Role of Extracellular Vesicles in Viral and Bacterial Infections: Pathogenesis, Diagnostics, and Therapeutics

Abstract

Extracellular vesicles (EVs), or exosomes, are nanovesicles of endocytic origin that carry host and pathogen-derived protein, nucleic acid, and lipid cargos. They are secreted by most cell types and play important roles in normal cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses and pathophysiological processes. New research is beginning to decipher how EVs influence viral and bacterial pathogenesis. In this review, we will describe how EVs influence viral and bacterial pathogenesis by spreading pathogen-derived factors and how they can promote and inhibit the immune response to these pathogens. We will also discuss the emerging potential of EVs as diagnostic and therapeutic tools.

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2018; 8(10): 2709-2721. doi: 10.7150/thno.20576
Review
Role of Extracellular Vesicles in Viral and Bacterial
Infections: Pathogenesis, Diagnostics, and Therapeutics
Meryl Rodrigues1, 2, Jia Fan1, Christopher Lyon1, Meihua Wan3, Ye Hu1, 2
1. Virginia G. Piper Biodesign Center for Personalized Diagnostics, Arizona State University Biodesign Institute, Tempe, Arizona, 85287
2. School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona, 85287
3. Department of Integrated Traditional Chinese and Western Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan, China, 610041
 Corresponding author: wanmh@scu.edu.cn and tyhu@asu.edu
© Ivyspring International Publisher. This is an open access article distributed under the terms of the Creat ive Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2017.04.14; Accepted: 2018.01.15; Published: 2018.04.09
Abstract
Extracellular vesicles (EVs), or exosomes, are nanovesicles of endocytic origin that carry host and
pathogen-derived protein, nucleic acid, and lipid cargos. They are secreted by most cell types and
play important roles in normal cell-to-cell communications but can also spread pathogen- and
host-derived molecules during infections to alter immune responses and pathophysiological
processes. New research is beginning to decipher how EVs influence viral and bacterial
pathogenesis. In this review, we will describe how EVs influence viral and bacterial pathogenesis by
spreading pathogen-derived factors and how they can promote and inhibit the immune response to
these pathogens. We will also discuss the emerging potential of EVs as diagnostic and therapeutic
tools.
Key words: extracellular vesicles, exosomes, pathogenesis, immune system, diagnostic, therapeutic
Introduction
Extracellular vesicles (EVs), particularly
exosomes, have gained attention for their potential as
disease biomarkers and therapeutic agents. Exosomes
are EVs released by the endocytic pathway that range
from 30-100 nm in diameter, and contain host (and
pathogen)-derived nucleic acid, protein and lipid
cargos. Microvesicles (also known as shedding
vesicles, ectosomes or microparticles), are a distinct
type of EV that forms by the outward budding of the
plasma membrane and are 100-1000 nm in diameter.
These EV types are distinct in their subcellular site of
origin and physical parameters, but the size overlap of
exosomes and microvesicles and differences in EV
isolation and handling procedures can lead to
confusion, despite ongoing efforts by the International
Society for Extracellular Vesicles (ISEV) to
standardize and harmonize these methods [1, 2]. This
review will focus on the role of exosomes in chronic
viral and bacterial disease, but due to the potential for
confusion between EV types among studies we will
refer to vesicles analyzed in all cited studies, except
those focusing on the exosome biosynthesis pathway
itself, as EVs to adhere to the ISEV nomenclature
guidelines.
Most cells secrete EVs, but EVs produced during
pathogen infections can reveal differences in their
composition to reflect their origin from infected cells
and the overall state of the infection. Studies have
revealed multiple ways by which viruses and bacteria
can manipulate EV synthesis to enhance their
transmission and pathogenesis (reviewed in [3]).
Conversely, EVs produced by immune cells play an
important role in host responses to infection. One
early example of this role was the finding that EVs
from B lymphocytes contained class II major
histocompatibility (MHCII)-antigen complexes that
could activate CD4+ T cells in an antigen-specific
manner [4]. Subsequent studies found that EVs of
dendritic cells contained class I major histocom-
patibility (MHCI)-peptide complexes that could
stimulate cytotoxic CD8+ T cell responses, identified
cell-dependent and independent mechanisms for the
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antigen presenting activity, and identified receptor
and cytokine/chemokine effects to regulate multiple
cells involved in the adaptive immune response [5-7].
Notably, EVs derived from cells infected with either
viral or bacterial pathogens demonstrate several
mechanisms to mediate the immune system,
including effects to inhibit host EV effects to promote
adaptive immune responses, indicating that better
understanding of these mechanisms is important to
improve therapeutic approaches used to treat these
pathogens. EVs carrying pathogen-derived factors are
also of interest as biomarkers of infection, since these
factors should be more stable than soluble factors in
circulation that are exposed to circulating hydrolase
activities. EVs are also stable in circulation, are
capable of packaging a broad array of biomolecules
and small molecule drugs, and exhibit potential as
selective targeted biogenic carriers [8-14]. Based on
these properties, this review describes current
knowledge of EV actions to promote disease and
regulate host immunity, and the potential of these
EVs as disease biomarkers and future therapeutic
agents.
