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Glycosylation of recombinant proteins is a post-translational modification that affects multiple physicochemical and biological properties of proteins. As such, it is a critical quality attribute that must be carefully controlled during protein production in the pharmaceutical industry. Glycosylation can be modulated by various conditions, including the composition of production media and feeds. In this study, the N-glycosylation-modulating effects of numerous compounds, including metal enzyme cofactors, enzyme inhibitors, and metabolic intermediates, were evaluated. Chinese hamster ovary cells producing three different IgG antibodies were cultivated in a fed-batch mode. First, a one-factor-at-a-time experiment was performed in 24-well deep well plates to identify the strongest modulators and appropriate concentration ranges. Then, a full response surface experiment was designed to gauge the effects and interactions of the 14 most effective hit compounds in an Ambr® 15 bioreactor system. A wide range of glycoform content was achieved, with an up to eight-fold increase in individual glycoforms compared to controls. The resulting model can be used to determine modulator combinations that will yield desired glycoforms in the final product.

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The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the cell will shut down protein translation and ultimately induce apoptosis. We co-overexpressed HsQSOX1b and survivin proteins in the antibody-producing cell line CHO-PAb to obtain a new cell line, CHO-PAb-QS. Compared with CHO-PAb cells, the survival time of CHO-PAb-QS cells in batch culture was extended by 2 days, and the antibody accumulation and productivity were increased by 52% and 45%, respectively. The proportion of (HC-LC)2 was approximately doubled in the CHO-PAb-QS cells, which adapted to the accelerated disulfide bond folding capacity by upregulating the UPR's strength and increasing the ER content. The results of the apoptosis assays indicated that the CHO-PAb-QS cell line exhibited more excellent resistance to apoptosis induced by ER stress. Finally, CHO-PAb-QS cells exhibited mild oxidative stress but did not significantly alter the redox status. This study demonstrated that strategies based on HsQSOX1b and survivin co-overexpression could facilitate protein disulfide bond folding and anti-apoptosis ability, enhancing antibody production efficiency in CHO cell lines.

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Rapid and efficient cell line development (CLD) process is essential to expedite therapeutic protein development. However, the performance of widely used glutamine-based selection systems is limited by low selection efficiency, stringency, and the inability to select multiple genes. Therefore, an AND-gate synthetic selection system is rationally designed using split intein-mediated protein ligation of glutamine synthetase (GS) (SiMPl-GS). Split sites of the GS are selected using a computational approach and validated with GS-knockout Chinese hamster ovary cells for their potential to enable cell survival in a glutamine-free medium. In CLD, SiMPl-GS outperforms the wild-type GS by selectively enriching high producers. Unlike wild-type GS, SiMPl-GS results in cell pools in which most cells produce high levels of therapeutic proteins. Harnessing orthogonal split intein pairs further enables the selection of four plasmids with a single selection, streamlining multispecific antibody-producing CLD. Taken together, SiMPl-GS is a simple yet effective means to expedite CLD for therapeutic protein production.

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Mucosal immunity is the main defense line against respiratory disease pathogens. Newcastle disease and avian infectious bronchitis are common respiratory diseases in poultry. However, the mucosal immune response is not sufficiently activated and thus fails to achieve the ideal immune protection. Therefore, it is important to develop a suitable mucosal immune adjuvant to enhance the immune response of live vaccines. Here, the bursal-derived peptide BP7, ß-glucan, and hyaluronic acid were selected as the adjuvant to be assembled into the composite nanopolypeptide adjuvant (CNPB7) with ultrasonic dispersion technology. The results showed that after optimizing assembly conditions, the optimal average particle size of nanoparticle CNPB7 was 514.9 nm and PDI was 0.298. To evaluate the non-specific immune responses of nanoparticle CNPB7, the chickens were immunized only with nanoparticle CNPB7. It was confirmed that nanoparticle CNPB7 enhanced the expression of CD3, CD4, CD80, and CD86 factors in the spleen lymphocyte from the chicken immunized with nanoparticle CNPB7. To investigate the mucosal immune response of nanoparticle CNPB7, the chickens were orally immunized with Newcastle disease virus (NDV)-infectious bronchitis virus (IBV) dual vaccines and CNPB7. The results proved that the levels of immunoglobulin SIgA, IL-4, IFN-γ, and IL-13 in the mucus samples from the respiratory and digestive tract in chicken immunized with nanoparticle CNPB7 and vaccines were significantly increased, compared to that of vaccine control. Finally, it was observed that nanoparticle CNPB7 promoted specific increased antibody productions against NDV and IBV in the immunized chicken. These results proved that the assembled nanoparticle CNPB7 could enhance the vaccination efficacy in chicken, which provided the experimental basis for the development of new adjuvants, and offered technical support for preventing virus transmission of avian diseases.

