Humanized Research Articles

Oncolytic activity of a chimeric influenza a virus carrying a human CTLA4 antibody in hepatocellular carcinoma

Oncolytic activity of a chimeric influenza a virus carrying a human CTLA4 antibody in hepatocellular carcinoma

Calotropis procera: A double edged sword against glioblastoma, inhibiting glioblastoma cell line growth by targeting histone deacetylases (HDAC) and angiogenesis

Despite substantial investments in anti-glioblastoma (GBM) drug discovery over the last decade, progress is limited to preclinical stages, with clinical studies frequently encountering obstacles. Angiogenic and histone deacetylase inhibitors (HDACi) have shown profound results in pre-clinical studies. Investigating a multicomponent anti-cancer remedy that disrupts the tumor angiogenic blood vessels and simultaneously disrupts HDACs, while inducing minimal side effects, is critically needed. The crude extracts derived from medicinal plants serve as a renewable reservoir of anti-tumor drugs, exhibiting reduced toxicity compared to chemically synthesized formulations. Calotropis procera is a traditional medicinal plant, and its anticancer potential against many cancer cell lines has been reported, however its antiangiogenic and HDAC inhibitory action is largely unknown. The anticancer activity of methanol leaf extract of C. procera was tested in three types of human glioblastoma cell lines. Wild-type and transgenic zebrafish embryos were used to evaluate developmental toxicity and angiogenic activity. A human angiogenic antibody array was used to profile angiogenic proteins in the U251 GM cell line. A real-time reverse transcriptase polymerase chain reaction (RT PCR) assay was used to detect the differential expression of eleven HDAC genes in U251 cells treated with C. procera extract. The extract significantly reduced the proliferation of all three types of GBM cell lines and the cytotoxicity was found to be more pronounced in U251 GM cells, with an IC50 value of 2.63 ± 0.23 μg/ml, possibly by arresting the cell cycle at the G2/M transition. The extract did not exhibit toxic effects in zebrafish embryos, even at concentrations as high as 1000 μg/ml. The extract also inhibited angiogenic blood vessel formation in the transgenic zebrafish model in a dose-dependent manner. The results from the angiogenic antibody array have suggested novel angiogenesis targets that can be utilized to treat GBM. Real-time RT PCR analysis has shown that C. procrea extract caused an upregulation of HDAC5, 7, and 10, while the mRNA of HDAC1, 2, 3 and 8 (Class I HDACs), and HDAC4, 6, and 9 (Class II) were downregulated in U251 GM cells. The cytotoxicity of the C. procera extract on GBM cell lines could be due to its dual action by regulation of both tumor angiogenesis and histone deacetylases enzymes. Through this study, the C. procera leaf extract has been suggested as an effective remedy to treat GBM with minimal toxicity. In addition, various novel angiogenic and HDAC targets has been identified which could be helpful in designing better therapeutic strategies to manage glioblastoma multiforme in human patients.

Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

Designing stable humanized antibodies.

Designing stable humanized antibodies.

Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

Fully Human Herpesvirus-Specific Neutralizing IgG Antibodies Generated by EBV Immortalization of Splenocytes-Derived from Immunized Humanized Mice.

Antiviral neutralizing antibodies (nAbs) are commonly derived from B cells developed in immunized or infected animals and humans. Fully human antibodies are preferred for clinical use as they are potentially less immunogenic. However, the function of B cells varies depending on their homing pattern and an additional hurdle for antibody discovery in humans is the source of human tissues with an immunological microenvironment. Here, we show an efficient method to pharm human antibodies using immortalized B cells recovered from Nod.Rag.Gamma (NRG) mice reconstituting the human immune system (HIS). Humanized HIS mice were immunized either with autologous engineered dendritic cells expressing the human cytomegalovirus gB envelope protein (HCMV-gB) or with Epstein-Barr virus-like particles (EB-VLP). Human B cells recovered from spleen of HIS mice were efficiently immortalized with EBV in vitro. We show that these immortalized B cells secreted human IgGs with neutralization capacities against prototypic HCMV-gB and EBV-gp350. Taken together, we show that HIS mice can be successfully used for the generation and pharming fully human IgGs. This technology can be further explored to generate antibodies against emerging infections for diagnostic or therapeutic purposes.

