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HIV persists through continued transmission Antiretroviral therapy suppresses, but does not eradicate, HIV infection. Low-level viraemia continues for life because of the persistence of treatment-resistant reservoirs of the virus. Various different types of reservoir are thought to exist. David Baltimore and colleagues use a combination of mathematical modelling and a cell culture model of HIV infection and drug treatment to propose that ongoing HIV replication can occur in the presence of drugs if the cells become infected through cell-to-cell transmission. They propose that cell-to-cell spread of virus could be a source of localized and intermittent ongoing replication, which may show little evolution, and which could contribute to replenishment of the virus reservoir and virus persistence. Latency and ongoing replication 1 have both been proposed to explain the drug-insensitive human immunodeficiency virus (HIV) reservoir maintained during antiretroviral therapy. Here we explore a novel mechanism for ongoing HIV replication in the face of antiretroviral drugs. We propose a model whereby multiple infections 2 , 3 per cell lead to reduced sensitivity to drugs without requiring drug-resistant mutations, and experimentally validate the model using multiple infections per cell by cell-free HIV in the presence of the drug tenofovir. We then examine the drug sensitivity of cell-to-cell spread of HIV 4 , 5 , 6 , 7 , a mode of HIV transmission that can lead to multiple infection events per target cell 8 , 9 , 10 . Infections originating from cell-free virus decrease strongly in the presence of antiretrovirals tenofovir and efavirenz whereas infections involving cell-to-cell spread are markedly less sensitive to the drugs. The reduction in sensitivity is sufficient to keep multiple rounds of infection from terminating in the presence of drugs. We examine replication from cell-to-cell spread in the presence of clinical drug concentrations using a stochastic infection model and find that replication is intermittent, without substantial accumulation of mutations. If cell-to-cell spread has the same properties in vivo , it may have adverse consequences for the immune system 11 , 12 , 13 , lead to therapy failure in individuals with risk factors 14 , and potentially contribute to viral persistence and hence be a barrier to curing HIV infection.

NK cells are an important subset of innate immune effectors with antiviral activity. However, NK cell development and immune responses in different tissues during acute and chronic HIV infection in vivo have been difficult to study due to the impaired development and function of NK cells in conventional humanized mouse models. In this issue of the JCI, Sangur et al. report on a transgenic MISTRG-6-15 mouse model with human IL-6 and IL-15 knocked into the previously constructed MISTRG mice. The predecessor model was deficient in Rag2 and γ chain (γc) with knock-in expression of human M-CSF, IL-3, GM-CSF, and TPO, and transgenic expression of human SIRPα. The researchers studied tissue-specific NK cell immune responses during HIV infection and clearly show that the endogenous human NK cells in the humanized mouse model suppressed HIV-1 replication in vivo. These findings provide insight into harnessing the innate immune response for clinical antiviral therapies.

HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir. Here, Kim et al. describe a new kick and kill strategy utilizing a single administration of a protein kinase C modulator and latency reversing agent in combination with injections of allogeneic peripheral blood natural killer cells diminishes the HIV reservoir in HIV-infected humanized mice.

The HIV reservoir consists of infected cells in which the HIV-1 genome persists as provirus despite effective antiretroviral therapy (ART). Studies exploring HIV cure therapies often measure intact proviral DNA levels, time to rebound after ART interruption, or ex vivo stimulation assays of latently infected cells. This study utilizes barcoded HIV to analyze the reservoir in humanized mice. Using bulk PCR and deep sequencing methodologies, we retrieve 890 viral RNA barcodes and 504 proviral barcodes linked to 15,305 integration sites at the single RNA or DNA molecule in vivo. We track viral genetic diversity throughout early infection, ART, and rebound. The proviral reservoir retains genetic diversity despite cellular clonal proliferation and viral seeding by rebounding virus. Non-proliferated cell clones are likely the result of elimination of proviruses associated with transcriptional activation and viremia. Elimination of proviruses associated with viremia is less prominent among proliferated cell clones. Proliferated, but not massively expanded, cell clones contribute to proviral expansion and viremia, suggesting they fuel viral persistence. This approach enables comprehensive assessment of viral levels, lineages, integration sites, clonal proliferation and proviral epigenetic patterns in vivo. These findings highlight complex reservoir dynamics and the role of proliferated cell clones in viral persistence. HIV reservoir dynamics are still incompletely understood. Here, the authors use barcoded HIV in a humanized mouse model to show that cell clones linked to viremia are likely eliminated, while proliferated cell clones contribute to viremia, are likely more resistant to elimination and might fuel viral persistence.

