Enhanced Antibody Production Research Articles

Safety and Effectiveness of the Pfizer-BioNTech COVID-19 Vaccine among Health Staff in Sulaimani City, Iraq: A Prospective Cohort Study

A cohort study was conducted among 210 health staff members in Sulaimani city to assess the safety and effectiveness of the Pfizer-BioNTech vaccine. They were divided into two groups: vaccinated and unvaccinated. The vaccinated group received two doses of the Pfizer vaccine, while the unvaccinated group did not receive any vaccines during this study. Vaccine reactogenicity was assessed using a self-report form. Whereas vaccine immunogenicity was assessed by testing the anti-receptor binding domain IgG (anti-RBD IgG) antibody. Several adverse effects were observed with each dose. The most frequent adverse effects were pain at the inoculation site, tiredness, myalgia and fever. The average adverse effects per person in the first and second doses were 5.3 (SD 3.3) and 6.3 (SD 3.5) respectively (p = 0.005). The immunized group's anti-RBD IgG antibody levels were greatly improved after taking the first and second doses (32.50 and 44.92 binding antibody units (BAU)/mL respectively). After eight months of taking the two doses, the antibody level dropped to 17.00 BAU/mL. This study indicates that the vaccine can be safe and effective for enhancing antibody production.

6479 Acute Estradiol and Progesterone Therapy In Hospitalized Adults With Moderate COVID-19: A Randomized Control Trial

Abstract Disclosure: D. Lovre: Research Investigator; Self; Novo Nordisk, Corcept Therapeutics. K.M. Bateman: None. L.Y. Saltzman,: None. M. Qadir: None. F. Mauvais-Jarvis: None. Background and Objective: COVID-19 outcomes are less severe in women vs men. Experimental studies show that estradiol (E2) and progesterone (P4) produce anti-inflammatory immune responses and immune tolerance and enhance antibody production. The effect of E2 and P4 combination on COVID-19 severity is unknown. The aim of the study was to evaluate the efficacy of a short systemic E2/P4 combination in mitigating COVID-19 severity in hospitalized men and women. Methods: Phase 2, double blind, placebo-equivalent controlled, single center trial of ten participants (men and women) who were hospitalized for COVID-19, with scores 3 to 5 on the 9-point World Health Organization (WHO) ordinal scale for clinical improvement. Participants were randomized into two arms and treated for five days: 1) E2 Cypionate (5 mg IM once) and micronized P4 (200mg, PO daily); and 2) Placebo-equivalent (normal saline (1ml, IM once) and folate (400mcgs, PO daily), in addition to standard of care (SOC). The primary outcome was the proportion of patients improving to scores 1 or 2 on the WHO scale on the day of discharge. Secondary outcomes included length of hospital stay (LOS), days on oxygen therapy (DOT), readmission rates (RR), adverse events (AEs) and change in inflammatory biomarkers assessed by untargeted proteomics. Findings: There were no significant differences between the treatment groups in COVID-19 severity by WHO score, LOS, DOT, RR or AEs. Patients in the E2/P4 arm exhibited a decrease in inflammatory biomarkers of respiratory and gastrointestinal disease (GI) and significant change in immune and inflammatory responses. Conclusions: Use of short-term systemic E2/P4 is associated with decrease in biomarkers of respiratory and GI disease inflammation and change in biomarkers of immune and inflammatory response. Presentation: 6/3/2024

Enhancing the Antibody Production Efficiency of Chinese Hamster Ovary Cells through Improvement of Disulfide Bond Folding Ability and Apoptosis Resistance.

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.

Evaluation of NAD+ precursors for improved metabolism and productivity of antibody-producing CHO cell.

In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD+) under the hypothesis that NAD+ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD+ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD+ precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD+ biosynthesis was verified, and then the effect of NAD+ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD+ levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD+ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD+ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD+ precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD+ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD+ precursors as medium and feed components for the biopharmaceutical production.

Comparative Analysis of Viral Load and Cytokines during SARS-CoV-2 Infection between Pregnant and Non-Pregnant Women.

