Exosomes Therapy in Zoonoses

Abstract

Exosomes are smaller extracellular vesicles (EVs) involved in complex intercellular communication, which were first discovered in sheep reticulocytes. Exosomes include two subpopulations, large (Exo-L, 90–120 nm) and small (Exo-S, 60–80 nm) exosome vesicles. Recently studies of RNA viruses including SARS-CoV-2 have demonstrated that exosomes release regulatory factors from infected cells and deliver other functional host genetic elements to neighbouring cells, and these functions are involved in the infection process and modulate the cellular responses. This review provides an overview of the biogenesis, composition, and some of the most striking functions of exosome secretion in zoonoses and identifies physiological/pathological areas in need of further research as well as potential therapeutic agents in zoonotic disease.

1. Introduction

Zoonotic diseases are estimated to cause 60% of infections in humans. Treatments are mainly based on the use of antibiotics and antimicrobials; this creates resistant strains, searching for new therapeutic approaches of paramount importance [1]. Cell-based therapy, and especially stem cell therapy, has become a promising therapeutic field in which many see opportunities to treat incurable diseases. Among the various cell types, MSCs have attracted attention due to the source of origin, a high proliferation rate, low-invasive and ethically unproblematic procurement procedure [2].

The therapeutic effect of MSCs seems to rely on the modifications they exert on their microenvironment through paracrine interactions, secretion of soluble factors, cytokines, and trophic factors. It is possible to simplify treatment by using only the components secreted by MSCs, constituting the so-called secretome, which includes immunoregulatory factors (e.g., IL-6 and IL-10), prostaglandin E2, hepatocyte growth factor, indolamine 2,3-dioxygenase, nitric oxide, TGF-ß, and human leukocyte antigen, as well as extracellular vesicles. In fact, these cells can be considered potent pharmacotherapeutics that release biologically active substances [3].

Once released, EVs and soluble proteins interact with target cells (by ligand-receptor interaction or by internalisation) and modulate cellular responses. The secretome can activate endogenous stem cells and progenitor cells, suppress apoptosis, regulate the inflammatory response, stimulate extracellular matrix remodelling and angiogenesis, reduce fibrosis and mediate chemoattraction [4].

Exosomes are nanoscale extracellular vesicles with a lipid bilayer of endocytic origin and are secreted by almost all cell types and physiological states [5]. They are small with a diameter of 40–100 nm [6] and can be permeable to biological barriers.

Exosome formation takes place by internal plasma membrane budding to form multivesicular bodies (MVB) [7], which can also originate from the trans-Golgi network [8]. Within the MVB, exosomes are formed by further internal budding and then released by fusion of the MVB membrane with the plasma membrane [9].

Exosomes contain a large number of parental cell proteins, and their membrane is abundant with proteins of the tetraspanin family (CD9, CD63, CD81, and CD82), which are widely used as exosome markers. They also have adhesion surface proteins to facilitate their internalisation by other cells [10].

They have a defined composition, depending on cell type, pathology, and activation state, thus exerting their role on specific target cells to release their protein content, mRNA, and, importantly, specific microRNA molecules [11].

Exosomes are considered safe because they lack the potential for endogenous tumor formation as they cannot self-replicate, have low immunogenicity and, when injected intravenously, lead to low embolism formation [12]. The application of exosomes in therapy also has technological advantages: they can be managed and stored easily, and they are a suitable product for use in emergency interventions [13].

Exosomes can be isolated from cell culture supernatant by different methods (each with different limitations). Current techniques are based on exosome size (30–100 nm), density (1.13–1.21 g/ml), and exosome-specific markers (such as CD63). The general method is based on ultracentrifugation of the cell culture supernatant. However, this method often entrains impurities from microvesicles or other cellular detritus. In addition, the high centrifugal force and duration of centrifugation can cause damage to the exosomes [14].

Summarising, with a conglomerate of cytokines, growth factors, mRNAs, and microRNAs having anti-inflammatory, immunomodulatory and regenerative functions; exosomes play a role as paracrine and endocrine mediators; a fact that, together with their safety profile, stability, and scalability, make them a valuable treatment option for zoonotic diseases either as vehicles or as vaccines themselves (see Table 1) [15].

