Distribution Of Antigens Research Articles

A Highly Sensitive Immunoassay to Enable Quantification of a Nanobody-Based Imaging Agent in Human Serum.

Immuno-positron emission tomography (i-PET) is a non-invasive imaging technique that combines the specificity of monoclonal antibodies with the sensitivity of positron emission tomography to visualize and quantify the distribution of target antigens in vivo, providing detailed spatial information about the presence and localization of specific biomarkers. Due to the crucial role that cytotoxic T cells play in antitumor responses, a new tracer consisting of a humanized anti-CD8 nanobody labeled with fluorine-18 was developed to inform immuno-oncology treatments and support medical decision-making. Nanobodies are single-domain antibodies with low molecular weight and fast peripheral blood clearance, both of which are advantageous properties for same-day imaging. However, these unique characteristics pose bioanalytical challenges when developing clinical pharmacokinetic (PK) assays, including the need for high assay sensitivity. This manuscript focuses on overcoming bioanalytical challenges related to sensitivity and matrix interference during the development of a method to quantify this novel anti-CD8 nanobody tracer in human serum. Out of the three immunoassay platforms evaluated (ELISA, SMCxPRO™ and Gyrolab®), a Gyrolab method was ultimately selected due to its superior sensitivity, equal detectability of both conjugated and unconjugated forms of the nanobody and its ability to minimize matrix interference. By selecting the right assay format, along with the appropriate critical reagents for capture and detection, matrix interference was diminished. This novel PK method was successfully qualified demonstrating acceptable performance across all parameters. The acquired bioanalytical insights gained could be applied to nanobody-derived conjugates or other modalities that require high sensitivity in the clinical settings.

Real-world clinical usage and efficacy of apalutamide in men with nonmetastatic castration-resistant prostate cancer: a multi-institutional study in the CsJUC.

To evaluate the real-world clinical usage and effectiveness of apalutamide in men with nonmetastatic castration-resistant prostate cancer (nmCRPC). We retrospectively reviewed the data of 186 men who received apalutamide across 17 institutions. The primary outcomes were the clinical usage of apalutamide for nmCRPC: prior usage of other androgen receptor signaling inhibitors (ARSIs), prior radical treatment, and the distribution of the prostate-specific antigen (PSA) doubling time (PSA-DT) at the initial administration of apalutamide. The secondary outcomes were the efficacy of apalutamide: PSA response (50% or 90% decline), progression-free survival, and skin-adverse events (AEs). We identified 75 patients with nmCRPC. A total of 31 (41.3%) patients received prior treatment with other ARSIs. A total of 42 men (56%) did not receive any prior radical treatment. The PSA-DT was <3.0, 3.0-5.9, 6.0-10, and>10months in 34.7%, 40%, 14.7%, and 10.6% of the patients, respectively. Patients receiving prior treatment with other ARSIs showed a significantly lower PSA response (PSA 50% decline, 88.4% vs. 18.8%; PSA 90% decline, 60.5% vs. 6.2%, P<.001, respectively) and significantly shorter progression-free survival (median: 37months vs. 4months; log-rank P<.001) than those without prior ARSI treatment, although cancer status did not differ between the groups. Skin-AEs were observed in 42.7%. This real-world study revealed that apalutamide was used for the treatment after other ARSIs in >40% of patients with nmCRPC and showed limited efficacy in this context, although the effectiveness of apalutamide without prior other ARSI treatment was comparable with that reported in clinical trial results.

Biodistribution and mechanisms of action of MF59 and MF59-like adjuvants.

Recently, adjuvants have received increasing attention as essential vaccine components. Nearly 100years have passed since Gaston Roman introduced the concept of adjuvants in 1925, during which numerous preclinical and clinical studies related to vaccine adjuvants have been conducted. However, to date, only a few adjuvants have been successfully used in marketed vaccines. This low clinical translational efficiency is mainly owing to the lack of comprehensive and in-depth understanding of the mechanisms of action of adjuvants in complex biological systems. In fact, MF59 is the first non‑aluminum adjuvant applied in approved human vaccines and is still used today; however, many unknowns regarding its mechanism of action remain. Therefore, in this review, the current status of the mechanism of action of MF59 and MF59-like adjuvants is discussed based on evidence-based temporal and spatial processes of immune-regulatory events. Moreover, the key factors of MF59 and MF59-like adjuvants that regulate humoral and cellular immune responses have been summarized. In addition, studies on the distribution and elimination of both antigens and adjuvants were analyzed based on published studies to gain mechanistic insights into the safety of MF59-adjuvanted vaccines. This review will not only benefit future prospects for the development of novel squalene-based adjuvants and their use in clinical applications but will also be valuable for future mechanistic investigations on other vaccine adjuvants.

