Unveiling the role of extracellular vesicles in HIV infection: molecular mechanisms, viral persistence, and therapeutic opportunities

Abstract

Human immunodeficiency virus (HIV) infection remains a major global health challenge despite the success of antiretroviral therapy, largely due to the persistence of viral reservoirs and chronic immune dysregulation. Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication during viral infection, operating at the interface between viral and host cellular pathways. Depending on their cellular origin and molecular cargo, EVs can exert context-dependent effects, with studies suggesting roles in both viral dissemination and persistence, as well as in the modulation of antiviral immune responses. This review examines the molecular mechanisms through which EVs have been proposed to contribute to HIV infection, focusing on host–virus interactions, immune regulation, chronic inflammation, and viral latency. We further discuss emerging therapeutic strategies targeting EV biology, including approaches aimed at modulating EV biogenesis and cargo composition, as well as the development of engineered EVs as platforms for drug delivery, gene editing, and immune modulation. Overall, this study highlights the emerging roles of EVs in HIV infection, emphasizing both their potential relevance and the limitations that currently complicate the interpretation of EV-associated effects.

Extracellular vesicles in HIV infection: from pathogenesis to therapeutic exploitation. This schematic overview illustrates the dual role of extracellular vesicles (EVs) during HIV infection, acting as both potential mediators of viral pathogenesis and platforms for therapeutic intervention.On the left, EVs derived from infected cells are proposed to promote viral dissemination through the transfer of viral RNA, proteins, and co-receptors to CD4⁺ T cells, to contribute to immune evasion by shielding infected cells from antibody recognition, and to drive inflammatory reprogramming associated with neuroinflammation. On the right, EVs are exploited as therapeutic tools, serving as delivery systems for antiviral drugs, RNA-based therapeutics, and gene editing strategies, as well as platforms for latency targeting approaches and vaccine-induced immune activation. Together, these mechanisms highlight the dual functional nature of EVs in HIV infection. Created with Biorender.comUnderstanding how extracellular vesicles shape HIV persistence may help to bridge current knowledge of viral pathogenesis with next-generation therapeutic strategies.

Introduction

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles released by most nucleated cells under both physiological and pathological conditions (Zaborowski et al. 2015). EVs carry a complex molecular cargo, comprising proteins, enzymes, lipids, metabolites, and multiple classes of coding and non-coding RNAs, which can reflect the functional state and cellular origin of the producing cell (Thèry et al. 2002, Robbins and Morelli 2014, Lee et al. 2024). Through receptor-ligand interactions, membrane fusion, and endocytic uptake, EVs contribute to intercellular communication and have been implicated in processes such as proliferation, differentiation, migration, immune regulation, tissue repair, and maintenance of homeostasis (Xie et al. 2019).

Viral infections continue to represent a major global health burden, as exemplified by HIV, hepatitis B and C viruses, influenza viruses, and emerging pathogens such as SARS-CoV-2, collectively accounting for substantial morbidity, mortality, and socioeconomic impact (Liu et al. 2025). Disease progression in viral infections reflects dynamic and reciprocal interactions between viruses and host cells, which shape immune responses, influence viral persistence, and affect therapeutic outcomes (Luo and Gao 2020, Li et al. 2024). Despite remarkable advances in antiviral therapy and vaccination strategies, persistent challenges, including viral genetic variability, drug resistance, immune escape, and unequal access to treatment, underscore the need to better understand host cellular communication pathways that regulate viral spread and immune control (Zannella et al. 2021, Talla et al. 2026).

In this context, EVs have emerged as important mediators of host–pathogen interactions during viral infection. Their biological effects appear to be highly context-dependent: EVs have been proposed to contribute to antiviral responses through the transfer of immunoregulatory molecules, while in other settings they may facilitate viral dissemination or persistence by transferring viral proteins or RNA, modulating cytokine signaling, or influencing inflammatory pathways (Dreux et al. 2012, Ramakrishnaiah et al. 2013, Moulin et al. 2023). However, many of these mechanisms are supported primarily by in vitro studies or indirect evidence, and their relevance in vivo remains to be fully established.

Among human viral infections, HIV-1 represents one of the most extensively studied models for investigating EV-virus interactions (Boucher et al. 2025a). A growing body of work suggests that EVs may influence viral persistence and disease progression, although definitive mechanistic evidence in people living with HIV is still limited. HIV-1 primarily targets CD4⁺ T cells and establishes lifelong infection through integration of the proviral genome and the formation of latent reservoirs. Even during clinically controlled phases, residual replication and chronic immune activation can persist, and viral rebound following antiretroviral therapy (ART) interruption underscores the need to better understand the cellular mechanisms that sustain persistence and immune imbalance, including those potentially involving EVs (Bandera et al. 2019, Tang et al. 2024).

Several studies suggest that HIV infection may alter EV biogenesis and release, potentially affecting both the quantity and composition of EV populations as well as the cellular pathways governing their secretion (György et al. 2015, Kemunto et al. 2026). These changes have been proposed to influence key pathogenic processes, including immune signaling, maintenance of viral latency, stability of viral reservoirs, and neuronal integrity. At the same time, EVs are being explored as potential therapeutic targets and delivery platforms for antiviral, immunomodulatory, and reservoir-directed strategies, although their translational applicability remains under investigation (Kumar et al. 2024).

In this review, we synthesize current knowledge on the multifaceted roles of EVs in HIV infection, with particular emphasis on proposed mechanisms underlying EV-associated viral dissemination, immune dysregulation, and latency. We also discuss emerging therapeutic and vaccine-oriented approaches, highlighting both their potential and the current limitations of the field.

Extracellular vesicle biogenesis and diversity

EVs are released by organisms across all domains of life, including bacteria and eukaryotes, highlighting their evolutionary conservation as a fundamental mechanism of intercellular communication (Liu et al. 2021a,b). In eukaryotic systems, EVs are widely recognized as mediators of intercellular signaling and are commonly described in terms of three major subtypes, exosomes, microvesicles, and apoptotic bodies, based on their biogenesis, size range, and molecular composition. However, these categories partially overlap and are best considered a simplified framework within a broader and heterogeneous EV population (Diaz-Garrido et al. 2021, Fang et al. 2022, Wang et al. 2024). In line with current MISEV2023 guidelines, the term “extracellular vesicles (EVs)” is used throughout the manuscript as the primary designation, while terms such as “exosomes” and “microvesicles” are retained only when referring to specific biogenetic pathways or experimental contexts where their origin is supported. For this reason, the following sections focus on the main mechanistic routes of EV biogenesis in eukaryotic cells, particularly relevant in the context of viral infections.