Exosomes
Exosome Biogenesis: Exosomes are formed in a
multi-step process, where regulated invagination of
early endosome membranes results in the
accumulation of intraluminal vesicles (ILVs) and the
formation of multivesicular bodies (MVBs) that
eventually fuse with the plasma membrane to release
exosomes into the extracellular compartment (Figure
1). Soluble factors (e.g., nucleic acids, proteins,
carbohydrates, and other factors), are captured from
the cytosol during endosomal membrane
invagination, but these components can be
preferentially enriched by interaction with endosomal
membrane factors, including the endosomal sorting
complexes required for transport (ESCRTs) [15],
which recognize ubiquitinylated proteins. Despite
much progress, there are still significant gaps in our
understanding of the mechanisms responsible for
sorting proteins into internal vesicles of
multivesicular compartments and, hence, to
exosomes. Exosome and microvesicle production are,
thus, fundamentally different in that microvesicles
bud directly from the plasma membrane (Figure 1)
[16].
Exosome composition: Mature exosomes range
from 30-100 nm in diameter, contain host- and
pathogen-derived proteins and nucleic acids, and
play important roles in health and disease [13, 17, 18]).
Exosomes primarily contain endosome-related
proteins: annexins involved in intracellular
membrane fusion and transport, lipid raft-associated
proteins, and ESCRT accessory proteins (Table 1)
(reviewed in [3, 19, 20]). Exosome membranes are
enriched for several broadly expressed tetraspanin
Figure 1. (A) Exosome biogenesis begins with the invagination of the plasma membrane to generate early endosomes. These endosomes can then invaginate to form intraluminal
vesicles (ILVs). This process creates multi-vesicular bodies (MVBs) that can then fuse with th
e plasma membrane to release mature ILVs, now called exosomes, into the
extracellular space. (B) Exosomes contain proteins, nucleic acids (including mRNAs, miRNAs, and DNA fragments), and lipids, a
nd these cargos can reflect selective
incorporation during exosome formation in a process controlled by lipid raft proteins, ESCRT accessory proteins (e.g., ALIX and TSG101) and tetraspanin proteins. The
cytosolic release of these contents upon exosome uptake can alter the phenotype of the recipient cells.

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proteins, including CD9, CD63, and CD81, which
interact with multiple proteins (e.g., integrins and
MHC molecules) to regulate the organization of large
molecular complexes and membrane subdomains.
Cell-specific exosome enrichment for several trans-
membrane proteins—including α- and β-integrins,
immunoglobulin family members, and cell-surface
peptidases—may also regulate preferential exosome
interactions with their target cells (reviewed in [21]).
Exosomes also contain RNA cargos, and while
exosome RNA content reflects the cell of origin and its
physiological state, evidence indicates that exosome
RNA content is also influenced by selective packaging
mechanisms that can, in some cases, enrich specific
RNA species in mature exosomes (Figure 1) [22].
Table 1. A list of the different proteins presents on the EV surface
and its cargos.
Class
Proteins
Function
Tetraspanins
CD9, CD63,
CD81
Cell signaling and cell adhesion
Histocompatibility complexes
MHCI, MHCII
Antigen presentation
Annexins
Annexin I, II, IV,
V, VI
Intracellular membrane fusions
and transport
Heat shock proteins
HSP 70, HSP90
Mediates folding
Ubiquitylated cargo-binding
proteins
TSG101
ESCRT pathway and tumor
suppressor
Lipid raft protein
FLOT1
Scaffolding protein for vesicle
formation
PDCD6-interacting protein
ALIX
ESCRT pathway
Transmembrane pr oteins
Integrin-α,
integrin-β
Cell adhesion
Exosome trafficking and uptake: Secreted
exosomes can specifically interact with cells close to
their release site, or at distant sites after transport
through the circulation, in a process regulated by
exosome membrane factors (e.g., integrins, annexins,
galectins and intercellular adhesion molecule 1) [16].
Exosome uptake can alter cell expression via RNA
transfer (mRNAs, miRNAs, and lncRNAs), cytosolic
proteins and receptors, and cell-specific adhesion
molecules to influence cell function and later exosome
uptake [23]. Exosome-derived factors can directly
affect gene transcription and translation, influence
signaling cascades to alter transcript and protein
modifications, and regulate protein localization and
key enzymatic reactions, with interactions among
factors supplied by the exosome and recipient cell
determining which molecular mechanisms
predominate [16].
EV regulation of immune responses. EVs
derived from immune cells carry proteins that can
regulate important aspects of host immunity,
including T-cell activation (e.g., MHCI and MHCII,
lymphocyte function-associated antigen-1, and
intercellular adhesion molecule-1, depending on the
parent cell) [24]. MHC I and MHC II and
immunomodulatory proteins are enriched on EVs of
antigen-presenting cells (APCs; e.g., dendritic cells
(DCs) and macrophages) [4] and these EVs appear
capable of activating T cells by transferring antigens
or MHC-antigen complexes to conventional APCs, or
by directly presenting MHC-antigen complexes to T
cells as APC surrogates [5, 25-28].
For the first mechanism, evidence suggests that
immature DCs that do not support robust immune
responses secrete EVs that can transfer MHC-antigen
complexes, or antigens, to mature DCs to activate
CD4+ and CD8+ T-cell responses [28, 29]. In the
so-called “cross-dressing” model, intact MHC-antigen
complexes are transferred from inactive to active DC
populations [27-30], whereas in the “cross-presen-
tation” mechanism, mature DCs present peptides
derived from captured EVs on their own MHC
molecules [25, 26].