Localization of Lesions in Autoimmune Blistering Diseases Is Independent of Site-Specific Target Antigen Expression

Autoimmune blistering diseases (AIBDs) involve autoantibodies targeting proteins in the epidermal/epithelial desmosome (pemphigus) or basement membrane zone (pemphigoid). Despite widespread antigen distribution, lesions exhibit a scattered involvement pattern. This study maps the frequency/severity of AIBD lesions on various body parts and investigates whether differential antigen expression contributes to specific predilection sites. We analyzed affected sites presenting blisters/erosions, erythematous/urticarial lesions, and mucosal lesions in bullous pemphigoid (BP-cohort 1, n = 65; BP-cohort 2, n = 119), pemphigus vulgaris (PV, n = 67), and pemphigus foliaceus (PF, n = 20) patients. To assess antigen expression, we conducted indirect immunofluorescence (IF) staining of 11 AIBD antigens from 13 anatomical sites of 10 body donors without AIBD. In BP, blisters/erosions and erythematous/urticarial lesions predominantly affected arms and legs, while PV/PF patients exhibited frequent involvement of buccal mucosa and back, respectively. IF staining identified significant regional differences in BP180, BP230, and integrin β4 expression, although these variations did not correlate with a higher lesion frequency/severity. Other antigens showed consistent expression across all regions. Our findings suggest that predilection sites for BP and PV/PF are largely unaffected by regional variations in antigen expression but may be influenced by factors like microbiota, mechanical stress, sunlight exposure, local immunity, or genetics.

Vaccine Specifically for Immunocompromised Individuals against Superbugs.

Immunocompromised populations, including cancer patients, elderly individuals, and those with chronic diseases, are the primary targets of superbugs. Traditional vaccines are less effective due to insufficient or impaired immune cells. Inspired by the "vanguard" effect of neutrophils (NE) during natural infection, this project leverages the ability of NE to initiate the NETosis program to recruit monocytes and DC cells, designing vaccines that can rapidly recruit immune cells and enhance the immune response. The PLGA microsphere vaccine platform (MSV) with a high level of safety contains whole-bacterial antigens both internally and externally, providing initial and booster effects through programmed distribution and release of antigens after a single injection. Experimental data indicate that immunizing mice with a mixture of MSV and NE induces the formation of spontaneous gel-like neutrophil extracellular traps (NETs) at the inoculation site. These NETs recruit immune cells and prevent the diffusion of vaccine components, thereby reducing damage from bacterial toxins and enhancing vaccine biosafety. This strategy shows excellent efficacy against MRSA-induced infections in not only healthy but also immunocompromised mice.

Renshen (Radix Ginseng) polysaccharide promotes repair of the mice intestinal mucosa through regulatory mechanisms based on polyamine and human antigen R.

To investigate the mechanism and effect of Renshen (Radix Ginseng) polysaccharide on the migration of intestinal epithelial cell line 6 (IEC-6), as well as the repair mechanism of Renshen (Radix Ginseng) polysaccharide on colonic injury induced by dextran sulfate sodium (DSS) in mice. Mice were fed 3% (w/v) DSS for 6 d to create colonic lesions. A cell-migration model was created using cell scratching. mRNA expression, protein expression, translation efficiency of mRNA, and nucleoplasmic distribution of human antigen R (HuR) were determined by real-time reverse transcription-quantitative polymerase chain reaction, western blotting, a dual luciferase reporter system, and immunofluorescence staining, respectively. Renshen (Radix Ginseng) polysaccharide promoted the migration of IEC-6 cells and affected expression of stromal interaction molecule 1 (STIM1) and cell division cycle 42 (Cdc42) at transcriptional and post-transcriptional levels. Renshen (Radix Ginseng) polysaccharide-induced repair of intestinal mucosal injury may be mediated by increased cell migration via polyamine-based regulatory mechanisms. In vitro and in vivo experiments suggest that Renshen (Radix Ginseng) polysaccharide-induced post-transcriptional regulation of STIM1 and Cdc42 may be related to differences in the regulation of different target genes by HuR. Taken together, these data provide a reference for further exploration of the protective effect of Renshen (Radix Ginseng) on the intestinal mucosa.