To better understand the vulnerabilities of pregnant women during the COVID-19 pandemic, we conducted a comprehensive, retrospective cohort study to assess differences in immune responses to SARS-CoV-2 infection between pregnant and non-pregnant women. Nasopharyngeal swabs and serum specimens from 90 pregnant and 278 age-matched non-pregnant women were collected from 15 March 2020 to 23 July 2021 at NewYork-Presbyterian Queens Hospital in New York City. Multiplex reverse transcription polymerase chain reaction, neutralizing antibody, and cytokine array assays were used to assess the incidence, viral load, antibody titers and profiles, and examine cytokine expression patterns. Our results show a lower incidence of SARS-CoV-2 infection in pregnant women compared with non-pregnant women. Pregnant women infected with SARS-CoV-2 exhibited a substantially lower viral load. In addition, the levels of both anti-spike protein receptor-binding domain IgG neutralizing antibodies and anti-N Protein IgG were elevated in pregnant women. Finally, cytokine profiling revealed differential expression of leptin across cohorts. These findings suggest that pregnancy is associated with distinct immune and virological responses to SARS-CoV-2 infection, characterized by lower infection rates, substantially lower viral loads, and enhanced antibody production. Differential cytokine expression indicates unique immune modulation in pregnant women.

Glycolipid antigen recognition by invariant natural killer T cells and its role in homeostasis and antimicrobial responses.

Due to the COVID-19 pandemic, the importance of developing effective vaccines has received more attention than ever before. To maximize the effects of vaccines, it is important to select adjuvants that induce strong and rapid innate and acquired immune responses. Invariant natural killer T (iNKT) cells, which constitute a small population among lymphocytes, bypass the innate and acquired immune systems through the rapid production of cytokines after glycolipid recognition; hence, their activation could be used as a vaccine strategy against emerging infectious diseases. Additionally, the diverse functions of iNKT cells, including enhancing antibody production, are becoming more understood in recent years. In this review, we briefly describe the functional subset of iNKT cells and introduce the glycolipid antigens recognized by them. Furthermore, we also introduce novel vaccine development taking advantages of iNKT cell activation against infectious diseases.

Lactobacilli-derived adjuvants combined with immunoinformatics-driven multi-epitope antigens based approach protects against Clostridium perfringens in a mouse model

Clostridium perfringens is ubiquitously distributed and capable of secreting toxins, posing a significant threat to animal health. Infections caused by Clostridium perfringens, such as Necrotic Enteritis (NE), result in substantial economic losses to the livestock industry annually. However, there is no effective commercial vaccine available. Hence, we set out to propose an effective approach for multi-epitope subunit vaccine construction utilizing biomolecules. We utilized immunoinformatics to design a novel multi-epitope antigen against C. perfringens (CPMEA). Furthermore, we innovated novel bacterium-like particles (BLPs) through thermal acid treatment of various Lactobacillus strains and selected BLP23017 among them. Then, we detailed the structure of CPMEA and BLPs and utilized them to prepare a multi-epitope vaccine. Here, we showed that our vaccine provided full protection against C. perfringens infection after a single dose in a mouse model. Additionally, BLP23017 notably augmented the secretion of secretory immunoglobulin A (sIgA) and enhanced antibody production. We conclude that our vaccine possess safety and high efficacy, making it an excellent candidate for preventing C. perfringens infection. Moreover, we demonstrate our approach to vaccine construction and the preparation of BLP23017 with distinct advantages may contribute to the prevention of a wider array of diseases and the novel vaccine development.

