Variant-specific IgA protects against Omicron infection.

BACKGROUND: The emergence of rapidly evolving SARS-CoV-2 variants, coupled with waning vaccine-induced immunity, has contributed to the rise of vaccine breakthrough infections. It is crucial to understand how vaccine-induced protection is mediated. METHODS: We examined two prospective cohorts of mRNA-vaccinated-and-boosted individuals during the Omicron wave of infection in Singapore. RESULTS: We found that, individuals, who remain uninfected over the follow-up period, had a higher variant-specific IgA, but not IgG, antibody response at 1-month post booster vaccination, compared with individuals who became infected. CONCLUSIONS: We conclude that IgA may have a potential contributory role in protection against Omicron infection.

Transcutaneous Bilirubin Monitoring in Preterm Infants of 23 to 34 Weeks' Gestation.

OBJECTIVE: The study aimed to evaluate the validity of transcutaneous bilirubin (TcB) measurements at three sites in premature infants born at 230/7 to 346/7 weeks' gestational age (GA) compared with total serum bilirubin (TSB) measurements. STUDY DESIGN: A prospective study was conducted at Banner - University Medical Center Phoenix, where informed consent was obtained from the infant's parent or legally authorized representative. Cohort A was comprised of infants 230/7 to 286/7 weeks' GA and Cohort B contained subjects 290/7 to 346/7 weeks' GA. Baseline TSB measurements were collected at approximately 24 hours of life, as the standard of care and the TcB measurements were obtained from the sternum, interscapular, and buttock areas at approximately ± 30 minutes from collection of the TSB. Statistical analysis of measurements including sensitivity, specificity, positive, and negative predictive values, and the area under the receiver operator characteristic curve (AUROC) were performed. RESULTS: A total of 166 infants were included in the study population. Cohort A consisted of 41 subjects and Cohort B contained 125 subjects. The results showed that baseline TcB measurements from the interscapular area were the most sensitive and specific with TSB levels >5.0 mg/dL in Cohort A. Baseline TcB measurements from the sternum demonstrated greatest sensitivity and specificity when the TSB level was >8.0 mg/dL in Cohort B. In general, each of the three sites in both cohorts demonstrated excellent AUROCs and negative predictive values. CONCLUSION: The use of a TcB meter in preterm infants can be a reliable noninvasive screening tool for hyperbilirubinemia, and it may be beneficial in decreasing painful stimuli and iatrogenic blood loss when used as an adjunct to TSB monitoring. KEY POINTS: · Interscapular TcB is sensitive/specific in 23 to 29 weeks' GA.. · Sternal TcB is sensitive/specific in 29 to 35 weeks' GA.. · TcB readings are reliable in preterm infants.. · TcB is reliable when serum bilirubin is >5.0 mg/dL..

Efficient recall of SARS-CoV-2 variant-reactive B cells and T responses in the elderly upon heterologous mRNA vaccines as boosters.

Waning antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of variants of concern highlight the need for booster vaccinations. This is particularly important for the elderly population, who are at a higher risk of developing severe coronavirus disease 2019 (COVID-19) disease. While studies have shown increased antibody responses following booster vaccination, understanding the changes in T and B cell compartments induced by a third vaccine dose remains limited. We analyzed the humoral and cellular responses in subjects who received either a homologous messenger RNA(mRNA) booster vaccine (BNT162b2 + BNT162b2 + BNT162b2; ''BBB") or a heterologous mRNA booster vaccine (BNT162b2 + BNT162b2 + mRNA-1273; ''BBM") at Day 0 (prebooster), Day 7, and Day 28 (postbooster). Compared with BBB, elderly individuals (≥60 years old) who received the BBM vaccination regimen display higher levels of neutralizing antibodies against the Wuhan and Delta strains along with a higher boost in immunoglobulin G memory B cells, particularly against the Omicron variant. Circulating T helper type 1(Th1), Th2, Th17, and T follicular helper responses were also increased in elderly individuals given the BBM regimen. While mRNA vaccines increase antibody, T cell, and B cell responses against SARS-CoV-2 1 month after receiving the third dose booster, the efficacy of the booster vaccine strategies may vary depending on age group and regimen combination.