Endosomal pathways and exosome formation

Exosomes are nanosized vesicles (∼ 30–150 nm) generated within the endosomal system as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and released upon fusion of MVBs with the plasma membrane (Fig. 1). Their biogenesis relies on coordinated membrane remodeling and cargo selection processes driven by both ESCRT-dependent and ESCRT-independent mechanisms (van Niel et al. 2018). The ESCRT machinery (ESCRT-0, -I, -II, and -III) contributes to cargo recognition, membrane invagination, and vesicle scission, while accessory proteins and lipids, including tetraspanins, ceramide, and phosphatidic acid, influence membrane curvature and microdomain organization (Guix et al. 2017, Martins and Alves 2020). The ATPase VPS4 disassembles ESCRT complexes and deubiquitinates cargo proteins to complete ILV formation (Vietri et al. 2020, Han et al. 2022).

Figure 1.

Biogenesis and heterogeneity of EVs. EVs originate from multiple cellular pathways. In the endosomal pathway, early endosomes mature into multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs), formed via ESCRT-dependent and -independent mechanisms. MVBs can either fuse with lysosomes for degradation or with the plasma membrane to release EVs. Alternatively, EVs may form by direct outward budding from the plasma membrane, involving cytoskeletal remodeling and lipid redistribution. During apoptosis, cells release apoptotic bodies through membrane blebbing and fragmentation, which are cleared by phagocytes via efferocytosis. EV populations are heterogeneous and overlapping, and current terminology reflects biogenesis rather than discrete categories. Created with Biorender.com

Neutral sphingomyelinase-2 (nSMase-2) catalyzes the conversion of sphingomyelin into ceramide, promoting negative membrane curvature (Trajkovic et al. 2008, Goñi and Alonso 2009). Ceramide metabolism may also influence intracellular signaling pathways linked to vesicle trafficking (Goñi and Alonso 2009). Additional regulators of trafficking includephospholipase D2, which generates phosphatidic acid and facilitates membrane invagination (Ghossoub et al. 2014), as well as Rab GTPases (RAB27a/b, RAB35, and RAB39) and SNARE complexes, which coordinate MVB transport and membrane fusion events (Hsu et al. 2010, D’Souza-Schorey and Schorey 2018, Parton et al. 2020, Borchers et al. 2021, Matsui et al. 2022, Arya et al. 2024). These interconnected pathways enable selective enrichment of proteins, lipids, and nucleic acids, thereby shaping EV composition and functional heterogeneity (Anel et al. 2019, Lindenbergh et al. 2020, Shukla et al. 2023).

Importantly, several enveloped viruses, including HIV, can exploit components of the endosomal and membrane trafficking machinery involved in EV biogenesis. This overlap has led to the “Trojan exosome” hypothesis, which proposes shared features between viral particles and EVs in their biogenesis and trafficking (Gould et al. 2003, Pawliczek and Crump 2009). It remains debated whether HIV-infected patients with well-controlled infection exhibit accelerated aging associated with extracellular vesicle (EV) expression. Some studies suggest that EVs can be involved in HIV cognitive disorders and may serve as reliable biomarkers for the early detection and management of HIV-associated neurocognitive disorders (HAND) (Luo et al. 2025). However, the extent and functional consequences of this overlap remain context-dependent and are not yet fully defined.

Plasma membrane shedding and microvesicle release

Microvesicles (MVs), also known as ectosomes, are larger vesicles (100–1000 nm) that originate directly from outward budding of the plasma membrane (Fig. 1) (Lim et al. 2021). Their formation involves coordinated phospholipid redistribution, cytoskeletal remodeling, and actomyosin contraction regulated by ARF6 signaling, MAPK/ERK activation, and Ca²⁺-dependent enzymes (Meldolesi 2021).

Both ESCRT-associated and ESCRT-independent mechanisms may contribute to membrane budding, suggesting partial mechanistic convergence among EV subtypes. The ESCRT-0 complex identifies the transmembrane proteins within the endosomal membrane. The ESCRT-I and -II subunits are involved in the recruitment of transmembrane proteins, lipids, and RNA molecules within membrane lipid rafts, facilitating membrane deformation and the formation of buds (Gurunathan et al. 2021, Sun et al. 2021), while the ESCRT-III complexes are responsible for the final detachment of macrovesicles from the cell surface (Clancy et al. 2021). Membrane remodeling also involves lipid raft reorganization and cytoskeletal rearrangements mediated by small GTPases, as well as scramblases and flippases (Hankins et al. 2015, Cocucci and Meldolesi 2015, Sakuragi and Nagata 2023). Due to their plasma membrane origin, MVs often retain surface proteins and receptors from the parent cell, which may influence intercellular signaling and microenvironmental interactions (Muralidharan-Chari et al. 2009, Zhou et al. 2025). In the context of viral infections, MV-mediated transfer of host or viral components has been proposed to modulate cellular susceptibility and immune responses, although the physiological relevance of these processes is still being clarified.

Apoptotic bodies

Apoptotic bodies (ApoBDs) are the largest EV-associated structures (1–5 μm) and are generated during programmed cell death through membrane blebbing and cellular fragmentation (Fig. 1) (Atkin-Smith et al. 2015, van Niel et al. 2018, Sanwlani and Gangoda 2021, Yu et al. 2023). They may contain intact organelles, genomic DNA, and cytoplasmic components, and are rapidly cleared by phagocytes through efferocytosis, thereby contributing to tissue homeostasis and limiting inflammatory responses (Liu et al. 2020, Li et al. 2022, Xiong et al. 2025). A hallmark of their biogenesis is the externalization of phosphatidylserines, which serves as an important signal for recognition by specific receptors, including annexin V-binding systems and TIM family proteins. This process enables efficient clearance by macrophages, epithelial cells, and Sertoli cells through a process known as “efferocytosis”, which is essential for preventing the release of potentially harmful intracellular contents and maintaining tissue integrity (Furuta and Zhou 2023, Mesa et al. 2015, Trzeciak et al. 2021). Although historically considered a byproduct of cell death, apoptotic bodies are increasingly implicated in intercellular communication and may contribute to antigen presentation and immune modulation in pathological contexts, including viral infections. However, their functional relevance in vivo remains incompletely defined.

