Exosomes are important vehicles of intercellular communication that shape host responses to physiologic, tumorigenic, and pathogenic conditions. The composition and function of exosomes are dynamic and depends on the state and condition of the cellular source. In prior work, we found that semen exosomes (SE) from healthy donors who do not use illicit drugs potently inhibit HIV-1. Following semen donation, specimens are either used immediately or frozen for use at a later time. It has been shown that short-term freezing of semen has no effect on SE-mediated HIV-1 inhibition. However, the effect of illicit drugs and prolonged freezing on SE bioactivity is unknown. Here, we show preservation of SE physical properties, (morphology, concentration, intensity/size) irrespective of illicit drug use or duration of semen freezing. Interestingly, illicit drugs and prolonged freezing decreased the levels of SE-bound CD63/CD9 and acetylcholinesterase activity respectively. Furthermore, we show differential effects of illicit drug use and prolonged freezing on SE-mediated HIV-1 inhibition. Our results highlight the importance of the source of SE and condition of semen storage on SE content and function. In-depth evaluation of donor drug-use and duration of semen storage on SE cargo and bioactivity will advance our understanding of SE composition and function.

HIV is most commonly transmitted sexually, and semen is the primary vector. Despite progress in studies of HIV pathogenesis and the success of combination antiretroviral therapy in controlling viral replication, current therapy cannot completely control sexual transmission. Thus, there is a need to identify effective methods of controlling HIV replication and transmission. Recently, it was shown that human semen contains exosomes that protect against HIV infection in vitro . In this study, we identified a mechanism by which semen exosomes inhibited HIV-1 RNA expression. We found that semen exosomes inhibit recruitment of transcription factors NF-κB and Sp1, as well as RNA Pol II, to the promoter region in the 5′ long terminal repeat (LTR) of HIV-1. The HIV-1 early protein transcriptional activator (Tat) was a target of semen exosomes, and semen exosomes inhibited the binding and recruitment of Tat to the HIV-1 LTR.

A total of 340 million sexually transmitted infections (STIs) are acquired each year. Antimicrobial agents that target multiple infectious pathogens are ideal candidates to reduce the number of newly acquired STIs. The antimicrobial and immunoregulatory properties of GML make it an excellent candidate to fit this critical need. Previous studies established the safety profile and antibacterial activity of GML against both Gram-positive and Gram-negative bacteria. GML protected against high-dose SIV infection and reduced inflammation, which can exacerbate disease, during infection. We found that GML inhibits HIV-1 and other human-pathogenic viruses (yellow fever virus, mumps virus, and Zika virus), broadening its antimicrobial range. Because GML targets diverse infectious pathogens, GML may be an effective agent against the broad range of sexually transmitted pathogens. Further, our data show that reutericyclin, a GML analog expressed by some lactobacillus species, also inhibits HIV-1 replication and thus may contribute to the protective effect of Lactobacillus in HIV-1 transmission.

Blood and semen are important body-fluids that carry exosomes for bioinformation transmission. Therefore, characterization of their proteomes is necessary for understanding body-fluid-specific physiologic and pathophysiologic functions. Using systematic multifactorial proteomic profiling, we characterized the proteomes of exosomes and exosome-free fractions from autologous blood and semen from three HIV-uninfected and three HIV-infected participants (total of 24 samples). We identified exosome-based protein signatures specific to blood and semen along with HIV-induced tissue-dependent proteomic perturbations. We validated our findings with samples from 16 additional donors and showed that unlike blood exosomes (BE), semen exosomes (SE) are enriched in clusterin. SE but not BE promote Protein·Nucleic acid binding and increase cell adhesion irrespective of HIV infection. This is the first comparative study of the proteome of autologous BE and SE. The proteins identified may be developed as biomarkers applicable to different fields of medicine, including reproduction and infectious diseases. Graphical Abstract Highlights SMPP enabled analysis of autologous blood and semen proteomes of HIV- and HIV+ participants. Common and distinct exProteins from autologous semen and blood are associated with exosomes. HIV-induced perturbations were less frequent in semen compared to blood. Semen but not blood exosomes is enriched in nucleic acids and protein binding factors.

The terms extracellular vesicles, microvesicles, oncosomes, or exosomes are often used interchangeably as descriptors of particles that are released from cells and comprise a lipid membrane that encapsulates nucleic acids and proteins. Although these entities are defined based on a specific size range and/or mechanism of release, the terminology is often ambiguous. Nevertheless, these vesicles are increasingly recognized as important modulators of intercellular communication. The generic characterization of extracellular vesicles could also be used as a descriptor of enveloped viruses, highlighting the fact that extracellular vesicles and enveloped viruses are similar in both composition and function. Their high degree of similarity makes differentiating between vesicles and enveloped viruses in biological specimens particularly difficult. Because viral particles and extracellular vesicles are produced simultaneously in infected cells, it is necessary to separate these populations to understand their independent functions. We summarize current understanding of the similarities and differences of extracellular vesicles, which henceforth we will refer to as exosomes, and the enveloped retrovirus, HIV-1. Here, we focus on the presence of these particles in semen, as these are of particular importance during HIV-1 sexual transmission. While there is overlap in the terminology and physical qualities between HIV-1 virions and exosomes, these two types of intercellular vehicles may differ depending on the bio-fluid source. Recent data have demonstrated that exosomes from human semen serve as regulators of HIV-1 infection that may contribute to the remarkably low risk of infection per sexual exposure.

Although extracellular vesicle (EV) surface electrostatic properties (measured as zeta potential, ζ-potential) have been reported by many investigators, the biophysical implications of charge and EV origin remains uncertain. Here, we compared the ζ-potential of human blood EVs (BEVs) and semen EVs (SEVs) from 26 donors that were HIV-infected (HIV+, n = 13) or HIV uninfected (HIV-, n = 13). We found that, compared to BEVs that bear neutral surface charge, SEVs were significantly more negatively charged, even when BEVs and SEVs were from the same individual. Comparison of BEVs and SEVs from HIV- and HIV+ groups revealed subtle HIV-induced alteration in the ζ-potential of EVs, with the effect being more significant in SEVs (∆ζ-potential = −8.82 mV, p-value = 0.0062) than BEVs (∆ζ-potential = −1.4 mV, p-value = 0.0462). These observations were validated by differences in the isoelectric point (IEP) of EVs, which was in the order of HIV + SEV ≤ HIV-SEV ≪ HIV + BEV ≤ HIV-BEV. Functionally, the rate and efficiency of SEV internalization by the human cervical epithelial cell line, primary peripheral blood lymphocytes, and primary blood-derived monocytes were significantly higher than those of BEVs. Mechanistically, removal of sialic acids from the surface of EVs using neuraminidase treatment significantly decreased SEV’s surface charge, concomitant with a substantial reduction in SEV’s internalization. The neuraminidase effect was independent of HIV infection and insignificant for BEVs. Finally, these results were corroborated by enrichment of glycoproteins in SEVs versus BEVs. Taken together, these findings uncover fundamental tissue-specific differences in surface electrostatic properties of EVs and highlight the critical role of surface charge in EV/target cell interactions.