treated with and without immunosuppressive therapy. The study consisted of 108 patients with IBD [76 with ulcerative colitis (UC) and 32 with Crohn's disease (CD)] from the gastroenterology outpatient clinic at the Hospital of the Kyoto Prefectural University of Medicine who underwent anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. The control group included 64 healthy subjects who received the anti-SARS-CoV-2 vaccine. When 10 AU/ml of neutralizing antibodies was used as cut-off value, the positive rates of neutralizing antibodies of patients with UC, patients with DC, and the control group were 97.3%, 84.3%, and 100%, respectively. The neutralizing antibody titer showed no difference between patients treated with and without immunosuppressive therapy. These results indicate that COVID-19 vaccination may be useful in patients with IBD, treated with or without immunosuppressive therapy.

Fluctuations of antibody serum titers for Toxoplasma gondii and Neospora caninum in naturally infected crossbred cows during gestation.

This study aimed to assess the fluctuations of antibody serum titers for Toxoplasma gondii and Neospora caninum in naturally infected crossbred cows during gestation and to investigate transplacental transmission of T. gondii; 51 cows were monitored during pregnancy by monthly serologic testing by indirect fluorescent antibody test. 33 cows were seronegative for both N. caninum and T. gondii, 10 were seropositive only for N. caninum, 5 were seropositive only for T. gondii, and 3 were seropositive for both N. caninum and T. gondii. In both protozoan infections, great variation in antibody levels in pregnant cows was observed, and there was significant increase (p<0.05) in the comparison between the averages of serological titration per trimester. There was a significant correlation (p<0.05) between month and probability of seropositivity for T. gondii. We conclude that pregnancy influences antibody titers of crossbred cows naturally infected with N. caninum and/or T. gondii, and that serologic testing for T. gondii in pregnant cows from the sixth month of gestation onward may decrease the number of false negative results.

Local and Systemic Antibody Responses in Beef Calves Vaccinated with a Modified-Live Virus Bovine Respiratory Syncytial Virus (BRSV) Vaccine at Birth following BRSV Infection.

Maternal antibodies interfere with BRSV vaccine responses and efficacy in young calves. The objective of this study was to determine if vaccination before the complete absorption of colostral antibodies results in adequate immune priming and clinical protection of beef calves. Within 6 h of life, calves were randomly assigned to 2 different treatment groups. Group Vacc (n = 25) received a single dose of a modified-live virus (MLV) BRSV vaccine intranasally (IN) and group Control (n = 25) received 2 mL of 0.9% saline IN. At approximately 3 months of age, all calves were experimentally challenged with BRSV. Serum and nasal secretion samples were collected before and after challenge for BRSV real-time RT-PCR and antibody testing. Respiratory signs were not observed before challenge. After challenge, respiratory scores were similar between groups. On the challenge day, >40% of calves in each group were febrile. The mean serum and nasal BRSV-specific antibody titers indicated natural BRSV exposure before the experimental challenge in both groups. All calves tested positive for BRSV and had a similar duration of shedding after challenge. Based on these results, vaccination at birth does not offer advantages for immune priming or clinical protection for beef calves in BRSV-endemic cow-calf herds.

Detection of human Metapneumovirus and respiratory Syncytial Virus associated with asthmatic patients using direct fluorescent assay and Real time – PCR

This study aimed to determine the incidence of human respiratory syncytial virus (hRSV) and metapneumovirus (hMPV) occurrence in asthmatic patients in Wasit province. Blood samples were collected for measure the total and differential white blood cells count (WBCs). Correlation results of total and differential WBCs count for neutrophils, lymphocytes, eosinophils and basophils count among studied groups were significant (P &lt;0.001). Enzyme linked immunosorbent assay (ELISA) technique has been applied for detection of total-IgE antibodies. Results revealed that the highest total-IgE antibodies titer in sera were significantly difference (p &lt; 0.01). A total of 80 nasopharyngeal swabs were immediately dipped in transport media and stored until using for the detection of suspected hRSV and hMPV patients group by direct fluorescent assay the results appeared 13 (16.25 %) and 11 (13.75 %) samples were given positive results for hRSV and hMPV, respectively. Results of hRSV in asthmatic patients were subjected to real time – polymerase chain reaction (RT-PCR) appeared 15 samples out of 80 samples (18.75 %) were gave positive result for this test.

