Antibody Secretion Research Articles

Comparative Analysis of IMT-P8 and LDP12 Cell-Penetrating Peptides in Increasing Immunostimulatory Properties of HIV-1 Nef-MPER-V3 Antigen.

There have been great efforts in vaccine design against HIV-1 since 1981. Various approaches have been investigated, including optimized delivery systems and effective adjuvants to enhance the efficacy of selective antigen targets. In this study, we evaluated the efficiency of IMT-P8 and LDP12 cell penetrating peptides in eliciting immune responses against HIV-1 Nef-MPER-V3 fusion protein as an antigen candidate. Moreover, the potency of HP91 and HSP27 was compared as an adjuvant in female BALB/c mice through different regimens. For this purpose, the recombinant Nef-MPER-V3, IMT-P8-Nef-MPER-V3 and LDP-Nef- MPER-V3 proteins were generated on a large scale. After mice immunization with different regimens, the secretion of antibodies, cytokines and granzyme B was evaluated by ELISA. Our results demonstrated that immunized mice receiving the Nef-MPER-V3 linked to IMT-P8 exhibited significantly higher levels of IgG compared to other groups. The IMT-P8-Nef- MPER-V3 with the Hp91 group showed the highest level of humoral response, which was significantly stronger than the LDP12 formulation using the same antigen (LDP-Nef-MPER-V3). Additionally, the combination of IMT-P8-Nef-MPER-V3 with either Hp91 or Hsp27 resulted in robust induction of IFN-γ compared to the LDP-Nef-MPER-V3 group. Furthermore, cytotoxic T lymphocyte (CTL) activation and proliferation assays indicated that IMT-P8 served as a more effective CPP, particularly when used in conjunction with the Hp91 adjuvant Conclusion: Altogether, the data indicated that Nef-MPER-V3 antigen in different formulations was effective in eliciting immune responses. This fusion protein has the high potency to induce both immunity arms, specifically when incorporated with IMT-P8, which showed priority to LDP12. Moreover, HP91 resulted in a greater humoral and cellular immune activation compared to HSP27. These findings suggest the potential of IMT-P8 as a superior delivery system for enhancing immune responses in vaccine development.

Minimalist Adjuvant-Free Nano-Vaccine Based on Antigen Self-Assembled Amyloid-Like Fibrils to Induce Potent Immune Response.

The development of cancer vaccines is at the forefront of cancer immunotherapy. Most existing strategies to induce an efficient anti-tumor immune response rely on molecular adjuvants and the incorporation of complex synthetic vectors into vaccine formulations. In contrast, this study introduces a one-step engineering technique to assemble the model antigen, Ovalbumin (OVA), into amyloid aggregates, leveraging biomimetic folding and aggregation to create non-fibrillar OVA globular aggregates and OVA amyloid-like fibrils as single-component, adjuvant-free vaccines. Notably, the OVA amyloid-like fibrils induced stronger immune responses compared to the native form, as evidenced by robust humoral immune reactions and the establishment of immune memory. These enhanced responses can be attributed to the self-adjuvant effect of the unique assembled structure, which preserves antigenic epitopes, improves antigen stability, facilitates antigen internalization, prolongs retention at the injection site, enhances antigen trafficking to the lymphoid organs, and promotes increased secretion of antibodies and cytokines. Furthermore, the efficacy of the vaccine was validated in a high OVA-expressing tumor model, demonstrating the potential of OVA amyloid-like fibrils as an effective vaccine for cancer immunoprevention. This minimalist self-adjuvant vaccine strategy holds promising implications for cancer immunotherapy and can inform the design of other protein antigen-based vaccines.

Effectiveness and safety study of formula containing probiotics, prebiotics, synbiotics on fullterm infants' growth - a systematic review and meta-analysis of randomized controlled study.

