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The introduction of more effective and selective mRNA delivery systems is required for the advancement of many emerging biomedical technologies including the development of prophylactic and therapeutic vaccines, immunotherapies for cancer and strategies for genome editing. While polymers and oligomers have served as promising mRNA delivery systems, their efficacy in hard-to-transfect cells such as primary T lymphocytes is often limited as is their cell and organ tropism. To address these problems, considerable attention has been placed on structural screening of various lipid and cation components of mRNA delivery systems. Here, we disclose a class of charge-altering releasable transporters (CARTs) that differ from previous CARTs based on their beta-amido carbonate backbone (bAC) and side chain spacing. These bAC-CARTs exhibit enhanced mRNA transfection in primary T lymphocytes in vitro and enhanced protein expression in vivo with highly selective spleen tropism, supporting their broader therapeutic use as effective polyanionic delivery systems. Polymers are promising for mRNA delivery, but can have limited efficacy in hard to transfect cells. Here, the authors report charge-altering releasable transporters for improved mRNA transfection in primary T-lymphocytes and enhanced and selective protein expression in vivo.

Dendritic cells can capture and transfer retroviruses in vitro across synaptic cell-cell contacts to uninfected cells, a process called trans-infection. Whether trans-infection contributes to retroviral spread in vivo remains unknown. Here, we visualize how retroviruses disseminate in secondary lymphoid tissues of living mice. We demonstrate that murine leukemia virus (MLV) and human immunodeficiency virus (HIV) are first captured by sinus-lining macrophages. CD169/Siglec-1, an I-type lectin that recognizes gangliosides, captures the virus. MLV-laden macrophages then form long-lived synaptic contacts to trans-infect B-1 cells. Infected B-1 cells subsequently migrate into the lymph node to spread the infection through virological synapses. Robust infection in lymph nodes and spleen requires CD169, suggesting that a combination of fluid-based movement followed by CD169-dependent trans-infection can contribute to viral spread.

Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous, and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer’s patches to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood-, and milk-borne retroviruses spanning three routes was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue-resident sentinel macrophages for establishing infection.