Methodological limitations in EV studies in HIV infection

A major limitation in EV research in the context of HIV infection is the substantial overlap between EVs and viral particles in terms of size, density, and biophysical properties. Consequently, complete separation of EVs from HIV virions with current isolation methods is not achievable. Common techniques, including differential ultracentrifugation, density gradient centrifugation, and size-exclusion chromatography, often lead to the co-isolation of EVs with HIV particles and other extracellular components, such as lipoproteins and non-vesicular nanoparticles. This complicates the interpretation of molecular and functional studies, as detected EV-associated signals may be partially influenced by co-purifying contaminants.

Although enrichment strategies can improve sample definition, they do not fully resolve this issue. Therefore, conclusions regarding the roles of EVs in HIV pathogenesis, immune modulation, and viral persistence should be interpreted with caution, as much of the available evidence comes from indirect observations or in vitro systems that may not fully reflect in vivo conditions.

Extracellular vesicles in viral pathogenesis

Viral transmission and immune modulation

Viruses have evolved multiple strategies to exploit EV pathways, potentially contributing to dissemination and modulation of host immunity. Both enveloped and non-enveloped viruses have been reported to hijack EV populations, including small EVs, microvesicles, and autophagosome-derived vesicles, to facilitate non-lytic cell-to-cell communication and possibly evade immune surveillance by mimicking physiological intercellular signaling (Bello-Morales et al. 2020, Cortes-Galvez et al. 2023).

During infection, viral proteins, nucleic acids, and host factors can be incorporated into EVs originating from multivesicular bodies, the plasma membrane, or autophagosomal compartments. This process involves host biogenesis machinery, including ESCRT components, tetraspanins, and lipid-raft microdomains, which may be co-opted by viruses to support the production of vesicle-associated material (Meckes and Raab-Traub 2011, Rodrigues et al. 2018, Bello-Morales et al. 2020, Fang et al. 2022, Moulin et al. 2023, Martin et al. 2024).

Once released, EV-associated cargo has been proposed to contribute to viral spread through several non-exclusive mechanisms (Table 1): i) Immune shielding, whereby host-derived membranes cloak viral cargo, reducing exposure to neutralizing antibodies and complement-mediated clearance (Blackwell et al. 1981, Maacha et al. 2019); ii) expansion of viral tropism, as EVs can transfer functional receptors or co-receptors, such as CCR5, CXCR4, or ACE2, to previously non-permissive cells, thereby broadening the range of susceptible targets (Mack et al. 2000); iii) receptor-independent entry, as EV-associated viruses can bypass canonical receptor-mediated pathways, providing alternative routes for infection (Gould et al. 2003); iv) cellular reprogramming, whereby EVs deliver viral and host regulatory molecules that reshape recipient cells toward a pro-viral state (Bello-Morales et al. 2018, Bello-Morales et al. 2020, Handala et al. 2020, Yu et al. 2023, Martin et al. 2024). EV uptake occurs through multiple endocytic routes, including clathrin-dependent and caveolin-mediated internalization, macropinocytosis, and phagocytosis (Mulcahy et al. 2014, Ragni 2025) and is modulated by surface ligands such as integrins, lectins, and heparan sulfate proteoglycans (Yáñez-Mó et al. 2015, Hallal et al. 2022). Following internalization, vesicles traffic through the endosomal system, where they may undergo lysosomal degradation, recycling, or cargo release into the cytoplasm (Piper and Katzmann 2007, Joshi et al. 2020). These processes could amplify vesicle-mediated transfer of host components, although direct evidence of their contribution to productive infection in vivo remains limited (Grewal et al. 2025).

Table 1.

Mechanisms by which extracellular vesicles promote viral dissemination and modulate host immune responses.

Mechanism	Description	Key molecular features	Outcome	References
Immune shielding	Viral components are enclosed within host-derived EV membranes, reducing immune recognition	Host lipid bilayer; ESCRT machinery; tetraspanins; absence of exposed viral antigens.	Immune evasion and increased viral stability.	(Blackwell et al. 1981, Maacha et al. 2019)
Expanded tropism	EVs transfer viral receptors to previously non-permissive cells.	Transfer of CCR5, CXCR4, and ACE2 via EV membranes.	Increased susceptibility of target cells.	(Mack et al. 2000)
Receptor-independent infection	EVs deliver infectious viral cargo independently of canonical receptor-mediated entry.	Direct membrane fusion; clathrin-independent uptake; macropinocytosis.	Maintenance of infection despite receptor blockade.	(Gould et al. 2003)
Host-environment modulation	EV cargo reprograms recipient cells to create a pro-viral environment.	Viral and host miRNAs; cytokines; regulatory proteins.	Suppression of antiviral responses and enhanced viral replication or latency.	(Yu et al 2023)
Multi-virion packaging	Multiple virions are packaged and transported within a single vesicle.	Autophagosome-derived vesicles; protective membrane structures.	Increased infectivity and potential alternative transmission routes.	(Santiana et al. 2018)

In some viral systems, EV-like particles have been reported to encapsulate multiple virions, potentially increasing environmental stability and enabling transmission under adverse conditions (Santiana et al. 2018). Collectively, these observations recognize EVs as potential modulators of viral persistence, immune evasion, and host-range diversification, although their relative contribution in vivo is still being defined.

Extracellular vesicles in HIV infection

Among viral pathogens, HIV-1 represents one of the most extensively studied models of EV–virus interplay. HIV-1 and EVs exploit overlapping biogenetic machinery, remodel host-cell membranes, and engage in extensive functional crosstalk that may influence viral transmission, immune evasion, and persistence.

HIV-1 particles and EVs share multiple structural and molecular features, including enrichment in lipid rafts, tetraspanins, MHC molecules, and heat-shock proteins. However, while virions carry viral genomic and structural components, EVs predominantly transport host-derived cargo. This partial overlap creates both experimental challenges in vesicle discrimination and biological opportunities for viral exploitation of EV pathways.

Shared biogenesis and molecular composition

Exosomes and HIV-1 particles share similarities in membrane composition and vesicle trafficking pathways. Exosomes originate from intraluminal budding within multivesicular bodies, whereas HIV-1 assembly mainly occurs at the plasma membrane. Despite these spatial differences, both processes rely on shared host machinery, particularly ESCRT components and tetraspanins, as shown in Fig. 2 (Pant et al. 2012, Dias et al. 2018, Welch et al. 2019, Lu et al. 2025).

Figure 2.