Probiotics, prebiotics, and synbiotics, are hot topics of research and have been shown to improve the body's disease state and promote health. Analysis of whether infant formula containing probiotcs, prebiotics, synbiotics is beneficial to infant and child growth. We systematically searched multiple electronic databases (PubMed, Web of Science, The Cochrane Library, Embase) to identify eligible studies published from 1966 to December 25, 2022. Included studies were randomized controlled trials (RCTs) studying the influence of milk powder containing probiotcs, prebiotics, synbiotics on infants and children's growth. RevMan 5.4 was used to analyze the data. A total of 55 RCTs with a total sample size of 8868 participants met the inclusion criteria. Milk powder with probiotics, prebiotics, synbiotics does not significantly improve the growth of infants and children (Weight, height, BMI, and Head Circumference); The incidence of minor adverse events (OR 0.88, 95% CI 0.70-1.11 P = 0.28) and serious adverse events (OR 0.92, 95% CI 0.62-1.36 P = 0.67) was also comparable to the control group; The intestinal microbial diversity of infants consuming probiotcs, prebiotics, synbiotics supplemented formula was lower than that of infants consuming formula without probiotcs, prebiotics, synbiotics (SMD -0.88, 95% CI -1.66- -0.1 P = 0.03), but the abundance of individual beneficial flora was increased. (SMD 1.62, 95%CI 0.61-2.62 P = 0.002). In particular, the abundance of Lactobacillus (SMD 1.62, 95% CI 0.61-2.62 P = 0.002). For metabolites, synbiotics increased fecal antibody concentrations (SMD 0.47, 95% CI 0.08-0.86 P = 0.02), but fecal short-chain fatty acid concentrations remained balanced in both groups (SMD 0.05 95% CI -0.17-0.28 P = 0.64). Compared to the control group, infants who consumed formula with prebiotics had softer stools (SMD -1.47, 95% CI -2.23 to -0.7 P = 0.002) and lower stool pH (SMD -0.82, 95% CI -1.15- -0.5 P < 0.00001), there is also more frequency of bowel movements (SMD 0.27, 95% CI 0.09-0.44 P = 0.002). Probiotcs, prebiotics, synbiotics supplemented formulas significantly increased abundance of individual probiotics, alter intestinal antibody secretion, and improve bowel movements. Incidence of adverse reactions did not differ between the two groups. So we can choose formula-supplemented probiotcs, prebiotics, synbiotics to maintain the intestinal health of infants.

Repeated COVID-19 mRNA-based vaccination contributes to SARS-CoV-2 neutralizing antibody responses in the mucosa.

To prevent infection by respiratory viruses and consequently limit virus circulation, vaccines need to promote mucosal immunity. The extent to which the currently used messenger RNA (mRNA)-based COVID-19 vaccines induce mucosal immunity remains poorly characterized. We evaluated mucosal neutralizing antibody responses in a cohort of 183 individuals. Participants were sampled at several time points after primary adenovirus vector-based or mRNA-based COVID-19 vaccination and after mRNA-based booster vaccinations. Our findings revealed that repeated vaccination with mRNA boosters promoted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in nasal secretions. Nasal and serum neutralizing antibody titers of both IgG and IgA isotypes correlated to one another. We investigated the source of these mucosal antibodies in a mouse model wherein mice received repeated mRNA vaccines for SARS-CoV-2. These experiments indicated that neutralizing antibody-producing cells reside in the spleen and bone marrow, whereas no proof of tissue homing to the respiratory mucosa was observed, despite the detection of mucosal antibodies. Serum transfer experiments confirmed that circulating antibodies were able to migrate to the respiratory mucosa. Collectively, these results demonstrate that, especially upon repeated vaccination, the currently used COVID-19 mRNA vaccines can elicit mucosal neutralizing antibodies and that vaccination might also stimulate mucosal immunity induced by previous SARS-CoV-2 infection. Moreover, migration of circulating antibodies to the respiratory mucosa might be a main mechanism. These findings advance our understanding of mRNA vaccine-induced immunity and have implications for the design of vaccine strategies to combat respiratory infections.

Enhanced tumoricidal activity of PD-1 antibody-secreting c-Met CAR-T cells against pancreatic cancer cells