A Lentiviral Pseudotype System to Characterize SARS-CoV-2 Glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 causes worldwide COVID-19 pandemic and poses agreat threat to global public health. Due to its high pathogenicity and infectivity, live SARS-CoV-2 is classified as a BSL-3 agent and has to be handled in BSL-3 condition. Nevertheless, entry of SARS-CoV-2 is mediated by viral spike (S) glycoprotein, and pseudovirus with SARS-CoV-2S protein can mimic every entry step of SARS-CoV-2 virus and be studied in BSL-2 settings. In this chapter, we describe a detailed protocol of production of lentivirus-based SARS-CoV-2S pseudovirus and its application in study of virus entry and determination of neutralizing antibody titer of human sera against SARS-CoV-2.

1039. Clinical and Economic Impact of COVID-19 and Serologic Protection among Farm Workers: Results from the Guatemala Agricultural Workers and Respiratory Illness Impact (AGRI) Study

Abstract Background In the Guatemala AGricultural workers and Respiratory Impact (AGRI) study, we evaluated the clinical and socioeconomic burdens of respiratory disease in a cohort of Guatemalan banana farm workers. Methods All eligible workers were offered enrollment from June 15–December 30, 2020, and annually, then followed for influenza-like illnesses (ILI) through: 1) self-reporting to study nurses, 2) sentinel surveillance at health posts, and 3) absenteeism. Workers with ILI submitted nasopharyngeal swabs for influenza, RSV, and SARS-CoV-2 testing, then completed surveys at days 0, 7, and 28. Enrollment and acute-illness serum samples were tested for anti-SARS-CoV-2 nucleocapsid IgG (anti-N, Roche Elecsys®), and neutralizing antibodies (NAb) were tested in a subset using a lentivirus-based pseudovirion assay. Results Through October 10, 2021, 1,833 workers were enrolled. The majority were male (84%), young (mean 31 years), and healthy (&amp;lt; 13% had comorbidity). Through October 10, 2021, 1,833 workers developed 169 ILIs (12.0/100 person-years) and 43 (25.4%) of these ILIs were laboratory-confirmed SARS-CoV-2 (3.1/100 person-years). Workers with SARS-CoV-2-positive ILI reported more anosmia (p&amp;lt; 0.01), dysgeusia (p&amp;lt; 0.01), difficulty concentrating (p=0.01), and irritability (p=0.01), and greater clinical and well-being severity scores (Flu-iiQ) than test-negative ILIs (Fig 1); they also had greater absenteeism (p&amp;lt; 0.01) and lost income (median US$127.1, p&amp;lt; 0.01). Among 1334 workers enrolled in 2020, 616 (46.2%) had anti-N IgG suggestive of prior SARS-CoV-2 infection. COVID-19 incidence density for IgG-seropositive workers was 0.4/100 Person ̶ Years (P ̶ Y), lower than those who were seronegative (2.3/100 P ̶ Y) (Fig 2). At enrollment, anti-N IgG titers in serum correlated with neutralizing antibody titers (R2=0.26, p&amp;lt; 0.0001). Notably, in &amp;lt; 6 months from enrollment, most workers with follow-up NAb testing (65/77, 84%) exhibited a 95% decrease in neutralizing antibody titers. Conclusion Guatemalan farm workers suffered a significant burden of COVID-19, including more severe clinical and economic outcomes than other respiratory illnesses. Ongoing vaccination programs and longitudinal serology will provide additional insight into long-term immunity. Disclosures Daniel Olson, MD, Pfizer: Grant/Research Support|Roche: Grant/Research Support Diva M Calvimontes, MD, Pfizer: Grant/Research Support Molly Lamb, PhD, Pfizer: Grant/Research Support Edwards Kathryn, MD, Bionet: Advisor/Consultant|IBM: Advisor/Consultant|Merck: Data Monitoring Committee|Moderna: Data Monitoring Committee|Pfizer: Data Monitoring Committee|Roche: Data Monitoring Committee|Sanofi: Data Monitoring Committee|Seqirus: Data Monitoring Committee|X-4 Pharma: Data Monitoring Committee Edwin J. Asturias, MD, Curevac: DSMB Member|Fundacion para la Salud Integral de los Guatemaltecos: Board Member|Inovio: DSMB Member|Merck: Honoraria|Pfizer: Grant/Research Support.

