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RNF43 is an important negative regulator of β-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of β-catenin. RNF43 has also been suggested to regulate β-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/β-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 vaccination campaign began in February 2021 and achieved a high rate of 62.7% of the total population fully vaccinated by August 16, 2021, in Mongolia. We aimed to assess the initial protective antibody production after two doses of a variety of types of SARS-CoV-2 vaccines in the Mongolian pre-vaccine antibody-naïve adult population. This prospective study was conducted from March-April to July-August of 2021. All participants received one of the four government-proposed COVID-19 vaccines including Pfizer/BioNTech (BNT162b2), AstraZeneca (ChAdOx1-S), Sinopharm (BBIBP-CorV), and Sputnik V (Gam-COVID-Vac). Before receiving the first shot, anti-SARS-CoV-2 S-RBD human IgG titers were measured in all participants (n = 1833), and titers were measured 21–28 days after the second shot in a subset of participants (n = 831). We found an overall average protective antibody response of 84.8% (705 of 831 vaccinated) in 21–28 days after two doses of the four types of COVID-19 vaccines. Seropositivity and titer of protective antibodies produced after two shots of vaccine were associated with the vaccine types, age, and residence of vaccinees. Seropositivity rate varied significantly between vaccine types, 80.0% (28 of 35) for AstraZeneca ChAdOx1-S; 97.0% (193 of 199) for Pfizer BNT162b2; 80.7% (474 of 587) for Sinopharm BBIBP-CorV, and 100.0% (10 of 10) for Sputnik V Gam-COVID-Vac, respectively. Immunocompromised vaccinees with increased risk for developing severe COVID-19 disease had received the Pfizer vaccine and demonstrated a high rate of seropositivity. A high geometric mean titer (GMT) was found in vaccinees who received BNT162b2, while vaccinees who received ChAdOx1-S, Sputnik V, and BBIBP-CorV showed a lower GMT. In summary, we observed first stages of the immunization campaign against COVID-19 in Mongolia have been completed successfully, with a high immunogenicity level achieved among the population with an increased risk for developing severe illness. [ABSTRACT FROM AUTHOR]

As the COVID-19 pandemic progresses, widespread community transmission of SARS-CoV-2 has ushered in a volatile era of viral immune evasion rather than the much-heralded stability of "endemicity" or "herd immunity." At this point, an array of viral strains has rendered essentially all monoclonal antibody therapeutics obsolete and strongly undermined the impact of vaccinal immunity on SARS-CoV-2 transmission. In this work, we demonstrate that antibody escape resulting in evasion of pre-existing immunity is highly evolutionarily favored and likely to cause waves of short-term transmission. In the long-term, invading strains that induce weak cross-immunity against pre-existing strains may co-circulate with those pre-existing strains. This would result in the formation of serotypes that increase disease burden, complicate SARS-CoV-2 control, and raise the potential for increases in viral virulence. Less durable immunity does not drive positive selection as a trait, but such strains may transmit at high levels if they establish. Overall, our results draw attention to the importance of inter-strain cross-immunity as a driver of transmission trends and the importance of early immune evasion data to predict the trajectory of the pandemic. [ABSTRACT FROM AUTHOR]

Background: Biosensor-based detection provides a rapid and low-cost alternative to conventional analytical methods for revealing the presence of the contaminants in water as well as solid matrices. Although important to be detected, small analytes (few hundreds of Daltons) are an issue in biosensing since the signal they induce in the transducer, and specifically in a Quartz-Crystal Microbalance, is undetectable. A pesticide like parathion (M = 292 Da) is a typical example of contaminant for which a signal amplification procedure is desirable. Methods/Findings: The ballasting of the analyte by gold nanoparticles has been already applied to heavy target as proteins or bacteria to improve the limit of detection. In this paper, we extend the application of such a method to small analytes by showing that once the working surface of a Quartz-Crystal Microbalance (QCM) has been properly functionalized, a limit of detection lower than 1 ppb is reached for parathion. The effective surface functionalization is achieved by immobilizing antibodies upright oriented on the QCM gold surface by a simple photochemical technique (Photonic Immobilization Technique, PIT) based on the UV irradiation of the antibodies, whereas a simple protocol provided by the manufacturer is applied to functionalize the gold nanoparticles. Thus, in a non-competitive approach, the small analyte is made detectable by weighing it down through a “sandwich protocol” with a second antibody tethered to heavy gold nanoparticles. The immunosensor has been proved to be effective against the parathion while showing no cross reaction when a mixture of compounds very similar to parathion is analyzed. Conclusion/Significance: The immunosensor described in this paper can be easily applied to any small molecule for which polyclonal antibodies are available since both the functionalization procedure of the QCM probe surface and gold nanoparticle can be applied to any IgG, thereby making our device of general application in terms of target analyte. [ABSTRACT FROM AUTHOR]