Histopathological and Immunohistochemical Evaluation of African Swine Fever Pigs in Bali Province

African Swine Fever (ASF) is a disease affecting pigs, caused by a double-stranded DNA virus that is not transmissible to humans or other animals. It leads to significant economic losses due to high morbidity and mortality rates, particularly in densely populated pig regions like Bali Province. During the ASF outbreak from June to December 2023, this study conducted histopathological research on clinical samples. Biological materials from twelve pigs confirmed positive for ASF via qPCR examination were histopathologically analyzed. Tissue samples from various organs underwent processing and examination using hematoxylin and eosin staining. Immunohistochemistry (IHC) was employed to detect the ASF virus’s p54 protein. Clinical symptoms, anatomical pathology, and histological examination revealed characteristic acute ASF lesions. Immunohistochemistry consistently showed p54 viral antigen distribution in mononuclear cells/macrophages across various organs, with the spleen and lymph nodes being dominant sites in 12 pigs (100%). This comprehensive study demonstrates the effectiveness of IHC in detecting the ASF virus and characterizing its histopathology.Keywords: ASF; histopathology; immunohistochemistry

Analytical Sensitivity Analysis and Clinical Impact Modeling of Rapigen Rapid Diagnostic Tests for Malaria.

Laboratory benchmarking allows objective analysis of the analytical performance of malaria rapid diagnostic tests (RDTs). We present the analytical detection limits of the Rapigen BIOCREDIT Malaria Ag Pf/Pv (pLDH/pLDH), the Rapigen BIOCREDIT Malaria Ag Pf (pLDH/HRPII), and two best-in-class WHO-prequalified comparator RDTs, generated using standardized panels containing recombinant antigen, invitro cultured parasites, international standards, and clinical samples. Detection limit antigen concentrations of HRP2, PfLDH, and PvLDH were determined for the Rapigen and comparator RDTs. Detection of antigens in international units (IU)/mL was also evaluated. The Rapigen Ag Pf (pLDH/HRPII) detected 3.9 and 3.9 IU/mL for PfLDH and HRP2, respectively, and the Ag Pf/Pv (pLDH/pLDH) detected 3.9 and 5.0 IU/mL for PfLDH and PvLDH, respectively. The comparator HRP2/PfLDH and HRP2/PvLDH detected 15.6 and 31.3 IU/mL for HRP2 and PfLDH and 15.6 and 50.0 IU/mL for HRP2 and PvLDH, respectively. The RDT clinical sensitivity was predicted through application of analytical detection limits to antigen concentration distributions from clinical symptomatic and asymptomatic cases. Febrile cases would be detected in a majority by both standard and Rapigen RDTs, but incremental increases in sensitivity in the Rapigen RDTs may be important for clinical cases currently missed by microscopy. Rapigen RDTs were predicted to have improved detection of asymptomatic cases and infections with parasites carrying hrp2 deletions through more sensitive PfLDH detection. Through the benchmarking and simulation of clinical sensitivity, a method for rapidly assessing the ability of new RDTs to meet clinical needs using high-sensitivity antigen distribution data is presented.

Spatial Engineering of Heterotypic Antigens on a DNA Framework for the Preparation of Mosaic Nanoparticle Vaccines with Enhanced Immune Activation against SARS‐CoV‐2 Variants