Exploring the multifaceted effects of Ammi visnaga: subchronic toxicity, antioxidant capacity, immunomodulatory, and anti-inflammatory activities

ABSTRACT Ammi visnaga (A. visnaga) is an annual herb that has been used in traditional medicine to treat various ailments attributed to the presence of its bioactive compounds. The purpose of this study was to identify and examine the phytochemical properties of the hydroalcoholic extract of A. visnaga using in vitro and in vivo models. Our findings demonstrated that the extract contained a variety of beneficial components, including phenols, flavonoids, tannins, coumarins, saponins, khellin, and visnagin. The total polyphenolic content and total flavonoid content were 23.26 mg/GAE/g dry weight and 13.26 mg/GAE/g dry weight, respectively. In vitro tests demonstrated that the extract possessed antioxidant properties as evidenced by the ability to scavenge free radicals, including DPPH, ABTS, nitric oxide (NO), phosphomolybdate, and ferric-reducing antioxidant power (FRAP). Further, the extract was found to inhibit hydrogen peroxide (H2O2)-induced hemolysis. In a 90-d in vivo study, female Wistar rats were administered 1 g/kg of A. visnaga extract orally resulting in a significant increase in total white blood cell count. Although morphological changes were observed in the liver, no marked alterations were noted in kidneys and spleen. In a female Swiss albino mice model of acetic acid-induced vascular permeability, A. visnaga significantly inhibited extravasations of Evans blue at doses of 0.5 or 1 g/kg with inhibition percentages of 51 and 65%, respectively, blocking tissue necrosis. The extract also demonstrated potential immunomodulatory properties in mice by enhancing antibody production in response to antigens. In silico molecular docking studies demonstrated a strong affinity between khellin or visnagin and immunomodulatory proteins, NF-κB, p52, and TNF-α. These findings suggest that A. visnaga may be considered a beneficial antioxidant with immunomodulatory properties and might serve as a therapeutic agent to combat certain diseases.

The non-ionizing electromagnetic stimulation enhanced antibody production (NESEAP) effect - Discovery and technological applications.

The rise of biological therapeutics in the global pharmaceuticals market has escalated the demand for quality monoclonal antibodies for healthcare and scientific applications. Reducing costs while enhancing production yields without compromising quality are the main challenges to the growth of this industry today. Over the last two decades non-ionizing radiation has been demonstrated to elicit targeted biological responses in a frequency and dose dependent manner. We hypothesize and design a millimeter wave radiation procedure to enhance the yields of antibody-producing hybridoma cell lines. We demonstrate this method enhances the production of IgA and IgG antibodies from MOPC315.BM and U13.6 cells by a factor of 24.05±3.32 and 1.41±0.03 respectively relative to untreated cells. No treatment associated cytotoxicity was observed in either cell line corroborating physiological viability of irradiated cells. Our results demonstrate proof-of-concept of a novel technique to significantly enhance antibody yields from hybridoma cells which could lead to a reduction in antibody production costs. Further studies will focus on scaling up of this technology and employment of non-contact, tuned electromagnetic stimulation of biological systems for targeted responses.

Increased inflammatory cytokines and oxidative stress enhanced antibody production in breast and prostate cancer patients with COVID-19 related depression.

Cancer management is highly dependent on the immune status of the patient. During the COVID-19 pandemic, a large number of people suffered from anxiety and depression, especially cancer patients. The effect of depression on breast cancer (BC) and prostate cancer (PC) patients, during the pandemic has been analyzed in this study. Levels of proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) and oxidative stress markers malondialdehyde (MDA) and carbonyl content (CC) were estimated in patients' serum samples. Serum antibodies against in vitro hydroxyl radical (•OH) modified pDNA (•OH-pDNA-Abs) were estimated using direct binding and inhibition ELISA. Cancer patients showed increased levels of proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) and oxidative stress markers (MDA and CC levels), which were further significantly enhanced in cancer patients with depression compared to normal healthy (NH) individuals. Increased levels of •OH-pDNA-Abs were detected in breast cancer (0.506 ± 0.063) and prostate cancer (0.441 ± 0.066) patients compared to NH subjects. Serum antibodies were found to be significantly elevated in BC patients with depression (BCD) (0.698 ± 0.078) and prostate cancer patients with depression (PCD) (0.636 ± 0.058). Inhibition ELISA also exhibited significantly high percent inhibition in BCD (68.8% ± 7.8%) and PCD (62.9% ± 8.3%) subjects compared to BC (48.9% ± 8.1%), and PC (43.4% ± 7.5%) subjects. Cancer is characterized by enhanced oxidative stress and increased inflammation, which may be exaggerated with COVID-19 related depression. High oxidative stress and compromised antioxidant homeostasis exerts alterations in DNA, leading to formation of neo-antigens, subsequently leading to the generation of antibodies. COVID-19 pandemic related depression needs to be addressed globally for improved cancer patient care and cancer disease management.