Evaluation of the safety and immunogenicity of different COVID-19 vaccine combinations in healthy individuals: study protocol for a randomized, subject-blinded, controlled phase 3 trial [PRIBIVAC].

BACKGROUND: Over 2021, COVID-19 vaccination programs worldwide focused on raising population immunity through the primary COVID-19 vaccine series. In Singapore, two mRNA vaccines (BNT162b2 and mRNA-1273) and the inactivated vaccine CoronaVac are currently authorized under the National Vaccination Programme for use as the primary vaccination series. More than 90% of the Singapore population has received at least one dose of a COVID-19 vaccine as of December 2021. With the demonstration that vaccine effectiveness wanes in the months after vaccination, and the emergence of Omicron which evades host immunity from prior infection and/or vaccination, attention in many countries has shifted to how best to maintain immunity through booster vaccinations. METHODS: The objectives of this phase 3, randomized, subject-blinded, controlled clinical trial are to assess the safety and immunogenicity of heterologous boost COVID-19 vaccine regimens (intervention groups 1-4) compared with a homologous boost regimen (control arm) in up to 600 adult volunteers. As non-mRNA vaccine candidates may enter the study at different time points depending on vaccine availability and local regulatory approval, participants will be randomized at equal probability to the available intervention arms at the time of randomization. Eligible participants will have received two doses of a homologous mRNA vaccine series with BNT162b2 or mRNA-1273 at least 6 months prior to enrolment. Participants will be excluded if they have a history of confirmed SARS or SARS-CoV-2 infection, are immunocompromised, or are pregnant. Participants will be monitored for adverse events and serious adverse events by physical examinations, laboratory tests and self-reporting. Blood samples will be collected at serial time points [pre-vaccination/screening (day - 14 to day 0), day 7, day 28, day 180, day 360 post-vaccination] for assessment of antibody and cellular immune parameters. Primary endpoint is the level of anti-SARS-CoV-2 spike immunoglobulins at day 28 post-booster and will be measured against wildtype SARS-CoV-2 and variants of concern. Comprehensive immune profiling of the humoral and cellular immune response to vaccination will be performed. DISCUSSION: This study will provide necessary data to understand the quantity, quality, and persistence of the immune response to a homologous and heterologous third booster dose of COVID-19 vaccines. This is an important step in developing COVID-19 vaccination programs beyond the primary series. TRIAL REGISTRATION: ClinicalTrials.gov NCT05142319 . Registered on 2 Dec 2021.

Antibody Response of Heterologous vs Homologous Messenger RNA Vaccine Boosters Against the Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant: Interim Results from the PRIBIVAC Study, a Randomized Clinical Trial.

BACKGROUND: Waning antibody levels post-vaccination and the emergence of variants of concern (VOCs) capable of evading protective immunity have raised the need for booster vaccinations. However, which combination of coronavirus disease 2019 (COVID-19) vaccines offers the strongest immune response against the Omicron variant is unknown. METHODS: This randomized, participant-blinded, controlled trial assessed the reactogenicity and immunogenicity of different COVID-19 vaccine booster combinations. A total of 100 BNT162b2-vaccinated individuals were enrolled and randomized 1:1 to either homologous (BNT162b2 + BNT162b2 + BNT162b2; "BBB") or heterologous messenger RNA (mRNA) (BNT162b2 + BNT162b2 + mRNA-1273; "BBM") booster vaccine. The primary end point was the level of neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wild-type and VOCs at day 28. RESULTS: A total of 51 participants were allocated to BBB and 49 to BBM; 50 and 48, respectively, were analyzed for safety and immunogenicity outcomes. At day 28 post-boost, mean SARS-CoV-2 spike antibody titers were lower with BBB (22 382 IU/mL; 95% confidence interval [CI], 18 210 to 27 517) vs BBM (29 751 IU/mL; 95% CI, 25 281 to 35 011; P = .034) as was the median level of neutralizing antibodies: BBB 99.0% (interquartile range [IQR], 97.9% to 99.3%) vs BBM 99.3% (IQR, 98.8% to 99.5%; P = .021). On subgroup analysis, significant higher mean spike antibody titer, median surrogate neutralizing antibody level against all VOCs, and live Omicron neutralization titer were observed only in older adults receiving BBM. Both vaccines were well tolerated. CONCLUSIONS: Heterologous mRNA-1273 booster vaccination compared with homologous BNT123b2 induced a stronger neutralizing response against the Omicron variant in older individuals. CLINICAL TRIALS REGISTRATION: NCT05142319.