IgMAT: immunoglobulin sequence multi-species annotation tool for any species including those with incomplete antibody annotation or unusual characteristics

BackgroundThe advent and continual improvement of high-throughput sequencing technologies has made immunoglobulin repertoire sequencing accessible and informative regardless of study species. However, to fully map dynamic changes in polyclonal responses precise framework and complementarity determining region annotation of rearranging genes is pivotal. Most sequence annotation tools are designed primarily for use with human and mouse antibody sequences which use databases with fixed species lists, applying very specific assumptions which select against unique structural characteristics. For this reason, data agnostic tools able to learn from presented data can be very useful with new species or with novel datasets.ResultsWe have developed IgMAT, which utilises a reduced amino acid alphabet, that incorporates multiple HMM alignments into a single consensus to automatically annotate immunoglobulin sequences from most organisms. Additionally, the software allows the incorporation of user defined databases to better represent the species and/or antibody class of interest. To demonstrate the accuracy and utility of IgMAT, we present analysis of sequences extracted from structural data and immunoglobulin sequence datasets from several different species.ConclusionsIgMAT is fully open-sourced and freely available on GitHub (https://github.com/TPI-Immunogenetics/igmat) for download under GPLv3 license. It can be used as a CLI application or as a python module to be integrated in custom scripts.

Spesolimab for the Treatment of Generalized Pustular Psoriasis.

Generalized pustular psoriasis (GPP) is a rare but severe skin inflammatory disorder characterized by the eruption of widespread sterile neutrophilic pustules, often accompanied by systemic inflammation. Given its life-threatening potential, GPP requires prompt accurate diagnosis and effective treatment, but its rarity and relapsing-remitting nature pose challenges in performing large-scale randomized controlled clinical trials. Established international guidelines are currently lacking and management guidance often follows that for plaque psoriasis. However, while it can co-exist with plaque psoriasis and has traditionally been classified as a most severe form of psoriasis, GPP is now recognized as a distinct entity, with its own clinicopathological, autoinflammatory, immunologic and genetic features. Research conducted over the past decade revealed that an imbalance of interleukin (IL)-36 signaling favoring the proinflammatory activity is the central driver of the pathogenesis of GPP, thereby laying the groundwork for the development of targeted therapies for the disease. This article reviews the evidence thus far on spesolimab, a selective humanized antibody against the IL-36 receptor that was recently licensed in Europe and the United States for the treatment of GPP flares in adults. In phase II, randomized controlled clinical trials, spesolimab led to rapid and effective skin clearance in patients experiencing a GPP flare and demonstrated superiority to placebo in preventing flares for up to 48 weeks with maintenance treatment, with reassuring safety and tolerability profiles. Spesolimab is considered to be a first-in-class medication establishing itself as the standard of care for the treatment of GPP flares, thus changing the paradigm of the management of GPP to a new era of scientifically- and evidence-based targeted therapy for this distinctive disease.

Safety and Pharmacokinetic Assessment of the FIC CLDN18.2/4-1BB Bispecific Antibody in Rhesus Monkeys.

Gastric cancer is one of the most common cancers worldwide, particularly in China, with over half a million new cases and over 400 thousand deaths in 2022. Zolbetuximab, a first-in-class investigational monoclonal antibody (mAb) targeting tumor-associated antigen CLDN18.2 which is highly expressed on gastric cancer cells, was recently reported to meet the primary endpoint in Phase III trial as first-line treatment in CLDN18.2 positive and HER2-negative gastric cancers. In the present study, we developed a humanized bispecific antibody (bsAb) CLDN18.2/4-1BB named PM1032. PM1032 activates immune cells via CLDN18.2 mediated crosslinking of 4-1BB, a potent stimulator of T/NK cells. It induced strong immunological memory in multiple tumor-bearing animal models, indicating significant potential as an effective treatment for CLDN18.2 positive cancers such as gastric cancer. Since liver and gastrointestinal (GI) related toxicities were reported in 4-1BB and CLDN18.2 targeting programs during the clinical development, respectively, extensive pharmacokinetics (PK) and safety profile characterization of PM1032 was performed in rhesus monkeys. PM1032 had a half-life comparable to a conventional IgG1 mAb, and serum drug concentration increased in a dose-dependent pattern. Furthermore, PM1032 was generally well tolerated, with no significant abnormalities observed in toxicity studies, including the liver and stomach. In summary, PM1032 demonstrated good PK and an exceptional safety profile in rhesus monkeys supporting further investigation in clinical studies.