AbstractMosaic nanoparticle vaccines with heterotypic antigens exhibit broad‐spectrum antiviral capabilities, but the impact of antigen proportions and distribution patterns on vaccine‐induced immunity remains largely unexplored. Here, we present a DNA nanotechnology‐based strategy for spatially assembling heterotypic antigens to guide the rational design of mosaic nanoparticle vaccines. By utilizing two aptamers with orthogonal selectivity for the original SARS‐CoV‐2 spike trimer and Omicron receptor‐binding domain (RBD), along with a DNA soccer‐ball framework, we precisely manipulate the spacing, stoichiometry, and overall distribution of heterotypic antigens to create mosaic nanoparticles with average, bipolar, and unipolar antigen distributions. Systematic in vitro and in vivo immunological investigations demonstrate that 30 heterotypic antigens in equivalent proportions, with an average distribution, lead to higher production of broad‐spectrum neutralizing antibodies compared to the bipolar and unipolar distributions. Furthermore, the precise assembly utilizing our developed methodology reveals that a mere increment of five Omicron RBD antigens on a nanoparticle (from 15 to 20) not only diminishes neutralization against the Omicron variant but also triggers excessive inflammation. This work provides a unique perspective on the rational design of mosaic vaccines by highlighting the significance of the spatial placement and proportion of heterotypic antigens in their structure–activity mechanisms.

Distribution of ABO and D antigen expression in Yogyakarta, Java Island: a pioneer large-scale study in Indonesia

ObjectiveHere, we sought to report ABO and D antigen distribution in blood donors from Yogyakarta, Java Island, Indonesia. Phenotype data (ABO/D) from donors who donated blood between January 1, 2018, and December 31, 2023, at the Yogyakarta Blood Donor Unit were extracted from the blood donor registry, and phenotype frequency was calculated subsequently.ResultsIn the 245,307 blood donors collected over six years, ABO phenotype frequency: O (frequency: 38.5%) > B (29.4%) > A (24.1%) > AB (8.0%). The D-positive phenotype was far more common (99.5%) than the D-negative phenotype (0.5%). The phenotypic pattern globally is similar to previous reports in Southeast Asia. The D antigen distribution is similar to world distribution as the most common blood group. For the first time in Indonesia, this distribution of ABO and D phenotype is reported in a large-scale study. This work is a pioneer in the coordinated optimization of transfusion guidelines at the national level.

P.115: Human leukocyte antigen distribution among kidney transplant donors and recipients: Data from central Indian region.

P.115: Human leukocyte antigen distribution among kidney transplant donors and recipients: Data from central Indian region.

Analysis of The Results of The Rhesus System Blood Group Phenotyping Examination in Routine Type O Blood Donors

The Rhesus (Rh) system is the second most important blood group system after ABO, has the highest immunogenicity and tends to produce alloantibodies that cause most transfusion reactions. In the Rh blood group system, there are more than 55 antigens, but the main ones are D, C, E, c, and e. Currently, only the D antigen is carried out without examining four other important Rh antigens. Still scarce, studies on the proportion of Rh antigens, except D, in Indonesia; in this study we have determined Rh antigens and phenotypes among a population of routine blood donors. The purpose of this study was to determine the phenotypic variation and frequency distribution of the main antigens of the Rhesus blood group system in routine blood type O blood donors at UDD PMI Depok City. This study was descriptive and observational with a cross-sectional design. Primary data are obtained from the Rhesus system antigen examination results on donor blood samples. The sample was 140 blood samples from 140 routine donors. A rhesus phenotyping examination was carried out using the gel method. The study was conducted on 140 routine donor blood samples at UDD PMI Depok City. The characteristics of the subjects were male (69.29%) and female (30.71%), with an average age of 38 years. Samples are analyzed for five major Rhesus antigens. Rhesus antigen test shows D antigen (100%), e antigen (97.14%), C antigen (95.71%), c antigen (36.43%) and E antigen (27.86%). Rhesus phenotype results were R1R1 (DCe/Dce) (62.86%), R1R2 (DCe/DcE) (22.86%), R1r (DCe/dce) (9.29%), R2R2 (DcE/DcE) (2.86%), R2r (DcE/dce) (1.43%), and R1RZ (DCe/DCE) (0.71%). In conclusion, the frequency of phenotypic changes in the Rh blood group system of regular blood donors at UDD PMI Depok City is classified as the most common types: DCe/DCe, DCe/DcE, DCe/dce, DcE/DcE, DcE/dce and DCe/DCE.

Histopathologic Features and Viral Antigen Distribution of H5N1 Highly Pathogenic Avian Influenza Virus Clade 2.3.4.4b from the 2022-2023 Outbreak in Iowa Wild Birds.