Retroviruses are infectious agents that cause cancer and immunodeficiency in many animal species. The early events during retroviral infection are critical to the establishment of viral infection in primary draining lymphoid tissue as well as the subsequent systemic spread of the viruses in the host. In our previous study, with murine leukemia virus (MLV), we illustrated the early events of retroviral infection in its natural host. After the entry into the host, lymph- and blood-borne MLV gets captured rapidly at primary draining lymph node by subcapsular sinus (SCS) macrophages and at spleen by metallophilic macrophages, respectively. The interaction of MLV particles with both populations of macrophages is mediated by the gangliosides incorporated into the virions and CD169/Siglec-1 expressed by the macrophages. MLV-laden CD169+ macrophages subsequently trans-infect B-1 cells in the lymph node, which facilitates the establishment of MLV infection. However, the role of CD169+ macrophages in systemic spread as well as in pathogenesis of murine retroviruses is not known. In addition, why MLV initially infects B-1 cells is unknown. In this thesis, studying a pathogenic splenomegaly-inducing murine retrovirus, the Friend virus complex (FVC), we have demonstrated that CD169+ macrophages play a protective role against retroviral pathogenesis. CD169+ macrophages in primary draining lymph node efficiently absorbed lymph-borne FVC and reduced the systemic spread of cell-free virus into blood and spleen. At the spleen, CD169+ macrophages captured blood-borne FVC and limited its spread to erythroblasts in red pulp, where FVC manifests its pathogenesis. Retrovirus-laden CD169+ macrophages interacted with conventional dendritic cell 1 (cDC1s) and promoted the cross-priming of cytotoxic CD8+ T cells, which is critical for the clearance of FVC-infected cells. Functional blockade of CD169 in primary draining lymph node accelerated the death in a susceptible mouse strain after FVC challenge. Collectively, we have illustrated a protective role of CD169 against pathogenic FVC infection both in limiting viral spread into erythroblast niche and in efficiently mounting cell-mediated immune response. The second part of my thesis is devoted to understanding why B-1 cells in draining lymph nodes are highly susceptible to FrMLV infection following s.c. infections. We used a mouse strain that is deficient in B-1 cell population (bumble, IkBNS-/-) to study the function of B-1 cells. FrMLV infection in bumble mice was significantly reduced compared to wild-type mice. An adoptive transfer of wild-type B-1 cells but not Tlr7-/- or Ifnar1-/- B-1 cells into bumble mice rescued FrMLV infection, suggesting that TLR7-mediated sensing as well as intrinsic type I IFN signaling are critical to the susceptibility of B-1 cells to FrMLV infection. The reduced FrMLV infection in bumble mice was mainly due to a compromised spread of FrMLV into B-2 cell population. Infected B-1 cell migrated to medullary cord where they were found in proximity to infected B-2 cells. These data revealed that retroviruses can exploit the innate-immune sensing in target cells to facilitate its infection. It also suggests that retroviruses can utilize an intrinsically susceptible cell type for the propagation of infection in lymphoid tissue. Together my work illustrates how CD169+ macrophages cooperate with either cDC1s or B-1 cells to promote protective CD8+ T cell responses or viral dissemination.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Natural killer (NK) cells respond rapidly in early HIV-1 infection. HIV-1 prevention and control strategies harnessing NK cells could be enabled by mechanistic understanding of how NK cells recognize HIV-infected T cells. Here, we profiled the phenotype of human primary NK cells responsive to autologous HIV-1-infected CD4 T cells in vitro. We characterized the patterns of NK cell ligand expression on CD4 T cells at baseline and after infection with a panel of transmitted/founder HIV-1 strains to identify key receptor-ligand pairings. CRISPR editing of CD4 T cells to knockout the NKp30 ligand B7-H6, or the NKG2D ligands MICB or ULBP2 reduced NK cell responses to HIV-infected cells in some donors. In contrast, overexpression of NKp30 or NKG2D in NK cells enhanced their targeting of HIV-infected cells. Collectively, we identified receptor-ligand pairs including NKp30:B7-H6 and NKG2D:MICB/ULBP2 that contribute to NK cell recognition of HIV-infected cells.

Targeting the HIV-infected reservoir in lymphoid tissues (LT) will be critical to developing a cure for people living with HIV (PLWH). LT explants used to study HIV infection enable the evaluation of human-specific disease progression and treatment response; however, their short lifespan makes it challenging to assess long-term treatment interventions. We therefore established an immune organoid model of HIV infection using human tonsil or spleen cells, demonstrating productive HIV infection and viral integration into CD4+ T cells. Treatment with a protease inhibitor fully suppressed ongoing viral production, with virologic rebound occurring within days of treatment interruption. The transfer of healthy allogeneic NK cells to target the reservoir upon treatment interruption reduced the number of infected cells, intact viral genomes, and production of de novo infectious viral particles. Adoption of this immune organoid platform will accelerate the evaluation of cure-based strategies to eliminate the HIV reservoir in tissues for PLWH.Competing Interest StatementJ.A.Z is a co-founder of CDR3 Therapeutics. C.A.B is a member of the Scientific Advisory Boards of ImmuneBridge and DeepCell, Inc. on topics unrelated to this project.Funder Information DeclaredNIH, R01 AI161803, R01 AI183381, T32AI007290, T32 AI007502NCRR, 1S10OD032300-01

Abstract Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of the retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer’s Patch to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood- and milk-borne retroviruses spanning three routes, was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue resident sentinel macrophages for establishing infection. Competing Interest Statement The authors have declared no competing interest.

ABSTRACT Our understanding of protective vs. pathologic immune responses to SARS-CoV-2, the virus that causes Coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses reveal widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, with the most profound disturbances including a prominent neutrophil hyperactivation signature and monocytes with anti-inflammatory features. We further demonstrate that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention. One Sentence Summary Single-cell profiling demonstrates multifarious dysregulation of innate immune phenotype associated with COVID-19 severity. Competing Interest Statement C.A.B. is a member of the Scientific Advisory Board of Catamaran Bio. W.J.G. is a consultant for 10x Genomics and Guardant Health, Co-founder of Protillion Biosciences, and is named on patents describing ATAC-seq.