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Echis ocellatus is one of the commonest snakes responsible for envenomation in Nigeria. Antivenom is the only effective treatment, but the country suffers from a limited supply of effective antivenom. This study therefore aimed to explore the feasibility of effective, mono-specific antibodies production through immunization in rabbits using the venom of Echis ocellatus from Nigeria. The World Health Organization guide on antivenom production was employed in the immunization and the resultant antibodies were purified using protein A agarose column chromatography. Antibody titer reached a high plateau by 2-month immunization, and SDS PAGE of the sera suggests the presence of intact immunoglobulins accompanied with the heavy (50 kDa) and light (25 kDa) chains. The venom has an intravenous LD50 of 0.35 mg/kg in mice, and the venom lethality at a challenge dose of 2 LD50 was effectively neutralized by the antibodies with a potency value of 0.83 mg venom per g antibodies. The antibodies also neutralized the procoagulant activity of the venom with an effective dose (ED) of 13 ± 0.66 µl, supporting its use for hemotoxic envenomation. The study establishes the feasibility of developing effective, mono-specific antibodies against the Nigerian Carpet viper.

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B cells and the antibodies they produce are critical in host defense against pathogens and contribute to various immune-mediated diseases. B cells responding to activating signals in vitro release extracellular vesicles (EV) that carry surface antibodies, yet B cell production of EVs that express antibodies and their function in vivo is incompletely understood. Using transgenic mice expressing the Cre recombinase in B cells switching to IgG1 to induce expression of fusion proteins between emerald green fluorescent protein (emGFP) and the EV tetraspanin CD63 as a model, we identify emGFP expression in B cells responding to foreign antigen in vivo and characterize the emGFP+ EVs they release. Our data suggests that emGFP+ germinal center B cells undergoing immunoglobulin class switching to express IgG and their progeny memory B cells and plasma cells, also emGFP+, are sources of circulating antigen-specific IgG+ EVs. Furthermore, using a mouse model of influenza virus infection, we find that IgG+ EVs specific for the influenza hemagglutinin antigen protect against virus infection. In addition, crossing the B cell Cre driver EV reporter mice onto the Nba2 lupus-prone strain revealed increased circulating emGFP+ EVs that expressed surface IgG against nuclear antigens linked to autoimmunity. These data identify EVs loaded with antibodies as a novel route for antibody secretion in B cells that contribute to adaptive immune responses, with important implications for different functions of IgG+ EVs in infection and autoimmunity.

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AIM: To compare antibody responses after vaccinations between patients with rheumatoid arthritis (RA) and patients with metabolic disorders (MD). The study places special emphasis on understanding how common diseases affect antibody responses in individuals with RA within real-world settings. METHODS: The participants were 117 patients with RA (66 with RA only and 51 with RA and MD) and 37 patients with MD who received both the primary series of vaccinations and a booster. Antibody titers were compared after the primary series of vaccinations and a booster, and factors influencing the antibody response were assessed. RESULTS: Following the primary series of vaccinations, a significant reduction in antibody titers was observed in patients with longer days between vaccination and antibody measurement, the use of IL-6 inhibitors, selective T cell co-stimulation modulators, and methotrexate. Comorbid MD did not exhibit significant influences on antibody response in RA. Notably, the presence of RA itself was not significant in multivariate linear regression analysis. After the administration of the booster, however, day between vaccination and antibody measurement, the use of IL-6 inhibitor, and methotrexate no longer remained significant. Only the use of selective T cell co-stimulation modulators retained its significance. CONCLUSIONS: MD did not exhibit a significant impact on antibody responses in RA patients. The reduced antibody response following the primary series in RA patients appeared to be attributed more to specific RA medications rather than to the disease itself. Booster vaccines are vital in restoring the antibody response in RA.

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In December 2019, Corona Virus Disease 2019 (COVID-19) was first identified and designated as a pandemic in March 2020 due to rapid spread of the virus globally. At the beginning of the pandemic, only a few treatment options, mainly focused on supportive care and repurposing medications, were available. Due to its effects on immune system, vitamin D was a topic of interest during the pandemic, and researchers investigated its potential impact on COVID-19 outcomes. However, the results of studies about the impact of vitamin D on the disease are inconclusive. In the present narrative review, different roles of vitamin D regarding the COVID-19 have been discussed to show that vitamin D supplementation should be recommended carefully.