Pathogen type	Pathogen	Role of exosome	What causes	Reference
Virus	Rabie	Intercellular communication	Viral infection process	[16]
		Vehicle	Bind acetylcholine receptor	[17, 18, 19]
		Intercellular communication	Inhibit replication and actívate immune system	[20]
	Ebola	Intercellular communication	Cell death and apoptosis	[21, 22]
		Vehicle	Increase in the levels of cytotoxic T-lymphocyte response	[23]
	HIV	Vehicle	Viral suppression	[24]
		Vehicle	Reactivation of HIV-infected CD4 T cells	[25]
	SARS-CoV-2	Intercellular communication	General improvement of the body	[26, 27, 28]
		Vehicle	General improvement of the body	[29]
		Intercellular communication	Nervous system recovery	[30, 31, 32]
	Influenza	Vehicle	That virus replication inhibited	[33]
		Intercellular communication	Release of pro-inflammatory cytokines and the overexpression of type I interferon	[34]
		Intercellular communication	Restore the permeability lung epithelial	[35]
Bacteria	Brucella	Vehicle	Immune response	[36, 37]
		Intercellular communication	Enhance immune system response	[38]
	Salmonella	Intercellular communication	Inflammatory reaction	[39]
		Intercellular communication	Innate and adaptive response	[40]
	M. tuberculosis	vehicle and adjuvant	Intensify the immune response	[41, 42]
		Intercellular communication	Exert an immune response via Th1 and Th2	[43]
Parasite	T. gondii	Intercellular communication	Response Th1 secrete IL-2 and IFN-gamma, activate macrophages and NK cells	[44]
		Intercellular communication	It increases fertility and this is because the immune response is type 2	[45]
Fungi	C. neoformans	Intercellular communication	Complement system is activated, leading to inflammation, phagocytosis and cell lysis	[46]
		Intercellular communication	Immune response	[47]

Table 1.

Overview of the different pathogens and how exosomes work as therapy.

This table describes the pathogens and their origin, as well as the function of exosomes in the organism. Source: Compilation based.

In view of the above, the objectives of this chapter are as follows:

1. To review zoonotic diseases of public health importance, as well as their current treatments.

2. To evaluate the therapeutic capacity of exosomes in infections of zoonotic origin.

   1. Possibility of altering target cells by acting as molecules in intercellular communication.

   2. Potential to act as a vehicle for the delivery of desired therapeutical targets.

2. Methodology

The study presented here follows a qualitative methodology, as it pursues the description and interpretative understanding of exosome therapy for diseases of zoonotic origin. Specifically, we will stick to documentary research to approach the aforementioned object of study. Thus, both review and research articles, as well as governmental websites will be used. In all cases, the types of documents are electronic. As this is documentary research of an exploratory nature, the aim of this exploratory documentary research is fitting the above-mentioned objectives.

The studies have been reviewed in different databases:

• Google Scholar: Google’s tool for finding books and journals, bibliographic references with abstracts, full texts, or personal and institutional websites.

• PubMed: digital storage system that integrates biomedical scientific journals, with access to abstracts, bibliographic references, tables and figures or complete articles.

• medRxiv/bioRxiv: search engines that allow for the consultation of unpublished scientific articles in the health field that are not peer-reviewed.

• WHO website: because it is the specialised body in charge of management, prevention, and decision-making in the field of global health issues.

• Website of the Spanish government: for consultation of updated COVID-19 data to updated COVID-19 data at the global level.

The search for documents was carried out based on keywords such as zoonosis, exosome, vaccine, infection, intercellular communication, rabies, Ebola, HIV, SARS-CoV-2, influenza, brucella, salmonella, mycobacterium tuberculosis, toxoplasma gondii, and cryptococcus neoformans.

For the development of the documentary work, a total of 108 bibliographic sources were found, based on: (1) year of publication, except for the study by Pan BT (1985), and (2) relevance of the study associated with our objectives, as some of them, despite addressing the subject, did so from a more general perspective.

For all these reasons, the sample includes information of both a qualitative and quantitative nature, which allows us to have a bigger knowledge range to argue properly our work.

3. Exosome-based therapy for zoonotic diseases

3.1 Rabies

Rabies virus is a single-stranded RNA belonging to the Rhabdovirus family. They are bullet-shaped, with a flat and a rounded end, an envelope with spicules surrounding the genome, and a helical capsid. The Rhabdovirus family includes the genus Vesiculovirus, which causes vesicular stomatitis virus, and the genus Lyssavirus, which causes rabies [48].

The receptor of the rabies virus is the nicotinic acetylcholine receptor. It enters the cell by endocytosis and replication takes place in the cytoplasm. It has RNA-dependent RNA polymerase. Transcription of the viral RNA produces five individual mRNAs that will give rise to five proteins, a process that takes place in the Negri corpuscles. Negri bodies are cytoplasmic inclusion bodies that correspond to aggregates of viral nucleocapsids and are formed as a result of the replication process (diagnostically useful) [49, 50].

Rabies is a zoonosis whose reservoir is wild animals, mainly bats, and dogs (in 99% of cases) [48]. The virus is excreted in the saliva of an infected animal and enters other animals through bite wounds or scratches. The virus is very labile, so transmission through contaminated objects is rare. Human-to-human transmission is not confirmed [51].