NEC-zero recommendations from scoping review of evidence to prevent and foster timely recognition of necrotizing enterocolitis.

BACKGROUND: Although decades have focused on unraveling its etiology, necrotizing enterocolitis (NEC) remains a chief threat to the health of premature infants. Both modifiable and non-modifiable risk factors contribute to varying rates of disease across neonatal intensive care units (NICUs). PURPOSE: The purpose of this paper is to present a scoping review with two new meta-analyses, clinical recommendations, and implementation strategies to prevent and foster timely recognition of NEC. METHODS: Using the Translating Research into Practice (TRIP) framework, we conducted a stakeholder-engaged scoping review to classify strength of evidence and form implementation recommendations using GRADE criteria across subgroup areas: 1) promoting human milk, 2) feeding protocols and transfusion, 3) timely recognition strategies, and 4) medication stewardship. Sub-groups answered 5 key questions, reviewed 11 position statements and 71 research reports. Meta-analyses with random effects were conducted on effects of standardized feeding protocols and donor human milk derived fortifiers on NEC. RESULTS: Quality of evidence ranged from very low (timely recognition) to moderate (feeding protocols, prioritize human milk, limiting antibiotics and antacids). Prioritizing human milk, feeding protocols and avoiding antacids were strongly recommended. Weak recommendations (i.e. "probably do it") for limiting antibiotics and use of a standard timely recognition approach are presented. Meta-analysis of data from infants weighing <1250 g fed donor human milk based fortifier had reduced odds of NEC compared to those fed cow's milk based fortifier (OR = 0.36, 95% CI 0.13, 1.00; p = 0.05; 4 studies, N = 1164). Use of standardized feeding protocols for infants <1500 g reduced odds of NEC by 67% (OR = 0.33, 95% CI 0.17, 0.65, p = 0.001; 9 studies; N = 4755 infants). Parents recommended that NEC information be shared early in the NICU stay, when feedings were adjusted, or feeding intolerance occurred via print and video materials to supplement verbal instruction. DISCUSSION: Evidence for NEC prevention is of sufficient quality to implement. Implementation that addresses system-level interventions that engage the whole team, including parents, will yield the best impact to prevent NEC and foster its timely recognition.

Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital.

This study investigated the clinical features of anaerobic bacteraemia in an acute-care hospital, and evaluated the antimicrobial susceptibility of these isolates to commonly available antibiotics. Microbiological and epidemiological data from 2009 to 2011were extracted from the laboratory information system and electronic medical records. One hundred and eleven unique patient episodes consisting of 116 anaerobic isolates were selected for clinical review and antibiotic susceptibility testing. Susceptibilities to amoxicillin-clavulanate, clindamycin, imipenem, metronidazole, moxifloxacin, penicillin and piperacillin-tazobactam were performed using Etest strips with categorical interpretations according to current CLSI breakpoints. Metronidazole-resistant and carbapenem-resistant anaerobic Gram-negative bacilli were screened for the nim and cfiA genes. Clinical data was obtained retrospectively from electronic medical records. During the 3 year period, Bacteroides fragilis group (41%), Clostridium species (14%), Propionibacterium species (9%) and Fusobacterium species (6%) were the most commonly isolated anaerobes. Patients with anaerobic bacteraemia that were included in the study were predominantly above 60 years of age, with community-acquired infections. The most commonly used empiric antibiotic therapies were beta-lactam/beta-lactamase inhibitor combinations (44%) and metronidazole (10%). The crude mortality was 25%, and appropriate initial antibiotic therapy was not significantly associated with improved survival. Intra-abdominal infections (39%) and soft-tissue infections (33%) accounted for nearly three-quarters of all bacteraemia. Antibiotics with the best anaerobic activity were imipenem, piperacillin-tazobactam, amoxicillin-clavulanate and metronidazole, with in-vitro susceptibility rates of 95%, 95%, 94% and 92% respectively. Susceptibilities to penicillin (31%), clindamycin (60%) and moxifloxacin (84%) were more variable. Two multidrug-resistant isolates of Bacteroides species were positive for nim and cfiA genes respectively, while another two imipenem-resistant Fusobacterium species were negative for cfiA genes. This study demonstrated that anaerobic bacteraemia in our patient population was predominantly associated with intra-abdominal and soft-tissue infections. Overall antibiotic resistance was high for penicillin and clindamycin, and the presence of emerging resistance to carbapenems and metronidazole warrants further monitoring.