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Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.

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Antibiotic resistance and re-emergence of highly resistant pathogens is a grave concern everywhere and this has consequences for all kinds of human activities. Herein, we showed that N-palmitoylethanolamine-derived cationic lipid (cN16E) had a lower minimum inhibitory concentration (MIC) against both Gram-positive and Gram-negative bacteria when it was loaded with Butea monosperma seed lectin (BMSL). The analysis using lectin-FITC conjugate labelling indicated that the improved antibacterial activity of BMSL conjugation was due to bacterial cell surface glycan recognition. Live and dead staining experiments revealed that the BMSL-cN16E conjugate (BcN16E) exerts antibacterial activity by damaging the bacterial membrane. BcN16E antimicrobial activity was demonstrated using an infected zebrafish animal model because humans have 70 % genetic similarity to zebrafish. BcN16E therapeutic potential was established successfully by rescuing fish infected with uropathogenic Escherichia coli (UPEC). Remarkably, the rescued infected fish treated with BcN16E prevented reinfection without further therapy, indicating BcN16E immunomodulatory potential. Thus, the study examined the expression of immune-related genes, including tnfα, ifnγ, il-1ß, il-4, il-10, tlr-2, etc. There was a significant elevation in the expression of all these genes compared to control and fish treated with BMSL or cN16E alone. Interestingly, when the rescued zebrafish were reinfected with the same pathogen, the levels of expression of these genes were many folds higher than seen earlier. Radial immune diffusion analyses (RIA) using zebrafish serum revealed antibody production during the initial infection and treatment. Interestingly, reinfected fish had significant immunoprecipitation in RIA, a feature absent in the groups treated with cN16E, BMSL, and control. These results clearly show that the BcN16E complex not only rescued infected zebrafish but also conferred long-lasting protection in terms of immunomodulation that protects against multiple reinfections. The findings support that BcN16E has immense potential as a novel immunostimulant for various biomedical applications.

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Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

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Antibodies are a valuable research tool, with uses including detection and quantification of specific proteins. By using peptide fragments to raise antibodies, they can be designed to differentiate between structurally similar proteins, or to bind conserved motifs in divergent proteins. Peptide sequence selection and antibody validation are crucial to ensure reliable results from antibody-based experiments. This chapter describes the steps for the identification of peptide sequences to produce protein- or isoform-specific antibodies using recombinant technologies as well as the subsequent validation of such antibodies. The photosynthetic protein Rubisco activase is used as a case study to explain the various steps involved and key aspects to take into consideration.

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cµ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cµ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.

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The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

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Aframomum melegueta K Schum (A. melegueta), an herbaceous plant renowned for its medicinal seeds, was investigated for its potential immunomodulatory effects in vitro and in vivo using ethanolic and methanolic extracts. The immunomodulatory effect was evaluated by measuring antibody titers using the agglutination technique, while anti-inflammatory activity was assessed in a carrageenan-induced mouse paw edema model. In vitro immunomodulatory activity was measured by lysozyme release from neutrophils. Additionally, white blood cell counts were analyzed post-extracts treatment. The MTT assay was employed to determine cytotoxicity, and the biochemical parameters of liver toxicity were evaluated. Remarkably, both extracts exhibited a dose-dependent reduction in paw edema (p < 0.001), with the most significant reduction observed at 1 g/kg (78.13 and 74.27% for ethanolic and methanolic extracts, respectively). Neutrophil degranulation was significantly inhibited in a dose-dependent manner (p < 0.003), reaching maximal inhibition at 100 µg/mg (60.78 and 39.7% for ethanolic and methanolic extracts, respectively). In comparison to the control group, both antibody production and white blood cell counts were reduced. Neither of the extracts showcased any cytotoxicity or toxicity. These findings suggest that A. melegueta extracts exhibit immunosuppressive and anti-inflammatory activities due to the presence of various biomolecules.