To construct c-Met CAR-T cells secreting PD-1 antibodies to reduce immune inhibitory effect of tumor cells and enhance the efficacy of CAR-T cell therapy against pancreatic cancer. Kaplan-Meier Plotter, GEPIA, and Timer 2.0 bioinformatics databases were used to analyze c-Met expression in pancreatic cancer and its correlation with survival and immune infiltration status. In clinical samples of pancreatic cancer and pancreatic cancer Aspc-1 cells, c-Met and PD-L1 expressions were detected using immunohistochemistry or flow cytometry. Using gene editing technology, PD-1 secretory antibodies and HIS tags were linked to second-generation c-Met CAR molecules to construct PD-1/c-Met CAR plasmids, which were then packaged into lentiviruses for infection of activated T cells. The positive rate and cell subset distribution of CAR-T cells were analyzed with flow cytometry, and secretory PD-1 antibodies in cell supernatants were detected using Western blotting. The target cell killing efficiency and proliferative activity of the modified CAR-T cells were evaluated after activation, and cytokine secretion was analyzed using ELISA. The expression of c-Met was significantly higher in pancreatic cancer than in normal tissues, and its expression level was negatively correlated with the patients' survival and positively correlated with immune cell infiltration. The clinical samples of pancreatic cancer tissues expressed significantly higher levels of c-Met and PD-L1 than the adjacent tissues, and 90.7% and 57.7% of Aspc-1 cells were positive for c-Met and PD-L1, respectively. The constructed PD-1/c-Met CAR-T cells were capable of secreting PD-1 antibodies and showed a significantly higher killing efficiency against tumor cells than c-Met CAR-T cells at an effector-to-target ratio of 20: 1, with also a higher proliferative activity after target cell stimulation and higher levels of IL-2 and TNF-α secretin. PD-1/c-Met CAR-T cells have higher killing efficiency against pancreatic cancer cells with also higher proliferative activity than c-Met CAR-T cells.

NF-κB pathway activation by Octopus peptide hydrolysate ameliorates gut dysbiosis and enhances immune response in cyclophosphamide-induced mice

Cyclophosphamide (CTX) is an anticancer medication that suppresses host immunity as well as adversely affects mucosal inflammation and gut microflora dysbiosis. The gut microflora is recognized as a substantial factor in host metabolism and immunological homeostasis. To improve immunity and inhibit cytotoxic and homeostatic imbalances triggered by CTX, it is essential to monitor immunoregulators. In this research, we assessed the impact of Octopus peptide hydrolysate (OPH) on immune modulation, intestinal integrity, and gut microbial composition in CTX-induced immune-deficient mice. The results revealed that OPH increased body weight, and immunological organ indices, and improved the histological changes in the colon, thymus, and spleen. The OPH stimulated the secretion of cytokines (IL-1β, IL-6, and TNF-α) and antibodies (IgM and IgA) while reducing the ratio of lipopolysaccharide (LPS) and diamine oxidase (DAO) in the serum. OPH further enhanced goblet cell and mucus production, upregulated the expression of gut tight-junction proteins (Occludin, Zonula Occludin-1, Mucin-2, and Claudin-1), and activated the TLR4/NF-κB cascade (p-IκBα, P65/p-p65). In addition, OPH treatment declined the Bacteroidetes/Firmicutes ratio, enhanced the relative ratio of Alistipes/Lachnospiraceae, and reversed the ecological equilibrium of the gut microflora. The findings revealed that OPH serves as a prebiotic to prevent CTX-mediated disruption in the intestinal barrier and boosts gut mucosal immunity by attenuating gut microflora imbalance, implying that OPH could be used as an immunological ingredient in nutritious foods to regulate the immune system and protect the gut from inflammatory diseases.

Bovine Respiratory Syncytial Virus Nanovaccine Induces Long-Lasting Humoral Immunity in Mice.

With limited therapies and vaccines available, human respiratory syncytial virus (HRSV) has a significant negative health impact on all age groups but particularly on infants, young children, and older adults. Bovine respiratory syncytial virus (BRSV) is pathogenically and antigenically similar to HRSV. Building upon previous studies using a BRSV nanovaccine coencapsulating multiple proteins, this work demonstrates the development and comparative evaluation of a coencapsulated nanovaccine to a cocktail nanovaccine formulation composed of polyanhydride nanoparticles encapsulating BRSV postfusion (F) glycoprotein and CpG ODN 1668 coadjuvant delivered simultaneously with nanoparticles encapsulating BRSV attachment glycoprotein (G) and CpG ODN 1668. These nanovaccine formulations were administered to C57BL/6 mice by one of two prime-boost regimens (i.e., intranasal/intranasal or intranasal/subcutaneous) followed by assessment of humoral immunity. The cocktail nanovaccine induced sustained anti-F and anti-G serum IgG antibody responses for 12 weeks postprimary immunization. Using polyanhydride particles to deliver G protein in a prime-boost regime also significantly induced serum anti-G antibodies compared to protein and coadjuvant alone. Serum IgG induced by the nanovaccine demonstrated virus-neutralizing capability from 42 to 119 days postprimary immunization. Further, anti-F IgG antibodies were detected in the bronchoalveolar lavage fluid of vaccinated animals. Finally, the nanovaccine induced long-lived anti-F antibody secreting plasma cells that were detectable in the bone marrow 205 days postprimary immunization. Overall, the BRSV nanovaccine(s) successfully induced long-lived humoral immune responses capable of virus neutralization, making this a promising vaccine candidate for further evaluation in other relevant animal models.