Quantifying Antibody Persistence After a Single Dose of COVID-19 Vaccine Ad26.COV2.S in Humans Using a Mechanistic Modeling and Simulation Approach.

Understanding persistence of humoral immune responses elicited by vaccination against coronavirus disease 2019 (COVID-19) is critical for informing the duration of protection and appropriate booster timing. We developed a mechanistic model to characterize the time course of humoral immune responses in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-seronegative adults after primary vaccination with the Janssen COVID-19 vaccine, Ad26.COV2.S. The persistence of antibody responses was quantified through mechanistic modeling-based simulations. Two biomarkers of humoral immune responses were examined: SARS-CoV-2 neutralizing antibodies determined by wild-type virus neutralization assay (wtVNA) and spike protein-binding antibodies determined by indirect spike protein enzyme-linked immunosorbent assay (S-ELISA). The persistence of antibody responses was defined as the period of time during which wtVNA and S-ELISA titers remained above the lower limit of quantification. A total of 442 wtVNA and 1,185 S-ELISA titers from 82 and 220 participants, respectively, were analyzed following administration of a single dose of Ad26.COV2.S (5 × 1010 viral particles). The mechanistic model adequately described the time course of observed wtVNA and S-ELISA serum titers and its associated variability up to 8 months following vaccination. Mechanistic model-based simulations show that single-dose Ad26.COV2.S elicits durable but waning antibody responses up to 24 months following immunization. Of the estimated model parameters, the production rate of memory B cells was decreased in older adults relative to younger adults, and the antibody production rate mediated by long-lived plasma cells was increased in women relative to men. A steeper waning of antibody responses was predicted in men and in older adults.

GABABR IgG Associated Encephalitis: Clinical Presentations and Measures to Improve Diagnostic Assay Specificity

Objective To review the clinical/oncological presentations of gamma aminobutyric acid-B receptor (GABABR)-IgG and evaluate the clinical specificity of antibody testing methodologies. Background GABABR-IgG is an intermediate-risk paraneoplastic autoantibody commonly associated with encephalitis and/or seizures. Design/Methods GABABR-IgG positive patients tested at Mayo Clinic Neuroimmunology Laboratory were identified. Available archived sera were retested by cell-based assay (CBA) at 1:10 and 1:100 dilutions. Results 105 GABABR-IgG seropositive patients with clinical details were identified (females, n = 56; median age 63 [range 8-82]). Most patients had one of three anti-GABABR encephalitis/seizure presentations: focal-onset seizures with altered mental status (n = 39), new-onset refractory status epilepticus (NORSE [n = 23]) or rapidly progressive dementia (n = 14). Fifty-three of 72 patients (74%) with available data had associated malignancy, primarily small cell lung cancer (SCLC). Two P/Q type VGCC-IgG positive patients had isolated Lambert-Eaton Myasthenic Syndrome. Additionally, 27 patients presented with non-specific symptoms or alternate diagnoses (atypical presentation); none had underlying SCLC (13 with malignancy data). Most patients with anti-GABABR encephalitis/seizure presentations had GABABR-IgG detected in CSF (95%, 52/55; of 3 who were negative, 1 had NMDA-IgG and another AMPA-IgG and CRMP5-IgG) and/or GABABR-IgG detected by CBA in serum at a dilution of 1:100 (76%, 31/41). Five encephalitis/seizure patients with GABABR-IgG detected in CSF were CBA positive only at 1:10 dilution in serum. Forty-eight of 76 patients had samples positive on tissue IFA (serum = 17/42, CSF = 40/49). Among 27 patients with atypical presentations, most did not have GABABR-IgG CSF CBA positivity (91%, 21/23) and/or GABABR-IgG 1:100 serum titer by CBA (0/17). None of 27 cases were tissue IFA positive (serum = 0/27, CSF = 0/21). Conclusions GABABR-IgG is associated with autoimmune encephalitis/seizure presentations. Our study highlights the importance of evaluating CSF and testing serum at both 1:10 and 1:100 dilutions, as seropositivity at only the 1:10 dilution by CBA without CSF GABABR-IgG positivity has poor clinical specificity.