There is also evidence that APC-derived EVs can
directly activate CD4+ and CD8+ T cells [31-33], and
stimulate both previously activated and memory T
cells [13, 34]. APC-derived EVs are also able to
directly activate naïve CD8+ T cells in vitro [5],
although they appear to be 10- to 20-fold less efficient
than APCs, suggesting that EVs may not have a direct
effect on naïve T cell activation in vivo. Similar studies
indicate that EVs of B cells can also directly present
antigens to induce T cell responses [13], although with
less efficiency than their parental B cells. Receptor
aggregation between interacting T cells and DCs also
creates an extended “immunological synapse” where
DC-derived EVs can directly interact with adjacent T
cells in a LFA-1 (lymphocyte function-associated
antigen 1) dependent manner to promote their
activation [6].
However, while evidence indicates that EVs can
directly and indirectly regulate in vitro CD4+ and
CD8+ T cell responses, it is unclear to what extent
they affect these responses in vivo.
EV roles in infectious diseases: EVs released by
infected cells contain pathogen- and host-derived
factors, and play key roles in pathogen-host
interactions, including pathogen uptake and
replication and regulation of the host immune
response (reviewed in [7, 35]). For example, studies
have shown that multiple viruses—including human
immunodeficiency virus 1 (HIV-1), hepatitis viruses
B, C and E (HBV, HCV, and HEV), and multiple
members of the human herpesvirus (HHV)
family—utilize exosome ESCRT machinery for viral
transmission [36, 37]. HIV-1, in particular, has
developed several exosome-mediated strategies to
manipulate the behavior of its target cells [38],
including a Nef-regulated mechanism that alters EV
protein trafficking in CD4+ T cells. Hepatitis A virus
(HAV), HCV and HEV employ the exosome

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biogenesis machinery to produce enveloped virions
that allow the virus to avoid immune surveillance
(reviewed in [39]). HHVs, which are responsible for a
broad range of important pathologies, employ
endosomes to evade anti-viral immune responses
through several distinct mechanisms that differ
among these viruses [37]. In the following sections,
we describe how several viruses subvert the exosome
biogenesis machinery for their replication and
infectivity. Much less is known about how bacteria
employ the EVs of their host to favor their growth and
survival, and most studies focus on the behavior of
Mycobacterium tuberculosis (Mtb), an important
intracellular pathogen. We will therefore summarize
current knowledge on how Mtb regulates cellular and
systemic processes to favor active and latent Mtb
infections, and how these processes overlap with
those of other bacterial pathogens (Figure 2).
EVs facilitate viral and bacterial pathogenesis
Viral and bacterial pathogens can subvert
exosome functions to promote pathogen replication,
survival, or pathology. Cells employ EVs to transfer
regulatory factors that modulate the response of local
and distant cells and systemic responses. In cells with
active viral or bacterial infections, the exosome
machinery can also package pathogen-derived factors
that alter the phenotype of EV recipient cells. Many
pathogen factors that are packaged into EVs interact
with ESCRT proteins or related factors, suggesting
that pathogens have evolved to exploit this
intercellular transport and signaling pathway, using it
to promote infection and repress anti-pathogen host
responses. We discuss several examples of these
interactions in the following sections.
Viruses
Functional overlaps between exosome biogenesis
and viral budding: Mechanisms involved in exosome
and enveloped virus budding share common features.
Many retroviruses are reported to interact with
ESCRT complex and ESCRT-related proteins involved
in exosome biogenesis through conserved protein
motifs, referred to as late domains since their deletion
or mutation leads to the arrest of virus assembly at
late stages of virion synthesis [36]. For example, HIV-1
virion interactions with TSG101, ALIX, and other host
proteins are similar to those employed to package
host proteins during exosome formation [40].
Similarities between the exosome and HIV-1
packaging mechanisms led to the statement of “the
Trojan exosome hypothesis”, which proposes that
HIV-1 evolved to utilize exosome biogenesis proteins
to package its capsid, while also exploiting exosome
uptake mechanisms to allow cell infection in the
absence of viral envelope proteins that normally
Figure 2. Overview of the EV incorporation of pathogen-derived factors by the EVs of their host cells, including pathogen re ceptors and regulatory factors, to promote infection
and pathogenesis. (A) EVs of HIV-infected c ells express the HIV receptor target proteins CCR5 and CXCR4 and reg ulatory factors, including the HIV protein Nef and TAR RNA,
among others. (B) EVs of HCV-infected cells express E2 and CD81, which promote HCV uptake, as well as viral RNA and host proteins (e.g., CD63) that promote HCV
infections. (C) EVs of HBV-infected cells contain EBV RNA and proteins (e.g., LMP1) and host proteins (e.g., EGFR and FGF2) that promote EBV infectivity and pathogenesis. (D)
EVs of Mtb-infected cells contain Mtb-derived glycolipids (LAM) and lipoproteins (LpqH) that regulate innate and acquired immune responses to promote infection.

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direct HIV-1 uptake [40]. Mounting evidence suggests
that several human viruses hijack proteins involved in
exosome biogenesis to package their capsids. Human
herpesvirus (HHV) family members exploit proteins
that regulate exosome biogenesis for their virion
production. HHV-1 (herpes simplex virus 1; HSV-1)
structural proteins contain potential TSG101 (ESCRT-I
complex) and ALIX (ESCRT-I associated) binding
motifs. Dominant-negative and siRNA expression
studies also indicate that HHV-1 does not employ
TSG101 or ALIX, but requires functional expression of
CHMP (ESCRT-III complex) and VPS4 (ESCRT-III
associated) proteins for the formation of its virion
envelope [36, 37]. HHV-5 (human cytomegalovirus;
HCMV) also appears to utilize a similar packaging
mechanism, since inhibition of either CHMP1A or
VPS4, but not ALIX, interferes with its virion
packaging. Neither HHV-4 (Epstein-Barr virus; EBV)
nor HHV-8 (Kaposi Sarcoma-associated herpes-
viruses) appear to require exosomal protein
interactions for their secretion [37].
