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Journal of Dairy Science Jun 2024Mycoplasmosis (due to infection with Mycoplasma bovis) is a serious disease of beef and dairy cattle that can adversely impact health, welfare and productivity (Maunsell...
Estimation of sensitivity and specificity of bulk tank milk PCR and 2 antibody ELISA tests for herd-level diagnosis of Mycoplasma bovis infection using Bayesian latent class analysis.
Mycoplasmosis (due to infection with Mycoplasma bovis) is a serious disease of beef and dairy cattle that can adversely impact health, welfare and productivity (Maunsell et al. (2011)). Mycoplasmosis can lead to a range of often severe, clinical presentations. Mycoplasma bovis (M. bovis) infection can present either clinically or subclinically, with the potential for recrudescence of shedding in association with stressful periods. Infection can be maintained within herds because of intermittent shedding (Calcutt et al., 2018, Hazelton et al., 2018). M. bovis is recognized as poorly responsive to treatment which represents a major challenge for control in infected herds. Given this, particular focus is needed on biosecurity measures to prevent introduction into uninfected herds in the first place. A robust and reliable laboratory test for surveillance is important both for herd-level prevention and control. The objective of this study was to estimate the sensitivity and specificity of 3 diagnostic tests (one PCR and 2 ELISA tests) on bulk tank milk, for the herd-level detection of M. bovis using Bayesian latent class analysis. In autumn 2018, bulk tank milk samples from 11,807 herds, covering the majority of the main dairy regions in Ireland had been submitted to the Department of Agriculture testing laboratory for routine surveillance were made available. A stratified random sample approach was used to select a cohort of herds for testing from this larger sample set. A final study population of 728 herds had bulk tank milk samples analyzed using a Bio-X ELISA (ELISA 1), an IDvet ELISA (ELISA 2) and a PCR test. A Bayesian latent class analysis (BLCA) was conducted to estimate the sensitivity (Se) and specificity (Sp) of the 3 diagnostic tests applied to bulk tank milk (BTM) for the detection of the herd-level infection. An overall LCA was conducted on all herds within a single population (a 3-test, 1-population model). The herds were also split into 2 populations based on herd size (small herds had < 82 cattle) (a 3-test, 2-population model) and separately into 3 regions in Ireland (Leinster, Munster and Connacht/Ulster) (a 3-test, 3-population model). The latent variable of interest was the herd-level M. bovis infection status. In total, 363/728 (50%) were large herds, 7 (1.0%) were positive on PCR, 88 (12%) positive on ELISA 1, and 406 (56%) positive on ELISA 2. Based on the 2-population model, the sensitivity (95% Bayesian credible interval (BCI) was 0.03 (0.02, 0.05), 0.22 (0.18, 0.27), 0.94 (0.88, 0.98) for PCR, ELISA 1 and ELISA 2 respectively. The specificity (95% BCI) was 0.99 (0.99, 1.0), 0.97 (0.95, 0.99), and 0.92 (0.86, 0.97) for PCR, ELISA 1 and ELISA 2 respectively. The herd-level true prevalence was estimated at 0.43 (BCI 0.35, 0.5) for smaller herds. The true prevalence was estimated at 0.62 (BCI 0.55, 0.69) for larger herds. The true prevalence was estimated at 0.56 (BCI 0.49, 0.463) in the 1-population model. For the 3-population model, the sensitivity (95% BCI) was 0.03 (0.02, 0.05), 0.24 (0.18, 0.29), 0.95 (0.9, 0.98) for PCR, ELISA 1 and ELISA 2 respectively. The specificity (95% BCI) was 0.99 (0.99, 1.0), 0.98 (0.96, 0.99), and 0.88 (0.79, 0.95) for PCR, ELISA 1 and ELISA 2 respectively. The herd-level true prevalence (95% BCI) was estimated at 0.65 (0.56, 0.73), 0.38 (0.28, 0.46) and 0.53 (0.4, 0.65) for population 1, 2, 3 respectively. Across all 3 models, the range in true prevalence was 38% to 65% of Irish dairy herds infected with M. bovis. The operating characteristics vary substantially between tests. The IDvet ELISA had a relatively high Se (the highest Se of the 3 tests studied) but it was estimated at 0.95 at its highest in 3-test, 3-population model. This test may be an appropriate test for herd-level screening or prevalence estimation within the context of the endemically infected Irish dairy cattle population. Further work is required to optimize this test and its interpretation when applied at herd-level to offset concerns related to the lower than optimal test Sp.
PubMed: 38851575
DOI: 10.3168/jds.2023-24590 -
MAbs 2024In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in...
In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.
Topics: Antibodies, Bispecific; Humans; CD47 Antigen; Immunoglobulin Fab Fragments; B7-H1 Antigen; Crystallography, X-Ray; Immunoglobulin Light Chains; Models, Molecular
PubMed: 38849989
DOI: 10.1080/19420862.2024.2362432 -
Poultry Science May 2024Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of heterodimeric cytokines that play a critical role in regulating T helper cell function....
Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of heterodimeric cytokines that play a critical role in regulating T helper cell function. IL-12 and IL-23 share a common p40 subunit, but differ in their p35 and p19 subunits, respectively. This difference in subunit composition results in distinct signaling pathways and biological functions for IL-12 and IL-23. Here, we report the functional characterization and immunomodulatory properties of chicken IL-12 and IL-23 using the panels of newly developed mouse anti-IL-12p40, IL-12p35-α and IL-23p19 monoclonal antibodies (mAbs). Western blot and indirect ELISA analysis demonstrated that the anti-chicken IL-12p40 mAbs (chIL-12p40; #10G10F4 and #10D8G2) bound to both recombinant proteins (IL-12 and IL-23), the anti-chicken IL-12p35 mAb (chIL-12p35; #2F1) specifically recognized recombinant IL-12, and the anti-chicken IL-23p19 mAb (chIL-23p19; #15A3) exhibited specificity for recombinant IL-23, without any cross-reactivity. Two ELISAs detecting specific chicken IL-12 (#10G10F4 and #2F1) or IL-23 (#10D8G2 and #15A3) were developed using newly developed mAb combinations, #10G10F4/ #2F1 and #10D8G2/#15A3 for IL-12 and IL-23, respectively, identified through a pairing assay. The levels of IL-12 and IL-23 in Resiquimod-848 stimulated-HD11 chicken macrophage cells were monitored over time using antigen-capture sandwich ELISA developed in this study. Furthermore, the levels of chicken IL-12 and IL-23 in the circulation of Eimeria maxima (E. maxima) and Eimeria tenella (E. tenella)-infected chickens were determined. Notably, the anti-chIL-12p40 mAbs (#10G10F4 and #10D8G2) neutralized the function of both chIL-12 and chIL-23 proteins, which share the p40 subunit, while the anti-chIL-23p19 mAb (#15A3) specifically neutralized chIL-23 protein in HD11 cells in vitro. The anti-chIL-12p35 mAb (#2F1), which is specific to the p35 subunit of IL-12, showed a partial neutralizing effect on chIL-12 protein. Collectively, our study validates the specificity and significance of 2 newly developed antigen-capture immunoassays for chIL-12 and chIL-23 which will expand our understanding of the functional characteristics of IL-12 and IL-23 and their association in normal and diseased chickens. These mAbs for each subunit, anti-chIL-12p35, anti-chIL-12p40 and anti-chIL-23p19, will serve as valuable immune reagents to elucidate host immune responses against disease pathogenesis in both fundamental and applied studies of avian species.
PubMed: 38848631
DOI: 10.1016/j.psj.2024.103872 -
Cell Reports Jun 2024The development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. We screened the circulating B...
The development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees to isolate over 9,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. Among the recovered antibodies was TXG-0078, an N-terminal domain (NTD)-specific neutralizing mAb that recognizes diverse alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy-chain-dominant binding pattern seen in other NTD-supersite-specific neutralizing Abs with much narrower specificity. We also report CC24.2, a pan-sarbecovirus neutralizing antibody that targets a unique receptor-binding domain (RBD) epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 shows protection in vivo, suggesting their potential use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.
PubMed: 38848216
DOI: 10.1016/j.celrep.2024.114307 -
Journal of Clinical Immunology Jun 2024Despite advancements in genetic and functional studies, the timely diagnosis of common variable immunodeficiency (CVID) remains a significant challenge. This exploratory...
Despite advancements in genetic and functional studies, the timely diagnosis of common variable immunodeficiency (CVID) remains a significant challenge. This exploratory study was designed to assess the diagnostic performance of a novel panel of biomarkers for CVID, incorporating the sum of κ+λ light chains, soluble B-cell maturation antigen (sBCMA) levels, switched memory B cells (smB) and the VISUAL score. Comparative analyses utilizing logistic regression were performed against established gold-standard tests, specifically antibody responses. Our research encompassed 88 subjects, comprising 27 CVID, 23 selective IgA deficiency (SIgAD), 20 secondary immunodeficiency (SID) patients and 18 healthy controls. We established the diagnostic accuracy of sBCMA and the sum κ+λ, achieving sensitivity (Se) and specificity (Spe) of 89% and 89%, and 90% and 99%, respectively. Importantly, sBCMA showed strong correlations with all evaluated biomarkers (sum κ+λ, smB cell and VISUAL), whereas the sum κ+λ was uniquely independent from smB cells or VISUAL, suggesting its additional diagnostic value. Through a multivariate tree decision model, specific antibody responses and the sum κ+λ emerged as independent, signature biomarkers for CVID, with the model showcasing an area under the curve (AUC) of 0.946, Se 0.85, and Spe 0.95. This tree-decision model promises to enhance diagnostic efficiency for CVID, underscoring the sum κ+λ as a superior CVID classifier and potential diagnostic criterion within the panel.
