Seminal exosomes and HIV‐1 transmission

Abstract Exosomes are endosomal‐derived membrane‐confined nanovesicles secreted by many (if not all) cell types and isolated from every human bodily fluid examined up to now including plasma, semen, vaginal secretions and breast milk. Exosomes are thought to represent a new player in cell‐to‐cell communication pathways and immune regulation, and be involved in many physiological and pathological processes. Susceptibility to HIV‐1 infection can be impacted by exosomes, while HIV‐1 pathogenesis can alter exosomal function and composition. Exosomes isolated from semen and vaginal fluid of healthy individuals can inhibit HIV‐1 infection and/or potently block viral transfer in vitro. However, the role of exosomes in HIV‐1 transmission and progression is not fully understood yet and some studies show conflicting results, mainly for exosomes isolated from plasma and breast milk. Determining the composition of exosomes from infected donors and studying their interaction with HIV‐1 in vitro compared to exosomes isolated from uninfected donors will provide insights into the role exosomes play in HIV‐1 transmission during sexual intercourse and breastfeeding.

In this review, we will briefly summarise how exosomes derived from multiple biological matrices compared to seminal exosomes modulate HIV-1 infection, with a focus on regulation of immune responses.

| E XOSOME S AND HIV-1: CHAR AC TERIS TI C S AND IMPAC T
HIV-1 and exosomes are generally similar in their size (Gentile et al., 1994) and density range (Cimarelli & Luban, 2000), making it difficult to purify exosomes from HIV-1 infected cells and fluids. Moreover, retroviruses such as HIV-1 and exosomes share common molecular properties in addition to morphological similarities (Cantin, Diou, Belanger, Tremblay, & Gilbert, 2008). Both exosomes and retroviruses contain major histocompatibility complex class II (MHC-II) molecules, cell surface molecules such as integrins (CD11a, CD18), co-stimulatory molecules (CD28, CD54) and complement neutralising molecules (CD55, CD59). However, HIV-1 particles could be separated from exosomes by using an iodixanol (Optiprep™) velocity gradient as HIV-1 and exosomes sedimentation velocities are significantly different (Cantin et al., 2008). Exosomes may impact retroviral infection based on biological similarities (Teow, Nordin, Ali, & Khoo, 2016), either through HIV-1 originating in the same intracellular system as exosomes and using the exosome biogenesis pathway to assemble, disseminate and ultimately infect other cells, or via retroviruses hijacking the mechanism of intercellular communication, including exosomes, to spread and infect other cells. Several additional mechanisms by which exosomes can affect HIV-1 infection have also been proposed in a more recent review (Ellwanger et al., 2017). HIV-1 can either release its viral proteins and RNA into exosomes or release virus particles surrounded by exosomes, or viral proteins can interfere with the exosomal release pathway. For example, Nef protein has been shown to reduce the expression of CD4 antigens in exosomes isolated from infected cells, in turn promoting HIV-1 replication (Arenaccio, Chiozzini, Columba-Cabezas, Manfredi, Affabris, et al., 2014;de Carvalho et al., 2014).
CD4 molecules were present in exosomes secreted from CD4 + T cells, and exosomes released by CD4 + T cells but not by CD4 − T cells were able to efficiently inhibit HIV-1 infection in vitro. It has been suggested that CD4 molecules displayed on the exosome surface can bind to envelop proteins of HIV-1, masking the virus interaction with target cells and preventing infection. The authors show that HIV-1 accessory protein Nef highly expressed in HIV-1-infected cells is associated with depletion of CD4 molecules both on cells and released exosomes due to the mechanism, by which Nef promotes targeting of CD4 molecules to multivesicular bodies and then to lysosomes for degradation.
Notably, CD4 + cells expressing Nef secreted exosomes with weakened inhibitory ability against HIV-1 infection. While evidence supports that depending on the cellular source and the protocol of isolation/purification, exosomes can either stimulate or suppress immune responses (Hosseini, Fooladi, Nourani, & Ghanezadeh, 2013;Teow et al., 2016;Thery, Ostrowski, & Segura, 2009;Zhang & Grizzle, 2011) and facilitate or inhibit HIV-1 infection/ transmission (Table 1), the exact role that exosomes play in HIV-1 infection has not been clearly elucidated. Furthermore, accumulating data suggest that exosomes may impact multiple stages of HIV-1 pathogenesis by modulating either target immune responses that alter infection, or by activating the latent viral reservoirs (Poveda & Freeman, 2017). These results suggest that plasma exosomes from patients using antiretrovirals may have pro-inflammatory effects promoting immune activation and oxidative stress and contributing to the chronic inflammation associated with HIV-1 disease progression and comorbidities even with suppressed viral loads. Other published data have shown that plasma exosomes enhance HIV-1 infection in human immune cell lines, with no indication on the patients' serology (Sims et al., 2017(Sims et al., , 2018. In contrast, plasma exosomes isolated from healthy,

