Exosome may be the next generation of promising cell-free vaccines

ABSTRACT

Traditional vaccines have limits against some persistent infections and pathogens. The development of novel vaccine technologies is particularly critical for the future. Exosomes play an important role in physiological and pathological processes. Exosomes present many advantages, such as inherent capacity being biocompatible, non-toxic, which make them a more desirable candidate for vaccines. However, research on exosomes are in their infancy and the barriers of low yield, low purity, and weak targeting of exosomes limit their applications in vaccines. Accordingly, further exploration is necessary to improve these problems and subsequently facilitate the functional studies of exosomes. In this study, we reviewed the origin, classification, functions, modifications, separation and purification, and characterization methods of exosomes. Meanwhile, we focused on the role and mechanism of exosomes for cancer and COVID-19 vaccines.

KEYWORDS:

• Exosome
• vaccine
• engineering
• isolation
• purification
• cancer
• SARS-CoV-2

Introduction

In the last century, vaccinations have been one of the greatest public health achievements. Vaccines are critical tools to reduce morbidity and mortality caused by communicable diseases. It has been reported that vaccines save at least 2–3 million lives annually around the world. Smallpox has been eradicated and polio has almost disappeared worldwide, owing to the use of vaccines.¹ Traditional vaccine technologies have been widely used for bacterial and viral pathogens, yet they failed to cure some persistent infections and pathogens with high sequence variability, complex viral antigens, and emerging pathogens.² It is essential for developing novel vaccine technology. Exosomes not only can effectively deliver vaccine ingredients in a range of communicable diseases,³ but also transfer genes, lipids, proteins and RNA between cells, as well as antigen presentation. They also regulate physiological and pathological processes. Meanwhile, their inherent capacity being biocompatible, ^4–8 non-toxic,⁹ as well as possessing the ability to cross biological barriers (such as the blood–brain barrier)^10,11 and penetrate into several tissues through their surface ligands and receptors.¹² The findings of these studies suggest that exosomes modified by different methods will have the potential as biomarkers, vaccines, and drug carriers.¹³ Exosomes are superior to other nanoparticles in terms of biosafety, and currently increasing evidences suggesting that exosomes would serve as the basis for the creation of cell-free cancer vaccines in the future.^14,15

Composition and biogenesis of exosomes

Exosomes, which are extracellular vesicles of about 30–150 nm are secreted by almost all mammalian cells.¹⁶ Exosomes originate from the initial endosomes. The first invagination of the cell membrane forms the early sorting endosomes, which form late sorting endosomes under the action of endocytic sorting complexes and associated transport proteins. A second invagination of late sorting endosomes results in the formation of multivesicular bodies. Subsequently, exosomes are released into the extracellular environment once the multivesicular bodies fuse with the plasma membrane.^7,17 Exosomes are taken up by various cells after being released into the extracellular environment and bind to target cells via receptor–ligand interactions or lipids adhesion to cells, and then they are fully internalized by endocytosis, pinocytosis, phagocytosis or fuse to the cell surface membrane,¹⁸ thereby exosomes release cargo into target cells and exerting biological functions.³ The cargo carried by exosomes contain abundant enzymes, transcription factors, heat shock proteins, major histocompatibility complexes, cytoskeletal components, signal transduction proteins, four transmembrane proteins, lipids, RNA, and DNA.^19,20 It is reported that exosomes are involved in clearance of unnecessary proteins or molecules in the cells, the substance exchange between cells, intercellular communication, pathogen transmission, the regulation of immune system, and antigen presentation.²¹

Exosomes are categorized into natural exosomes and artificial exosomes depending on whether they are artificially modified or not. Natural exosomes are subdivided into animal-derived and plant-derived. Almost all types of animal cells secrete exosomes,^7,22 including tumor cells, mast cells, dendritic cells, mesenchymal stem cells, B cells, T cells, macrophages, natural killer cells, neurons, adipocytes, endothelial cells, and epithelial cells.^23,24 Plant-derived exosomes emerge as a new therapeutic option²⁵ and are currently promising,⁷ such as ginger exosome-like nanoparticles (GELN-RNA) which improve colitis in mice via IL-22-dependent mechanisms,²⁶ and GELN microRNA which eliminates exosomes^Nsp12Nsp13–mediated inflammation in the lungs.²⁷ In addition, bacteria-derived exosomes are involved in the disease process. For instance, mycobacterium tuberculosis-derived exosomes may play a significant role in the pathogenesis of tuberculosis by delivering mycobacterium tuberculosis components to the recipient cells.²⁸