Refractory Pediatric NMDA Receptor Encephalitis: A Case Series

Objective To characterize clinical features of our institution's refractory pediatric NMDA receptor encephalitis (NMDARE) patients, in the hopes of identifying predictive risk factors and specific treatment escalation targets. Background Management protocols for pediatric NMDARE increasingly recommend anti-CD-20 agents following first-line treatment with high-dose corticosteroids, intravenous immunoglobulin (IVIg), and/or plasma exchange therapy (PLEX). Even with early, aggressive treatment, some patients exhibit refractory disease or recurrences. Identification of clinical predictors for refractory disease course may allow earlier, targeted treatment escalation in these patients. Design/Methods We performed IRB-approved retrospective, descriptive review of patients in our institutional NMDARE database (2011-2021). Refractory disease was defined as lack of neurological improvement within 1-3 months, or recurrent relapse, after standardized treatment protocol (steroids, IVIg or PLEX, and two doses of rituximab 500 mg/m2). Demographics, clinical information, and diagnostic results from refractory patients were collected. Results 8/73 (10.9%) patients met criteria for refractory NMDARE (median age 10.0 years, IQR 8.3-14.0. 2 male, 6 female). Median days from symptom onset to first treatment was 12.5 (IQR 8.5-18.8), to detection of +NMDA Ab was 20.5 (17.8-24.0), and to rituximab was 27.5 (26.5-31.0). All were critically ill at disease onset, with seizures, encephalopathy, and respiratory failure. 2 (25%) had associated ovarian teratoma. Oligoclonal bands were tested in 6, with 4 resulting positive (67%). 7 had confirmed B-cell depletion after rituximab. Post-rituximab NMDA titers persisted in serum in 5 (63%) and CSF in 8 (100%). Immune therapy escalation was varied, and included repeat rituximab, mycophenolate, cyclophosphamide, and tocilizumab. Conclusions Severe initial presentation is a consistent feature among our refractory patients. The contribution of other factors such as oligoclonal bands and time to diagnosis/treatment are less clear, and warrant inter-group comparison with non-refractory patients. Persistence of serum and CSF titers despite B-cell depletion may suggest utility in targeting CD-20-negative mature plasma cells which may continue to produce disease-causing antibodies.

Active Immunization Against NMDA NR1 Subunit as a Model of Autoimmune Encephalitis

Objective To identify and develop the optimal active immunization induction method for NMDAR encephalitis in rodents. Background Encephalitis is a devastating neurologic disorder with high morbidity and mortality. Many cases are autoimmune. N-Methyl-D-aspartic acid receptor (NMDAR) encephalitis (NMDARE), characterized by antibodies against the NMDAR in the blood and spinal fluid of patients, is the most common form of autoimmune encephalitis (AE). A translational rodent model of NMDARE would allow for in-depth studies into AE pathophysiology, leading to advances in the diagnosis and treatment of this debilitating neuropsychiatric disorder. Design/Methods 7-week-old female C57BL/6J mice were injected subcutaneously with an emulsion of complete Freund's adjuvant, attenuated Mycobacterium tuberculosis (TB), and a 30 amino acid peptide flanking the NMDAR NR1 subunit N368/G369 residue targeted by antibodies in NMDARE patients. Three different induction methods were tested by varying the amount and injection method of pertussis toxin, subcutaneous injection sites, reimmunization, and amounts of TB. Mice were bled biweekly and sacrificed at 2, 4, 6, 8, and 14 weeks. Serum and CSF NMDAR antibody titer; mouse behavior; hippocampal NMDAR protein and cluster density; and brain immune cell entry and cytokine content were examined. Results Immunized mice had serum and CSF NMDAR antibodies. Mice exhibited behavioral changes, altered hippocampal NMDAR protein, brain immune cell entry, and elevated cytokines in their brains. Titers were higher and changes were sustained in reimmunized mice. Conclusions Active immunization against the portion of the NMDAR targeted in patients with NMDARE resulted in robust production of NMDAR antibodies in the blood and spinal fluid, changes in hippocampal NMDAR protein, elevations in brain immune cells and cytokines, and behavioral changes in mice. Reimmunization was needed to sustain the responses. Active immunization therefore holds potential as a translational model of NMDARE, allowing for the creation of a novel generation of diagnostics and therapeutics.

