Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization.

Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

Safety and Efficacy of SAB-185 for Non-hospitalized Adults with COVID-19: A Randomized Clinical Trial.

BACKGROUND: To address the need for novel COVID-19 therapies, we evaluated the fully-human polyclonal antibody product SAB-185 in a phase 3 clinical trial. METHODS: Non-hospitalized high-risk adults within 7 days of COVID-19 symptom onset were randomized 1:1 to open-label SAB-185 3,840 units/kg or casirivimab/imdevimab 1200 mg. Non-inferiority comparison was undertaken for the pre-Omicron population (casirivimab/imdevimab expected to be fully active) and superiority comparison for the Omicron population (casirivimab/imdevimab not expected to be active). Primary outcomes were the composite of all-cause hospitalizations/deaths and grade ≥3 treatment-emergent adverse events (TEAEs) through day 28. Secondary outcomes included time to sustained symptom improvement and resolution. RESULTS: Enrollment was terminated early due to low hospitalization/death rates upon Omicron emergence. 733 adults were randomized, 255 included in pre-Omicron and 392 in Omicron analysis populations. Hospitalizations/deaths occurred in 6 (5.0%) and 3 (2.2%) of pre-Omicron SAB-185 and casirivimab/imdevimab arms, respectively (absolute difference [95% CI] 2.7% [-2.3%, 8.6%]), inconclusive for non-inferiority; and 5 (2.5%) versus 3 (1.5%) (absolute difference 1.0% [-2.3%, 4.5%]) for Omicron. Risk ratios for grade ≥3 TEAEs were 0.94 [0.52, 1.71] (pre-Omicron) and 1.71 [0.96, 3.07] (Omicron). Time to symptom improvement and resolution were shorter for SAB-185, median 11 vs 14 (pre-Omicron) and 11 vs 13 days (Omicron) (symptom improvement), and 16 vs 24 days and 18 vs >25 days (symptom resolution), p<0.05 for symptom resolution for Omicron only. CONCLUSIONS: SAB-185 had an acceptable safety profile with faster symptom resolution in the Omicron population. Additional studies are needed to characterize its efficacy for COVID-19.

A countrywide survey of hrp2/3 deletions and kelch13 mutation co-occurrence in Ethiopia.

Malaria elimination relies on detection of Plasmodium falciparum Histidine-Rich Proteins 2/3 (HRP2/3) through rapid diagnostic tests (RDTs) and treatment with artemisinin-combination therapies (ACTs). Data from the Horn of Africa suggest increasing hrp2/3 gene deletions and ACT partial resistance kelch13 (k13) mutations. To assess this, 233 samples collected during a national survey from 7 regions of Ethiopia were studied for hrp2/3 deletions by droplet digital dPCR and k13 mutations by DNA sequencing. Approximately 22% of the study population harbored complete hrp2/3 deletions by ddPCR. Thirty-two of 42 of k13 SNPs identified were R622I associated with ACT partial resistance. Both hrp2/3 deletions and k13 mutations associated with ACT partial resistance appear to be co-occurring especially in Northwest Ethiopia. Ongoing national surveillance relying on accurate laboratory methods are required to fully elaborate the genetic diversity of P. falciparum to inform public health policy makers.

Idiopathic multicentric Castleman disease (iMCD)-idiopathic plasmacytic lymphadenopathy: A distinct subtype of iMCD-not otherwise specified with different clinical features and better survival.

Idiopathic multicentric Castleman disease (iMCD) is subclassified into iMCD-thrombocytopenia, anasarca, reticulin fibrosis, renal dysfunction, organomegaly (TAFRO) and iMCD-not otherwise specified (NOS) according to the Castleman Disease Collaborative Network (CDCN) consensus criteria. With a deeper understanding of iMCD, a group of patients with iMCD-NOS characterised by polyclonal hypergammaglobulinaemia, plasmacytic/mixed-type lymph node histopathology and thrombocytosis has attracted attention. This group of patients has been previously described as having idiopathic plasmacytic lymphadenopathy (IPL). Whether these patients should be excluded from the current classification system lacks sufficient evidence. This retrospective analysis of 228 patients with iMCD-NOS identified 103 (45.2%) patients with iMCD-IPL. The clinical features and outcomes of patients with iMCD-IPL and iMCD-NOS without IPL were compared. Patients with iMCD-IPL showed a significantly higher inflammatory state but longer overall survival. No significant difference in overall survival was observed between severe and non-severe patients in the iMCD-IPL group according to the CDCN severity classification. Compared with lymphoma-like treatments, multiple myeloma-like and IL-6-blocking treatment approaches in the iMCD-IPL group resulted in significantly higher response rates and longer time to the next treatment. These findings highlight the particularities of iMCD-IPL and suggest that it should be considered a new subtype of iMCD-NOS.