The Role of Macrophage Migration Inhibitory Factor (MIF) and D-Dopachrome Tautomerase (D-DT/MIF-2) in Infections: A Clinical Perspective.

Macrophage migration inhibitory factor (MIF) and its homolog, D-dopachrome tautomerase (D-DT), are cytokines that play critical roles in the immune response to various infectious diseases. This review provides an overview of the complex involvement of MIF and D-DT in bacterial, viral, fungal, and parasitic infections. The role of MIF in different types of infections is controversial, as it has either a protective function or a host damage-enhancing function depending on the pathogen. Depending on the specific role of MIF, different therapeutic options for MIF-targeting drugs arise. Human MIF-neutralizing antibodies, anti-parasite MIF antibodies, small molecule MIF inhibitors or MIF-blocking peptides, as well as the administration of exogenous MIF or MIF activity-augmenting small molecules have potential therapeutic applications and need to be further explored in the future. In addition, MIF has been shown to be a potential biomarker and therapeutic target in sepsis. Further research is needed to unravel the complexity of MIF and D-DT in infectious diseases and to develop personalized therapeutic approaches targeting these cytokines. Overall, a comprehensive understanding of the role of MIF and D-DT in infections could lead to new strategies for the diagnosis, treatment, and management of infectious diseases.

Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system

BackgroundRNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production.Methods and resultsBL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp′)-OmpA′, SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34.ConclusionThe functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.

Direct Comparison of the Hinge-Cleaving Proteases IgdE and BdpK for LC-MS-Based IgG1 Clonal Profiling.

Human antibodies are heterogeneous molecules primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, such as ELISA, lack the power to resolve abundances at the clonal level. Recently, we introduced an LC-MS-based approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the constant Fc region of the antibody. Here, we explore an alternative IgG1 hinge-cleaving protease, BdpK (FabDELLO), and compare it directly to IgdE for use in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest all IgG1s from the same set of plasma samples. Both proteases cleave IgG1 specifically in the hinge, albeit via different mechanisms and at two distinct cleavage sites. Notwithstanding these differences, the Fab fragments generated by IgdE or BdpK produced highly similar clonal repertoires. However, IgdE required ∼16 h of incubation to digest plasma IgG1s, while BdpK required ∼2 h. We authenticated the similarity of the clones by top-down proteomics using electron transfer dissociation. We conclude that BdpK performs very well in digesting polyclonal plasma IgG1s and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge-digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in plasma.

Generation and Characterization of SORT1-Targeted Antibody-Drug Conjugate for the Treatment of SORT1-Positive Breast Tumor.

Antibody-drug conjugates (ADCs) have greatly improved the outcomes of advanced breast tumors. However, the treatment of breast tumors with existing ADCs is still hindered by many issues, such as tumor antigen heterogeneity and drug resistance. Therefore, ADCs against new targets would provide options for the treatment of these challenges. Sortilin-1 (SORT1) may be a promising target for ADC as it is upregulated in breast cancer. To evaluate the possibility of SORT1 as an ADC target, a humanized antibody_8D302 with high affinity against SORT1 was generated. Additionally, 8D302 was conjugated with MMAE and DXd to generate two ADCs_8D302-MMAE and 8D302-DXd, respectively. Both 8D302-MMAE and 8D302-DXd showed effective cytotoxicity against SORT1 positive breast tumor cell lines and induced bystander killing. Consequently, 8D302-MMAE showed relatively better anti-tumor activity than 8D302-DXd both in vitro and in vivo, but 8D302-DXd had superior safety profile and pharmacokinetics profile over 8D302-MMAE. Furthermore, SORT1 induced faster internalization and lysosomal trafficking of antibodies and had a higher turnover compared with HER2. Also, 8D302-DXd exhibited superior cell cytotoxicity and tumor suppression over trastuzumab-DXd, a HER2-targeted ADC. We hypothesize that the high turnover of SORT1 enables SORT1-targeted ADC to be a powerful agent for the treatment of SORT1-positive breast tumor.