EVALUATION OF THE EFFICIENCY OF THE SEROLOGICAL HAI METHOD FOR THE DETECTION OF ANTIBODIES TO THE EGG DROP SYNDROME VIRUS

The article describes the features of the course of the disease of laying hens with Egg drop syndrome (EDS). This is a contagious viral disease of commercial and breeding laying hens, which is characterized by damage to the reproductive organs, a sharp decrease in laying capacity, changes in the pigmentation and shape of laid eggs, the appearance of effusions, thinning, deformations, and in some places the complete absence of a shell, a decrease in the percentage of hatching of chickens from hatching eggs and a deterioration in their viability. Morbidity is 10–70 %, mortality is 1–10 %. The disease in chickens occurs during the egg-laying period, until then no clinical signs of infection are observed. Three forms of EDS have been described: classic, endemic, and sporadic. The diagnosis of EDS is established on the basis of the results of laboratory tests, taking into account epizootological data, clinical signs and pathological anatomical changes. At the same time, special attention is paid to the decrease in egg production and the appearance of deformed and shelles eggs in clinically healthy chickens, the death of embryos and day-old chicks. Laboratory diagnostics is based on express, virological and retrospective methods. One of the most common methods of diagnosing EDS is retrospective diagnosis, which consists in detecting a diagnostic (minimum 4-fold) increase in the antibody titer in paired blood sera of chickens by the methods of HAI, IHA or ELISA. HAI is recommended for mass serological examinations of poultry farms. The detection of Anti-Hemagglutinin Antibody (Anti-HA) in the studied blood sera at a titer of 1:16 (4 log2) and higher in farms where specific prevention of EDS is not carried out indicates the circulation of the field strain of EDS virus among poultry. It should be noted that chickens that do not yet lay deformed eggs can be infected with the virus, but do not give a humoral immune response to latent infection. Thus, in flocks of chickens that were infected transovarially, antibodies are not synthesized during the breeding period. A negative serological test in chickens under the age of 20 weeks does not guarantee the absence of EDS infection. Differential diagnosis requires the exclusion of infectious bronchitis, infectious laryngotracheitis, Newcastle disease and respiratory mycoplasmosis. An analysis of 20 inactivated vaccines from various manufacturing companies registered in Ukraine for the specific prevention of EDS was carried out, including monovalent – 3 and associated, namely 3-component – 10 and 4-component – 7. During serological monitoring, it was established, that the average titers in the sera of laying hens to the EDS virus from batches of different ages due to different terms after vaccination with inactivated vaccines ranged from 7.0 log2 to 13.2 log2. The average titers to the EDS virus above the basic norm were 16 % among birds of 4-8 batches (280, 310 and 460 days). It was established that the percentage of protective antibodies to the EDS virus in 25 batches of laying hens ranged from 78 to 100. The use of inactivated vaccines against EDS stimulates the formation of an active immune response in laying hens in 84 % of the studied batches. Carrying out constant monitoring of the effectiveness of inactivated vaccines against by means of serological monitoring of blood sera from birds in the HAI will allow, depending on the epizootic situation, to timely adjust the vaccination scheme and ensure stable economic indicators in commercial and breeding flocks of laying hens.

Batch-to-batch consistency trial of an adenovirus type-5 vector-based COVID-19 vaccine in adults aged 18 years and above

ABSTRACT Background The demonstration of batch-to-batch consistency is indispensable for quality control of vaccines. Methods We conducted a randomized, double-blind, parallel-controlled trial to evaluate the immunogenicity consistency of a single shot of Ad5-nCoV in healthy adults who had not previously received any COVID-19 vaccine. All eligible participants were randomly assigned equally to receive one of the three consecutive batches of Ad5-nCoV (5 × 1010 viral particles/vial, 0.5 mL). The primary endpoint was geometric mean titers (GMTs) of serum SARS-CoV-2 receptor-binding domain (RBD)-specific IgG on day 28 post-vaccination. Results One thousand fifty participants were enrolled, with 350 (33%) participants per group. On day 28 post-vaccination, GMTs in three groups were 78.3 binding antibody units (BAU)/mL (95% CI 70.3–87.3), 82.9 BAU/mL (73.9–92.9), and 78.8 BAU/mL (70.2–88.4), respectively. The two-sided 95% CIs for the GMT ratios between each pair of batches were all between 0.67 and 1.5. The highest incidence of solicited adverse reactions within 7 days post-vaccination was reported by batch 3 recipients (23.1% versus 15.1% in batch 1 recipients and 14.6% in bath 2 recipients; p = 0.0039). None of the serious adverse events were related to vaccination. Conclusions Immunogenicity consistency between consecutive batches of Ad5-nCoV was well established in adults. Clinical trial registration This trial was registered with ClinicalTrials.gov (NCT05313646).