Dominant epitopes of cross-reactive anti-domain III human antibody response change from early to late convalescence of infection with dengue virus.

Cross-neutralizing activity of human antibody response against Dengue virus complex (DENV) changes importantly over time. Domain III (DIII) of the envelope protein of DENV elicits a potently neutralizing and mostly type-specific IgG response. We used sera from 24 individuals from early- or late convalescence of DENV1 infection to investigate the evolution of anti-DIII human IgG with the time lapse since the infection. We evaluated the correlation between the serotype-specific reactivity against recombinant DIII proteins and the neutralization capacity against the four serotypes, and examined its behavior with the time of convalescence. Also, we use a library of 71 alanine mutants of surface-exposed amino acid residues to investigate the dominant epitopes. In early convalescence anti-DIII titers and potency of virus neutralization were positively associated with correlation coefficients from 0.82 to 1.0 for the four serotypes. For late convalescence, a positive correlation (r = 0.69) was found only for DENV1. The dominant epitope of the type-specific response is centered in the FG-loop (G383, E384, and K385) and includes most of the lateral ridge. The dominant epitope of the anti-DIII cross-reactive IgG in secondary infections shifts from the A-strand during early convalescence to a site centered in residues E314-H317 of the AB-loop and I352-E368 of the DI/DIII interface, in late convalescence. An immunoassay based on the detection of IgG anti-DIII response can be implemented for detection of infecting serotype in diagnosis of DENV infection, either primary or secondary. Human dominant epitopes of the cross-reactive circulating antibodies change with time of convalescence.

Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination.

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Development of a competitive ELISA based on the LSDV A33 antigen.

Goatpoxvirus (GTPV), sheeppoxvius (SPPV), and the Lumpy skin disease virus (LSDV) is a Capripoxvirus belonging to the family poxviridae. They can cause significant economic losses in countries where this disease are endemic. However, effective and convenient diagnostic tools against sera antibody are not readily available until now. Toward this goal, a polyclonal antibody competitive enzyme-linked immunosorbent assay (c-ELISA) of detecting serogroup-specific antibody is established based on major LSDV antigen A33. Serum samples (n = 605) were collected to optimize the c-ELISA from different areas. The cut-off value for the c-ELISA was estimate using percent inhibition (PI) values. The diagnostic performance of test including sensitivity (sn) and specificity (sp) were obtained by receiver operator characteristic (ROC) analysis. Among these analysis, > 57.61% PI value was accepted as cut-off of the c-ELISA, the diagnostic sn an diagnostic sp were reached to 96.4% and 98.5%, at > 95% confidence interval. These results show that the developed competitive ELISA is sensitive, specific, and reliable, which make it appropriate for serological investigation.

Induction therapy in heart transplantation: A systematic review and network meta-analysis for developing evidence-based recommendations.

INTRODUCTION: Induction therapy (IT) utility in heart transplantation (HT) remains contested. Commissioned by a clinical-practice guidelines panel to evaluate the effectiveness and safety of IT in adult HT patients, we conducted this systematic review and network meta-analysis (NMA). METHODS: We searched for studies from January 2000 to October 2022, reporting on the use of any IT agent in adult HT patients. Based on patient-important outcomes, we performed frequentist NMAs separately for RCTs and observational studies with adjusted analyses, and assessed the certainty of evidence using the GRADE framework. RESULTS: From 5156 publications identified, we included 7 RCTs and 12 observational studies, and report on two contemporarily-used IT agents-basiliximab and rATG. The RCTs provide only very low certainty evidence and was uninformative of the effect of the two agents versus no IT or one another. With low certainty in the evidence from observational studies, basiliximab may increase 30-day (OR 1.13; 95% CI 1.06-1.20) and 1-year (OR 1.11; 95% CI 1.02-1.22) mortality compared to no IT. With low certainty from observational studies, rATG may decrease 5-year cardiac allograft vasculopathy (OR .82; 95% CI .74-.90) compared to no IT, as well as 30-day (OR .85; 95% CI .80-.92), 1-year (OR .87; 95% CI .79-.96), and overall (HR .84; 95% CI .76-.93) mortality compared to basiliximab. CONCLUSION: With low and very low certainty in the synthetized evidence, these NMAs suggest possible superiority of rATG compared to basiliximab, but do not provide compelling evidence for the routine use of these agents in HT recipients.