Bronchoalveolar cytokine profile differentiates Pulmonary Langerhans cell histiocytosis patients from other smoking-related interstitial lung diseases

BackgroundPulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease (ILD) associated with smoking, whose definitive diagnosis requires the exclusion of other forms of ILD and a compatible surgical lung biopsy. Bronchoalveolar lavage (BAL) is commonly proposed for the diagnosis of ILD, including PLCH, but the diagnostic value of this technique is limited. Here, we have analyzed the levels of a panel of cytokines and chemokines in BAL from PLCH patients, in order to identify a distinct immune profile to discriminate PLCH from other smoking related-ILD (SR-ILD), and comparing the results with idiopathic pulmonary fibrosis (IPF) as another disease in which smoking is considered a risk factor.MethodsBAL samples were collected from thirty-six patients with different ILD, including seven patients with PLCH, sixteen with SR-ILD and thirteen with IPF. Inflammatory profiles were analyzed using the Human Cytokine Membrane Antibody Array. Principal component analysis (PCA) was performed to reduce dimensionality and protein–protein interaction (PPI) network analysis using STRING 11.5 database were conducted. Finally, Random forest (RF) method was used to build a prediction model.ResultsWe have found significant differences (p < 0.05) on thirty-two cytokines/chemokines when comparing BAL from PLCH patients with at least one of the other ILD. Four main groups of similarly regulated cytokines were established, identifying distinct sets of markers for each cluster. Exploratory analysis using PCA (principal component analysis) showed clustering and separation of patients, with the two first components capturing 69.69% of the total variance. Levels of TARC/CCL17, leptin, oncostatin M (OSM) and IP-10/CXCL10 were associated with lung function parameters, showing positive correlation with FVC. Finally, random forest (RF) algorithm demonstrates that PLCH patients can be differentiated from the other ILDs based solely on inflammatory profile (accuracy 96.25%).ConclusionsOur results show that patients with PLCH exhibit a distinct BAL immune profile to SR-ILD and IPF. PCA analysis and RF model identify a specific immune profile useful for discriminating PLCH.

Therapeutic efficacy of an alpha-particle emitter labeled anti-GD2 humanized antibody against osteosarcoma-a proof of concept study.

Current treatments for osteosarcoma (OS) have a poor prognosis, particularly for patients with metastasis and recurrence, underscoring an urgent need for new targeted therapies to improve survival. Targeted alpha-particle therapy selectively delivers cytotoxic payloads to tumors with radiolabeled molecules that recognize tumor-associated antigens. We have recently demonstrated the potential of an FDA approved, humanized anti-GD2 antibody, hu3F8, as a targeted delivery vector for radiopharmaceutical imaging of OS. The current study aims to advance this system for alpha-particle therapy of OS. The hu3F8 antibody was radiolabeled with actinium-225, and the safety and therapeutic efficacy of the [225Ac]Ac-DOTA-hu3F8 were evaluated in both orthotopic murine xenografts of OS and spontaneously occurring OS in canines. Significant antitumor activity was proven in both cases, leading to improved overall survival. In the murine xenograft's case, tumor growth was delayed by 16-18days compared to the untreated cohort as demonstrated by bioluminescence imaging. The results were further validated with magnetic resonance imaging at 33days after treatment, and microcomputed tomography and planar microradiography post-mortem. Histological evaluations revealed radiation-induced renal toxicity, manifested as epithelial cell karyomegaly and suggestive polyploidy in the kidneys, suggesting rapid recovery of renal function after radiation damage. Treatment of the two canine patients delayed the progression of metastatic spread, with an overall survival time of 211 and 437days and survival beyond documented metastasis of 111 and 84days, respectively. This study highlights the potential of hu3F8-based alpha-particle therapy as a promising treatment strategy for OS.

Human VH4-34 antibodies derived from B1 cells are more frequently autoreactive than VH4-34 antibodies derived from memory cells.