Background: Common variable immunodeficiency disorders (CVID), the most common form of primary antibody deficiency, are rare conditions associated with considerable morbidity and mortality. The clinical benefit of immunoglobulin replacement therapy (IgGRT) is substantial: timely treatment with appropriate doses significantly reduces mortality and the incidence of CVID-complications such as major infections and bronchiectasis. Unfortunately, CVID-patients still face a median diagnostic delay of 4 years. Their disease burden, expressed in annual loss of disability-adjusted life years, is 3-fold higher than in the general population. Hurdles to treatment access and reimbursement by healthcare payers may exist because the value of IgGRT is poorly documented. This paper aims to demonstrate cost-effectiveness and cost-utility (on life expectancy and quality) of IgGRT in CVID., Methods and Findings: With input from a literature search, we built a health-economic model for cost-effectiveness and cost-utility assessment of IgGRT in CVID. We compared a mean literature-based dose (≥450mg/kg/4wks) to a zero-or-low dose (0 to ≤100 mg/kg/4wks) in a simulated cohort of adult patients from time of diagnosis until death; we also estimated the economic impact of diagnostic delay in this simulated cohort. Compared to no or minimal treatment, IgGRT showed an incremental benefit of 17 life-years (LYs) and 11 quality-adjusted life-years (QALYs), resulting in an incremental cost-effectiveness ratio (ICER) of €29,296/LY and €46,717/QALY. These results were robust in a sensitivity analysis. Reducing diagnostic delay by 4 years provided an incremental benefit of six LYs and four QALYs compared to simulated patients with delayed IgGRT initiation, resulting in an ICER of €30,374/LY and €47,495/QALY., Conclusions: The health-economic model suggests that early initiation of IgGRT compared to no or delayed IgGRT is highly cost-effective. CVID-patients' access to IgGRT should be facilitated, not only because of proven clinical efficacy, but also due to the now demonstrated cost-effectiveness., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: both EdV and PvW received funding from PPTA to perform the work. EdV has received a research grant from Shire/Takeda for performing independent research on unclassified antibody deficiency. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: COVID-19 pandemic resulted in about 165 million infections and 3.4 million deaths all over the world across 15 months. The most severe clinical presentation of COVID-19 diseases is interstitial pneumonia. Methods: In this paper we describe clinical outcomes based on radiological features as well as the pattern of haematochemical parameters and IgG/IgM antibodies in 75 patients hospitalized due to COVID-related interstitial pneumonia not requiring intensive care assistance. Each patient underwent routine laboratory tests, including inflammatory markers and coagulation profile at baseline. Computed Tomography (CT) was performed at baseline and after 3 months to assess the persistence of radiological sequelae. A Generalized Linear Model (GLM) was used to test for each patient the association between individual haematochemical parameters at the time of hospital admission and the subsequent radiological features after three months. The presence of IgG antibodies was quantitatively determined in 70 patients at the time of hospital admission and after 3 months. A subgroup of 49 and 21 patients underwent additional dosage of IgG after 6 and 12 months, respectively. IgM serological antibodies were available for 17 patients at baseline and 61 at T3, with additional follow-up for 51 and 20 subjects after 6 and 12 months, respectively. Results: Only 28 out of 75 patients discharged from the hospital were totally healed after 3 months, while 47 patients (62.7%) still presented radiological sequelae. According to the GLM model, specific haematochemical baseline parameters—such as IL-6, GPT, platelets and eosinophil count—showed a statistically significant association with the presence of radiological sequelae at month 3 highlighting an OR = 0.5, thus meaning that subjects completely healed after 3 months presented half levels of IL-6 at baseline compared to patients with sequelae. In general, IgG serum levels were always higher than IgM at the time of hospitalization (75% at T0; n = 12 out of 16 patients with data available in both visits), after 3 months (72.1%; n = 44 out of 61 pts.), after 6 months (56.8%; 25 out of 44 pts.), and one year after hospitalization (60%; 12 out of 20 pts.). Overall, IgG and IgM serum levels presented a statistically significant decreasing trend from the baseline to month 3, 6 and 12. One patient presented an increase in IgM between baseline and month 3 but negative PCR test for SARS-COV2 on throat swab. Conclusions: As supported by our findings on 75 patients, COVID-related interstitial pneumonia triggers early IgG levels (higher than IgM) that gradually decrease over 12 months. Mid-term sequelae are still detectable at lung Computed Tomography after 3 months from the hospital admission. Occasionally, it is possible to observe increase of IgM levels in presence of low concentrations of IgG and negative PCR ELISA tests for SARS-COV2 RNA. Baseline levels of IL-6 could be proposed as predictor of radiological mid/long-term sequelae after COVID-related interstitial pneumonia. [ABSTRACT FROM AUTHOR]

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors' blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors. [ABSTRACT FROM AUTHOR]

Background: Plasma-derived intravenous immunoglobulin (IVIg) products contain a dynamic spectrum of immunoglobulin (Ig) G reactivities reflective of the donor population from which they are derived. We sought to model the concentration of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG which could be expected in future plasma pool and final-product batches of CSL Behring's immunoglobulin product Privigen. Study design and methods: Data was extracted from accessible databases, including the incidence of coronavirus disease 2019 and SARS-CoV-2 vaccination status, antibody titre in convalescent and vaccinated groups and antibody half-life. Together, these parameters were used to create an integrated mathematical model that could be used to predict anti-SARS-CoV-2 antibody levels in future IVIg preparations. Results: We predict that anti-SARS-CoV-2 IgG concentration will peak in batches produced in mid-October 2021, containing levels in the vicinity of 190-fold that of the mean convalescent (unvaccinated) plasma concentration. An elevated concentration (approximately 35-fold convalescent plasma) is anticipated to be retained in batches produced well into 2022. Measurement of several Privigen batches using the Phadia™ EliA™ SARS-CoV-2-Sp1 IgG binding assay confirmed the early phase of this model. Conclusion: The work presented in this paper may have important implications for physicians and patients who use Privigen for indicated diseases. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus-2), cause of COVID-19 (Coronavirus Disease of 2019), represents a significant risk to people living with pre-existing conditions associated with exacerbated inflammatory responses and consequent dysfunctional immunity. In this paper, we have evaluated the influence of obesity, a condition associated with chronic systemic inflammation, on the secretion of SARS-CoV-2-specific IgG antibodies in the blood of COVID-19 patients. Our hypothesis is that obesity is associated with reduced amounts of specific IgG antibodies. Results have confirmed our hypothesis and have shown that SARS-CoV-2 IgG antibodies are negatively associated with Body Mass Index (BMI) in COVID-19 obese patients, as expected based on the known influence of obesity on humoral immunity. Antibodies in COVID-19 obese patients are also negatively associated with serum levels of pro-inflammatory and metabolic markers of inflammaging and pulmonary inflammation, such as SAA (serum amyloid A protein), CRP (C-reactive protein), and ferritin, but positively associated with NEFA (nonesterified fatty acids). These results altogether could help to identify an inflammatory signature with strong predictive value for immune dysfunction. Inflammatory markers identified may subsequently be targeted to improve humoral immunity in individuals with obesity and in individuals with other chronic inflammatory conditions. [ABSTRACT FROM AUTHOR]

Public health emergency of SARS-CoV-2 has facilitated diagnostic testing as a related medical countermeasure against COVID-19 outbreak. Numerous serologic antibody tests have become available through an expedited federal emergency use only process. This paper highlights the analytical characteristic of an ELISA based assay by AnshLabs and three random access immunoassay (RAIA) by DiaSorin, Roche, and Abbott that have been approved for emergency use authorization (EUA), at a tertiary academic center in a low disease-prevalence area. The AnshLabs gave higher estimates of sero-prevalence, over the three RAIA methods. For positive results, AnshLabs had 93.3% and 100% agreement with DiaSorin or Abbott and Roche respectively. For negative results, AnshLabs had 74.3% and 78.3% agreement with DiaSorin and Roche or Abbott respectively. All discrepant samples that were positive by AnshLabs and negative by RAIA tested positive by all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens Advia Centaur XPT analyzer. None of these methods, however, are useful in early diagnosis of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored. [ABSTRACT FROM AUTHOR]