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BACKGROUND: People with overweight/obesity generally have impaired immune responses, resulting among others in increased risk of severe complaints and hospitalization after infections with severe acute respiratory syndrome coronavirus 2 (COVID-19), as well as decreased antibody production after vaccinations. Plant stanol ester previously increased the combined IgM/IgG antibody titers toward a hepatitis A vaccination in patients with allergic asthma, but the underlying mechanism is unknown. OBJECTIVES: We evaluated whether plant stanol ester consumption improved the immune response in subjects with overweight/obesity after a COVID-19 vaccination. METHODS: A double-blind, randomized, placebo-controlled trial was performed. Thirty-two subjects with overweight/obesity consumed products with added plant stanols (4 g/d; provided as plant stanol ester) or control ≥2 wk before receiving their COVID-19 vaccination until 4 wk after vaccination. Antibody titers were analyzed weekly and statistically analyzed using mixed models. Serum metabolic markers and cytokine profiles were also analyzed. RESULTS: IgM concentrations against the COVID-19 Spike protein were increased in the plant stanol ester group compared with the control group, with the largest difference observed 2 wk after vaccination [31.2 (0.43, 62.1) BAU/mL, or +139%; Group × Time: P = 0.031]. Subjects that produced very low IgM antibodies produced, as expected, hardly any IgG antibodies. In those with IgG seroconversion, IgG Spike concentrations were also increased in the plant stanol ester group compared with the control group [71.3 (2.51, 140.1) BAU/mL; Group P = 0.043]. Stimulated cytokine concentrations decreased in the plant stanol ester group compared with the control group in all 3 cytokine domains (that is, proinflammatory, T helper [Th1]/Th17, and Th2/regulatory T cells). Between-group differences in serum LDL cholesterol or other metabolic markers were not observed. CONCLUSIONS: Consuming plant stanols (4 g/d) affects immune responses to COVID-19 vaccinations, translating into increased serum anti-COVID-19 IgM concentrations in subjects with overweight/obesity. Only in IgG seroconverted subjects, serum anti-COVID-19 IgG concentrations also increase. These effects are independent of reductions in LDL cholesterol. These results suggest that this high-risk group for COVID-19 complications could benefit from plant stanol consumption. This trial was registered at clinicaltrials.gov as NCT04844346.

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Vaccination is important for reducing disease incidence in the poultry industry. To enhance immunity and vaccine efficacy, chicken cytokines associated with antibody production must be identified. In this study, we focused on interleukin-5 (IL-5), involved in antibody production in mice, measuring its expression and effects on antibody production. Concanavalin A-stimulated splenocytes were used for RT-PCR to clone IL5 cDNAs. Recombinant IL-5 was prepared from the clone and administered to chickens with antigen via the ocular-topical route twice every alternate week. IL-5 enhanced antigen-specific IgY and inhibited antigen-specific serum IgA production in serum. Our findings suggest that IL-5 plays an important role in chicken antibody production, with possible unique functions.

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Autoreactive B cells play a critical role in rheumatoid arthritis (RA). These cells differentiate into long-living memory B cells and autoantibody-producing plasma cells, and also present autoantigens to T cells to amplify misdirected immune responses. The therapeutic benefit of B-cell-deleting depleting therapies suggests that B cells are emerging as important factors in the pathogenesis of RA. Aiming at evaluation of the function of B cells, which are usually derived from peripheral blood of RA patients and healthy donors, it is possible to conduct a series of experiments, such as in vitro assessment of antibody production and BCR-mediated cytokine release. These techniques can also be applied for in vivo application.

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Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.

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Antibodies are potent biopharmaceuticals used to treat severe diseases, including cancers. During the past decade, more complex modalities have been developed including bispecific T-cell engager (BiTE®) molecules, e.g. by Amgen. However, non-natural and complex molecule formats often prove to be difficult-to-express (DTE), which is the case for BiTE® molecules. Due to the growing importance of multispecific modalities such as half-life extended (HLE) BiTE® and HLE dual-targeting bispecific T-cell engager (dBiTE) molecules, this artificial class of therapeutic proteins was investigated for molecular bottlenecks in stable production cell lines, by analyzing all relevant steps of recombinant protein production. As a result, drastically reduced intracellular BiTE® molecule-encoding mRNA levels were identified as a potential production bottleneck. Using in vitro transcription (IVT), the transcription rate of the BiTE® molecule-encoding mRNA was identified as the root cause for reduced amounts of intracellular mRNA. In an attempt to improve the transcription rate of a BiTE® molecule, it could be demonstrated that the artificial and special structure of the BiTE® molecule was not the rate limiting step for reduced IVT rate. However, modulation of the primary DNA sequence led to significant improvement of IVT rate. The analyses presented provide insight into the HLE BiTE® / HLE d(BiTE®) class of DTE proteins and perhaps into other classes of DTE proteins, and therefore may lead to identification of further production bottlenecks and optimization strategies to overcome manufacturability challenges associated with various complex therapeutics.

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