Exosome engineering

The appropriate properties of exosomes make them well suited as a drug delivery vehicle, but their clinical application faces several challenges, such as a limited half-life, and low solubility.²⁹ Natural exosomes lack particular cell or tissue targeting properties. In order to optimize the targeting of exosomes and overcome the limitations of using autologous, hence numerous studies showed that engineered exosomes enhanced anticancer immunogenicity by expressing specific antigenic molecules.^30,31 The engineered exosome present multiple antigens and can be designed in two ways, parental cell-based modification and post-isolation modification.³¹ In contrast to parental cell-based exosomes, post-isolated exosomes are technically less complex.³¹ Typical manipulations which include electroporation,^32,33 extrusion, ultrasound, incubation, and freeze-thaw³⁴ can be directly applied to post-isolated exosome engineering.

Parental cell-based exosome modification occurs before exosomes are isolated from parental cells.³¹ The most common modification approach is to transfer proteins to the exosome surface exploiting exosome signal peptides. Another approach is to load molecules into the lumen of the exosomes, which is accomplished by recruiting molecular sorting modules (MSMs) that bind to proteins or RNAs of interest and drives them to the exosome to sort proteins and RNA entering the lumen. Moreover, RNA loading methods are also used in parental cell-based exosome engineering. There is a device for loading mRNAs called EXOtic (Exosomal Transfer Into Cells). In the EXOtic device, there is also a cytoplasmic delivery assistance system and a targeting module in addition to the RNA packaging system. These systems work together to enable the loading of therapeutic mRNAs into exosomes.³¹

Parental cells can be transfected by vectors to generate surface-modified exosomes that can express targeting moieties via a natural biosynthesis process.³⁵ The indirect exosome modification possesses some advantages over direct exosome modification in terms of expression and stability displayed targeting moieties of engineered exosome.³⁶ Next, we discussed some details about genetically engineered exosomes for targeting cancer. For example, the C1C2 domain of lactadherin can be used to express anti-Her2 single-chain variable fragments (scFv) on the surface of therapeutic exosomes. Parental cells were genetically engineered to generate exosomes of Her2-targeting by expressing fusion proteins of anti-Her2 scFv that was fused to the C-terminus on the C1C2 domain of lactadherin, and N-terminus used signal peptide to introduce fusion proteins into the secretory pathway thus inducing it to bind to extramembrane of the exosome. Compared to non-engineered exosomes, engineered exosomes exhibited the better uptake by breast cancer cells expressing HER2 gene, showing two-fold and two to three-fold higher accumulation in vivo.³⁷

Direct exosome gene modifications can be categorized as membrane protein-based gene modifications and lipid–protein interaction-based gene modifications. Many membrane proteins of exosomes such as Lamp2b and tetraspanins, which can be applied to display targeting moieties.³⁸ For example, a study described that exosomes modified by the insertion of ApoB between amino acids 170–171 of CD9 facilitated BBB penetration via hijacking receptor-mediated transcytosis³⁹ and the engineered exosome prolonged the residence in the brain after intravenous administration.³⁸

Another approach to direct exosome engineering is chemical modification, which loads various types of ligands onto the exosome membrane by means of conjugation and hydrophobic insertion.²⁹ Exosome membranes are composed of lipids and proteins, which can be chemically modified by lipid insertion, chemical ligation, affinity binding, and enzymatic conjugation. The exosome membrane allows hydrophobic insertion of lipids and lipid-labeled molecules, and targeted peptides labeled with lipid fragments and aptamers can be inserted into the exosome membrane by a simple mixing and incubation.³⁸ DSPE-PEG is a widely used module to anchor targeting molecules on the surface of exosomes. A study showed that exosomes modified with the application of DSPE-PEG overcame the BBB and targeted delivery drugs to treat glioblastoma.⁴⁰ Chemical ligation is a way based on reactive groups on lipids or proteins of exosome membrane that can react with reactive fragment-labeled peptides thereby allowing the target peptide to modify the exosome. Affinity binding is a method by which targeting moieties are attached to affinity molecules on proteins or lipids of exosomal membrane. Enzymatic conjugation is an approach based on protein ligase, and surface modifications of exosomes, which is achieved by enzymatic reactions between exosomal membrane proteins and target proteins/peptides.³⁸