EV biogenesis and contribution to HIV transmission and replication. This figure illustrates the shared biogenetic pathways between EVs and HIV-1, and their role in viral transmission and replication. (1) EV biogenesis involves both the endosomal pathway, where early endosomes mature into multivesicular bodies (MVBs) that release exosomes upon fusion with the plasma membrane, and the direct outward budding of microvesicles. These processes are regulated by the ESCRT machinery and associated proteins, including TSG101, Alix, and CD63, which are also exploited by HIV-1 during viral assembly and budding. (2) EVs derived from HIV-infected cells can transfer viral RNA, proteins, and host factors to recipient cells, enhancing viral transmission and promoting intracellular signaling pathways that support viral replication. This functional overlap highlights the convergence between EV biology and HIV-1 life cycle dynamics. Created with Biorender.com

HIV-1 virions incorporate multiple host-derived proteins, including HLA molecules and tetraspanins, reflecting the composition of EVs released from T lymphocytes, macrophages, and dendritic cells (Poon et al. 2000, Thali 2009, Jankovičová et al. 2020, Gurung et al. 2021). Proteomic analyses in macrophages further demonstrate the presence of canonical EV markers within HIV-1 particles, underscoring the close relationship between viral budding and EV biogenesis (Benaroch et al. 2010, Iordanskiy et al. 2013). Functional studies indicate that tetraspanins play a dual role in HIV replication and spread. While their overexpression can restrict viral infectivity, HIV-1 actively modulates their expression to optimize cell-to-cell transmission (Gordòn-Alonso et al. 2006, Krementsov et al. 2009).

Similarly, both EV formation and HIV-1 budding depend on ESCRT-I (TSG101), ALIX, and ESCRT-III, with HIV-1 uniquely exploiting ESCRT-II through GAG late-domain motifs (PTAP, YPXL, PPXY) that mimic host sorting signals. Knockout of the ESCRT-II subunit EAP45 in T cells impairs viral budding, with rescue dependent on cargo size and the YPXL-ALIX pathway (Irie and Harty 2005, Barnes and Wilson 2019, Meng et al. 2020, Ju et al. 2021). EV biogenesis engages similar routes, including ALIX-TSG101 via syntenin/syndecan and ceramide-dependent ILV formation (Baietti et al. 2012, Friand et al. 2015, Stoorvogel 2015, Sette et al. 2016, Wei et al. 2021).

Overall, these observations highlight a strong convergence between EV biogenesis and HIV budding pathways, supporting the concept that HIV-1 exploits host vesicular trafficking machinery. This overlap also provides a mechanistic basis for the interplay between EVs and HIV during intercellular communication.

EV-mediated HIV transmission and replication

Although HIV-1 is classically described as spreading via cell-free virions, a large body of evidence indicates that direct cell-to-cell transmission represents a major mode of viral dissemination, particularly within densely packed lymphoid tissues. In this context, EVs may contribute to intercellular communication alongside established mechanisms such as direct cell-to-cell transmission (Bracq et al. 2018). EVs exert both antiviral and proviral effects in HIV infection, with outcomes determined by vesicle origin, molecular cargo, and the physiological state of the producing cell (Lee et al. 2023). EVs can transfer restriction factors, antiviral RNAs, or immune-modulatory signals, that limit HIV replication (Martin et al. 2024). A prominent example is APOBEC3G (A3G), which, when delivered via EVs, suppresses HIV replication through both deaminase-dependent and independent mechanisms (Wang et al. 2012). EV-mediated delivery of restriction factors may therefore enhance antiviral defenses in recipient cells, although this mechanism remains incompletely understood (de Carvalho et al. 2014).

EVs may also function as decoys, intercepting virions before they reach susceptible targets. CD4-bearing EVs derived from T cells can bind circulating HIV-1 particles, while EVs from non-permissive cells can deliver interferon-stimulated genes (e.g. ISG15, ISG56, Mx2) to macrophages (Sun et al. 2016). Additional antiviral effects have been reported for EVs present in breast milk, semen, and components of the microbiota, as well as circulating EVs from individuals receiving antiretroviral therapy (Näslund et al. 2014, Welch et al. 2020).

Conversely, HIV-1 can exploit EVs pathways to enhance viral spread and immune evasion. As shown in Fig. 2, infected cells release EVs enriched in viral proteins and RNAs, including Nef, Tat, and TAR, which can reprogram recipient cells (Tang et al. 2024). As demonstrated by Lenassi et al., Nef-containing EVs have been detected in patient serum and have been associated with apoptosis of bystander CD4⁺ T cells (Lenassi et al. 2010). According to da Silva-Januàrio et al., these vesicles may also contribute to downregulation of CD4 and MHC-I expression, potentially weakening immune recognition (de Carvalho et al. 2014). Within the central nervous system, EV-associated Tat and Nef have been implicated in neuroinflammation and neuronal injury, promoting microglial activation, neuronal apoptosis, and β-amyloid accumulation (Cabrera-Pastor 2024, Andràs 2020). Notably, EVs enriched in TAR RNA persist even under effective antiretroviral therapy, suggesting a possible contribution to chronic inflammation and to HIV-associated neurocognitive disorders (Sampey et al. 2016).

EV-mediated HIV latency and reactivation

EVs are increasingly recognized as regulators of HIV latency, acting through defined signaling pathways, inflammatory networks, and chromatin remodeling mechanisms that influence both latency maintenance and viral reactivation. Importantly, several studies provide direct mechanistic evidence linking EV-associated cargo to latency control.

Barclay et al. demonstrated that EVs carrying phosphorylated c-Src activate the EGFR-PI3K-AKT-mTOR axis, leading to STAT3 and SRC-1 recruitment and subsequent NF-κB- and p300-dependent chromatin remodeling. This signaling cascade promotes HIV reactivation in both cell-line models and primary CD4⁺ T cells, even in the presence of latency inhibitors, indicating that EV-associated signaling molecules can override pharmacological suppression (Barclay et al. 2020).

EV-mediated latency regulation can also occur indirectly through inflammatory signaling. Rubione et al. showed that EVs derived from plasma of people living with HIV stimulate macrophages to release galectin-1, which in turns binds glycan residues on latently infected CD4⁺ T cells and induces NF-κB-dependent viral reactivation (Rubione et al. 2022). Similarly, Hong et al. reported that circulating exosomes preferentially interact with activated CD4⁺ T cells and can trigger viral transcription in resting CD4⁺ T cells, suggesting that EV-mediated intercellular communication may function as a physiological latency-reversing mechanism (Hong et al. 2017).

Within the central nervous system, vesicle-like extracellular particles further contribute to latency regulation. Naushad et al. demonstrated that extracellular condensates isolated from the basal ganglia enhance HIV transcription in latency models, whereas condensates from Δ⁹-tetrahydrocannabinol-treated animals suppress reactivation and counteract TNF-α-mediated activation, indicating that vesicular signaling in the CNS is responsive to both inflammatory and pharmacological modulation (Naushad et al. 2026).