Immunometabolic effects of lactate on humoral immunity in healthy individuals of different ages

Aging is characterized by chronic systemic inflammation and metabolic changes. We compare the metabolic status of B cells from young and elderly donors and found that aging is associated with higher oxygen consumption rates, and especially higher extracellular acidification rates, measures of oxidative phosphorylation and of anaerobic glycolysis, respectively. Importantly, this higher metabolic status, which reflects age-associated expansion of pro-inflammatory B cells, is found associated with higher secretion of lactate and autoimmune antibodies after in vitro stimulation. B cells from elderly individuals induce in vitro polarization of CD4+ T cells from young individuals into pro-inflammatory CD4+ T cells through metabolic pathways mediated by lactate, which can be inhibited by targeting lactate enzymes and transporters, as well as signaling pathways supporting anaerobic glycolysis. Lactate also induces immunosenescent B cells that are glycolytic, express transcripts for multiple pro-inflammatory molecules, and are characterized by a higher metabolic status. These results altogether may have relevant clinical implications and suggest alternative targets for therapeutic interventions in the elderly and patients with inflammatory conditions and diseases.

B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

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.

The Structural Characterization of a Polysaccharide from the Dried Root of Salvia miltiorrhiza and Its Use as a Vaccine Adjuvant to Induce Humoral and Cellular Immune Responses.

In order to supplement the research gap concerning Salvia miltiorrhiza polysaccharide extracted from Danshen in NMR analysis, and to clarify its immune enhancement effect as an adjuvant, we isolated and purified SMPD-2, which is composed of nine monosaccharides such as Ara, Gal, and Glc from Danshen. Its weight average molecular weight was 37.30 ± 0.096 KDa. The main chain was mainly composed of →4)-α-D-Galp-(1→, →3,6)-β-D-Glcp-(1→ and a small amount of α-L-Araf-(1→. After the subcutaneous injection of SMPD-2 as an adjuvant to OVA in mice, we found that it enhanced the immune response by activating DCs from lymph nodes, increasing OVA-specific antibody secretion, stimulating spleen lymphocyte activation, and showing good biosafety. In conclusion, SMPD-2 could be a promising candidate for an adjuvant.

New Therapies for Highly Sensitized Patients on the Waiting List.

Exposure to HLA alloantigens through pregnancy, blood products, and previous transplantations induce powerful immunologic responses that create an immunologic barrier to successful transplantation. This is commonly detected through screening for HLA antibodies using Luminex beads coated with HLA antigens at transplant evaluation. Currently accepted approaches to desensitization include plasmapheresis/low-dose or high-dose intravenous Ig plus anti-CD20. However, these approaches are often unsuccessful because of the inability to remove high titer circulating HLA antibodies and limit rebound responses by long-lived anti-HLA antibody secreting plasma cells (PCs) and memory B cells (B MEM ). This is especially significant for patients with a calculated panel reactive antibody of 99%-100%. Newer desensitization approaches, such as imlifidase (IgG endopeptidase), rapidly inactivate IgG molecules and create an antibody-free zone by cleaving IgG into F(ab'2) and Fc fragments, thus eliminating complement and cell-mediated injury to the graft. This represents an important advancement in desensitization. However, the efficacy of imlifidase is limited by pathogenic antibody rebound, increasing the potential for antibody-mediated rejection. Controlling antibody rebound requires new strategies that address the issues of antibody depletion and inhibition of B MEM and PC responses. This will likely require a combination of agents that effectively and rapidly deplete pathogenic antibodies and prevent immune cell activation pathways responsible for antibody rebound. Here, using anti-IL-6 receptor (tocilizumab) or anti-IL-6 (clazakizumab) could offer long-term control of B MEM and PC donor-specific HLA antibody responses. Agents aimed at eliminating long-lived PCs (anti-CD38 and anti-B-cell maturation antigen×CD3) are likely to benefit highly HLA sensitized patients. Complement inhibitors and novel agents aimed at inhibiting Fc neonatal receptor IgG recycling will be important in desensitization. Administering these agents alone or in combination will advance our ability to effectively desensitize patients and maintain durable suppression post-transplant. After many years of limited options, advanced therapeutics will likely improve efficacy of desensitization and improve access to kidney transplantation for highly HLA sensitized patients.

