Introduction. In 2022, 1.5 million people acquired the Human Immunodeficiency Virus (HIV), and an estimated 37.7 million individuals lived with HIV-1 (PLWH) worldwide. While combination antiretroviral therapy (cART) effectively suppresses viral replication in PLWH, it does not entirely silence viral transcription. Our research and others have identified the presence of HIV-1 products, including non-coding viral RNA and viral proteins, enclosed within extracellular vesicles (EVs), measuring approximately 50 nm to 150 nm in size. These EVs are not infectious and can be isolated from cell culture supernatants of HIV-1 chronically infected cell lines and patient biofluids. Recently, we expanded a previously accepted and extensively used sequential differential ultracentrifugation (DUC) method by employing higher g-force with longer spin times to recover smaller EVPs (<100 nm) and have found presence of virus in both large and very small EVPs.

Methods. We isolated EVPs larger and smaller than the current accepted size for HIV-1 which carry viral protein and nucleic acid cargoes. These included infectious large (2K), medium (normal HIV-1 size; 100K), and very small (167K/18hrs.) viral particles that we collectively refer to as EVPs. Furthermore, we designed a modified virus recovery assay (VRA) from a previously described method, which indicates that both the collected EVPs were infectious, including the novel small-sized EVPs under 100 nm. The standard assays for EV characterizations were used to validate each prep.  Also, the data was further validated using filtrations, and other methods of EV purification. Viral and EV markers were used to quantify each category.

Results. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation. All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EVPs with an average size well below 100 nm. Synchronized and released cultures contained large infectious EVPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EVPs positive for CD63, HSP-70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope glycoprotein colocalized on the same Frac-E particles. Surprisingly, Frac-E EVPs were infectious, and infectivity was significantly reduced by immunodepletion with anti-CD63, indicating the presence of this protein on the surface of infectious EVPs in Frac-E.

Discussion. Here, we show the existence of unique, infectious particles (both very large and very small) than the currently accepted size for HIV-1. The methodology can potentially be employed for various viruses originating from mammalian cells and other infectious agents in situations where EVPs may significantly impact disease progression by transmitting highly replicating virulent nucleic acids.

The mission of the talk is to identify immune damaging participants involved in antiviral immunoinflammatory lesions. We argue these could be targeted and their activity changed selectively by maneuvers that at the same time may not diminish the impact of components that help resolve lesions. Ideally, we need to identify therapeutic approaches that can reverse ongoing lesions that lack unwanted side effects and are affordable to use. By understanding the delicate balance between immune responses that cause tissue damage and those that aid in resolution, novel strategies can be developed to target detrimental immune components while preserving the beneficial ones. Some strategies involve rebalancing the participation of immune components through various approaches, such as removing or blocking proinflammatory T cell products, expanding regulatory cells, restoring lost protective cell function, using monoclonal antibodies to counteract inhibitory molecules, and exploiting metabolic differences between inflammatory and immuno-protective responses. These strategies can help reverse ongoing viral infections. We explain various approaches, from model studies and some clinical evidence that achieve innate and adaptive immune rebalancing offering insights into potential application to controlling chronic viral induced lesions.