Innate immune pathways provide an additional mechanistic link between EVs and latency. DeMarino et al. showed that HIV-infected myeloid cells release TAR-enriched EVs that activate TLR3 signaling in recipient cells, promoting downstream inflammation responses (DeMarino et al. 2024). Pharmacological targeting of TAR-TLR3 interactions reduced this effect, supporting a direct contribution of EV-associated RNA cargo to latency-related signaling pathways, as shown in Fig. 3 (DeMarino et al. 2024).

Figure 3.

Extracellular vesicle-mediated regulation of HIV latency, reactivation, and host immune responses. This figure summarizes the role of EVs in modulating HIV latency, viral reactivation, and immune responses, including both pathogenic and protective effects. (3) EVs carrying viral components, such as TAR RNA and viral proteins, can be internalized by recipient cells, where they activate signaling pathways including NF-κB and PI3K/Akt, leading to transcriptional activation of the provirus and contributing to viral reactivation from latency. (4) In parallel, EVs present in biological fluids may exert protective and immunomodulatory functions by delivering host-derived molecules, including specific microRNAs (e.g. miR-29a, miR-128) and proteins, which can inhibit viral replication and modulate immune activation. Together, these findings highlight the dual role of EVs in both promoting HIV persistence and contributing to antiviral defense mechanisms. Created with Biorender.com

Collectively, these findings indicate that EVs may contribute to the regulation of HIV latency through discrete, mechanistically defined pathways involving both protein and RNA cargo. Notably, current evidence suggests that these effects are driven by a restricted subset of functionally active molecules rather than global EV composition, supporting a model in which EV-mediated latency control is selective and context-dependent.

Immunomodulatory roles of EVs in HIV infection

Beyond their direct effects on viral replication, EVs have been proposed as potential regulators of immune homeostasis during HIV infection, potentially influencing both innate and adaptive immune responses (Bello-Morales et al. 2020). However, most of the available evidence derives from in vitro systems or ex vivo systems, and direct evidence supporting a causal role for EVs in immune dysregulation in people living with HIV-1 remains limited.

Neyrinck-Leglantier et al. reported that circulating EVs from individuals receiving suppressive therapy are enriched in immunomodulatory molecules such as PD-L1, TGF-β1, CCR5, and MHC components, suggesting a possible role in sustaining chronic immune activation while simultaneously dampening antiviral T-cell responses (Neyrinck-Leglantier et al. 2024). Similarly, Chettimada et al. identified EV-associated miRNAs (e.g. miR-21–5p and miR-146a-5p) showing increased levels in HIV-positive individuals and correlating with metabolic markers of immune activation. However, the functional relevance of EV-mediated miRNA transfer remains uncertain. Only a limited subset of miRNAs displays consistent differences between HIV-positive and HIV-negative individuals, and many reported differences are not retained after multiple testing correction (Chettimada et al. 2020). In addition, EV-associated miRNAs are often present at low levels, and it remains unclear whether they are delivered in sufficient amounts to exert functional effects under physiological conditions (Chettimada et al. 2020).

Conversely, EVs derived from mucosal tissues or microbiota have been proposed to exert protective effects. For instance, breast milk-derived EVs have been reported to inhibit HIV entry by binding DC-SIGN on dendritic cells and modulating downstream pro-inflammatory signaling (Näslund et al. 2014). More direct experimental evidence has been obtained in epithelial cell models: Xu et al. showed that EV-enriched fractions released from TLR3-activated cervical epithelial cells are associated with reduced HIV replication in macrophages, and depletion of EVs from conditioned supernatants attenuated this antiviral effect (Xu et al. 2021). However, these findings rely on in vitro depletion approaches and do not exclude contributions from co-isolated extracellular components, including viral particles or non-vesicular material. The reported delivery of antiviral miRNAs (e.g. miR-29 family, miR-125b, miR-150) and interferon-stimulated gene products (ISG56, OAS1, MxA, Mx2) suggests a potential role in modulating innate immune responses, although their relative contribution compared to soluble factors remains unclear (Mansouri et al. 2025).

EV-mediated regulation of inflammation appears to be highly context-dependent. Mansouri et al. highlighted that controlled TLR2 activation promotes transient physiological inflammation, whereas excessiveTLR4 signaling is associated with pathological inflammation and tissue damage. Although a direct role for EV-mediated delivery was not demonstrated in this context, EV-associated miRNAs have been proposed to target TLR signaling pathways, suggesting a possible regulatory layer linking EV signaling to innate immune balance (Mansouri et al. 2025).

Bacterial EVs further illustrate this duality. Vesicles derived from commensal species such as Lactobacillus are associated with immune tolerance and mucosal homeostasis, whereas EVs from dysbiotic bacteria (e.g. Gardnerella vaginalis and Mobiluncus mulieris) promote pro-inflammatory responses that may increase susceptibility to infection (Liu et al. 2024, Joseph et al. 2024). However, these observations are largely based on correlative data, and direct evidence demonstrating a causal contribution of EV-associated cargo is still limited.

In the context of HIV infection, EVs have been proposed to contribute to immune crosstalk by transporting viral RNAs and proteins capable of engaging pattern-recognition receptors, including TLRs and STING pathways, thereby inducing cytokine production and type I interferon responses (Kouwaki et al. 2017, Peng et al. 2023, Gao et al. 2025). For example, HIV-derived RNA species such as vmiR88, vmiR99, and TAR RNA have been shown to activate TLR8/NF-κB signaling in macrophages, contributing to persistent immune activation (Bernard et al. 2014), whereas other EV populations have been reported to deliver cGAMP and activate STING-dependent antiviral pathways (Zhang et al. 2024). However, an important caveat is that the amount of HIV-derived RNA present in EVs, and the number of EVs required to deliver sufficient RNA to recipient cells to reach the threshold necessary for TLR8/NF-κB activation, remain poorly defined. Thus, although these findings support a potential role for EV-associated viral RNA in innate immune activation, the physiological relevance of such effects remains difficult to assess. Moreover, similar innate immune responses can be triggered by free viral RNA or protein complexes, further complicating the attribution of these effects specifically to EV-associated cargo.

These pathways are counterbalanced by viral immune evasion strategies. Some studies have reported that lentiviral proteins can interfere with innate sensing pathways including cGAS-STING signaling, highlighting the dynamic interplay between host immune activation and viral immune evasion. However, these findings are largely based on models involving Vpx, which is encoded by HIV-2 and SIV rather than HIV-1, and should therefore be interpreted with caution in the context of HIV-1 infection e (Su et al. 2019).