Polynucleotides Compensate for Impaired T-Dependent Antibody Production Induced in C57Bl/6 Mice by a Nucleotide-Free Diet both in Vivo and in Vitro, but a Mononucleotide-Nucleoside Mixture Is Effective Only In Vivo

Actions of nucleotides on in vitro humoral immune responses were studied in mice fed a nucleotide-free diet, a nucleotide-free diet plus a mononucleotide-nucleoside mixture or a nucleotide-free diet plus yeast RNA (polynucleotides). Cultured spleen cells from mice fed a nucleotide-free diet produced fewer numbers of antibody-secreting cells in response to a T-dependent antigen, compared with those from controls fed nucleotide-supplemented diets. Immunoglobulin M concentrations in these supernatants were significantly lower in cultured cells from mice fed the nucleotide-free diet or the nucleotide-free diet plus the mononucleotide/nucleoside mixture compared with concentrations in cells from mice fed the nucleotide-free diet plus RNA. Concanavalin A-potentiated cytokine (interleukin-4 and interleukin-5) production by purified T helper cells was also lower in cultured cells from mice fed a nucleotide-free diet than in those from mice fed nucleotide-supplemented diets. In vivo supplementation with the mononucleotide/nucleoside mixture restored impaired in vitro antibody and concanavalin A-potentiated cytokine production in mice fed a nucleotide-free diet. However, addition of RNA to the culture enhanced antibody production in spleen cells from mice of all diet groups. Supplementing the culture with RNA did not enhance mitogen-potentiated cytokine production. This in vitro action of RNA was retained after the removal of oligonucleotides (molecular weight <1000), but was reduced by modification of bases and cleavage of phosphodiester bonds of RNA. Thus the in vitro action of RNA is mainly attributed to polynucleotides, indicating their potential role in modulation of local humoral immune responses in the body.

Duck IL-7 as a novel adjuvant improves the humoral immune response to an inactivated duck tembusu virus vaccine

Duck tembusu virus (DTMUV), belonging to the Flavivirus genus, Flaviviridae family, has caused huge economic losses in the duck industry. However, the inactivated DTMUV vaccine requires multiple immunizations and has incomplete effectiveness. The humoral immune response is a key factor in the control of DTMUV infection. IL-7 derived from mammals has the ability to enhance antibody production. Whether duck IL-7 (duIL-7) possesses the ability to improve the humoral immunity of inactivated DTMUV vaccine has not yet been declared. Here, a beta-propiolactone (BPL)-inactivated DTMUV vaccine was employed to characterize the adjuvant property of duIL-7 in humoral immune responses. Intramuscular injection of DTMUV inactivated vaccine with or without duIL-7 was administered twice to the ducks. The results showed that duIL-7 was able to promote rapid antibody responses and enhance DTMUV-specific IgG and neutralizing antibody production to the vaccine. T follicular helper (Tfh) cells play a key role in assisting long humoral immunity. It was found that duIL-7 upregulated duIl-6 and duIl-21 gene expression at 3 w post first vaccination, which encode Tfh cell differentiation-related cytokines duIL-6 and duIL-21, respectively. This may be the reason that duIL-7 could prolong the humoral immune response to the inactivated DTMUV vaccine. Next, the ability of duIL-7 to simplify the immunization procedure of the inactivated DTMUV vaccine was tested. When ducks were immunized once, the titers of neutralizing antibodies in ducks from the inactivated DTMUV vaccine supplemented with duIL-7 group were significantly higher than those of ducks from the inactivated DTMUV vaccine group (P < 0.05). In addition, duIL-7 could assist the inactivated DTMUV vaccine in maintaining neutralizing antibodies at high levels during the whole experimental period. The viral titers in the ducks immunized with the inactivated DTMUV vaccine and duIL-7 were lower than those in the ducks immunized with the inactivated DTMUV vaccine alone at 3 days post infection (3 dpi, P < 0.05). Overall, duIL-7 possessed the ability to promote and prolong humoral immune responses to the inactivated DTMUV vaccine, even at one dose. This study provides a new efficient adjuvant for inactivated DTMUV vaccine development.