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 μg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague. [ABSTRACT FROM AUTHOR]

Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-dependent enhancement (ADE) of disease as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral/ocular) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7–10 days post-infection. To evaluate the potential for ADE, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2. [ABSTRACT FROM AUTHOR]

New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries—two synthetic and two immunized—against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics. [ABSTRACT FROM AUTHOR]

Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagents are expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via N-hydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve. [ABSTRACT FROM AUTHOR]

Background: Health personnel (HP) are on the frontlines during response to public health emergencies like COVID-19. This risk of exposure suggests the need for safety in responding to any pandemic. Therefore, to ascertain the rate of SARS-CoV-2 infection and immunity, and their determinants amongst HP become relevant. Methods: A cross sectional health facility-based study was carried-out amongst HP in the Centre Region of Cameroon from 1st February to 30th June 2021. Characteristics and access to preventive tools were collected using face-to-face administered questionnaire. Nasopharyngeal swabs and whole blood were collected for PCR, IgG and IgM testing respectively. STATA version 17 software was used for data analysis. Determinants of COVID-19 infection were explored by estimating crude and adjusted Odd Ratio. Results: Out of 510 HP reached, 458 were enrolled with mean age of 35 (±10) years. Thirty-four (7.4%) were PCR-positive to SARS-CoV-2 with 73.5% being clinicians versus 9 (26.4%) non-clinicians (p = 0.05). Sero-positivity to SARS-CoV-2 IgG/IgM was 40.2% (184/458), with 84.2% being clinicians versus 29 (15.8%) non-clinicians (p = 0.733). Amongst the 34 HP with PCR-positivity, 16 (47%) had no antibodies, while, 15 (44%) were IgG only. An estimate of HP (43.7%) had at least an evidence of PCR, IgG or IgM contact to COVID-19. Determinants of PCR-positivity was being clinical staff (AOR = 0.29, P = 0.039); and that of IgG/IgM were being non clinical staff (AOR = 0.41, p = 0.018) and regular use of face masks (AOR = 0.44, p = 0.001). HP trained on IPC (24%) were mainly from peripheral level (74.7%, p = 0.002). Conclusion: Active infections were within the range of pandemic control (<10%). However, around two-fifths of participants have had contact with the virus, indicating that HP remains a population at risk of COVID-19 and other similarly-transmitted epidemic prone diseases, and also an important source of transmission. There is need of vaccine to achieve protectiveness, and optimal response also requires capacity building to improve the health system when challenged by a future pandemic. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) continue to pose a huge threat to public health, and their prevention and treatment remain major international issues. Neuraminidase (NA) is the second most abundant surface glycoprotein on influenza viruses, and antibodies to NA have been shown to be effective against influenza infection. In this study, we generated a monoclonal antibody (mAb), named FNA1, directed toward N1 NAs. FNA1 reacted with H1N1 and H5N1 NA, but failed to react with the NA proteins of H3N2 and H7N9. In vitro, FNA1 displayed potent antiviral activity that mediated both NA inhibition (NI) and blocking of pseudovirus release. Moreover, residues 219, 254, 358, and 388 in the NA protein were critical for FNA1 binding to H1N1 NA. However, further validation is necessary to confirm whether FNA1 mAb is indeed a good inhibitor against NA for application against H1N1 and H5N1 viruses. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the seroprevalence and spatial and temporal clustering of SARS-CoV-2 antibodies in household cats within 63 counties in Illinois from October 2021 to May 2023. The analysis followed a stepwise approach. First, in a choropleth point map, we illustrated the distribution of county-level seroprevalence of SARS-CoV-2 antibodies. Next, spatial interpolation was used to predict the seroprevalence in counties without recorded data. Global and local clustering methods were used to identify the extent of clustering and the counties with high or low seroprevalence, respectively. Next, temporal, spatial, and space-time scan statistic was used to identify periods and counties with higher-than-expected seroprevalence. In the last step, to identify more distinct areas in counties with high seroprevalence, city-level analysis was conducted to identify temporal and space-time clusters. Among 1,715 samples tested by serological assays, 244 samples (14%) tested positive. Young cats had higher seropositivity than older cats, and the third quarter of the year had the highest odds of seropositivity. Three county-level space-time clusters with higher-than-expected seroprevalence were identified in the northeastern, central-east, and southwest regions of Illinois, occurring between June and October 2022. In the city-level analysis, 2 space-time clusters were identified in Chicago's downtown and the southwestern suburbs of Chicago between June and September 2022. Our results suggest that the high density of humans and cats in large cities such as Chicago, might play a role in the transmission and clustering of SARS-CoV-2. Our study provides an in-depth analysis of SARS-CoV-2 epidemiology in Illinois household cats, which will aid in COVID-19 control and prevention. [ABSTRACT FROM AUTHOR]

The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures. [ABSTRACT FROM AUTHOR]

Clostridial dermatitis (CD), caused by Clostridium septicum, is an emerging disease of increasing economic importance in turkeys. Currently, there are no effective vaccines for CD control. Here, two non-toxic domains of C. septicum alpha toxin, namely ntATX-D1 and ntATX-D2, were identified, cloned, and expressed in Escherichia coli as recombinant subunit proteins to investigate their use as potential vaccine candidates. Experimental groups consisted of a Negative control (NCx) that did not receive C. septicum challenge, while the adjuvant-only Positive control (PCx), ntATX-D1 immunization (D1) and ntATX-D2 immunization (D2) groups received C. septicum challenge. Turkeys were immunized subcutaneously with 100 μg of protein at 7, 8 and 9 weeks of age along with an oil-in-water nano-emulsion adjuvant, followed by C. septicum challenge at 11 weeks of age. Results showed that while 46.2% of birds in the PCx group died post-challenge, the rate of mortality in D1- or D2-immunization groups was 13.3%. The gross and histopathological lesions in the skin, muscle and spleen showed that the disease severity was highest in PCx group, while the D2-immunized birds had significantly lower lesion scores when compared to PCx. Gene expression analysis revealed that PCx birds had significantly higher expression of pro-inflammatory cytokine genes in the skin, muscle and spleen than the NCx group, while the D2 group had significantly lower expression of these genes compared to PCx. Peripheral blood cellular analysis showed increased frequencies of activated CD4+ and/or CD8+ cells in the D1 and D2-immunized groups. Additionally, the immunized turkeys developed antigen-specific serum IgY antibodies. Collectively, these findings indicate that ntATX proteins, specifically the ntATX-D2 can be a promising vaccine candidate for protecting turkeys against CD and that the protection mechanisms may include downregulation of C. septicum-induced inflammation and increased CD4+ and CD8+ cellular activation. [ABSTRACT FROM AUTHOR]