In addition, structural alteration is another strategy to improve the effectiveness of exosomes. Exosome-liposome hybridization is used to optimize the surface characteristics of exosomes, and the hybridization strategy can modify immunogenicity, improve colloidal stability, increase circulation time in the blood, and elevate target cell uptake.^36,41 The hybridization through PEG can avoid the attack of immune cells by forming a hydration layer, therefore, engineered exosomes achieve a higher stability and longer circulation time.⁴² In addition, exosomes modified with magnetic compounds can be effectively applied for exosome detection, isolation and targeted drug delivery. For example, a novel therapeutic platform was established by combining magnetic targeting characteristics and drug delivery capabilities of magnetic nanoparticles with BBB penetration capabilities and siRNA encapsulation properties of engineered exosomes for the treatment of Gloiblastoma.⁴³

Although cell resources are crucial for biodistribution of exosomes in vivo, a large proportion of injected exosomes are distributed in the reticuloendothelial system, including the lungs, liver, spleen, and gastrointestinal tract, and exosomes are eliminated by them, resulting in a short half-life circulation. In addition, macrophage capture also plays a major role in exosome clearance.^29,44 Targeting of exosomes, which not only increases the effectiveness of exosome delivery, but also reduces out-of-target side effects.⁴⁵ Further clinical applications of exosomes should focus on their manipulation to increase their lifetime in circulation while reducing immune clearance. A study showed that ‘invisible’ exosomes to the immune system through the incorporation of different anti-phagocytic molecules (including CD47, CD24, CD44, etc.) on the exosomes’ surface allow for better systemic bioavailability due to longer residence time in circulation.⁴⁶ For instance, the exosomes containing CD47 achieve protection against phagocytosis by interacting with α-ligand signal-regulating protein.⁴⁷

Exosome isolation and characterization

Exosome isolation

Exosome isolation techniques have developed dramatically in recent decades. Currently, methods of exosome isolation and purification usually include ultracentrifugation, ultrafiltration, size-exclusion chromatography (SEC), polymer precipitation, immunoaffinity capture techniques, and microfluidic techniques, which can be combined to achieve better results.⁴⁸ Table 1 shows the comparative advantages and disadvantages of exosome isolation methods.

Table 1. Advantages and disadvantages of exosome isolation methods.

Methods	Advantages	Disadvantages
Ultracentrifugation	① Low cost ② Large number of samples can be extracted at once	① Impurities ② Time-consuming ③ Expensive equipment ④ Low recovery and purity ⑤ Poor reproducibility ⑥ Damage to exosome
Density gradient centrifugation	① High purity	① Cumbersome operation ② Time-consuming ③ Require preliminary preparation
Ultrafiltration	① Simple and quick ② Cheap equipment and chemicals	① Pressure may deform or rupture exosome ② Pore clogging ③ Contamination
SEC	① High yield and purity ② Stability of exosomes in structure and biological activity	① Cannot distinguish the same size vesicles of exosomes ② Special columns and packings required
Polymer precipitation	① High yield ② Large-scale isolation of exosomes is possible	① Low Purity
Immunoaffinity capture	① High purity ② High specificity	① Only the positive labeled exosome subtypes are included ② High cost ③ Require time for antigen-antibody binding
Microfluidic	① High-throughput ② High specificity ③ High purity and recovery	① High cost and complex ② Low yield