Human B1 cells produce natural antibodies characterized by overutilization of heavy chain variable region VH4-34 in comparison to other B cell populations. VH4-34-containing antibodies have been reported to be autoreactive and to be associated with lupus and other autoimmune dyscrasias. However, it has been unclear to what extent VH4-34 antibodies manifest autoreactivity in B1 cells or other B cell populations-in other words, are VH4-34 containing antibodies autoreactive wherever found, or mainly within the B1 cell population? To address this issue we sort purified single human B1 and memory B cells and then amplified, sequenced, cloned and expressed VH4-34-containing antibodies from 76 individual B cells. Each of these antibodies was tested for autoreactivity by HEp-2 IFA and autoantigen ELISA. Antibodies were scored as autoreactive if positive by either assay. We found VH4-34 antibodies rescued from B1 cells were much more frequently autoreactive (14/48) than VH4-34 antibodies rescued from memory B cells (2/28). Among B1 cell antibodies, 4 were HEp-2+, 6 were dsDNA+ and 4 were positive for both. Considering only HEp-2+ antibodies, again these were found more frequently among B1 cell VH4-34 antibodies (8/48) than memory B cell VH4-34 antibodies (1/28). We found autoreactivity was associated with greater CDR3 length, as expected; however, we found no association between autoreactivity and a previously described FR1 "hydrophobic patch". Our results indicate that autoreactive VH4-34-containing antibodies tend to reside within the human B1 cell population.

“In House” assays for the quantification of Annexin V and its autoantibodies in patients with recurrent pregnancy loss and in vitro fertilisation failures

Several studies have been shown that Annexin V (ANXV) autoantibodies concentrations are associated with both early recurrent pregnancy losses (RPLs) or in vitro fertilization failure (IVFf). We investigated the association between ANXV autoantibodies and ANVX levels in RPL, IVFf and normal group women. The study was conducted on 22 female patients with RPLs, 66 patients with IVFf, and 16 normal samples from women who had given birth. ANXV autoantibodies were measured using an ELISA test developed by fixing a homemade recombinant ANXV protein and examined with labeled human antibodies, while ANXV concentrations were measured by a competitive ELISA using a homemade anti ANXV polyclonal antibody. The results showed a clear relationship between the high levels of ANXV autoantibodies and the recurrent abortion. On the other hand, ANXV measurement in those patients showed decreased concentrations compared to normal samples. Negative correlation between ANXV and its autoantibodies levels was reported in almost all patients’ samples. Our data supports the possibility that ANXV autoantibodies are a risk factor for reproductive failures associated with both RPLs and/or IVFf and the significant role for ANXV in the maintenance of pregnancy.

Enhancement of SARS-CoV-2 Infection via Crosslinking of Adjacent Spike Proteins by N-Terminal Domain-Targeting Antibodies.

The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)-NTD complexes, supported by molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection.

Enzymatic comparison and expression pattern of pig B4GALNT2 and B4GALNT2-like proteins

Abstract Objectives The final step in the production of the human Sd(a) antigen is catalyzed by beta-1,4-N-acetyl-galactosamine transferase 2 (B4GALNT2). This is done by adding a N-acetylgalactosamine residue via a beta-1,4 linkage to a subterminal galactose residue that has been substituted with an alpha-2,3-linked sialic acid. The final stage of the production of the Cad antigen is also catalyzed by B4GALNT2. Knocking out pig B4GALNT2 gene decreased human serum antibodies binding to pig cells, which greatly reduces the immunological rejection in clinical xenotransplantation trials. Interestingly, gene region LOC110255214 (hereafter named B4GALNT2-like) showed high similarity with the B4GALNT2 gene in the pig genome in our previous work, but whether B4GALNT2-like shares similar biological properties like B4GALNT2 remains to be elucidated, whether B4GALNT2-like is a potential immune gene in xenotransplantation remains to be determined. Methods In this study, we compared the tissue expression pattern of B4GALNT2-like and B4GALNT2 in Bama pigs. Results We found the expression of B4GALNT2-like was significantly higher in the duodenum, but lower in the heart, spleen, lung, kidney, comparing to B4GALNT2. Applied the Escherichia coli recombinant expression, we obtained 768 and 1,300 μg protein for B4GALNT2 and B4GALNT2-like from 1 L culture, respectively. Using the expressed recombinant proteins, the enzymatic activity of the two proteins was determined and compared. Conclusions The enzymatic assay showed that B4GALNT2-like has comparable catalytic activity with B4GALNT2 (58.7 % of B4GALNT2), addressing an important question whether B4GALNT2-like is a new immunological rejection gene.

Deciphering the human antibody response against Burkholderia pseudomallei during melioidosis using a comprehensive immunoproteome approach.

The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients. In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio. Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.