Many oncology antibody-drug conjugates (ADCs) have failed to demonstrate efficacy in clinic because of dose-limiting toxicity caused by uptake into healthy tissues. We developed an approach that harnesses ADC affinity to broaden the therapeutic index (TI) using two anti-mesenchymal-epithelial transition factor (MET) monoclonal antibodies (mAbs) with high affinity (HAV) or low affinity (LAV) conjugated to monomethyl auristatin E (MMAE). The estimated TI for LAV-ADC was at least 3 times greater than the HAV-ADC. The LAV- and HAV-ADCs showed similar levels of anti-tumor activity in the xenograft model, while the 111In-DTPA studies showed similar amounts of the ADCs in HT29 tumors. Although the LAV-ADC has ~2-fold slower blood clearance than the HAV-ADC, higher liver toxicity was observed with HAV-ADC. While the SPECT/CT 111In- and 124I- DTPA findings showed HAV-ADC has higher accumulation and rapid clearance in normal tissues, intravital microscopy (IVM) studies confirmed HAV mAb accumulates within hepatic sinusoidal endothelial cells while the LAV mAb does not. These results demonstrated that lowering the MET binding affinity provides a larger TI for MET-ADC. Decreasing the affinity of the ADC reduces the target mediated drug disposition (TMDD) to MET expressed in normal tissues while maintaining uptake/delivery to the tumor. This approach can be applied to multiple ADCs to improve the clinical outcomes. [ABSTRACT FROM AUTHOR]

This paper models the prevalence of antibodies to Borrelia burgdorferi in domestic dogs in the United States using climate, geographic, and societal factors. We then use this model to forecast the prevalence of antibodies to B. burgdorferi in dogs for 2016. The data available for this study consists of 11,937,925 B. burgdorferi serologic test results collected at the county level within the 48 contiguous United States from 2011-2015. Using the serologic data, a baseline B. burgdorferi antibody prevalence map was constructed through the use of spatial smoothing techniques after temporal aggregation; i.e., head-banging and Kriging. In addition, several covariates purported to be associated with B. burgdorferi prevalence were collected on the same spatio-temporal granularity, and include forestation, elevation, water coverage, temperature, relative humidity, precipitation, population density, and median household income. A Bayesian spatio-temporal conditional autoregressive (CAR) model was used to analyze these data, for the purposes of identifying significant risk factors and for constructing disease forecasts. The fidelity of the forecasting technique was assessed using historical data, and a Lyme disease forecast for dogs in 2016 was constructed. The correlation between the county level model and baseline B. burgdorferi antibody prevalence estimates from 2011 to 2015 is 0.894, illustrating that the Bayesian spatio-temporal CAR model provides a good fit to these data. The fidelity of the forecasting technique was assessed in the usual fashion; i.e., the 2011-2014 data was used to forecast the 2015 county level prevalence, with comparisons between observed and predicted being made. The weighted (to acknowledge sample size) correlation between 2015 county level observed prevalence and 2015 forecasted prevalence is 0.978. A forecast for the prevalence of B. burgdorferi antibodies in domestic dogs in 2016 is also provided. The forecast presented from this model can be used to alert veterinarians in areas likely to see above average B. burgdorferi antibody prevalence in dogs in the upcoming year. In addition, because dogs and humans can be exposed to ticks in similar habitats, these data may ultimately prove useful in predicting areas where human Lyme disease risk may emerge. [ABSTRACT FROM AUTHOR]

Background: Increasingly, vaccine efficacy studies are being recommended in low-and-middle-income countries (LMIC), yet often facilities are unavailable to take and store infant blood samples correctly. Dried blood spots (DBS), are useful for collecting blood from infants for diagnostic purposes, especially in low-income settings, as the amount of blood required is miniscule and no refrigeration is required. Little is known about their utility for antibody studies in children. This systematic review aims to investigate the correlation of antibody concentrations against infectious diseases in DBS in comparison to serum or plasma samples that might inform their use in vaccine clinical trials. Methods and findings: We searched MEDLINE, Embase and the Cochrane library for relevant studies between January 1990 to October 2020 with no language restriction, using PRISMA guidelines, investigating the correlation between antibody concentrations in DBS and serum or plasma samples, and the effect of storage temperature on DBS diagnostic performance. We included 40 studies in this systematic review. The antibody concentration in DBS and serum/plasma samples reported a good pooled correlation, (r2 = 0.86 (ranged 0.43 to 1.00)). Ten studies described a decline of antibody after 28 days at room temperature compared to optimal storage at -20°C, where antibodies were stable for up to 200 days. There were only five studies of anti-bacterial antibodies. Conclusions: There is a good correlation between antibody concentrations in DBS and serum/plasma samples, supporting the wider use of DBS in vaccine and sero-epidemiological studies, but there is limited data on anti-bacterial antibodies. The correct storage of DBS is critical and may be a consideration for longer term storage. [ABSTRACT FROM AUTHOR]