Ultracentrifugation is the most commonly used exosome separation techniques, which is suitable for the majority of samples and is regarded as the ‘gold standard’ for exosome isolation because of its mature technology and low-cost.⁴⁹ Differential ultracentrifugation (DUC) and density gradient ultracentrifugation (DGUC) are the two alternative isolation techniques.⁵⁰ Differential ultracentrifugation is low-cost. The drawbacks are too time-consuming, the equipment is costly, low recovery and purity,⁵¹ and poor reproducibility. Furthermore, repeated centrifugation may also cause some damage to the exosome.^52,53 The purity of extracellular products obtained by density gradient ultracentrifugation is higher than that by differential ultracentrifugation,⁵⁴ but its clinical applicability is confined due to the preliminary preparation required, low yield, cumbersome operation, and long centrifugation time (>16 h).^52,55 Ultrafiltration is a separation method based on molecular size. It works by concentrating exosomes from a large volume of material to a small volume for further purification, which is simple and rapid, and the exosomes obtained are of intermediate yield.^50,56 However, exosomes isolated by ultrafiltration may be deformed or break due to pressure. Additionally, it is prone to pore clogging.^53,57 If this method is employed exclusively, exosomes are contaminated by non-exosomal free-floating humoral peptides such as albumin and α-1-antitrypsin.⁵⁷ Size-exclusion chromatography is a separation method based on variations in molecule size. This approach produces exosomes with high yield and purity,⁵⁸ and preserves the integrity and biological activity of exosomes since it utilizes gravity flow.⁵⁹ However, exosomes cannot be distinguished from vesicles of the same size. Polymer precipitation produces low purity exosomes with a high yield.⁶⁰ The exosomes extracted by this technique are susceptible to contamination by lipoproteins or virus particles to impact future analyses (such as proteomics and mass spectrometry). Recently, many commercial kits relying on precipitation technology have been used for exosome isolation, and the ExoQuick kit is widely used.⁵⁰ The immunoaffinity technique using magnetic beads offers a higher capture efficiency and greater sensitivity.⁶¹ The immunoaffinity method is unaffected by exosome morphology and has the characteristics of high specificity and purity, intermediate yield.⁶⁰ However, the exosomes isolated by this method are only subtypes with positive markers and do not include all types of exosomes.⁶² This method is time-consuming because of the time required for antigen–antibody binding. In addition, extra steps are required to isolate and purify the exosomes after antibody binding.⁶³ Microfluidics are considered a promising approach that integrate sample processing, analysis, monitoring, and other processes on a chip, resulting in miniaturized, high-throughput capabilities which is less time-consuming.⁶⁴ It is able to distinguish, capture, enrich, and separate particles of very similar shape and size.⁶⁵ This separation technology has the advantages of minimal feed volumes, high throughput, and quick sample processing, along with high purity and sensitivity.⁵² Nevertheless, the procedure is costly, complex⁵⁰ and unsuitable for large-scale production.⁶³

Characterization of exosomes

EVs are similar in size and morphology and it is indispensable to distinguish different EVs subtypes by using some characterization methods after EVs are isolated. In particular, some certain markers are utilized to determine whether the extracted material is an exosome or not, so as to verify the method of isolation of exosomes. Based on these characteristics, the methods for characterizing exosomes can be briefly classified as quantitative exosome analysis methods, qualitative exosome characterization methods, and single vesicle characterization methods.⁶⁶

Total exosome count, protein count, lipid count, and DNA/RNA count are used for quantitative characterization of exosomes. Total exosome counts are frequently detected via nanoparticle tracking analysis (NTA), flow cytometry (FCM), dynamic light scattering (DLS), and tunable resistive pulse sensing (TRPS). Lipid count has been achieved by using techniques, such as fluorescence microscopy and fourier-transform infrared spectroscopy (FTIR).⁶⁶ Mass spectrometry, western blot (WB), and enzyme-linked immunosorbent analysis (ELISA) are used for protein count. For RNA counting, polymerase chain reaction (PCR), microarray analysis, and next-generation sequencing (NGS) are utilized.^67,68 Protein markers, lipid markers and DNA/RNA markers are used for qualitative characterization of exosomes. Protein markers are often identified using mass spectrometry, flow cytometry (FCM), and enzyme-linked immunosorbent analysis (ELISA). For the recognition of DNA/RNA markers, PCR, microarray analysis, and next-generation sequencing (NGS) are employed. Additionally, mass spectrometry is used to detect lipid markers. When evaluating individual vesicles, structure, size, and chemical composition are prominent aspects to consider. The structure of exosomes has been visualized by applying scanning electron microscope (SEM), transmission electron microscope (TEM), cryo-electron microscope (cryo-EM), and atomic force microscope (AFM).⁶⁹ The size of exosome has been measured via nanoparticle tracking analysis (NTA), flow cytometry (FCM), dynamic light scatterin (DLS), and tunable resistive pulse sensing (TRPS). Raman Spectroscopy is used to determine chemical composition.⁶⁶ There are also a number of individual or combined methods used to perform exosome assays, such as surface plasmonic biosensors, microchip-based technologies, electrochemical techniques, fluorescence, and colorimetry.⁵⁶ Figure 1 shows the common exosome characterization methods.

