Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Emerging trends of edible vaccine therapy for combating human diseases especially COVID-19: Pros, cons, and future challenges.

The researchers are still doing efforts to develop an effective, reliable, and easily accessible vaccine candidate to protect against COVID-19. As of the August 2020, nearly 30 conventional vaccines have been emerged in clinical trials, and more than 200 vaccines are in various development stages. Nowadays, plants are also considered as a potential source for the production of monoclonal antibodies, vaccines, drugs, immunomodulatory proteins, as well as used as bioreactors or factories for their bulk production. The scientific evidences enlighten that plants are the rich source of oral vaccines, which can be given either by eating the edible parts of plants and/or by oral administration of highly refined proteins. The use of plant-based edible vaccines is an emerging trend as it possesses minimum or no side effects compared with synthetic vaccines. This review article gives insights into different types of vaccines, the use of edible vaccines, advantages of edible vaccines over conventional vaccines, and mechanism of action of edible vaccines. This review article also focuses on the applications of edible vaccines in wide-range of human diseases especially against COVID-19 with emphasis on future perspectives of the use of edible vaccines.

Quantitative Analysis of Vaccine Antigen Adsorption to Aluminum Adjuvant Using an Automated High-Throughput Method.

Aluminum-containing adjuvants have been widely used in vaccine formulations to safely and effectively potentiate the immune response. The examination of the extent of antigen adsorption to aluminum adjuvant is always evaluated during the development of aluminum adjuvant containing vaccines. A rapid, automated, high-throughput assay was developed to measure antigen adsorption in a 96-well plate format using a TECAN Freedom EVO® (TECAN). The antigen adsorption levels at a constant adjuvant concentration for each sample were accurately measured at 12 antigen/adjuvant (w/w) formulation ratios. These measurements were done at aluminum adjuvant concentrations similar to normal vaccine formulations, unlike previous non-automated and automated adjuvant adsorption studies. Two high-sensitivity analytical methods were used to detect the non-absorbed antigens. The antigen-to-adjuvant adsorption curves were fit to a simple Langmuir adsorption model for quantitatively analyzing the antigen to the adjuvant adsorption level and strength. The interaction of two model antigens, bovine serum albumin and lysozyme, with three types of aluminum adjuvant, were quantitatively analyzed in this report. Automated, high-throughput methodologies combined with sensitive analytical methods are useful for accelerating practical vaccine formulation development.LAY ABSTRACT: Vaccines are probably the most effective public health method to prevent epidemics of many infectious diseases. Many of the most effective vaccines contain aluminum adjuvant. This report describes novel technology that can be used to better optimize the efficacy and stability of aluminum adjuvant-containing vaccines.

Preparation of the Multifunctional Liposome-Containing Microneedle Arrays as an Oral Cavity Mucosal Vaccine Adjuvant-Delivery System.

Recently, the multifunctional liposome-constituted microneedle arrays (LiposoMAs) have been proven to be an interesting vaccine adjuvant-delivery system (VADS) that are stable and can be vaccinated via oral cavity mucosal route. When given to mice at oral mucosa, the LiposoMAs can effectively eliminate the ingredient loss caused by chewing, swallowing, and saliva flowing and can, thus, elicit robust systemic as well as mucosal immunoresponses against the loaded antigens. In addition, the LiposoMAs can induce a mixed Th1/Th2 immunoresponse and strong cellular/humoral immunity due to special adjuvanticity and targeting delivery functions of the nanoparticulate VADS. In this chapter, the preparation, characterization as well as mucosal vaccination of the LiposoMAs are introduced. In addition, the methods for sampling mouse organs, tissues, and cells and for evaluation of the immunization efficacy are mainly included.

Design and evaluation of surface and adjuvant modified PLGA microspheres for uptake by dendritic cells to improve vaccine responses.

Designing strategies for targeting antigens to dendritic cells is a major goal in vaccinology. Here, PLGA (poly lactic-co-glycolic acid) microspheres and with several surface modifications that affect to their uptake by human blood primary dendritic cells and monocytes have been evaluated. Higher uptake was found by all the cell types when cationic microspheres (PLGA modified with polyethylene imine) were used. These cationic particles were in vivo evaluated in mice. In addition, MPLA(1) or poly(I:C)(2) and α-GalCer(3) were also encapsulated to address their adjuvant effect. All the microspheres were able to produce humoral immune responses, albeit they were higher for cationic microspheres. Moreover, surface charge seemed to have a role on biasing the immune response; cationic microspheres induced higher IFN-γ levels, indicative of Th1 activation, while unmodified ones mainly triggered IL4 and IL17A release, showing Th2 activation. Thus, we have shown here the potential and versatility of these MS, which may be tailored to needs.

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8(+) T cell responses in mice.

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration "cold chain". Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8(+) T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8(+) T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. FROM THE CLINICAL EDITOR: This paper reports that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize into a biocompatible adjuvant in a single step, enabling distributed and on-demand vaccine production and eliminating the need for refrigeration of vaccines. The findings highlight the possibility of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines.

Biodistribution of vaccines comprised of hydrophobically-modified poly(γ-glutamic acid) nanoparticles and antigen proteins using fluorescence imaging.

Fluorophores-modified nanoparticles comprised of poly(γ-glutamic acid)-phenylalanine (γ-PGA-Phe-633) and ovalbumin (OVA-750) termed NPs-633/OVA-750 were prepared to assess their biodistribution using an in vivo fluorescence imager. Dynamic light scattering measurements indicated that NPs-633/OVA-750 were about 200nm in diameter. The release of encapsulated OVA from NPs-633 in PBS was negligible (∼10%) for a week. When subcutaneously injected, the localization period of OVA-750-encapsulated into NPs-633 at the site of injection (SOI) was much longer than that of free OVA-750, but was shorter as compared to a mixture with aluminum hydroxide. The NPs-633 disappeared at the SOI and major organs within 1month after administration. Moreover, intravenously and intraperitoneally administered NPs-633 were mainly observed at the liver, and there was more rapid clearance from all organs as compared with non-biodegradable NPs. These fast clearance and degradation characteristics of γ-PGA-Phe NPs will be important not only for avoiding undesired adverse effects, but also for inducing a strong vaccine effect.

The immunobiology of aluminium adjuvants: how do they really work?

Aluminium adjuvants potentiate the immune response, thereby ensuring the potency and efficacy of typically sparingly available antigen. Their concomitant critical importance in mass vaccination programmes may have prompted recent intense interest in understanding how they work and their safety. Progress in these areas is stymied, however, by a lack of accessible knowledge pertaining to the bioinorganic chemistry of aluminium adjuvants, and, consequently, the inappropriate application and interpretation of experimental models of their mode of action. The objective herein is, therefore, to identify the many ways that aluminium chemistry contributes to the wide and versatile armoury of its adjuvants, such that future research might be guided towards a fuller understanding of their role in human vaccinations.

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome.

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.