Background: M-type phospholipase A2 receptor (PLA2R) is the major autoantigen in adult idiopathic membranous nephropathy (IMN). Although reactive epitopes in the PLA2R domains have been identified, the clinical value of these domains recognized by anti-PLA2R antibodies remains controversial. Accordingly, this study aimed to quantitatively detect changes in the concentrations of different antibodies against epitopes of PLA2R in patients with IMN before and after treatment to evaluate the clinical value of epitope spreading. Methods: Highly sensitive time-resolved fluorescence immunoassay was used to quantitatively analyze the concentrations of specific IgG and IgG4 antibodies against PLA2R and its epitopes (CysR, CTLD1, CTLD6-7-8) in a cohort of 25 patients with PLA2R-associated membranous nephropathy (13 and 12 in the remission and non-remission groups, respectively) before and after treatment, and the results were analyzed in conjunction with clinical biochemical indicators. Results: The concentration of specific IgG (IgG4) antibodies against PLA2R and its epitopes (CysR, CTLD1 and CTLD6-7-8) in non-remission group was higher than that in remission group. The multipliers of elevation of IgG (IgG4) antibody were 5.6(6.2) fold, 3.0(24.3) fold, 1.6(9.0) fold, and 4.2(2.6) fold in the non-remission/remission group, respectively. However, the difference in antibody concentrations between the two groups at the end of follow-up was 5.6 (85.2), 1.7 (13.1), 1.0 (5.1), and 1.5 (22.3) times higher, respectively. When detecting concentrations of specific IgG antibodies against PLA2R and its different epitopes, the remission rate was 66.67% for only one epitope at M0 and 36.36% for three epitopes at M0. When detecting concentrations of specific IgG4 antibodies against PLA2R and its different epitopes, the remission rate was 100.00% for only one epitope at M0 and 50.00% for three epitopes at M0. A trivariate logistic regression model for the combined detection of eGFR, anti-CTLD678 IgG4, and urinary protein had an AUC of 100.00%. Conclusion: Low concentrations of anti-CysR-IgG4, anti-CTLD1-IgG4, and anti-CTLD6-7-8-IgG4 at initial diagnosis predict rapid remission after treatment. The use of specific IgG4 against PLA2R and its different epitopes combined with eGFR and urinary protein provides a better assessment of the prognostic outcome of IMN. [ABSTRACT FROM AUTHOR]

Background: Helicobacter pylori infections are generally acquired during childhood and affect half of the global population, but its transmission route remains unclear. It is reported that H. pylori can be internalized into Candida, but more evidence is needed for the internalization of H. pylori in human gastrointestinal Candida and vaginal Candida. Methods: Candida was isolated from vaginal discharge and gastric mucosa biopsies. We PCR-amplified and sequenced H. pylori-specific genes from Candida genomic DNA. Using optical and immunofluorescence microscopy, we identified and observed bacteria-like bodies (BLBs) in Candida isolates and subcultures. Intracellular H. pylori antigen were detected by immunofluorescence using Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies. Urease activity in H. pylori internalized by Candida was detected by inoculating with urea-based Sabouraud dextrose agar, which changed the agar color from yellow to pink, indicating urease activity. Results: A total of 59 vaginal Candida and two gastric Candida strains were isolated from vaginal discharge and gastric mucosa. Twenty-three isolates were positive for H. pylori 16S rDNA, 12 were positive for cagA and 21 were positive for ureA. The BLBs could be observed in Candida cells, which were positive for H. pylori 16S rDNA, and were viable determined by the LIVE/DEAD BacLight Bacterial Viability kit. Fluorescein isothiocyanate (FITC)-conjugated antibodies could be reacted specifically with H. pylori antigen inside Candida cells by immunofluorescence. Finally, H. pylori-positive Candida remained positive for H. pylori 16S rDNA even after ten subcultures. Urease activity of H. pylori internalized by Candida was positive. Conclusion: In the form of BLBs, H. pylori can internalize into gastric Candida and even vaginal Candida, which might have great significance in its transmission and pathogenicity. [ABSTRACT FROM AUTHOR]

Antibody drug conjugates (ADCs) have emerged as a highly promising class of cancer therapeutics, comprising antibodies, effector molecules, and linkers. Among them, DS-8201a with DXd as the effector molecule, has shown remarkable anti-tumor efficacy against solid tumors, sparking a surge of interest in ADCs with camptothecin derivatives as ADC effector molecules. In this study, we introduced and successfully constructed quaternary ammonium ADCs utilizing camptothecin derivatives WL-14 and CPTS-1 for the first time. All four ADCs displayed excellent stability under physiological conditions and in plasma, facilitating their prolonged circulation in vivo. Moreover, the four ADCs, employing Val-Cit or Val-Ala dipeptide linkers effectively achieved complete release of the effector molecules via cathepsin B. Although, the in vitro antitumor activity of these ADCs was comparatively limited, the development of quaternary ammonium ADCs based on novel camptothecin derivatives as effector molecules is still a viable and promising strategy. Significantly, our study provides valuable insights into the crucial role of linker optimization in ADCs design. [ABSTRACT FROM AUTHOR]

Detection of infections in wildlife species is increasingly important to reduce the risk of spreading zoonotic and economically important parasites, understand disease epidemiology and promote the conservation of wildlife species. Serological tests are key in disease diagnosis and surveillance by detecting immunoglobulins against infectious agents. However, the need for species-specific reagents has limited the application of serological tests in wildlife species. This study evaluated the serum immunoglobulin-binding capability of polyclonal anti-kangaroo antibody and two non-species-specific reagents, including protein A/G and protein L, with the largest range of Australian marsupial species so far, including 32 species representing three major marsupial orders. Immunoglobulin-binding capability was assessed using immunoblotting, enzyme-linked immunosorbent assay and Western blot techniques. Variation in immunoglobulin-binding capability was observed between the three reagents and across the species tested, both across but also within taxonomic groups. Taxonomic distance was thus not always a good predictor of immunoglobulin-binding affinity, emphasizing the need to validate these reagents for each species separately. However, all three reagents bound with the serum immunoglobulins of most marsupial species tested. The findings of this study provide a valuable reference for species differences in affinity to protein A/G, protein L and anti-kangaroo antibody, assisting in the selection of appropriate reagents and the development of sero-immunological assays in Australian marsupials. [ABSTRACT FROM AUTHOR]

Background: Observational studies have indicated that both Helicobacter pylori infection and the presence of Helicobacter pylori antibodies may increase the risk of gastroesophageal reflux disease (GERD). However, the exact association between Helicobacter pylori antibodies and the occurrence of GERD remains largely unresolved. Therefore, this two-sample Mendelian randomization (MR) study aims to investigate the causal relationship between Helicobacter pylori infection and GERD. Methods: This study encompassed seven different specific protein antibodies targeting Helicobacter pylori and utilized a genome-wide association study (GWAS) on GERD. MR analysis was conducted to assess the causal relationship between Helicobacter pylori antibodies and the development of GERD. Results: Genetically predicted serum levels of Helicobacter pylori IgG antibodies were positively associated with an increased risk of GERD (odds ratio [OR] = 1.001, 95% CI 1.000–1.003, P = 0.043). No causal relationship was found between other Helicobacter pylori antibodies and gastroesophageal reflux disease. Conclusion: The outcomes derived from our two-sample Mendelian randomization analysis demonstrate a discernible link between the levels of Helicobacter pylori IgG antibodies and an augmented susceptibility to GERD. However, it is imperative to expand the sample size further in order to corroborate the correlation between Helicobacter pylori infection and GERD. [ABSTRACT FROM AUTHOR]