Ultrafiltration of highly concentrated antibody solutions: Experiments and modeling for the effects of module and buffer conditions.

Although ultrafiltration is currently used for the concentration and formulation of nearly all biotherapeutics, obtaining the very high target concentrations for monoclonal antibody products is challenging. The objective of this work was to examine the effects of the membrane module design and buffer conditions on both the filtrate flux and maximum achievable protein concentration during the ultrafiltration of highly concentrated monoclonal antibody solutions. Experimental data were obtained using both hollow fiber and screened cassettes and in the presence of specific excipients that are known to alter the solution viscosity. Data were compared with predictions of a recently developed model that accounts for the complex thermodynamic and hydrodynamic behavior in these systems, including the effects of back-filtration arising from the large pressure drop through the module due to the high viscosity of the concentrated antibody solutions. Model calculations were in good agreement with experimental data in hollow fiber modules with very different fiber length and in screened cassettes having different screen geometries. These results provide important insights into the key factors controlling the filtrate flux and maximum achievable protein concentration during ultrafiltration of highly concentrated antibody solutions as well as a framework for the development of enhanced ultrafiltration processes for this application. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:692-701, 2016.

Anaerobic bacteraemia revisited: species and susceptibilities.

INTRODUCTION: This retrospective study was performed to evaluate the frequency of anaerobic bacteraemia over a 10-year period, and to provide updated antibiotic susceptibilities for the more clinically relevant anaerobes causing blood stream infection. MATERIALS AND METHODS: Data were retrieved from the laboratory information system for the period 2003 to 2012. During this time, blood cultures were inoculated in Bactec™ Plus vials (BD, USA) and continuously monitored in the Bactec™ 9000 blood culture system (BD, USA). Anaerobic organisms were identified using commercial identification kits, predominantly API 20 A (bioMérieux, France) supplemented with Vitek ANC cards (bioMérieux, France) and AN-Ident discs (Oxoid, United Kingdom). A representative subset of isolates were retrieved from 2009 to 2011 and antimicrobial susceptibilities to penicillin, amoxicillin-clavulanate, clindamycin, imipenem, moxifloxacin, piperacillin-tazobactam and metronidazole were determined using the Etest method. RESULTS: Anaerobes comprised 4.1% of all positive blood culture with 727 obligate anaerobes recovered over the 10-year period, representing a positivity rate of 0.35%. The only significant change in anaerobe positivity rates occurred between 2003 and 2004, with an increase of 0.2%. The Bacteroides fragilis group (45%) were the predominant anaerobic pathogens, followed by Clostridium species (12%), Propioniobacterium species (11%) and Fusobacterium species (6%). The most active in vitro antibiotics were imipenem, piperacillin-tazobactam, amoxicillin-clavulanate and metronidazole, with susceptibilities of 95.0%, 93.3%, 90.8% and 90.8% respectively. Resistance was high to penicillin, clindamycin and moxifl oxacin. However, there were apparent differences for antibiotic susceptibilities between species. CONCLUSION: This study indicates that the anaerobes comprise a small but constant proportion of bloodstream isolates. Antibiotic resistance was high to some antibiotics, but metronidazole, the beta-lactam/beta-lactamase inhibitors and carbapenems retained good in vitro activity.