Identification of cocoonase and cocoonase like protein using polyclonal antibody of Antheraea mylitta cocoonase.

BACKGROUND: Cocoonase is a proteolytic enzyme released by silk moths during pupal adult emergence. Without damaging the silk fibroin, this enzyme dissolves the shell of the tasar cocoon by exclusively targeting the protein sericin. Prior to this study, there was no available antibody against Antheraea mylitta cocoonase to identify or screen out similar variants or cocoonase like protein. RESULTS: In the present study, naturally secreted A. mylitta cocoonase was purified and used to immunize New Zealand white rabbits. The developed polyclonal antibody of cocoonase was purified and its specific interaction with cocoonase was determined using Indirect ELISA. The confirmation of its specificity and immuno-reactivity was evaluated by western blot using native cocoonase of tasar silkworm A. mylitta. The efficacy and specificity of the polyclonal antibody were further verified and confirmed by western blot which was performed to detect ten different ecotypes of A. mylitta cocoonase. CONCLUSION: The developed antibody successfully detected the cocoonase of different ecotypes. Thus, in future this antibody can serve as one of the molecular detection method for cocoonase and cocoonase-like proteins.

Development of a label-free photoelectrochemical immunosensor for novel astrovirus detection.

Since 2017, an infectious goose gout disease characterized by urate precipitation in viscera, mainly caused by novel goose astrovirus (GoAstV) infection, has emerged in the main goose-producing region of China. The current challenge in managing goose gout disease is largely due to the absence of a rapid and efficient detection method for the GoAstV pathogen. Notably, the potential application of immunosensors in detecting GoAstV has not yet been explored. Herein, a label-free PEC immunosensor was fabricated by using purchased TiO2 as the photoactive material and antibody against GoAstV P2 proteins as the specific recognition element. First, we successfully expressed the capsid spike domain P2 protein of ORF2 from GoAstV CHSH01 by using the pET prokaryotic expression system. Meanwhile, the polyclonal antibody against GoAstV capsid P2 protein was produced by purified protein. To our knowledge, this is the first establishment and preliminary application of the label-free photoelectrochemical immunosensor method in the detection of AstV. The PEC immunosensor had a linear range of 1.83 fg mL-1 to 3.02 ng mL-1, and the limit of detection (LOD) was as low as 0.61 fg mL-1. This immunosensor exhibited high sensitivity, great specificity, and good stability in detecting GoAstV P2 proteins. To evaluate the practical application of the immunosensor in real-world sample detection, allantoic fluid from goose embryos was collected as test samples. The results indicated that of the eight positive samples, one false negative result was detected, while both negative samples were accurately detected, suggesting that the constructed PEC immunosensor had good applicability and practical application value, providing a platform for the qualitative detection of GoAstV.

The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research.

Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7ß1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.

Searching for EGF Fragments Recreating the Outer Sphere of the Growth Factor Involved in Receptor Interactions.

The aims of this study were to determine whether it is possible to use peptide microarrays obtained using the SPOT technique (immobilized on cellulose) and specific polyclonal antibodies to select fragments that reconstruct the outer sphere of proteins and to ascertain whether the selected peptide fragments can be useful in the study of their protein-protein and/or peptide-protein interactions. Using this approach, epidermal growth factor (EGF) fragments responsible for the interaction with the EGF receptor were searched. A library of EGF fragments immobilized on cellulose was obtained using triazine condensing reagents. Experiments on the interactions with EGFR confirmed the high affinity of the selected peptide fragments. Biological tests on cells showed the lack of cytotoxicity of the EGF fragments. Selected EGF fragments can be used in various areas of medicine.