A high-throughput neutralizing assay for antibodies and sera evaluation against Epstein-Barr virus

BackgroundEpstein-Barr virus (EBV) is a wide-spread human herpesvirus that is highly associated with infectious mononucleosis and several malignancies. Evaluation of EBV neutralizing antibody titers is important for serological studies, vaccine development and monoclonal antibody screening. The traditional method based on antibody inhibition of EBV transformation of B cells is very time-consuming. A more practical flow cytometry-based (FCM) approach to evaluate neutralizing titers is not amenable to achieving high-throughput evaluation of large-scale samples. A high-throughput approach is urgently needed.ResultsHere, we present a rapid and high-throughput method based on high content imaging system (HCIS) analysis. EBV titers determined by the HCIS-based assay were similar to those obtained by the FCM-based assay. Neutralizing titers of sera and monoclonal antibodies measured by the HCIS-based assay strongly correlated with titers measured by the FCM-based assay. HCIS assays showed a strong correlation between B cell infection neutralizing titers and the anti-gp350 IgG titers in healthy EBV carriers and monkey sera. Finally, anti-gHgL IgG titers from sera of healthy EBV carriers significantly correlated with epithelial cell infection neutralizing titers.ConclusionsThis HCIS-based assay is a high-throughput assay to determine viral titers and evaluate neutralizing potentials of sera and monoclonal antibodies. This HCIS-based assay will aid the development of vaccines and therapeutic monoclonal antibody against EBV.

Neuromyelitis optica: Clinical course and potential prognostic indicators

BackgroundNeuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune neurological disorder associated with antibodies to aquaporin-4 (AQP4). NMOSD has been thought to follow a progressive disease course, with step-wise accumulation of disability over time, even in patients undergoing immunosuppressive/immunomodulatory therapy. The influence of factors such as AQP4 seropositivity, AQP4 serum titer levels, and administration of plasmapheresis on NMOSD prognosis is, as yet, unclear. MethodsWe performed a retrospective chart review of 53 persons with NMOSD at Duke University Hospital—collecting data on longitudinal disease course, imaging, demographics, and serum AQP4 titers (measured using the ELISA or FACS method). Most patients in our cohort were treated with high-dose corticosteroids and, following diagnosis, received maintenance immunosuppressive/immunomodulatory therapies. Longitudinal data on EDSS scores were used to calculate the slope of disability over time for each participant. We additionally investigated the correlation between initial AQP4 seropositivity, initial AQP4 serum titer levels, and treatment with plasmapheresis on disability progression for each participant. ResultsContrary to current views on NMOSD disease course, the majority of our participants showed either no change (31.9%) or improvement (27.1%) in disability over time. Our results additionally revealed no significant association between clinical prognosis and initial AQP4 seropositivity (p = 0.830), initial AQP4 serum titer levels (p = 0.338), or administration of plasmapheresis (p = 0.1149). ConclusionsOur study presents a contemporary view of the clinical course of NMOSD and shows a more favorable view of its disease course than prior studies (performed before high-efficacy disease modifying therapies became widely-used for this patient population). Most patients in this study received treatment with high-dose corticosteroids following NMOSD flares, as well as a variety of maintenance immunosuppressive therapies. The results of this study cannot shed light on the disease course of untreated NMOSD. Our findings additionally challenge the theory that AQP4 seropositivity or serum titer levels at time of diagnosis may be used to effectively predict NMOSD prognosis. While we were unable to find evidence supporting a favorable effect of plasmapheresis administration on disease outcomes, further research is needed to determine the role plasmapheresis ought to play in the treatment of NMOSD.