The osmotic pressure of highly concentrated monoclonal antibody solutions: effect of solution conditions.

The behavior of monoclonal antibodies at high concentrations is important in downstream processing, drug formulation, and drug delivery. The objective of this study was to evaluate the osmotic pressure of a highly purified monoclonal antibody at concentrations up to 250 g/L over a range of pH and ionic strength, and in the presence of specific excipients, using membrane osmometry. Independent measurements of the second virial coefficient were obtained using self-interaction chromatography, and the net protein charge was evaluated using electrophoretic light scattering. The osmotic pressure at pH 5 and low ionic strength was >50 kPa for antibody concentrations above 200 g/L. The second virial coefficients determined from the oncotic pressure (after subtracting the Donnan contribution) were in good qualitative agreement with those determined by self-interaction chromatography. The second virial coefficient decreased with increasing ionic strength and increasing pH due to the reduction in intermolecular electrostatic repulsion. The third virial coefficient was negative under all conditions, suggesting that multi-body interactions in this system are attractive. The virial coefficients were essentially unaffected by addition of sucrose or proline. These results have important implications for the analysis of protein-protein interactions in downstream processing at high protein concentrations.

Improved method for evaluating the dead volume and protein-protein interactions by self-interaction chromatography.

Self-interaction chromatography (SIC) is a well-established method for studying protein-protein interactions. The second virial coefficient in SIC is evaluated directly from the measured retention coefficient for the protein using a column packed with resin on which the same protein has been immobilized on the pore surface. One of the challenges in determining the retention coefficient is the evaluation of the dead volume, which is the retention volume that would be measured for a noninteracting solute with the same effective size as the protein of interest. Previous studies of SIC have used a "dead column" packed with the same resin but without the immobilized protein to evaluate the dead volume, but this creates several experimental and theoretical challenges. We have developed a new approach using a dextran standard with effective size equal to that of the protein (as determined by size exclusion chromatography). The second virial coefficient was evaluated for a monoclonal antibody over a range of buffer conditions using this new approach. The data were in good agreement with independent measurements obtained by membrane osmometry under conditions dominated by repulsive interactions. The simplicity and accuracy of this method should facilitate the use of self-interaction chromatography for quantifying protein-protein interactions.

Recovery modeling of tangential flow systems.

The demand for increased formulation concentrations for protein therapeutics puts a significant strain on already existing tangential flow filtration (TFF) systems that were constructed with lower protein concentration targets as part of their design criteria. TFF is commonly used to buffer exchange and concentrate the product to the appropriate drug substance concentration. Analyzing the ability of an existing TFF system to process under conditions outside its original design specifications can be challenging. In this analysis, we present a systematic approach to assess the operational limits of a TFF process with consideration of system performance parameters for changing process targets. In two new engineering diagrams, the recovery efficiency diagram and the operating space plot, all relevant operational constraints and parameters are related to allow rapid process fit evaluation. The engineering assessment of TFF systems presented in this article allows a rational review of system limitations during process fit evaluations of existing TFF systems. It also provides a rational basis for targeted system upgrades and setting system design specifications for the design of new systems if existing systems are found inadequate.

Involvement of neuronal nitric oxide synthase in ongoing fetal brain injury following near-term rabbit hypoxia-ischemia.

Neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) are implicated in neuronal injury following acute hypoxia-ischemia (HI). Our hypothesis was that NO from nNOS is responsible for ongoing mitochondrial dysfunction in near-term fetal HI. Recently, we synthesized new selective nNOS inhibitors that prevent the cerebral palsy phenotype in our animal model. We tested the efficacy of a selective nNOS inhibitor (JI-8) in fetal brains after in utero HI in our rabbit model. Brain slices at 29 days gestation were obtained after in utero HI, and immediately cultured in medium containing JI-8 or saline for 3-6 days. Mitochondrial membrane integrity and function were determined by flow cytometry using rhodamine 123 and JC-1, and cell death by using propidium iodide. JI-8 decreased NO production in brain slices and also showed significant preservation of mitochondrial function at both 3 and 6 days (p < 0.05) when compared with saline and inducible NOS inhibitor 1400W. There was no difference in cell death. In conclusion, nNOS is involved in ongoing mitochondrial dysfunction after in utero HI. The subacute brain slice model could be a tool for studying the mechanisms involved in ongoing neuronal injury, and for rapidly assessing potential neuroprotectants.