Pre-exposure prophylaxis is based on an inactivated vaccine in at-risk personnel. As the incubation period is long, post-exposure vaccination is feasible. The original vaccine designed by Pasteur was an attenuated virus vaccine (obtained from neural tissue of infected animals) and had the disadvantage of producing adverse reactions with allergic encephalomyelitis. Post-exposure prophylaxis is based on wound lavage, passive immunisation with human rabies immune globulin and active immunisation with inactivated vaccine (five intramuscular doses) [52].

It is known that infected cells release exosomes into the environment [53, 54]; however, the role of exosomes in infections is not entirely clear. Therefore, Wang et al. evaluated the role of exosomes in rabies virus infection. After isolation of exosomes by density gradient, they characterised them by transmission electron microscopy and western blotting. The results were that, after infection, the release of exosomes increased. They also used treatment with two inhibitors of exosome secretion, GW4869, and si-Rab27a, and found not only that exosome secretion decreased, but also that the presence of intra- and extracellular viral RNA was reduced. This finding provides a better understanding of the role of exosomes in rabies virus infection and proposes it as a new research target for the development of therapeutic strategies [16].

Research groups such as Yang et al., Alvarez-Erviti et al. and Kumar et al. highlight the role of rabies virus glycoprotein-modified exosomes (RVG-modified exosomes) for, in addition to being loaded with the desired content to tackle the condition, the ability to cross the blood-brain barrier and bind specifically to the acetylcholine receptor of neurons [17, 18, 19].

Yang et al. conducted a study with the human diploid cell line Medical Research Council-5 (MRC-5), which is used to develop vaccines. In the case of rabies, a vaccine based on this cell line exists, but with low efficacy due to the limitation of infection on the cells. These researchers demonstrated that, following rabies virus infection in the MRC-5 cell line, exosomes have a dual function: (I) to interfere with the type I interferon signalling pathway to increase its production and (II) through this regulation, to inhibit rabies virus replication [20].

3.2 Ebola

Ebola virus is a filamentous, enveloped, helical nucleocapsid, single-stranded negative RNA genome. It belongs to the Filovirus family where the Marburg virus is also found. The genome has the information to encode seven structural proteins that form the virion. It replicates in the cytoplasm similar to Rhabdoviruses [55].

The Ebola disease outbreak in West Africa, which began in March 2014, was the largest viral haemorrhagic epidemic in history. Nearly 40% of people who contracted Ebola during this outbreak died. The natural reservoir is unknown. Transmission is by direct contact with infected blood and secretions; there is no airborne transmission. There is no vaccine or specific treatment. Favipiravir, an antiviral against RNA viruses, has been used; in addition, passive immunisation is carried out by the administration of sera [56].

Exosomes released by the Ebola virus, called VP40+, cause cell death and apoptosis, eventually destroying immune cells [21]. Because exosomes play a role in intercellular communication and influence cellular responses, they may be targeted for therapy development, specifically for vaccine production [22].

CD8 T cells play a central role in antiviral immunity and are also involved in the immune response generated against other intracellular pathogens, such as intracellular bacteria. Destruction of virus-infected cells requires the T-lymphocyte receptor to recognise peptides associated with MHC class I molecules. This triggers the activation of lymphocytes to (I) destroy infected cells and (II) produce inflammatory cytokines [57]. This immune response has been seen in Ebola survivors [58] and in non-human primates [59].

Exosomes are vesicles released by all cell types of the organism [60, 61]. By developing intramuscular DNA vector vaccines, namely NEFmut, scientists Anticoli S. et al. incorporated exosomes to study the immune response against Ebola virus (based on those discussed above). They were able to see an increase in the levels of cytotoxic T-lymphocyte response and cope with the disease [23].

3.3 HIV

Human Immunodeficiency Virus (HIV) belongs to the retrovirus family. It is a virus with two identical single-stranded RNA molecules enveloped. They are called Retroviridae because they possess an enzyme with reverse transcriptase activity (retrotranscriptase) that allows RNA to be transcribed into DNA, making it an RNA-dependent DNA polymerase. There are four families of retroviruses: oncornavirus (human T-lymphotropic virus), spumavirus (only in animals), alpharetrovirus (Rous sarcoma virus), and lentivirus (HIV) [62].

Replication begins with the binding of viral glycoproteins to the primary receptor, the CD4 molecule, and to the co-receptors CCR5 or CXCR4. They infect CD4 T cells and other cells that express the CD4 molecule on their surface, such as monocytes, macrophages, or dendritic cells. They penetrate the host cell by fusion of the envelope with the cell membrane. Binding to the co-receptor brings the envelope into contact with the plasma membrane and the glycoprotein gp41 promotes membrane fusion. The genome is released into the cytoplasm and undergoes a reverse transcription process, which uses the transfer RNA as a primer [63].