Topics: Humans; Common Variable Immunodeficiency; Biomarkers; Male; Female; Adult; Middle Aged; Logistic Models; Young Adult; Adolescent; Aged; Immunoglobulin kappa-Chains; Sensitivity and Specificity; B-Lymphocytes; Immunoglobulin lambda-Chains; Memory B Cells
PubMed: 38847936
DOI: 10.1007/s10875-024-01746-1 -
MBio Jun 2024Herpes B virus (BV) is a zoonotic virus and belongs to the genus , the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in...
Herpes B virus (BV) is a zoonotic virus and belongs to the genus , the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus , the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection.
PubMed: 38847539
DOI: 10.1128/mbio.01092-24 -
Se Pu = Chinese Journal of... Jun 2024Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability,... (Review)
Review
Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.
Topics: Antibodies; Antibodies, Monoclonal; Chromatography; Chromatography, Affinity; Chromatography, Ion Exchange; Hydrophobic and Hydrophilic Interactions
PubMed: 38845514
DOI: 10.3724/SP.J.1123.2023.12010 -
PloS One 2024Viral Load (VL) monitoring is a crucial component of patient care during antiretroviral therapy (ART) but is not routinely available in many resource-constrained...
INTRODUCTION
Viral Load (VL) monitoring is a crucial component of patient care during antiretroviral therapy (ART) but is not routinely available in many resource-constrained settings, where millions of patients will require care for decades to come. We hypothesise a serologic 'recent infection' test (Sedia LAg assay) which has a high dynamic range for detecting antigen-driven antibody response can provide informative proxies for VL trajectories.
METHODS
A retrospective study where we analysed data linked via specimens in a well-described repository for recent infection test benchmarking (CEPHIA collaboration). Patient panels were comprised of 1) observations straddling ART start; 2) observations from a period of stable viral suppression; 3) observations straddling rebound after a period of viral suppression. We analysed an individual's Sedia LAg ELISA normalised optical density (ODn) trends within these categories. Using groups 2) and 3) we evaluated the specificity and sensitivity of a proposed proxy for "the latest observation is at a time of VL rebound"; proxy was defined as follows: we estimated patient-specific mean-previous-ODn for all observations with at least two preceding virally suppressed observations. We considered various thresholds to define both "VL suppression" and "ODn uptick".
RESULTS
In regression analysis by category: 1) ODn gradients are statistically significantly negative just after ART-start (p = 0.010); 2) During periods of stable viral suppression, ODn tended to decline, but not statistically significantly, for a range of clinically meaningful "VL suppression" thresholds; 3) comparing ODn values just before, versus at, "VL rebound", ODn changes were statistically significantly increasing at rebound (p = 0.001). In the analysis comparing groups 2) and 3), at a Z score threshold of 0.8, the proposed proxy for a first viral rebound had an observed specificity and sensitivity both close to 90%.
CONCLUSION
The high dynamic range of serological tests previously investigated for defining 'recent infection' has potential, as demonstrated using the Sedia LAg ELISA, to provide meaningful information about the success of ART, during treatment initiation, at times of stable suppression, and to flag possible viral rebound. It should be investigated how this can be combined with patient management workflows and (clinical and) other data, to provide efficiencies in long-term monitoring viral control in resource-limited settings.
Topics: Humans; Viral Load; HIV Infections; Retrospective Studies; Male; Female; Adult; Enzyme-Linked Immunosorbent Assay; Sensitivity and Specificity; Middle Aged; Anti-Retroviral Agents; HIV-1
PubMed: 38843247
DOI: 10.1371/journal.pone.0303393 -
Scientific Reports Jun 2024Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on...
Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.
Topics: Animals; Dogs; Dirofilariasis; Dirofilaria repens; Dog Diseases; Antibodies, Helminth; Helminth Proteins; Antigens, Helminth; Larva; Antibody Formation
PubMed: 38839868
DOI: 10.1038/s41598-024-63523-9 -
The Journal of Veterinary Medical... Jun 2024Immunoglobulin A (IgA) is notable for its broad specificity toward multiple bacteria. Phosphorylcholine (PC) plays a role in the infection of pathogenic bacteria...
Immunoglobulin A (IgA) is notable for its broad specificity toward multiple bacteria. Phosphorylcholine (PC) plays a role in the infection of pathogenic bacteria carrying PC and in the induction of IgA responses in the host immune system. The commercially available mouse monoclonal IgA, TEPC15-IgA, is a distinctive antibody with specificity for PC, warranting further exploration of its response to PC-bearing enteric bacteria. In this study, using 17 different enteric bacteria, including 3 aerobic and 14 anerobic bacteria that could be cultured in vitro, we confirmed that TEPC15-IgA recognizes 4 bacterial species: Lactobacillus taiwanensis, Limosilactobacillus frumenti, Streptococcus infantis, and Escherichia coli, although reactivity varied. Interestingly, TEPC15-IgA did not react with four of six Lactobacillus species used. Moreover, distinct target molecules associated with PC in L. taiwanensis and L. frumenti were evident, differing in molecular weight. These findings suggest that the natural generation of PC-specific IgA could prevent PC-mediated infections and potentially facilitate the formation of a microflora rich in indigenous bacteria with PC, particularly in the gastrointestinal tract.
PubMed: 38839348
DOI: 10.1292/jvms.23-0441