In 2022, a new epornitic of H5N1 highly pathogenic avian influenza (HPAI) virus clade 2.3.4.4b emerged in U.S. domestic poultry with high prevalence in wild bird populations. We describe pathological findings of HPAI H5N1 in nine wild birds encompassing eight different species, including Accipitriformes (red-tailed hawk, bald eagle), Cathartiforme (turkey vulture), Falconiforme (peregrine falcon), Strigiforme (one adult great-horned owl, one juvenile great-horned owl), Pelecaniforme (American white pelican), and Anseriformes (American green-winged teal, trumpeter swan). All these birds died naturally (found dead, or died in transit to or within a rehabilitation center), except for the bald eagle and American green-winged teal, which were euthanized. Gross lesions were subtle, characterized by meningeal congestion observed in the turkey vulture, bald eagle, and adult great-horned owl. Histologically, encephalitis was observed in all cases (9/9, 100%). Leukocytoclastic and fibrinoid vasculitis with necrotizing encephalitis was observed in the red-tailed hawk, great-horned owls, and American white pelican (5/9, 55.6%), and perivascular lymphohistiocytic encephalitis was seen in the turkey vulture, peregrine falcon, green-winged teal, and bald eagle (4/9, 44.4%). Coagulative necrosis or lymphohistiocytic/lymphoplasmacytic inflammation was identified in the kidney (6/8, 75%), liver (6/9, 66.7%), heart (5/9, 55.6%), and lung (2/9, 22.2%). Immunopositive signals against Influenza virus A nucleoprotein were predominantly detected within the brain (9/9, 100%), air sac (7/9, 77.8%), lung (7/9, 77.8%), kidney (6/8, 75%), heart (6/9, 66.7%), and liver (5/9, 55.6%). Additionally, other organs, such as the pancreas, spleen, intestines, gonads, and adrenals occasionally exhibited positive viral protein signals. In these organs, in addition to parenchymal cells, viral protein signals were often identified in endothelial cells. Our results suggest that the 2022-2023 HPAIV H5N1 clade 2.3.4.4b replicated systemically in all examined birds, with brain lesions being the most prevalent and associated with a subset of birds displaying clinical signs observed perimortem.

Stereoscopic Imaging of Single Molecules at Plasma Membrane of Single Cell Using Photoreduction-Assisted Electrochemistry.

Stereoscopic imaging of single molecules at the plasma membrane of single cell requires spatial resolutions in 3 dimensions (x-y-z) at 10-nm level, which is rarely achieved using most optical super-resolution microscopies. Here, electrochemical stereoscopic microscopy with a detection limit down to a single molecule is achieved using a photoreduction-assisted cycle inside a 20-nm gel electrolyte nanoball at the tip of a nanopipette. On the basis of the electrochemical oxidation of Ru(bpy)3 2+ into Ru(bpy)3 3+ followed by the reduction of Ru(bpy)3 3+ into Ru(bpy)3 2+ by photogenerated isopropanol radicals, a charge of 1.5 fC is obtained from the cycling electron transfers involving one Ru(bpy)3 2+/3+ molecule. By using the nanopipette to scan the cellular membrane modified with Ru(bpy)3 2+-tagged antibody, the morphology of the cell membrane and the distribution of carcinoembryonic antigen (CEA) on the membrane are electrochemically visualized with a spatial resolution of 14 nm. The resultant stereoscopic image reveals more CEA on membrane protrusions, providing direct evidence to support easy access of membrane CEA to intravenous antibodies. The breakthrough in single-molecule electrochemistry at the cellular level leads to the establishment of high-resolution 3-dimensional single-cell electrochemical microscopy, offering an alternative strategy to remedy the imperfection of stereoscopic visualization in optical microscopes.

Frequency of Duffy, Kidd, Lewis, and Rh Blood Group Antigens and Phenotypes Among Donors in the Al-Ahsa Region, Saudi Arabia.