SARS-CoV-2 delta (B.1.617.2) spike protein adjuvanted with Alum-3M-052 enhances antibody production and neutralization ability.

Optimizing adjuvant is one of the critical methods to improve the vaccine. 3M-052, a novel TLR7/8 agonist which was designed for slow dissemination at the injection site, has a potential as adjuvant, but its performance as a vaccine adjuvant for SARS-CoV-2 (B.1.617.2) spike protein has not been studied. The present study aimed to evaluate the effect of Alum-3M-052 as an adjuvant to improve mice serum antibody titers and pseudovirus neutralization efficiency. Female Balb/c mice were immunized 3 times at day 0, 7 and 21 intramuscularly with SARS-CoV-2 (B.1.617.2) spike protein and adjuvant (Alum or Alum-3M-052). Mice serum was collected weekly since day 7. Antibody titers of mice serum anti-SARS-CoV-2 (B.1.617.2) IgG and IgM were detected by ELISA. Inhibition rates of mice serum blocking SARS-CoV-2 (B.1.617.2) spike protein binding to ACE2 were detected by SARS-CoV-2 (B.1.617.2) Inhibitor Screening Kit. Neutralization efficiencies of mice serum against both SARS-CoV-2 (BA.2.12.1) pseudovirus and SARS-CoV-2 (B.1.617.2) pseudovirus were detected by pseudovirus neutralizing assay. Serum of mice immunized by SARS-CoV-2 (B.1.617.2) spike protein adjuvanted with Alum-3M-052 had highest antibody titers and higher neutralization efficiency against both SARS-CoV-2 (BA.2.12.1) pseudovirus and SARS-CoV-2 (B.1.617.2) pseudovirus. Besides, neutralization efficiency of anti-SARS-CoV-2 (B.1.617.2) spike protein antibody against SARS-CoV-2 (BA.2.12.1) pseudovirus was lower than that of SARS-CoV-2 (B.1.617.2) pseudovirus. Alum-3M-052 rapidly increased the titer of anti-SARS-CoV-2 (B.1.617.2) spike protein neutralizing antibodies and enhanced the neutralization ability against pseudoviruses and variants. This study provided evidence for the application of Alum-3M-052 as an adjuvant in COVID-19 vaccines production.

Role of oxidative stress in modulating CHO cell culture performance: Impact on titer and quality attributes of a monoclonal antibody therapeutic

AbstractBACKGROUNDThe enormous success of antibody therapeutics has resulted in attempts to enhance protein expression which requisites high energy demand which leads to oxidative stress as characterized by significant levels of intracellular reactive oxygen species (ROS), disturbing many cellular functions. This study delineates the mechanistic role of oxidants and ROS scavengers in relation to cell viability, apoptosis and mitochondrial membrane potential (MMP), as well as critical quality attributes such as charge heterogeneity.RESULTSAddition of ROS scavengers such as glutathione, N‐acetylcysteine (NAC) and ascorbic acid to culture result in decreased levels of ROS (52%), increased MMP (47%) and viable cell populations (42%), as well as reduced cell apoptosis (69%) and a decrease in lysosome activity (38%). These effects rescue cells from damage caused by oxidative stress and DNA fragmentation during the production phase. Cells use the peroxiredoxin reductive pathway for regulation of ROS levels in response to oxidative stress environments. Enzymes involved in ROS reduction were found to be significantly high in antioxidant‐treated cells. Treatment with oxidants resulted in decreased titer (47%) and an increase in acidic species (18%), whereas addition of antioxidants resulted in enhancement of titer (38%) as well as abundance in basic variants (24%) of recombinant IgG1.CONCLUSIONAntioxidants improve cell culture performance by alleviating the viable cell pool through activating the intracellular reductive mechanism for maintaining high ∆ᴪm, thus reducing apoptosis and enhancing antibody production. Media supplementation with a combination of antioxidants during the production phase is likely to enhance antibody titer and quality. © 2022 Society of Chemical Industry (SCI).