Beyond innate sensing, EVs have also been implicated in the modulation of adaptive immune responses, including effects on dendritic cell maturation, antigen presentation, and NK cell activity (Teer et al. 2025). For example, Mercier et al. reported that microvesicle-enriched fractions co-isolated with HIV particles can enhance dendritic cell activation through mechanisms involving HSP90α/β and Nef. However, given the well-recognized challenges in separating EVs from virions, these findings should be interpreted with caution (Mercier et al. 2013).

Overall, while EVs have been implicated in multiple aspects of immune regulation during HIV infection, their causal contribution and relative importance in vivo remain to be clearly established (Wen et al. 2017). Further studies using improved isolation strategies and in vivo models will be required to clarify the extent to which EVs contribute to immune dysregulation in people living with HIV.

Therapeutic targeting of EV pathways in HIV infection

Inhibition of EV release and modulation of EV cargo

Given the involvement of EVs in viral dissemination, chronic inflammation, and immune dysregulation, modulation of EV pathways has been explored as a promising adjunctive strategy in HIV therapy (Table 2). However, the therapeutic relevance of these approaches remains primarily based on experimental models, and their applicability in people living with HIV is incompletely defined. One approach involves targeting EV biogenesis pathways. DeMarino et al. demonstrated that cannabidiol significantly reduces EV secretion from HIV-infected myeloid cells and is associated with decreased levels of viral components, including Nef, gp120, TAR, and env RNA, across multiple experimental systems such as primary macrophages, neurospheres, and plasma (DeMarino et al. 2022). Similarly, Zhu et al. showed that inhibition of neutral sphingomyelinase 2, a key regulator of ceramide-dependent EV formation, reduces brain-derived EV release and modulates miRNA signatures in EcoHIV-infected mice, with reported improvements in cognitive and behavioral parameters (Zhu et al. 2022).

Table 2.

Pharmacological strategies targeting extracellular vesicle biogenesis and cargo to modulate HIV-associated pathogenesis.

Strategy	Molecule	Mechanism of action	Outcomes	References
Inhibition of EV biogenesis	Cannabidiol (CBD)	Restores autophagy and reduces EV secretion and associated viral cargo (e.g. Nef, gp120, TAR RNA, env RNA).	Decreased EV release in HIV-infected myeloid cells and reduced pathogenic cargo.	(DeMarino et al. 2022)
	nSMase2 inhibitor (PDDC)	Blocks ceramide-dependent EV formation.	Reduced brain EV levels; restoration of miRNA profiles, and improved cognitive outcomes in EcoHIV mouse models.	(Zhu et al. 2022)
Inhibition of ITIM/lectin-mediated EV release	DCIR antagonism	Blocks HIV binding and internalization and inhibits ITIM-dependent exosome secretion	Reduced release of pro-apoptotic EVs, decreased miR-155–enriched EV pathogenicity, and partial immune restoration.	(Boucher et al. 2025)
Modulation of EV cargo (HIV-induced alterations)	Kinase inhibitors	Prevent incorporation of kinases (e.g. CDK10, GSK3β, MAPK8) into EVs.	Normalization of EV cargo composition and reduced impact on recipient cell cycle regulation.	(Mensah et al. 2025)
	Targeting spliceosome-related pathways	Interferes with U6 snRNA and associated proteins exported into EVs.	Highlights infection-driven nuclear and EV remodeling processes.	(Huang et al. 2025)
Blockade of EV-dependent nuclear trafficking	Itraconazole triazole analogues	Inhibit the VOR complex (VAP-A/ORP3/Rab7), blocking nuclear entry of late endosomes.	Reduced nuclear delivery of EV cargo and decreased HIV infection with lower toxicity.	(Carbone et al. 2024)
Enhancement of EV uptake/clearance	Proton pump inhibitors (PPIs)	Promote macropinocytosis via v-ATPase relocalization.	Reduced circulating EV levels (shown in non-HIV models).	(Lu et al. 2024)
Modulation of EV cargo via PDZ-domain interference	PDZ inhibitors (HTLV-1 model)	Disrupt Tax-1–syntenin-1 interaction, affecting ESCRT-dependent cargo loading	Enrichment of antiviral miRNAs and reduction of viral cargo; potential relevance to HIV.	(Puttemans et al. 2025)

Host receptors involved in EV production have also been investigated as potential targets. The dendritic cell immunoreceptor (DCIR) has been implicated in both HIV internalization and EV release, through its ITIM motif. Inhibition of DCIR signaling was reported to reduce the production of EVs enriched in pro-apoptotic factors and to partially restore immune function in humanized mouse models (Mfunyi et al. 2015). In addition, EVs-associated miR-155 has been linked to enhanced viral replication and immune dysfunction (Boucher et al. 2025b). However, given the complexity of DCIR signaling and its broader immunological roles, the specific contribution of EV modulation to these effects remains difficult to isolate.

HIV infection has also been associated with changes in EV cargo composition, including alterations in signaling pathways and RNA processing. Mensah et al. reported that EVs derived from latently infected cells are enriched in active kinases (CDK10, GSK3β, MAPK8) capable of influencing cell-cycle regulation in recipient cells, and that pharmacological inhibition of these pathways alters EV cargo and downstream effects (Mensah et al. 2025). Complementary observations suggest that retroviral infection may promote EV-mediated export of spliceosomal components, suggesting that targeting RNA-processing pathways could mitigate EV-driven disease mechanisms in vitro (Huang et al. 2025).

Targeting intracellular trafficking pathways required for EV-mediated transport represents an additional strategy. Carbone et al. identified the VAP-A/ORP3/Rab7 (VOR) complex as a host machinery involved in late endosome trafficking and reported that triazole analogues derived from itraconazole selectively inhibit the VOR complex, thereby reducing the nuclear delivery of EV-associated material and HIV-1 infection in experimental systems (Carbone et al. 2024). Nevertheless, these pathways are also involved in fundamental cellular processes, raising potential concerns regarding specificity and toxicity.

In addition to reducing EV release, enhancing EV clearance has been proposed as a complementary approach. Lu et al. demonstrated that proton pump inhibitors promote EV uptake via macropinocytosis, leading to reduced circulating EV levels (Lu et al. 2024).

Evidence from other retroviral systems further supports the potential to pharmacologically modulate EV cargo. For example, Puttemans et al. found that disrupting the interaction between the HTLV-1 protein Tax-1 and the ESCRT-associated protein syntenin-1 alters EV composition, suggesting conserved host–virus interactions that may be therapeutically exploitable across retroviral infections (Puttemans et al. 2025).