In Al-Ahsa, Saudi Arabia, the high consanguinity rates contribute to the prevalence of inherited hemoglobinopathies such as sickle cell disease and thalassemia, which frequently require blood transfusions. These transfusions carry the risk of alloimmunization, necessitating a precise blood component matching to mitigate health risks. Local antigen frequency data is vital for optimizing transfusion practices and enhancing the safety of these medical procedures for the Al-Ahsa population. This study investigated the distribution of Duffy, Kidd, Lewis, and Rh blood group antigens in 1,549 individuals from the region; comparing the frequencies with global data. Serological analyses revealed a high prevalence of the Fy(a+b-) and Jk(a+b+) phenotypes in the Duffy and Kidd blood groups, respectively, with Jk(a-b-) being notably scarce. The Lewis blood group exhibited a significant presence of Le(a-b+) and Le(a+b-) phenotypes, whereas Le(a+b+) was less common. In the Rh system, the D antigen was most prevalent, with other antigens following in descending order of frequency. The study underscores the regional variation in antigen frequencies, emphasizing the need for local blood banks to adapt their screening and matching practices to mitigate the risk of alloimmunization and enhance transfusion safety. These findings are pivotal for refining transfusion strategies and understanding the immunohematology landscape in Al-Ahsa.

T-Cell Epitope Vaccine Prediction Analysis Targeting Phosphoprotein (P) Rabies Virus Based on the Presence of HLA-I Alleles A, B, and C Loci Throughout Southeast Asia: An Immunoinformatics Study

T cell immunity, like responses of CD4+and CD8+ T-cell, plays an important role to fight against viral infections and pathological harm. Several previous studies have shown the results that rabies virus (RABV) protein can act as an ideal receptor for rabies neuroseptic vaccine by inducing a response of T-cell. In this research, we evaluated possible vaccine epitopes based on the Rabies virus sequence and human lymphocyte antigen (HLA) distribution. First, this study used the rabies virus protein P sequence obtained from the NCBI database. Next, we predicted rabies CTL protein epitopes based on the frequency of HLA-I distribution allele locus A, B, and C in Southeast Asia region (&gt; 1%) using Immune Epitope Database and Analysis Resource (iedb.org). Our results predict the presence of 12 epitopes of the protein P RABV. A cluster analysis of epitopes shows that seven P-protein clusters cover 97.47% of the Southeast Asian population. After a conservative epitope analysis, 8 epitopes of protein P showed protection in 11 different types of isolated Rabies virus. Finally, 4 epitopes (SQTVEEIISY119-128; RSSGIFLYNF218-227, ASGPPALEW178-186, IISYVTVNF125-133) were used to vaccinate 97.47% of people in Southeast Asia. Our results suggest that both single and combined CTL epitopes which were predicted in this study can be used as a more effective alternative vaccine against rabies virus’ infections and development

Specific antigens in malignancy-associated membranous nephropathy.

Membranous nephropathy (MN) is a glomerular disease mediated by autoimmune complex deposition, with approximately 30% of cases attributed to secondary causes. Among them, malignant tumors are a significant cause of secondary MN. Recent advancements in the identification of MN-specific antigens, such as THSD7A and NELL-1, suggest a potential association with malignant tumors, yet definitive proof of this relationship remains elusive. Therefore, this article aims to review the distribution of MN-specific antigens in patients with MN caused by malignant tumors and the possible role of these antigens in the pathogenesis of the disease.

Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Abstract 3147: DXC025, a novel anti-MUC1/EGFR bispecific antibody-tubulysin conjugate with a function linker, exhibits potent anti-tumor efficacy