Dendritic cell (DC)-to-T cell transmission is an example of infection in trans, in which the cell transmitting the virus is itself uninfected. During this mode of DC-to-T cell transmission, uninfected DCs concentrate infectious virions, contact T cells and transmit these virions to target cells. Here, we investigated the efficiency of DC-to-T cell transmission on the number of cells infected and the sensitivity of this type of transmission to the antiretroviral drugs tenofovir (TFV) and raltegravir (RAL). We observed activated monocyte-derived and myeloid DCs amplified T cell infection, which resulted in drug insensitivity. This drug insensitivity was dependent on cell-to-cell contact and ratio of DCs to T cells in coculture. DC-mediated amplification of HIV-1 infection was efficient regardless of virus tropism or origin. The DC-to-T cell transmission of the T/F strain CH077.t/2627 was relatively insensitive to TFV compared to DC-free T cell infection. The input of virus modulated the drug sensitivity of DC-to-T cell infection, but not T cell infection by cell-free virus. At high viral inputs, DC-to-T cell transmission reduced the sensitivity of infection to TFV. Transmission of HIV by DCs in trans may have important implications for viral persistence in vivo in environments, where residual replication may persist in the face of antiretroviral therapy.

Background New COVID-19 treatments are desperately needed as case numbers continue to rise and emergent strains threaten vaccine efficacy. Cell therapy has revolutionized cancer treatment and holds much promise in combatting infectious disease, including COVID-19. Invariant natural killer T (iNKT) cells are a rare subset of T cells with potent antiviral and immunoregulatory functions and an excellent safety profile. Current iNKT cell strategies are hindered by the extremely low presence of iNKT cells, and we have developed a platform to overcome this critical limitation. Methods We produced allogeneic HSC-engineered iNKT ( Allo HSC-iNKT) cells through TCR engineering of human cord blood CD34 + hematopoietic stem cells (HSCs) and differentiation of these HSCs into iNKT cells in an Ex Vivo HSC-Derived iNKT Cell Culture. We then established in vitro SARS-CoV-2 infection assays to assess Allo HSC-iNKT cell antiviral and anti-hyperinflammation functions. Lastly, using in vitro and in vivo preclinical models, we evaluated Allo HSC-iNKT cell safety and immunogenicity for off-the-shelf application. Results We reliably generated Allo HSC-iNKT cells at high-yield and of high-purity; these resulting cells closely resembled endogenous human iNKT cells in phenotypes and functionalities. In cell culture, Allo HSC-iNKT cells directly killed SARS-CoV-2 infected cells and also selectively eliminated SARS-CoV-2 infection-stimulated inflammatory monocytes. In an in vitro mixed lymphocyte reaction (MLR) assay and an NSG mouse xenograft model, Allo HSC-iNKT cells were resistant to T cell-mediated alloreaction and did not cause GvHD. Conclusions Here, we report a method to robustly produce therapeutic levels of Allo HSC-iNKT cells. Preclinical studies showed that these Allo HSC-iNKT cells closely resembled endogenous human iNKT cells, could reduce SARS-CoV-2 virus infection load and mitigate virus infection-induced hyperinflammation, and meanwhile were free of GvHD-risk and resistant to T cell-mediated allorejection. These results support the development of Allo HSC-iNKT cells as a promising off-the-shelf cell product for treating COVID-19; such a cell product has the potential to target the new emerging SARS-CoV-2 variants as well as the future new emerging viruses.

Human immunodeficiency virus (HIV) has infected millions of people worldwide and continues to be a major global health problem. Scientists required a small animal model to study HIV pathogenesis and immune responses. To this end, humanized mice were created by transplanting human cells and/or tissues into immunodeficient mice to reconstitute a human immune system. Thus, humanized mice have become a critical animal model for HIV researchers, but with some limitations. Current conventional humanized mice are prone to death by graft versus host disease induced by the mouse signal regulatory protein α and CD47 signaling pathway. In addition, commonly used humanized mice generate low levels of human cytokines required for robust myeloid and natural killer cell development and function. Here, we describe recent advances in humanization procedures and transgenic and knock-in immunodeficient mice to address these limitations.

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60(o)C.

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide–major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide–MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy.Trogocytosis, the uptake of membrane proteins by an antigen-presenting cell from its cognate T cell, allows the identification of neoepitopes targeted by T cell receptors with high sensitivity.