Figure 1. The common exosome characterization techniques.

ELISA, enzyme-linked immunosorbent analysis; PCR, polymerase chain reaction; NGS, next-generation sequencing; SEM, scanning electron microscope; TEM, transmission electron microscope; cryo-EM, cryo-electron microscope; AFM, atomic force microscope; NTA, nanoparticle tracking analysis; DLS, dynamic light scatterin; TRPS, tunable resistive pulse sensing; WB, western blot; FTIR, fourier-transform infrared spectroscopy; ddPCR, droplet digital PCR.

Exosomes and vaccines

Exosomes have attracted great interest for their ability to stimulate dendritic cells recognizing and killing cancer cells.¹⁴ In addition, exosomes transfer antigens into DC for action, causing the initiation and amplification of specific immune responses.⁷⁰ These characteristics make it a desirable candidate for cancer vaccines. In order to improve the targeting of exosomes, many studies have genetically engineered exosomes to express specific antigenic molecules or target cancer cells.³⁰ To achieve genetic modification of exosomes, a novel exosome platform named Synthetic Multivalent Antibody Retargeted Exosome (SMART-Exo) was designed and developed.^71,72 Table 2 describes the benefits and disadvantages of available vaccines and exosome-based vaccines.

Table 2. Advantages and disadvantages of vaccine types.

Vaccine types	Advantages	Disadvantages
Inactivated vaccine	① Mature technology ② Relatively simple to construct ③ Inactive ingredient	① Booster immunization is needed
Adenoviral vector vaccine	① Mass production ② Multiple epitopes can be induced simultaneously ③ Strong immunogenicity	① Relatively complex to construct ② Pre-existing anti-adenovirus immune responses with potential adverse events
DNA vaccine	① Vectors can be constructed to encode different antigens in a single vaccine ② Mass production, low cost, high stability of production and storage ③ No active ingredients, no risk of vaccine-triggering disease	① Weak immune response ② Delivery agent is required to reach the nucleus
RNA vaccine	① Rapid development ② Novel mRNA sequences can be quickly assembled into existing vaccine formulations ③ No risk of integration with host cell genes ④ No active ingredients, no risk of vaccine-triggering disease	① High cost ② Lipid nanoparticles must be stored at ultra-low temperature ③ Prone to severe and rare allergic reactions
Recombinant protein vaccine	① Mature technology ② Mass production ③ No active ingredients ④ Relatively stable	① Adjuvants required ② Elicit weak immune response ③ Only protein fragments are expressed, not all proteins ④ Screening for the best combination of antigens take time
EV-based Vaccine	① Self-presenting antigen ② Crossing the blood-brain barrier ③ High absorptivity	① Mass production is difficult ② Immune response to disease need to be further explored

Currently, there are two ways to load proteins onto exosomes. One way is feeding antigens to dendritic cells (DCs) and then collecting the supernatants to separate exosomes for indirect loading of peptides, which ensures that antigen peptide binds to MHC molecules. MHC molecules are crucial for the interaction between DCs and T cells. However, the limitation of this strategy is that the type of antigen must be recognized by the DCs.⁷³ The other way is that any soluble antigen can be attached to the exosome and can be used to modify exosomes with additional functions, such as adding adhesion molecules to exosomes, which can be targeted to certain sites.⁷⁴

Exosomes and cancer vaccines

Exosomes can not only stimulate anti-tumor immune responses, but also inhibit anti-tumor immune responses to promote tumor growth through the ability of intercellular signaling and antigen presentation.⁷⁵ An appealing topic of study is how the tumor microenvironment is impacted by normal-derived and tumor-derived exosomes, which provides chances for the enhancement of antitumor immune responses, the development of exosomal vaccines, and the use of exosomes as drug nanocarriers.¹⁷

In cancer immunotherapy, tumor-derived exosomes (TEX) which are rich in tumor antigens can be presented by APC to induce T and B cell responses. Tumor-derived exosomes (TEX) have emerged as a promising cell-free therapeutic vaccine in cancer immunotherapy.⁷⁶ TEXs are enriched with proteins such as major histocompatibility complex I and II (MHCI and MHCII), CD81, CD54, and CD63, which can be used for exosome binding and uptake by related proteins on DCs.⁷⁶ Meanwhile, heat shock proteins are presented on TEX, including 71 kDa heat shock protein, 70 kDa heat shock protein 4, and HSP90 α & β.⁷⁷ The heat shock protein (HSP) targets and activates antigen-presenting cells including dendritic cells (DCs), thus providing a natural link between the innate and adaptive immune responses.⁷⁸ HSPs have powerful adjuvant capabilities that enhance the immunogenicity of TEXs and improve the effectiveness of cancer vaccines.⁷⁶