Finally, immunomodulatory nanotherapeutic approaches targeting EV-related pathways have also been explored. Poly(I)-loaded nanoparticles combined with immune checkpoint blockade have been shown to restore NK cell cytotoxicity, limit CD4⁺ T-cell persistence, and promote TRAIL-mediated killing through TLR3 activation, promoting antiviral responses (Sánchez-Cerrillo et al. 2025).

Collectively, these studies suggest that EV dynamics can be targeted at multiple levels, including biogenesis, cargo loading, intracellular trafficking, and systemic clearance, supporting the development of EV-focused adjunctive strategies alongside antiretroviral therapy, although their clinical relevance remains to be further defined.

Environmental and pathological factors exacerbating EV-associated alterations in HIV infection

In addition to pharmacological modulation, several environmental and comorbidity-associated factors have been reported to influence EV release and composition during HIV infection, potentially contributing to tissue dysfunction and disease progression. However, in most cases, the available evidence does not distinguish EV-specific effects from broader cellular stress responses.

Alcohol exposure represents one of the most studied modifiers of EV dynamics. Dagur et al. showed that in HIV-infected hepatocyte models and humanized mice, alcohol exposure is associated with increased EV release, alongside lysosomal function, reduced LAMP1 expression, and oxidative stress, ultimately contributing to liver injury (Dagur et al. 2021).

Substance use has also been linked to alterations in EV cargo composition (Caobi et al. 2023). Caobi et al. demonstrated that morphine exposure in HIV-infected PBMCs is associated with changes in EV-associated miRNAs, including upregulation of miR-1246, which has been implicated in pathways related to neuronal survival, calcium signaling, autophagy, and inflammatory responses (Caobi et al. 2023). These findings highlight how comorbid conditions can reprogram EV signaling and exacerbate HIV-associated neuropathology, although their functional relevance in vivo remains to be fully established.

Additional evidence indicates that viral proteins and local microenvironmental stressors may further influence EV production and composition. Chemparathy et al. reported that HIV-1 Tat can enhance astrocyte-derived EV release by stabilizing HIF-1α, a transcription factor activated under hypoxic and inflammatory conditions (Chemparathy et al. 2024). EVs released under these conditions were enriched in miRNAs associated with neurotoxicity and were linked to reduced neuronal viability and altered dendritic morphology in cellular and organoid models.

Overall, these studies suggest that environmental factors, substance abuse, and viral proteins may influence EV biogenesis and cargo composition during HIV infection. However, whether EVs act as primary drivers of pathology or mainly reflect underlying cellular stress remains unclear, and further in vivo studies are required to clarify their mechanistic contribution.

Engineered EVs as therapeutic delivery platforms

The intrinsic ability of EVs to transport biomolecules across cellular barriers has prompted interest in their use as potential therapeutic delivery platforms.

Naturally derived EVs, particularly those originating from mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), have attracted increasing interest due to their well-established immunomodulatory and regenerative properties. MSC-derived EVs can regulate both innate and adaptive immune responses, including macrophage polarization, dendritic cell activation, and T and NK cell function, and have shown therapeutic potential by modulating inflammation, enhancing antimicrobial responses, and promoting tissue repair (Liu et al. 2023a; Manzoor et al. 2023). In parallel, stem cell-derived EVs have been reported to exert neuroprotective and anti-inflammatory effects in iPSC-derived neural models of HIV infection, supporting their potential application in mitigating virus-induced cellular damage (Branscome et al. 2022).

Their intrinsic biocompatibility, low immunogenicity, and ability to cross biological barriers, including the blood-brain barrier, have been proposed as advantageous features for targeting HIV reservoirs and HIV-associated comorbidities. However, most current applications remain at the preclinical proof-tof-concept stage, and their translational potential in humans has not yet been established. Several engineering strategies have been explored to enhance EV targeting and cargo delivery, as shown in Table 3. Zou et al. proposed the use of exosomes displaying the broadly neutralizing antibody 10E8 scFv for selective recognition of Env-expressing cells, enabling delivery of curcumin or pro-apoptotic miR-143 (Zou et al. 2019). This approach reduced p24 levels in infected cell models and showed biodistribution in humanized mice (Zou et al. 2019).

Table 3.

Engineered extracellular vesicle platforms for therapeutic delivery in HIV research.

Platform	Cargo/mechanism	Therapeutic goal	Model	Main findings	References
10E8-scFv–engineered exosomes	Curcumin or miR-143; Env-targeting scFv	Targeting and elimination of Env⁺ reservoir cells	ACH2 cells, PBMCs from ART-treated individuals, humanized mice	Selective cytotoxicity in Env⁺ cells, reduced p24 levels, accumulation in Env⁺ tissue, and minimal toxicity	(Zou et al. 2019)
NanoMEDIC (virus-like EVs)	Cas9–sgRNA ribonucleoproteins	Genome editing (potentially adaptable to HIV)	iPSC-derived muscle cells, mice	Efficient delivery of CRISPR RNPs and robust exon skipping	(Watanabe et al. 2023)
Rapamycin-inducible CD63⁺ EVs (FKBP/FRB system)	Inducible loading of cytosolic proteins; fusogenic Syncytin-1	Controlled intracellular protein delivery (potentially adaptable to HIV)	HeLa cells	∼7.5-fold increase in cytosolic delivery and targeted cytotoxicity when loaded with toxins	(Bui et al. 2023)
MELT (magnetoelectric nanoparticle–EV hybrid)	Tat inhibitor ZL0580 in EVs combined with MMAE-loaded nanoparticles	Suppression of microglial latency and elimination of residual infected cells	HC69 microglia; blood-brain barrier model	High drug loading (∼80%), partial BBB crossing (∼20%), and reduced LTR activity	(Andre et al. 2025)
EXOtic-engineered EVs	mRNA encoding ZPAMt (ZFP–DNMT3A fusion)	Block-and-lock strategy via LTR CpG methylation	HIV-infected cells; hu-PBMC and hu-CD34⁺ mice	Reduced viremia, targeted LTR methylation, BBB penetration, and suppression of CNS HIV	(Kojima et al. 2018, Anticoli et al. 2018)

EV-based platforms have also been adapted as vehicles for gene-editing delivery. Gee et al. introduced the NanoMEDIC system, an approach capable of transiently delivering CRISPR-Cas ribonucleoproteins via virus-like EVs. Cells expressing HIV-1 Gag and the vesicular stomatitis virus glycoprotein G (VSV-G) generated virus-like EVs incorporating Cas9-sgRNA complexes through a chemically inducible dimerization mechanism. These engineered vesicles achieved robust exon skipping in human iPSC-derived muscle cells and in vivo mice models (Watanabe et al. 2023).