Despite these functional interactions, it is not
clear how all of these viruses employ exosome
proteins in their packaging and secretion. While
components of multiple viruses have been shown to
associate with MVBs, there is little evidence that these
viruses localize within MVBs and are secreted by
MVB fusion with the plasma membrane. Only one
HHV study appears to provide data consistent with
an MVB release mechanism, reporting that HHV-6
virions localize within MVBs in infected cells [37],
although these virions (~200 nm) would be much
larger than exosomes (30-100 nm).
All members of the hepatitis virus family are
reported to employ exosome-related proteins to form
enveloped virions. HBV envelope proteins colocalize
with MVB proteins ALIX and VPS4B, and
dominant-negative versions of either of these proteins
block the release of enveloped HBV virions [41]. HCV
interacts with Hrs (ESCRT-0 complex) to promote
apparent MVB uptake of viral capsids [42] and EVs
isolated from HCV-infected hepatoma cell lines and
sera of patients with chronic HCV infections contain
HCV core and envelope proteins and full-length HCV
RNA [43]. HAV and HEV are shed as naked viral
particles in feces but circulate as membrane-enclosed
virions, which are less infectious but are masked by
the host’s immune response [37]. Recent evidence
suggests that production of these circulating
enveloped virions requires interaction with the
exosomal sorting components CHMP2a (ESCRT-III
complex), ALIX, and VSP4 (ESCRT-I and -III
associated) for HAV [44, 45] or Hrs (ESCRT-0
complex) for HEV [46]. Similar to HHV, however,
there is scarce evidence for MVB-mediated release of
hepatitis family viruses, with only one study
indicating that ~50 nm enveloped virions are
detectable in MVBs of HEV-infected cells [46].
EVs can alter virus antigenicity and infectivity:
Results suggest that some persistent viruses (e.g.,
HCV and HAV) employ EVs as a strategy to escape
negative selective pressure from neutralizing
antibodies and other immune responses that act to
promote viral clearance [47-49]. MVB-mediated
encapsulation may also allow a virus to spread
beyond its normal range of cell hosts through the
normal EV uptake process, as demonstrated by the
ability of EVs containing HAV capsids to infect target
cells using EV surface proteins instead of the
EV-masked viral receptor proteins (reviewed in [3]).
EVs can spread viral docking receptors to
promote viral infectivity: HIV normally binds to CD4
and the chemokine receptors CCR5 or CXCR4 on
target cells to mediate infection, and cells lacking
these receptors, or with receptor mutations, are
resistant to HIV infection. EVs secreted by HIV-
infected cells contain CCR5 or CXCR4, however, and
their uptake by cells lacking these receptors facilitates
HIV infection of these otherwise HIV-resistant cells
[50, 51]. The widespread EV markers CD81 and CD63
colocalize with subgenomic HCV RNA and appear to
promote its packaging into EVs. The HCV envelope
glycoprotein E2 also colocalizes with CD81 and cells
that internalize EVs containing this complex are more
susceptible to HCV infection [49, 52]. Interaction with
this complex may also promote HCV uptake by EVs
[53, 54]. Studies suggest that EV proteins may
facilitate viral-receptor-independent transmission of
HCV and HAV, and presumably other EV-enveloped
viruses, to uninfected cells [43, 49].
Regulatory actions of virus-associated EVs on
host cells: EVs derived from virus-infected cells can
also transfer viral proteins to influence viral
pathogenesis. EVs from HIV-infected cells contain the
HIV-1 protein Nef, which regulates endocytosis,
cytoskeletal rearrangement, and organelle trafficking
to increase the number of EVs released from
HIV-infected cells [55, 56], and may thus promote
EV-mediated HIV infectivity. Nef-induction of
EV-associated ADAM17 also appears to promote
HIV-infection of resting CD4+ T cells [57], while
ADAM17 and TNFα together can activate latent
HIV-1 infections in primary CD4+ T lymphocytes and
macrophages [17]. Finally, EVs carrying Nef appear to
exert complex effects to regulate HIV-1 infection and
pathogenesis through actions on uninfected cells
[58-60], including the ability to alter the functions of
import immune responses.
Human gammaherpesviruses, such as EBV,
have complex effects to promote both viral infection

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and cancer. EVs derived from EBV-infected cells
exploit the endosomal-exosomal pathway to enclose
both EBV- and host-derived regulatory factors [61,
62]. In EBV-infected cells, an interaction between the
EV protein CD63 and the viral protein LMP1 appears
to promote LMP1 incorporation into EVs [63, 64] and
EVs that contain LMP1 can deliver important
signaling proteins to uninfected cells [62]. LMP1 also
induces the expression of both epidermal growth
factor receptor (EGFR) and fibroblast growth factor 2
(FGF2), which are also packaged in LMP-1-marked
EVs [65, 66], suggesting that EV-mediated transfer of
these receptors may stimulate the growth of recipient
cells with a potential to promote EBV-mediated tumor
development.
Effects of virus-associated EVs to inhibit
anti-viral responses: Viruses employ several
EV-mediated strategies to attenuate host immune
responses. EVs of HIV-infected macrophages deliver
Nef to recipient cells to alter their immune function.