The design protocol of the effective cancer vaccine must be able to trigger strong and sustained immunity mediated by T cells.⁷⁹ TEX contains tumor-associated antigens and participates in antigen presentation either directly or indirectly, triggering CD8⁺ T cell responses for cross-initiating immunotherapy.^80,81 Nonetheless, TEXs-induced immunosuppression and limited TEXs immunogenicity, and frequent application of TEXs alone in vivo leads to unsatisfactory antitumor immune effects. Therefore, several strategies have been used to improve the effectiveness of TEXs vaccines. TEXs can be genetically or non-genetically modified to enrich tumor antigens, microRNAs, and immunostimulatory molecules on TEXs to directly enhance tumor cell death or to be killed by immune cells. Another strategy is to improve TEX vaccination by DC loading, TEX-loaded DCs promote the transformation of naïve CD8⁺T cells into mature antigen-specific CTL cells, induce the activation of NF-κB in macrophages, and participate in tumor cytotoxicity via the release of tumor necrosis factor (TNF).⁷⁶

In addition to tumor-derived exosomes, dendritic cell-derived exosomes (DCexos) also have the potential to be cell-free therapeutic vaccines due to their resistance to tumor immunosuppression, significant bioavailability, and biostability.⁸² DCexos which are inert vesicles have a longer half-life in vivo and may be stored for a longer period of time. It is reported that DCexos are also more resistant to immunomodulation by the tumor or tumor microenvironment (TME) than DCs,⁸³ thus supporting their clinical application as tumor vaccines.^83–86

DCexos are enriched in MHC class I and II complexes, heat shock proteins (HSPs), and costimulatory molecules such as CD86, which stimulate the activation of CD4⁺ helper T cells and CD8⁺ CTLs and induce potent antitumor effects.^87–89 Some functional molecules such as MHC-I, MHC-II, CD40, CD80, CD86, and the FasL, TRAIL, and NKG2D (natural killer group 2D) ligands on the surface of DCexos contribute to the enhancement of both innate and adaptive anti-tumor immune responses.⁹⁰ In the antitumor response, DCexos also induce other immune cells such as NK cells.⁹¹ NK cell effector function enhanced in clinical trials using modified DCexos to treat cancer, which suggest that DCexos may induce NK cell function in vivo.⁹¹

It is reported that DCexos dramatically increased CD8⁺ T lymphocytes, resulting in elevating IFN-γ and IL-2, decreasing CD25⁺Foxp3⁺ Treg cells, and decreasing IL-10 and TGF-β at tumor site, thus improving the tumor microenvironment.⁹² While in the tumor microenvironment, DCexos contain TNF, Fas ligand (FasL) and TNF-related apoptotic ligand (TRAIL) can cause apoptosis in tumor cells.⁹³ Meanwhile, DCexos activate natural killer cells through TNF superfamily ligands, thereby presenting an inhibitory effect on tumors.⁹⁴

Despite the fact that we have concentrated on DCexos as a tumor vaccine, exosomes derived from other cell such as macrophages, NK cells, B cells, T cells, mesenchymal stem cells, and tumor cells, have also been employed in the study of tumor vaccines.^93,95 Normally, exosomes derived from immune cells promote the proliferation of T cells.⁹⁶ Exosomes are usually presented by mature DCs to elicit T cell responses because the antigens on exosomes must be taken up by DCs to activate T lymphocytes, which avoids the use of adjuvants to some extent, and exosomes can directly activate T lymphocytes.⁹⁷