Controlling protein transport in ultrafiltration using small charged ligands.

Previous studies have demonstrated that protein transport during ultrafiltration can be strongly influenced by solution pH and ionic strength. The objective of this study was to examine the possibility of controlling protein transmission using a small, highly charged ligand that selectively binds to the protein of interest. Experiments were performed using bovine serum albumin and the dye Cibacron Blue. Protein sieving data were obtained with essentially neutral and negatively charged versions of a composite regenerated cellulose membrane to examine the effects of electrostatic interactions. The addition of only 1 g/L of Cibacron Blue to an 8 g/L BSA solution reduced the BSA sieving coefficient through the negatively-charged membrane by more than two orders of magnitude, with this effect being largely eliminated at high salt and with the neutral membrane. Protein sieving data were in good agreement with model calculations based on the partitioning of a charged sphere in a charged pore accounting for the change in net protein charge due to ligand binding and the increase in solution ionic strength due to the free ligand in solution.

In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci.

UNLABELLED: Shed cells or disrupted parts of the biofilm may enter the circulation causing serious and very hard to treat biofilm-associated infections. The activity of antimicrobial agents against the shed cells/disrupted biofilms is largely unknown. METHODS: We studied the in vitro susceptibility of intact and disrupted biofilms of thirty clinical isolates of methicillin-resistant and methicillin-susceptible Staphylococcus aureus (MRSA and MSSA) and Staphylococcus epidermidis to vancomycin, quinupristin/dalfopristin, and linezolid and compared it to that of the suspended (planktonic) cells. RESULTS: Bacteria in the disrupted biofilms were as resistant as those in the intact biofilms at the minimum inhibitory concentrations of the antibiotics. At higher concentrations, bacteria in the disrupted biofilms were significantly (P < 0.001) less resistant than those in the intact biofilms but more resistant than the planktonic cells. Quinupristin/dalfopristin showed the best activity against cells of the disrupted biofilms at concentrations above MICs and vancomycin, at 500 and 1,000 microg/ml, was significantly more active against the biofilms of MRSA and S. epidermidis CONCLUSION: The difficulty of treating biofilm-associated infections may be attributed not only to the difficulty of eradicating the biofilm focus but also to the lack of susceptibility of cells disrupted from the biofilm to antimicrobial agents.

Three putative N-glycosylation sites within the murine 5-HT3A receptor sequence affect plasma membrane targeting, ligand binding, and calcium influx in heterologous mammalian cells.

The serotonin type 3(A) receptor (5-HT3(A)R) is a ligand-gated ion channel (LGIC) that modulates a diverse set of cognitive and physiological functions. The 5-HT3(A)R, as with other LGICs, is a pentameric ion channel comprising five glycoprotein subunits. Although the N-terminal of the 5-HT3(A)R contains three putative N-linked glycosylation sites, the importance of each glycosylation site has not yet been established. To address this question, we used tunicamycin treatment and site-directed mutagenesis to inhibit selectively N-linked glycosylation at each site and then examined the effects of these treatments on receptor expression and function in transiently transfected heterologous cells. We show that the murine 5-HT3(A)R is glycosylated and that each N-linked glycosylation site plays a role in receptor regulation. Our findings suggest that N109 is necessary for receptor assembly, whereas N174 and N190 are important for plasma membrane targeting and ligand binding. Furthermore, we demonstrate that each site is necessary for 5-HT3(A)R-mediated Ca(2+) influx. We conclude that N-glycosylation is a critical step in the maturation, trafficking, and function of the murine 5-HT3(A)R.