[Plasmablastic lymphoma presenting with plasmacytosis and polyclonal hypergammopathy].

A 72-year-old woman presented with generalized lymphadenopathies and plasmacytosis accompanied by polyclonal hypergammopathy. 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) showed FDG accumulation in the systemic lymph nodes, spleen, and multiple bones. Human immunodeficiency virus antibody was negative. Lymph node histologic findings showed a monotonous population of plasma cells with a starry-sky appearance. The cells were positive for CD19, λ, and Epstein-Barr virus-encoded RNA, and negative for CD20 and CD56. The MIB-1 index was 80%. A diagnosis of plasmablastic lymphoma with plasmacytosis and polyclonal hypergammopathy was made, and complete metabolic response was achieved after six cycles of dose-adjusted-EPOCH therapy (etoposide, prednisolone, vincristine, cyclophosphamide, and doxorubicin).

Phase 2 Safety and Antiviral Activity of SAB-185, a Novel Polyclonal Antibody Therapy for Nonhospitalized Adults With COVID-19.

BACKGROUND: SAB-185, a novel fully human IgG polyclonal immunoglobulin product, underwent phase 2 evaluation for nonhospitalized adults with mild-moderate coronavirus disease 2019 (COVID-19). METHODS: Participants received intravenous SAB-185 3840 units/kg (low-dose) or placebo, or 10 240 units/kg (high-dose) or placebo. Primary outcome measures were nasopharyngeal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA < lower limit of quantification (LLOQ) at study days 3, 7, and 14, time to symptomatic improvement, and safety through day 28. RESULTS: Two-hundred thirteen participants received low-dose SAB-185/placebo (n = 107/106) and 215 high-dose SAB-185/placebo (n = 110/105). The proportions with SARS-CoV-2 RNA < LLOQ were higher for SAB-185 versus placebo at days 3 and 7 and similar at day 14, and significantly higher at day 7 for high-dose SAB-185 versus placebo only, relative risk 1.23 (95% confidence interval, 1.01-1.49). At day 3, SARS-CoV-2 RNA levels were lower with low-dose and high-dose SAB-185 versus placebo: differences in medians of -0.78 log10 copies/mL (P = .08) and -0.71 log10 copies/mL (P = .10), respectively. No difference was observed in time to symptom improvement: median 11/10 days (P = .24) for low-dose SAB-185/placebo and 8/10 days (P = .50) for high-dose SAB-185/placebo. Grade ≥3 adverse events occurred in 5%/13% of low-dose SAB-185/placebo and 9%/12% of high-dose SAB-185/placebo. CONCLUSIONS: SAB-185 was safe and generally well tolerated and demonstrated modest antiviral activity in predominantly low-risk nonhospitalized adults with COVID-19. Clinical Trials Registration. NCT04518410.

Genetic and nongenetic mechanisms for colorectal cancer evolution.

The stepwise accumulation of key driver mutations is responsible for the development and malignant progression of colorectal cancer in primary sites. Genetic mouse model studies have revealed combinations of driver gene mutations that induce phenotypic changes in tumors toward malignancy. However, cancer evolution is regulated by not only genetic alterations but also nongenetic mechanisms. For example, certain populations of metastatic cancer cells show a loss of malignant characteristics even after the accumulation of driver mutations, and such cells are eliminated in a negative selection manner. Furthermore, a polyclonal metastasis model has recently been proposed, in which cell clusters consisting of genetically heterogeneous cells break off from the primary site, disseminate to distant organs, and develop into heterogenous metastatic tumors. Such nongenetic mechanisms for malignant progression have been elucidated using genetically engineered mouse models as well as organoid transplantation experiments. In this review article, we discuss the role of genetic alterations in the malignant progression of primary intestinal tumors and nongenetic mechanisms for negative selection and polyclonal metastasis, which we learned from model studies.

Optimum timing of antithymocyte globulin in relation to adoptive regulatory T cell therapy.