Abstract Antibody-drug conjugates (ADCs) have become a promised drug class in cancer therapy. Armed with three main components-an antibody, a linker molecule, and a cytotoxic agent (“payload”), plus sites of the antibody conjugation and the distribution of tumor-associate antigens, nowadays ADCs have proved the unique ability of the precision of targeted therapy, a great leap forward from traditional chemotherapeutic approaches that cause widespread effects without specificity. Both epidermal growth factor receptor (EGFR) and mucin 1 (MUC1) are tumor-associated antigens (TAA) that are co-expressed in many solid tumors, such as, colon cancer, non-small cell lung cancer (NSCLC), esophageal cancer, epidermal cancer, and pancreatic cancer. In particular, MUC1, a glycoprotein essential for the formation of the epithelial mucous barrier, is hypoglycosylated and dimerizes with EGFR, a potent oncoprotein, in transformed cells. For our anti-Muc1 ADC clinical program, for treatment of colon and pancreatic cancers, it was found out that the highly glycosylated transmembrane Muc1 had much more turning over rates on the luminal surface of epithelial cells. Here, we first designed and generated bispecific Fab/scFv antibodies targeting both MUC1 and EGFR, using the knob-into-hole technology. The addition of the affinity- and internalization-optimized anti-EGFR arm to the other single Muc1 arm was aimed to broaden tumor selectivity and to reduce on-target toxicity, for the treatment of gastrointestinal tumors in comparison to individual full IgG1-ADC, either EGFR or Muc1-ADC. Indeed, many of generated anti- MUC1/EGFR bispecific antibodies (BsAbs) showed strong binding affinity in MUC1modetateEGFRmodetate cells, in addition to both MUC1 and EGFR highly expressed cells. Internalization assays demonstrated that the BsAbs were endocytosed in tumor cells co-expressing EGFR and MUC1 more efficiently than monoclonal antibodies targeting MUC1. The selected BsAbs were subsequently conjugated with tubulysin B analogs with varieties of Tubulysin B analog payloads and function peptidyl spacer/linkers to generate bispecific ADC (DXC025) candidates. DXC025 candidates exhibited very potent cytotoxicity in vitro against epidermal cancer, lung adenocarcinoma, and pancreatic cancer cell lines with IC50 of single or tens digital pM. In in vivo cell-derived xenograft (CDX) models, DXC025 candidates demonstrated much superior efficacies of tumor growth inhibition in comparison with parental (monoclonal), either Muc1- or EGFR- ADC for the treatment of gastrointestinal tumors. These results indicated that the MUC1/EGFR-ADC (DXC025) would be a promising ADC candidate for targeted treatment of either Muc1- or EGFR-expressing gastrointestinal tumors. Citation Format: Xingyan Jiang, Junxiang Jia, Huihui Guo, Xiangfei Kong, Yongfang Xu, Sishi Ye, Yong Du, Zhicang Ye, Qikuang Cen, Lingli Zhang, Yongxiang Chen, Gaituo Chen, Lu Bai, Yunxia Zheng, Wei Zheng, Jun Zheng, Juan Wang, Wenjun Li, Yuanyuan Huang, Linyao Zhao, Yifang Xu, Mengmeng Liu, Binbin Chen, Meng Dai, Miaomiao Chen, Zhixiang Guo, Qingliang Yang, Robert Y. Zhao. DXC025, a novel anti-MUC1/EGFR bispecific antibody-tubulysin conjugate with a function linker, exhibits potent anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3147.

Natural Infection with H5N1 Highly Pathogenic Influenza (HPAI) Virus in 5- and 10-Day-Old Commercial Pekin Ducklings (Anas platyrhynchos domesticus).

Highly pathogenic avian influenza (HPAI) has resulted in catastrophic economic losses globally in poultry. This case report describes the diagnostic detection and pathology of HPAI H5N1 in 5-day-old commercial ducklings, which is an atypical age for detection of natural infection of HPAI in poultry. The pathology observed at 5 days of age was also compared to lesions observed in ducklings from the same flock evaluated at 10 days of age before depopulation. The California Animal Health and Food Safety (CAHFS) Laboratory, Tulare, received ten 5-day-old Pekin duckling (Anas platyrhynchos domesticus) carcasses for diagnostic evaluation due to mortality that started increasing at 3 days of age. The most common gross findings included bilateral pulmonary edema with congestion and enlarged, mottled livers and spleens. Microscopically, cerebral neuronophagia, pancreatic necrosis, and interstitial pneumonia with pulmonary edema were observed in the 5-day-old ducklings. Oropharyngeal and cloacal swabs were positive for avian influenza virus (AIV) by real-time reverse transcriptase PCR. The AIV was typed as HPAI, EA/AM 2.3.4.4b H5N1 goose/Guangdong clade lineage by the National Veterinary Services Laboratory. Ducks at the affected premises were depopulated 4 days after the 5-day-old ducklings were submitted to the CAHFS lab, at which time additional tissue samples were collected for comparison to 10-day-old ducklings on the same premises. Differences in microscopic lesions and AIV tissue distribution were observed between the 5-day and 10-day tissues collected. Notably, microscopic lesions were more severe in the brain and pancreas at 10 days of age. Findings in 10-day-old ducklings included cerebral lymphoplasmacytic perivascular cuffing, gliosis, neuronal degeneration, and pancreatic necrosis. AIV antigen distribution and intensity was greatest in the cerebral tissue of the brains at 10 days and in the lungs at 5 days of age. To the authors' knowledge, published studies are limited on AIV natural infection in domestic ducks less than 9 days of age.