Nef is associated with intracellular sorting and
trafficking pathways that promote the lysosomal
degradation of CD4 and MHCI to reduce their surface
expression [59, 67], rendering cells that express Nef
less susceptible to cytotoxic immune responses.
Evidence also indicates that Nef+ EVs facilitate HIV
pathogenesis by conditioning their target cells to
undergo apoptosis, promoting CD4+ T cell depletion
and HIV-mediated immune suppression to reduce
immune clearance of HIV-infected cells [60, 68, 69].
The major EBV oncoprotein LMP1, which is
carried by EVs of EBV-infected cells, plays an
important role in EBV infection (reviewed in [3]).
LMP1 expression has an important function to
activate B cells; however, recent work suggests that
EVs carrying LMP1 may also promote B cell activation
and proliferation [70] and can inhibit proliferation of
T cells and the ability of natural killer (NK) cells to
exert cytotoxic effects. [61, 65]. EBV also encodes a
number of miRNAs that can modify the transcriptome
of infected cells, and non-infected cells via EV transfer
[71, 72]. EBV-infected cells release EVs containing
EBV miRNAs that suppress EBV target genes,
including CXCL11, an immunoregulatory gene
involved in antiviral activity [73]. EVs released by
EBV-infected cells also contain the host-derived
protein galectin-9, which is known to induce
apoptosis of EBV-specific CD4+ T cells through an
interaction with immunoglobulin mucin-3, and to
negatively regulate both macrophage and T cell
activation [74].
Viruses thus appear to employ multiple
EV-based mechanisms to suppress the clearance of
their host cells by the immune system to promote
continued viral infections; however, the in vivo
relevance of these mechanisms is not clear.
Viral transfer through immune cell EVs: In
addition to actions to inhibit the antiviral activity of
immune cells, virus-derived EVs can also use these
cells to promote viral transfer to new host cells. HIV-1
virions captured by immature DCs and exocytosed in
association with the DC cell’s EVs can trans-infect
CD4+ T cells [75]. The Trojan horse hypothesis of HIV-1
trans-infection [40, 76] takes this further, suggesting
that HIV-1 virions are retained in the MVB
compartment of mature DCs and trans-infect CD4+ T
cells in lymph nodes by following the same trafficking
pathway that DC EVs use to disseminate antigens [40,
76, 77].
Bacteria
Regulatory actions of bacterial-associated EVs
on host cells: Bacterial pathogens can be classified
based on the nature of their interactions with their
host, including whether they prefer or require an
intracellular or extracellular niche to initiate and
maintain active infections. Both extracellular and
intracellular bacteria can display complicated
lifecycles, but intracellular bacteria have several
unique options to subvert cellular processes,
including the EV pathway, to promote their growth
and survival. Mycobacterium tuberculosis is perhaps the
best studied of these pathogens with respect to its EV
effects due to its significant worldwide impact on
public health, although several other intracellular
pathogens are responsible for significant human
diseases. M. tuberculosis (Mtb) evades the innate
immune response by stably infecting phagocytic cells,
such as macrophages, which are primarily responsible
for clearance of microbial pathogens. Mtb lipoproteins
and lipoglycans inhibit phagosome maturation,
generating a stable intracellular niche for the engulfed
Mtb bacilli and blocking MHCII-antigen complex
cycling to the cell surface to inhibit the host response
to Mtb-derived antigens [78, 79]. Mtb-derived factors
can also promote EV release and it is hypothesized
that some mycobacterial proteins contain signals that
direct them to MVBs to promote their incorporation
into EV [80].
Mtb-related EVs play important roles in
regulating the phenotypes of both infected and
uninfected cells and likely contribute to the overall
pathogenesis of Mtb infections [78, 79]. EVs of
Mtb-infected macrophages can stimulate non-infected
macrophages to secrete chemokines to induce the
migration of naïve T-cells and macrophages [81]. Mice
intranasally injected with EVs from Mtb and M. bovis
BCG, revealed increased TNFα and IL-12 production,
as well as the recruitment of macrophages and
neutrophils to the lung [82], suggesting that these EVs

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could recruit non-infected target cells to promote
disease progression. Macrophages infected with M.
avium also revealed increased EV secretion, which
lead to a pro-inflammatory response in non-infected
macrophages while simultaneously downregulating a
number of IFN-γ-inducible genes in naïve cells to
inhibit the inducible expression of MHC-II and the
CD64 immunoglobulin receptor [83]. Macrophages
infected with M. avium and M. smegmatis exhibited
increased EV secretion and increased EV expression
of HSP70 to promote in vitro macrophage activation
and TNFα expression [84]. EVs of Mtb-infected cells
also contain the 19 kDa lipoprotein LpqH, which can
promote inflammation and stimulate in vitro
macrophage activation and TNF-α expression via the
Toll-like receptor/MyD88 pathway (reviewed in [85]).
These results suggest that EVs from mycobacterium-
infected cells can both activate and recruit immune
cells, and may therefore influence innate and acquired
immune responses during mycobacterial infection
[82], although the relative impact of such putative
effects on the overall immune response is not clear.
Effects of bacterial-associated EVs to inhibit
anti-bacterial immune responses: EVs released by
macrophages infected with Mtb bacilli contain protein
cargos that regulate both innate and adaptive immune
responses. EVs from Mtb-infected macrophages
contain the glycolipid lipoarabinomannan (LAM) that
inhibits T cell receptor signaling and T cell activation
responses, which may induce immune suppressive
mechanisms that promote the survival of Mtb-infected
cells to maintain active Mtb infections [86, 87]. EVs
from Mtb-infected cells can partially suppress the
ability of macrophages to respond to INF-γ to inhibit
macrophage APC function [88]. EVs of macrophages
infected with M. avium can also downregulate a
number of IFN-γ-inducible genes in naïve cells to
inhibit the inducible expression of MHC-II and the
CD64 immunoglobulin receptor [83].