Repeated serological testing tells about the change in the overall infection in a community. This study aimed to evaluate changes in antibody prevalence and kinetics in a closed cohort over six months in different sub-populations in India. The study included 10,000 participants from rural and urban areas in five states and measured SARS-CoV-2 antibodies in serum in three follow-up rounds. The overall seroprevalence increased from 73.9% in round one to 90.7% in round two and 92.9% in round three. Among seropositive rural participants in round one, 98.2% remained positive in round two, and this percentage remained stable in urban and tribal areas in round three. The results showed high antibody prevalence that increased over time and was not different based on area, age group, or sex. Vaccinated individuals had higher antibody prevalence, and nearly all participants had antibody positivity for up to six months. [ABSTRACT FROM AUTHOR]

For rapid screening and quantification of an antisera antibody, a nanometer bithiophene-based conductive biolinker can enhanced signal performance and can be used to verify the interaction of an anti-IFN-γ antibody with an IFN-γ protein. The experimental measurements take a generic approach which takes advantage of the functionality of thiophene-based linkers for biosensors. Effects associated with using bithiophene as a biolinker for surface plasmon resonance (SPR) spectroscopy are examined in this paper. By using an atomic force microscope (AFM), it was observed that the morphology of the bithiophene modified gold sensor surface became smoother than the original gold surface. We compared the response and concentration of the anti-IFN-γ antibody on a bithiophene-coated and dextran-coated biochip as well as on different thickness-modified surfaces under SPR relevant conditions. The results indicate that a response to IFN-γ molecules immobilized on a sensor using a bithiophene biolinker improved more than 8-fold when compared to that of a sensor using a dextran biolinker. Furthermore, the regeneration ability of the sensor surface shows good repeatability as only less than a 1% decrease was found after repeating the experimental work over 6 cycles. The characteristics provided us with a good platform for rapid screening, real-time monitoring and quantitative concentration of the autoimmune antibody activities. [ABSTRACT FROM AUTHOR]

Huntington’s disease (HD) cellular pathology is characterised by the aggregation of mutant huntingtin (mHTT) protein into inclusion bodies. The present paper compared the sensitivity of five widely used mHTT antibodies (S830; MW8; EM48; 1C2; ubiquitin) against mice from five commonly used HD mouse models (R6/1; YAC128; HdhQ92; B6 HdhQ150; B6 x129/Ola HdhQ150) at two ages to determine: the most sensitive antibodies for each model; whether mHTT antibody binding differed depending on aggregation stage (diffuse versus frank inclusion); the role of ubiquitin during aggregation as the ubiquitin proteosome system has been implicated in disease development. The models demonstrated unique profiles of antibody binding even when the models varied only by background strain (HdhQ150). MW8 was highly sensitive for detecting frank inclusions in all lines whereas EM48, ubiquitin and 1C2 demonstrated consistent staining in all models irrespective of age or form of mHTT. MW8 and S830 were the most sensitive antibodies with 1C2 the least. Ubiquitin levels were stable for each model regardless of age. Ubiquitin was particularly sensitive in young YAC128 mice that demonstrate an absence of inclusions until ~12 months of age suggesting high affinity to mHTT in its diffuse form. The data indicate that generalisations across models regarding the quantification of aggregations may not be valid and that mHTT antibody binding is unique to the mouse model and sensitive to changes in inclusion development. [ABSTRACT FROM AUTHOR]

Many approaches have been designed to extract brain effective connectivity from functional magnetic resonance imaging (fMRI) data. However, few of them can effectively identify the connectivity network structure due to different defects. In this paper, a new algorithm is developed to infer the effective connectivity between different brain regions by combining artificial immune algorithm (AIA) with the Bayes net method, named as AIAEC. In the proposed algorithm, a brain effective connectivity network is mapped onto an antibody, and four immune operators are employed to perform the optimization process of antibodies, including clonal selection operator, crossover operator, mutation operator and suppression operator, and finally gets an antibody with the highest K2 score as the solution. AIAEC is then tested on Smith’s simulated datasets, and the effect of the different factors on AIAEC is evaluated, including the node number, session length, as well as the other potential confounding factors of the blood oxygen level dependent (BOLD) signal. It was revealed that, as contrast to other existing methods, AIAEC got the best performance on the majority of the datasets. It was also found that AIAEC could attain a relative better solution under the influence of many factors, although AIAEC was differently affected by the aforementioned factors. AIAEC is thus demonstrated to be an effective method for detecting the brain effective connectivity. [ABSTRACT FROM AUTHOR]

Enhancement or inhibition of cytokine signaling and corresponding immune cells responses are critical factors in various disease treatments. Cytokine signaling may be inhibited by cytokine-neutralizing antibodies (CNAs), which prevents further activation of cytokine receptors. However, CNAs may result in enhanced—instead of inhibitory—cytokine signaling (an “agonistic effect”) in various in vitro and in vivo experiments. This may lead to lack of efficacy or adverse events for cytokine-inhibiting based medicines. Alternatively, cytokine-antibody complexes may produce stronger signaling vs. cytokine alone, thereby increasing the efficacy of stimulating cytokine-based drugs, at equal or lower cytokine doses. In this paper, the effect of cytokine signaling enhancement by a CNA was studied in a generic mathematical model of interleukin-4 (IL-4) driven T-cell proliferation. The occurrence of the agonistic effect depends upon the antibody-to-cytokine binding affinity and initial concentrations of antibody and cytokine. Model predictions were in agreement with experimental studies. When the cytokine receptor consists of multiple subunits with substantially differing affinities (e.g., IL-4 case), the choice of the receptor chain to be blocked by the antibody is critical, for the agonistic effect to appear. We propose a generic mechanism for the effect: initially, binding of the CNA to the cytokine reduces free cytokine concentration; yet, cytokine molecules bound within the cytokine-CNA complex—and released later and over time—are “rescued” from earlier clearance via cellular internalization. Hence, although free cytokine-dependent signalling may be less potent initially, it will also be more sustained over time; and given non-linear dynamics, it will lead ultimately to larger cellular effector responses, vs. the same amount of free cytokine in the absence of CNA. We suggest that the proposed mechanism is a generic property of {cytokine, CNA, receptor} triads, both in vitro and in vivo, and can occur in a predictable fashion for a variety of cytokines of the immune system. [ABSTRACT FROM AUTHOR]