During antigen presentation, B cell-derived exosomes first interact with antigen and then regulate T cell activation and cytokine secretion.^98,99 Additionally, it is reported that cytotoxic T lymphocyte (CTL)-derived exosomes contain CTL surface membrane molecules (CD3, CD8 and TCR) that cause tumor cell death through TCR-MHC/antigen interactions.¹⁰⁰ NK cell-derived exosomes play a significant therapeutic function, which produce cytotoxic effects on melanomas by presenting perforin and FasL and participate in apoptosis in vivo and in vitro.¹⁰¹ Exosomes from M1-polarized macrophage can enhance the activity of anti-tumor vaccines and promote CTL-related immune responses.^102,103 Exosomes secreted by M1-polarized macrophages can be taken up by local macrophages and DCs in lymph nodes (LNs), increasing the release of Th1 cytokines and enhancing the CTL response. In melanoma, macrophage-derived exosomes enhance the efficacy of tumor polypeptide vaccines and significantly inhibit tumor growth.¹⁰⁴ Figure 2 shows the mechanism of action of tumor-derived exosomes and immune cell-derived exosomes on tumor cells.

Figure 2. The mechanism of action of tumor-derived exosomes and immune cell-derived exosomes on tumor cells.

(a) TEXs is modified to enrich tumor antigens, microRNAs, and immunostimulatory molecules on TEXs to enhance tumor cell death or to be killed by immune cells. (b) TEX is presented to CD8⁺ T cells by DC, ultimately leading to tumor cell death through cytotoxicity. (c) TEX activates the B cell response to produce antibodies, which eventually causes the tumor cells are phagocytosed by macrophages. (d) TEX-loaded DCs with activate macrophages, which participate in cytotoxicity by releasing TNF. (e) DCexo induces NK cell function and NK cell-derived exosome presents cytotoxic effects via perforin and FasL. (f) CTL cell-derived exosomes cause tumor cell death through TCR-MHC/antigen interactions.

Exosomes and COVID-19 vaccines

At present, the outbreak of the COVID-19 pandemic has accelerated the research on EVs, especially the development of EV-based vaccines.¹⁰⁵ As exosomes are essential intercellular communication molecules with immunogenicity, high biocompatibility and inherent cargo-loading capacity, they offer new opportunities for the development of COVID-19 vaccines and may be crucial in restricting the COVID-19 pandemic.¹⁰⁶ Extracellular vesicles (EVs) have several biological advantages over virus-vectors, such as endogenous origin, the ability to cross biological barriers, high stability, immunogenicity, and low toxicity. Several companies are experimenting and testing COVID-19 vaccines based on the EVs platform.¹⁰⁷ For example, a biologic company had researched two different COVID-19 vaccines.¹⁰⁶ One type of vaccine comprises human HEK293 cells transfected with vectors expressing four structural proteins (S, E, M and N proteins) of the SARS-CoV-2. The other type is an mRNA vaccine. EVs are loaded with five mRNAs encoding modified SARS-CoV-2 spike, nucleocapsid protein, membrane protein, cytosolic proteins, and full-length spike protein from the wuhan-1 isolate.¹⁰⁵ Another biologic company developed a vaccine in which induced pluripotent stem cell line were transfected with different mRNAs encoding SARS-CoV-2 antigen proteins. These cells produce a large amount of EVs that carry the viral mRNAs and corresponding antigen proteins. This type of vaccine possesses multiple mRNAs and is better than single mRNA vaccines. Another exoVACC^TM vaccine is a modular vaccine system that takes advantages of EVs, which can simultaneously deliver specific antigens and immune-stimulating adjuvants to antigen-presenting cells (APCs) to elicit cellular and humoral immune responses. Recent reports have shown that exosomal vaccines loaded with mRNAs encoding SARS-CoV-2 spikes and nucleocapsid proteins induced long-lasting cellular and humoral immune responses and produced fewer adverse effects than the currently available COVID-19 vaccines.¹⁰⁸ Accoding to Kuate et al., the mice were initially immunized with an exosomal vaccine containing the SARS-S protein and then were immunized with an adenovirus vector vaccine expressing the S protein, their neutralizing antibody titers were higher than those in the serum of SARS patients. Moreover, both vaccines induced neutralizing antibody titers in mice.¹⁰⁹ Therefore, EV-based vaccine design protocols can be applied to the treatment of SARS-CoV-2.