EVs as diagnostic markers: EV expression of
pathogen-derived factors and changes in the EV
abundance of specific host-derived factors can serve
as diagnostic biomarkers, indicators of disease
progression, and/or capture targets for the
enrichment of pathogen-derived EVs for further
analysis. Changes in EV composition during disease
progression make them excellent biomarker
candidates. The first blood-based EV test for cancer
diagnosis became commercially available in the US in
January 2016, marking a major step in the maturation
of EVs as diagnostic factors [89]. The study of EVs for
diagnosis of infectious disease is relatively new but
shows great promise, particularly for intracellular
bacterial pathogens, such as mycobacteria. Diagnosis
of these pathogens normally requires culture or
molecular analysis of pathogen samples derived from
the site of infection and can misdiagnose patients with
low pathogen loads. EVs containing pathogen-
derived factors are actively secreted from most cells to
accumulate in the circulation; some studies indicate
that infection increases EV release rates [81, 90]
although it is not clear if this increase is common to all
infections. Most current approaches that diagnose
active tuberculosis cases use sputum samples as the
primary diagnostic specimen. However, there are
limitations associated with sputum diagnostics and
the World Health Organization has issued a call for
new approaches that can diagnose active tuberculosis
cases using minimally invasive patient samples, such
as peripheral blood samples [91].
Several studies have now indicated the potential
of EVs from minimally or non-invasive biological
samples to detect such infections. Serum EV
concentrations in mice infected with M. bovis BCG
correlated and exhibited similar kinetics with M. bovis
BCG mycobacterial load, suggesting the potential
utility of serum EVs as diagnostic biomarkers for
disease burden [81]. A subsequent study used
liquid-chromatography and tandem mass spectro-
metry to identify 41 mycobacterial proteins in EVs
derived from Mtb-infected J774 cells [80] and in 2014,
analysis of serum EVs isolated from patients with
active tuberculosis cases detected numerous
mycobacterial proteins, indicating that Mtb-derived
EVs can function as markers of active disease [92].
Mycobacterial RNA was also detected in EVs derived
from Mtb-infected macrophages [93], implying the
potential for analyzing Mtb RNA in EVs as a
diagnostic marker for active tuberculosis cases.
Recognition and quantitation of pathogen-
derived EVs: Despite increasing scientific and clinical
interest in the potential of EVs for disease diagnosis,
there are few standard procedures for their isolation,
detection, characterization and quantification. The
ISEV has emphasized the development and harmon-
ization of standard protocols for specimen handling,
isolation and analysis to facilitate comparison of
results achieved within this fast-growing field [1, 2].
EVs are too small to analyze by conventional optical
detection methods, and their low refractive index and
heterogeneous size and composition complicate such
analyses, but recent advances now allow nanoparticle
quantitation, which is useful for general EV analyses.
Nanoparticle tracking analysis (NTA) employs a
laser beam to illuminate all vesicles in a sample
suspension, a light microscope to record the scattered
light, and software to measure vesicle sizes as
determined by the Brownian motion track of each
particle [94, 95]. These instruments are commercially
available and can measure the number and absolute

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2716
size distribution of vesicles in a solution, and
quantitate EVs based on their unique size profile.
Resistive pulse sensing (RPS) can determine the
absolute size distribution of vesicles in sample
suspensions via the Coulter principle [96], and at least
one company has developed an instrument to exploit
this approach. This system consists of two fluid cells
divided by a non-conductive nanoporous membrane.
A particle moving through one of these nanopores in
response to a voltage applied across the cell
membrane alters the ion flow, resulting in a brief
“resistive pulse”, which is recorded for calculation
against a reference standard made with beads of
known diameter and concentration [94, 96]. Finally,
alternating current electrokinetic (ACE) microarrays
can isolate EVs from plasma samples to allow on-chip
immunofluorescent detection of EV proteins and to
provide mRNA for RT-PCR analysis [97], providing a
potential means to isolate and analyze total EV
populations without a separate EV isolation step
(Figure 3). None of these approaches can quantitate
disease-specific EV populations from the general EV
population.
Transmission electron microscopy can visualize
EVs and analyze size and morphology, but this
method is labor intensive and requires the use of
procedures that are expensive and impractical for
clinical use. More importantly, while it can potentially
visualize specific EV sub-types after immune-gold
staining, it cannot cope with the challenge of
identifying and quantitating disease-associated EVs
amidst the diverse population of serum EVs,
particularly during early disease progression when
these EVs should be extremely rare in the highly
abundant circulating EV population.
Other optical approaches have attempted to
address this issue. Nanoparticles are too small for
direct detection by conventional flow cytometry, but
one company has developed a high-resolution flow
cytometer that can directly detect EVs. This
specialized machine requires high-power lasers and
high-performance photomultiplier tubes, detection of
light scattering at customized angles, and the
application of fluorescence-based thresholding to
distinguish particles of interest from noise [98, 99].
Standard flow cytometers can, however, analyze
multiplex bead-based platforms to detect and analyze
aggregate signal derived from multiple EVs after they
are bound to micrometer polystyrene capture beads
[100]. Stimulated emission depletion (STED)
microscopy can also be used to measure multiple
markers on single EVs, but this approach does not
appear suitable for the analysis of rare EV populations
without a prior isolation step.