To avoid the development of HIV resistance, a combination of three drugs from two different classes is recommended. The recommended antiretroviral treatment in naïve patients (those who have not had previous antiretroviral treatment) is outlined below [64, 65]:

• 2 NRTIs (nucleoside reverse transcriptase inhibitors) + 1 NNRTI (non-nucleoside reverse transcriptase inhibitor).

• 2 NRTIs (nucleoside reverse transcriptase inhibitors) + 1 PI (protease inhibitor).

• 2 NRTIs (nucleoside reverse transcriptase inhibitors) + 1 INI (integrase inhibitor).

The above-mentioned therapy has side effects such as decreased life expectancy and the virus may develop resistance. Shrivastava et al. developed an HIV promoter targeting the ZPZ-362 protein that fuses with DNA methyltransferase domains capable of long-term HIV suppression. This promoter is introduced into exosomes acting as a transport medium and allowing delivery to act in epigenetic regulation. Analysing the results, they found that this transport medium performed its function perfectly, as once inside, the methylation machinery acted, leading to viral suppression [24].

More than half of HIV-infected people suffer from cognitive problems due to prolonged inflammation and disruption of the blood-brain barrier (BBB). The problem is that not all treatments are able to cross the BBB. Therefore, exosomes have been proposed as drug delivery vehicles since, among their many characteristics, is the ability to cross the BBB [66].

Among the essential genes for viral replication that form a regulatory protein is Tat (Trans-Activator of Transcription). It increases the level of HIV double-stranded RNA transcription; in its absence, there is little RNA transcription. On this premise, Tang et al. introduced the Tat protein into exosomes, enabling the reactivation of HIV-infected CD4 T cells. This makes it possible to deal with latently infected cells [25].

3.4 SARS-CoV-2

SARS-CoV-2 belongs to the coronavirus (CoV) family, named after its characteristic morphology, which resembles a solar corona when viewed under the electron microscope. CoVs are classified into four different genera according to their phylogeny and genomic structure: alpha, beta, gamma, and delta [67]. They are a family of viruses that can cause multiple systemic infections or damage in various animal species [68]. Genomic analyses show that SARS-CoV-2 is from the same beta coronavirus (βCoV) family as SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV), and they have high sequence homology [69]. In 2003 SARS-CoV-1 caused an epidemic with 8273 cases and 775 deaths [70]. The MERS-CoV epidemic, however, caused 1139 cases and 431 deaths in 2013 [71].

Coronaviruses are composed of a single-stranded positive-sense RNA, with a genome ranging from 26.2 to 31.7 kb [67]. It has mainly three viral proteins in the virion envelope: spike protein (S), membrane protein (M), and envelope protein (E). The spike protein mediates virus entry and determines the range of potential hosts, cell tropism, and pathogenic disease [72].

SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor to infect bronchial epithelial ciliated cells and type II pneumocytes [73], which explains the severity of lung involvement. In addition to the lungs, the receptor is widely distributed in the gastrointestinal, hepatic, renal, nervous, cardiovascular, and other systems. This can lead to pathologies beyond the respiratory system [74]. After binding to the receptor, the viruses fuse their envelope with the host cell membrane and the nucleocapsids reach the target cell [75].

At the end of 2019, several cases of acute respiratory infection were discovered in the Chinese province of Wuhan. Those affected had in common pneumonia of unknown aetiology and exposure to the Wuhan market [76]. Due to its virological similarity and clinical picture with Severe Acute Respiratory Syndrome (SARS) caused by the SARS-CoV-1 coronavirus, the World Health Organisation (WHO) named the coronavirus SARS-CoV-2 and the disease it causes COVID-19 [77].

On 11 March 2020, the WHO declared the disease a pandemic because of the increase in the number of people affected since the discovery of the virus [78]. At the time of writing (25 March 2022) and according to the Ministry of Health, the total number of COVID-19 cases worldwide is 47,465,969,674 [79], affecting more than 200 countries. It has therefore become a global health problem and many researchers are currently seeking to eradicate it.

Fortunately, scientists have succeeded in developing vaccines to tackle COVID-19. These are composed of mRNA or adenoviral vectors that encode the virus and are enveloped by lipids [80]. Millions of lives have been saved thanks to these vaccines, but issues such as the use of booster doses, the origin of SARS-CoV-2 variants, and whether the vaccines can cope with the side effects of the disease remain unresolved.

Multiple preclinical studies have demonstrated favourable therapeutic effects of intravenously administered MSC-derived exosomes in animal models of acute lung injury [26], acute respiratory distress syndrome (ARDS) [27], asthma, and other inflammatory diseases, with analyses revealing reduced alveolar inflammation, enhanced oedema clearance, restoration of permeable epithelium, membranes and other sequelae of cytokine storm (Figure 1) [28].