Reducing the recipient's T cell repertoire is considered to increase the efficacy of regulatory T cell (Treg) therapy. This necessitates timing the administration of antithymocyte globulin (ATG) early enough before adoptive cell therapy (ACT) so that residual serum ATG does not deplete the transferred Tregs. The optimum time point in this regard has not been defined. Herein, we report the effects of residual serum ATG on the viability of an in vitro expanded Treg cell product used in a clinical trial of ACT in kidney transplant recipients (NCT03867617). Patients received ATG monotherapy (either 6 or 3 mg/kg body weight) without concomitant immunosuppression 2 to 3 weeks before transplantation and Treg transfer. An anti-ATG immunoglobulin G (IgG) immune response was elicited in all patients within 14 days. In turn, the elimination of total and Treg-specific ATG was accelerated substantially over control patients receiving the same dose of ATG with concomitant immunosuppression. However, ATG serum concentrations of <1 µg/mL, which had previously been reported as subtherapeutic threshold, triggered apoptosis of Tregs in vitro. Therefore, ATG levels need to decline to lower levels than those previously thought for efficacious Treg transfer. In 5 of 6 patients, such low levels of serum ATG considered safe for Treg transfer were reached within 2 weeks after ATG administration.

Cloning, expression of porcine GSDME and identification of its site cleaved by caspase-3.

As a member of the gasdermin family, gasdermin E (GSDME) is specifically cleaved by caspase-3, resulting in pyroptosis. To date, the biological characteristics and functions of human and mouse GSDME have been extensively studied; however, little is known of porcine GSDME (pGSDME). In this study, the full-length pGSDME-FL was cloned, which encodes 495 amino acids (aa) that have closely evolutionary relationships to the homolog of camelus, aquatic mammals, cattle and goat. Moreover, pGSDME was detected at different levels of expression in 21 tissues and 5 pig-derived cell lines tested by qRT-PCR, with the highest expression levels in mesenteric lymph nodes and PK-15 cell lines. Anti-pGSDME polyclonal antibody (pAb) with good specificity was generated by expressing the truncated recombinant protein pGSDME-1-208 and immunizing the rabbits. By western blot analysis using highly specific anti-pGSDME polyclonal antibody (pAb) prepared as primary antibody, it was not only confirmed that paclitaxel and cisplatin were positive stimuli to pGSDME cleavage and caspase-3 activation, but also identified the aspartate (D268) at position 268th of pGSDME as a cleavage site of caspase-3, and the overexpressed pGSDME-1-268 possesses cytotoxicity to HEK-293T cells, indicating that pGSDME-1-268 may contain active domains and involve pGSDME-mediated pyroptosis. These results lay a foundation for further investigating the function of pGSDME, especially its role in pyroptosis and its interaction with pathogens.

Effect of duck interferon-α and an anti-cap protein polyclonal antibody against duck circovirus.

Duck circovirus (DuCV) is one of the most prevalent viruses in the duck breeding industry, and causes persistent infection and severe immunosuppression. Currently, there is a serious lack of prevention and control measures and no commercial vaccine against DuCV. Therefore, effective antiviral drugs are important for treating DuCV infection. Interferon (IFN) is an important component of antiviral innate immunity, but it remains unclear whether duck IFN-α has a clinical effect against DuCV. Antibody therapy is an important way to treat viral infections. The DuCV structural protein (cap) is immunogenic, and it remains to be determined whether an anti-cap protein antibody can effectively block DuCV infection. In this study, the duck IFN-α gene and the DuCV structural protein cap gene were cloned, expressed and purified in Escherichia coli to prepare duck recombinant IFN-α and the cap protein. Then, rabbits were immunized with the recombinant cap protein to prepare a rabbit polyclonal antibody. This study investigated the antiviral effect of duck recombinant IFN-α and the anti-cap protein antibody and their combined effect on Cherry Valley ducks infected with DuCV. The results showed that the treatment significantly alleviated the clinical symptoms of immune organ atrophy and immunosuppression compared with the control. The histopathological damage of the target organs was alleviated, and replication of DuCV in the immune organs was significantly inhibited. The treatment also reduced the damage caused by DuCV to the liver and immune function, and increased the level of the DuCV antibody in the blood, thereby improving antiviral activity. Notably, the combination of duck IFN-α and the polyclonal antibody completely blocked DuCV infection after 13 days under the experimental conditions, showing a better inhibitory effect on DuCV infection than single treatments. These results showed that duck recombinant IFN-α and the anti-cap protein antibody can be used as antiviral drugs to clinically treat and control DuCV infection, particularly the vertical transmission of the virus in breeding ducks.