Figure 3. Methods used to detect and quantify EVs. (A) Nanoparticle tracking analysis (NTA) analyzes laser scattering in purified vesicle suspensions to track the Brownian
motion of the vesicles and determine their absolute size (reproduced with permission from [95]
, copyright 2011 Elsevier). (B) Resistive pulse sensing (RPS) measures the
“resistive pulse” rate produced when purified vesicle suspensions migrate across a membrane in response to applied voltage to determine vesicle concentration relative to a
reference standard (adapted with permission from [96], copyright 2012 American Chemical Society). (C) Alternating current electrokinetic (ACE) microarrays use the electrical
properties of EVs to isolate these particle s from plasma sample and then detect the immunofluorescent signal from target EV proteins. The schematic shows a cross-section and
a top-down view of a single electrode well on the array (reproduced with permission from [97], copyright 2017 American Chem ical Society).

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Several groups have recently reported robust
on-chip isolation and detection methods to study and
profile EVs. In 2014, a group reported the
development of a nanoplasmonic exosome (nPLEX)
sensor consisting of an affinity ligand-modified gold
film that contained an array of periodic nanoholes, in
which EV binding produced a spectral shift
proportional to the number of targeted EVs bound on
the array [101]. In 2015, a second group reported the
development of an immunomagnetic exosome RNA
(iMER) platform for on-chip EV enrichment, RNA
isolation, reverse transcription and real-time analysis
of distinct RNA targets, which they used for
treatment-induced mRNA in glioblastoma
multiforme patients [102]. In 2017, we published a
nanoplasmonic enhanced scattering (nPES) method
where EVs are bound to a chip by a pan-specific EV
antibody, and hybridized with antibody-labeled
nanoparticles specific to a second common EV protein
and a disease-specific EV marker so that target EVs
produce a shifted nPES signal in direct proportion to
their number [103]. All these technologies should
allow one to modify the EV targets analyzed by
changing the affinity of the detection antibody or
ligand, and thus should be readily adaptable for any
disease for which there is a disease-specific EV
biomarker available (Figure 4). None of these
approaches are yet available for clinical applications,
but they demonstrate the potential of new chip
technologies to rapidly profile disease-specific EVs
from human samples after minimal sample
preparation.
Potential roles for EVs as disease therapeutics:
Many of the EV features that allow EVs to regulate the
pathogenesis of infectious pathogens may also allow
them to act as effective agents for the development of
novel therapeutic approaches. For example, the ability
of EVs to selectively deliver molecules to specific
recipient cell types has significant potential for the
delivery of small molecules and other therapeutic
agents. There are a number of advantages associated
with using EVs as the basis for a pathogen-specific
therapeutic system (reviewed in [35, 104-107]). EVs
can incorporate various pathogen-derived factors,
including receptors involved in cell targeting,
suggesting it should be possible to modify EVs to
Figure 4. On-chip designs for isolation and detection of disease-specific exosomes. (A) An image of the nanoplasmonic exo some (nPLEX
) sensor chip integrated into a
multichannel microfluidic cell for independent and parallel analysis (adapted with permission from [101], copyright 2014 Nature America). This device uses specific affinity ligands
to capture EVs on an array containing nanoholes, which are blocked by EV binding to produce a spectral shift that indicates the number of target EVs bound to the array. (B)
Photograph of the microfluidic immunomagnetic exosomal RNA (iMER) platform iMER prototype with a schematic of its input material (adapted with permission from [102],
copyright 2015 Macmillan Publishers Limited). The iMER chip binds antibody-labeled magnetic beads reacted with serum EVs, isolates their RNA, and performs RT-PCR to
analyze target mRNAs. (C) Schematic and data from a nanoplasmonic-enhanced scattering analysis (nPES) assay (adapted with permission from [103], copyright 2017 Macmillan
Publishers Limited). Serum EVs bound to the nPES chip by an EV-specific antibody produce red or green light when they bind only the EV- or the disease-specific nPES probe
(lower left and middle images; single probe controls), but produce an intense yellow signal when they bind both nanoprobes (lower right image; experimental well).

Theranostics 2018, Vol. 8, Issue 7
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2718
target specific cells of interest via incorporation of
receptors that target a cell type or cell population of
interest. EVs are also efficiently internalized by APCs,
implying that they may be useful in the direct
delivery of antigens or MHC-antigen complexes and
costimulatory molecules to directly promote robust
immune responses against a pathogen. EVs package
various materials and maintain the integrity of these
materials as they freely circulate, suggesting that EVs
can be employed to package and shuttle various
therapeutic agents (e.g., siRNAs, small molecule
therapeutics, and other agents) with similar efficiency
(reviewed in [108]).
APC-derived EVs can exhibit anti-pathogen
activities, as previously discussed, through their
ability to prime immune responses. Mice injected with
EVs from macrophages treated with Mtb culture
filtrate protein produced antigen-specific CD4+ and
CD8+ T cell activation responses similar to those of
mice immunized with M. bovis BCG, the only accepted
means of vaccination for Mtb [109]. Notably, however,
the mice injected with Mtb-derived EVs did not
exhibit an antigen-specific increase in TH2 cells that
can limit the effectiveness of a vaccine response,
unlike the M. bovis BCG-immunized mice. This
finding matched results from previous studies
reporting that antigenic EVs primarily induced
cytotoxic TH1 immune responses that supported the
clearance of intracellular pathogens [110]. Mice
vaccinated with Mtb-derived EVs and M. bovis BCG
revealed similar protection when subsequently
challenged with a Mtb inoculation. Taken together
these data indicate that vaccination with Mtb-derived
EVs was as efficient as vaccination with M. bovis BCG
bacilli, if not more so, indicating the potential for
pathogen-free vaccine approaches using non-
infectious EVs containing pathogen-derived antigens
[109]. However, it is not clear if the MHC of such EVs
must match the MHC of the vaccine recipient to
promote an optimal pathogen clearance response and
avoid inducing autoimmune responses that might
occur when a small number of cells incorporate
EV-derived MHC complexes that do not match those
of the vaccine recipient. This would present a
significant challenge to large-scale production and
administration of an EV-based vaccine.