Figure 1.

Pathogenesis of SARS-CoV-2 and stem-cell-based therapy and exosomes. (1) SARS-CoV-2 enters the human body via droplets. In human cells, SARS-CoV-2 binds with the ACE2 receptors present on host cells and produces a cytokine storm. This storm results in a severe lung injury. (2) MSCs. (3) MSCs-Exo or EVs are considered a possible future treatment due to many of their properties, such as ACE2—(lack of ACE2 receptors) which prevents a cytokine storm and immune modulation and restoration of damaged cells due to their essential growth factors and metabolites. Source: Compilation based.

Taking advantage of the benefits provided by exosomes, scientists Jiang et al. studied the feasibility of an exosome-based vaccine. They introduced the spike protein-binding domain into Salmonella typhimurium exosomes. Once the animals were vaccinated (first group), they tested the control group (unvaccinated animals infected with the Delta variant). The first group had less virus replication, less alveolar damage, a slight or no decrease in body mass, and a high titre of IgG antibodies measured in serum and bronchoalveolar lavage compared to the control. This demonstrates the feasibility of an exosome-based vaccine against COVID-19 [29].

One of the side effects of COVID-19 is how it can affect the nervous system. There are several reasons for this. First, because of the cytokine storm producing neuroinflammation which is the basis of numerous neurological diseases such as Alzheimer’s, Parkinson’s, multiple sclerosis [30], or even psychiatric conditions [31]. Secondly, the direct neuroinvasive capacity of ACE2 receptors, which are also found in the nervous system and are considered potential targets [32]. The third is the indirect action on the nervous system due to the already demonstrated relationship between the intestinal microbiota and the nervous system, as the virus invades the intestinal mucosa and causes inflammatory processes that can lead to neurological damage [31]. Therefore, exosomes could be used as a therapy for this side effect, as intranasal administration not only offers the benefits mentioned above but is also a direct route of entry into the nervous system and also has the ability to cross the blood-brain barrier [29].

3.5 Influenza

The Orthomyxovirus family has a helical capsid, is enveloped and the negative single-stranded RNA is segmented into eight fragments. Influenza viruses or influenza viruses stand out. There are three types: A, the most important; B, the endemic type; C, very rare [81].

The influenza virus transcribes and replicates its genome in the nucleus of the target cell. Replication begins with the binding of haemagglutinin to the sialic acid of cell surface glycoproteins. They replicate in the nucleus because they cannot synthesise their own mRNA. Transcriptase uses cellular RNA as a primer to initiate viral mRNA synthesis (cap-snatching) [82]. The mRNA is synthesised using 5′-CAP ends of heterologous nuclear RNA as a primer (mRNA cap-stealing). It is assembled in the cytoplasm and exits by budding through the plasma membrane [83].

Virus transmission is airborne via respiratory secretions. Its genetic diversity is based on its segmented genome structure and its ability to infect and replicate in humans and many animal species, such as birds and pigs. Influenza virus type A is characterised by high antigenic variability, much higher than in type B and C [84].

Antigenic variations can be of two types:

• Most commonly, minor changes (antigenic drifts, antigenic drift, or antigenic slippage), involve a change in the surrounding strain within the same virus type. They are due to point mutations in haemagglutinin and neuraminidase. They are responsible for epidemics [85].

• Major changes (antigenic shifts, antigenic jumping, or antigenic conversion) show a change in the surrounding virus subtype and are responsible for periodic influenza A virus pandemics. This antigenic shift occurs due to the exchange between the gene segments of different influenza virus strains (both human and animal) when the two strains infect the same cell. Such recombination only occurs in the pig and occurs between human and avian viruses. It should be noted that this process only occurs in influenza A virus [85].

For at-risk personnel, possible immunoprophylaxis is a vaccination with inactivated viruses. Other therapeutic options include amantadine, oseltamivir, and zanamivir, which should be administered early within 48 h of symptom onset [81].

miRNAs are single-stranded RNAs that are approximately 22 nucleotides in length and have the ability to regulate the expression of various genes that play a role in development, cancer, defence against organisms, immunity, homeostasis, etc. Scientists Liu et al. studied the role of miRNAs in influenza virus, focusing their research on the functional role of hsa-miR-1975 in the defence of the infected organism. The methodology was based on assessing the antiviral effects of this miRNA by RT-PCR, Western Blot, and plaque assays. The results obtained were not only that virus replication is inhibited, but also that the release of hsa-miR-1975 occurs via exosomes [33]. This indicates evidence of the importance of investigating the therapeutic possibilities of exosomes due to their ability to harbour content that modifies cellular communication [10].