Varied immune responses of HBV-specific B cells in patients undergoing pegylated interferon-alpha treatment for chronic hepatitis B

The changes in HBV-specific B cells in patients with chronic hepatitis B (CHB) undergoing pegylated interferon-α (PEG-IFNα) treatment and achieving functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B cells. We included 39 nucleos(t)ide analogue-treated patients with CHB who received sequential combination therapy with PEG-IFNα and eight treatment-naïve patients. HBV-specific B cells were characterized exvivo using fluorescently labeled hepatitis B surface and core antigens (HBsAg and HBcAg). The frequency, phenotype, and subsets of HBV-specific B cells and follicular helper T cells (Tfh cells) were detected using flow cytometry. The functionality of HBV-specific B cells was quantified through ELISpot assays. During treatment, the fraction of activated memory B cells (MBCs) among HBsAg-specific B cells and the expression of IgG, CXCR3, and CD38 increased. The antibody-secretion capacity of HBsAg-specific B cells was only restored in patients achieving a functional cure after treatment and it positively correlated with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts among HBsAg-specific B cells. HBcAg-specific B cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBC after treatment, irrespective of functional cure. The number of CD40L+ Tfh cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. After PEG-IFNα treatment, HBsAg- and HBcAg-specific B cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh cells is associated with the active recovery of HBsAg-specific B cells. HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of patients with chronic hepatitis B offers a promising therapeutic strategy for viral clearance, e.g. via therapeutic vaccination. We analyzed the alterations in HBV-specific B cells in patients treated with pegylated interferon-α and identified novel pathways for immunotherapeutic boosting of B cell immunity.

Chitosan-alginate/R8 ternary polyelectrolyte complex as an oral protein-based vaccine candidate induce effective mucosal immune responses

Vaccination is the most effective method for preventing infectious diseases. Oral vaccinations have attracted much attention due to the ability to boost intestinal and systemic immunity. The focus of this study was to develop a poly (lactide-co-glycolide) acid (PLGA)-based ternary polyelectrolyte complex (PEC) with chitosan, sodium alginate, and transmembrane peptides R8 for the delivery of antigen proteins. In this study, the antigen protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis (MAP) antigens HBHA, Ag85B, and Bfra, was combined with R8 to generate self-assembled conjugates. The results showed that PEC presented a cross-linked reticular structure to protect the encapsulated proteins in the simulated gastric fluid. Then, the nanocomposite separated into individual nanoparticles after entering the simulated intestinal fluid. The ternary PEC with R8 promoted the in vivo uptake of antigens by intestinal lymphoid tissue. Moreover, the ternary PEC administered orally to mice promoted the secretion of specific antibodies and intestinal mucosal IgA. In addition, in the mouse models of MAP infection, the ternary PEC enhanced splenic T cell responses, thus reducing bacterial load and liver pathology score. These results suggested that this ternary electrolyte complex could be a promising delivery platform for oral subunit vaccine candidates, not limited to MAP infection.