| PL A S MA E XOSOME S AND HIV-1 INFEC TI ON
HIV-1-negative donors did not impact HIV-1 infection of monocytederived dendritic cells or HIV-1 transfer from those cells to CD4 + T cells (Naslund et al., 2014). This suggests that exosomes derived from the same source (plasma) isolated from seronegative and in-
Analyses of semen of 12 different healthy donors revealed more than 10 12 particles per ejaculate with a range between 4.7 × 10 11 and 3.12 × 10 13 SE/ml. Seminal exosome (SE) size is ranging between 50 and 200 nm, a size a little bigger than general exosomes size.
Transmission electron microscopy analysis of SE samples has shown that they are composed of lipid bilayer particles in the characteristic size range of exosomes and nanovesicles from other cellular sources.
SE have also been shown to express the universal exosome markers heat shock protein (HSP)-70 and CD63, but not ER calnexin indicating that the vesicles are coming from non-endoplasmic reticulum sources (Vojtech et al., 2014). These data together with other pub-

| Role in HIV-1 infection
Semen is known to have both immunostimulatory and immunosuppres-  (Carlsson, Pahlson, Bergquist, Ronquist, & Stridsberg, 2000) and measles virus infection (Kitamura et al., 1995). Since there is considerable overlap in the cellular generation of enveloped viruses (including HIV-1) and exosomes (Gould, Booth, & Hildreth, 2003;, another hypothesis is that HIV-1 triggers alterations in SE composition and signalling. These phenotypic changes may promote a cascade of functional effects, such as increased cytokine expression and target cell activation, with decreased DC maturation, which collectively compromise the induction of a protective immune response and promote HIV-1 infection in the female genital mucosa. SE isolated from the semen of healthy donors are generally immunosuppressive, inhibiting the activation of macrophages, NK cells and T cells (Kelly & Critchley, 1997;Kelly et al., 1991;Skibinski, Kelly, Harkiss, & James, 1992;Tarazona et al., 2011;Vojtech et al., 2014). SE may therefore work in tandem with other components of semen, such as prostaglandins, to render the female reproductive tract less responsive to the presence of sperm antigens. This immune response favours conditions for fertilisation but could impact susceptibility to viral infection.
Exosomes from seronegative human semen have been shown to inhibit HIV-1 cellular entry and transmission in vitro. In TZM-bl cells, HIV-1 inhibition was more pronounced when SE were first preincubated with HIV-1 at 37°C, before exposure to the target cells (Madison et al., 2014), showing a direct effect of SE on the virus.

Madison et al. showed that human vaginal epithelial cells V248 inter-
nalised SE by endocytosis and direct fusion with the cellular plasma membrane (Madison, Jones, & Okeoma, 2015). Once internalised, SE are able to efficiently prevent transmission of HIV-1 infection from vaginal epithelial cells to CD4 + CCR5 + monocytic cells and CD4 + CXCR4 + T lymphocytes, as well as peripheral blood mononuclear cells . In vivo, SE can block intravaginal replication of murine AIDS (mAIDS) virus and limit the disease progression in mice . infections, they have no effect on HSV types 1 and 2 replication (Madison et al., 2014), suggesting that the antiviral activity of SE is restricted to HIV-1 or more generally to retroviruses. However, HSV, perhaps similarly to HIV-1, seems to hijack exosomes in different compartments modifying their composition and utilising them to disseminate to and infect other cells (Heikkila, Ryodi, & Hukkanen, 2016;Kalamvoki & Deschamps, 2016;Sadeghipour & Mathias, 2017). The properties of SE isolated from HIV-1-infected patients have not been fully explored, and this represents a gap in the clear understanding of the role of exosomes in HIV-1 transmission.

| Vaginal fluid exosomes
Similar to human semen, human vaginal fluid contains exosomes which have been found to reduce HIV-1 vector transmission by 60% in 293 T/17 cells (Smith & Daniel, 2016). Integrated viral DNA and total viral DNA were both decreased by 47% and 58.4%, respectively, compared to the controls, in the presence of vaginal fluid exosomes (VFE). However, the efficiency of HIV-1 vector entry into target cells was not significantly different in the presence or absence of these exosomes (Smith & Daniel, 2016

| Breast milk exosomes
Conflicting reports have been published on exosomes isolated from human breast milk from healthy donors. Indeed, pre-exposure to exosomes purified from human breast milk inhibits HIV-1 infection mediated by dendritic cells (DC) by binding to DC-SIGN, and blocking transport and transfer of virus to target CD4 + T cells (Naslund et al., 2014). This might suggest that exosomes from milk may be protective against HIV-1 transmission from mother to child through breastfeeding. Two more recent papers (Sims et al., 2017(Sims et al., , 2018, however, show that exosomes isolated from human breast milk can enhance HIV-1 entry when exosomes and HIV-1 are added at the same time to target cells (human CD4 + lymphoblastoid T and macrophage-like cell lines) and co-incubated for 3 days. These contradictory data may be the result of differences in exosome collection and purification or experimental design. This caveat also applies to many of the discrepant results reported on the functional role of exosomes.
The impact of SE, VFE, plasma, and breast milk exosomes on HIV-1 infection/transmission is summarised in Table 1.  .