One notable function of exosomes is in cell-cell communication in the immune system, aiding the body’s defence against pathogens. Exosomes carry proteins, and mRNA and, as mentioned above, can carry miRNA. An investigation was carried out to look at the performance of exosomes against influenza virus infection. Among the miRNAs studied, miR-483-3p emphasised its role in defense. The researchers were able to demonstrate its capabilities in the release of pro-inflammatory cytokines and the overexpression of type I interferon. They were able to demonstrate the role of exosomes in the inflammatory reaction when a microorganism is present in the body [34].

Influenza virus is acquired via the respiratory tract through inhalation of aerosolized viral particles. Initially, local infection of the upper respiratory tract is established and can lead to acute lung injury. MSCs, specifically umbilical cord-derived MSCs (UC-MSCs) and their derived exosomes, which mediate paracrine transport, have the ability to restore the lung microenvironment. They restore the permeability that characterises lung epithelial cells and removes alveolar fluid from impaired cells [35].

3.6 Brucella

The most clinically relevant species are Brucella abortus, Brucella melitensis, and Brucella suis. They are responsible for classical zoonoses and the most prominent reservoirs are cattle and wild animals; cows (B. abortus), goats (B. melitensis), and pigs (B. suis) [86]. The zoonosis in humans is known as brucellosis, Bang’s disease, Malta fever, or undulant fever [87].

They are Gram- coccobacilli, non-flagellating (hence not motile). They grow only under aerobic conditions, and are non-fermenting and slow-growing. They are facultative intracellular pathogens [88].

Brucellosis is a globally distributed zoonosis, is an occupational disease, and is a reportable disease in most countries of the world. B. melitensis infections are more frequent in Mediterranean countries, Latin America and Asia. The main prophylactic measure is to avoid exposure and prevent consumption of unpasteurised dairy products. Isolation of infected persons is not necessary, as the infection is not transmitted from person to person [87].

Attenuated B. abortus and melitensis vaccines have been successful in preventing infection in cattle [88]. The absence of an effective human vaccine is a cause for concern, because Brucella could be used as an agent of biological terrorism. Current treatment in humans is based on doxycycline with streptomycin or gentamicin; the alternative is co-trimoxazole. Antibiotic therapy should be prolonged for 3–4 weeks. For children and pregnant women, co-trimoxazole and rifampicin are used [87].

As mentioned above, exosomes can function as vehicles and deliver various molecules. This ability is exploited by macrophages to transport interferon-induced transmembrane protein 3 (IFITM3) from uninfected to infected macrophages once the organism is exposed to the Brucella pathogen [36]. IFITM3 is a critical protein for the immune system as it prevents the replication of foreign agents when they enter the body [37]. In this study by Yi et al., they found, in addition to the transmembrane protein mentioned above, that exosomes transport multiple proteins after infection including those involved in the immune response. This ability of exosomes represents a breakthrough in the development of a vaccine to tackle brucellosis [36].

In the research and development of an effective vaccine for Malta fever, a group of scientists led by Solanki KS treated mice inoculated with a virulent strain of B. abortus S544 with exosomes. The exosomes were outer membrane vesicles extracted from B. abortus S19Δper an attenuated strain, OMVs S19 Δper. After analysis of the results and comparison between the control group (inoculated with the strain, but not treated) and the vaccinated group (inoculated with the strain and vaccinated with OMVs S19) they were able to conclude that there was an increase in the Th1 and Th2 immune system response, with an increase in IgG antibody titres, and cytokines such as IL-2, IL-4, TNF, among others [38].

3.7 Salmonella

Typhoid salmonellosis is caused by Salmonella typhi and Salmonella paratyphi serotypes A, B, and C. S. typhi has a capsular antigen that does not form a true capsule because it does not completely cover the cell wall. Adherence of the bacteria to jejunal cells (M cells) facilitates invasion by endocytosis-transfer-exocytosis. Dissemination via blood and lymph from the intestinal tract occurs. Cases of typhoid salmonellosis in Europe are imported by travellers and occur as a circumstantial epidemic. Humans are the only reservoir; they can survive in the gallbladder, generating asymptomatic carriers [89, 90].

Enteric salmonellosis is caused by Salmonella enteritidis and Salmonella typhimurium. The bacteria gain access to the gastrointestinal tract through food (animal reservoir). Adhesion to enterocytes of the ileum and colon facilitates mucosal invasion mediated by bacterial surface invasions. Endotoxin production is the main cause of food-borne toxic-infection. Enteric salmonellosis occurs both endemically and epidemically. The main focus of infection is livestock; it is a zoonosis [91, 92].