Conclusions

Although EV-based vaccines present many advantages, there are still a large number of obstacles needed to be overcome in clinical applications. For example, production and isolation of exosomes on a large scale remains a great challenge. In terms of targeted therapies, the original structure of exosomes can be modified in a variety of methods, such as genetic engineering, chemical procedures, physical techniques, and microfluidic technology, which make additional cargo load on exosomes to expand the biomedical applications of exosomes.¹¹⁰ In conclusion, the development of exosome modification techniques, automated manufacturing processes and strict quality control systems are pivotal to produce engineered exosomes of high purity on a large scale. A large number of cells are required to secrete sufficient exosomes for large-scale production. Bioreactors produce an enormous number of cells in a short period of time and the most popular bioreactors currently available are hollow fiber bioreactors and stirred tank bioreactors.⁶³ Exosome isolation can be achieved by different methods. Though conventional centrifugation technology is low-cost, it takes a long time. In addition, exosome extracted by this method are low purity. Ultrafiltration does not require expensive equipment and can be utilized for large-scale production. Nevertheless, isolation of exosomes from impurity proteins needs to be optimized. Size-exclusion chromatography technique preserves the integrity of exosomes, while it cannot distinguish between vesicles of the same density and size. Polymer precipitation method is simple to operate and easy to scale up. However, the polymer precipitation method is prone to be contaminated. Immunoaffinity isolation technique is highly specific, but requires extra isolation and purification steps after binding the antibody and is not suitable for large-scale production. In contrast, microfluidic technology, which takes the advantage of precise nanoscale liquid and particle control capabilities is faster and more efficient than the current techniques for exosome isolation. However, it is not appropriate for large-scale production. Methods of exosome isolation should ensure the integrity, sensitivity and specificity. The methods of exosome isolation or combinations should be optimized to obtain high-purity exosomes.

Exosome-based vaccines demonstrate impressive outcomes in the cancer and viral infectious diseases. Inactivated vaccines have no active ingredients and are technically mature, but a single dose is not sufficient to induce long-lasting immunity. Adenoviral vector vaccines induce multiple epitopes and can be produced on a large scale, but pre-existing anti-adenovirus immune responses have potential adverse events. DNA vaccines allow the construction of vectors to encode different antigens in a single vaccine, which can be mass-produced with high stability in production and storage. However, DNA vaccines can only elicit a weak immune response and require a delivery agent to reach the nucleus. RNA vaccines can quickly assemble novel mRNA sequences into existing vaccine formulations and there is no risk of integration with host cell genes. However, RNA vaccines are prone to rare and severe allergic reactions. Recombinant protein vaccines require adjuvants and are easy to produce on a large scale, however, they can only express protein fragments. Conversely, exosomes are effective carriers of viral antigens, which present antigens in their natural state and are able to cross the blood–brain barrier and generate a protective immune response. Compared to other carriers, such as lipid-based nanoparticles and viral carriers, exosomes have lower immunogenicity and higher absorptivity and are safely and efficiently employed in vaccines.¹⁰⁶

This review summarizes methods for large-scale production, isolation, and characterization of exosomes, and focuses on the anti-tumor mechanism of tumor-derived exosomes and immune cell-derived exosomes. These studies provide a pivotal basis for the development of exosome-based vaccines for cancer. As cell-free vaccines, exosomes have limitations in terms of therapeutic efficacy. Tumor-derived exosomes may carry immunosuppressive molecules and induce pro-tumor effect, which limits their role in anti-tumor immunotherapy. To address this issue, tumor-derived exosomes need to be modified to improve their anti-tumor immune response. More studies are needed to evaluate different modification techniques, screen out the optimal combinations of antigens and adjuvants, and evaluate the adverse molecular responses of EV in recipient cells, which will benefit to improving therapeutic efficacy and safety of exosomes in vaccines. In the future, exosome-based vaccines are expected to become a significant breakthrough in the field of cancer immunization and infectious diseases with increased research and technological advances.

Authors’ contribution

Conceptualization: Ruiru Cai; Hong Zeng; Hailian Zhu; Youwei Dou

Design: Shibo Sun

Writing-original draft: Zelan Dai

Writing-review: Shibo Sun

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [grant no. 82160007], the Yunnan Provincial Science and Technology Department [grant no. 2022201AY070001-074], Yunnan Province young and middle-aged academic and technical leaders reserve talent project [grant no. 202305AC160017], 535 Talent Project of First Affiliated Hospital of Kunming Medical University [grant no. 2023535D12], and Student Innovation Fund project of Kunming Medical University [grant no. 2023CXD077, 2023CXD091, 2023CXD195, 2023CXD247, 2023CXD257, 2023CXD270].