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19×anti-CD3 (blinatumomab) or anti-CD33×anti-CD3 bispecific antibodies mediate Tcell activation and direct Tcell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines invitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited invivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous Tcells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing Tcells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Purification of secretory IgA monoclonal antibodies enriched fraction directly from cell culture medium using aqueous two-phase systems

Secretory immunoglobulin A [sIgA] is a promising candidate for enteric therapeutics applications, and several sIgA-based constructs are currently being developed by groups utilizing clarified Chinese hamster ovary [CHO] cell culture supernatants. To the monoclonal antibody downstream processing typically entails chromatography-based purification processes beginning with Protein A chromatography. In this paper, aqueous two-phase systems [ATPS] were employed for the preliminary purification of secretory immunoglobulin A [sIgA] monoclonal antibody [mAb] from clarified CHO-cell culture supernatants. A 24 full factorial design was utilized. The influence of various process parameters such as pH, PEG molecular weight [MPEG], PEG concentration [CPEG], and phosphate salt concentration [CPHO], on the sIgA partition coefficient [K sIgA] and the recovery index [Y] in the PEG phase were evaluated. The Elisa assay revealed that, in the ATPS conditions tested, sIgA mAb was mostly detected in PEG upper phase. Run 14 with the highest sIgA activity exhibited the following conditions: MPEG 8.000 g/mol, CPEG 12,5 %, pH 7,0 and CPHO 10 %, and a sIgA K of 94.50 and a recovery index [Y] of 33.52 %. The proposed platform provides straightforward implementation, yields comparable results, and offers significantly improved economics for manufacturing sIgA mAb biotherapeutics.

Oxidized ATP Suppresses B Lymphocyte Activity to Attenuate Antibody-mediated Rejection of Kidney Allografts in Mice

Background. Antibody-mediated rejection (AMR) is a major cause of renal allograft dysfunction and loss. Targeting B cells and/or donor-specific antibody removal using plasma exchange and anti-CD20 antibodies are increasingly used in clinical practice, but the efficacy remains limited. Recent studies suggest that targeting purinergic P2X7 receptor/ATP axis can have profound immune regulatory effects in transplant models, but the mechanisms involved remain incompletely defined. Methods. Purified B cells were isolated from the spleen of Balb/C mice and cultured with oxidized ATP at different concentrations. Proliferation and differentiation of B cells were examined. Effects of oxidized ATP were examined in a presensitized animal model where kidney allograft rejection mimics aspects of clinical AMR. Histopathology was assessed at the time of rejection or on day 5 after kidney transplantation. Infiltrating immune cells in renal allografts were detected by flow cytometry. Results. Oxidized ATP inhibited B-cell activation and proliferation in vitro, significantly attenuated histological signs of graft injury and prolonged kidney allograft survival. Mechanistically, oxidized ATP inhibited antibody secretion by activated B cells in response to lipopolysaccharide stimulation and markedly suppressed the production of donor-specific antibody in kidney allograft recipients. Oxidized ATP also reduced graft infiltration by other inflammatory cells. Conclusions. These findings provide evidence for the involvement of the purinergic P2X7 receptor pathway in AMR and suggest that targeting this pathways may have important clinical implications.

PCK2 maintains intestinal homeostasis and prevents colitis by protecting antibody-secreting cells from oxidative stress.

Maintaining intracellular redox balance is essential for the survival, antibody secretion, and mucosal immune homeostasis of immunoglobulin A (IgA) antibody-secreting cells (ASCs). However, the relationship between mitochondrial metabolic enzymes and the redox balance in ASCs has yet to be comprehensively studied. Our study unveils the pivotal role of mitochondrial enzyme PCK2 in regulating ASCs' redox balance and intestinal homeostasis. We discover that PCK2 loss, whether globally or in B cells, exacerbates dextran sodium sulphate (DSS)-induced colitis due to increased IgA ASC cell death and diminished antibody production. Mechanistically, the absence of PCK2 diverts glutamine into the TCA cycle, leading to heightened TCA flux and excessive mitochondrial reactive oxygen species (mtROS) production. In addition, PCK2 loss reduces glutamine availability for glutathione (GSH) synthesis, resulting in a decrease of total glutathione level. The elevated mtROS and reduced GSH expose ASCs to overwhelming oxidative stress, culminating in cell apoptosis. Crucially, we found that the mitochondria-targeted antioxidant Mitoquinone (Mito-Q) can mitigate the detrimental effects of PCK2 deficiency in IgA ASCs, thereby alleviating colitis in mice. Our findings highlight PCK2 as a key player in IgA ASC survival and provide a potential new target for colitis treatment.

Stability Engineering of Recombinant Secretory IgA.