Typhoidal salmonellosis should be treated with antibiotics. Third-generation cephalosporins are recommended. Other antibiotics such as ciprofloxacin or co-trimoxazole may be treatment alternatives. In typhoid salmonellosis, elimination of chronic carriers presents a problem but can be achieved with high doses of antibiotics (4-quinolones or ampicillin) [89, 93].

In enteric salmonellosis, symptomatic treatment is sufficient, loperamide to decrease intestinal activity and fluid and electrolyte replacement. Antibiotic treatment is reserved for exceptional cases [91, 94].

Macrophages or mononuclear phagocytes are cells of the immune system, derived from blood monocytes that migrate to tissues where they differentiate into mature macrophages that can live for years. Their main function is phagocytosis of pathogens and cellular debris, although they also participate in inflammatory processes by chemotaxis of other cells of the immune system. They are antigen-presenting cells (APCs) and possess an extraordinary cytokine-producing capacity (TNF-α, IL-1, IL-6, IL-12, etc.) in response to pathogen-associated molecular patterns (PAMPs) [95].

Hui WW et al. studied the ability of infected macrophages to trigger immune responses and thus “alert” inactivated macrophages. This communication is carried out by exosomes; this allows macrophages to perform their function and, in addition, due to the possession of lipopolysaccharides by the exosomes, causes an inflammatory reaction to be exerted [39].

Years later, this researcher and his group, due to the lack of an approved vaccine for humans, delved deeper into macrophage exosomes. They studied the proteome of the exosomes of Salmonella-infected macrophages. They observed that, once the exosomes are administered, the body’s innate and adaptive response is triggered, leading to a release of the aforementioned cytokines and an increase in the production of IgG antibodies. Likewise, when exosomes are administered intranasally, mucosal defences are activated [40].

3.8 Mycobacterium tuberculosis

The genus Mycobacterium is characterised by aerobic, non-sporulating, non-motile bacilli. The most important bacteria in this group are Mycobacterium tuberculosis and M. bovis, the aetiological agents of tuberculosis, and M. leprae, the aetiological agent of leprosy [96].

Many of the special characteristics of TB bacteria are due to their cell wall chemistry. It has a murein coat, numerous lipids such as glycolipids (cord factor being trehalose dimycolate), mycolic acids, mucosides, and waxes [97].

Primary TB infection is localised in the lungs. In most cases, pathogens enter the lung via respiratory droplets (airborne transmission), where they are phagocytosed by alveolar macrophages (facultative intracellular pathogen). Bacteria are able to reproduce in macrophages due to their ability to inhibit phagolysosome formation (prevents fusion of the phagosome with lysosomes). In about 10% of cases, the primary infection can reactivate in immunocompromised individuals and progress after months or years to a secondary stage characterised by tissue necrosis [98].

TB is an endemic disease. Although it is much less common in developed countries, from a global perspective it remains a priority medical problem. The major reservoir is the human (except for M. bovis). The main source of infection is the human carrier; there are no healthy carriers. Transmission of the disease is usually direct, via respiratory droplets [98, 99].

An attenuated vaccine is available that reduces the risk of infection by 5% and contains Bacillus Calmette-Guerin (BCG), which is an attenuated strain of M. bovis. Vaccination provides temporary immunity, for a period of 5–10 years. In persons at risk of developing the disease who are: contact tuberculin-positive, immunocompromised tuberculin-positive, or radiologically confirmed residual tuberculosis, isoniazid prophylaxis for 6 months is recommended [98, 99].

The exosomes shed by the cells into the medium, which allows intercellular communication, can be used as potent immunotherapeutic agents to tackle TB. First, it is possible to add immune cell exosomes to the current vaccine against Mycobacterium tuberculosis to enhance the immune response and thus make the defence against subsequent infections effective. This possibility as an adjuvant has been tested in hepatitis and has given good results [41]. And secondly, the ability of exosomes to harbour molecules inside them in order to administer the desired drugs, which are unable to cross biological membranes on their own [42].

In the study conducted by Cheng and Schorey, the main objective was to evaluate the therapeutic capabilities of macrophage-derived exosomes in mice infected with tuberculosis bacteria. The researchers were confronted with the fact that, for the vaccine that exists, the protective efficacy decreases over time and the lungs are exposed to the infection. They concluded that both exosomes applied as vaccines, as a booster with the currently available vaccine, exert an immune response via Th1 and Th2, although the latter is more limited. Finally, an increase in IFN-γ and IL-2 levels in lung cells after vaccination was determined by ELISA [43].

3.9 Toxoplasma gondii

It is an obligate intracellular parasite that causes toxoplasmosis. It has several life forms:

Tachyzoites: the result of asexual multiplication by endopolygeny or multiple fusion. They are arranged in clusters or pseudocysts. They characterise the acute phase of infection in which rapid multiplication of the parasite occurs [100].