Liposomes are currently a favored drug delivery
system to carry therapeutics to target tissues and cells,
but have several drawbacks. These can include poor
in vivo stability and retention; problems with drug
loading, leakage and release; and difficulty directing
therapeutic liposomes to specific cell and tissue
targets (reviewed in [105]). By contrast, EVs can
selectively package diverse factors during their
biogenesis, and can selectively target multiple
different cells and tissue types by virtue of specific
membrane factor interactions (reviewed in [111, 112]).
Selective tagging of therapeutic agents to utilize
packing factors involved in EV biogenesis or simple
mass action may allow cultured cells to efficiently
package therapeutic agents into EVs for therapeutic
applications. Producing therapeutic EVs in cells with
known cell specificities or modifying EVs to carry
cell-specific targeting factors may also yield better
specificity and uptake rates than current liposomes
and nanocarriers [113, 114], although EVs can still
demonstrate some nonspecific accumulation in highly
vascularized tissues, including the lung and liver
[114], likely due to EV size, as previously observed for
other particles [115].
EVs have already been used for in vitro delivery
of exogenous nucleic acids to target cells. For
example, one study found that EVs released by
cultured THP-1 monocytes efficiently transferred
miR150 to recipient cells, where it regulated gene
expression and cell function [116]. Researchers have
also exploited the RNA transport capacity of EVs to
deliver short interfering RNAs (siRNA) to
post-translationally silence recipient cell target genes
during in vitro cancer studies, and one in vivo study
pre-transfected EV donor cells with suicide genes and
injected the resulting EVs into an orthotopic mouse
cancer model to target schwannoma tumors [117].
Most studies using EVs as delivery vehicles have
concentrated on cancer models, so the potential for EV
therapy in infectious diseases is still unclear; however,
similar approaches should be feasible for the
treatment of chronically infected cells once
researchers have identified or designed EV factors
that exhibit specific interactions with infected host
cells.
Conclusion
Mounting evidence indicates pathogen-derived
EV factors play important roles in several human
diseases, and a better understanding of these
mechanisms may provide new insights for future
therapeutic development. Several reports indicate
that immunomodulatory molecules present in or on
EVs can affect pathogen responses through actions to
activate or suppress immune responses, and it is
possible that increased knowledge of these
mechanisms will improve pathogen treatment
approaches, including the potential use of EVs to
develop more effective vaccines and immuno-
therapies. Pathogen-specific EV factors are also of
great interest as novel disease biomarkers, due to their
close association with disease and their potential for
greater diagnostic sensitivity and specificity due to
their stability in blood and urine. Specific EV

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biomarkers and means to analyze specific EV subsets
have been lacking to date, but new research, including
the recent development of several approaches to
analyze specific EV populations, appear poised to
allow the clinical translation of EV diagnostic assays.
The stability, cell-targeting and packaging properties
of EVs also recommend them as vectors to deliver
new therapeutics, although there are still several
questions that must be addressed to allow their
translation to therapeutic applications. These include
how to control the purity of EV preparations, how to
evaluate and control the co-expression of different
molecules on these EVs, and what are the best
administration routes to achieve targeted delivery
and desired effects for different applications. Further
research is needed to address these and other issues
and to evaluate whether specific EVs can be used for
diagnostic and therapeutic approaches, but the
inherent properties of these particles appear likely to
lend themselves to these approaches.
Abbreviations
APCs: antigen-presenting cells; DCs: dendritic
cells; EBV: Epstein-Barr virus; EGFR: epidermal
growth factor receptor; ESCRTs: endosomal sorting
complexes required for transport; EVs: extracellular
vesicles; FGF2: fibroblast growth factor 2; HAV, HBV,
HCV, and HEV: hepatitis viruses A, B, C and E;
HCMV: human cytomegalovirus; HHV: human
herpesvirus; HIV-1: human immunodeficiency
virus-1; HSV-1: herpes simplex virus 1; ILVs:
intraluminal vesicles; iMER: immunomagnetic
exosome RNA; ISEV: International Society for
Extracellular Vesicles; LAM: lipoarabinomannan;
LFA-1: lymphocyte function-associated antigen 1;
lncRNAs: long non-coding RNAs; MHCI and MHCII:
class I and II major histocompatibility complexes;
miRNAs: microRNAs; mRNAs: messenger RNAs;
Mtb: Mycobacterium tuberculosis; MVBs: multivesicular
bodies; NK: natural killer; nPLEX: nPES:
nanoplasmonic enhanced scattering; nPLEX:
nanoplasmonic exosome; NTA: nanoparticle tracking
analysis; RPS: resistive pulse sensing; ACE:
alternating current electrokinetic; siRNA: short
interfering RNAs; STED: stimulated emission
depletion.
Competing Interests
The authors have declared that no competing
interest exists.
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