Single Nucleotide Polymorphisms (SNPs) found in Genome-Wide Association Study (GWAS) mainly influence the susceptibility of complex diseases, but they still could not comprehensively explain the relationships between mutations and diseases. Interactions between SNPs are considered so important for deeply understanding of those relationships that several strategies have been proposed to explore such interactions. However, part of those methods perform poorly when marginal effects of disease loci are weak or absent, others may lack of considering high-order SNPs interactions, few methods have achieved the requirements in both performance and accuracy. Considering the above reasons, not only low-order, but also high-order SNP interactions as well as main-effect SNPs, should be taken into account in detection methods under an acceptable computational complexity. In this paper, a new pairwise (or low-order) interaction detection method IG (Interaction Gain) is introduced, in which disease models are not required and parallel computing is utilized. Furthermore, high-order SNP interactions were proposed to be detected by finding closely connected function modules of the network constructed from IG detection results. Tested by a wide range of simulated datasets and four WTCCC real datasets, the proposed methods accurately detected both low-order and high-order SNP interactions as well as disease-associated main-effect SNPS and it surpasses all competitors in performances. The research will advance complex diseases research by providing more reliable SNP interactions. [ABSTRACT FROM AUTHOR]

Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds. [ABSTRACT FROM AUTHOR]

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10–45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure. [ABSTRACT FROM AUTHOR]

Studies suggest perinatal infection with SARS-CoV-2 can induce adverse birth outcomes, but studies published to date have substantial limitations. We therefore conducted an observational study of 211 births occurring between January 2020-September 2021 in three urban cohorts participating in the Environmental Influences on Child Health Outcomes Program. Serology was assessed for IgG, IgM and IgA antibodies to nucleocapsid, S1 spike, S2 spike, and receptor-binding domain. There were no differences in gestational age (GA), birth weight, preterm birth (PTB) or low birth weight (LBW) among seropositive mothers. However, the few (n = 9) IgM seropositive mothers had children with lower BW (434g, 95% CI: 116–752), BW Z score-for-GA (0.73 SD, 95% CI 0.10–1.36) and were more likely to deliver preterm (OR 8.75, 95% CI 1.22–62.4). Though there are limits to interpretation, the data support efforts to prevent SARS-CoV-2 infections in pregnancy. [ABSTRACT FROM AUTHOR]

Background: Despite the increasing number of cases of secondary antibody deficiency (SAD) and immunoglobulin (Ig) utilization, there is a paucity of data in the literature on clinical and patient-reported outcomes in this population. Objective: To describe immunoglobulin utilization patterns, clinical and patient-reported outcomes in patients with SAD on immunoglobulin replacement therapy (IgRT). Methods: A cross-sectional study of patients with secondary antibody deficiency enrolled in the Ontario Immunoglobulin Treatment (ONIT) Case Registry from June 2020 to September 2022 was completed. Demographics, comorbidities, indications for immunoglobulin treatment, clinical infections at baseline and post IgRT, and patient-reported outcomes were collected and analyzed. Results: There were 140 patients (58 males; 82 females; median age 68) with SAD during the study period; 131 were on subcutaneous Ig (SCIG) and 9 were on intravenous Ig (IVIG). The most common indication was chronic lymphocytic leukemia (CLL) (N = 52). IgRT reduced the average annual number of infections by 82.6%, emergency room (ER) visits by 84.6%, and hospitalizations by 83.3%. Overall, 84.6% of patients reported their health as better compared to before IgRT. Among those patients who switched from IVIG to SCIG (N = 35), 33.3% reported their health as the same, and 62.9% reported their health as better. Conclusions: This study demonstrates that IgRT significantly improved clinical outcomes and patient-reported general health state in patients with SAD. This study also further supports the use of SCIG in patients with SAD. [ABSTRACT FROM AUTHOR]

Objectives: To investigate maternal antibody levels to varicella in infants <12 months of age in Ontario, Canada. Study design: In this study, we included specimens from infants <12 months of age, born at ≥37 weeks gestational age, who had sera collected at The Hospital for Sick Children (Toronto, Canada) between 2014–2016. We tested sera using a glycoprotein-based enzyme-linked immunosorbent assay (gpELISA). We measured varicella susceptibility (antibody concentration <150mIU/mL) and mean varicella antibody concentration, and assessed the probability of susceptibility and concentration between one and 11 months of age using multivariable logistic regression and Poisson regression. Results: We found that 32% of 196 included specimens represented infants susceptible to varicella at one month of age, increasing to nearly 80% at three months of age. At six months of age, all infants were susceptible to varicella and the predicted mean varicella antibody concentration declined to 62 mIU/mL (95% confidence interval 40, 84), well below the threshold of protection. Conclusions: We found that varicella maternal antibody levels wane rapidly in infants, leaving most infants susceptible by four months of age. Our findings have implications for the timing of first dose of varicella-containing vaccine, infection control measures, and infant post-exposure prophylaxis recommendations. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has created an urgent need for effective therapeutic and diagnostic strategies to manage the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the emergence of numerous variants of concern (VOCs) has made it challenging to develop targeted therapies that are broadly specific in neutralizing the virus. In this study, we aimed to develop neutralizing nanobodies (Nbs) using computational techniques that can effectively neutralize the receptor-binding domain (RBD) of SARS-CoV-2 VOCs. We evaluated the performance of different protein-protein docking programs and identified HDOCK as the most suitable program for Nb/RBD docking with high accuracy. Using this approach, we designed 14 novel Nbs with high binding affinity to the VOC RBDs. The Nbs were engineered with mutated amino acids that interacted with key amino acids of the RBDs, resulting in higher binding affinity than human angiotensin-converting enzyme 2 (ACE2) and other viral RBDs or haemagglutinins (HAs). The successful development of these Nbs demonstrates the potential of molecular modeling as a low-cost and time-efficient method for engineering effective Nbs against SARS-CoV-2. The engineered Nbs have the potential to be employed in RBD-neutralizing assays, facilitating the identification of novel treatment, prevention, and diagnostic strategies against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Healthcare workers (HCWs) are disproportionately infected with SARS-CoV-2 when compared to members of the general public; estimating the seroprevalence of SARS-CoV-2 antibody and SARS-CoV-2 infection rate among HCWs is therefore crucial. This study was carried out in four health facilities in Lagos Nigeria to determine the prevalence of IgG antibodies (seroprevalence) and SARS-CoV-2 active infection rate via a positive rtPCR result, the cross-sectional study was conducted between December 2020 and July 2021. Nasopharyngeal and blood samples were collected from HCWs and screened for SARS-CoV-2 infection using the rtPCR technique and antibody using the Abbott anti-SARS-CoV-2 IgG CMIA assay, respectively. Demographic and occupational exposures data were obtained and analysed using descriptive and inferential statistics, variables significant via inferential statistics were subjected to a multivariate analysis. A total of 413 participants were enrolled, with a mean age in years of 38.4±11.0. The seroprevalence was 30.9% (115/372) while 63/395 (15.9%) were actively infected with the virus. HCWs whose job role had direct contact with patients had a higher percentage of SARS-CoV-2 infection when compared with those not in direct contact, also being a health care worker was significantly associated with getting a positive COVID-19 PCR result. In conclusion the SARS-CoV-2 seroprevalence seen in this study was higher than national serosurvey estimates indicating HCWs are at higher risk of COVID-19 infection when compared to the general public. Vaccination and effective implementation of infection control measures are important to protect HCWs. [ABSTRACT FROM AUTHOR]