Secretory IgA (SIgA) presents a promising avenue for mucosal immunotherapy yet faces challenges in expression, purification, and stability. IgA exists in two primary isotypes, IgA1 and IgA2, with IgA2 further subdivided into two common allotypes: IgA2m(1) and IgA2m(2). The major differences between IgA1 and IgA2 are located in the hinge region, with IgA1 featuring a 13-amino acid elongation that includes up to six O-glycosylation sites. Furthermore, the IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and IgA2m(2). While IgA1 demonstrates superior epitope binding and pathogen neutralization, IgA2 exhibits enhanced effector functions and stability against mucosal bacterial degradation. However, the noncovalent linkage in the IgA2m(1) allotype raises production and stability challenges. The introduction of distinct single mutations aims to facilitate an alternate disulfide bond formation to mitigate these challenges. We compare four different IgA2 versions with IgA1 to further develop secretory IgA antibodies against SARS-CoV-2 for topical delivery to mucosal surfaces. Our results indicate significantly improved expression levels and assembly efficacy of SIgA2 (P221R) in Nicotiana benthamiana. Moreover, engineered SIgA2 displays heightened thermal stability under physiological as well as acidic conditions and can be aerosolized using a mesh nebulizer. In summary, our study elucidates the benefits of stability-enhancing mutations in overcoming hurdles associated with SIgA expression and stability.

Molecular Events in Immune Responses to Sublingual Influenza Vaccine with Hemagglutinin Antigen and Poly(I:C) Adjuvant in Nonhuman Primates, Cynomolgus Macaques.

Sublingual vaccines offer the benefits of inducing mucosal immunity to protect against respiratory viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and influenza, while also enabling needle-free self-administration. In a previous study, a sublingual SARS-CoV-2 vaccination was created by combining a recombinafigureCoV-2 spike protein receptor-binding domain antigen with a double strand RNA Poly(I:C) adjuvant. This vaccine was tested on nonhuman primates, Cynomolgus macaques. This study examined the immune and inflammatory responses elicited by the sublingual influenza vaccine containing hemagglutinin (HA) antigen and Poly(I:C) adjuvants, and assessed the safety of this vaccine in nonhuman primates. The Poly(I:C)-adjuvanted sublingual vaccine induced both mucosal and systemic immunities. Specifically, the sublingual vaccine produced HA-specific secretory IgA antibodies in saliva and nasal washings, and HA-specific IgA and IgG were detected in the blood. This vaccine appeared to be safe, as judged from the results of blood tests and plasma C-reactive protein levels. Notably, sublingual vaccination neither increased the production of inflammation-associated cytokines-IFN-alpha, IFN-gamma, and IL-17-in the blood, nor upregulated the gene expression of proinflammatory cytokines-IL12A, IL12B, IFNA1, IFNB1, CD69, and granzyme B-in white blood cells. Moreover, DNA microarray analyses revealed that sublingual vaccination evoked both enhancing and suppressing expression changes in genes associated with immune-related responses in cynomolgus monkeys. Therefore, the sublingual vaccine with the Poly(I:C) adjuvant is safe, and creates a balanced state of enhancing and suppressing the immune-related response.

In vivo tracking of ex vivo generated 89 Zr-oxine labeled plasma cells by PET in a non-human primate model.

B cells are an attractive platform for engineering to produce protein-based biologics absent in genetic disorders, and potentially for the treatment of metabolic diseases and cancer. As part of pre-clinical development of B cell medicines, we demonstrate a method to collect, ex vivo expand, differentiate, radioactively label, and track adoptively transferred non-human primate (NHP) B cells. These cells underwent 10- to 15-fold expansion, initiated IgG class switching, and differentiated into antibody secreting cells. Zirconium-89-oxine labeled cells were infused into autologous donors without any preconditioning and tracked by PET/CT imaging. Within 24 hours of infusion, 20% of the initial dose homed to the bone marrow and spleen and distributed stably and equally between the two. Interestingly, approximately half of the dose homed to the liver. Image analysis of the bone marrow demonstrated inhomogeneous distribution of the cells. The subjects experienced no clinically significant side effects or laboratory abnormalities. A second infusion of B cells into one of the subjects resulted in an almost identical distribution of cells, suggesting a non-limiting engraftment niche and feasibility of repeated infusions. This work supports the NHP as a valuable model to assess the potential of B cell medicines as potential treatment for human diseases.