TGF-β3 in differentiation and function of Tph-like cells and its relevance to disease activity in patients with systemic lupus erythematosus.

T peripheral helper (Tph) cells have major roles in pathological processes in SLE. We sought to clarify the mechanisms of Tph cell differentiation and their relevance to clinical features in patients with SLE. Phenotypes and functions of Tph cell-related markers in human CD4+ T cells purified from volunteers or patients were analysed using flow cytometry and quantitative PCR. Renal biopsy specimens from patients with LN were probed by multicolour immunofluorescence staining. Among multiple cytokines, transforming growth factor (TGF)-β3 characteristically induced programmed cell death protein 1 (PD-1)hi musculoaponeurotic fibrosarcoma (MAF)+, IL-21+IL-10+ Tph-like cells with a marked upregulation of related genes including PDCD-1, MAF, SOX4 and CXCL13. The induction of Tph-like cells by TGF-β3 was suppressed by the neutralization of TGF-β type II receptor (TGF-βR2). TGF-β3-induced Tph-like cells efficiently promoted the differentiation of class-switch memory B cells into plasmocytes, resulting in enhanced antibody production. The proportion of Tph cells in the peripheral blood was significantly increased in patients with SLE than in healthy volunteers in concordance with disease activity and severity of organ manifestations such as LN. TGF-β3 was strongly expressed on macrophages, which was associated with the accumulation of CD4+ C-X-C chemokine receptor (CXCR5)-PD-1+ Tph cells, in the renal tissue of patients with active LN. The induction of Tph-like cells by TGF-β3 mainly produced from tissue macrophages plays a pivotal role in the pathological processes of active LN by enhancing B-cell differentiation in patients with SLE.

LAMP-1 Chimeric to HIV-1 p55Gag in the Immunization of Neonate Mice Induces an Early Germinal Center Formation and AID Expression.

Neonates have a limited adaptive response of plasma cells, germinal center (GC) B cells, and T follicular helper cells (TFH). As neonatal vaccination can be an important tool for AIDS prevention, these limitations need to be overcome. Chimeric DNA vaccine encoding p55Gag HIV-1 protein conjugated with lysosomal-associated membrane protein 1 (LAMP-1) has been described as immunogenic in the neonate period. Herein, we investigated the immunologic mechanisms involved in neonatal immunization with a LAMP-1/p55Gag (LAMP/Gag) DNA vaccine in a C57BL/6 mouse background. Neonatal LAMP/Gag vaccination induced strong Gag-specific T-cell response until adulthood and elevated levels of anti-Gag IgG antibodies. We also demonstrated for the first time that the immunogenicity of the neonatal period with LAMP/Gag is due to the induction of high-affinity anti-p24 IgG antibodies and long-term plasma cells. Together with that, there is the generation of early TFH cells and the formation of GC sites with the upregulation of activation-induced cytidine deaminase (AID) enzyme mRNA and protein expression in draining lymph nodes after neonatal LAMP/Gag vaccination. These findings underscore that the LAMP-1 strategy in the chimeric vaccine could be useful to enhance antibody production even in the face of neonatal immaturity, and they contribute to the development of new vaccine approaches for other emerging pathogens at an early stage of life.

Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies

Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 125 randomly selected phage clones. Several DC-binding phage clones were selected using the specificity assay. Phage-54, -74, -16 and -121 bound not only with chicken DCs, but also with duck and goose DCs. In vitro, confocal microscopy observation demonstrated that phage-54 and phage-74 efficiently adsorbed onto DCs within 15 min compared to T7-wt. The pull-down assay, however, did not detect any of the previously reported proteins for chicken DCs that could have interacted with the nanobodies displayed on phage-54 and phage-74. Nonetheless, Specified pathogen-free chickens immunized with phage-54 and phage-74 displayed higher levels of anti-p10 antibody than the T7-wt, indicating enhanced antibody production by nanobody mediated-DC targeting. Therefore, this study identified two avian (chicken, duck and goose) DC-specific binding nanobodies, which may be used for the development of DC-targeting vaccines.