In a complementary approach, Bui et al. developed a rapamycin-inducible cargo-loading system, the EXOtic platform, to enhance EV controlled incorporation of cytosolic proteins and mRNA, without altering vesicle morphology or release (Bui et al. 2023). These systems have been used for experimental delivery of cytotoxic proteins or regulatory factors, resulting in cell-specific effects in model systems. For example, when loaded with diphtheria toxin, these EV induced selective target-cell killing, underscoring the versatility of EV-based cytosolic protein delivery and its potential application to antiviral restriction factors and genome-editing strategies.

Hybrid nanotechnology approaches have further expanded EV-based strategies. Andre et al. developed a magnetoelectric nanoparticle–EV hybrid system (MELT) designed to deliver Tat inhibitors across blood-brain barrier models and target latent reservoirs, suppressing viral reactivation (Andre et al. 2025). In addition, EV-mediated epigenetic silencing strategies have been proposed. Shrivastava et al. engineered exosomes to deliver mRNA encoding a zinc-finger protein fused to the DNA-methyltransferase (DNMT3A), inducing CpG methylation of the HIV-1 LTR and suppressing viral transcription in experimental models (Shrivastava et al. 2021). This suppression of HIV replication and reduced viremia in humanized mice supported the feasibility of “block-and-lock” strategies mediated by EV delivery however (Kojima et al. 2018). Collectively, engineered EV platforms represent a versatile set of experimental tools for targeted delivery of therapeutic molecules, including drugs, nucleic acids, and genome-editing systems. While these approaches have shown encouraging results in preclinical models, further word is needed to address challenges related to scalability, targeting specificity, biodistribution, and safety, and to evaluate their potential to complement existing antiretroviral therapies.

EV-based vaccine strategies

A growing body of preclinical evidence has explored engineered EVs as potential vaccine platforms capable of inducing robust antiviral immune responses. Compared with conventional delivery systems, EVs have been proposed to facilitate efficient antigen presentation and CD8⁺ T-cell activation. One of the most studied approaches is the Nefmut-based platform developed by Anticoli et al., in enla viral antigens are fused to a non-oncogenic Nef mutant highly enriched in EVs (Anticoli et al. 2018). A wide range of heterologous antigens, including HPV E7, Ebola virus VP24/VP40/NP, West Nile virus NS3, have been shown to remain stable when fused to Nefmut and to be efficiently incorporated into EVs in vivo following intramuscular DNA delivery. In experimental models, this approach is associated with the induction of antigen-specific cytotoxic CD8⁺ T-cell responses against multiple viral antigens and may be adaptable to target conserved or latency-associated HIV proteins (Anticoli et al. 2018).

A second strategy involves dendritic cell-derived EVs incorporating Nefmut–Tat fusion proteins. Pordanjani et al. reported that these EVs can induce both humoral and cellular immune responses in experimental models, promoting increased IgG and IgG2a levels and Th1-associated cytokines, such as IFN-γ, TNF-α, and granzyme B. Co-administration with Hsp70-containing EVs further amplified these responses, yielding the strongest immunity following exposure to single-cycle replicable HIV (Pordanjani et al. 2024)).

Similarly, EV-based approaches such as Texo platforms have been explored for antigen delivery. In this context, gp120-loaded Texo cells have been reported to induce antigen-specific CD8⁺ T-cell responses capable of eliminating antigen-pulsed target cells in vivo and to confer preventive and therapeutic effects in experimental models (Santos and Almeida 2021). Notably, this system appears to function independently of CD4⁺ T-cell help, which may represent a clear advantage in the setting of HIV-associated CD4⁺ T-cell depletion (Santos and Almeida 2021).

Texo-based vaccines strategies have also been extended from model antigens to HIV-relevant targets. In experimental models, ovalbumin (OVA)-Texo vaccination has been associated with reversal of CD8⁺ T-cell exhaustion and partial restoration of cytotoxic function, as indicated by reduced PD-1/LAG-3 expression and improved IFN-γ production. This approach was subsequently adapted to generate an HIV-specific Gag-Texo platform, which was associated with the induction of Gag-specific immune responses in experimental models (Wang et al. 2017). Together, these findings suggest that Texo-based systems may offer a framework for modulating dysfunctional antiviral immunity in persistent HIV infection.

Concluding remarks and future perspectives

EVs have been increasingly implicated in multiple aspects of HIV infection, acting at the interface between viral persistence, immune dysregulation, chronic inflammation, and tissue damage. Accumulating evidence suggests that EV-associated communication may contribute to processes such as viral dissemination, reservoir dynamics, and the development of long-term comorbidities, including those affecting immune-privileged sites such as the central nervous system. Much of the current knowledge derives from in vitro systems or indirect observations, and the extent to which EVs play a causal role in these processes in people living with HIV remains to be fully established.

In parallel, EVs are being explored as potential therapeutic tools. Engineered EVs have been developed as delivery platforms for antiviral agents, nucleic acids, and gene-editing systems, with encouraging results reported in preclinical models. Similarly, EV-based vaccine approaches have demonstrated the capacity to induce antigen-specific immune responses and modulate dysfunctional antiviral immunity in experimental settings.

Despite these advances, several challenges must be addressed before EV-based strategies can be effectively translated into clinical practice. These include the need for standardized methods for EV isolation and characterization, improved strategies to distinguish EVs from co-isolated viral and non-vesicular components, scalable manufacturing processes, and a better understanding of EV biodistribution and targeting specificity in vivo. In addition, the safety and long-term effects of EV-based interventions require careful evaluation.

Future studies integrating multi-omics approaches with advanced in vitro and in vivo models will be important to better define EV-associated signatures linked to viral persistence and treatment response. Such efforts may support the identification of EV-based biomarkers and inform the rational design of next-generation therapeutic strategies.

Overall, EVs represent a complex and context-dependent component of HIV biology, with potential roles in both pathogenesis and therapeutic intervention. A more precise definition of their functional contribution in vivo will be essential to determine whether EV-targeted approaches can complement existing strategies aimed at achieving durable viral control. In this context, EV-based interventions may open new avenues.

Conflicts of interest

The authors report no conflict of interest.

Funding

This work was supported by the University of Salerno through the project “Personalization and optimization of care for patients with HIV: the pathogen/host interplay” (project code: 300397FAC23CONTI_01).