Bradyzoites: asexual reproductive forms formed by endodiogeny or binary fission. They group together to form spherical tissue cysts. They constitute the chronic (latent) phase of infection [100].

Oocysts: form after sexual multiplication in cells of the host (cat) intestinal epithelium. They are excreted in the faeces and to be infective they need to continue sporulation outdoors, which, depending on external conditions, will occur between two and 21 days. In a warm, humid climate, oocysts can persist viable for more than a year [100].

Humans can become infected by ingesting the tissue cysts contained in the meat of infected animals or by ingesting mature oocysts in contaminated food and water. When a woman’s primary infection coincides with her pregnancy, the parasite can cross the placental barrier and infect the foetus, resulting in congenital infection [101, 102].

Pyrimethamine, sulphadiazine, and folinic acid are generally used as treatments. In children with congenital toxoplasmosis, pyrimethamine alone is used. In cases of HIV with toxoplasmic encephalitis, if sensitivity to sulphonamides develops, clindamycin is given instead of sulphadiazine. For HIV patients, trimethoprim and sulfamethoxazole (co-trimoxazole) are used as prophylaxis. In pregnant women spiramycin [103, 104, 105].

Dendritic cells immunised with Toxoplasma gondii producing antigens are effective in coping with the infection and producing an effective immune response. Based on this premise, Aline F and her team developed a vaccine based on exosomes derived from dendritic cells that had been exposed to T. gondii. They were able to prove that the response generated is mediated by Th1 lymphocytes. These secrete IL-2 and IFN-gamma, activate macrophages and NK cells. They also promote the function of cytotoxic T-lymphocytes that activate the cell-mediated immune response to intracellular pathogens such as Toxoplasma gondii [44].

A woman infected with T. gondii is capable of transmitting it to her foetus. In the study by Beauvillain et al., they focus on how the use of exosomes for this pathology can protect the offspring. They tested both parasite-exposed and non-parasite-exposed dendritic cells and exosomes as vaccines. They concluded that, in addition to triggering an effective immune response, it increases fertility, and this is because the immune response is type 2 (Th1-mediated response leads to non-viability of pregnancy due to poor implantation and maintenance of the placenta) [45].

3.10 Cryptococcus neoformans

It is a unicellular yeast with a size of 3–5 μm surrounded by a polysaccharide capsule. It inhabits soils rich in organic substances. The most common route of entry in humans is respiratory; organisms are inhaled and reach the lungs. Pulmonary cryptococcosis may develop [106].

From the primary pulmonary focus, pathogens spread via the blood to other organs. C. neoformans shows a major affinity for the central nervous system, where it develops very severe meningoencephalitis. The treatment of choice for this disease is amphotericin B and 5-fluorocytosine [106, 107, 108].

Oliveira et al. investigated the role of C. neoformans exosomes upon entry into the organism. They were able to prove in vitro that these vesicles play a modulating role in infection and the generation of the immune response. The complement system is activated, leading to inflammation, phagocytosis, and cell lysis. Furthermore, when exosomes come into contact with macrophages, they release antimicrobial compounds such as TGF, TNF, and IL-10 [46].

Due to the lack of knowledge about fungal exosomes, they wanted to study them in-depth. They used advanced techniques such as cryogenic electron microscopy and tomography, proteomics (random access liquid chromatography in tandem with mass spectrometry), and nanoscale flow cytometry. They were able to conclude that exosomes can function as stand-alone vaccines and elicit an immune response to confront the pathogen [47].

4. Conclusions and future perspective

Exosome therapies are a major breakthrough in preventive and therapeutic medicine. There are currently 259 ongoing studies involving exosomes according to the clinical trials website ClinicalTrials.gov. In this review, exosomes derived from different cell types are proposed as a therapy for zoonotic infections. The rationale for the use of exosomes, the benefits involved and the fact that MSCs are not used have been outlined above. Due to their immunomodulatory capacity, exosomes will help the immune system to cope with pathologies. They can also be used as vehicles by introducing the desired molecules inside them due to their permeability to biological barriers.

Delivering exosomes intranasally provides faster action, allows smaller doses to achieve the same effect as injection therapy, is non-invasive, avoids the pain typically associated with parenteral therapy, and the potential for side effects is minimal.

Since exosomes trigger the immune system’s defence against a variety of zoonotic diseases, further development of their use as vaccines would be important for clinical trials. If finally viable, one option to consider is the creation of a bank of exosomes so that they can be available for both prevention and disease control.

Finally, one option to study would be the possibility that exosomes could fight not just one disease but a spectrum of diseases by activating the immune system and thus boosting the body’s defenses. This would make it a universal tool for tackling different pathologies.

Acknowledgments

We want to thank Ramón Wahl for his contribution to the performance of figure and table.

Conflict of interest

The authors declare no conflict of interest.