Bioactivity of orange-spotted grouper (Epinephelus coioides) interleukin-6 in innate immunity: Inducing inflammation, antimicrobial peptides, and innate immune molecular gene expression as well as activating phagocytosis of leukocytes and increasing survival under Vibrio and NNV infection

Interleukin-6 (IL-6) is a multifunctional cytokine involved in innate and acquired immunity. In mammals, IL-6 can induce the secretion of acute-phase proteins (APPs) and antimicrobial peptides, activate macrophages and neutrophils, and regulate the expression of inflammatory cytokines in innate immunity, resulting in enhanced resistance to invading pathogens. In teleost fish, the functions of IL-6 are still unclear. In our previous studies using orange-spotted grouper (Epinephelus coioides) as an animal model, we demonstrated that IL-6 induces the Th2 pathway in acquired immunity and enhances antibody production. In the current study, we evaluated in greater detail the mechanisms underlying the functions of IL-6 in innate immunity. In gene expression assays, the expression of fish inflammatory cytokine genes (e.g., ifnγ, ccl4, il1β, tnfα 1, and tnfα 2), antimicrobial peptide genes (e.g., epinecidin 1, hepcidin 1, and hepcidin 2), and genes related to innate immunity (e.g., mx, crp, and complement C3) was higher in fish injected with recombinant IL-6 (rIL-6). The serum from those same fish also presented higher antibacterial activity and higher phagocytosis activity of leukocytes. In a pathogen challenge involving Vibrio campbellii and nervous necrosis virus, fish injected with rIL-6 presented a higher survival rate and lower pathogen count in the body. These results demonstrate that the administration of rIL-6 can activate innate immune responses and enhance resistance against invading bacterial or viral pathogens.

Role of fibrosarcoma-induced CD11b+ myeloid cells and tumor necrosis factor-α in B cell responses

The role of B cells in the anti-tumor immune response remains controversial. An increase in the number of B cells in the peripheral blood of some tumor patients has been associated with poor immunotherapy efficacy. However, the mechanism leading to the generation of these cells is not well-described. Using a fibrosarcoma model, we show that intraperitoneal administration of a xenogeneic antigen in tumor-bearing mice evokes large increases in antigen-specific serum immunoglobulin formation compared to tumor-naïve mice. An inability of tumor-bearing mice to induce enhanced antibody production after myeloid cell depletion suggests the antibody responses are CD11b+ myeloid cell-dependent. In vitro, CD11b+ myeloid cells promoted B cell proliferation, activation, and survival. High levels of tumor necrosis factor (TNF)-α were produced by CD11b+ cells, and TNF-α blockade inhibited B cell responses. CD11b+ cells appear to be important promoters of B cell responses and targeting B cells may increase the efficacy of immunotherapy in tumor-bearing hosts.

Protein antigen conjugated with cholesteryl amino-pullulan nanogel shows delayed degradation in dendritic cells and augmented immunogenicity

Carriers that augment delivery, immunogenicity or both are crucial in the development of vaccines especially component vaccines as components of pathogens are often poorly immunogenic. Cholesteryl pullulan (CHP) that forms nano-sized hydrogel (nanogel) and encapsulates proteins was shown to be useful in the delivery of vaccines. Here we demonstrate that subcutaneous immunization of mice with bovine serum albumin (BSA) chemically conjugated to NH2-CHP nanogel induces strong antibody production. This augmented antibody production requires covalent conjugation between BSA and CHP, but does not require nanogel formation. Conjugation of NH2-CHP nanogel induces persistence of BSA in dendritic cells (DCs) in vivo. As resistance to lysosomal degradation was previously shown to augment antigen presentation by DCs, conjugation of antigens with CHP nanogel may enhance antibody production to antigens by delaying lysosomal degradation. Therefore, delayed degradation of antigens by covalent conjugation with nanoparticles may be a good strategy for the development of effective vaccines.