Prior to the age of measles vaccination, infants are believed to be protected against measles by passively transferred maternal antibodies. However, the quantity and quality of such protection have not been well established in the Indian setting. We undertook this study to characterize the transfer and decline in maternal anti-measles antibodies among infants, and determine their susceptibility to measles. In this population-based, birth-cohort study, we enrolled pregnant women and their newborn infants, from a catchment area of 30 Anganwadis in Chandigarh, India. We collected maternal blood at delivery, and infant blood samples at birth, and 3, 6, and 9 months of age. Anti-measles IgG antibodies were measured using quantitative ELISA. We assessed antibody decline using log-linear models. In total, 428 mother-infant dyads were enrolled, and data from 413 dyads were analyzed. At birth, 91.5% (95% CI: 88.8, 94.2) of infants had protective antibody levels, which declined to 26.3% (95% CI: 21.0%, 31.9) at 3 months, 3.4% (95% CI: 0.9, 5.9) at 6 months, and 2.1% (95% CI: 0.1, 4.1) at 9 months. Younger mothers transferred lower levels of antibodies to their infants. We concluded that the majority of infants are susceptible to measles as early as three months of age, much earlier than their eligibility to receive measles vaccination. [ABSTRACT FROM AUTHOR]

FcγRI (CD64) is the only high-affinity Fcγ receptor found on monocytes, macrophages, eosinophils, neutrophils and dendritic cells. It binds immunoglobulin G (IgG) antibody-antigen complexes at its Fc region to trigger key immune responses. CD64 contains three immunoglobulin-fold extracellular domains (D1, D2 and D3) and a membrane-spanning region. Despite the importance of CD64, no solution structure for this is known to date. To investigate this, we used analytical ultracentrifugation, small-angle X-ray scattering, and atomistic modelling. Analytical ultracentrifugation revealed that CD64 was monomeric with a sedimentation coefficient s020,w of 2.53 S, together with some dimer. Small-angle X-ray scattering showed that its radius of gyration RG was 3.3–3.4 nm and increased at higher concentrations to indicate low dimerization. Monte Carlo modelling implemented in the SASSIE-web package generated 279,162 physically-realistic trial CD64 structures. From these, the scattering best-fit models at the lowest measured concentrations that minimised dimers revealed that the D1, D2 and D3 domains were structurally similar to those seen in three CD64 crystal structures, but showed previously unreported flexibility between D1, D2 and D3. Despite the limitations of the scattering data, the superimposition of the CD64 solution structures onto crystal structures of the IgG Fc-CD64 complex showed that the CD64 domains do not sterically clash with the IgG Fc region, i.e. the solution structure of CD64 was sufficiently compact to allow IgG to bind to its high-affinity Fcγ receptor. This improved understanding may result in novel approaches to inhibit CD64 function, and opens the way for the solution study of the full-length CD64-IgG complex. [ABSTRACT FROM AUTHOR]

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94–233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays. [ABSTRACT FROM AUTHOR]

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal. [ABSTRACT FROM AUTHOR]

Antibody development is still associated with substantial risks and difficulties as single mutations can radically change molecule properties like thermodynamic stability, solubility or viscosity. Since antibody generation methodologies cannot select and optimize for molecule properties which are important for biotechnological applications, careful sequence analysis and optimization is necessary to develop antibodies that fulfil the ambitious requirements of future drugs. While efforts to grab the physical principles of undesired molecule properties from the very bottom are becoming increasingly powerful, the wealth of publically available antibody sequences provides an alternative way to develop early assessment strategies for antibodies using a statistical approach which is the objective of this paper. Here, publically available sequences were used to develop heuristic potentials for the framework regions of heavy and light chains of antibodies of human and murine origin. The potentials take into account position dependent probabilities of individual amino acids but also conditional probabilities which are inevitable for sequence assessment and optimization. It is shown that the potentials derived from human sequences clearly distinguish between human sequences and sequences from mice and, hence, can be used as a measure of humaness which compares a given sequence with the phenotypic pool of human sequences instead of comparing sequence identities to germline genes. Following this line, it is demonstrated that, using the developed potentials, humanization of an antibody can be described as a simple mathematical optimization problem and that the in-silico generated framework variants closely resemble native sequences in terms of predicted immunogenicity. [ABSTRACT FROM AUTHOR]

Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4) in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication. [ABSTRACT FROM AUTHOR]

Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×106 cfu/3 µg and 2×109 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy. [ABSTRACT FROM AUTHOR]

Background: Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients. Methodology/Principal Findings: In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass. Conclusions: The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers. [ABSTRACT FROM AUTHOR]

Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems. [ABSTRACT FROM AUTHOR]